AU2016202153B2 - Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells - Google Patents
Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells Download PDFInfo
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Abstract
The present invention relates nucleic acid constructs for the production of recombinant parvoviral (e.g. adeno-associated viral) vectors in insect cells, to insect cells comprising 5 such constructs and to methods wherein the cells are used to produce recombinant parvoviral virions. The insect cells preferably comprise a first nucleotide sequence encoding the parvoviral rep proteins whereby the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon that effects partial exon skipping upon expression in insect cells. The insect cell further comprises a second 10 nucleotide sequence comprising at least one parvoviral (AAV) inverted terminal repeat (ITR) nucleotide sequence and a third nucleotide sequence comprising a sequences coding for the parvoviral capsid proteins.
Description
Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells
The present application is a divisional application of Australian Application No. 2013254897, which is incorporated in its entirety herein by reference. 5 Field of the invention
The present invention relates to the production of adeno-associated virus in insect cells and to adeno-associated virus with improvements in expression and stability of the viral rep proteins that increase the productivity of adeno-associated viral vectors in insect cells. 10 Background of the invention
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
Adeno-associated virus (AAV) may be considered as one of the most promising 15 viral vectors for human gene therapy. AAV has the ability to efficiently infect dividing as well as non-dividing human cells, the AAV viral genome integrates into a single chromosomal site in the host cell's genome, and most importantly, even though AAV is present in many humans it has never been associated with any disease. In view of these advantages, recombinant adeno-associated virus (rAAV) is being evaluated in gene 20 therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, and other diseases.
Host cells that sustain AAV replication in vitro are all derived from mammalian cell types. Therefore, rAAV for use in gene therapy has thus far mainly been produced on mammalian cell lines such as e.g. 293 cells, COS cells, HeLa cells, KB cells, and 25 other mammalian cell lines (see e.g. US 6,156,303, US 5,387,484, US 5,741,683, US 5,691,176, US 5,688,676, US 20020081721, WO 00/47757, WO 00/24916, and WO 96/17947). rAAV vectors are typically produced in such mammalian cell culture systems by providing DNA plasmids that contain the therapeutic gene flanked by the origin of AAV replication (inverted terminal repeats or ITRs), genes for AAV replication proteins 30 Rep78, Rep68, Rep52, and Rep40, and genes for virion or structural proteins VP1, VP2, and VP3. In addition, a plasmid containing early genes from adenovirus (E2A, E40RF6, VARNA) is provided to enhance the expression of the AAV genes and improve vector yield (see e.g. Grimm et al, 1998, Hum. Gene Ther. 9: 2745-2760). However, in most of these mammalian cell culture systems, the number of AAV particles generated per cell is in the order of 104 particles (reviewed in Clark, 2002, Kidney Int. 61_(Suppl. 1): 9-15). Fomic^ panicles of rAAV may be required. To produce ibis number of rAAV particles, transfect km and culture with approximately ,!0'u cultured human 293 cells, the equivalent of 5,000 175-enf flasks of cells, would be required,, which means transfecting up to 10* * 293 cells. Therefore, large scale production of rAAV using mammalian cell culture systems to obtain material for elimeal trials has already proven to be problematic, production at commercial scale may not even be feasible. Furthermore there is always the risk, that a vector for elimeal use that is produced in a mammalian ceil culture will be contaminated with undesirable, perhaps pathogenic, material present in the nmniniali.au. host cell.
To overcome these problems of mammalian productions systems, recently, an AAV production system has been, developed using insect cells (Urabe ei af, 2002, Hum, Gene Ther. JL3; 1935-1943; US 20030148506 and US 20040197895). For production of AAV in insect ceils some modifications were necessary in order to achieve the correct stoichiometry of the three AAV capsid proteins (VF1, VP2 and VP3), which relies on a comhination of alternate usage of two splice acceptor sites and the suboptimai utilization of an ACG initiation codon for YP2 that is not accurately reproduced hy insect cells. To mimic the correct stoichiometry of the capsid proteins in insect cells Urabe el al. (2002, supra) use a construct that is transcribed into a single polycisitonic messenger that is able fo express ail three VP proteins without requiring splicing and wherein the most upstream initiator codon is replaced hy the sohoptimal initiator codon ACG. in co-pending application (PCT/NL2005/0500I.8) the present inventors have-further improved the Infeetlvity of haculovirus-produced rAAV vectors based production by tardier optimisation of the stoichiometry of AAV capsid proteins in insect cells,
For expression of the AAV Rep proteins in the AAV insect cel! expression system as initially developed by Urabe ef al, (2002, mpra'% a recombinant bacnloviras construct is used that harbours two independent Rep expression units (one for R.ep78 and one for Rep5.2), each under the control of a separate insect cell promoter, the ΛΙΒ1 and PoIH promotel's, respectively. In this system, the ΔΙΕ.1 promoter, a. much weaker promoter than the Poll:! promoter, was chosen for driving Rep78 expression since it is known that in mammalian ceils a less abundant expression of Rep78 as compared, to Rep52 favours high vector yields (Li ei ah, 1.997,1 Virol, 7R 5236-43; Grimm et ah, 1998, supra).
More recently however, Kohlbrenner et al. (2005, Mol. Ther. 12: 1217-25) reported that the baculovirus construct for expression of the two Rep protein, as used by Urabe et ah, suffers from an inherent instability. By splitting the palindromic orientation of the two Rep genes in Urabe's original vector and designing two separate baculovirus vectors for expressing Rep52 and Rep78, Kohlbrenner et al. (2005, supra) increased the passaging stability of the vector. However, despite the consistent expression of Rep78 and Rep52 from the two independent baculovirus-Rep constructs in insect cells over at least 5 passages, rAAV vector yield is 5 to 10fold lower as compared to the original baculovirus-Rep construct designed by Urabe et al. (2002, supra).
There is thus still a need to overcome the above serious limitations of large scale (commercial) production of AAV vectors in insect cells. The present invention relates to means and methods that allow for stable and high yield (large scale) production of AAV vectors in insect cells.
It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.
Summary of the Invention
According to a first aspect of the invention there is provided a first nucleic acid construct comprising a first nucleic acid sequence which encodes both Rep78 and Rep52 proteins from a Rep78 nucleotide sequence; the first nucleic acid sequence being operably linked to an expression control sequence that includes a promoter that is active in insect cells and is constructed such that both Rep78 and Rep52 are produced upon expression in an insect cell.
According to a second aspect of the invention there is provided an insect cell comprising the nucleic acid construct according to the invention, wherein said nucleic acid construct expresses parvoviral Rep78 and Rep52 proteins.
According to a third aspect of the invention there is provided a recombinant AAV virion produced by: (i) culturing the insect cell of the invention under conditions that permit production of the recombinant AAV virion; and (ii) recovering the recombinant AAV virion.
According to a fourth aspect of the invention there is provided a method for producing a recombinant parvoviral virion in an insect cell, comprising: (a) culturing the insect cell of the invention under conditions such that recombinant parvoviral virion is produced; and, (b) recovering the recombinant parvoviral virion from the culture.
According to a fifth aspect of the invention there is provided a recombinant parvoviral virion produced by the method according to the invention.
Description of the invention
Definitions
Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.
As used herein, the term “operably linked” refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame. "Expression control sequence" refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked. An expression control sequence is "operably linked" to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signal for introns, and stop codons. The term "expression control sequence" is intended to include, at a minimum, a sequence whose presence are designed to influence expression, and can also^ advantageous components. For example, leader .sequences and fusion partner sequences are expression control sequences. The term can also Include the design of die nucleic acid sequence such, that undesirable, potential initiation codons in and out of frame, are removed .from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or poiyadenylation sequences (pAj which direct the addition of a poiyA tall, fe„ a string of adenine residues at the 3 «end of a mifNA, sequences .inferred to. as pofyA sequences. It also can be designed to enhance rnRNA stability. Expression control sequences which affect the transcription amt translation stability,, e.g., promoters, as well as sequences which effect the translation, e,g., Kozak sequences, are known in insect cells. Expression control sequences can be of such nat ure as to modulate the nucleotide sequence to which it is operaMy linked such that lower expression levels or higher expression, levels are achieved.
As used herein, the term “promoter” or 'transcription regulatory sequence* refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for 'DNA-dependent RNA polymerase, transcription initiation, sites and any other DNA sequences, including, but not limited ίο transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in dm art to act directly or indirectly to regulate the amount of transcription from the promoter. A ‘'constitutive* promoter is a promoter that is active in. most tissues under most physiological and developmental conditions, An “inducible*' promoter is a promoter that is physio logically or developmenially regulated, e.g, by the application of a chemical inducer. A “tissue specific” promoter is only acti ve in specific types of tissues or cells.
