WO1998058536A1 - Lapins transgeniques exprimant la cd4 et un recepteur de chimiokine - Google Patents

Lapins transgeniques exprimant la cd4 et un recepteur de chimiokine Download PDF

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WO1998058536A1
WO1998058536A1 PCT/US1998/012935 US9812935W WO9858536A1 WO 1998058536 A1 WO1998058536 A1 WO 1998058536A1 US 9812935 W US9812935 W US 9812935W WO 9858536 A1 WO9858536 A1 WO 9858536A1
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rabbit
transgenic
human
hiv
cells
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PCT/US1998/012935
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Mark A. Goldsmith
Israel F. Charo
John M. Taylor
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J. David Gladstone Institutes
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Priority to AU81582/98A priority patent/AU8158298A/en
Priority to EP98931458A priority patent/EP0989802A1/fr
Publication of WO1998058536A1 publication Critical patent/WO1998058536A1/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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/107—Rabbit
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0337—Animal models for infectious diseases

Definitions

  • CCR5 additional host cell cof ctors, such as CCR5 , for primary macrophage-tropic strains of HIV (Deng et al . Nature 381:661-666 (1996)) and CXCR4 for T cell tropic isolates (Feng et al . Science 272:872-877 (1996)).
  • One aspect of the invention is a transgenic rabbit or rabbit cell expressing a human chemokine receptor and human CD4.
  • a further aspect of the invention is a method of generating a transgenic rabbit or rabbit cell comprising (a) introducing a transgene comprising a human chemokine receptor into a fertilized rabbit pronucleus;
  • a further aspect of the invention is a method of generating a transgenic rabbit or rabbit cell comprising
  • FIG. 1 Distinct coreceptor utilization maps to individual amino acids within the V3 hypervariable loop of gpl20. Coreceptor utilization assessed by the transfection-infection assay was determined for a panel of matched chimeric HIV-1 variants with similar V3 regions in the backbone of NL4-3. Cellular tropism (monocyte-derived macrophages and HeLa cells) and syncytium- inducing (SI) versus non-syncytium-inducing (NSI) phenotype determined by MT-2 co-cultivation assay are described in detail elsewhere and are summarized here for reference. All HIV-1 variants grew well in PBMC.
  • Figure 2 Enhanced HIV-1 infection of CHO cells in the presence of human chromosome 12.
  • Parental CHO and CHO- 12 (carrying chromosome 12) cells were transiently transfected with vectors encoding human CD4 and human CCR5 were analyzed for intracellular expression of p24 after culturing with or without HIV-1 BaL. Cells positive for both CD4 and p24 (indicated by the box marker) were markedly increased in the CHO-12 cells.
  • FIG. 3 Transfected rabbit SIRC cells are permissive for infection by specific steps in the HIV viral life cycle.
  • the SIRC cells were transfected with plasmids encoding CD4 with or without HIV-1 Nef and the relative cell surface expression of CD4 was measured by FACS .
  • SIRC cells, NIH-3T3 cells, and HeLa cells were co-transfected by an LTR reporter (CAT) construct with and without a plasmid encoding HIV-1 Tat, and the results shown in Figure 3B.
  • SIRC cells, NIH-3T3 cells, and HeLa cells also were co-transfected by a Rev-dependent reporter (CAT) construct with and without a plasmid encoding HIV-1 Rev, and the results are shown in Figure 3C.
  • CAT Rev-dependent reporter
  • FIG. 4 Total cellular mRNA was extracted from HeLa and SIRC cell lines expressing human CD4 and human CCR5 , which cell lines were cultured in the presence or absence of HIV-1 YU-2, and the mRNA analyzed for the presence of unspliced (9KB) and partially spliced (4KB) viral mRNA species. The results are shown in Figure 4. Figure 5. Transfected rabbit SIRC cells are permissive for infection by HIV-1. The indicated cell lines were stained for intracellular p24 and assessed by FACS after culturing with or without HIV-1 BaL.
  • FIG. 7 HeLa cells, SIRC cells, and NIH-3T3 cells were inoculated with various strains of HIV-1 (BaL, YU-2, JR- CSF and NL4-3) and the culture supernatants were tested for p24 content by an ELISA. Open bars represent cells encoding human CD4 and solid bars represent cells encoding both CD4 and CCR5.
