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WO1992010574A1 - Inhibition du virus de l'immunodeficience humaine a l'aide d'un gene viral a association adeno des cellules humaines - Google Patents

Inhibition du virus de l'immunodeficience humaine a l'aide d'un gene viral a association adeno des cellules humaines Download PDF

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WO1992010574A1
WO1992010574A1 PCT/US1991/009008 US9109008W WO9210574A1 WO 1992010574 A1 WO1992010574 A1 WO 1992010574A1 US 9109008 W US9109008 W US 9109008W WO 9210574 A1 WO9210574 A1 WO 9210574A1
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gene
hiv
rep
lentivirus
recombinant plasmid
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PCT/US1991/009008
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English (en)
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Barrie J. Carter
Beth Ann Antoni
Arnold B. Rabson
Irving L. Miller
James P. Trempe
Nor Chejanovsky
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The United States Of America, As Represented By The Secretary, U.S. Department Of Commerce
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Publication of WO1992010574A1 publication Critical patent/WO1992010574A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • AIDS immunodeficiency syndrome
  • HAV human immunodeficiency virus
  • T4 + T cells Reviewed in Gallo, R.C., 1987, Sci. Am. 256:47-56; Fauci, A.S., 1988, Science 239;616- 622
  • Development of AIDS almost always leads to death and generation of effective therapies against this virus are essential. HIV infection disrupts immune responses and development of AIDS vaccines is rendered extremely difficult.
  • HIVl is the prototype human lentivirus representing a group of pathogenic retroviruses which induce chronic disease.
  • the current state of our understanding indicates that the life cycle of HIV is controlled by a complex system of trans-acting gene regulation mediated by several HIV genes.
  • permissive cells such as activated T-cells
  • infection by HIV leads to a complex cascade of gene regulation which occurs in two stages.
  • All the HIV genes are transcribed from a single RNA transcription promoter located in the 5" terminal LTR (long terminal repeat) region. In the early stage of expression, a low level of transcription leads to expression of spliced RNAs that express several HIV genes including tat, rev and nef. These genes are trans-acting regulators which alter the
  • the most dominant HIV gene in the regulation scheme is tat.
  • the Tat protein recognizes and element, TAR, in the 5' end of all HIV mRNAs and activates transcription and perhaps translation of its own gene and all other HIV genes (Hauber, et al, 1987, Proc. Natl. Acad. Sci. USA, ji4_:6363; Ja obovitz et al. , 1988, Mol. Cell. Biol. 8.:2555: Berkhout et al ., 1989 Cell 59:273).
  • Tat represents a positive feedback trans-activation system and expression of Tat is critical both for replication (Dayton et al., 1986, Cell 44:941; Fisher et al., 1986 Nature 320:367) and for high level gene expression of HIV (Sodroski et al., 1985, Science 229:74; Arya, et al., 1985, Science 229:69; Rosen, et al., 1985, Cell 4_1:813; Cullen, 1986, Cell 46:973; Peterlin, et al., 1986, Proc. Natl. Acad. Sci.
  • Tat represents a potential target for therapeutic intervention.
  • expression of this interfering agent is induced by the tat gene so that it could function as a negative feedback inhibitor of Tat and thus of HIV growth.
  • the invention is based upon the concept of using another trans-acting gene product or protein to inhibit the trans-acting regulation of HIV. More specifically, the invention is based upon use of a trans-acting gene of
  • SUBSTITUTESHEET a heterologous virus to inhibit trans-regulation in HIV.
  • a variety of trans-acting genes of heterologous viruses have been reported to activate HIV gene expression (for example Gendelman et al., 1986, Proc. Natl. Acad. Sci. USA. .82:9759) generally by enhancing transcription directed by the HIV-LTR.
  • No trans-acting gene of heterologous viruses have been reported to inhibit HIV gene expression.
  • a trans-acting gene from a human parvovirus adeno-associated virus type 2 (AAV2) can inhibit tat-mediated activation of gene expression and that it can block production of infectious HIV particles in human cells.
  • Our invention is based on a concept different from that based on trans-dominant mutant phenotypes as described recently.
  • Herskowitz (1987, Nature 329:219- 222) discussed the usefulness of generation of mutations in genes that create a dominant negative phenotype in studying and analysing gene function. A number of groups have used this approach and for instance Friedman et al.
