CA2264482A1 - An inducible method for production of recombinant adeno-associated viruses utilizing t7 polymerase - Google Patents

An inducible method for production of recombinant adeno-associated viruses utilizing t7 polymerase Download PDF

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CA2264482A1
CA2264482A1 CA002264482A CA2264482A CA2264482A1 CA 2264482 A1 CA2264482 A1 CA 2264482A1 CA 002264482 A CA002264482 A CA 002264482A CA 2264482 A CA2264482 A CA 2264482A CA 2264482 A1 CA2264482 A1 CA 2264482A1
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James M. Wilson
Nancie Chen
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University of Pennsylvania Penn
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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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

101520253035W0 98/ 10088CA 02264482 1999-03-03PCT/US97/ 15716AN INDUCIBLE METHOD FOR PRODUCTION OF RECOMBINANTADENO-ASSOCIATED VIRUSES UTILIZING T7 POLYMERASEBackground of the InventionAdeno-associated virus is a replication-deficient parvovirus, the genome of which is about 4.6 kbin length, including 145 nucleotide inverted terminalrepeats (ITRs). The single-stranded DNA genome of AAVcontains genes responsible for replication (rep) andformation of virions (cap).When this nonpathogenic human virus infects ahuman cell, the viral genome integrates into chromosome19 resulting in latent infection of the cell. Productionof infectious virus and replication of the virus does notoccur unless the cell is coinfected with a lytic helpervirus such as adenovirus or herpesvirus. Upon infectionwith a helper virus, the AAV provirus is rescued andamplified, and both AAV and helper virus are produced.AAV possesses unique features that make itattractive as a vector for delivering foreign DNA tocells. Various groups have studied the potential use ofAAV in the treatment of disease states.However, an obstacle to the use of AAV fordelivery of DNA is the lack of highly efficient methodsfor encapsidation of recombinant genomes. See, R. Kotin,Hum. Gene Ther., §:793—801 (1994).gene product is toxic to cells and thus cannot beFurthermore, the repexpressed at high levels. For example, previously knownmethods employ transfection of host cells with a rAAVgenome 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 absenceof contaminating wild-type AAV or helper adenovirus isAnd,in the absence of the AAV rep gene product, integrationassociated with little recombinant gene expression.is inefficient and not directed to chromosome 19.WO 98/100881015202530CA 02264482 1999-03-03PCTVUS97fl57l62Bacteriophage T7 RNA polymerase (T7 P01) is theproduct of T7 gene 1, which can recognize its responsivepromoter sequence specifically and exhibit a hightranscriptase activity [M. Chamberlin et al, Nat re,;;§:227-231 (1970); J. Dunn and F. Studier, J. Mol.Biol., ;§§:447—535 (1983); and B. Moffatt et al, ggll,g2:221-227 (1987)].expression of proteins in E. coli [S. Tabor and C.Richardson, Natl. Acad. Sci. USA, §;:1074-1078(1985); F. Studier and B. Moffatt, J. Mol. Biol.,;§g:113—13o (l986)],infected eukaryotic cells [T. Fuerst et al, Proc. Natl.Acad. Sci. USA, §;:8122-8126 (1986); A. Ramsey-Ewing andJ. Biol. Chem., g1;:16962-16966 (1996)],mammalian cells [A. Lieber et al, Nucl. Acids Res.,;1:8485-8493 (1989)].What is needed is an efficient method forIt has been used for heterologousPIOC.in recombinant vaccinia virus-B. Moss, and inproduction of rAAV which avoids the problems associatedwith rep toxicity for the packaging cell.Summary of the InventionThe present invention provides an induciblemethod for efficient production of rAAV which makes useof T7 polymerase. T7 P01 is derived from lambda phageThus,expression of rep/cap can be controlled by placing theseand its promoter is not active in mammalian cells.genes under control of the T7 promoter and providing theT7 P01 in trans or under the control of an inducibleThus,rep which rendered prior art methods of producing rAAVpromoter. this method avoids the toxic effects ofinefficient. The method of the invention is particularlysuitable for large scale production of rAAV, which isdesired for rAAV vectors to be used in gene therapy.In one aspect, the invention provides a methodof producing rAAV which utilizes three vectors. A firstWO 98/10088101520253035CA 02264482 1999-03-03PCTIUS97/157163vector is capable of expressing T7 polymerase in the hostcell following transfection or infection. A secondvector comprises the AAV rep and cap genes under theThe thirdvector comprises a cassette containing 5' and 3' AAVcontrol of T7 promoter sequences (T7/rep/cap).inverted terminal repeats (ITRs) flanking a selectedtransgene. A host cell containing these three vectors iscultured under conditions which permit replication andpackaging of a recombinant AAV, and the rAAV isrecovered.In another aspect, the invention provides amethod in which a host cell is stably transfected withone of the three components of the system used in thetriple infection system. The remaining components areintroduced into the host cell, as described above.In one embodiment, the invention providesmethod in which a vector containing T7/rep/cap and avector containing a cassette comprising a selectedminigene flanked by 5' and 3' AAV ITRs are introducedThe host cellis then cultured under conditions which permit productionof rAAV.a method which utilizes a host cell stably transfectedinto a host cell expressing T7 polymerase.In another embodiment, this invention provideswith a plasmid containing T7/rep/cap. A vectorcontaining T7 pol and a vector containing a cassettecomprising 5' AAV inverse terminal repeat (ITR), aselected