WO1998010088A1 - An inducible method for production of recombinant adeno-associated viruses utilizing t7 polymerase - Google Patents

Abstract

Methods for efficient production of recombinant AAV are described. In one aspect, three vectors are introduced into a host cell. A first vector directs expression of T7 polymerase. A second vector carries rep and cap under the control of the T7 promoter. A third vector contains a rAAV cassette which contains a minigene flanked by AAV ITRs. In a second aspect, the host cell is stably transfected to contain a plasmid bearing one of the required vector components and the host cell is double transfected/infected.

Description

AN INDUCIBLE METHOD FOR PRODUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRUSES UTILIZING T7 POLYMERASE

Background of the Invention

Adeno-associated virus is a replication- deficient parvovirus, the genome of which is about 4.6 kb in length, including 145 nucleotide inverted terminal repeats (ITRs) . The single-stranded DNA genome of AAV contains genes responsible for replication (rep) and formation of virions (cap) . When this nonpathogenic human virus infects a human cell, the viral genome integrates into chromosome 19 resulting in latent infection of the cell. Production of infectious virus and replication of the virus does not occur unless the cell is coinfected with a lytic helper virus such as adenovirus or herpesvirus. Upon infection with a helper virus, the AAV provirus is rescued and amplified, and both AAV and helper virus are produced.

AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells. Various groups have studied the potential use of AAV in the treatment of disease states.

However, an obstacle to the use of AAV for delivery of DNA is the lack of highly efficient methods for encapsidation of recombinant genomes. See, R. Kotin, Hum. Gene Ther.. 5:793-801 (1994). Furthermore, the rep gene product is toxic to cells and thus cannot be expressed at high levels. For example, previously known methods employ transfection of host cells with a rAAV genome which lacks rep and cap genes followed by co- infection with wild-type AAV and adenovirus. However, this method leads to unacceptably high levels of wild- type AAV. Incubation of cells with rAAV in the absence of contaminating wild-type AAV or helper adenovirus is associated with little recombinant gene expression. And, in the absence of the AAV rep gene product, integration is inefficient and not directed to chromosome 19. Bacteriophage T7 RNA polymerase (T7 Pol) is the product of T7 gene 1, which can recognize its responsive promoter sequence specifically and exhibit a high transcriptase activity [M. Chamberlin et al, Nature - ££8_:227-231 (1970); J. Dunn and F. Studier, J. Mol.

Biol.. 166:447-535 (1983); and B. Moffatt et al, Cell. 49_:221-227 (1987)]. It has been used for heterologous expression of proteins in E. coli [S. Tabor and C. Richardson, Proc. Natl. Acad. Sci. USA. 82:1074-1078 (1985); F. Studier and B. Moffatt, J. Mol. Biol..

189:113-130 (1986)], in recombinant vaccinia virus- infected eukaryotic cells [T. Fuerst et al, Proc. Natl. Acad. Sci. USA. 1:8122-8126 (1986); A. Ramsey-Ewing and B. Moss, J. Biol. Chem.. 271:16962-16966 (1996)], and in mammalian cells [A. Lieber et al, Nucl. Acids Res.. 17:8485-8493 (1989)].

What is needed is an efficient method for production of rAAV which avoids the problems associated with rep toxicity for the packaging cell.

Summary of the Invention

The present invention provides an inducible method for efficient production of rAAV which makes use of T7 polymerase. T7 Pol is derived from lambda phage and its promoter is not active in mammalian cells. Thus, expression of rep/cap can be controlled by placing these genes under control of the T7 promoter and providing the T7 Pol in trans or under the control of an inducible promoter. Thus, this method avoids the toxic effects of rep which rendered prior art methods of producing rAAV inefficient. The method of the invention is particularly suitable for large scale production of rAAV, which is desired for rAAV vectors to be used in gene therapy.

In one aspect, the invention provides a method of producing rAAV which utilizes three vectors. A first vector is capable of expressing T7 polymerase in the host cell following transfection or infection. A second vector comprises the AAV rep and cap genes under the control of T7 promoter sequences (T7/rep/cap) . The third vector comprises a cassette containing 5 • and 3 ' AAV inverted terminal repeats (ITRs) flanking a selected transgene. A host cell containing these three vectors is cultured under conditions which permit replication and packaging of a recombinant AAV, and the rAAV is recovered.

In another aspect, the invention provides a method in which a host cell is stably transfected with one of the three components of the system used in the triple infection system. The remaining components are introduced into the host cell, as described above. In one embodiment, the invention provides method in which a vector containing T7 /rep /cap and a vector containing a cassette comprising a selected minigene flanked by 5¹ and 3' AAV ITRs are introduced into a host cell expressing T7 polymerase. The host cell is then cultured under conditions which permit production of rAAV. In another embodiment, this invention provides a method which utilizes a host cell stably transfected with a plasmid containing T7 /rep/cap. A vector containing T7 pol and a vector containing a cassette comprising 5' AAV inverse terminal repeat (ITR), a selected minigene, and 3' AAV ITR are introduced into the host cell. The host cell is cultured under conditions which permit production of rAAV. In still another embodiment, the invention provides a method which utilizes a host cell stably transfected with a rescuable rAAV cassette. A vector containing T7 pol and a vector containing T7 /rep/ cap are introduced into the host cell. The host cell is cultured under conditions which permit production of rAAV. In yet another aspect, the present invention provides a method which utilizes a host cell stably transfected with two of the three components of the system used in the triple infection system. The remaining component is then introduced into the host cell, as described above.

In a further aspect, the present invention provides a method which utilizes a host cell stably transfected with the three components of the system used in the triple infection system. In this aspect, the T7 Pol is controlled by an inducible promoter.

In still a further aspect, the invention provides a rAAV produced according to the method of the invention. Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings

Fig. 1 provides a schematic illustration of the construction of a recombinant adenovirus containing the T7 polymerase gene.

Fig. 2 provides a schematic illustration of the construction of a recombinant plasmid containing the AAV rep/cap genes under control of a T7 promoter. Fig. 3 provides a schematic illustration of the construction of a recombinant adenovirus containing the rep/cap genes under control of a T7 promoter.

Fig. 4 provides a schematic illustration of the construction of a recombinant hybrid Ad/AAV virus.

Detailed Description of the Invention

The invention provides an inducible method for efficient production of recombinant AAV vectors useful particularly for gene delivery and transfer. Specifically, the invention provides methods of AAV production in which expression of the toxic but necessary rep gene is controlled by the T7 promoter.

