WO2023215851A2 - Plasmid optimized for packaging of aav vectors - Google Patents

Abstract

The present invention includes an AAV vector packaging platform. The invention further includes helper plasmids useful for the production of AAV vector particles, as well as methods of producing and using AAV vector particles to introduce transgenes into target cells and treat diseases in subjects in need thereof.

Description

TITLE OF THE INVENTION Plasmid Optimized for Packaging of AAV Vectors

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/339,338, filed May 6, 2022, which is hereby incorporated by reference in its entirety herein.

SEQUENCE LISTING

The present application contains a Sequence Listing in XML format and is herein incorporated by reference in its entirety. Said XML file, created on April 26, 2023, is named 371276 7000WOI 00018 SequenceListingST26.xml and is 61,674 bytes in size.

BACKGROUND OF THE INVENTION

Adeno-associated viruses (AAV) are relatively small members of the parvoviridae family that infect humans and closely related primate species. Normally replicationdefective, these nonenveloped, smgle-stranded DNA viruses require co-infection of the host cell with a helper virus, typically an adenovirus, which provides the viral genes necessary for replication. The AAV genome is approximately 4.7-kilobases in size and is flanked by inverted terminal repeat (ITR) sequences which are required for genome replication and efficient encapsidation. AAVs are attractive candidates for use as gene therapy vectors for a number of reasons including their minimal pathogenicity, ability to infect non-dividing cells, and stable integration largely at a specific locus (AAVS1) in the human genome, though most vector-adapted AAVs are no longer capable of integration due to the lack of a rep (replication) gene in the vector sequence.

Clinical trials demonstrating the efficacy of AAV vectors for correcting smgle- gene disorders and diseases has greatly increased demand for AAV production systems capable of high efficiency production of vector particles at lower costs under good manufacturing practices (GMP) conditions safe for human use. As such, there is a need for AAV vector packaging platforms, including modified helper plasmids capable of producing AAV particles with a variety of capsid serotypes at a scale and efficiency necessary for widespread clinical use. The current invention addresses these needs. SUMMARY OF THE INVENTION

As described herein, the present disclosure relates to adeno-associated virus (AAV) based vector packaging platform. The present disclosure also includes helper plasmids useful for the production of AAV vector particles, as well as methods of producing and using AAV vector particles to introduce transgenes into target cells and treat diseases in subjects in need thereof.

As such, in one aspect, the invention includes An AAV vector packaging platform comprising a helper plasmid comprising a 3 ’ untranslated region (3 ’ UTR) from a naturally occurring AAV serotype, a natural or recombinant serotype AAV capsid (cap) gene, an SV40 origin of replication, a replication (rep) gene comprising a human collagen intron, adenoviral helper factors E2, E4orf6, and VA RNA, and a selectable marker.

In certain embodiments, the SV40 origin of replication is flanked by a p5 promoter located at the 5’ end and an IFNbeta S/MAR element located at the 3’ end.

In certain embodiments, the location of the p5 promoter results in reduced levels of rep protein production.

In certain embodiments, the location of the p5 promoter results in increased packing efficiency of full-length genomes.

In certain embodiments, the location of the p5 promoter results in reduced levels of backbone packaging.

In certain embodiments, the p5 promoter/SV40 origin of replication/IFNbeta S/MAR element comprises a nucleotide sequence set forth in SEQ ID NO: 3.

In certain embodiments, the 3 ’ UTR improves vector production.

In certain embodiments, the rep gene comprising a human collagen intron reduces the ability to form replication competent AAV by recombination.

In certain embodiments, the AAV cap gene is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrhlO, AAVrh25, Anc80L65, LK03, rhl8, rh74, rh32.33, rh39, rh43, OligoOOl, PHP-B, and SparklOO.

In certain embodiments, the inclusion of the adenoviral helper factors improve transfection conditions.

In certain embodiments, the AAV vector packaging platform of the above embodiments or aspects or any embodiment or aspect disclosed herein further comprises a transgene plasmid. In certain embodiments, the transgene plasmid comprises a 3’ inverted repeat (ITR) sequence, a transgene payload, and a 5’ inverted repeat (ITR) sequence.

In certain embodiments, the selectable marker is selected from the group consisting of an antibiotic resistance gene and anon-antibiotic system.

In certain embodiments, the non-antibiotic system is selected from the group consisting of an -NP sugar selection backbone, a synthetic doggybone system, an auxotrophic gene system, and a ccdB-based system.

In certain embodiments, the antibiotic resistance gene is a non-P-lactam resistance gene.

In certain embodiments, the non-P-lactam resistance gene is kanamycin.

In certain embodiments, the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 1.

In certain embodiments, the rep gene comprises a nucleic acid sequence set forth in SEQ ID NO: 2.

In certain embodiments, the helper plasmid comprises an AAV cap gene is derived from AAV3b.

In certain embodiments, the helper plasmid comprises a nucleic acid sequence set forth in SEQ ID NO: 4.

In certain embodiments, the helper plasmid comprises an AAV cap gene is derived from AAVrhlO.

In certain embodiments, the helper plasmid comprises a nucleic acid sequence set forth in SEQ ID NO: 5.

In another aspect, the invention includes an isolated polynucleotide comprising an AAV helper plasmid, said helper plasmid comprising a 3’ untranslated region (3’ UTR) from a naturally occurring AAV serotype, a natural or recombinant serotype AAV capsid (cap) gene, an SV40 origin of replication, a replication (rep) gene comprising a human collagen intron, adenoviral helper factors E2, E4orf6, and VA RNA, and a selectable marker.

In certain embodiments, the isolated polynucleotide of any of the above aspects for embodiments or any embodiment or aspect disclosed herein further comprises an SV40 origin of replication flanked by a p5 promoter located at the 5’ end and an IFNbeta S/MAR element at the 3 ' end.

In certain embodiments, the p5 promoter/SV40 origin of replication/IFNbeta S/MAR element comprises a nucleotide sequence set forth in SEQ ID NO: 3. In certain embodiments, the AAV cap gene is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrhlO, AAVrh25, Anc80L65, LK03, rhl8, rh74, rh32.33, rh39, rh43, OligoOOl, PHP-B, and SparklOO.

