AU722624B2

AU722624B2 - An inducible method for production of recombinant adeno-associated viruses utilizing T7 polymerase

Info

Publication number: AU722624B2
Application number: AU41833/97A
Authority: AU
Inventors: Nancie Chen; James M. Wilson
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 1996-09-06
Filing date: 1997-09-04
Publication date: 2000-08-10
Anticipated expiration: 2017-09-04
Also published as: CA2264482A1; WO1998010088A1; EP0931158A1; JP2001500015A; AU4183397A; IL128780A0

Description

it 1I WO 98/10088 PCT/US97/15716 AN INDUCIBLE METHOD FOR PRODUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRUSES UTILIZING T7 POLYMERASE Background of the Invention Adeno-associated virus is a replicationdeficient 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 coinfection with wild-type AAV and adenovirus. However, this method leads to unacceptably high levels of wildtype 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.

Ih ft WO 98/10088 PCT/US97/15716 2 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 Chamberlin et al, Nature, 228:227-231 (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 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 virusinfected eukaryotic cells Fuerst et al, Proc. Natl.

Acad. Sci. USA, 83:8122-8126 (1986); A. Ramsey-Ewing and B. Moss, J. Biol. Chem., 271:16962-16966 (1996)], and in mammalian cells 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 WO 98/10088 PCTIUS97/15716 3 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 consisting essentially of 5' and 3' AVV 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 prpvides 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 15 introduced into the host cell, as described above.

SIn one embodiment, the invention provides a method in which a vector comprising T7 rep cap and a vector comprising a cassette consisting essentially of a selected minigene flanked by 5' 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 25 comprising T7 pol and a vector consisting essentially of 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 comprising T7 pol and a vector comprising T7 rep cap are R4{- ontroduced into the host cell.

Ehost cell is cultured under conditions which permit 1V 35 piduction of rAAV.

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WO 98/10088 PCT/US97/15716 4 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.

WO 98/10088 PCT/US97/15716 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 WO 98/10088 PCT/US97/15716 6 carrying the required elements of this system, 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/Transfection 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 Strauss, Nucleic Acid Res., 17:8485-8493 (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 WO 98/10088 PCT/US97/15716 7 gene is linked to a nuclear localization signal, such as that described in Dunn, Gene, 68:259-266 (1988), using conventional techniques.

Desirably, T7 Pol is under the control of a cytomegalovirus (CMV) immediate early promoter/enhancer [see, 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, T. Shenk, J.

Virol., 61:3096-3101 (1987), which provides the AAV2 genome within the plasmid psub201; E. W. Lusby et al, J.

Virol., 41:518-526 (1982) and J. Smuda and B.J. Carter, Virologv, 184:310-318 (1991).

Similarly, the T7 promoter sequences 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 WO 98/10088 PCTIUS97/15716 8 promoter. The inventors believe this sequence increases expression of the gene 5- to 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, 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, 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 WO 98/10088 PCT/US97/15716 9 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 betagalactosidase (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, 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 noninherited genetic defect or treat an epigenetic disorder or disease. The selection of the transgene sequence is not a limitation of this invention.

WO 98/10088 PCT/US97/15716 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, 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 adenoviral sequences) which permit it to infect the selected host WO 98/10088 PCT/US97/15716 11 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/Transfection 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 WO 98/10088 PCT/US97/15716 12 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, 293 cells or other packaging cell lines expressing required adenoviral genes, with a plasmid containing the desired gene, 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/Transfection 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 WO 98/10088 PCT/US97/15716 13 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 cotransfection techniques are employed, CaPO 4 WO 98/10088 PCT/US97/15716 14 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, F. L. Graham and L. Prevec, Methods Mol. Biol., 1:109-128 (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 Lieber et al, Nucl. Acids Res., 17:8485-8493 (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 BglII and PvuII restriction enzymes and the fragments separated on an agarose gel. The BglII/PvuII T7 Pol DNA fragment was ligated to the BglII/EcoRV cleaved vector pAd.CMV.link.l to form pAd.CMV.T7N. pAd.CMV.link.1 is a WO 98/10088 PCT/US97/15716 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 cotransfected with Cla I linearized Addel327 backbone using Cellphate kit (Pharmacia). Approximately 1 week posttransfection, the T7 Pol adenovirus can be isolated from the plaques for further purification.

Example 2 Cell Lines Expressin T7 Pol A cell line stably expressing T7 Pol is established by co-transfection of plasmids pMTT7N and pMTCB6+ (which provides a selective marker) H. Choo et al, DNA, 5:529-538; Eur. J. Biochem., 174:417-424] into 293 cell at a ratio of 10:1 using calcium phosphate precipitation Graham and A. van der Eb, Virol., 52:456-467 (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 BglII/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 WO 98/10088 PCT/US97/15716 16 with Hygromycin at a concentration of 400 pg/ml. Each positive clone is analyzed for the presence of T7 Pol by its ability to produce expression of T7-LacZ or T7rep/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 Construction The plasmid pTM1 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 SacI site and subcloned into the pTMI 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 SacI 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 NcoI 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., 61:3096-3101 (1987)] containing the right end of the AAV genome (without ITR sequence), the c- WO 98/10088 PCT/US97/15716 17 terminal portion of rep and full-length cap sequence.

