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WO1999061641A1 - Production de vecteur-aav (virus adeno-associe) - Google Patents

Production de vecteur-aav (virus adeno-associe) Download PDF

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Publication number
WO1999061641A1
WO1999061641A1 PCT/GB1999/001634 GB9901634W WO9961641A1 WO 1999061641 A1 WO1999061641 A1 WO 1999061641A1 GB 9901634 W GB9901634 W GB 9901634W WO 9961641 A1 WO9961641 A1 WO 9961641A1
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WO
WIPO (PCT)
Prior art keywords
adeno
producer cell
gene
rep
cell according
Prior art date
Application number
PCT/GB1999/001634
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English (en)
Inventor
Robert James Anderson
Hugh Grant Prentice
Ian Duncan Macdonald
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University College London
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
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Publication of WO1999061641A1 publication Critical patent/WO1999061641A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to an improved adeno-associated viral producer cell and to a method for the production of an adeno-associated viral vector.
  • Adeno-associated virus is a vector which has shown promise in virally-mediated gene therapy protocols.
  • AAV is a small, 20-22nm, replication defective, non-pathogenic parvo virus.
  • Six serotypes of AAV are currently known (types 1 to 6) , of which type 2 is the most extensively studied.
  • AAV type 2 has a single stranded DNA genome of 4.7kB which encodes both structral proteins through a cap gene and non- structural proteins through a rep gene.
  • the virus has a pair of 146bp inverted terminal repeats (ITRs) which are the only cis acting elements in AAV required for replication and packaging .
  • ITRs inverted terminal repeats
  • recombinant AAV is produced by transfection of a permissive cell line with a mix of two plasmids .
  • a first plasmid supplies the AAV functions in trans and contains only the AAV rep and cap genes .
  • a second plasmid contains the gene or genes of interest (transgenes) between viral ITRs.
  • Recombinant AAV is produced from the transfected cells after infection with an appropriate helper virus, usually adenovirus . The AAV is then purified from both the supernatant and the lysed cell pellet. This system is inefficient in AAV production, due in part to the low probability of all cells receiving both plasmids; a prerequisite for the production of recombinant AAV.
  • An improvement to this method has been described by Clark et al in Hum . Gen . Ther . 1995; 6:1329-1341.
  • An AAV producer cell line was constructed using a single plasmid which carries the transgene between viral ITRs, together with the rep and cap genes and a selectable marker. After appropriate selection, all the cells will have the necessary sequences to create recombinant AAV. These cells are infected with helper virus and the recombinant AAV purified from the resulting cell pellet and cell supernatant.
  • AAV producer cell can be constructed which will produce AAV vectors at higher efficiencies than have hitherto been possible.
  • the present invention provides an adeno-associated viral producer cell which comprises a stable transfectant containing at least one rep gene and at least two cap genes, wherein the number of cap genes exceeds the number of rep genes .
  • each rep gene encodes a rep mRNA which has reduced efficiency of translation as compared with wild-type rep mRNA.
  • the ratio of cap genes: rep genes in the producer cell is at least 2:1, preferably at least 3:1, more preferably around 3 to 4:1.
  • the rep mRNA of the viral producer cells according to the present invention has a mutant initiation codon which reduces the efficiency of translation. Wild-type initiation codons are AUG whereas mutant initiation codons may be UUG, CUG, or GUG, preferably ACG.
  • the rep and cap genes are associated with a gene encoding a selectable marker so as to enable producer cells containing the rep and cap genes to be readily identified and maintained.