In certain embodiments, the selectable marker is a kanamycin resistance gene.

In certain embodiments, the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: I.

In certain embodiments, the rep gene comprises a nucleic acid sequence set forth in SEQ ID NO: 2.

In certain embodiments, the AAV cap gene is derived from AAV3b.

In certain embodiments, the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 4.

In certain embodiments, the AAV cap gene is derived from AAVrhlO.

In certain embodiments, the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 5.

In another aspect, the invention includes a composition compnsing the AAV vector packaging platform of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein.

In another aspect, the invention includes a method for producing an AAV vector particle, said method comprising contacting a cell with the AAV vector packaging platform of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein, culturing the cell to produce the AAV vector particle, and harvesting and purifying the AAV vector particle.

In certain embodiments, the AAV vector packaging platform comprises a helper plasmid and a transgene plasmid.

In certain embodiments, the cell is an immortalized cell.

In certain embodiments, the immortalized cell is selected from the group consisting of a HEK 293T cell, a HEK293 cell lacking a large T element, a HeLa cell, a CHO cell, and a hTERT-immortahzed human cell.

In another aspect, the invention includes a composition comprising AAV vector particles, wherein said AAV vector particles are produced by the method of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein.

In another aspect, the invention includes a method of introducing a transgene into a target cell, comprising contacting an immortalized cell with the AAV vector packaging platform of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein comprising the transgene, thereby producing a packaging cell, culturing the packaging cell to produce AAV vector particles comprising the transgene, isolating and purifying the AAV vector particles, and contacting the target cell with an effective amount of the AAV vector particles, thereby introducing the transgene into the target cell.

In another aspect, the invention includes a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of the above aspects or embodiments or any aspect or embodiment disclosed herein and a pharmaceutically acceptable diluent or excipient.

In certain embodiments, the AAV vector particles comprise a transgene.

In certain embodiments, expression of the transgene corrects the dysfunction of an endogenous gene.

In certain embodiments, the disease is related to a dysfunction of an endogenous gene.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1 is a map illustrating the features and layout of the pR2CrhlO_KanV2 plasmid.

FIG. 2 is a linear map of the pR2Crh!0_KanV2 plasmid.

FIG. 3 is a map illustrating the features and layout of the pR2C3b-KanV2 plasmid.

FIG. 4 is a linear map of the pR2C3b-KanV2 plasmid.

DETAILED DESCRIPTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles “a”, “an”, and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “AAV vector” as used herein refers to a polynucleotide vector comprising one or more genes of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). AAV vectors can be produced and packaged into infectious viral particles when present in a host cell that has been transfected with one or more helper plasmids encoding and expressing rep and cap proteins and one or more proteins from adenovirus open reading frame E4orf6. The AAV vectors may be operably linked to promoter and enhancer sequences that can regulate the expression of the protein encoded by the AAV vector.

The terms “AAV virion” or “AAV viral particle” or “AAV vector particle” as used herein refers to a viral particle composed of capsid proteins from at least one AAV serotype surrounding a polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector”. Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.

The term “Packaging” as used herein refers to intracellular process by which viral virions or particles (e.g. AAV virions or particles), especially viral vector particles or virions are assembled in a host cell. “Packaging” cells comprise the polynucleotide (e g. helper plasmids) and protein components necessary to assemble functional viral virions.

A “biomarker” or “marker” as used herein generally refers to a nucleic acid molecule, clinical indicator, protein, or other analyte that is associated with a disease. In certain embodiments, a nucleic acid biomarker is indicative of the presence in a sample of a pathogenic organism, including but not limited to, viruses, viroids, bacteria, fungi, helminths, and protozoa. In various embodiments, a marker is differentially present in a biological sample obtained from a subject having or at risk of developing a disease (e.g., an infectious disease) relative to a reference. A marker is differentially present if the mean or median level of the biomarker present in the sample is statistically different from the level present in a reference. A reference level may be, for example, the level present in an environmental sample obtained from a clean or uncontaminated source. A reference level may be, for example, the level present in a sample obtained from a healthy control subject or the level obtained from the subject at an earlier timepoint, i.e., prior to treatment. Common tests for statistical significance include, among others, t-test, ANOVA, Kruskal -Wallis, Wilcoxon, Mann- Whitney and odds ratio. Biomarkers, alone or in combination, provide measures of relative likelihood that a subject belongs to a phenotypic status of interest. The differential presence of a marker of the invention in a subject sample can be useful in characterizing the subject as having or at risk of developing a disease (e.g., an infectious disease), for determining the prognosis of the subject, for evaluating therapeutic efficacy, or for selecting a treatment regimen.

By “agent” is meant any nucleic acid molecule, small molecule chemical compound, antibody, or polypeptide, or fragments thereof.

By “alteration” or “change” is meant an increase or decrease. An alteration may be by as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%, or even by as much as 70%, 75%, 80%, 90%, or 100%.

By "biologic sample" is meant any tissue, cell, fluid, or other material derived from an organism.

By "capture reagent" is meant a reagent that specifically binds a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.

As used herein, the terms “determining”, “assessing”, “assaying”, “measuring” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase “determining a level” of an analyte or “detecting” an analyte is used.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

“Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

By "fragment" is meant a portion of a nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides.

“Homologous” as used herein, refers to the subunit sequence identity between two polymeric molecules, e.g, between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleotides that pair through the formation of hydrogen bonds.

“Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity' or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular matenal, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity' are typically determined using analytical chemistry' techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "marker profile" is meant a characterization of the signal, level, expression or expression level of two or more markers (e.g., polynucleotides).