Thus, in one aspect, the method of the invention for production of rAAV involves introducing into a host cell the AAV rep and cap genes under control of a T7 promoter, and a recombinant adeno-associated virus (rAAV) cassette containing a selected minigene flanked by AAV ITRs. Upon introduction of a gene encoding T7 pol, high level expression of rep protein from the T7/rep/cap construct is induced and cells may be grown on a large scale. When rep expression is desired, the cells are caused to express the T7 polymerase which acts on the T7 promoter. This facilitates the efficient replication and packaging of rAAV carrying a gene of interest.

A host cell may be triple transfected (or infected) with vectors containing the above elements. Alternatively, a host cell which expresses one or more of the required elements and may be transfected/infected with the remaining elements is utilized. In another alternative, a host cell is utilized which stably expresses all three elements of the system, and the T7 pol is placed under the control of an inducible promoter, which permits rep/cap expression to be controlled and the avoidance of toxic effects to the cell.

For each of the vector components used in the method of the invention, adenoviral constructs are currently preferred. However, using the information provided herein and known techniques, one of skill in the art could readily construct a different viral (adenoviral or non-adenoviral) or a plasmid vector which is capable of driving expression of the desired genes in the host cell. For example, although less preferred because of their inability to infect non-dividing cells, vectors carrying the required elements of this system, e.g., the T7 polymerase, may be readily constructed using retroviruses. Therefore, this invention is not limited by the virus or plasmid selected for purposes of introducing the T7 pol, T7/rep/cap, or AAV cassette into the host cell. Desirably, at least one of the vectors is a virus which provides the necessary helper functions to enable packaging. Alternatively, the helper functions may be provided by a co-transfected adenovirus or herpesvirus. Suitable techniques for introducing these vectors into the host cell are discussed below and are known to those of skill in the art. As used herein, a "host cell" is any cell (cell line) , preferably mammalian, which permits expression of the T7 pol and T7/rep/cap and packaging of the rAAV containing the cassette, under the conditions described herein. Suitable packaging cells are known, and may be readily selected by the skilled artisan.

A. Triple Infection /Trans fection As stated above, a host cell used for assembly and packaging of recombinant AAV may be transfected with plasmid vectors or infected with viral vectors containing the required components of the system.

1. T7 Pol Vectors In a preferred embodiment, a first vector contains the T7 Pol gene under the control of a suitable promoter. In example 5 below, the nuclear localized T7 Pol gene is obtained from a publicly available plasmid [M. Strauss, Nucleic Acid Res.. 12:8485-8493 (1989)]. However, the gene may alternatively be obtained from other commercial and academic sources, including the American Type Culture Collection (pTF7-3, Accession No. 484944). See, also GenBank accession number M30308. Desirably, the T7 pol gene is linked to a nuclear localization signal, such as that described in Dunn, Gene. 6JJ:259-266 (1988), using conventional techniques.

Desirably, T7 Pol is under the control of a cytomegalovirus (CMV) immediate early promoter/enhancer [see, e.g., Boshart et al, Cell. 41:521-530 (1985)]. However, other suitable promoters may be readily selected by one of skill in the art. Useful promoters may be constitutive promoters or regulated (inducible) promoters, which will enable control of the amount of the transgene to be expressed. For example, another suitable promoter includes, without limitation, the Rous sarcoma virus LTR promoter/enhancer. Still other promoter/enhancer sequences may be selected by one of skill in the art.

In addition, the vector also includes other conventional regulatory elements necessary to drive expression of T7 Pol in a cell transfected with the vector. Such regulatory elements are known to those of skill in the art.

2. T7 /Rep/Cap Vectors

The second vector component of this system contains the rep and cap genes under control of a T7 promoter. The rep and cap genes can be obtained from a variety of known sources. See, e.g., T. Shenk, J. Virol.. 61:3096-3101 (1987), which provides the AAV2 genome within the plasmid psub20l; E. W. Lusby et al, J. Virol. , 4_l: 518-526 (1982) and J. Smuda and B.J. Carter, Virology. 1 :310-318 (1991). Similarly, the T7 promoter sequences

[J. J. Dunn and F.W. Studier, J. Mol. Biol.. 166:477-535 (1983) may be obtained from a variety of commercial and academic sources. In a preferred embodiment, the vector further contains the sequence of untranslated region (UTR) of encephalomyocarditis (EMCV) downstream of the T7 promoter. The inventors believe this sequence increases expression of the gene 5- to 10-fold.

In addition, the vector also includes conventional regulatory elements necessary to drive expression of the rep/cap in a cell transfected with the vector. Such regulatory elements are known to those of skill in the art.

3. rAAV Cassette (Template)

The third vector component contains a rAAV cassette containing a minigene flanked by AAV ITRs. As discussed in more detail below, such a minigene contains a suitable transgene, a promoter, and other regulatory elements necessary for expression of the transgene. The AAV sequences employed are preferably limited to the cis-acting 5 ' and 3 ' inverted terminal repeat (ITR) sequences [See, e.g., B. J. Carter, in "Handbook of Parvoviruses" , ed. , P. Tijsser, CRC Press, pp.155-168 (1990)]. Desirably, substantially the entire 143 bp sequences encoding the ITRs are used in the vectors. Some degree of minor modification of these sequences is expected to be permissible for this use. The ability to modify these ITR sequences is within the skill of the art. See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual.", 2d edit.. Cold Spring Harbor Laboratory, New York (1989) . Alternatively, it may be desirable to use functional fragments of the ITRs. Such fragments may be determined by one of skill in the art. The AAV ITR sequences may be obtained from any known AAV, including presently identified human AAV types. Similarly, AAVs known to infect other animals may also be employed in the vector constructs of this invention. The selection of the AAV is not anticipated to limit the following invention. A variety of AAV strains, types 1-4, are available from the American Type Culture Collection or available by request from a variety of commercial and institutional sources. In the following exemplary embodiment an AAV-2 is used for convenience.

The 5 • and 3 • AAV ITR sequences flank a minigene which is made up of a selected transgene sequence and associated regulatory elements. The transgene sequence of the vector is a nucleic acid sequence heterologous to the AAV sequence, which encodes a polypeptide or protein of interest. The transgene is operatively linked to regulatory components in a manner which permits transgene transcription.

The composition of the transgene sequence will depend upon the use to which the resulting vector will be put. For example, one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include without limitation an E. coli beta- galactosidase (LacZ) cDNA, an alkaline phosphatase gene and a green fluorescent protein gene. These sequences, when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, e.g., ultraviolet wavelength absorbance, visible color change, etc. A more preferred transgene sequence includes a therapeutic gene which expresses a desired gene product in a host cell. These therapeutic nucleic acid sequences typically encode products which may be administered to a patient in vivo or ex vivo to replace or correct an inherited or non- inherited genetic defect or treat an epigenetic disorder or disease. The selection of the transgene sequence is not a limitation of this invention. In addition to the major elements identified above, the minigene also includes conventional regulatory elements necessary to drive expression of the transgene in a cell transfected with the vector carrying the AAV cassette. Thus the minigene contains a selected promoter which is linked to the transgene and located within the minigene, between the AAV ITR sequences of the vector.

