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WO1997015679A1 - Virus recombines contenant des elements genetiques mobiles et procedes d'utilisation desdits virus en therapie genique - Google Patents

Virus recombines contenant des elements genetiques mobiles et procedes d'utilisation desdits virus en therapie genique Download PDF

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WO1997015679A1
WO1997015679A1 PCT/US1996/017176 US9617176W WO9715679A1 WO 1997015679 A1 WO1997015679 A1 WO 1997015679A1 US 9617176 W US9617176 W US 9617176W WO 9715679 A1 WO9715679 A1 WO 9715679A1
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virus
sequence
cell
gene
recombinant
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WO1997015679A9 (fr
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William Mark Kelley
James M. Wilson
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The Trustees Of The University Of Pennsylvania
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Definitions

  • the present invention relates to the field of somatic gene therapy and to the development of novel recombinant adenoviruses for use as gene transfer vehicles.
  • Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a therapeutic or reporter transgene to a variety of cell types.
  • Human adenoviruses are comprised of a linear, approximately 36 kb double-stranded DNA genome, which is divided into 100 map units (m.u.), each of which is approximately 360 bp in length.
  • the DNA contains short inverted terminal repeats (ITR) at each end of the genome that are required for viral DNA replication.
  • the gene products are organized into early (El through E4) and late (Ll through L5) regions, based on expression before or after the initiation of viral DNA synthesis [see, e.g., M. S.
  • Recombinant adenoviruses are capable of providing extremely high levels of transgene delivery to virtually all cell types, regardless of the mitotic state.
  • the efficacy of this system in delivering a therapeutic transgene in vivo that complements a genetic imbalance has been demonstrated in animal models of various disorders [K. F. Kozarsky et al, Somatic Cell Mol. Genet.. 19:449-458 (1993) ("Kozarsky et al I”); K. F. Kozarsky et al, J. Biol. Chem.. 2.69:13695-13702 (1994) ("Kozarsky et al II) ; Y. atanabe, Atherosclerosis. 16:261-268 (1986); K.
  • the first-generation recombinant, replication- deficient adenoviruses which have been developed for gene therapy contain deletions of the entire Ela and part of the Elb regions.
  • This replication-defective virus is grown on an adenovirus-transformed, complementation human embryonic kidney cell line containing a functional adenovirus Ela gene which provides a trans-acting Ela protein, the 293 cell [ATCC CRL1573].
  • the resulting virus is capable of infecting many cell types and can express the introduced gene (providing it carries its own promoter) , but cannot replicate in a cell that does not carry the El region DNA unless the cell is infected at a very high multiplicity of infection.
  • adenoviruses remain attractive gene therapy vectors due to the ease of production of large quantities of virus and the subsequent concentration of the virions into very high titer stocks, high in vivo transduction efficiencies and the ability to infect fully differentiated and quiescent cells.
  • the invention provides a recombinant replication defective virus, preferably an adenovirus, which contains (a) the DNA of, or corresponding to, at least a portion of the genome of the virus, capable of infecting a mammalian cell; and (b) a first expression sequence comprising a selected gene operatively linked to regulatory sequences directing its expression, the gene and regulatory sequences in operative association with the necessary cis-acting sequences of a transposon, which cis-acting sequences are capable of mediating transposition or retrotransposition.
  • This entire expression sequence of (b) is flanked by the DNA of (a) .
  • This recombinant virus is capable of infecting a mammalian cell and capable of expressing the selected gene and transferring it to the chromatin of the infected cell in vivo or in vitro, when in the presence of a transposase.
  • a recombinant replication defective virus which contains, in addition to the components recited above, a second expression sequence comprising a suitable trans-acting transposase operatively linked to regulatory sequences directing its expression, the expression sequence flanked by the DNA of (a).
  • the invention provides a recombinant trans-acting replication defective virus, preferably an adenovirus, comprising (a) the DNA of, or corresponding to, at least a portion of the genome of a virus, capable of infecting a mammalian cell; and (b) an expression sequence comprising a suitable trans-acting transposase gene operatively linked to regulatory sequences directing its expression, the expression sequence flanked by the DNA of (a) .
  • This trans-acting recombinant virus is capable of infecting a mammalian cell and capable of expressing the transposase in the cell in vivo or in vitro.
  • the invention provides a method for delivering and stably integrating a gene into host cell chromatin by infecting the cell with the first above-described recombinant replication defective virus and providing a transposase to the cell.
  • the invention provides a method for delivering and stably integrating a gene into host cell chromatin by infecting the cell with the first above-described cis-acting recombinant replication defective virus and co-infecting the cell with an above- described trans-acting recombinant replication defective virus.
  • the invention provides a method for delivering and stably integrating a gene into host cell chromatin by infecting the cell with a recombinant virus which contains both the cis-acting and trans-acting sequences as above-described.