minigene, and 3' AAV ITR are introduced into thehost cell.which permit production of rAAV.The host cell is cultured under conditionsIn still anotherembodiment, the invention provides a method whichutilizes a host cell stably transfected with a rescuablerAAV cassette.containing T7/rep/cap are introduced into the host cell.A vector containing T7 pol and a vectorThe host cell is cultured under conditions which permitproduction of rAAV.WO 98/100881015202530CA 02264482 1999-03-03PCT/US97ll 57164In yet another aspect, the present inventionprovides a method which utilizes a host cell stablytransfected with two of the three components of theTheremaining component is then introduced into the hostsystem used in the triple infection system.cell, as described above.In a further aspect, the present inventionprovides a method which utilizes a host cell stablytransfected with the three components of the system usedIn this aspect, the T7Pol is controlled by an inducible promoter.in the triple infection system.In still a further aspect, the inventionprovides a rAAV produced according to the method of theinvention.other aspects and advantages of the presentinvention are described further in the following detaileddescription of the preferred embodiments thereof.Brief Description of the DrawingsFig. 1 provides a schematic illustration of theconstruction of a recombinant adenovirus containing theT7 polymerase gene.Fig. 2 provides a schematic illustration of theconstruction of a recombinant plasmid containing the AAVrep/cap genes under control of a T7 promoter.Fig. 3 provides a schematic illustration of theconstruction of a recombinant adenovirus containing therep/cap genes under control of a T7 promoter.Fig. 4 provides a schematic illustration of theconstruction of a recombinant hybrid Ad/AAV virus.Detailed Description of the InventionThe invention provides an inducible method forefficient production of recombinant AAV vectors usefulparticularly for gene delivery and transfer.T’ T H I}W0 98/ 10088101520253035CA 02264482 1999-03-03PCT/US97/157165Specifically, the invention provides methods of AAVproduction in which expression of the toxic but necessaryrep gene is controlled by the T7 promoter.Thus, in one aspect, the method of theinvention for production of rAAV involves introducinginto a host cell the AAV rep and cap genes under controlof a T7 promoter, and a recombinant adeno-associatedvirus (rAAV) cassette containing a selected minigeneflanked by AAV ITRs.encoding T7 pol, high level expression of rep proteinUpon introduction of a genefrom the T7/rep/cap construct is induced and cells may begrown on a large scale. When rep expression is desired,the cells are caused to express the T7 polymerase whichacts on the T7 promoter. This facilitates the efficientreplication and packaging of rAAV carrying a gene ofinterest.A host cell may be triple transfected (orinfected) with vectors containing the above elements.Alternatively, a host cell which expresses one or more ofthe required elements and may be transfected/infectedwith the remaining elements is utilized. In anotheralternative, a host cell is utilized which stablyexpresses all three elements of the system, and the T7pol is placed under the control of an inducible promoter,which permits rep/cap expression to be controlled and theavoidance of toxic effects to the cell.For each of the vector components used in themethod of the invention, adenoviral constructs arecurrently preferred. However, using the informationprovided herein and known techniques, one of skill in theart could readily construct a different viral (adenoviralor non-adenoviral) or a plasmid vector which is capableof driving expression of the desired genes in the hostcell.their inability to infect non-dividing cells, vectorsFor example, although less preferred because ofWO 98/100881015202530CA 02264482 1999-03-03PCT/US97/157166carrying the required elements of this system, e.g., theT7 polymerase, may be readily constructed usingretroviruses. Therefore, this invention is not limitedby the virus or plasmid selected for purposes ofintroducing the T7 pol, T7/rep/cap, or AAV cassette intothe host cell.a virus which provides the necessary helper functions toDesirably, at least one of the vectors isenable packaging. Alternatively, the helper functionsmay be provided by a co-transfected adenovirus orherpesvirus. Suitable techniques for introducing thesevectors into the host cell are discussed below and areknown to those of skill in the art. As used herein, a"host cell" is any cell (cell line), preferablymammalian, which permits expression of the T7 pol andT7/rep/cap and packaging of the rAAV containing thecassette, under the conditions described herein.Suitable packaging cells are known, and may be readilyselected by the skilled artisan.A. Triple Infection/TransfectionAs stated above, a host cell used forassembly and packaging of recombinant AAV may betransfected with plasmid vectors or infected with viralvectors containing the required components of the system.1. T7 Pol VectorsIn a preferred embodiment, a firstvector contains the T7 Pol gene under the control of asuitable promoter. In example 5 below, the nuclearlocalized T7 Pol gene is obtained from a publiclyavailable plasmid [M. Strauss, Nucleic Acid Res.,_2l_7:8485-8493 (1989) ] .alternatively be obtained from other commercial andHowever, the gene mayacademic sources, including the American Type CultureCollection (pTF7—3, Accession No. 484944). See, alsoGenBank accession number M30308. Desirably, the T7 polWO 98/10088101520253035CA 02264482 1999-03-03PCT/US97/157167gene is linked to a nuclear localization signal, such asthat described in Dunn, gggg, §§:259-266 (1988), usingconventional techniques.Desirably, T7 Pol is under thecontrol of a cytomegalovirus (CMV) immediate earlypromoter/enhancer [see, e.g., Boshart et al, Qgll,g;:521—53o (1935)).may be readily selected by one of skill in the art.However, other suitable promotersUseful promoters may be constitutive promoters orregulated (inducible) promoters, which will enablecontrol of the amount of the transgene to be expressed.For example, another suitable promoter includes, withoutlimitation, the Rous sarcoma virus LTR promoter/enhancer.Still other promoter/enhancer sequences may be selectedby one of skill in the art.In addition, the vector also includesother conventional regulatory elements necessary to driveexpression of T7 Pol in a cell transfected with thevector. Such regulatory elements are known to those ofskill in the art.2. T7/Rep/Cap VectorsThe second vector component of thissystem contains the rep and cap genes under control of aT7 promoter. The rep and cap genes can be obtained fromSee, e.g., T. Shenk, Q;Virol., §;:3096-3101 (1987), which provides the AAV2genome within the plasmid psubzol; E. W. Lusby et al, Q;Virol., g;:518-526 (1982) and J. Smuda and B.J. Carter,Virology, ;§g:310-318 (1991).Similarly, the T7 promoter sequences[J. J. Dunn and F.W. Studier, J. Mol. §iol., l§§:477-535(1983) may be obtained from a variety of commercial anda variety of known sources.academic sources. In a preferred embodiment, the vectorfurther contains the sequence of untranslated region(UTR) of encephalomyocarditis (EMCV) downstream of the T7W0 98/ 10088101520253035CA 02264482 1999-03-03PCT/US97/157168promoter. The inventors believe this sequence increasesexpression of the gene 5- to 10-fold.In addition, the vector also includesconventional regulatory elements necessary to driveexpression of the rep/cap in a cell transfected with thevector. Such regulatory elements are known to those ofskill in the art.3. rAAV Cassette (Template)The third vector component contains arAAV cassette containing a minigene flanked by AAV ITRS.As discussed in more detail below, such a minigenecontains a suitable transgene, a promoter, and otherregulatory elements necessary for expression of thetransgene.The AAV sequences employed arepreferably limited to the cis-acting 5' and 3' invertedterminal repeat (ITR) sequences [See, e.g., B. J. Carter,in "Handbook of Parvoviruses", ed., P. Tijsser, CRCPress, pp.155-168 (1990)].entire 143 bp sequences encoding the ITRs are used in theDesirably, substantially thevectors. Some degree of minor modification of thesesequences is expected to be permissible for this use.The ability to modify these ITR sequences is within theskill of the art.al, "Molecular Cloning.See, e.g., texts such as Sambrook etA Laboratory Manual.", 2d edit.,Cold Spring Harbor Laboratory, New York (1989).Alternatively, it may be desirable to use functionalfragments of the ITRs. Such fragments may be determinedby one of skill in the art.The AAV ITR sequences may be obtainedfrom any known AAV, including presently identified humanAAV types.may also be employed in the vector constructs of thisSimilarly, AAVs known to infect other animalsinvention. The selection of the AAV is not anticipatedto limit the following invention. A variety of AAVWO 98/100881015202530CA 02264482 1999-03-03PCT/US97/157169strains, types 1-4, are available from the American TypeCulture Collection or available by request from a varietyIn thefollowing exemplary embodiment an AAV-2 is used forof commercial and institutional sources.convenience.The 5' and 3' AAV ITR sequences flanka minigene which is made up of a selected transgenesequence and associated regulatory elements. Thetransgene sequence of the vector is a nucleic acidsequence heterologous to the AAV sequence, which encodesa polypeptide or protein of interest. The transgene isoperatively linked to regulatory components in a mannerwhich permits transgene transcription.The composition of the transgenesequence will depend upon the use to which the resultingvector will be put. For example, one type of transgenesequence includes a reporter sequence, which uponexpression produces a detectable signal. Such reportersequences include without limitation an E. coli beta-galactosidase (Lacz) cDNA, an alkaline phosphatase geneand a green fluorescent protein gene. These sequences,when associated with regulatory elements which drivetheir expression, provide signals detectable byconventional means, e.g., ultraviolet wavelengthabsorbance, visible color change, etc. A more preferredtransgene sequence includes a therapeutic gene whichexpresses a desired gene product in a host cell. Thesetherapeutic nucleic acid sequences typically encodeproducts which may be administered to a patient in vivoor ex vivo to replace or correct an inherited or non-inherited genetic defect or treat an epigenetic disorderor disease. The selection of the transgene sequence isnot a limitation of this invention., .. ...............,....u..............,.... . . WO 98/100881015'20253035CA 02264482 1999-03-03PCT/US97/1571610In addition to the major elementsidentified above, the minigene also includes conventionalregulatory elements necessary to drive expression of thetransgene in a cell transfected with the vector carryingthe AAV cassette.promoter which is linked to the transgene and locatedThus the minigene contains a selectedwithin the minigene, between the AAV ITR sequences of thevector.Selection of the promoter whichmediates expression of the transgene is a routine matterand is not a limitation of the vector. Useful promotersinclude those which are discussed above in connectionwith the first vector component.The minigene will also desirablycontain