Selection of the promoter which mediates expression of the transgene is a routine matter and is not a limitation of the vector. Useful promoters include those which are discussed above in connection with the first vector component.

The minigene will also desirably contain heterologous nucleic acid sequences including sequences providing signals required for efficient polyadenylation of the transcript and introns with functional splice donor and acceptor sites. A common poly-A sequence which is employed in the exemplary vectors of this invention is that derived from the papovavirus SV-40. The poly-A sequence generally is inserted following the transgene sequences and before the 3* AAV ITR sequence. A common intron sequence is also derived from SV-40, and is referred to as the SV-40 T intron sequence. A minigene of the present invention may also contain such an intron, desirably located between the promoter/enhancer sequence and the transgene. Selection of these and other common vector elements are conventional and many such sequences are available [see, e.g., Sambrook et al, and references cited therein].

The rAAV vector containing the AAV ITRs flanking the minigene may be carried on a plasmid backbone and used to transfect a selected host cell or may be flanked by viral sequences (e.g., adenoviral sequences) which permit it to infect the selected host cell. Suitable Ad/AAV recombinant viruses may be produced in accordance with known techniques. See, e.g., WO 96/13598, WO 95/23867, and WO 95/06743, which are incorporated by reference herein. B. Double Infection /Trans fection

A cell line which stably expresses T7 pol may be constructed, and then double transfected (or infected) with a vector containing T7/rep/cap and a vector containing a rAAV cassette, as illustrated in the following table (Inf = infection and Txf = transfection) .

T7 rep/cap rAAV

System A Inf Inf

System B Inf Txf

System C Txf Inf System D Txf Txf

Alternatively, a cell line stably transfected with T7 rep/cap may be double transfected (infected) with a vector carrying T7 pol and a vector carrying the rAAV cassette, as illustrated in the following table.

T7 Pol rAAV

System E Inf Inf

System F Inf Txf

System G Txf Inf System H Txf Txf

In still another alternative, a cell line which contains a rescuable rAAV cassette may be double transfected (infected) with a vector containing T7 Pol and a vector containing T7/rep/cap, as illustrated in the following table.

T7 Pol T7 rep/cap System I Inf Inf

System J Inf Txf

System K Txf Inf System L Txf Txf The plasmid and viral vectors used in double transfection/infection steps are as described above in connection with the triple transfection and/or infection system. A stable cell line of the invention can be produced by transfection of a desired cell, e.g., 293 cells or other packaging cell lines expressing required adenoviral genes, with a plasmid containing the desired gene, e.g., T7 Pol, using conventional techniques and selected via an accompanying resistant marker gene. Depending upon whether inducible or constitutive expression is desired, an appropriate promoter may be selected. For example, if a host cell inducibly expressing T7 Pol is desired, the cell may be transfected with a plasmid containing T7 Pol under control of a metallothionein promoter. Alternatively, if a host cell constitutively expressing T7 Pol is desired, it may be inserted under control of a RSV or CMV promoter. Similar techniques may be used for providing a host cell containing the T7/rep/cap and a host cell containing a rescuable rAAV. The examples below describe production of stable cell lines. However, one of skill in the art could readily produce such cell lines using other conventional techniques. See, generally, Ausubel et al, Current Protocols in Molecular Biology (Wiley Interscience 1987) .

C. Single Infection /Trans fection

A cell line which stably expresses two of the components of this system may be constructed, and then transfected (or infected) with a vector containing the remaining component of the system, as described above. For example, using the techniques described herein, a cell line is utilized which is stably transfected with the T7/rep/cap and a rescuable rAAV. The cell line is then transfected or infected with a vector containing the T7 pol. As another example, the cell line is stably transfected with the T7 pol and a rescuable rAAV. The cell line is then transfected or infected with a vector containing the T7 rep/cap. D. Cell Line Containing T7 Pol, rAAV and

T7 /rep /cap

A cell line which stably expresses all three of the components of this system may be constructed and utilized in the method of the invention. Using known techniques, a suitable packaging cell line is constructed which contains the rAAV, the T7/rep/cap and the T7 pol. In this embodiment, the T7 Pol is placed under the control of an inducible promoter. Suitable inducible promoters are known to those of skill in the art and are discussed herein. For example, T7 Pol may be placed under control of a metallothionein promoter. In this manner, expression of the T7 Pol, and thus the rep/cap, which are under control of the T7 promoter can be regulated and toxic effects to the cell avoided. E. Production of Vectors and rAAV

Assembly of the selected DNA sequences of the adenovirus, AAV and the reporter genes or therapeutic genes and other vector elements into the vectors described above utilize conventional techniques. Such techniques include cDNA cloning such as those described in texts [Sambrook et al, cited above], use of overlapping oligonucleotide sequences of the adenovirus or AAV genome, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.

Whether using the three vector system, or stably infected cells, introduction of the vectors into the host cell is accomplished using known techniques. Where appropriate, standard transfection and co- transfection techniques are employed, e.g. , CaP0₄ transfection techniques using the complementation human embryonic kidney (HEK) 293 cell line (a human kidney cell line containing a functional adenovirus Ela gene which provides a transacting Ela protein) . Other conventional methods employed in this invention include homologous recombination of the viral genomes, plaquing of viruses in agar overlay, methods of measuring signal generation, and the like.

Following infection/transfection, the host cell is then cultured under standard conditions, to enable production of the rAAV. See, e.g., F. L. Graham and L. Prevec, Methods Mol. Biol.. 7:109-128 (1991). Desirably, once the rAAV is identified using conventional techniques, it may be isolated using standard techniques and purified.

These examples illustrate the preferred methods of the invention. These examples are illustrative only and do not limit the scope of the invention.

Example 1 - Construction of a T7 Pol Adenovirus Figure 1 provides a schematic of the construction of the recombinant adenovirus carrying the T7 polymerase.