  • Yet a further aspect of this invention is a mammalian cell which stably expresses a selected gene integrated into its chromatin, produced by the methods above.
  • Another aspect of this invention is a method of generating recombinant retroviruses.
  • This method includes the steps of infecting a retrovirus packaging cell line containing a transposase gene with a cis-acting recombinant replication defective virus as above described, wherein the resulting producer cell line produces recombinant retrovirus containing the selected gene.
  • Fig. 1 is a schematic drawing of plasmid pAdCMVga g-pol, which contains in clockwise order from the top center: an adenovirus serotype 5 sequence of map units 0 to 1 (Ad5 0-1 mu, black bar) , followed by a cytomegalovirus (CMV) promoter (CMV, striped arrow) , an SV-40 intron sequence, the Mo-MLV gag gene (striped bar), the Mo-MLV pol gene (cross-hatched bar) with a splice acceptor (SA) ; a polyadenylation sequence (pA, shaded bar) , Ad5 9 to 16 mu (black bar) , with the remainder of the plasmid obtained from pBR322-based plasmid pAT153 (open bar) . Restriction endonuclease enzyme recognition sites are indicated by their conventional names.
  • Figs. 2A - 2C are the DNA sequence of the pAdCMVgag-pol plasmid [SEQ ID NO: 1], in which nucleotides 1 to 360 span Ad5 mu 0-1; nucleotides 361 to 974 span the CMV promoter; nucleotides 975-1122 span the SV-40 intron sequence, nucleotides 1208-2878 span Mo-MLV gag; nucleotides 2862-6462 span Mo-MLV pol; nucleotides 6726 - 6933 span the pA sequence; nucleotides 6934-9419 span Ad5 map units 9.3 to 16.1, with the remainder of the sequence nucleotides 9420-12135 from pAT153.
  • Fig. 3 is a schematic drawing of plasmid pAdNeo-int, which contains in clockwise order from the top center: an Ad5 0 to 1 mu (black bar) , followed by a pA site (open bar), a 3' portion of a neomycin resistance gene (neo, shaded bar) , a splice donor sequence (SD) , a ⁇ globin intron (black bar), an SA site, the 5' portion of the neo gene (shaded bar) , a counterclockwise directed thymidine kinase promoter (tk, striped arrow) , an enhancer (enh, striped bar) , Ad5 9-16 mu (black bar) , with the remainder of the plasmid obtained from pAT153 (black line) .
  • Figs. 4A - 4B are the DNA sequence of the pAdNeo-int plasmid [SEQ ID NO: 2], in which nucleotides l - 360 span Ad mu 0-1; nucleotides 365 to 428 span the pA site; nucleotides 429 - 1043 span the 3' neo sequence; nucleotides about 1044 to 1947 span the intron; nucleotides 1948 to 2143 span the 5- neo sequence; nucleotides 2144 to 2424 span the tk promoter/enhancer; nucleotides 2452 to 4907 span the Ad5 map units 9.3 to 16.1, with the remainder of the sequence from pAT153.
  • Fig. 5 is a schematic drawing of a plasmid pAdMLVNeo-int, which contains in clockwise order from the top center: Ad5 0-1 mu (black bar) , followed by a rat genomic sequence (striped bar) ; a counterclockwise directed Mo-MLV LTR (open bar with arrow) , an enh (shaded bar) , a clockwise directed tk promoter sequence (striped arrow); a 5' portion of neo (shaded bar), an SA sequence, a ⁇ globin intron (black bar), an SD site, the 3 1 portion of the neo gene (shaded bar) , a pA sequence (open bar) , a ⁇ sequence (shaded bar) , a counterclockwise directed Mo-MLV LTR (open bar with arrow) , a rat genomic sequence (striped bar) ; Ad5 9-16 mu (black bar) with the remainder of the plasmid obtained from pAT153 (
  • Figs. 6A - 6C are the DNA sequence of the pAdMLVNeo-int plasmid [SEQ ID NO: 3], in which nucleotides 1 to 360 span Ad mu 0-1; nucleotides 361 to 1076 span the irrelevant rat genomic sequence (nucleotides unknown indicated by N) ; nucleotides 1077 - 1610 span the leftward Mo-MLV LTR, nucleotides 1817-2097 span the rightward enhancer/tk promoter sequence; nucleotides 2110- 2293 span the 5' neo gene sequence; nucleotides 2294 to 3197 span the ⁇ globin intron, nucleotides 3198-3815 span the 3' portion of the neo gene, nucleotides 3817-3871 span the pA site; nucleotides 4298-4892 span the rightward Mo-MLV LTR, nucleotides 4893-5393 span the irrelevant rat genomic sequence; nucle
  • Fig. 7A is a schematic linear map of recombinant cis-acting adenovirus H5.020TKneo-int[LTR] .