heterologous nucleic acid sequences includingsequences providing signals required for efficientpolyadenylation of the transcript and introns withfunctional splice donor and acceptor sites. A commonpoly-A sequence which is employed in the exemplaryvectors of this invention is that derived from thepapovavirus SV—40. The poly-A sequence generally isinserted following the transgene sequences and before the3' AAV ITR sequence.derived from SV-40, and is referred to as the SV—40 Tintron sequence.A common intron sequence is alsoA minigene of the present invention mayalso contain such an intron, desirably located betweenthe promoter/enhancer sequence and the transgene.Selection of these and other common vector elements areconventional and many such sequences are available [see,e.g., Sambrook et al, and references cited therein].The rAAV vector containing the AAVITRs flanking the minigene may be carried on a plasmidbackbone and used to transfect a selected host cell ormay be flanked by viral sequences (e.g., adenoviralsequences) which permit it to infect the selected host101520253035WO 98/10088CA 02264482 1999-03-03PCT/US97/1571611cell.produced in accordance with known techniques. See, e.g.,WO 96/13598, WO 95/23867, and WO 95/06743, which areincorporated by reference herein.Suitable Ad/AAV recombinant viruses may beB. Double Infection/TransfectionA cell line which stably expresses T7 polmay be constructed, and then double transfected (orinfected) with a vector containing T7/rep/cap and avector containing a rAAV cassette, as illustrated in thefollowing table (Inf = infection and Txf = transfection).T7 re c gggySystem A Inf InfSystem B Inf TxfSystem C Txf InfSystem D Txf TxfAlternatively, a cell line stablytransfected with T7 rep/cap may be double transfected(infected) with a vector carrying T7 pol and a vectorcarrying the rAAV cassette, as illustrated in thefollowing table.T7 0 rggySystem E Inf InfSystem F Inf TxfSystem G Txf InfSystem H Txf TxfIn still another alternative, a cell linewhich contains a rescuable rAAV cassette may be doubletransfected (infected) with a vector containing T7 Poland a vector containing T7/rep/cap, as illustrated in thefollowing table.T7 0 T7 re caSystem I Inf InfSystem J Inf TxfSystem K Txf InfSystem L Txf Txf. M.,........,.............._.............».» ,,..,,.....l....l,.. , .W0 98I10088101520253035CA 02264482 1999-03-03PCT/US97/1571612The plasmid and viral vectors used indouble transfection/infection steps are as describedabove in connection with the triple transfection and/orinfection system.A stable cell line of the invention can beproduced by transfection of a desired cell, e.g., 293cells or other packaging cell lines expressing requiredadenoviral genes, with a plasmid containing the desiredgene, e.g., T7 Pol, using conventional techniques andselected via an accompanying resistant marker gene.Depending upon whether inducible or constitutiveexpression is desired, an appropriate promoter may beselected. For example, if a host cell induciblyexpressing T7 Pol is desired, the cell may be transfectedwith a plasmid containing T7 Pol under control of aif a host cellconstitutively expressing T7 Pol is desired, it may bemetallothionein promoter. Alternatively,inserted under control of a RSV or CMV promoter. Similartechniques may be used for providing a host cellcontaining the T7/rep/cap and a host cell containing arescuable rAAV. The examples below describe productionof stable cell lines. However, one of skill in the artcould readily produce such cell lines using otherconventional techniques. See, generally, Ausubel et al,Current Protocols in Molecular Biology (WileyInterscience 1987).C. Single Infection/TransfectionA cell line which stably expresses two ofthe components of this system may be constructed, andthen transfected (or infected) with a vector containingthe remaining component of the system, as describedabove. For example, using the techniques describedherein, a cell line is utilized which is stablytransfected with the T7/rep/cap and a rescuable rAAV.The cell line is then transfected or infected with a101520253035W0 98/10088CA 02264482 1999-03-03PCT/US97/1571613vector containing the T7 pol. As another example, thecell line is stably transfected with the T7 pol and arescuable rAAV. The cell line is then transfected orinfected with a vector containing the T7 rep/cap.D. Cell Line Containing T7 Pol, rAAV andT7/rep/capA cell line which stably expresses allthree of the components of this system may be constructedand utilized in the method of the invention. Using knowntechniques, a suitable packaging cell line is constructedwhich contains the rAAV, the T7/rep/cap and the T7 pol.In this embodiment, the T7 Pol is placed under thecontrol of an inducible promoter. Suitable induciblepromoters are known to those of skill in the art and arediscussed herein. For example, T7 Pol may be placedIn thismanner, expression of the T7 P01, and thus the rep/cap,under control of a metallothionein promoter.which are under control of the T7 promoter can beregulated and toxic effects to the cell avoided.E. Production of Vectors and rAAVAssembly of the selected DNA sequences ofthe adenovirus, AAV and the reporter genes or therapeuticgenes and other vector elements into the vectorsSuchtechniques include cDNA cloning such as those describeddescribed above utilize conventional techniques.in texts [sambrook et al, cited above], use ofoverlapping oligonucleotide sequences of the adenovirusor AAV genome, polymerase chain reaction, and anysuitable method which provides the desired nucleotidesequence.Whether using the three vector system, orstably infected cells, introduction of the vectors intothe host cell