  • the marker comprises a drug- resistance marker, such as an antibiotic resistance marker which enables the cells to be grown in medium containing the relevant drug or antibiotic.
  • the selectable marker may be a cell surface marker such as a receptor which can be specifically targeted by a complimentary molecule such as an antibody thereto.
  • One such cell surface marker is nerve growth factor (NGF) .
  • the at least one rep gene is associated with a first gene encoding a selectable marker and the at least two cap genes are associated with a second gene encoding a selectable marker, which is different from the first selectable marker.
  • the at least two cap genes are associated with a second gene encoding a selectable marker, which is different from the first selectable marker.
  • the at least one rep gene is spaced sufficiently apart from the cap genes to prevent simultaneous recombination of rep and cap.
  • the viral producer cells must be transfected or infected with adeno- associated viral DNA which would normally be cap " rep " .
  • the AAV would necessarily contain inverted terminal repeats and so a problem arises in producing AAV vectors because there is a low but significant frequency of homologous recombination.
  • the AAV producer cells contain a rep gene contiguous with a cap gene, homologous recombination events result in contaminating amounts of wild-type AAV being formed. This problem is avoided where the cap and rep genes are spaced sufficiently apart to prevent such simultaneous recombination of rep and cap.
  • the at least one rep gene and/or the at least two cap genes are plasmid-borne so as to avoid contaminating homologous recombination. More preferably, both rep and cap genes are plasmid-borne.
  • the viral producer cell is formed from a single rep-containing plasmid which carries a single rep gene and multiple copies of a cap-containing plasmid preferably each carrying a single cap gene.
  • each cap-containing plasmid has an origin of replication and the producer cell is capable of producing a replication initiator for the origin of replication of the cap-containing plasmid. In this way, multiple copies of the cap-containing plasmid are made in the cell.
  • Various combinations of replication initiator and origin of replication are known.
  • a particularly useful combination is where the cell is a COS cell and the origin of replication is SV40 ori .
  • COS cells produce T antigen as a replication initiator for SV40 ori.
  • EBNA1 as a replication initiator for oriP.
  • EBNA1 and oriP are derived from the Epstein Barr virus which is capable of infecting a variety of cells, including human cell lines.
  • E4 0RF6 needs to be present as a polypeptide to facilitate conversion of the AAV single stranded genome to the double stranded replicative form.
  • E4 0RF6 a polypeptide encoded by the adenovirus E4 region open reading frame 6 (E4 0RF6) was responsible for increasing the levels of expression of an AAV transgene (see J. Virol .70 (5) :3772-3234, 1996 and J.Virol. 70 (1) :520-532 , 1996) although the precise nature of the polypeptide is unknown.
  • the present invention provides the adeno-associated viral producer cell further comprising a gene encoding a protein with adenovirus E4 ORF6 activity, which gene is under the control of an inducible promoter.
  • adeno-associated viral producer cell further comprising a gene encoding a protein with adenovirus E4 ORF6 activity, which gene is under the control of an inducible promoter.
  • E4 ORF6 constitutive expression of E4 ORF6 in the AAV producer cell tends to have an adverse effect on the cell. This was unexpected. However, by putting the gene under control of an inducible promoter the producer cell is able to tolerate the presence of the protein.
  • Any inducible promoter may be used, such as the MMTV-LTR (Mouse Mammary Tumour Virus-Long Terminal Repeat) promoter, which is inducible by dexamethasone .
  • the inducer may be added to the medium in which the cells grow or, alternatively, may be produced upon infection of the cell by AAV or helper virus for AAV.