This T7 promoter-driven rep/cap construct is named pT7-crep/cap.

The first 535 bp sequence of rep was removed from the pCR-N-Rep plasmid by SacI digestion and subcloned into pT7-C-rep/cap, which has similarly been digested with SacI 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., 271:3639-3646 (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 ClaI/SalI digestion, after filling in the sticky ends of EagI and SalI 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.de1327 backbone DNA into 293 cell for the generation of T7 rep/cap adenovirus.

WO 98/10088 PCT/US97/15716 18 Example 4 Cell Line Expressing rep/caD 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 B-galactosidase from a CMV promoter. The linear arrangement of AV.CMVLacZ includes: the 5' AAV ITR (bp 1-173) obtained by PCR using pAV2 A. Laughlin et al, Gene, 23: 65-73 (1983)] as template [nucleotide numbers 365-538 of SEQ ID NO:1]; a CMV immediate early enhancer/promoter [Boshart et al, Cell, 41:521-530 (1985); nucleotide numbers 563-1157 of SEQ ID NO:1], an SV40 intron (nucleotide numbers 1178- 1179 of SEQ ID NO:1), E. coli beta-galactosidase cDNA (nucleotide numbers 1356 4827 of SEQ ID NO:1), 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:1) and 3'AAV ITR, obtained from pAV2 as a SnaBI- BglII fragment (nucleotide numbers 5053 5221 of SEQ ID NO:1).

WO 98/10088 PCT/US97/15716 19 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.

ExamDle 6 Cell line containihn rescuable, integrated rAAV template tp293 cells are transfected/infected with pAV.CMVLacZ/rAAV Ad hybrid virus to generate cell line 15 that has incorporated rAAV, as determined by analysis of the genomic 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 20 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.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

WO 98/10088 PCT/US97/15716 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Trustees of the University of Pennsylvania Wilson, James M.

Chen, Nancie N.

(ii) TITLE OF INVENTION: An Inducible Method for Production of Recombinant Adeno-Associated Viruses Utilizing T7 Polymerase (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Howson and Howson STREET: Spring House Corporate Cntr, PO Box 457 CITY: Spring House STATE: Pennsylvania COUNTRY: USA ZIP: 19477 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 60/024,699 FILING DATE: 06-SEP-1996 (viii) ATTORNEY/AGENT INFORMATION: NAME: Kodroff, Cathy A.

REGISTRATION NUMBER: 33,980 REFERENCE/DOCKET NUMBER: GNVPN.022CIP1PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 215-540-9200 TELEFAX: 215-540-5818 INFORMATION FOR SEQ ID NOil: SEQUENCE CHARACTERISTICS: LENGTH: 10398 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA WO 98/10088 WO 9810088PCTIUS97/15716 21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GAATTCGCTA