The terms “substantially Identical”, “substantial identity” or “essentially similar” or “essential similarity” means that two peptide or two nucleotide sequences, when optimally aligned, such as by the programs GAP or BESTF.IT using default parameters, share at least a certain percentage of sequence identity as defined elsewhere herein. GAP uses the Needlema» and Wunseh global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps, Generally, the GAP default parameters are used, with a gap creation penalty ™ 50 (nucleotides) / 8 (proteins) and gap extension -penalty ~ 3 (nucleotides) / 2 (protoins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is B!osum62 (HenikolT & Hemkoffi 1992, PNAS 89, 9.15-919), it is clear than when RMA sequences are said to be essemiai)> similar or have a certain degree of sequence identity with UNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such, as the GCO Wisconsin Package, Version .10,3, available from A.ccelrys Inc., 9685 Scranton Road, San Diego, CA 92.121-3752 USA or the open-source software Emboss for Windows (current version 2,7,1-4/7), Alternatively percent similarity or identity may be deienTjined by searching against databases such as PASTA, BLAST, etc.
Nucleotide sequences encoding pamwiral Rep proteins of the invention, may also be defined by their capability to hybridise with the nucleotide sequence of SEQ ID NO, 10, respectively, under moderate, or preferably under stringent hybridisation conditions. Stringent hybridisation conditions are herein, defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridise at a temperature of about 65*0 in a solution comprising about I M salt, pretcrabiy 6 x SBC or any other solid ion having a comparable ionic strength, and washing at 65 X in a solution comprising about 0,1 M salt, or less, preferably 0,2 x SBC or any other solution having a. comparable ionic strength. Preferably, the'hybridisation is performed overnight, i.e, at feast for 10 hoars and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific, hybridisation of sequences having about 90% or more sequence identity.
Moderate conditions are herein defined, as conditions that allow a. nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridise at a temperature of about 45!!C in a. solution comprising about I M salt, preferably 6 x SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 x SSC or any other solution having a comparable ionic strength.. Preferably, the hybridisation is performed overnight, be. at least .for 10 hours, sad preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions-will usually allow the specific hybridisation of sequences hawing up to 50% sequence identity, The poison skilled in the aft. will fee able to modify these hybridisation conditions in order u> specifically- identity sequences varying in identity between 50% and 90%,
The present invention relates the. use of animal parvoviruses, in particular depertdeviruses such as infectious human or simian AAV, and the components thereof (e.g.j an animal parvovirus genome) for use as vectors for introduction and/or expression of nucleic acids iu mammalian cells, in particular, the invention relates to improvements hi productivity of such parvovirai vectors when produced in insect ceils.
Viruses of the Parvoviridae family are small DMA animal viruses, The family Paxvoviridae may be divided 'between, two subfamilies: the Parvovmnae, which, infect vertebrates, and the Densovirinac, which infect insects. Members of the subfamily ParvovMnae are herein referred to as the parvoviruses and include the: genus Dependovims. As may he deduced from the name of their genus, members of the Dependoviras are unique in that they usually require coinfection with a. helper virus such as .adenovirus or herpes \ for productive infection in cell culture, The genus Dependovims includes AAV, which normally infects humans (e.g., serotypes 1, 2,3A, SB, 4, 5, and 6} or primates (e.g,, serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e,g„ bovine, canine, equine, and ovine adeno-assoeiated viruses). Further information on parvoviruses and other members of the Parvoviridao is described in Kenneth I, Bern·', 'Turvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virokup, ted Ftl 1996), For convenience the present invention is further exemplified and described herein by reference to AAV, it is however understood that, the invention is not limited to AAV but may equally be applied to other parvoviruses.
The genomic organization of ail known AAV serotypes is very similar, The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (at) in length, inverted terminal repeats (111½) Hank the unique coding nucleotide sequences for the non-simctural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VP!, -2 and -3) form, the capsid. The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wtAAV infection in mammalian cells the Rep genes (i.e. Rep78 and Rep52) are expressed from the P5 promoter and the P19 promotor, respectively and both Rep proteins have a function in the replication of the viral genome. A splicing event in the Rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also in insect cells the Rep78 and Rep52 proteins suffice for AAV vector production. A "recombinant parvoviral or AAV vector" (or "rAAV vector") herein refers to a vector comprising one or more polynucleotide sequences of interest, genes of interest or "transgenes" that are flanked by parvoviral or AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger nucleic acid construct (e.g. in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), then the rAAV vector is typically referred to as a "pro-vector" which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.
In a further aspect the invention relates to a nucleotide sequence comprising an open reading frame comprising nucleotide sequences encoding animal parvoviruses Rep proteins, wherein the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon. The suboptimal initiation codon preferably is an initiation codon that effects partial exon skipping. Partial exon skipping is herein understood to mean that at least part of the ribosomes do not initiate translation at the suboptimal initiation codon of the Rep78 protein but at an initiation codon further downstream, whereby preferably the initiation codon further downstream is the initiation codon of the Rep52 protein. The suboptimal initiation codon preferably effects partial exon skipping upon expression of the nucleotide sequence in an insect cell. Preferably, the suboptimal initiation codon effects partial exon skipping in an insect cell so as to produce- in the insect cell a molar ratio of Rep78 to Rep$2 in the range of 1:10 to 10:1, 1:5 to 5:1, or 1:3 to 3:1, preferably at about 20 - 40 hours post infection, more preferably at about 30 - 40 hours post infection, using a bacidovinis expression. The .molar ration ofthe Rep /S and Rep52 may be detemrmed by means o f Western blotting as described in Example 1,1.3, preferably using a monoclonal antibody that recognizes a common epitope of both Rep7S and Rep.5.2, or using die antibody described in Example 1,13,
The term "snbopiimal initiation codon" herein not only refers to die trinucleotide i.ntlllation codon itself but also to its context. Thus, a subopdma! initiation codon may consist of an "optimal" ATG codon, in a sithoptitnal context, e.g. a non-Kozak context, However, more preferred are suboplitnal initiation codons wherein the tri-nucleotide imitiation codon itself is suboptitnal, i.e, is not ATG. Snboptitnal is herein understood to mean, that the codon is less efficient in the inititiation of translation in an otherwise identical context as compared to the normal ATG codon. Preferably, the efficiency of suboptima! codon is less than 90, SO, 60, 40. or 20% of the efficiency of the normal ATG codon in an. otherwise identical context. Methods for comparing die relative efficiency of inMtiation of translation are known per sc to the skilled, person. Preferred suhoptimal initiation codons may be selected from ACG, TTG, CTO, and 6TG. More preferred is ACG. A nucleotide sequence encoding animal parvoviruses Rep proteins, is herein understood as a nucleotide sequence encoding the noo-structural Rep proteins that are required and sufficient for parvovlral vector production in insect ceils such the Rep78 and Rep52 proteins. The animal parvovirus nucleotide sequence preferably is from a dependovirus, more preferably from a human or simian ad eno-associated virus (AAV) and most preferably from an AAV which normally infects humans (e.g·. serotypes 1, 2, 3.A, 3B, 4, 5, and 6) or primates (e.g,, serotypes 1 and 4), An example of a nucleotide sequence encoding animal parvoviruses Rep proteins is gi ven in SEQ ID No, 10, which depicts a part of the AAV serotype-2 sequence genome -encoding the Rep proteins. The Rep78 coding sequence comprises nucleotides 11 - 1876 and the Kep55 coding sequence comprises nucleotides 683 - .1876, It is understood that the exact molecular weights of the Rcp78 and Rep52 proteins, as well as the exact positions of the translation initiation codons may differ between, different parvoviruses. However, the skilled person will know how to identify the corresponding position in nucleotide sequence from oilier parvoviruses fern AAV-2, A nucleotide sequence encoding animal parvoviruses Rep proteins may thus also be defined as a nucleotide sequence: a) that encodes a polypeptide comprising an amino add sequence that has at least 50, 60, 70, 80, 88, 89, 90, 95, 97, 98, or 99% sequence identity with the amino acid sequence of'SEQ ID NO, 11; b) that has at least 50, 90, 70, 80, 8.1, 82, 85, 90, 95, 97, 98, or 99% sequence identity with the nucleotide sequence of positions 11 - 1876 of SEQ ID NO. 10; e) the complementary strand of which hybridises to a 'nucleic acid molecule sequence of (a) or (b): d) nucleotide- sequences the sequence of which differs from the sequence of a nucleic add molecule of (¢) due to the degeneracy of the genetic code. Preferably, the nucleotide sequence encodes animal parvoviruses Rep proteins that are required and sufficient for parvovira! vector production in insect cells. A further preferred nucleotide sequence of the invention.eomprises an. expression control sequence that comprising a nine nucleotide sequence of SEQ. I D NO: 7 or a nucleotide sequence substantially homologous to SEQ. ID NO: 7, upstream of the initiation codon of the nucleotide sequence encoding the parvovira! Rep?8 protein, A sequence with substantial identity to the nucleotide sequence of SEQ. ID NO: 7 and that will help increase expression of the parvovira! Rep78 protein is e,g. a sequence which, has at least 69%, 70%, 89% or 90% Identity to the nine nucleotide sequence of SEQ ID NO: 7.