  • FIG. 8 The BaL and YU-2 culture supernatants from Figure 7 were serially transferred onto PHA-blasted human PBMC and the p24 content measured by an ELISA. As shown in Figure 8, SIRC-CD4-CCR5 cells produced functional virions .
  • Figure 9 Primary rabbit peripheral blood lymphocytes were isolated and transfected with HIV clone YU-2. Culture supernatants were harvested and used to inoculate PHA- blasted human PBMC. The p24 content was measured by an ELISA, and the results shown in Figure 9.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence "TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA” .
  • nucleic acid sequence has at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, and preferably at least about 95 percent sequence identity as compared to a reference sequence.
  • the percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence.
  • the reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.
  • the reference sequence is at least 18 nucleotides long, typically at least about 30 nucleotides long, and preferably at least about 50 to 100 nucleotides long.
  • substantially complementary refers to a sequence that is complementary to a sequence that substantially corresponds to a reference sequence.
  • heterologous gene or “heterologous CD4" is defined in relation to the transgenic nonhu an organism producing such a gene product.
  • a heterologous polypeptide also referred to as a xenogeneic polypeptide, is defined as a polypeptide having an amino acid sequence or an encoding DNA sequence corresponding to that of a cognate gene found in an organism not consisting of the transgenic nonhuman animal.
  • a transgenic mouse harboring a human CD4 gene can be described as harboring a heterologous lymphocyte transduction gene.
  • a transgene containing various gene segments encoding a heterologous protein sequence may be readily identified, e.g.
  • telomere sequences may be detected in the transgenic nonhuman animals of the invention with antibodies specific for human CD4 epitopes encoded by human CD4 gene segments.
  • a cognate heterologous gene refers to a corresponding gene from another species; thus, if murine CD4 is the reference, human CD4 is a cognate heterologous gene (as is porcine, ovine, or rat CD4 , along with CD4 genes from other species) .
  • Oligonucleotides can be synthesized, for example, on an Applied Bio Systems oligonucleotide synthesizer according to specifications provided by the manufacturer.
  • transgenic rabbits can be produced using the techniques of, for example Snyder et al . (Mol . Rep. Dev. 40:419-28 (1995)), Fan et al . (Proc. Natl . Acad. Sci . U.S.A. 91:8724-8728 (1994)), and Taylor et al . (Frontiers in Bioscience 2:d298-308 (1997)).
  • Specific pathogen-free (SPF) New Zealand White rabbits are preferably used. They are preferably housed in an SPF barrier facility for at least 2 weeks before use.
  • embryos from 5-6 female donors are collected for microinjection to enable 2-3 recipients to be implanted, and approximately 6 microinjection days are planned per construct to achieve at least 3 founder animals.
  • the gestation period is 30 days and the average litter size from implanted recipients is typically 4-6 pups (the normal nontransgenic litter size is 8 pups) .
  • the pups are screened with DNA isolated from an ear biopsy.
  • approximately 10% of the recipients pups are transgene-positive . They are typically ready to mate after 5 months.
  • transgenic rabbits are generated as follows. For superovulation, 50 units of pregnant mare serum (PMS) is injected . v. to each of 5 potential embryo donors at 5 months of age, and 4 days later 50 units of human chromic gonadotropin are injected i . v. At that time, the embryo donors are mated with a proven fertile male. On the following morning, the donors are euthanized with sodium pentobarbital , and the oviducts are flushed with sterile culture medium to recover embryos: an average of 15- 20 embryos per donor are obtained. Fertilized embryos are microinj ected with a DNA solution containing the construct of interest, then they are incubated for 2-3 h to monitor survival.
  • PMS pregnant mare serum
  • microinj ected embryos are implanted through the infundibulum into the oviducts of a recipient that had been mated with a vasectomized male in the previous day.
  • 2-3 recipients are prepared per experiment for implanting 10-20 embryos in each oviduct, and at least 8 recipients (usually requiring 4 or more microinjection days, depending upon embryo yield) are planned per construct .