GGGGGTGGAG

GGCGGAAGTG

TGACGTTTTT

GGATGTTGTA

GAATAAGAGG

AGGGAGATCT

GGGCGACCTT

ACTCCATCAC

TTCGAGCTTG

CCGCCCAACG

ATAGGGACTT

GTACATCAAG

CCCGCCTGGC

TACGTATTAG

GGATAGCGGT

TTGTTTTGGC

ACGCAAATGG

AACCGTCAGA

GACCGATCCA

GTTAACTGGT

CAAATCAAAG

TTACTTCTGC

GAGCCTGCTA

TTTTCGTTGC

ATCCCGTCGT

TTGCAGCACA

CTTCCCAACA

AAGCGGTGCC

GCATCATCAA

TTTGTGACGT

TGATGTTGCA

GGTGTGCGCC

GTAAATTTGG

AAGTGAAATC

GCTGCGCGCT

TGGTCGCCCG

TAGGGGTTCC

CATGCCTGCA

ACCCCCGCCC

TCC&TTGACG

TGTATCATAT

ATTATGCCCA

TCATCGCTAT

TTGACTCACG

ACCAAAATCA

GCGGTAGGCG

TCGCCTGGAG

GCCTCCGGAC

AAGTTTAGTC

A&CTGCTCCT

TCTAAAAGCT

AAGCAAAAAA

CGGTCTGGGA

TTTACAACG7

*TCCCCCTTTC

*GTTGCGCAGC

GGAAAGCTGG

TAATATACCT

GGCGCGGGGC

AGTGTGGCGG

GGTGTACACA

GCGTAACCGA

TGAATAATTT

CGCTCGCTCA

GCCTCAGTGA

TTGTAGTTAA

GGTCGTTACA

ATTGACGTCA

TCAATGGGTG

GCCAAGTACG

GTACATGACC

TACCATGGTG

GGG;ATTTCCA

ACGGGACTTT

TGTACGGTGG

ACGCCATCCA

TCTAGAGGAT

TTTTTGTCTT

CAGTGGATGT

GCGGAATTGT

GAAGTCACCA

*GGCATTGGTC

*CGTGACTGGG

GCCAGCTGGC

CTGAATGGCG

TATTTTGGAT

GTGGGAACGG

AACACATGTA

GGAAGTGPICA

GTAAGATTTG

TGTGTTACTC

CTGAGGCCGC

GCGAGCGAGC

TGATTAACCC

TAACTTACGG

ATAATGACGT

GAGTATTTAC

CCCCCTATTG

TTATGGGACT

ATGCGGTTTT

AGTCTCCACC

CCAAAATGTC.

GAGGTCTATA

CGCTGTTTTG

CCGGTACTCG

TTATTTCAG

TGCCTTTACT

ACCCGCGGCC

TGTCGTTTAC

TGGACACCAG

AAAACCCTGG

GTAATAGCGA

AATGGCGCTT

TGAAGCCAAT ATGATAATGA

GGCGGGTGAC

AGCGACGGAT

ATTTTCGCGC

GCCATTTTCG

ATAGCGCGTA

CCGGGCAAAG

GCGCAGAGAG

GCCATGCTAC

TAAATGGCCC

ATGTTCCCAT

GGTAAACTGC

ACGTCAATGA

TTCCTACTTG

GGCAGTACAT

CCATTGACGT

GTAACAACTC

TAAGCAGAGC

ACCTCCATAG

AGGAACTGAA

TCCCGGATCC

TCTAGGCCTG

GCAATTCCCG

TTTGACCAAC

CAAGGAGCTG

CGTTACCCAA

AGAGGCCCGC

TGCCTGGTTT

GGCCGATACT

GTAGTAGTGT

GTGGCAAAAG

GGTTTTAGGC

CGGGAAAACT

ATATTTGTCT

CCCGGGCGTC

GGAGTGGCCA

TTATCTACAA

GCCTGGCTGA

AGTAACGCCA

CCACTTGGCA

CGGTAAATGG

GCAGTACATC

CAATGGGCGT

CAATGGGAGT

CGCCCCATTG

TCGTTTAGTG

AAGACACCGG

AAACCAGAAAL

GGTGGTGGTG

TACGGAAGTG

GGGATCGAAA

AAGAACGTGA

CTCAAGCGCG

CTTAATCGCC

ACCGATCGCC

CCGGCACCAG

GTCGTCGTCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 CTGGAGTGCG ATCTTCCTGA CCTCAAACTG GCAGATGCAC GGTTACGATG CGCCCATCTA CACCAACGTA ACCTATCCCA WO 98/10088 WO 9810088PCTIUS97/15716 22 TTACGGTCAA TCCGCCGTTT GTTCCCACGG AGARTCCGhC GGGTTGTTAC TCGCTCACAT

TTARTGTTGA

ACTCGGCGTT

TGCCGTCTGA

TGGTGCTGCG

GCATTTTCCG

TTGCCACTCG

GCGGCGAGTT

TCGCCAGCGG

ATCGCGTCAC

ATCTCTATCG

CCTGCGATGT

AGCCGTTGCT

TGYGATGAGCA

TGCGCTGTTC

TGTATGTGGT

CCGATGATCC

ATCGTAATCA

ATCACGACGC

AAGGCGGCGG

ATGAAGACCA

CTGGAGAGAC

GCGGTTTCGC

TCTGGGACTG

CTTACGGCGG

TCTTTGCCGA

TCCAGTTCCG

GCGATAACGA

AAGTGCCTCT

AGCCGGAGAG

CATGGTCAGA

TGAAAGCTGG

TCATCTGTGG

ATTTGACCTG

TTGGAGTGAC

TGACGTCTCG

CTTTAATGAT

GCGTGACTAC

CACCGCGCCT

ACTACGTCTG

TGCGGTGGTT

CGGTTTCCGC

GATTCGAGGC

GACGATGGTG

GCATTATCCG

GGATGAAGCC

GCGCTGGCTA

CCCGAGTGTG

GCTGTATCGC

AGCCGACACC

GCCCTTCCCG

GCGCCCGCTG

TAAATACTGG

GGTGGATCAG

TGATTTTGGC

CCGCACGCCG

TTTATCCGGG

GCTCCTGCAC

GGATGTCGC7

CGCCGGGCAA

AGCCGGGCAC

CTACAGGAAG

TGCAACGGGC

AGCGCATTTT

GGCAGTTATC

TTGCTGCATA

GATTTCAGCC

CTACGGGTAA

TTCGGCGGTG

AACGTCGAAA

GAACTGCACA

GAGGTGCGGA

GTTAACCGTC

CAGGPUTATCC

AACCATCCGC

AATATTGAAA

CCGGCGATGA

ATCATCTGGT

TGGATCAAAT

ACGGCCACCG

GCTGTGCCGA

ATCCTTTGCG

CAGGCGTTTC

TCGCTGATTA

GATACGCCGA

CATCCAGCGC

CAAACCATCG

TGGATGGTGG.

