CA2265522A1 - Retroviral vectors capable of transducing non-dividing cells - Google Patents

Abstract

In accordance with the present invention, methods have been developed to modify retroviral vectors derived from viruses which are not known to be pathogenic in humans (e.g., MLV), so that such vectors are rendered capable of transducing heterologous sequences into non-dividing cells. Thus, it has been discovered that retroviruses can be rendered capable of infecting non-dividing cells by introducing into the viral particle one of several specifically defined modifications. For example, an element which is recognized by the nuclear import machinery of a non-dividing cell can be associated with the nucleoprotein complex of the retrovirus. Alternatively, at least one protein encoded by viral gag or pol nucleic acid is modified so as to be recognized by the nuclear import machinery of a non-dividing cell.

Description

?W0 98/12314101520CA 02265522 l999-03- l5PCT/US97/15934Retroviral Vectors Capable of TransducinqNon—dividing CellsFIELD OF THE INVENTIONThe present invention relates generally to thefield of viral vectors. aspect, theinvention relates to novel recombinant retroviruses whichIn a specificare useful for the transfer and expression of nucleic acidsequences in non—dividing cells.BACKGROUND OF THE INVENTIONThe development of genetic vectors has heraldedthe fast—growing field of somatic gene transfer. Vectorsbased on simple retroviruses, such as the Moloney Leukemia(MLV) ,integrate into the genome of the target cell.Virus are often selected because they efficientlyIntegrationis thought to be a prerequisite for long-term expression ofthe transduced gene. However,currently availableretroviral vectors can only integrate in actively dividingcells. This feature severely limits the use of retroviralvectors for in vivo gene transfer. Non—dividing cells arethe predominant, long—lived cell type in the body,andaccount for most desirable targets of gene transfer,including liver, muscle, and brain. Even protocolsattempting the transduction of hematopoietic stem cellsrequire demanding ex vivo procedures to trigger division ofthese cells prior to infection.?W0 98ll23141015202530CA 02265522 l999-03- l5PCT/US97/ 159342In the early steps of infection, retrovirusesdeliver their nucleoprotein core into the cytoplasm of thetarget cell.Here, reverse transcription of the viraltheThe complex must reach the nucleusto achieve integration of the viral DNA into the host cellchromosomes.genome takesplace while core matures into apreintegration complex.For simple retroviruses (oncoretroviruses),this step requires the dissolution of the nuclear membraneat mitotic prophase, most likely because the bulky size ofthe preintegration complex prevents its passive diffusionthrough the nuclear pores.While retroviral vectors are useful for manykinds of in vitro gene transfer studies, problems includinglow titers limit their use for some in vitro and most inyiyg studies. Further, another problem is that integrationof retroviral vectors into the host genome was thought toThus,although retroviral vectors capable of infecting a broadbe restricted to cells undergoing DNA replication.class of cell types are known, cell division is necessaryThisretrovirusfor the proviral integration of these vectors.the efficient use ofThus,been utilized to introduce genes into non—dividing or post-effectively restrictsvectors to replicating cells. retroviruses have notmitotic cells.HIV and otherlentiviruses have the ability to infect non—dividing cells,In contrast with oncoretroviruses,such as terminally differentiated macrophages and quiescentT lymphocytes. This property is likely crucial for HIVtransmission, spread and persistence in the body, as wellas for AIDS induction.(MA)HIV-1 nucleoprotein complex (NPC) through the nucleopore ofMatrix and. Vpr govern. the import of thethe target cells in the absence of the breakdown of thenuclear envelope, allowing integration of the viral genome?WO 981123141015202530CA 02265522 l999-03- l5PCTlUS97/ 159343into the host cell chromosome. MA has intrinsickaryophilic properties conferred. by a highly conservedstretch of basic which nuclearAt the time of virus assembly,residues acts as alocalization signal (NLS).a subset of MA molecules undergo phosphorylation on theirC-terminal tyrosine. Tyrosinenphosphorylated MA then binds(IN) and becomes a component of the NPC inwhich its karyophilic potential is revealed by interactingwith one component of the cell nuclear import machinery,karyopherin a.to integraseVpr is another component of the viral NPC thatexhibits karyophilic properties. However, Vpr does notcontain a canonical NLS. Instead, the domain critical fornuclear localization of Vpr has recently been mapped to thetwo putative N—terminal a—helices thereof.therefore play a crucial roleVpr couldin HIV-1 nuclear importthrough a pathway independent from MA; an NLS—independentpathway.In corroboration of this hypothesis, it has beenfound that NLS peptide affects MA, but not Vpr,localization,nuclearand interferes with MA, but not Vpr—mediatedHIV-1 nuclear import1Q:lO27—l032 (1996)).distinct yet convergent pathways most probably ensures the(see Gallay et al. in J. VirologyThe availability of these twoability of HIV-1 to infect non—dividing cells under avariety of conditions.Mutant viruses lacking both MA NLS and Vpr cannotgrow efficiently in terminally differentiated macrophages.In addition, an.2HIV~1—based. vector containing the samemutations displayed lower ig_y;yg levels of transduction ofin nonmitotic such asheterologous sequences Cells.neurons, compared with the wild—type vector, confirming thekey roles of MA and Vpr in HIV-1 infection of non-dividingcells.?WO 981123141015202530ICA 02265522 l999-03- 15PCT/US97/ 159344A retroviral vector system was recently derivedfrom HIV (Naldini et al., Science g1g:263—267 (1996)). Incontrast to a murine leukemia virus—based counterpart, theHIV vector could transduce heterologous sequences into avariety of growth-arrested cells in vitro, as well as intoAdditionally, the HIV vectorcould mediate stable in vivo gene transfer into terminallydifferentiated neurons. The ability of HIV—based viralvectors to deliver genes ig_y;yg into non—dividing cellshuman primary macrophages.could increase the applicability of retroviral vectors ingene therapy. However, a major limitation of these vectorsis in their potential biohazard, as HIV is a major pathogenin humans.Accordingly, there is still a need in the art forimproved methods for the introduction of nucleic acids intonon—dividing cells. In addition, retroviral constructs areneeded which facilitate such methods.BRIEF DESCRIPTION OF THE INVENTIONIn accordance with. the present invention, theabove—described need in the art has been satisfied by thedevelopment of methods to modify retroviral vectors derivedfrom viruses which are not known to be pathogenic in humans(e.g., MLV),transducing heterologous sequences into non~dividing cells.Thus,renderedso that such vectors are rendered capable ofit has been discovered that retroviruses can. bebyseveralcapable of infecting non—dividing cellsthespecifically defined modifications.introducing into viral particle one ofFor example, anelement which is recognized by the nuclear import machineryof a non—dividing cell and which can associate with thenucleoprotein complex