  • Adeno-associated virus Adeno-associated virus
  • Parvovirdae or parvoviruses Adeno-associated virus
  • AAV-2 human adeno-associated virus type 2
  • AAV is distinguished from other parvoviruses in that it grows efficiently only in cells coinfected by a helper virus.
  • Helper viruses include adenoviruses, herpes virus and pox viruses. In the absence of helper virus, the AAV genome is integrated into the host-cell chromosome at high efficiency but there is little or on AAV gene expression from the integrated provirus.
  • AAV exhibits several inhibitory functions such as inhibition of tumor induction in hamsters by oncogenic adenoviruses (Kirschstein et al., 1968, Proc. Soc. Exp. Biol. Med. 128:670; de la Maza and Carter, 1981, J. Natl. Cancer Inst. j67:1323), decrease in tumorigenicity of cells transformed by a viral or cellular on ⁇ ogene (Ostrove et al., 1981, Virology, 113:521; Katz and Carter, 1986, Cancer Research. 4 ⁇ :3023) and inhibition of growth of the helper virus (Carter and Laughlin 1984, the Parvoviruses, Plenum Press, pp. 67-123) .
  • AAV inhibition of adenovirus-induced tumors in hamsters required only terminal regions of AAV DNA which contained the replication origin but no gene coding regions (de al Maza and Carter, 1981) . More recently an AAV gene, rep, has been discovered which has some trans-acting inhibitory properties.
  • Rep78 and rep68 are coded by mRNA transcribed from the p 5 promoter and Rep52 and Rep40 are coded by MRNA transcribed from the p 1g promoter.
  • the AAV rep gene mediates both complex positive and negative regulatory effects (reviewed in Carter et al., 1990 Handbook of Parvoviruses. CRC Press, Vol. I pp. 227- 254) .
  • Rep is auto-regulated and shows strong negative regulation of its own synthesis (Chejanovsky and Carter, 1990, J. Virol, in press; Antoni et al., manuscript in preparation) .
  • rep may activate expression of AAV genes (Tratschin et al., 1986, Mol. Cell. Biol. 6.:2883; Labow et al., 1986, J. Virol.. j5():251; Trempe and Carter 1988, J. Virol.
  • Rep may negatively regulate expression of heterologous prokaryotic genes from AAV promoters (Tratschin et al., 1986, Mol Cell. Biol. €5:2883; Trempe and Carter, 1988, J. Virol. jS2:68; Mendelson et al., 1988, Virology 166:154 and Virology 166:612) or from heterologous promoters (Labow et al., 1987. Mol. Cell. Biol. 7:1320).
  • AAV promoters Tratschin et al., 1986, Mol Cell. Biol. €5:2883; Trempe and Carter, 1988, J. Virol. jS2:68; Mendelson et al., 1988, Virology 166:154 and Virology 166:612
  • heterologous promoters Labow et al., 1987. Mol. Cell. Biol. 7:1320.
  • Rep protein in the present invention we modify expression of rep to overcome the otherwise strong negative autoregulation by Rep protein on its own synthesis.
  • Rep proteins can inhibit growth of HIV and production of infectious HIV in human cells.
  • the modification of rep expression leads to increased Rep protein and this leads to more efficient inhibition of HIV than for Rep expressed from an AAV genome background.
  • Rep gene or the rep gene product i.e. Rep protein
  • Rep protein can be used to inhibit production of infectious HIV and this forms the basis of a novel approach to therapeutic intervention in treatment of HIV mediated disease.
  • AAV adeno-associated virus
  • Figure 1 shows a schematic diagram of construction of a recombinant plasmid, pHIVrep DNA to express the AAV rep gene according to the present invention.
  • the CAT gene was removed from pBennCAT by cleaving with the enzymes Hindlll and BamH-I.
  • Figure 2 shows construction of a plasmid pHIVrepam having an amber (nonsense) mutation in the AAV rep gene.
  • pNTC3 which contains an AAV genome having an amber mutation (am) in the rep gene, was made by converting nucleotide 1033 from C to A (Chejanovsky and Carter, 1988, Virology. 171:2391).
  • pNTC3 was cleaved with Sstl and Hindlll to yield a 1 kb fragment spanning the region of the AAV genome containing the amber mutation.
  • pHIVrep was cleaved with Sstl and Hindlll to yield a large fragment, a 1 kb Sstl/Hindlll fragment and a 600 bp Sstl fragment.