Elimination of possible false translation initiation sites in the Rep protein coding sequences, other than the Rep78 and Rep52 translation initiation sites, of other parvoviruses will be well understood by an artisan of skill in the art, as will be the elimination of putative splice sites that may be recognised in insect cells. The various modifications of the wild-type parvovira! sequences for proper expression In insect cells is achieved by application of well-known genetic engineering techniques such, as described e.g. hi Samhrook. and Russell (2901} ’’Molecular Cloning; A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York, Various further modifications of Rep protein coding regions are know» to the skilled artisan which could increase yield of Rep protein. These modifications are within the scope of the present invention. M a further aspect the invention relates to a nucleic acid construct comprising a nucleotide sequence encoding parvovlral Rep proteins. as defined above. Preferably, in the construct, the nucleotide sequence encoding the parvoviral Rep proteins is operabty linked to expression control sequences for expression, in. an insect cell. These expression control sequences will at least include a. promoter that is active in insect cells. Techniques known to one skilled in. the art for expressing foreign genes in insect host cells can be used to practice the invention. Methodology for -molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith. 1986, A Manual of Methods for Baeuiovirus Vectors and insect Culture Procedures, Texas Agricultural Experimental Station Bull. No, 7555, College Station, Tex,; Luckow, 1991, In firokop et al, Cloning and Expression of Heterologous Genes in Insect Cells with Baeuiovirus Vectors* Recombinant. .DMA Technology and Applications, 97-152; King, L, A, and R. D, Possee, 1992, The baeuiovirus expression system. Chapman arid Hall, United Kingdom; O'Reilly, D, R„ L. K., Miller, V. A. Luckow, '1992, Baeuiovirus Expression Vectors: A Laboratory Manual, New York; W. H, Freeman and Richardson, C, D„ 1995, Baeuiovirus Expression Protocols, Methods in Molecular Biology, volume 39; US 4,745,051; US2003148506; and WO 03/074714, A particularly suitable promoter for transcription of the nucleotide sequence of the invention encoding of the par, u\ hal Rep proteins is e.g, the polyhedron promoter. However, other promoters that are active m insect cells are known in the art, e,g. the plO, p35, 1E-.1 or Δ1Ε-1 promoters and further promoters described in the above references.
Preferably the nucleic acid construct for expression of the parvoviral Rep proteins in insect, cells is an insect cell-compatible vector. An ''insect cell-compatible vector" or ’Vector" is understood to a nucleic add molecule capable of productive transfbnnation or transfection of an insect or insect cell. Exemplary biological vectors Include plasmids, linear nucleic acid molecules, and .recombinant viruses. Any vector can be employed as long as it is insect eelhcompatlbie. The· vector may integrate into the insect cells genome but the presence of the vector in the insect cell need not be permanent and transient episomal vectors are also included. The vectors can be introduced by .any means known, for example by chemical treatment of the cells, electroporation, or infection. In. a preferred embodiment, the vector is a baeuiovirus, a viral-vector, or a plasmid. In a more preferred embodiment, the vector is a baeuiovirus. ie, the construct is a tmnilo viral vector. Baeuloviral vectors and methods for their use are described in the above cited inferences on molecular engineering of insect cells.
In another aspect the invention relates to an insect cell that comprises no more than one type of nucleotide, sequence comprising a single open reading frame encoding a parvoviral Rep protein. Preferably the single open reading frame encodes one or more of the parvoviral Rep proteins, more preferably the open reading frame encodes all of the parvoviral Rep proteins, most preferably the open reading frame encodes the full-length. Rep 78 protein from which preferably at least both Hep 52 and Rep 78 proteins may be expressed in the insect ceil. It is understood herein, that the insect cell, may comprise more than one copy of the single type of nucleotide sequence, e.g, in a multicopy episonml vector, but that these are multiple copies of essentially one and the same nucleic acid molecule, or at least .nucleic acid molecules that encode one and the same Rep amino acid sequence, e.g, nucleic .acid molecules that only differ between each, other due to the degeneracy of the genetic code. The presence of only a smgle type of nucleic acid molecule encoding, the parvoviral Rep proteins avoids recombination between homologous sequences as may be present in different types of vectors comprising Rep sequences, which may give rise to defective Rep expression constructs that affect (stability of) parvoviral production levels in insect cells. Preferably, in the insect cell, the nuefroride sequence comprising.the single open, reading frame encoding one or more parvo viral Rep proteins is part of a nucleic acid construct wherein the «.uclcoti.de sequence is operabiy linked to expression control sequences for expression in an insect ceil. A further preferred insect cell comprises as a ‘forsf’ nucleotide sequence a nucleotide sequence as defined above encoding parvoviral Rep' proteins, preferably a coding sequence with a suboptimai initiation codes as defined above, or a nucleic acid construct'as defined above or the insect cell comprises as a '‘first” nucleic acid construct a nucleic acid, construct as defined above comprising such nucleotide sequences.
Any insect cell which allo ws for replication of a recombinant parvoviral (rAAV) vector and which can be maintained in culture can be used in accordance with the present invention. For example, the cell line used can be from Spodoptera fmglperds, drosophila cell lines, or mosquito cell lines, e,g., Aedes alhopictus derived cell lines. Preferred insect cells or cell lines are cells from the insec? species which are susceptible to baeulovims Infection, including e.g, Se30L SelZT)2109, SeUCRl, SIR, SiBOCff, SOL BT1-TN-5BM, MG-1,1x1368, HzArai, Ha2302, Hx2E5, High Five (Invitrogen, CA, USA) and expresW^ (US 6,.103,526; Protein Sciences Corp., CT, USA). A preferred insect cell according to the invention, in addition to the above described "first" nucleotide sequence or a nucleic acid construct, further comprises: a) a second nucleotide sequence comprising at least one parvoviral. inverted terminal repeat (1TR) nucleotide sequence; and, b) a third nucleotide sequence comprising parvoviral Cap protein coding sequences operably linked io expression control sequences for expression in an insect ceil.
In the context of the invention "at least one parvoviral ITR nucleotide sequence” is understood to mean, a palindromic sequence, comprising mostly complementary, symmetrically arranged sequences also referred to as "A," "B," and "€" regions. The ITR functions- as an origin, of replication, a site: having a "cis” role m replication, i.e., being a meogoihoa site for teas acting replication proteins such as e,g. Rep 78 (or RepbS) which recognize the palindrome and specific sequences interna! to die palindrome. One exception to the symmetry of the ITR sequence is the ”D” region of the ITR, It is unique (not having a complement, within one ITR). Nicking of singlestranded DNA occurs at the: junction between, the A and D regions. It is the region where new DNA. synthesis initiates. The D region normally sits to one side of the palindrome and provides directionality to the nucleic acid replication step. An parvovirus replicating in a mammalian cell typically has two ITR sequences. It is, however, possible to engineer an ITR so that binding sites arc on both strands of the A regions and D regions are located symmetrically, one on each side of the palindrome. On a double-stranded circular DNA template (e.g., a plasmid), the Rcp?8- or Rep68~ assisted nucleic acid replication then proceeds in both directions and a single ITR suffices for parvoviral replication of a circular vector. Thus, one ITR nucleotide sequence can be used in the context of the present invention. Preferably, however, two or another even cumber of regular ITRs are used. Most preferably, two ITR sequences are used, A preferred parvoviral HR is an AAV ITR. For safety reasons it may be desirable to construct a recombinant parvoviral (rAAV) vector that Is unable to further propagate after initial introduction into a cell Such a safety mechanism for limiting undesirable vector propagation, in a recipient nmy be provided by using rAAV with a chimeric ITR as described in IJS20CB148506,
The number of nucleic acid constructs employed in t he insect ceil for the production, of the recombinant' paryoviral frAAV) vector is not limiting in the invention. For example, one, two, three, four, five, or more separate constructs can fee employed to produce rAAV in insect cells in accordance with, the methods of the present invention, if live constructs are employed, one construct encodes AAV VP 1, another construct encodes AAV VP2, yet another construct encodes AAV VP3, still yet another construct encodes the Rep protein as defined above and' a final construct comprises at least one AAV ITR, If fewer than five, eonstructs are used, the constructs can. comprise various combinations of the at least one AAV ITR. and the Vfil, VP2, VP3, and. the Rep protein coding sequences. Preferably, two constructs or three constructs are used, with two constructs being more preferred as described above. If two constructs' are used, preferably the insect cell comprises; (a) a first nucleic acid construct for expression of the Rep proteins as defined above, which construct further comprises the third nucleotide sequences as defined in (b) above (comprising parvovimi Cap protein coding sequences operabiy linked to at least one expression control sequence for expression in an insect cell; see: also below); and (c) a second nucleic add construct comprising the second nucleotide sequence as defined, in (a) above, (comprising at least one parvoviral/AAV ITR nucleotide sequence). If three constructs- are used, preferably the same configuration as used for two constructs is used except that, separate constructs are used for expression of the capsid proteins and for expression of the Rep proteins. The sequences on each construct can be in any order relative to each other. For example, if one construct comprises ITRs and an- ORF comprising nucleotide sequences encoding VP capsid proteins, tire VP ORF can be located on the construct .such that, upon replication of the DNA between ITR sequences, the VP ORF is replicated or not replicated. For another example, the Rep coding sequences and/or the ORF comprising nucleotide sequences encoding YP capsid proteins can be in any order on a. construct. In is understood that also the second, third and further nucleic acid construct^) preferably are an insect cell-compatible vectors, preferably a baciilo viral vectors as described above, Alternatively, in the insect cell of the invention, one or more of foe first nucleotide sequence, second .nucleotide sequence, third nucleotide sequence, and fourth nucleotide sequence and optional further nncieotide sequences may be stably integrated in the genome of the insect cell. One of ordinary skill in the art knows bow to stably introduce a nucleotide sequence into the-insect genome and how to identify a cell having such a nucleotide sequence in the genome. The ·incorporation into the genome may be aided by, toe example, hie use of a vector comprising nucleotide sequences highly homologous to regions of the insect genome. The use of specific sequences, such as transposons, is another way to introduce a nucleotide sequence into a genome.