of the retrovirus can be introducedinto a retrovirus.?WO 98/123141015202530CA 02265522 l999-03- l5PCT/US97/159345In accordance with the present invention, it hasbeen discovered.that integrase (IN), the enzyme responsiblefor inserting the viral DNA into the host cell chromosomes,appears to play a dual role in HIV-1 infection ofnon-dividing cells. First, by binding to the C—terminalphosphotyrosine of matrix protein (MA), IN mediates theincorporation of the karyophilic properties of MA into theHIV-1 nucleoprotein complex (NPC). Second, IN facilitatesthe migration of the viral genome to the nucleopore byinteracting with one component of the cell nuclear importmachinery, karyopherin a (presumably through the INNLS(s)). Thus, IN has been identified. as a. preferred"element" for use in the practice of the present invention.In another embodiment of the present invention,at least one protein encoded by viral gag or pol nucleicacid is modified so as to be recognized by the nuclearimport machinery of a non-dividing cell.BRIEF DESCRIPTION OF THE FIGUREFigure 1 illustrates the ability of HIV-1integrase to enhance the transduction of non-dividing cellsby an MLV vector.DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, thereare provided recombinant retroviruses capable of infectingnon-dividing cell(s). Invention retroviruses comprise thefollowing components:a viral GAG,a viral POL,a viral ENV,an element which associates with thenucleoprotein complex of said retrovirus,wherein said element is recognized by the?ICA 02265522 l999-03- 15W0 98ll23l4 PCT/US97Il59346nuclear import machinery of said non-dividing cell,an heterologous nucleic acid operably linked toa regulatory sequence, and5 cis—acting‘ nucleic acid. necessary for reversetranscription and integration, andoptionally, packaging of said retrovirus.In accordance with another embodiment of thepresent invention, there are provided viral particles10 produced by the above—described recombinant retroviruses.In accordance with yet another embodiment of thepresent invention, there are provided methods of producingthe above—described recombinant retrovirus(es). Inventionmethods comprise transfecting a suitable packaging host15 cell with one or more vectors comprising:a nucleic acid encoding a viral gag,a nucleic acid encoding a viral pol,a nucleic acid encoding a viral env,a nucleic acid encoding an element which20 associates with the nucleoproteincomplex of said retrovirus, whereinsaid element is recognized by thenuclear import machinery of said non-dividing cell, and25 a nucleic acid encoding a packaging signalflanked by cis—acting nucleic acidsnecessary for reverse transcription andintegration, and. a cloning site forintroduction of a heterologous nucleic30 acid, operably linked to a regulatorynucleic acid.Optionally, invention method further comprisesrecovering the recombinant virus produced by the above-described transfected host cell.?W0 98/121514101520253035CA 02265522 l999-03- l5PCT/U S97/ 159347Retroviruses are RNA viruses wherein the viralgenome is RNA. When a host cell is infected with aretrovirus, the genomic RNA is reverse transcribed into aDNA intermediate which is integrated very efficiently intothe chromosomal DNA of infected cells. This integrated DNAintermediate is referred to as a provirus. Transcriptionof the provirus and assembly into infectious virus occursin a cell line containing appropriate sequences enablingencapsidation, or, if necessary, in the presence of anappropriate helper virus. A helper virus is not requiredfor the production of the recombinant retrovirus of thepresentinvention, since thesequences required forencapsidation are co—transfection withprovided byappropriate vectors.The retroviral genome and the proviral DNA havethree genes: the ggg, the pgl, and the egg, which areflanked by two long terminal repeat (LTR) ?equences. Theggg gene encodes the internal structural (matrix, capsid,and nucleocapsid) proteins; the pg; gene encodes integraseand the RNA—directed.DNA polymerase (reverse transcriptase)and the egg gene encodes viral envelope glycoproteins. The5’ and 3’ LTRs serve to promote transcription and p-The LTRs contain allother g;§—acting sequences necessary for viral replication.olyadenylation of the virion RNAs.Lentiviruses have additional genes including vif, vpr, tat,rev, vpu, nef, and vpx (in HIV—1, HIV-2 and/or SIV).Adjacent to the 5' LTR are sequences necessaryfor reverse transcription of the genome (the tRNA primerbinding site) and for efficient encapsidation of viral RNAinto particles (the Psi site). If the sequences necessaryfor encapsidation (or packaging of retrovirual RNA intoinfectious virions) are missing from the viral genome, theresult is 21 gig defect which prevents encapsidation ofgenomic RNA. However, the resulting mutant is stillcapable of directing the synthesis of all virion proteins.?W0 98/12314101520253035ICA 02265522 l999-03- 15PCTIU S97/ 159348Invention retroviruses can be produced. by avariety of methods, e.g., by transfecting a suitable hostcell with one or more vectors comprising:a nucleic acid encoding a viral gag;a nucleic acid encoding a viral pol;a nucleic acid encoding a viral env;anucleic acid encoding an element whichassociates with the nucleoprotein complex of saidretrovirus, wherein said element is recognized by thenuclear import machinery of said non—dividing cell, anda nucleic acid sequence encoding a packagingsignal flanked by gi§—acting nucleic acid sequences forreverse transcription and integration, and. providing acloning site for introduction of a heterologous gene,operably linked to a regulatory nucleic acid sequence.A presently preferred method for the productionof retroviruses according to the invention involves the useof a combination of a minimum of four vectors in order toproduce a recombinant virion or recombinant retrovirus. Afirst vector provides a nucleic acid encoding a viral gagand a viral pol. These sequences encode a group specificantigenq reverse transcriptase, integrase ammi protease-enzymes necessary for reverse transcription, integrationand maturation. Such sequences can be obtained from avariety of viral sources, e.g.,Moloney murine leukemiavirus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), gibbon ape leukemiavirus (GaLV), human immunodeficiency virus (HIV), RousSarcoma Virus (RSV), and the like.A second vector employed in the practice of thepresent invention provides a nucleic acid encoding a viral(env).envelope The env gene can be derived from anyvirus, including retroviruses. The env may be amphotropicenvelope jprotein which allows transduction. of cells ofhuman and other species, or may" be ecotropic envelope?CA 02265522 l999-03- 15W0 98/ 12314 PCT/US97/ 159349protein, which is able to transduce only mouse and ratcells. Further, it may be desirable to target therecombinant virus by linkage of the envelope protein withan antibody or a particular ligand for targeting to a5 receptor of a particular cell—type. By inserting asequence (including regulatory region) of interest into theviral vector, along with another gene which encodes theligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral10 vectors can. be made target specific by inserting, forexample, a glycolipid, or a protein. Targeting is oftenaccomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or canreadily ascertain without undue