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WO1990011359A1 - Procede intracellulaire d'inhibition de l'hiv dans des cellules de mammiferes - Google Patents

Procede intracellulaire d'inhibition de l'hiv dans des cellules de mammiferes Download PDF

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Publication number
WO1990011359A1
WO1990011359A1 PCT/US1990/001266 US9001266W WO9011359A1 WO 1990011359 A1 WO1990011359 A1 WO 1990011359A1 US 9001266 W US9001266 W US 9001266W WO 9011359 A1 WO9011359 A1 WO 9011359A1
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Prior art keywords
hiv
cells
construct
infected
dna
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PCT/US1990/001266
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English (en)
Inventor
David Baltimore
Elaine A. Dzierzak
Richard C. Mulligan
Xiao-Hong Sun
Didier Trono
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Whitehead Institute For Biomedical Research
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Publication of WO1990011359A1 publication Critical patent/WO1990011359A1/fr

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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32611—Poliovirus
    • C12N2770/32622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • HIV Human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • DPA D-penicillamine
  • the present invention relates to a method of reducing the effects of HIV in an HIV-infected individual by means of a recombinant nucleic acid sequence, or recombinant construct, expressed in HIV-infected cells, which encodes a product whose expression in such cells results in inhibition of the HIV, either directly or indirectly.
  • the present invention also relates to the recombinant construct and a composition which includes the construct.
  • the portion generally includes all or a segment of the HIV promoter (i.e., a sufficient segment for the
  • the recombinant construct occurs only in HIV-infected cells, is toxic only to HIV-infected cells, or both.
  • the recombinant construct additionally comprises DNA of non-HIV origin or mutated or altered HIV DNA.
  • the recombinant construct can be present in cells prior to their infection by HIV and/or can be introduced into cells after they have become
  • a recombinant construct which comprises a portion of the HIV genome, or an equivalent nucleotide
  • the recombinant construct is referred to as an
  • the DNA of non-HIV origin can encode 1) a protein or
  • the second agent will generally be a drug selected for its ability to act in conjunction with the encoded protein ox polypeptide to cause cell death. In either case, only HIV-infected cells are killed.
  • a nucleotide sequence referred to as a mutated or altered construct, which encodes a mutated HIV gag gene, or other mutated HIV gene encoding another protein provided in trans, is introduced into mammalian cells.
  • Cells containing the mutated gag construct express HIV at lower levels than HIV-infected cells in which the mutated gag construct is not present. As a result, less virus is released from infected cells and further infection of cells is less than it would otherwise be.
  • the present invention provides a means by which the effects of HIV can be reduced (diminished or eliminated) through the activity of a gene product produced in HIV-infected cells. This intracellular production method makes it possible to kill
  • HIV-infected cells selectively and to reduce or eliminate the production in HIV-infected cells of infectious virus.
  • Figure 1 is a schematic representation of the construction of the HIV/2A gene and its derivatives, with a restriction map of HIV/2A.
  • the HIV-1 LTR is shown by the first box.
  • the coding sequence of HIV/2A or its derivatives is shown by the second box, in which the CAT sequence, the poliovirus sequence and the random amino acids are represented by the hatched, shaded and black portions,
  • polyadenylation site is indicated.
  • FIG. 1 shows the results of in vitro
  • Figure 3 demonstrates the inhibitory effect of 2A protein on mRNA translation.
  • Panel a shows results of experiments in which HeLa cells were cotransfected with equal molar amounts of pRSVCAT, pSVETA or carrier DNA, as indicated, plus one of the following: carrier DNA (lanes 1 and 2); pHIV/2A (lanes 3 and 4); pHIV/2A-1 (lanes 5 and 6);
  • Panel c is a representation of the rationale of the experimental design.
  • Figure 4 demonstrates the cleavage of P220 in 2A-producing HeLa cells.