  • Implanted recipients are placed on a warm blanket until they have recovered from anesthesia. They are monitored daily during subsequent housing: in the fourth week, each pregnant recipient is provided with a nesting box having breeder bedding.
  • rabbit embryos Compared to the mouse, rabbit embryos have twice the diameter, a tougher and thicker cell membrane layer, a slightly more granular cytosol, and similar-sized pronuclei . Thus, preferably, twofold greater DNA concentrations are used at higher purity for rabbit embryo microinjection than for the mouse .
  • FI males are mated to several nontransgenic females whenever possible to increase the number of hemizygotes.
  • hemizygous FI males are cross-bred with FI females.
  • Candidate F2 rabbits are tested by backcrossing against nontransgenic animals.
  • Proven homozygous F2 males and females are mated to maintain an F2 line.
  • Embryos from independent transgenic rabbit lines are typically frozen for long-term storage and 2-3 males and one female are maintained as breeder stock for each construct line. At least 150 embryos are collected and placed in 1.5 M glycerol in microtubes. The tubes are cooled quickly to -7°C, cooled further to -35°C at 0.5°C/min, then plunged into liquid nitrogen. Embryos are stored in two separate liquid nitrogen tanks .
  • Rabbits are quite sensitive to noise, excessive room activity, and improper handling; and breeding efficiency, as well as recovery from surgery, can be reduced significantly in animals harboring pathogens. When rabbits are stressed, they readily absorb or abort fetuses and often ignore newborn pups. The use of SPF rabbits in barrier facility having restricted access, a nursery for pregnant and nursing females, and a temporary holding room to quarantine recently shipped rabbits while their health status is checked minimizes these problems.
  • Genotyping is performed on DNA extracted from ear punch samples as described above. Since typically up to 25% of transgene-positive founders fail to express the desired protein, preferably 3 independent founder animals (F0) for each transgene construct are obtained in order to achieve 2 expressors. These transgene-positive F0 animals are mated at sexual maturity directly with human CD4 transgenic rabbits to produce FI animals carrying both transgenes at an expected rate of 25% of the pups (based on Mendelian transmission) . Transmission of the CD4 gene in the stable transgenic rabbit line is monitored by flow cytometry of PBMC isolated from peripheral blood samples obtained by ear venipuncture .
  • Transgene expression patterns are assessed by flow cytometry and immunohistochemistry with a thorough survey of hematolymphoid (thymus, lymph nodes, spleen, peripheral blood lymphocytes and monocytes, peritoneal macrophages) and non-hematopoietic (all major organs, including the central nervous system) tissues; anti-CCR5 and anti-CXCR4 monoclonal antibodies for this purpose are now available from several sources. Animal lines with tissue-appropriate expression are expanded for studies with HIV-1.
  • rabbit cells expressing human CD4 and human CCR5 or CXCR4 are provided. These cells can be engineered by conventional transfection to express human CD4 and human CCR5 or CXCR4 or may be freshly-isolated primary rabbit lymphocytes from the transgenic rabbits of the invention.
  • the transgene DNA used for the construction of transfected cells or transgenic animals may comprise cDNA or human genomic DNA. If cDNA, a transgene is typically provided as a construct in which the transgene is placed under the control of a heterologous promoter with other appropriate elements for expression, such an enhancer sequences.
  • the coding sequences for CD4 , CCR5 , CCR3 , CXCR4 , and other chemokine receptors are well characterized in the art. Expression constructs are well known in the art and are exemplified in the Experimental Examples. If genomic DNA, typically the transgene is provided as part of a genomic DNA clone, such as in a PI or BAC clone. Such clones typically provide the transgene under the control of regulatory elements native to the transgene.
  • the CD4 and chemokine receptor transgene of choice can be separately used to generate transgenic rabbits, from which progeny transgenic rabbits can be mated to generate rabbits doubly transgenic, i.e., transgenic for both CD4 and a chemokine receptor such as CCR5.