  • the largest Sstl/Hindlll fragment of pHIVrep was ligated with the 1 kb Sstl/Hindlll fragment from pNTC3 to yield pHIVrepamdlS (pNT39) .
  • This latter plasmid was cleaved with Sstl and ligated with the 600 bp Sstl fragment from pHIV to yield PHIVrepam.
  • FIG. 3 summarizes the structure of the AAV genome and the relevant features of pHIVrep and pHIVrepam.
  • Stippled boxes indicate terminal repeats (replication origins) and solid circles indicate transcription promoters, (p 5 , p 19 , and p 40 ) .
  • the poly(A) site is at map position 96.
  • RNAs from AAV promoters are shown as heavy arrows with the introns indicated by the caret.
  • the coding regions for the four rep proteins (Rep78, Rep68, Rep52, and Rep40) and for the viral capsids (VP1, VP2, and VP3) are shown with open boxes.
  • pAV2 contains an entire AAV2 genome and pJDT269 contains a deletion in the rep gene shown by the gap.
  • p DT95 contains a deletion of a Hindi fragment (Hc2/Hc3) in the capsid gene region.
  • pHIVLTR contains 553 nucleotides of an HIV LTR region (cross hatching) extending to nucleotide +80 relative to the RNA transcription, start site (heavy arrow) .
  • the TAR region (+1 to +44) is shown by the open box.
  • the vertically shaded region is about 500 base pairs of cellular DNA sequence derived from the original proviral clone pB2.
  • pHIVrep contains the same AAV region as in pJDT95 i.e. from AAV2 nucleotide 263 to 4678 but containing the deletion of the capsid genes. AAV RNA transcription start sites are shown by the light arrows.
  • pHIVrepam contains an amber mutation at AAV nucleotide 1033. pHIVrep has the region between two Sail sites inverted.
  • Figure 4 Effect of AAV rep gene on tat-mediated activation of expression of CAT from pHIVcat in human cells.
  • Cultures of human 293 cells (10 6 cells per culture) were transfected, using the calcium phosphate procedure, with 1 ⁇ g of pBenncat together with 10 ⁇ g of either pJDT269, pAHP, pAV2 or pHIVrep as indicated on the figure and varying amounts of a pARtat plasmid as indicated on the abscissa.
  • the transfected cells were grown at 37°C for 48 hours then lysates were prepared and CAT activity was measured.
  • FIG. 5 Western Analysis of Tat induction of Rep proteins in 293 cells. Plasmids pAV2 and pHIVrep were each cotransfected with increasing amounts of pHIVtat into 293 human cells. Panels A and B represent transfection of 0.1 or 1.0 ⁇ g of pHIVrep, respectively. Panels C and D represent transfection 0.1 or 1.0 ⁇ g of pAV2, respectively. In panels A, B, C and D lanes 1 to 4 represent cultures in which increasing amounts of pARtat plasmid (0, 0.01, 0.05 and 0.1 ⁇ g, respectively were cotransfected). Panel E, lanes 5 to 7, represents control transfections. Figure 6 Northern analysis of Tat induction of rep RNA transcription.
  • Plasmids pHIVrep and pHIVrepam were each cotransfected with increasing amounts of the pARtat plasmid into cultures of 293 cells.
  • L and H refer to transfection of 0.1 ⁇ g or 1.0 ⁇ g respectively, of pHIVrep or pHIVrepam.
  • Panels A, B, and C refer to, respectively, 0, 0.01 or 0.10 ⁇ g, respectively, of cotransfected pARtat.
  • FIG. 7 Induction of Rep by Tat in HeLa Cells.
  • Cultures of 10 6 HeLa cells were transfected by the calcium phosphate method including a glycerol-shock, with 5 ⁇ g (L) or 10 ⁇ g (H) of either pHIV rep or pAV2 or 10 ⁇ g of control carrier plasmid, pGEM4Z.
  • pARtat was cotransfected at 2.5 ⁇ g (lanes 2, 5, 8, 11 and 14) or 5.0 ⁇ g (lanes 3, 6, 9, 12 and 15).
  • Cells were harvested after 48 hours and the nuclear fractions were analyzed by SDS-PAGE and immunoblotting with an antibody to Rep. The Rep proteins were visualized by incubation with radiolabeled goat anti-rabbit IgG and autoradiography.
  • FIG 8 Effect of Rep of HIV production.