  • the abbreviations are defined in the figures above; the restriction sites and locations are marked.
  • the symbol 9-100 mu ( ⁇ E3) represents Ad sequence from Ad5dl7001 which has a 3 kb deletion in the E3 gene.
  • Fig. 7B is a schematic linear map of recombinant trans-acting adenovirus H5.020CMVgag-pol. The abbreviations are defined in the figures above; the restriction sites and locations are marked.
  • Fig. 7C is a schematic linear map of recombinant adenovirus H5.020TKneo-int. The abbreviations are defined in the figures above; the restriction sites and locations are marked.
  • Figs. 8A - 81 are the DNA sequence of recombinant cis-acting adenovirus H5.020TKneo-int[LTR] [SEQ ID NO: 4] .
  • Figs. 9A - 91 are the DNA sequence of recombinant trans-acting adenovirus H5.02OCMVgag-pol [SEQ ID NO: 5].
  • Figs. 10A - 10H are the DNA sequence of recombinant adenovirus H5.020TKneo-int [SEQ ID NO: 6].
  • the present invention provides a novel method and compositions for generating a recombinant virus capable of integrating a transgene into the chromatin of a target host cell.
  • the use of these methods and compositions can increase the persistence of transgene expression in a gene therapy protocol.
  • the present invention involves generating a recombinant virus or pair of viruses, preferably adenoviruses, that contains sufficient transposon elements to permit transposition or retrotransposition of a transgene from the recombinant adenovirus into the target cell chromatin.
  • Transposition is the movement of DNA from one DNA strand to another or from one locus on a DNA strand to another. This can occur by replication or independent of replication. "Retrotransposition” defines the movement of DNA from one site to another through an RNA intermediate.
  • the transposition or retrotransposon system of this invention was initially designed of two recombinant adenoviruses, a cis-acting and a trans-acting virus. However, one can readily place both functions on a single adenovirus depending on the adenovirus genes deleted. For ease of understanding, the cis-acting and trans ⁇ acting functions are discussed and exemplified on different recombinant adenoviruses.
  • One recombinant virus contains a minigene comprising a transposition or retrotransposition- dependent reporter gene and/or a selectable marker and/or therapeutic transgene in association with by cis-acting elements from a selected transposon.
  • This minigene is constructed in a plasmid and cloned into a selected deletion in a replication defective adenovirus.
  • the selectable marker is a neomycin resistance gene (neo) and the cis elements are transposon LTRs and the ⁇ sequence from Mo-MLV. See Figs. 5, 6, 7A and 8 for the sequences of these components.
  • a second recombinant virus contains a second minigene, or expression cassette, that codes for the respective trans-acting transposase under the control of a suitable heterologous promoter.
  • This second minigene is constructed in a plasmid and cloned into a selected deletion in a replication defective adenovirus.
  • the transposase elements are the Mo-MLV gag-pol open reading frames and the promoter is a cytomegalovirus promoter. See Figs. 1, 2, 7B and 9 for the sequences of these components.
  • Co-infection of a host cell with the cis-acting and trans-acting recombinant adenoviruses brings the cis- acting elements which are in association with the transgene in contact with the trans-acting transposase elements within a single nuclei, where transposition or retrotransposition can occur.
  • Transposition or retrotransposition enables the cis-acting minigene cloned into the cis-acting recombinant adenovirus to be rescued from the adenoviral genome and integrated into the host cell chromatin.
  • the "first minigene" of the cis-acting recombinant adenoviruses is defined as an expression cassette containing a selected transgene under the control of suitable regulatory elements.
  • the first minigene may have a transgene in the size in the range of several hundred base pairs up to about 8.3 kb including the necessary cis elements.
  • the "second minigene” of the trans-acting recombinant adenovirus is defined as an expression cassette containing a transposase under the control of suitable regulatory elements.
  • transposons A “transposon” may be defined as a discrete piece of DNA which can move either through replication or independent of replication from one DNA strand to another DNA strand or another locus on the same DNA strand.
  • the transposon DNA sequence is composed of a trans acting transposase gene and the cis acting elements necessary for transposition.
  • trans-acting transposon sequences or transposase is meant the necessary trans elements capable of mediating transposition or retrotransposition.
  • transposase transposase
  • transposase the necessary trans elements capable of mediating transposition or retrotransposition.
  • transposase transposase
  • cis-acting transposon sequence is meant the necessary cis elements capable of mediating transposition or retrotransposition.
  • the cis sequences include, but are not necessarily limited to, the following: Inverted Terminal Repeats (ITRs) , Long Terminal Repeats (LTRs) , Terminal Repeats (TRs) , and ⁇ sequences.
  • ITRs Inverted Terminal Repeats
  • LTRs Long Terminal Repeats
  • TRs Terminal Repeats
  • ⁇ sequences The cis and trans elements of most transposons may be separated onto different DNA strands and remain functional in the transposition of the cis sequences.
