RU2816963C2 - Modified closed circular dna (ccdna) containing symmetric modified inverted end repeated sequences - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a group of inventions comprising a non-viral capsid-free DNA vector with covalently closed ends (ccDNA vector) for delivering nucleic acid, a ccDNA expression construct which codes ccDNA, a host cell for producing the ccDNA expression construct, method of producing a ccDNA vector, using a composition comprising a ccDNA vector for treating, preventing, relieving, monitoring or diagnosing a disease or disorder in a subject, a method of delivering a therapeutic protein to a subject, a kit for delivering a nucleic acid and a kit for producing a ccDNA vector. In one embodiment, the ccDNA vector comprises a nucleic acid sequence flanked by two symmetric ITRs which are not wild-type ITRs.

EFFECT: invention extends the range of means for delivering nucleic acid.

56 cl, 29 dwg, 7 tbl, 9 ex

Description

RELATED APPLICATIONS

[0001] This application claims benefit from U.S. Provisional Patent Application No. 62/757872, filed November 9, 2018, and U.S. Provisional Patent Application No. 62/757892, filed November 9, 2018, the contents of each of which are incorporated in their entirety in this document by reference.

TECHNICAL FIELD

[0002] The present invention relates to the field of gene therapy, including the delivery of closed-end DNA-based regulatory switches to a target cell, tissue, organ or organism.

LIST OF SEQUENCES

[0003] The sequence listing for this application was submitted electronically via EFS-Web as a sequence listing in ASCII format with a file name of "05320 Sequence_Listing.txt", a creation date of November 8, 2019, and a size of 204 KB (209,626 bytes) . This application includes a sequence listing that was filed electronically, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE ART

[0004] The goal of gene therapy is to improve clinical outcomes in patients suffering from either genetic mutations or acquired diseases caused by aberrations in the gene expression profile. Gene therapy includes the treatment or prevention of medical conditions caused by defective genes or abnormal regulation or expression, such as underexpression or overexpression, that can lead to a disorder, disease, cancer, or the like. For example, treating, preventing, or ameliorating a disease or disorder caused by a defective gene may be accomplished by delivering corrective genetic material to a patient, resulting in therapeutic expression of said genetic material in said patient. Gene therapy is based on the provision of a transcription cassette with an active gene product (sometimes called a transgene), for example, which can lead to a positive gain-of-function effect, a negative loss-of-function effect, or another outcome, such as, for example, an oncolytic effect. Gene therapy can also be used to treat a disease or malignancy caused by other factors. Treatment of monogenic disorders in humans can be achieved by delivering a normal gene to target cells and expressing it in the target cells. Delivery and expression of the corrective gene into the target cells (target cells) of the patient can be carried out using numerous methods, including the use of genetically engineered viruses and viral vectors for gene delivery. Among the many viral-derived vectors available (eg, recombinant retrovirus, recombinant lentivirus, recombinant adenovirus, etc.), recombinant adeno-associated virus (rAAV) is gaining popularity as a versatile vector for gene therapy. Moreover, as technology improves and highly efficient gene transfer and expression is achieved, the ability to regulate such expression at both the temporal and spatial levels becomes increasingly important.

[0005] Adeno-associated viruses (AAVs) belong to the Parvoviridae family; more specifically, they constitute the genus Dependoparvovirus. The AAV genome consists of a linear, single-stranded DNA molecule that contains approximately 4.7 kilobases (kb) and consists of two major open reading frames (ORFs) encoding the nonstructural protein Rep (replication) and the structural protein Cap (capsid). A second ORF has been identified in the cap gene that encodes assembly-activating protein (AAP). The DNA flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences of approximately 145 nucleotides in length, with discontinuous palindromic sequences that can fold into energetically stable hairpin structures that function as primers for DNA replication. Typically, the wild-type sequences are identical but inverted relative to each other. In addition to their role in DNA replication, these ITR sequences have been shown to be involved in the integration of viral DNA into the cellular genome, rescue from the host genome or plasmid, and encapsidation of the viral nucleic acid in mature virions (Muzyczka, (1992) Curr. Top Micro Immunol 158:97–129).

[0006] AAV-derived vectors (i.e., recombinant AAV (rAVV) or AAV vectors) are promising for the delivery of genetic material because (i) they are capable of infecting (transducing) a wide range of nondividing and dividing cell types, including myocytes and neurons; (ii) they lack viral structural genes, which reduces host cell responses to viral infection, such as interferon-mediated responses; (iii) wild-type viruses are considered non-pathogenic to humans; (iv) unlike wild-type AAV, which is capable of integration into the host cell genome, replication-defective AAV vectors lack the rep gene and usually persist as episomes, which accordingly limits the risk of insertional mutagenesis or genotoxicity; and (v) compared to other vector systems, AAV vectors are generally considered to be weaker immunogens, and accordingly they do not trigger a significant immune response (see (ii)), thus allowing vector DNA persistence and potentially long-term expression therapeutic transgenes. AAV vectors can also be prepared and formulated at high titers and delivered by intra-arterial, intravenous, or intraperitoneal injection, allowing vector distribution and gene transfer to relevant muscle regions through a single injection in rodents (Goyenvalle et al., 2004; Fougerousse et al., 2007; Koppanati et al., 2010; Wang et al., 2009) and dogs. In a clinical trial for the treatment of spinal muscular dystrophy type 1, AAV vectors were delivered systemically to target the brain, resulting in apparent clinical improvements.

[0007] However, the use of AAV particles as a vector for gene delivery has a number of serious disadvantages. One significant disadvantage associated with rAAV is the limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010). As a result, the use of AAV vectors has been limited to protein coding capacity of less than 150,000 Da. The second disadvantage is that, due to the prevalence of wild-type AAV infection in the population, candidates for rAAV gene therapy must be screened for the presence of neutralizing antibodies that eliminate the vector from the patient. A third disadvantage relates to the immunogenicity of the capsid, which precludes re-administration in patients who have not been withdrawn from initial treatment. The patient's immune system may respond to the vector, which essentially acts as a "booster" shot, stimulating the immune system to generate high titers of anti-AAV antibodies that preclude future treatment. Some recent reports have raised concerns regarding immunogenicity in situations involving high doses. Another notable disadvantage is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA before expression of the heterologous gene. Although attempts have been made to circumvent this problem by constructing double-stranded DNA vectors, this strategy further limits the size of the transgene expression cassette that can be integrated into the AAV vector (McCarty, 2008; Varenika et al., 2009; Foust et al., 2009) .

[0008] In addition, standard AAV capsid virions are produced by introducing a plasmid or plasmids containing the AAV genome, rep genes, and cap genes (Grimm et al., 1998). When these helper plasmids are introduced into the trans position, the AAV genome is “rescued” (i.e., released followed by amplification) from the host genome and further enclosed in the capsid (viral capsids) to obtain biologically active AAV vectors. However, such capsid-enclosed viral AAV vectors have been found to be ineffective at transducing certain cell and tissue types. These capsids also induce an immune response.

[0009] Accordingly, the use of adeno-associated virus vectors (AAV vectors) for gene therapy is limited due to pre-existing immunity in certain patients, limited to single administration to patients who were not previously immune (due to the patient's established immune response), limited range of transgenic genetic material suitable for delivery in AAV vectors due to the minimal viral packaging capacity (approximately 4.5 kb) of the associated AAV capsid, as well as slow AAV-mediated gene expression. The application of clinical gene therapy using rAAV is further complicated by variability among patients, which cannot be predicted based on dose response in syngeneic mouse models or other types of models.

[0010] Recombinant capsidless AAV vectors can be produced as an isolated linear nucleic acid molecule containing transgene expression regions and promoter regions flanked by two wild-type AAV inverted terminal repeat (ITR) sequences, including Rep binding sites and terminal resolution sites. These recombinant AAV vectors do not contain AAV capsid protein coding sequences and may be single-stranded, double-stranded, or duplex, where one or both ends are covalently linked through two palindromic wild-type ITR sequences (eg, WO2012/123430, US Pat. No. 9,598,703). They avoid many of the problems of AAV-mediated gene therapy due to significantly higher transgene capacity, rapid onset of transgene expression, and lack of the features of AAV-based vectors that typically lead to the development of immunity and rapid clearance of these vectors. However, persistent transgene expression may be undesirable in some cases.

[0011] There is still a significant unmet need for controlled recombinant DNA vectors with improved production and/or expression properties.

BRIEF DESCRIPTION OF THE INVENTION

[0012] The present invention provides generally non-viral capsidless DNA vectors with covalently closed ends (referred to herein as a “closed end DNA vector” or “ccDNA vector”). The cccDNA vectors described herein are capsidless linear duplex DNA molecules formed by a continuous strand of complementary DNA with covalently closed ends (linear, contiguous, and non-capsidized structure) that contain a 5'-terminal inverted repeat (ITR) sequence and a 3' ITR, identical, but reverse complementary to each other, i.e. said sequences are symmetrical or substantially symmetrical. In some embodiments, the cccDNA vector comprises at least one heterologous nucleotide sequence operably located between two flanking inverted terminal repeat (ITR) sequences, all or a portion of the heterologous nucleotide sequence being under the control of at least one regulatory switch. In other embodiments, the technology described herein provides a cccDNA vector containing two modified AAV inverted terminal repeat (ITR) sequences, wherein the modified ITRs are symmetrical to each other and flank the transgene to be expressed. The cccDNA vectors as described herein can be produced in eukaryotic cells and are therefore free of prokaryotic DNA modifications and bacterial endotoxin contamination in insect cells.

[0013] In one aspect, non-viral capsidless DNA vectors with covalently closed ends are preferably linear duplex molecules and can be derived from a vector polynucleotide that encodes a heterologous nucleic acid operably located between two symmetrical modified inverted terminal repeat sequences (e.g., mod- ITR) (for example, AAV ITR). Thus, both ITRs have the same sequence, but are reverse complementary (inverted) to each other. In alternative embodiments, a pair of modified ITRs are substantially symmetrical as defined herein, i.e. said pair of modified ITRs may have different sequences but a similar or identical symmetrical three-dimensional shape. For example, one modified ITR may belong to one serotype and another modified ITR may belong to a different serotype, but they contain the same mutation (eg, insertion, deletion, or nucleotide substitution) in the same region. That is, for illustrative purposes only, the 5' mod-ITR may be taken from AAV2 and include a deletion in the C region, and the 3' mod-ITR may be taken from AAV5 and contain a corresponding deletion in the C' region; provided that said 5' mod-ITR and 3' mod-ITR have the same or symmetrical three-dimensional spatial organization, they are provided as a modified ITR pair for use according to the present invention.

[0014] In some embodiments, the pair of modified ITRs comprises at least one deletion, insertion or substitution, or any combination thereof, relative to a wild-type ITR sequence from an AAV of the same serotype. Additions, deletions, or substitutions in a symmetric ITR are the same but are inversely complementary to each other. For example, the insertion of 3 nucleotides into the C-region of the 5'-ITR will correspond to the insertion of three reverse complementary nucleotides into the corresponding region of the C-region of the 3'-ITR. For illustration purposes only, if the addition in the 5'-ITR is AACG, then the addition in the 3'-ITR is CGTT at the corresponding site. For example, if the sense strand of the 5' ITR is ATCGATCG, adding AACG between G and A results in the sequence ATCGAACGATCG. The corresponding sense strand of the 3' ITR is CGATCGAT (reverse complement ATCGATCG) with the addition of CGTT (i.e. reverse complement AACG) between T and C, resulting in the sequence CGATCG7TCGAT (reverse complement ATCGA4CGATCG), i.e. mirror image of the 5' ITR modification. In some embodiments, the modified ITRs comprise a terminal resolution site and a replication protein binding site (RPS) (sometimes referred to as a replication protein binding site), such as a Rep binding site. In some embodiments, the modified ITRs do not contain a terminal resolution site and/or a replication protein binding site (RPS), such as a Rep binding site.

[0015] In some embodiments, the cccDNA vector comprises: (1) an expression cassette containing a cis-regulatory element, a promoter, and at least one transgene; or (2) a promoter operably linked to at least one transgene, and (3) two self-complementary symmetrical sequences, for example symmetrical modified ITRs, flanking said expression cassette, wherein said cDNA vector is not associated with a capsid protein.

[0016] In one embodiment, said cccDNA vector contains additional components for regulating transgene expression, such as cis-regulatory elements and/or regulatory switches, which are described in more detail herein under the heading "Regulatory Switches", to control and regulation of transgene expression, for example, a regulatory switch, for example, a “safety switch”, allowing for controlled death of the cell containing the cccDNA vector. Said cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a miR-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, the ITR may act as a promoter for the transgene.

[0017] In some embodiments, the two self-complementary sequences may be modified ITR sequences from any known parvovirus, such as a dependovirus such as AAV (eg, AAV1-AAV12). Any AAV serotype may be used, including, but not limited to, a modified AAV2 ITR sequence that retains the Rep binding site (RBS), such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), and the terminal resolution (trs), along with a variable palindromic sequence that ensures the formation of a secondary hairpin structure. In some embodiments, the modified ITR is a synthetic ITR sequence that may retain a functional Rep binding site (RBS), such as 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), and a terminal resolution site (TRS), along with a variable palindromic sequence that ensures the formation of a secondary hairpin structure. In some examples, modified ITR sequences retain the RBS sequence, trs, and the structure and position of the Rep binding element forming the terminal loop region of one of the hairpin ITR secondary structures from the corresponding wild-type ITR sequence, e.g., AAV1, 2, 3, 4, 5 , 6, 7, 8, 9, and 10, 11 and 12.

[0018] Examples of modified ITR sequences for use in cccDNA vectors containing symmetric modified ITRs are shown in Table 4 herein, which shows ITR pairs (modified 5' ITR and symmetric modified 3' ITR). Examples of modified ITR sequences for use in cccDNA vectors are selected from any of the following modified ITR pairs: SEQ ID NO: 484 (ITR-33, left) and SEQ ID NO: 469 (ITR-18, right); SEQ ID NO: 485 (ITR-34, left) and SEQ ID NO: 95 (ITR-51, right); SEQ ID NO: 486 (ITR-35, left) and SEQ ID NO: 470 (ITR-19, right); SEQ ID NO: 487 (ITR-36, left) and SEQ ID NO: 471 (ITR-20, right); SEQ ID NO: 488 (ITR-37, left) and SEQ ID NO: 472 (ITR-21, right); SEQ ID NO: 489 (ITR-38, left) and SEQ ID NO: 473 (ITR-22, right); SEQ ID NO: 490 (ITR-39, left) and SEQ ID NO: 474 (ITR-23, right); SEQ ID NO: 491 (ITR-40, left) and SEQ ID NO: 475 (ITR-24, right); SEQ ID NO: 492 (ITR-41, left) and SEQ ID NO: 476 (ITR-25, right); SEQ ID NO: 493 (ITR-42, left) and SEQ ID NO: 477 (ITR-26, right); SEQ ID NO: 494 (ITR-43, left) and SEQ ID NO: 478 (ITR-27, right); SEQ ID NO: 495 (ITR-44, left) and SEQ ID NO: 479 (ITR-28, right); SEQ ID NO: 496 (ITR-45, left) and SEQ ID NO: 480 (ITR-29, right); SEQ ID NO: 497 (ITR-46, left) and SEQ ID NO: 481 (ITR-30, right); SEQ ID NO: 498 (ITR-47, left) and SEQ ID NO: 482 (ITR-31, right); SEQ ID NO: 499 (ITR-48, left) and SEQ ID NO: 483 (ITR-32, right).

[0019] In some embodiments, examples of modified ITR sequences for use in cccDNA vectors comprise partial sequences of an ITR selected from the following sequence pairs: SEQ ID NO: 101 and SEQ ID NO: 102; SEQ ID NO: 103 and SEQ ID NO: 96; SEQ ID NO: 105 and SEQ ID NO: 106; SEQ ID NO: 545 and SEQ ID NO: 116; SEQ ID NO: 111 and SEQ ID NO: 112; SEQ ID NO: 117 and SEQ ID NO: 118; SEQ ID NO: 119 and SEQ ID NO: 120; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 107 and SEQ ID NO: 108; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO: 125 and SEQ ID NO: 126; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 129 and SEQ ID NO: 130; SEQ ID NO: 131 and SEQ ID NO: 132; SEQ ID NO: 133 and SEQ ID NO: 134; SEQ ID NO: 547 and SEQ ID NO: 546, also shown in FIG. 6V-21V.

[0020] In some embodiments, the cccDNA vector may contain an ITR with a modification in each ITR corresponding to any of the modifications in the ITR sequences or partial ITR sequences listed in Tables 2, 3, 4, 5, 6, 7, 8, 9, or 10A-10B in PCT/US 18/49996, filed September 7, 2018, wherein the ITR flanking sequence is symmetrical (e.g., reverse complementary) thereto or substantially symmetrical as defined herein, i.e., the ITRs have a symmetrical three-dimensional spatial organization.

[0021] In a non-limiting exemplary example, the present invention provides a closed-end DNA vector containing a promoter operably linked to a transgene, with or without a regulatory switch, wherein said cccDNA is devoid of capsid proteins and: (a) is produced from cccDNA -plasmid (e.g., see Fig. 1A-B) that encodes symmetrical ITRs, each modified ITR containing the same number of base pairs in intramolecular duplexes in a secondary hairpin configuration (preferably excluding division of any terminal AAA or TTT loop in said configuration relative to the reference sequences indicated), and (b) identified as cccDNA using the cccDNA identification assay using agarose gel electrophoresis on a native gel and under denaturing conditions in Example 1.

[0022] In one embodiment, the flanking modified ITRs are substantially symmetrical to each other. In this embodiment, the modification is identical—the addition, substitution, or deletion is the same, but the ITRs are not identical reverse complements. In this embodiment, said pair of modified ITRs are substantially symmetrical, having a symmetrical three-dimensional spatial organization, but do not contain identical reverse complementary nucleotide sequences. That is, in some embodiments, the ITRs of a mod-ITR pair may contain different reverse complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization—that is, both ITRs contain mutations that result in the same overall three-dimensional organization. forms. For example, one ITR (eg, 5' ITR) in a mod-ITR pair may be from one serotype and the other ITR (eg, 3' ITR) from a different serotype, but both may contain the same corresponding mutation (eg, if 5 ' ITR contains a deletion in region C, a cognate modified 3' ITR from another serotype contains a deletion at the corresponding position in region C'), such that the pair of modified ITRs has the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR in a pair of modified ITRs may be from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as in a combination AAV2 and AAV6, with a modification in one ITR reflected at a similar position in a cognate ITR from another serotype. In one embodiment, a pair of substantially symmetrical modified ITRs is referred to as a modified ITR (mod-ITR) pair, provided that the nucleotide sequence differences between the ITRs do not affect the properties or overall shape and they have substantially the same shape in three-dimensional space. For example, a mod-ITR that is at least 95%, 96%, 97%, 98%, or 99% sequence identical to the canonical mod-ITR as assessed using standard methods well known in the art, such as BLAST ( Basic Local Alignment Search Tool), BLASTN with default parameters, and also have a symmetrical 3D spatial organization so that their 3D structure has the same shape in geometric space. An essentially symmetric mod-ITR pair contains identical loops A, C-C' and B-B' in three-dimensional space, for example, if the modified ITR in an essentially symmetric mod-ITR pair contains a deletion in the C-C' arm, then the cognate mod -ITR contains a corresponding deletion in the C-C' loop and also has a similar three-dimensional structure of the remaining A and B-B' loops with the same shape in geometric space as the cognate mod-ITR.

[0023] In some embodiments, the ITR structural element can be any structural element that is involved in the functional interaction of the ITR with a large Rep protein (eg, Rep 78 or Rep 68). In some embodiments, the structural element provides selectivity for the interaction of the ITR with the large Rep protein, i.e., at least partially determines which Rep protein functionally interacts with the ITR. In other embodiments, said structural element physically interacts with a large Rep protein upon binding of said Rep protein to the ITR. Each structural element may be, for example, an ITR secondary structure, an ITR nucleotide sequence, an intermediate sequence between two or more elements, or a combination of any of the above. In one embodiment, said structural elements are selected from the group consisting of arm A and arm A', arm B and arm B', arm C and arm C', arm D, a Rep binding site (RBE) and RBE' (i.e. ., complementary to the RBE sequence), and a terminal resolution site (trs).

[0024] In particular, said ITR may be structurally modified. For example, the nucleotide sequence of the structural element may be modified relative to the wild-type ITR sequence. In one embodiment, said structural element (e.g., arm A, arm A', arm B, arm B', arm C, arm C', arm D, RBE, RBE', and trs) of the ITR may be removed and replaced with the structural element wild type from another parvovirus. For example, the replacement structure may be taken from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus or insect AAV. For example, the ITR may be an AAV2 ITR, and arm A or A', or the RBE may be replaced by a structural element from AAV5. In another example, said ITR may be an AAV5 ITR, and the C or C', RBE and trs arms may be replaced by a structural element from AAV2. In another example, the AAV ITR may be an AAV5 ITR in which arms B and B' are replaced by arms B and B' of the AAV2 ITR.

[0025] As a non-limiting example, Table 3 provides examples of modifications to at least one nucleotide (e.g., deletion, insertion, and/or substitution) in modified ITR regions, where X indicates modification of at least one nucleic acid (e.g., deletion, insertion and/or substitution) in the indicated fragment relative to the corresponding wild-type ITR. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in any of the C and/or C' regions and/or B and/or B' regions preserves three consecutive nucleotides T (ie, TTT) in at least one terminal loop. For example, if the modification results in any of: an ITR with a single arm (for example, a single arm C-C' or a single arm B-B') or a modified arm C-B' or C'-B, or A two-arm ITR comprising at least one truncated arm (e.g., a truncated arm C-C' and/or a truncated arm B-B'), then in at least said single arm, or in at least one of the ITR arms with two arms (where one arm may be truncated), three consecutive T nucleotides (i.e., TTT) are retained in at least one terminal loop. In some embodiments, the C-C' truncated arm and/or the B-B' truncated arm contains three consecutive T nucleotides (ie, TTT) in the terminal loop.

[0026] The technology described herein also relates to a cccDNA vector that is capable of delivering and encoding one or more transgenes in a target cell, for example, if the ccDNA vector contains a multicistronic sequence, or if the transgene and its natural genomic context (eg, transgene, introns and endogenous untranslated regions) are included together in said cDNA vector. These transgenes may be protein-coding transcripts, non-coding transcripts, or both. Said cccDNA vector may contain multiple coding sequences and a non-canonical translation initiation site or more than one promoter for the expression of protein-coding transcripts, non-coding transcripts, or both. The transgene may contain a sequence encoding more than one protein, or may be a non-coding transcript sequence. Said expression cassette may contain, for example, more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any number in the range of about 4,000 to 10,000 nucleotides, or from 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides. cccDNA vectors have no restrictions on the size of capsided AAV vectors, which allows the delivery of a significant size expression cassette to ensure efficient transgene expression. In some embodiments, the cDNA vector lacks prokaryote-specific methylation.

[0027] The expression cassette may also contain an internal ribosome entry site (IRES) and/or a 2A element. Cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a miR-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, the ITR may act as a promoter for a transgene. In some embodiments, the cccDNA vector contains additional components for regulating transgene expression. For example, the additional regulatory component may be a regulatory switch as described herein, including, but not limited to, a “kill switch” that is capable of killing a cccDNA-infected cell, if necessary, and other inducible and/or repressible elements.

[0028] The technology described herein also provides new methods for delivering and efficiently selectively expressing one or more transgenes using these cccDNA vectors. The cccDNA vector can be taken up by host cells and also transported into the nucleus in the absence of the AAV capsid. In addition, the cccDNA vectors described herein do not have a capsid and, accordingly, avoid the immune response that may occur in response to capsid-containing vectors.

[0029] Aspects of the present invention relate to methods for producing the cDNA vectors described herein. Other embodiments relate to a cccDNA vector obtained using the method of the present invention. In one embodiment, said non-capsid non-viral DNA vector (ccDNA vector) is derived from a plasmid (referred to herein as a "ccDNA plasmid") containing a template polynucleotide expression construct comprising, in this order: a first 5' inverted terminal repeat (e.g. AAV ITR); expression cassette; and 3' ITR (e.g., AAV ITR), wherein the 5' and 3' ITR are modified ITRs relative to the wild-type ITR, and said modifications are identical to each other, i.e., said ITRs are symmetrical (i.e., are mirror image structures) relative to each other.

[0030] The cccDNA vector as described herein can be produced in several ways that will be apparent to one of ordinary skill in the art upon reading this document. For example, the template polynucleotide expression construct used to produce cccDNA vectors according to the present disclosure may be a ccDNA plasmid (eg, see Table 7 or Fig. 1B or 6B), a ccDNA bacmid, and/or a ccDNA baculovirus. In one embodiment, said cccDNA plasmid contains a restriction cloning site (e.g., SEQ ID NO: 7) operably located between the ITR, into which an expression cassette can be inserted, containing, for example, a promoter operably linked to a transgene, such as a reporter gene and /or therapeutic gene). In some embodiments, cccDNA vectors are derived from a polynucleotide template (e.g., ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus) containing modified symmetrical 5' and 3' ITRs flanking the expression cassette, wherein the modification is either or more of a deletion, insertion and/or substitution relative to wild-type AAV2 or AAV3 ITR sequences.

[0031] In a permissive host cell, in the presence of, for example, Rep, a polynucleotide template containing at least one modified ITR is replicated to produce cccDNA vectors. The production of a cccDNA vector occurs in two stages: first, excision (“release”) of the template from the template backbone (for example, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) through Rep proteins; and secondly, Rep-mediated replication of the excised cccDNA vector. Rep proteins and Rep binding sites in various AAV serotypes are well known to those skilled in the art. One skilled in the art will appreciate how to select a serotype Rep protein that binds to and replicates a nucleic acid sequence based on at least one functional ITR. For example, if a replication-competent modified ITR is taken from an AAV2 serotype, the corresponding Rep will be taken from an AAV serotype that works with that serotype, e.g., an AAV2 ITR for Rep AAV2 or AAV4, but not for Rep AAV5, which will not work. After replication, the cccDNA vector with covalently closed ends continues to accumulate in permissive cells, and the cccDNA vector is preferably sufficiently stable over time in the presence of the Rep protein under standard replication conditions, for example, to accumulate in an amount of at least 1 pg/cell, preferably at least 2 pg/cell, preferably at least 3 pg/cell, more preferably at least 4 pg/cell, even more preferably at least 5 pg/cell.

[0032] Accordingly, one aspect of the present invention relates to a method comprising the following steps: a) incubating a population of host cells (e.g., insect cells) bearing said template polynucleotide expression construct (e.g., ccDNA plasmid, ccDNA bacmid, and/or cDNA baculovirus) that does not contain viral capsid coding sequences, in the presence of Rep protein under effective conditions and for a period of time sufficient to induce production of the cccDNA vector in host cells, wherein said host cells do not contain viral capsid coding sequences ; and b) collecting and isolating said cDNA vector from the host cells. The presence of the Rep protein induces replication of the vector polynucleotide with a modified ITR to produce a cccDNA vector in the host cell. However, expression of viral particles (eg, AAV virions) does not occur. Thus, there are no size restrictions imposed by vir ions.

[0033] The presence of a cDNA vector isolated from host cells can be confirmed by digesting DNA isolated from a host cell with a restriction enzyme having a single recognition site on the cDNA vector and analyzing the digested DNA material on denaturing and non-denaturing gels for confirming the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[0034] According to one aspect of the present invention, there is provided a non-viral capsidless DNA vector with covalently closed ends (ccDNA vector), characterized in that the specified cccDNA vector contains at least one heterologous nucleotide sequence operably located between two flanking sequences of symmetrical inverted ends repeats (symmetrical ITRs), wherein said symmetrical ITRs are not wild-type ITRs and each of the flanking ITRs contains the same symmetrical modification. In accordance with one embodiment, said symmetrical ITR sequences are synthetic. In some embodiments, said ITRs are selected from any of those listed in Table 4. In some embodiments, each of said symmetrical ITRs is modified by a deletion, insertion, and/or substitution in at least one of the ITR regions selected from A, A', B, B ', C, C', D and D'. In some embodiments, the deletion, insertion, and/or substitution results in the deletion of all or part of the stem-loop structure, typically formed by regions A, A', B, B'C, or C'. In some embodiments, the symmetrical ITR is modified by a deletion, insertion, and/or substitution that results in the deletion of all or part of the stem-loop structure, typically formed by regions B and B'. In some embodiments, the symmetrical ITR is modified by a deletion, insertion, and/or substitution that results in the deletion of all or part of the stem-loop structure, typically formed by regions C and C'. In some embodiments, the symmetrical ITR is modified by a deletion, insertion, and/or substitution that results in the deletion of a portion of the stem-loop structure typically formed by regions B and B' and/or a portion of the stem-loop structure, usually formed by regions C and C'. In some embodiments, the symmetrical ITR comprises a single stem-loop structure in a region that typically contains a first stem-loop structure defined by regions B and B' and a second stem-loop structure, formed by areas C and C'. In some embodiments, the symmetrical ITR comprises a single stem and two loops in a region that typically contains a first loop-on-stem structure formed by regions B and B' and a second loop-on-stem structure formed by regions C and WITH'. In some embodiments, the symmetrical ITR comprises a single stem and a single loop in a region that typically contains a first loop-on-stem structure formed by regions B and B' and a second loop-on-stem structure formed by regions C and WITH'. In some embodiments, the symmetrical ITR is a modified AAV2 ITR comprising nucleotide sequences selected from: the ITR of FIG. 7A-22B or Table 4 herein, and an ITR that is at least 95% sequence identical to the ITRs listed in Table 4 or shown in FIGS. 7A-22B. In some embodiments, all or a portion of the heterologous nucleotide sequence is under the control of at least one regulatory switch.

[0035] According to another aspect of the present invention, there is provided a non-viral capsidless DNA vector with covalently closed ends (ccDNA vector), characterized in that the specified cccDNA vector contains at least one heterologous nucleotide sequence operably located between two flanking inverted terminal repeat sequences wild type (WT-ITR), wherein all or part of the heterologous nucleotide sequence is under the control of at least one regulatory switch. In some embodiments, the WT-ITR sequences are symmetric WT-ITR sequences or substantially symmetric WT-ITR sequences. In some embodiments, the WT-ITR sequences are selected from any combination of WT-ITRs listed in Table 1. In some embodiments, the flanking WT-ITR sequence is at least 95% identical to the ITR listed in Table 1 or Table 2, and all the substitutions are conservative nucleic acid substitutions that do not affect the structure of the WT-ITR. In some embodiments, the at least one control switch is selected from any of the control switches or a combination of control switches listed in Table 5 or the section entitled “Regulator Switches” herein. In some embodiments, the cccDNA vector, when digested with a restriction enzyme having a single recognition site on the ccDNA vector and analyzed by native and denaturing gel electrophoresis, exhibits characteristic bands of linear and continuous DNA compared to control linear and discontinuous DNA. In some embodiments, the ITR sequences are based on sequences from a virus selected from parvovirus, dependovirus, and adeno-associated virus (AAV). In some embodiments, said ITRs are based on sequences from adeno-associated virus (AAV). In some embodiments, said ITRs are based on sequences from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, said vector is contained in a nanocarrier. In some embodiments, the nanocarrier comprises a lipid nanoparticle (LNP).

[0036] In accordance with certain aspects and embodiments of the present invention, said cccDNA vector is produced by a method comprising the following steps: (a) incubating a population of insect cells bearing the ccDNA expression construct in the presence of at least one Rep protein, wherein said expression construct the cccDNA construct encodes the ccDNA vector, under effective conditions and for a period of time sufficient to induce the production of the ccDNA vector in insect cells; and (b) isolating said cDNA vector from insect cells. In some embodiments, the ccDNA expression construct is selected from ccDNA plasmid, ccDNA bacmid, and ccDNA baculovirus. In some embodiments, the insect cell expresses at least one Rep protein. In some embodiments, the at least one Rep protein is from a virus selected from parvovirus, dependovirus, and adeno-associated virus (AAV). In some embodiments, the at least one Rep protein is from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0037] According to some embodiments of the present invention, there is provided a cccDNA expression construct that encodes a cccDNA vector according to any aspects and embodiments of the present invention. In some embodiments, the construct is a cDNA plasmid, ccDNA bacmid, or ccDNA baculovirus.

[0038] In some embodiments, the present invention provides a host cell comprising a cccDNA expression construct according to any aspects or embodiments described herein. In some embodiments, the host cell expresses at least one Rep protein. In some embodiments, the at least one Rep protein is from a virus selected from parvovirus, dependent virus, and adeno-associated virus (AAV). In some embodiments, the at least one Rep protein is from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. In some embodiments, said host cell is an insect cell. In some embodiments, said insect cell is an Sf9 cell.

[0039] According to some embodiments of the present invention, there is provided a method of producing a cccDNA vector, comprising (a) incubating a host cell according to any aspects and embodiments of the present invention under effective conditions and for a period of time sufficient to induce production of the cccDNA vector; and (b) isolating said cccDNA from the host cells.

[0040] According to some embodiments of the present invention, there is provided a method of treating, preventing, ameliorating, monitoring, or diagnosing a disease or disorder in a subject, the method comprising: administering to a subject in need thereof a composition comprising an cccDNA vector in accordance with any aspects and embodiments of the present invention, characterized in that said at least one heterologous nucleotide sequence is selected for the treatment, prevention, amelioration, diagnosis or monitoring of a disease or disorder. In some embodiments, the at least one heterologous nucleotide sequence, when transcribed or translated, corrects an abnormal amount of endogenous protein in a subject. In some embodiments, the at least one heterologous nucleotide sequence, when transcribed or translated, corrects the abnormal function or activity of an endogenous protein or pathway in a subject. In some embodiments, the at least one heterologous nucleotide sequence encodes or contains a nucleotide molecule selected from the group consisting of RNAi, siRNA, siRNA, lncRNA, and an antisense oligo- or polynucleotide. In some embodiments, the at least one heterologous nucleotide sequence encodes a protein. In some embodiments, the protein is a marker protein (eg, a reporter protein). In some embodiments, the at least one heterologous nucleotide sequence encodes an agonist or antagonist of an endogenous protein or pathway associated with the disease or disorder. In some embodiments, the at least one heterologous nucleotide sequence encodes an antibody. In some embodiments, the disease or disorder is selected from the group consisting of a metabolic disease or disorder, a CNS disease or disorder, an eye disease or disorder, a blood disease or disorder, a liver disease or disorder, an immune disease or disorder, an infectious disease, a disease or disorder muscles, cancer; and a disease or disorder based on an abnormal level and/or function of a gene product. In some embodiments, said metabolic disease or disorder is selected from the group consisting of diabetes, lysosomal storage disorder, mucopolysaccharide disorder, urea cycle disease or disorder, and glycogen storage disease or disorder. In some embodiments, the lysosomal storage disorder is selected from the group consisting of Gaucher disease, Pompe disease, metachromatic leukodystrophy (MLD), phenylketonuria (PKU), and Fabry disease. In some embodiments, the disease or urea cycle disorder is ornithine transcarbamylase (OTC) deficiency. In some embodiments, the mucopolysaccharide disorder is selected from the group consisting of Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome, Morquio syndrome, and Maroteaux-Lamy syndrome. In some embodiments, said CNS disease or disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's syndrome, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease , muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma caused by damage to the spinal cord or head, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, mental disorders, schizophrenia, drug or drug addiction, neuroses, psychoses , dementia, paranoia, attention deficit disorder, sleep disorders, pain disorders, eating or weight disorders, as well as cancers and tumors of the central nervous system. In some embodiments, said ocular disease or disorder is selected from the group consisting of an ophthalmic disorder affecting the retina, posterior tract, and/or optic nerve. In some embodiments, said ophthalmic disorder affecting the retina, posterior tract, and/or optic nerve is selected from the group consisting of diabetic retinopathy, macular degeneration, including age-related macular degeneration, geographic atrophy, and vascular or “wet” form of macular degeneration, glaucoma, uveitis, retinitis pigmentosa, Stargardt disease, Leber congenital amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE), X-chromosomal retinitis pigmentosa (XLRP), X-chromosomal retinal dissection (XLRS), choroideremia, congenital optic neuropathy Leber's (LHON), achromatopsia, rod-cone dystrophy, Fuchs' endothelial corneal dystrophy, diabetic macular edema and eye cancer and tumors. In some embodiments, the blood disease or disorder is selected from the group consisting of hemophilia A, hemophilia B, thalassemia, anemia, and blood cancer. In some embodiments, the liver disease or disorder is selected from the group consisting of progressive familial intrahepatic cholestasis (PFIC) and liver cancer, as well as tumors. In some embodiments, the disease or disorder is cystic fibrosis. In some embodiments, the cDNA vector is administered in combination with a pharmaceutically acceptable carrier.

[0041] According to some embodiments of the present invention, there is provided a method of delivering a therapeutic protein to a subject, the method comprising administering to the subject a composition comprising a cccDNA vector according to any aspects and embodiments of the present invention, wherein said at least one heterologous nucleotide sequence encodes therapeutic protein. In some embodiments, the therapeutic protein is a therapeutic antibody. In some embodiments, the therapeutic protein is selected from the group consisting of an enzyme, erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, cytokine, cystic fibrosis transmembrane conductance regulator (CFTR), peptide growth factor, and hormone.

[0042] According to some embodiments of the present invention, there is provided a kit comprising a cccDNA vector according to any aspects and embodiments of the present invention and a nanocarrier packaged in a container with a package insert.

[0043] In accordance with some aspects of the present invention, there is provided a kit for producing a cccDNA vector, characterized in that the kit contains an expression construct containing at least one restriction site for insertion of at least one heterologous nucleotide sequence or regulatory switch, or a first and a second, wherein said at least one restriction site is operatively located either between (i) symmetrical inverted terminal repeat (symmetric ITR) sequences, wherein said symmetrical ITRs are not wild-type ITRs, or between (ii) two inverted terminal repeat sequences wild-type terminal repeats (WT-ITR). In some embodiments, the kit is suitable for producing a cccDNA vector in accordance with any of the aspects and embodiments of the present invention. In some embodiments, the kit further comprises a population of insect cells that does not contain viral capsid coding sequences that, in the presence of the Rep protein, can induce production of the cccDNA vector. In some embodiments, the kit further comprises a vector containing a polynucleotide sequence that encodes at least one Rep protein, wherein the vector is suitable for expressing the at least one Rep protein in an insect cell.

[0044] These and other aspects of the present invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In FIG. Figure 1A shows an example of the structure of a cDNA vector. According to the specified embodiment, the specified example cccDNA vector contains an expression cassette containing a CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a transgene (eg, luciferase) is inserted into the cloning site (R3/R4) between the CAG promoter and the WPRE. The expression cassette is flanked by two symmetrical inverted terminal repeats (ITRs) - that is, the modified 5' ITR and the modified 3' ITR flanking the expression cassette are symmetrical to each other.

[0046] In FIG. 1B shows an example structure of a cccDNA vector (or the corresponding sequence present in an example cDNA plasmid) with an expression cassette containing an enhancer/promoter, an open reading frame (ORF) for transgene insertion, a post-transcriptional element (WPRE) and a poly(A) signal . The open reading frame (ORF) allows the transgene to be inserted into the cloning site between the CAG promoter and the WPRE. Said expression cassette is flanked by two symmetrical inverted terminal repeats (ITRs) - that is, a modified ITR upstream (at the 5' end) and a modified ITR downstream (at the 3' end) of said expression cassette, both 5' ITR and 3' ITR are modified ITRs, but contain the same modifications (i.e., they are mirror images of each other, i.e., symmetrical to each other).

[0047] In FIG. Figure 2A shows an example of the structure of a cDNA vector. According to the specified embodiment, the specified example cccDNA vector contains an expression cassette containing a CAG promoter, WPRE and BGHpA. An open reading frame (ORF) encoding a transgene (eg, luciferase) is inserted into the cloning site (R3/R4) between the CAG promoter and the WPRE. Said expression cassette is flanked by two wild type inverted terminal repeats (WT-ITRs), that is, 5' WT-ITR and 3' WT-ITR flanking said expression cassette.

[0048] In FIG. 2B shows an example structure of a cccDNA vector (or the corresponding sequence present in an example ccDNA plasmid) with an expression cassette containing an enhancer/promoter, an open reading frame (OKF) for transgene insertion, a post-transcriptional element (WPRE), and a poly(A) signal . The open reading frame (OKF) allows the transgene to be inserted into the cloning site between the CAG promoter and the WPRE. Said expression cassette is flanked by two WT inverted terminal repeats (WT-ITR) - that is, WT-ITR upstream (at the 5' end) and WT-ITR downstream (at the 3' end) of said expression cassette, said 5' WT The ITR and 3' WT-ITR may be from the same serotype or from different serotypes.

[0049] In FIG. 3A shows the T-shaped stem-loop structure of the wild-type left ITR from AAV2 (SEQ ID NO: 538) with the A-A' arm, B-B' arm, C-C' arm, two Rep binding sites indicated (RBE and RBE'); the terminal resolution site (trs) is also shown. RBE contains a series of 4 duplex tetramers believed to interact with either Rep 78 or Rep 68. In addition, RBE' is also believed to interact with the Rep complex assembled on a wild-type ITR or a mutated ITR in the construct. Regions D and D' contain transcription factor binding sites and other conserved structures. In fig. 3B shows putative Rep-catalyzed nicking and ligation activity in the left wild-type ITR (SEQ ID NO: 539), including the T-shaped stem-loop structure of the left wild-type ITR of AAV2, indicating the A-A' arm, arm B-B', C-C' arm, two Rep binding sites (RBE and RBE'); also shown is the terminal resolution site (trs), and regions D and D' containing several transcription factor binding sites and other conserved structures.

[0050] In FIG. 4A shows the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the RBE-containing portions of the A-A' arm, C-C' arm, and B-B' arm of the left ITR of wild-type AAV2 (SEQ ID NO: 540). In fig. 4B shows an example sequence of a mutated ITR (also called a modified ITR) for the left ITR. Shown is the primary structure (left) and predicted secondary structure (right) of the RBE portion of arm A-A', arm C, and arm B-B' from an example mutated left ITR (ITR-1, left) (SEQ ID NO: 113). In fig. 4C shows the primary structure (left) and secondary structure (right) of the RBE-containing portion of the A-A' loop, and arms B-B' and C-C' of the right wild-type AAV2 ITR (SEQ ID NO: 541). In fig. 4D shows the primary structure (left) and secondary structure (right) of the RBE portion of the A-A loop, B-B' arm, and C-C' arm of the right wild-type AAV2 ITR (SEQ ID NO: 541). In fig. 4E shows an example of a right modified ITR. Shown are the primary structure (left) and predicted secondary structure (right) of the RBE-containing portion of the A-A' arm, B-B' arm, and C arm of an example mutant right ITR (ITR-1, right) (SEQ ID NO: 114). Any combination of left and right ITRs (eg, AAV2 ITR from a different viral serotype or synthetic ITRs) can be used as long as the left ITR is symmetrical or inversely complementary to the right ITR. Each of the polynucleotide sequences in FIG. 3A-3D refers to the sequence used in the plasmid or bacmid/baculovirus genome used to produce cccDNA as described herein. Also in each of FIGS. 4A-4E include the corresponding cccDNA secondary structures derived from the configurations of the cccDNA vector in the plasmid or bacmid/baculovirus genome and predicted Gibbs free energy values.

[0051] In FIG. 5A is a schematic representation of the upstream process for producing baculovirus-infected insect cells (BICs) that are suitable for producing cccDNA in the manner schematically illustrated in FIG. 5V. In fig. 5B is a schematic representation of an example of a method for obtaining cccDNA, and FIG. 5C illustrates the biochemical method and process for confirming the production of the cccDNA vector. In fig. 5D and FIG. 5E is a schematic diagram describing a method for identifying the presence of cccDNA in DNA collected from cell pellets obtained in the cccDNA production process of FIG. 4B. In fig. 5E shows DNA having a discontinuous structure. cccDNA can be cut by a restriction endonuclease, which has one recognition site on the ccDNA vector, to form two DNA fragments of different sizes (1 kb and 2 kb) under both neutral and denaturing conditions. In fig. 5E also shows cccDNA having a linear and continuous structure. This cccDNA vector can be cut by a restriction endonuclease to form two DNA fragments, which migrate as 1 kb and 2 kb fragments under neutral conditions, however, under denaturing conditions, these chains remain connected and form single strands, which migrate and form single strands, which migrate in the form of fragments of 2 kb and 4 kb. In fig. 5D schematically shows the expected bands for an example of cccDNA either left undigested or digested with a restriction endonuclease and then subjected to electrophoresis on a native gel or a denaturing gel. The diagram on the far left shows a native gel and several bands indicating that, in duplex and uncut form, cccDNA exists in at least monomeric and dimeric states, which appear as a smaller faster-migrating monomer and a slower-migrating dimer with a size of 2 times greater than that of the monomer. The second schematic from the left demonstrates that when cccDNA is cut with a restriction endonuclease, the original bands disappear and faster migrating bands (eg, smaller ones) appear that correspond to the expected sizes of fragments remaining after cleavage. Under denaturing conditions, the original duplex DNA is single-stranded and migrates as an object twice the size of that observed on the native gel because the complementary strands are covalently linked. Accordingly, in the second diagram from the right, digested ccDNA exhibits a band distribution similar to that observed on the native gel, however, these bands migrate as fragments that are twice the size of their equivalents on the native gel. The diagram on the far right shows that uncut cccDNA under denaturing conditions migrates as a single-stranded open ring and, accordingly, the bands observed are twice as large as those observed under native conditions when the ring is not opened. In this figure, "kb" is used to indicate the relative size of nucleotide molecules, based, depending on the context, on either the length of the nucleotide chain (for example, for single-stranded molecules observed under denaturing conditions) or the number of base pairs (for example, for double-stranded molecules observed under native conditions).

[0052] In FIG. 6A shows an example of a Rep bacmid in the plasmid pFBDLSR containing the nucleic acid sequences for the Rep proteins Rep52 and Rep78. This example Rep bacmid contains: a fragment of the IE1 promoter (SEQ ID NO: 66); the nucleotide sequence of Rep78, including the Kozak sequence (SEQ ID NO: 67), the polyhedrin promoter sequence of Rep52 (SEQ ID NO: 68), and the nucleotide sequence of Rep58, starting with the Kozak sequence gccgccacc) (SEQ ID NO: 69). In fig. 6B is a schematic diagram of an example cccDNA plasmid with a modified left ITR (L-modified ITR), a CAG promoter, a luciferase transgene, a WPRE, and a polyadenylation sequence, and a modified right ITR (R-modified ITR), wherein said L-modified ITR and R-modified ITRs are symmetrical to each other. In fig. 6C is a schematic diagram of an example cccDNA plasmid with left WT-ITR (L-WT-ITR), CAG promoter, luciferase transgene, WPRE and polyadenylation sequence, and right WT-ITR (R-WT ITR).

[0053] In FIG. 7A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR (“ITR-33 (Left)” SEQ ID NO: 101); in fig. 7B shows a cognate symmetric right ITR (ITR-18, right) with the predicted lowest energy structure of the RBE-containing portion of the A-A' arm and the portions C-C' and B-B' of an example modified right ITR ("ITR-18 (right) " SEQ ID NO: 102). Both ITR-33 (left) and ITR-18 (right) are predicted to form a single-arm structure (ie, single arm C-C').

[0054] In FIG. 8A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-34 (Left)" SEQ ID NO: 103); in fig. 8B shows a cognate symmetric right ITR (ITR-51, Right) with the predicted lowest energy structure of the RBE-containing portion of the A-A' arm and the portions C-C' and B-B' of an example modified right ITR ("ITR-51 (Right) " SEQ ID NO: 96). Both ITR-34 (left) and ITR-51 (right) are predicted to form a single-arm structure (ie, single arm B-B').

[0055] In FIG. 9A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-35 (Left)" SEQ ID NO: 105), and FIG. 9B shows a cognate symmetric right ITR (ITR-19, right) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR ("ITR-35 (right) " SEQ ID NO: 105). Both ITR-35 (left) and ITR-19 (right) are predicted to form a single-arm structure (ie, single arm C-C').

[0056] In FIG. 10A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-36 (Left)" SEQ ID NO: 454), and FIG. 10B shows a cognate symmetric right ITR (“ITR-20 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR ("ITR-20 (Right)" SEQ ID NO: 116). Both ITR-36 (left) and ITR-20 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm C-C' is truncated).

[0057] In FIG. 11A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-37 (Left)" SEQ ID NO: 111), and FIG. 11B shows a cognate symmetric right ITR (“ITR-21 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR ("ITR-21 (Right)" SEQ ID NO: 112). Both ITR-37 (left) and ITR-21 (right) are predicted to form a single-stem structure (e.g., a single loop-on-stem structure in a region that would normally contain the first loop-on-stem structure, formed by regions B and B', and a second stem-loop structure formed by regions C and C').

[0058] In FIG. 12A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-38 (Left)" SEQ ID NO: 117), and FIG. 12B shows a cognate symmetric right ITR (“ITR-22 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR (“ITR-22 (Right)" SEQ ID NO: 118). Both ITR-38 (left) and ITR-22 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm B-B' is truncated).

[0059] In FIG. 13A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-39 (Left)" SEQ ID NO: 119), and FIG. 13B shows a cognate symmetric right ITR (“ITR-23 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR (“ITR-23 (Right)" SEQ ID NO: 120). Both ITR-39 (left) and ITR-23 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm B-B' is truncated).

[0060] In FIG. 14A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-40 (Left)" SEQ ID NO: 121), and FIG. 14B shows a cognate symmetric right ITR (“ITR-24 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR (“ITR-24 (Right)" SEQ ID NO: 122). Both ITR-40 (left) and ITR-24 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm B-B' is truncated).

[0061] In FIG. 15A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-41 (Left)" SEQ ID NO: 107), and FIG. 15B shows a cognate symmetric right ITR (“ITR-25 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR (“ITR-25 (Right)" SEQ ID NO: 108). Both ITR-41 (left) and ITR-25 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm B-B' is truncated).

[0062] In FIG. 16A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-42 (Left)" SEQ ID NO: 123), and FIG. 16B shows a cognate symmetric right ITR (“ITR-26 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR (“ITR-26 (Right)" SEQ ID NO: 124). Both ITR-42 (left) and ITR-26 (right) are predicted to form a structure with two arms, one of which is elongated (eg, arm C-C' is truncated).

[0063] In FIG. 17A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-43 (Left)" SEQ ID NO: 125), and FIG. 17B shows a cognate symmetric right-handed ITR (“ITR-27 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right-handed ITR (“ITR-27 (Right)" SEQ ID NO: 126). Both ITR-43 (left) and ITR-27 (right) are predicted to form a structure with two arms, one of which is elongated (eg, arm C-C' is truncated).

[0064] In FIG. 18A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-44 (Left)" SEQ ID NO: 127), and FIG. 18B shows a cognate symmetric right ITR (“ITR-28 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR (“ITR-28 (Right)" SEQ ID NO: 128). Both ITR-44 (left) and ITR-28 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm C-C' is truncated).

[0065] In FIG. 19A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-45 (Left)" SEQ ID NO: 129), and FIG. 19B shows a cognate symmetric right ITR (“ITR-29 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR (“ITR-29 (Right)" SEQ ID NO: 130). Both ITR-45 (left) and ITR-29 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm C-C' is truncated).

[0066] In FIG. 20A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-46 (Left)" SEQ ID NO: 131), and FIG. 20B shows a cognate symmetric right ITR (“ITR-30 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR ("ITR-30 (Right)" SEQ ID NO: 132). Both ITR-46 (left) and ITR-30 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm C-C' is truncated).

[0067] In FIG. 21A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-47 (Left)" SEQ ID NO: 133), and FIG. 21B shows a cognate symmetric right ITR (“ITR-31 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR ("ITR-31 (Right)" SEQ ID NO: 134). Both ITR-47 (left) and ITR-31 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm C-C' is truncated).

[0068] In FIG. 22A shows the predicted lowest energy structure of the RBE-containing arm portion A-A' and the portions C-C' and B-B' of an example modified left ITR ("ITR-48 (Left)" SEQ ID NO: 547), and FIG. 22B shows a cognate symmetric right ITR (“ITR-32 (right)”) with the predicted lowest energy structure of the RBE-containing arm portion A-A' and portions C-C' and B-B' of an example modified right ITR ("ITR-32 (Right)" SEQ ID NO: 546). Both ITR-48 (left) and ITR-32 (right) are predicted to form a structure with two arms, one of which is truncated (eg, arm C-C' is truncated).

[0069] In FIG. Figure 23 shows the luciferase activity of Sf9 GlycoBac insect cells transfected with 16 cccDNA variants of the symmetric mutant ITRs from Table 4 (also see Table 7 for complete constructs). The cDNA vector contained the luciferase gene flanked by symmetrical mutant ITRs selected from Table 4. “Mock” conditions are represented by transfection reagents only, without donor DNA.

[0070] In FIG. 24A and FIG. 24B shows GFP activity in Sf9 GlycoBac insect cells transfected with the WT/WT ITR construct described in Example 4. The gccDNA vector contained the GFP gene flanked by wild type ITR (WT ITR). In fig. 24A is a fluorescence microscopy image at 40x magnification of Sf9 cells transfected with a cccDNA vector with WT/WT ITR GFP and then infected with Rep virus. In fig. Figure 24B shows a bright field image at 40x magnification of the same cells as in image "A". In fig. 24C shows a bright field image at 40x magnification of control cells transfected with WT/WT ITR but not infected with Rep virus. These cells did not generate any fluorescent signal.

[0071] In FIG. Figure 25 shows a native agarose gel (1% agarose) of putative ccDNA with wild-type ITR. Lane 1 shows the 1 kb Plus DNA ladder, and lane 2 shows ccDNA produced using a plasmid containing the wild-type ITR cassette, both lanes taken from the same gel. Monomeric GFP cccDNAs are expected to be approximately 4 kb in size and a dimer of approximately 8 kb.

[0072] In FIG. Figure 26 shows a denaturing gel of cccDNA containing wild-type ITR. Lane 1 shows the 1 kb Plus DNA ladder, lane 2 shows wild-type ccDNA, which was not cut with endonuclease, and lane 3 shows the same wild-type ccDNA, but cut with the enzyme ClaI restriction endonuclease. All samples are taken from the same gel.

[0073] In FIG. 27 shows the potential secondary structure of symmetrical ITRs from construct-388 (mutant) and construct-393 (wild-type AAV2).

[0074] In FIG. 28 shows changes in body weight of CD-1 mice treated with LNP + poly(C) (control); LNP + asymmetric cccDNAs produced in Sf9 containing the WT AAV2 ITR on the left and a truncated ITR on the right; LNP + synthetic cccDNA containing WT AAV2 ITR, symmetrical on both sides, left and right; LNP + synthetic cccDNAs containing asymmetric ITRs, WT AAV2 ITR on the left and truncated ITR on the right; cccDNAs produced in Sf9 with mutant ITRs are symmetrical on both sides, left and right (design-388); and cccDNA containing wild-type AAV2 ITR, symmetrical on both sides of the construct, left and right (construct-393; produced in Sf9).

[0075] In FIG. 29A and 29B show images of in vivo luciferase expression on day 14 in CD-1 mice treated with: (1) LNP + poly(C) (control; top left); (2) LNP + cccDNA with mutant ITRs located symmetrically on both the left and right sides of the construct (Construct-388) produced in Sf9 (top right); and cccDNAs containing symmetric wild-type AAV2 ITRs produced in Sf9 (Construct-393; lower middle panel).

DETAILED DESCRIPTION OF THE INVENTION

[0076] One of the biggest challenges in developing therapeutics, particularly for rare diseases, is the large number of individual conditions. About 350 million people worldwide live with a rare disease, which is defined by the National Institutes of Health as a disorder or condition diagnosed in fewer than 200,000 people. Approximately 80 percent of these rare disorders are genetic in origin, and approximately 95 percent of them have no FDA-approved treatment.

[0077] Among the advantages of the cccDNA vectors described herein are the provision of a method that can be rapidly adapted for a variety of diseases, and in particular for rare monogenic diseases, which could significantly change the current state of the art of treating many genetic disorders or diseases. In addition, the cccDNA vectors described herein contain a regulatory switch that allows control of gene expression after delivery.

I. Definitions

[0078] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have standard meanings known to those skilled in the art to which this invention relates. It should be understood that the present invention is not limited to the specific methodology, protocols and reagents, etc. described herein and, accordingly, is subject to various variations. The terminology herein serves solely to describe specific embodiments and does not limit the scope of the present invention, which is defined solely by the claims. Definitions of standard terms in the field of immunology and molecular biology can be found in the following sources: The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.). Fields Virology, 6th Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.). Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology, Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al.. Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons. Inc., 2003 (ISBN 0471142735, 9780471142737), contents each of which is incorporated by reference herein in its entirety.