  • Cultures of 10 6 HeLa cells were transfected by the calcium phosphate method with 10 ⁇ g of HIV proviral clone (pNL432) and the amounts of pHIVrep of pHIVrepam as indicated. All transfection mixes were adjusted to the same amount of DNA using a carrier plasmid (pUC18) .
  • the mock transfection contained only pUCl ⁇ DNA.
  • the medium from each culture was collected 48 hours after transfection and assayed for the HIV p24 (gag) antigen using an antigen capture assay (Dupont-NEN Research Products) .
  • Figure 9 Western analysis of HIV inhibition by Rep.
  • lane 1 represents the mock transfection using only pGEM4Z
  • lane 2 represents transfection with pNL432 alone
  • lanes 3, 5, and 7 represent cotransfection of pNL432 with 10 ⁇ g, 3.0 ⁇ g and 1.0 ⁇ g of pHIVrep, respectively
  • lanes 4 and 6 represent cotransfection of pNL432 with 10 ⁇ g and 3.0 ⁇ g of pHIVrepA, respectively.
  • All human cell lines were grown in monolayers at 37°C in 5% C0 2 in Dulbecco's minimal essential medium supplemented with fetal bovine serum (10% v/v) and the antibiotics penicillin and streptomycin.
  • 293 cells are human embryonic kidney cells transformed by adenovirus type 5 (Graham et al., 1977, J. Gen. Virol. 36:59) obtained originally from N. Jones (Purdue University, Indiana) .
  • HeLaJW cells are a subclone of the human cervical carcinoma cell line, HeLa, obtained from J. Rose (NIH) .
  • SW480 cells are a human colon carcinoma cell line obtained from the American Type Culture Collection at passage number 100.
  • Plasmids pAV2 contains an entire infectious AAV2 genome inserted into pAHP, a pBR322 based plasmid (Laughlin et al., 1983, Gene. 23.:65).
  • pJDT269 a rep derivative of pAV2
  • JDT95 is a cap derivative of pAV2 (Tratschin et al., 1984, J. Virol. 51:611).
  • pNL43 contains an infectious HIV proviral genome (Adachi et al., 1986, J. Virol. 59:284) and pARtat (pHIVtat) contains only the first exon of the Tat coding sequence expressed from the HIVLTR region of pNL43
  • pSVtat contains the first exon of the Tat coding sequence but expressed from a SV40 early region promoter (Jeang et al., 1988 J. Virol. 62.:3874).
  • pNL432 also contains the same infectious HIV plasmid genome as pNL43 and was derived from pNL43 by deletion of flanking cellular sequences. Thus pNL432 contains only 1.5 kb of flanking cell sequence whereas pNL43 contains 9 to 12 kb of flanking sequence on either side.
  • pHIV-CAT plasmids expressing chloramphenicol acetyltransferase (cat) from an HIV-LTR promoter were used.
  • pBennCat (Gendelman et al., 1986, Proc. Natl. Acad. Sci. USA 83.:9759) the HIV-LTR was obtained from an HIV provirus pB2 (Benn et al., 1985, Science 230:949) as described in Figure 1.
  • pUCCat the HIV-LTR-cat region of pBennCat was inserted into a pUC18 vector.
  • pBennCat and pUCCat are functionally equivalent and collectively referred to as pHIV-cat.
  • pHIVrep contains the AAV rep gene expressed from an HIV-LTR ( Figure 1) .
  • An American Type Culture Collection deposit under the Budapest Treaty of E ⁇ coli containing the plasmid pHIVrep was accepted by the ATCC on June 20, 1990 and given the accession no. 68342.
  • pHIVrepam contains an amber (nonsense) mutation in the rep gene ( Figure 2) .
  • pHIVrep contains a rearrangement in the rep gene ( Figure 1) .