  • Transposons that involve an RNA intermediate during transposition are a subset of transposons and are specifically referred to as "retrotransposons".
  • Moloney murine leukemia retrovirus Mo-MLV [K. Harbers et al, Proc. Natl. Acad. Sci.. USA. 28_:7609-7613 (1981)]
  • Transposons have been found in a wide range of evolutionarily diverse species and among different kingdoms. These organisms include bacteria, insects, yeast, nematodes, maize, mice and humans. Any RNA transposon may be used in the present invention.
  • the necessary cis elements are the LTRs and ? sequence of MoMLV.
  • Trans elements or transposase include the gag and pol gene sequences of the MoMLV. These sequences can be readily isolated from the MoMLV sequence by conventional molecular engineering techniques [see, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual.”, 2d edit., Cold Spring Harbor Laboratory, New York (1989) and references cited therein].
  • the MoMLV LTRs and ⁇ sequence for use in the cis-acting recombinant virus were isolated with rat genomic flanking sequence, which appears in Fig. 7A as a striped bar. This sequence is irrelevant to the function of the LTRs and ⁇ sequence and can be eliminated if desired.
  • the transgene sequence of the cis- acting virus is contained within the first minigene.
  • This transgene is defined as a nucleic acid sequence or reverse transcript thereof, heterologous to the adenovirus sequence, which encodes a polypeptide or protein of interest.
  • the composition of the transgene sequence will depend upon the use to which the resulting recombinant adenovirus will be put.
  • one type of transgene sequence includes a reporter sequence, which upon expression produces a detectable signal.
  • reporter sequences include, without limitation, an E. coli beta- galactosidase (LacZ) cDNA, a human placental alkaline phosphatase gene and a green fluorescent protein gene.
  • transgene sequences when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, e.g. , ultraviolet wavelength absorbance, visible color change, etc.
  • Another type of transgene sequence includes a selectable marker gene, which permits selection of the gene in transfected cells when grown in media with ingredients which can select for those cells which contain the marker gene.
  • selectable markers are usually antibiotic resistance genes, such as neomycin resistance gene, ampicillin resistance gene, or puromycin resistance genes, among others. These genes may be selected by one of skill in the art.
  • transgene sequence includes a therapeutic gene which expresses a desired gene product in a host cell.
  • therapeutic nucleic acid sequences typically encode products for administration and expression in a patient in vivo or ex vivo to replace or correct an inherited or non-inherited genetic defect or treat an epigenetic disorder or disease.
  • therapeutic genes which are desirable for the performance of gene therapy include, without limitation, a normal cystic fibrosis transmembrane regulator (CFTR) gene [Riordan et al, Science. 2 5.:1066- 1073 (1989)], a low density lipoprotein (LDL) gene [T. Yamamoto et al, Cell. 19:27-28 (Nov.
  • heterologous promoter and enhancer for the trans-acting minigene and for the cis-acting minigene is a routine matter and is not a limitation of the virus or plasmid vector itself.
  • Useful promoters may be constitutive promoters or regulated (inducible) promoters, which will enable control of the amount of the transgene to be expressed.
  • a desirable promoter is that of the cytomegalovirus immediate early promoter/enhancer [see, e.g., Boshart et al, Cell. 11:521-530 (1985)].
  • CMV enhancer/chicken ⁇ -actin promoter is also useful, e.g., a CMV enhancer/chicken ⁇ -actin promoter. Still other promoter/enhancer sequences may be selected by one of skill in the art.
  • the first and second minigenes also include conventional regulatory elements necessary to drive expression of the transgene or the transposase in a cell transfected with the cis- acting or trans-acting viruses.
  • Most desirable are nucleic acid sequences heterologous to the adenovirus sequences and the transposon sequences that provide signals required for efficient polyadenylation of the transcript.
  • a common poly-A sequence is that derived from the papovavirus SV-40.
  • poly-A sequences from the growth hormone gene terminator and other sources The particular poly A sequences used in the exemplified minigenes are illustrated in Figs. 2, 4, 6, 8 and 9. The poly-A sequence follows the transgene sequences or transposase sequences.
  • sequences useful in the minigenes of this invention are introns with functional splice donor and/or acceptor sites (SD/SA) .
  • a common intron sequence is also derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • An intron used in the examples, for the purpose of demonstrating the efficacy of this invention, is the G ⁇ -globin intron of Figs. 3 and 4.
  • Other intron sequences may be obtained by one of skill in the art for similar use. Such introns may be located desirably between the promoter/enhancer sequence and the transgene or transposase.
  • the first (cis-acting) and second (trajis-acting) minigenes described above are inserted into respective plasmid vectors between flanking virus sequences.
  • both the cis-acting and trans-acting minigenes may be incorporated into a single plasmid flanked by viral sequences.