CCACAAGGT;

CTCTGGCTCZ

ATCAGCGCCI1

GCCAGACGCG

GCTGGGTCGG

TACGCGCCGG

TGGAAGATCA

AACCGACTAC

GCGCTGTACT

CAGTTTCTTT

AAATTATCGA

ACCCGAAACT

CCGCCGACGG

TTGAAAATGG

ACGAGCATCA

TGCTGATGAA

TGTGGTACAC

CCCACGGCAT

GCGAACGCGT

CGCTGGGGAA

CTGTCGATCC

ATATTATTTG

AATGGTCCAT

AATACGCCCA

GTCAGTATCC

AATATGATGA

ACGATCGCCA

TGACGGPJAGC

AAGTGACCAG-

r CGCTGGATGC

LAACAGTTGAI

kCAGTACGCGI1

?GGCAGCAGTC

AATTATTTTT

TTACGGCCAG

AGAAAACCGC

GGATATGTGG

ACAAATCAGC

GGAGGCTGAJ

ATGGCPLGGGT

TGAGCGTGGT

GTGGAGCGCC

CACGCTGATT

TCTGCTGCTG

TCCTCTGCAT

GCAGAACAAC

GCTGTGCGAC

GGTGCCAPLTG

AACGCGAATG

TGAATCAGGC

TTCCCGCCCG

CCCGATGTAC

CAAAAAATGG

CGCGATGGGT

CCGTTTACAG

MAACGGCAAC

GTTCTGTATG

AAAACACCAG

CGAATACCTG

ITAAGCCGCTG

TGAACTGCC7

AGTGCAACCG-

GCGTCTGGCC

GATGGCGTTA

GACAGTCGTT

CTCGCGGTGA

CGGATGAGCG

GATTTCCATG

GTTCAGATGT

GAAACGCAGG

GGTTATGCCG

GAAATCCCGA

GAAGCAGAP*G

CTGAACGGCA

GGTCAGGTCA

TTTAACGCCG

CGCTACGGCC

AATCGTCTGA

GTGCAGCGCG

CACGGCGCTA

GTGCAGTATG

GCGCGCGTGG

CTTTCGCTAC

AACAGTCTTG

GGCGGCTTCG

CCGTGGTCGG

'AACGGTCTGG

CAGCAGTTTT

TTCCGTCATA

GCAAGCGGTG

GAACTACCGC

AACGCGACCG

GAAAACCTCA

1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 WO 98/10088 WO 9810088PCT/US97/15716