The plasmid pMTT7N was obtained from Dr. Michael Strauss [A. Lieber et al, Nucl. Acids Res.. 12:8485-8493 (1989)]. pMTT7N contains a N-terminal nuclear location signal of SV40 large T antigen fused to the T7 Pol gene (T7N Pol) which is linked to the polyadenylation sequence of SV40. Expression is driven by the inducible mouse metallothionein promoter. The pMTT7N plasmid DNA was digested with Bglll and PvuII restriction enzymes and the fragments separated on an agarose gel. The Bglll/PvuII T7 Pol DNA fragment was ligated to the Bglll/EcoRV cleaved vector pAd.CMV.link.l to form pAd.CMV.T7N. pAd.CMV.link.l is a plasmid containing the adenoviral sequences 0 to 16 map units deleted of Ela and Elb into which a CMV promoter-polylinker cassette was cloned. This is described in X. Ye et al, J. Biol. Chem.. 271:3639-3646 (1996).

In pAd.CMV.T7N, the expression unit of T7 Pol is directed by the CMV promoter. The promoter for the T7 Pol gene is linked to a PolyA tail as a cassette within the sequence of adenovirus 0-1 map unit (mu) and 9-16 mu. The pAd.CMV.T7N is linearized by Nhe I digestion and co- transfected with Cla I linearized Addel327 backbone using Cellphate kit (Pharmacia) . Approximately 1 week post- transfection, the T7 Pol adenovirus can be isolated from the plaques for further purification.

Example 2 - Cell Lines Expressing T7 Pol

A cell line stably expressing T7 Pol is established by co-transfection of plasmids pMTT7N and pMTCB6+ (which provides a selective marker) [K. H. Choo et al, SNA, 5:529-538; Eur. J. Biochem.. 124:417-424] into 293 cell at a ratio of 10:1 using calcium phosphate precipitation [F. Graham and A. van der Eb, Virol.. 5_2:456-467 (1973)]. Colony cloning is carried out by Geneticin selection at a concentration of 1 mg/ml. Each clone obtained is transfected with pT7 rep/cap plasmid [see. Example 3 below] and analyzed for its ability to induce the expression of Rep protein upon induction by supplementation with Zn⁺⁺.

To establish a stable cell line that constitutively expresses the T7 Pol, the T7N Pol (obtained by Bglll/PvuII digestion of pMTT7N, as described above) was subcloned downstream of RSV promoter at the cloning sites of BamHI and PvuII in the vector of pEBVhis [Invitrogen] . The resulting plasmid, designated pEBVhisT7N, was transfected into 293 cells and selected with Hygromycin at a concentration of 400 μg/ml. Each positive clone is analyzed for the presence of T7 Pol by its ability to produce expression of T7-LacZ or T7- rep/cap in cells transfected with these plasmids.

Example 3 - Production of T7 rep/cap Adenovirus

The production of this recombinant adenoviral vector is illustrated schematically in Figs. 2 and 3. A. Plasmid Constiruction

The plasmid pTMl [B. Moss et al, Nature. 348:91-92 (1990)], designed for expressing genes under control of the T7 promoter/EMCV UTR (untranslated region of encephalomyocarditis) , was used as the vector for expressing AAV rep/cap. The entire coding sequence of rep/cap was separated into two portions by the unique Sad site and subcloned into the pTMl plasmid as described below.

Because there is no appropriate restriction enzyme existing between the initiation site of rep and its natural promoter, p5, the left end of the rep sequence (N-rep) was first amplified by PCR. The sequence of the upper primer was SEQ ID NO: 2: TATTTAAGCCCGAGTGAGCT. (from position of 255 to 274) which introduced a nucleotide substitution A->T at position 274 (underlined) . A Sad site was then generated to permit the cloning of N-rep into pTMl and in-frame expression of Rep protein from the EMCV UTR preferred initiation site (within the Ncol site) . The PCR product (739 bp in length) was directly cloned into pCR2.1 vector (Invitrogen) and named pCR-N-rep. The pTM-1 plasmid was digested with Sad and Stu I restriction enzymes and ligated with a 3.7 kb SacI/SnaBI fragment from psub201 [Samulski et al, J. Virol. ■ .51:3096-3101 (1987)] containing the right end of the AAV genome (without ITR sequence), i.e., the c- terminal portion of rep and full-length cap sequence. This T7 promoter-driven rep/cap construct is named pT7-c- rep/cap.

The first 535 bp sequence of rep was removed from the pCR-N-Rep plasmid by Sad digestion and subcloned into pT7-C-rep/cap, which has similarly been digested with Sad and subjected to alkaline phosphatase treatment to prevent self-ligation of the vector. The final construct was named pT7 rep/cap which contains the full length coding sequence of rep/cap downstream of T7 promoter/EMCV UTR, followed by the T7 terminating sequence.

B. Production T7 rep/ cap Adenovirus pAd.link is a construct similar to pAd.CMV. link, a plasmid containing the adenoviral sequences 0 to 16 map units deleted of Ela and Elb as described in the other adenovirus vectors into which a CMV promoter-polylinker cassette was cloned and described in X. Ye et al, J. Biol. Chem.. 221:3639-3646 (1996). However, pAd.link contains no CMV promoter or polyA tail sequence.

The entire region including the T7 promoter, EMCV UTR, rep/cap and T7 terminating sequence was excised from pT7 rep/cap by digestion with Clal and EagI, and then subcloned into the adenoviral sequences of pAd.link, which had previously been subjected to Clal/Sail digestion, after filling in the sticky ends of EagI and Sail by Klenow polymerase. The resulting plasmid is designated pAd.T7 rep/cap. The pAd.T7 rep/cap is co-transfected with the Clal linearized Ad.del327 backbone DNA into 293 cell for the generation of T7 rep/cap adenovirus. Example 4 - Cell Line Expressing rep/cap

A cell line stably transfected with pT7 rep/cap is established by transfection of pMTCB6+ into 293 cell at ratio of 10:1 and selected with Geneticin. Each clone is analyzed for the presence of rep protein by transfection with T7 Pol expressing plasmid.

Example 5 - Production of Recombinant AAV Hybrid Vector Plasmid pAV.CMVLacZ serves as a template for rAAV to be replicated and packaged in the presence of AAV non-structural and capsid proteins.