  • the immunoblot experiment was performed using an antiserum against P220, essentially as described by Bernstein et al., except that an alkaline phosphatase conjugated anti-rabbit IgG (Promega) was used as the second antibody according to the vendor's instructions. Extracts were prepared 48 hours post- transfection from:
  • lanes 1 and 2 HeLa cells transfected with or without pSVETA; lanes 3 and 4, 2A-38 cells
  • Extracts were also prepared from HeLa cells infected with wildtype (lane 6) or 2A-1 mutant virus (lane 5) at
  • Figure 5 is a schematic representation of recombinant retroviral constructs comprising a portion of the HIV promoter and a gene of non-HIV origin (here, the Herpes simplex virus thymidine kinase (TK) gene).
  • TK Herpes simplex virus thymidine kinase
  • Figure 6 is a photograph of Southern blots showing the correct structure of integrated proviral DNAs in infected 143 osteosarcoma cells or 143 osteosarcoma cells containing the tat gene sequence.
  • Figure 7 is a photograph showing effects of acyclovir (ACV) at two concentrations on recombinant HIV-TK retrovirally infected 143 tat cells (left, O ⁇ M ACV; middle 10 ⁇ M ACV; right, 100/ ⁇ M ACV).
  • ACCV acyclovir
  • Figure 8 is a schematic representation of mutated constructs used to transfect mammalian cells.
  • Figure 9 is a schematic representation of the genomic organization of HIV-1 (top panel) and of various gag and rev mutants. In the top panel, the gag region is enlarged underneath, with nucleotide numbers indicating the initiation codon, the
  • I-VII the various mutants are represented.
  • I and II contain a stop codon in the p17 and p24 sequence, respectively.
  • Ill and IV have a three- and
  • V has a four amino-acid insertion in the Rev coding sequence, as well as a stop codon in the tat gene.
  • VI and VII have an in- frame deletion in the gag precursor.
  • Figure 10 is a schematic representation of constructs used to create HT4( ⁇ E-dhfr) cell lines.
  • the broken arrow refers to the env translational frameshift, the thick line upstream of the 3' LTR to the sequence coding for a mutant dihydrofolate reductase.
  • the dotted line in I- ⁇ E-dhfr indicates the presence of a stop codon in the p17 coding sequence
  • the dark triangles in III- ⁇ E-dhfr and V-dhfr represent linker insertions in the p24 and Rev coding sequences, respectively
  • the broken line in VI- ⁇ E-dhfr corresponds to the deletion described in Figure 9.
  • FIG 11 shows results of immunoblot analysis of cytoplasmic extracts from HT4( ⁇ E-dhfr) cells. Lanes: 1, HT4-6C (negative control); 2,
  • HT4(R7-dhfr) infected with a replication competent version of HIV
  • Figure 12 is a graphic representation of p24 activity in the supernatant from HIV-infected
  • Figure 13 is a graphic representation of virus yield produced by HIV-infected HT4( ⁇ E-dhfr) cells. Detailed Description of the Invention
  • the present invention is based on the discovery that the adverse effects of HIV on mammalian cells infected with the virus can be reduced (inhibited or eliminated) by expression in such cells of a
  • the portion of the HIV genome in the cells can be present in a recombinant construct which also includes DNA of non-HIV origin or altered or mutated HIV DNA.
  • the portion of the HIV genome present in the recombinant construct includes all or a portion of the HIV long terminal repeat (LTR).
  • the LTR or portion thereof present in such a recombinant construct includes, in turn, all or a portion of the HIV promotor; if only a portion of the HIV promoter is present in the construct, it is a sufficient segment for the promoter to be functional.
  • the recombinant construct additionally comprises 1) DNA of non-HIV origin which encodes a product whose expression in HIV-infected cells is toxic to (causes death of) the cells or 2) altered or mutated HIV DNA whose expression reduces
  • HIV-infected cells infectious virus by HIV-infected cells.
  • these two components are present in recombinant constructs of this invention, in such a relationship to one another that the HIV LTR or LTR portion controls expression or activation of the non-HIV DNA or o f the al te red or mutated HIV DNA .