GTGTGACGCT_

TTTGCATCGAi

AGATGTGGAT

CACCGCTGGA

TCGAACGCTG

CAGATACACT

AAACCTTATT

CCGTTGATGT

AGCTGGCGCA

CCGACCGCCT

CCCCGTACGT

GCCCACACCA

TGATGGAAAC

ACGGTTTCCA

TACAGCTGAG

CGGGCAGGCC

ACACAAACTT

ACTTCCCGTT

GTATTATTTT

TTTCTGACAA

ATTGATGAGT

ATTTGTGATG

AACAATTGCA

CCTCTAGAGT

AGTGATGGAG

AAAGGTCGCC

CAGATCTGGA

GGCGGTAAAC

GATCACTTGG

TGAGGTACTG

CCCCGCCGCG

GCTGGGTAAT

TGGCGATAA

TAACGACATT

GAAGGCGGCG4

TGCTGATGCG

TATCAGCCGG

TGAAGTGGCG

GGTAGCAGAG

TACTGCCGCC

CTTCCCGAGC

GTGGCGCGGC

CAGCCATCGC

TATGGGGATT

CGCCGGTCGC

ATGTCTGCCC

TTGGATGTTC

TTTCCCGATT

TGCCGCTATT

ACTCGGCCTC

TTGGACAAAC

CTATTGCTTT

TTCATTTTAT

CGAGTAGATA

TTGGCCACTC

CGACGCCCGG

AGGTGCTGAG

ATATTAGGAA

TGCTGGCCTG

AAATGTGTGG

rcccAcGccA kAGCGTTGGC PiARCAACTGC

GGCGTAAGTG

GGCCATTACC

GTGCTGATTA

AAAACCTACC

AGCGATACAC

CGGGTAAACT

TGTTTTGACC

GAAAACGGTC

GACTTCCAGT

CATCTGCTGC

GGTGGCGACG

TACCATTACC

GTATTTCGCG

GGTTTATTCT

TGGCTACATG

TCTCTGTTCT

GACTCTAGGC

CACAACTAGA

ATTTGTAACC

GTTTCAGGTT

AGTAGCATGG

CCTCTCTGCG

GCTTTGCCCG

GTACGATGAG

CCAGCCTGTG

CACCCGCGCI

GCGTGGCTTP

TCCCGCATCT

AATTTAACCG

TGACGCCGCT

AAGCGACCCG

AGGCCGAAGC

CGACCGCTCA

GGATTGATGG

CGCATCCGGC

GGCTCGGATT

GCTGGGATCT

TGCGCTGCGG

TCAACATCAG

ACGCGGAAGA

ACTCCTGGAG

AGTTGGTCTG

TAAGGAAATC

TTTTCTTTTA

ACATCAACCA

CGCTATTATT

GGCCGCGGGG

ATGCAGTGAA

ATTATAAGCT

CAGGGGGP&GG

CGGGTTAATC

CGCTCGCTCG

GGCGGCCTCA

ACCCGCACCA

ATGCTGGATG

GAGTTTGGC!I

AGGGTGGGAP

GACCACCAGC

CCAGTCAGGC

GCGCGATCAG

CATTGACCCT

AGCGTTGTTG

CGCGTGGCAG

TAGTGGTCAA

GCGGATTGGC

AGGGCCGCAA

GCCATTGTCA

GACGCGCGAA

CCGCTACAGT

AGGCACATGG

CCCGTCAGTA

GTGTCAAAAA

CATTATGTAC

CTTTTTTATC

TATCAGCAAA

CCAACCGCTG

ATCCAGACAT

AAAAATGCTT

GCAATAAACA

TGTGGGAGGT

ATTAACTACA

CTCACTGAGG

GTGAGCQAGC

GGTGCAGACC

TGACCGAGGA

CTAGCGATGA

LAGAATATATA

GAAATGGATT

TTTCTTTCAC

TTCACCCOTG

AACGCCTGGG

CAGTGCACGG

CATCAGGGGA

ATGGCGATTA

CTGAACTGCC

GAAPJACTATC

GACATGTATA

TTGAATTATG

CAACAGCAAC

CTGAATATCG

TCGGCGGAAT

TAATAATAAC

TATTTAAAAA

ATGGGAGCCT

AGTGATACGG

TTTGGTCTGC

GATAAGATAC

TATTTGTGAA

AGTTAACAALC

TTTTTCGGAT

AGGAACCCCT

CCGGGCGACC

GAGCGCGCAG

CTGCGAGTGT

GCTGAGGCCC

AGATACAGAT

.AGGTGGGGGT

3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 CTTATGTAGT TTTGTATCTG TTTTGCAGCA GCCGCCGCCG CCATGAGCAC CAACTCGTTT wo 98/10088 WO 9810988PCT1US97/15716