is accomplished using known techniques.Where appropriate, standard transfection and co-transfection techniques are employed, e.g., CaPO4WO 98/100881015202530CA 02264482 1999-03-03PC1VUS97fl57l614transfection techniques using the complementation humanembryonic kidney (HEK) 293 cell line (a human kidney cellline containing a functional adenovirus Ela gene whichprovides a transacting E1a protein). other conventionalmethods employed in this invention include homologousrecombination of the viral genomes, plaguing of virusesin agar overlay, methods of measuring signal generation,and the like.Following infection/transfection, the hostcell is then cultured under standard conditions, toenable production of the rAAV. e.g., F. L. Grahamand L. Prevec, Methods Mol. Biol., 1:109-128 (1991).Desirably, once the rAAV is identified using conventionalSee,techniques, it may be isolated using standard techniquesand purified.These examples illustrate the preferred methodsof the invention. These examples are illustrative onlyand do not limit the scope of the invention.Example 1 - Construction of a T7 Pol AdenovirusFigure 1 provides a schematic of theconstruction of the recombinant adenovirus carrying theT7 polymerase.The plasmid pMTT7N was obtained from Dr.Michael Strauss [A. Lieber et al, uggl. Acids Res,,17:8485-8493 (1989)]. pMTT7N contains a N-terminalnuclear location signal of SV40 large T antigen fused tothe T7 Pol gene (T7N Pol) which is linked to thepolyadenylation sequence of SV40. Expression is drivenby the inducible mouse metallothionein promoter.The pMTT7N plasmid DNA was digested with BglIIand PvuII restriction enzymes and the fragments separatedThe BglII/PvuII T7 Pol DNA fragmentwas ligated to the BglII/EcoRV cleaved vectorpAd.CMV.link.1 to form pAd.CMV.T7N. pAd.CMV.link.1 is aon an agarose gel.1015202530W0 98/10088CA 02264482 1999-03-03PCT/US97/1571615plasmid containing the adenoviral sequences 0 to 16 mapunits deleted of Ela and Elb into which a CMVpromoter-polylinker cassette was cloned. This isdescribed in X. Ye et al, J. giol. Ch§m., g1;:3639-3646(1996).In pAd.CMV.T7N, the expression unit of T7 Polis directed by the CMV promoter. The promoter for the T7Pol gene is linked to a PolyA tail as a cassette withinthe 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 usingCellphate kit (Pharmacia).transfection, the T7 Pol adenovirus can be isolated fromApproximately 1 week post-the plaques for further purification.Example 2 - Cell Lines Expressing T7 PolA cell line stably expressing T7 Pol isestablished by co-transfection of plasmids pMTT7N andpMTCB6+ (which provides a selective marker) [K. H. Chooet al, _D_L, §:529-538; Eur. J. Bioghem., ;_7_g:417—424]into 293 cell at a ratio of 10:1 using calcium phosphateprecipitation [F. Graham and A. van der Eb, Virol.,§g:456-467 (1973)).Geneticin selection at a concentration of 1 mg/ml.Colony cloning is carried out byEachclone obtained is transfected with pT7 rep/cap plasmid[see, Example 3 below] and analyzed for its ability toinduce the expression of Rep protein upon induction bysupplementation with Zn**.To establish a stable cell line thatconstitutively expresses the T7 Pol, the T7N Pol(obtained by BglII/PvuII digestion of pMTT7N, asdescribed above) was subcloned downstream of RSV promoterat the cloning sites of BamHI and PvuII in the vector ofpEBVhis [Invitrogen].pEBVhisT7N, was transfected into 293 cells and selectedThe resulting plasmid, designated.,............‘,..................-...........-.....»...‘..m.M .‘ ....... vl . mWO 98/100881015202530CA 02264482 1999-03-03PCT/US97/1571616Eachpositive clone is analyzed for the presence of T7 Pol bywith Hygromycin at a concentration of 400 pg/ml.its ability to produce expression of T7-Lacz or T7-rep/cap in cells transfected with these plasmids.Example 3 — Production of T7 replcap AdenovirusThe production of this recombinant adenoviralvector is illustrated schematically in Figs. 2 and 3.A. Plasmid ConstructionThe plasmid pTM1 [B. Moss et al, Nature,;A§:91-92 (1990)], designed for expressing genes undercontrol of the T7 promoter/EMCV UTR (untranslated regionof encephalomyocarditis), was used as the vector forexpressing AAV rep/cap. The entire coding sequence ofrep/cap was separated into two portions by the uniqueSacI site and subcloned into the pTM1 plasmid asdescribed below.Because there is no appropriaterestriction enzyme existing between the initiation siteof rep and its natural promoter, p5, the left end of therep sequence (N-rep) was first amplified by PCR. Thesequence of the upper primer was SEQ ID NO:2:TATTTAAGCCCGAGTGAGCE (from position of 255 to 274) whichintroduced a nucleotide substitution A—>T at position 274(underlined). A SacI site was then generated to permitthe cloning of N-rep into pTM1 and in-frame expression ofRep protein from the EMCV UTR preferred initiation site(within the NcoI site). The PCR product (739 bp inlength) was directly cloned into pCR2.1 vector(Invitrogen) and named pCR—N—rep.The pTM-1 plasmid was digested with SacIand Stu I restriction enzymes and ligated with a 3.7 kbSacI/SnaBI fragment from psub201 [Samulski et al, Q;V'rol., §;:3096-3101 (1987)] containing the right end ofthe AAV genome (without ITR sequence), i.e., the c-1015202530WO 98/10088CA 02264482 1999-03-03PCT/US97/1571617terminal 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 wasremoved from the pCR-N—Rep plasmid by SacI digestion andsubcloned into pT7-C-rep/cap, which has similarly beendigested with SacI and subjected to alkaline phosphatasetreatment to prevent self-ligation of the vector. Thefinal construct was named pT7 rep/cap which contains thefull length coding sequence of rep/cap downstream of T7promoter/EMCV