  • an AAV inducible promoter was used; the P5 promoter. In this case, upon infection with both adeno-virus and AAV, the P5 promoter is induced so as to induce the E4 ORF6 protein.
  • the gene encoding adenovirus E4 0RF6 protein may be conveniently introduced into the producer cell on a plasmid.
  • the E4 0RF6 gene may be readily obtained from an adenovirus such as adenovirus type 2, which is deposited as ATCC VR846, the sequence of which is published by EMBL at locus HACG. See also Ohman et al in Virology 194 (1) , 50-58, 1993 where cloning of E4 ORF is reported. From the EMBL sequence the E4 region spans nucleotides 32801-35609 and E4 ORF6 from Ohman et al spans nucleotides 33193-34115.
  • the protein with E4 ORF6 activity has a nuclear localisation sequence which is typically situated outside of the functional part of the E4 ORF6 protein, typically at or near the N- or C-terminal sequence of the protein.
  • the nuclear localisation sequence confers on the protein an important property.
  • the present invention provides a method for the production of an adeno-associated viral vector, which comprises introducing into the viral producer cell DNA comprising a transgene flanked by adeno-associated virus inverted terminal repeats, allowing formation of adeno- associated viral vectors in the viral producer cell, and isolating the adeno-associated viral vectors; wherein the DNA is cap " rep " .
  • the DNA may be introduced into the producer cell by transfection, for example as part of a suitable plasmid, in the presence of an adeno-associated helper virus, or by infection as part of a suitable virus.
  • the DNA may be integrated into the genome of an adeno- associated helper virus which infects the producer cell or the DNA may comprise the genome of an adeno-associated virus which is infected into the producer cell in the presence of an adeno-associated helper virus.
  • the helper virus comprises an adenovirus, such as adenovirus type 5.
  • This Example compares titre of AAV (type 2) from 293 cells (ATCC CRL 1573) , COS 7 cells (ATCC CRL 1651) and COS cells according to the present invention.
  • a cell line containing multiple copies of the AAV cap gene was created as follows.
  • pUC 18 was modified by the insertion of a pair of linkers : - 5 ' CAT CGA TGG CCA GAT CTG ATA TCG ATG 3 ' and 5 ' CAT GGT AGC TAC CGG TCT AGA CTA TAG CTA CCT AG 3 ' between the Kpn I/Bam
  • AAV was obtained from the ATCC. After digest with Bgl II, the resulting AAV fragment was cloned into the modified pUC18 to give pUC/AAV.
  • the AAV cap gene was excised from pUC/AAV by digestion with SnaBI and HinD III and ligated into HinD III/ Sma I digested pBK-CMV (Stratagene) to yield pCMV-cap.
  • a COS cell stable transfectant cell line was created by introducing 2 ⁇ g pCMV- cap DNA into exponentially growing COS 7 cells (ATCC CRL 1651 ) using Lipofectin following the manufacturers' instructions. This gave COS/cap.
  • p3 ' SS (Stratagene) was digested with Pfl MI and Nsi I to release a 1.8kbp Hygromycin resistance cassette. This was blunted with T4 DNA polymerase .
  • the AUG initiation codon of rep68/78 in pUC/AAV/Neo was mutated by standard techniques to give an ACG codon. This was verified by sequencing.
  • This construct was digested with Apa I and Nde I to remove the cap gene and the ends blunted with T4 DNA polymerase .
  • the hygromycin cassette was ligated to the mutated rep gene cassette to give pUC/ACGrep/Hygro .
  • COS cell stable transfectant cell line was created by introducing 2 ⁇ g pUC/ACGrep/Hygro DNA into exponentially growing COS/cap cells using Lipofectin following the manufacturers' instructions. This gave COS/rep/cap.
  • COS/rep/cap or COS/cap or parental COS cells were grown to 80% confluency in 92mm tissue culture plates (Nunclon) in DMEM supplemented with 10% FCS .
  • 4.6 ⁇ g of AAV ⁇ -galactosidase (AAV ⁇ -gal) and in the case of parental COS cells, 9.4 ⁇ g of rep/cap plamid were addded to a 17 x 120mm conical polystyrene sterile tube (Falcon) , together with lOO ⁇ l of serum free medium (Opti-MEM - Life Sciences) .
  • 56 ⁇ l of lipofectin was added to a second polystyrene tube together with another lOO ⁇ l of Opti-MEM.