In the invention, die third nucleotide sequence comprising parvoviral capsid (Cap) protein coding sequences is herein understood to comprises sequences encoding each of the three parvoviral capsid proteins, VP!, -2 and ~3. The third nucleotide sequence comprising the capsid protein coding sequences may be present In various forms. Eg, separate coding sequences for each of the capsid proteins VPI, «2 and -3 may used, whereby each coding sequence is operably linked to expression control sequences for expression in an insect ceil. More preferably, however, the third nucleotide sequence comprises a single open reading frame encoding all three of the animal parvoviral (AAV) VPI, VP2, and VPS capsid proteins, wherein the initiation codon ibr translation -of the VPI capsid protein is asuboplknal initiation codon that is not ATG as e.g, described by Urabe ct al. (2002, Mipra). A suboptimai initiation codon for the VPI capsid protein may be as defined above for the Rep78 protein. More preferred suboptinial initiation codons for the VPI capsid protein may he selected from AGO, TTG, CTO and GIG, of which CTG and GIG are most preferred. A preferred third nucleotide sequence lor the expression of the capsid proteins further comprises an expression control sequence comprising a nine nucleotide sequence of SEQ. ID NO: 7 or a nucleotide sequence substantially homologous to SEQ. ID NO: 7, upstream of the initiation, codon of the nucleotide sequence encoding the VPI capsid protein. A sequence with substantial ideality to the nucleotide sequence of SEQ. ID NO: 7 and that will, help increase expression of VPI is e.g. a sequence which has at least 60%, 71)%, 80% or 90% identity to'die tune: nucleotide sequence of SEQ ID NO: 7. A further preferred third nucleotide sequence for expression of the capsid proteins further preferably comprises at least one modification of the nucleotide sequence encoding the VPI capsid protein selected from among a € at nucleotide position 12, an A at nucleotide position 21, and a € at nucleotide position 24 (with reference to position 1. being the first nucleoiide of the translation initiation codon; sec SEQ ID NO.!). Elimination of possible false initiation codons tor translation of VP I of other serotypes will he well understood by an artisan of skill, in the art, as will be the elimination of putative splice sites that may be recognised in insect cells. Various further modifications of VP coding regions are known to -the skilled artisan which could either increase yield of VP and virion or have other desired effects, such as altered tropism or reduce antigenicity of the virion. These modifications are within the scope of the present invention. Preferably the. nucleotide sequence of the invention encoding the parvovirai capsid proteins is operably linked to expression, control sequences for expression in an insect cell which will at least include a promoter that is active in insect cells. Such, control sequences and further techniques and materials (e.g, vectors) tor expressing parvoviral capsid proteins in insect host ceils are already described above for the Rep proteins.
In a preferred embodiment of the invention, the second nucleotide sequence present in the insect cells of the invention, be. the sequence comprising at least one parvovtral (AAV) ITR, further comprises at least, one nucleotide sequence encoding a gene product of interest, whereby preferably the at least one nucleotide sequence encoding a gene product of interest becomes incorporated into the genome of a recombinant parvoviral (rAAV) vector produced in the insect cell. Preferably, at least one nucleotide sequence encoding a gene product of interest is a sequence for expression in a mammalian cell, Preferably, the second nucleotide sequence comprises two parvoviral (AAV) ITR. nncleoti.de sequences and wherein the at: least one nucleotide sequence encoding a gene product, of interest is located between the two parvoviral (AAV) ITR nucleotide sequences. Preferably, the nucleotide sequence encoding a gene product of interest (for expression in the mammalian cell) will be incorporated into dm recombinant parvovimi (rAAV) vector produced in ibe insect cell if it is located between two regular ITRs, or is located on either side of an ITR engineered with two D regions.
The second nucleotide sequence defined herein above may thus comprise a nucleotide sequence encoding at least one "gene product of interest" for expression in a mammalian cell, located such that it will be incorporated Into an recombinant parvoviral (rAAV) veetoi leplieated in the insect cell. Any nucleotide sequence can be incorporated for later expression in a mammalian cell, transfected with the recombinant parvovtral (rAAV) vector produced in accordance with the present invention. The nucleotide sequence may e.g. encode a protein it may express an. RMAi agent, i.e, an RNA molecule that is capable of RNA interference such as e.g. a shRNA (short haitpmRNA) or an siKNIA (short interfering RNA). "siRNA" means a small interfering RNA. that is a short-length double-stranded RN A that are not toxic in mammalian cells (Elbashir et a.L 2001, Nature 411: 494-98; Caplen et al, 2001, Proe, Natl. Acad. Scf USA 98: 9742-47), In a preferred embodiment, the second nucleotide sequence may comprise two nucleotide sequences and each encodes one gene product of interest lor expression in a mammalian cell Each of the two nucleotide sequences encoding a. product of interest is located such that it will be incorporated into a recombinant parvoviral (rAAV) vector replicated in the insect cell,
The product of interest for expression in a mammalian cell may he a therapeutic gene product, A therapeutic gene product can be a polypeptide, or an RNA molecule (siRNA), or other gene product that, when expressed in a target cell, provides a desired therapeutic effect such as e,g. ablation of an undesired activity, e,g. the ablation of an infected cell, or the complementation of a genetic defect, e.g, causing a deficiency in an en/ymaUc activity. Examples of therapeutic polypeptide gene products include CFTR, Factor IX, Lipoprotein lipase (LPL, preferably LPL S447X; see WO 01/00220), Apolipoprolein Al, Uridine Diphosphate GlUcuronosyitransferase (UGT), Retinitis Pigmentosa GTPase Regulator Interacting Protein (RP-GR1P), and cytokines or interleukins like e.g, iL~10.
Alternatively, or in addition, as a second gene product, second nucleotide sequence defined herein above may comprise a nucleotide sequence encoding a polypeptide that serve as marker proteins to assess cell transformation and expression. Suitable marker proteins lor this purpose ate e.g, the fluorescent protein OFF, and die selectable marker genes HSV thymidine kinase (lor selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on. hygromyein BY Tn5 aminoglycoside phosphotransferase (tor selection on. ¢34.18.), and dihydrofolste reductase (DMFR) (for selection on methotrexate), CD20, the bw affinity nerve growth factor gene. Sources for obtaining these marker genes and methods for their use are provided in Sambrook and Russel (2001). "Molecular Cloning: A. Laboratory Manual (3Λ' edition), Gold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York. Furthermore, second nucleotide sequence defined herein above may comprise a nucleotide sequence encoding a polypeptide that may serve as a fail-safe mechanism that allows to care a subject from cells transduced with the recombinant parvovirai (rAAV) vector of the invention, if deemed necessary. Such a nucleotide sequence, often referred to as a suicide gene, encodes a protein ihai is capable of converting a prodrug into a toxic substance that is capable of killing the transgenic cells in which the protein is expressed. Suitable examples of such suicide genes include e.g, the E.co'li cytosine deaminase gene or one of .the thymidine kinase genes from Herpes Simplex Vims, Cytomegalovirus and Varicella-Zoster virus, in which case ganciclovir may he used as prodrag to kill the transgenic ceils in the subject (see e,g. Clair et al„, 1987, Aniimierob, Agents Chemother. 31: 844-849), .In another embodiment one of the gene products of interest can he an AAV protein, in particular, a Rep protein, such as Rep? 8 or Rep68, or a functional fragment thereof A nucleotide sequence encoding a Rep78 and/or a RepbS, if present on the genome of a. .recombinant parvoviral (rAAV) vector of the invention and expressed in a mammalian cell transduced with the vector, allows for integration of the recombinant parvoviral (rAAV) vector into the genome of the transduced mammalian cell. Expression ofRep'78 aud/or Repf>8 in an. rAAV-iransduced or infected mammalian cell can provide an advantage for certain uses of the recombinant parvoviral (rAAV) vector, by allowing long term or permanent expression of any other gene product of interest introduced, in the cell by the vector.