UTR, followed by the T7 terminatingsequence.B. Production T7 rep/cap AdenoviruspAd.link is a construct similar topAd.CMV.link, a plasmid containing the adenoviralsequences 0 to 16 map units deleted of Ela and Elb asdescribed in the other adenovirus vectors into which aCMV promoter-polylinker cassette was cloned and describedin X. Ye et al, J. Biol. Chem., ;1;:3639-3646 (1996).However, pAd.link contains no CMV promoter or polyA tailsequence.The entire region including the T7promoter, EMCV UTR, rep/cap and T7 terminating sequencewas excised from pT7 rep/cap by digestion with ClaI andEagI, and then subcloned into the adenoviral sequences ofpAd.link, which had previously been subjected toC1aI/SalI digestion, after filling in the sticky ends ofEagI and Sa1I by Klenow polymerase. The resultingplasmid is designated pAd.T7 rep/cap.The pAd.T7 rep/cap is co-transfected withthe ClaI linearized Ad.del327 backbone DNA into 293 cellfor the generation of T7 rep/cap adenovirus.W0 98/100881015202530CA 02264482 1999-03-03PCTIUS97/1571618Example 4 - Cell Line Expressing repzcgpA cell line stably transfected with pT7 rep/capis established by transfection of pMTCB6+ into 293 cellat ratio of 10:1 and selected with Geneticin. Each cloneis analyzed for the presence of rep protein bytransfection with T7 Pol expressing plasmid._;amole 5 - Production of Recombinant AAV Hybrid VectorPlasmid pAV.CMVLacZ serves as a template forrAAV to be replicated and packaged in the presence of AAVnon-structural and capsid proteins.Plasmid AV.CMVLacz is a rAAV cassette in whichrep and cap genes are replaced with a minigene expressingB-galactosidase from a CMV promoter. The lineararrangement of AV.CMVLacZ includes:(a) the 5' AAV ITR (bp 1-173) obtained by PCRusing pAV2 [C. A. Laughlin et al, gggg, ;_: 65-73 (1983)]as template [nucleotide numbers 365-538 of SEQ ID NO:1];(b) a CMV immediate early enhancer/promoter[Boshart et al, ggll, g;:521—530 (1985); nucleotidenumbers 563-1157 of SEQ ID N0:1],(c) an SV40 intron (nucleotide numbers 1178-1179 of SEQ ID N021),(d) E. coli beta-galactosidase CDNA(nucleotide numbers 1356 - 4827 of SEQ ID NO:1),(e)BamHI-BclI restriction fragment containing thean SV40 polyadenylation signal (a 237cleavage/poly—A signals from both the early and latetranscription units; nucleotide numbers 4839 - 5037 ofSEQ ID NO:1) and(f) 3'AAV ITR, obtained from pAV2 as a SnaBI-BglII fragment (nucleotide numbers 5053 - 5221 of SEQ IDNO:1).101520W0 98/ 10088CA 02264482 1999-03-03PCT/US97/1571619Where desired, the Lacz gene can be replacedwith a desired therapeutic or other transgene for theSee, Fig. 4. Thesequence including CMV directed Lacz reporter cassette inpurpose of generating new rAAV.between two AAV ITR sequences is excised frompAV.CMV.LacZ by PvuII digestion. This fragment isligated with the EcoRV treated pAd.1ink to generate theplasmid pAd.AV.CMVLacZ. This plasmid is co-transfectedwith ClaI linearized Addel327 backbone DNA to generate anadeno-rAAV hybrid virus.Example 6 — Cell line containing rescuable. integratedrAAV template293 cells are transfected/infected withpAV.CMVLacZ/rAAV Ad hybrid virus to generate cell linethat has incorporated rAAV, as determined by analysis ofthe genomic DNA by Southern blot.for the rescue of rAAV template by transfection/infectionThe clone is examinedwith rep/cap expressing constructs.Numerous modifications and variations of thepresent invention are included in the above-identifiedspecification and are expected to be obvious to one ofskill in the art.the processes of the present invention are believed to beSuch modifications and alterations toencompassed in the scope of the claims appended hereto.W0 98/ 10088CA 02264482 1999-03-03PCTIU S97/ 1571620SEQUENCE LISTING(1) GENERAL INFORMATION:(i)(ii)(iii)(iV)(V)(Vi)(vii)(viii)(ix)APPLICANT: Trustees of the University of PennsylvaniaWilson, James M.Chen, Nancie N.TITLE OF INVENTION: An Inducible Method for Production ofRecombinant Adeno-Associated viruses Utilizing T7PolymeraseNUMBER OF SEQUENCES: 2CORRESPONDENCE 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: 19477COMPUTER READABLE FORM:(A) MEDIUM TYPE: Floppy disk(B) COMPUTER: IBM PC compatible(C) OPERATING SYSTEM: PC-DOS/MS-DOS(D) SOFTWARE: Patentln Release #1.0, Version #1.3OCURRENT APPLICATION DATA:(A) APPLICATION NUMBER: wo(B) FILING DATE:(C) CLASSIFICATION:PRIOR APPLICATION DATA:(A) APPLICATION NUMBER: US 60/024,699(B) FILING DATE: O6-SEP-1996ATTORNEY/AGENT INFORMATION:(A) NAME: Xodroff, Cathy A.(B) REGISTRATION NUMBER: 33,980(C) REFERENCE/DOCKET NUMBER: GNVPN.O22CIP1PCTTELECOMMUNICATION INFORMATION:(A) TELEPHONE: 215-540-9200(B) TELEFAX: 215-540-5818(2) INFORMATION FOR SEQ ID NO:1:(i)(ii)SEQUENCE CHARACTERISTICS:(A) LENGTH: 10398 base pairs(8) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: unknownMOLECULE TYPE: CDNAWO 98110088CA02264482 1999-03-0321(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GAATTCGCTAGGGGGTGGAGGGCGGAAGTGTGACGTTTTTGGATGTTGTAGAATAAGAGGAGGGAGATCTGGGCGACCTTACTCCATCACTTCGAGCTTGCCGCCCAACGAIAGGGACTTGTACATCAAGCCCGCCTGGCTACGTATTAGGGATAGCGGTTTGTTTTGGCACGCAAATGGAACCGTCAGAGACCGATCCAGTTAACTGGTCAAATCAAAGTTACTTCTGCGAGCCTGCTATTTTCGTTGCATCCCGTCGTTTGCAGCACACTTCCCAACAAAGCGGTGCCCCTCAAACTGGCATCATCAATTTGTGACGTTGATGTTGCAGGTGTGCGCCGTAAATTTGGAAGTGAAATCGCTGCGCGCTTGGTCGCCCGTAGGGGTTCCCATGCCTGCAACCCCCGCCCTCCATTGACGTGTATCATATATTATGCCCATCATCGCTATTTGACTCACGACCAAAATCAGCGGTAGGCGTCGCCTGGAGGCCTCCGGACAAGTTTAGTCAACTGCTCCTTCTAAAAGCTAAGCAAAAAACGGTCTGGGATTTACAACGTTCCCCCTTTCGTTGCGCAGCGGAAAGCTGGGCAGATGCACTAATATACCTGGCGCGGGGCAGTGTGGCGGGGTGTACACAGCGTAACCGATGAATAATTTCGCTCGCTCAGCCTCAGTGATTGTAGTTAAGGTCGTTACAATTGACGTCATCAATGGGTGGCCAAGTACGGTACATGACCTACCATGGTGGGGATTTCCAACGGGACTTTTGTACGGTGGACGCCATCCATCTAGAGGATTTTTTGTCTTCAGTGGATGTGCGGAATTGTGAAGTCACCAGGCATTGGTCCGTGACTGGGGCCAGCTGGCCTGAATGGCGCTGGAGTGCGGGTTACGATGTATTTTGGATGTGGGAACGGAACACATGTAGGAAGTGACAGTAAGATTTGTGTGTTACTCCTGAGGCCGCGCGAGCGAGCTGATTAACCCTAACTTACGGATAATGACGTGAGTATTTACCCCCCTBTTGTTATGGGACTATGCGGTTTTAGTCTCCACCCCAAAATGTCGAGGTCTATACGCTGTTTTGCCGGTACTCGTTATTTCAGGTGCCTTTACTACCCGCGGCCTGTCGTTTACTGGACACCAGAAAACCCTGGGTAATBGCGAAATGGCGCTTATCTTCCTGACGCCCATCTATGAAGCCAATGGCGGGTGACAGCGACGGATATTTTCGCGCGCCATTTTCGATAGCGCGTACCGGGCAAAGGCGCAGAGAGGCCATGCTACTAAATGGCCCATGTTCCCBTGGTAAACTGCACGTCAATGATTCCTACTTGGGCAGTACATCCATTGACGTGTAACAACTCTAAGCAGAGCACCTCCATAGAGGAACTGAATCCCGGATCCTCTAGGCCTGGCAATTCCCGTTTGACCAACCAAGGAGCTGCGTTACCCAAAGAGGCCCGCTGCCTGGTTTGGCCGATACTCACCAACGTAPCT/US97/15716ATGATAATGA 