Plasmid AV.CMVLacZ is a rAAV cassette in which rep and cap genes are replaced with a minigene expressing β-galactosidase from a CMV promoter. The linear arrangement of AV.CMV acZ includes: (a) the 5¹ AAV ITR (bp 1-173) obtained by PCR using pAV2 [C. A. Laughlin et al, Gene. 23: 65-73 (1983)] as template [nucleotide numbers 365-538 of SEQ ID NO:l];

(b) a CMV immediate early enhancer/promoter [Boshart et al, Cell. 41:521-530 (1985); nucleotide numbers 563-1157 of SEQ ID NO:l],

(c) an SV40 intron (nucleotide numbers 1178- 1179 of SEQ ID N0:1) ,

(d) E. coli beta-galactosidase cDNA (nucleotide numbers 1356 - 4827 of SEQ ID N0:1), (e) an SV40 polyadenylation signal (a 237

BamHI-BclI restriction fragment containing the cleavage/poly-A signals from both the early and late transcription units; nucleotide numbers 4839 - 5037 of SEQ ID NO:l) and (f) 3'AAV ITR, obtained from pAV2 as a SnaBI-

Bglll fragment (nucleotide numbers 5053 - 5221 of SEQ ID N0:l) . Where desired, the LacZ gene can be replaced with a desired therapeutic or other transgene for the purpose of generating new rAAV. See, Fig. 4. The sequence including CMV directed LacZ reporter cassette in between two AAV ITR sequences is excised from pAV.CMV.LacZ by PvuII digestion. This fragment is ligated with the EcoRV treated pAd.link to generate the plasmid pAd.AV.CMVLacZ. This plasmid is co-transfected with Clal linearized Addel327 backbone DNA to generate an adeno-rAAV hybrid virus.

Example 6 - Cell line containing rescuable. integrated rAAV template

293 cells are transfected/infected with pAV.CMVLacZ/rAAV Ad hybrid virus to generate cell line that has incorporated rAAV, as determined by analysis of the geno ic DNA by Southern blot. The clone is examined for the rescue of rAAV template by transfection/infection with rep/cap expressing constructs.

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Truβteeβ of the University of Pennsylvania Wilson, James M. Chen, Nancie N.

(ii) TITLE OF INVENTION: An Inducible Method for Production of Recombinant Adeno-Aβsociated Viruβeβ Utilizing T7 Polymerase

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Howson and Howson

(B) STREET: Spring House Corporate Cntr, PO Box 457

(C) CITY: Spring House

(D) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19477

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(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 60/024,699

(B) FILING DATE: 06-SEP-1996

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kodroff, Cathy A.

(B) REGISTRATION NUMBER: 33,980

(C) REFERENCE/DOCKET NUMBER: GNVPN.022CIP1PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-540-9200

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(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10398 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAATTCGCTA GCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT ATGATAATGA 60

GGGGGTGGAG TTTGTGACGT GGCGCGGGGC GTGGGAACGG GGCGGGTGAC GTAGTAGTGT 120

GGCGGAAGTG TGATGTTGCA AGTGTGGCGG AACACATGTA AGCGACGGAT GTGGCAAAAG 180

TGACGTTTTT GGTGTGCGCC GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC 240

GGATGTTGTA GTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATTTTCG CGGGAAAACT 300

GAATAAGAGG AAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA ATATTTGTCT 360

AGGGAGATCT GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC 420

GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA 480

ACTCCATCAC TAGGGGTTCC TTGTAGTTAA TGATTAACCC GCCATGCTAC TTATCTACAA 540

TTCGAGCTTG CATGCCTGCA GGTCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 600

CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 660

ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA 720

GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG 780

CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC 840

TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 900

GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 960

TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG 1020

ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG 1080

AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG 1140

GACCGATCCA GCCTCCGGAC TCTAGAGGAT CCGGTACTCG AGGAACTGAA AAACCAGAAA 1200

GTTAACTGGT AAGTTTAGTC TTTTTGTCTT TTATTTCAGG TCCCGGATCC GGTGGTGGTG 1260

CAAATCAAAG AACTGCTCCT CAGTGGATGT TGCCTTTACT TCTAGGCCTG TACGGAAGTG 1320

TTACTTCTGC TCTAAAAGCT GCGGAATTGT ACCCGCGGCC GCAATTCCCG GGGATCGAAA 1380

GAGCCTGCTA AAGCAAAAAA GAAGTCACCA TGTCGTTTAC TTTGACCAAC AAGAACGTGA 1440

TTTTCGTTGC CGGTCTGGGA GGCATTGGTC TGGACACCAG CAAGGAGCTG CTCAAGCGCG 1500

ATCCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC 1560

TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC 1620

CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGCGCTT TGCCTGGTTT CCGGCACCAG 1680

AAGCGGTGCC GGAAAGCTGG CTGGAGTGCG ATCTTCCTGA GGCCGATACT GTCGTCGTCC 1740

CCTCAAACTG GCAGATGCAC GGTTACGATG CGCCCATCTA CACCAACGTA ACCTATCCCA 1800 TTACGGTCAA TCCGCCGTTT GTTCCCACGG AGAATCCGAC GGGTTGTTAC TCGCTCACAT 1860