In the recombinant parvoviral (rAAV) vectors of the invention the at least one nucleotide sequence* s) encoding a gene product of interest lhr expression, in a mammalian coll, preferably is/are operafely linked to at least one mammalian cell-compatible expression, control sequence, e.g,, a promoter. Many such promoters are known in the art (see Satnbrook and Russel, 2001, supra). Coniituiivc promoters that are broadly expressed in many cell-types, such, as the CMV promoter may he used. However, more preferred will be promoters that arc-inducible, tissue-specific, cell-type-· specific, or cell cycle-specific. For example, for liver-specific expression a promoter may be selected from an «1-anti-trypsin promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, LFS (thyroxine-binding globlin) promoter, HCR-ApoCil hybrid promoter, HCR-bAAX hybrid promoter and an ape lipoprotein E promoter, Other examples Include the E2F promoter Ibr tumor-selective, and, in particular*, neurological cell tumor-selective .expression (Parr et. a).,, 1997, Nat, Med, 3:1145-9) or the 1L-2 promoter ibr use in mononuclear blood cells (Bagenbaugb et ah, .! 997,1 Exp Med; 185: 2101-10). AAV is able to Meet a number of mammalia» cells. See, e,g., Tratschin et a!, (1985, Mol, Ceil Biol 5:3251-3260) and Grimm et at (1999, Bum, Gene Ther. 10:2445-2450), However, AAV transduction of human synovial fibroblasts is sigaificastly- more efficient than in similar murine cedis, jemiiugs et ah, Arthritis Res, 3:1 (2001), and the cellular tropicity of AAV differs among serotypes. See, e.g., Davidson et al, (2000, Proe, Natl Acad, See USA, 97:3428-3432), who discuss differences among AAV2, AAV4, and AAV5 with respect to mammalian CHS cell tropism and transduction efficiency, AAV sequences that may be used in, the present invention for the production of recombinant AAV vectors in insect cells can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the .amino acid and the nucleic acid levels, provide an identical set.of genetic functions, produce virions which are: essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e.g. GeuEauk Accession number 'US9790; GenBank Accession number JO 1901; GenBank Accession number AF043303; GenBank Accession number AF085716; Chlorim et al. (1997, l Vir, 71: 6823-33); Srivastava et al. (1983, J, Vir, 45:555-64); Chlmini et al, (1999, J. Vir, 73:1309-1319); Rutledge et al. (1998, J, Vir. 72:309-319); and Wtt et al (2000, j. Vir, 74: 8635-47), AAV serotypes 1, 2, 3,4 and. 5 are preferred source of AAV nucleotide sequences for use in Hie context of the present invention. Preferably the AAV 1TR sequences for use in the context of the present invention, are derived from AAV),, AAV2, and/or AAY4, Likewise, the Rep (Rep78 and Rep$2) coding sequences are preferably derived from AAV I, AAV2, and/or AAY4, The sequences coding for the VP!, VP2, and VP3 capsid proteins for use In the context of the present invention may however be taken .from any of the: known 42 serotypes, more preferably from AAV.!, AAV2. AAV3, AAV4, AAV.5, AAV6, AAV7, AAV'8 or AA* 79 or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries, AAV Rep and ITR sequences are particularly conserved among most serotypes. The Rep78 proteins of various AAV serotypes are e.g·.. more than 89"··;? identical and the total nucleoli.de sequence Identity at the genome level, between AA.V2, A.AV3A, AAV3B, and AAV6 is around 8256 fBantci.-Sch.aa! et al, 1999, 3. Virol., -/312-:939-947). Moreover, the Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (fe,5 functionally substitute) corresponding sequences from other serotypes in production of AAV particles in mammalian cells, US20O3148506 reports that AAV Rep and ITR. sequences afro efficiently crosscomplement other AAV Rep and ITR sequences in insect cells.
The AAV VP proteins are known to determine the cellular tropieity of the AAV virion. The V'P protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped. rAAV particles comprising the capsid proteins of a serotype {e,g,s AAV3) and the Rep and/or ITR. sequences of another AAV serotype ie.g,, AAV2). Such pseudotyped rAAV particles are a part of the present invention.
Modified "AAV" sequences also can be used In the context of the present invention, e.g. for the production of rAAV vectors in insect cells. Such, modified sequences e.g. include sequences having at least about 70%, at least about 75%, at least abont 80%, at least about 85%,· at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity (e.g., a. sequence having about 75-99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAVS., A.A.V6, AAV 7, AAVS or AAV9 ITR, Rep. or VP can be used in place of wild-type AAV ITR, Rep, or VP sequences.,
Although similar to other AAV serotypes in many respects, AAVS differs from other human and simian AAV serotypes more than other known Iranian and simian serotypes. In view thereof the production of tAAVS can. differ from production of other serotypes in insect cells. Where methods of the invention are employed to produce rAAVS, it is preferred that one or more constructs comprising, collectively in the ease of more than one construct, a nucleotide sequence comprising an. AAVS ITR, a nucleotide sequence comprises an AAV.5 Rep coding sequence· (i.e, a nucleotide sequence comprises aa AAVS Rep78). Such ITR and Rep sequences can be modified as desired to editor! efficient, production of rAAVS or pseudotyped rAAVS vectors in insect cells, E.g,, the start codon of the Rep sequences can be modified, VI5 splice sites can be modified or eliminated, and/or the VP 1start codon and nearby nucleotides can be modified to improve the production of rAAVS vectors in ihc insect cell. la another aspect the invention thus relates to a .method, for producing a recombinant parvovirai (rAAV'} virion (comprising a recombinant parvovirai (rAAV) vector as defined above) in an insect cell. Preferably, the method comprises the steps of; (a) culturing an insect cell as de.tln.ed in herein above under conditions such that recombinant parvovirai (rAAV) vector is produced; and, (b) recovery of the recombinant parvovirai (rAAV) vector, It is understood here that the recombinant parvovirai (rAAV) vector produced in the method preferably is .an infectious parvovirai or AAV virion that comprise the recombinant 'parvovirai (rAAV) vector nucleic acids, Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art and described e,g. in the above cited references on molecular engineering of insects cells.
Preferably the method further comprises the step of affinity-purification of the (virions comprising the) recombinant parvovirai (rAAV) vector using an anti-AAV antibody, preferably an Immobilised antibody. The anti-AAV antibody preferably is an monoclonal. antibody, A .particularly suitable antibody is a single chain eamelid antibody or a .fragment thereof as e,g, obtainable from camels or llamas (see e.g, Muyldermans, 2001, Biotecbnol. 74: 277-302). The antibody for affinity-purification of rAAV preferably is an antibody that specifically binds an epitope on a AAV capsid protein, whereby preferably the epitope is an epitope that is present on capsid protein of more than one AAV serotype, E,g< the antibody may be raised or selected on the basis of specific binding to AAV2 capsid but at the same time also it may also specifically bind to AAV!, AAV3 and AAV5 capsids.