  • both transgenes are introduced to the same embryo or host cell, either as parts of separate DNA molecules or as part of the same DNA molecule.
  • Viral gene expression and production of infectious virions in these cells can be, for example, measured as follows. Cultures are inoculated with several doses of infectious virus (quantitated by conventional endpoint dilution/TCID50 analysis) of representative strains (e.g.,
  • BaL, NL4-3, YU-2, ADA successful infection/expression is quantitated simultaneously by: (1) intracellular staining for p24 expression and FACS to determine the proportion of infected cells at each dose; (2) conventional quantitative - ELISA of secreted p24 to measure gene expression; and (3) quantitation of infectious virion production by harvesting supernatants and performing reinfection endpoint analyses on both the human and rabbit cell lines.
  • Tat and Rev functions are assessed directly and quantitatively in rabbit cells and compared with these functions in human and murine cells.
  • Rev function is assessed by an established SI nuclease assay in which Rev-defective proviruses are introduced into the target cells in the presence or absence of wild type Rev; the spliced and unspliced transcripts are then measured by nuclease protection using HIV-1-specific DNA probes (Malim, M. et al . , Mol . Cell. Biol . 13:6180-6189 (1993)).
  • Tat function is assessed by an established method involving cotransfection of an HIV- 1-LTR construct (linked to a reporter gene) with or without a Tat expression vector (Newstein, M. et al . , J Virol .
  • transgenic rabbits are provided that express human CD4 and selected human chemokine receptors (CCR5 and CXCR4 , alone and in combination) selectively in tissues that reflect the expression pattern in humans .
  • genomic constructs containing the appropriate endogenous signals for expression.
  • bacteriophage PI or BAC human genomic clones are used to encompass the necessary elements.
  • Candidate clones are obtained from a commercial vendor (for example, Genome Systems, Inc.,) and analyzed by a combination of conventional restriction mapping, PCR and Southern hybridization to identify suitable clones.
  • a CCR5 clone that contains the CCR3 gene as well is used since this receptor has been implicated in tropism of HIV-1 for the central nervous system.
  • the increasing availability of several anti-CCR5 and anti-CXCR4 antibodies now makes it possible to assess surface expression of native, untagged proteins expressed from these constructs .
  • PBMC are isolated from CCR5+/CD4+ animals, CXCR4+/CD4+, CD4+, and non-transgenic animals by venipuncture and conventional purification using density centrifugation with Ficoll-Hypaque; initial work will focus on the highest expressor lines for each transgene. Both blasted (treated with phytohemagglutin in for 24 hrs .
  • non-activated cells are cultured in recombinant human interleukin-2 (IL-2).
  • IL-2 human interleukin-2
  • Peritoneal monocyte-derived macrophages are harvested for infection studies by intraperitoneal injection of thioglycollate followed several days later by lavaging of the abdominal cavity with sterile saline.
  • Thymocytes are harvested by surgical isolation of the thymus followed by mechanical dispersion of the cells and density gradient centrifugation. Following each method of cell extraction, flow cytometry is performed with anti-CD4, anti-CD8 or anti-CDllb (macrophage) antibodies (and others as needed) to determine the purity of the cell fractions.
  • All virus stocks used for in vitro infections are preferably titrated by endpoint dilution on human activated PBMC.
  • Viral infection and spread are preferably monitored by several methods such as (1) visual inspection of the cultures via microscope for cytopathic effects and syncytium formation; (2) quantitative ELISA of secreted p24 antigen in culture medium harvested serially (every 2 days) over a 21-day period (may be narrowed or expanded as needed); and (3) intracellular staining for p24 antigen after fixing and permeabilizing the cells.
  • Cells are also preferably monitored for downregulation of surface CD4 expression (rabbit and human CD4) , which typically accompanies intracellular expression of viral gpl60, Nef and other proteins.
  • viruses are serially passaged on transgenic rabbit PBMC to verify that viral infectivity is preserved.
  • Experiments will also be performed to test the efficacy of selected human chemokines at various concentrations (MlP-l ⁇ , MIP-1 and RANTES for CCR5 ;