  • the DNA/lipofectin mixture was mixed gently and incubated for 15 minutes at room temperature.
  • the cells were washed twice with HBSS and after the final aspiration the DNA/lipofectin mixture was layered onto the cells with fresh Opti-MEM to ensure the cells were adequately covered.
  • the cells were then incubated for 5 hours at 37°C and 5% C0 2 , after which the serum free medium was replaced with fresh DMEM containing 10% FCS and adenovirus type 5 at a multiplicity of infection (MOI) of 5. Incubation was then continued until full cytopathic effect of the adenovirus was seen, typically after 48 to 72 hours.
  • MOI multiplicity of infection
  • the infected cells were scraped off and collected together with the supernatant. Trypsin was added to a final concentration of 0.02% and deoxycholate to a final concentration of 0.5%. The sample was then incubated at 37°C for 2 hours and then subjected to three cycles of freeze-thawing to release any virus remaining within the cells. Following the final freeze cycle, the sample was incubated at 56°C for 1 hour to inactivate the heat-labile adenovirus. The sample was spun at 2100 xG for 10 minutes to pellet the cellular debris and the supernatant containing the recombinant AAV was decanted into a separate 50ml tube (Falcon) .
  • Falcon 50ml tube
  • the concentrated viral preparation was desalted using a Pharmacia Biotech pD-10 Sephadex desalt column. The desalted viral preparation was then further concentrated using a Centriprep 30 concentrator to give a final volume of approximately 500 ⁇ l.
  • the viral sample Prior to infecting cells, the viral sample was sterilised free of bacteria using a 0.2 ⁇ m filter (Sartorius - Centrisart C4). The viral concentrate was then either analysed immediately, used to infect target cells or stored at -70°C.
  • Viral titre was quantitated using a dot-blot assay as follows. Hybond-N nylon membrane (Amersham-Life Science) was prewetted in 6x SSC, and then inserted in the dot blot apparatus (Bio-Rad) .
  • the reference control DNA and the recombinant AAV preparation were diluted in lOx SSC to the following ratios, neat, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048 and immediately prior to loading, the samples were denatured by heating at 100°C for 5 minutes . The samples were then loaded onto the nylon membrane and sucked through under negative pressure. The nylon filter was then removed from the dot blot apparatus and dried by incubation for 2 hours at 80°C. After drying both sides of the filter were UV irradiated (Stratagene UV Stratalinker) .
  • Prehybridisation buffer (24 mis 20x SSC, 8 mis 50x Denharts, 4mls 10% SDS, 80 ⁇ l (lOng/ml) salmon sperm DNA) , was warmed to 60°C and added to the filter present in a hybridisation bottle. Prehybridisation was allowed to continue for 4 hours at 65°C. After this time, the prehybridisation buffer was then discarded and replaced with hybridisation buffer (9 mis 20x SSC, 3mls 50x Denharts, 1.5mls 10% SDS, 300 ⁇ l EDTA 0.5M, 30 ⁇ l salmon sperm DNA (lOng/ml) , 16.2ml H 2 0) containing a radiolabelled probe.
  • the probe used was the CMV promoter present in both the viral preparation and the reference plasmid.
  • the CMV promoter probe was radiolabelled using the rediprime DNA labelling system (Amersham Life Science) following the manufacturers' instructions. After the labelling reaction had completed, unincorporated radio-label was removed using a Qiagen desalt column following the manufacturers instructions.
  • the probe Prior to the addition of the probe to the hybridisation mix and the filter, the probe was denatured by heating to 100°C for 5 minutes. Hybridisation was allowed to proceed for 18 hours at 65°C. Post hybridisation, the filter was washed twice in 2x SSC and 0.1% SDS at 37°C for 10 minutes, followed by two washes in 2x SSC and 0.1% SDS at 65°C for 10 minutes.
  • Titres of virus, as determined by the viral dot-blot, obtained from the COS rep/cap cells were higher than those obtained using parental cells and the two plasmid system. No virus was produced from the COS/cap cells.
  • All titres refer to the number of genomes obtained per ml. of virus as assessed by dot-blot hybridisation.