[0079] As used herein, the terms “administration,” “administered,” and variations thereof refer to the administration of a composition or agent (e.g., nucleic acids, particularly cctDNA) to a subject and include simultaneous and sequential administration of one or more compositions or one or more agents . “Administration” may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also includes in vitro and ex viva treatment. Administration of the composition or agent to a subject is accomplished by any suitable route, including orally, pulmonaryly, intranasally, parenterally (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, lymphatic, intratumoral, or topical. Administration includes self-administration and administration by another person. Administration may be accomplished by any suitable route. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the appropriate route is intravenous, the composition is administered by administering said composition or agent into a vein of the subject.

[0080] As used herein, the term "antibody" is used in the broadest sense to cover a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they demonstrate the required antigen-binding activity. "Antibody fragment" refers to a molecule, other than an intact antibody, that contains a portion of the intact antibody that binds the same antigen to which the intact antibody binds. In one embodiment, the antibody or fragment thereof comprises an immunoglobulin chain or antibody fragment and at least one immunoglobulin variable domain sequence. Examples of antibodies or fragments thereof include, but are not limited to, Fv, scFv, Fab fragment, Fab', F(ab') ₂ , Fab'-SH, single domain antibody (dAb), heavy chain, light chain, heavy and light chain , complete antibody (e.g., including all of Fc, Fab, heavy chains, light chains, variable regions, etc.), bispecific antibody, diabody, linear antibody, single chain antibody, intrabody, monoclonal antibody, chimeric antibody, multispecific antibody or multimeric antibody. The antibody or fragment thereof may belong to any class, including, but not limited to, IgA, IgD, IgE, IgG and IgM, and any subclass thereof, including, but not limited to, IgG1, IgG2, IgG3, IgG4 , IgA1 and IgA2. In addition, the antibody can be obtained from any mammal, such as primates, humans, rats, mice, horses, goats, etc. In one embodiment, said antibody is a human antibody or a humanized antibody. In some embodiments, the antibody is a modified antibody. In some embodiments, the antibody components may be expressed separately such that the antibody self-assembles upon expression of the protein components. In some embodiments, the antibody is "humanized" to reduce immunogenic reactions in humans. In some embodiments, the antibody has a desired function, such as interacting with and inhibiting a protein of interest, to treat a disease or symptom of a disease. In one embodiment, the antibody or antigen-binding fragment thereof comprises a framework region or Fc region.

[0081] As used herein, the term “antigen-binding domain” of an antibody molecule refers to the portion of an antibody molecule, such as an immunoglobulin (Ig) molecule, that is involved in binding an antigen. In embodiments, said antigen binding site is formed by amino acid residues of the variable (V) regions of the heavy (H) and light (L) chains. Three highly heterogeneous regions in the variable regions of the heavy and light chains, called hypervariable regions, are located between more conserved flanking stretches called “framework regions” (FR). FRs are amino acid sequences that naturally occur between and adjacent to hypervariable regions in immunoglobulins. In embodiments, in an antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are located relative to each other in three-dimensional space such that they form an antigen-binding surface complementary to the three-dimensional surface of the antigen being bound. These three hypervariable regions of each of the heavy and light chains are called “complementarity determining regions” or “CDRs”. The framework region and CDR have been defined and described, for example, in: Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (eg, variable heavy chain and variable light chain) typically consists of three CDRs and four FRs arranged in amino acid sequence, from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3 and FR4.

[0082] As used herein, the expression “anti-therapeutic immune response against a nucleic acid”, “anti-vector immune response against a vector vector”, “immune response against a therapeutic nucleic acid”, “immune response against a vector vector” or the like refers to to any unwanted immune response against a therapeutic nucleic acid, viral or non-viral in origin. In some embodiments, the unwanted immune response is an antigen-specific immune response against the viral vector itself. In some embodiments, the immune response is specific for a vector vector, which may be double-stranded DNA, single-stranded RNA, or double-stranded RNA. In other embodiments, said immune response is specific to the sequence of the vector vector. In other embodiments, said immune response is specific to the CpG content of the vector vector.

[0083] As used herein, “carrier” includes all possible solvents, dispersion media, bases, coatings, diluents, antibacterial and antimycotic agents, isotonic and absorption retarding agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Also, auxiliary active ingredients may be included in these compositions. The expression "pharmaceutically acceptable" refers to molecular entities and compositions that do not result in a toxic, allergic or similar adverse reaction when administered to a host.

[0084] As used herein, the term “ccDNA” refers to capsid-free closed linear double-stranded (ds) duplex DNA for non-viral gene transfer, synthetic or otherwise. A detailed description of the cDNA is provided in international application PCT/US2017/020828, filed March 3, 2017, the contents of which are expressly incorporated herein by reference in their entirety. Some methods for producing a cccDNA vector containing various sequences and inverted terminal repeat (ITR) configurations using cellular methods are described in example 1 in international application PCT/US 18/49996, filed September 7, 2018, and international application PCT/US2018 /064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Some methods for producing synthetic cccDNA vectors containing different ITR sequences and configurations are described, for example, in international application PCT/US2019/14122, filed January 18, 2019, the contents of which are incorporated herein by reference in their entirety. In this document, the terms “ccDNA vector” and “ccDNA” are used interchangeably. In some embodiments, the cccDNA is linear closed-end duplex (CELiD) CELiD DNA. In some embodiments, said cccDNA is a DNA-based minicircle. In some embodiments, the cccDNA is a minimalistic vector for immunologically defined gene expression (MIDGE). In some embodiments, said cccDNA is service DNA. In some embodiments, the cccDNA is a dumbbell-shaped linear duplex DNA with closed ends containing two ITR hairpin structures at the 5' and 3' ends of the expression cassette. In some embodiments, the cccDNA is Doggybone™ DNA.

[0085] As used herein, the term “ccDNA bacmid” refers to an infectious baculovirus genome containing the cccDNA genome as an intermolecular duplex that is capable of propagation in E. coli as a plasmid, and accordingly can serve as a shuttle vector for the baculovirus.

[0086] As used herein, the term “ccDNA baculovirus” refers to a baculovirus that contains the cccDNA genome as an intermolecular duplex within the baculovirus genome.

[0087] As used herein, the terms “ccDNA-baculovirus-infected insect cell” and “ccDNA-BIIC” are used interchangeably and refer to an invertebrate animal host cell (including, without limitation, an insect cell (e.g., an Sf9 cell)) infected with cccDNA-BIIC. baculovirus.

[0088] As used herein, the term “ccDNA genome” refers to an expression cassette that further includes at least one inverted terminal repeat region. The cDNA genome may further comprise one or more spacer regions. In some embodiments, the cDNA genome is included as an intermolecular duplex DNA polynucleotide in a plasmid or viral genome.

[0089] As used herein, the term “ccDNA plasmid” refers to a plasmid that contains the cccDNA genome as an intermolecular duplex.

[0090] As used herein, the terms “closed-end DNA vector,” “ccDNA vector,” and “ccDNA” are used interchangeably and refer to a non-viral, capsid-free DNA vector with at least one covalently end-closed end (i.e., with an intramolecular duplex). In some embodiments, the cccDNA comprises two covalently closed ends.

[0091] As used herein, the term “ccDNA spacer region” refers to an intervening sequence that separates functional elements in a specified ccDNA vector or ccDNA genome. In some embodiments, cccDNA spacer regions hold two functional elements at a desired distance for optimal functionality. In some embodiments, cDNA spacer regions provide or enhance the genetic stability of the cDNA genome within, for example, a plasmid or baculovirus. In some embodiments, cccDNA spacer regions facilitate prepared genetic manipulation of the cccDNA genome by providing convenient locations for cloning sites and the like. For example, in certain aspects, an oligonucleotide "polylinker" containing multiple restriction endonuclease sites or non-open reading frame sequence, designed to not contain known protein (e.g., transcription factor) binding sites, can be placed in the cccDNA genome for separation cis-acting factors, for example, by insertion of a 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 86-mer, 176-mer, etc. between the terminal resolution site and the 5' transcriptional regulatory element. Likewise, a spacer can be inserted between the polyadenylation signal sequence and the 3' end resolution site.

[0092] As used herein, the expression "effective amount" or "therapeutically effective amount" of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect, for example, inhibition of expression of the target sequence compared to the level of expression , detectable in the absence of therapeutic nucleic acid. Suitable assays for measuring the expression of a target gene or target sequence include, for example, examining protein or RNA levels using techniques known to those skilled in the art, such as dot blot, Northern blot, in situ hybridization, ELISA, immunoprecipitation, functional enzyme assays, as well as phenotypic assays known to those skilled in the art.

[0093] As used herein, the term “exogenous” refers to a substance present in a cell other than its natural source. The term “exogenous” as used herein may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced through a process involving human activity into a biological system, such as a cell or organism, in which it is not normally present. are detected and introduction of the nucleic acid or polypeptide into such a cell or organism is desired. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that has been introduced through a process involving human activity into a biological system, such as a cell or organism, where it is present in relatively minor quantities, and increasing the amount of the nucleic acid or polypeptide in cell or organism is desired, for example, to obtain ectopic expression or levels. Conversely, as used herein, the term “endogenous” refers to a substance that is native to a biological system or cell.

[0094] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, either, for example, in the form of a reporter peptide, or, more preferably, in the form of a polypeptide that kills a cell, such as a toxin or cell imparting agent. sensitivity to killing when exposed to a specific agent or in the absence of a specific agent. Effector proteins include any protein or peptide that directly targets or damages the DNA and/or RNA of a host cell. For example, effector proteins may include, but are not limited to, a restriction endonuclease that targets a host cell DNA sequence (genomic or extrachromosomal element), a protease that cleaves a target polypeptide essential for cell survival, a DNA gyrase inhibitor, and a ribonuclease-type toxin. In some embodiments, expression of an effector protein controlled by a synthetic biological chain as described herein can participate as a factor in another synthetic biological chain, thereby expanding the range and complexity of the response of the biological chain system.

[0095] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear stretch of nucleic acids that contains a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but does not contain capsid coding sequences, other vector sequences or inverted terminal repeat regions. The expression cassette may further comprise one or more upstream sequences (eg, promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0096] As used herein, the term “flanking” refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Typically, in the ABC sequence, B is flanked by A and C. The same is true for the arrangement of AxBxC. Accordingly, the flanking sequence precedes or follows the flanking sequence, but need not be adjacent to or immediately adjacent to the flanking sequence. In one embodiment, the term “flanking” refers to the terminal repeats at each end of a linear, single-stranded synthetic AAV vector.

[0097] As used herein, the term “full-length antibody” refers to an immunoglobulin (Ig) molecule (eg, an IgG antibody), for example, naturally occurring and formed through normal recombination processes of immunoglobulin gene fragments.

[0098] As used herein, the term “functional antibody fragment” refers to a fragment that binds to the same antigen as recognized by an intact (eg, full-length) antibody. The terms "antibody fragment" or "functional fragment" also include isolated fragments consisting of variable regions, such as "Fv" fragments consisting of heavy and light chain variable regions, or recombinant single chain polypeptide molecules in which light and heavy chains the variable regions are linked by a peptide linker (“scFv proteins”). In some embodiments, the antibody fragment does not contain portions of the antibodies without antigen-binding activity, such as Fc fragments or individual amino acid residues.

[0099] As used herein, the terms “gap” and “break” are used interchangeably and refer to the interrupted portion of synthetic DNA vectors as described herein, creating a stretch of a portion of single-stranded DNA in an otherwise double-stranded ccDNA. The length of the gap can be from one 1 base pair to 100 base pairs in one strand of duplex DNA. Typical gaps designed and created using the methods described herein, and synthetic vectors obtained by these methods, may have a length of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 base pairs . Examples of gaps herein may range in length from 1 bp to 10 bp, from 1 to 20 bp, from 1 to 30 bp.

[00100] As used herein, the term “gene” is used broadly to refer to any nucleic acid segment associated with the expression of specific RNA or protein, in vitro or in vivo. Accordingly, genes include regions encoding expressed RNAs (which typically include sequences encoding polypeptides) and, often, regulatory sequences necessary for their expression. Genes may be obtained from a variety of sources, including cloning from a source of interest or synthesis based on known or predicted sequence information, and may include sequences designed to have specific desired parameters.

[00101] As used herein, the expression “genetic disease” or “genetic disorder” refers to a disease caused in part or in whole, directly or indirectly, by one or more abnormalities in the genome, including and in particular a condition present from birth. The abnormality may be a mutation, insertion or deletion in a gene. This anomaly may affect the coding sequence of the gene or its regulatory sequence.

[00102] As used herein, the term “heterologous” refers to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively.

[00103] As used herein, the terms “heterologous nucleotide sequence” and “transgene” are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that is inserted into a cccDNA vector and can be delivered and expressed by a cccDNA vector. vector as described in this document. A heterologous nucleic acid sequence can be combined with a naturally occurring nucleic acid sequence (or variant thereof) (eg, by genetic engineering) to form a chimeric nucleotide sequence encoding a chimeric polypeptide. A heterologous nucleic acid sequence can be joined to a polypeptide variant (eg, by genetic engineering) to form a nucleotide sequence encoding a polypeptide fusion variant. Transgenes of interest include, but are not limited to, nucleic acids encoding polypeptides, preferably therapeutic (eg, for medical, diagnostic or veterinary purposes) or immunogenic polypeptides (eg, for vaccines). In some embodiments, the nucleic acids of interest include nucleic acids that are transcribed into therapeutic RNA. Transgenes contemplated for use in cccDNA vectors herein include, but are not limited to, transgenes that express or encode one or more polypeptides, peptides, ribozymes, aptamers, peptide nucleic acids, siRNAs, RNAi, siRNAs, lncRNAs, antisense oligos - or polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[00104] As used herein, the term “homology” or “homologous” refers to the percentage of nucleotide residues in a homology arm that are identical to nucleotide residues in the corresponding sequence on the target chromosome after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment to determine percent homology of a nucleotide sequence can be achieved in various ways known in the art, for example, using open access computer software such as BLAST, BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR). Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment throughout the sequences being compared. In some embodiments, a nucleic acid sequence (e.g., a DNA sequence), such as a homology arm of a repair template, is considered “homologous” if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, according to is at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (eg, genomic sequence) of the host cell.

[00105] As used herein, the term “host cell” refers to any cell type that is susceptible to transformation, transfection, transduction, or the like, by therapeutic nucleic acids as described herein. In non-limiting examples, the host cell may be an isolated primary cell, pluripotent stem cells, CD34 ⁺ cells, induced pluripotent stem cells, or any of a number of immortalized cell lines (eg, HepG2 cells). Alternatively, the host cell may be an in situ or in vivo cell in a tissue, organ or organism. In addition, the host cell may be a target cell, for example, a mammalian subject (eg, a human patient in need of gene therapy).

[00106] As used herein, “immunoglobulin variable domain sequence” refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may comprise all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N- or C-terminal amino acids, or may include other changes that are compatible with the formation of the protein structure.

[00107] As described herein, an “inducible promoter” is a promoter that is characterized by initiation or enhancement of transcriptional activity in the presence of an inducer or inducing agent, under the influence of an inducer or inducing agent, or contact with an inducer or inducing agent. An "inducer" or "inducing agent" as defined herein may be an endogenous or normally exogenous compound or protein that is administered such that it is active in inducing the transcriptional activity of an inducible promoter. In some embodiments, said inducer or inducing agent, i.e., a chemical, compound, or protein, may itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer may be an inducer protein expressed by another component or module ), which itself may be under the control or be an inducible promoter. In some embodiments, the inducible promoter is induced in the absence of certain agents, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothioneine, ecdysone, mammalian viruses (eg, adenoviral late promoter; murine mammary tumor virus long terminal repeat (MMTV-LTR)) and other steroid-responsive promoters, rapamycin-responsive promoters and so on.

[00108] As used herein, an “entry agent responsive domain” is a transcription factor domain that binds or otherwise responds to a condition or input agent such that the linked DNA binding fusion domain acquires the ability to respond to the presence of the condition or input agent. In one embodiment, the presence of said condition or input agent results in a conformational change in the input agent response domain, or in the protein to which it is fused, that modifies the transcription modulating activity of the transcription factor.

[00109] The term "in vivo" refers to assays or methods that are performed on or in an organism, such as a multicellular animal. In certain aspects described herein, the method or use may be stated to be "in vivo" if a single cell organism such as a bacterium is used. The term "ex vivo" refers to methods and uses that are carried out using a living cell with an intact membrane located outside the body of a multicellular animal or plant, for example, explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissues or cells, including but not limited to blood cells.

[00110] The term "in vitro" refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cell extracts, and may refer to the introduction of a programmable synthetic biological circuit in a cell-free system, such as a cell-free environment. systems such as cell extracts.

[00111] As used herein, the term “local delivery” refers to the delivery of an active agent, such as interfering RNA (eg, siRNA), directly to a target site within an organism. For example, the agent may be locally delivered by direct injection into a disease site such as a tumor or other target site such as an inflammatory site or a target organ such as the liver, heart, pancreas, kidney, and the like.

[00112] As used herein, the expressions "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably herein and refer to an ITR that contains a mutation in at least one or more nucleotides compared to the WT-ITR from the same serotype. The mutation may result in a change in one or more of regions A, C, C', B, B' in the ITR, and may result in a change in three-dimensional spatial organization (i.e., three-dimensional structure in geometric space) compared to three-dimensional spatial organization by the WT-ITR organization from the same serotype.

[00113] As used herein, the term “ncDNA” or “nicked cccDNA” refers to closed-ended DNA containing a break or gap of 1-100 base pairs in the stem region or 5' spacer region located upstream of the open reading frame (e.g. , promoter and transgene for expression).

[00114] As used herein, the term “nucleic acid” refers to a polymer containing at least two nucleotides (i.e., a deoxyribonucleotide or ribonucleotide) in single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof. The DNA may be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. The DNA may be in the form of a minicircle, plasmid, bacmid, minigene, utility DNA (linear covalently closed DNA vector), closed end linear duplex DNA (CELiD or cccDNA), Doggybone™ DNA, dumbbell DNA, minimalistic vector for immunologically defined gene expression (MIDGE), viral vector or non-viral vectors. The RNA may be in the form of short interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof . Nucleic acids include nucleic acids containing known nucleotide analogues or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, that have binding properties similar to those of the reference nucleic acid. Examples of such analogues and/or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate-morpholine oligomer (morpholino), phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methyl ribonucleotides, locked nucleic acid (LNA™) and peptide nucleic acids ( PNK). Except as specifically stated, the term covers known analogues of natural nucleotides that have binding properties similar to those of the reference nucleic acid. Unless otherwise indicated, the specific nucleic acid sequence is also intended to include conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as explicitly stated sequence.

[00115] As used herein, “nucleotides” contain the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are connected to each other by the indicated phosphate groups.

[00116] As used herein, the terms “nucleic acid therapeutic,” “nucleic acid therapeutic,” and “TNA” are used interchangeably and refer to any therapeutic option using nucleic acids as the active component of a therapeutic agent for treating a disease or disorder. As used herein, these expressions refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, RNA interfering (RNAi), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (eg, lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-end linear duplex DNA (ccDNA/CELiD), plasmids, bacmids, Doggybone DNA vectors ™, minimalistic immunologically defined gene expression (MIDGE) vector, non-viral utility DNA vector (linear covalently closed DNA vector) or dumbbell minimal DNA vector (“dumbbell DNA”).

[00117] The term “promoter” as used herein refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of said nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Pro motors can be constitutive, inducible, repressible, tissue specific, or any combination of these. A promoter is a control region of a nucleic acid sequence that controls the initiation and rate of transcription of the rest of the nucleic acid sequence. A promoter may also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. In some embodiments of aspects described herein, a promoter may drive the expression of a transcription factor that controls the expression of the promoter itself, or the expression of another promoter used in another modular component of the synthetic biological circuits described herein. The promoter sequence contains the transcription initiation site, as well as protein-binding domains responsible for binding RNA polymerase. Eukaryotic promoters often, but not always, contain TATA boxes and CAT boxes. Various promoters, including inducible promoters, can be used to drive the expression of transgenes in cDNA vectors as described herein.

[00118] The term “enhancer” as used herein refers to a cis-acting regulatory sequence (e.g., 50-1500 base pairs in size) that binds one or more proteins (e.g., activator proteins or transcription factor ) to enhance transcriptional activation of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs 5' from the start site of the gene or 3' from the start site of the gene they regulate. The enhancer may be located within an intronic region or within an exonic region of an unrelated gene.

[00119] A promoter may be said to drive the expression or drive the transcription of the nucleic acid sequence that it regulates. As used herein, “operably linked” refers to a combination whose components so described are interrelated to enable them to function in the intended manner. For example, a promoter is operably linked to a coding sequence if said promoter affects its transcription or expression. The expressions "operably linked", "operably located", "operably linked", "under control" and "under transcriptional control" indicate that the promoter is in the correct functional position and/or orientation with respect to the nucleic acid sequence that it regulates. to control transcriptional initiation and/or expression of the specified sequence. "Inverted promoter" as used herein refers to a promoter in which the nucleic acid sequence is in reverse orientation such that a strand that was a sense strand becomes a non-coding strand and vice versa. Inverted promoter sequences can be used, according to various embodiments, to regulate the state of a switch. Additionally, in various embodiments, a promoter may be used in combination with an enhancer.

[00120] A promoter can be a promoter naturally associated with a gene or sequence, and can also be obtained by isolating 5' non-coding sequences located 5' in the direction of the coding segment and/or exon of a particular gene or sequence. Such a promoter may be called "endogenous". Likewise, in some embodiments, an enhancer may be an enhancer naturally associated with a nucleic acid sequence located either in the 5'-3' or 3'-5' direction of the sequence.

[00121] In some embodiments, the coding segment of the nucleic acid is located under the control of a “recombinant promoter” or “heterologous promoter”; both of these terms refer to a promoter not normally associated with the encoded nucleic acid sequence to which the promoter is operably associated in the natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a particular nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell; and synthetic promoters or enhancers that are not “naturally occurring,” i.e., contain other elements of other transcriptional regulatory regions, and/or mutations that alter expression obtained using genetic engineering methods known in the art . In addition to obtaining promoter and enhancer nucleic acid sequences synthetically, promoter sequences can be obtained using recombinant cloning and/or nucleic acid amplification technology, including PCR, in relation to synthetic biological circuits and modules as described herein (see, for example, US Pat. No. 4,683,202, US Pat. No. 5,928,906, each of which is incorporated herein by reference). In addition, it is also possible to use control sequences that direct transcription and/or expression of sequences in non-nuclear organelles such as mitochondria, chloroplasts, and the like.

[00122] As used herein, the terms “Rep binding site,” “Rep binding element,” “RBE,” and “RBS” are used interchangeably and refer to the binding site for a Rep protein (e.g., AAV Rep 78 or AAV Rep 68) that, upon binding by the protein Rep allows the Rep protein to perform site-specific endonuclease activity against a sequence including the RBS. The RBS sequence and its reverse complement together form one RBS. RBS sequences are known in the art and include, for example, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), the RBS sequence identified in AAV2. Any known RBS sequence may be used in embodiments of the present invention, including other known AAV RBS sequences and other known naturally occurring or synthetic RBS sequences. Without wishing to be bound by any theory, the nuclease domain of the Rep protein is believed to bind to the duplex nucleotide sequence GCTC, and accordingly, the two known AAV Rep proteins directly bind to the duplex oligonucleotide 5'-(GCGC)(GCTC)(GCTC)(GCTC) -3' (SEQ ID NO: 531), and provide stable assembly on it. In addition, soluble aggregated conformers (i.e., an indefinite number of interconnected Rep proteins) dissociate and bind to oligonucleotides that contain Rep binding sites. Each Rep protein interacts with both nitrogenous bases and phosphodiester backbones on each chain. Interactions with nitrogenous bases provide sequence specificity, whereas interactions with the phosphodiester backbone are nonspecific or less sequence specific and stabilize the protein-DNA complex.

[00123] As used herein, the term “reporter” refers to a protein that can be used to produce a detectable indicator. Reporters typically generate a measurable signal, such as fluorescence, color, or luminescence. Reporter protein coding sequences encode proteins whose presence in a cell or organism can be easily observed. For example, fluorescent proteins cause a cell to fluoresce when excited by light of a specific wavelength, luciferases cause a cell to catalyze a reaction that generates light, and enzymes such as β-galactosidase convert a substrate into a colored product. Examples of reporter polypeptides suitable for experimental or diagnostic use include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP), and other fluorescent proteins , chloramphenicol acetyltransferase (CAT), luciferase and others well known in the art.

[00124] As used herein, a “repressor protein” (“repressor protein”) or “inducer protein” is a protein that binds to a regulatory sequence element and represses or activates, respectively, the transcription of sequences operably linked to said regulatory sequence element . Preferred repressor and inducer proteins as described herein are sensitive to the presence or absence of at least one input agent or environmental factor. Preferred proteins as described herein are modular in form, containing, for example, separable DNA-binding and entry agent-binding or response elements or domains.

[00125] As used herein, the term “sequence identity” refers to the similarity between two nucleotide sequences. For the purposes of the present invention, the degree of sequence identity of two deoxyribonucleotide sequences is determined using the Needleman Wunsch algorithm (Needleman and Wunsch, 1970, supra), implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, above), preferably version 3.0.0 or later. Optional parameters used: pass opening penalty 10, pass extension penalty 0.5, and substitution matrix EDNAFULL (EMBOSS version from NCBI NUC4.4). The Needle output called "longest identical fragment length" (obtained using the "-nobrief" option) is used as percent identity and is calculated as follows: (Identical deoxyribonucleotides x 100)/(Alignment length Total number of gaps in alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[00126] As used herein, the terms “sense” and “antisense” refer to the orientation of a structural element in a polynucleotide. The semantic and antisense variants of the element are inversely complementary to each other.

[00127] As used herein, the term “spacer region” refers to an intervening sequence that separates functional elements in a vector or genome. In some embodiments, the AAV spacer regions keep two functional elements at a desired distance for optimal functionality. In some embodiments, the spencer regions provide or enhance the genetic stability of the vector or genome. In some embodiments, spacer regions facilitate prepared genetic manipulation of the genome by providing convenient locations for cloning sites and skipping the estimated number of base pairs. For example, in certain aspects, an oligonucleotide "polylinker" or "multiple cloning site" containing multiple restriction endonuclease sites, or a non-open reading frame sequence designed to not contain binding sites for a known protein (e.g., a transcription factor), may be placed in a vector or genome to separate cis-acting factors, for example, a 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 86-mer, 176-mer, etc. can be inserted.

[00128] The term “subject” as used herein refers to a human or animal subject to treatment, including prophylactic treatment, with a cccDNA vector in accordance with the present invention. Typically, said animal is a vertebrate such as, but not limited to, a primate, rodent, pet or game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, felines such as domestic cats, canids such as dogs, foxes, wolves, bird species such as chicken, emu, ostrich, and fish such as trout, catfish and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject may be male or female. In addition, the subject may be an infant or child. In some embodiments, the subject may be a newborn or unborn subject, for example, the subject is in utero. Preferably, said subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but these examples are not limiting. Non-human mammals may be usefully used as subjects in animal models of diseases and disorders. In addition, the methods and compositions described herein can be used for domesticated animals and/or companion animals. The human subject may be of any age, gender, race or ethnicity, such as Caucasian (White), Asian, African, Black, African American, Afro-European, Hispanic, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.

[00129] As used herein, the term “essentially symmetrical modified ITRs” or “essentially symmetrical mod-ITR pair” refers to a pair of modified ITRs within a single cccDNA genome or ccDNA vector, both of which are reverse complementary in sequence throughout their entire length. . For example, a modified ITR may be considered substantially symmetrical if it contains some nucleotide sequences that differ from the reverse complementary sequence, provided that such changes do not affect the properties and overall shape. One non-limiting example is a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the canonical sequence (as assessed by BLAST using default parameters), and has a symmetrical three-dimensional spatial organization with respect to the cognate modified ITR, such that their three-dimensional structures have the same shape in geometric space. In other words, a pair of essentially symmetrical modified ITRs contains loops A, C-C' and B-B' that are identically organized in three-dimensional space. In some embodiments, the ITRs of a mod-ITR pair may contain different reverse complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization, that is, both ITRs contain mutations that result in the same overall three-dimensional shape. For example, one ITR (eg, 5' ITR) in a mod-ITR pair may be from one serotype, and the other ITR (eg, 3' ITR) may be from a different serotype, yet both may have the same corresponding mutation ( for example, if the specified 5' ITR contains a deletion in the C region, a cognate modified 3' ITR from another serotype contains a deletion at the corresponding position in the C region), so that the specified pair of modified ITRs has the same symmetrical three-dimensional spatial organization. In such embodiments, each ITR in a pair of modified ITRs may be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) e.g., combinations of AAV2 and AAV6, wherein a modification in one ITR is reflected in a similar position in a cognate ITR from another serotype. In one embodiment, a substantially symmetric pair of modified ITRs is referred to as a modified ITR (mod-ITR) pair, provided that the nucleotide sequence differences between the ITRs do not affect the properties or overall shape and they have substantially the same shape in three-dimensional space. A non-limiting example is provided by a mod-ITR that is at least 95%, 96%, 97%, 98%, or 99% sequence identical to the canonical mod-ITR as assessed using standard methods well known in the art, such as BLAST ( Basic Local Alignment Search Tool, or BLASTN, at its default settings, also has a symmetrical three-dimensional spatial organization, so that their three-dimensional structure has the same shape in geometric space. An essentially symmetric mod-ITR pair contains identical loops A, C-C' and B-B' in three-dimensional space, for example, if the modified ITR in an essentially symmetric mod-ITR pair contains a deletion of the C-C' arm, then the cognate mod- The ITR contains a corresponding deletion of the C-C' arm and also has a similar three-dimensional structure of the remaining loops, A and B-B', the same shape in geometric space as the cognate mod-ITR.

[00130] As used herein, the term “substantially symmetrical WT-ITRs” or “substantially symmetrical WT-ITRs pair” refers to a pair of WT-ITRs within a single cccDNA genome or ccDNA vector, where both ITRs are wild-type ITRs , which have a reverse complementary sequence along their entire length. For example, an ITR may be considered a wild-type sequence even if it contains one or more nucleotides that differ from nucleotides in the canonical naturally occurring sequence, provided that such changes do not affect the properties and overall three-dimensional structure of the sequence. In some aspects, said differing nucleotides represent conservative sequence changes. A non-limiting example is a sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the canonical sequence (as assessed, for example, by BLAST using default parameters), and also has a symmetrical three-dimensional spatial organization relative to another WT-ITR such that their 3D structure has the same shape in geometric space. This substantially symmetrical WT-ITR contains the same loops A, C-C' and B-B' in three-dimensional space. An essentially symmetrical WT-ITR can be functionally confirmed to be WT by determining whether it contains a functional Rep binding site (RBE or RBE') and a terminal resolution site (trs) that pairs with the corresponding Rep protein. Others may optionally be tested functions, including transgene expression under permissive conditions.

[00131] As used herein, the term “suppress,” “reduce,” “influence,” “inhibit,” and/or “reduce” (and other similar terms) generally refers to the act of reducing, directly or indirectly, a concentration, level, function, activity, behavior relative to the natural, expected, average, or relative to the control state.

[00132] As used herein, the term “symmetrical ITR” refers to a pair of ITRs within a single cccDNA genome or cccDNA vector that are mutated or modified relative to wild-type dependoviral ITR sequences and are reverse complementary throughout their entire length. Neither ITR is the sequence of the wild-type AAV2 ITR (ie, they are a modified ITR, also called a mutant ITR), and may contain sequence differences from the wild-type ITR due to addition, deletion, substitution, truncation, or point mutations of nucleotides. For convenience, herein the ITR located 5' upstream of the expression cassette in the cccDNA vector is referred to as the "5' ITR" or "left ITR", and the ITR located 3' downstream of the expression cassette in the cccDNA vector. -vector, is called the “3' ITR” or “right ITR”.

[00133] As used herein, the terms “synthetic AAV vector” and “synthetic production of AAV vector” refer to the AAV vector and methods for producing it synthetically in a completely cell-free environment.

[00134] As used herein, the term “terminal repeat” or “TR” includes any viral terminal repeat or any synthetic sequence that contains at least one minimum required origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence (“RBS”) (also called the RBE (Rep binding element)) and the terminal resolution site (“TRS”) together form the “minimum required origin of replication” and, accordingly, the TR contains at least one RBS and at least at least one TRS. Each of the TRs that are inversely complementary to each other within a specific stretch of polynucleotide sequence is generally referred to as an “inverted terminal repeat” or “ITR.” In the context of a virus, ITRs mediate viral replication, packaging, integration, and proviral rescue. As has been unexpectedly discovered herein, ITRs other than wild-type dependoviral ITRs are still capable of performing the traditional functions of wild-type ITRs, and accordingly, the term ITR as used herein refers to a TR in a cccDNA genome or a cccDNA vector. which is capable of mediating the replication of the cDNA vector. One skilled in the art will appreciate that in complex cDNA vector configurations, more than two ITRs or symmetrical pairs of ITRs may be present. Said ITR may be an AAV ITR or a non-AAV ITR, or may be derived from an AAV ITR or a non-AAV ITR. For example, the ITR may be from the Parvoviridae family, which includes parvoviruses and dependoviruses (eg, canine parvovirus, bovine parvovirus, murine parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as an origin of replication SV40 may be used as an ITR, which may further be modified by truncation, substitution, deletion, insertion and/or addition. The Parvoviridae family of viruses consists of two subfamilies: Parvovirmae, which infect vertebrates, and Densovirinae, which infect invertebrates. Typically, these ITRs consist of approximately 145 nucleotides and are naturally inverted relative to each other. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that are capable of replication in vertebrate hosts, including, but not limited to, humans, primates, cattle, canines, horses, and sheep.

[00135] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably and refer to the region in which Rep forms a tyrosine phosphodiester bond with a 5'-thymidine, forming a 3' OH that serves as a substrate for DNA extension by cellular DNA polymerase, for example DNA polymerase delta or DNA polymerase epsilon. Alternatively, the Rep-thymidine complex may participate in a coordinated ligation reaction. In some embodiments, the TRS spans at least an unpaired thymidine base. In some embodiments, the nicking efficiency of the TRS can be controlled at least in part by the distance between it and the RBS within a single molecule. If the acceptor substrate is a complementary ITR, the resulting product is an intramolecular duplex. TRS sequences are known in the art and include, for example, 5'-GGTTGA-3' (SEQ ID NO: 45), a hexanucleotide sequence identified in AAV2. Any known TRS sequence may be used in embodiments of the present invention, including other known AAV TRS sequences and other known natural or synthetic TRS sequences such as AGTT (SEQ ID NO: 46), GGTTGG (SEQ ID NO: 47), AGTTGG (SEQ ID NO: 48), AGTTGA (SEQ ID NO: 49), and other motifs such as RRTTRR (SEQ ID NO: 50).

[00136] As used herein, the term “transcriptional regulator” refers to transcriptional activators and repressors that either activate or repress transcription of a gene of interest. Promoters are regions of nucleic acid that initiate transcription of a particular gene. Transcription activators typically bind in the vicinity of transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind to transcriptional promoters and sterically hinder the initiation of transcription by RNA polymerase. Other transcriptional regulators can serve as either activators or repressors depending on their site of binding and cellular and environmental conditions. Non-limiting examples of classes of transcriptional regulators include, but are not limited to, homeodomain proteins, zinc finger proteins, Forkhead proteins, and leucine zipper proteins.

[00137] As used herein, the terms “treat” and/or “treating” include suppressing, substantially inhibiting, slowing or reversing the progression of a condition, substantially alleviating clinical symptoms of a condition, or substantially preventing the onset of clinical symptoms of a condition, obtaining favorable or desired clinical results . Treatment also refers to achieving one or more of: (a) reducing the severity of a disorder; (b) limiting the development of symptoms characteristic of the disorder or disorders being treated; (c) limiting the worsening of symptoms characteristic of the disorder or disorders being treated; (d) limiting the recurrence of the disorder or disorders in patients who previously had the disorder or disorders; and (f) limiting the recurrence of symptoms in patients who have not previously had symptoms of the specified disorder or disorders.

[00138] Beneficial or desired clinical results, such as pharmacological and/or physiological effects, include, but are not limited to, preventing the onset of a disease, disorder or condition in a subject who may be predisposed to, but does not yet experience, the disease, disorder or condition. symptoms or does not yet exhibit symptoms of a specified disease (preventative treatment), relief of symptoms of a specified disease, disorder or condition, reduction in the severity of a disease, disorder or condition, stabilization (i.e., no worsening) of a specified disease, disorder or condition, prevention the spread of the specified disease, disorder or condition, the delay or slowing of the specified disease, disorder or condition, the alleviation or temporary relief of the specified disease, disorder or condition, and combinations of the above, and the increase in the duration of survival compared to expected survival without treatment.

[00139] As used herein, the terms “therapeutic amount,” “therapeutically effective amount,” “effective amount,” or “pharmaceutically effective amount” of an active agent (e.g., a cccDNA lipid particle as described herein) are used interchangeably and refer to the amount sufficient to provide the intended benefit of treatment. Dosage levels are based on various factors, including the type of injury, age, body weight, gender, medical condition of the patient, severity of the condition, route of administration, and the specific active agent used. Accordingly, the dosage regimen may vary significantly, but may determined in the usual way by the attending physician using standard methods. In addition, the terms “therapeutic amount”, “therapeutically effective amount” and “pharmaceutically effective amount” include prophylactic or prophylactic amounts of the compositions of the present invention. In prophylactic or prophylactic use of the present invention, pharmaceutical compositions or medicaments are administered to a patient susceptible to or otherwise at risk of developing a disease, disorder or condition, in sufficient quantities to eliminate or reduce the risk of, reduce the severity of, or delay the onset of said disease, disorder or condition, including biochemical, histological and/or behavioral symptoms of the specified disease, disorder or condition, its complications, and intermediate pathological phenotypes that occur during the development of the specified disease, disorder or condition. It is usually preferable to use the maximum dose, that is, the highest safe dose according to some medical opinion. The terms “dose” and “dosage” are used interchangeably herein.

[00140] As used herein, the term “therapeutic effect” refers to the effects of treatment, the results of which are considered desirable and beneficial. The therapeutic effect may include, directly or indirectly, stopping, reducing or eliminating the disease. The therapeutic effect may also include, directly or indirectly, stopping, reducing or eliminating the progression of disease manifestations.

[00141] For any therapeutic agent, a therapeutically effective amount described herein may initially be determined based on preliminary in vitro studies and/or animal models. The therapeutically effective dose may also be determined based on human data. The dose used may be adjusted based on the relative bioavailability and potency of the compound administered. Dosage adjustments to achieve maximum effectiveness based on the methods described above and other well known methods are within the ability of one skilled in the art. The general principles for determining therapeutic efficacy, which can be found in Chapter 1 of the manual: Goodman and Gilman, The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.

[00142] Pharmacokinetic principles provide the basis for modifying dosing regimens to obtain the desired degree of therapeutic efficacy with a minimum of unacceptable adverse events. In situations where plasma drug concentrations can be measured and related to the therapeutic window, additional recommendations for dosage modification may be provided.

[00143] As used herein, the terms “vector” or “expression vector” refer to a replicon, such as a plasmid, bacmid, phage, virus, virion or cosmid, to which another DNA segment can be attached, i.e. "insertion" of a "transgene" or "expression cassette" in such a way as to allow expression or replication of the attached segment ("expression cassette") in the cell. A vector may be a nucleic acid construct designed for delivery into a host cell or for transfer between different host cells. According to the present invention, the vector may be viral or non-viral in origin in the final form. For the purposes of the present invention, "vector" generally refers to a synthetic AAV vector or a nicked cccDNA vector. Accordingly, the term “vector” includes any genetic element that is capable of replication when associated with appropriate control elements and that can transfer gene sequences into cells. In some embodiments, the vector may be a recombinant vector or an expression vector.

[00144] As used herein, the term “recombinant vector” refers to a vector that includes a heterologous nucleic acid sequence, or “transgene,” that is capable of expression in vivo. It should be understood that the vectors described herein may, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a method of maintaining a nucleotide sequence of interest in a subject in a high copy number of extrachromosomal DNA, thereby eliminating potential effects of chromosomal integration.

[00145] As used herein, “wild-type ITR” or “WT-ITR” refers to a sequence from a naturally occurring ITR sequence in an AAV or other dependovirus that retains, for example, Rep binding activity and Rep nicking ability. The WT-ITR nucleotide sequence from any AAV serotype may differ slightly from the canonical naturally occurring sequence due to genetic code degeneracy or drift, and accordingly, WT-ITR sequences provided for use herein include WT-ITR sequences derived from as a result of naturally occurring changes that occur during the production process (for example, replication errors).

[00146] As used herein, the term “comprising” or “comprising” is used to refer to compositions, methods, and their respective component(s) essential to the specified method or composition, but capable of including unspecified elements, significant or not.

[00147] As used herein, the term “consisting essentially of” refers to elements required for a particular implementation. The specified term allows for the presence of elements that do not significantly affect the basic and new or functional characteristic(s) of the specified embodiment.

[00148] The term “consisting of” refers to compositions, methods and their respective components as described herein, which do not include any elements not specified in the above description of an embodiment.

[00149] As used herein and in the accompanying claims, terms in the singular include their plural equivalents unless the context clearly indicates otherwise. Accordingly, for example, “method” includes one or more methods and/or steps of the type described herein and/or those that would be apparent to those skilled in the art upon reading the present specification, and the like. Likewise, the term “or” includes “and” unless the context clearly requires otherwise. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. The expression “for example” as used herein indicates a non-limiting example. Accordingly, the expression “for example” is synonymous with the expression “for example”.

[00150] As used herein, the terms “such as,” “for example,” and the like are used. are exemplary embodiments and are not intended to limit the scope of the present invention.

[00151] Unless otherwise specified, all technical and scientific terms used herein have their usual meaning known to one skilled in the art to which the invention relates. Although any methods and materials similar or equivalent to those described herein may be used in the practice and testing of the present invention, the preferred materials and methods are those described herein.

[00152] Except in the worked examples or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified by the term “about” in all cases. The term "about" when used in relation to a percentage may mean ±1%. The present invention is further explained in detail by the following examples, but they are not intended to limit the scope of the present invention.

[00153] It should be understood that the present invention is not limited to the specific techniques, protocols and reagents, etc. described herein and, thus, may vary. The terminology used herein is intended solely to describe specific embodiments, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

II. Closed-end DNA vectors (ccDNA)

[00154] The present invention provides novel non-viral capsidless ccDNA molecules with covalently closed ends (ccDNA). cccDNA vectors as described herein do not have packaging restrictions imposed by limited space within the viral capsid. cccDNA vectors represent a promising eukaryotic-produced alternative to prokaryotic-produced plasmid DNA vectors other than encapsulated AAV genomes. This allows for the insertion of control elements, eg, regulatory switches as described herein, large transgenes, multiple transgenes, etc., and the incorporation of native genetic regulatory elements into a transgene, if desired.

[00155] There are many structural features of cDNA vectors that distinguish them from plasmid-based expression vectors. cccDNA vectors may have one or more of the following characteristics: the absence of original (i.e., not inserted) bacterial DNA, the absence of a prokaryotic origin of replication, autonomy, i.e., they do not require any sequences other than two ITRs, including Rep binding sites and terminal resolution sites (RBS and TRS), and exogenous sequence between ITRs, the presence of ITR sequences that form hairpins are of eukaryotic origin (i.e., they are produced in eukaryotic cells), and the absence of bacterial-type DNA methylation or , essentially any other methylation perceived as abnormal by the mammalian host. In general, it is preferred that the inventive vectors do not contain any prokaryotic DNA, but it is contemplated that certain prokaryotic DNA may be inserted as an exogenous sequence, according to a non-limiting example, in a promoter or enhancer region. Another important feature that distinguishes cccDNA vectors from plasmid expression vectors is that cccDNA vectors are single-stranded linear DNA with closed ends, while plasmids are always double-stranded DNA.

[00156] The use of a cccDNA vector as described herein provides several advantages over plasmid-based expression vectors. Such advantages include, but are not limited to, the following: 1) Plasmids contain bacterial DNA sequences and are subject to specific prokaryotic methylation, such as 6-methyl-adenosine and 5-methyl-cytosine methylation, whereas the capsidless AAV vector sequences are of eukaryotic origin and do not undergo specific prokaryotic methylation; as a result, capsidless AAV vectors are less likely to induce inflammatory and immune responses compared to plasmids; 2) plasmids require the presence of a resistance gene during production, but cccDNA vectors do not; 3) a circular plasmid is not delivered to the nucleus when introduced into a cell and requires excessive dosing to avoid degradation by cellular nucleases, while cDNA vectors contain viral cis-elements, i.e., modified ITRs, conferring resistance to nucleases, and can be designed to target and deliver to the nucleus. It has been proposed that the minimal defining elements required for ITR function are the Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) for AAV2) and the terminal resolution site (TRS; 5'-AGTTGG-3 ' (SEQ ID NO: 48) for AAV2) plus a variable palindromic sequence providing hairpin formation. In contrast, transduction with capsidless AAV vectors as described herein can effectively target cell and tissue types that are difficult to transduce with standard AAV virions using different delivery reagents.

[00157] cccDNA vectors preferably have a linear and continuous structure rather than a discontinuous structure, as assessed using restriction enzyme digestion and electrophoretic analysis (Figure 5D). A linear and continuous structure is thought to be more resistant to attack by cellular endonucleases and is also less likely to undergo recombination and cause mutagenesis. Accordingly, a cccDNA vector having a linear and continuous structure is a preferred embodiment. A continuous linear single-stranded cccDNA vector with an intramolecular duplex may have covalently linked ends without AAV capsid protein coding sequences. These cDNA plasmids are structurally different from plasmids (including the cDNA plasmids described herein), which are circular duplex nucleic acid molecules of bacterial origin. The complementary strands of plasmids can be separated after denaturation to produce two nucleic acid molecules, while, on the contrary, cccDNA vectors containing complementary strands are represented by a single DNA molecule and, accordingly, even after denaturation remain as a single molecule. In some embodiments, cccDNA vectors as described herein can be produced without prokaryotic DNA base methylation, unlike plasmids. Accordingly, cccDNA vectors and ccDNA plasmids differ both in structure (in particular, linear or circular), as well as in the methods used to obtain and purify these different objects (see below), as well as in DNA methylation, which occurs in a prokaryotic manner in in the case of cDNA plasmids and eukaryotic type in the case of cDNA vector.

[00158] The present invention provides non-viral capsidless ccDNA molecules with covalently closed ends (ccDNA). These non-viral, capsid-free cccDNA molecules can be produced in permissive host cells from an expression construct (e.g., a cccDNA plasmid, ccDNA bacmid, ccDNA baculovirus, or an integrated cell line) containing a heterologous gene (transgene) located between two symmetrical inverted terminal repeat (ITR) sequences, wherein said ITRs are not wild-type ITRs and are symmetrical with respect to each other. Thus, these ITRs are modified ITRs, and the 3' ITR sequence is the reverse complement of the 5' ITR sequence, and vice versa. In some embodiments, the ITR is modified by deletion, insertion, and/or substitution relative to the wild-type ITR sequence (eg, AAV ITR). In some embodiments, the modified ITR contains a functional terminal resolution site (trs) and a Rep binding site. The cccDNA vector is preferably duplex, eg, self-complementary, over at least part of the molecule, such as an expression cassette (eg, the ccDNA is not a double-stranded circular molecule like a plasmid). The cccDNA vector has covalently closed ends and is thus resistant to exonuclease digestion (eg, exonuclease I or exonuclease III digestion), for example, for an hour at 37°C.

[00159] In one aspect, the cccDNA vector comprises, in the 5' to 3' direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR. According to one embodiment, said first ITR (5' ITR) and second ITR (3' ITR) are asymmetrical to each other, that is, they have different three-dimensional spatial configurations. In an example embodiment, the first ITR may be a wild-type ITR and the second ITR may be a mutated or modified ITR, or vice versa, where the first ITR may be a mutated or modified ITR and the second ITR may be a wild-type ITR. In one embodiment, the first ITR and the second ITR are both mutated or modified but are different sequences, or contain different modifications, or are non-identical mutated or modified ITRs, and have different three-dimensional spatial configurations. In other words, a cccDNA vector with asymmetric ITRs contains ITRs that differ in that any changes in one ITR relative to the WT-ITR are not reflected in the other ITR; or, alternatively, that the asymmetric ITRs in a pair of mutated or modified asymmetric ITRs may have a different sequence and a different three-dimensional shape.

[00160] In some embodiments, the cccDNA vector comprises, in the 5' to 3' direction: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, where the first ITR and second ITR are symmetrical to each other, that is, they are the same sequence, but are reverse complementary to each other. That is, the cccDNA vector may contain ITR sequences that have a symmetrical three-dimensional spatial organization such that their structure has the same shape in geometric space, or contains the same A, C-C' and B-B' loops in three-dimensional space. In such an embodiment, the pair of symmetrical ITRs or the pair of substantially symmetrical ITRs may be mutated or modified ITRs that are not wild-type ITRs. In an example embodiment, the first ITR may be a wild-type ITR and the second ITR may be a mutated or modified ITR, or vice versa, where the first ITR may be a mutated or modified ITR and the second ITR a wild-type ITR. In one embodiment, both of the ITRs, the first ITR and the second ITR, are modified but are different sequences, or contain different modifications, or are non-identical modified ITRs, and have different three-dimensional spatial configurations. In another example embodiment, the first ITR (or 5 TTR) may be a modified ITR, e.g., SEQ ID NO: 484 (i.e., ITR-33, left), and the second ITR (or 3' ITR) may be a mutated or a modified ITR, for example, SEQ ID NO: 469 (ie, ITR-18, right). In other words, a cccDNA vector with asymmetric ITRs contains ITRs that differ in that any changes in one ITR relative to the WT-ITR are not reflected in the other ITR; or, alternatively, where the asymmetric ITRs in a pair of modified asymmetric ITRs may have a different sequence and a different three-dimensional shape. The ITRs in a pair of mutated or modified ITRs may have the same sequence, which contains one or more modifications relative to the wild type ITR, and be reverse complementary (inverted) to each other. According to one embodiment of the claims, the ITRs in a pair of modified ITRs are substantially symmetrical as defined herein, that is, the ITRs in a pair of modified ITRs may have a different sequence but a similar or identical symmetrical three-dimensional shape. In some embodiments, the symmetrical ITRs or substantially symmetrical ITRs are wild-type ITRs (WT-ITRs) as described herein. Accordingly, both ITRs have wild-type sequence but do not necessarily have to be WT-ITRs from the same AAV serotype. In one embodiment, one WT-ITR can be taken from one AAV serotype, and the other WT-ITR can be taken from a different AAV serotype. In such an embodiment, the ITRs in a WT-ITR pair are substantially symmetrical as defined herein, that is, they may contain a modification of one or more conserved nucleotides while maintaining a symmetrical three-dimensional spatial organization.

[00161] In some embodiments, the cccDNA vector comprises, in the 5' to 3' direction: a wild-type adeno-associated virus (AAV) first inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein ) and a second AAV ITR, wherein the first WT-ITR and the second WT-ITR are from the same AAV serotype or from different AAV serotypes. In an example embodiment, the first WT-ITR (or 5' WT-ITR) may be taken from AAV 2, and the second WT-ITR (or 3' WT-ITR) may be taken from AAV6. Examples of WT-ITR in a cDNA vector and for use in making a cDNA plasmid are presented below in the section entitled “ITR” and in Table 1 herein.

[00162] Wild-type ITR sequences of the present invention are DNA sequences included in an expression construct (eg, ccDNA plasmid, ccDNA baculovirus) to produce a ccDNA vector.

[00163] In some embodiments, a cccDNA vector described herein comprises an expression cassette with a transgene, which may be, for example, a regulatory sequence, a nucleic acid coding sequence (e.g., such as a miR or antisense sequence), or a nucleic acid coding sequence. polypeptide (eg, such as a transgene). In one embodiment, the transgene may be operably linked to one or more regulatory sequences that provide or control expression of the transgene. In one embodiment, said polynucleotide comprises a first WT-ITR sequence and a second WT-ITR sequence, wherein the nucleotide sequence of interest is flanked by said first and second WT-ITR sequences.

[00164] Examples of ITRs are discussed below in the section entitled "ITR" and in Tables 2 and 5 herein, or in Tables 2, 3, 4, 5, 6, 7, 8, 9 or 10A-10B of the PCT/ US 18/49996, filed September 7, 2018, where the ITR flanking sequences are symmetrical (eg, reverse complementary) or substantially symmetrical.

[00165] The ITR sequences of the present invention are DNA sequences included in an expression construct (eg, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus) to produce a ccDNA vector. Accordingly, the ITR sequences actually contained in a cccDNA vector produced from a cccDNA plasmid or other expression construct may or may not be identical to the ITR sequences of the present invention as a result of naturally occurring variations (including conserved and non-conservative modifications) occurring during production (for example, as a result of a replication error).

[00166] In some embodiments, a cccDNA vector described herein containing a transgene expression cassette that is a therapeutic nucleic acid sequence may be operably linked to one or more regulatory sequences that provide or control expression of the transgene. In one embodiment, said polynucleotide comprises a first ITR sequence and a second ITR sequence, characterized in that the nucleotide sequence of interest is flanked by first and second ITR sequences, and said first and second ITR sequences are asymmetric to each other or symmetric to each other.

[00167] In one embodiment, the expression cassette is located between two ITRs and contains, in that order, any one or more of the following: a promoter operably linked to the transgene, a post-transcriptional regulatory element; and polyadenylation and termination signal. In one embodiment, said promoter is regulated - inducible or repressible. A promoter can be any sequence that facilitates transcription of a transgene. In one embodiment, said promoter is a CAG promoter (eg, SEQ ID NO: 03) or a variant thereof. Said post-transcriptional regulatory element is a sequence that modulates the expression of a transgene, in a non-limiting example, any sequence forming a tertiary structure that enhances the expression of a transgene that is a therapeutic nucleic acid molecule.

[00168] In one embodiment, said post-transcriptional regulatory element comprises a WPRE (eg, SEQ ID NO: 8). In one embodiment, said polyadenylation and termination signal comprises BGHpolyA (eg, SEQ ID NO: 9). Additionally, any cis-regulatory element known in the art, or a combination thereof, can be used, for example, the SV40 late polyadenylation signal 5' enhancer sequence (USE) or other post-transcriptional processing elements, including, but not limited to, the gene thymidine kinase of herpes simplex virus or hepatitis B virus (HBV). In one embodiment, the length of the expression cassette in the 5'-3' direction is greater than the known maximum length of sequence enclosed in the AAV virion capsid. In one embodiment, the length is greater than 4.6 kilobases, or greater than 5 kilobases, or greater than 6 kilobases, or greater than 7 kilobases. Various examples of expression cassettes are presented herein.

[00169] Said expression cassette may contain more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any number of nucleotides ranging from about 4,000 to 10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, said expression cassette may comprise a transgene (eg, a therapeutic nucleic acid sequence) having a length in the range of 500 to 50,000 nucleotides. In some embodiments, said expression cassette may comprise a transgene (eg, a therapeutic nucleic acid sequence) having a length ranging from 500 to 75,000 nucleotides in length. In some embodiments, said expression cassette may comprise a transgene (eg, a therapeutic nucleic acid sequence) having a length ranging from 500 to 10,000 nucleotides. In some embodiments, said expression cassette may comprise a transgene (eg, a therapeutic nucleic acid sequence) having a length in the range of 1000 to 10,000 nucleotides. In some embodiments, said expression cassette may comprise a transgene (eg, a therapeutic nucleic acid sequence) having a length in the range of 500 to 5,000 nucleotides. These cccDNA vectors do not have the size restrictions characteristic of capsid-enclosed AAV vectors, which allows delivery of a significant size expression cassette to ensure efficient transgene expression. In some embodiments, the cDNA vector does not contain prokaryote-specific methylation.

[00170] In some embodiments, the expression cassette may also comprise an internal ribosome entry site (IRES) and/or a 2A element. Cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a miR-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, the ITR may act as a promoter for the transgene. In some embodiments, the cccDNA vector contains additional components that regulate the expression of the transgene, for example, a regulatory switch described herein in the section entitled "Regulatory Switches" for controlling and regulating the expression of the transgene, and may contain, if desired, a regulatory switch. a switch that provides a “kill switch” to ensure controlled death of the cell containing the cccDNA vector.

[00171] In FIG. 1A-1B are schematic representations of non-limiting examples of cccDNA vectors or corresponding ccDNA plasmid sequences. cccDNA vectors are capsidless and can be derived from a plasmid encoding, in this order: a first ITR, an expressed transgene cassette, and a second ITR, wherein the first and second ITR sequences are mutated relative to the corresponding wild-type AAV2 ITR sequence, and the mutations are the same (i.e. i.e. modified ITRs are symmetrical). The expressed transgene cassette preferably includes one or more of the following elements, in this order: an enhancer/promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (eg, WPRE) and a polyadenylation and termination signal (eg, poly(A) BGH).