60GTAGTAGTGT 120GTGGCAAAAG 180GGTTTTAGGC 240CGGGAAAACT 300ATATTTGTCT 360CCCGGGCGTC 420GGAGTGGCCA 480TTATCTACAA 540GCCTGGCTGA 600AGTAACGCCA 660CCACTTGGCA 720CGGTAAATGG 780GCAGTACATC 840CAATGGGCGT 900CAATGGGAGT 960CGCCCCATTG 1020TCGTTTAGTG 1080AAGACACCGG 1140AAACCAGAAA 1200GGTGGTGGTG 1260TACGGAAGTG 1320GGGATCGAAA 1380AAGAACGTGA 1440CTCAAGCGCG 1500CTTAATCGCC 1560ACCGATCGCC 1620CCGGCACCAG 1680GTCGTCGTCC 1740ACCTATCCCA 1800.,-.....h 1......-.......................w.,u......‘ .. .,... ,.W0 98/ 10088TTACGGTCAATTAATGTTGAACTCGGCGTTTGCCGTCTGATGGTGCTGCGGCATTTTCCGTTGCCACTCGGCGGCGAGTTTCGCCAGCGGATCGCGTCACATCTCTATCGCCTGCGATGTAGCCGTTGCTTGGATGAGCATGCGCTGTTCTGTATGTGGTCCGATGATCCATCGTAATCAATCACGACGCAAGGCGGCGGATGAAGACCACTGGAGAGACGCGGTTTCGCTCTGGGACTGCTTACGGCGGTCTTTGCCGATCCAGTTCCGGCGATAACGAAAGTGCCTCTAGCCGGAGAGCATGGTCAGATCCGCCGTTTTGAAAGCTGGTCATCTGTGGATTTGACCTGTTGGAGTGACTGACGTCTCGCTTTAATGATGCGTGACTACCACCGCGCCTACTACGTCTGTGCGGTGGTTCGGTTTCCGCGATTCGAGGCGACGATGGTGGCATTATCCGGGATGAAGCCGCGCTGGCTACCCGAGTGTGGCTGTATCGCAGCCGACACCGCCCTTCCCGGCGCCCGCTGTAAATACTGGGGTGGATCAGTGATTTTGGCCCGCBCGCCGTTTATCCGGGGCTCCTGCACGGATGTCGCTCGCCGGGCAAAGCCGGGCACCAGTTCCCACGGCTACAGGAAGTGCAACGGGCAGCGCATTTTGGCAGTTATCTTGCTGCATBGATTTCAGCCCTACGGGTAATTCGGCGGTGAACGTCGAAAGAACTGCACAGAGGTGCGGAGTTAACCGTCCAGGATATCCAACCATCCGCAATATTGAAACCGGCGATGAATCATCTGGTTGGATCAAATACGGCCACCGGCTGTGCCGAATCCTTTGCGCAGGCGTTTCTCGCTGATTAGATACGCCGACATCCAGCGCCAAACCATCGTGGATGGTGGCCACAAGGTACTCTGGCTCAATCAGCGCCT02264482 1999-03-0322AGAATCCGACGCCBGACGCGGCTGGGTCGGTACGCGCCGGTGGAAGATCAAACCGACTACGCGCTGTACTCAGTTTCTTTAAATTATCGAACCCGAAACTCCGCCGACGGTTGABAATGGACGAGCATCATGCTGATGABTGTGGTACACCCCACGGCATGCGAACGCGTCGCTGGGGAACTGTCGATCCATATTATTTGAATGGTCCATAATACGCCCAGTCAGTATCCAATATGATGAACGATCGCCATGACGGAAGCAAGTGACCAGCGCTGGATGGAACAGTTGATCAGTACGCGTGGCAGCAGTGGGGTTGTTACAATTBTTTTTTTACGGCCAGAGAAABCCGCGGATATGTGGACAAATCAGCGGAGGCTGAAATGGCAGGGTTGAGCGTGGTGTGGAGCGCCCACGCTGATTTCTGCTGCTGTCCTCTGCATGCAGAACAACGCTGTGCGACGGTGCCAATGAACGCGAATGTGAATCAGGCTTCCCGCCCGCCCGATGTACCAAAAAATGGCGCGATGGGTCCGTTTACAGAAACGGCAACGTTCTGTATGAAAACACCAGCGAATACCTGTAAGCCGCTGTGAACTGCCTAGTGCABCCGGCGTCTGGCGPCT/US97/15716TCGCTCACAT 1860GATGGCGTTA 1920GACAGTCGTT 1980CTCGCGGTGA 2040CGGATGAGCG 2100GATTTCCATG 2160GTTCAGATGT 2220GAAACGCAGG 2280GGTTATGCCG 2340GAAATCCCGA 2400GAAGCAGAAG 2460CTGAACGGCA 2520GGTCAGGTCA 2580TTTAACGCCG 2640CGCTACGGCC 2700BATCGTCTGA 2760GTGCAGCGCG 2820CACGGCGCTA 2880GTGCAGTATG 2940GCGCGCGTGG 3000CTTTCGCTAC 3060AACAGTCTTG 3120GGCGGCTTCG 3180CCGTGGTCGG 3240AACGGTCTGG 3300CAGCAGTTTT 3360TTCCGTCATA 3420GCAAGCGGTG 3480GAACTACCGC 3540AACGCGACCG 3600GAAAACCTCA 3660T IW0 98/ 10088GTGTGACGCTTTTGCATCGAAGATGTGGATCACCGCTGGATCGAACGCTGCAGATACACTAAACCTTATTCCGTTGATGTAGCTGGCGCACCGACCGCCTCCCCGTACGTGCCCACACCATGATGGAAACACGGTTTCCATACAGCTGAGCGGGCAGGCCACACAAACTTACTTCCCGTTGTATTATTTTTTTCTGACAAATTGATGAGTATTTGTGATGAACAATTGCACCTCTAGAGTAGTGATGGAGAAAGGTCGCCCAGATCTGGAGGCGGTAAACGATCACTTGGTGAGGTACTGCTTATGTAGTCCCCGCCGCGGCTGGGTAATTGGCGATAAATAACGACATTGAAGGCGGCGTGCTGATGCGTATCAGCCGGTGAAGTGGCGGGTAGCAGAGTACTGCCGCCCTTCCCGAGCGTGGCGCGGCCAGCCATCGCTATGGGGATTCGCCGGTCGCATGTCTGCCCTTGGATGTTCTTTCCCGATTTGCCGCTATTACTCGGCCTCTTGGACAAACCTATTGCTTTTTCATTTTATCGAGTAGATATTGGCCACTCCGACGCCCGGAGGTGCTGAGATATTAGGAATGCTGGCCTGAAATGTGTGGTTTGTATCTGCATCCCACGCCAAAGCGTTGGCAAACAACTGCGGCGTAAGTGGGCCATTACCGTGCTGATTAAAAACCTACCAGCGATACACCGGGTAAACTTGTTTTGACCGAAAACGGTCGACTTCCAGTCATCTGCTGCGGTGGCGACGTACCATTACCGTATTTCGCGGGTTTATTCTTGGCTACATGTCTCTGTTCTGACTCTAGGCCACAACTAGAATTTGTAACCGTTTCAGGTTAGTAGCATGGCCTCTCTGCGGCTTTGCCCGGTACGATGAGCCAGCCTGTGCACCCGCGCTGCGTGGCTTATTTTGCAGCA02264482 1999-03-0323TCCCGCATCTAATTTABCCGTGACGCCGCTAAGCGACCCGAGGCCGAAGCCGACCGCTCAGGATTGATGGCGCATCCGGCGGCTCGGATTGCTGGGATCTTGCGCTGCGGTCAACATCAGACGCGGAAGAACTCCTGGAGAGTTGGTCTGTAAGGAAATCTTTTCTTTTAACATCAACCACGCTATTATTGGCCGCGGGGATGCAGTGAAATTATAAGCTCAGGGGGAGGCGGGTTAATCCGCTCGCTCGGGCGGCCTCAACCCGCACCAATGCTGGATGGAGTTTGGCTAGGGTGGGAAGCCGCCGCCGGACCACCAGCCCAGTCAGGCGCGCGATCBGCATTGACCCTAGCGTTGTTGCGCGTGGCAGTAGTGGTCBAGCGGATTGGCAGGGCCGCAAGCCATTGTCAGACGCGCGAACCGCTACAGTAGGCACATGGCCCGTCAGTBGTGTCAAAAACATTATGTACCTTTTTTATCTATCAGCAAACCAACCGCTGATCCAGACATAAAAATGCTTGCAATAAACATGTGGGAGGTATTAACTACACTCACTGAGGGTGAGCGAGCGGTGCAGACCTGACCGAGGACTAGCGATGAAGAATATATACCATGAGCACPCT/US97/ 15716GAAATGGATT 3720TTTCTTTCAC 3780TTCACCCGTG 3840AACGCCTGGG 3900CAGTGCACGG 3960CATCAGGGGA 4020ATGGCGATTA 4080CTGAACTGCC 4140GAAAACTATC 4200GACATGTATA 4260TTGAATTATG 4320CAACAGCAAC 4380CTGAATATCG 4440TCGGCGGAAT 4500TAATAATAAC 4560TATTTAAAAA 4620ATGGGAGCCT 4680AGTGATACGG 4740TTTGGTCTGC 4800GATAAGATAC 4860TATTTGTGAA 4920AGTTAACAAC 4980TTTTTCGGAT 5040AGGAACCCCT 5100CCGGGCGACC 5160GAGCGCGCAG 5220CTGCGAGTGT 5280GCTGAGGCCC 5340AGATACAGAT 5400BGGTGGGGGT 5460CAACTCGTTT 5520 WO 