  • SDF-1 for CXCR4 to suppress viral spread in these cultures, since this may represent another important characteristic and disease determinant during typical human infections.
  • a range of virus strains is used in these studies.
  • One of ordinary skill in the art would appreciate that the initial choices of viruses to be used in vivo is driven by the results of these studies in cell culture, and that a representative CCR5 -dependent virus and a representative CXCR4-dependent virus are used in the early studies.
  • Seroconversion is monitored by serial collection of small peripheral blood samples (50-200 ⁇ l , every two weeks initially) and analysis using commercially available HIV-1 antibody assays adapted for rabbit serum by use of anti-rabbit-IgG antisera as the secondary antibody.
  • Successful infection is expected to result in the development of a detectable humoral response to HIV-1 within 3-4 weeks.
  • Detection of proviral DNA in lysed PBMCs and tissues is performed using DNA PCR of the highly conserved HIV-1 gag p26 segment and quantified using serial dilution, or if greater precision is required, by inclusion of an internal DNA template competitor.
  • the PCR conditions for HIV gag p26 amplification have been determined to detect reliably the presence of 10 DNA copies per reaction.
  • the small volumes of plasma available from rabbits precludes the use of standardized HIV-1 RNA load assays in their approved format. Nevertheless, the Chiron HIV-1 RNA bDNA assay is being adapted for use with small volumes (50 ⁇ l and 200 ⁇ l) to facilitate studies of human infants and small animals.
  • An ultrasensitive HIV-1 RNA QC-PCR assay adapted from previously reported assays (Piatak, M. J. et al . , Science 259:1749-1754 (1993); Piatak, M. J. et al .
  • Methods of RNA extraction based on RNA binding to activated silica are used with rabbit tissues to assure purification of viral RNA from contaminating substances that may inhibit PCR.
  • Infectious virus from HIV-1 infected rabbits is obtained by cocultivation of primary PBMCs from infected rabbits with uninfected human donor cells readily available from the Irwin Memorial Blood Bank in San Francisco. HIV-1 cocultivation is performed quantitatively or qualitatively as needed. In addition, virus is transferred from one animal to another as definitive evidence of productive infection in vivo.
  • the effects of viral infection on the animals can also be assessed in several ways.
  • Third, representative animals demonstrating robust infections with detectable changes in peripheral lymphocyte counts are sacrificed and subjected to thorough postmortem examinations and immuno-histochemistry of major hematolymphoid organs (e.g., thymus, spleen, bone marrow and lymph nodes) with anti-HIV antibodies to detect viral infection and disruptions in tissue architecture or cell morphology.
  • transgenic rabbits and cells of the invention are especially useful as animal models of HIV infection and in the screening of anti-HIV pharmaceuticals.
  • EXAMPLE 1 Structure/function analyses of HIV coreceptors A transient transfection/infection assay system was developed that is useful for distinguishing between permissivity and nonpermissivity to infection by HIV-1. Using this assay we found that the murine form of CCR5 (Boring, L. et al., J. Biol. Chem. 271:7551-7558 (1996)) exhibited virtually no detectable capacity to support infection by macrophage-tropic HIV-1, defining as least one key basis for the failure of transgenic mice expressing human CD4 to serve as permissive hosts for HIV (see for example, (Lores, P. et al., AIDS Res and Human Retroviruses . 8:2063-2071 (1992)).
  • chimeric human/mouse CCR5 receptors were prepared and evaluated for HIV-1 coreceptor function. Extensive experiments with selective substitutions demonstrated that multiple elements distributed throughout the extracellular segments contribute to viral entry. Similar observations recently have been reported by others (Bieniasz, P. et al., EMBO J. (1997)). Further studies with chimeric receptors revealed that viral coreceptor activity is dissociable from ligand-dependent signaling responses (Atchison, R. E. et al . , Science 274:1924-1926 (1996)). Additional studies involved mutating human CCR5 within the highly conserved aspartate-arginine-tyrosine (DRY) sequence - that is thought to be critical for G-protein coupling.
  • DRY aspartate-arginine-tyrosine
  • CCR5 mediates viral entry into macrophages