[00172] In FIG. 2A-2B are schematic representations of non-limiting examples of cccDNA vectors or corresponding ccDNA plasmid sequences. cccDNA vectors are capsidless and can be derived from a plasmid encoding, in this order: a first WT-ITR, an expressed transgene cassette, and a second WT-ITR. In some embodiments, the first and second ITR sequences are the wild-type AAV2 ITR sequence. In some embodiments, said first and second ITR sequences are selected from any combination of WT-ITRs presented in Table 1. Said expressed transgene cassette preferably includes, in that order, any one or more of the following: an enhancer/promoter or a regulatory switch, ORF reporter (transgene), post-transcriptional regulatory element (eg, WPRE), and polyadenylation and termination signal (eg, poly(A) BGH).

[00173] In FIG. 1A-1C from international application PCT/US2018/050042, filed September 7, 2018 and incorporated herein by reference in its entirety, provides a schematic representation of a non-limiting example of ccDNA vectors or corresponding ccDNA plasmid sequence. cccDNA vectors are capsidless and can be derived from a plasmid encoding, in that order: a first ITR, an expressed transgene cassette, and a second ITR, wherein at least one of the first and/or second ITR sequences is mutated relative to the corresponding wild-type AAV2 ITR sequence. Said expressed transgene cassette preferably includes, in that order, one or more of the following: an enhancer/promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., poly(A) BGH).

Therapeutic nucleic acids

[00174] Said expression cassette may contain any transgene of interest. Transgenes of interest include, but are not limited to, polypeptide-encoding nucleic acids or non-coding nucleic acids (e.g., RNAi, miR, etc.), preferably therapeutic (e.g., for medical, diagnostic, or veterinary applications) or immunogenic polypeptides (for example, for vaccines) polypeptides. In some embodiments, the transgenes in the expression cassette encode one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof.

[00175] Exemplary therapeutic nucleic acids of the present invention may include, but are not limited to, minigenes, plasmids, minicircles, short interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, double-stranded DNA (eg , cccDNA, CELiD, linear covalently closed DNA ("utility"), DNA doggybone™, protelomeric DNA with closed ends or dumbbell-shaped linear DNA), dsRNA Dicer substrate, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (siRNA), mRNA, tRNA, rRNA and viral DNA vectors, viral RNA vector; and any combination of the above.

[00176] siRNAs or siRNAs that can down-regulate intracellular levels of specific proteins through a process called RNA interference (RNAi) are also contemplated by the present invention as nucleic acid-based therapeutics. Once the siRNA or siRNA is introduced into the cytoplasm of the host cell, these double-stranded RNA constructs bind to a protein called RISC. The RISC complex removes the sense strand of miRNA or siRNA. The RISC complex, in combination with complementary mRNA, cleaves the mRNA and releases the cut strands. RNAi is caused by the specific destruction of mRNA, which leads to down-regulation of the corresponding protein.

[00177] Antisense oligonucleotides (ASOs) and ribozymes that inhibit translation of mRNA into protein can be nucleic acid-based therapeutics. In the case of antisense constructs, these single-stranded deoxynucleic acids have a sequence complementary to the mRNA sequence of the target protein and are capable of binding to the mRNA through Watson-Crick base pairing. This binding prevents translation of the target mRNA and/or triggers the destruction of the mRNA transcript by RNase H. As a result, the antisense oligonucleotide has increased specificity of action (ie, downregulation of a specific disease-associated protein).

[00178] In any of the methods provided herein, the therapeutic nucleic acid may be a therapeutic RNA. The therapeutic RNA may be an mRNA translation inhibitor, an RNA interference (RNAi) agent, a catalytically active RNA molecule (ribozyme), transfer RNA (tRNA), or RNA that binds an mRNA transcript (ASO), protein, or other molecular ligand (aptamer). . In any of the methods provided herein, the RNAi agent may be double-stranded RNA, single-stranded RNA, micro RNA, short interfering RNA, short hairpin RNA, or triplex oligonucleotide.

[00179] In some embodiments, the transgene is a therapeutic gene or marker protein. In some embodiments, the transgene is an agonist or antagonist. In some embodiments, said antagonist is an antibody mimetic or antibody fragment, or an antigen-binding fragment thereof, e.g., a neutralizing antibody or antibody fragment, or the like. In some embodiments, the transgene encodes an antibody, including a full-length antibody or antibody fragment as defined herein. In some embodiments, the antibody is an antigen binding domain or an immunoglobulin variable domain sequence as defined herein.

In particular, the transgene may encode one or more therapeutic agents, including, but not limited to, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, and also variants and/or active moieties thereof for use in the treatment, prevention and/or alleviation of one or more symptoms of diseases, dysfunctions, injuries and/or disorders. Examples of transgenes are described herein in the section entitled "Method of Treatment".

III. Inverted terminal repeats (ITRs)

[00180] As described herein, in one embodiment, said cccDNA vectors are capsidless linear duplex DNA molecules formed by a continuous strand of complementary DNA with covalently closed ends (linear, contiguous, and non-capsidated structure) that contain an inverted 5' terminal repeat (ITR) sequence and an inverted 3' ITR sequence that are different or asymmetrical. In some embodiments, the ITR sequences are wild-type ITR sequences. In some embodiments, the ITR sequences are not wild-type ITR sequences. In some embodiments, the ITR sequences are modified ITR sequences.

[00181] In some embodiments, the cccDNA vectors contain a heterologous gene located between two wild-type inverted terminal repeat (WT-ITR) flanking sequences that are either reverse complementary (inverted) to each other, or, alternatively, substantially are symmetrical - that is, the ITRs in the WT-ITR pair have a symmetrical three-dimensional spatial organization. In some embodiments, the wild-type ITR sequence (e.g., AAV WT-ITR) contains functional Rep binding sites (RBS; 5′-CCCCGCTCGCTCGCTC-3′ for AAV2, SEQ ID NO: 531) and a functional terminal resolution site (TRS; e.g. 5'-AGTT-3', SEQ ID NO: 46).

[00182] In some embodiments, the cccDNA vectors comprise a heterologous gene located between two modified inverted terminal repeat (mod-ITR) flanking sequences that are reverse complementary (inverted) to each other, or substantially symmetrical as defined herein (i.e. contain similar modifications) and are not wild-type ITRs. These reverse complementary ITRs are called symmetric ITRs. These ITRs are modified from existing naturally occurring wild-type parvovirus ITRs (eg, AAV ITRs) by deletion, insertion and/or substitution; and may include a functional Rep binding site (RBS; e.g. 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: 531) and a functional terminal resolution site (TRS; e.g. 5'-AGTT-3', SEQ ID NO: 46) .

[00183] In some embodiments, the ITR sequence may be taken from viruses of the Parvoviridae family, which includes two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect insects. The subfamily Parvovirinae (called parvoviruses) includes the genus Dependovirus, members of which, in most cases, require coinfection with a helper virus, such as adenovirus or herpes virus, for productive infection. The genus Dependovirus includes adeno-associated virus (AAV), which commonly infects humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g. adeno-associated viruses of cattle, canines, horses, sheep). For a general description of parvoviruses and other members of the family Parvoviridae, see Kenneth I. Bems, "Parvoviridae: The Viruses and Their Replication," chapter 69 of FIELDS VIROLOGY (3rd edition, 1996). One skilled in the art will appreciate that the ITR of any known parvovirus can be used in the compositions and methods as described herein. In some embodiments, said ITRs are from a dependovirus such as AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ genome, and AAV -DJ8 For example, NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), chimeric ITRs or ITRs from any synthetic AAV. In some embodiments, the AAV is capable of infecting warm-blooded animals, such as avian adeno-associated viruses (AAAV), bovine adeno-associated virus (BAAV), canine, equine, and sheep. In some embodiments, the ITR is derived from parvovirus B19 (GenBank accession no. NC 000883), mouse small virus (MVM) (GenBank accession no. NC 001510); goose parvovirus (GenBank NC accession number 001701); snake parvovirus 1 (GenBank accession number NC 006148).

[00184] In some embodiments, the selection of a serotype may be based on the tissue tropism of the specified serotype. AAV2 has broad tissue tropism, AAV1 predominantly targets neurons and skeletal muscle, and AAV5 predominantly targets neurons, retinal pigment epithelium, and photoreceptors. AAV6 predominantly targets skeletal muscle and lungs. AAV8 predominantly targets tissues of the liver, skeletal muscle, heart, and pancreas. AAV9 predominantly targets liver, skeletal, and lung tissue. In some embodiments, the serotype is AAV2.

[00185] One skilled in the art will recognize that ITR sequences have a general structure containing a double-stranded Holliday structure, which is typically a T-shaped or Y-shaped hairpin structure (see, for example, FIG. 3A and FIG. 4A), with each ITR formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), and a single-stranded sequence D (wherein the order of these palindromic sequences determines the orientation of the ITR), and accordingly, modified ITR sequences from any AAV serotype can be constructed for use in a ccDNA vector or ccDNA plasmid based on the exemplary AAV2 ITR sequences of the present invention. See, for example, structural analysis and comparison of ITR sequences from different AAV serotypes (AAV1-AAV6) described in Grimm et al., J. Virology, 2006; 80(1); 426-439; Yan et al., J. Virology, 2005; 364-379; Duan et al., Virology 1999; 261; 8-14.

[00186] Accordingly, although the AAV2 ITR is used as examples of ITRs (e.g., wild-type (WT) ITRs or modified ITRs) in the cccDNA vectors described herein, the cccDNA vector as described herein can be prepared with an ITR or based on the ITR of any known AAV serotype, including, for example, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12). One skilled in the art will be able to determine the corresponding sequence in other serotypes by known methods. The present invention also provides populations and sets of cccDNA vectors containing ITRs from a combination of different AAV serotypes—that is, one ITR (e.g., wild-type (WT) ITR or modified ITR) may be from one AAV serotype and another ITR (e.g. , wild-type (WT) ITR or modified ITR) may be from a different serotype. Without being bound by any theory, in one embodiment, one ITR (e.g., a wild-type (WT) ITR or a modified ITR) may be taken from or based on an AAV2 ITR sequence, and the other ITR (e.g., a wild-type ITR (WT) or modified ITR) cccDNA vector may be taken from or based on any one or more sequences of AAV ITR serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

[00187] Described herein are specific changes and mutations in the WT-ITR whereby the cccDNA may include WT-ITRs that are substantially symmetrical, that is, have a symmetrical three-dimensional spatial organization such that their structure has the same shape in geometric space. This may occur if the G-C pair is modified, for example, by replacing it with a C-G pair or vice versa, or if the A-T pair is modified by replacing it with a T-A pair, or vice versa. Accordingly, using the above illustrative example of a 5' WT-ITR containing the sequence ATCGATCG and a 3' WT-ITR containing CGATCGAT (i.e., the reverse complement of ATCGATCG), said WT-ITRs would be substantially symmetrical if, for example, a 5' WT-ITR would have the sequence ATCGA4CG, with T modified by substitution for A, and a substantially symmetric 3' WT-ITR would have the sequence GCATCGAT, without modification of A with substitution for T. In some embodiments, the ITR in such a pair WT-ITRs are essentially symmetrical in that they have a symmetrical three-dimensional spatial organization.

[00188] Specific changes and mutations in the ITR are described in detail herein, however, in the context of an ITR, “altered,” “mutated,” or “modified” means that nucleotides have been inserted, deleted, and/or replaced relative to the wild-type or occurring ITR sequence the nature of the ITR sequence, where the two ITRs flanking the transgene or heterologous nucleic acid contain the same modifications or are substantially symmetrical as defined herein, i.e. have the same three-dimensional spatial organization, so that their structure has the same shape in geometric space. The altered or mutated ITR may be an engineered ITR. As used herein, “constructed” refers to a feature that is subject to human manipulation. For example, a polypeptide is considered “engineered” if at least one feature of the polypeptide, such as its sequence, has been manipulated by humans to produce a feature that differs from what occurs naturally.

[00189] In some embodiments, the ITR (eg, wild-type (WT) ITR or modified ITR) may be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from more than one AAV serotype. In another embodiment, the synthetic ITR does not include AAV-based sequences. In yet another embodiment, the synthetic ITR retains the ITR structure described above although it contains only part or no AAV-derived sequence. In some aspects, the synthetic ITR may preferentially interact with wild-type Rep or Rep of a particular serotype, or in some cases, it is not recognized by wild-type Rep but is recognized only by mutated Rep. In some embodiments, the ITR is a synthetic ITR sequence that retains functional Rep binding sites (RBS), such as 5'-GCGCGCTCGCTCGCTC-3', and a terminal resolution site (TRS), along with a variable palindromic sequence that provides hairpin secondary structure. In some examples, the modified ITR sequence retains the RBS sequence, trs, and the structure and position of the Rep binding element forming the terminal loop portion of one of the ITR hairpin secondary structures as the corresponding wild-type AAV2ITR sequence. Examples of ITR sequences for use in cccDNA vectors are disclosed in Tables 2-9, 10A and 10B, SEQ ID NOs: 2, 52, 101-449 and 545-547, and partial ITR sequences are presented in FIG. 26A - 26B from PCT application No. PCT/US18/49996, filed September 7, 2018. In some embodiments, the cccDNA vector may contain an ITR with a modification in the ITR corresponding to any of the modifications in the ITR sequences or partial ITR sequences present in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A and 10B from PCT Application No. PCT/US18/49996, filed September 7, 2018.

[00190] In one embodiment, the flanking ITRs (eg, wild-type (WT) ITRs or modified ITRs) are substantially symmetrical to each other. If the ITRs are modified ITRs the modifications are identical represent the same addition, substitution or deletion, but the ITRs are not identical reverse complements. For example, said ITRs (e.g., wild-type (WT) ITRs or modified ITRs) may be from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12) , for example, are represented by a combination of AAV2 and AAV6, with a modification in a similar position.

[00191] In one embodiment, said substantially symmetrical ITRs, where one ITR is inverted relative to the other, are at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical. identical, at least 97% identical, at least 98% identical, or at least 99% identical, or % identical is anything in between. Homology can be determined by standard methods well known in the art, for example, using BLAST (“Basic Local Alignment Search Tool”), BLASTN with default parameters. ITRs are considered substantially symmetrical if the overall geometry of their loops A B and C are similar. In some embodiments, the ITRs in a WT-ITR pair are substantially symmetrical in that they have a symmetrical three-dimensional spatial organization, for example, having the same three-dimensional organization of the arms A C-C'. B-B' and D. In one embodiment, the ITRs in a pair of substantially symmetrical WT-ITRs are inverted with respect to each other and are at least 95% identical, at least 96%... 97%... 98%... 99%... 99.5% identical to each other, or % identity is anywhere in between, and one of the WT-ITRs retains the Rep binding site (RBS) 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and the terminal resolution site (trs) . In some embodiments, a substantially symmetrical WT-ITR pair is inverted with respect to each other and is at least 95% identical, at least 96%...97%...98%...99%...99.5% identical to each other, or % identity matches any intermediate value, and one of the WT-ITRs retains a Rep binding site (RBS) 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) and a terminal resolution site (trs), along with a variable palindromic sequence providing the formation of a hairpin secondary structure.

[00192] In one embodiment, a pair of substantially symmetrical modified ITRs is referred to as a pair of modified ITRs (mod-ITRs) provided that the difference in nucleotide sequences of the ITRs does not affect the properties or overall shape and they have substantially the same shape in three-dimensional space . For example, a mod-ITR whose sequence is at least 95%, 96%, 97%, 98%, or 99% identical to the canonical mod-ITR sequence as assessed using standard methods well known in the art, for example, using BLAST (“Basic Local Alignment Search Tool”), BLASTN using default parameters, also has a symmetrical 3D spatial organization so that their 3D structure has the same shape in geometric space. An ITR in a pair of essentially symmetric mod-ITRs contains identical loops A C-C' and B-B' in three-dimensional space, for example, if a modified ITR in a pair of essentially symmetric mod-ITRs contains a division in the C-C arm, then the cognate mod -ITR contains a corresponding deletion in the C-C arm and also has a similar three-dimensional structure of the remaining A and B-B' loops, taking the same shape in geometric space as the cognate mod-ITR. In a non-limiting example, substantially symmetrical ITRs may have symmetrical stereochemical characteristics such that their structure takes on the same shape in geometric space. This can occur, for example, if the G-C pair is modified, for example, replaced by a CG pair or vice versa, or the A-T pair is modified and replaced by a T-A pair, or vice versa. Accordingly, in the above illustrative example of a modified 5' ITR with the sequence ATCGAACGATCG, and a modified 3' ITR with the sequence CGATCGTTCGAT (i.e., the reverse complement of ATCGAACGATCG), said modified ITRs still remain symmetrical, provided that, for example, the 5' The ITR had the sequence ATCGLAAC C ATCG, where the G is further modified with a replacement for C, and the substantially symmetrical 3' ITR has the sequence CGATCGTTCGAT, without a similar modification of T in addition to A. In some embodiments, the ITRs in such a pair of modified ITRs are substantially symmetrical, since the specified a pair of modified ITRs have symmetrical stereochemical characteristics.

[00193] In some embodiments, the ITRs of a mod-ITR pair may have different reverse complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization—that is, both ITRs contain mutations that result in the same overall three-dimensional shape . For example, one ITR (eg, 5' ITR) in a mod-ITR pair may be from one serotype, and the other ITR (eg, 3' ITR) may be from a different serotype, however, both ITRs may contain the same corresponding mutation (eg , in the event that a 5'ITR contains a deletion in region C, a cognate modified 3'ITR from another serotype contains a deletion at a similar position in region C), such that the ITRs in a pair of modified ITRs have the same symmetrical three-dimensional spatial organization. A pair of essentially symmetric mod-ITRs contains identical loops A, C-C' and B-B' in three-dimensional space. In a non-limiting example, for example, if a modified ITR in a substantially symmetric mod-ITR pair contains a deletion in the C-C arm, then the cognate mod-ITR contains a corresponding deletion in the C-C arm and also has a similar three-dimensional structure of the remaining loops A and B-B', taking the same form in geometric space as the cognate mod-ITR. In another example, if the 5' mod-ITR contains a deletion of one or more nucleic acids in the B region, the cognate modified 3'-ITR contains a deletion at the corresponding nucleotide position in the B' arm.

[00194] It is well known to one skilled in the art that if a 5' mod-ITR has a modification in region A, the related 3'ITR will have a modification at the corresponding position in region A'; if a 5' mod-ITR contains a modification in region C, a cognate 3'ITR will contain a modification at the corresponding position in region C, or vice versa, if a 5' mod-ITR contains a modification in region C, a cognate 3' ITR will contain a modification in the corresponding position position in area C; if a 5' mod-ITR contains a modification in region B, a cognate 3'ITR will contain a modification at the corresponding position in region B', or vice versa, if a 5' mod-ITR contains a modification in region B', a cognate 3'ITR will contain a modification in the corresponding position in area B; and if a 5' mod-ITR contains a modification in region A', a cognate 3'mod-ITR will contain a modification at a corresponding position in region A, such that said mod-ITRs are symmetrical or substantially symmetrical as defined herein, respectively, a pair of modified ITRs have the same symmetrical three-dimensional spatial organization.

[00195] In some embodiments, when the symmetric modified ITRs are substantially symmetric, the difference in the nucleotide sequence of the flanking symmetric modified ITR relative to another ITR is that the change is a substitution of one nucleic acid for a conserved nucleic acid. In some embodiments, when the symmetric modified ITRs are substantially symmetric, the difference in the nucleotide sequence of the flanking symmetric modified ITR relative to another ITR is that the change is a substitution of one, or two, or three nucleic acids for a complementary nucleic acid, wherein said changes may be located sequentially, or alternatively, dispersed throughout the ITR or located in the ITR in a discontinuous manner. In some embodiments, when the ITRs in a pair of symmetric modified ITRs are substantially symmetric as defined herein, the ITR may comprise the substitution: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleic acids into complementary nucleic acids relative to the flanking substantially symmetric modified ITR, provided that said difference in nucleotide sequences of the two substantially symmetric modified ITRs does not affect properties or general shape, and that they have essentially the same shape in three-dimensional space.

[00196] Any parvovirus ITR (eg, wild-type (WT) ITR or modified ITR) can be used as the ITR or as the base ITR for modification. Preferably, said parvovirus is a dependovirus. More preferably, said parvovirus is AAV. The choice of serotype may be based on the tissue tropism of the specified serotype. AAV2 has broad tissue tropism, AAV1 predominantly targets neurons and skeletal muscle, and AAV5 predominantly targets neurons, retinal pigment epithelium and photoreceptors. AAV6 predominantly targets skeletal muscle and lungs. AAV8 predominantly targets tissues of the liver, skeletal muscle, heart, and pancreas. AAV9 predominantly targets liver, skeletal, and lung tissue. In one embodiment, said modified ITR is based on the AAV2 ITR.

[00197] In some embodiments, the vector polynucleotide comprises a WT-ITR pair selected from the group presented in Table 1. Table 1 provides examples of WT-ITR combinations from the same serotype or different serotypes or different parvoviruses. The order given does not indicate the position of the ITR, for example, “AAV1, AAV2” indicates that cccDNA may contain the WT-AAV1 ITR at the 5' position, and the WT-AAV2 ITR at the 3' position, or vice versa, the WT-AAV2ITR at the 5' position , a WT-AAV1 ITR at position 3'. Abbreviations: AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7) , AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12); AAVrh8, AAVrh10, AAV-DJ and AAV-DJ8 genome (For example, NCBI: NC 002077; NC 001401; NC001729; NC001829; NC006152; NC 006260; NC 006261), ITR of warm-blooded animals (avian AAV (AAAV), bovine AAV ( BAAV ), canine, equine and ovine AAV), ITR from parvovirus B19 (GenBank accession no. NC 000883), small mouse virus (MVM) (GenBank accession no. NC 001510); goose: goose parvovirus (GenBank accession no. NC 001701); snake: snake parvovirus 1 (GenBank accession no. NC 006148).

[00198] In some embodiments, said vector polynucleotide or said non-viral non-capsid DNA vectors with covalently closed ends comprise a pair of WT-ITRs selected from the WT-ITRs listed in Table 2. In a non-limiting example, Table 2 provides example WT-ITR sequences from different AAV serotypes.

[00199] In some embodiments of the present invention, the cccDNA vector does not contain a WT-ITR consisting of a nucleotide sequence selected from any of the sequences in Table 2. In some embodiments, the ccDNA vector does not contain a WT-ITR consisting of a nucleotide sequence a sequence selected from any of: SEQ ID NO: 558-567, SEQ ID NO: 51 or SEQ ID NO: 1.

[00200] According to alternative embodiments of the present invention, if the cccDNA vector contains a WT-ITR containing a nucleotide sequence selected from any of: SEQ ID NO: 558,567, SEQ ID NO: 51, or SEQ ID NO: 1, flanking The ITR is also a WT ITR and the cccDNA contains a regulatory switch, for example, as described herein. In some embodiments, the cccDNA vector comprises a regulatory switch as described herein, and the selected WT-ITR comprises any nucleotide sequence selected from the group consisting of: SEQ ID NO: 558-567, SEQ ID NO:51, or SEQ ID NO: 1.

[00201] The cctDNA vector described herein may include WT-ITR structures that retain the functional portions of RBE, trs, and RBE'. In fig. 3A and FIG. Figure 3B shows, using wild-type ITR as an example, one possible mechanism for the functioning of the trs site as part of the wild-type ITR structure of a cccDNA vector. In some embodiments, the cccDNA vector comprises one or more functional WT-ITR polynucleotide sequences that contain a Rep binding site (e.g., RBS; 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531) for AAV2) and a terminal resolution site ( TRS; e.g. 5'-AGTT (SEQ ID NO: 46)). In some embodiments, the at least one WT-ITR is functional. In alternative embodiments, if the cccDNA vector contains two WT-ITRs that are substantially symmetrical to each other, at least one WT-ITR is functional and at least one WT-ITR is non-functional. In alternative embodiments, when the cccDNA vector contains two modified ITRs that are symmetrical to each other, at least one modified ITR is functional and at least one modified ITR is non-functional. Said cccDNAs may comprise a regulatory switch, for example, as described herein and further detailed in Section V below.

[00202] In some embodiments, the cccDNA vector does not contain a modified ITR selected from any sequence consisting of or substantially consisting of: SEQ ID NO: 500-529 as described herein. In some embodiments, the cccDNA vector does not contain an ITR that is selected from any sequence selected from SEQ ID NO: 500-529.

[00203] In some embodiments, the cccDNA vector comprises an ITR pair selected from the group consisting of: SEQ ID NO: 484 (ITR-33, left) and SEQ ID NO: 469 (ITR-18, right); SEQ ID NO: 485 (ITR-34, left) and SEQ ID NO: 95 (ITR-51, right); SEQ ID NO: 486 (ITR-35, left) and SEQ ID NO: 470 (ITR-19, right); SEQ ID NO: 487 (ITR-36, left) and SEQ ID NO: 471 (ITR-20, right); SEQ ID NO: 488 (ITR-37, left) and SEQ ID NO: 472 (ITR-21, right); SEQ ID NO: 489 (ITR-38, left) and SEQ ID NO: 473 (ITR-22, right); SEQ ID NO: 490 (ITR-39, left) and SEQ ID NO: 474 (ITR-23, right); SEQ ID NO: 491 (ITR-40, left) and SEQ ID NO: 475 (ITR-24, right); SEQ ID NO: 492 (ITR-41, left) and SEQ ID NO: 476 (ITR-25, right); SEQ ID NO: 493 (ITR-42, left) and SEQ ID NO: 477 (ITR-26, right); SEQ ID NO: 494 (ITR-43, left) and SEQ ID NO: 478 (ITR-27, right); SEQ ID NO: 495 (ITR-44, left) and SEQ ID NO: 479 (ITR-28, right); SEQ ID N0:496 (ITR-45, left) and SEQ ID N0:480 (ITR-29, right); SEQ ID NO: 497 (ITR-46, left) and SEQ ID NO: 481 (ITR-30, right); SEQ ID NO: 498 (ITR-47, left) and SEQ ID NO: 482 (ITR-31, right); SEQ ID NO: 499 (ITR-48, left) and SEQ ID NO: 483 (ITR-32, right).

[00204] In one embodiment of each of these aspects, said cccDNA vector or said non-viral capsidless DNA vectors with covalently closed ends comprise a pair of symmetrical ITRs selected from ITRs containing partial ITR sequences shown in FIG. 6B - 21B, or selected from combinations of: SEQ ID NO: 101 and SEQ ID NO: 102; SEQ ID NO: 103 and SEQ ID NO: 96; SEQ ID NO: 105 and SEQ ID NO: 106; SEQ ID NO: 545 and SEQ ID NO: 116; SEQ ID NO: 111 and SEQ ID NO: 112; SEQ ID NO: 117 and SEQ ID NO: 118; SEQ ID NO: 119 and SEQ ID NO: 120; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 107 and SEQ ID NO: 108; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO: 125 and SEQ ID NO: 126; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 129 and SEQ ID NO: 130; SEQ ID NO: 131 and SEQ ID NO: 132; SEQ ID NO: 133 and SEQ ID NO: 134; SEQ ID NO: 547 and SEQ ID NO: 546.

[00205] In some embodiments, the cccDNA vector may contain an ITR with a modification in the ITR corresponding to any of the modifications in the ITR sequences or partial ITR sequences presented in Table 5, or the sequences presented in FIG. 7A - 22B herein or in Tables 2, 3, 4, 5, 6, 7, 8, 9 or 10A-10B in PCT/US18/49996 filed September 7, 2018, wherein the ITR flanking sequence is symmetric ( for example, inversely complementary) to it or essentially symmetrical.

[00206] In some embodiments, the ccDNA may form an intramolecular duplex secondary structure. In a non-limiting example, the secondary structure of the first ITR and the symmetric second ITR is presented in the context of wild-type ITR (see, for example, Figs. 2A, 2B, 4C) and/or modified ITR structures (see, for example, Figs. 7A to 22B). Secondary structures are inferred or predicted based on the ITR sequences of the plasmid used to generate the cccDNA vector. Examples of secondary structures of pairs of modified symmetrical ITRs in which part of the stem-loop structure has been removed are shown in FIG. 7A - 22V. Examples of secondary structures of modified ITRs containing a single stem and two loops are shown in Fig. 10A - 10B, 12A-12V, 13A-13V, 13A-22V. Examples of the secondary structure of a modified ITR with a single stem and a single loop are shown in FIG. 7A - 7B (eg with a single loop CC) and FIGS 8A - 8B (eg with a single loop BB'). Examples of the secondary structure of a modified ITR with a single stem instead of two loops are shown in FIG. 11A - 11B. In some embodiments, said secondary structure can be inferred, as shown herein, using thermodynamic methods based on nearest neighbor rules, which predict structure stability as quantified by changes in folding free energy. For example, the specified structure can be predicted by finding the structure with the lowest free energy. In some embodiments, an algorithm described in Reuter, J.S., & Mathews, D.H. may be used to predict the ITR structure. (2010) RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics. 11, 129, and implemented in the RNAstructure software (available on the Internet at: "rna.urmc.rochester.edu/RNAstructureWeb/index.html"). The specified algorithm can also include both parameters for the change in free energy at 37°C, and enthalpy change parameters obtained from the experimental literature, allowing the prediction of conformational stability at arbitrary temperatures.Using RNA structure analysis software, some of the modified ITR structures can be predicted as modified T-shaped loop-on-stem structures " with the calculated Gibbs free energy (ΔC) for unfolding under physiological conditions presented in Figures 7A - 22B. Using RNA structure analysis software, three types of modified ITRs are predicted to have a higher Gibbs free energy for unfolding than the wild type AAV2 ITR (-92.9 kcal/mol); said ITRs are as follows: (a) modified ITRs with a single arm/single unpaired loop structure according to the present invention, predicted to have a Gibbs free energy for unfolding in the range of -85 to -70 kcal /mol. (b) the modified ITRs having a single hairpin structure described herein are predicted to have a Gibbs free energy for unfolding in the range of -70 to -40 kcal/mol. (c) the modified ITRs having the two-arm structure described herein are predicted to have a Gibbs free energy for unfolding in the range of -90 to -70 kcal/mol. Without wishing to be bound by any theory, structures with a higher Gibbs free energy will be more easily unfolded for replication by the Rep 68 or Rep 78 replication proteins. Thus, modified ITRs have a higher Gibbs free energy for unfolding - e.g., a single arm/single arm structure unpaired loop, single hairpin structure, truncated structure - show a tendency to replicate more efficiently than wild type ITR.

[00207] In one embodiment, the left ITR of the cccDNA vector a is modified or mutated relative to the structure of the wild-type AAV ITR, and the right ITR is symmetrical (reverse complementary) to it and contains the same mutations. In one embodiment, the right ITR of the cccDNA vector is modified relative to the structure of the wild-type AAV ITR, and the left ITR is symmetrical (reverse complementary) to it and contains the same mutations. In such an embodiment, a modification of the ITR (eg, left or right ITR) can be achieved by deletion, insertion, or substitution of one or more nucleotides of a wild-type ITR derived from the AAV genome.

[00208] The ITRs used in the present invention may be resolvable or non-resolvable, and the ITRs selected for use in cDNA vectors are preferably AAV sequences, with sequences from serotypes 1, 2, 3, 4, 5, 6, 7, 8 being preferred and 9. The permitted AAV ITRs are not necessarily represented by a wild-type ITR sequence (e.g., an endogenous AAV ITR sequence or a wild-type AAV ITR sequence may be altered by insertion, deletion, truncation, and/or introduction of missense mutations), provided that the terminal the repeat mediates required functions, e.g., replication, packaging, viral integration and/or provirus rescue, etc. Typically, but not necessarily, ITRs are taken from the same AAV serotype, e.g., both ITR sequences of a cDNA vector are taken from AAV2. As described above, in some embodiments, the ITRs in a pair of modified ITRs are substantially symmetrical, that is, have a symmetrical three-dimensional spatial organization, but do not contain identical reverse complementary nucleotide sequences. In such embodiments, both ITRs in a modified ITR pair may be from different serotypes (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), e.g., combinations of AAV2 and AAV6 , with the modification of one ITR reflected in the corresponding position of a cognate ITR from another serotype. In some embodiments, the modified ITRs may be synthetic sequences that function as AAV inverted terminal repeats, such as the “DD sequence” as described in US Pat. No. 5,478,745 to Samulski et al.

[00209] In one embodiment, the cccDNA may include an ITR structure that is mutated relative to one of the wild-type ITRs as described herein, but the mutant or modified ITR retains a functional Rep binding site (RBE or RBE') and a terminal resolution site ( trs). In one embodiment, the mutant ITR cDNA includes a functional replication protein site (RPS-1) and is produced using a replication competent protein that binds the RPS-1 site.

[00210] In one embodiment, at least one of the ITRs is a defective ITR with respect to Rep binding and/or Rep nicking. In one embodiment, the defect is represented by at least 30%, in other embodiments, by at least 35% ..., 50%.., 65%..., 75%..., 85%..., 90%..., 95%..., 98%..., insufficiency of a function relative to wild-type ITR or its complete absence, or insufficiency of a function corresponding to any intermediate value. The host cells do not express viral capsid proteins and the polynucleotide vector template does not contain any viral capsid coding sequences. In one embodiment, said polynucleotide vector templates and host cells that do not contain AAV capsid genes and resulting protein also do not encode or express capsid genes of other viruses. In addition, in a particular embodiment, said nucleic acid molecule also does not contain sequences encoding the AAV Rep protein.

[00211] In some embodiments, the ITR structural element can be any structural element that is involved in the functional interaction of the ITR with the large Rep protein (eg, Rep 78 or Rep 68). In some embodiments, the structural element provides selectivity for the interaction of the ITR with the large Rep protein, i.e., determines at least in part which Rep protein functionally interacts with the ITR. In other embodiments, said structural element physically interacts with a large Rep protein upon binding of said Rep protein to the ITR. Each structural element may be, for example, an ITR secondary structure or an ITR nucleotide sequence. an intermediate sequence between two or more elements; or a combination of any of the above. In one embodiment, said structural elements are selected from the group consisting of arm A and arm A', arm B and arm B', arm C and arm C, arm D, a Rep binding site (RBE), and RBE' (i.e. sequence complementary to RBE) and a terminal resolution site (trs).

[00212] More specifically, said ITR may be structurally modified. For example, the nucleotide sequence of the structural element may be modified compared to the wild-type ITR sequence. In one embodiment, a structural element (e.g., arm A, arm A', arm B, arm B', arm C, arm C, arm D. RBE, RBE', and trs) of the ITR can be removed and replaced with a wild-type structural element from another parvovirus. For example, the structure to be replaced may be taken from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus or insect AAV. For example, the ITR may be an AAV2 ITR and the A or A' arm or RBE may be replaced by a structural element from AAV5. In another example, said ITR may be an AAV5 ITR and the C or C arms, RBE and trs may be replaced by a building block from AAV2. In another example, said AAV ITR may be an AAV5 ITR where arms B and B' are replaced by arms B and B' of an AAV2 ITR.

[00213] By way of example only, Table 3 shows exemplary modifications of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in modified ITR regions wherein X indicates modification of at least one nucleic acid (e.g., deletion, insertion, and /or substitution) in the indicated region relative to the corresponding wild-type ITR. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in any of the C and/or C and/or B and/or B' regions preserves three consecutive nucleotides T (t ie TTT) in at least one terminal loop. For example, if said modification results in any of: an ITR with a single arm (e.g., a single C-C' arm or a single B-B' arm), or a modified C-B' arm or a C'-B arm, or a two-arm ITR comprising at least one truncated arm (e.g., a truncated arm C-C' and/or a truncated arm B-B'), then in at least said single arm, or in at least one of the arms A two-arm ITR (in which one arm may be truncated) retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In some embodiments, the C-C' truncated arm and/or the B-B' truncated arm contains three consecutive T nucleotides (ie, TTT) in the terminal loop.

[00214] Table 3 provides examples of combinations of modifications to at least one nucleotide (e.g., deletions, insertions, and/or substitutions) in different regions or arms B-B' and C-C' of the ITR (X indicates a nucleotide modification, e.g. addition, deletion or substitution of at least one nucleotide in the specified region).

[00215] In some embodiments, a modified ITR for use herein may comprise any of the combinations of modifications presented in Table 3 and also modification of at least one nucleotide in any one or more regions selected from: the region between A' and C , between C and C', between C and B, between B and B', and between B' and A. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in region C , or C', or B, or B', the terminal loop of the stem-loop structure is still retained. In some embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' retains three consecutive T nucleotides (i.e., TTT) of at least at least in one terminal loop. In alternative embodiments, any modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) between C and C' and/or B and B' retains at least three consecutive nucleotides A (i.e., AAA) at least in one terminal loop. In some embodiments, a modified ITR for use in accordance with the present invention may comprise any of the combinations of modifications presented in Table 3, as well as modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in any one or more regions selected of: A', A, and/or D. For example, in some embodiments, a modified ITR for use herein may contain any of the combinations of modifications presented in Table 3, as well as a modification of at least one nucleotide (e.g., deletion, insertion and/or substitution) in region A. In some embodiments, a modified ITR for use herein may contain any of the combinations of modifications presented in Table 3, as well as a modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution ) in area A'. In some embodiments, a modified ITR for use herein may comprise any of the combinations of modifications presented in Table 3, as well as a modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in the A and/or A' region . In some embodiments, a modified ITR for use herein may comprise any of the combinations of modifications presented in Table 3, as well as a modification of at least one nucleotide (e.g., deletion, insertion, and/or substitution) in region D.

[00216] In one embodiment, the nucleotide sequence of the building block may be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16 , 17, 18, 19, or 20 or more nucleotides, or any number of nucleotides in between) to produce a modified structural element. In one embodiment, examples of specific modifications to symmetrical ITRs are provided herein (e.g., the pairs of symmetrical modified ITRs identified in Table 4.

[00217] In some embodiments, the ITR may be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any number of nucleotides in between). In other embodiments, the ITR sequence may be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, is at least 98%, at least 99% or more sequence identical to one of the modified ITRs from Table 4 (e.g., or the RBE-containing portion of arm A-A' and arms C-C' and B-B' of symmetrical modified pairs selected from the group consisting of, for example, SEQ ID NO: 101 and SEQ ID NO: 102; SEQ ID NO: 103 and SEQ ID NO: 96; SEQ ID NO: 105 and SEQ ID NO: 106; SEQ ID NO: 545 and SEQ ID NO: 116; SEQ ID NO: 111 and SEQ ID NO: 112; SEQ ID NO: 117 and SEQ ID NO: 118; SEQ ID NO: 119 and SEQ ID NO: 120; SEQ ID NO: 121 and SEQ ID NO: 122; SEQ ID NO: 107 and SEQ ID NO: 108; SEQ ID NO: 123 and SEQ ID NO: 124; SEQ ID NO: 125 and SEQ ID NO: 126; SEQ ID NO: 127 and SEQ ID NO: 128; SEQ ID NO: 129 and SEQ ID NO: 130; SEQ ID NO: 131 and SEQ ID NO: 132; SEQ ID NO: 133 and SEQ ID NO: 134; SEQ ID NO: 547 and SEQ ID NO: 546.

[00218] In some embodiments, the modified ITR may, for example, include removal or deletion of a particular arm in its entirety, such as complete or partial removal or deletion of the A-A' arm, or complete or partial removal or deletion of the B-B' arm, or complete or partial deletion or deletion of the C-C' arm, or, alternatively, deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs forming the stem of the loop, provided that the resulting loop is retained the stem (e.g., single arm) terminates (e.g., see ITR-6). In some embodiments, the modified ITR may include removing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, the modified ITR may include removing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm. In some embodiments, the modified ITR may include deleting 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and deleting 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from arm B-B'. Any combination of base pair deletions is possible, for example, 6 base pairs may be deleted in the C-C' arm and 2 base pairs in the B-B' arm. An illustrative example is a modified ITR with a deletion of at least 7 base pairs in both the C region and the C region, a nucleotide substitution in the loop between the C and C' regions, and a deletion of at least one base pair in both the B region. and in the region B' so that the modified ITR contains two arms, and at least one arm (for example, C-C') is truncated. Note that in this example, since the modified ITR contains at least one base pair deletion in each of the B and B' regions, the B-B' arm is also truncated compared to the WT ITR.

[00219] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more complementary base pairs are removed from both the C portion and the C' portion of the C-C' arm, such such that the shoulder C-C' becomes truncated. In other words, when a base is removed from the C portion of the C-C' arm, the complementary base pair is removed from the C' portion, thereby truncating the C-C' arm. In such embodiments, 2, 4, 6, 8, or more base pairs are removed from the C-C' arm such that the C-C' arm is truncated. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the C portion of the C-C' arm such that only the C' portion of the arm is retained. Alternative embodiments remove 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs from the C portion of the C-C arm such that only the C portion of the arm is retained.

[00220] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more complementary base pairs are removed from both the B portion and the B' portion of the B-B' arm such that , so that the shoulder B-B' becomes truncated. In other words, when a base is removed from the B portion of the B-B' arm, the complementary base pair is removed from the B' portion, thereby truncating the B-B' arm. Such embodiments remove 2, 4, 6, 8, or more base pairs from the B-B' arm such that the B-B' arm is truncated. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the B portion of the B-B' arm such that only the B portion of the arm is retained. In alternative embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more base pairs are removed from the B' portion of the B-B' arm such that only the B portion of the arm is retained.

[00221] In some embodiments, the modified ITR may contain from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide deletions relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITR may contain from 1 to 30 nucleotide deletions relative to the full-length WT ITR sequence. In some embodiments, the modified ITR contains 2 to 20 nucleotide deletions relative to the full-length wild-type ITR sequence.

[00222] In some embodiments, the modified ITR defines two opposing, longitudinally symmetrical "loops-on-stem", eg, the C-C' loop has a different length than the B-B' loop. In some embodiments, one of the opposed longitudinally symmetrical stem-loops of the modified ITR comprises a C-C' and/or B-B' stem portion with a length ranging from 8 to 10 base pairs, and a loop portion (e.g., between C -C' or between B-B'), which contains from 2 to 5 unpaired deoxyribonucleotides. In some embodiments, one of the longitudinally symmetrical stem-loops of the modified ITR comprises a C-C and/or B-B' stem portion less than 8 bases in length or less than 7, 6, 5, 4, 3, 2, 1 base pairs and a loop part (for example, between C-C or between B-B'), which contains 0 5 nucleotides. In some embodiments, the modified longitudinally asymmetric stem-loop ITR comprises a C-C and/or B-B' stem portion less than 3 base pairs in length.

[00223] In some embodiments, the modified ITR does not contain any nucleotide deletions in the RBE-containing portion of the A or A' regions so as not to interfere with DNA replication (eg, Rep protein binding to the RBE, or nicking at the end resolution site). In some embodiments, a modified ITR provided for use herein contains one or more deletions in the B, B', C, and/or C region as described herein. Several non-limiting examples of modified ITRs are shown in FIG. 6V - 21V.

[00224] In some embodiments, the modified ITR may include a deletion of the B-B' arm, thereby retaining the C-C' arm, for example, see the examples ITR-33 (left) and ITR-18 (right) shown in FIG. . 7A - 7B, or ITR-35 (left) and ITR-19 (right) (Fig. 9A - 9B). In some embodiments, the modified ITR may include a deletion of the C-C' arm, thereby retaining the B-B' arm, for example, see examples ITR-34 (left) and ITR-51 (right) shown in FIG. 8A - 8B. In some embodiments, the modified ITR may include deletion of the B-B' arm and the C-C arm such that a single stem-loop is maintained, for example, see examples ITR-37 (left) and ITR-21 (right) shown in Fig. 11A - 11B. In some embodiments, the modified ITR may include a deletion of the C region such that the truncated C-loop and arm B-B' are retained, for example, see the examples ITR-36 (left) and ITR-20 (right) shown in FIG. . 10A - 10V; ITR-42 (left) and ITR-26 (right) (Fig. 16A - 16B); ITR-43 (left) and ITR-27 (right) (FIGS. 17A - 17B); ITR-44 (left) and ITR-28 (right) (FIGS. 18A - 18B); ITR-45 (left) and ITR-29 (right) (Fig. 19A - 19B); ITR-46 (left) and ITR-23 (right) (FIGS. 20A - 20B); ITR-47 (left) and ITR-31 (right) (Fig. 21A - 21B); ITR-48 (left) and ITR-32 (right) (Fig. 22A - 22B). Likewise, in some embodiments, the modified ITR may include a deletion of the B region such that the truncated B-loop and C-C' arm are retained, for example, see examples ITR-38 (left) and ITR-22 (right), shown in Fig. 12A - 12V; ITR-39 (left) and ITR-23 (right) (FIGS. 13A - 13B); ITR-40 (left) and ITR-24 (right) (Figs. 14A - 14B) and ITR-41 (left) and ITR-25 (right) (Figs. 15A - 15B).

[00225] In some embodiments, the modified ITR may include a base pair deletion in any one or more of the C-part, the C'-part, the B-part, or the B'-part, such that between the C-B' and parts C'-B paired complementary bases to obtain a single arm, for example, see ITR-10 (right) and ITR-10 (left).

[00226] In some embodiments, in addition to modification to one or more nucleotides in the C, C', B, and/or B' region, a modified ITR for use herein may comprise a modification (e.g., deletion, substitution, or addition) of at least at least 1, 2, 3, 4, 5, 6 nucleotides in any one or more regions selected from: the region between A' and C, between C and C', between C' and B, between B and B' and between B ' and A. For example, nucleotide A between B' and C in a modified right ITR may be replaced by G, C or A or deleted, or one or more nucleotides may be added; the T nucleotide between C' and B in the modified left ITR may be replaced by G, C or A or deleted, or one or more nucleotides may be added.

[00227] In some embodiments of the present invention, the cccDNA vector does not contain a modified ITR consisting of a nucleotide sequence selected from any of: SEQ ID NO: 550,557. In some embodiments of the present invention, the cccDNA vector does not contain a modified ITR, containing a nucleotide sequence selected from any of: SEQ ID NO: 550-557.

[00228] In some embodiments, said cccDNA vector comprises a regulatory switch as described herein and a selected modified ITR having a nucleotide sequence selected from any sequence from the group consisting of: SEQ ID NO: 550-557.

[00229] According to another embodiment, the structure of the structural element may be modified. For example, in the specified structural element the height of the stem and/or the number of nucleotides in the loop are changed. For example, the stem height may be approximately 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides or more, or any number of nucleotides in between. In one embodiment, the stem height can be from about 5 nucleotides to about 9 nucleotides and operably interacts with Rep. In another embodiment, the stem may be approximately 7 nucleotides in height and operably interacts with Rep. In another example, a loop may contain 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any number of nucleotides in between.

[00230] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites within an RBE or extended RBE may be increased or decreased. According to one example, an RBE or extended RBE may contain 1, 2, 3, 4, 5 or 6 or more GAGY binding sites, or any number within the range of these values. Each GAGY binding site may independently be the exact GAGY sequence or a sequence similar to GAGY, provided that the sequence is sufficient for binding of the Rep protein.

[00231] In another embodiment, the intervening sequence between two elements (such as, but not limited to, an RBE and a hairpin) may be changed (eg, increased or decreased) to alter the functional interaction with the large Rep protein. For example, said intermediate sequence may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or any number of nucleotides within the range specified.

[00232] Table 4 provides examples of symmetric modified ITR pairs (ie, modified left ITRs and symmetric right modified ITRs). The portion of the sequences highlighted in bold (red) corresponds to the partial ITR sequences (ie, loop sequences A-A', C-C and B-B'), also shown in FIG. 7A - 22V. These examples of modified ITRs may comprise RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 531), spacer ACTGAGGC (SEQ ID NO: 532), spacer complement GCCTCAGT (SEQ ID NO: 535) and RBE' (i.e., complement RBE ) GAGCGAGCGAGCGCGC (SEQ ID NO: 536).

[00233] In embodiments of the present invention, the cccDNA vector as described herein does not contain modified ITRs having a nucleotide sequence selected from any of SEQ ID Nos: 550, 551, 552, 553, 553, 554, 555, 556 and 557.

IV. Regulatory elements

[00234] CCDNA vectors can be derived from expression constructs that further contain a specific combination of cis-regulatory elements. Said cis-regulatory elements include, but are not limited to, a promoter, a riboswitch, an insulator, a miR-regulated element, a post-transcriptional regulatory element, a tissue-specific and cell-specific promoter, and an enhancer. In some embodiments, the ITR may act as a promoter for the transgene. In some embodiments, the cccDNA vector contains additional components for regulating expression of the transgene, such as regulatory switches as described herein to regulate expression of the transgene, or a “kill switch” that is capable of killing a cell containing the cccDNA vector.

[00235] These cccDNA vectors can be derived from expression constructs that further contain a specific combination of cis-regulatory elements, such as WHP post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 8) and poly(A) BGH (SEQ ID NO: 9). Suitable expression cassettes for use in expression constructs do not have the packaging restrictions imposed by the viral capsid.

[00236] Promoters: The expression cassettes of the present invention include a promoter that can influence overall expression levels as well as cell specificity. If expressed transgenely, they may include a highly active immediate early promoter of viral origin. Expression cassettes may contain tissue-specific eukaryotic promoters to restrict transgene expression to specific cell types and reduce toxic effects and immune responses caused by unregulated ectopic expression.

[00237] Suitable promoters, including those described above, can be derived from viruses and. accordingly, may be called viral promoters, or they may originate from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (eg, pol I, pol II, pol III). Examples of promoters include, but are not limited to, the SV40 early promoter, the murine mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early (CMVIE) promoter region, Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6, e.g. SEQ ID NO: 18) ( Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1;31(17)), human H1 promoter (H1) ( eg SEQ ID NO: 19), CAG promoter. human alpha-1 antitrypsin (hAAT) promoter (eg, SEQ ID NO: 21); and so on. In embodiments, said promoters are modified at the nitrone-containing 3' end to include one or more nuclease cleavage sites. In some embodiments, the DNA containing the nuclease cleavage site(s) is foreign to the promoter DNA.

[00238] In some embodiments, a promoter may contain one or more specific transcriptional control sequences to further enhance expression, and/or to alter expression spatially and/or temporally. The promoter may also include distal enhancer or repressor elements, which may be located several thousand base pairs away from the transcription start site. The promoter can come from sources including viruses, bacteria, fungi, plants, insects and animals. A promoter may regulate expression of a gene component constitutively or differentially with respect to the cell, tissue or organ where expression occurs, or with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions or inducing agents. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator/promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and CMV promoter IE, as well as the promoters listed below. Such promoters and/or enhancers can be used to express any gene of interest, for example, gene editing molecules, donor sequence, therapeutic proteins, etc. For example, the vector can contain a promoter that is operably linked to a nucleic acid sequence encoding a therapeutic protein. Said promoter operably linked to the coding sequence of the therapeutic protein may be a simian virus 40 (SV40) promoter, a murine mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, such as a long terminal repeat (LTR) virus promoter bovine immunodeficiency virus (BIV) promoter, Moloney virus promoter, avian leukemia virus (ALV) promoter, cytomegalovirus (CMV) promoter such as CMV early promoter, Epstein-Barr virus (EBV) promoter or Rous sarcoma virus (RSV) promoter. In some embodiments, the promoter may also be a human gene promoter, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a tissue-specific promoter, such as a liver-specific promoter, such as the human alpha-1 antitrypsin (HAAT) promoter, natural or synthetic. In one embodiment, delivery to the liver can be achieved using specific targeting of endogenous ApoE by a cccDNA vector-containing composition that is directed to hepatocytes by a low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[00239] In one embodiment, the promoter used is the native promoter of a gene encoding a therapeutic protein. The promoters and other regulatory sequences of the corresponding genes encoding therapeutic proteins are known and have been characterized. The promoter region used may further include one or more additional regulatory sequences (eg, native), such as enhancers (eg, SEQ ID NO: 22 and SEQ ID NO: 23).

[00240] Non-limiting examples of suitable promoters for use in accordance with the present invention include the CAG promoter, for example (SEQ ID NO: 3), the hAAT promoter (SEQ ID NO: 21), the human EF1-α promoter (SEQ ID NO: 6) or a fragment of the promoter EF1a (SEQ ID NO: 15), IE2 promoter (eg, SEQ ID NO: 20) and rat EF1-α promoter (SEQ ID NO: 24).

[00241] In some embodiments, the expression cassette may comprise a synthetic regulatory element, such as a CAG promoter (SEQ ID NO: 3). The CAG promoter contains (i) the cytomegalovirus (CMV) early enhancer element, (ii) the promoter, first exon, and first intron of the chicken beta-actin gene, and (iii) the splice acceptor of the rabbit beta-globin gene. Alternatively, the expression cassette may contain an hAAT promoter (SEQ ID NO: 21), an alpha-1-antitrypsin (AAT) promoter (SEQ ID NO: 4 or SEQ ID NO: 74), a liver-specific (LP1) promoter (SEQ ID NO: 5 or SEQ ID NO: 16), human elongation factor 1 alpha (EF1α) promoter or fragment thereof (e.g., SEQ ID NO: 6 or SEQ ID NO: 15), rat EF1α promoter (SEQ ID NO: 24), or IE2 promoter (SEQ ID NO: 20). In some embodiments, said expression cassette includes one or more constitutive promoters, e.g., a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer) or a cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer, e.g., SEQ ID NO: 22). Alternatively, an inducible promoter, a promoter native to the transgene, a tissue-specific promoter, or various promoters known in the art may be used.

[00242] Polyadenylation Sequences: A sequence encoding a polyadenylation sequence may be included in a cccDNA vector to stabilize the mRNA expressed from the ccDNA vector and promote nuclear export and translation. In one embodiment, the cccDNA vector does not contain a polyadenylation sequence. In other embodiments, said vector includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50 or more adenine dinucleotides. In some embodiments, the polyadenylation sequence comprises about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any number of nucleotides anywhere in between.

[00243] Expression cassettes may include a polyadenylation sequence known in the art, or a variant thereof, for example, a naturally occurring sequence isolated from bovine BGHpA (e.g., SEQ ID NO: 74) or SV40pA virus (e.g., SEQ ID NO: 10), or a synthetic sequence (eg, SEQ ID NO: 27). Some expression cassettes also include the SV40 5' late poly(A) signal enhancer (USE) sequence. In some embodiments, these USEs may be used in combination with SV40pA or a heterologous poly(A) signal.

[00244] These expression cassettes may also include a post-transcriptional element to enhance expression of the transgene. In some embodiments, a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to enhance transgene expression (eg, SEQ ID NO: 8). Other post-transcriptional processing elements may be used, such as a post-transcriptional element of the herpes simplex virus or hepatitis B virus (HBV) thymidine kinase gene. Secretory sequences, such as VH-02 and VK-A26 sequences, such as SEQ ID NO: 25 and SEQ ID NO: 26, may be associated with the transgenes.

[00245] In one embodiment, the host cells do not express viral capsid proteins and the polynucleotide vector array does not contain any viral capsid coding sequences. In one embodiment, the polynucleotide vector array contains neither AAV capsid genes nor capsid genes from other viruses. In one embodiment, said nucleic acid molecule also does not contain sequences encoding the AAV Rep protein. Accordingly, in some embodiments, the nucleic acid molecule of the present invention does not contain functional cap AAV or rep AAV genes.

V. Regulatory switches

[00246] A molecular regulatory switch is a switch that generates a measurable change of state in response to a signal. Such regulatory switches can be advantageously combined with the cDNA vectors described herein to control the output of the cDNA vector. In some embodiments, the cccDNA vector contains a regulator that serves to fine-tune the expression of the transgene. For example, it may serve to provide a biocontainment function to the cDNA vector. In some embodiments, the switch is a “two-way” switch that is designed to start or stop (ie, stop) expression of a gene of interest in cccDNA in a controlled and regulated manner. In some embodiments, the switch may include a "kill switch" that may instruct a cell containing the cccDNA vector to initiate programmed cell death upon activation of the switch.

A. Binary Regulator Switches

[00247] In some embodiments, the cDNA vector contains a regulator that can serve to controllably modulate transgene expression. In such an embodiment, said expression cassette located between the ITR of the cccDNA vector may further comprise a regulatory region, such as a promoter, cis-element, repressor, enhancer, etc., operably linked to the gene of interest, wherein said regulatory region regulates one or more cofactors or exogenous agents. Accordingly, in one embodiment, transcription and expression of the gene of interest from the cccDNA vector will occur only when said one or more cofactors or exogenous agents are present in the cell. In another embodiment, one or more cofactors or exogenous agents can be used to derepress transcription and expression of a gene of interest.