experimentation, specific15 methods to achieve delivery of a retroviral vector to aspecific target.Examples of retroviral—derived env genes includeMoloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV),20 gibbon ape leukemia virus (GaLV), human immunodeficiencyvirus (HIV), Rous Sarcoma Virus (RSV), and the like. Otherenv genes such as Vesicular stomatitis virus (VSV) (ProteinG) can also be used.The vector providing the viral env nucleic acid25 sequence is operably associated with regulatory sequence,g;g., a promoter or enhancer. Preferably, the regulatorysequence is a viral promoter. The regulatory sequence canbe any eukaryotic promoter or enhancer, including forexample, the Moloney murine leukemia virus promoter-30 enhancer element, the human cytomegalovirus enhancer (asused in the illustrative example), or the vaccinia P7.5promoter. In some cases, such as the Moloney murineleukemia virus promoter—enhancer element, these promoter-enhancer elements are located within or adjacent to the LTR35 sequences.?W0 98ll23l41015202530ICA 02265522 l999-03- 15PCT/U S97/ 1593410A third vector contemplated for use in thepractice of the present invention provides the cis—actingviral sequences necessary for the viral life cycle. Suchsequences the reversetranscription signals, integration signals, viral promoter,include w packaging sequence,enhancer, and polyadenylation sequences. Such sequencescan be obtained from a variety of viral sources, e.g.,MoMuLV, Hamusv, MuMTV, GaLV, HIV, RSV, and the like.The third vector also contains a cloning site forThe(either containing the heterologous nucleicintroduction of a heterologous nucleic acid sequence.cloning siteacid sequence therein, or absent any insert therein) canthen be transferred to a non—dividing cell. Theheterologous nucleic acid sequence is typicallyincorporated into this vector prior to incorporation ofThus,example, this third vector can be used for the preparationproducts encoded thereby into a viral particle. forof a viral particle containing the heterologous nucleicacid sequence, thereby facilitating the direct introductionof the heterologous nucleic acid sequence into anon—dividing cell.A fourth vector contemplated. for use in thepractice of the present invention provides the elementwhich associates with the nucleoprotein complex of saidretrovirus.For example, as illustrated in Example 8,_HIV—l integrase is expressed under the control of the HIV-1promoter.Since recombinant retroviruses produced bystandard methods in the art are defective, they requireassistance in order to produce infectious vector particles.Typically, this assistance is provided, for example, byusing a helper cell line that provides the missing viralfunctions. a nucleotideThese plasmids are missingsequence which enables the packaging mechanism to recognize?W0 98/123141015202530, .........._......................._..———..-..............._ ..... MCA 02265522 l999-03- l5PCT/US97/159341 J.an RNA transcript for encapsidation. Examples of helpercell lines which have deletions of the packaging signalinclude T2, PA3l7, PA12, and the like. Suitable cell linesproduce empty virions, since no genome is packaged. If aretroviral vector is introduced into such cells in whichthe packaging signal is intact, but the structural genesare replaced by other genes of interest, the vector can bepackaged and vector virion produced.The invention method for ofrecombinant retrovirus employs different constructs thanare employed in the standard helper virus/packaging cellline method described above.productionThe four or more individualvectors used to co~transfect a suitable packaging cell linecollectively contain all of the required genes forproduction of a recombinant virus for infection andtransfer of nucleic acid to a non—dividing cell.Consequently, there is no need for use of a helper virus.As readily recognized by those of skill in theart, a variety of different packaging cell lines can beprepared in accordance with the present invention. Thus,for example, a stable packaging cell line containingseveral of the above—described vectors can be prepared,such that one only need introduce a vector containing theheterologous nucleic acid sequence in order to produce avirion which is capable of infecting non—dividing cells andhence nucleic aciddelivering heterologous sequencesthereto.Thus,the invention,in accordance with another embodiment ofthere are provided stable packaging celllines containing:a nucleic acid encoding a viral gag,a nucleic acid encoding a viral pol,"a nucleic acid encoding a viral env, and?CA 02265522 l999-03- 15WO 98112314 PCT/US97/159341 2a nucleic acid encoding an element whichassociates with the nucleoprotein complex ofa retrovirus, wherein said element isrecognized by the nuclear import machinery5 of a non-dividing cell.1015202530The above—described cell lines are especially useful forthe rapid introduction of heterologous nucleic acidsequences into a host. Thus, a vector encoding:a nucleic acid encoding a packaging signalflanked by cis—acting nucleic acidsnecessary for reverse transcription andintegration, and.a heterologous nucleic acidoperably linked to a regulatory nucleic acidis introduced into the above—described stable packagingcell thenparticles. The resulting viral particles are useful forline, which is induced to produce viralthe introduction of heterologous nucleic acid sequencesinto non-dividing cells.In accordance with yet another embodiment of theinvention, there are provided stable packaging cell linescontaining:a first nucleic acid encoding a viral gag,a second nucleic acid encoding a viral pol,wherein at least one protein encoded bysaid first or second nucleic acid ismodified so as to be recognized by thenuclear import machinery of a non-dividingcell,a nucleic acid encoding a viral env.andThe above—described cell lines are similarly useful for therapid introduction of heterologous nucleic acid sequencesinto a host, as described hereinabove.?W0 98/12314101520253035~« CA 02265522 l999-03- l5PCT/US97/15934l 3Elements which associate with the nucleoproteincomplex of a retrovirus (either directly or indirectly)Iand which are recognized by the nuclear import machinery ofa non-dividing cell, as contemplated for use in thepractice of the present invention include viral proteinswhich are recognized by the nuclearmachinery of a non-dividing cell, such as, for example,matrix protein (MA), integrase (IN), and the like, as wellas viral proteins which are indirectly recognized by thenuclear import machinery of cell (byassociating with the nucleoprotein complex and an agentwhich is recognized by the nuclear import machinery of acell), reverse(RT), and the like.A presently preferred viral protein contemplated for useherein is a lentiviral integrase, with HIV integrase beingparticularly preferred.directly importa non-dividingnon-dividing such as, for example,transcriptase nucleocapsid, protease,Additional elements contemplated for use in thethesignals,practice of nuclearincludesaid NLS is operablyassociated with the nucleoprotein complex of a retrovirus.Those of skill identify NLSssuitable for use herein. See, the numerousNLS sequences described by Dingwall and Laskey in TIBS;§:478—481 (1991) and Goerlich and Mattaj_2__'Z;:l5l3—l5l8 (1996).derived from HIV-1exhibitspresent inventionlocalization whereinin the art can readilyfor example,in Sciencea suitable