  • CXCR4 mediates entry into many CD4 -positive transformed T-cell lines.
  • CD4 -positive transformed T-cell lines Although virtually all primary HIV-1 isolates replicate in primary CD4 -positive T-lymphocytes, certain variants ( "macrophage-tropic ) fail to infect transformed T-cell lines, whereas other strains ("T-cell tropic ) replicate well in these cell lines but not in macrophages. Changes in cellular tropism by HIV-1 strains seem to be a key event in the pathogenesis of HIV-1 disease (Gouilleux, F. et al., EMBO J. 14:2005-2013 (1995); Koot , M. et al . , J. Infect. Pis.
  • Native rabbit T cells are partially susceptible to infection by high-titer HIV-1 stocks in vitro and in vivo (Filice, G. et al . , Nature 335:366-369 (1988); Gordon, M.R. et al . , Annals of the New York Academy of Sciences 616:270-280 (1990); Reina, S. et al . , J. Virol. 67 (9) : 5367-5374 (1993); Kulaga, H. et al . , Proc . Natl . Acad. Sci . U.S.A. 85:4455-4459(1988)).
  • a transfectable rabbit epithelial cell line (SIRC) was transfected by conventional methods with plasmids encoding human CD4 with or without HIV-1 Nef, and the relative cell surface expression of CD4 was measured by fluorescence-activated cell sorting (FACS) ; as observed previously in human cells, the presence of HIV-1 Nef markedly attenuated the surface expression of human CD4 in rabbit cells as shown qualitatively by FACS profiles (Fig. 3A, left) and by statistical analysis of the FACS histograms (Fig. 3A, right) . Therefore, rabbit cells are permissive for the downregulation activity of HIV-1 Nef, which is one its key recognized functions.
  • FACS fluorescence-activated cell sorting
  • HIV-1 Tat in promoting expression of viral genes via the HIV-1 LTR was tested in SIRC cells by co-transfection of an LTR reporter (CAT) construct with and without a plasmid encoding HIV-1 Tat. While Tat only modestly augmented LTR activity in murine (NIH- 3T3) cells compared to its robust activity in human (HeLa) cells, Tat -dependent expression of the reporter gene was readily detected in rabbit cells (Fig. 3B) . Therefore, rabbit cells (but not mouse cells) support HIV-1 Tat function.
  • CAT Rev-dependent reporter
  • rabbit cells support HIV-1 Rev function. Further analysis of the permissivity of rabbit cells for HIV infection and for Rev function per se was performed using stable transfectants of the SIRC and HeLa cell lines expressing human CD4 and human CCR5. Total cellular RNA extracted from cells cultured in the presence or absence of the HIV-1 strain YU-2 was analyzed by Northern blotting for the presence of unspliced (9kB) and partially spliced (4kB) viral mRNA species.
  • cDNA constructs #1 and #2 in established vectors intended to promote broad tissue expression: one utilizes the murine major histocompatibility complex (MHC) class I promoter region, which drives expression in many hematolymphoid and other tissues; the second utilizes a promoter/enhancer derived from cytomegalovirus (CMV) , which also drives relatively unselective tissue expression.
  • MHC murine major histocompatibility complex
  • CMV cytomegalovirus
  • construct 1 contains promoter region from murine Major Histocompatibility Complex (MHC) class I genes upstream of the human CCR5 cDNA and a rabbit ⁇ -globin polyadenylation sequence downstream of the CCR5 cDNA.
  • the CCR5 sequence encodes an epitope-tagged variant of human CCR5.
  • Construct 2 contains promoter/enhancer region from cytomegalovirus upstream of the human CCR5 cDNA and a rabbit ⁇ -globin polyadenylation sequence downstream of the CCR5 cDNA.
  • CCR5 from this construct is not epitope-tagged.
  • MO human chromosome 3
  • CCR2B and CCR5 have been shown to be approximately 20 kb apart (Raport et al . , J. Biol. Chem. 271:17101 (1996)).
  • the three PI clones were tested for human CCR5 by PCR analysis and for length of the 3 ' UT region by PCR on all three clones using vector specific primers (T7 and Sp6) and primers from the 3' UT region of the hCCR5 cDNA.
  • clone 2426 and 2427 contained hCCR5 and greater than 3 kb of 3 ' UT of hCCR5 in each clone.
  • Some preliminary Southerns have been done on the 2426 clone showing an insert of approximately 80 kb by pulsed field gel electrophoresis.
  • An Miu I fragment of 2426 which contains the entire insert plus 2 kb of 5' vector sequence and 5 kb of 3' vector sequence has been isolated and injected into mice, as described by Linton et al . , (Linton et al., J. Clin. Invest. 92 (6) : 3029-3037 (1993)).