  • the encoded product produced when the DNA is expressed may itself be toxic to cells, or the product may require the presence of another substance to be toxic to cells. In either of these cases, HIV-infected cells are killed selectively, as described in greater detail below, and non-HIV infected cells are not
  • the recombinant construct includes all or a portion of the HIV genome containing a mutation or an
  • alteration, expression of the construct in cells reduces (inhibits or eliminates) the production of infectious HIV, thus reducing further infection of other cells.
  • a construct comprising a portion of the HIV genome and all or a portion of a gene of non-HIV origin which encodes a product capable of interfering with HIV function in infected cells is introduced into cells.
  • This recombinant construct is referred to as an
  • HIV/non-HIV construct can include the HIV long terminal repeat (LTR) or a portion thereof and DNA encoding a protein or polypeptide which is toxic to cells when expressed in sufficient quantities.
  • LTR long terminal repeat
  • DNA encoding a protein or polypeptide which is toxic to cells when expressed in sufficient quantities.
  • HIV/non-HIV recombinant construct can include the HIV LTR and DNA encoding all or a portion of the poliovirus protein 2A; the HIV LTR serves as the controlling element for expression of protein 2A.
  • This construct is introduced into mammalian cells using known techniques. In HIV-infected cells in which the transactivator (tat) gene is present, the LTR is activated and the poliovirus protein 2A is produced, with the result that protein synthesis in the cells is blocked and cell death occurs.
  • the HIV LTR/poliovirus protein 2A gene construct can be present in HIV-infected cells and HIV-free cells, it will be activated only in HIV-infected cells (because of the role of tat in its activation). Thus, it provides a means for selectively killing HIV-infected cells.
  • pHIV/2A a construct (designated pHIV/2A) was produced to express the poliovirus 2A protein in mammalian cells.
  • pHIV/2A includes a 695 BstEII fragment of poliovirus (PV) cDNA (Mahoney strain), which is in frame with the coding sequence of a bacterial chloramphenicol acetyl transferase (CAT) gene, whose expression is under the control of the HIV-1 LTR.
  • the coding sequence of the fusion gene includes: 73 N- terminal amino acids of CAT; a poliovirus sequence including 50
  • fusion genes which contain mutations in the 2A-encoding sequence, were also constructed, as described in Example 1.
  • One fusion gene, designated pHIV/2A-1 contains a single amino acid insertion in 2A, which results in a mutant poliovirus (2A-1) defective in shutting off host protein synthesis upon infection.
  • the other fusion gene, designated pHIV/2APX has a 53 amino acid deletion in 2A.
  • a recombinant construct such as pHIV/2A, in which the HIV LTR controls the expression of a protein (e.g. the poliovirus protein 2A), vhich inhibits (reduces or eliminates) translation of cellular mRNA when HIV is present, is introduced into cells as a means of countering the effects of HIV.
  • a recombinant construct can be introduced into cells prior to infection by HIV (e.g., in anticipation of exposure to/infection by HIV) or after infection has occurred. Only upon activation of the HIV LTR, which will occur in cells containing HIV, will expression of the nucleotide sequence of the recombinant construct occur, resulting in production of poliovirus 2A. Poliovirus 2A will block mRNA translation (protein synthesis) only in HIV-infected cells and, thus, will be toxic to (cause cell death) HIV-infected cells only.
  • a protein e.g. the poliovirus protein 2A
  • an HIV/non-HIV construct can include all or a portion of the HIV promoter and DNA encoding a product, which alone is not toxic to cells in which it is expressed, but in the presence of a selected substance (i.e., in conjunction with a selected substance or agent) is toxic to cells.
  • the non-HIV DNA encoding such a product is present in the recombinant construct in such a manner that its expression is controlled by the HIV promoter or promoter portion.
  • TK thymidine kinase
  • expression of the Herpes simplex TK gene is under the control of a minimal promoter element of the HIV LTR.
  • a recombinant construct comprising the minimal promoter and the TK gene is introduced into cells by known techniques (e.g., by transfection, by means of a retrovirus or other suitable vector).
  • the HIV promoter will function only in the presence of transactivator protein (tat), which is present only in HIV-infected cells.