GATGGAAGCA

CAGAATGTGA

TTGACCTACG

GCCGCTGCAG

AGCAGTGCAG

TTGGATTCTT

CAGGTTTCTG

CCAGACTCTG

CGCGCGCGGT

AGGACGTGGT

TGGAGGTAGC

TAGCAGGAGC

GGCAGGCCCT

GATATGAGAT

CGGGGATTCA

TCATGT.AGCT

TTTTCCATGC

ATATTTCTGG

TTTACAAAGC

GCGTAGTTAC

TCTACCTGCG

AGCAGGTTCC

ACCGGGTGCA

ACTTCGTTAA

TCGCCGCCCA

TCCGCCGTAG

GTCACCTGC7

TTTCGCTGTA

GGCGCAGGG7

CGCTGGCCAC-

TTGTGAGCTC

TGGGCTCCAG

AGACCGTGTC

CCACCGCCCG

CTTCCCGTTC

TGACCCGGGA

CCCTGAAGGC

TTTGGATTTG

AGGCCCGGGA

AI4AGGTGACT

ACCACTGCAG

GCTGGGCGTG

TGGTGTAAGT

GCATCTTGGA

TGTTGTGCAG

TAGAAGGAAA

ATTCGTCCAT

GATCACTAAC

GCGGGCGGAG

CCTCACAGAT

GGGCGATGAA

TGAGCAGCTG

ACTGGTAGTT

GCATGTCCC7

*GCGATAGCAG

*GCATGCTTTI

CTACGGCATC

CGGCAGTAGIJ

CCTCGTCAGC

GGTGCGCTTC

ATATTTGACA

CATTGATGGT

TGGAAICGCCG

CGGGATTGTG

ATCCGCCCGC

ACTTAATGTC

TTCCTCCCCT

GATCAAGCAA

CCAGCGGTCT

CTGGATGTTC

AGCTTCATGC

GTGCCTAAAA

GTTTACAAAG

CTGTATTTTT

AACCACCAGC

TGCGTGGAAG

AATGATGGCA

GTCATAGTTG

GGTGCCAGAC

TTGCATTTCC

GAAAACGGT7

CGACTTACCG

AALGAGAGCTC

GACTCGCATC

TTCTTGCAAC

GAGCGTTTGI

TCGATCCAG(

CGGTGCTCGI

GTAGTCTGG(

AGGCTGGTC(

ACGCGCATGC

CGCCCCGTCC

TTGGAGACTG

ACTGACTTTG

GATGACAAGT

GTTTCTCAGC

CCCAATGCGG

GTGTCTTGCT

CGGTCGTTGA

AGATACATG

TGCGGGOTGG

ATGTCTTTCA

CGGTTAAGCT

AGGTTGGCTA

ACAGTGTATC

AACTTGGAGA

ATGGGCCCAC

TGTTCCAGGA

TGCGGTATAA

CACGCTTTGA

TCCGGGGTAG

CAGCCGGTGG

ICAGCTGCCGT

TTTTCCCTG1A GAAGCAAAG7 iCCPAGCAGT~I

IATATCTCCTC

rCCAGACGGGC

TCACGGTGAI

STGCTGGTGC']

CCCCATGGGC

TGCCCGCAAA

CAGCCTCCGC

CTTTCCTGAG

TGACGGCTCT

AGCTGTTGGA

TTTAAAACAT

GTCTTTATTT

GGGTCCTGTG

GCATAAGCCC

TGTTGTAGAT

GTAGCAAGCT

GGGATGGGTG

TGTTCCCAGC

CGGTGCACTT

CGCCCTTGTG

GGGCGGCGGC

TGAGATCGTC

TGGTTCCATC

GTTCAGATGG

GGGAGATCAG

GCCCGTAAAT

CATCCCTGAG

CCAPAATCCGC

TTTTCAACGG

CCAGGCGGTC

GTTTCGCGGG

CAGGGTCATG

GGGGTGCGCT

GAAGCGCTGC

CGGGGTGCGT

CTCTACTACC

CGCCGCTTCA

CCCGCTTGCA

TTTGGCACAA

TCTGCGCCAG

AAATAAAAAA

AGGGGTTTTG

TATTTTTTCC

GTCTCTGGGG

GATCCAGTCG

GATTGCCAGG

CATACGTGGG

CATATCCCTC

GGGAAATTTG

ACCTCCAAGA

CTGGGCGAAG

ATAGGCCATT

CGGCCCAGGG

GGGGATCATG

CTGGGAAGAA

CACACCTATT

CAGGGGGGCC

CAGAAGGCGC

TTTGAGACCG

CCACAGCTCG

TTGGGGCGGC

TCTTTCCACG

CCGGGCTGCG

CGGTCTTCGC

5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 CCTGCGCGTC GGCCAGGTAG CATTTGACCA TGGTGTCATA GTCCAGCCCC TCCGCGGCGT 1. WO 98/10088 WO 9810088PCTIUS97/15716