[00248] Any nucleic acid regulatory regions known to one skilled in the art may be used in a cccDNA vector designed to contain a regulatory switch. In a non-limiting example, regulatory regions may modulate small molecule switches or inducible or repressible promoters. Non-limiting examples of inducible promoters include hormone inducible or metal inducible promoters. Other examples of inducible promoter/enhancer elements include, but are not limited to, the RU486 inducible promoter, the ecdysone inducible promoter, the rapamycin inducible promoter, and the metallothionein promoter. The use of classical switches based on tetracycline or other antibiotics, including those described in the source: (Fussenegger et al., Nature Biotechnol. 18: 1203-1208 (2000)) is envisaged.

B. Small molecule regulatory switches

[00249] Various small molecule regulatory switches known in the art are known in the art and can be combined with cccDNA vectors as described herein to form a regulatory switch-controlled cccDNA vector. In some embodiments, said regulatory switch may be selected from any one or combination of the following: an orthogonal ligand/nuclear receptor pair, e.g., retinoid receptor variant/LG335 and GRQCIMFI, along with an artificial promoter controlling expression of an operably linked transgene, e.g. as described in: Taylor, et al. BMC Biotechnology 10 (2010): 15; engineered steroid receptors, such as a modified C-terminal truncated progesterone receptor that is unable to bind progesterone but binds RU486 (mifepristone) (US Pat. No. 5,364,791); Drosophila ecdysone receptor and their ecdysteroid ligands (Saez, et al., PNAS, 97(26)(2000), 14512-14517; or the antibiotic trimethoprim (TMP) switch as described in Sando R 3 ^rd ; Nat Methods 2013, 10(11): 1085-8.

[00250] Other small molecule regulatory switches known to one of ordinary skill in the art are also contemplated for use in controlling transgene expression of cccDNA and include, but are not limited to, those described in Buskirkef al., Cell; Chem and Biol., 2005; 12(2); 151-161; abscisic acid sensitive positive switch; such as described in: Liang, F.-S., et al., (2011) Science Signaling, 4(164); exogenous L-arginine-sensitive positive switches, such as those described in Hartenbach, et al. Nucleic Acids Research, 35(20), 2007, synthetic bile acid-sensitive positive switches such as those described in: Rossger et al., Metab Eng. 2014, 21: 81-90; biotin-sensitive positive switches such as those described in Weber et al., Metab. Eng. Mar 2009; 11(2): 117-124; dual-input benzoate/vanillin sensing regulatory switches such as those described in Xie et al., Nucleic Acids Research, 2014; 42(14); e116; 4-hydroxytamoxifen sensitive switches such as those described in: Giuseppe et al., Molecular Therapy, 6(5), 653-663; and flavonoid (phloretin)-sensitive regulatory switches such as those described in Gitzinger et al., Proc. Natl. Acad. Sci. USA. 2009 Jun 30; 106(26): 10638-10643.

[00251] In some embodiments, the regulatory switch for controlling the transgene or expressed by the cccDNA vector is a switch for activating prodrugs, such as those described in US Pat. Nos. 8,771,679 and 6,339,070.

[00252] Examples of regulatory switches for use in cDNA vectors include, but are not limited to, those presented in Table 5.

C. Regulatory switches “with access code”

[00253] In some embodiments, said control switch may be a "passcode switch" or a "passcode circuit." Switches with an access code allow you to fine-tune the control of transgene expression from a cDNA vector when specific conditions arise, that is, a combination of conditions must be met to carry out transgene expression and/or repression. For example, for transgene expression to occur, at least conditions A and B must occur. A regulatory access code switch may require any number of conditions to be met, for example, for transgene expression to occur, at least 2 conditions must occur, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or more conditions. In some embodiments, at least 2 conditions must occur (eg, conditions A B); in some embodiments, at least 3 conditions must occur (eg, A B and C, or A B and D). According to a non-limiting example, to carry out gene expression with cccDNA containing a regulatory switch with the access code "ABC", conditions A, B and C must be met. Conditions A, B and C may be as follows; condition A represents the presence of a condition or disease, condition B represents a hormonal response, and condition C represents a response to transgene expression. In a non-limiting exemplary example, where the transgene is insulin, condition A is satisfied if the subject has diabetes, condition B is high blood sugar, and condition C is the level of endogenous insulin when it is underexpressed. Once the sugar level drops or the required insulin level is reached, the transgene (eg insulin) is “turned off” until the above 3 conditions occur again, “turning it on” again. In another exemplary example, if the transgene is EPO, condition A is the presence of chronic kidney disease (CKD), condition B is satisfied when the subject's kidneys are hypoxic, condition C is satisfied when the recruitment of erythropoietin-producing cells (EPC) to the kidneys is impaired ; or, alternatively, impaired HIF-2 activation. Once the oxygen level increases or the required EPO level is reached, the transgene (eg EPO) is “turned off” until the above 3 conditions occur again to “turn it on” again.

[00254] Access code regulatory switches are suitable for fine tuning transgene expression from a cccDNA vector. For example, a passcode regulatory switch may be modular, that is, it contains multiple switches, such as a tissue-specific inducible promoter that is turned on only in the presence of a certain level of a metabolite. In this embodiment, to achieve transgene expression from a cccDNA vector, the inducible agent must be present (condition A) in the desired cell type (condition B), and the level of the metabolite must be equal to, above, or below a certain threshold value (condition C). In alternative embodiments, said regulatory passcode switch may be designed such that transgene expression is "on" when conditions A and B are met, but "off" when condition C is met. This embodiment is useful when meeting condition C is a direct result expression of the transgene, that is, condition C is used as positive feedback to turn off transgene expression from the cDNA vector after said transgene has had sufficient of the desired therapeutic effect.

[00255] In some embodiments, a passcode regulatory switch for use in a cccDNA vector is disclosed in WO 2017/059245, which describes a switch called a “Passcode switch” or “passcode circuit.” "Passcode circuit" or "Passcode kill switch", which is a synthetic biological circuit that uses hybrid transcription factors (TFs) to engineer complex environmental requirements for cell survival. The access code regulatory switches described in WO 2017/059245 are particularly suitable for use in cccDNA vectors because they are modular and tunable, both in terms of the environmental conditions that control circuit activation and the output modules that control cell fate. . In addition, the access code strand in particular is suitable for use in cccDNA vectors because, without proper access code molecules, it allows transgene expression to occur only when the necessary predefined conditions are met. In the event that something unexpected happens to the cell, or if transgene expression is no longer needed for some reason, an appropriate “safety switch” (i.e., blocking switch) can be activated.

[00256] In some embodiments, a passcode regulatory switch or “passcode circuit” provided for use in a cccDNA vector comprises hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to set biocontainment conditions. Unlike a blocking switch, which triggers cell death when a predetermined condition is met, a passcode circuit ensures cell survival or transgene expression in the presence of a specific passcode and can be easily reprogrammed to allow transgene expression and/or survival cells only when a predetermined environmental condition is met or an access code is present.

[00257] In one aspect, a passcode system that restricts cell growth to the presence of a predetermined set of at least two selected agents includes one or more nucleic acid constructs encoding expression modules, including: i) a toxin expression module that encodes a toxin that is toxic to the host cell, characterized in that the sequence encoding the toxin is functionally linked to the P1 promoter, the repression of which occurs upon binding of the first hybrid repressor protein hRP1; ii) an expression module of the first hybrid repressor protein, which encodes the first hybrid repressor protein hRP1, characterized in that the expression of hRP1 is controlled by a logical AND element formed by two hybrid transcription factors hTF1 and hTF2, the binding or activity of which depends on agents A1 and A2, respectively , such that both agents A1 and A2 are required for hRP1 expression, and in the absence of A1 or A2, hRP1 expression is insufficient to repress the P1 toxin promoter module and produce toxin, so that the host cell dies. In this system, all hybrid factors, hTF1, hTF2 and hRP1, contain an environment-sensitive module from one transcription factor and a DNA recognition module from another transcription factor, which makes the binding of the corresponding regulatory switch to the access code sensitive to the presence of an environmental agent, A1 or A2, which is different from the agent to which the corresponding subunits typically bind in nature.

[00258] Accordingly, the cccDNA vector may contain a “passcode regulatory circuit” that requires the presence and/or absence of specific molecules to activate the output module. In some embodiments, when genes encoding cellular toxins are placed in an output module, the access coded regulatory circuit can be used not only to regulate transgene expression, but also to create a "safety switch" mechanism in which the circuit kills cell if the specified cell behaves in an undesirable manner (for example, leaves a particular environment defined by sensory domains, or differentiates into a different cell type). In one non-limiting example, the modular structure of hybrid transcription factors, circuit architecture, and output module allow the circuit to be reconfigured to sense other environmental signals, respond to environmental signals in a different manner, and control other functions in the cell, along with inducible cell death, as is known in the art.

[00259] For a regulatory switch with an access code as described herein, any combination of regulatory switches as described herein can be used, for example, small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional switches transgene regulation, post-translational regulators, radiation-controlled switches, hypoxia-mediated switches, and other regulatory switches known to those skilled in the art. Regulatory switches provided for the application are also described in the review article Kis et al., J R Soc Interface. 12:20141000 (2015) and summarized in Table 1 from the Kis paper. In some embodiments, the control switch for use in the passcode system may be selected from any of the switches in Table 5 or a combination thereof.

D. Nucleic acid-based regulatory switches to control transgene expression.

[00260] In some embodiments, the regulatory switch for controlling the transgene expressed by the cccDNA is based on a nucleic acid-based control mechanism. Examples of nucleic acid-based control mechanisms are known in the art and are contemplated for use. For example, such mechanisms include riboswitches such as those described, for example, in US 2009/0305253, US 2008/0269258. US 2017/0204477, WO 2018026762 A1, US patent US 9222093 and application EP 288071, and also disclosed in the review work of Villa JK et al., Microbiol Spectr. May 2018; 6(3). Also included are transcriptional biosensors that respond to metabolites, such as those described in WO 2018/075486 and WO 2017/147585. Other mechanisms known in the art for use include transgene silencing using an miRNA or RNAi molecule (e.g., miR shRNA). For example, the cccDNA vector may contain a regulatory switch that encodes an RNAi molecule complementary to the transgene expressed by the cccDNA vector. When such RNAi is expressed, even if the cDNA vector expresses the transgene, its silencing will occur under the action of a complementary RNAi molecule, and if RNAi is not expressed when the transgene is expressed by the cDNA vector, silencing of the transgene under the influence of RNAi will not occur. Such an example of an RNAi molecule controlling gene expression, or used as a regulatory switch, is described in US 2017/0183664. In some embodiments, the regulatory switch comprises a repressor that blocks expression of the transgene from the cccDNA vector. In some embodiments, said positive/negative switch is a small transcription activating RNA (STAR) switch, such as those described in Chappell J. et al., Nat Chem Biol. Mar 2015; 11(3):214-20; and Chappell et al., Microbiol Spectr. May 2018; 6(3. In some embodiments, said control switch is a toehold switch such as described in US2009/0191546, US2016/0076083, WO 2017/087530, US2017/0204477, WO 2017/075486 and Green et al , Cell, 2014;159(4):925-939.

[00261] In some embodiments, said regulatory switch is a tissue-specific self-inactivating regulatory switch, for example, as described in US 2002/0022018, characterized in that it deliberately disables transgene expression at a site where transgene expression would otherwise be detrimental. In some embodiments, the regulatory switch is a recombinase-reversible gene expression system, for example, as described in US 2014/0127162 and US Pat. No. 8,324,436.

[00262] In some embodiments, the regulatory switch for controlling a transgene of interest or a gene expressed by a cccDNA vector is a hybrid of a nucleic acid-based control mechanism and a small molecule regulatory system. Such systems are well known to those skilled in the art and are provided for use herein. Examples of such regulatory switches include, but are not limited to, the LTRi system or the Lac-Tet-RNAi system, for example, as described in US 2010/0175141, Deans T. et al., Cell., 2007, 130(2) ; 363-372, WO 2008/051854 and US Patent 9388425.

[00263] In some embodiments, a regulatory switch for controlling a transgene of interest or a gene expressed by a cccDNA vector includes a circular permutation as described in US Pat. No. 8,338,138. In such an embodiment, the molecular switch is multistable, i.e. capable of switching between at least two states, or alternatively bistable, i.e. is either in an "off" or "on" state, such as being able or unable to emit light, able or unable to bind or not, able or unable to catalyze, able or unable to transfer electrons, and so on. According to another aspect, a molecular switch uses a fusion molecule, whereby the switch is capable of switching between more than two states. For example, in response to a particular threshold state exhibited by an insertion sequence or an acceptor sequence, a corresponding other fusion molecule sequence may exhibit a range of states (eg, a range of binding activity, a range of enzyme catalysis, etc.). Accordingly, rather than switching between an “on” and “off” state, the fusion molecule may exhibit a differential response to a stimulus.

[00264] In some embodiments, the nucleic acid regulatory switch may be selected from any switch or combination of switches from Table 5.

E. Post-transcriptional and post-translational regulatory switches.

[00265] In some embodiments, the regulatory switch for controlling a transgene of interest or a gene expressed by a cccDNA vector is a post-transcriptional modification system. For example, such a regulatory switch may be an aptazyme riboswitch sensitive to tetracycline or theophylline, as described in US 2018/0119156, GB 201107768, WO 2001/064956A3, patent EP 2707487 and Beilstein et al., ACS Synth. Biol., 2015, 4 (5), pp 526-534; Zhong et al., Elife. 2016 Nov 2; 5. pii: e18858. In some embodiments, it is contemplated that one skilled in the art can encode both the transgene and an inhibitory siRNA that contains a ligand-sensitive aptamer (negative switch), thereby generating a positive ligand-sensitive switch.

[00266] In some embodiments, the regulatory switch for controlling a transgene of interest or a gene expressed by a cccDNA vector is a post-translational modification system. In alternative embodiments, the gene or protein of interest is expressed as a proprotein or pre-proprotein, or contains a signal response element (SRE) or destabilizing domain (DD) attached to the expressed protein, which prevents the correct folding and/or activity of the protein until until post-translational modification occurs. In the case of a destabilizing domain (DD) or SRE, said destabilizing domain is post-translationally cleaved in the presence of an exogenous agent or small molecule. One skilled in the art will be able to use such control methods as described in US Pat. No. 8,173,792 and PCT Application WO 2017180587. Other post-transcriptional control switches contemplated for use in a cDNA vector to control the activity of a functional transgene are disclosed in: Rakhit et al., Chem. Biol. 2014; 21 (9): 1238-52 and Navarro et al., ACS Chem Biol. 2016; 19; 11 (8): 2101-2104A.

[00267] In some embodiments, the regulatory switch for controlling a transgene or gene of interest expressed by a cccDNA vector is a post-translational modification system that inserts ligand-responsive inteins into the coding sequence of the transgene such that the transgene or expressed protein remains inhibited until splicing. For example, this has been demonstrated using 4-hydroxytamoxifen and thyroid hormone (see, for example, US Patents 7541450, 9200045; 7192739, Buskirk, et al, Proc Natl Acad Sci USA. 2004 Jul 20; 101(29): 10505-10510 ; ACS Synth Biol. 2016 Dec 16; 5(12): 1475-1484; and 2005 Feb; 14(2): 523 532. In some embodiments, the post-transcriptional regulatory switch may be selected from any or combination thereof of Table 5.

F. Other examples of control switches

[00268] Any known regulatory switch can be used in a cccDNA vector to control gene expression of a transgene expressed by the cccDNA vector, including switches triggered by environmental changes. Additional examples include, but are not limited to: Suzuki et al.'s BOC method (Suzuki et al., Scientific Reports 8; 10051 (2018); genetic code expansion and non-physiological amino acid; radiation- or ultrasound-controlled positive/negative switches (see, for example, Scott S et al., Gene Ther. 2000 Jul;7(13):1121-5; US Patents 5612318; 5571797; 5770581; 5817636; and WO 1999/025385 Al. In some embodiments, said regulatory switch is controlled by an implantable system, e.g. according to US patent 7840263; US 2007/0190028 A1, where gene expression is controlled by one or more forms of energy, including electromagnetic energy, which activates promoters operably linked to the transgene in the cDNA vector.

[00269] In some embodiments, a the regulatory switch provided for use in the cccDNA vector is a hypoxia-mediated or stress-activated switch, such as those described in WO 1999060142 A2, US Pat. No. 5,834,306; 6218179; 6709858; US 2015/0322410; Greco et al, (2004) Targeted Cancer Therapies 9, S3 68, as well as FROG, TOAD and NRSE elements and condition-inducible silencing elements, including hypoxia response elements (HRE), inflammatory response elements (IRE) and shear-activated voltage elements (SSAE), for example, as described in US Pat. No. 9,394,526. This embodiment is suitable for “turning on” transgene expression from a cDNA vector after ischemia or in ischemic tissues and/or tumors.

[00270] In some embodiments, the regulatory switch provided for use in the cccDNA vector is an optogenetic (eg, light-controlled) regulatory switch, such as one of the switches discussed in: Poleskaya et al., BMC Neurosci . 2018; 19 (Suppl 1): 12, and are also provided for use according to the present invention. In such embodiments, the cccDNA vector may contain genetic elements that are light-sensitive and can regulate transgene expression in response to light in the visible wavelength range (eg, blue light, near-infrared). cccDNA vectors containing optogenetic regulatory switches are suitable for transgene expression in locations in the body accessible to such light sources, such as skin, eyes, muscles, etc., and can also be used to express cccDNA vectors in internal organs and tissues where the light signal can be delivered in a suitable manner (eg, using an implantable device as described herein). Such optogenetic regulatory switches include the use of light-sensitive elements or light-inducible transcription effectors (LITEs) (eg, as described in 2014/0287938), the Light-On system (eg, as described in Wang et al., Nat Methods. 2012 Feb 12 ; 9(3):266-9; which has been reported to allow in vivo control of the expression of the insulin transgene, the Cru2/CIB1 system (for example, as described in Kennedy et al., Nature Methods; 7, 973-975 (2010 ); and the FKF1/GIGANTEA system (eg, as described in Yazawa et al., Nat Biotechnol. 2009 Oct; 27(10):941-5).

G. "Emergency switches"

[00271] Other embodiments of the present invention provide a cccDNA vector containing a "kill switch". A "kill switch" as described herein allows the cell containing the specified cccDNA vector to be killed or undergo programmed cell death as a method of permanently removing the introduced cccDNA vector from the subject's system. One skilled in the art will appreciate that the use of "kill switches" in the cccDNA vectors of the present invention typically involves targeting the cccDNA vector to a limited number of cells that the subject can tolerate losing, or to a cell type that is apoptotic. desirable (for example, on cancer cells). In all aspects, the "kill switch" as described herein is designed to provide rapid and reliable killing of a cell containing the cccDNA vector in the absence of an incoming survival signal or other specified condition. In other words, the "safety switch" encoded by the cccDNA vector herein may limit the survival of the cell containing the cccDNA vector to an environment defined by specific input signals. Such "kill switches" serve to provide a biological containment function if it is necessary to either remove the cccDNA vector from a subject or ensure that it does not express the encoded transgene. Accordingly, “kill switches” are synthetic biological circuits in the cDNA vector that couple environmental signals to the conditional survival of the cell containing said cDNA vector. In some embodiments, different cDNA vectors may be designed to have different "kill switches". This provides the ability to control which cells expressing the transgene are killed when using cocktails of cccDNA vectors.

[00272] In some embodiments, the cccDNA vector may comprise a “kill switch” that is a modular biological containment circuit. In some embodiments, a "safety switch" provided for use in a cccDNA vector is described in WO 2017/059245, which describes a switch called a "safety switch" that contains at least two repressible sequences arranged in a mutually inhibitory manner such that an environmental signal represses the activity of a second molecule in the construct (eg, a small molecule binding transcription factor is used to promote a "survival" state by repressing toxin production). In cells containing a cccDNA vector containing a "safety blocker", after loss of the environmental signal, the circuit finally switches to a "blocked" state in which derepression of the toxin occurs, leading to the production of the toxin, which kills the cell. Another embodiment provides a synthetic biological circuit, referred to as a passcode circuit or a passcode kill switch, that uses hybrid transcription factors (TFs) to engineer complex environmental requirements for cell survival. “Emergency interlocks” and “access code emergency switches” described in WO 2017/059245 are particularly suitable for use in cccDNA vectors because they are modular and tunable, both with respect to the environmental conditions controlling circuit activation and and in relation to output modules that control cell fate. With the proper selection of toxins, which include, but are not limited to, an endonuclease such as EcoRI, the "access code strands" present in the cDNA vector can be used not only to kill the host cell containing the cDNA vector, but also to destroy it genome and accompanying plasmids.

[00273] Other "kill switches" known to one skilled in the art are provided for use in a cccDNA vector as described herein, for example, as described in US 2010/0175141; US 2013/0009799; US 2011/0172826; US2013/0109568, as well as the “kill switches” described in Jusiak et al, Reviews in Cell Biology and Molecular Medicine; 2014; 1-56; Kobayashi et al, PNAS, 2004; 101; 8419-9; Marchisio et al, Int. Journal of Biochem and Cell Biol., 2011; 43; 310-319; and Reinshagen et al, Science Translational Medicine, 2018, 11.

[00274] Accordingly, in some embodiments, the cccDNA vector may comprise a "kill switch" nucleic acid construct that contains a nucleic acid encoding an effector toxin or reporter protein, wherein expression of the effector toxin (e.g., death protein) or reporter protein is controlled by a predetermined condition. For example, a predetermined condition may be the presence of an environmental agent, such as an exogenous agent, without which the cell will by default express an effector toxin (eg, a death protein) and die. In alternative embodiments, the predetermined condition is the presence of two or more environmental agents, for example, the cell will survive only when supplied with two or more required exogenous agents, and without any of them, the cell containing the cccDNA vector will die.

[00275] In some embodiments, the cccDNA vector is modified to include a "kill switch" to kill cells containing the cccDNA vector to effectively stop in vivo expression of a transgene expressed by the cccDNA vector (e.g., a therapeutic gene, protein or peptide, etc.). In particular, the cccDNA vector is also genetically engineered to express a switch protein that does not function in mammalian cells under normal physiological conditions. Cells expressing the switch protein will only be killed upon administration of a drug or environmental condition specifically targeting said switch protein, and accordingly, expression of the therapeutic protein or peptide will then cease. For example, it has been reported that cells expressing HSV thymidine kinase can be killed by the administration of drugs such as ganciclovir and cytosine deaminase. See, for example, Dey and Evans, Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK), in Targets in Gene Therapy, edited You (2011); and Beltinger et al, Proc. Natl. Acad. Sci. USA 96(15):8699–8704 (1999). In some embodiments, the cccDNA vector may contain a siRNA “kill switch” called DISE (Death Induced by Survival Gene Elimination) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[00276] In some aspects, an "emergency blocker" is a biological circuit or system that renders a cellular response sensitive to a predetermined condition, such as the absence of an agent in the environment in which the cells grow, such as an exogenous agent. Such a chain or system may contain a nucleic acid containing expression modules that form an emergency regulatory circuit sensitive to a predetermined condition, wherein said construct comprises:

i) a first repressor protein expression module, characterized in that said first repressor protein binds a nucleic acid of a first repressor protein binding element, and represses transcription from a coding sequence containing said first repressor protein binding element, wherein the repressor activity of the first repressor protein is sensitive to inhibition the first exogenous agent, the presence or absence of which determines a predetermined condition;

ii) a second repressor protein expression module, characterized in that the second repressor protein binds a nucleic acid of a second repressor protein binding element and represses transcription from a coding sequence containing said second repressor protein binding element, wherein the second repressor protein is different from the first repressor protein; And

iii) an effector expression module comprising a nucleic acid sequence encoding an effector protein operably linked to a genetic element comprising a second repressor protein binding element, such that expression of the second repressor protein causes repression of effector expression from the effector expression module, wherein the second expression module comprises the element , which binds the nucleic acid of the first repressor protein, which provides transcriptional repression of the second repressor protein if the specified element is bound by the first repressor protein; the corresponding modules form a regulatory circuit such that, in the absence of a first exogenous agent, a first repressor protein is produced from the expression module of the first repressor protein and represses transcription from the expression module of the second repressor protein, such that the inhibition of effector expression by the second repressor protein is eliminated, allowing expression of the effector protein , but in the presence of the first exogenous agent, the activity of the first repressor protein is inhibited, which allows expression of the second repressor protein, which maintains the expression of the effector protein in the "off" state, thus the first exogenous agent in the chain is required to maintain expression of the effector protein in the "off" state , and removal or absence of the first exogenous agent results in expression of the effector protein by default.

[00277] In some embodiments, the effector is a toxin or protein that induces a cell death program. Any protein that is toxic to the host cell can be used. In some embodiments, the toxin kills only those cells in which it is expressed. In other embodiments, the toxin also kills other cells of the same host organism. Any of a variety of products that cause cell death are suitable for use in a "kill blocker". In particular, it is advisable to use agents that inhibit DNA replication, protein translation or other processes or, for example, destroy the nucleic acid of the host cell. To identify an effective mechanism for killing host cells upon circuit activation, several toxin genes that directly damage host cell DNA or RNA were tested. The endonuclease ecoRI ²¹ , the DNA gyrase inhibitor ccdB ²² , and the ribonuclease-type toxin mazF ²³ were tested because they are well characterized, are native to E. coli, and provide a range of killing mechanisms. To increase the reliability of the chain and provide a chain-independent mode of cell death, the system can be further adapted to express, for example, a targeted protease or nuclease that further interacts with a repressor that keeps the death gene in an "off" state. When the survival signal is lost or abolished, repression of the death gene is eliminated even more effectively due to, for example, active degradation of the repressor protein or its transcript. In a non-limiting example, mƒ-Lon protease was used not only to degrade LacI, but also to target important proteins for their degradation. The mƒ-Lon degradation tag pdt#1 can be attached to the 3′ end of five important genes whose protein products, in particular, are sensitive to mƒ-Lon degradation ²⁰ ; Cell viability was measured after ATc removal. Among the important target genes tested, the peptidoglycan biosynthesis gene murC provided the most pronounced and rapid cell death phenotype (proportion of surviving cells < 1 × 10 ^-4 after no more than 6 hours).

[00278] As used herein, the term “predetermined input signal” refers to an agent or condition that is known to influence the activity of a transcription factor polypeptide. Typically, such agents are capable of binding a transcription factor polypeptide and/or changing its conformation, thereby modifying the activity of said transcription factor polypeptide. Examples of predetermined inputs include, but are not limited to, environmental inputs not required for the survival of a particular host organism (ie, in the absence of a synthetic biological circuit as described herein). Conditions that may provide a predetermined input signal include, for example, temperature, for example if the activity of one or more factors is temperature sensitive, the presence or absence of light, including light at wavelengths of a particular spectrum, and the concentration of a gas, salt, metal or mineral. Environmental inputs include, for example, small molecule, biological agents such as pheromones, hormones, growth factors, metabolites, nutrients, etc., and analogs thereof; concentrations of chemicals, environmental byproducts, metal ions and other such molecules or agents; light levels; temperature; mechanical stress or pressure; or electrical signals such as currents and voltages.

[00279] In some embodiments, reporters are used to quantify the strength or activity of a signal received by modules or programmable synthetic biological circuits of the present invention. In some embodiments, reporters can be fused in-frame to other coding sequences to identify the location of a protein in a cell or organism. Luciferases can be used as effector proteins in various embodiments of the present invention, for example, to alter low levels of gene expression, since cells typically have little or no background luminescence in the absence of luciferase. In other embodiments, enzymes that produce colored substrates can be quantified using spectrophotometers or other absorbance-measuring instruments, including plate readers. Like luciferases, enzymes such as β-galactosidase can be used to measure low levels of gene expression because they tend to amplify weak signals. In some embodiments, the effector protein may be an enzyme that can degrade or otherwise destroy a particular toxin. In some embodiments, the effector protein may be an odorant enzyme that converts the substrate into an odor-bearing product. In some embodiments, the effector protein may be an enzyme that phosphorylates or dephosphorylates either small molecules or other proteins, or an enzyme that methylates or demethylates other proteins or DNA.

[00280] In some embodiments, the effector protein may be a receptor, a ligand, or a lytic protein. Receptors typically have three domains: an extracellular domain for binding ligands such as proteins, peptides or small molecules, a transmembrane domain, and an intracellular or cytoplasmic domain, which can often be involved in some signal transduction phenomenon such as phosphorylation. In some embodiments, transporter, channel, or pump gene sequences are used as effector proteins. Non-limiting examples and sequences of effector proteins for use with "kill switches" as described herein can be found in the Registry of Standard Biological Parts on the World Wide Web at parts.igem.org.

[00281] As used herein, a “modulator protein” is a protein that modulates expression from a target nucleic acid sequence. Modulator proteins include, for example, transcription factors, including transcription activators and repressors, as well as proteins that bind or modify a transcription factor and influence its activity. In some embodiments, the modulator protein includes, for example, a protease that degrades a protein factor involved in regulating expression of a target nucleic acid sequence. Preferred modulator proteins include modular proteins in which, for example, the DNA binding and entry agent binding or response elements or domains are separated and can be transferred so that, for example, the DNA binding domain of the first modulator protein is fused to the entry agent response domain the second modulator protein leads to the formation of a new protein that binds the DNA sequence recognized by the first protein, but is sensitive to the input agent to which the second protein normally responds. Accordingly, as used herein, the term “modulator polypeptide,” more specifically “repressor polypeptide,” includes, along with said polypeptides such as “LacI (repressor) polypeptide,” variants or derivatives of such polypeptides that respond to a different input agent or variant entry agent. Accordingly, in the case of the LacI polypeptide, LacI mutants or variants are provided that bind to agents other than lactose or IPTG. A wide variety of such agents are known in the art.

[00282] Table 5. Examples of Regulatory Switches ^b Can be activated by the effector; different from removing the effector that provides the off state. ^c Possibility of switching off by the effector; different from removing the effector that provides the on state. ^d A ligand or other physical stimulus (e.g., temperature, electromagnetic radiation, electricity) that stabilizes the switch in either the on or off state. ^e Refers to the reference number given in: Kis et al., JR Soc Interface. 12:20141000 (2015), the contents of which, together with references cited, are incorporated herein by reference.

VI. Preparation of cccDNA vector

A. Receipt in general case

[00283] The preparation of the cccDNA vector is described in section IV of PCT/US18/49996, filed September 7, 2018, incorporated herein by reference in its entirety. As described herein, said cccDNA vector can be produced using a method comprising the following steps: a) incubating a population of host cells (eg, insect cells) carrying a template polynucleotide expression construct (eg, ccDNA plasmid, ccDNA bacmid, and /or cDNA baculovirus) that does not contain sequences encoding the viral capsid, in the presence of Rep protein under conditions effective to induce and for a time sufficient to induce the production of said cDNA vector in host cells, wherein said host cells do not contain sequences encoding the viral capsid; and b) collecting and isolating the cDNA vector from the host cells. The presence of the Rep protein induces replication of the vector polynucleotide with a modified ITR to produce a cccDNA vector in the host cell. However, viral particles (eg AAV virions) are not expressed. Thus, there are no size restrictions such as those naturally present in AAV vectors or other viral vectors.

[00284] The presence of a cccDNA vector isolated from host cells can be confirmed by digesting DNA isolated from a host cell with a restriction enzyme having a single recognition site on the ccDNA vector and analyzing the digested DNA material on a non-denaturing gel for confirmation the presence of characteristic bands of linear and continuous DNA compared to linear and discontinuous DNA.

[00285] Some embodiments of the present invention provide the use of host cell lines that have stably integrated a polynucleotide DNA expression vector template (ccDNA template) into their own genome to produce a non-viral DNA vector, for example, as described in Lee, L. et al. (2013) Plos One 8(8): e69879. Preferably, Rep is added to host cells at an MOI (multiplicity of infection) of approximately 3. If the host cell line is a mammalian cell line, for example, HEK293 cells, then said cell lines may contain a stably integrated polynucleotide vector array, and a second vector, for example, herpesvirus, can be used to introduce the Rep protein into cells, which ensures the excision and amplification of cctDNA in the presence of Rep and a helper virus.

[00286] In one embodiment, the host cells used to produce the cccDNA vectors described herein are insect cells, and to deliver both the polynucleotide that encodes the Rep protein and the template polynucleotide expression construct of the non-viral DNA vector for cccDNA is used by baculovirus, for example as described in FIG. 4A - 4C and in Example 1. In some embodiments, the host cell is modified to express the Rep protein.

[00287] The cccDNA vector is then collected and isolated from the host cells. The timing of collection and recovery of the cccDNA vectors described herein from cells can be selected and optimized to ensure that the cccDNA vectors are obtained in high yield. For example, the collection time may be selected based on cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are cultured under sufficient conditions and harvested sufficiently after infection with the baculovirus to produce cccDNA vectors, but before the majority of the cells begin to die due to toxicity of the baculovirus. These DNA vectors can be isolated using plasmid purification kits, such as Endo-Free Plasmid Kits from Qiagen. Other methods developed for isolating plasmids can also be adapted for DNA vectors. In general, any method for purifying nucleic acids can be used.

[00288] These DNA vectors can be purified by any DNA purification methods known to those skilled in the art. In one embodiment, purified cDNA vectors are obtained as DNA molecules. In another embodiment, purified cccDNA vectors are obtained in the form of exosomes or microparticles.

[00289] The presence of a cccDNA vector can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme having a single recognition site on the DNA vector and analyzing both digested and uncleaved DNA material using gel electrophoresis to confirm the presence characteristic bands of linear and continuous DNA, different from those of linear and discontinuous DNA. Fig. 4C and FIG. 4D illustrate one embodiment for identifying the presence of end-closed cccDNA vectors produced by the methods described herein.

B. cDNA plasmid

[00290] A cDNA plasmid is a plasmid used to subsequently produce a cDNA vector. In some embodiments, the cccDNA plasmid can be constructed using known methods to provide at least the following functionally related components in the direction of transcription: (1) a 5' ITR sequence as described herein (e.g., a wild-type or 5' ITR sequence modified); (2) an expression cassette containing a cis-regulatory element, for example, a promoter, an inducible promoter, a regulatory switch, an enhancer, etc.; and (3) a 3'-ITR sequence as described herein (eg, a wild-type or modified 3'-ITR sequence), wherein the 3'-ITR sequence is symmetrical to the 5'-ITR sequence. In some embodiments, the ITR-flanked expression cassette contains a cloning site for introducing an exogenous sequence. This expression cassette replaces the rep and cap coding regions of the AAV genomes.

[00291] In one aspect, a cccDNA vector is derived from a plasmid, referred to herein as a “ccDNA plasmid,” encoding, in this order: an adeno-associated virus (AAV) first inverted terminal repeat (ITR), an expression cassette containing at least a transgene, and a regulatory switch, and a mutated or modified AAV ITR, wherein the cDNA plasmid does not contain sequences encoding an AAV capsid protein. In alternative embodiments, said ccDNA plasmid encodes, in the specified order: a first (or 5') modified or mutated AAV ITR expression cassette containing at least a transgene and a regulatory switch, and a second (or 3') modified AAV ITR, wherein said ccDNA - the plasmid does not contain sequences encoding the AAV capsid protein, and the 5'- and 3'-ITRs are symmetrical to each other. In alternative embodiments, said cccDNA plasmid encodes, in said order: a first (or 5') modified or mutated AAV ITR expression cassette containing at least a transgene and a regulatory switch, and a second (or 3') mutated or modified AAV ITR, wherein the specified cDNA plasmid does not contain sequences encoding the AAV capsid protein, and the modified 5'- and 3'-ITRs contain the same modifications (i.e., they are reverse complementary or symmetrical with respect to each other). In alternative embodiments, said ccDNA plasmid encodes, in the specified order: a first (or 5') modified or mutated AAV ITR expression cassette containing at least a transgene and a regulatory switch, and a second (or 3') WT AAV ITR, wherein said ccDNA - the plasmid does not contain sequences encoding the AAV capsid protein, and the 5'- and 3'-ITRs are symmetrical to each other. In alternative embodiments, said ccDNA plasmid encodes, in said order: a first (or 5') WT AAV ITR expression cassette containing at least a transgene and a regulatory switch, and a second (or 3') WT AAV ITR, wherein said ccDNA plasmid does not contains sequences encoding the AAV capsid protein, and the 5'- and 3'-ITRs are symmetrical to each other.

[00292] In a further embodiment, the cDNA plasmid system does not contain viral capsid protein coding sequences (i.e., does not contain AAV capsid genes as well as capsid genes of other viruses). Additionally, in a particular embodiment, the ccDNA plasmid also does not contain sequences encoding the AAV Rep protein. Accordingly, in a preferred embodiment, the cccDNA plasmid lacks the functional AAV cap and AAV rep genes, GG-3' for AAV2), as well as the variable palindromic hairpin sequence.

[00293] The cDNA plasmid of the present invention can be produced using natural nucleotide sequences from the genomes of any AAV serotypes well known in the art. In one embodiment, the cccDNA plasmid backbone is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV 5, AAV7, AAV8, AAV9, AAV 10, AAV 11, AAV 12, AAVrh8, AAVrh10, the AAV-DJ genome, and AAV-DJ8 . For example, NCBI: NC 002077; NC001401; NC 001729; NC 001829; NC 006152; NC 006260; NC 006261; Kotin and Smith, The Springer Index of Viruses, available at the email address maintained by Springer (web address:

oesys.springer.de/viruses/database/mkchapter.asp?virID=42.04.) (note: references to an email posting address or database refer to the content available at the posting email address or database as of the actual filing date of this application). In a particular embodiment, the cDNA plasmid backbone is derived from the AAV2 genome. In another specific embodiment, the cDNA plasmid backbone is a synthetic backbone engineered to include ITRs at the 5' and 3' ends derived from one of the AAV genomes.

[00294] The cccDNA plasmid may optionally include a selectable or selectable marker for use in creating a cell line producing the cccDNA vector. In one embodiment, said selectable marker may be inserted downstream (ie, in the 3' direction) of the 3'ITR sequence. In another embodiment, said selectable marker may be inserted upstream (ie, in the 5' direction) of the 5'ITR sequence. Suitable selectable markers include, for example, markers that confer drug resistance. Selection markers can be, for example, genes for resistance to blasticidin S, kanamycin, geneticin, etc. In a preferred embodiment, the selectable marker for selecting for drug sensitivity is a blasticidin S resistance gene.

[00295] An example of cccDNA (eg, rAAVO) is produced from the rAAV plasmid. A method for producing an rAAV vector may include: (a) delivering an rAAV plasmid to a host cell as described above, wherein both the host cell and the plasmid do not contain genes encoding a capsid protein, (b) culturing said host cell under conditions , ensuring the production of the cDNA genome; and (c) collecting cells and isolating the AAV genome produced by said cells.

C. An example of a method for obtaining cDNA vectors from cDNA plasmids

[00296] This document also provides methods for producing capsidless cccDNA vectors, in particular, a method with sufficiently high yield to provide sufficient quantities of vector for in vivo experiments.

[00297] In some embodiments, a method for producing a cccDNA vector includes the following steps: (1) introducing a nucleic acid construct containing an expression cassette and two symmetrical ITR sequences into a host cell (for example, Sf9 cells), (2) optionally generating a clonal line cells, for example, using a selectable marker present on a plasmid, (3) introducing a Rep-encoding gene (by transfection or infection with a baculovirus carrying the gene) into said insect cell; and (4) cell collection and cDNA vector purification. The nucleic acid construct containing the expression cassette and the two ITR sequences described above to produce a capsidless AAV vector may be in the form of a cfAAV plasmid, or a bacmid or baculovirus derived from a cfAAV plasmid as described below. Said nucleic acid construct can be introduced into a host cell by transfection, viral transduction, stable integration, or other methods known in the art.

D. Cell lines

[00298] The host cell lines used to produce the cccDNA vector may include insect cell lines derived from Spodoptera frugiperda, such as Sf9 Sf21 cells or Trichoplusia ni cells, or cell lines of other invertebrates, vertebrates or other eukaryotes, in including mammalian cells. Other cell lines known to one skilled in the art may also be used, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1 180, monocytes, as well as mature and immature dendritic cells. Host cell lines can be transfected to stably express the cccDNA plasmid to produce a ccDNA vector in high yield.

[00299] In some embodiments, cccDNA plasmids can be introduced into Sf9 cells by transient transfection using reagents (eg, liposomal, calcium phosphate) or physical methods (eg, electroporation) known in the art. Alternatively, stable Sf9 cell lines can be generated that have stably integrated the ccDNA plasmid into the genome. Such stable cell lines can be created by incorporating a selectable marker into a cccDNA plasmid as described above. If the cccDNA plasmid used to transfect a cell line includes a selectable marker, such as an antibiotic, cells that have been transfected with the ccDNA plasmid and have integrated the ccDNA plasmid into the genome can be selected by adding the antibiotic to the cell growth medium. Stable cell clones can then be isolated using single cell expansion or colony transfer methods and expanded.

E. Isolation and purification of cDNA vectors

[00300] Examples of a method for preparing and isolating cccDNA vectors are presented in FIG. 4A - 4E and in specific examples below. cccDNA vectors as described herein can be derived from producer cells expressing AAV Rep protein(s) further transformed with a ccDNA plasmid, ccDNA bacmid, or ccDNA baculovirus. Plasmids suitable for the production of cccDNA vectors include those shown in FIG. 9A (suitable for obtaining Rep BIIC), FIG. 9B (plasmid used to obtain the cDNA vector).

[00301] In some embodiments, the polynucleotide encodes an AAV Rep protein (Rep 78 68) delivered to the producing cell in a plasmid (Rep-plasmid), bacmid (Rep-bacmid), or baculovirus (Rep-baculovirus). Rep plasmid, Rep bacmid and Rep baculovirus can be produced by the methods described above.

[00302] Methods for producing a cccDNA vector, which is an example of a ccDNA vector, are described herein. Expression constructs used to produce cccDNA vectors of the present invention may be a plasmid (eg, ccDNA plasmids), bacmid (eg, ccDNA-baculovirus), and/or baculovirus (eg, ccDNA-baculovirus). In a non-limiting example, the cDNA vector can be obtained from cells coinfected with cDNA baculovirus and Rep baculovirus. Rep proteins produced from Rep baculovirus can replicate cccDNA baculovirus to produce cccDNA vectors. Alternatively, cccDNA vectors can be generated from cells stably transfected with a construct containing the AAV Rep protein coding sequence (Rep78/52) delivered in Rep plasmids, Rep bacmids or Rep baculovirus. cccDNA Baculovirus can transiently transfect cells, can be replicated by the Rep protein, and can produce cccDNA vectors.

[00303] Permissive insect cells such as Sf9, Sf21, Tni (Trichoplusia ni), High Five cells can be transfected with a bacmid (e.g., cccDNA bacmid), and said bacmid produces a cccDNA baculovirus, which is a recombinant baculovirus comprising sequences containing symmetrical ITRs and an expression cassette. The insect cells can then be infected with the cDNA baculovirus to produce a new generation of recombinant baculovirus. Optionally, this step can be repeated one or more times to obtain more recombinant baculovirus.

[00304] The timing for harvesting the cccDNA vectors described herein from cells can be selected and optimized to achieve high yield production of cccDNA vectors. For example, the collection time may be selected based on cell viability, cell morphology, cell growth, and the like. Typically, cells can be harvested after sufficient time has passed after baculovirus infection to produce cDNA vectors (eg, cDNA vectors) but before most cells begin to die due to viral toxicity. cccDNA vectors can be isolated from Sf9 cells using plasmid purification kits such as the ENDO-FREE PLASMID® kits from Qiagen. Other methods developed for plasmid isolation can also be adapted for cDNA vectors. In general, any nucleic acid purification methods known in the art, as well as commercially available DNA extraction kits, can be used.

[00305] Alternatively, purification can be accomplished by alkaline lysis of the cell pellet, centrifuging the resulting lysate, and performing chromatographic separation. In one non-limiting example, this process can be accomplished by loading the supernatant onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acids, followed by elution (e.g., 1.2 M NaCl) and further chromatographic purification on a gel filtration column (for example, on 6 Fast Flow GE). The capsidless AAV vector is then isolated, for example by sedimentation.

[00306] In some embodiments, cccDNA vectors may also be purified in the form of exosomes, or microparticles. It is known in the art that many cell types release not only soluble proteins, but also complex protein/nucleic acid cargos by shedding membrane microvesicles (Cocucci et al, 2009; EP 10306226.1). Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles); both the first and second contain proteins and ROAR as cargo. Microvesicles are formed by direct budding from the plasma membrane, and exosomes are released into the extracellular environment upon fusion of multivesicular endosomes with the plasma membrane. Accordingly, cccDNA vector-containing microvesicles and/exosomes can be isolated from cells transduced with the ccDNA plasmid, or bacmid, or baculovirus produced using the ccDNA plasmid.

[00307] Microvesicles can be isolated by filtering or ultracentrifuging the culture medium at 20,000 x g, and exosomes at 100,000 x g. The optimal duration of ultracentrifugation can be determined experimentally and depends on the specific cell type from which the vesicles are isolated. Preferably, the culture medium is first cleared by low-speed centrifugation (eg at 2000 x g for 5-20 minutes) and centrifuged using, for example, an AMICON® spin column (Millipore, Watford, UK). Microvesicles and exosomes can be further purified by FACS or MACS using specific antibodies that recognize specific surface antigens present on microvesicles and exosomes. Other methods for purifying microvesicles and exosomes include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. After purification, the vesicles are washed, for example, with phosphate-buffered saline. One advantage of using microvesicles or exosomes to deliver cccDNA-containing vesicles is that these vesicles can be targeted to different cell types by incorporating proteins into their membranes that are recognized by specific receptors on the relevant cell types. (See also EP 10306226)

[00308] Another aspect of the present invention relates to methods for purifying cccDNA vectors from host cell lines that have stably integrated the cccDNA construct into the genome. In one embodiment, the cDNA vectors are purified as DNA molecules. In another embodiment, cccDNA vectors are purified as exosomes or microparticles.

[00309] In FIG. 5 of PCT/US18/49996 shows a gel confirming the production of cccDNA from several ccDNA plasmid constructs using the method described in the examples section. The presence of cccDNA confirms the characteristic gel banding pattern described herein.

VII. Pharmaceutical compositions

[00310] In another aspect, pharmaceutical compositions are provided. Said pharmaceutical composition contains a cccDNA vector as described herein and a pharmaceutically acceptable carrier or diluent.

[00311] DNA vectors as described herein can be included in pharmaceutical compositions suitable for administration to a subject for in vivo delivery to cells, tissues or organs of the subject. Typically, said pharmaceutical composition comprises a cccDNA vector as described herein and a pharmaceutically acceptable carrier. For example, the cccDNA vectors described herein may be included in a pharmaceutical composition suitable for the desired route of therapeutic administration (eg, parenteral administration). Passive tissue transduction by intravenous or intra-arterial high-pressure infusion, as well as intracellular injection such as intranuclear microinjection or

intracytoplasmic injection. Pharmaceutical compositions for therapeutic purposes can be formulated in a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high concentration of cDNA vector. Sterile injection solutions can be prepared by introducing the cccDNA vector compound in the required amount into an appropriate buffer with one or a combination of ingredients listed above, if necessary, followed by sterilizing filtration.

[00312] Pharmaceutically active compositions containing a cccDNA vector can be formulated to deliver a transgene in nucleic acid into recipient cells, resulting in therapeutic expression of the transgene therein. The composition may also include a pharmaceutically acceptable carrier.

[00313] The cccDNA vector as described herein can be formulated into a pharmaceutical composition suitable for local, systemic, intra-amniotic, intrathecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, interstitial (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (eg, extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrastromal, intracameral and intravitreal), intracochlear and mucosal (eg, oral, rectal, nasal) administration . Passive tissue transduction via high-pressure intravenous or intra-arterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated.

[00314] Pharmaceutical compositions for therapeutic purposes generally must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated into a solution, microemulsion, dispersion, liposome or other ordered structure suitable for high concentration of cDNA vector. Sterile injectable solutions can be prepared by incorporating the cccDNA vector in the required amount into an appropriate buffer along with one or a combination of ingredients listed above, if necessary, followed by sterilizing filtration.

[00315] Various methods and methods for delivering nucleic acids into cells are known in the art. For example, nucleic acids such as cccDNA can be formulated into lipid nanoparticles (LNPs), lipidoids, liposomes, lipid nanoparticles, lipoplexes or core/shell nanoparticles. Typically, LNPs consist of a nucleic acid molecule (e.g., cccDNA), one or more ionizable or cationic lipids (or salts thereof), one or more nonionic or neutral lipids (e.g., a phospholipid), a molecule that prevents aggregation (e.g., PEG or PEG-lipid conjugate), and optionally a sterol (eg cholesterol).

[00316] Another method of delivering nucleic acids, such as cctDNA, into a cell is by conjugating the nucleic acid to a ligand that is internalized by the cell. For example, the ligand may bind to a cell surface receptor and be internalized through endocytosis. Said ligand may be covalently linked to a nucleotide in the nucleic acid. Examples of conjugates for delivering nucleic acids into cells are described, for example, in WO 2015/006740, WO 2014/025805, WO 2012/037254, WO 2009/082606, WO 2009/073809, WO 2009/018332, WO 2006/11 2872, WO 2004 /090108, WO 2004/091515 and WO 2017/177326.

[00317] Nucleic acids, such as cctDNA, can also be delivered into a cell by transfection. Suitable transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include, but are not limited to, TurboFect Transfection Reagent (Thermo Fisher Scientific), Pro-Ject Reagent (Thermo Fisher Scientific), TRANSPASS™ P Protein Transfection Reagent (New England Biolabs), CHARIOT™ Protein Delivery Reagent (Active Motif), Protejuice ™ Protein Transfection Reagent (EMD Millipore), 293Fectin reagent, lipofectamine ™ 2000, Lipofectamine ™ 3000 (Thermo Fish Fish Er Scientific), Lipofectamine ™ (Thermo Fisher Scientific), Lipofectin ™ lipostrinum (Thermo Fisher Scientific , DMPJE-C, CELLFECTIN™ (Thermo Fisher Scientific), OLIGOFECTAMINE™ (Thermo Fisher Scientific), LIPOFECT ACE™, FUGENE™ (Roche, Basel, Switzerland), FUGENE™ HD (Roche), TRANSFECTAM™ (transfectam, Promega, Madison , Wisconsin), TFX-10™ (Promega), TFX-20™ (Promega), TFX-50™ (Promega), TRANSFECTIN™ (BioRad, Hercules, CA), SILENTFECT™ (Bio-Rad), Effectene™ (Qiagen , Valencia, CA), DC-chol (Avanti Polar Lipids), GENEPORTER™ (Gene Therapy Systems, San Diego, CA), DHARMAFECT 1™ (Dharmacon, Lafayette, CO), DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, MO), and ESCORT™ IV (Sigma Chemical Co.). Nucleic acids such as cctDNA can also be delivered into cells using microfluidics techniques known to those skilled in the art.

[00318] Methods for non-viral delivery of nucleic acids in vivo or ex vivo include electroporation, lipofection (see US Pat. Nos. 5,049,386; 4,946,787 and commercially available reagents such as Transfectam™ and Lipofectin™), microinjection, biolistics, virosomes, liposomes (see For example, sources: Crystal, Science 270:404-410 (1995); Blaese et al, Cancer Gene Ther. 2:291-297 (1995); Behr et al, Bioconjugate Chem. 5:382-389 (1994); Remy et al, Bioconjugate Chem. 5:647-654 (1994); Gao et al, Gene Therapy 2:710-722 (1995); Ahmad et al, Cancer Res. 52:4817-4820 (1992); US patents, #4186183, #4217344, #4235871, #4261975, #4485054, #4501728, #4774085, #4837028 and #4946787), immunoliposomes, microbubbles (Frenkel et al. Ultrasound Med. Biol. (2002) 28(6): 817 -22; Tsutsui et al, Cardiovasc. Ultrasound (2004) 2:23), polycation or lipid nucleic acid conjugates, deproteinized DNA and agent-enhanced DNA uptake. Sonoporation using, for example, the Sonitron 2000 system (Rich-Mar) can also be used to deliver nucleic acids.

[00319] cccDNA vectors as described herein can also be administered directly into the body to transduce cells in vivo. Administration is by any of the routes commonly used to bring the molecule into maximum contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable routes for administering such nucleic acids are available and well known to those skilled in the art, and although more than one route may be used to administer a particular composition, a particular route can often provide a faster and more efficient response than another route of administration.

[00320] Methods of administering a cDNA nucleic acid vector as described herein may include delivery to hematopoietic stem cells, for example, by methods described, for example, in US Pat. No. 5,928,638.

[00321] Various delivery methods known in the art, or modifications thereof, can be used to deliver cccDNA vectors in vitro or in vivo. For example, in some embodiments, cccDNA vectors are delivered by transiently permeating the cell membrane using mechanical, electrical, ultrasonic, hydrodynamic, or laser energy to facilitate DNA entry into target cells. For example, the cccDNA vector can be delivered by transiently disrupting the cell membrane by forcing the cell through a channel of limited size or other methods known in the art. In some cases, the cccDNA vector is directly injected individually as deproteinized DNA into skin, thymus, cardiac, skeletal muscle, or liver cells.

[00322] In some cases, the cccDNA vector is delivered using a gene gun. Spherical gold or tungsten particles (1-3 µm in diameter) coated with capsidless AAV vectors can be accelerated to high speed using pressurized gas to penetrate target tissue cells.

[00323] In some embodiments, electroporation is used to deliver cccDNA vectors. Electroporation causes temporary destabilization of the cell membrane of target tissue cells by introducing a pair of electrodes into the tissue, so that DNA molecules from the environment around the destabilized membrane can penetrate into the cytoplasm and nucleoplasm of the specified cell. Electroporation has been used in vivo for many tissue types such as skin, lung, and muscle.

[00324] In some cases, the cccDNA vector is delivered by hydrodynamic injection, which is a simple and highly effective method for direct intracellular delivery of any water-soluble compounds and particles to the internal organs and skeletal muscles throughout the limb.

[00325] In some cases, cccDNA vectors are delivered using ultrasound, creating nanoscopic pores in the membrane to facilitate intracellular delivery of DNA particles into internal organ or tumor cells, so the size and concentration of plasmid DNA is of great importance for the effectiveness of the system.. In some cases, cccDNA - vectors are delivered by magnetofection using magnetic fields to concentrate particles containing nucleic acid into target cells.

[00326] In some cases, chemical delivery systems can be used, for example, using nanometer complexes that involve the compaction of negatively charged nucleic acid with polycationic nanometer particles, formulated in cationic liposomes/micelles or cationic polymers. Cationic lipids used for the delivery method include, but are not limited to, monovalent cationic lipids. polyvalent cationic lipids, guanidine-containing compounds, cholesterol derivatives, cationic polymers (eg, poly(ethylenimine), poly-L-lysine, protamine, other cationic polymers) and lipid-polymer hybrid.

A. Exosomes

[00327] In some embodiments, a cccDNA vector as described herein is delivered by packaging into an exosome. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after the fusion of multivesicular bodies with the plasma membrane. Their surface consists of the lipid bilayer of the cell membrane of the donor cell, they contain cytosol from the cell that produced the exosome, and they expose membrane proteins of the parent cell on the surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MCs), and dendritic cells (DCs). In some embodiments, exosomes with a diameter of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, or 50 to 100 nm are provided for use. Exosomes can be isolated for delivery to target cells either using donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing capsidless AAV vectors according to the present invention.

B. Microparticles/Nanoparticles

[00328] In some embodiments, a cccDNA vector as described herein is delivered on a lipid nanoparticle. Typically, lipid nanoparticles contain an ionizable aminolipid (e.g., heptatriakonta-6, 9, 28, 31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3 -phosphocholine, DSPC), cholesterol and envelope lipid (polyethylene glycol-dimyristoylglycerol, PEG-DMG), for example, as described in Tam et al. (2013). Advances in Lipid Nanopartides for siRNA delivery. Pharmaceuticals 5(3): 498 -507.

[00329] In some embodiments, the lipid nanoparticle has an average diameter of from about 10 nm to about 1000 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 300 nm. In some embodiments, the lipid nanoparticle has a diameter of from about 10 nm to about 300 nm. In some embodiments, the lipid nanoparticle has a diameter of less than 200 nm. In some embodiments, the lipid implementation of the nanoparticle has a diameter of from about 25 to about 200 nm. In some embodiments, the lipid nanoparticle composition (eg, a composition comprising a plurality of lipid nanoparticles) has a size distribution with an average particle size (eg, diameter) of from about 70 nm to about 200 nm; more often the average size is approximately 100 nm or less.

[00330] Various lipid nanoparticles known in the art can be used to deliver the cDNA vector as described herein. For example, various delivery methods using lipid nanoparticles are described in US patents No. 9404127, No. 9006417 and No. 9518272.