98/10088GATGGAAGCACAGAATGTGATTGACCTACGGCCGCTGCAGAGCAGTGCAGTTGGATTCTTCAGGTTTCTGccacacrcracccccccccrAGGACGTGGTTGGAGGTAGCTAGCAGGAGCcqcacccccrGATATGAGATCGGGGATTCATCATGTAGCTTTTTCCATGCATATTTCTGGTTTACAAAGCGCGTAGTTACTCTACCTGCGAGCAGGTTCCACCGGGTGCAACTTCGTTAATCGCCGCCCATCCGCCGTAGGTCACCTGCTTTTCGCTGTAGGCGCAGGGTcccrccccacCCTGCGCGTCTTGTGAGCTCTGGGCTCCAGAGACCGTGTCCCACCGCCCGCTTCCCGTTCTGACCCGGGACCCTGAAGGCTTTGGATTTGAGGCCCGGGABAAGGTGACTACCACTGCAGGCTGGGCGTGTGGTGTAAGTGCATCTTGGATGTTGTGCAGTAGAAGGAAAATTCGTCCATGATCACTAACGCGGGCGGAGCCTCACAGATGGGCGATGAATGAGCAGCTGBCTGGTAGTTGCATGTCCCTGCGATAGCBGGCATGCTTTTCTACGGCATCCGGCAGTAGTCCTCGTCAGCGGTGCGCTTGGGCCAGGTAGCAATATTTGACBCATTGATGGTTGGAACGCCGCGGGATTGTGATCCGCCCGCACTTAATGTCTTCCTCCCCTGATCAAGCAACCAGCGGTCTCTGGATGTTCAGCTTCATGCGTGCCTAAAAGTTTACAAAGCTGTATTTTTAACCACCAGCTGCGTGGAAGAATGATGGCAGTCATAGTTGGGTGCCAGACTTGCATTTCCGAAAACGGTTCGACTTACCGAAGAGAGCTGGACTCGCATGTTCTTGCAAGGAGCGTTTGATCGATCCBGCCGGTGCTCGTGTAGTCTGGGAGGCTGGTCCCATTTGACCA24ACGCGCATGCCGCCCCGTCCTTGGAGACTGACTGACTTTGGATGACAAGTGTTTCTCAGCCCCAATGCGGGTGTCTTGCTCGGTCGTTGAAGATACATGGTGCGGGGTGGATGTCTTTCACGGTTAAGCTAGGTTGGCTAACAGTGTATCAACTTGGAGAATGGGCCCACTGTTCCAGGATGCGGTATAACACGCTTTGATCCGGGGTAGCAGCCGGTGGCAGCTGCCGTTTTTCCCTGAGAAGCAAAGTCCAAGCAGTTATATCTCCTCCCAGACGGGCTCACGGTGAATGCTGGTGCTTGGTGTCATA02264482 1999-03-03CCCCATGGGCTGCCCGCAAACAGCCTCCGCCTTTCCTGAGTGACGGCTCTAGCTGTTGGATTTAAAACATGTCTTTATTTGGGTCCTGTGGCATBAGCCCTGTTGTAGATGTAGCAAGCTGGGATGGGTGTGTTCCCAGCCGGTGCACTTCGCCCTTGTGGGGCGGCGGCTGAGATCGTCTGGTTCCATCGTTCAGATGGGGGAGATCAGGCCCGTAAATCATCCCTGAGCCAAATCCGCTTTTCAACGGCCAGGCGGTCGTTTCGCGGGCAGGGTCATGGGGGTGCGCTGAAGCGCTGCGTCCAGCCCCPCT/US97/ 15716CGGGGTGCGT 5580CTCTACTACC 5640CGCCGCTTCA 5700CCCGCTTGCA 5760TTTGGCACAA 5820TCTGCGCCAG 5880AAATAAAAAA 5940AGGGGTTTTG 6000TATTTTTTCC 6060GTCTCTGGGG 6120GATCCAGTCG 6180GATTGCCAGG 6240CATACGTGGG 6300CATATCCCTC 6360GGGAAATTTG 6420ACCTCCAAGA 6480CTGGGCGAAG 6540ATAGGCCATT 6600CGGCCCAGGG 6660GGGGATCATG 6720CTGGGAAGAA 6780CACACCTATT 6840CAGGGGGGCC 6900CAGAAGGCGC 6960TTTGAGACCG 7020CCACAGCTCG 7080TTGGGGCGGC 7140TCTTTCCACG 7200CCGGGCTGCG 7260CGGTCTTCGC 7320TCCGCGGCGT 7380WO 98/10088GGCCCTTGGCTGAGGGCGTAAGGCCCCGCAAAACCAGGTTGTCCACGCTCCCTCGACCGAATGACTATCGCCGGCAGCGCGGCCTGTCGCCCCGCCACCACTGGGCTACGCTTCTCGCTTGATGACGACCATCACTGGACTTGGCATGGAGCATGGAGCCCCACTCCAAGCTTGGCAGAAGCAGCGTTGGTAGGCTGGCGGAAGCGACTGTCCGTGTTTCTCTGCATCGCCTTTCTCAATGGCTGTGTGCCTTGAGTCCAATTAGCAGAGGGCTACACTAAAAAGAGTTGGTTTGCAAGCTCTACGGGGTGCGCAGCTTGGAGCTTGGGCGACGGTCTCGTCCCCCATGCGGTGACGAAATGCCCTTGAGTCGCCGCACTTCTGGGTCATTTGCGGTATTAACGTTTCGGTCTTGCTGGCCCGGCGGCATATCAGGGACACGCTGATCGTTTGTAGGCGCGGGCCACCTCAATTGGAGCCCATATCCATCGTCCTGGCCAGGGTTGCCTTCTGCTGCAAAGTAAAGTCTGAGGATGCTGCGCTCACGCTGACGAACCCCCACCCGGTAAGCGAGGTATGTGAAGGACAGTGTAGCTCTTGAGCAGATTACCTGACGCTCACACCCTTGGAGGGCGAGAAATACATTCCACGATTTTTGATGCAGGCTGTCCGAGCCTTCAACTATGACTGTCTTTCGGCGAGCGGAATCTTGCGAGAAGCAGGTTCGCGACGCGGGATGCCCGCTTCAAGGACACGGCGATTCGCCCTATACGACCTGAATGAATCAATTCTGCGTCCGCCACGGGTGCGCAACTGGTTAGCACGTCTGCGAGAAACGCGGATGGCTACCCTTAGGTATCTCCGTTCAGCCCACACGACTTAAGGCGGTGCTATTTGGTATCATCCGGCAAAGCGCAGAAAAGTGGAACGAA02264482 1999-03-0325AGGCGCCGCACCGATTCCGGGCCAGGTGAGGTTTCTTACCTGTCCCCGTACCAGTCAGCTTTCTTTATCBGACCGCTTTCCACGCCCTCGGCCATTATCGCGAGGCTGGAGCGTTGCAGGTCGCTCGCGGTATGCCGCCTCTTGTCTGCCGAAGCCGGCGTGCGGAGAACTCTCCAGCAGTGATCGTGCTAGAATGAATCCCTGAGCAACAGTCAGCGCCGTGGAACACCAGTTCGGTGTGACCGCTGCGTCGCCACTGGACAGBGTTCTTGCGCTCTGCCAAACCACCGAAAGGATCTCAACTCACGTTCGAGGGGCAGGGAGTAGGCACTCTGGCCGTTCTGGTTTCCTACAGACTTGCCTTCCGGTGTGCAACTCGTGCTGGAGCGCCTCAAGCCTTCCGGCATGGCTGGCCTTCCCCCATGCTGTCCTCTTACCAGCGGCGAGCACTCCCCGCGTTGCACCTCGCTTGTGAATGCGCCGCACGCGGCCTGTCGTTGACCGATACGCAACATGAATGCTGCACCATTTACATCTGTAAGGTCGTTCGCCTTATCCGGCBGCAGCCACTGAAGTGGTGTGAAGCCAGTCTGGTAGCGGAAGAAGATCCAAGGGATTTTI’CT/US97/ 15716TGCAGACTTT 7440TCCGCGCCGC 7500TCGGGGTCAR 7560ATGAGCCGGT 7620AGAGGCCTGT 7680GGCGCGGGGC 7740AGGACAGGTG 7800GACGATOATC 7860CGTCACTGGT 7920GGCCGACGCG 7980CATTATGATT 8040CAGGCAGGTA 8100CCTAACTTCG 8160ATGGAACGGG 8220GCGTCGCGGT 8280AACGGATTCA 8340CAAACCAACC 8400CGCATCTCGG 8460AGGACCCGGC 8520GAGCGAACGT 8580GTCTTCGGTT 8640ATGTTCCGGA 8700TTAACGAAGC 8760CTCCAAGCTG 8820TAACTATCGT 8880TGGTAACAGG 8940GCCTAACTAC 9000TACCTTCGGA 9060TGGTTTTTTT 9120TTTGATCTTT 9180GGTCATGAGA 9240WO 98/10088TTATCAAAAATAAAGTATATATCTCAGCGAACTACGATACCGCTCACCGGAGTGGTCCTGGTAAGTAGTTGTGTCACGCTGTTACATGATGTCAGAAGTACTTACTGTCATTCTGAGAATACCGCGCCACAAACTCTCAAABCTGATCTTCAAAATGCCGCTTTTTCAATGAATGTATTTCCTGACGTCTAGGCCCTTTCGGATCTTCACATGAGTAAACTCTGTCTATTGGGAGGGCTTCTCCAGATTTCAACTTTATCCGCCAGTTAACGTCGTTTGGCCCCCATGTTAGTTGGCCGCTGCCATCCGTAGTGTATGCGATAGCAGAACGGATCTTACCCAGCATCTTTCAABAAAGGGATTATTGAAGAGAAAAATAAAAGAAACCATGTCTTCAACACTAGATCCTTTTGGTCTGACTCGTTCATCCACCATCTGGCATCAGCAATACGCCTCCATCTAGTTTGCGCTATGGCTTCAGTGCAAAAAAAGTGTTATCAAAGATGCTTTGCGACCGAGTTTTAAAAGTGGCTGTTGAGATACTTTCACCAATAAGGGCGCATTTATCAGACAAATAGGGTATTATCATG(2) INFORMATION FOR SEQ ID NO:2:02264482 1999-03-0326TTAAATTAAAAGTTACCAATATAGTTGCCTCCCAGTGCTGABCCAGCCAGCBGTCTATTAAACGTTGTTGTTCAGCTCCGGCGGTTAGCTCTCATGGTTATCTGTGACTGTGCTCTTGCCCTCATCATTGTCCAGTTCGAAGCGTTTCTGACACGGBAATGGTTATTGTCGTTCCGCGCAACATTAACCT(i) SEQUENCE CHARACTERISTICS:(A)(B)(C)(13)(ii) MOLECULE TYPE: other nucleicTYPE:unknownLENGTH: 20 base pairsnucleic acidSTRANDEDNESS: singleTOPOLOGY:acid(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:TATTTAAGCC CGAGTGAGCTAATGAAGTTTGCTTAATCAGGACTCCCCGTCAATGATACCCCGGBAGGGCATTGTTGCCGCCATTGCTGCGTTCCCAACGCCTTCGGTCCTGGCAGCACTGTGAGTACTCCGGCGTCAACGAAAACGTTCTGTAACCCACGGTGAGCAABGTTGBATACTTCATGAGCGGCATTTCCCCGATAAAAATAGPCT7US97HS7l6TAAATCAATC 9300TGAGGCACCT 9360CGTGTAGATA 9420GCGAGACCCA 9480CGAGCGCAGA 9540GGAAGCTAGA 9600AGGCATCGTG 9660ATCAAGGCGA 9720TCCGATCGTT 9780GCATAATTCT 9840AACCAAGTCA 9900ACGGGATAAT 9960TTCGGGGCGA 10020TCGTGCACCC 10080AACAGGAAGG 10140CATACTCTTC 10200ATACATATTT 10260AAAAGTGCCA 10320GCGTATCACG 103801039820T I"W

Claims (26)

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 5' 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.
CA002264482A 1996-09-06 1997-09-04 An inducible method for production of recombinant adeno-associated viruses utilizing t7 polymerase Abandoned CA2264482A1 (en)

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