GGCCCTTGGC

TGAGGGCGTA

AGGCCCCGCA

AAACCAGGTT

GTCCACGCTC

CCTCGACCGA

ATGACTATCG

CCGGCAGCGC

GGCCTGTCGC

CCCGCCACCA

CTGGGCTACG

CTTCTCGCTT

GATGACGACC

ATCACTGGAC

TTGGCATGGA

GCATGGAGCC

CCACTCCAAG

CTTGGCAGAA

GCAGCGTTGG

TAGGCTGGCG

GAAGCGACTO

TCCGTGTTTC

TCTGCATCGC

CTTTCTCAAT

GGCTGTGTGC

CTTGAGTCCA

ATTAGCAGAG

GGCTACACTA

AAAAGAGTTG

GTTTGCAAGC

TCTACGGGGT

GCGCAGCTTG

GAGCTTGGGC

GACGGTCTCG

TCCCCCATGC

GGTGACGAAA

TGCCCTTGAG

TCGCCGCACT

TCTGGGTCAT

TTGCGGTATT

AACGTTTCGG

TCTTGCTGGC

CCGGCGGCAT

ATCAGGGACA

CGCTGATCGT

TTGTAGGCGC

GGGCCACCTC

AATTGGAGCC

CATATCCATC

GTCCTGGCCA

GGGTTGCCTT

CTGCTGCAAA

GTAAAGTCTG

AGGATGCTGC

GCTCACGCTG

ACGAACCCCC

ACCCGGTAAiG

CGAGGTATGT

GAAGGACAGT

GTAGCTCTTG

AGCAGATTAC

CTGACGCTCA

CCCTTGGAGG

GCGAGAAATA

CATTCCACGA

TTTTTGATGC

AGGCTGTCCG

AGCCTTCAAC

TATGACTGTC

TTTCGGCGAG

CGGAATCTTG

CGAGAAGCAG

GTTCGCGACG

CGGGATGCCC

GCTTCAAGGA

CACGGCGATT

CGCCCTATAC

GACCTGAATG

AATCAATTCT

GCGTCCGCCA

CGGGTGCGCA

ACTGGTTAGC

ACGTCTGCGA

GAAACGCGGA

TGGCTACCCT

TAGGTATCTC

CGTTCAGCCC

ACACGACTTA

AGGCGGTGCT

ATTTGGTATC

ATCCGGCAAA

GCGCAGAAAA

GTGGAACGAA

AGGCGCCGCA

CCGATTCCGG

GCCAGGTGAG

GTTTCTTACC

TGTCCCCGTA

CCAGTCAGCT

TTCTTTATCA

GACCGCTTTC

CACGCCCTCG

GCCATTATCG

CGAGGCTGGA

GCGTTGCAGG

TCGCTCGCGG

TATGCCGCCT

CTTGTCTGCC

GAAGCCGGCG

TGCGGAGAAC

TCTCCAGCAG

TGATCGTGCT

AGAATGAATC

CCTGAGCAAC

AGTCAGCGCC

GTGGAACACC

AGTTCG4GTGT

GACCGCTGCG

TCGCCACTGG

ACAGAGTTCT

TGCGCTCTGC

CAAACCFICCG

AAAGGATCTC

AACTCACGTT

CGAGGGGCAG

GGAGTAGGCA

CTCTGGCCGT

TCTGGTTTCC

TACAGACTTG

CCTTCCGGTG

TGCAACTCGT

GCTG4GAGCGC

CTCAAGCCTT

CCGGCATGGC

TGGCCTTCCC

CCATGCTGTC

CTCTTPLCCAG

CGGCGAGCAC

TCCCCGCGTT

GCACCTCGCT

TGTGAATGCG

CCGCACGCGG

CCTGTCGTTG

ACCGATACGC

AACATGAATG

CTGCACCATT

TACATCTGTA

AGGTCGTTCG

CCTTATCCGG

CAGCAGCCAC

TGAAGTGGTG

TGAAGCCAGT

CTG4GTAGCGG

AAGAAGATCC

AAGGGATTTT

TGCAGACTTT

TCCGCGCCGC

TCGGGGTCAA

ATGAGCCGGT

AGAGGCCTGT

GGCGCGGGGC

AGGACAGGTG

GACGATGATC

CGTCACTGOT

GGCCGACGCG

CATTATGATT

CAGGCAGGTA

CCTAACTTCG

ATGGAACGGG

GCGTCGCGGT

AACGGATTCA

CAAACCAACC

CGCATCTCGG

AGGACCCGGC

GAGCGAACGT

GTCTTCGGTT

ATGTTCCGGA

TTAACGAAGC

CTCCAAGCTG

TAACTATCGT

TGGTAACAGG

GCCTAACTAC

TACCTTCGGA

TGGTTTTTTT

TTTGATCTTT

GGTCATGAGA

7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 WO 98/10088 WO 9810088PCTIUS97/15716