By the term “microbe” is meant any and all organisms classed within the commonly used term “microbiology,” including but not limited to, bacteria, viruses, fungi and parasites

By the term “microarray” is meant a collection of nucleic acid probes immobilized on a substrate. As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or doublestranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that specifically binds a target nucleic acid (e.g., a nucleic acid biomarker). Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subj ect. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

“Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

By "reference" is meant a standard of comparison. As is apparent to one skilled in the art, an appropriate reference is where an element is changed in order to determine the effect of the element. In one embodiment, the level of a target nucleic acid molecule present in a sample may be compared to the level of the target nucleic acid molecule present in a clean or uncontaminated sample. For example, the level of a target nucleic acid molecule present in a sample may be compared to the level of the target nucleic acid molecule present in a corresponding healthy cell or tissue or in a diseased cell or tissue (e.g., a cell or tissue derived from a subject having a disease, disorder, or condition).

As used herein, the term “sample” includes a biologic sample such as any tissue, cell, fluid, or other material derived from an organism.

By “specifically binds” is meant a compound (e.g., nucleic acid probe or primer) that recognizes and binds a molecule (e.g., a nucleic acid biomarker), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYB OX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'³ and e'¹⁰⁰ indicating a closely related sequence.

By the term “substantially microbial hybridization signature” is a relative term and means a hybridization signature that indicates the presence of more microbes in a tumor sample than in a reference sample. By the term “substantially not a microbial hybridization signature” is a relative term and means a hybridization signature that indicates the presence of less microbes in a reference sample than in a tumor sample.

By "subject" is meant a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, feline, mouse, or monkey. The term “subject” may refer to an animal, which is the object of treatment, observation, or experiment (e.g., a patient).

By "target nucleic acid molecule" is meant a polynucleotide to be analyzed. Such polynucleotide may be a sense or antisense strand of the target sequence. The term "target nucleic acid molecule" also refers to amplicons of the original target sequence. In various embodiments, the target nucleic acid molecule is one or more nucleic acid biomarkers.

A “target site” or “target sequence” refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

By the term “tumor tissue sample” is meant any sample from a tumor in a subject including any solid and non-solid tumor in the subject.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention is based on the unexpected observation that certain modifications can be made to AAV helper plasmids which significantly improve the efficiency of both transfection of packaging cells and the production of functional AAV vector particles. These modified helper plasmids can be used in AAV vector packaging platforms which can be used with a variety of capsid proteins, based on the desired tropism and function of the resulting AAV vector particles. As such, the present invention includes AAV viral vector packaging platforms comprising helper plasmids useful for the efficient production of AAV viral particles. Also included are isolated nucleic acids comprising the helper plasmids and methods of producing AAV vector particles and introducing transgenes into target cells, both methods comprising the AAV packaging platform of the invention.

AAV Vectors

AAV are relatively small, non-enveloped viruses with a ~4 kb genome that is flanked by inverted terminal repeats (ITRs). The genome contains two open reading frames, one of which provides proteins necessary for replication and the other provides components required for construction of the viral capsid. Wild-type AAV is typically found in the presence of adenovirus as the adenoviruses provide helper proteins that are essential for packaging of the AAV genome into virions. Therefore, AAV production piggy-backs on co-infection with adenovirus and relies on three key elements: the ITR- flanked genome, the open-reading frames, and adeno-helper genes. Due to their non- pathogenic ability to readily infect human cells, AAV is well-studied as a vector for gene delivery. AAV may be readily obtained and their use as vectors for gene delivery has been described in, for example, Muzyczka, 1992; U.S. Patent No. 4,797,368, and PCT Publication WO 91/18088. Construction of AAV vectors is described in a number of publications, including Lebkowski et al., 1988; Tratschin et al., 1985; Hermonat and Muzy czka, 1984; vectors is described in a number of publications, including Lebkowski et al., 1988; Tratschin et al., 1985; Hermonat and Muzyczka, 1984.

AAV-based vector systems typically separate the viral AAV genes, Adenovirus- derived helper genes, and the transgene payload onto two or three separate plasmids. Three plasmid systems consist of an AAV helper plasmid comprising the rep (replication) and cap (capsid) genes, an adenoviral helper plasmid comprising at least the E2a gene, E4 gene, and VA (viral associated) RNA, and a payload plasmid comprising the transgene and associated promoters and enhancers flanked by ITR sequences. The helper plasmid or plasmids do not comprise ITRs in order to prevent packaging of a functional, infectious viral genome.

Two plasmid systems combine the AAV rep and cap genes and adenoviral helper genes onto a single plasmid and simplify viral vector production by reducing the number of transfected plasmids. Often, a dedicated packaging cell line is used which is engineered to express AAV/helper genes prior to introduction of the payload plasmid.

In certain embodiments, the AAV vector packing platform of the current invention comprises a helper plasmid comprising adenoviral helper genes or factors as well as rep and cap genes. In certain embodiments, the adenoviral helper genes comprise an E2 gene, an E4orf6 gene, and VA RNA. The protein product of the E2 gene participates in viral DNA replication and late genes, while the E4orf6 gene inhibits host cell p53. The VA or viral associated RNA is anon-coding RNA used by adenoviruses to regulate translation of viral genes and increase the stability of ribosome-bound transcripts, as well as preventing the activation of dsRNA-degrading innate cellular defense mechanisms. In certain embodiments, the inclusion of adenoviral helper factors on the same helper plasmid as the rep and cap genes acts to improve transfection conditions.

Successful gene therapies require efficient infection of target tissues and establishment of long term gene expression, and previous studies have demonstrated that AAV vectors can successfully infect and transduce a broad variety of cell and tissue ty pes, such as brain, liver, muscle, among others, and has the ability to infect both dividing and quiescent cells. Additionally, AAV-mediated transduction of tissues has been demonstrated to result in long term gene expression greater than 1.5 years in animal models including canine, murine and hamster.

The tissue tropism of AAV vector particles is influenced by the serotype of the capsid protein, though the receptors and co-receptors that the capsid proteins bind to are often poorly understood and can be expressed by multiple tissue types. For example, AAV2, one of the most well-studied serotypes, has a binding affinity largely for heparan sulfate proteoglycan (HSPG) and as such has a tropism in humans for eye, brain, lung, liver, muscle, and joint tissues. Likewise, AAVs 1, 4, 5, and 6 have a binding affinity largely for sialic acid and a tropism for neuronal tissues and AAVs 5 and 8 which share a tropism for skeletal muscle cell. In this way, the serotype of the AAV capsid protein can be selected to target the pay load nucleic acid of the AAV vector to a specific tissue or cell type. Alteration or modification of capsid protein structure can also alter the tissue or cellular tropism and affinity of the resulting AAV vector particles.