In a further aspect the invention, relates to a rAAV virion produced In the above described methods of the invention, using the nucleic acid constructs and cells as defined above. Preferably the rAAV virion comprises in its genome at least one nucleotide sequence encoding a gene product of interest, whereby the at least one nucleotide sequence is not a native AAV nucleotide sequence, and whereby in the stoichiometry of the AAV VP 1., VP2, and VP3 capsid proteins the amount of VPb (a) is at least 100, 105, 110,120, 150, 200 or 400% of the amount of YP2; or (b) is at least 8, .10, 10.5, 1.1, 12, .15,. 20 or 40% of the amount of VP3; or (c) is at least as defined in both (a) and (b), Preferably, the amount of VPL VP2 and VP3 is determined using an antibody recognising an epitope that is common to each of Vfil,. VP2 and VP3, Various immunoassays arc available In the art that will allow quantify the relative amounts of VPI» VP2 and/or VP3 (sec e,g. Using Antibodies, B, Harlow and D. Lane, '1999, Cold Spring Harbor Laboratory Press, New York). An. suitable antibody recognising an epitope that is common to each of the three- capsid proteins is e.g. the mouse antLCap Bl antibody (as is commercially available from P.roge.u, Germany). A preferred rAAV virion according to the invention is a virion comprising in its genome at least one nucleotide sequence encoding a gene product of interest, whereby the at least one nucleotide sequence is not a native AAV nucleotide sequence, and whereby the AAV virion comprises a VP! capsid protein comprises a leucine or & valine at amino add position 1, A more preferred AA.V virion according to the invention has the ratio's of capsid proteins as defined above and comprises a VP1 capsid protein comprises a leucine or a valine at amino acid position .!,
In this document and in its claims, the verb "to comprise5* and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a” or "an” does not exclude the possibility that more than one of the element is present, unless the context dearly requires that there be: one and only one of the elements. The indefinite article "a" or "an” thus usually means "at least one".
Description of the figures
Figure 1: A) Organisation of Rep expression in. the wild type AAV genome. The Rep7S and Rep 52 genes are expressed from respectively the PS and Pi 9 promoter. Expression of RepbS and Rep40 (which are the spliced variants ofreap. Rep?8 and. Rep52) are not shown. Both expression units contain a ATG-mitiailon site, B) The construct of the invention has the Rep ORF under the control of a single promoter (e.-g. the polyhedron (Poll !} promoter). This promoter drives the expression, of both. Rep78 and Rep52 because the Rep78 initiation codon ATG is converted to the alternate ACG initiation codon and partially skipped by the ribosome. C) The. original construct by Urabe et ah (2002, supra) drives Rep78 and Rep52 independently from two different, promoters (reap, A1E1 and poll-!}.
Figure. _2: Western blot analysis of Rep pmieurs expressed from recombinant, b&culo virus that was passaged 5 times on insect cells. Tbs original baenlovirus designed by Urabe et ah, 2002 (original. R.E!VRae~to-Bae} results in a slow decrease of Rep7'8/52 expression over 5 passages. Tbs expression unit lor Rep78 and 52 designed by Urabe et al., 2002 inserted m baeuie virus backbone PSC (original REF / FSC) also results in a decrease of Rep?8/S2 expression following passaging on insect ceils. However, the baculovirus with die REP expresrich unit containing die ACG initiation codon in the PSC backbone (REP-ACG / PSC) results in stable expression of Rep'7 8/52 over at least 5 passages.. Western blot analysis wasperformed as described in Example 1,1,3.
Figure 3: Results of Table I plotted in. a graph.
Figure 4; Comparison of die 'Stabilities of various rAAV constructs in insect cells. rAAV production in SR- cells was performed as described above in Example .!, For all productions the ITR containing baculovirus and the capsid gene containing baculovirus were identical, die passage number was the same as foe Rep gene containing baculoviruses. 4 different Rep gene containing haeulovirases were used: 1) The REP» ACG / PSC, 2) SLR: the original construct by Urabe et al. (2002, supra), 3) Rep32 + Rep78(B2B); Two separate Bac-fo-Bae baeulo viruses, one containing the Rep 78 gene and the other one containing the Rep 52 gene. 4) RepS2 f Rep78(PSC): Two separate pi-mein sciences bacnlovimses one containing the Rep 78 gene and foe other one containing the Rep 52 gene.
Figure 5: Stability of the REP-ACG / PSC baculovirus constructs up to passage 8, rAAV productions in SP->- cells were performed as described In Example I.
Figure 6; Comparison .of the effect of passage effect, -cm rep protein expression of foe original construct from Urabe et al. (2002, supra) with a REP-ACG / ESC construct in accordance with the invention. The baculovirus passages and the western blot were done as described in Example .1. During a normal passage of the rep bacnloviruscs, samples were taken at 40 hours after addition of the b&culo viruses to foe SF cells and western blot was performed.
Examples .1,1.1 Baculo virus ninvnrid construction la order to express Rep?ti and Rep32 1m a sole bleistrorde messenger RNA, the ATG initiation codon of Rbp?8 situated on the expression vector pFastBaeDuaISLR {'Urabe et al, 2002, supra) was converted to ACG. The upstream primer used was;
BaasHI 5' "CgcggatcctgttaagiiCGGGGGGGTTTTACGiiGATTGTGETTAitGGTC---3^ (SEQ ID GO.8} PRIMER SEQUENCE forward
The 30primer that was used in the FOR reaction was flanking the REP78 gene and contains a Xbal she (TCTAGA):
Xbal 5 s~AGGCTCTAGATTCGAG. AGCGGCCCG~3' {SEQ ID GO.9} PRIMER SEQUENCE reverse
The sequence between the above-mentioned primer set was amplified by PCR (reaction volume 50 μ}; lx Fix Amp, Buffer,. 0.3mM dNTPX, ImM. MgS<)4, ISClrnM primer forw., ISOrnM' primer rev,, 2x enhancer solution, template 50n.g fpFasiBaeDoalSLR), I U Platinum Fix (luvitrogen, Carlsbad, CA, USA) using the following protocol; 1 cycle of 95T, 5 min; 35 cycles of 95°€, 15 see; 55°C, 30 sec; 72°C. 2 min; 1 cycle of 72°€, 10 min; 4*C> for ever). The PCR product was cloned in PCR “blunt IUTOPO using the Zero Blunt ΧΟΡΟ PCR. cloning kit (Invitrogen). The Rep78 was subeloned into pEastBaeDua! (luvitrogen) using the restriction sites Spei and Xbal. The mutated Rep expression cassette was finally cloned (using restriction enzymes BsiZlTl and Avrll) into the baeulov?ru> expression construct (cut open with EeoRV and Xbal) pPSC'lO (Protein Sciences Corporation, Meriden, €T, USA|, The sequence analysis of the construct was verified by Based ear, Leiden, the Netherlands. I > 1,2 Reecmibmant baeulovirus production
Recombinant bacufoviruses derived from the Autographa cahibrnlca nuclear polyhydrosis virus (AeNPV) were produced using the GeneXpress BacaloKlT (Protein Sciences Corporation). Transfection, was performed as follows: in a round bottom.! 4ml tube 200 μΐ GRACE medium was mixed with 6 μ! cellfeetine (Invitrogen), and in a. eppendorf tube 200 μΐ GRACH medium was mixed with 50 μ! viral DMA (protein sciences) and 2 p,g transfer plasmid (REP), The contents from the eppendorf tube were added to the tube and mixed carefully, After an incubation period of 30 minutes at RT .! ,300 pi GRACE was added to the transfection mix. Insect cells in a T25 flask were washed with GR ACE medium, and the transfection mixture was added dropwise to the coll layer. After an. incubation of 6 hours at 28°C SF9O0II serum supplemented with 10% PBS was added carefully and the T25 flask was put. in a 28°C stove for 5 days after which the recombinant baeulovirus was harvested. JJJJSegMMaLMal»
Insect cells (SFt) were infected with hacuiovirus~REP. At 16, 40, and 64 hours posi-infection cells a sample was taken and cells wore lysed by adding CUV 10 x TRiS lysis buffer (1.5M NaCi, 0.5M TRIS, 0,01 M MgCI, 1% TRITON XG00, pH8,5, Idler sterilised) and incubated at 2S°C for 30 minutes in a shaker (Junova 44, New Brunswick). Free DMA and RNA was degraded by incubation with benxonase at 37°C for 30 minutes. Cell lysate was centrifuged (1,900 x g; 15 min; 4,3C). NuPAGE LDS sample buffer (4x, invitrogen) was added to a sample of the supernatant and was loaded onto a 4» 12% Bis~Tris gel (120V). Proteins were blotted onto a. FVDF membrane (BioRad) for 30 minutes, 10V (Semidry blotting). Western immnnoehemistry was performed by blocking the membrane with Superbloek-PBS blocking buffer (PIERCE) and subsequent: incubation with, mouse anti-Rep (303.9, Progen, Germany; dilution 1;50) and rabbit ami-mouse.......HR? (D.AKO, dilution 1:5.00). The Rep-proteins were visualized by chemoluminescent staining with lumi-Iight plus Western-blotting substrate (Roche),
El.....RabyJG
The performance of the newly designed Rep-construct: of the invention {REP-ACG / PSC) was compared with the original Rep construct s in both 1) PSC baeulo virus backbone and in 2) Bae-io-Bac baeulovirus backbone (Ura.be et a!.,2002). Ail three constructs were serially passaged until passage 5, AAVl-LPL production experiments were performed using die passage 2, 3, 4 and. 5 Rep-constructs in combination with an AAV-L'PL and a AAV-Cap recombinant baculo virus of respectively passage 2, 3, 4 and 5 (AAV-LPL and AAV-Cap recombinant Baculo virus used here are described, below in Example 2). AAVI-LPL production yields were determined by qPCR arid are shown in Tabic .1. The original baculoviras designed by Ombe.et ah, 200:2 (original REF/Bae-to-Bae) results in. a fast decrease of AAV production over 5 passages. The expression unit .for Rep designed by Urabe ei aL, 2002 inserted in baeulovims backbone PSC (original REP / PSC) also results in a decrease o f AAV production following passaging on insect cells- However- the baeulovims with the RHP expression unit containing the ACG initiation codon in the PSC backbone (REP-ACG PSC) results in stable AAV production over at least .5 passages. Therefore, reproducible production yields of AAV-LPL over several passages (e.g. 2 to 5) were only obtained using baeolovirases containing the REP-ACG construct.