  • pAHP, pUC18, pUC19, or pGEM4Z Promega Corp., Madison, Wisconsin
  • Plasmids were constructed and grown and DNA was purified using standard techniques as generally described in Sambrook et al., 1989 Molecular Cloning. Cold Spring Harbor Laboratory Press. Transfection of DNA in Cells
  • CAT activity was measured by acetylation of 14 C- chloramphenicol in reactions containing l to 40 ⁇ l of cell extract as described (Garman et al., 1982, Mol. Cell. Biol. 2.:1044). All extracts were assayed in the linear range i.e. not more than 50% acetylation of the substrate. Results are expressed in arbitrary units of CAT activity where 1 unit results in acetylation of 1% of the substrate in 1 hr at 39 C. In some transfection experiments the levels of CAT activity were than expressed as a percentage of the control. Analysis of RNA
  • Proteins were extracted from cells and analysed by gel electrophoresis and immunoblotting (Western blotting) as described using an antibody to AAV rep protein raised against a synthetic oligopeptide (Mendelson, et al., 1986, J. Virol. .60:823) or serum from an HIV patent to detect the HIV p24 (gag) protein and its precursor p55. Analysis of HIV production
  • the production of infectious HIV was determined by assaying portions of cell culture medium for the p24 gag protein using the antigen capture assay kit (Dupont NEN Research Products) and for viral reverse transcriptase using a 32 p-TTP based assay (Willey et al., 1987, J. Virol. 67:139).
  • Structure of HIV rep and related lasmids pHIVrep was constructed in order to replace the AAV p 5 promoter with the HIV transcription promoter comprising the HIV LTR and sequences extending to the Hindlll site at +80 nucleotides downstream of the HIV mRNA start site (Fig. 1,3).
  • the pHIVrep construct contains AAV sequences beginning from nucleotide 263 immediately downstream from the p 5 TATA box.
  • AAV p 5 promoter sequences retains the normal start side of the AAV mRNA (p 5 mRNA) coding for rep78 and rep68 in addition to the start site of the HIV LTR promoter. As shown below only the HIV LTR start site is used in transcription from this plasmid. Because HIVrep retains the TAR region (nucleotides +1 to +44) of HIV LTR it was designed to be inducible by tat. pHIVrepam has a nonsense mutation that prevents expression of any functional rep proteins except under special conditions in the presence of a suppressor tRNA as described in Chejanovsky and Carter (1989, Virology 171:2391 and U.S.
  • pHIVrepam is identical to pHIVrep.
  • pHIVLTR contains the HIV LTR but has no inserted AAV sequence.
  • pHIVrep has a rearranged rep gene and also expresses no functional rep protein. Inhibition of tat function bv pHIVrep
  • pHIVrepam or pHIVrep probably reflects competition for factors binding to the HIVLTR or TAR region because pHIVLTR showed a similar modest inhibition. Similar results were seen in both 293 cells and SW480 cells. Thus, the inhibition by pHIVrep in 293 cells is not accounted for by inhibition of the adenovirus transcriptional activator EIA present in these cells but not in SW480 cells. Similar results to those shown in Fig. 4 and Table I were obtained in other experiments when Tat was supplied from an SV40 promoter using pSVtat (B. Antoni, unpublished) or when lipofection was used rather than calcium phosphate precipitation to introduce DNA into cells (I. Miller, unpublished) . Expression of Rep proteins and RNA from pHIV Rep
  • Infectious HIV particles can be efficiently generated in HeLa cells transfected with an HIV proviral clone, pNL432. Accumulation of HIV into the medium from transfected HeLa cells can be measured by a viral reverse transcriptase assay (Table II) or by detection of the HIV p24 gag protein in an antigen capture immunoassay (Fig. 8) .
  • a viral reverse transcriptase assay Table II
  • Fig. 8 antigen capture immunoassay
  • the level of viral reverse transcriptase was inhibited at least 50 fold whereas there was little or no inhibition (less than 2-fold) with pHIVrepam. Furthermore the inhibition was dose-dependent with respect to rep.
  • Assay of p24 antigen in the cell medium showed a similar strong and dose- dependent inhibition by pHIVrep but not by pHIVrepam.
  • Rep52 and Rep40 are also expressed at low levels from HIVrep from RNA transcripts originating from the p t9 promoter but this promoter is not induced by Tat. It is now rendered obvious by our invention that, even if the inhibition of HIV is mediated mainly by Rep78, the other Rep proteins might also have an inhibitory function. A more general likelihood, which is an obvious extension of the present invention, is that the domain or portion of the Rep proteins responsible for inhibition of HIV could be identified by appropriate mutations or alteration by standard techniques obvious to anyone skilled in the art. Thus the recombinant construction pHIVrep is one embodiment only of the invention.
  • the AAV Rep protein or Rep proteins could be introduced into cells in a variety of alternative ways as a means of inhibiting HIV production.
  • One embodiment shown here is by introduction of the rep gene as a recombinant plasmid using an HIV LTR to direct expression.