[00331] In some embodiments, a cccDNA vector as described herein is delivered on a gold nanoparticle. Typically, the nucleic acid may be covalently associated with the gold nanoparticle or non-covalently associated with the gold nanoparticle (eg, associated by charge-charge interaction), for example, as described in Ding et al. (2014). Gold Nanoparticles for Nucleic Acid Delivery. Mol. Ther. 22(6); 1075-1083. In some embodiments, gold nanoparticle-nucleic acid conjugates are prepared using methods described, for example, in US Pat. No. 6,812,334.

C. Conjugates

[00332] In some embodiments, a cccDNA vector as described herein is conjugated (e.g., covalently linked to an agent that enhances cellular uptake. "Agent that enhances cellular uptake" is a molecule that facilitates the transport of a nucleic acid across a lipid membrane. For example, the nucleic acid may be conjugated to a lipophilic compound (e.g., cholesterol, tocopherol, etc.), cell penetrating peptide (CPP) (e.g., penetratin, TAT, Syn1B, etc.), and polyamines (e.g., spermine Additional examples of agents that enhance cellular uptake are disclosed, for example, in: Winkler (2013), Oligonucleotide conjugates for therapeutic applications, Ther. Deliv. 4(7): 791-809.

[00333] In some embodiments, the cccDNA vector as described herein is conjugated to a polymer (eg, a polymer molecule) or a folate molecule (eg, a folic acid molecule). In general, delivery of nucleic acids conjugated to polymers is known in the art, for example, described in WO 2000/34343 and WO 2008/022309. In some embodiments, the cccDNA vector as described herein is conjugated to a poly(amide) polymer, for example, as described in US Pat. No. 8,987,377. In some embodiments, the nucleic acid described herein is conjugated to a folic acid molecule as described in US Pat. No. 8,507,455.

[00334] In some embodiments, the cccDNA vector as described herein is conjugated to a carbohydrate, for example, as described in US Pat. No. 8,450,467.

D. Nanocapsule

[00335] Alternatively, cDNA vector nanocapsule formulations as described herein may be used. Generally, nanocapsules can entrap substances in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (about 0.1 μm in size) must be constructed using polymers that are degradable in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles that meet the specified requirements are provided for use.

E. Liposomes

[00336] cccDNA vectors in accordance with the present invention can be added to liposomes for delivery to a cell or target organ of a subject. Liposomes are vesicles having at least one lipid bilayer. Liposomes are often used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They work by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds containing a phosphatidylcholine group, although these compositions may also include other lipids.

[00337] The preparation and use of liposomes is generally known to those skilled in the art. Liposomes with improved serum stability and circulatory half-life have been developed (US Patent No. 5,741,516). In addition, various methods for preparing liposomal and liposomal-like formulations as potential drug carriers have been described (US Pat. No. 5,567,434; US Pat. No. 5,552,157; US Pat. No. 5,565,213; US Pat. No. 5,738,868 and US Pat. No. 5,795,587).

[00338] Liposomes have been used successfully with certain cell types that are generally resistant to transfection using other procedures. In addition, liposomes are free of DNA length restrictions typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into various cultured cell lines and animals. In addition, several successful clinical trials have been completed to study the effectiveness of liposome-mediated drug delivery.

[00339] Liposomes are formed from phospholipids that are dispersed in an aqueous environment and spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLV). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUV) with a diameter ranging from 200 to 500 angstroms containing an aqueous solution in the core.

[00340] In some embodiments, the liposome contains cationic lipids. The term "cationic lipid" includes lipids and synthetic lipids having both polar and nonpolar domains that may be positively charged at or near physiological pH, and that bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids to cells. In some embodiments, cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. In some embodiments, the cationic lipids include linear, branched alkyl, alkenyl groups, or any combination of the above. In some embodiments, cationic lipids contain from 1 to about 25 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, or 25 carbon atoms. In some embodiments, cationic lipids contain more than 25 carbon atoms. In some embodiments, straight-chain or branched alkyl or alkene groups contain six or more carbon atoms. Cationic the lipid may also contain, in some embodiments, one or more alicyclic groups. Non-limiting examples of alicyclic groups include cholesterol and other steroid groups. In some embodiments, political lipids using one or more counterions. Examples of counterions (anions) include, but are not limited to , Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite and nitrate.

[00341] In some aspects of the present invention, the liposomal formulation includes one or more compounds with a polyethylene glycol (PEG) functionality (so-called "PEGylated compounds"), which can reduce immunogenicity/antigenicity, provide hydrophilicity and hydrophobicity of the compound(s), and reduce dosing frequency . The liposomal formulation may also simply include a polyethylene glycol (PEG) polymer as an additional component. In such aspects, the molecular weight of the PEG or PEG functional group can range from 62 Da to about 5000 Da.

[00342] According to some aspects of the present invention, there is provided a liposomal formulation that delivers an API with an extended release or controlled release profile over a period of hours to weeks. In some related aspects, the liposomal composition may include aqueous chambers that are connected by lipid bilayers. In other related aspects, the liposomal formulation encapsulates the API with components that undergo a physical transition at elevated temperature, thereby releasing the API over a period of hours to weeks.

[00343] In some aspects, the liposomal composition contains sphingomyelin and one or more lipids as described herein. In some aspects, the liposomal composition contains optisomes.

[00344] According to some aspects of the present invention, there is provided a liposomal formulation that includes one or more lipids selected from: N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl -sn-glycero-phosphoethanolamine), conjugated to MPEG (methoxypolyethylene glycol) lipid a, HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleylphosphatidylserine); POPC (palmitoyloleylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxy polyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dierucoylphosphatidylcholine); DOPE (dioleyl-sn-glycero-phosphoethanolamine), cholesterol sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleyl-sn-glycero-phosphatidylcholine) or any combination of the above.

[00345] According to some aspects of the present invention, there is provided a liposomal formulation comprising a phospholipid, cholesterol and a PEGylated lipid in a molar ratio of 56:38:5. In some aspects, the total lipid content of the liposomal formulation is 2-16 mg/ml. According to some aspects of the present invention, there is provided a liposomal composition comprising a phosphatidylcholine-functional lipid, an ethanolamine-functional lipid, and a PEGylated lipid. In some aspects, the present invention provides a liposomal formulation comprising a phosphatidylcholine-functional lipid, an ethanolamine-functional lipid, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. According to some aspects of the present invention, there is provided a liposomal composition comprising a lipid containing a phosphatidylcholine functionality, cholesterol and a pegylated lipid. According to some aspects of the present invention, there is provided a liposomal composition comprising a lipid containing a phosphatidylcholine functionality and cholesterol. In some aspects, said PEGylated lipid is PEG-2000-DSPE. In some aspects, the present invention provides a liposomal formulation comprising DPPG, soy PC, MPEG-DSPE lipid conjugate, and cholesterol.

[00346] In some aspects of the present invention, there is provided a liposomal formulation comprising one or more phosphatidylcholine-functional lipids and one or more ethanolamine-functional lipids. In some aspects, the present invention provides a liposomal formulation comprising any one or more of phosphatidylcholine-functional lipids, ethanolamine-functional lipids, and sterols, such as cholesterol. In some aspects, the liposomal formulation comprises DOPC/DEPC; and DOPE.

[00347] According to some aspects of the present invention, there is provided a liposomal formulation further containing one or more pharmaceutical excipients, for example, sucrose and/or glycine.

[00348] According to some aspects of the present invention, there is provided a liposomal composition having a unilamellar or multilamellar structure. According to some aspects of the present invention, there is provided a liposomal formulation that contains multivesicular particles and/or foam-based particles. According to some aspects of the present invention, there is provided a liposomal formulation whose relative particle size is larger than that of conventional nanoparticles, ranging from approximately 150 to 250 nm. In some aspects, the liposomal formulation is a lyophilized powder.

[00349] In some aspects of the present invention, a liposomal formulation is provided that is prepared and loaded with cccDNA vectors disclosed or described herein by adding a weak base to a mixture containing the isolated cccDNA outside the liposome. This addition raises the pH outside the liposomes to about 7 ,3 and stimulates the entry of API into the liposome. According to some aspects of the present invention, there is provided a liposomal composition having pH values that are acidic within the liposomes. In such cases, the pH value within the liposomes may be 4-6.9, and more preferably pH 6.5. According to other aspects of the present invention, a liposomal formulation is provided using intraliposomal drug stabilization technology. In such cases, polymeric or non-polymeric highly charged anions and intraliposomal entrapment agents, such as polyphosphate or sucrose octasulfate, are used.

[00350] According to other aspects of the present invention, there is provided a liposomal composition containing phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.

[00351] Non-limiting examples of cationic lipids include star dendrimers of polyethylenimine and polyamidoamine (PAMAM), lipofectin (combination of DOTMA and DOPE), lipofectase, lipofectamine (LIPOFECTAMTNE™, e.g. LIPOFECTAMINE™ 2000), DOPE, cytofectin (Gilead Sciences, Foster City, California) and eupectins (JBL, San Luis Obispo, California). Examples of cationic liposomes can be prepared from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl] -N,N,N-trimethylammonium methyl sulfate (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamido) ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide; And

Dimethyldioctadecylammonium bromide (DDAB). Nucleic acids (eg, CELiD) may also be complexed with, for example, poly(L-lysine) or avidin, and lipids, such as steryl-poly(L-lysine), may or may not be included in the mixture.

[00352] In some embodiments, the cccDNA vector as described herein is delivered using a cationic lipid described in US Pat. No. 8,158,601, or a polyamine compound or lipid as described in US Pat. No. 8,034,376.

F. Examples of compositions with liposomes and lipid nanoparticles (LNPs)

[00353] cccDNA vectors in accordance with the present invention can be added to liposomes for delivery to a cell in need of gene editing, for example, in need of a donor sequence. Liposomes are vesicles that contain at least one lipid bilayer. Liposomes are often used as carriers for drug/therapeutic delivery in the context of pharmaceutical development. They work by fusing with the cell membrane and rearranging its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Liposomal compositions for such delivery consist of phospholipids, in particular compounds containing a phosphatidylcholine group, although these compositions may also include other lipids.

[00354] According to some aspects of the present invention, there is provided a liposomal formulation that includes one or more polyethylene glycol (PEG) functional compounds (so-called “PEGylated compounds”) that can reduce immunogenicity/antigenicity, provide hydrophilicity and hydrophobicity to the compound(s) and reducing the frequency of its dosing. Alternatively, said liposomal formulation simply comprises a polyethylene glycol (PEG) polymer as an additional component. In such aspects, the molecular weight of the PEG or PEG functional group can range from 62 Da to about 5000 Da.

[00355] According to some aspects of the present invention, a liposomal formulation is provided that delivers an API according to an extended release or controlled release profile over a period of hours to weeks. In some related aspects, the liposomal composition may include aqueous chambers that are connected by lipid bilayers. In other related aspects, the liposomal formulation encapsulates the API with components that undergo a physical transition at elevated temperature that releases the API over a period of hours to weeks.

[00356] In some aspects, the liposomal composition comprises sphingomyelin and one or more lipids as described herein. In some aspects, the liposomal composition contains optisomes.

[00357] According to some aspects of the present invention, there is provided a liposomal formulation that includes one or more lipids selected from: N-(carbonyl-methoxypolyethylene glycol-2000)-1, 2-distearoyl-c-glycero-3-phosphoethanolamine, sodium salt (distearoyl -sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol)-conjugated lipid. HSPC (hydrogenated soy phosphatidylcholine); PEG (polyethylene glycol); DSPE (distearoyl-sn-glycero-phosphoethanolamine); DSPC (distearoylphosphatidylcholine); DOPC (dioleylphosphatidylcholine); DPPG (dipalmitoylphosphatidylglycerol); EPC (egg phosphatidylcholine); DOPS (dioleylphosphatidylserine); POPC (palmitoyloleylphosphatidylcholine); SM (sphingomyelin); MPEG (methoxy polyethylene glycol); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DSPG (distearoylphosphatidylglycerol); DEPC (dierucoylphosphatidylcholine); DOPE (dioleyl-sn-glycero-phosphoethanolamine), cholesterol sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleyl-sn-glycero-phosphatidylcholine) or any combination of the above.

[00358] According to some aspects of the present invention, there is provided a liposomal formulation comprising a phospholipid, cholesterol and a PEGylated lipid in a molar ratio of 56:38:5. In some aspects, the total lipid content of the liposomal formulation is 2-16 mg/ml. According to some aspects of the present invention, there is provided a liposomal composition comprising a phosphatidylcholine-functional lipid, an ethanolamine-functional lipid, and a PEGylated lipid. According to some aspects of the present invention, there is provided a liposomal composition comprising a phosphatidylcholine-functional lipid, an ethanolamine-functional lipid, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. According to some aspects of the present invention, there is provided a liposomal composition comprising a lipid containing a phosphatidylcholine functionality, cholesterol and a pegylated lipid. According to some aspects of the present invention, there is provided a liposomal composition comprising a lipid containing a phosphatidylcholine functionality and cholesterol. In some aspects, said PEGylated lipid is PEG-2000-DSPE. In some aspects, the present invention provides a liposomal formulation comprising DPPG, soy PC, MPEG-DSPE lipid conjugate, and cholesterol.

[00359] According to some aspects of the present invention, there is provided a liposomal formulation comprising one or more phosphatidylcholine-functional lipids and one or more ethanolamine-functional lipids. In some aspects, the present invention provides a liposomal formulation comprising any one or more of phosphatidylcholine-functional lipids, ethanolamine-functional lipids, and sterols, such as cholesterol. In some aspects, the liposomal formulation comprises DOPC/DEPC; and DOPE.

[00360] According to some aspects of the present invention, there is provided a liposomal formulation further comprising one or more pharmaceutical excipients, for example, sucrose and/or glycine.

[00361] According to some aspects of the present invention, a liposomal composition with a unilamellar or multilamellar structure is provided. According to some aspects of the present invention, there is provided a liposomal formulation that contains multivesicular particles and/or foam-based particles. According to some aspects of the present invention, there is provided a liposomal formulation whose relative particle size is larger than that of conventional nanoparticles and is approximately 150 to 250 nm. In some aspects, the liposomal formulation is a lyophilized powder.

[00362] In some aspects of the present invention, a liposomal formulation is provided that is prepared and loaded with cccDNA vectors disclosed or described herein by adding a weak base to a mixture containing isolated cccDNA outside of a liposome. This addition increases the pH outside the liposomes to approximately 7.3 and stimulates the entry of the API into the liposome. According to some aspects of the present invention, there is provided a liposomal composition having pH values that are acidic within the liposomes. In such cases, the pH value within the liposomes may be 4-6.9, and more preferably pH 6.5. According to other aspects of the present invention, a liposomal formulation is provided using intraliposomal drug stabilization technology. In such cases, polymeric or non-polymeric highly charged anions and intraliposomal entrapment agents, such as polyphosphate or sucrose octasulfate, are used.

[00363] According to other aspects of the present invention, there is provided a liposomal composition containing phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine. In some embodiments, the liposomal formulation is one described in Table 6 below.

[00364] According to some aspects of the present invention, a lipid nanoparticle comprising cccDNA and an ionizable lipid is provided. For example, a lipid nanoparticle formulation that is prepared and loaded with cccDNA produced by the method described in international application PCT/US2018/050042, filed September 7, 2018, which is incorporated herein. This can be accomplished by vigorously mixing ethanol solutions of lipids with an aqueous solution of ccDNA at low pH, which protonates the ionizable lipid and provides favorable energetic characteristics for ccDNA/lipid binding and particle nucleation. The particles can be further stabilized by diluting with water and removing the organic solvent. The particles can be concentrated to the desired level.

[00365] Typically, lipid particles are prepared with a ratio (mass or weight) of total lipid to cccDNA of about 10:1 to 30:1. In some embodiments, said lipid to cccDNA ratio (mass ratio; weight ratio) may range from about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids and cccDNA can be adjusted to provide the desired N/P ratio, for example, an N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Typically, the total content of lipid particles in the composition can vary from about 5 mg/ml to about 30 mg/ml.

[00366] An ionizable lipid is typically used to condense a nucleic acid cargo, such as cccDNA, at low pH and to promote membrane binding and fusogenicity. Typically, ionizable lipids are lipids containing at least one amino group that is positively charged or becomes protonated under acidic conditions, such as pH 6.5 or lower. Ionizable lipids are also referred to as cationic lipids herein.

[00367] Examples of ionizable lipids are described in PCT patent publications WO2015/095340, WO2015/199952, WO2018/011633, WO2017/049245, WO2015/061467, WO2012/040184, WO2012/000104, W O2015/074085, WO2016/081029, WO2017/004143 , WO2017/075531, WO2017/117528, WO2011/022460, WO2013/148541, WO2013/116126, WO2011/153120, WO2012/044638, WO2012/054365, WO2011/090 965, WO2013/016058, WO2012/162210, WO2008/042973, WO2010 /129709, WO2010/144740, WO2012/099755, WO2013/049328, WO2013/086322, WO2013/086373, WO2011/071860, WO2009/132131, WO2010/048536, WO20 10/088537, WO2010/054401, WO2010/054406, WO2010/054405 , WO2010/054384, WO2012/016184, WO2009/086558, WO2010/042877, WO2011/000106, WO2011/000107, WO2005/120152, WO2011/141705, WO2013/126 803, WO2006/007712, WO2011/038160, WO2005/121348, WO2011 /066651, WO2009/127060, WO2011/141704, WO2006/069782, WO2012/031043, WO2013/006825, WO2013/033563, WO2013/089151, WO2017/099823, WO20 15/095346, and WO2013/086354, as well as US patent publications US2016 /0311759, US2015/0376115, US2016/0151284, US2017/0210697, US2015/0140070, US2013/0178541, US2013/0303587, US2015/0141678, US2015/0239926, US20 16/0376224, US2017/0119904, US2012/0149894, US2015/0057373 , US2013/0090372, US2013/0274523, US2013/0274504, US2013/0274504, US2009/0023673, US2012/0128760, US2010/0324120, US2014/0200257, US2015/0203 446, US2018/0005363, US2014/0308304, US2013/0338210, US2012 /0101148, US2012/0027796, US2012/0058144, US2013/0323269, US2011/0117125, US2011/0256175, US2012/0202871, US2011/0076335, US2006/0083780, US20 13/0123338, US2015/0064242, US2006/0051405, US2013/0065939 , US2006/0008910, US2003/0022649, US2010/0130588, US2013/0116307, US2010/0062967, US2013/0202684, US2014/0141070, US2014/0255472, US2014/0039 032, US2018/0028664, US2016/0317458, and US2013/0195920, the contents of each of which are incorporated herein by reference in their entirety.

[00368] In some embodiments, said ionizable lipid is MCZ (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6, 9, 28, 31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3- DMA or MC3), having the following structure:

[00369] The DLin-MC3-DMA lipid is described in Jayaraman et al, Angew. Chem. Int. Ed Engl. (2012), 51(34): 8529-8533, the contents of which are incorporated herein by reference in their entirety.

[00370] In some embodiments, said ionizable lipid is an ATX-002 lipid having the following structure:

[00371] Lipid ATX-002 is described in WO2015/074085, the contents of which are incorporated herein by reference in their entirety.

[00372] In some embodiments, said ionizable lipid is (13Z, 16Z)-N,N-dimethyl-3-nonyldocose-13,16-dien-1-amine (compound 32), having the following structure:

[00373] Compound 32 is described in WO2012/040184, the contents of which are incorporated herein by reference in their entirety.

[00374] In some embodiments, said ionizable lipid is compound 6 or compound 22, having the following structure:

[00375] Compounds 6 and 22 are described in WO2015/199952, the contents of which are incorporated herein by reference in their entirety.

[00376] Without limitation, the ionizable lipid may comprise 20-90 mol% of the total lipid content present in the lipid nanoparticle. For example, the ionizable lipid molar content may be 20-70 mol%, 30-60 mol%, or 40-50 mol% of the total lipid content present in the lipid nanoparticle. In some embodiments, the ionizable lipid contains from about 50 mol. % to approximately 90 mol. % of the total lipid content present in the lipid nanoparticle.

[00377] In some aspects, said lipid nanoparticle may further comprise a non-cationic lipid. Nonionic lipids include amphipathic lipids, neutral lipids and anionic lipids. Accordingly, said non-cationic lipid may be a neutral, uncharged, zwitterionic or anionic lipid. Non-cationic lipids are usually used to enhance fusogenicity.

[00378] Examples of non-cationic lipids include, but are not limited to, distearoyl-sn-glycerophosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleylphosphatidylethanolamine (DOPE ), palmitoyloleylphosphatidylcholine ( RORS), palmitoiloleil phosphatidydanolamine (Pope), dioleil phosphateanolamine 4- (n-maleimidomethyl)-cyclogxan-1-carboxylate (Dope-MAL), dipalmitoilphospatinolamine (DPPE), dimiristoilphosphotanomine (DMPE), DISTR Aroilfospatidanomine, monomethyl phosphatidydidanomin such as 16- O-monomethyl-PE), dimethylphosphatidylethanolamine (such as 16-O-dimethyl-PE), 18-1-trans-PE, 1-stearoyl-2-oleoyl-phosphatidylcholine-ethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), diopeipphosphatidipserin (DOPS), sphingomyelin (SM), dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPC), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleylphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE) ), lecithin, phosphatidylethanolamine, pisolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg ophingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, lysophosphatidylcholine, dilinoleylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in said lipids are preferably acyl groups derived from fatty acids containing C ₁₀ -C ₂₄ carbon chains, for example lauroyl, myristoyl, papimitoyl, stearoyl or oleoyl.

[00379] Other examples of non -caped lipids suitable for use in these lipid nanoparticles are pecking in lipid phosphorus, such as, for example, stearlamine, milkmetic, hexadecylamine, acylpalmitate, glyceryricinoleate, hexadecylsterata, amphothelmiristitis, amphotherapy. Polymers, triatanolamine-lauryl sulfate, alkylaryl sulfate polyethyloxylated fatty acid amides, dnoctadecyldimethylammonium bromide, ceramide, sphingomyelin, etc.

[00380] In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, said non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some preferred embodiments, said non-cationic lipid is DPSC.

[00381] Examples of non-cationic lipids are described in PCT Publication W02017A099823 and US Patent Publication US 2018/0028664, the contents of each of which are incorporated herein by reference in their entirety. In some examples, said non-cationic lipid is oleic acid or a compound of formula (I),

, formulas (II) or formula (IV), , as defined in US 2018/0028664, the contents of which are incorporated herein by reference in their entirety.

[00382] Said non-cationic lipid may comprise 0-30 mol% of the total lipid content present in the lipid nanoparticle. For example, the non-cationic lipid content is 5-20 mol% or 10-15 mol% of the total lipid content present in the lipid nanoparticle. In various embodiments, the molar ratio of ionizable lipid to neutral lipid ranges from about 2:1 to about 8:1.

[00383] In some embodiments, the lipid nanoparticles do not contain any phospholipids.

[00384] In some aspects, the lipid nanoparticle may further comprise a component such as a sterol to provide membrane integrity.

[00385] One example of a sterol that is suitable for use in such a lipid nanoparticle is cholesterol and its derivatives. Non-limiting examples of cholesterol derivatives include polar analogues such as 5α-cholestanol, 5α-coprostanol, cholesterol-(2'-hydroxy)-ethyl ether, cholesterol-(4'-hydroxy)-butyl ether and 6-ketocholestanol; non-polar analogues such as 5α-cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone and cholesteryl decanoate; and mixtures thereof. In some embodiments, said cholesterol derivative is a polar analogue such as cholesteryl (4'-hydroxy)-butyl ether.

[00386] Examples of cholesterol derivatives are described in PCT Publication WO2009/127060 and Patent Publication CIIIAUS2010/0130588, the contents of each of which are incorporated herein by reference in their entirety.

[00387] The membrane integrity component, such as a sterol, may contain 0-50 mol% of the total lipid content present in the lipid nanoparticle. In some embodiments, such a component constitutes 20-50 mol.% 30-40 mol.% of the total lipid content of the lipid nanoparticle.

[00388] In some aspects, said lipid nanoparticle may further comprise polyethylene glycol (PEG) or a conjugated lipid molecule. Typically, they are used to inhibit the aggregation of lipid nanoparticles and/or provide steric stabilization. Examples of conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic polymer lipid (CPL) conjugates; and mixtures of the above. In some embodiments, said conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxypolyethylene glycol)-conjugated lipid.

[00389] Examples of PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxypolyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG- phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG-succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-( w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG-dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1, 2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture of the above Additional examples of PEG-lipid conjugates are described, for example, in US 5, 885, 613, US 6, 287, 591, US200 3/0077829, US 2003/007 7829, US2005/0175682, US 2008/002005 8, US2011/0117125 , US2010/0130588, US2016/0376 224 and US2017/0119904, the contents of each of which are incorporated herein by reference in their entirety.

[00390] In some embodiments, the PEG-lipid conjugate is a compound of formula (III), formulas (III-a-I), formulas (Ш-а-2), formula (III-b-1), formula (III-b-2), or formula (V) as defined in US2018/0028664, the contents of which are incorporated herein by reference in their entirety.

[00391] In some embodiments, the PEG lipid is a compound of formula (II), , as defined in US20150376115 or US2016/0376224, the contents of each of which are incorporated herein by reference in their entirety

[00392] The PEG-DAA conjugate may be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. Said PEG lipid may be any one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol( 1-[8'-(cholest-5-en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4 -ditetradecoxybenzyl-[omega]-methyl-poly(ethylene glycol)ether) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, said PEG lipid may be selected from the group consisting of PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000],

[00393] Lipids conjugated to a molecule other than PEG can also be used in place of PEG lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (CPL) conjugates can be used instead of or in conjunction with PEG-lipid.

[00394] Examples of conjugated lipids, i.e., PEG lipids, POZ lipid conjugates, ATTA-lipid conjugates, and cationic polymer lipid conjugates are described in published PCT patent publications W01996/010392, WO1998/051278, WO2002/08754 1 , WO2005/026372, WO2008/147438, WO2009/086558, WO2012/000104, WO2017/117528, WO2017/099823, WO2015/199952, WO2017/004143, WO2015/095 346, WO2012/000104, WO2012/000104, and WO2010/006282, published US patent applications US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2013/0303587, US2018/0028664, US2015/0376115, US2016/0376224, US201 6/0317458, US2013/0303587, US2013/0303587, and US20110123453, and US Patents US5885613, US6287591, US6320017 and US6586559, each of which is incorporated herein by reference in its entirety.

[00395] Said PEG or conjugated lipid may comprise 0-20 mol% of the total lipid content present in the lipid nanoparticle. In some embodiments, the PEG or conjugated lipid content is 0.5-10% or 2-5 mol% of the total lipid content present in the lipid nanoparticle.

[00396] The molar ratios of ionizable lipid, non-cationic lipid, sterol and PEG/conjugated lipid may vary depending on the circumstances. For example, said lipid particle may contain 30-70% ionizable lipid by molar content or total weight of the composition, 0-60% cholesterol by molar content or total weight of the composition, 0-30% non-cationic lipid by molar content or total weight of the composition and 1-10% of the conjugated lipid by molar content or by total weight of the composition. Preferably, said composition contains 30-40% ionizable lipid by molar content or total weight of the composition, 40-50% cholesterol by molar content or total weight of the composition and 10-20% non-cationic lipid by molar content or total weight of the composition. According to some other embodiments, the composition contains 50-75% ionizable lipid by molar content or total weight of the composition, 20-10% cholesterol by molar content or total weight of the composition; and 5-10% non-cationic lipid by molar content or total weight of the composition, and 1-10% conjugated lipid by molar content or total weight of the composition. Said composition may contain 60-70% ionizable lipid by molar content or total weight of the composition, 25-35% cholesterol by molar content or total weight of the composition, and 5-10% non-cationic lipid by molar content or total weight of the composition. Said composition may also contain up to 90% ionizable lipid by molar content or total weight of the composition, and 2-15% non-cationic lipid by molar content or total weight of the composition. The composition may also be a lipid nanoparticle formulation, for example, containing 8-30% ionizable lipid by molar content or total weight of the composition, 5-30% non-cationic lipid by molar content or total weight of the composition, and 0-20% cholesterol from the molar content or from the total weight of the composition; 4-25% ionizable lipid by molar content or total composition weight, 4-25% non-cationic lipid by molar content or total composition weight, 2-25% cholesterol by molar content or total composition weight, 10-35% lipid conjugate by molar content or total weight of the composition, and 5% cholesterol by molar content or total weight of the composition; or 2-30% ionizable lipid by molar content or total weight of the composition, 2-30% non-cationic lipid by molar content or total weight of the composition, 1-15% cholesterol by molar content or total weight of the composition, 2-35% conjugate lipid by molar content or total weight of the composition, and 1-20% cholesterol by molar content or total weight of the composition; or even up to 90% ionizable lipid by molar content or total weight of the composition and 2-10% non-cationic lipids by molar content or total weight of the composition, or even 100% cationic lipid by molar content or total weight of the composition. In some embodiments, the lipid particulate composition comprises an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particulate composition contains an ionizable lipid, cholesterol, and a PEGylated lipid in a molar ratio of 60:38.5:1.5.

[00397] In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid, wherein the lipid molar ratio ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40 -60%, non-cationic lipid mole percentage ranges from 0 to 30 with a target of 0-15%, sterol mole percentage ranges from 20 to 70% with a target of 30-50%, and PEGylated lipid mole percentage ranges from 1 to 6% with a target of 2-5 %.

[00398] Lipid nanoparticles (LNPs) containing cccDNA are disclosed in international application PCT/US2018/050042, filed September 7, 2018, which is incorporated herein in its entirety, and are provided for use in the methods and compositions described herein.

[00399] The size of lipid nanoparticles can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK), and their diameter is approximately 50-150 nm, approximately 55-95 nm, or approximately 70-90 nm.

[00400] The pKa of cationic lipids in formulations can be compared with the efficiency of LNPs for delivering nucleic acids (see sources: Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010), both of which are incorporated by reference herein in their entirety). The preferred pKa range is from about 5 to about 7. The pKa of each cationic lipid in the lipid nanoparticles is determined using an assay based on 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) fluorescence. Lipid nanoparticles containing cationic lipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol%) in PBS at a total lipid concentration of 0.4 mM can be prepared using the in-line method as described in this document and elsewhere. TNS can be prepared as a 100 µM stock solution in distilled water. Vesicles can be diluted to a lipid concentration of 24 μM in 2 ml buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where pH varies from 2.5 to 11. An aliquot of TNS solution can be added to achieving a final concentration of 1 µM; after vortex mixing, fluorescence intensity is measured at room temperature on an SLM Aminco Series 2 spectrophotometer. Luminescence at excitation and emission wavelengths of 321 nm and 445 nm. The best fit method using the sigmoid function can be used to analyze fluorescence data; pKa is measured as the pH value, obtaining the half-maximal fluorescence intensity.

[00401] Relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration by tail vein injection. Activity at doses of 0, 3 and 1.0 mg cccDNA/kg was compared and expressed as ng luciferase per gram of liver measured 4 hours after administration.

[00402] Without limitation, the lipid nanoparticle of the present invention includes a lipid composition that can be used to deliver a capsid-free non-viral DNA vector to a target site of interest (eg, a cell, tissue, organ, etc.). Typically, said lipid nanoparticle contains a capsid-free non-viral DNA vector and an ionizable lipid or a salt thereof.

[00403] In some embodiments, the lipid particle comprises an ionizable lipid/non-cationic lipid/sterol/conjugated lipid in a molar ratio of approximately 50:10:38.5:1.5.

[00404] In some embodiments, the lipid particle comprises an ionizable lipid/non-cationic lipid/sterol/conjugated lipid in a molar ratio of approximately 50.0:7.0:40.0:3.0.

[00405]

[00406] According to other aspects of the present invention, there is provided a lipid nanoparticle formulation containing phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.

[00407] In some embodiments, one or more additional connections may also be included. These compounds may be administered individually or these additional compounds may be included in the lipid nanoparticles of the present invention. In other words, said lipid nanoparticles may contain other compounds in addition to the cDNA, or at least a second cDNA different from the first. Without limitation, other additional compounds may be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, analogs and derivatives nucleic acids, extract prepared from biological materials, or any combination of the above.

[00408] In some embodiments, the one or more additional compounds may be a therapeutic agent. Said therapeutic agent may be selected from any class suitable for use for therapeutic purposes. In other words, said therapeutic agent may be selected from any class suitable for use for therapeutic purposes. In other words, said therapeutic agent may be selected in accordance with the therapeutic purpose and the desired biological effect. For example, if the cccDNA within the LNP is useful for treating cancer, the additional compound may be an anticancer agent (e.g., a chemotherapeutic agent, a targeted anticancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate) In another example, if the LNP containing cccDNA is suitable for treating an infection, the additional compound may be an antimicrobial agent (eg, an antibiotic or an antiviral compound).In yet another example, if the LNP containing cccDNA is suitable for treating an immune disease or disorder, said additional compound may be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways).In some embodiments, the compositions and methods of the present Other cocktails with other lipid nanoparticles containing other compounds, such as cctDNA encoding another protein or another compound, such as a therapeutic agent, may be used in the invention.

[00409] In some embodiments, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressant. In some embodiments, the additional compound is immunostimulatory.

[00410] The present invention also provides a pharmaceutical composition containing said lipid nanoparticle and a pharmaceutically acceptable carrier or excipient.

[00411] According to some aspects of the present invention, a lipid nanoparticle formulation is provided, further comprising one or more pharmaceutical excipients. In some embodiments, the lipid nanoparticle formulation further comprises sucrose, Tris, trehalose, and/or glycine.

[00412] Typically, the lipid nanoparticles of the present invention have an average diameter selected to provide the intended therapeutic effect. Accordingly, in some aspects, said lipid nanoparticle has an average diameter of from about 30 nm to about 150 nm, more often from about 50 nm to about 150 nm, more often from about 60 nm to about 130 nm, more often from about 70 nm to about 110 nm, most commonly from about 85 nm to about 105 nm, and preferably about 100 nm. According to some aspects of the present invention, lipid particles are provided that have a relative size larger than standard nanoparticles, ranging from approximately 150 to 250 nm. The size of lipid nanoparticles can be determined by quasi-elastic light scattering using, for example, the Malvern Zetasizer Nano ZS system (Malvern, UK).

[00413] Depending on the intended use of the lipid particles, the proportions of the components may vary and the delivery efficiency of a particular formulation may be measured using, for example, an endosome release parameter (ERP) assay.

[00414] The cccDNA can be complexed with the lipid portion of the particle or encapsulated at a lipid position in a lipid nanoparticle. In some embodiments, the cccDNA may be completely encapsulated at a lipid position in a lipid nanoparticle, thereby protecting it from degradation by a nuclease, for example, in an aqueous solution. In some embodiments, the cccDNA in the lipid nanoparticle is not substantially degraded after exposing the lipid nanoparticle to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes. In some embodiments, said cccDNA in a lipid nanoparticle is not substantially degraded after incubation of said particle in serum at 37° C. for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6. 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 hours.

[00415] In some embodiments, the lipid nanoparticles are substantially non-toxic to a subject, such as a mammal such as a human. [00416] In some aspects, the lipid nanoparticle formulation is a lyophilized powder.

[00417] In some embodiments, lipid nanoparticles are solid core particles that contain at least one lipid bilayer. In other embodiments, said lipid nanoparticles have a non-bilayer structure, i.e., a non-lamellar (ie, non-bilayer) morphology. Without limitation, non-bilayer morphologies may include, for example, three-dimensional tubes, rods, structures with cubic symmetry, and the like. Non-lamellar morphology (ie, non-bilayer structure) of lipid particles can be determined using analytical techniques known and used by those skilled in the art. Such techniques include, but are not limited to, transmission cryo-electron microscopy (“cryo-TEM”), differential scanning calorimetry (“DSC”), X-ray diffraction, and the like. For example, the morphology of said lipid nanoparticles (lamellar or non-lamellar) can be readily assessed and characterized using, for example, cryo-TEM analysis as described in US2010/0130588, the contents of which are incorporated herein by reference in their entirety.

[00418] In some further embodiments, the non-lamellar morphology lipid nanoparticles are electron dense.

[00419] According to some aspects of the present invention, a lipid nanoparticle with a unilamellar or multilamellar structure is provided. According to some aspects of the present invention, there is provided a lipid nanoparticle formulation that contains multivesicular particles and/or foam particles.

[00420] By controlling the composition and content of lipid components, it is possible to control the rate at which the lipid conjugate is released from the lipid particle and, in turn, the rate at which the lipid nanoparticle becomes fusogenic. In addition, other parameters, including, for example, pH, temperature or ionic strength, can be used to change and/or control the rate at which the lipid nanoparticle becomes fusogenic. Other methods that can be used to control the rate at which the specified lipid nanoparticle becomes fusogenic will be obvious to those skilled in the art after reading the above description. It is also apparent that it is possible to control the size of lipid particles by controlling the composition and concentration of the lipid conjugate.

[00421] The pKa of cationic lipids in formulations can be compared with the efficiency of LNPs for delivering nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28 , 172-176 (20 1 0), both of which are incorporated by reference herein in their entirety). The preferred pKa range is from about 5 to about 7. The pKa of each cationic lipid in the lipid nanoparticles is determined using an assay based on 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) fluorescence.

[00422] Encapsulation of cccDNA in lipid particles can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye whose fluorescence increases upon binding to a nucleic acid, such as the Oligreen ^® or PicoGreen ^® assay. Typically, encapsulation is determined by adding a dye to a specified lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed when a small amount of nonionic detergent is added. Detergent-mediated disruption of the lipid bilayer releases the encapsulated cccDNA, allowing it to interact with the membrane-impermeable dye. The encapsulation of cccDNA can be calculated using the formula E=(I ₀ -I)/I ₀ , where I and I ₀ refer to the fluorescence intensity before and after the addition of detergent.

VIII. Methods for delivering cccDNA vectors

[00423] In some embodiments, the cccDNA vector can be delivered to a target cell in vitro or in vivo by various suitable methods. The cccDNA vectors can be used or introduced separately. cccDNA vectors can be delivered into cells without the aid of a transfection reagent or other physical method. Alternatively, cccDNA vectors can be delivered using any transfection reagent known in the art or other physical method known in the art that facilitates DNA entry into the cell, e.g., liposomes, alcohols, polylysine-rich compounds, arginine-rich compounds , calcium phosphate, microvesicles, microinjections, electroporation, etc.

[00424] In contrast, transduction with capsidless AAV vectors as described herein can provide effective targeting of cell and tissue types that are difficult to transduce with standard AAV virions using different delivery reagents.

[00425] In another embodiment, the cDNA vector is administered to the CNS (eg, the brain or eye). The specified cDNA vector can be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, cerebrum, including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, striatum, cerebrum and inferior colliculus. The specified cDNA vector can also be delivered to other areas of the eye, such as the retina, cornea and/or optic nerve. The specified cDNA vector can be delivered into the cerebrospinal fluid (for example, by lumbar puncture). Also, the specified cDNA vector can be introduced into the CNS by intravascular injection in situations where the blood-brain barrier is disrupted (for example, a brain tumor or cerebral infarction).

[00426] In some embodiments, the cccDNA vector may be administered to the desired region(s) of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., presence of sugar such as mannitol). intranasal, intraauricular, intraocular (eg, intravitreous, subretinal, anterior chamber) and periocular (eg, sub-Tenon's region) delivery, as well as intramuscular delivery with retrograde delivery to motor neurons.

[00427] In some embodiments, the cccDNA vector is administered as a liquid formulation by direct injection (eg, stereotactic injection) into the desired region or compartment of the CNS. In other embodiments, the cccDNA vector may be administered by topical application to the desired area or by intranasal administration of an aerosol formulation. Administration to the eyes can be accomplished by topical application of liquid drops. As a further alternative, the cDNA vector can be administered as a sustained release solid formulation (see, for example, US Pat. No. 7,201,898). In additional embodiments, said cccDNA vector can be used for retrograde transport to treat, alleviate, and/or prevent motor neuron-related diseases and disorders (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc. .). For example, said cDNA vector can be delivered to muscle tissue, from which it can migrate into neurons.

IX. Additional applications of cccDNA vectors

[00428] The compositions and cccDNA vectors described herein can be used to deliver a transgene for a variety of purposes. In some embodiments, the transgene encodes a protein or functional RNA that is intended to be used for research purposes, for example, to create a somatic model of transgenic animals carrying the transgene, for example, to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended for use in creating an animal model of a disease. In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating, preventing, or alleviating disease states or disorders in a mammalian subject. The transgene may be transferred into a subject (eg, expressed in the subject) in an amount sufficient to treat a disease associated with decreased expression, underexpression, or dysfunction of the gene. In some embodiments, the transgene may be transferred into a subject (e.g., expressed in the subject) in an amount sufficient to treat a disease associated with increased expression, activity of a gene product, or inappropriate stimulatory regulation of a gene that suppresses or otherwise reduces the expression of said transgene. transgene In some embodiments, the transgene is used to knock out an endogenous gene.

X. Directions for use

[00429] The cDNA vector of the present invention can also be used in a method for delivering a nucleotide sequence of interest to a target cell. The method may in particular be a method of delivering a therapeutic gene of interest to a cell of a subject in need thereof. The present invention allows for in vivo expression of a polypeptide, protein or oligonucleotide encoded by a therapeutic exogenous DNA sequence in cells of a subject such that the polypeptide, protein or oligonucleotide is expressed at therapeutic levels. These results are observed both in vivo and in vitro delivery modes of the cDNA vector.

[00430] A method of delivering a nucleic acid of interest to a cell of a subject may include administering to the subject a cccDNA vector of the present invention containing said nucleic acid of interest. In addition, the present invention provides a method for delivering a nucleic acid of interest to a cell of a subject in need thereof, comprising repeatedly administering a cccDNA vector of the present invention containing said nucleic acid of interest. Since the cDNA vector of the present invention does not induce an immune response, such a multiple administration strategy will not disrupt the host immune system response against the cDNA vector of the present invention, unlike the situation observed with encapsidated vectors.

[00431] The cccDNA vector nucleic acid(s) are administered in sufficient quantities to transfect cells of the desired tissue and to ensure sufficient levels of gene transfer and expression without undesirable side effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intravenous (eg, in a liposomal formulation), direct delivery to an organ of choice (eg, intraportal delivery to the liver), intramuscular, and other parenteral routes of administration. Routes of administration can be combined if required.

[00432] The cccDNA vector is not limited to one type of cccDNA vector. Therefore, in another aspect, multiple cDNA vectors containing different exogenous DNA sequences can be delivered simultaneously or sequentially to a target cell, tissue, organ, or organism of a subject. Accordingly, this strategy can ensure the expression of multiple genes. Delivery can also be done multiple times and, importantly for gene therapy in a clinical setting, with subsequent increases or decreases in doses given the lack of host immune response against the capsid due to the absence of the viral capsid.

[00433] The present invention also provides a method of treating a disease in a subject, comprising administering to a target cell in need thereof (particularly a muscle cell or tissue) of said subject a therapeutically effective amount of a cccDNA vector, optionally with a pharmaceutically acceptable carrier. Although said cDNA vector can be introduced in the presence of a carrier, such a carrier is not necessary. The proposed cccDNA vector contains a nucleotide sequence of interest suitable for treating a disease. In particular, said cDNA vector may contain a desired exogenous DNA sequence operably linked to control elements capable, when administered to a subject, of directing transcription of the desired polypeptide, protein or oligonucleotide encoded by the exogenous DNA sequence. The specified cDNA vector can be introduced by any suitable route, as described above and elsewhere in this document.

XI. Treatment methods

[00434] The technology described herein also demonstrates methods for producing, as well as methods for using, the disclosed cccDNA vectors in a variety of ways, including, for example, uses, methods, diagnostic procedures and/or gene therapy regimens ex situ, in vitro and in vivo.

[00435] The present invention provides a method of treating a disease or disorder in a subject, comprising administering to a target cell in need thereof (e.g., a muscle cell or tissue, or other type of diseased cell) of the subject a therapeutically effective amount of a cccDNA vector, optionally with a pharmaceutically acceptable carrier. . Although said cDNA vector can be introduced in the presence of a carrier, such a carrier is not necessary. The proposed cccDNA vector contains a nucleotide sequence of interest suitable for treating a disease. In particular, said cDNA vector may contain a desired exogenous DNA sequence operably linked to control elements capable of directing transcription of the desired polypeptide, protein or oligonucleotide encoded by said exogenous DNA sequence when administered to a subject. The specified cDNA vector can be introduced by any suitable route, as described above and elsewhere in this document.

[00436] Any transgene can be delivered using cDNA vectors as described herein. Transgenes of interest include polypeptide-encoding nucleic acids or non-coding nucleic acids (e.g., RNAi, miR, etc.), preferably therapeutic (e.g., for medical, diagnostic or veterinary use) or immunogenic (e.g., for vaccines) polypeptides.

[00437] In some embodiments, the transgenes to be expressed by the cccDNA vectors described herein express or encode one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNA, RNAi, antisense oligonucleotides, antisense polynucleotides. antibodies, antigen-binding fragments, or any combination of these.

[00438] In particular, the transgene may encode one or more therapeutic agents, including, but not limited to, protein(s), polypeptide(s), peptide(s), enzyme(s), antibodies, antigen binding fragments, as well as variants and/or active fragments of the above, agonists, antagonists, mimetics for use in the treatment, prevention and/or alleviation of one or more symptoms of a disease, dysfunction, injury and/or disorder. In one aspect, the disease, dysfunction, injury, injury, and/or disorder is a disease, dysfunction, injury, injury, and/or disorder in a human.

[00439] As noted herein, the transgene may encode a therapeutic protein or peptide, or a therapeutic nucleic acid sequence or therapeutic agent, including, but not limited to, one or more agonists, antagonists, antiapoptotic factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietic agents, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, nerve growth factors, neuroactive peptides, neuroactive peptide receptors, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors , enzymes, receptor binding proteins, transport proteins or one or more inhibitors thereof, serotonin receptors or one or more serotonin uptake inhibitors, serpins, serpin receptors, tumor suppressors, diagnostic molecules, chemotherapeutic agents, cytotoxins, or any combination of the above.

[00440] In some embodiments, a transgene in an expression cassette, expression construct, or cDNA vector described herein may be codon optimized for a host cell. As used herein, the term “codon-optimized” or “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in vertebrate cells of interest, e.g., mouse or human (e.g., humanized sequences), by replacing at least one, more than one or a significant number of codons of a native sequence (eg, a prokaryotic sequence) to the codons that are most or most frequently used in the genes of the specified vertebrate. Different species exhibit a specific systematic preference for certain codons for specific amino acids. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined using, for example, the Gene Forge® codon optimization and customized gene synthesis platform from Aptagen (Aptagen, Inc.) or other publicly available database.

[00441] In some embodiments, the cDNA vector expresses the transgene in the host cell of interest. In some embodiments, the host cell in question is a human host cell, including, for example, blood cells, stem cells, hematopoietic cells, CD34 ⁺ cells, liver cells, cancer cells, vascular cells, muscle cells, pancreatic cells, nerve cells, eye cells or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts or any other cells derived from a mammalian body, including, without limitation, hepatocytes (i.e. liver cells), lung cells, heart cells, cells pancreas, intestinal cells, diaphragmatic cells, renal cells (ie, kidney cells), nerve cells, blood cells, bone marrow cells, or any one or more selected tissues of the subject in which gene therapy is to be performed. In one aspect, the host cell in question is a human host cell.

[00442] Disclosed herein are compositions and formulations of cccDNA vectors that include one or more ccDNA vectors of the present invention along with one or more pharmaceutically acceptable buffers, diluents, or excipients. Such compositions may be included in one or more diagnostic or therapeutic kits for diagnosing, preventing, treating, or alleviating one or more symptoms of a disease, injury, disorder, injury, or dysfunction. In one aspect, said disease, injury, disorder, injury, or dysfunction is a disease, injury, disorder, injury, or dysfunction in a human.

[00443] Another aspect of the technology described herein provides a method of providing a diagnostically or therapeutically effective amount of a cccDNA vector to a subject in need thereof, the method comprising introducing into a cell, tissue or organ of the subject in need thereof an amount of the cccDNA vector as described herein herein for a period of time allowing the transgene to be expressed from a cccDNA vector, thereby providing the subject with a diagnostically or therapeutically effective amount of the protein, peptide, nucleic acid expressed by said cccDNA vector. According to a further aspect, said subject is a human.

[00444] According to another aspect of the technology described herein, there is provided a method of diagnosing, preventing, treating, or alleviating at least one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or injury in a subject. In a general and broad sense, the method includes at least the step of administering to a subject in need thereof one or more disclosed cccDNA vectors, in an amount and for a period of time sufficient to diagnose, prevent, treat, or alleviate one or more symptoms of a disease, disorder , dysfunction, damage, abnormal condition, or injury in the subject. According to a further aspect, said subject is a human.

[00445] Another aspect is provided by the use of said cccDNA vector as a tool for treating or reducing one or more symptoms of a disease or disease state. There are a number of hereditary diseases for which defective genes are known and which are generally divided into two classes: states of deficiency, usually enzyme deficiencies, which are usually inherited in a recessive manner, and states of imbalance, which may involve regulatory or structural proteins, and which are usually, but not always, inherited in a dominant manner. For diseases with deficiency conditions, cccDNA vectors can be used to deliver transgenes to introduce the normal gene into diseased tissues for replacement therapy, and, in some embodiments, to create animal models of the disease using antisense mutations. For disease states of imbalance, cccDNA vectors can be used to create a disease state in a model system that can subsequently be used to combat said disease state. Accordingly, cccDNA vectors and methods as described herein allow the treatment of genetic diseases. According to the present document, treatment of a disease state is carried out by partially or completely eliminating the deficiency or imbalance that causes the disease or makes it more severe.

[00446] In general, a cccDNA vector as described herein can be used to deliver any transgene to treat, prevent, or alleviate symptoms associated with any gene expression disorder. Illustrative examples of disease conditions include, but are not limited to: cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (eg, Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic defects, degenerative retinal diseases (and other eye diseases), mitochondria (eg, Leber hereditary optic neuropathy (LHON) ), Leigh's syndrome and subacute sclerosing encephalopathy), myopathies (eg, facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of whole organs (eg, brain, liver, kidneys, heart); and so on. In some embodiments, the cccDNA vectors described herein can be advantageously used in the treatment of individuals with metabolic disorders (eg, ornithine transcarbamylase deficiency).

[00447] In some embodiments, a cccDNA vector described herein can be used to treat, alleviate, and/or prevent a disease or disorder caused by a mutation in a gene or gene product. Examples of diseases or disorders that can be treated using these cccDNA vectors include, but are not limited to, metabolic diseases or disorders (eg, Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage disease); diseases or disorders of the urea cycle (eg, or nitine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (eg, metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII; Gunther syndrome)); liver diseases or disorders (eg, progressive familial intrahepatic cholestasis (PFIC); diseases or disorders of the blood system (eg, hemophilia (A and B), thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (eg, cystic fibrosis ).

[00448] In yet another additional aspect, a cccDNA vector as described herein can be used to deliver a heterologous nucleotide sequence in situations where regulation of the level of transgene expression (for example, the expression of transgenes encoding hormones or growth factors, as described herein) is required. . In one example, the transgene may inhibit a pathway that controls the expression or activity of a target gene. In another example, the transgene may enhance the activity of a pathway that controls the expression or activity of a target gene.

[00449] Accordingly, in some embodiments, a cccDNA vector described herein can be used to correct an abnormal level and/or function of a gene product (eg, a missing or defective protein) that results in a disease or disorder. Said cccDNA vector may produce a functional protein and/or modify levels of said protein to alleviate or reduce symptoms resulting from or provide an advantage to a particular disease or disorder caused by the absence or defect of the protein. For example, treatment of OTC deficiency can be achieved by producing a functional OTC enzyme; treatment of hemophilia A and B can be achieved by modifying the levels of factor VIII, factor IX and factor X; treatment of PKU can be achieved by modifying levels of the enzyme phenylalanine hydroxylase; treatment of Fabry disease or Gaucher disease can be achieved by producing functional alpha-galactosidase or beta-glucocerebrosidase, respectively; treatment of MLD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively; treatment of cystic fibrosis can be carried out by producing a functional regulator of transmembrane conductance in cystic fibrosis; treatment of glycogen storage disease can be carried out by restoring the function of the functional enzyme glucose-6-phosphatase; and treatment of PFIC can be achieved by obtaining functional ATP8B1, ABCB11, ABCB4 or TJP2 genes.

[00450] In alternative embodiments, cccDNA vectors as described herein can be used to provide antisense nucleic acid to a cell in vitro or in vivo. For example, if the transgene is an RNAi molecule. expression of an antisense nucleic acid or RNAi in a target cell reduces the level of expression of a particular protein by the specified cell. Accordingly, transgenes, which are RNAi molecules or antisense nucleic acids, can be introduced to reduce the level of expression of a particular protein in a subject in need thereof. Antisense nucleic acids can also be introduced into cells in vitro to regulate cell physiology, for example to optimize cell or tissue culture systems.

[00451] In some embodiments, examples of transgenes encoded by the cccDNA vector include, but are not limited to: lysosomal enzymes (e.g., hexosaminidase A associated with Tay-Sachs disease or iduronate sulfatase associated with Gunther syndrome /MPS II), erythropoietin , angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, and cytokines (eg, interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, interleukin 12, granulocyte-macrophage colony-stimulating factor, lymphotoxin and etc.), peptide growth factors and hormones (for example, somatotropin, insulin, insulin-like growth factor 1 and 2, platelet-derived growth factor (TGF), epidermal growth factor EGF), fibroblast growth factor (FGF), nerve growth factor (NGF) ), neurotrophic factors 3 and 4, brain-derived neurotrophic factor (BDNF), glial growth factor (GDNF), transforming growth factors α and β, etc.), receptors (eg, tumor necrosis factor receptor). In some example embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some example embodiments, the cDNA vector encodes more than one transgene. In some example embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments, the transgene encodes an antibody, including a full-length antibody or antibody fragment as defined herein. In some embodiments, the antibody is an antigen binding domain or immunoglobulin variable domain sequence as defined herein. Other illustrative examples of transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins conferring anticancer drug resistance, and tumor suppressor gene products.

[00452] In an exemplary embodiment, a transgene expressed by said cccDNA vector can be used to treat muscular dystrophy in a subject in need thereof, the method comprising: administering an effective amount of the cccDNA vector described herein to treat, alleviate, or prevent, said cDNA vector contains a heterologous nucleic acid encoding dystrophins, minidystrophin, microdystrophin, myostatin propeptide, follistatin, soluble type II activin receptor, IGF-1, anti-inflammatory polypeptides, such as dominant mutant I-kappa-B, sarcospan, utrophin, microdystrophin, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, antibody or antibody fragment against myostatin or myostatin propeptide, and/or RNAi directed against myostatin. In specific embodiments, the cccDNA vector can be introduced into skeletal, diaphragmatic, and/or cardiac muscle as described herein.

[00453] In some embodiments, the cccDNA vector can be used to deliver a transgene to skeletal, cardiac, or diaphragmatic muscle to produce a polypeptide (e.g., enzyme) or functional RNA (e.g., RNAi, microRNA, antisense RNA) that normally circulates in the blood, or for systemic delivery to other tissues for the treatment, amelioration, and/or prevention of a disorder (eg, a metabolic disorder such as diabetes (eg, insulin), hemophilia (eg, VIII), and a mucopolysaccharidosis disorder (eg, Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome A, B, C, D. Morquio syndrome, Maroteaux-Lamy syndrome, etc.) or a lysosomal storage disorder (such as Gaucher disease [glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase] or Fabry disease [α-galactosidase A]) or glycogen storage disorders (such as Pompe disease [lysosomal acid α-glucosidase]). Other suitable proteins for the treatment, amelioration and/or prevention of metabolic disorders are described above.

[00454] In other embodiments, a cccDNA vector as described herein can be used to deliver a transgene in a method of treating, ameliorating, and/or preventing a metabolic disorder in a subject in need thereof. Illustrative examples of metabolic disorders and transgenes encoding polypeptides are described herein. Optionally, the polypeptide is secreted (e.g., a polypeptide that is a naturally occurring secreted polypeptide, or a polypeptide that is engineered to be secreted, e.g., by achieving functional association with a secretory signal sequence, as is known to those skilled in the art ).

[00455] In another aspect, the present invention provides a method of treating, ameliorating, and/or preventing congenital heart failure or peripheral arterial disease (PAD) in a subject in need thereof, the method comprising administering a cccDNA vector as described herein to the subject - a mammal, wherein said cDNA vector contains a transgene encoding, for example, Ca ²⁺ -ATPase of the sarcoplasmic endoreticulum (SERCA2a), an angiogenic factor, an inhibitor of phosphatase I (1-1), RNAi to phospholamban; phospholamban inhibitory or dominant negative molecule such as phospholamban S16E, a zinc finger protein that regulates the phospholamban gene, β2-adrenergic receptor, beta-2-adrenergic receptor kinase (βARK), PI3-kinase, calsarcan, beta-adrenergic receptor inhibitor kinase (βARKct), protein phosphatase 1 inhibitor 1, S100A1, parvalbumin, adenylyl cyclase type 6, a molecule that knocks down G protein-coupled receptor kinase type 2, such as truncated constitutively active βARKct, Pim-1, PGC-1α, SOD- 1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206 and/or mir-208.