TTAATGTTGA TGAAAGCTGG CTACAGGAAG GCCAGACGCG AATTATTTTT GATGGCGTTA 1920

ACTCGGCGTT TCATCTGTGG TGCAACGGGC GCTGGGTCGG TTACGGCCAG GACAGTCGTT 1980

TGCCGTCTGA ATTTGACCTG AGCGCATTTT TACGCGCCGG AGAAAACCGC CTCGCGGTGA 2040

TGGTGCTGCG TTGGAGTGAC GGCAGTTATC TGGAAGATCA GGATATGTGG CGGATGAGCG 2100

GCATTTTCCG TGACGTCTCG TTGCTGCATA AACCGACTAC ACAAATCAGC GATTTCCATG 2160

TTGCCACTCG CTTTAATGAT GATTTCAGCC GCGCTGTACT GGAGGCTGAA GTTCAGATGT 2220

GCGGCGAGTT GCGTGACTAC CTACGGGTAA CAGTTTCTTT ATGGCAGGGT GAAACGCAGG 2280

TCGCCAGCGG CACCGCGCCT TTCGGCGGTG AAATTATCGA TGAGCGTGGT GGTTATGCCG 2340

ATCGCGTCAC ACTACGTCTG AACGTCGAAA ACCCGAAACT GTGGAGCGCC GAAATCCCGA 2400

ATCTCTATCG TGCGGTGGTT GAACTGCACA CCGCCGACGG CACGCTGATT GAAGCAGAAG 2460

CCTGCGATGT CGGTTTCCGC GAGGTGCGGA TTGAAAATGG TCTGCTGCTG CTGAACGGCA 2520

AGCCGTTGCT GATTCGAGGC GTTAACCGTC ACGAGCATCA TCCTCTGCAT GGTCAGGTCA 2580

TGGATGAGCA GACGATGGTG CAGGATATCC TGCTGATGAA GCAGAACAAC TTTAACGCCG 2640

TGCGCTGTTC GCATTATCCG AACCATCCGC TGTGGTACAC GCTGTGCGAC CGCTACGGCC 2700

TGTATGTGGT GGATGAAGCC AATATTGAAA CCCACGGCAT GGTGCCAATG AATCGTCTGA 2760

CCGATGATCC GCGCTGGCTA CCGGCGATGA GCGAACGCGT AACGCGAATG GTGCAGCGCG 2820

ATCGTAATCA CCCGAGTGTG ATCATCTGGT CGCTGGGGAA TGAATCAGGC CACGGCGCTA 2880

ATCACGACGC GCTGTATCGC TGGATCAAAT CTGTCGATCC TTCCCGCCCG GTGCAGTATG 2940

AAGGCGGCGG AGCCGACACC ACGGCCACCG ATATTATTTG CCCGATGTAC GCGCGCGTGG 3000

ATGAAGACCA GCCCTTCCCG GCTGTGCCGA AATGGTCCAT CAAAAAATGG CTTTCGCTAC 3060

CTGGAGAGAC GCGCCCGCTG ATCCTTTGCG AATACGCCCA CGCGATGGGT AACAGTCTTG 3120

GCGGTTTCGC TAAATACTGG CAGGCGTTTC GTCAGTATCC CCGTTTACAG GGCGGCTTCG 3180

TCTGGGACTG GGTGGATCAG TCGCTGATTA AATATGATGA AAACGGCAAC CCGTGGTCGG 3240

CTTACGGCGG TGATTTTGGC GATACGCCGA ACGATCGCCA GTTCTGTATG AACGGTCTGG 3300

TCTTTGCCGA CCGCACGCCG CATCCAGCGC TGACGGAAGC AAAACACCAG CAGCAGTTTT 3360

TCCAGTTCCG TTTATCCGGG CAAACCATCG AAGTGACCAG CGAATACCTG TTCCGTCATA 3420

GCGATAACGA GCTCCTGCAC TGGATGGTGG CGCTGGATGG TAAGCCGCTG GCAAGCGGTG 3480

AAGTGCCTCT GGATGTCGCT CCACAAGGTA AACAGTTGAT TGAACTGCCT GAACTACCGC 3 40

AGCCGGAGAG CGCCGGGCAA CTCTGGCTCA CAGTACGCGT AGTGCAACCG AACGCGACCG 3600

CATGGTCAGA AGCCGGGCAC ATCAGCGCCT GGCAGCAGTG GCGTCTGGCG GAAAACCTCA 3660 GTGTGACGCT CCCCGCCGCG TCCCACGCCA TCCCGCATCT GACCACCAGC GAAATGGATT 3720

TTTGCATCGA GCTGGGTAAT AAGCGTTGGC AATTTAACCG CCAGTCAGGC TTTCTTTCAC 3780

AGATGTGGAT TGGCGATAAA AAACAACTGC TGACGCCGCT GCGCGATCAG TTCACCCGTG 3840

CACCGCTGGA TAACGACATT GGCGTAAGTG AAGCGACCCG CATTGACCCT AACGCCTGGG 3900

TCGAACGCTG GAAGGCGGCG GGCCATTACC AGGCCGAAGC AGCGTTGTTG CAGTGCACGG 3960

CAGATACACT TGCTGATGCG GTGCTGATTA CGACCGCTCA CGCGTGGCAG CATCAGGGGA 4020

AAACCTTATT TATCAGCCGG AAAACCTACC GGATTGATGG TAGTGGTCAA ATGGCGATTA 4080

CCGTTGATGT TGAAGTGGCG AGCGATACAC CGCATCCGGC GCGGATTGGC CTGAACTGCC 4140

AGCTGGCGCA GGTAGCAGAG CGGGTAAACT GGCTCGGATT AGGGCCGCAA GAAAACTATC 4200

CCGACCGCCT TACTGCCGCC TGTTTTGACC GCTGGGATCT GCCATTGTCA GACATGTATA 4260

CCCCGTACGT CTTCCCGAGC GAAAACGGTC TGCGCTGCGG GACGCGCGAA TTGAATTATG 4320

GCCCACACCA GTGGCGCGGC GACTTCCAGT TCAACATCAG CCGCTACAGT CAACAGCAAC 4380

TGATGGAAAC CAGCCATCGC CATCTGCTGC ACGCGGAAGA AGGCACATGG CTGAATATCG 4440

ACGGTTTCCA TATGGGGATT GGTGGCGACG ACTCCTGGAG CCCGTCAGTA TCGGCGGAAT 4500

TACAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA TAATAATAAC 4560

CGGGCAGGCC ATGTCTGCCC GTATTTCGCG TAAGGAAATC CATTATGTAC TATTTAAAAA 4620

ACACAAACTT TTGGATGTTC GGTTTATTCT TTTTCTTTTA CTTTTTTATC ATGGGAGCCT 4680

ACTTCCCGTT TTTCCCGATT TGGCTACATG ACATCAACCA TATCAGCAAA AGTGATACGG 4740

GTATTATTTT TGCCGCTATT TCTCTGTTCT CGCTATTATT CCAACCGCTG TTTGGTCTGC 4800

TTTCTGACAA ACTCGGCCTC GACTCTAGGC GGCCGCGGGG ATCCAGACAT GATAAGATAC 4860

ATTGATGAGT TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA 4920

ATTTGTGATG CTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC 4980

AACAATTGCA TTCATTTTAT GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTCGGAT 5040

CCTCTAGAGT CGAGTAGATA AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT 5100

AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC 5160

AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG 5220

CAGATCTGGA AGGTGCTGAG GTACGATGAG ACCCGCACCA GGTGCAGACC CTGCGAGTGT 5280

GGCGGTAAAC ATATTAGGAA CCAGCCTGTG ATGCTGGATG TGACCGAGGA GCTGAGGCCC 5340

GATCACTTGG TGCTGGCCTG CACCCGCGCT GAGTTTGGCT CTAGCGATGA AGATACAGAT 5400

TGAGGTACTG AAATGTGTGG GCGTGGCTTA AGGGTGGGAA AGAATATATA AGGTGGGGGT 5460

CTTATGTAGT TTTGTATCTG TTTTGCAGCA GCCGCCGCCG CCATGAGCAC CAACTCGTTT 5520 GATGGAAGCA TTGTGAGCTC ATATTTGACA ACGCGCATGC CCCCATGGGC CGGGGTGCGT 5580