Table .! ; Production, of r.AAV virions using the baculo virus constructs of several passages· Mb cells were infected with three recombinant baculoviruses encoding a LPL-veotor unit of passage 2, 3, 4 or 5 , a Rep-expression unit of passage 2, 3, 4 or 5 and a Cap-expression unit of passage 2, 3, 4 or 5, After three days cells were harvested and AAV yields (vector genomes per ml; vg/ml) were determined by qPCR,
Table 2: Q-PCR performed on the various Bae~Rep constructs following passaging on insect ceils ; Passage 2-5),
Table 2 shows the results of a quantitative PCR (Q-PCR) assay that was designed tor the Rep-expression unit in the recombinant bacakmmses and for a Ranking baeulovims ORP (gene copies per mi: ge's/mi). The ratio between the Q-PCR values determines the presence of deletions in the Rep-baculovirus, A ratio of .1 theoretically means that all hacuioviruses in the batch contain a recombinant Rep78 or 52-sequence, The original bacnlovims designed by Urabe et ak; 2002 (original REP/Bac-io-Bac) shows significant amounts of the recombinant baeulovims at passage 5 have deletions in the Rep sequences. The expression unit for Rep7S and 52 designed by Urabe et a!,, 2002 inserted in baeuiovims backbone PSC (original REP / PSC) shows a very early amt dramatic loss of recombinant, haeulovirus, However, the haeulovirus with the REP expression unit containing the ACG initiation codon in the PSC backbone (REP-ACG / PSC) (clone €4 and A3) show stable recombinant b&cufoviruses over at least 5 passages. 2.1.1 Bacufovirasplasmid construction.
In order to express VP.1,2,3 from a sole poi.yidsiror.de messenger RNA, the baeuiovirus-AAV-Cap construct was designed as described by (Umbe et at, 2002, supra). Bristly, the ATG initiation codon of VP1 was mutated to ACXT A potential ATG initiation codon at position 1! has been changed to ACG, The splice acceptor site downstream of the VP1 initiation codon was destroyed (mutation at position 21 and 24), The mutated Cap expression cassette was cloned into a'bacuiovirux expression construct: pFastBacDual tpFBDAAVIVPm11) with. BamHI/StuI restriction sites. This plasmid CpPBDAAVI VPml.! ) was the starting materia! for introduction of alternate initiation codons for VPi. The forward primer used, by tlrabe et ai (2002, supra) in order to introduce the mentioned mutations was:
BamRT i 11 21 24 5' ··'cgeggatcctgttaac}ACGGCTGCCGACGGTϊATC2:ACCCGATϊGGCTC·.3,' [SEQ ID NO, 1}
The following forward primers were used to make the expression constructs using pFBDAAVlVPml 1 (Urabe et aL, 2002,-supra) as starting material: 5' --'CgcggatcctattaagTTGGClGCCGACGGTTAlCmCCCGATTGGCsrC-3f (SEQ ID NO.2). to "CgcggatcccgttaagATTGCTGCC;3ACGGTTATCTACeCGATTGGeTC~3i (SEQ ID MO.3} .to -cgcggatcctgttaagGTGGCEGCCGACGGTEATCTACCCGATTGGCTC~-3f (SEQ ID MO. 4) 5' "-cgcggatcCtgttaagCTGGCTGCCGACGGTTATCTACCCGATTGGCTC-S^ (SEQ ID NO. 5}
The backward-primer that was used in the PCR reactions with the above forward primers was directed to position ~ 230 bp downstream of the VP.1. initiation codon and contains a unique Stu 1 site (AGGCCT). 5 * “G!tcQTA.GGCCTTGTCGTGCTCGAGGGCGGC"'3> 1SBQ ID GO, 6)
Fragments were amplified with the above-mentioned sets of forward and backward primer pairs by PCR, Following· digestion of PCR products with BamHIi and Siul the PCR products were subcloned into the: BarnHI / StaJ sites of pFBDAAVivpmi I resulting in the various to. be tested bacuiovirns-AAV-C-ap constructs. DMA constructs were verified by sequence analysis at Baseclear, Leiden, the Netherlands. 2,1,2 Recombinant bacuiovirns production 'Recombinant becuto viruses derived .from the Autographs califbmica nuclear polyhydrosis virus (AcNPV) were produced using the Bac-to-Bac bacuiovirns expression system (hrvitrogen). rBac-Cap was amplified by infecting 2x1 if SIP cells per ml at an moi of 0,1. Three days after inter.!ion five cells were spun down, and the supernatant containing the virus recovered, 2J.,3..mct>mh<»3ni ·\Α\ nrqdtie|fon rAAV batches were produced using three recombinant bacnlovimses according to Urabe et ai„ 2002, However, lor this study one bacuiovirns harboured an expression construct for the LPL^'^ ••••transgeae. The therapeutically active agent, expressed from the transgene is a naturally occurring, variant of human lipoprotein lipase, a single chain polypeptide of 448 ammo acids. The LFLS44iX variant has a deletion of two amino acids at the C-temiimis of the protein, The second bacuiovirns harboured an. expression construct' for the AAV replication genes, Rep 78 and Rep 52. The third bacuiovirns harboured the AAVI capsid sequence with, either au ACG or a TTG, CTG, GTG initiation, codon for "VP.1.
Mammahan-rAAV batches produced with the plasmid-transfection system were produced according to Grimm et a!,, 1998 (Novel tools for production and purification of recombinant adcno-associated virus vectors. Hum Gene Ther. 1998 Dee 10;9(18):2745-60),
Insect cells were infected with baeulovirus-Cap. At three days post-infection cells were centrifuged (3,000 g; 15 min). The supernatant was filtered through a. 0.22am Mi Hex filter. NuPAGE LDS sample buffer (firvitrogert) was added to a sample of the supernatant and was loaded onto a 4-12% Bis-Iris gel. The gel was run at tOOV. Proteins were blotted onto a nitrocellulose membrane (BioRad) for I hr, 100V, 350mA. Western immunochemistry was perforated by blocking the membrane with !% marvel, dried skimmed milk and subsequently incubation with mouse anti-Cap (B.l from Progen. Germany; dilution 1:50) and rabbit anti-mouse—HRP (DAKO, dilution 1:.100). VP I ,2 and 3: were visualized by chemoluminescent staining with hind-light plus Western-blotting substrate (Roche),
Human ΐΡΒ^ίΛ.activity was assayed as previously described using a radioactive triolcoylglye-erof emulsion substrate (Mlsson-Ehle and Scholia, 1976), Human l.Pi./v':'x immunoreaeiive mass was assayed rising a sandwich ELISA with chicken IgY and mouse SD2 anti-hLPL antibodies (Lin el ah, 2000). Plasma triglyceride: levels were measured by using commercial kits following manufacturer protocols (Boehrrager Mannheim, #450032). "A_Iksiryiri
In order to introduce different alternate initiation codons for VP.1. expression in the baeulovirus plasmid designed by llrabe et ah (2002, supra) a series of upstream primers were designed containing a BarnHl restriction site and either a TTG, ATT, GTG or CTG codon in place of the ACG initiation codon of VPL PGR using these primers in combination with a downstream primer containing a StuI site resulted in amplified fragments that were subcloned info the BaraEO/Stui site of pFBDVPmll (Bac-Cap). The resulting haeufovims plasmids were used for the preparation of recombinant bacuioviruses using the Bae-to-Bac baculovirus expression system. The prepared recombinant baeuloviruses were infected on. insect celts In order to produce AAV capsids. At three days following infection viral protein expression of the different baculovirus batches were determined on Western Mots, From the Western blots it became clear that the haculoYirus construct containing the TTG initiation codon for VP I expressed, t his protein to a: higher level compared to the pre viously used. ACG initiation codon. The ratio between VPl and VP2 using the TTG codon was found to be 1:1 which is similar to what is reported for wild type AAV (not shown). in order to investigate the mibefiviiy of the AAV capsids derived from recombinant bacnfo viruses with the TTG initiation codon rAAV was generated. Also a. rAAV batch was generated by plasmid transfection on mammalian HEK293 ceils. A vector genome titer of both rAAV hatches was determined by qPCR. This titer was used to Meet HER 293 cells in a microtiter plate: at an increasing moi. At two days •following infection an. quantitative assay (LPIf“+'i'>· “mass assay i for the transgene product. (LP1?44:X:) was performed on the medium, of the imeeicd cells. The assay showed that the amount of ΕΡ1ο*ΐ!Λ produced by baeuio virus-produced rAAV was similar to the LPL produced by the plasmid-produced rAAV (not shown). 2,2,3 injection of rAAV batches in mice
The rAAV batches produced with the baculovirus-production system.and with the conventional mammalian plasmid-production system were injected intramuscularly in mice to follow LPLS44iX-pn>tein activity and triglyceride .fasting in vivo. At 3 days, 7 days and at 2 weeks following ioieotion blood samples were taken and. evaluated. Between. .3 and 7 days post virus administration blood-plasma sampled from' both mice injected with mammalian-rAAV and one mouse Injected with hacufo-rAAV was turned from milky to completely clear. Blood plasma derived from one baeuIo-rAAVrinjeeted mouse remained relatively milky however fat level was dearly reduced. Triglyceride levels were lowered, respectively of all treated mice (not shown). On day 14 TG levels in both raammabau-AAV and bacu{ovi.rus-(TTG)~AAV treated mice TG levels were reduced for 96%. Plasma samples taken at two weeks after virus administration showed that the Ι,ΡΐΤ”" 'χ -activity o f the mice treated with baculovi.nts-AAV and mammalian-AAV was similar (not shown).