In certain preferred embodiments, the current invention comprises helper plasmids comprising a capsid protein derived from AAV3b and variants thereof. AAV3b and capsid proteins based on AAV3b have a tropism for liver tissues including hepatocytes and are useful for directing therapeutic nucleic acids to those tissues. In certain embodiments, the AAV3b capsid protein is encoded by the nucleic acid set forth in SEQ ID NO: 9. In certain embodiments, the AAV3b capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 10.

In certain preferred embodiments, the current invention comprises helper plasmids comprising a capsid protein derived from AAVrhlO and variants thereof. AAVrhlO and capsid proteins based on AAVrhlO have a tropism for central nervous system (CNS) tissue and are useful for delivering therapeutic nucleic acids to the brain, spinal cord, and peripheral nerve tissue. In certain embodiments, the AAVrhlO capsid protein is encoded by a nucleic acid set forth in SEQ ID NO: 11. In certain embodiments, the AAVrhlO capsid protein is encoded by a nucleic acid sequence set forth in SEQ ID NO: 12.

It is contemplated that the AAV vector platform of the invention can be used with any naturally occurring, modified, hybrid, or engineered AAV capsid protein including, but not limited to AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrhlO, AAVrh25, Anc80L65, LK03, AAVrhl8, AAVrh74, AAVrh32.33, AAVrh39, AAVrh43, OligoOOl, PHP-B, and SparklOO among others. The skilled artisan would be able to select an appropriate capsid protein for use with the invention based on the desired target tissue or cell type.

In certain embodiments, the AAV vector platform of the invention comprises a helper plasmid comprising a replication or rep gene comprising an intron sequence. The AAV rep gene typically encodes four regulatory proteins called Rep78, Rep68, Rep52 and Rep40 which are produced from alternative splicing and, in wildtype virus, two promoters. The term “intron sequence” refers to a nucleotide sequence which is normally removed from mature mRNA by splicing. The inclusion of an intron sequence in the rep gene reduces the possibility of replication-competent AAV particles being formed through recombination events, and as such improves the safety profile of the AAV vector particles produced by the AAV vector platform and methods of the invention. The intron sequence also reduces the risk that the resulting AAV vector will gain the ability to efficiently integrate into the host cell genome through the inclusion of the rep gene into the vector dunng a recombination event. In certain preferred embodiments, the mtron is a human collagen intron. In certain embodiments, the rep gene comprising a human collagen intron comprises the nucleic acid sequence set forth in SEQ ID NO: 8. It is also possible that any intron sequence can be used to provide the safety features of the invention. Thus, it is also contemplated that any human intron sequence can be used with the rep gene of the helper plasmid of the invention.

In certain embodiments, the AAV vector platform of the invention comprises a helper plasmid comprising a 3 ’ untranslated region (3 ’ UTR) from a naturally occurring AAV serotype. The purpose of this region is to improve vector production. In certain embodiments, the 3’ UTR is derived from AAV1. In certain embodiments, the 3’ UTR is derived from AAV7. In certain preferred embodiments, the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 1. It is also contemplated that a 3’ UTR sequence derived from any naturally occurring AAV serotype can be used with the invention in order to provide improved vector production.

In certain embodiments, the AAV vector platform of the invention provides a helper plasmid comprising an SV40 origin of replication. In certain embodiments, the SV40 origin of replication is flanked by a p5 promoter on the 5’ end and an IFNbeta S/MAR element located at the 3’ end. The S/MAR or scaffold/matrix attachment region supports replication function of the helper plasmid. S/MAR regions derived from the human beta-interferon gene have been shown to support efficient episome and transgene expression in mammalian cells, including the host cells of the invention. The p5 promoter is an AAV-derived promoter that drives expression of the Rep gene transcripts (Rep78, Rep68, and Rep58). In certain embodiments of the current invention, the location of the p5 promoter results in reduced production levels of rep proteins. In certain embodiments, the location of the p5 promoter results in increased packaging efficiency of full-length genomes comprising the payload polynucleotide. In certain embodiments, the location of the p5 promoter results in reduced levels of backbone packaging into the resulting viral vector particles. In certain embodiments, the SV40 origin of replication with flanking 5’ p5 promoter and 3’ IFNbeta S/MAR element comprises the nucleic acid sequence set forth in SEQ ID NO: 7.

In certain embodiments, the AAV vector platform of the invention comprises a helper plasmid comprising a selectable marker. As used herein, the term “selectable marker” refers to a gene that encodes a protein that confers the ability of a host cell to grow under conditions which the host cell would not normally grow. In certain embodiments, the selectable marker may confer resistance to an antibiotic or drug to the host cell in which the selectable marker is expressed. The purpose of the selectable marker is to select for host cells which successfully express the helper plasmid during production of the AAV vector particles.

In certain embodiments, the helper plasmid of the invention comprises a selectable marker appropriate for clinical use. In certain embodiments, the selectable marker is an antibiotic resistance gene. Recent studies have demonstrated the desirability for non- - lactam antibiotic resistance markers in order to avoid allergy or sensitivity in patients to residual antibiotic. In certain preferred embodiments, the non- -lactam antibiotic resistance gene is kanamycin. Kanamycin is a member of the aminoglycoside class of antibiotics which functions by inhibiting ribosomal translocation and thereby inhibiting protein translation.

It is also contemplated that the selectable marker used in the helper plasmid of the invention could be a non-antibiotic system. Several non-antibiotic selection systems are known in the art and include, but are not limited to an -NP sugar selection backbone, a synthetic doggybone system, an auxotrophic gene system, and a ccdB-based system among others. It is to be understood that the invention is not limited by the type of selectable marker used. Table 1: Sequences used in the invention

Nucleic Acids and Vectors

The present disclosure provides an isolated nucleic acid encoding a polypeptide.