TTATCAAAAA

TAAAGTATAT

ATCTCAGCGA

ACTACGATAC

CGCTCACCGG

AGTGGTCCTG

GTAAGTAGTT

GTGTCACGCT

GTTACATGAT

GTCAGAAGTA

CTTACTGTCA

TTCTGAGAAT

ACCGCGCCAC

AAACTCTCAA

AACTGATCTT

CAAAATGCCG

CTTTTTCAAT

GAATGTATTT

CCTGACGTCT

AGGCCCTTTC

GGATCTTCAC

ATGAGTAAAC

TCTGTCTATT

GGGAGGGCTT

CTCCAGATTT

CAACTTTATC

CGCCAGTTAA

CGTCGTTTGG

CCCCCATGTT

AGTTGGCCGC

TGCCATCCGT

AGTGTATGCG

ATAGCAGAAC

GGATCTTACC

CAGCATCTTT

CAAAAAAGGG

ATTATTGAAG

AGAAAAATAA

AAGAAACCAT

GTCTTCAAi

CTAGATCCTT

TTGGTCTGAC

TCGTTCATCC

ACCATCTGGC

ATCAGCAATA

CGCCTCCATC

TAGTTTGCGC

TATGGCTTCA

GTGCAAAAAA

AGTGTTATCA

AAGATGCTTT

GCGACCGAGT

TTTAAAAGTG

GCTGTTGAGA

TACTTTCACC

AATAAGGGCG

CATTTATCAG

ACAAATAGGG

TATTATCATG

TTAAATTAAA

AGTTACCAMT

ATAGTTGCCT

CCCAGTGCTG

AACCAGCCLG

CAGTCTATTA

AACGTTGTTG

TTCAGCTCCG,

GCGGTTAGCT

CTCATGGTTA

TCTGTGACTG

TGCTCTTGCC

CTCATCATTG

TCCAGTTCGA

AGCGTTTCTG

ACACGGAAAT

GGTTATTGTC

GTTCCGCGCA

ACATTAACCT

AATGAAGTTT

GCTTAATCAG

GACTCCCCGT

CAATGATACC

CCGGAAGGGC

ATTGTTGCCG

CCATTGCTGC

GTTCCCAACG

CCTTCGGTCC

TGGCAGCACT

GTGAGTACTC

CGGCGTCAAC

GAAAACGTTC

TGTAACCCAC

GGTGAGCAAA

GTTGAATACT

TCATGAGCGG

CATTTCCCCG

ATAAAAATAG

TAAATCAATC!

TGAGGCACCT

CGTGTAGATA

GCGAGACCCA

CGAGCGCAGA

GGAAGCTAGA

AGGCATCGTG

ATCAAGGCGA

TCCGATCGTT

GCATAATTCT

AACCAAGTCA

ACGGGATAAT

TTCGGGGCGA

TCGTGCACCC

AACAGGAAGG

CATACTCTTC

ATACATATTT

AAAAGTGCCA

GCGTATCACG

9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10398 INFORMATION FOR SEQ ID NOs2: SEQUENCE CHARACTERISTICSt LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TATTTAAGCC CGAGTGAGCT

Claims

1. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of: 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 a cassette consisting essentially of a 5' AAV inverted terminal repeat (ITR), a selected minigene, and a 3' AAV ITR; culturing the host cell under conditions which permit replication and packaging of recombinant AAV; and 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. The method according to claim 1 wherein the third vector further comprises adenoviral sequences flanking the cassette. WO 98/10088 PCT/US97/15716 28

6. The method according to any of claims 1 to 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: providing a host cell which expresses T7 polymerase; introducing into the host cell a first vector which comprises AAV rep and cap genes under control of T7 promoter sequences; 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 culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

9. The method according to claim 8 wherein step comprises the step of transfecting the host cell with a vector comprising the T7 promoter and the AAV rep and cap genes. The method according to claim 8 wherein step comprises the step of infecting the host cell with a recombinant adenovirus comprising the T7 promoter sequences, and the AAV rep and cap genes. WO 98/10088 PCTIUS97/15716 29

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

12. The method according to claim 8 wherein step 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: providing a host cell stably I transfected with AAV rep and cap genes under control of T7 promoter sequences; introducing into the host cell a vector comprising T7 polymerase; introducing into the host cell a vector comprising a cassette consisting essentially of a 5' AAV inverse terminal repeat (ITR), a selected minigene, and a 3' AAV ITR; and culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

14. The method according to claim 13 wherein step comprises the step of transfecting the host cell with a vector comprising the T7 polymerase gene. The method according to claim 13 wherein step comprises the step of infecting the host cell with a recombinant adenovirus comprising the T7 polymerase gene under control of regulatory sequences SRA4'controlling expression thereof. lco uj WO 98/10088 PCT/US97/15716

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

17. The method according to claim 13 wherein step 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: providing a host cell comprising a cassette consisting essentially of 5' AAV inverse terminal repeats (ITR), a selected minigene, and a 3' AAV ITR; introducing into the host cell a vector comprising AAV rep and cap genes under control of T7 promoter sequences; introducing into the host cell a vector comprising the T7 polymerase; and culturing the host cell under conditions which permit replication and packaging of a recombinant AAV.

19. The method according to claim 18 wherein step comprises the step of transfecting the host cell with a plasmid vector. The method according to claim 18 wherein step comprises the step of infecting the host cell with a recombinant adenoviral vector. WO 98/10088 PCT/US97/15716 31

21. The method according to claim 18 wherein step 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 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: providing a host cell stably transfected with a cassette consisting essentially of 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; introducing into the host cell a vector comprising the T7 polymerase; and 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; providing a host cell stably transfected with a cassette consisting essentially of AAV inverse terminal repeats (ITR), a selected minigene, and a 3' AAV ITR and a plasmid comprising T7 polymerase; introducing into the host cell a vector comprising AAV rep and cap genes under control of T7 promoter sequences; and WO 98/10088 PCTIUS97115716 32 culturing the host cell under conditions which permit replication and packaging of a recombinant AAV. A method for production of recombinant adeno-associated virus (AAV) comprising the steps of: 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; su, r (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 inducing expression of said T7 promoter.

26. A recombinant adeno-associated virus (AAV) produced according to the method of any one of claims 1-25.

27. Methods for the production of a recombinant adeno-associated virus (AAV) or a recombinant adeno-associated virus produced therefrom substantially as herein described with reference to the examples and accompanying figures. DATED this sixteenth day of May, 2000. Si 1tRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA SIts Patent Attorneys *AVIES COLLISON CAVE