[00456] The cccDNA vectors as described herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles containing the cccDNA vectors, which is inhaled by the subject. Said respirable particles may be liquid or solid. Aerosols of liquid particles containing cDNA vectors can be produced by any suitable methods. for example, using a pneumatic aerosol nebulizer or an ultrasonic nebulizer, as is known to those skilled in the art. See, for example, US Pat. No. 4,501,729. Particulate aerosols containing cccDNA vectors can similarly be produced using any particulate drug aerosol generator using methods known in the pharmaceutical art.

[00457] In some embodiments, these cDNA vectors can be introduced into CNS tissue (eg, brain, eye). In specific embodiments, cccDNA vectors as described herein can be administered to treat, alleviate, or prevent CNS diseases, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Illustrative examples of CNS diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger disease, spinal cord or head injury, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, psychiatric disorders, including mood disorders (eg, depression, bipolar disorder, persistent affective disorder disorder, secondary mood disorder), schizophrenia, drug dependence (eg, alcoholism and substance abuse), neuroses (eg, anxiety, obsessive disorder, somatoform disorder, dissociative disorder, grief, postpartum depression), psychosis (eg, hallucinations and delirium), dementia, paranoia, attention deficit disorder, psychosexual disorders, sleep disorders, pain disorders, eating or weight disorders (eg, obesity, cachexia, anorexia nervosa and bulimia), and cancers and tumors of the central nervous system (eg, tumors pituitary gland).

[00458] Ocular disorders that may be treated, ameliorated, or prevented by the cccDNA vectors of the present invention include ophthalmic disorders affecting the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis , age-related macular degeneration, glaucoma). Many ophthalmic diseases and disorders are associated with one or more of three types of features: (1) angiogenesis, (2) inflammation, and (3) degeneration. In some embodiments, a cccDNA vector as described herein can be used to deliver antiangiogenic factors; anti-inflammatory factors; factors that slow down cell degeneration, promote cell preservation or promote cell growth, and combinations of the above. Diabetic retinopathy, for example, is characterized by angiogenesis. Treatment of diabetic retinopathy can be achieved by delivering one or more antiangiogenic factors either intraocularly (eg, into the vitreous) or periocularly (eg, into the sub-Tenon's space). One or more neurotrophic factors may also be delivered together, either intraocularly (eg, intravitreally) or periocularly. Additional ocular diseases that may be treated, ameliorated, or prevented by the cccDNA vectors of the present invention include geographic atrophy, vascular or "wet" macular degeneration, Stargardt disease, Leber congenital amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE) , X-linked retinitis pigmentosa (XLRP), X-linked retinal dissection (XLRS), choroideremia, Leber congenital optic neuropathy (LCON), achromatopsia, rod-cone dystrophy, Fuchs' corneal endothelial dystrophy, diabetic macular edema; eye cancer and tumors.

[00459] In some embodiments, inflammatory diseases or disorders of the eye (eg, uveitis) can be treated, alleviated, or prevented using the cccDNA vectors of the present invention. One or more anti-inflammatory factors may be expressed following intraocular administration (eg, into the vitreous chamber or anterior chamber) of a cccDNA vector as described herein. In other embodiments, ocular diseases or disorders characterized by retinal degeneration (eg, retinitis pigmentosa) can be treated, alleviated, or prevented using the cccDNA vectors of the present invention. Intraocular (eg, vitreal) administration of a cccDNA vector described herein encoding one or more neurotrophic factors can be used to treat such retinal degeneration-related diseases. In some embodiments, diseases or disorders that involve both angiogenesis and retinal degeneration (eg, age-related macular degeneration) can be treated using the cccDNA vectors of the present invention. Treatment of age-related macular degeneration may be accomplished by intraocular (eg, intravitreal) administration of a cccDNA vector as described herein encoding one or more neurotrophic factors, and/or intraocular or periocular (eg, sub-Tenon's space) administration of a cccDNA vector. as described herein, encoding one or more antiangiogenic factors. Glaucoma is characterized by increased eye pressure and loss of retinal ganglion cells. Treatment options for glaucoma include administration of one or more neuroprotective agents that protect cells from excitotoxic damage using a cccDNA vector as described herein. Accordingly, such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines and neurotrophic factors, which can be delivered intraocularly, optionally intravitreally, using a cccDNA vector as described herein.

[00460] In other embodiments, a cccDNA vector as described herein can be used to treat seizures, for example, to reduce the occurrence, frequency, or severity of seizures. The effectiveness of therapeutic treatment of seizures can be assessed based on behavioral signs (eg, trembling, eye or oral tics) and/or electrographic methods (most seizures are characterized by distinctive electrographic abnormalities). Thus, the cccDNA vector described herein can also be used for the treatment of epilepsy, which is characterized by multiple seizures over time. In one exemplary embodiment, somatostatin (or an active fragment thereof) is administered to the brain using a cccDNA vector as described herein for the treatment of a pituitary tumor. In this embodiment, a cccDNA vector as described herein encoding somatostatin (or an active fragment thereof) is introduced into the pituitary gland by microinfusion. Similarly, this treatment can be used to treat acromegaly (abnormal secretion of growth hormone by the pituitary gland). The nucleic acid sequence (eg, GenBank Accession No. J00306) and amino acid sequence (eg, GenBank Accession No. P01166; contains processed active peptides somatostatin-28 and somatostatin-14) of somatostatins are known to those skilled in the art. In specific embodiments, the cccDNA vector may encode a transgene that contains a secretory signal as described in US Pat. No. 7,071,172.

[00461] Another aspect of the present invention relates to the use of a cccDNA vector as described herein to produce antisense RNA, RNAi, or other functional RNA (eg, a ribozyme) for systemic delivery to a subject in vivo. Accordingly, in some embodiments, the cccDNA vector may contain a transgene that encodes an antisense nucleic acid, a ribozyme (e.g., as described in US Pat. No. 5,877,022), RNAs that affect spliceosome-mediated trans-splicing (see Puttaraju et al. , (1999) Nature Biotech. 17:246; US Patent No. 6013487; US Patent No. 6083702), interfering RNAs (RNAi) mediating gene silencing (see Sharp et al., (2000) Science 287:2431), or others untranslated RNAs such as guide RNAs (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; US Pat. No. 5,869,248 to Yuan et al.); and so on.

[00462] In some embodiments, the cccDNA vector may further also contain a transgene that encodes a reporter polypeptide (eg, an enzyme such as green fluorescent protein or alkaline phosphatase). In some embodiments, the transgene that encodes a reporter protein suitable for experimental or diagnostic purposes is selected from any of the following transgenes: β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase ( CAT), luciferase and other genes well known in the art. In some aspects, cccDNA vectors containing a transgene encoding a reporter polypeptide can be used for diagnostic purposes or as markers of cccDNA vector activity in a subject to which they are administered.

[00463] In some embodiments, the cccDNA vector may contain a transgene or heterologous nucleotide sequence that has homology to and recombines with a locus on the host chromosome. This approach can be used to correct a genetic defect in a host cell.

[00464] In some embodiments, the cccDNA vector may contain a transgene that can be used to express an immunogenic polypeptide in a subject, such as for vaccination. The transgene may encode any immunogen of interest known in the art, including, without limitation, human immunodeficiency virus immunogens, influenza virus immunogens, gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, and the like.

XII. Introduction

[00465] In certain embodiments, more than one administration (e.g., two, three, four, or more administrations) may be administered to achieve the desired level of gene expression over a period of time at intervals of varying lengths, such as daily, weekly, monthly, annually, and etc.

[00466] Examples of routes for administering the cccDNA vector described herein include oral, rectal, transmucosal, intranasal, inhalation (e.g., aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular [including skeletal, diaphragmatic, and/or cardiac], intrapleural, intracerebral, and intraarticular), local (e.g., on the surface of the skin and mucous membranes, including the surface of the respiratory tract, and percutaneous administration), intralymphatic administration, etc., as well as direct injection into a tissue or organ (for example, into the liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain).

[00467] Administration of said cccDNA vector to a subject may be at any site, including, without limitation, a site selected from the group consisting of brain, skeletal muscle, smooth muscle, heart, diaphragm, respiratory epithelium, liver, kidney, spleen , pancreas, skin and eyes. The cDNA vector may also be introduced into a tumor (eg, into or near a tumor or lymph node). The most appropriate route in any given case depends on the nature and severity of the condition being treated, ameliorated and/or prevented, and the nature of the particular cccDNA vector used. In addition, cccDNA allows the introduction of more than one transgene in a single vector or in several cccDNA vectors (for example, in a cccDNA cocktail).

[00468] Administration of the cccDNA vector as described herein into skeletal muscle in accordance with the present invention includes, but is not limited to, administration into skeletal muscle of the extremities (e.g., shoulder, forearm, thigh, and/or calf), back, neck, head ( e.g. tongue), chest, abdomen, pelvis/perineum and/or fingers. The cccDNA vector as described herein can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion (optionally, isolated limb, leg and/or arm perfusion; see, for example, Arruda et al., ( 2005) Blood 105: 3458-3464) and/or direct intramuscular injection. In specific embodiments, the cccDNA vector described herein is administered to a limb (arm and/or leg) of a subject (e.g., a subject with a muscular dystrophy, such as Duchenne muscular dystrophy (DMD)) by perfusion of the limb, optionally by isolated limb perfusion (for example, by intravenous or intra-articular administration). In embodiments, a cccDNA vector as described herein can be administered without the use of “hydrodynamic” methods.

[00469] Administration of a cccDNA vector as described herein into cardiac muscle includes administration into the left atrium, right atrium, left ventricle, right ventricle, and/or septum. The cccDNA vector as described herein can be delivered to cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, by direct injection into the heart (eg, left atrium, right atrium, left ventricle, right ventricle), and/or by perfusion of the coronary artery. Administration into the diaphragmatic muscle may be accomplished by any suitable route, including intravenous administration, intra-arterial administration, and/or intraperitoneal administration. Administration into smooth muscle can be carried out by any suitable route, including intravenous administration, intra-arterial administration and/or intraperitoneal administration. In one embodiment, administration may be performed into, near and/or on endothelial cells present in smooth muscle.

[00470] In some embodiments, the cccDNA vector of the present invention is administered to skeletal muscle, diaphragm muscle, and/or cardiac muscle (e.g., to treat, alleviate, and/or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart disease) insufficiency).

A. Ex vivo treatment

[00471] In some embodiments, cells are removed from a subject, injected with a cDNA vector, and then returned to the subject. Methods for extracting cells from a subject for treatment ex vivo and then reintroducing them back into the subject are known in the art (see, for example, US Pat. No. 5,399,346; the contents of which are incorporated herein in their entirety). Alternatively, the cccDNA vector is introduced into cells obtained from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.

[00472] The cells transduced with the cccDNA vector are preferably administered to a subject in a "therapeutically effective amount" in combination with a pharmaceutical carrier. Those skilled in the art will appreciate that therapeutic effects do not have to be complete or provide a cure, as long as they are beneficial to the subject in some way.

[00473] In some embodiments, the cccDNA vector may encode a transgene (sometimes referred to as a heterologous nucleotide sequence) that is any polypeptide that is desired to be produced in a cell in vitro, ex vivo, or in vivo. For example, in contrast to the use of cccDNA vectors in a treatment method as described herein, in some embodiments, the cccDNA vectors can be introduced into cultured cells and the expressed gene product isolated therefrom, for example, to produce antigens or vaccines.

[00474] These cccDNA vectors can be used in both veterinary and human medicine. Suitable subjects for ex vivo gene delivery methods as described above include both birds (e.g., chickens, ducks, geese, quail, turkeys, and pheasants) and mammals (e.g., humans, cattle, sheep, goats, horses, felines, canids and lagomorphs), and mammals are preferred. Human subjects are most preferred. Human subjects include newborns, infants, children and adolescents, and adults.

[00475] One aspect of the technology described herein relates to a method for delivering a transgene into a cell. Typically, in in vitro methods, the cccDNA vector can be introduced into a cell using the methods disclosed herein as well as other methods known in the art. The cDNA vectors disclosed herein are preferably introduced into the cell in a biologically effective amount. If said cDNA vector is introduced into a cell in vivo (eg, a subject), a biologically effective amount of said cDNA vector is an amount sufficient to ensure transduction and expression of said transgene in the target cell.

B. Dose ranges

[00476] Optionally, in vivo and/or in vitro assays may be used to determine optimal dosage ranges for use. The exact dosage to be used in the formulation also depends on the route of administration and the severity of the condition and should be determined based on the judgment of one skilled in the art and taking into account the circumstances of each subject. Effective doses can be extrapolated from dose response curves obtained in in vitro test systems or animal models.

[00477] The cccDNA vector is administered in sufficient quantities to transfect cells of the desired tissue and provide sufficient levels of gene transfer and expression without excessive adverse effects. Standard and pharmaceutically acceptable routes of administration include, but are not limited to, those described below in the Introduction section, such as direct delivery to the target organ (e.g., intraportal delivery to the liver), oral delivery, inhalation (including intranasal and intratracheal delivery), intraocular intravenous, intramuscular, subcutaneous, intradermal, intratumoral delivery; and other parenteral routes of administration. Routes of administration can be combined if required.

[00478] The dosage amount of cccDNA vector required to achieve a given "therapeutic effect" will vary depending on several factors, including, but not limited to: the route of administration of the nucleic acid, the level of gene or RNA expression required to achieve therapeutic effect, the specific disease or disorder being treated, and the stability of the gene(s), RNA product(s), or resulting protein(s) expressed. One skilled in the art will readily be able to determine a dosage range of a cccDNA vector for treating a patient having a particular disease or disorder based on the above factors as well as other factors well known in the art.

[00479] The dosage regimen may be adjusted to ensure optimal therapeutic response. For example, the oligonucleotide may be administered repeatedly, for example, multiple doses may be administered daily, or the dose may be proportionally reduced based on the therapeutic situation dictated. One skilled in the art will readily be able to determine suitable dosages and administration schedules for the oligonucleotides of the present invention, both for administration to cells and for administration to subjects.

[00480] A "therapeutically effective dose" is within a relatively wide range of values, which can be determined in clinical trials and depends on the specific application (nerve cells will require very small amounts, whereas systemic injections will require significant amounts). For example, for direct in vivo injection into human skeletal or cardiac muscle, a therapeutically effective dose would be approximately 1 μg - 100 g cDNA vector a. If exosomes or microparticles are used to deliver the cccDNA vector, the therapeutically effective dose can be determined experimentally but is expected to deliver between 1 μg and approximately 100 g of vector.

[00481] The preparation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosage and treatment regimens for use of the specific compositions described herein in various treatment regimens.

[00482] For in vitro transfection, the effective amount of ccDNA vector to be delivered into cells (1×10 ⁶ cells) is on the order of 0.1-100 μg of ccDNA vector, preferably from 1 to 20 μg, and more preferably from 1 up to 15 µg, or 8 to 10 µg. Larger cccDNA vectors require higher doses. If exosomes or microparticles are used, the effective in vitro dose can be determined experimentally, but should generally deliver the same amount of cccDNA vector.

[00483] Treatment may include administration of a single dose or multiple doses. In some embodiments, more than one dose may be administered to a subject; in fact, doses can be administered multiple times as needed since the cDNA vector does not induce an immune response against the capsid in the host due to the absence of a viral capsid. Therefore, a person skilled in the art can easily determine the appropriate number of doses. The number of doses administered may be, for example, about 1-100, preferably 2-20 doses.

[00484] Without wishing to be bound by any particular theory, the absence of a typical antiviral immune response upon administration of a cccDNA vector as described herein (ie, the absence of capsid components) allows the cccDNA vector to be administered to a host repeatedly. In some embodiments, the number of times the heterologous nucleic acid is administered to a subject is from 2 to 10 times (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In some embodiments, the cDNA vector is delivered to a subject more than 10 times.

[00485] In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once per calendar day (eg, in a 24-hour period). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once every 2, 3, 4, 5, 6, or 7 calendar days. In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once per calendar week (eg, 7 calendar days). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once every two weeks (eg, once every two calendar weeks). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once per calendar month (eg, once every 30 calendar days). In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once every six calendar months. In some embodiments, a dose of the cccDNA vector is administered to a subject no more than once per calendar year (eg, 365 days or 366 days in a leap year).

C. Unit dosage forms

[00486] In some embodiments, these pharmaceutical compositions may be presented in a unit dosage form. The unit dosage form is typically adapted for one or more specific routes of administration of the pharmaceutical composition. In some embodiments, said unit dosage form is adapted for administration by inhalation. In some embodiments, the unit dosage form is adapted for administration by a vaporizer. In some embodiments, the unit dosage form is adapted for administration by a nebulizer. In some embodiments, the unit dosage form is adapted for administration using an aerosol generator. In some embodiments, the unit dosage form is adapted for oral administration, for buccal administration, or for sublingual administration. In some embodiments, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments, the unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments, the pharmaceutical composition is a formulation for topical administration. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form is usually that amount of said compound that provides a therapeutic effect.

XIII. Various Applications

[00487] The cccDNA vectors and compositions described herein can be used to introduce a nucleic acid sequence (eg, a therapeutic nucleic acid sequence) into a host cell. In some embodiments, cccDNA vectors and compositions as described herein can be used to introduce a nucleic acid sequence (eg, a therapeutic nucleic acid sequence) into a host cell, wherein the expression of said therapeutic nucleic acid sequence is under the control of a regulated switch. In one embodiment, the introduction of a nucleic acid sequence into a host cell using cccDNA vectors as described herein can be monitored using suitable biomarkers from treated patients to assess gene expression.

[00488] In some embodiments, the cccDNA vectors can be used in a variety of ways, including, for example, ex situ, in vitro, and in vivo gene therapy applications, methods, diagnostic procedures, and/or gene therapy regimens.

[00489] In some embodiments, the compositions and cDNA vectors of the present invention can be used to deliver a transgene for various purposes, as described above. In some embodiments, the transgene encodes a protein or functional RNA that is intended to be used for research purposes, for example, to create a somatic model of a transgenic animal carrying the transgene, for example, to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA that is intended for use in creating an animal model of a disease.

[00490] In some embodiments, the transgene encodes one or more peptides, polypeptides, or proteins that are useful for treating, ameliorating, or preventing disease states in a mammalian subject. Said transgene may be transferred into a patient's body (eg, expressed in the patient) in an amount sufficient to treat a disease associated with decreased expression, absence of expression, or dysfunction of said gene.

[00491] Some embodiments provide for the use of these cccDNA vectors in diagnostic and screening methods where the transgene is transiently or stably expressed in a cell culture system or, alternatively, in a transgenic animal model.

[00492] In another aspect, the technology described herein provides a method for transducing a population of mammalian cells. In a general and broad sense, the method includes at least the step of introducing into one or more cells of a population a composition that contains an effective amount of one or more cccDNAs as described herein.

[00493] In addition, the present invention provides compositions, as well as therapeutic and/or diagnostic kits, that include one or more disclosed cccDNA vectors or one or more disclosed cccDNA compositions, in formulations prepared using one or more additional ingredients, or prepared together with one or more instructions for their use.

[00494] In some embodiments, the cell for intended administration of the cccDNA vector as described herein may be any type of cell, including, but not limited to, neural cells (including cells of the peripheral and central nervous system, in particular cells brain), lung cells, retinal cells, epithelial cells (eg, intestinal and respiratory epithelial cells), muscle cells, dendritic cells, pancreatic cells (including islet cells), liver cells, myocardial cells, bone cells (eg, stem cells bone marrow), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. Alternatively, said cell may be any progenitor cell. In a further embodiment, said cell may be a stem cell (eg, neural stem cell, liver stem cell). In yet another embodiment, the cell may be a cancer or tumor cell. In addition, said cells may come from any species as stated above.

EXAMPLES

[00495] The following examples are intended to be illustrative and not limiting. EXAMPLE 1: Construction of cDNA vectors

[00496] The preparation of cDNA vectors using a polynucleotide construct template is described in Example 1 of PCT/US18/49996. For example, the template polynucleotide construct used to produce the ccDNA vectors of the present invention may be a ccDNA plasmid, a ccDNA bacmid, and/or a ccDNA baculovirus. Without being limited by theory, in a permissive host cell in the presence of, for example, Rep, the template a polynucleotide construct containing two symmetrical ITRs and an expression construct where at least one of the ITRs is modified relative to the wild-type ITR sequence is replicated to produce cccDNA vectors. Obtaining a cccDNA vector takes place in two stages: first, excision (“rescue”) of the template from the matrix backbone (for example, ccDNA plasmid, ccDNA bacmid, ccDNA baculovirus genome, etc.) using Rep proteins, and, second, Rep-mediated replication of the excised cccDNA vector.

[00497] An example of a method for producing cccDNA vectors is a ccDNA plasmid as described herein. In FIGS. 1A and 1B, the polynucleotide construct template of each of the cDNA plasmids includes both a left modified ITR and a right modified ITR with the following sequences located between said ITRs: (i) enhancer/promoter; (ii) transgene cloning site; (iii) a post-transcriptional response element (eg, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)); and (iv) a polyadenylation signal (eg, from the bovine growth hormone gene (BGHpA). Unique restriction endonuclease recognition sites (R1-R6) were also introduced between all components (shown in Fig. 1A and Fig. 1B) to facilitate the introduction of new genetic components into specific sites of the construct. The enzyme sites R3 (PmeI) GTTTAAAC (SEQ ID NO: 7) and R4 (PacI) TTAATTAA (SEQ ID NO: 542) are inserted into the cloning site to introduce the open reading frame of the transgene. These sequences are cloned into the plasmid pFastBac HT B obtained from ThermoFisher Scientific.

[00498] Briefly, a number of cccDNA vectors were prepared from the ccDNA plasmid constructs shown in Table 7 using the method shown in FIG. 5A-5C, and the ITR sequences shown in Table 4. Table 7 indicates the number of corresponding polynucleotide sequences for each component, including sequences active as a replication protein site (RPS) (eg, a Rep binding site) at either end of the promoter, functionally associated with a transgene. The numbers in Table 7 refer to the SEQ ID NOs herein corresponding to the sequences of each component. Plasmids from Table 7 were constructed with a WPRE containing SEQ ID NO: 8 followed by BGHpA containing SEQ ID NO: 9 in the 3' untranslated region between the transgene and the right ITR.

[00499] In other embodiments, a series of cccDNA vectors are generated from ccDNA plasmid constructs containing AAV2 5' and 3' WT-ITR using the method presented in FIG. 5A-5C. In some embodiments, the construct for producing cccDNA vectors comprises a promoter that is a regulatory switch as described herein, such as an inducible promoter.

Preparation of cccDNA-bacmid

[00500] As shown in FIG. 5A, DHlOBac competent cells (MAX EFFICIENCY® DH10Bac™ Competent Cells, Thermo Fisher) were transformed with either test or control plasmids according to the protocol according to the manufacturer's instructions. To obtain recombinant cccDNA bacmids, recombination between the plasmid and the baculovirus shuttle vector was induced in DH10Bac cells. Recombinant bacmids were selected by positive selection screening based on blue-white screening in E. coli (marker Ф80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG with antibiotics to select transformants and maintenance of bacmids and transposase plasmids. White colonies resulting from transposition that disrupts the β-galactoside indicator gene were selected and cultured in 10 ml of medium.

[00501] Recombinant cccDNA bacmids were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adherent Sf9 or Sf21 insect cells were cultured in 50 ml of medium in T25 flasks at 25°C. After 4 days, the culture medium (containing the P0 virus) was separated from the cells and filtered through a filter with a pore size of 0.45 μm, separating infectious baculovirus particles from the cells or cellular debris.

[00502] Optionally, the first generation of baculovirus (P0) was amplified by infecting untreated Sf9 or Sf21 insect cells in 50-500 ml of medium. Cells were maintained in suspension cultures in an orbital shaker incubator at 130 rpm and 25°C, with cell diameter and viability monitored until the cells reached a diameter of 18-19 nm (as opposed to the untreated cell diameter of 14-15 nm). nm) and a density of approximately 4.0E+6 cells/ml. At 3 to 8 days postinfection, baculovirus P1 particles in the medium were collected after centrifugation to remove cells and debris, followed by filtration through a 0.45-μm pore size filter.

[00503] The cDNA baculovirus containing the test constructs was collected and the infectivity or titer of the baculovirus was determined. In particular, 4x20 ml of Sf9 cell culture with a density of 2.5E+6 cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000. 1/10000, 1/50000, 1/100000, and incubated at 25-27°C. Infectivity was determined daily for 4-5 days by the increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[00504] As shown in FIG. 5A, a “Rep plasmid” was obtained according to, for example, FIG. 9A in the pFASTBAC™-Dual expression vector (ThermoFisher) containing both Rep78 (SEQ ID NO: 13) or Rep68 (SEQ ID NO: 12) and Rep52 (SEQ ID NO: 14) or Rep40 (SEQ ID NO: 11 ).

[00505] The Rep plasmid was transformed into DHlOBac competent cells (MAX EFFICIENCY® DH10Bac™ Competent Cells (Thermo Fisher)) following the protocol provided by the manufacturer. Recombination was induced between the Rep plasmid and the baculovirus shuttle vector in DH10Bac cells to produce recombinant bacmids (“Rep bacmids”). Recombinant bacmids were selected by positive selection, which included blue-white screening in E. coli (marker Ф80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG. Isolated white colonies were collected and inoculated into 10 ml of selective medium (kanamycin, gentamicin, tetracycline in LB broth). Recombinant bacmids (Rep bacmids) were isolated from E. coli, and Sf9 or S121 insect cells were transfected with these Rep bacmids to produce infectious baculovirus.

[00506] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days and an infectious recombinant baculovirus (“Rep-baculovirus”) was isolated from the culture. Optionally, first generation Rep baculovirus (P0) was amplified by infecting untreated insect cells with Sf9 or Sf21 and culturing in 50-500 ml of medium. 3-8 days after infection, baculovirus P1 particles in the medium were collected by separating the cells by centrifugation or filtration, or using another fractionation method. Rep-baculovirus was collected and the infectivity of the baculovirus was determined. In particular, 4x20 ml of Sf9 cell culture with a density of 2.5x10 ⁶ cells/ml was treated with baculovirus P1 in the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined daily for 4-5 days by the increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

Obtaining and describing the characteristics of the cDNA vector

[00507] As shown in FIG. 5B, then Sf9 insect cell culture medium containing either (1) a sample containing cccDNA bacmid or cccDNA baculovirus or (2) Rep baculovirus described above was added to a fresh Sf9 cell culture (2.5E+ 6 cells/ml, 20 ml) in a ratio of 1:1000 and 1:10000, respectively. Cells were then cultured at 130 rpm at 25°C. Cell diameter and viability were detected 4–5 days after coinfection. When the cell diameter reached 18-20 nm with a viability of approximately 70-80%, the cell cultures were centrifuged, the medium was removed and the cell pellets were collected. Cell pellets were first resuspended in a sufficient volume of aqueous medium, either water or buffer. The cccDNA vector was isolated from cells and purified using the Qiagen MIDI PLUS™ purification protocol (Qiagen, treatment of 0.2 mg cell pellet weight per column).

[00508] Yield values for cccDNA vectors produced and purified from Sf9 insect cells were initially determined based on UV absorbance at 260 nm.

[00509] Evaluation of cccDNA vectors can be accomplished by identification using agarose gel electrophoresis under native or denaturing conditions, as illustrated in FIG. 5D, where (a) the presence of characteristic bands migrating as fragments of twice the size on denaturing gels compared to native gels after restriction endonuclease digestion and gel electrophoretic analysis, and (b) the presence of monomer and dimer bands (2×) on denaturing gels Undigested material is characterized by the presence of a cDNA vector.

[00510] The structures of the isolated cccDNA vectors were further analyzed by digesting DNA obtained from coinfected Sf9 cells (as described herein) with restriction endonucleases selected based on a) the presence of only one cut site in the ccDNA vectors, and b) obtaining the resulting fragments large enough to be clearly visible when fractionated on a 0.8% denaturing agarose gel (>800 i.u.). As shown in FIG. 5D and 5E. linear discontinuous DNA vectors and linear and continuous cccDNA vectors can be distinguished by the size of the reaction products - for example, a discontinuous DNA vector is expected to produce 1 kb fragments. and 2 kb, and the cDNA vector with a continuous structure fragments of 2 kb in size. and 4 kb.

[00511] Thus, in order to qualitatively demonstrate that the isolated cccDNA vectors have covalently closed ends as required by their definition, samples were digested with a restriction endonuclease identified in the context of the specific DNA vector sequence as a single site restriction endonuclease , preferably producing two cleavage products of unequal size (eg, 1000 bp and 2000 bp). After digestion and electrophoresis on a denaturing gel (which separates two complementary DNA strands), linear DNA that is not covalently closed will be separated into 1000 bp fragments. and 2000 bp, while covalently closed DNA (i.e. cDNA vector) will be divided into fragments 2 times larger (2000 bp and 4000 bp), since two chains The DNA is linked, and after unfolding it will be twice as long (although single-stranded). In addition, when the monomeric, dimeric, and n-mer forms of DNA vectors are digested, they will all separate into fragments of equal sizes due to the interconnected ends of the multimeric DNA vectors (see Fig. 5D).

[00512] As used herein, the phrase “assay for identifying DNA vectors by agarose gel electrophoresis under native and denaturing gel conditions” refers to an assay for assessing the presence of closed ends of cccDNA by performing restriction endonuclease digestion followed by electrophoretic assessment of the digestion products. One example of such an analysis is given below, although one skilled in the art will appreciate that many variations of this example known in the art can be performed. The restriction endonuclease is selected to be the enzyme that makes a single cut of the cDNA vector of interest to produce products that are approximately 1/3× and 2/3× the length of the DNA vector. This ensures band separation on both native and denaturing gels. It is important to remove the buffer from the sample before denaturation. The Qiagen PCR Product Purification Kit or desalting “spun columns” such as the GE HEALTHCARE ILUSTRA™ MICROSPIN™ G-25 columns are examples of endonuclease digestion agents known in the art. This analysis involves, for example, i) digestion of DNA with suitable restriction endonuclease(s), 2) addition, for example, to a Qiagen PCR purification kit, eluting with distilled water, iii) addition of a 10x denaturing solution (10×=0.5 M NaOH, 10 mM EDTA), adding 10x unbuffered dye solution and running the analysis together with DNA ladders prepared by adding 10x denaturing solution to a 4x, 0.8-1.0% gel pre-incubated with 1 mM EDTA and 200 mM NaOH to ensure uniform NaOH concentration in the gel and gel chamber, and run the gel in the presence of 1x denaturing solution (50 mM NaOH, 1 mM EDTA). One skilled in the art will understand what voltage should be used to perform electrophoresis depending on the size and desired time to obtain results. After electrophoresis, the gels are dried and neutralized in 1x TBE or TAE and transferred to distilled water or 1x TBE/TAE with 1x SYBR Gold. The bands can then be visualized, for example, using Thermo Fisher's SYBR® Gold Nucleic Acid Gel Stain (10,000X concentrate in DMSO) and epifluorescent light (blue) or UV (312 nm).

[00513] The purity of the resulting cccDNA vector can be assessed using any method known in the art. In one non-limiting example of a method, the contribution of the ccDNA plasmid to the total UV absorbance of the sample can be calculated by comparing the fluorescence intensity of the ccDNA vector and the standard. For example, if based on UV absorbance it is determined that 4 μg of ccDNA vector is loaded on the gel, and the fluorescence intensity of the ccDNA vector is equivalent to a 2 kb band. with a known mass of 1 μg, then the mass of the ccDNA vector is 1 μg, and the ccDNA vector constitutes 25% of the total amount of UV absorbing material. The intensity of the band on the gel is then plotted as a function of the calculated input amount to which the band corresponds. For example, if the total quantity of the cDNA vector corresponds to 8 kb, and the excised comparative band corresponds to 2 kb, then the intensity of the indicated band on the graph will correspond to 25% of the total amount administered, which in this case will be 0.25 mcg when 1.0 mcg is administered. Using titration of the cccDNA vector plasmid to construct a standard curve, the quantity for the cccDNA vector band is calculated from the regression line equation, which can then be used to determine the percentage of the total input amount represented by the cccDNA vector, or the percentage purity.

Preparation of cccDNA-bacmid

[00514] DHlOBac competent cells (MAX EFFICIENCY® DH10Bac™ Competent Cells, Thermo Fisher) were transformed with either the test or control plasmid according to the protocol from the manufacturer's instructions. Recombination between the plasmid and the baculovirus shuttle vector was induced in DH10Bac cells to produce recombinant cccDNA bacmids. Recombinant bacmids were selected by positive selection based on blue-white screening in E. coli (marker F80dlacZAM15 provides a-complementation of the α-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG with antibiotics for selection transformants and maintenance of bacmid and transposase plasmids. White colonies resulting from transposition that disrupts the beta-galactoside indicator gene were collected and cultured in 10 ml of medium.

[00515] Recombinant cccDNA bacmids were isolated from E. coli and transfected into Sf9 or Sf21 insect cells using FugeneHD to produce infectious baculovirus. Adhesive Sf9 or Sf21 insect cells were cultured in 50 ml of medium in T25 flasks at 25°C. After 4 days, the culture medium (containing P0 virus) was separated from the cells and filtered through a 0.45-μm filter to separate infectious baculovirus particles from cells or cellular debris.

[00516] Optionally, the first generation of baculovirus (P0) was amplified by infecting untreated Sf9 or Sf21 insect cells in 50-500 ml of medium. Cells were maintained in suspension cultures in an orbital shaker incubator at 130 rpm and 25°C, with cell diameter and viability monitored until the cells reached a diameter of 18-19 nm (as opposed to the untreated cell diameter of 14-15 nm). nm) and a density of about 4.0E+6 cells/ml. At 3 to 8 days postinfection, baculovirus P1 particles in the medium were collected after centrifugation to remove cells and debris, followed by filtration through a 0.45-μm pore size filter.

[00517] The cDNA baculovirus containing the test constructs was collected and the infectivity, or titer, of the baculovirus was determined. In particular, 4x20 ml of Sf9 cell culture with a density of 2.5E+6 cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated at 25-27°C . Infectivity was determined daily for 4-5 days by the increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

[00518] The "Rep plasmid" was produced in the pFASTBAC™-Dual expression vector (ThermoFisher) containing both Rep78 or Rep68 and Rep52 or Rep40. The indicated Rep plasmid was transformed into competent DH10Bac cells (MAX EFFICIENCY® DH10Bac™ Competent Cells (Thermo Fisher)), according to the protocol provided by the manufacturer. Recombination between the Rep plasmid and the baculovirus shuttle vector was induced in DH10Bac cells to produce recombinant bacmids (“Rep bacmid”). Recombinant bacmids were selected by positive selection, which included blue-white screening in E. coli (marker Ф80dlacZΔM15 provides α-complementation of the β-galactosidase gene from the bacmid vector) on a bacterial agar plate containing X-gal and IPTG. Isolated white colonies were collected and inoculated into 10 ml of selective medium (kanamycin, gentamicin, tetracycline in LB broth). Recombinant bacmids (Rep bacmids) were isolated from E. coli, and Sf9 or Sf21 insect cells were transfected with these Rep bacmids to produce infectious baculovirus.

[00519] Sf9 or Sf21 insect cells were cultured in 50 ml of medium for 4 days and an infectious recombinant baculovirus (“Rep-baculovirus”) was isolated from the culture. Optionally, first generation Rep baculovirus (P0) was amplified by infecting naïve insect cells with S19 or Sf21 and culturing in 50-500 ml of medium. At 3 to 8 days postinfection, baculovirus P1 particles in the medium were collected either by cell separation by centrifugation, filtration, or other fractionation method. Rep-baculovirus was collected and the infectivity of the baculovirus was determined. In particular, 4x20 ml of Sf9 cell cultures at a density of 2.5x10 ⁶ cells/ml were treated with baculovirus P1 in the following dilutions: 1/1000, 1/10000, 1/50000, 1/100000, and incubated. Infectivity was determined daily for 4-5 days by the rate of increase in cell diameter and cell cycle arrest, as well as changes in cell viability.

EXAMPLE 2: Preparation of synthetic cccDNA by excision from a double-stranded DNA molecule

[00520] The synthetic production of cccDNA vectors is described in Examples 2-6 of International Application PCT/US19/14122, filed January 18, 2019, which is incorporated herein by reference in its entirety. One example of a method for producing a cDNA vector using a synthetic method involves cutting out a double-stranded DNA molecule. Briefly, a cccDNA vector can be produced using a double-stranded DNA construct, for example, see FIG. 7A-8E from PCT/US19/14122. In some embodiments, the double-stranded DNA construct is a ccDNA plasmid, for example, see FIG. 6 in international patent application PCT/US2018/064242, filed December 6, 2018).

[00521] In some embodiments, the design for producing the cDNA vector includes a regulatory switch as described herein.

[00522] For purposes of illustration, Example 1 describes the preparation of cccDNA vectors as an example of closed-end DNA vectors generated using this method. However, although cccDNA vectors are described in this example to illustrate synthetic in vitro production methods to produce a closed-end DNA vector by excision of a double-stranded polynucleotide containing an ITR and an expression cassette (e.g., a heterologous nucleic acid sequence), followed by ligation of the free 3' and 5' ends as described herein, one skilled in the art will know that, as shown above, a double-stranded DNA polynucleotide molecule can be modified to produce any desired closed-end DNA vector, including, without limitation, service DNA, Doggybone™ DNA, dumbbell DNA, etc. Examples of cccDNA vectors for producing transgenes and therapeutic proteins can be produced using the synthetic production method described in Example 2.

[00523] The method comprises (i) excision of an expression cassette coding sequence from a double-stranded DNA construct, and (ii) formation of hairpin structures in one or more ITRs; and (iii) joining the free 5' and 3' ends by ligation, for example, using T4 DNA ligase.

[00524] Said double-stranded DNA construct contains, in the direction from the 5' to the 3' end: a first restriction endonuclease site; 5' ITR; expression cassette; 3' ITR; and a second restriction endonuclease site. Said double-stranded DNA construct is then contacted with one or more restriction endonucleases to create double-strand breaks at both restriction endonuclease sites. Both sites can be targeted by a single endonuclease, or each site can be targeted by a separate endonuclease, provided that the restriction sites are not included in the template for the cDNA vector. This results in the excision of the sequence between the endonuclease restriction sites from the rest of the double-stranded DNA construct (see Fig. 9 of PCT/US19/14122). After ligation, a DNA vector with closed ends is formed.

[00525] One or both ITRs used in the method may be wild-type ITRs. Modified ITRs may also be used, which modification may include deletion, insertion or substitution of one or more nucleotides from the wild-type ITR in the sequences forming arms B and B' and/or arms C and C (see, for example, FIG. 6 -8 and 10, Fig. 11B from PCT/US19/14122), and may contain two or more hairpin loops (see, for example, Fig. 6-8, Fig. 11B from PCT/US 19/14122) or a single hairpin loop (see, for example, Fig. 10A-10B, Fig. 11B from PCT/US 19/14122). A modified hairpin loop ITR can be produced by genetic modification of an existing oligonucleotide or by biological and/or chemical de novo synthesis.

EXAMPLE 3: Preparation of ccDNA by designing oligonucleotides

[00526] Another example of a method for producing a cccDNA vector using a synthetic method that involves the assembly of various oligonucleotides is presented in Example 3 of PCT/US 19/14122, where the ccDNA vector is prepared by synthesizing a 5' oligonucleotide and a 3' ITR oligonucleotide and ligating said ITR oligonucleotides to a double-stranded polynucleotide containing an expression cassette. In fig. 11B from PCT/US 19/14122 shows an example of a method for ligating a 5'-ITR oligonucleotide and a 3'-ITR oligonucleotide to a double-stranded polynucleotide containing an expression cassette.

[00527] In some embodiments, the design for producing the cDNA vector includes a regulatory switch described herein.

[00528] ITR oligonucleotides may contain WT-ITR as described herein or may include modified ITRs as described herein. Modified ITRs may include a deletion, insertion, or substitution of one or more nucleotides relative to the wild-type ITR in the sequences forming arms B and B' and/or arms C and C. ITR oligonucleotides containing WT-ITR or mod-ITR as described herein, intended for use in cell-free synthesis, can be obtained by genetic modification or biological and/or chemical synthesis. As described herein, the ITR oligonucleotides in Examples 2 and 3 may contain WT-ITR or modified ITR (mod-ITR) in symmetric or asymmetric configurations, as discussed herein.

EXAMPLE 4: Preparation of cccDNA using a single-stranded DNA molecule [00529] Another example of a method for producing a cccDNA vector using the synthetic method presented in Example 4 of PCT/US 19/14122 uses single-stranded linear DNA containing two sense ITRs that flank sense sequence of the expression cassette and covalently attached to two antisense ITRs that flank the antisense expression cassette, and the ends of said single-stranded linear DNA are then ligated to form a single-stranded end-locked molecule. One non-limiting example involves synthesizing and/or producing a single-stranded DNA molecule, annealing portions of said molecule to form a single linear DNA molecule that contains one or more base-paired secondary structure regions, and then ligating the free 5' and 3' ends together with the formation of a closed single-chain molecule.

[00530] In some embodiments, the design for producing a cccDNA vector includes a regulatory switch as described herein.

[00531] ITR oligonucleotides may contain WT-ITR as described herein, or may contain modified ITRs as described herein.

[00532] An example of a single-stranded DNA molecule for producing a cDNA vector contains, in a 5' to 3' direction: a sense first ITR; semantic sequence of the expression cassette; semantic second ITR; antisense second ITR; antisense sequence of the expression cassette; and antisense first ITR.

[00533] The single-stranded DNA molecule for use in the exemplary method of Example 4 can be prepared by any DNA synthesis method described herein. for example, DNA synthesis in vitro, or obtained by cleaving a DNA construct (for example, a plasmid) with nucleases and melting the resulting dsDNA fragments to produce ssDNA fragments.

[00534] Annealing can be accomplished by reducing the temperature to a level below the calculated melting temperature of the sense and antisense sequence pairs. The melting point depends on the content of the specific nucleic bases and the characteristics of the solution used, for example, on the salt concentration. Melting points for any particular combination of sequence and solution can be easily calculated by one skilled in the art.

[00535] The free 5' and 3' ends of the annealed molecule can be ligated to each other or ligated to a hairpin molecule to form a cccDNA vector. Suitable examples of ligation methods and hairpin molecules are described in Examples 2 and 3.

EXAMPLE 5: cDNA vectors express the luciferase transgene in vitro

[00536] Constructs were obtained by introducing an open reading frame encoding a luciferase reporter gene into the cloning site of the cDNA plasmid constructs: construct-15-30 (see Table 7 above), or constructs containing the AAV2 WT-ITR, including the coding luciferase sequence. HEK293 cells were cultured and transfected with 100 ng, 200 ng or 400 ng of 15-30 plasmid constructs using FUGENE® (Promega Corp.) as the transfection agent. Luciferase expression from each plasmid was determined based on the luciferase activity in each cell culture, confirming that the luciferase activity was the result of gene expression from the plasmids.

EXAMPLE 6: Expression of transgenic luciferase protein in vivo from cDNA vectors.

[00537] In vivo transgene protein expression from cccDNA vectors produced from the constructs described above was assessed in mice. cccDNA vectors derived from cccDNA plasmid constructs were tested and demonstrated long-lasting and sustained transgenic luciferase expression in a mouse model following hydrodynamic injection of the cccDNA construct without liposomes and repeated dosing (at day 28), and longevity (to day 42) of exogenous cccDNA firefly luciferase. In other experiments, luciferase expression from selected cccDNA vectors is assessed in vivo, wherein said cccDNA vectors contain a luciferase transgene and: i) a modified 5' ITR and a symmetric modified 3TTR selected from any of the symmetric pairs presented in Table 4 or the pair of modified ITRs presented in fig. 7A-22V; or ii) AAV2 5' WT-ITR and AAV2 3' WT-ITR.

[00538] In vivo transgene protein expression from cccDNA vectors produced from AAV2 WT-ITR constructs as described above is assessed in mice. An cccDNA vector derived from cccDNA plasmid constructs was tested and demonstrated long-lasting and sustained transgenic luciferase expression in a mouse model following hydrodynamic injection of the cccDNA construct without liposomes and repeated dosing (at day 28) and longevity (to day 42) of exogenous luciferase cccDNA fireflies. In other experiments, luciferase expression from selected ccDNA vectors is assessed in vivo, said ccDNA vectors containing the luciferase transgene, AAV2 5' WT-ITR and AAV2 3' WT-ITR.

[00539] In vivo luciferase expression: Male CD-I IGS mice, 5-7 weeks of age (Charles River Laboratories), are administered 0.35 mg/kg of cccDNA vector expressing luciferase in a volume of 1.2 ml by intravenous (i.v.) c) hydrodynamic tail vein injection on day 0. Luciferase expression was assessed by IVIS imaging on days 3, 4, 7, 14, 21, 28, 31, 35 and 42. Briefly, mice were injected intraperitoneally with 150 mg/kg luciferin substrate. and then assess whole-body luminescence using IVIS® imaging.

[00540] IVIS imaging is performed on day 3, day 4, day 7, day 14, day 21, day 28, day 31, day 35 and day 42, and harvested organs are imaged ex vivo after sacrifice on day 42.

[00541] Animals are weighed daily throughout the study and their general health and well-being are monitored. At sacrifice, the blood of each animal is collected by terminal cardiac puncture, divided into two parts and processed to obtain 1) plasma and 2) serum, with the plasma flash frozen and the serum used for a panel of liver enzyme tests, after which it is flash frozen. In addition, livers, spleens, kidneys, and inguinal lymph nodes (LNs) are collected and imaged ex vivo using IVIS.

[00542] Liver luciferase expression is assessed using the MAXDISCOVERY® Luciferase ELISA assay kit (BYO Scientific/PerkinElmer), qPCR luciferase of liver samples, histopathology of liver samples and/or a panel of serum liver enzymes (VetScanVS2; Abaxis Preventive Care Profile Plus).

EXAMPLE 7: Generation and Analysis of WT/WT cfDNA

[00543] The ITR of wild type AAV type II was used to study the formation of cccDNA and the ability to express the transgene encoded by the specified ccDNA. Vector construction, analysis of cccDNA production, and assessment of transgene expression of cccDNA in human cell culture are described in more detail below.

Design of WT/WT ITR

[00544] A wild-type AAV type II ITR cassette plasmid was developed in silico and then evaluated in insect Sf9 cells. This cassette contained a green fluorescent protein (GFP) reporter gene under the control of the p10 promoter sequence for expression in insect cells.

[00545] Sf9 suspension cultures were maintained in Sf900 III medium (Gibco) in vented 200 mL tissue culture flasks. Cultures were subcultured every 48 hours, and before each subculture, cell numbers and growth rates were measured using a ViCell Counter (Beckman Coulter). Cultures were maintained under shaking conditions (1 arc second swing, 130 rpm) at 27°C.

[00546] cccDNA vectors were prepared and constructed as described in Example 1 above. Briefly, as shown in FIG. 4B, Sf9 cells transduced with the plasmid construct were allowed to grow adhesively for 24 hours under steady-state conditions at 27°C. After 24 hours, transfected Sf9 cells were infected with the Rep vector via baculovirus-infected insect cells (BIIC). BIICs were pre-assayed to determine infectivity characteristics and used at a final dilution of 1:2000. BIICs diluted 1:100 in Sf900 insect cell medium were added to each well of pre-transfected cells. BIIC without Rep vector was added to a subset of wells as a negative control. The tablet contents were mixed by gentle shaking on a plate shaker for 2 minutes. The cells were then cultured for an additional 48 hours at 27°C under steady-state conditions. All experimental and control designs were analyzed in triplicate.

[00547] After 48 hours, the 96-well plate was removed from the incubator, briefly equilibrated to room temperature, and visually examined for GFP expression using fluorescence microscopy. Fluorescence and bright-field images were captured at 40× magnification. As expected, the negative control (sample that was treated in the absence of baculovirus-containing Rep cells) did not show significant GFP expression. Robust GFP expression was observed in the WT/WT ITR GFP vector sample, indicating successful transfection with the cccDNA-encoded transgene. The results are presented in Fig. 24A-24B. As expected, the negative control (sample that was treated in the absence of baculovirus-containing Rep cells) did not show significant GFP expression. Robust GFP expression was observed in the wild-type vector sample, indicating successful transfection and expression of the cccDNA-encoded transgene.

Analysis of cccDNA production

[00548] To ensure that the cccDNA obtained in the previous study had the expected closed-end structure, experiments were performed to obtain a sufficient amount of ccDNA that could subsequently be tested for the proper structure. Briefly, Sf9 suspension cultures were transfected with WT/WT ITR DNA. Cultures were seeded at a density of 1.25×10 ⁶ cells/ml in Erlenmeyer culture flasks with limited gas exchange. DNA:lipid transfection complexes were prepared using FuGene® transfection reagent according to the manufacturer's instructions. Mixtures of complexes were prepared and incubated in the same manner as described above for plate assays, with volumes increasing in proportion to the number of cells transfected. As in the case of the reporter gene assay, a ratio of 4.5:1 (reagent volume/DNA mass) was used. Mock-transfected (transfection reagents only) and untreated control cultures were prepared in parallel with the experimental cultures. After adding transfection reagents, cultures were allowed to recover for 10–15 minutes at room temperature with gentle rotation before being transferred to a shaking incubator at 27°C. After 24 hours of incubation with shaking, cell counts were performed and growth rates were measured for all vials (experimental and control) using a ViCell counter (Beckman Coulter). All vials (except for the growth control) were infected with BIIC containing the Rep vector at a final dilution of 1:5000. A positive control was also obtained using an established VIS double infection procedure to obtain cccDNA. For a dual infection culture, a number of cells equal to the average number of viable cells in all experimental cultures were seeded. The dual infection control culture was infected with VIS with Rep and reporter gene at a final dilution of 1:5000 for each sample, respectively. After infection, the cultures were placed back into the incubator under the previously described shaking conditions. Cell counts, growth rates, and viability rates were measured daily for all vials for 3 days postinfection. Measurements at time T=0 were performed after newly infected cultures had been allowed to recover for approximately 2 hours under shaking incubation conditions. After 3 days, cells were collected by centrifugation for 15 minutes. The supernatant was discarded, the weight of the pellet was recorded, and the pellet was frozen at −80°C until DNA extraction.

[00549] Putative crude cccDNA was extracted from all vials (experimental and control) using the Qiagen Plasmid Plus Midi Purification Kit (Qiagen) according to the manufacturer's protocol for “high yield.” Quantification of eluates was performed using absorbance data obtained from NanoDrop OneC (ThermoFisher). The resulting cccDNA extracts were stored at 4°C.

[00550] The above cccDNA extracts were run on a native agarose gel (1% agarose, 1x TAE buffer) prepared using a 1:10,000 dilution of SYBR Safe Gel Stain (ThermoFisher Scientific) along with a 1 kb Tracklt Plus DNA ladder. The gel was then imaged using a Gbox Mini Imager under UV/blue light. As previously described, cccDNA samples run on native gels are expected to have two primary bands: a band at approximately 4000 bp, representing monomeric material, and a band at approximately 8,000 bp, representing dimeric material. The wild-type sample was tested and showed the expected monomer and dimer bands on a native agarose gel. The results for a representative sample of these designs are presented in Fig. 25. Putative crude ccDNA and small-scale control extracts were further analyzed using paired restriction digestion and denaturing agarose gel to confirm double-stranded DNA structure indicative of ccDNA. Wild-type cccDNA is expected to have a single Clal restriction site; therefore, if properly formed, it forms two characteristic fragments when Clal is cleaved. The high-fidelity restriction endonuclease Clal (New England Biolabs) was used to digest the putative cccDNA extract according to the manufacturer's instructions. Extracts from mock-transfected and control cultures were not analyzed because spectrophotometric quantitation using NanoDrop (ThermoFisher) as well as native agarose gel analysis indicated that there was no detectable ccDNA/plasmid-like product in the eluates. Digested material was purified using a Qiagen PCR kit. Clean-up Kit (Qiagen) according to the manufacturer's instructions, except that the purified digest material was eluted in nuclease-free water instead of the Qiagen elution buffer. Alkaline agarose gel (0.8% alkaline agarose) was equilibrated in equilibration buffer (1 mM EDTA, 200 mM NaOH) overnight at 4°C. A 10x denaturing solution (50 mM NaOH, 1 mM EDTA) was added to samples of purified digested ccDNA and corresponding uncleaved ccDNA (1 μg total), and the samples were heated at 65°C for 10 min. A 10x loading dye (bromophenol blue, 50% glycerol) was added to each denatured sample and mixed. Plus DNA Tracklt 1 kb Ladder (ThermoFisher Scientific) was also loaded onto the gel as reference DNA. The gel was run for ~18 hours at 4°C and constant voltage (25 V), followed by washing with deionized _H2O and neutralization in 1xTAE buffer (Tris-acetate, EDTA), pH 7.6, for 20 minutes at stirring gently. The gel was then transferred to a 11x TAE/1x SYBR Gold solution for approximately 1 hour with gentle stirring. The gel was then imaged using a Gbox Mini Imager (Syngene) under UV/blue light. Uncut denatured samples were expected to migrate at about 9000 bp, and ClaI-treated samples were expected to produce two bands, one at about 2000 bp and the other at about 6000 bp.

[00551] Two significant bands were visible in each lane with the ClaI-digested samples migrating on the denaturing gel at the expected sizes, in stark contrast to the uncleaved sample, which migrated at the expected size of approximately 9000 bp. In fig. Figure 26 shows the results for a representative sample, where two bands are visible above background values for the digested sample, in contrast to the single band observed for the undigested sample. Accordingly, cccDNA apparently formed correctly in this sample.

Functional expression in human cell culture

[00552] To evaluate the functionality of WT/WT ITR cccDNA produced in a small scale production process, HEK293 cells are transfected with WT/WT cccDNA samples. Actively dividing HEK293 cells were seeded into 96-well microtiter plates at a density of 3 x 10 ⁶ cells per well (80% confluent) and incubated for 24 hours under previously described conditions for adherent HEK293 cultures. After 24 hours, transfection was carried out with 200 ng of crude, small-scale produced cccDNA using Lipofectamine (Invitrogen, ThermoFisher Scientific). Transfection complexes were prepared according to the manufacturer's instructions, and the transfection complex was used to transfect previously plated HEK293 cells in a total volume of 10 μl. All experimental designs and controls were analyzed in triplicate. Transfected cells are incubated under previously described conditions for 72 hours. After 72 hours, the 96-well plate is removed from the incubator and allowed to equilibrate briefly to room temperature. The GFP expression assay was performed as described above. Total luminescence is determined using a SpectraMax M Series microplate reader. Data for replicates are averaged. Expression of GFP in human cell culture shows that cccDNA was correctly generated and expressed for each sample in the context of human cells.

EXAMPLE 8: Run-through screening for symmetrical ITR mutants

[00553] Additional analyzes were performed on the relationship of ITR structure to cccDNA production. A series of mutants were constructed to study the effect of specific structural changes on the formation of cccDNA and the ability to express the transgene encoded by the specified ccDNA. Construction of mutants, analysis of cctDNA formation, and assessment of transgenic expression of cccDNA in human cell culture were performed using methods similar to those described above.

Construction of Mutant ITRs [00554] A library of 16 plasmids with unique type II symmetric mutant AAV ITR cassettes was developed in silico and then evaluated in S19 insect cells and human embryonic kidney (HEK293) cells. Each ITR cassette contained either a luciferase (LUC) or green fluorescent protein (GFP) reporter gene under the control of a p10 promoter sequence for expression in insect cells, and a CAG promoter sequence for expression in mammalian cells. Mutations in the ITR sequence were created symmetrically in the right and left ITR regions. The library contained 16 right-handed double mutants as described in Table 4 herein; the predicted structures are shown in Fig. 7A-22B.

[00555] Sf9 suspension cultures were maintained in Sf900 III medium (Gibco) in vented 200 mL tissue culture flasks. Cultures were subcultured every 48 hours, and before each subculture, cell numbers and growth rates were measured using a ViCell Counter (Beckman Coulter). Cultures were maintained under shaking conditions (1 arc second swing, 130 rpm) at 27°C. Adherent cultures of HEK293 cells were maintained in GlutiMax DMEM (Dulbecco's modified Eagle's medium, Gibco) with 1% fetal bovine serum and 0.1% penicillin-streptomycin (PenStrep) in 250 ml culture flasks at 37°C with 5% CO ₂ . Cultures were treated with trypsin and subcultured every 96 hours. For each subculture, a 1:10 dilution from a 90-100% confluent vial was used.