The nucleic acid of the present disclosure may comprise a polynucleotide sequence encoding any one of the AAV packaging platforms, helper plasmids, and constituent features disclosed herein.

In certain embodiments, AAV vector packaging system comprises a helper plasmid comprising a 3’ untranslated region (3’ UTR) derived from a naturally occurring AAV serotype. In certain embodiments, the 3’ UTR is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO: I.

In certain embodiments, the 3’ untranslated region is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO: 1.

In certain embodiments, the 3’ untranslated region is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO: 1.

In certain embodiments, the AAV vector packaging system comprises a helper plasmid comprising a replication (rep) gene comprising a human collagen intron. In certain embodiments, the rep gene is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 2.

In certain embodiments, the rep gene is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO: 2.

In certain embodiments, the rep gene is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO: 2.

In certain embodiments, the AAV vector packaging system comprises a helper plasmid comprising a SV40 origin of replication is flanked by a p5 promoter located at the 5‘ end and an IFNbeta S/MAR element located at the 3’ end. In certain embodiments, the region comprising the SV40 origin of replication, p5 promoter, and 3’ IFNbeta S/MAR element is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 3.

In certain embodiments, the region comprising the SV40 origin of replication, p5 promoter, and 3’ IFNbeta S/MAR element is encoded by a nucleic acid comprising the polynucleotide sequence set forth in SEQ ID NO: 3.

In certain embodiments, the region comprising the SV40 origin of replication, p5 promoter, and 3’ IFNbeta S/MAR element is encoded by a nucleic acid consisting of the polynucleotide sequence set forth in SEQ ID NO: 3. In certain embodiments, the AAV vector packaging system comprising a helper plasmid. In certain embodiments, the helper plasmid is encoded by a nucleic acid comprising a polynucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 96%, 97%, 98%, 99% identity to SEQ ID NOs: 4-6.

In certain embodiments, the AAV vector packaging system comprising a helper plasmid is encoded by a nucleic acid comprising a polynucleotide sequence set forth in SEQ ID NOs: 4-6.

In certain embodiments, the AAV vector packaging system comprising a helper plasmid is encoded by a nucleic acid consisting of a polynucleotide sequence set forth in SEQ ID NOs: 4-6.

Also provided is a host cell comprising any of the nucleic acids disclosed herein. The host cell may be of eukaryotic, prokaryotic, mammalian, or bacterial origin. A method of producing an AAV vector particle is also provided herein, wherein the method comprises culturing the host cell.

In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is an immortalized cell. Non-limiting examples of immortalized cells suitable for use as host cells for the nucleic acids of the invention include but are not limited to HEK 293T cells, HEK293 cells lacking a large T element, HeLa cells, CHO cells, and hTERT-immortalized human cells, among others. The skilled artisan would be able to select an immortalized cell suitable for use as a host cell based on the desired application of the nucleic acids of the invention.

Methods of Use

In another aspect, the invention provides a method for producing an AAV vector particle, said method comprising: contacting a cell with the AAV vector packaging platform of the invention, culturing the cell to produce the AAV vector particle, and harvesting and purifying the AAV vector particle. In certain embodiments, the AAV vector packaging platform comprises a helper plasmid and a transgene plasmid. In certain embodiments, the transgene plasmid comprises a payload gene operably linked to a promoter sequence and flanked by AAV ITR sequences. In certain embodiments, the helper plasmid comprises any of the helper plasmids of the current invention or disclosed herein.

In another aspect, the invention also provides a method of introducing a transgene into a target cell, comprising contacting an immortalized cell with the AAV vector packaging platform of the invention, thereby producing a packaging cell, culturing the packaging cell to produce AAV vector particle comprising the transgene, isolating and purifying the AAV vector particles, and contacting the target cell with an effective amount of the AAV vector particles.

In certain embodiments, the cell comprising the AAV vector packaging platform of the invention is an immortalized cell or cell line and comprises a packaging cell. In certain embodiments, the immortalized cell is selected from the group consisting of a HEK293 cell, a HEK293 cell lacking a large T element, a HeLa cell, a CHO cell, and a hTERT-immortalized cell. It is also contemplated that the AAV vector packing platform of the invention can be used with any human cell or cell line that can be transduced or transfected to express the helper and transgene plasmids of the invention. One who is skilled in the art would be able to select a cell or cell line appropriate for use as a packaging cell with the AAV vector packaging platform of the invention.

In certain embodiments, the method of the invention comprises harvesting and purifying the AAV vector particles. A number of techniques are known in the art for the efficient purification of AAV particles including, but not limited to centrifugation over a gradient and chromatography. In certain embodiments, the centrifugation occurs at very high speeds and is also known as ultracentrifugation or ultrahigh speed centrifugation. Purification methods employing centrifugation often include the use of a gradient in which the separation of viral particles is based on the density of the particles in a medium of varying density. A number of different density mediums are known in the art including but not limited to cesium chloride (CsCl), sepharose-based medium, and iodixanol including OptiPrep™. Chromatography-based purification methods include, but are not limited to affinity based, heparin based, and ion exchange techniques.

Affinity based techniques make use of antibodies specific for the assembled AAV capsid. Heparin-based techniques take advantage that AAV particles bind to heparin sulfate proteoglycan with high affinity. In certain embodiments, the purification method involves a chemical partitioning technique including an aqueous two-phase system. In certain embodiments, the purification technique involves any combination of these techniques.

In another aspect, the invention includes a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising AAV vector particles, wherein said vector particles are produced by any of the methods of the invention disclosed herein. Pharmaceutical Compositions

Certain embodiments of the disclosure are directed to therapeutically treating an individual in need thereof. As used herein, the term "therapeutically" includes, but is not limited to, the administration of a treatment comprising an AAV vector particle to a subject who displays symptoms or signs of pathology, disease, or disorder, in which treatment is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of pathology, disease, or disorder.