NLS can beAnother protein thatand hence isFor example,integrase.karyophilic properties, usefulherein, is Vpr. In addition, consensus NLSS have also beenidentified in the art, characterized as comprising acontiguous sequence of seventeen amino acids, wherein thefirst two amino acids are basic amino acids, followed by aspacer region of any ten amino acids, followed by a basiccluster in which at least three of the next five aminoacids are basic.?W0 98/123141015202530CA 02265522 l999-03- l5PCTIUS97/1593414In addition, numerous specific NLSS have beenidentified in the art, e.g.:the amino acid sequence KRKQ (SEQ ID NO:l),the amino acid sequence KELQKQ (SEQ IDNO:2),the amino acid sequence KRKGGIG (SEQ IDNO:3),the amino acid sequence PKKKRKV (SEQ IDNO:4),the amino acid sequence AAFEDLRVLS (SEQ IDNO:5),the yeast GAL4 targeting signal,and the like.Heterologous nucleic acid sequence(s) employed inthe practice of the present invention are operably linkedto a regulatory nucleic acid sequence. As used herein, theterm "heterologous" nucleic acid sequence refers to asequence that originates from a foreign species, or, iffrom the same species, it may be substantially modifiedfrom its original form. Alternatively, an unchangednucleic acid sequence that is not normally expressed in acell is aa heterologous nucleic acid sequence. As usedherein, the term "operably linked" refers to a functionallinkage between the regulatory sequence and theheterologous nucleic acid sequence. Preferably, theheterologous sequence is linked to a promoter, resulting ina chimeric gene. The heterologous nucleic acid sequence ispreferably under control of either the viral LTR promoter—enhancer signals or of an internal promoter. Retainedsignals within the retrovirual LTR can still bring aboutefficient integration of the vector into the host cellgenome.Theheterologous to the desired gene sequence.promoter sequence may be homologous orA wide range of?W0 98/123141015202530...........................................;........‘..-........ .CA 02265522 l999-03- l5PCTIUS97ll 5934l 5promoters are suitable for use in the practice of thepresent invention, including viral or mammalian promoters.Cell or tissue specific promoters can be utilized to targetexpression of gene sequences in specific cell populations.Mammalian and.'viral promoters suitable for use in thepractice of the present invention are well knwn and readilyavailable in the art.Preferably during the cloning stage, the nucleicacid construct referred to as the transfer vector, havingthe packaging signal and the heterologous cloning site,will also contain a selectable marker gene. Marker genesare utilized to assay for the presence of the vector, andthus, The presenceof this marker gene ensures the growth of only those hostcells which express the inserted DNA. Typical selectiongenes encode proteins that confer resistance to antibioticsto confirm infection and integration.and other toxic substances,§.g., histidinol, puromycin,hygromycin, neomycin, methotrexate, and the like. Othermarker systems commmonly used in the art includeB—galactosidase (Lacz)systems, which can conveniently be monitored visually.and luciferase reporter or markerRecombinant viruses of the invention are capableof transferring nucleic acid sequence(s) into non-dividingcells. The term nucleic acid sequence refers to anypreferably DNA.may be derived from a variety ofCDNA, synthetic DNA, RNA, or combinationsSuch nucleic acid sequences may comprise genomicnucleic acid molecule, The nucleic acidmolecule sources,including DNA,thereof.DNA which may or may not include naturally occurringintrons. Moreover, such genomic DNA may be obtained inassociation with promoter regions, introns, or poly AGenomic DNA may be extracted and purified fromcells by well themessenger RNA (mRNA) can be isolated fromsequences.suitable means known in art.Alternatively,?W0 98/123141015202530ICA 02265522 l999-03- 15PCTIUS97I 159341 6cells and used to produce CDNA by reverse transcription orother means.The phrase "non—dividing" cell refers to a cellthat does not go through mitosis. Non—dividing cells maybe blocked at any point in the cell cycle, (§;g., QJGV(3/S, Cg/M), as long as the cell is not actively dividing.For ex vivo infection, a dividing cell can be treated toblock cell division by standard techniques used by those ofskillaphidocolinin the art, such as, for example, irradiation,treatment, serum starvation, contactinhibition, and the like. it should be understoodthat ex vivo infection is often performed without blockingHowever,the cells since many cells are already arrested (e.g., stemcells).Recombinant retrovirus vectors according to thepresent invention are capable of infecting any non—dividingcell, of the mechanism. used to block celldivision or the point in the cell cycle at which the cellis blocked.the bodyhematopoietic stenl cells,regardlessExamples of pre—existing non—dividing cells ininclude neurons, myocytes, hepatocytes,lymphocytes, dendritic cells,and the like,Recombinantepithelial cells, macrophages, as well asderivatives thereof. retrovirus vectorsaccording to the present invention can be used for both invivo gene delivery (for example, by injection), as well asex vivo gene delivery.The method of the present invention contemplatesthe use of at least three vectors which provide all of thefunctions required for packaging of recombinant virions asdiscussed above, plus a fourth vector which provides thewith thethereby facilitating infection ofelement which is capable of associatingnucleoprotien complex,non—dividing cells. Invention method also contemplatestransfection of vectors including viral genes such as vpr,?CA 02265522 l999-03- 15W0 98/12314 PCT/US97/1593417vif, nef, vpx, tat, rev, and vpu. Some or all of thesegenes can be included, for example, on the packagingconstruct vector, or, alternatively, they may reside on101520253035individual vectors. There is no limitation to the numberof vectors which can be utilized, as long as they are co-transfected to the packaging cell line in order to producea single recombinant retrovirus. For example, one couldput the env nucleic acid sequence on the same construct asthe gag and pol.Viral vectors contemplated for use herein areintroduced via transfection or infection into a packagingcell The cell line producesparticles that contain the vector genome.line. packaging viralMethods fortransfection or infection are well known by those of skillin the art. After co—transfection of the at least fourvectors to the packaging cell line, the recombinant virusis recovered from the culture media and titered by standardmethods used by those of skill in the art.In accordance with. yet another aspect of thepresent invention, there is provided another class ofrecombinant retroviruses which are capable of infectingnon—dividing cell(s). This class of invention retrovirusescomprises the following components:a viral GAG,a viral POL,a viral ENV,an heterologous nucleic acid operably linked toa regulatory sequence, andcis-acting nucleic acid. necessary" for reversetranscription and integration,with thenucleoprotein complex of said retrovirus iswherein a protein associatedmodified so as to be recognized. by thenuclear import machinery of said non—dividing cell.?CA 02265522 l999-03- 15W0 98/ 12314 PCT/US97/1593418In accordance with another embodiment of thepresent invention, there