[00556] cccDNA vectors were prepared and constructed as described in Example 1 above. Briefly, as shown in FIG. 5B, Sf9 cells transduced with plasmid constructs were allowed to grow adhesively for 24 hours under steady-state conditions at 27°C. After 24 hours, transfected Sf9 cells were infected with the Rep vector via baculovirus-infected insect cells (BIIC). BIIC was pre-analyzed to determine infectivity characteristics and used at a final dilution of 1:2000. BIIC at a 1:100 dilution in Sf900 insect cell medium was added to each well of pre-infected cells. BIIC without Rep vector was added to a subset of wells as a negative control. The tablet contents were mixed by gentle shaking on a plate shaker for 2 minutes. The cells were then cultured for an additional 48 hours at 27°C under steady-state conditions. All experimental and control designs were analyzed in triplicate.

[00557] After 48 hours, the 96-well plate was removed from the incubator, allowed to equilibrate briefly to room temperature, and luciferase expression was analyzed (OneGlo Luciferase Assay (Promega Corporation)). Total luminescence was measured using a SpectraMax M Series microplate reader. Data for replicates were averaged. Luciferase activity for the symmetric mutant ITR constructs from Table 7 is shown in FIG. 23. As expected, three negative control samples (medium only, mock transfection without donor DNA, sample that was treated in the absence of Rep-containing baculovirus cells) showed no significant luciferase expression. Robust luciferase expression was observed in each of the mutant samples, indicating that for each sample, transfection with the cDNA-encoded transgene was successful and it was expressed regardless of the mutation.

EXAMPLE 9: In vivo assessment of luciferase expression from cccDNA constructs with symmetric mutant ITRs

CD-1 mice (N=30, male, approximately 4 weeks old) were treated with various types of cccDNA produced in Sf9, as well as the synthetic routes described above. Specifically, the in vivo expression of a cccDNA construct containing mutant ITRs on both the left and right sides in a symmetrical configuration (Construct-388) (see FIG. 27; left panel) was compared with that of a cccDNA construct containing wild-type AAV ITR both on the left and on the right side (Design-393) (see Fig. 27; right panel). These constructs were produced in Sf9 cells as described above and introduced into LNP. In addition, cccDNAs produced in Sf9 containing the wild-type asymmetric AAV2ITR ITR configuration on the left side and a truncated mutant ITR on the right side of the construct were also incorporated into the LNP and used as a control. In addition, synthetically produced cccDNAs containing either a symmetric or asymmetric ITR configuration were also tested for expression levels.

Animals in cages were examined daily; clinical observations were made at 1, 6 and 24 hours after dosing. The body weight of all animals was recorded at specified time points (see Fig. 28), cctDNA was administered at a dose of 5 ml/kg on day 0 by intravenous injection through the lateral tail vein. Animals received a dose of luciferin 150 mg/kg (60 mg/ml) by intraperitoneal (i.p.) injection in a volume of 2.5 ml/kg. IVIS imaging and measurements were performed in all animals within 15 minutes of luciferin administration. As shown in FIG. 28, the percentage change in body weight in animals dosed with construct-388 was similar to the percentage in animals treated with construct-393 on day 6 (Fig. 28). The IVIS measurement result, which reflects luciferase expression levels, is presented in FIG. 29A and FIG. 29V. Surprisingly, in vivo expression of cccDNA containing mutant ITRs on the left and right sides of the construct (i.e., construct-388) was observed at levels similar to those observed for the cccDNA construct containing the wild-type AAV ITR on both the left. and on the right side of the structure (i.e., structure-393) (see Figs. 29A and 29B). These data suggest that an intact ITR may not be required for functional localization and expression of cccDNA. Additionally, synthetic cccDNA produced by the cell-free methods described herein also exhibited robust expression levels as expected (see FIG. 29A).

SOURCES

All sources listed and disclosed herein and in the examples, including patents, patent applications, international patent applications and publications are incorporated herein in their entirety by reference.

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LIST OF SEQUENCES

<110> GENERATION BIO CO.

<120> MODIFIED CLOSED-END DNA (ccDNA) CONTAINING

SYMMETRICAL

MODIFIED INVERTED TERMINAL REPEATS

<130> 131698-05320

<140> PCT/US2019/060395

<141> 2019-11-08

<150> 62/757872

<151> 2018-11-09

<150> 62/757,892

<151> 2018-11-09

<160> 574

<170> PatentIn, version 3.5

<210> 1

<211> 141

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 1

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc

120

gagcgcgcag ctgcctgcag g

141

<210> 2

<211> 130

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 2

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc

120

tgcctgcagg

130

<210> 3

<211> 1923

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 3

tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta

60

ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc

120

aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg

180

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc

240

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat

300

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc

360

ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga

420

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg

480

gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg

540

cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta

600

attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg

660

gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg

720

cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg

780

aagcgcgcgg cgggcgggag tcgctgcgac gctgccttcg ccccgtgccc cgctccgccg

840

ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc

900

gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt

960

ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc gggggggagc

1020

ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc

1080

ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg

1140

aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag

1200

gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg gcggtcgggc

1260

tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc ttcgggtgcg

1320

gggctccgta cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg

1380

ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga ggggcgcggc

1440

ggcccccgga gcgccggcgg ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg

1500

taatcgtgcg agagggcgca gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct

1560

gggaggcgcc gccgcacccc ctctagcggg cgcggggcga agcggtgcgg cgccggcagg

1620

aaggaaatgg gcggggaggg ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc

1680

cagcctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg

1740

ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tttagccttc

1800

ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcatttgtcg

1860

acagaattcc tcgaagatcc gaaggggttc aagcttggca ttccggtact gttggtaaag

1920

cca

1923

<210> 4

<211> 1272

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 4

aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc

60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc

120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc

180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc

240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt

300

ggtttaggta gtgtgagagg gtccgggttc aaaaccactt gctgggtggg gagtcgtcag

360

taagtggcta tgccccgacc ccgaagcctg tttccccatc tgtacaatgg aaatgataaa

420

gacgcccatc tgatagggtt tttgtggcaa ataaacattt ggtttttttg ttttgttttg

480

ttttgttttt tgagatggag gtttgctctg tcgcccaggc tggagtgcag tgacacaatc

540

tcatctcacc acaaccttcc cctgcctcag cctcccaagt agctgggatt acaagcatgt

600

gccaccacac ctggctaatt ttctattttt agtagagacg ggtttctcca tgttggtcag

660

cctcagcctc ccaagtaact gggattacag gcctgtgcca ccacacccgg ctaatttttt

720

ctatttttga cagggacggg gtttcaccat gttggtcagg ctggtctaga ggtaccggat

780

cttgctacca gtggaacagc cactaaggat tctgcagtga gagcagaggg ccagctaagt

840

ggtactctcc cagagactgt ctgactcacg ccaccccctc caccttggac acaggacgct

900

gtggtttctg agccaggtac aatgactcct ttcggtaagt gcagtggaag ctgtacactg

960

cccaggcaaa gcgtccgggc agcgtaggcg ggcgactcag atcccagcca gtggacttag

1020

cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca gcagcctccc

1080

ccgttgcccc tctggatcca ctgcttaaat acggacgagg acagggccct gtctcctcag

1140

cttcaggcac caccactgac ctgggacagt gaatccggac tctaaggtaa atataaaatt

1200

tttaagtgta taatgtgtta aactactgat tctaattgtt tctctctttt agattccaac

1260

ctttggaact ga

1272

<210> 5

<211> 547

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 5

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc

60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag

180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag

240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct

300

gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt

360

gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca

420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa

480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg

540

gaactga

547

<210> 6

<211> 1179

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 6

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg

60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt

120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca

180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc

240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt

300

acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg

360

gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg

420

cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct

480

ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg

540

caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc

600

gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga

660

gcgcggccac cgagaatcgg acggggggtag tctcaagctg gccggcctgc tctggtgcct

720

ggtctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca

780

gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg

840

acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg

900

tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat

960

tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg

1020

gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa

1080

ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca

1140

gtggttcaaa gtttttttct tccatttcag gtgtcgtga

1179

<210> 7

<211> 8

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 7

gtttaaac

8

<210> 8

<211> 581

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 8

gagcatctta ccgccattta ttcccatatt tgttctgttt ttcttgattt gggtatacat

60

ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg atatgtaatt

120

actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg ttatttacgc

180

tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tgatattctt

240

aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct gtatctagct

300

attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt gctgtctctt

360

ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac

420

gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg gactttcgct

480

ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg ctgctggaca

540

ggggctaggt tgctgggcac tgataattcc gtggtgttgt c

581

<210> 9

<211> 225

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 9

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct

60

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct

120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg

180

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggc

225

<210> 10

<211> 213

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 10

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta

60

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag

120

ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt

180

tttaaagcaa gtaaaacctc tacaaatgtg gta

213

<210> 11

<211> 1260

<212> DNA/DNA

<213> Adeno-associated virus - 2

<400> 11

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag

60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag

120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg

180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc

300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg

360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt

420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc

480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa

540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg

600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg

660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag

720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc

780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag

840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc

900

aactacgcag acaggtacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg

960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga

1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc

1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataaatgatt

1200

taaatcaggt atggctgccg atggttatct tccagattgg ctcgaggaca ctctctctga

1260

<210> 12

<211> 1932

<212> DNA/DNA

<213> Adeno-associated virus - 2

<400> 12

atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc

60

ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat

120

tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag

180

cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct tttctttgtg

240

caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg

300

aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt

360

taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc

420

gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa

480

acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg

540

aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag

600

gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact

660

tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag

720

cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg

780

tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc

840

cccgactacc tggtggggcca gcagcccgtg gaggacatt ccagcaatcg gatttataaa

900

attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc

960

acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag

1020

accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc

1080

aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg

1140

aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc

1200

gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc

1260

aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg

1320

ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag

1380

gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg

1440

gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca

1500

gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg

1560

gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg

1620

aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc

1680

ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt

1740

tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg

1800

cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa

1860

caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga

1920

cactctctctga

1932

<210> 13

<211> 1876

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 13

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacgg

60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt

120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga

180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct

240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac

300

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat

360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac

420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt

480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag

540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc

600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag

660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac

720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc

780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac

840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg

900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct

960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac

1020

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt

1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg

1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag

1200

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca

1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga

1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt

1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc

1500

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca

1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc

1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc

1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat

1800

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg

1860

catctttgaa caataa

1876

<210> 14

<211> 1194

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 14

atggagctgg tcgggtggct cgtggacaag gggattacct cggagaagca gtggatccag

60

gaggaccagg cctcatacat ctccttcaat gcggcctcca actcgcggtc ccaaatcaag

120

gctgccttgg acaatgcggg aaagattatg agcctgacta aaaccgcccc cgactacctg

180

gtgggccagc agcccgtgga ggacatttcc agcaatcgga tttataaaat tttggaacta

240

aacgggtacg atccccaata tgcggcttcc gtctttctgg gatgggccac gaaaaagttc

300

ggcaagagga acaccatctg gctgtttggg cctgcaacta ccgggaagac caacatcgcg

360

gaggccatag cccacactgt gcccttctac gggtgcgtaa actggaccaa tgagaacttt

420

cccttcaacg actgtgtcga caagatggtg atctggtggg aggaggggaa gatgaccgcc

480

aaggtcgtgg agtcggccaa agccattctc ggaggaagca aggtgcgcgt ggaccagaaa

540

tgcaagtcct cggcccagat agacccgact cccgtgatcg tcacctccaa caccaacatg

600

tgcgccgtga ttgacgggaa ctcaacgacc ttcgaacacc agcagccgtt gcaagaccgg

660

atgttcaaat ttgaactcac ccgccgtctg gatcatgact ttgggaaggt caccaagcag

720

gaagtcaaag actttttccg gtgggcaaag gatcacgtgg ttgaggtgga gcatgaattc

780

tacgtcaaaa agggtggagc caagaaaaga cccgccccca gtgacgcaga tataagtgag

840

cccaaacggg tgcgcgagtc agttgcgcag ccatcgacgt cagacgcgga agcttcgatc

900

aactacgcag accgctacca aaacaaatgt tctcgtcacg tgggcatgaa tctgatgctg

960

tttccctgca gacaatgcga gagaatgaat cagaattcaa atatctgctt cactcacgga

1020

cagaaagact gtttagagtg ctttcccgtg tcagaatctc aacccgtttc tgtcgtcaaa

1080

aaggcgtatc agaaactgtg ctacattcat catatcatgg gaaaggtgcc agacgcttgc

1140

actgcctgcg atctggtcaa tgtggatttg gatgactgca tctttgaaca ataa

1194

<210> 15

<211> 141

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 15

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt

60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc

120

tgcggcgcgc gcagcacctt t

141

<210> 16

<211> 556

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 16

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc

60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg tggtttaggt

180

agtgtgagag gggaatgact cctttcggta agtgcagtgg aagctgtaca ctgcccaggc

240

aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact tagcccctgt

300

ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc

360

ccctctggat ccactgctta aatacggacg aggacactcg agggccctgt ctcctcagct

420

tcaggcacca ccactgacct gggacagtga atccggacat cgattctaag gtaaatataa

480

aatttttaag tgtataattt gttaaactac tgattctaat tgtttctctc ttttagattc

540

caacctttgg aactga

556

<210> 17

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 17

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 18

<211> 241

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 18

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag

60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga

120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat

180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga

240

c

241

<210> 19

<211> 215

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 19

gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa

60

cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc

120

tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg

180

gatttgggaa tcgtataaga actgtatgag accac

215

<210> 20

<211> 150

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 20

ataaacgata acgccgttgg tggcgtgagg catgtaaaag gttacatcat tatcttgttc

60

gccatccggt tggtataaat agacgttcat gttggttttt gtttcagttg caagttggct

120

gcggcgcgcg cagcaccttt gcggccatct

150

<210> 21

<211> 546

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 21

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc

60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc

120

cactcgaccc cttggaattt ttcggtggag aggagcagag gttgtcctgg cgtggtttag

180

gtagtgtgag aggggaatga ctcctttcgg taagtgcagt ggaagctgta cactgcccag

240

gcaaagcgtc cgggcagcgt aggcgggcga ctcagatccc agccagtgga cttagcccct

300

gtttgctcct ccgataactg gggtgacctt ggttaatatt caccagcagc ctcccccgtt

360

gcccctctgg atccactgct taaatacgga cgaggacagg gccctgtctc ctcagcttca

420

ggcaccacca ctgacctggg acagtgaatc cggactctaa ggtaaatata aaatttttaa

480

gtgtataatg tgttaaacta ctgattctaa ttgtttctct cttttagatt ccaacctttg

540

gaactg

546

<210> 22

<211> 317

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 22

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt

60

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca

120

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa

180

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag

240

aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag

300

gcctaggctt ttgcaaa

317

<210> 23

<211> 576

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 23

tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa

60

cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata

120

atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag

180

tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc

240

cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta

300

tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg

360

cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt

420

ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca

480

aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag

540

gtctatataa gcagagctgg tttagtgaac cgtcag

576

<210> 24

<211> 1313

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 24

ggagccgaga gtaattcata caaaaggagg gatcgccttc gcaaggggag agcccaggga

60

ccgtccctaa attctcacag acccaaatcc ctgtagccgc cccacgacag cgcgaggagc

120

atgcgcccag ggctgagcgc gggtagatca gagcacacaa gctcacagtc cccggcggtg

180

gggggagggg cgcgctgagc gggggccagg gagctggcgc ggggcaaact gggaaagtgg

240

tgtcgtgtgc tggctccgcc ctcttcccga gggtggggga gaacggtata taagtgcggt

300

agtcgccttg gacgttcttt ttcgcaacgg gtttgccgtc agaacgcagg tgagtggcgg

360

gtgtggcttc cgcgggcccc ggagctggag ccctgctctg agcgggccgg gctgatatgc

420

gagtgtcgtc cgcagggttt agctgtgagc attcccactt cgagtggcgg gcggtgcggg

480

ggtgagagtg cgaggcctag cggcaacccc gtagcctcgc ctcgtgtccg gcttgaggcc

540

tagcgtggtg tccgccgccg cgtgccactc cggccgcact atgcgttttt tgtccttgct

600

gccctcgatt gccttccagc agcatgggct aacaaaggga gggtgtgggg ctcactctta

660

aggagcccat gaagcttacg ttggatagga atggaagggc aggaggggcg actggggccc

720

gcccgccttc ggagcacatg tccgacgcca cctggatggg gcgaggcctg tggctttccg

780

aagcaatcgg gcgtgagttt agcctacctg ggccatgtgg ccctagcact gggcacggtc

840

tggcctggcg gtgccgcgtt cccttgcctc ccaacaaggg tgaggccgtc ccgcccggca

900

ccagttgctt gcgcggaaag atggccgctc ccggggccct gttgcaagga gctcaaaatg

960

gaggacgcgg cagcccggtg gagcgggcgg gtgagtcacc cacacaaagg aagagggcct

1020

tgcccctcgc cggccgctgc ttcctgtgac cccgtggtct atcggccgca tagtcacctc

1080

gggcttctct tgagcaccgc tcgtcgcggc ggggggaggg gatctaatgg cgttggagtt

1140

tgttcacatt tggtgggtgg agactagtca ggccagcctg gcgctggaag tcattcttgg

1200

aatttgcccc tttgagtttg gagcgaggct aattctcaag cctcttagcg gttcaaaggt

1260

attttctaaa cccgtttcca ggtgttgtga aagccaccgc taattcaaag caa

1313

<210> 25

<211> 19

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 25

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 26

<211> 19

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 26

Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala

1 5 10 15

Ser Arg Gly

<210> 27

<211> 7

<212> PRT/Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

peptide

<400> 27

Pro Lys Lys Lys Arg Lys Val

15

<210> 28

<400> 28

000

<210> 29

<400> 29

000

<210> 30

<400> 30

000

<210> 31

<400> 31

000

<210> 32

<400> 32

000

<210> 33

<400> 33

000

<210> 34

<400> 34

000

<210> 35

<400> 35

000

<210> 36

<400> 36

000

<210> 37

<400> 37

000

<210> 38

<400> 38

000

<210> 39

<400> 39

000

<210> 40

<400> 40

000

<210> 41

<400> 41

000

<210> 42

<400> 42

000

<210> 43

<400> 43

000

<210> 44

<400> 44

000

<210> 45

<211> 6

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 45

ggttga

6

<210> 46

<211> 4

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 46

agtt

4

<210> 47

<211> 6

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 47

ggttgg

6

<210> 48

<211> 6

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 48

agttgg

6

<210> 49

<211> 6

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 49

agttga

6

<210> 50

<211> 6

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 50

rrttrr

6

<210> 51

<211> 141

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 51

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca

120

actccatcac taggggttcc t

141

<210> 52

<211> 130

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 52

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt

60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact

120

aggggttcct

130

<210> 53

<211> 3123

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 53

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcc

60

cgggcgcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg

120

gttcctgaac agagaaacag gagaatatgg gccaaacagg atatctgtgg taagcagttc

180

ctgccccggc tcagggccaa gaacagttgg aacagcagaa tatgggccaa acaggatatc

240

tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gatggtcccc agatgcggtc

300

ccgccctcag cagtttctag agaaccatca gatgtttcca gggtgcccca aggacctgaa

360

atgaccctgt gccttatttg aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc

420

ttctgctccc cgagctctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga

480

gacgccatcc acgctgtttt gacttccata gaaggccgcc accatggaag acgccaaaaa

540

cataaagaaa ggcccggcgc cattctatcc gctggaagat ggaaccgctg gagagcaact

600

gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta cagatgcaca

660

tatcgaggtg gacatcactt acgctgagta cttcgaaatg tccgttcggt tggcagaagc

720

tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg aaaactctct

780

tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg cgcccgcgaa

840

cgacatttat aatgaacgtg aattgctcaa cagtatgggc atttcgcagc ctaccgtggt

900

gttcgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc tcccaatcat

960

ccaaaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt cgatgtacac

1020

gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtgc cagagtcctt

1080

cgatagggac aagacaattg cactgatcat gaactcctct ggatctactg gtctgcctaa

1140

aggtgtcgct ctgcctcata gaactgcctg cgtgagattc tcgcatgcca gagatcctat

1200

ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat tccatcacgg

1260

ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg tcttaatgta

1320

tagatttgaa gaagagctgt ttctgaggag ccttcaggat tacaagattc aaagtgcgct

1380

gctggtgcca accctattct ccttcttcgc caaaagcact ctgattgaca aatacgattt

1440

atctaattta cacgaaattg cttctggtgg cgctcccctc tctaaggaag tcggggaagc

1500

ggttgccaag aggttccatc tgccaggtat caggcaagga tatgggctca ctgagactac

1560

atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg gtaaagttgt

1620

tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg gcgttaatca

1680

aagaggcgaa ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa acaatccgga

1740

agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca tagcttactg

1800

ggacgaagac gaacacttct tcatcgttga ccgcctgaag tctctgatta agtacaaagg

1860

ctatcaggtg gctcccgctg aattggaatc catcttgctc caacacccca acatcttcga

1920

cgcaggtgtc gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg ccgttgttgt

1980

tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg ccagtcaagt

2040

aacaaccgcg aaaaagttgc gcggagggagt tgtgtttgtg gacgaagtac cgaaaggtct

2100

taccggaaaa ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca agaagggcgg

2160

aaagatcgcc gtgtaagagc atcttaccgc catttattcc catatttgtt ctgtttttct

2220

tgatttgggt atacatttaa atgttaataa aacaaaatgg tggggcaatc atttacattt

2280

ttagggatat gtaattacta gttcaggtgt attgccacaa gacaaacatg ttaagaaact

2340

ttcccgttat ttacgctctg ttcctgttaa tcaacctctg gattacaaaa tttgtgaaag

2400

attgactgat attcttaact atgttgctcc ttttacgctg tgtggatatg ctgcttttata

2460

gcctctgtat ctagctattg cttcccgtac ggctttcgtt ttctcctcct tgtataaatc

2520

ctggttgctg tctcttttag aggagttgtg gcccgttgtc cgtcaacgtg gcgtggtgtg

2580

ctctgtgttt gctgacgcaa cccccactgg ctggggcatt gccaccacct gtcaactcct

2640

ttctgggact ttcgctttcc ccctcccgat cgccacggca gaactcatcg ccgcctgcct

2700

tgcccgctgc tggacagggg ctaggttgct gggcactgat aattccgtgg tgttgtctgt

2760

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga

2820

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag

2880

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga

2940

agacaatagc aggcatgctg gggatgcggt gggctctatg gcaggaaccc ctagtgatgg

3000

agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg

3060

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc

3120

agg

3123

<210> 54

<211> 3117

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 54

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca

120

actccatcac taggggttcc tgaacagaga aacaggaa tatgggccaa acaggatatc

180

tgtggtaagc agttcctgcc ccggctcagg gccaagaaca gttggaacag cagaatatgg

240

gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg

300

tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg

360

ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg

420

cttctgttcg cgcgcttctg ctccccgagc tctatataag cagagctcgt ttagtgaacc

480

gtcagatcgc ctggagacgc catccacgct gttttgactt ccatagaagg ccgccaccat

540

ggaagacgcc aaaaacataa agaaaggccc ggcgccattc tatccgctgg aagatggaac

600

cgctggagag caactgcata aggctatgaa gagatacgcc ctggttcctg gaacaattgc

660

ttttacagat gcacatatcg aggtggacat cacttacgct gagtacttcg aaatgtccgt

720

tcggttggca gaagctatga aacgatatgg gctgaataca aatcacagaa tcgtcgtatg

780

cagtgaaaac tctcttcaat tctttatgcc ggtgttgggc gcgttattta tcggagttgc

840

agttgcgccc gcgaacgaca tttataatga acgtgaattg ctcaacagta tgggcatttc

900

gcagcctacc gtggtgttcg tttccaaaaa ggggttgcaa aaaattttga acgtgcaaaa

960

aaagctccca atcatccaaa aaattattat catggattct aaaacggatt accagggatt

1020

tcagtcgatg tacacgttcg tcacatctca tctacctccc ggttttaatg aatacgattt

1080

tgtgccagag tccttcgata gggacaagac aattgcactg atcatgaact cctctggatc

1140

tactggtctg cctaaaggtg tcgctctgcc tcatagaact gcctgcgtga gattctcgca

1200

tgccagagat cctatttttg gcaatcaaat cattccggat actgcgattt taagtgttgt

1260

tccattccat cacggttttg gaatgtttac tacactcgga tatttgatat gtggatttcg

1320

agtcgtctta atgtatagat ttgaagaaga gctgtttctg aggagccttc aggattacaa

1380

gattcaaagt gcgctgctgg tgccaaccct attctccttc ttcgccaaaa gcactctgat

1440

tgacaaatac gatttatcta atttacacga aattgcttct ggtggcgctc ccctctctaa

1500

ggaagtcggg gaagcggttg ccaagaggtt ccatctgcca ggtatcaggc aaggatatgg

1560

gctcactgag actacatcag ctattctgat tacacccgag ggggatgata aaccgggcgc

1620

ggtcggtaaa gttgttccat tttttgaagc gaaggttgtg gatctggata ccgggaaaac

1680

gctgggcgtt aatcaaagag gcgaactgtg tgtgagaggt cctatgatta tgtccggtta

1740

tgtaaacaat ccggaagcga ccaacgcctt gattgacaag gatggatggc tacattctgg

1800

agacatagct tactgggacg aagacgaaca cttcttcatc gttgaccgcc tgaagtctct

1860

gattaagtac aaaggctatc aggtggctcc cgctgaattg gaatccatct tgctccaaca

1920

ccccaacatc ttcgacgcag gtgtcgcagg tcttcccgac gatgacgccg gtgaacttcc

1980

cgccgccgtt gttgttttgg agcacggaaa gacgatgacg gaaaaagaga tcgtggatta

2040

cgtcgccagt caagtaacaa ccgcgaaaaa gttgcgcgga ggagttgtgt ttgtggacga

2100

agtaccgaaa ggtcttaccg gaaaactcga cgcaagaaaa atcagagaga tcctcataaa

2160

ggccaagaag ggcggaaaga tcgccgtgta agagcatctt accgccattt attcccatat

2220

ttgttctgtt tttcttgatt tgggtataca tttaaatgtt aataaaacaa aatggtgggg

2280

caatcattta catttttagg gatatgtaat tactagttca ggtgtattgc cacaagacaa

2340

acatgttaag aaactttccc gttatttacg ctctgttcct gttaatcaac ctctggatta

2400

caaaatttgt gaaagattga ctgatattct taactatgtt gctcctttta cgctgtgtgg

2460

atatgctgct ttatagcctc tgtatctagc tattgcttcc cgtacggctt tcgttttctc

2520

ctccttgtat aaatcctggt tgctgtctct ttttagaggag ttgtggcccg ttgtccgtca

2580

acgtggcgtg gtgtgctctg tgtttgctga cgcaaccccc actggctggg gcattgccac

2640

cacctgtcaa ctcctttctg ggactttcgc tttccccctc ccgatcgcca cggcagaact

2700

catcgccgcc tgccttgccc gctgctggac aggggctagg ttgctgggca ctgataattc

2760

cgtggtgttg tctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc

2820

ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc

2880

atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa

2940

gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcagg

3000

aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg

3060

cccgggaaac ccgggcgtgc gcctcagtga gcgagcgagc gcgcagctgc ctgcagg

3117

<210> 55

<400> 55

000

<210> 56

<400> 56

000

<210> 57

<211> 3912

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 57

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcc

60

cgggcgcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg

120

gttcctggct cagaggctca gaggcacaca ggagtttctg ggctcaccct gcccccttcc

180

aacccctcag ttcccatcct ccagcagctg tttgtgtgct gcctctgaag tccacactga

240

acaaacttca gcctactcat gtccctaaaa tgggcaaaca ttgcaagcag caaacagcaa

300

acacacagcc ctccctgcct gctgaccttg gagctggggc agaggtcaga gacctctctg

360

ggcccatgcc acctccaaca tccactcgac cccttggaat ttcggtggag aggagcagag

420

gttgtcctgg cgtggtttag gtagtgtgag agggtccggg ttcaaaacca cttgctgggt

480

ggggagtcgt cagtaagtgg ctatgccccg accccgaagc ctgtttcccc atctgtacaa

540

tggaaatgat aaagacgccc atctgatagg gtttttgtgg caaataaaca tttggttttt

600

ttgttttgtt ttgttttgtt ttttgagatg gaggtttgct ctgtcgccca ggctggagtg

660

cagtgacaca atctcatctc accacaacct tcccctgcct cagcctccca agtagctggg

720

attacaagca tgtgccacca cacctggcta attttctatt tttagtagag acgggtttct

780

ccatgttggt cagcctcagc ctcccaagta actgggatta caggcctgtg ccaccacacc

840

cggctaattt tttctatttt tgacagggac ggggtttcac catgttggtc aggctggtct

900

agaggtaccg gatcttgcta ccagtggaac agccactaag gattctgcag tgagagcaga

960

gggccagcta agtggtactc tcccagagac tgtctgactc acgccacccc ctccaccttg

1020

gacacaggac gctgtggttt ctgagccagg tacaatgact cctttcggta agtgcagtgg

1080

aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag gcgggcgact cagatcccag

1140

ccagtggact tagcccctgt ttgctcctcc gataactggg gtgaccttgg ttaatattca

1200

ccagcagcct cccccgttgc ccctctggat ccactgctta aatacggacg aggacagggc

1260

cctgtctcct cagcttcagg caccaccact gacctgggac agtgccgcca ccatggaaga

1320

cgccaaaaac ataaagaaag gcccggcgcc attctatccg ctggaagatg gaaccgctgg

1380

agagcaactg cataaggcta tgaagata cgccctggtt cctggaacaa ttgcttttac

1440

agatgcacat atcgaggtgg acatcactta cgctgagtac ttcgaaatgt ccgttcggtt

1500

ggcagaagct atgaaacgat atgggctgaa tacaaatcac agaatcgtcg tatgcagtga

1560

aaactctctt caattcttta tgccggtgtt gggcgcgtta tttatcggag ttgcagttgc

1620

gcccgcgaac gacatttata atgaacgtga attgctcaac agtatgggca tttcgcagcc

1680

taccgtggtg ttcgtttcca aaaaggggtt gcaaaaaatt ttgaacgtgc aaaaaaagct

1740

cccaatcatc caaaaaatta ttatcatgga ttctaaaacg gattaccagg gatttcagtc

1800

gatgtacacg ttcgtcacat ctcatctacc tcccggtttt aatgaatacg attttgtgcc

1860

agagtccttc gatagggaca agacaattgc actgatcatg aactcctctg gatctactgg

1920

tctgcctaaa ggtgtcgctc tgcctcatag aactgcctgc gtgagattct cgcatgccag

1980

agatcctatt tttggcaatc aaatcattcc ggatactgcg attttaagtg ttgttccatt

2040

ccatcacggt tttggaatgt ttactacact cggatatttg atatgtggat ttcgagtcgt

2100

cttaatgtat agatttgaag aagagctgtt tctgaggagc cttcaggatt acaagattca

2160

aagtgcgctg ctggtgccaa ccctattctc cttcttcgcc aaaagcactc tgattgacaa

2220

atacgattta tctaatttac acgaaattgc ttctggtggc gctcccctct ctaaggaagt

2280

cggggaagcg gttgccaaga ggttccatct gccaggtatc aggcaaggat atgggctcac

2340

tgagactaca tcagctattc tgattacacc cgagggggat gataaaccgg gcgcggtcgg

2400

taaagttgtt ccattttttg aagcgaaggt tgtggatctg gataccggga aaacgctggg

2460

cgttaatcaa agaggcgaac tgtgtgtgag aggtcctatg attatgtccg gttatgtaaa

2520

caatccggaa gcgaccaacg ccttgattga caaggatgga tggctacatt ctggagacat

2580

agcttactgg gacgaagacg aacacttctt catcgttgac cgcctgaagt ctctgattaa

2640

gtacaaaggc tatcaggtgg ctcccgctga attggaatcc atcttgctcc aacaccccaa

2700

catcttcgac gcaggtgtcg caggtcttcc cgacgatgac gccggtgaac ttcccgccgc

2760

cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa gagatcgtgg attacgtcgc

2820

cagtcaagta acaaccgcga aaaagttgcg cggagggagtt gtgtttgtgg acgaagtacc

2880

gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga gagatcctca taaaggccaa

2940

gaagggcgga aagatcgccg tgtaagagca tcttaccgcc atttattccc atatttgttc

3000

tgtttttctt gatttgggta tacatttaaa tgttaataaa acaaaatggt ggggcaatca

3060

tttacatttt tagggatatg taattactag ttcaggtgta ttgccacaag acaaacatgt

3120

taagaaactt tcccgttatt tacgctctgt tcctgttaat caacctctgg attacaaaat

3180

ttgtgaaaga ttgactgata ttcttaacta tgttgctcct tttacgctgt gtggatatgc

3240

tgctttatag cctctgtatc tagctattgc ttcccgtacg gctttcgttt tctcctcctt

3300

gtataaatcc tggttgctgt ctcttttaga ggagttgtgg cccgttgtcc gtcaacgtgg

3360

cgtggtgtgc tctgtgtttg ctgacgcaac ccccactggc tggggcattg ccaccacctg

3420

tcaactcctt tctgggactt tcgctttccc cctcccgatc gccacggcag aactcatcgc

3480

cgcctgcctt gcccgctgct ggacaggggc taggttgctg ggcactgata attccgtggt

3540

gttgtctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt

3600

gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca

3660

ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga

3720

ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg caggaacccc

3780

tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac

3840

caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca

3900

gctgcctgca gg

3912

<210> 58

<211> 3906

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 58

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca

120

actccatcac taggggttcc tggctcagag gctcagaggc acacaggagt ttctgggctc

180

accctgcccc cttccaaccc ctcagttccc atcctccagc agctgtttgt gtgctgcctc

240

tgaagtccac actgaacaaa cttcagccta ctcatgtccc taaaatgggc aaacattgca

300

agcagcaaac agcaaacaca cagccctccc tgcctgctga ccttggagct ggggcagagg

360

tcagagacct ctctgggccc atgccacctc caacatccac tcgacccctt ggaatttcgg

420

tggagaggag cagaggttgt cctggcgtgg tttaggtagt gtgagagggt ccgggttcaa

480

aaccacttgc tgggtgggga gtcgtcagta agtggctatg ccccgacccc gaagcctgtt

540

tccccatctg tacaatggaa atgataaaga cgcccatctg atagggtttt tgtggcaaat

600

aaacatttgg tttttttgtt ttgttttgtt ttgttttttg agatggaggt ttgctctgtc

660

gcccaggctg gagtgcagtg acacaatctc atctcaccac aaccttcccc tgcctcagcc

720

tcccaagtag ctgggattac aagcatgtgc caccacacct ggctaatttt ctatttttag

780

tagagacggg tttctccatg ttggtcagcc tcagcctccc aagtaactgg gattacaggc

840

ctgtgccacc acacccggct aattttttct atttttgaca gggacggggt ttcaccatgt

900

tggtcaggct ggtctagagg taccggatct tgctaccagt ggaacagcca ctaaggattc

960

tgcagtgaga gcagagggcc agctaagtgg tactctccca gagactgtct gactcacgcc

1020

accccctcca ccttggacac aggacgctgt ggtttctgag ccaggtacaa tgactccttt

1080

cggtaagtgc agtggaagct gtacactgcc caggcaaagc gtccgggcag cgtaggcggg

1140

cgactcagat cccagccagt ggacttagcc cctgtttgct cctccgataa ctggggtgac

1200

cttggttaat attcaccagc agcctccccc gttgcccctc tggatccact gcttaaatac

1260

ggacgaggac agggccctgt ctcctcagct tcaggcacca ccactgacct gggacagtgc

1320

cgccaccatg gaagacgcca aaaacataaa gaaaggcccg gcgccattct atccgctgga

1380

agatggaacc gctggagagc aactgcataa ggctatgaag agatacgccc tggttcctgg

1440

aacaattgct tttacagatg cacatatcga ggtggacatc acttacgctg agtacttcga

1500

aatgtccgtt cggttggcag aagctatgaa acgatatggg ctgaatacaa atcacagaat

1560

cgtcgtatgc agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat

1620

cggagttgca gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat

1680

gggcatttcg cagcctaccg tggtgttcgt ttccaaaaag gggttgcaaa aaattttgaa

1740

cgtgcaaaaa aagctcccaa tcatccaaaa aattattatc atggattcta aaacggatta

1800

ccagggattt cagtcgatgt acacgttcgt cacatctcat ctacctcccg gttttaatga

1860

atacgatttt gtgccagagt ccttcgatag ggacaagaca attgcactga tcatgaactc

1920

ctctggatct actggtctgc ctaaaggtgt cgctctgcct catagaactg cctgcgtgag

1980

attctcgcat gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt

2040

aagtgttgtt ccattccatc acggttttgg aatgtttact acactcggat atttgatatg

2100

tggatttcga gtcgtcttaa tgtatagatt tgaagaagag ctgtttctga ggagccttca

2160

ggattacaag attcaaagtg cgctgctggt gccaacccta ttctccttct tcgccaaaag

2220

cactctgatt gacaaatacg atttatctaa tttacacgaa attgcttctg gtggcgctcc

2280

cctctctaag gaagtcgggg aagcggttgc caagaggttc catctgccag gtatcaggca

2340

aggatatggg ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa

2400

accgggcgcg gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac

2460

cgggaaaacg ctgggcgtta atcaaagagg cgaactgtgt gtgagaggtc ctatgattat

2520

gtccggttat gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct

2580

acattctgga gacatagctt actgggacga agacgaacac ttcttcatcg ttgaccgcct

2640

gaagtctctg attaagtaca aaggctatca ggtggctccc gctgaattgg aatccatctt

2700

gctccaacac cccaacatct tcgacgcagg tgtcgcaggt cttcccgacg atgacgccgg

2760

tgaacttccc gccgccgttg ttgttttgga gcacggaaag acgatgacgg aaaaagagat

2820

cgtggattac gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt

2880

tgtggacgaa gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat

2940

cctcataaag gccaagaagg gcggaaagat cgccgtgtaa gagcatctta ccgccattta

3000

ttcccatatt tgttctgttt ttcttgattt gggtatacat ttaaatgtta ataaaacaaa

3060

atggtggggc aatcatttac atttttaggg atatgtaatt actagttcag gtgtattgcc

3120

acaagacaaa catgttaaga aactttcccg ttatttacgc tctgttcctg ttaatcaacc

3180

tctggattac aaaatttgtg aaagattgac tgatattctt aactatgttg ctccttttac

3240

gctgtgtgga tatgctgctt tatagcctct gtatctagct attgcttccc gtacggcttt

3300

cgttttctcc tccttgtata aatcctggtt gctgtctctt ttagaggagt tgtggcccgt

3360

tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac gcaaccccca ctggctgggg

3420

cattgccacc acctgtcaac tcctttctgg gactttcgct ttccccctcc cgatcgccac

3480

ggcagaactc atcgccgcct gccttgcccg ctgctggaca ggggctaggt tgctgggcac

3540

tgataattcc gtggtgttgt ctgtgccttc tagttgccag ccatctgttg tttgcccctc

3600

ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga

3660

ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca

3720

ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc

3780

tatggcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca

3840

ctgaggccgc ccgggaaacc cgggcgtgcg cctcagtgag cgagcgagcg cgcagctgcc

3900

tgcagg

3906

<210> 59

<400> 59

000

<210> 60

<400> 60

000

<210> 61

<400> 61

000

<210> 62

<400> 62

000

<210> 63

<211> 126

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 63

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcc

60

cgggcgcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg

120

gttcct

126

<210> 64

<211> 120

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 64

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgcccggga aacccgggcg tgcgcctcag tgagcgagcg agcgcgcagc tgcctgcagg

120

<210> 65

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 65

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct

60

cagtgagcga gcgagcgcgc

80

<210> 66

<211> 141

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 66

aataaacgat aacgccgttg gtggcgtgag gcatgtaaaa ggttacatca ttatcttgtt

60

cgccatccgg ttggtataaa tagacgttca tgttggtttt tgtttcagtt gcaagttggc

120

tgcggcgcgc gcagcacctt t

141

<210> 67

<211> 1876

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 67

cgcagccacc atggcggggt tttacgagat tgtgattaag gtccccagcg accttgacga

60

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt

120

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga

180

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct

240

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac

300

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat

360

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac

420

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt

480

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag

540

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc

600

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag

660

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac

720

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc

780

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac

840

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg

900

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct

960

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac

1020

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt

1080

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg

1140

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag

1200

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat

1260

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca

1320

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga

1380

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt

1440

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc

1500

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac

1560

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca

1620

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc

1680

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc

1740

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat

1800

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg

1860

catctttgaa caataa

1876

<210> 68

<211> 129

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 68

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc

60

gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca

120

tcgggcgcg

129

<210> 69

<211> 1203

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 69

gccgccacca tggagttggt gggctggctc gtggacaaag gcattacttc ggaaaagcag

60

tggattcagg aggatcaggc atcttacatc tcattcaacg ctgccagtaa ctcgaggtcc

120

cagatcaagg cagcgctgga caacgcggga aagattatga gtctgaccaa aactgctcca

180

gactacctcg ttggtcagca accggtggaa gatatctcca gcaacaggat ctacaagatt

240

ctggagctca acggctacga ccctcaatac gctgcctcag tgttcttggg ttgggccacc

300

aagaaattcg gcaagagaaa cactatctgg ctgttcggcc ccgctaccac tggaaagaca

360

aacatcgcag aagcgattgc tcacacggtg ccattctacg gctgcgtcaa ctggacaaac

420

gagaacttcc cgttcaacga ctgtgtcgat aagatggtta tctggtggga ggaaggaaag

480

atgacggcca aagtggtcga aagcgccaag gcaattctgg gtggctctaa agtgcgcgtc

540

gaccagaagt gcaaatcttc agctcaaatc gatcctaccc ccgttattgt gacatcaaac

600

acgaacatgt gtgccgtgat cgacggaaac agtacaacgt tcgaacacca gcaacctctc

660

caggatcgta tgttcaagtt cgagctcacc cgccgtttgg accatgattt cggcaaggtc

720

actaaacaag aggttaagga cttcttccgc tgggctaaag atcacgttgt ggaggttgaa

780

catgagttct acgtcaagaa aggaggtgct aagaaacgtc cagccccgtc ggacgcagat

840

atctccgaac ctaagagggt gagagagtcg gtcgcacagc caagcacttc tgacgcagaa

900

gcttccatta actacgcaga taggtaccaa aacaagtgca gcagacacgt gggtatgaac

960

ttgatgctgt tcccatgccg ccagtgtgag cgtatgaacc aaaactctaa catctgtttc

1020

acacatggcc agaaggactg cctcgaatgt ttccctgtgt cagagagtca gcccgtctca

1080

gtcgttaaga aagcttacca aaagttgtgc tacatccacc atattatggg taaagtccct

1140

gatgcctgta ccgcttgtga tctggtcaac gtggatttgg acgactgtat tttcgagcaa

1200

taa

1203

<210> 70

<211> 388

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 70

gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca gttcctgccc

60

cggctcaggg ccaagaacag ttggaacagc agaatatggg ccaaacagga tatctgtggt

120

aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc

180

tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc

240

ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc

300

tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc

360

atccacgctg ttttgacttc catagaag

388

<210> 71

<211> 1662

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 71

gccgccacca tggaagacgc caaaaacata aagaaaggcc cggcgccatt ctatccgctg

60

gaagatggaa ccgctggaga gcaactgcat aaggctatga agagatacgc cctggttcct

120

ggaacaattg cttttacaga tgcacatatc gaggtggaca tcacttacgc tgagtacttc

180

gaaatgtccg ttcggttggc agaagctatg aaacgatatg ggctgaatac aaatcacaga

240

atcgtcgtat gcagtgaaaa ctctcttcaa ttctttatgc cggtgttggg cgcgttattt

300

atcggagttg cagttgcgcc cgcgaacgac atttataatg aacgtgaatt gctcaacagt

360

atgggcattt cgcagcctac cgtggtgttc gtttccaaaa aggggttgca aaaaattttg

420

aacgtgcaaa aaaagctccc aatcatccaa aaaattatta tcatggattc taaaacggat

480

taccagggat ttcagtcgat gtacacgttc gtcacatctc atctacctcc cggttttaat

540

gaatacgatt ttgtgccaga gtccttcgat agggacaaga caattgcact gatcatgaac

600

tcctctggat ctactggtct gcctaaaggt gtcgctctgc ctcatagaac tgcctgcgtg

660

agattctcgc atgccagaga tcctattttt ggcaatcaaa tcattccgga tactgcgatt

720

ttaagtgttg ttccattcca tcacggtttt ggaatgttta ctacactcgg atatttgata

780

tgtggatttc gagtcgtctt aatgtataga tttgaagaag agctgtttct gaggagcctt

840

caggattaca agattcaaag tgcgctgctg gtgccaaccc tattctcctt cttcgccaaa

900

agcactctga ttgacaaata cgatttatct aatttacacg aaattgcttc tggtggcgct

960

cccctctcta aggaagtcgg ggaagcggtt gccaagaggt tccatctgcc aggtatcagg

1020

caaggatatg ggctcactga gactacatca gctattctga ttacacccga gggggatgat

1080

aaaccgggcg cggtcggtaa agttgttcca ttttttgaag cgaaggttgt ggatctggat

1140

accgggaaaa cgctgggcgt taatcaaaga ggcgaactgt gtgtgagagg tcctatgatt

1200

atgtccggtt atgtaaacaa tccggaagcg accaacgcct tgattgacaa ggatggatgg

1260

ctacattctg gagacatagc ttactgggac gaagacgaac acttcttcat cgttgaccgc

1320

ctgaagtctc tgattaagta caaaggctat caggtggctc ccgctgaatt ggaatccatc

1380

ttgctccaac accccaacat cttcgacgca ggtgtcgcag gtcttcccga cgatgacgcc

1440

ggtgaacttc ccgccgccgt tgttgttttg gagcacggaa agacgatgac ggaaaaagag

1500

atcgtggatt acgtcgccag tcaagtaaca accgcgaaaa agttgcgcgg aggagttgtg

1560

tttgtggacg aagtaccgaa aggtcttacc ggaaaactcg acgcaagaaa aatcagagag

1620

atcctcataa aggccaagaa gggcggaaag atcgccgtgt aa

1662

<210> 72

<211> 581

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 72

gagcatctta ccgccattta ttcccatatt tgttctgttt ttcttgattt gggtatacat

60

ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg atatgtaatt

120

actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg ttatttacgc

180

tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tgatattctt

240

aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct gtatctagct

300

attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt gctgtctctt

360

ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac

420

gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg gactttcgct

480

ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg ctgctggaca

540

ggggctaggt tgctgggcac tgataattcc gtggtgttgt c

581

<210> 73

<211> 225

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 73

tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct

60

ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct

120

gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg

180

ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggc

225

<210> 74

<211> 1177

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 74

ggctcagagg ctcagaggca cacaggagtt tctgggctca ccctgccccc ttccaacccc

60

tcagttccca tcctccagca gctgtttgtg tgctgcctct gaagtccaca ctgaacaaac

120

ttcagcctac tcatgtccct aaaatgggca aacattgcaa gcagcaaaca gcaaacacac

180

agccctccct gcctgctgac cttggagctg gggcagaggt cagagacctc tctgggccca

240

tgccacctcc aacatccact cgaccccttg gaatttcggt ggagaggagc agaggttgtc

300

ctggcgtggt ttaggtagtg tgagagggtc cgggttcaaa accacttgct gggtggggag

360

tcgtcagtaa gtggctatgc cccgaccccg aagcctgttt ccccatctgt acaatggaaa

420

tgataaagac gcccatctga tagggttttt gtggcaaata aacatttggt ttttttgttt

480

tgttttgttt tgttttttga gatggaggtt tgctctgtcg cccaggctgg agtgcagtga

540

cacaatctca tctcaccaca accttcccct gcctcagcct cccaagtagc tgggattaca

600

agcatgtgcc accacacctg gctaattttc tatttttagt agagacgggt ttctccatgt

660

tggtcagcct cagcctccca agtaactggg attacaggcc tgtgccacca cacccggcta

720

attttttcta tttttgacag ggacggggtt tcaccatgtt ggtcaggctg gtctagaggt

780

accggatctt gctaccagtg gaacagccac taaggattct gcagtgagag cagaggcca

840

gctaagtggt actctcccag agactgtctg actcacgcca ccccctccac cttggacaca

900

ggacgctgtg gtttctgagc caggtacaat gactcctttc ggtaagtgca gtggaagctg

960

tacactgccc aggcaaagcg tccgggcagc gtaggcgggc gactcagatc ccagccagtg

1020

gacttagccc ctgtttgctc ctccgataac tggggtgacc ttggttaata ttcaccagca

1080

gcctcccccg ttgcccctct ggatccactg cttaaatacg gacgaggaca gggccctgtc

1140

tcctcagctt caggcaccac cactgacctg ggacagt

1177

<210> 75

<400> 75

000

<210> 76

<400> 76

000

<210> 77

<400> 77

000

<210> 78

<400> 78

000

<210> 79

<400> 79

000

<210> 80

<400> 80

000

<210> 81

<400> 81

000

<210> 82

<400> 82

000

<210> 83

<400> 83

000

<210> 84

<400> 84

000

<210> 85

<400> 85

000

<210> 86

<400> 86

000

<210> 87

<400> 87

000

<210> 88

<400> 88

000

<210> 89

<400> 89

000

<210> 90

<400> 90

000

<210> 91

<400> 91

000

<210> 92

<400> 92

000

<210> 93

<400> 93

000

<210> 94

<400> 94

000

<210> 95

<211> 122

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 95

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg

122

<210> 96

<211> 72

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 96

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc

60

aagcgagcgc gc

72

<210> 97

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 97

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 98

<211> 72

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 98

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 99

<211> 122

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 99

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg

122

<210> 100

<211> 130

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 100

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc

120

tgcctgcagg

130

<210> 101

<211> 70

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 101

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtgcgcct cagtgagcga

60

gcgagcgcgc

70

<210> 102

<211> 70

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 102

gcgcgctcgc tcgctcactg aggcgcacgc ccgggtttcc cgggcggcct cagtgagcga

60

gcgagcgcgc

70

<210> 103

<211> 72

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 103

gcgcgctcgc tcgctcactg aggccgtcgg gcgacctttg gtcgcccggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 104

<211> 72

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 104

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 105

<211> 72

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 105

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 106

<211> 72

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 106

gcgcgctcgc tcgctcactg aggccgacgc ccgggctttg cccgggcggc ctcagtgagc

60

gagcgagcgc gc

72

<210> 107

<211> 83

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 107

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg ctttgcccgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 108

<211> 83

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 108

gcgcgctcgc tcgctcactg aggccgggca aagcccgacg cccgggcttt gcccgggcgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 109

<211> 77

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 109

gcgcgctcgc tcgctcactg aggccgaaac gtcgggcgac ctttggtcgc ccggcctcag

60

tgagcgagcg agcgcgc

77

<210> 110

<211> 77

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 110

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgtt tcggcctcag

60

tgagcgagcg agcgcgc

77

<210> 111

<211> 51

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 111

gcgcgctcgc tcgctcactg aggcaaagcc tcagtgagcg agcgagcgcg c

51

<210> 112

<211> 51

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 112

gcgcgctcgc tcgctcactg aggctttgcc tcagtgagcg agcgagcgcg c

51

<210> 113

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 113

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct

60

cagtgagcga gcgagcgcgc

80

<210> 114

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 114

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 115

<211> 77

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 115

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgc

77

<210> 116

<211> 79

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 116

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggcgcctc

60

agtgagcgag cgagcgcgc

79

<210> 117

<211> 89

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 117

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgactttgtc

60

gcccggcctc agtgagcgag cgagcgcgc

89

<210> 118

<211> 89

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 118

gcgcgctcgc tcgctcactg aggccgggcg acaaagtcgc ccgacgcccg ggctttgccc

60

gggcggcctc agtgagcgag cgagcgcgc

89

<210> 119

<211> 87

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 119

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgattttcgc

60

ccggcctcag tgagcgagcg agcgcgc

87

<210> 120

<211> 87

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 120

gcgcgctcgc tcgctcactg aggccgggcg aaaatcgccc gacgcccggg ctttgcccgg

60

gcggcctcag tgagcgagcg agcgcgc

87

<210> 121

<211> 85

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 121

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgtttcgccc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 122

<211> 85

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 122

gcgcgctcgc tcgctcactg aggccgggcg aaacgcccga cgcccgggct ttgcccgggc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 123

<211> 89

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 123

gcgcgctcgc tcgctcactg aggccgcccg ggaaacccgg gcgtcgggcg acctttggtc

60

gcccggcctc agtgagcgag cgagcgcgc

89

<210> 124

<211> 89

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 124

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggtttccc

60

gggcggcctc agtgagcgag cgagcgcgc

89

<210> 125

<211> 87

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 125

gcgcgctcgc tcgctcactg aggccgcccg gaaaccgggc gtcgggcgac ctttggtcgc

60

ccggcctcag tgagcgagcg agcgcgc

87

<210> 126

<211> 87

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 126

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cggtttccgg

60

gcggcctcag tgagcgagcg agcgcgc

87

<210> 127

<211> 85

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 127

gcgcgctcgc tcgctcactg aggccgcccg aaacgggcgt cgggcgacct ttggtcgccc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 128

<211> 85

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 128

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgtttcgggc

60

ggcctcagtg agcgagcgag cgcgc

85

<210> 129

<211> 83

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 129

gcgcgctcgc tcgctcactg aggccgccca aagggcgtcg ggcgaccttt ggtcgcccgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 130

<211> 83

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 130

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc ctttgggcgg

60

cctcagtgag cgagcgagcg cgc

83

<210> 131

<211> 81

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 131

gcgcgctcgc tcgctcactg aggccgccaa aggcgtcggg cgacctttgg tcgcccggcc

60

tcagtgagcg agcgagcgcg c

81

<210> 132

<211> 81

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 132

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc tttggcggcc

60

tcagtgagcg agcgagcgcg c

81

<210> 133

<211> 79

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 133

gcgcgctcgc tcgctcactg aggccgcaaa gcgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgcgc

79

<210> 134

<211> 79

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 134

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgct ttgcggcctc

60

agtgagcgag cgagcgcgc

79

<210> 135

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 135

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagcc cgggctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 136

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 136

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacacc cgggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 137

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 137

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgac cggtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 138

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 138

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgca cgtgcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 139

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 139

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc atggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 140

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 140

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagac cggtctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 141

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 141

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaca cgtgtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 142

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 142

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgac atgtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 143

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 143

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagca cgtgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 144

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 144

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaacc cgggttgcct

60

cagtgagcga gcgagcgcgc

80

<210> 145

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 145

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagcc atggctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 146

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 146

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaac cggttggcct

60

cagtgagcga gcgagcgcgc

80

<210> 147

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 147

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacacc atggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 148

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 148

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgaa cgttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 149

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 149

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgca attgcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 150

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 150

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaac cggtttgcct

60

cagtgagcga gcgagcgcgc

80

<210> 151

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 151

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagaa cgttctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 152

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 152

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 153

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 153

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaaa cgtttggcct

60

cagtgagcga gcgagcgcgc

80

<210> 154

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 154

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgaa atttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 155

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 155

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 156

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 156

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaacc atggttgcct

60

cagtgagcga gcgagcgcgc

80

<210> 157

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 157

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaac atgttggcct

60

cagtgagcga gcgagcgcgc

80

<210> 158

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 158

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagac atgtctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 159

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 159

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaca attgtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 160

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 160

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaaa cgttttgcct

60

cagtgagcga gcgagcgcgc

80

<210> 161

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 161

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaac atgtttgcct

60

cagtgagcga gcgagcgcgc

80

<210> 162

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 162

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaca attgttgcct

60

cagtgagcga gcgagcgcgc

80

<210> 163

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 163

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaagaa atttctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 164

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 164

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacaaa attttggcct

60

cagtgagcga gcgagcgcgc

80

<210> 165

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 165

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgaaaaa atttttgcct

60

cagtgagcga gcgagcgcgc

80

<210> 166

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 166

gcgcgctcgc tcgctcgctg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cagcgagcga gcgagcgcgc

80

<210> 167

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 167

gcgcgctcgc tcgctcaatg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cattgagcga gcgagcgcgc

80

<210> 168

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 168

gcgcgctcgc tcgctcaccg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cggtgagcga gcgagcgcgc