As used herein, the term "subject" is intended to include living organisms such as mammals. Examples of subjects include, but are not limited to, horses, cows, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, mice, gerbils, non-human primates, humans and the like, non-mammals, including, e.g, non-mammalian vertebrates, such as birds (e.g. , chickens or ducks) fish or frogs (e.g. , Xenopus), and a non-mammalian invertebrates, as well as transgenic species thereof. Preferably, the subject is a human.

Pharmaceutical compositions of the present disclosure may comprise the therapeutic engineered AAV vector particles as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate- buffered saline (PBS) and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are preferably formulated for a number of administration routes including oral, inhalation, nasal, nebulization, intravenous injection, intramuscular injection, intrathecal injection, intrapleural injection, intracistemal magna injection, subcutaneous injection, and/or trans dermal injection. Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient’s disease, and the type and functional nature of the patient’s immune response to the phage particles, although appropriate dosages may be determined by clinical trials.

The therapeutic AAV vector particles of the disclosure can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Administration of the AAV vector particles of the disclosure may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

In certain embodiments, the effective dose range is measured in units known to a person of skill in the art to be suitable for the description of AAV vector particle doses. In some embodiments, the effective dose range for a vaccine or therapeutic compound of the disclosure is measured by transducing units (TU)/kg/dose or genome copies(GC)/kg/dose or particles/kg/dose. In some embodiments, the dosage provided to a patient is between about 10⁶ - 10¹⁴ TU/kg In some embodiments, the dosage provided to a patient is between about 10⁶ - 10¹⁴ GC/kg. In some embodiments, the effective dose range is measured by colony forming units (CFU), 50% tissue culture infectious dose (TCID50), and combinations thereof.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of a disease or disorder contemplated in the disclosure.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every' three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

Dosage size can be adjusted according to the weight, age, and stage of the disease of the subject being treated. AAV vector particles may also be administered multiple times at these dosages. The AAV vector particles can be administered by using infusion techniques that are commonly known in the art of immunotherapy or vaccinology. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

The administration of the AAV vector particle compositions of the disclosure may be carried out in any convenient manner known to those of skill in the art. The AAV vector particles of the present disclosure may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a subject or patient trans-arterially, subcutaneously, intranasally, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, or intraperitoneally. In other instances, the AAV vector particles of the disclosure are injected directly into a site of inflammation in the subject, a local disease site in the subject, a LN, an organ, a tumor, and the like. It should be understood that the method and compositions that would be useful in the present disclosure are not limited to the particular formulations set forth in the examples.

In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.

The carrier can be a solvent or dispersion medium containing, for example, saline, buffered saline, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it is advisable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.

Formulations can be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for any suitable mode of administration, known to the art. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They can also be combined where desired with other active agents, e.g., analgesic agents.

The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); 30 “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzy mology ” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and AAV particles of the disclosure, and, as such, may be considered in making and practicing the disclosure.

It should be understood that the method and compositions that would be useful in the present disclosure are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

EXPERIMENTAL EXAMPLES

The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the disclosure is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein. Example 1: A high-efficiency AAV helper plasmid system

FIGs. 1 and 2 are maps of the pR2CrhlO helper plasmid comprising an AAVrhlO capsid protein. FIG. 1 is a circular view and FIG. 2 is a linear view showing the location of various features.

FIGs. 3 and 4 are maps of the pR2C3b helper plasmid comprising an AAV3 capsid protein. FIG. 3 is a circular view and FIG. 4 is a linear view showing the location of various features.

Enumerated Embodiments

The following enumerated embodiments are provided, the numbering of which is not to be constmed as designating levels of importance.

Embodiment 1 provides an AAV vector packaging platform comprising a helper plasmid comprising: i. a 3’ untranslated region (3’ UTR) from a naturally occurring AAV serotype; ii. a natural or recombinant serotype AAV capsid (cap) gene; iii. an SV40 origin of replication iv. a replication (rep) gene comprising a human collagen intron; v. adenoviral helper factors E2, E4orf6, and VA RNA; and vi. a selectable marker.

Embodiment 2 provides the AAV vector packaging platform of embodiment 1, wherein the SV40 origin of replication is flanked by a p5 promoter located at the 5’ end and an IFNbeta S/MAR element located at the 3’ end.

Embodiment 3 provides the AAV vector packaging platform of embodiment 2, wherein the location of the p5 promoter results in reduced levels of rep protein production.

Embodiment 4 provides the AAV vector packaging platform of embodiment 2, wherein the location of the p5 promoter results in increased packing efficiency of full- length genomes.

Embodiment 5 provides the AAV vector packaging platform of embodiment 2, where in the location of the p5 promoter results in reduced levels of backbone packaging. Embodiment 6 provides the AAV vector packaging platform of embodiment 2, wherein the p5 promoter/SV40 origin of replication/IFNbeta S/MAR element comprises a nucleotide sequence set forth in SEQ ID NO: 3.

Embodiment 7 provides the AAV vector packaging platform of embodiment 1, wherein the 3 ’ UTR improves vector production.

Embodiment 8 provides the AAV vector packaging platform of embodiment 1, wherein the rep gene comprising a human collagen intron reduces the ability to form replication competent AAV by recombination.

Embodiment 9 provides the AAV vector packaging platform of embodiment 1, wherein the AAV cap gene is a serotype selected from the group consisting of AAV 1 , AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 12, AAV- Bl, AAV-DJ, AAV -Retro, AAVrh8, AAVrhlO, AAVrh25, Anc80L65, LK03, rhl8, rh74, rh32.33, rh39, rh43, OligoOOl, PHP-B, and SparklOO.

Embodiment 10 provides the AAV vector packaging platform of embodiment 1, wherein the inclusion of the adenoviral helper factors improve transfection conditions.

Embodiment 11 provides the AAV vector packaging platform of embodiment 1 , further comprising a transgene plasmid.

Embodiment 12 provides the AAV vector packaging platform of embodiment 11, wherein the transgene plasmid comprising a 3’ inverted repeat (ITR) sequence, a transgene payload, and a 5’ inverted repeat (ITR) sequence.

Embodiment 13 provides the AAV vector packaging platform of embodiment 1 , wherein the selectable marker is selected from the group consisting of an antibiotic resistance gene and a non-antibiotic system.