are provided viral particles1015202530produced by the above—described recombinant retroviruses.In accordance with still another embodiment ofthe presentinvention, thereare provided methods ofproducing the above—described recombinant retrovirus(es).According to this embodiment of the invention,methodpackaging host cell with one or more vectors comprising:inventioninfection comprises transfecting a suitablea first nucleic acid encoding a viral gag,a second nucleic acid encoding a viral pol,wherein at least one proteinencoded.by said first or second nucleicacid is modified so as to be recognizedby the nuclear import machinery of saidnon-dividing cell,SDnucleic acid encoding a viral env, and$1!nucleic acid encoding a packaging signalflanked by cis-acting nucleic acidsnecessary for reverse transcription andintegration, and. a cloning site forintroduction of a heterologous nucleicacid, operably linked to a regulatorynucleic acid.Optionally, invention method further comprisesrecovering the recombinant virus produced by the above-described transfected host cell.A variety of modifications of proteins associatedwith the nucleoprotein complex of said retrovirusarecontemplated. for use herein, such as, for example, bymutation of the protein associated with the nucleoproteincomplex of said retrovirus so as to be recognized by thecell.thenuclear import machinery ofthesaid non-dividingAlternatively, protein associated with?W0 98/123141015202530CA 02265522 l999-03- l5PCT/U S97/ 159341 9nucleoprotein complex of the retrovirus can be modified bythe addition of a karyophilic agent thereto.Examplary karyophilic agents contemplated for useherein include reverse transcriptase, matrix protein,nucleocapsid, protease, and the like.integrase,As yet another alternative, retrovirus accordinginvention can beto the prepared employing proteinassociated with the nucleoprotein complex thereof which hasbeen modified by theaddition thereto of a nuclearlocalization signal, as described hereinabove.In accordance with yet another embodiment of thepresent invention, there are provided methods for theintroduction and expression of heterologous nucleic acidscell(s). compriseinfecting non—dividing cell(s) with any of the recombinantin non—dividing Invention methodsviruses described herein, and expressing the heterologousnucleic acid in said non—dividing cell.It may be desirable to modulate the expression ofa gene regulating molecule in a cell by the introduction ofa molecule by the method. of the invention. The term"modulate" envisions the suppression of expression of agene when it isover—expressed, or augmentation ofexpression when it is under—expressed. Where a cellproliferative disorder is associated with the expression ofa gene, nucleic acid sequences that interfere with thegene's expression at the translational level can be used.This approach utilizes, antisense nucleicfor example,acid, ribozymes, or triplex agents to block transcriptionor translation of a specific mRNA, either by masking thatmRNA with an antisense nucleic acid or triplex agent, or bycleaving it with a ribozyme.?W0 98/123141015202530CA 02265522 l999-03- l5PCT/U S97] 1593420Antisense nucleic acids are DNA or RNA moleculesthat are complementary to at least a portion of a specificmRNA molecule 262:40,(Weintraub, Scientific American,1990). In the cell, the antisense nucleic acids hybridizeto the corresponding mRNA, forming a double—strandedmolecule. The antisense nucleic acids interfere with thetranslation of the mRNA, since the cell will not translatea mRNA that is double-stranded.about 15 nucleotides are preferred,Antisense oligomers ofsince they are easilysynthesized and are less likely to cause problems thanlarger molecules when introduced into the target cell. Themethods to inhibittranslation of genes is well known in the art;1g:289, 1988).use of antisensethe in vitro(Marcus-Sakura, Anal.Biochem.,The antisense nucleic acid can be used to blockexpression of a mutant protein or a dominantly active geneproduct, such as amyloid precursor protein that accumulatesin Alzheimer's disease.theParkinsonism, and other diseases.theproteins associated with toxicity.Such methods are also useful fortreatment of Huntington's disease, hereditaryAntisense nucleic acidsare also useful for inhibition of expression ofUse of an oligonucleotide to stall transcriptionis known as the triplex strategy since the oligomer windsaround double-helical DNA, forming a three—strand helix.Therefore, these triplex compounds can be designed torecognize a unique site on a chosen gene (Maher, et a1.,Antisense Res. and Dev., l§32:227, 1991; Helene, C.,Anticancer Drug Design, 6§6):569, 1991).Ribozymes are RNA molecules theability to specifically cleave other single—stranded RNA inpossessinga manner analogous to DNA restriction endonucleases.Through the modification of nucleotide sequences whichencode these RNAs, it is possible to engineer molecules?W0 98/ 123141015202530..,...¢................._...~..».,.............u........»...\,.CA 02265522 l999-03- l5PCT/US97/1 59342 1that recognize specific nucleotide sequences in an RNAmolecule and cleave it (Cech, J. Med, Assn,,ggg 3030, 1988). A major advantage of this approach isthat, because they are sequence—specific, only mRNAs withparticular sequences are inactivated.Amer .It may be desirable to transfer a nucleic acidencoding a biological response modifier. Included in thiscategory are immunopotentiating agents including nucleicacids encoding a rmmber of the cytokines classified as"interleukins". These include,1 through 12.necessarily working according to the same mechanisms, arefor example, interleukinsAlso included in this category, although notinterferons, and in particular gamma interferon (y—IFN),(TNF) and granulocyte—macrophage—colony stimulating factor (GM—CSF). It may be desirable todeliver to bonetumor necrosis factorsuch nucleic acidsmarrow cellsormacrophages to treat enzymatic deficienciesor immunedefects. Nucleic acids encoding growth factors, toxicpeptides, ligands, receptors, or other physiologicallyimportant proteins can also be introduced into specificnon~dividing cells.The recombinant retrovirus of the invention canbe used to treat an HIV infected cell (e.g.,macrophage) In addition,respiratory epithelium, for example, can be infected withT—cell orwith an anti—HIV molecule.a recombinant retrovirus of the invention having a gene forcystic fibrosis transmembrane conductance regulator (CFTR)for treatment of cystic fibrosis.The method of the invention may also be usefulfor neuronal or glial cell transplantation, or "grafting",which involves transplantation of cells infected with theof theinfection in vivo into the central nervous system or intorecombinant retrovirus invention ex vivo, orthe ventricular cavities or subdurally onto the surface of?WO 98112314101520253035ICA 02265522 l999-03- 15PCT/US97l 159342 2a host brain. Such methods for grafting will be known tothose skilled in the art and are described in NeuralQrafQing_in_th§_Mgmmg;i§g_g?