  • the rep gene might be introduced into said mammal in different ways either by using various virus vector systems or other delivery methods, such as liposomes, or by introduction of the gene into cells which are transplanted into the mammal.
  • a cell may be transfected with the recombinant plasmid according to the instant invention using various conventional transfer means, such as liposomes or viral vectors, e.g., vaccinia, SV-40, retroviral vectors, etc.
  • the Rep protein may be administered directly to a mammal or to said mammal's cells using a pharmaceutically acceptable carrier or a conventional means of administration known in the art, such as liposomes.
  • it could inhibit productive infections of other classes of human retroviruses such as HIV2, HTLVI and HTLVII.
  • the rep gene of AAV serotype 2 Another obvious development of the invention is that the rep gene or Rep protein from other AAV serotypes or species could be used in an analogous way to inhibit production of HIV or related viruses.
  • the mutant Ela gene product described by Ventura et al. differs from the wild-type Ela gene by virtue of a single point mutation. As such the mutant Ela gene that may inhibit HIV could readily mutate (i.e., revert) to a wild-type Ela gene that would then activate HIV.
  • our invention uses a parvovirus gene, rep, with no homology to HIV and because we use the wild-type rep gene coding sequence as the inhibitory gene it cannot revert to a form that activates HIV.
  • our invention is much more efficient at inhibiting HIV as measured by production of infectious HIV from an HIV plasmid transfected into HeLa cells. As measured by extracellular reverse transcriptase activity (Table I) or p24 antigen assay (Fig. 8) our invention inhibited HIV by 59 to 78 fold (that is to less than 1.5% of the control level).
  • the mutant adenovirus gene of Ventura et al. only inhibited extracellular p24 antigen at best by 3 fold (i.e. to 33% of the control). Therefore, our invention is much more effective and does not suffer from the disadvantages of reversion to wild- type as noted above for the invention of Ventura et al.

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Abstract

Plasmide recombiné comportant un promoteur de transcription apte à être induit par des produits génétiques exprimés par un lentivirus humain et par un gène non homologue de celui-ci. Le gène non homologue code un produit génétique qui inhibe la croissance lentivirale et la production de protéines lentivirales en parasitant l'action trans du système de régulation génique du lentivirus. Ledit promoteur de transcription accroît davantage l'expression du gène non homologue. On a également prévu un procédé d'inhibition chez les mammifères de la croissance lentivirale et de la production de protéines lentivirales, comprenant la transfection avec ledit plasmide recombiné de cellules prélevées sur des mammifères infectés par le lentivirus, ainsi qu'un procédé d'inhibition de la croissance et de la production lentivirales, comprenant l'administration à des mammifères infectés par le lentivirus de la protéine Rep codée par ledit gène non homologue dans un excipient pharmaceutiquement acceptable.
PCT/US1991/009008 1990-12-06 1991-12-06 Inhibition du virus de l'immunodeficience humaine a l'aide d'un gene viral a association adeno des cellules humaines WO1992010574A1 (fr)

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Cited By (2)

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EP0486917A2 (fr) * 1990-11-17 1992-05-27 BEHRINGWERKE Aktiengesellschaft Activité antivirale du gène rep codé par l'AAV-2
EP0733103A1 (fr) * 1993-11-09 1996-09-25 Targeted Genetics Corporation Production de titres eleves de vecteurs d'aav recombinants

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US4981790A (en) * 1984-05-25 1991-01-01 Dana Farber Cancer Institute Stable TatIII cell lines, TatIII gene products, and assay methods

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US4981790A (en) * 1984-05-25 1991-01-01 Dana Farber Cancer Institute Stable TatIII cell lines, TatIII gene products, and assay methods

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0486917A2 (fr) * 1990-11-17 1992-05-27 BEHRINGWERKE Aktiengesellschaft Activité antivirale du gène rep codé par l'AAV-2
EP0486917A3 (en) * 1990-11-17 1992-12-16 Behringwerke Aktiengesellschaft Antiviral activity from the rep gene of adenoassociated virus-2
EP0733103A1 (fr) * 1993-11-09 1996-09-25 Targeted Genetics Corporation Production de titres eleves de vecteurs d'aav recombinants
EP0733103A4 (fr) * 1993-11-09 1997-12-17 Targeted Genetics Corp Production de titres eleves de vecteurs d'aav recombinants

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