  • each plasmid sequence is transfected into a host cell which has been transfected with viral DNA which supplies any necessary viral sequence missing from the plasmid. Homologous recombination between the plasmid vector and the second virus results in recombinant viruses of this invention.
  • this retrotransposon system can be employed to generate any number of different recombinant viruses useful in gene therapy (e.g., retroviruses)
  • the present invention exemplifies the generation of recombinant adenoviruses.
  • adenoviruses have many desirable characteristics as gene therapy vehicles and thus are presently preferred.
  • the DNA sequences of a number of adenovirus types are available from Genbank, including type A 5 [Genbank Accession No. M73260].
  • the adenovirus sequences may be obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, and further including any of the presently identified 41 human types [see, e.g., Horwitz, cited above].
  • adenoviruses known to infect other animals may also be employed in the constructs of this invention.
  • the selection of the adenovirus serotype is not anticipated to limit the following invention.
  • a variety of adenovirus strains are available from the American Type Culture Collection, Rockville, Maryland, or available by request from a variety of commercial and institutional sources. In the following exemplary embodiment an adenovirus, type 5 (Ad5) is used for convenience.
  • adenovirus sequences are deleted of a substantial portion of the El region.
  • the entire El region falls between Ad5 mu 1 and 11.5. It is preferred to delete the sequences falling between mu 1 and about 9.
  • the cis-acting and trans-acting minigenes are preferably inserted into the El deletion site of the adenovirus.
  • the adenovirus nucleic acid sequences employed in the recombinant viruses are adenovirus genomic sequences from which all viral genes are deleted. More specifically, the adenovirus sequences may be only the cis-acting 5' and 3' inverted terminal repeat (ITR) sequences of an adenovirus (which function as origins of replication) and the native 5' packaging/enhancer domain, that contains sequences necessary for packaging linear Ad genomes and enhancer elements for the El promoter.
  • ITR inverted terminal repeat
  • the adenovirus 5' sequence containing the 5' ITR and packaging/enhancer region can be employed as the 5' adenovirus sequence in recombinant adenoviruses of this invention.
  • the 3' adenovirus sequences including the right terminal (3") ITR sequence of the adenoviral genome spanning about bp 35,353 - end of the adenovirus genome, or map units "98.4-100 may be desirably employed as the 3' sequence of the recombinant adenovirus, flanking the minigene. Any necessary gene products will then be supplied by helper viruses or packaging cell lines.
  • the cis-acting and/or trans-acting minigene(s) is placed directly after Ad5 mu 0-1.
  • the 3' end of the minigene is flanked in the shuttle vector with Ad sequences from mu about 9 or 9.2 to about 16.
  • the remainder of a suitable shuttle vector may be supplied by conventional plasmid sequence, e.g., from plasmid pAT153 [Twigg et al, Nature f 283:216-218 (1980)] or other plasmid sequences.
  • each plasmid is transfected into a cell line with another adenovirus, which via homologous recombination between Ad mu 9-16, generates a complete virus.
  • the second adenovirus which supplies the
  • 3* sequences of the recombinant adenovirus after homologous recombination with the plasmid can be selected from among many adenoviruses with deletions in other genes. See, for example, the adenoviruses described in J. F. Engelhardt, Hum. Gene Ther.. 5_:1217-1229 (Oct.
  • the adenovirus which participates in homologous recombination with the plasmid(s) of this invention may be deleted of all or a portion of the adenovirus delayed early gene E3 (which spans mu 76.6 to 86.2).
  • the E3 gene product is not necessary for the formation of a functioning virus.
  • the adenovirus may contain a temperature sensitive mutation in the E2a gene at nucleotide 125 and/or another mutation or deletion in the E4 gene.
  • the adenovirus may further contain other deletions or modifications.
  • the shuttle plasmids described below which contain the minigenes in the site of an ⁇ d El deletion between Ad5 mu 1 and 9 were homologously recombined with adenovirus Ad5 dl7001 [Engelhardt et al, Nat. Genet.. 1:27-34 (1993), incorporated by reference] which supplied the Ad5 sequences spanning mu 9 to 100, with a 3 kb deletion in E3.
  • any missing Ad genes may be supplied by packaging cell lines and/or helper viruses.
  • a desired host target cell is infected with the recombinant cis-acting and trans-acting adenoviruses described above.
  • the selection of the target cell depends upon the use of the recombinant virus, i.e., whether or not the transgene is to be replicated in vitro or ex vivo for production in a desired cell type for redelivery into a patient, or in vivo for delivery to a particular cell type or tissue.
  • Target cells may therefor be any mammalian cell (preferably a human cell) .
  • the recombinant virus in in vivo use, can target to any cell type normally infected by adenovirus, depending upon the route of administration, i.e., it can target, without limitation, neurons, hepatocytes, epithelial cells and the like.