§, Bjorklund and Stenevi, eds.,(1985), incorporated by reference herein. Proceduresinclude intraparenchymal transplantation, (i;§., within thehost brain) achieved by injection or deposition of tissuewithin the host brain so as to be apposed to the brainparenchyma at the time of transplantation.theselected regions of the recipient subject's brain may beAdministration of cells or virus intomade by drilling a hole and piercing the dura to permit theneedle of a microsyringe to be inserted. The cells orrecombinant retrovirus can injectedthe cellpreparation infected ex vivo, or the recombinant retrovirusalternatively beintrathecally into spinal cord region. Aof the invention, permits grafting of neuronal cells to anypredetermined site in the brain or spinal cord, and allowsmultiple grafting simultaneously in several different sitesusing the same cell suspension or viral suspension andcells from differentpermits mixtures ofanatomicalregions.Cells infected with a recombinant retrovirus ofthe invention, in vivo, or ex vivo, used for treatment ofa neuronal disorder for example, may optionally contain anexogenous gene, for example, a gene which encodes areceptor or a gene which encodes a ligand.whichnoradrenaline,Such receptorsGABA ,glutamate,include receptors respond to dopamine,adrenaline, serotonin,acetylcholine and other neuropeptides, as described above.Examples of ligands which may provide a therapeutic effectin a neuronal disorder includedopamine, adrenaline,noradrenaline, acetylcholine, gamma~aminobutyric acid andserotonin. The diffusion and uptake of a required ligandcell would bebeneficial in a disorder where the subject's neural cell isafter secretion by an infected donor?WO 98112314101520253035CA 02265522 l999-03- l5PCT/US97/159342 3defective in the production of such a gene product. A cellgenetically modified to secrete a neurotrophic factor, suchas nerve growth factor (NGF), might be used to preventdegeneration of cholinergic neurons that might otherwisedie without treatment. Alternatively, cells be graftedinto a subject with a disorder of the basal ganglia, suchas Parkinson's disease, can. be modified. to contain. anexogenous gene encoding L—DOPA, the precursor to dopamine.Parkinson's disease is characterized by a loss of dopamineneurons in the substantia—nigra of the midbrain, which havethe basal ganglia as their major target organ.Other neuronal disorders that can similarly betreated according to the method of the invention includeAlzheimer's disease, Huntington's disease, neuronal damagedue to stroke, and the like.Alzheimer's disease is characterized by degeneration of thedamage in the spinal cord,cholinergic neurons of the basal forebrain. Theneurotransmitter for these neurons is acetylcholine, whichis necessary for their survival. Engraftment ofcholinergic cells infected with a recombinant retrovirus ofthe invention containing an exogenous gene for a factorwhich would promote survival of these neurons can beaccomplished by the method of the invention. Following astroke, there is selective loss of cells in the CA1 of thehippocampus as well as cortical cell losswhich mayunderlie cognitive function and memory loss in thesepatients. Once identified, molecules responsible for CA1cell death can be inhibited by the methods of thisinvention. For example, or a geneencoding an antagonist can be transferred to a neuronalantisense sequences,cell and. implanted into the hippocampal region. of thebrain.The method of transferring nucleic acid alsocontemplates the grafting" of neuroblasts in combinationwith other therapeutic procedures useful in the treatment?W0 98/123141015202530ICA 02265522 l999-03- 15PCT/US97/159342 4of disorders of the CNS. For" example, the retroviralinfected cells can be co—administered with agents such asgrowth factors, gangliosides, antibiotics,neurotransmitters, neurohormones, toxins, neurite promotingmolecules and antimetabolites and precursors of thesemolecules such as the precursor of dopamine, L—DOPA.Further, there are a number of inheritedneurologic diseases in which defectivegeneslysosomal storage diseasesmay bereplaced. including: such asB—hexosaminidase or glucocerebrosidase;hypoxanthine phosphoribosyl transferasethose involvingdeficiencies inactivity (the "Lesch—Nyhan" syndrome); amyloidpolyneuropathies (prealbumin); Duchenne's -musculardystrophy, and retinoblastoma, for example.For diseases due to deficiency of a proteinproduct, gene transfer could introduce a normal gene intothe affected tissues for replacement therapy, as well as tocreate animal models forthe disease using antisensemutations. For example, it may be desirable to insert aFactor IX encoding nucleic acid into a retrovirus forinfection of a muscle or liver cell.The invention will now be described in greaterdetail by reference to the following non—limiting examples.Example 1Integrase (IN) contains multiple putative nuclear(NLSS). To determine the role of INin nuclear import of the HIV-1 nucleoprotein complex (NPC),localization signalsan HIV-1 mutant lacking IN, called AIN, was generated byintroducing a stop codon at the 3’ end of the reversetranscriptase (RT) coding sequence and then used to acutelyinfect irradiated HeLa cells. At 1 hour and 8 hours post-infection, cytoplasmic and nuclear extracts were analyzed?CA 02265522 l999-03- 15W0 98/12314 PCT /US97/ 159342 5for the presence of nucleocapsid (NC) and RT, othercomponents of the NPC. In wild—type (wt) infected cells,10152025NC or RT were predominantly associated with the cytoplasmiccompartment at 1. hour, whereas they’ were found. in thenucleus at 8 hours (about 50% of molecules).Similar results strains(AIN) or mutated in both Vpr and the MAIn contrast, no nuclear translocation ofwere observed usingdefective for INNLS (ANLS Avpr).NC or of RT was observed for a strain defective for all(MA ANLS Avpr AIN). As Control, thelocalization of HIV-1 (CA) wasCA remains associated. with. the cytoplasmicAltogether,that IN is capable of functioning as a mediator of HIV-1three determinantssubcellular capsidmonitored.compartment. these findings strongly suggestnuclear import in non—dividing cells.Example 2IN is a karyophilic proteinTo investigate whether IN contains karyophilicproperties, cells were transfected with a constructexpressing IN alone. Nuclear and cytoplasmic extracts werethen analyzed for their IN content. IN was foundessentially associated. with the nucleus of transfectedcells. In. addition, fluorescein—isothiocyanate (FITC)—labeled IN microinjected in the cytoplasm of cells was alsodetected predominantly in the nucleus of these cells,whereas FITC—BSA used as control remained associated withthe cytoplasm.that IN is aThese demonstratekaryophilic protein.results?CA 02265522 l999-03- l5wo 93/12314 PCT/US97ll59342 6Example 3IN as a component of HIV—l NPC interacts withcell nuclear import machineryThe interaction of IN with the cell nuclear5 import machinery was then investigated. To explore this1015202530issue, purified. HIV—1 NPC were immunoprecipitated withanti-karyopherin a antibodies,thenantibodies as detectors.and the bound material wasanti—IN, anti—MAin HIV- 1 NPCextracted with mild conditions (0.05% of detergent), IN co—analyzed by immunoblotusingInterestingly,Under the sameSince MA and INare associated in the HIV-1 pre—integration complex (PIC),immunoprecipitated with karyopherin a.conditions, MA also co—immunoprecipitated.it could not be ascertained that IN did not indirectly bindthrough MA. when HIV-1 PIC wereextracted with stringent conditions (0.5% of detergent), IN(but not MA)suggesting a direct association between the two molecules.karyopherin a, However,co—immunoprecipitated with karyopherin a,Example 4IN directly binds karyopherin a in anNLS-dependent mannerThe binding of karyopherin a with IN was theninvestigated in vitro. Recombinant GST—IN fusion proteincould capture one member of the karyopherin a family(karyopherin a—2), called Rch1.l, overexpressed intransfected cells. In contrast, GST‘ alone, GST—CA. orGST—Nef could not bind Rch1.1. Furthermore, NLS peptideblocked IN—Rch1.l complex formation, whereas reversepeptide has no effect. In addition, a GST-recombinant formof another member of the karyopherin a family (karyopherina—l), called hSRPl, These datademonstrate the NLS—dependent direct binding of IN tocould bind recombinant IN.karyopherin a.?W0 98ll23l41015202530..:...........