Embodiment 14 provides the AAV vector of embodiment 13, wherein the nonantibiotic system is selected from the group consisting of an -NP sugar selection backbone, a synthetic doggybone system, an auxotrophic gene system, and a ccdB-based system.

Embodiment 15 provides the AAV vector of embodiment 13, wherein the antibiotic resistance gene is a non-p-lactam resistance gene. Embodiment 16 provides the AAV vector packaging platform of embodiment 15, wherein the non-p-lactam resistance gene is kanamycin.

Embodiment 17 provides the AAV vector packaging platform of embodiment 1, wherein the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 1.

Embodiment 18 provides the AAV vector packaging platform of embodiment 1 , wherein the rep gene comprises a nucleic acid sequence set forth in SEQ ID NO: 2.

Embodiment 19 provides the AAV vector packaging platform of embodiment 1 , wherein the helper plasmid comprises an AAV cap gene is derived from AAV3b.

Embodiment 20 provides the AAV vector packaging platform of embodiment 18, wherein the helper plasmid comprises a nucleic acid sequence set forth in SEQ ID NO: 4.

Embodiment 21 provides the AAV vector packaging platform of embodiment 1 , wherein the helper plasmid comprises an AAV cap gene is derived from AAVrhlO.

Embodiment 22 provides the AAV vector packaging platform of embodiment 20, wherein the helper plasmid comprises a nucleic acid sequence set forth in SEQ ID NO: 5.

Embodiment 23 provides an isolated polynucleotide comprising an AAV helper plasmid, said helper plasmid comprising: i. a 3’ untranslated region (3’ UTR) from a naturally occurring AAV serotype; ii. a natural or recombinant serotype AAV capsid (cap) gene; hi. an SV40 origin of replication iv. a replication (rep) gene comprising a human collagen intron; v. adenoviral helper factors E2, E4orf6, and VA RNA; and vi. a selectable marker.

Embodiment 24 provides the isolated polynucleotide of embodiment 23, further comprising an SV40 origin of replication flanked by a p5 promoter located at the 5’ end and an IFNbeta S/MAR element at the 3’ end.

Embodiment 25 provides the isolated polynucleotide of embodiment 23, wherein the p5 promoter/SV40 origin of replication/IFNbeta S/MAR element comprises a nucleotide sequence set forth in SEQ ID NO: 3. Embodiment 26 provides the isolated polynucleotide of embodiment 23, wherein the AAV cap gene is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrhlO, AAVrh25, Anc80L65, LK03, rhl8, rh74, rh32.33, rh39, rh43, OligoOOl, PHP-B, and SparklOO.

Embodiment 27 provides the isolated polynucleotide of embodiment 23, wherein the selectable marker is a kanamycin resistance gene.

Embodiment 28 provides the isolated polynucleotide of embodiment 23, wherein the 3' UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 1.

Embodiment 29 provides the isolated polynucleotide of embodiment 23, wherein the rep gene comprises a nucleic acid sequence set forth in SEQ ID NO: 2.

Embodiment 30 provides the isolated polynucleotide of embodiment 23, wherein the AAV cap gene is derived from AAV3b.

Embodiment 31 provides the isolated polynucleotide of embodiment 30, wherein the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 4.

Embodiment 32 provides the isolated polynucleotide of embodiment 23, wherein the AAV cap gene is derived from AAVrhlO.

Embodiment 33 provides the isolated polynucleotide of embodiment 32, wherein the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 5.

Embodiment 34 provides a composition comprising the AAV vector packaging platform of any one of embodiments 1-22.

Embodiment 35 provides a method for producing an AAV vector particle, said method comprising: i. contacting a cell with the AAV vector packaging platform of any one of embodiments 1-21; ii. culturing the cell to produce the AAV vector particle; and iii. harvesting and purifying the AAV vector particle.

Embodiment 36 provides the method of embodiment 35, wherein the AAV vector packaging platform comprises a helper plasmid and a transgene plasmid. Embodiment 37 provides the method of embodiment 35, wherein the cell is an immortalized cell.

Embodiment 38 provides the method of embodiment 35, wherein the immortalized cell is selected from the group consisting of a HEK 293T cell, a HEK293 cell lacking a large T element, a HeLa cell, a CHO cell, and a hTERT-immortalized human cell.

Embodiment 39 provides a composition comprising AAV vector particles, wherein said AAV vector particles are produced by the method of any one of embodiments 35-38.

Embodiment 40 provides a method of introducing a transgene into a target cell, comprising: i. contacting an immortalized cell with the AAV vector packaging platform of any one of embodiments 1-21 comprising the transgene, thereby producing a packaging cell; ii. culturing the packaging cell to produce AAV vector particles comprising the transgene: iii. isolating and purifying the AAV vector particles; and iv. contacting the target cell with an effective amount of the AAV vector particles, thereby introducing the transgene into the target cell.

Embodiment 41 provides a method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the composition of embodiment 39 and a pharmaceutically acceptable diluent or excipient.

Embodiment 42 provides the method of embodiment 41, wherein the AAV vector particles comprise a transgene.

Embodiment 43 provides the method of embodiment 42, wherein expression of the transgene corrects the dysfunction of an endogenous gene.

Embodiment 44 provides the method of embodiment 41, wherein the disease is related to a dysfunction of an endogenous gene.

Other Embodiments The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. An AAV vector packaging platform comprising a helper plasmid comprising: i. a 3’ untranslated region (3’ UTR) from a naturally occurring AAV serotype; ii. a natural or recombinant serotype AAV capsid (cap) gene; hi. an SV40 ongin of replication iv. a replication (rep) gene comprising a human collagen intron; v. adenoviral helper factors E2. E4orf6, and VA RNA; and vi. a selectable marker.

2. The AAV vector packaging platform of claim 1, wherein the SV40 origin of replication is flanked by a p5 promoter located at the 5 ’ end and an IFNbeta S/MAR element located at the 3’ end.

3. The AAV vector packaging platform of claim 2, wherein the location of the p5 promoter results in reduced levels of rep protein production.