  • the cis acting elements e.g., the ITRs of MoMLV, promote transcription of the RNA transcript of the minigene. Note that transcription is driven in the direction of the arrows in the figures. As the RNA transcript is processed, e.g., introns, if any, are removed, by the cellular mechanisms.
  • the trans-acting virus expresses the transposase, in this case, the MoMLV gag and pol gene products.
  • the transposase mediates reverse transcription of the cis RNA transcript into DNA.
  • the DNA of the minigene i.e., the promoter, the transgene, and the pA sequence only
  • This integration of the cis-acting minigene provides expression of the accompanying transgene in the host cell.
  • Some transposon sequences e.g., those from MoMLV, require that the cell be dividing during the transposition or retrotransposition. Others do not.
  • the cell may be induced to divide by known means, such as the administration of various growth factors, if necessary.
  • This method thus enables the transgene to be inserted permanently into the host target cell, where it will be replicated in progeny cells.
  • the adenovirus sequences will eventually be degraded by the cell. Therefore, the use of the recombinant transposon system of this invention eliminates any need to constantly administer gene therapy vehicles to deliver a selected transgene to a patient.
  • novel recombinant viruses of this invention provide efficient gene transfer vehicles for somatic gene therapy.
  • these transgenes can be delivered to a patient in vivo or ex vivo to provide stable integration of the desired gene into a target cell.
  • these viruses can be employed to correct genetic deficiencies or defects, such as that characteristic of cystic fibrosis.
  • One of skill in the art can generate any number of other gene transfer vehicles by including a selected transgene for the treatment of other conditions or disorders.
  • the recombinant viruses of the present invention may be administered to a patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle.
  • a suitable vehicle includes sterile saline.
  • Other aqueous and non ⁇ aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.
  • the recombinant viruses of this invention may be administered in sufficient amounts to transfect the desired cells and provide sufficient levels of integration and expression of the selected transgene to provide a therapeutic benefit without undue adverse effects or with medically acceptable physiological effects which can be determined by those skilled in the medical arts.
  • Conventional and pharmaceutically acceptable parenteral routes of administration include direct delivery to the target organ, tissue or site, intranasal, intravenous, intramuscular, subcutaneous, intradermal and oral administration. Routes of administration may be combined, if desired. Dosages of the recombinant virus will depend primarily on factors such as the condition being treated, the selected gene, the age, weight and health of the patient, and may thus vary among patients.
  • a therapeutically effective human dosage of the viruses of the present invention is believed to be in the range of from about 20 to about 50 ml of saline solution containing concentrations of from about 1 x 10 7 to 1 x IO 10 pfu/ml virus of the present invention.
  • a preferred human dosage is about 20 ml saline solution at the above concentrations. The dosage will be adjusted to balance the therapeutic benefit against any side effects. The levels of expression of the selected gene can be monitored to determine the selection, adjustment or frequency of administration.
  • recombinant retroviruses are produced by transfecting a packaging cell line such as ⁇ Crip [Danos and Mulligan, Proc. Nat'l. Acad. Sci.. USA. £5.:6460-6464 (Sept. 1988)], which contains retrovirus gag, pol and env genes, with a plasmid containing a heterologous gene of interest and necessary cis elements.
  • the cell mechanism controlled by the retrovirus, will package an RNA transcript of the inserted gene, and produce a certain quantity of recombinant retroviruses carrying the inserted gene as well as a quantity of "empty" retroviral particles which do not contain the inserted gene.
  • the Ad-based cis-acting plasmid described above to transfect the cell, one can infect the cell with between lxlO 2 to lxlO 3 copies of the transgene sequence.
  • This increase in the number of transgene sequences in the producing cell enables the ratio of recombinant retrovirus to empty retrovirus to be skewed in favor of the recombinant retrovirus.
  • Example 1 Construction of plasmids for the production of recombinant adenoviruses used in the Mo-MLV retrotransposition system
  • pAdMLVneo-int TSEO ID NO: 31 The plasmid pEMB2 [Wilson et al, Science.
  • pEMB2 ⁇ RI contains a Bgl II site in place of the Eco RI site in pEMB2.
  • the 2968 bp Hind III/Bgl II fragment from PEMB2 ⁇ RI containing the Mo-MLV sequences was then isolated and ligated into the Hind III/Bgl II sites of pAdLinkl, an adenovirus based plasmid containing adenovirus map units 0-1, a multiple cloning site, Ad mu 9 through 16 and other pAT153 sequences (IGHT Vector Core) to create pAdMLVcis.
  • pAdMLVcis contains a unique Bam HI site located between the MoMLV LTRs into which the 2025 bp Bgl II fragment of pNeo-int (containing the retrotransposition dependant neo gene) was ligated to create pAdMLVneo-int [SEQ ID NO: 3].