—-.a.~......~..»......_.-................. .... . .CA 02265522 l999-03- l5PCT/U S97] 159342 '7Example 5The C—terminal domain of IN contains two NLSSrecoqnized by karyopherinTo map the IN domains recognized by karyopherina, truncated forms of GST-IN were generated and tested fortheir affinity to Rch1.1. Rchl was captured by a GST-INfusion protein lacking 50 N-terminal amino acids of IN (50-288) but not by variants deleted at the C—terminus (1—170or 50—185). it could be deduced thatthe IN domain recognized. by karyopherin a isbetween residues 185 and 288. In this region, two putativeNLSV located around positions 186-188(KRKQ) and NLS2, around positions 211-216 (KELQKQ). BothNLSs were mutated and the resulting GST—ANLS IN proteinswere tested for their affinity for Rchl.l.From these results,locatedNLS were noted;Mutationsintroduced in NLS, completely abrogated the IN—Rch1 complexformation, whereas mutations in NLS2 decreased (about 50%)the interaction between the two moles. These data suggestthat NLS, IN is the main site of recognition of karyopherina, although NLS2 IN appears to contribute to the stabilityof this interaction.Interestingly, NLS, IN (consensus:KRKGGIG) is highly conserved among lentiviruses (HIV—1,HIV-2, SIV, BIV, FIV, Visna, CAEV, EIAV), but is absent innon—lentiviruses (MLV, RSV, HTLV), suggesting an importantrole for this NLS motif in lentiviral infection.To confirnl the previous results, FITC—labeledGST—ANLS1 or GST—ANLS2 were microinjected in the cytoplasmof cells and their subcellular localization was monitoredby confocal microscopy. Both NLS mutants did not enterinside the nucleus, but instead remained associated withthe cytoplasmic compartment. These findings indicate thattwo IN C—terminal NLSs are likely involved in IN—na complexformation and IN nuclear import.?ICA 02265522 l999-03- 15wo 93/12314 PCT/US97/1593428Example 6Recruitment of karyopherin B and nucleoporinbv IN—Ka complexes in vitroThe events involved in IN—mediated HIV-1 nuclear5 import were recapitulated in vitro. Karyopherin a and1015202530karyopherin B were successively added to GST—IN to mimicthe docking of IN to the nucleopore.Karyopherin E bound to GST—IN—karyopherin a complexes butnot to GST—IN alone. No binding of components of the cellnuclear import machinery was observed when GST alone wasused as negative control. In addition, neither of the NLSIN mutants (i.e., GST-ANLSl IN and GST—ANLS2 IN) recruitedkaryopherin a (or any of the other components of the cellnuclear import machinery).sequentialThese findings corroborate themodel of nuclear import of NLS—bearing protein, in which anNLS—substrate, such as IN or MA, is first recognized by thecytosolic karyopherin a through its NLS, and then docked tothe nucleopore via karyopherin 3.Example 7IN qoverns HIV-1 nuclear import iny—irradiated CD4+ HeLa cells throuqh its NLSsTo determine the role of the karyophilic motifsof IN in HIV-1y—irradiated CD4+ HeLa cells were acutely infected with ayp;—defective strain mutated in both MA NLS and IN NLS,(Avpr MA ANLS IN ANLSI). at 1 hour and 8hours post—infection, cytoplasmic and nuclear extracts wereinfection. of non—dividing cells,As previously,analyzed for the presence of components of HIV-1 NPC; NCand RT.RT wasImportantly, no nuclear translocation of NC or ofobserved 8 hours post—infection for a straindefective for the three karyophilic viral determinants; theIN NLS1, the MA NLS and Vpr (MA ANLS Avpr IN ANLSQ.through its NLS,Thesefindings strongly suggest that IN, is a?W0 98/123141015202530CA 02265522 l999-03- l5PCT/U S97/ 159342 9main mediator of HIV-1 nuclear import in y~irradiated CD4+HeLa cells.Example 8HIV-1 Inteqrase induces infection ofnon~dividinq cells bv MLV—based vectorsHuman HeLa cells were arrested in G1—S stage byaphidicolin treatment, then infected with:an MLV—based vector(encoding the marker, beta~galactosidase), pseudotyped with the VSV Genvelope,an HIV—based vector (encoding the marker, beta-galactosidase), pseudotyped with the VSV Genvelope, ora "chimeric" vector according to the invention,wherein the above—described MLV—based vectoris supplemented with an additional vectorencoding HIV-1 integrase.For comparison, non—arrested (i.e.,dividing) HeLa cellswere infected with each of the above—described vectors.Transduction was scored by X—gal staining of thecultures 48 hours after infection. Results are presentedand are expressed as the number of blue cellsper 50 pl of supernatant.in Figure 1,Inspection of Figure 1 reveals that the HIV—basedvector is capable of infecting non-dividing cells as wellas dividing cells. In contrast, while the MLV—based vectoris capable of infecting dividing cells, this vector isvirtually incapable of infecting arrested cells. However,infection of non—dividing cells is surprisingly effectiveemploying a chimeric construct according to the invention.?CA 02265522 l999-03- l5wo 93/12314 PCT/US97/159343 0While the invention has been described in detailwith reference to certain preferred embodiments thereof, itwill be understood that modifications and variations arewithin the spirit and scope of that which is described and5 claimed.?CA 02265522 1999-03-15W0 98/12314 PCT/US97/ 1593431SEQUENCE LISTING(1) GENERAL INFORMATION(i) APPLICANT: Trono, Didier P.Gallay, Philippe A.(ii) TITLE OF THE INVENTION: RETROVIRAL VECTORS CAPABLEOF TRANSDUCING NON-DIVIDING CELLS(iii) NUMBER OF SEQUENCES: 5(iv) CORRESPONDENCE ADDRESS:(A) ADDRESSEE: Gray Cary Ware & Freidenrich(B) STREET: 4365 Executive Drive, Suite 1600(C) CITY: San Diego(D) STATE: CA(E) COUNTRY: USA(F) ZIP: 92121(v) COMPUTER READABLE FORM:(A) MEDIUM TYPE: Diskette(B) COMPUTER: IBM Compatible(C) OPERATING SYSTEM: DOS(D) SOFTWARE: FastSEQ for Windows DEMONSTRATION Version 2.0D(Vi) CURRENT APPLICATION DATA:(A) APPLICATION NUMBER: 08/715,318(B) FILING DATE: 17-SEP-1996(C) CLASSIFICATION:(vii) PRIOR APPLICATION DATA:(A) APPLICATION NUMBER:(B) FILING DATE:(viii) ATTORNEY/AGENT INFORMATION:(A) NAME: Reiter, Stephen E(B) REGISTRATION NUMBER: 31,192(C) REFERENCE/DOCKET NUMBER: P41 90295(ix) TELECOMUNICATION INFORMATION:(A) TELEPHONE: 619-677-l409(B) TELEFAX: 619-677-1465(C) TELEX:(2) INFORMATION FOR SEQ ID NO:l:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:?CA 02265522 1999-03-15W0 98/123143 2’Lys Arg Lys Gln1(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:Lys Glu Leu Gln Lys Gln1 5(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:Lys Arg Lys Gly Gly Ile Gly1 5(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 7 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:Pro Lys Lys Lys Arg Lys Val1 5(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:PCTIUS97/ 15934?CA 02265522 1999-03-15W0 98/ 12314 PCTIUS97/1593433Ala Ala Phe Glu Asp Leu Arg Val Leu Ser1 5 10