CAGAATGTGA TGGGCTCCAG CATTGATGGT CGCCCCGTCC TGCCCGCAAA CTCTACTACC 5640

TTGACCTACG AGACCGTGTC TGGAACGCCβ TTGGAGACTG CAGCCTCCGC CGCCGCTTCA 5700

GCCGCTGCAG CCACCGCCCG CGGGATTGTG ACTGACTTTG CTTTCCTGAG CCCGCTTGCA 5760

AGCAGTGCAG CTTCCCGTTC ATCCGCCCGC GATGACAAGT TGACGGCTCT TTTGGCACAA 5820

TTGGATTCTT TGACCCGGGA ACTTAATGTC GTTTCTCAGC AGCTGTTGGA TCTGCGCCAG 5880

CAGGTTTCTG CCCTGAAGGC TTCCTCCCCT CCCAATGCGG TTTAAAACAT AAATAAAAAA 5940

CCAGACTCTG TTTGGATTTG GATCAAGCAA GTGTCTTGCT GTCTTTATTT AGGGGTTTTG 6000

CGCGCGCGGT AGGCCCGGGA CCAGCGGTCT CGGTCGTTGA GGGTCCTGTG TATTTTTTCC 6060

AGGACGTGGT AAAGGTGACT CTGGATGTTC AGATACATGG GCATAAGCCC GTCTCTGGGG 6120

TGGAGGTAGC ACCACTGCAG AGCTTCATGC TGCGGGGTGG TGTTGTAGAT GATCCAGTCG 6180

TAGCAGGAGC GCTGGGCGTG GTGCCTAAAA ATGTCTTTCA GTAGCAAGCT GATTGCCAGG 6240

GGCAGGCCCT TGGTGTAAGT GTTTACAAAG CGGTTAAGCT GGGATGGGTG CATACGTGGG 6300

GATATGAGAT GCATCTTGGA CTGTATTTTT AGGTTGGCTA TGTTCCCAGC CATATCCCTC 6360

CGGGGATTCA TGTTGTGCAG AACCACCAGC ACAGTGTATC CGGTGCACTT GGGAAATTTG 6420

TCATGTAGCT TAGAAGGAAA TGCGTGGAAG AACTTGGAGA CGCCCTTGTG ACCTCCAAGA 6480

TTTTCCATGC ATTCGTCCAT AATGATGGCλ ATGGGCCCAC GGGCGGCGGC CTGGGCGAAG 6540

ATATTTCTGG GATCACTAAC GTCATAGTTG TGTTCCAGGA TGAGATCGTC ATAGGCCATT 6600

TTTACAAAGC GCGGGCGGAG GGTGCCAGAC TGCGGTATAA TGGTTCCATC CGGCCCAGGG 6660

GCGTAGTTAC CCTCACAGAT TTGCATTTCC CACGCTTTGA GTTCAGATGG GGGGATCATG 6720

TCTACCTGCG GGGCGATGAA GAAAACGGTT TCCGGGGTAG GGGAGATCAG CTGGGAAGAA 6780

AGCAGGTTCC TGAGCAGCTG CGACTTACCG CAGCCGGTGG GCCCGTAAAT CACACCTATT 6840

ACCGGGTGCA ACTGGTAGTT AAGAGAGCTG CAGCTGCCGT CATCCCTGAG CAGGGGGGCC 6900

ACTTCGTTAA GCATGTCCCT GACTCGCATG TTTTCCCTGA CCAAATCCGC CAGAAGGCGC 6960

TCGCCGCCCA GCGATAGCAG TTCTTGCAAG GAAGCAAAGT TTTTCAACGG TTTGAGACCG 7020

TCCGCCGTAG GCATGCTTTT GAGCGTTTGA CCAAGCAGTT CCAGGCGGTC CCACAGCTCG 7080

GTCACCTGCT CTACGGCATC TCGATCCAGC ATATCTCCTC GTTTCGCGGG TTGGGGCGGC 7140

TTTCGCTGTA CGGCAGTAGT CGGTGCTCGT CCAGACGGGC CAGGGTCATG TCTTTCCACG 7200

GGCGCAGGGT CCTCGTCAGC GTAGTCTGGG TCACGGTGAA GGGGTGCGCT CCGGGCTGCG 7260

CGCTGGCCAG GGTGCGCTTG AGGCTGGTCC TGCTGGTGCT GAAGCGCTGC CGGTCTTCGC 7320

CCTGCGCGTC GGCCAGGTAG CATTTGACCA TGGTGTCATA GTCCAGCCCC TCCGCGGCGT 7380 GGCCCTTGGC GCGCAGCTTG CCCTTGGAGG AGGCGCCGCA CGAGGGGCAG TGCAGACTTT 7440

TGAGGGCGTA GAGCTTGGGC GCGAGAAATA CCGATTCCGG GGAGTAGGCA TCCGCGCCGC 7500

AGGCCCCGCA GACGGTCTCG CATTCCACGA GCCAGGTGAG CTCTGGCCGT TCGGGGTCAA 7560

AAACCAGGTT TCCCCCATGC TTTTTGATGC GTTTCTTACC TCTGGTTTCC ATGAGCCGGT 7620

GTCCACGCTC GGTGACGAAA AGGCTGTCCG TGTCCCCGTA TACAGACTTβ AGAGGCCTGT 7680

CCTCGACCGA TGCCCTTGAG AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG GGCGCGGGGC 7740

ATGACTATCG TCGCCGCACT TATGACTGTC TTCTTTATCA TGCAACTCGT AGGACAGGTG 7800

CCGGCAGCGC TCTGGGTCAT TTTCGGCGAG GACCGCTTTC GCTGGAGCGC GACGATGATC 7860

GGCCTGTCGC TTGCGGTATT CGGAATCTTG CACGCCCTCG CTCAAGCCTT CGTCACTGGT 7920

CCCGCCACCA AACGTTTCGG CGAGAAGCAG GCCATTATCG CCGGCATGGC GGCCGACGCG 7980

CTGGGCTACG TCTTGCTGGC GTTCGCGACG CGAGGCTGGA TGGCCTTCCC CATTATGATT 8040

CTTCTCGCTT CCGGCGGCAT CGGGATGCCC GCGTTGCAGG CCATGCTGTC CAGGCAGGTA 8100

GATGACGACC ATCAGGGACA GCTTCAAGGA TCGCTCGCGG CTCTTACCAG CCTAACTTCG 8160

ATCACTGGAC CGCTGATCGT CACGGCGATT TATGCCGCCT CGGCGAGCAC ATGGAACGGG 8220

TTGGCATGGA TTGTAGGCGC CGCCCTATAC CTTGTCTGCC TCCCCGCGTT GCGTCGCGGT 8280

GCATGGAGCC GGGCCACCTC GACCTGAATG GAAGCCGGCG GCACCTCGCT AACGGATTCA 8340

CCACTCCAAG AATTGGAGCC AATCAATTCT TGCGGAGAAC TGTGAATGCG CAAACCAACC 8400

CTTGGCAGAA CATATCCATC GCGTCCGCCA TCTCCAGCAG CCGCACGCGG CGCATCTCGG 8460

GCAGCGTTGG GTCCTGGCCA CGGGTGCGCA TGATCGTGCT CCTGTCGTTG AGGACCCGGC 8520

TAGGCTGGCG GGGTTGCCTT ACTGGTTAGC AGAATGAATC ACCGATACGC GAGCGAACGT 8580

GAAGCGACTG CTGCTGCAAA ACGTCTGCGA CCTGAGCAAC AACATGAATG GTCTTCGGTT 8640

TCCGTGTTTC GTAAAGTCTG GAAACGCGGA AGTCAGCGCC CTGCACCATT ATGTTCCGGA 8700

TCTGCATCGC AGGATGCTGC TGGCTACCCT GTGGAACACC TACATCTGTA TTAACGAAGC 8760

CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 8820

GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 8880

CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 8940

ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 9000

GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 9060

AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 9120

GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 9180

TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 9240 TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 9300

TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 9360

ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA 9420

ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA 9480

CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 9540

AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 9600

GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC AGGCATCGTG 9660

GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA 9720

GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT 9780

GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT 9840

CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 9900

TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT 9960

ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA 10020

AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 10080

AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 10140

CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 10200

CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 10260

GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 10320

CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG 10380

AGGCCCTTTC GTCTTCAA 10398

( 2 ) INFORMATION FOR SEQ ID NO: 2 :

( i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

( B) TYPE : nucleic acid

( C) STRANDEDNESS : single

(D ) TOPOLOGY : unknown

(ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TATTTAAGCC CGAGTGAGCT 20

Claims

What is claimed is:

1. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of:

(a) introducing into a selected host cell a first vector comprising T7 polymerase under control of sequences which drive expression thereof, a second vector comprising AAV rep and cap genes under control of T7 promoter sequences which drive expression of rep and cap; and a third vector comprising from 5* to 3', a cassette consisting essentially of a 5' AAV inverted terminal repeat (ITR) , a selected minigene, and a 3 • AAV ITR;

(b) culturing the host cell under conditions which permit replication and packaging of recombinant AAV; and

(c) recovering the recombinant AAV.

2. The method according to claim 1 wherein at least one of the vectors is an adenovirus and the host cell is a 293 cell.

3. The method according to claim 1 wherein the first vector is a recombinant adenovirus.

4. The method according to claim 1 wherein the second vector is a recombinant adenovirus.

5. The method according to claim 1 wherein the third vector further comprises adenoviral sequences flanking the cassette.

6. The method according to any of claims 1 to 5 wherein the minigene contains a transgene which is a marker gene.

7. The method according to claim 6 wherein the minigene contains a transgene which is a therapeutic gene.

8. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of:

(a) providing a host cell which expresses T7 polymerase;

(b) introducing into the host cell a first vector which comprises AAV rep and cap genes under control of T7 promoter sequences;

(c) introducing into the host cell a second vector comprising a cassette consisting essentially of 5' AAV inverse terminal repeat (ITR), a selected minigene, and 3' AAV ITR; and

(d) culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

9. The method according to claim 8 wherein step (b) comprises the step of transfecting the host cell with a vector comprising the T7 promoter and the AAV rep and cap genes.

10. The method according to claim 8 wherein step (b) comprises the step of infecting the host cell with a recombinant adenovirus comprising the T7 promoter sequences, and the AAV rep and cap genes.

11. The method according to claim 8 wherein step (c) comprises transfecting the host cell with a vector comprising the cassette.

12. The method according to claim 8 wherein step (c) comprises infecting the host cell with a recombinant adenovirus comprising the cassette flanked by adenovirus sequences.

13. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of:

(a) providing a host cell stably transfected with AAV rep and cap genes under control of T7 promoter sequences;

(b) introducing into the host cell a vector comprising T7 polymerase;

(c) introducing into the host cell with vector comprising a cassette consisting essentially of a 5' AAV inverse terminal repeat (ITR), a selected minigene, and a 3' AAV ITR; and

(d) culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

14. The method according to claim 13 wherein step (b) comprises the step of transfecting the host cell with a vector comprising the T7 polymerase gene.

15. The method according to claim 13 wherein step (b) comprises the step of infecting the host cell with a recombinant adenovirus comprising the T7 polymerase gene under control of regulatory sequences controlling expression thereof.

16. The method according to claim 13 wherein step (c) comprises the step of transfecting the host cell with a vector comprising the cassette.

17. The method according to claim 13 wherein step (c) comprises the step of infecting the host cell with a recombinant adenovirus comprising the cassette flanked by adenovirus sequences.

18. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of:

(a) providing a host cell comprising a cassette consisting essentially of 5 ' AAV inverse terminal repeats (ITR), a selected minigene, and a 3' AAV ITR;

(b) introducing into the host cell a vector comprising AAV rep and cap genes under control of T7 promoter sequences;

(c) introducing into the host cell a vector comprising the T7 polymerase; and

(d) culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

19. The method according to claim 18 wherein step (b) comprises the step of transfecting the host cell with a plasmid vector.

20. The method according to claim 18 wherein step (b) comprises the step of infecting the host cell with a recombinant adenoviral vector.

21. The method according to claim 18 wherein step (c) comprises the step of transfecting the host cell with a plasmid vector containing the T7 polymerase under control of regulatory sequences which direct expression thereof .

22. The method according to claim 18 wherein step (c) comprises the step of infecting the host cell with a recombinant adenovirus comprising the T7 polymerase under control of regulatory sequences which direct expression thereof.

23. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of:

(a) providing a host cell stably transfected with a cassette consisting essentially of 5¹ AAV inverse terminal repeats (ITR) , a selected minigene, and a 3 ' AAV ITR and a plasmid comprising AAV rep and cap genes under control of T7 promoter sequences;

(b) introducing into the host cell a vector comprising the T7 polymerase; and

(c) culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

24. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of;

(a) providing a host cell stably transfected with a cassette consisting essentially of 5* AAV inverse terminal repeats (ITR) , a selected minigene, and a 3' AAV ITR and a plasmid comprising T7 polymerase;

(b) introducing into the host cell a vector comprising AAV rep and cap genes under control of T7 promoter sequences; and (c) culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

25. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of:

(a) providing a host cell stably transfected with

(i) a cassette consisting essentially of 5' AAV inverse terminal repeats (ITR), a selected minigene, and a 3' AAV ITR;

(ii) a plasmid comprising T7 polymerase under control of sequences which regulate expression thereof, said sequences comprising an inducible promoter; and

(iii) a plasmid AAV rep and cap genes under control of T7 promoter sequences; and

(b) inducing expression of said T7 promoter.

26. A recombinant adenovirus produced according to the method of any one of claims 1 - 25.