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Abstract

Cette invention se rapporte à des lapins transgéniques et à des cellules de lapins transgéniques exprimant la molécule CD4 et un récepteur de chimiokine humaine tel que CXR4 ou CCR5. Cette invention se rapporte également à des procédés de création de tels lapins transgéniques. On génère le double transgène soit en introduisant les deux entités transgéniques dans un pronucleus fécondé, soit en élevant deux lapins transgéniques possédant chacun une des entités transgéniques. Ce lapin peut être contaminé par le VIH.
PCT/US1998/012935 1997-06-23 1998-06-22 Lapins transgeniques exprimant la cd4 et un recepteur de chimiokine WO1998058536A1 (fr)

Priority Applications (3)

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CA002295059A CA2295059A1 (fr) 1997-06-23 1998-06-22 Lapins transgeniques exprimant la cd4 et un recepteur de chimiokine
AU81582/98A AU8158298A (en) 1997-06-23 1998-06-22 Transgenic rabbits expressing cd4 and chemokine receptor
EP98931458A EP0989802A1 (fr) 1997-06-23 1998-06-22 Lapins transgeniques exprimant la cd4 et un recepteur de chimiokine

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US5048097P 1997-06-23 1997-06-23
US60/050,480 1997-06-23

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000039316A1 (fr) * 1998-12-31 2000-07-06 The J. David Gladstone Institutes Rongeurs transgeniques et lignees cellulaires de rongeur exprimant des co-recepteurs du vih
WO2003000869A1 (fr) * 2001-06-25 2003-01-03 Geneticlab Co., Ltd. Animaux transgeniques et cellules exprimant des proteines necessaires pour la sensibilite a l'infection par vih
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6563014B2 (en) * 1999-12-14 2003-05-13 Albert Einstein College Of Medicine Of Yeshiva University Self-contained system for sustained viral replication
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000568A1 (fr) * 1992-06-19 1994-01-06 Transgene S.A. Lapin transgenique sensible au hiv, son usage a titre de modele animal, et son procede d'obtention
WO1996031242A1 (fr) * 1995-04-07 1996-10-10 Albert Einstein College Of Medicine Of Yeshiva University CELLULE β DE RECOMBINAISON ET SES UTILISATIONS
WO1997028258A1 (fr) * 1996-01-30 1997-08-07 The National Institutes Of Health Cellules exprimant les molecules humaines cd4 et cxcr4

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994000568A1 (fr) * 1992-06-19 1994-01-06 Transgene S.A. Lapin transgenique sensible au hiv, son usage a titre de modele animal, et son procede d'obtention
WO1996031242A1 (fr) * 1995-04-07 1996-10-10 Albert Einstein College Of Medicine Of Yeshiva University CELLULE β DE RECOMBINAISON ET SES UTILISATIONS
WO1997028258A1 (fr) * 1996-01-30 1997-08-07 The National Institutes Of Health Cellules exprimant les molecules humaines cd4 et cxcr4

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YU FENG ET AL: "HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor", SCIENCE, vol. 272, May 1996 (1996-05-01), XP002027999, DOI: doi:10.1126/science.272.5263.872 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6759519B2 (en) 1995-06-06 2004-07-06 Human Genome Sciences, Inc. Antibodies to human G-protein chemokine receptor HDGNR10 (CCR5receptor)
US6800729B2 (en) 1995-06-06 2004-10-05 Human Genome Sciences, Inc. Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor)
WO2000039316A1 (fr) * 1998-12-31 2000-07-06 The J. David Gladstone Institutes Rongeurs transgeniques et lignees cellulaires de rongeur exprimant des co-recepteurs du vih
US6563014B2 (en) * 1999-12-14 2003-05-13 Albert Einstein College Of Medicine Of Yeshiva University Self-contained system for sustained viral replication
WO2003000869A1 (fr) * 2001-06-25 2003-01-03 Geneticlab Co., Ltd. Animaux transgeniques et cellules exprimant des proteines necessaires pour la sensibilite a l'infection par vih

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