Claims (28)

That which is claimed is:

1. A recombinant retrovirus capable of infecting a non-dividing cell, said retrovirus comprising the following components:
a viral GAG, a viral POL, a viral ENV, an element which associates with the nucleoprotein complex of said retrovirus, wherein said element is recognized by the nuclear import machinery of said non-dividing cell, an heterologous nucleic acid operably linked to a regulatory sequence, and cis-acting nucleic acid necessary for reverse transcription and integration.

2. A retrovirus according to claim 1 wherein said element is selected from reverse transcriptase, matrix protein, nucleocapsid, protease or integrase.

3. A retrovirus according to claim 2 wherein said element is a lentiviral integrase.

4. A retrovirus according to claim 3 wherein said element is HIV integrase.

5. A retrovirus according to claim 1 wherein said element is a nuclear localization signal, operably associated with the nucleoprotein complex of said retrovirus.

6. A retrovirus according to claim 5 wherein said nuclear localization signal is derived from HIV-1 integrase.

7. A retrovirus according to claim 1 wherein the components of said retrovirus are selected based on the type of non-dividing cell targeted for introduction of said heterologous nucleic acid.

8. A retrovirus according to claim 7 wherein the components of said retrovirus are obtained from MoMuLV, HaMuSV, MuMTV, GaLV, HIV or RSV.

9. A retrovirus according to claim 7 wherein said non-dividing cell is a neuron, a myocyte, an hepatocyte, an hematopoietic stem cell, a lymphocyte, a dendritic cell, an epithelial cell or a macrophage.

10. A viral particle produced by the recombinant retrovirus of claim 1.

11. A recombinant retrovirus capable of infecting a non-dividing cell, said retrovirus comprising the following components:
a viral GAG, a viral POL, a viral ENV, an heterologous nucleic acid operably linked to a regulatory sequence, and cis-acting nucleic acid necessary for reverse transcription and integration, wherein a protein associated with the nucleoprotein complex of said retrovirus is modified so as to be recognized by the nuclear import machinery of said non-dividing cell.

12. A retrovirus according to claim 11 wherein said protein associated with the nucleoprotein complex of said retrovirus is mutated so as to be recognized by the nuclear import machinery of said non-dividing cell.

13. A retrovirus according to claim 11 wherein said protein associated with the nucleoprotein complex of said retrovirus is modified by the addition of a karyophilic agent thereto.

14. A retrovirus according to claim 13 wherein said karyophilic agent is matrix protein or integrase.

15. A retrovirus according to claim 13 wherein said karyophilic agent interacts with said nucleoprotein complex by association with reverse transcriptase, nucleocapsid or protease.

16. A retrovirus according to claim 11 wherein said protein associated with the nucleoprotein complex of said retrovirus is modified by the addition thereto of a nuclear localization signal.

17. A retrovirus according to claim 11 wherein the components of said retrovirus are selected based on the type of non-dividing cell targeted for introduction of said heterologous nucleic acid.

18. A retrovirus according to claim 17 wherein the components of said retrovirus are obtained from MoMuLV, HaMuSV, MuMTV, GaLV, HIV or RSV.

19. A retrovirus according to claim 17 wherein said non-dividing cell is a neuron, a myocyte, an hepatocyte, an hematopoietic stem cell, a lymphocyte, a dendritic cell, an epithelial cell or a macrophage.

20. A viral particle produced by the recombinant retrovirus of claim 11.

21. A method of producing a recombinant retrovirus capable of infecting a non-dividing cell, said method comprising transfecting a suitable packaging host cell with one or more vectors comprising:
a nucleic acid encoding a viral gag, a nucleic acid encoding a viral pol, a nucleic acid encoding a viral env, a nucleic acid encoding an element which associates with the nucleoprotein complex of said retrovirus, wherein said element is recognized by the nuclear import machinery of said non-dividing cell, and a nucleic acid encoding a packaging signal flanked by cis-acting nucleic acids necessary for reverse transcription and integration, and a cloning site for introduction of a heterologous nucleic acid, operably linked to a regulatory nucleic acid.

22. A method according to claim 21 further comprising recovering the recombinant virus produced by said transfected host cell.

23. A method of producing a recombinant retrovirus capable of infecting a non-dividing cell, said method comprising transfecting a suitable packaging host cell with one or more vectors comprising:
a first nucleic acid encoding a viral gag, a second nucleic acid encoding a viral pol, wherein at least one protein encoded by said first or second nucleic acid is modified so as to be recognized by the nuclear import machinery of said non-dividing cell, a nucleic acid encoding a viral env, and a nucleic acid encoding a packaging signal flanked by cis-acting nucleic acids necessary for reverse transcription and integration, and a cloning site for introduction of a heterologous nucleic acid, operably linked to a regulatory nucleic acid.

24. A method according to claim 23 further comprising recovering the recombinant virus produced by said transfected host cell.

25. A method for the introduction and expression of a heterologous nucleic acid in a non-dividing cell, said method comprising infecting said non-dividing cell with a recombinant virus according to claim 1, and expressing the heterologous nucleic acid in said non-dividing cell.

26. A method for the introduction and expression of a heterologous nucleic acid in a non-dividing cell, said method comprising infecting said non-dividing cell with a recombinant virus according to claim 11, and expressing the heterologous nucleic acid in said non-dividing cell.

27. A stable packaging cell line containing:
a nucleic acid encoding a viral gag, a nucleic acid encoding a viral pol, a nucleic acid encoding a viral env, and a nucleic acid encoding an element which associates with the nucleoprotein complex of a retrovirus, wherein said element is recognized by the nuclear import machinery of a non-dividing cell.

28. A stable packaging cell line containing:
a first nucleic acid encoding a viral gag, a second nucleic acid encoding a viral pol, wherein at least one protein encoded by said first or second nucleic acid is modified so as to be recognized by the nuclear import machinery of said non-dividing cell, and a nucleic acid encoding a viral env.