80

<210> 169

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 169

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 170

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 170

gcgcgctcgc tcgctcactg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc

60

cagtgagcga gcgagcgcgc

80

<210> 171

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 171

gcgcgctcgc tcgctcactg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt

60

cagtgagcga gcgagcgcgc

80

<210> 172

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 172

gcgcgctcgc tcgctcactg agaccgggcg accaaaggtc gcccgacgcc cgggcggtct

60

cagtgagcga gcgagcgcgc

80

<210> 173

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 173

gcgcgctcgc tcgctcgatg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 174

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 174

gcgcgctcgc tcgctcgcgg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

ccgcgagcga gcgagcgcgc

80

<210> 175

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 175

gcgcgctcgc tcgctcgcta aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 176

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 176

gcgcgctcgc tcgctcgctg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc

60

cagcgagcga gcgagcgcgc

80

<210> 177

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 177

gcgcgctcgc tcgctcgctg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt

60

cagcgagcga gcgagcgcgc

80

<210> 178

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 178

gcgcgctcgc tcgctcgctg agaccgggcg accaaaggtc gcccgacgcc cgggcggtct

60

cagcgagcga gcgagcgcgc

80

<210> 179

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 179

gcgcgctcgc tcgctcgagg aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

cctcgagcga gcgagcgcgc

80

<210> 180

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 180

gcgcgctcgc tcgctcgata aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

tatcgagcga gcgagcgcgc

80

<210> 181

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 181

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 182

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 182

gcgcgctcgc tcgctcgatg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt

60

catcgagcga gcgagcgcgc

80

<210> 183

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 183

gcgcgctcgc tcgctcgatg agaccgggcg accaaaggtc gcccgacgcc cgggcggtct

60

catcgagcga gcgagcgcgc

80

<210> 184

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 184

gcgcgctcgc tcgctcgaga aggccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

tctcgagcga gcgagcgcgc

80

<210> 185

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 185

gcgcgctcgc tcgctcgagg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc

60

cctcgagcga gcgagcgcgc

80

<210> 186

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 186

gcgcgctcgc tcgctcgagg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt

60

cctcgagcga gcgagcgcgc

80

<210> 187

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 187

gcgcgctcgc tcgctcgagg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc

60

cctcgagcga gcgagcgcgc

80

<210> 188

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 188

gcgcgctcgc tcgctcgagg aagccgggcg accaaaggtc gcccgacgcc cgggcggctt

60

cctcgagcga gcgagcgcgc

80

<210> 189

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 189

gcgcgctcgc tcgctcgagg agaccgggcg accaaaggtc gcccgacgcc cgggcggtct

60

cctcgagcga gcgagcgcgc

80

<210> 190

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 190

gcgcgctcgc tcgctcgaga gggccgggcg accaaaggtc gcccgacgcc cgggcggccc

60

tctcgagcga gcgagcgcgc

80

<210> 191

<400> 191

000

<210> 192

<400> 192

000

<210> 193

<400> 193

000

<210> 194

<400> 194

000

<210> 195

<400> 195

000

<210> 196

<400> 196

000

<210> 197

<400> 197

000

<210> 198

<400> 198

000

<210> 199

<400> 199

000

<210> 200

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 200

gcgcgctcgc tcgctcgaga aagccgggcg accaaaggtc gcccgacgcc cgggcggctt

60

tctcgagcga gcgagcgcgc

80

<210> 201

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 201

gcgcgctcgc tcgctcgaga agaccgggcg accaaaggtc gcccgacgcc cgggcggcct

60

tctcgagcga gcgagcgcgc

80

<210> 202

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 202

gcgcgctcgc tcgctcgaga gagccgggcg accaaaggtc gcccgacgcc cgggcggctc

60

tctcgagcga gcgagcgcgc

80

<210> 203

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 203

gcgcgctcgc tcgctcgaga ggaccgggcg accaaaggtc gcccgacgcc cgggcggtcc

60

tctcgagcga gcgagcgcgc

80

<210> 204

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 204

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 205

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 205

gcgcgctcgc tcgctcaaga gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc

60

tcttgagcga gcgagcgcgc

80

<210> 206

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 206

gcgcgctcgc tcgctcacga gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc

60

tcgtgagcga gcgagcgcgc

80

<210> 207

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 207

gcgcgctcgc tcgctcacta gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc

60

tagtgagcga gcgagcgcgc

80

<210> 208

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 208

gcgcgctcgc tcgctcactg gaaccgggcg accaaaggtc gcccgacgcc cgggcggttc

60

cagtgagcga gcgagcgcgc

80

<210> 209

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 209

gcgcgctcgc tcgctcactg aaaccgggcg accaaaggtc gcccgacgcc cgggcggttt

60

cagtgagcga gcgagcgcgc

80

<210> 210

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 210

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacgcc cgggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 211

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 211

gcgcgctcgc tcgctcaata aaaccgggcg accaaaggtc gcccgacgcc cgggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 212

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 212

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 213

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 213

gcgcgctcgc tcgctcactg aggccgagcg accaaaggtc gctcgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 214

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 214

gcgcgctcgc tcgctcactg aggccggacg accaaaggtc gtccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 215

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 215

gcgcgctcgc tcgctcactg aggccgggag accaaaggtc tcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 216

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 216

gcgcgctcgc tcgctcactg aggccgggca accaaaggtt gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 217

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 217

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 218

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 218

gcgcgctcgc tcgctcactg aggccgggcg aacaaagttc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 219

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 219

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 220

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 220

gcgcgctcgc tcgctcactg aggccaagcg accaaaggtc gcttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 221

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 221

gcgcgctcgc tcgctcactg aggccaaacg accaaaggtc gtttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 222

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 222

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 223

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 223

gcgcgctcgc tcgctcactg aggccaaaaa accaaaggtt ttttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 224

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 224

gcgcgctcgc tcgctcactg aggccaaaaa gccaaaggct ttttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 225

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 225

gcgcgctcgc tcgctcactg aggccaaaaa gacaaagtct ttttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 226

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 226

gcgcgctcgc tcgctcactg aggccaaaaa gaaaaattct ttttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 227

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 227

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaatttt ttttgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 228

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 228

gcgcgctcgc tcgctcactg aggccgaaaa aaaaaatttt tttcgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 229

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 229

gcgcgctcgc tcgctcactg aggccggaaa gaaaaattct ttccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 230

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 230

gcgcgctcgc tcgctcactg aggccgggaa gaaaaattct tcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 231

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 231

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 232

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 232

gcgcgctcgc tcgctcactg aggccgggcg gaaaaattcc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 233

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 233

gcgcgctcgc tcgctcactg aggccgggcg aaaaaatttc gcccgacgcc cgggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 234

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 234

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacacc cgggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 235

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 235

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgcc atggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 236

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 236

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgac atgtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 237

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 237

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgacgaa cgttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 238

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 238

gcgcgctcgc tcgctcactg aggccaggcg accaaaggtc gcctgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 239

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 239

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgacacc cgggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 240

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 240

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgacgcc atggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 241

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 241

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgacgac atgtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 242

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 242

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgacgaa cgttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 243

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 243

gcgcgctcgc tcgctcactg aggccgggcg gccaaaggcc gcccgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 244

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 244

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacacc cgggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 245

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 245

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgcc atggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 246

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 246

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgac atgtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 247

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 247

gcgcgctcgc tcgctcactg aggccgggcg acaaaatgtc gcccgacgaa cgttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 248

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 248

gcgcgctcgc tcgctcactg aggccggggcg acaaaatgtc gcccgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 249

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 249

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacacc cgggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 250

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 250

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgcc atggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 251

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 251

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgac atgtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 252

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 252

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgacgaa cgttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 253

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 253

gcgcgctcgc tcgctcactg aggccaaaag accaaaggtc ttttgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 254

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 254

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaatttt ttttgacacc cgggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 255

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 255

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaatttt ttttgacgcc atggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 256

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 256

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaatttt ttttgacgac atgtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 257

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 257

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaatttt ttttgacgaa cgttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 258

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 258

gcgcgctcgc tcgctcactg aggccaaaaa aaaaaatttt ttttgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 259

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 259

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacacc cgggtggcct

60

cagtgagcga gcgagcgcgc

80

<210> 260

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 260

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgcc atggcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 261

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 261

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgac atgtcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 262

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 262

gcgcgctcgc tcgctcactg aggccggggca gaaaaattct gcccgacgaa cgttcggcct

60

cagtgagcga gcgagcgcgc

80

<210> 263

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 263

gcgcgctcgc tcgctcactg aggccgggca gaaaaattct gcccgaagca attgctgcct

60

cagtgagcga gcgagcgcgc

80

<210> 264

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 264

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 265

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 265

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 266

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 266

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 267

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 267

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 268

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 268

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaatttt ttttgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 269

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 269

gcgcgctcgc tcgctcacta aggccgggca gaaaaattct gcccgacgcc cgggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 270

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 270

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 271

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 271

gcgcgctcgc tcgctcgatg gggccgggcg gccaaaggcc gcccgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 272

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 272

gcgcgctcgc tcgctcgatg gggccggggcg acaaaatgtc gcccgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 273

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 273

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 274

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 274

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaatttt ttttgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 275

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 275

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacgcc cgggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 276

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 276

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgcc cgggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 277

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 277

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgcc cgggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 278

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 278

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgcc cgggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 279

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 279

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgcc cgggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 280

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 280

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaatttt ttttgacgcc cgggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 281

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 281

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgcc cgggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 282

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 282

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgcc cgggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 283

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 283

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgcc cgggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 284

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 284

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacgcc cgggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 285

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 285

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgcc cgggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 286

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 286

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacgcc cgggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 287

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 287

gcgcgctcgc tcgctcgcgg gggccgggca gaaaaattct gcccgacgcc cgggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 288

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 288

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgcc cgggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 289

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 289

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgacgcc cgggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 290

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 290

gcgcgctcgc tcgctcaata aaaccgggcg acaaaatgtc gcccgacgcc cgggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 291

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 291

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgcc cgggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 292

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 292

gcgcgctcgc tcgctcaata aaaccaaaaa aaaaaatttt ttttgacgcc cgggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 293

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 293

gcgcgctcgc tcgctcaata aaaccgggca gaaaaattct gcccgacgcc cgggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 294

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 294

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgacacc cgggtggcct

60

tagtgagcga gcgagcgcgc

80

<210> 295

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 295

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgacgcc atggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 296

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 296

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgacgac atgtcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 297

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 297

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgacgaa cgttcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 298

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 298

gcgcgctcgc tcgctcacta aggccgggcg accaaaggtc gcccgaagca attgctgcct

60

tagtgagcga gcgagcgcgc

80

<210> 299

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 299

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacacc cgggtggcct

60

catcgagcga gcgagcgcgc

80

<210> 300

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 300

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacgcc atggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 301

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 301

gcgcgctcgc tcgctcgatg gggccggggcg accaaaggtc gcccgacgac atgtcggcct

60

catcgagcga gcgagcgcgc

80

<210> 302

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 302

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgacgaa cgttcggcct

60

catcgagcga gcgagcgcgc

80

<210> 303

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 303

gcgcgctcgc tcgctcgatg gggccgggcg accaaaggtc gcccgaagca attgctgcct

60

catcgagcga gcgagcgcgc

80

<210> 304

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 304

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacacc cgggtggttc

60

tctcgagcga gcgagcgcgc

80

<210> 305

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 305

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacgcc atggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 306

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 306

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacgac atgtcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 307

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 307

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgacgaa cgttcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 308

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 308

gcgcgctcgc tcgctcgaga gaaccgggcg accaaaggtc gcccgaagca attgctgttc

60

tctcgagcga gcgagcgcgc

80

<210> 309

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 309

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacacc cgggtggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 310

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 310

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacgcc atggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 311

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 311

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgacgac atgtcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 312

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 312

gcgcgctcgc tcgctcgcgg gggccggggcg accaaaggtc gcccgacgaa cgttcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 313

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 313

gcgcgctcgc tcgctcgcgg gggccgggcg accaaaggtc gcccgaagca attgctgccc

60

ccgcgagcga gcgagcgcgc

80

<210> 314

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 314

gcgcgctcgc tcgctcaata aaaccgggcg accaaaggtc gcccgacacc cgggtggttt

60

tattgagcga gcgagcgcgc

80

<210> 315

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 315

gcgcgctcgc tcgctcaata aaaccgggcg accaaaggtc gcccgacgcc atggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 316

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 316

gcgcgctcgc tcgctcaata aaaccgggcg accaaaggtc gcccgacgac atgtcggttt

60

tattgagcga gcgagcgcgc

80

<210> 317

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 317

gcgcgctcgc tcgctcaata aaaccgggcg accaaaggtc gcccgacgaa cgttcggttt

60

tattgagcga gcgagcgcgc

80

<210> 318

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 318

gcgcgctcgc tcgctcaata aaaccgggcg accaaaggtc gcccgaagca attgctgttt

60

tattgagcga gcgagcgcgc

80

<210> 319

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 319

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacacc cgggtggcct

60

tagtgagcga gcgagcgcgc

80

<210> 320

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 320

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgcc atggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 321

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 321

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgac atgtcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 322

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 322

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgacgaa cgttcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 323

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 323

gcgcgctcgc tcgctcacta aggccaggcg accaaaggtc gcctgaagca attgctgcct

60

tagtgagcga gcgagcgcgc

80

<210> 324

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 324

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacacc cgggtggcct

60

tagtgagcga gcgagcgcgc

80

<210> 325

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 325

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacgcc atggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 326

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 326

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacgac atgtcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 327

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 327

gcgcgctcgc tcgctcacta aggccgggcg gccaaaggcc gcccgacgaa cgttcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 328

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 328

gcgcgctcgc tcgctcacta aggccggggcg gccaaaggcc gcccgaagca attgctgcct

60

tagtgagcga gcgagcgcgc

80

<210> 329

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 329

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacacc cgggtggcct

60

tagtgagcga gcgagcgcgc

80

<210> 330

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 330

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacgcc atggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 331

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 331

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgacgac atgtcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 332

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 332

gcgcgctcgc tcgctcacta aggccggggcg acaaaatgtc gcccgacgaa cgttcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 333

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 333

gcgcgctcgc tcgctcacta aggccgggcg acaaaatgtc gcccgaagca attgctgcct

60

tagtgagcga gcgagcgcgc

80

<210> 334

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 334

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacacc cgggtggcct

60

tagtgagcga gcgagcgcgc

80

<210> 335

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 335

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgcc atggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 336

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 336

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgac atgtcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 337

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 337

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgacgaa cgttcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 338

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 338

gcgcgctcgc tcgctcacta aggccaaaag accaaaggtc ttttgaagca attgctgcct

60

tagtgagcga gcgagcgcgc

80

<210> 339

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 339

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaatttt ttttgacacc cgggtggcct

60

tagtgagcga gcgagcgcgc

80

<210> 340

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 340

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaatttt ttttgacgcc atggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 341

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 341

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaatttt ttttgacgac atgtcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 342

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 342

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaatttt ttttgacgaa cgttcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 343

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 343

gcgcgctcgc tcgctcacta aggccaaaaa aaaaaatttt ttttgaagca attgctgcct

60

tagtgagcga gcgagcgcgc

80

<210> 344

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 344

gcgcgctcgc tcgctcacta aggccggggca gaaaaattct gcccgacacc cgggtggcct

60

tagtgagcga gcgagcgcgc

80

<210> 345

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 345

gcgcgctcgc tcgctcacta aggccgggca gaaaaattct gcccgacgcc atggcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 346

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 346

gcgcgctcgc tcgctcacta aggccgggca gaaaaattct gcccgacgac atgtcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 347

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 347

gcgcgctcgc tcgctcacta aggccgggca gaaaaattct gcccgacgaa cgttcggcct

60

tagtgagcga gcgagcgcgc

80

<210> 348

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 348

gcgcgctcgc tcgctcacta aggccgggca gaaaaattct gcccgaagca attgctgcct

60

tagtgagcga gcgagcgcgc

80

<210> 349

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 349

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacacc cgggtggcct

60

catcgagcga gcgagcgcgc

80

<210> 350

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 350

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgcc atggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 351

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 351

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgac atgtcggcct

60

catcgagcga gcgagcgcgc

80

<210> 352

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 352

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgacgaa cgttcggcct

60

catcgagcga gcgagcgcgc

80

<210> 353

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 353

gcgcgctcgc tcgctcgatg gggccaggcg accaaaggtc gcctgaagca attgctgcct

60

catcgagcga gcgagcgcgc

80

<210> 354

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 354

gcgcgctcgc tcgctcgatg gggccgggcg gccaaaggcc gcccgacacc cgggtggcct

60

catcgagcga gcgagcgcgc

80

<210> 355

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 355

gcgcgctcgc tcgctcgatg gggccggggcg gccaaaggcc gcccgacgcc atggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 356

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 356

gcgcgctcgc tcgctcgatg gggccggggcg gccaaaggcc gcccgacgac atgtcggcct

60

catcgagcga gcgagcgcgc

80

<210> 357

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 357

gcgcgctcgc tcgctcgatg gggccggggcg gccaaaggcc gcccgacgaa cgttcggcct

60

catcgagcga gcgagcgcgc

80

<210> 358

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 358

gcgcgctcgc tcgctcgatg gggccgggcg gccaaaggcc gcccgaagca attgctgcct

60

catcgagcga gcgagcgcgc

80

<210> 359

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 359

gcgcgctcgc tcgctcgatg gggccggggcg acaaaatgtc gcccgacacc cgggtggcct

60

catcgagcga gcgagcgcgc

80

<210> 360

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 360

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgacgcc atggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 361

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 361

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgacgac atgtcggcct

60

catcgagcga gcgagcgcgc

80

<210> 362

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 362

gcgcgctcgc tcgctcgatg gggccggggcg acaaaatgtc gcccgacgaa cgttcggcct

60

catcgagcga gcgagcgcgc

80

<210> 363

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 363

gcgcgctcgc tcgctcgatg gggccgggcg acaaaatgtc gcccgaagca attgctgcct

60

catcgagcga gcgagcgcgc

80

<210> 364

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 364

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacacc cgggtggcct

60

catcgagcga gcgagcgcgc

80

<210> 365

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 365

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgcc atggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 366

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 366

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgac atgtcggcct

60

catcgagcga gcgagcgcgc

80

<210> 367

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 367

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgacgaa cgttcggcct

60

catcgagcga gcgagcgcgc

80

<210> 368

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 368

gcgcgctcgc tcgctcgatg gggccaaaag accaaaggtc ttttgaagca attgctgcct

60

catcgagcga gcgagcgcgc

80

<210> 369

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 369

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaatttt ttttgacacc cgggtggcct

60

catcgagcga gcgagcgcgc

80

<210> 370

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 370

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaatttt ttttgacgcc atggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 371

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 371

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaatttt ttttgacgac atgtcggcct

60

catcgagcga gcgagcgcgc

80

<210> 372

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 372

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaatttt ttttgacgaa cgttcggcct

60

catcgagcga gcgagcgcgc

80

<210> 373

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 373

gcgcgctcgc tcgctcgatg gggccaaaaa aaaaaatttt ttttgaagca attgctgcct

60

catcgagcga gcgagcgcgc

80

<210> 374

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 374

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacacc cgggtggcct

60

catcgagcga gcgagcgcgc

80

<210> 375

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 375

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacgcc atggcggcct

60

catcgagcga gcgagcgcgc

80

<210> 376

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 376

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacgac atgtcggcct

60

catcgagcga gcgagcgcgc

80

<210> 377

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 377

gcgcgctcgc tcgctcgatg gggccggggca gaaaaattct gcccgacgaa cgttcggcct

60

catcgagcga gcgagcgcgc

80

<210> 378

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 378

gcgcgctcgc tcgctcgatg gggccgggca gaaaaattct gcccgaagca attgctgcct

60

catcgagcga gcgagcgcgc

80

<210> 379

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 379

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacacc cgggtggttc

60

tctcgagcga gcgagcgcgc

80

<210> 380

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 380

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgcc atggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 381

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 381

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgac atgtcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 382

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 382

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgacgaa cgttcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 383

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 383

gcgcgctcgc tcgctcgaga gaaccaggcg accaaaggtc gcctgaagca attgctgttc

60

tctcgagcga gcgagcgcgc

80

<210> 384

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 384

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacacc cgggtggttc

60

tctcgagcga gcgagcgcgc

80

<210> 385

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 385

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgcc atggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 386

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 386

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgac atgtcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 387

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 387

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgacgaa cgttcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 388

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 388

gcgcgctcgc tcgctcgaga gaaccgggcg gccaaaggcc gcccgaagca attgctgttc

60

tctcgagcga gcgagcgcgc

80

<210> 389

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 389

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacacc cgggtggttc

60

tctcgagcga gcgagcgcgc

80

<210> 390

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 390

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgcc atggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 391

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 391

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgac atgtcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 392

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 392

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgacgaa cgttcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 393

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 393

gcgcgctcgc tcgctcgaga gaaccgggcg acaaaatgtc gcccgaagca attgctgttc

60

tctcgagcga gcgagcgcgc

80

<210> 394

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 394

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacacc cgggtggttc

60

tctcgagcga gcgagcgcgc

80

<210> 395

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 395

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgcc atggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 396

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 396

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgac atgtcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 397

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 397

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgacgaa cgttcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 398

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 398

gcgcgctcgc tcgctcgaga gaaccaaaag accaaaggtc ttttgaagca attgctgttc

60

tctcgagcga gcgagcgcgc

80

<210> 399

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 399

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaatttt ttttgacacc cgggtggttc

60

tctcgagcga gcgagcgcgc

80

<210> 400

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 400

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaatttt ttttgacgcc atggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 401

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 401

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaatttt ttttgacgac atgtcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 402

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 402

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaatttt ttttgacgaa cgttcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 403

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 403

gcgcgctcgc tcgctcgaga gaaccaaaaa aaaaaatttt ttttgaagca attgctgttc

60

tctcgagcga gcgagcgcgc

80

<210> 404

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 404

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacacc cgggtggttc

60

tctcgagcga gcgagcgcgc

80

<210> 405

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 405

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgcc atggcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 406

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 406

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgac atgtcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 407

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 407

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgacgaa cgttcggttc

60

tctcgagcga gcgagcgcgc

80

<210> 408

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 408

gcgcgctcgc tcgctcgaga gaaccgggca gaaaaattct gcccgaagca attgctgttc

60

tctcgagcga gcgagcgcgc

80

<210> 409

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 409

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacacc cgggtggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 410

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 410

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgcc atggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 411

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 411

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgac atgtcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 412

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 412

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgacgaa cgttcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 413

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 413

gcgcgctcgc tcgctcgcgg gggccaggcg accaaaggtc gcctgaagca attgctgccc

60

ccgcgagcga gcgagcgcgc

80

<210> 414

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 414

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacacc cgggtggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 415

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 415

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgcc atggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 416

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 416

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgac atgtcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 417

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 417

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgacgaa cgttcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 418

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 418

gcgcgctcgc tcgctcgcgg gggccgggcg gccaaaggcc gcccgaagca attgctgccc

60

ccgcgagcga gcgagcgcgc

80

<210> 419

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 419

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacacc cgggtggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 420

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 420

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacgcc atggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 421

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 421

gcgcgctcgc tcgctcgcgg gggccgggcg acaaaatgtc gcccgacgac atgtcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 422

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 422

gcgcgctcgc tcgctcgcgg gggccggggcg acaaaatgtc gcccgacgaa cgttcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 423

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 423

gcgcgctcgc tcgctcgcgg gggccggggcg acaaaatgtc gcccgaagca attgctgccc

60

ccgcgagcga gcgagcgcgc

80

<210> 424

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 424

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacacc cgggtggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 425

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 425

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgcc atggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 426

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 426

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgac atgtcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 427

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 427

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgacgaa cgttcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 428

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 428

gcgcgctcgc tcgctcgcgg gggccaaaag accaaaggtc ttttgaagca attgctgccc

60

ccgcgagcga gcgagcgcgc

80

<210> 429

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 429

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacacc cgggtggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 430

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 430

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacgcc atggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 431

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 431

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacgac atgtcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 432

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 432

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgacgaa cgttcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 433

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 433

gcgcgctcgc tcgctcgcgg gggccaaaaa aaaaaatttt ttttgaagca attgctgccc

60

ccgcgagcga gcgagcgcgc

80

<210> 434

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 434

gcgcgctcgc tcgctcgcgg gggccggggca gaaaaattct gcccgacacc cgggtggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 435

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 435

gcgcgctcgc tcgctcgcgg gggccgggca gaaaaattct gcccgacgcc atggcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 436

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 436

gcgcgctcgc tcgctcgcgg gggccgggca gaaaaattct gcccgacgac atgtcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 437

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 437

gcgcgctcgc tcgctcgcgg gggccgggca gaaaaattct gcccgacgaa cgttcggccc

60

ccgcgagcga gcgagcgcgc

80

<210> 438

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 438

gcgcgctcgc tcgctcgcgg gggccgggca gaaaaattct gcccgaagca attgctgccc

60

ccgcgagcga gcgagcgcgc

80

<210> 439

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 439

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacacc cgggtggttt

60

tattgagcga gcgagcgcgc

80

<210> 440

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 440

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgcc atggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 441

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 441

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgac atgtcggttt

60

tattgagcga gcgagcgcgc

80

<210> 442

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 442

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgacgaa cgttcggttt

60

tattgagcga gcgagcgcgc

80

<210> 443

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 443

gcgcgctcgc tcgctcaata aaaccaggcg accaaaggtc gcctgaagca attgctgttt

60

tattgagcga gcgagcgcgc

80

<210> 444

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 444

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgacacc cgggtggttt

60

tattgagcga gcgagcgcgc

80

<210> 445

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 445

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgacgcc atggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 446

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 446

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgacgac atgtcggttt

60

tattgagcga gcgagcgcgc

80

<210> 447

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 447

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgacgaa cgttcggttt

60

tattgagcga gcgagcgcgc

80

<210> 448

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 448

gcgcgctcgc tcgctcaata aaaccgggcg gccaaaggcc gcccgaagca attgctgttt

60

tattgagcga gcgagcgcgc

80

<210> 449

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 449

gcgcgctcgc tcgctcaata aaaccgggcg acaaaatgtc gcccgacacc cgggtggttt

60

tattgagcga gcgagcgcgc

80

<210> 450

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 450

gcgcgctcgc tcgctcaata aaaccgggcg acaaaatgtc gcccgacgcc atggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 451

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 451

gcgcgctcgc tcgctcaata aaaccgggcg acaaaatgtc gcccgacgac atgtcggttt

60

tattgagcga gcgagcgcgc

80

<210> 452

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 452

gcgcgctcgc tcgctcaata aaaccgggcg acaaaatgtc gcccgacgaa cgttcggttt

60

tattgagcga gcgagcgcgc

80

<210> 453

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 453

gcgcgctcgc tcgctcaata aaaccgggcg acaaaatgtc gcccgaagca attgctgttt

60

tattgagcga gcgagcgcgc

80

<210> 454

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 454

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacacc cgggtggttt

60

tattgagcga gcgagcgcgc

80

<210> 455

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 455

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgcc atggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 456

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 456

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgac atgtcggttt

60

tattgagcga gcgagcgcgc

80

<210> 457

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 457

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgacgaa cgttcggttt

60

tattgagcga gcgagcgcgc

80

<210> 458

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 458

gcgcgctcgc tcgctcaata aaaccaaaag accaaaggtc ttttgaagca attgctgttt

60

tattgagcga gcgagcgcgc

80

<210> 459

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 459

gcgcgctcgc tcgctcaata aaaccaaaaa aaaaaatttt ttttgacacc cgggtggttt

60

tattgagcga gcgagcgcgc

80

<210> 460

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 460

gcgcgctcgc tcgctcaata aaaccaaaaa aaaaaatttt ttttgacgcc atggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 461

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 461

gcgcgctcgc tcgctcaata aaaccaaaaa aaaaaatttt ttttgacgac atgtcggttt

60

tattgagcga gcgagcgcgc

80

<210> 462

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 462

gcgcgctcgc tcgctcaata aaaccaaaaa aaaaaatttt ttttgacgaa cgttcggttt

60

tattgagcga gcgagcgcgc

80

<210> 463

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 463

gcgcgctcgc tcgctcaata aaaccaaaaa aaaaaatttt ttttgaagca attgctgttt

60

tattgagcga gcgagcgcgc

80

<210> 464

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 464

gcgcgctcgc tcgctcaata aaaccgggca gaaaaattct gcccgacacc cgggtggttt

60

tattgagcga gcgagcgcgc

80

<210> 465

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 465

gcgcgctcgc tcgctcaata aaaccgggca gaaaaattct gcccgacgcc atggcggttt

60

tattgagcga gcgagcgcgc

80

<210> 466

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 466

gcgcgctcgc tcgctcaata aaaccgggca gaaaaattct gcccgacgac atgtcggttt

60

tattgagcga gcgagcgcgc

80

<210> 467

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 467

gcgcgctcgc tcgctcaata aaaccgggca gaaaaattct gcccgacgaa cgttcggttt

60

tattgagcga gcgagcgcgc

80

<210> 468

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 468

gcgcgctcgc tcgctcaata aaaccgggca gaaaaattct gcccgaagca attgctgttt

60

tattgagcga gcgagcgcgc

80

<210> 469

<211> 120

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 469

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

cgcacgcccg ggtttcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg

120

<210> 470

<211> 122

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 470

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca

120

gg

122

<210> 471

<211> 129

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 471

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gcgcctcagt gagcgagcga gcgcgcagct

120

gcctgcagg

129

<210> 472

<211> 101

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 472

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ctttgcctca gtgagcgagc gagcgcgcag ctgcctgcag g

101

<210> 473

<211> 139

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 473

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgaca aagtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga

120

gcgcgcagct gcctgcagg

139

<210> 474

<211> 137

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 474

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgaaa atcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc

120

gcgcagctgc ctgcagg

137

<210> 475

<211> 135

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 475

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgaaa cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc

120

gcagctgcct gcagg

135

<210> 476

<211> 133

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 476

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcaaag cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc

120

aggctgcctgc agg

133

<210> 477

<211> 139

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 477

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg gtttcccggg cggcctcagt gagcgagcga

120

gcgcgcagct gcctgcagg

139

<210> 478

<211> 137

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 478

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgg tttccgggcg gcctcagtga gcgagcgagc

120

gcgcagctgc ctgcagg

137

<210> 479

<211> 135

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 479

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgcccgt ttcgggcggc ctcagtgagc gagcgagcgc

120

gcagctgcct gcagg

135

<210> 480

<211> 133

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 480

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgccctt tgggcggcct cagtgagcga gcgagcgcgc

120

aggctgcctgc agg

133

<210> 481

<211> 131

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 481

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgccttt ggcggcctca gtgagcgagc gagcgcgcag

120

ctgcctgcag g

131

<210> 482

<211> 129

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 482

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgctttg cggcctcagt gagcgagcga gcgcgcagct

120

gcctgcagg

129

<210> 483

<211> 127

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 483

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgggcgacc aaaggtcgcc cgacgtttcg gcctcagtga gcgagcgagc gcgcagctgc

120

ctgcagg

127

<210> 484

<211> 120

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 484

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtgcg

60

cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct

120

<210> 485

<211> 122

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 485

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgt cgggcgacct ttggtcgccc

60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc

120

ct

122

<210> 486

<211> 122

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 486

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc

120

ct

122

<210> 487

<211> 129

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 487

cctgcaggca gctgcgcgct cgctcgctca ctgaggcgcc cgggcgtcgg gcgacctttg

60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta

120

ggggttcct

129

<210> 488

<211> 101

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 488

cctgcaggca gctgcgcgct cgctcgctca ctgaggcaaa gcctcagtga gcgagcgagc

60

gcgcagagag ggagtggcca actccatcac taggggttcc t

101

<210> 489

<211> 139

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 489

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgacttt gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac

120

tccatcacta ggggttcct

139

<210> 490

<211> 137

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 490

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgatttt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc

120

catcactagg ggttcct

137

<210> 491

<211> 135

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 491

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggcgtttcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttcct

135

<210> 492

<211> 133

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 492

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc

60

gggctttgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc

120

actaggggtt cct

133

<210> 493

<211> 139

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 493

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggaaacc cgggcgtcgg

60

gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac

120

tccatcacta ggggttcct

139

<210> 494

<211> 137

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 494

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccggaaaccg ggcgtcgggc

60

gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc

120

catcactagg ggttcct

137

<210> 495

<211> 135

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 495

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgaaacggg cgtcgggcga

60

cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca

120

tcactagggg ttcct

135

<210> 496

<211> 133

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 496

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccaaagggcg tcgggcgacc

60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc

120

actaggggtt cct

133

<210> 497

<211> 131

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 497

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc caaaggcgtc gggcgacctt

60

tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac

120

tagggggttcct

131

<210> 498

<211> 129

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 498

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc aaagcgtcgg gcgacctttg

60

gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta

120

ggggttcct

129

<210> 499

<211> 127

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 499

cctgcaggca gctgcgcgct cgctcgctca ctgaggccga aacgtcgggc gacctttggt

60

cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg

120

ggttcct

127

<210> 500

<211> 43

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 500

gcccgctggt ttccagcggg ctgcgggccc gaaacggggcc cgc

43

<210> 501

<211> 28

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 501

cgggcccgtg cgggcccaaa gggcccgc

28

<210> 502

<211> 28

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 502

gcccgggcac gcccgggttt cccgggcg

28

<210> 503

<211> 22

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 503

cgtgcgggcc caaagggccc gc

22

<210> 504

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 504

cgggcgacca aaggtcgccc g

21

<210> 505

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 505

cgcccgggct ttgcccgggc

20

<210> 506

<211> 42

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 506

cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gc

42

<210> 507

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 507

cgggcgacca aaggtcgccc g

21

<210> 508

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 508

cgcccgggct ttgcccgggc

20

<210> 509

<211> 34

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 509

cgggcgacca aaggtcgccc gacgcccggg cggc

34

<210> 510

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 510

cgggcgacca aaggtcgccc g

21

<210> 511

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 511

cgcccgggct ttgcccgggc

20

<210> 512

<211> 30

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 512

cggggcccga cgcccgggct ttgcccgggc

thirty

<210> 513

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 513

cgggcgacca aaggtcgccc g

21

<210> 514

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 514

cgcccgggct ttgcccgggc

20

<210> 515

<211> 29

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 515

cgggcccgac gcccggggctt tgcccggggc

29

<210> 516

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 516

cgggcgacca aaggtcgccc g

21

<210> 517

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 517

cgcccgggct ttgcccgggc

20

<210> 518

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 518

gcccgggcaa agcccgggcg

20

<210> 519

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 519

cgggcgacct ttggtcgccc g

21

<210> 520

<211> 42

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 520

gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cg

42

<210> 521

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 521

gcccgggcaa agcccgggcg

20

<210> 522

<211> 31

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 522

gcccgggcgt cgggcgacct ttggtcgccc g

31

<210> 523

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 523

gcccgggcaa agcccgggcg

20

<210> 524

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 524

cgggcgacct ttggtcgccc g

21

<210> 525

<211> 34

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 525

gccgcccggg cgacgggcga cctttggtcg cccg

34

<210> 526

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 526

gcccgggcaa agcccgggcg

20

<210> 527

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 527

cgggcgacct ttggtcgccc g

21

<210> 528

<211> 31

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 528

gcccgggcgt cgggcgacct ttggtcgccc g

31

<210> 529

<211> 21

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 529

cgggcgacct ttggtcgccc g

21

<210> 530

<211> 1653

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 530

atgccgccac cccggaccgg ccgaggcctt ctctggctgg gtctggttct gagctccgtc

60

tgcgtcgccc tcggatccga aacgcaggcc aactcgacca cagatgctct gaacgttctt

120

ctcatcatcg tggatgacct gcgcccctcc ctgggctgtt atggggataa gctggtgagg

180

tccccaaata ttgaccaact ggcatcccac agcctcctct tccagaatgc ctttgcgcag

240

caagcagtgt gcgccccgag ccgcgtttct ttcctcactg gcaggagacc tgacaccacc

300

cgcctgtacg acttcaactc ctactggagg gtgcacgctg gaaacttctc caccatcccc

360

cagtacttca aggagaatgg ctatgtgacc atgtcggtgg gaaaagtctt tcaccctggg

420

atatcttcta accataccga tgattctccg tatagctggt cttttccacc ttatcatcct

480

tcctctgaga agtatgaaaa cactaagaca tgtcgagggc cagatggaga actccatgcc

540

aacctgcttt gccctgtgga tgtgctggat gttcccgagg gcaccttgcc tgacaaacag

600

agcactgagc aagccataca gttgttggaa aagatgaaaa cgtcagccag tcctttcttc

660

ctggccgttg ggtatcataa gccacacatc cccttcagat accccaagga atttcagaag

720

ttgtatccct tggagaacat caccctggcc cccgatcccg aggtccctga tggcctaccc

780

cctgtggcct acaacccctg gatggacatc aggcaacggg aagacgtcca agccttaaac

840

atcagtgtgc cgtatggtcc aattcctgtg gactttcagc ggaaaatccg ccagagctac

900

tttgcctctg tgtcatattt ggatacacag gtcggccgcc tcttgagtgc tttggacgat

960

cttcagctgg ccaacagcac catcattgca tttacctcgg atcatgggtg ggctctaggt

1020

gaacatggag aatgggccaa atacagcaat tttgatgttg ctacccatgt tcccctgata

1080

ttctatgttc ctggaaggac ggcttcactt ccggaggcag gcgagaagct tttcccttac

1140

ctcgaccctt ttgattccgc ctcacagttg atggagccag gcaggcaatc catggacctt

1200

gtggaacttg tgtctctttt tcccacgctg gctggacttg caggactgca ggttccacct

1260

cgctgccccg ttccttcatt tcacgttgag ctgtgcagag aaggcaagaa ccttctgaag

1320

cattttcgat tccgtgactt ggaagaggat ccgtacctcc ctggtaatcc ccgtgaactg

1380

attgcctata gccagtatcc ccggccttca gacatccctc agtggaattc tgacaagccg

1440

agtttaaaag atataaagat catgggctat tccatacgca ccatagacta taggtatact

1500

gtgtgggttg gcttcaatcc tgatgaattt ctagctaact tttctgacat ccatgcaggg

1560

gaactgtatt ttgtggattc tgacccattg caggatcaca atatgtataa tgattcccaa

1620

ggtggagatc ttttccagtt gttgatgcct tga

1653

<210> 531

<211> 16

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 531

gcgcgctcgc tcgctc

16

<210> 532

<211> 8

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 532

actgaggc

8

<210> 533

<211> 22

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 533

cgggcgacca aaggtcgccc ga

22

<210> 534

<211> 10

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 534

cgcccgggcg

10

<210> 535

<211> 8

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 535

gcctcagt

8

<210> 536

<211> 16

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 536

gagcgagcga gcgcgc

16

<210> 537

<211> 20

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 537

gcccgggcaa agcccgggcg

20

<210> 538

<211> 165

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 538

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg

60

ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc

120

gagcgcgcag agagggagtg gccaactcca tcactagggg ttcct

165

<210> 539

<211> 140

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 539

cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccgcc

60

cggggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg

120

cgcagagaga tcactagggg

140

<210> 540

<211> 91

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 540

gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg

60

tcgcccggcc tcagtgagcg agcgagcgcg c

91

<210> 541

<211> 91

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 541

gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc

60

ccgggcggcc tcagtgagcg agcgagcgcg c

91

<210> 542

<211> 8

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 542

ttaattaa

8

<210> 543

<211> 80

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 543

gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct

60

cagtgagcga gcgagcgcgc

80

<210> 544

<211> 52

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 544

gcgcgctcgc tcgctcactg aggcctttgc ctcagtgagc gagcgagcgc gc

52

<210> 545

<211> 79

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 545

gcgcgctcgc tcgctcactg aggcgcccgg gcgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgcgc

79

<210> 546

<211> 81

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 546

ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg tttcggcctc

60

agtgagcgag cgagcgcgca g

81

<210> 547

<211> 81

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 547

ctgcgcgctc gctcgctcac tgaggccgaa acgtcgggcg acctttggtc gcccggcctc

60

agtgagcgag cgagcgcgca g

81

<210> 548

<211> 42

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 548

cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gc

42

<210> 549

<211> 42

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 549

gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cg

42

<210> 550

<211> 144

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 550

aggaacccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc

60

cgcccgggca aagcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg

120

agcgcgcaga gagggagtgg ccaa

144

<210> 551

<211> 43

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 551

gcccgctggt ttccagcggg ctgcgggccc gaaacggggcc cgc

43

<210> 552

<211> 28

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 552

cgggcccgtg cgggcccaaa gggcccgc

28

<210> 553

<211> 28

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 553

gcccgggcac gcccgggttt cccgggcg

28

<210> 554

<211> 22

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 554

cgtgcgggcc caaagggccc gc

22

<210> 555

<211> 43

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 555

gcgggccgga aacgggcccg ctgcccgctg gtttccagcg ggc

43

<210> 556

<211> 28

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 556

cgcccgggaa acccgggcgt gcccgggc

28

<210> 557

<211> 28

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 557

gggccgcccg ggaaacccgg gcgtgccc

28

<210> 558

<211> 143

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 558

ttgcccactc cctctctgcg cgctcgctcg ctcggtgggg cctgcggacc aaaggtccgc

60

agacggcaga ggtctcctct gccggcccca ccgagcgagc gacgcgcgca gagagggagt

120

gggcaactcc atcactaggg taa

143

<210> 559

<211> 143

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 559

ttaccctagt gatggagttg cccactccct ctctgcgcgc gtcgctcgct cggtggggcc

60

ggcagaggag acctctgccg tctgcggacc tttggtccgc aggccccacc gagcgagcga

120

gcgcgcagag agggagtggg caa

143

<210> 560

<211> 144

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 560

ttggccactc cctctatgcg cactcgctcg ctcggtgggg cctggcgacc aaaggtcgcc

60

agacggacgt gggtttccac gtccggcccc accgagcgag cgagtgcgca tagagggagt

120

ggccaactcc atcactagag gtat

144

<210> 561

<211> 144

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 561

atacctctag tgatggagtt ggccactccc tctatgcgca ctcgctcgct cggtggggcc

60

ggacgtggaa acccacgtcc gtctggcgac ctttggtcgc caggccccac cgagcgagcg

120

agtgcgcata gagggagtgg ccaa

144

<210> 562

<211> 127

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 562

ttggccactc cctctatgcg cgctcgctca ctcactcggc cctggagacc aaaggtctcc

60

agactgccgg cctctggccg gcagggccga gtgagtgagc gagcgcgcat agagggagtg

120

gccaact

127

<210> 563

<211> 127

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 563

agttggccac attagctatg cgcgctcgct cactcactcg gccctggaga ccaaaggtct

60

ccagactgcc ggcctctggc cggcagggcc gagtgagtga gcgagcgcgc atagagggag

120

tggccaa

127

<210> 564

<211> 166

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 564

tcccccctgt cgcgttcgct cgctcgctgg ctcgtttggg ggggcgacgg ccagaggggcc

60

gtcgtctggc agctctttga gctgccaccc ccccaaacga gccagcgagc gagcgaacgc

120

gacagggggg agagtgccac actctcaagc aagggggttt tgtaag

166

<210> 565

<211> 166

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 565

cttacaaaac ccccttgctt gagagtgtgg cactctcccc cctgtcgcgt tcgctcgctc

60

gctggctcgt ttgggggggt ggcagctcaa agagctgcca gacgacggcc ctctggccgt

120

cgccccccca aacgagccag cgagcgagcg aacgcgacag ggggga

166

<210> 566

<211> 144

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 566

ttgcccactc cctctaatgc gcgctcgctc gctcggtggg gcctgcggac caaaggtccg

60

cagacggcag aggtctcctc tgccggcccc accgagcgag cgagcgcgca tagagggagt

120

gggcaactcc atcactaggg gtat

144

<210> 567

<211> 144

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

polynucleotide

<400> 567

atacccctag tgatggagtt gcccactccc tctatgcgcg ctcgctcgct cggtggggcc

60

ggcagaggag acctctgccg tctgcggacc tttggtccgc aggccccacc gagcgagcga

120

gcgcgcatta gagggagtgg gcaa

144

<210> 568

<211> 12

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 568

atcgaacgat cg

12

<210> 569

<211> 12

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 569

cgatcgttcg at

12

<210> 570

<211> 12

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 570

atcgaaccat cg

12

<210> 571

<211> 39

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 571

ggccgccaaa ggcgtcgggc gacctttggt cgcccggcc

39

<210> 572

<211> 39

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 572

ggccgggcga ccaaaggtcg cccgacgcct ttggcggcc

39

<210> 573

<211> 47

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 573

gccgcccggg caaagcccgg gcgtcgggcg acctttggtc gcccggc

47

<210> 574

<211> 47

<212> DNA/DNA

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic

oligonucleotide

<400> 574

gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggc

47

<---

Claims (61)

1. A non-viral capsid-free DNA vector with covalently closed ends (ccDNA vector) for delivery of a nucleic acid, wherein said cccDNA vector contains at least one nucleic acid sequence located between two flanking symmetrical inverted terminal repeats (symmetrical ITRs), wherein said symmetrical ITRs are not wild-type ITRs, and wherein each of the flanking symmetrical ITRs is modified by a deletion, insertion and/or substitution in the A' and/or A region, and each flanking ITR contains the same symmetrical modification. 2. CctDNA vector according to claim 1, characterized in that said flanking symmetrical ITRs are synthetic. 3. The cccDNA vector according to any of the previous paragraphs, characterized in that said flanking symmetrical ITRs contain sequences selected from the group consisting of SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 486, SEQ ID NO: 487, SEQ ID NO:488, SEQ ID NO:489, SEQ ID NO: 490, SEQ ID NO:491, SEQ ID NO:492, SEQ ID NO:493, SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO: 498, SEQ ID NO: 499, SEQ ID NO: 469, SEQ ID NO: 95, SEQ ID NO: 470, SEQ ID NO: SEQ ID NO: 471, SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:474, SEQ ID NO:475, SEQ ID NO: 476, SEQ ID NO: 477, SEQ ID NO: 478, SEQ ID NO: 479, SEQ ID NO: 480, SEQ ID NO: 481, SEQ ID NO: 482, and SEQ ID NO: 483. 4. The cccDNA vector according to any of the previous paragraphs, characterized in that each of the specified flanking symmetrical ITRs is modified by deletion, insertion and/or substitution in at least one of the ITR regions selected from the group consisting of regions A, A', B, B', C, C, D and D'. 5. The cccDNA vector according to claim 4, characterized in that said deletion, insertion and/or substitution leads to the deletion of the entire stem-loop structure or part thereof formed by regions A, A', B, B'C or with'. 6. The cccDNA vector according to claim 4 or 5, characterized in that each of the flanking symmetrical ITRs is modified by deletion, insertion and/or substitution, which leads to the deletion of the entire stem-loop structure or part thereof formed by regions B and B'. 7. CctDNA vector according to any one of paragraphs. 4-6, characterized in that each of the flanking symmetrical ITRs is modified by a deletion, insertion and/or substitution that results in the deletion of all or part of the stem-loop structure formed by regions C and C'. 8. The cccDNA vector according to claim 6 or 7, characterized in that each of the flanking symmetrical ITRs is modified by deletion, insertion and/or substitution, which leads to the deletion of all or part of the “stem-loop” structure formed by regions B and B', and/or all or part of the stem-loop structure formed by regions C and C'. 9. The cccDNA vector according to claim 8, characterized in that each of the flanking symmetrical ITRs is modified by deletion of the entire stem-loop structure formed by regions B and B', and the entire stem-loop structure formed by areas C and C'. 10. CctDNA vector according to any one of paragraphs. 1-8, characterized in that each of the flanking symmetrical ITRs contains a single stem-loop structure in a region that, in the wild-type ITR, contains the first stem-loop structure formed by regions B and B' , and a second stem-loop structure formed by regions C and C'. 11. The cccDNA vector of claim 10, wherein each of the flanking symmetrical ITRs contains a single stem and two loops in a region which, in a wild-type ITR, contains a first stem-loop structure formed by regions B and B', and a second stem-loop structure formed by regions C and C'. 12. The cccDNA vector according to claim 10 or 11, characterized in that each of the flanking symmetrical ITRs contains a single stem and a single loop in a region which, in a wild-type ITR, contains a first stem-loop structure formed by the regions B and B', and a second stem-loop structure formed by regions C and C'. 13. CctDNA vector according to any one of paragraphs. 1-12, characterized in that said cDNA vector contains one or more aptamers. 14. CctDNA vector according to any one of paragraphs. 1-13, characterized in that said cDNA vector contains at least one regulatory switch. 15. The cccDNA vector according to any of the previous claims, characterized in that said ccDNA vector exhibits characteristic bands of linear and continuous DNA compared to control linear and discontinuous DNA when digested with a restriction enzyme having a single recognition site on the ccDNA vector, and analysis by native and denaturing gel electrophoresis. 16. The cccDNA vector according to any of the previous claims, characterized in that said flanking symmetrical ITRs are taken from a virus selected from the group consisting of parvovirus, dependovirus and adeno-associated virus (AAV). 17. The cccDNA vector according to claim 16, characterized in that said flanking symmetrical ITRs are taken from an adeno-associated virus (AAV). 18. The cccDNA vector according to claim 17, characterized in that said flanking symmetrical ITRs are taken from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , and AAV12. 19. CctDNA vector according to any one of paragraphs. 1-18, characterized in that the specified vector is located in a nanocarrier. 20. The cDNA vector according to claim 19, characterized in that said nanocarrier contains a lipid nanoparticle (LNP). 21. The cccDNA vector according to any of the previous paragraphs, characterized in that the specified ccDNA vector is obtained by a method including the following steps: (a) incubating a population of insect cells bearing a cccDNA expression construct in the presence of at least one Rep protein, wherein said cccDNA expression construct encodes said cccDNA vector, under effective conditions and for a period of time sufficient to induce production of the cccDNA vector in the cells insects; And (b) isolating said cDNA vector from insect cells. 22. The ccDNA vector according to claim 21, characterized in that said ccDNA expression construct is selected from the group consisting of a ccDNA plasmid, a ccDNA bacmid and a ccDNA baculovirus. 23. The cDNA vector according to claim 21 or 22, characterized in that said insect cell expresses at least one Rep protein. 24. The cDNA vector according to claim 23, characterized in that said at least one Rep protein is taken from a virus selected from the group consisting of parvovirus, dependovirus and adeno-associated virus (AAV). 25. The cctDNA vector according to claim 24, characterized in that said at least one Rep protein is taken from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. 26. A cDNA expression construct that encodes a ccDNA vector according to any one of claims. 1-25, wherein the expression construct contains at least one restriction site operably located between said flanking symmetrical ITRs. 27. The cDNA expression construct according to claim 26, which is a cDNA plasmid, ccDNA bacmid or ccDNA baculovirus. 28. A host cell for producing the cccDNA expression construct of claim 26 or 27. 29. The host cell of claim 28, which expresses at least one Rep protein. 30. The host cell of claim 29, wherein said at least one Rep protein is from a virus selected from the group consisting of parvovirus, dependovirus and adeno-associated virus (AAV). 31. The host cell according to claim 30, characterized in that said at least one Rep protein is taken from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. 32. Host cell according to any one of paragraphs. 28-31, characterized in that said host cell is an insect cell. 33. The host cell according to claim 32, wherein said insect cell is an Sf9 cell. 34. A method for obtaining a cDNA vector, comprising: (a) incubating a host cell according to any one of claims. 28-33 under effective conditions and for a period of time sufficient to induce production of the cDNA vector; and (b) isolating said cccDNA from the host cell. 35. Use of a composition containing a cccDNA vector according to any one of claims. 1-25 for treating, preventing, ameliorating, monitoring or diagnosing a disease or disorder in a subject, wherein the disease or disorder is selected from the group consisting of a metabolic disease or disorder, a CNS disease or disorder, an eye disease or disorder, a blood disease or disorder, a disease or a liver disorder, an immune disease or disorder, an infectious disease, a muscle disease or disorder, cancer, and a disease or disorder based on an abnormal level and/or function of a gene product, wherein: said metabolic disease or disorder is selected from the group consisting of diabetes, lysosomal storage disorder, mucopolysaccharide disorder, urea cycle disease or disorder; and glycogen storage diseases or disorders; said CNS disease or disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan's disease, Leigh's syndrome, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma caused by damage to the spinal cord or head, Tay-Sachs disease, Lesch-Nyhan disease, epilepsy, cerebral infarction, mental disorders, schizophrenia, drug or drug addiction, neuroses, psychoses, dementia, paranoia , attention deficit disorders, sleep disorders, pain disorders, eating or weight disorders, as well as cancers and tumors of the central nervous system; said blood disease or disorder is selected from the group consisting of hemophilia A, hemophilia B, thalassemia, anemia and blood cancer; said liver disease or disorder is selected from the group consisting of progressive familial intrahepatic cholestasis (PFIC), liver cancer, and tumors; or said eye disease or disorder is selected from the group consisting of an ophthalmic disorder affecting the retina, posterior tract and/or optic nerve. 36. The use of claim 35, wherein said at least one nucleic acid sequence, when transcribed or translated, modifies an abnormal amount of endogenous protein in a subject. 37. The use of claim 35, wherein said at least one nucleic acid sequence, when transcribed or translated, modifies the abnormal function or activity of an endogenous protein or pathway in a subject. 38. Application according to any one of paragraphs. 35-37, characterized in that said at least one nucleic acid sequence encodes or contains a nucleotide molecule selected from the group consisting of an aptamer, RNAi, siRNA, siRNA, lncRNA and an antisense oligo- or polynucleotide. 39. Application according to any one of paragraphs. 35-37, characterized in that said at least one nucleic acid sequence encodes a protein. 40. Use according to claim 39, characterized in that said protein is a marker protein. 41. Application according to any one of paragraphs. 35-40, wherein said at least one nucleic acid sequence encodes an agonist or antagonist of an endogenous protein or pathway associated with said disease or disorder. 42. Application according to any one of paragraphs. 35-41, characterized in that said cDNA vector additionally contains an aptamer. 43. Application according to any one of paragraphs. 35-41, characterized in that said at least one nucleic acid sequence encodes an antibody. 44. The use of claim 35, wherein said lysosomal storage disorder is selected from the group consisting of Gaucher disease, Pompe disease, metachromatic leukodystrophy (MLD), phenylketonuria (PKU) and Fabry disease. 45. Use according to claim 35, characterized in that said disease or urea cycle disorder is ornithine transcarbamylase (OTC) deficiency. 46. Use according to claim 35, characterized in that said mucopolysaccharide disorder is selected from the group consisting of Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome, Morquio syndrome and Maroteaux-Lamy syndrome. 47. The use of claim 35, characterized in that said ophthalmological disorder affecting the retina, posterior tract and/or optic nerve is selected from the group consisting of diabetic retinopathy, macular degeneration, including age-related macular degeneration, geographic atrophy, vascular or wet macular degeneration, glaucoma, uveitis, retinitis pigmentosa, Stargardt disease, Leber congenital amaurosis (LCA), Usher syndrome, pseudoxanthoma elasticum (PXE), X-chromosomal retinitis pigmentosa (XLRP), X-chromosomal retinal dissection (XLRS), choroideremia , congenital Leber optic neuropathy (LHON), achromatopsia, rod-cone dystrophy, Fuchs corneal endothelial dystrophy, diabetic macular edema; and cancer and tumors of the eyes. 48. Application according to any one of paragraphs. 35-43, characterized in that said disease or disorder is cystic fibrosis. 49. Application according to any one of paragraphs. 35-48, characterized in that said cDNA vector is administered in combination with a pharmaceutically acceptable carrier. 50. A method of delivering a therapeutic protein to a subject, said method comprising administering to said subject a composition containing a cccDNA vector according to any one of claims. 1-25. 51. The method according to claim 50, characterized in that said therapeutic protein is a therapeutic antibody. 52. The method according to claim 50, characterized in that said therapeutic protein is selected from the group consisting of factor VIII, factor IX, factor X, phenylalanine hydroxylase, enzyme, erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase , cytokine, cystic fibrosis transmembrane conductance regulator (CFTR), peptide growth factor and hormone. 53. A nucleic acid delivery kit containing a cccDNA vector according to any one of claims. 1-25 and nanocarrier, packaged in a container with instructions for use. 54. Kit for obtaining cDNA vector according to any one of paragraphs. 1-25, wherein said set comprises an expression construct containing at least one restriction site for insertion of at least one nucleic acid sequence or regulatory switch, or both the first and second, wherein said at least one restriction site operably located between flanking symmetric inverted terminal repeats (symmetric ITRs), wherein said flanking symmetric ITRs are not wild-type ITRs, and instructions for use. 55. The kit of claim 54, further comprising a population of insect cells that does not contain viral capsid coding sequences that, in the presence of the Rep protein, can induce production of a cccDNA vector. 56. Set according to any one of paragraphs. 54-55, further comprising a vector containing a polynucleotide sequence that encodes at least one Rep protein, wherein said vector is suitable for expressing said at least one Rep protein in an insect cell.