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Abstract

Cette cellule productrice de virus adéno-associé comporte au moins un gène rep et au moins deux gènes cap, le nombre de gènes cap dépassant celui des gènes rep.
PCT/GB1999/001634 1998-05-22 1999-05-21 Production de vecteur-aav (virus adeno-associe) WO1999061641A1 (fr)

Applications Claiming Priority (2)

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GB9811172.7 1998-05-22
GBGB9811172.7A GB9811172D0 (en) 1998-05-22 1998-05-22 Virus production

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007148971A3 (fr) * 2006-06-21 2008-02-07 Amsterdam Molecular Therapeutics Bv Vecteurs aav avec séquences de codage rep ameliorées pour une production dans des cellules d'insecte
AU2013254897B2 (en) * 2006-06-21 2016-01-28 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5354678A (en) * 1990-10-30 1994-10-11 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1996017947A1 (fr) * 1994-12-06 1996-06-13 Targeted Genetics Corporation Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants
WO1996022378A1 (fr) * 1995-01-20 1996-07-25 Rhone-Poulenc Rorer S.A. Cellules pour la production d'adenovirus recombinants

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5354678A (en) * 1990-10-30 1994-10-11 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1996017947A1 (fr) * 1994-12-06 1996-06-13 Targeted Genetics Corporation Lignees cellulaires d'encapsidation utilisees pour la generation de titres hauts de vecteurs aav recombinants
WO1996022378A1 (fr) * 1995-01-20 1996-07-25 Rhone-Poulenc Rorer S.A. Cellules pour la production d'adenovirus recombinants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIAO X. ET AL.: "Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus", JOURNAL OF VIROLOGY, vol. 72, no. 3, March 1998 (1998-03-01), pages 2224 - 2232 2232, XP002098258, ISSN: 0022-538X *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007148971A3 (fr) * 2006-06-21 2008-02-07 Amsterdam Molecular Therapeutics Bv Vecteurs aav avec séquences de codage rep ameliorées pour une production dans des cellules d'insecte
KR20090037401A (ko) * 2006-06-21 2009-04-15 암스테르담 몰레큘러 테라퓨틱스 비. 브이. 곤충세포 내 ａａｖ의 생산에 유용한 ａａｖ-ｒｅｐ78의 번역을 위한 변형된 개시 코돈을 갖는 벡터
JP2009540823A (ja) * 2006-06-21 2009-11-26 アムステルダム モレキュラー セラピューティクス ビー．ブイ． 昆虫細胞におけるａａｖの生成に有用なａａｖ−ｒｅｐ７８の翻訳の改変型開始コドンを有するベクター
RU2457252C2 (ru) * 2006-06-21 2012-07-27 Амстердам Молекьюла Терапьютикс (Амт) Ип Б.В. AAV ВЕКТОРЫ С УСОВЕРШЕНСТВОВАННЫМИ Rep-КОДИРУЮЩИМИ ПОСЛЕДОВАТЕЛЬНОСТЯМИ, ИСПОЛЬЗУЕМЫМИ В СИСТЕМАХ ПРОДУКЦИИ НА ОСНОВЕ КЛЕТОК НАСЕКОМЫХ
AU2007261806B2 (en) * 2006-06-21 2013-08-15 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells
US8512981B2 (en) * 2006-06-21 2013-08-20 Amsterdam Molecular Therapeutics B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
JP2014012013A (ja) * 2006-06-21 2014-01-23 Unicure Ip Bv 昆虫細胞におけるａａｖの生成に有用なａａｖ−ｒｅｐ７８の翻訳の改変型開始コドンを有するベクター
CN101506369B (zh) * 2006-06-21 2014-02-12 尤尼克尔生物制药股份有限公司 具有经修饰的用于在昆虫细胞中产生aav的aav-rep78翻译起始密码子的载体
CN103849629A (zh) * 2006-06-21 2014-06-11 尤尼克尔生物制药股份有限公司 具有经修饰的用于在昆虫细胞中产生aav的aav-rep78翻译起始密码子的载体
US8952144B2 (en) 2006-06-21 2015-02-10 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-REP78 useful for production of AAV
AU2013254897B2 (en) * 2006-06-21 2016-01-28 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells
KR101589259B1 (ko) 2006-06-21 2016-02-01 유니큐어 아이피 비.브이. 곤충세포 내 ａａｖ의 생산에 유용한 ａａｖ-ｒｅｐ78의 번역을 위한 변형된 개시 코돈을 갖는 벡터
EP3023500A1 (fr) * 2006-06-21 2016-05-25 UniQure IP B.V. Cellules d'insectes pour la production de vecteurs d'aav
CN103849629B (zh) * 2006-06-21 2017-06-09 尤尼克尔Ip股份有限公司 具有经修饰的用于在昆虫细胞中产生aav的aav‑rep78翻译起始密码子的载体
US9708627B2 (en) 2006-06-21 2017-07-18 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-REP78 useful for production of AAV
US9988645B2 (en) 2006-06-21 2018-06-05 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-REP78 useful for production of AAV
US10138496B2 (en) 2006-06-21 2018-11-27 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
US10533188B2 (en) 2006-06-21 2020-01-14 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV
EP3023500B1 (fr) 2006-06-21 2020-02-12 uniQure IP B.V. Cellules d'insectes pour la production de vecteurs d'aav
EP3705577A1 (fr) * 2006-06-21 2020-09-09 uniQure IP B.V. Vecteurs aav avec des séquences de codage rep améliorées pour la production de cellules d'insectes
US10865423B2 (en) 2006-06-21 2020-12-15 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-REP78 useful for production of AAV
US11613765B2 (en) 2006-06-21 2023-03-28 Uniqure Ip B.V. Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV

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