4. The AAV vector packaging platform of claim 2, wherein the location of the p5 promoter results in increased packing efficiency of full-length genomes.

5. The AAV vector packaging platform of claim 2, where in the location of the p5 promoter results in reduced levels of backbone packaging.

6. The AAV vector packaging platform of claim 2, wherein the p5 promoter/SV40 origin of replication/IFNbeta S/MAR element comprises a nucleotide sequence set forth in SEQ ID NO: 3.

7. The AAV vector packaging platform of claim 1, wherein the 3’ UTR improves vector production. The AAV vector packaging platform of claim 1, wherein the rep gene comprising a human collagen intron reduces the ability to form replication competent AAV by recombination. The AAV vector packaging platform of claim 1, wherein the AAV cap gene is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV- Retro, AAVrh8, AAVrhlO, AAVrh25, Anc80L65, LK03, rhl8, rh74, rh32.33, rh39, rh43, OligoOOl, PHP-B, and SparklOO. The AAV vector packaging platform of claim 1, wherein the inclusion of the adenoviral helper factors improve transfection conditions. The AAV vector packaging platform of claim 1, further comprising a transgene plasmid. The AAV vector packaging platform of claim 11, wherein the transgene plasmid comprising a 3’ inverted repeat (ITR) sequence, a transgene payload, and a 5’ inverted repeat (ITR) sequence. The AAV vector packaging platform of claim 1 , wherein the selectable marker is selected from the group consisting of an antibiotic resistance gene and a nonantibiotic system. The AAV vector of claim 13, wherein the non-antibiotic system is selected from the group consisting of an -NP sugar selection backbone, a synthetic doggybone system, an auxotrophic gene system, and a ccdB-based system. The AAV vector of claim 13, wherein the antibiotic resistance gene is a non- - lactam resistance gene. The AAV vector packaging platform of claim 15, wherein the non-P-lactam resistance gene is kanamycin. The AAV vector packaging platform of claim 1, wherein the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 1. The AAV vector packaging platform of claim 1, wherein the rep gene comprises a nucleic acid sequence set forth in SEQ ID NO: 2. The AAV vector packaging platform of claim 1, wherein the helper plasmid comprises an AAV cap gene is derived from AAV3b. The AAV vector packaging platform of claim 19, wherein the helper plasmid comprises a nucleic acid sequence set forth in SEQ ID NO: 4. The AAV vector packaging platform of claim 1, wherein the helper plasmid comprises an AAV cap gene is derived from AAVrhlO. The AAV vector packaging platform of claim 21, wherein the helper plasmid comprises a nucleic acid sequence set forth in SEQ ID NO: 5. An isolated polynucleotide comprising an AAV helper plasmid, said helper plasmid comprising: i. a 3’ untranslated region (3’ UTR) from a naturally occurring AAV serotype; ii. a natural or recombinant serotype AAV capsid (cap) gene; iii. an SV40 origin of replication iv. a replication (rep) gene comprising a human collagen intron; v. adenoviral helper factors E2, E4orf6, and VA RNA; and vi. a selectable marker. The isolated polynucleotide of claim 23, further comprising an SV40 origin of replication flanked by a p5 promoter located at the 5 ’ end and an IFNbeta S/MAR element at the 3’ end. The isolated polynucleotide of claim 23, wherein the p5 promoter/SV40 origin of replication/IFNbeta S/MAR element comprises a nucleotide sequence set forth in SEQ ID NO: 3. The isolated polynucleotide of claim 23, wherein the AAV cap gene is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV-B1, AAV-DJ, AAV-Retro, AAVrh8, AAVrhlO, AAVrh25, Anc80L65, LK03, rhl8, rh74, rh32.33, rh39, rh43, OligoOOl, PHP-B, and SparklOO. The isolated polynucleotide of claim 23, wherein the selectable marker is a kanamycin resistance gene. The isolated polynucleotide of claim 23, wherein the 3’ UTR comprises a nucleic acid sequence set forth in SEQ ID NO: 1. The isolated polynucleotide of claim 23, wherein the rep gene comprises a nucleic acid sequence set forth in SEQ ID NO: 2. The isolated polynucleotide of claim 23, wherein the AAV cap gene is derived from AAV3b. The isolated polynucleotide of claim 30, wherein the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 4. The isolated polynucleotide of claim 23, wherein the AAV cap gene is derived from AAVrhlO. The isolated polynucleotide of claim 32, wherein the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 5. A composition comprising the AAV vector packaging platform of any one of claims 1-22. A method for producing an AAV vector particle, said method comprising: i. contacting a cell with the AAV vector packaging platform of any one of claims 1-21; ii. culturing the cell to produce the AAV vector particle; and iii. harvesting and purifying the AAV vector particle. The method of claim 35, wherein the AAV vector packaging platform comprises a helper plasmid and a transgene plasmid. The method of claim 35, wherein the cell is an immortalized cell. The method of claim 37, wherein the immortalized cell is selected from the group consisting of a HEK 293T cell, a HEK293 cell lacking a large T element, a HeLa cell, a CHO cell, and a hTERT-immortalized human cell. A composition comprising AAV vector particles, wherein said AAV vector particles are produced by the method of any one of claim 34-37. A method of introducing a transgene into a target cell, comprising: i. contacting an immortalized cell with the AAV vector packaging platform of any one of claims 1-21 comprising the transgene, thereby producing a packaging cell; ii. culturing the packaging cell to produce AAV vector particles comprising the transgene; iii. isolating and purifying the AAV vector particles; and iv. contacting the target cell with an effective amount of the AAV vector particles, thereby introducing the transgene into the target cell. A method of treating a disease in a subject in need thereof, comprising administering to the subject an effective amount of the composition of claim 39 and a pharmaceutically acceptable diluent or excipient. The method of claim 41, wherein the AAV vector particles comprise a trans gene. The method of claim 42, wherein expression of the transgene corrects the dysfunction of an endogenous gene. The method of claim 41, wherein the disease is related to a dysfunction of an endogenous gene.