  • a plasmid was made containing the retrotransposition dependent selectable marker without the terminal repeats to generate a recombinant virus for use as a control.
  • This plasmid was designed as follows. The 2025 bp Bam Hi/Hind III fragment of pNeo-int [described by J. D. Freeman et al, Biotechniques. 12(1) :47-50 (1994) and incorporated by reference herein] was isolated and ligated into the Bgl II/Hind III sites of pAdLinkl to create pAdneo-int [SEQ ID NO: 2].
  • Example 2 Generation of Recombinant Adenoviruses
  • a series of recombinant adenoviruses were generated using the plasmids of Example 1, so that the recombinant adenoviruses incorporate all elements of recombinant Mo-MLV necessary to achieve specific retrotransposition of a representative transgene, e.g., a neomycin resistance gene, from the recombinant adenovirus into the target cell chromatin in vitro .
  • a representative transgene e.g., a neomycin resistance gene
  • A. H5.020TKneo-intrLTR1 TSEO ID NO: 41 pAdMLVneo-int [SEQ ID NO: 3] of Example 1 was linearized by partial digestion with Nhel.
  • An E3 deleted Ad5 DNA, Ad5dl7001, a variant that carries a 3 kb deletion between mu 78.4 through 86 in the nonessential E3 region was digested with Clal to remove the left end, i.e., 917 bp from the 5' end of the adenovirus sequence, rendering the DNA non-infectious.
  • the linearized pAdMLVneo-int was co-transfected into HEK 293 cells [ATCC CRL1573] with the Clal restricted Ad5dl7001. 293 cells contain and express the transforming genes of human Ad5 to allow replication of the adenovirus.
  • the transfected packaging cells were grown in DMEM with 10% FBS without HEPES buffer in a 5% C0 2 incubator.
  • Example 2 H5.020CMV ⁇ a ⁇ -D ⁇ l TSEO ID NO: 51 pAdCMVgag-pol [SEQ ID NO: 1] of Example 1 was linearized with Nhel. The linearized plasmid was co- transfected into HEK 293 cells with Clal restricted
  • Ad5dl700l The transfected packaging cells were grown in DMEM with 10% FBS without HEPES buffer in a 5% C0 2 incubator. Following the co-transfection, only products of homologous recombination which occurred between Ad mu 9-16 of the plasmid vector and the 5' deleted Ad5dl7001 could produce replicative Ad virus in 293 cells.
  • the resulting recombinant trans-acting adenovirus, H5.020CMVgag-pol is depicted in Fig. 7B, with its sequence [SEQ ID NO: 5] provided in Fig. 9.
  • Several recombinant viral plaques were harvested and purified by two rounds of plaque purification.
  • H5.020TKneo-int TSEO ID NO: 61 pAdneo-int [SEQ ID NO: 2] of Example 1 was linearized with Nhel and transfected with Ad5dl7001 as described in parts A and B above.
  • the resulting recombinant adenovirus, H5.020TKneo-int is depicted in Fig. 7C, with its sequence [SEQ ID NO: 6] provided in Fig. 10.
  • Several recombinant viral plaques were harvested and purified by two rounds of plaque purification.
  • HeLa cells are grown under standard conditions (DMEM supplemented with 10% Fetal calf serum at 37° in a 5% C0 2 incubator) .
  • HeLa cells are infected with one of the following combinations of viruses
  • the cells that are infected with the viruses in #1 have Geneticin resistant colonies representing retrotransposed neo genes resulting in neo transduced cells.
  • Cells infected with either conditions #2 or #3 have no Geneticin resistant colonies, indicating either an integration event independent of retrotransposition or loss of the neo gene.
  • total DNA is isolated from the expanded clones from condition #1 and is subjected to Southern blot analysis. When the blots are probed with neo sequences, all clones show a band migrating with the chromosomal DNA.

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Abstract

L'invention concerne des adénovirus recombinés, avec délétion de E1, qui contiennent un transgène associé aux éléments d'une séquence de transposon recombiné, par ex., provenant du virus de la leucémie murine de Moloney (Mo-MLV), nécessaire pour effectuer une rétrotransposition spécifique du transgène à partir de l'adénovirus recombiné dans la chromatine de la cellule cible. L'invention concerne également des procédés de production desdits adénovirus recombinés et leurs procédés d'utilisation.