Example 3: Stability of rAAV constructs with modified Rep 78 initiation codon in iaseclcelA M___ComgarimMlhejdaMliMgsM/m^ rAAV productions in SF-t- cells were performed as described above in Example 1, For all productions the 1TR. containing baculovirus and the capsid gene containing baeuiovims were identical., the passage number was the same as the Rep gene containing baeulo viruses, 4 different Rep gene containing haeufeviruses were used; 1) The REP-ACG / PSC, 2) SLR.: the original construct by lirabe et ah (2002, supmk 3) Rep52 r Rep78{82B): Two separate Bac~to~Bac Imeubviruses, one containing the Rep 7S gene and the other one containing the Rep 52 gene, 4) R.ep52 + Rep/8( PSC): Two separate protein sciences'baouhvirases one. containing the Rep 78 gene and the other one containing the Rep 52 gene.
Results are shown in Figure 4,: At fifth baculovirus passage rAAV production is already improved by more than a factor 10 using a REP-ACG / PSC in. accordance with invention as compared to the original Rep construct and compared to the split Rep constructs. M_SiaMMiEMihg, rAAV productions m SPr cells were performed, as described in Example 1, For all productions the: ITR containing baculovirus and the capsid gene containing baeubviras were identical, the passage number was the same as the REP-ACG / PSC bacubvims, Results are shown in. Figure 5. The REP-ACG / PSC baculovirus is stable to at least passage: 8. rAAV production titers of REP-ACG / PSC are stable up to at least -8th passage of the baculovirus.
The effect of passage number on the expression of Rep protein for the original construct from Urabe et al, (2002, $upm) was compared to a REP-ACG / PSC construct to accordance with the invention. The baculovirus passages and the western blot were done as described in Example 1. During a normal passage of the rep baeulo viruses, samples were taken at 40 hours after addition, of the baeulo viruses to the SF cells and western blot was performed. Figure 6 clearly shows diminished Rep expression in higher passages compared to earlier passages for the original Urabe construct (SLR.), while the Rep expression in the REP-ACG / PSC construct stays the same in the higher passages compared to the lower ones.
Claims (24)
- Claims1. A first nucleic acid construct comprising a first nucleic acid sequence which encodes both Rep78 and Rep52 proteins from a Rep78 nucleotide sequence; the first nucleic acid sequence being operably linked to an expression control sequence that includes a promoter that is active in insect cells and is constructed such that both Rep78 and Rep52 are produced upon expression in an insect cell.
- 2. The nucleic acid construct according to claim 1, wherein the promoter is selected from the group consisting of polyhedron promoter, plO promoter, p35 promoter, IE-1 promoter and delta-IEl promoter.
- 3. The nucleic acid construct according to claim 1 or claim 2 that comprises one polyadenylation sequence at the 3’ end of the first nucleic acid sequence.
- 4. An insect cell comprising the nucleic acid construct according to any one of claims 1 to 3, wherein said nucleic acid construct expresses parvoviral Rep78 and Rep52 proteins.
- 5. The insect cell according to claim 4, wherein the promoter is selected from the group consisting of polyhedron promoter, plO promoter, p35 promoter, IE-1 promoter and delta-IEl promoter.
- 6. The insect cell according to claim 4 or claim 5, wherein the nucleic acid construct comprises one polyadenylation sequence at the 3’ end of the first nucleic acid sequence.
- 7. The insect cell according to any one of claims 4 to 6, selected from the group consisting of Se301, Seizd2109, Seucrl, sf9, Sf900+, Sf21, Bti-Tn-5bl-4, Mg-1, Tn368, Hzaml, Ha2302, Hz2e5, High five and Express+.
- 8. The insect cell according to any one of claims 4 to 7, further comprising a second nucleotide sequence comprising at least one parvoviral inverted terminal repeat (ITR) nucleotide sequence.
- 9. The insect cell according to claim 8, further comprising a third nucleotide sequence comprising parvoviral Cap protein coding sequences operably linked to expression control sequences for expression of Cap proteins in the insect cell.
- 10. The insect cell according to claim 8 or claim 9, wherein the second nucleotide sequence comprises two parvoviral ITR nucleotide sequences.
- 11. The insect cell according to claim 9 , further comprising, in addition to the first nucleic acid construct, a second nucleic acid construct, wherein: (a) the third nucleotide sequence is comprised in the first nucleic acid construct, and (b) the second nucleotide sequence is comprised in the second nucleic acid construct.
- 12. The insect cell according to claim 9 or claim 10, further comprising, in addition to the nucleic acid construct comprising the first nucleic acid sequence which encodes both Rep78 and Rep52 proteins, a second and a third nucleic acid construct, wherein (a) the second nucleic acid construct comprises the second nucleotide sequence, and (b) the third nucleic acid construct comprises the third nucleotide sequence.
- 13. The insect cell according to any one of claims 8 to 12, wherein the second nucleotide sequence further comprises a fourth nucleotide sequence encoding a gene product of interest positioned either 3’ or 5’ to the ITR sequence or between two ITR sequences.
- 14. The insect cell according to claim 13, wherein the fourth nucleotide sequence encoding the gene product of interest is positioned between the two ITR sequences.
- 15. The insect cell according to claim 13 or claim 14, wherein the gene product of interest is a therapeutic gene product.
- 16. The insect cell according to claim 15, wherein the therapeutic gene product is selected from the group consisting of Cystic fibrosis transmembrane conductance regulator (CFTR), Factor IX, lipoprotein lipase (LPL), apolipoprotein Al, uridine diphosphate glucuronosyltransferase, Retinitis pigmentosa GTPase Regulator Interacting Protein, a cytokine and an interleukin.
- 17. The insect cell according to claim 16, wherein the LPL is LPL S447X.
- 18. The insect cell according to claim 16, wherein the interleukin is Interleukin 10.
- 19. The insect cell according to any one of claims 13 to 18, wherein the second nucleotide sequence comprises a nucleotide sequence encoding one or more of the following marker polypeptides: green fluorescent protein, Herpes Simplex Virus (HSV) thymidine kinase (TK), hygromycin B phosphotransferase, Tn5 aminoglycoside phosphotransferase, dihydrofolate reductase and CD20.
- 20. The insect cell according to any one of claims 13 to 18, wherein the second nucleotide sequence comprises one or more of the following suicide genes: Escherichia coli cytosine deaminase, HSV-TK, Cytomegalovirus TK or Varicella-Zoster TK.
- 21. The insect cell according to any one of claims 13 to 20, wherein the second nucleotide sequence comprises a nucleotide sequence encoding Rep78 and/or Rep68.
- 22. A recombinant AAV virion produced by: (i) culturing the insect cell as defined in any of claims 9 to 21 under conditions that permit production of the recombinant AAV virion; and (ii) recovering the recombinant AAV virion.
- 23. A method for producing a recombinant parvoviral virion in an insect cell, comprising: (a) culturing the insect cell as defined in any of claims 9 to 21 under conditions such that recombinant parvoviral virion is produced; and, (b) recovering the recombinant parvoviral virion from the culture.
- 24. A recombinant parvoviral virion produced by the method according to claim 23.
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