PCT/US1996/017176 1995-10-27 1996-10-24 Virus recombines contenant des elements genetiques mobiles et procedes d'utilisation desdits virus en therapie genique WO1997015679A1 (fr)

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Cited By (10)

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WO2000052201A2 (fr) * 1999-03-02 2000-09-08 Aventis Research & Technologies Gmbh & Co. Kg Systeme de test pour mettre en evidence une reaction d'epissure et utilisation dudit systeme
WO2000068399A2 (fr) * 1999-05-11 2000-11-16 Regents Of The University Of Minnesota Diffusion de sequences de transposon integratreur par des vecteurs
WO2001044280A2 (fr) * 1999-12-14 2001-06-21 Genovo, Inc. Methodes et compositions destinees a la fabrication d'adenovirus incompetents pour la replication
US6489458B2 (en) 1997-03-11 2002-12-03 Regents Of The University Of Minnesota DNA-based transposon system for the introduction of nucleic acid into DNA of a cell
EP1379281A2 (fr) * 2001-03-26 2004-01-14 The Board Of Trustees Of The Leland Stanford Junior University Systeme de vecteurs adenoviraux dependants d'un auxiliaire, et methodes d'utilisation
US6806080B2 (en) * 1999-01-15 2004-10-19 University Of Southern California Hybrid vectors for gene therapy
US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US7291604B2 (en) 2003-09-03 2007-11-06 The General Hospital Corporation Methods of treating restenosis
EP2441770A1 (fr) 2006-02-10 2012-04-18 The University of Cincinnati Protéine 1 d'inhibiteur de phosphatase en tant que régulateur de la fonction cardiaque
US8227432B2 (en) 2002-04-22 2012-07-24 Regents Of The University Of Minnesota Transposon system and methods of use

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WO1995023867A1 (fr) * 1994-03-03 1995-09-08 Rhone-Poulenc Rorer S.A. Adenovirus recombinants integratifs, leur preparation et leur utilisation therapeutique
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WO1995006743A2 (fr) * 1993-08-31 1995-03-09 Uab Research Foundation Procedes et compositions de production a grande echelle de virus adeno-associe recombine
WO1995023867A1 (fr) * 1994-03-03 1995-09-08 Rhone-Poulenc Rorer S.A. Adenovirus recombinants integratifs, leur preparation et leur utilisation therapeutique
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6489458B2 (en) 1997-03-11 2002-12-03 Regents Of The University Of Minnesota DNA-based transposon system for the introduction of nucleic acid into DNA of a cell
US7148203B2 (en) 1997-03-11 2006-12-12 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US7160682B2 (en) 1998-11-13 2007-01-09 Regents Of The University Of Minnesota Nucleic acid transfer vector for the introduction of nucleic acid into the DNA of a cell
US6806080B2 (en) * 1999-01-15 2004-10-19 University Of Southern California Hybrid vectors for gene therapy
WO2000052201A3 (fr) * 1999-03-02 2001-05-03 Aventis Res & Tech Gmbh & Co Systeme de test pour mettre en evidence une reaction d'epissure et utilisation dudit systeme
WO2000052201A2 (fr) * 1999-03-02 2000-09-08 Aventis Research & Technologies Gmbh & Co. Kg Systeme de test pour mettre en evidence une reaction d'epissure et utilisation dudit systeme
US6579681B1 (en) 1999-03-02 2003-06-17 Aventis Research And Technologies Gmbh & Co. Kg Test system for detecting a splicing reaction and use thereof
WO2000068399A2 (fr) * 1999-05-11 2000-11-16 Regents Of The University Of Minnesota Diffusion de sequences de transposon integratreur par des vecteurs
WO2000068399A3 (fr) * 1999-05-11 2001-01-25 Univ Minnesota Diffusion de sequences de transposon integratreur par des vecteurs
WO2001044280A2 (fr) * 1999-12-14 2001-06-21 Genovo, Inc. Methodes et compositions destinees a la fabrication d'adenovirus incompetents pour la replication
WO2001044280A3 (fr) * 1999-12-14 2001-11-29 Genovo Inc Methodes et compositions destinees a la fabrication d'adenovirus incompetents pour la replication
EP1379281A4 (fr) * 2001-03-26 2005-05-18 Univ Leland Stanford Junior Systeme de vecteurs adenoviraux dependants d'un auxiliaire, et methodes d'utilisation
US6943012B2 (en) 2001-03-26 2005-09-13 The Board Of Trustees Of The Leland Stanford Junor University Helper dependent adenoviral vector system and methods for using the same
EP1379281A2 (fr) * 2001-03-26 2004-01-14 The Board Of Trustees Of The Leland Stanford Junior University Systeme de vecteurs adenoviraux dependants d'un auxiliaire, et methodes d'utilisation
US8227432B2 (en) 2002-04-22 2012-07-24 Regents Of The University Of Minnesota Transposon system and methods of use
US7291604B2 (en) 2003-09-03 2007-11-06 The General Hospital Corporation Methods of treating restenosis
US8133878B1 (en) 2003-09-03 2012-03-13 The General Hospital Corporation Methods for treating restenosis
EP2441770A1 (fr) 2006-02-10 2012-04-18 The University of Cincinnati Protéine 1 d'inhibiteur de phosphatase en tant que régulateur de la fonction cardiaque

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