  • tat transactivator protein
  • HIV LTR promoter is
  • the HIV LTR promoter is functional and TK is expressed.
  • TK does not have adverse effects on cells.
  • acyclovir or analogues thereof e.g., Gancyclovir, FIAU
  • TK is toxic to cells.
  • HIV-infected cells will be eliminated by the
  • the construct component of non-HIV origin is the TK gene and the additional substance
  • the recombinant construct can be introduced, for example, into haematopoietic stem cells, which are the primary target of HIV infection. Blood cells, T- lymphocytes and monocytes/macrophages derive from haematopoietic stem cells, which can be purified prior to introduction of the construct.
  • bone marrow cells which contain haematopoietic stem cells, can be taken from an individual, for example, one thought to be
  • the total (mixed) cell population or a purified portion of the stem cells can be infected or transfected with the recombinant retroviral construct, or can be introduced by other means, such as transfection.
  • the individual's bone marrow can be partially cleared by irradiation or through use of a cytotoxic drug.
  • the retrovirus will have or be modified to include a surface protein that is stem-cell tropic, thus making it possible for it to be targeted to marrow cells.
  • a construct comprising mutated or altered HIV DNA encoding a mutated or altered HIV protein provided in trans is introduced into cells, prior to or following infection of the cells by HIV.
  • mutated or altered refers to a sequence (nucleic acid sequence or amino acid sequence) which differs from the corresponding HIV sequence (nucleic acid sequence) or HIV-encoded sequence (amino acid sequence) by one or more changes (additions,
  • deletions in its constituents (i.e., nucleic acid(s), amino acid(s)).
  • the mutated or altered HIV DNA is DNA encoding a mutated or altered HIV gag protein. Expression of the mutated construct results in reduction of the quantity of HIV released from infected cells and, thus, a reduction in HIV available to infect additional cells.
  • infected mammalian cells by conferring a dominant negative phenotype on cells in which the mutants are expressed.
  • a selected gag mutant When a selected gag mutant is expressed, its product dominantly interferes with the normal function of the product of the parent, or wild type, gene. It has been shown that such gag mutants interfere with the generation of infectious viral particles from cells in which they are cotransfected with a wild type proviral DNA. It has also been shown that cells which constitutively express such HIV gag mutants have an impaired ability to support HIV replication when infected with wild type virus.
  • a recombinant construct whose expression confers a dominant negative phenotype on the cells into which it is introduced (e.g., hemato-poietic stem cells).
  • the construct includes a selected HIV gag mutant and a functional HIV promoter, under the control of which the gag mutant is expressed.
  • Mutations can confer a dominant negative phenotype on cells: when a gene carrying such a mutation is expressed, its product can dominantly interfere with the function normally accomplished by the product of the parental gene. Herskowitz, I., Nature, 329:219-222 (1987). When the protein is multimeric, an effective dominant negative variant can be one making a monomer that is still capable of interacting with the wild type polypeptide chains but is otherwise defective, and that can recruit wild type monomers into non- functional multimers. In such a case, only a moderate level of expression of the dominant negative mutant might be sufficient to exert a strong inhibitory effect on the parental protein.
  • the generation of dominant negative mutations which has been called intracellular immunization, (Baltimore, D., Nature, 235:395-396 (1988)), has been shown to be a potential strategy for
  • HIV human immunodeficiency virus
  • hematopoietic elements can be reconstituted from cells all derived from such a precursor, then the spread of the virus might be prevented in this patient.
  • Such a scheme poses the same problems as any gene therapy procedure, but a preliminary challenge is to identify effective dominant negative variants of HIV genes.
  • HIV synthesizes its major internal structural proteins as a polypeptide precursor (Pr55 gag ). This precursor is both
  • HIV gag proteins are likely to play a role in viral assembly and release, in stabilization of the virion, in
  • HIV gag mutants can interfere with the generation of infectious viral particles from cells in which they were co- transfected with a wild tyee proviral DNA.
  • cells constitutively expressing such HIV gag variants have a severely impaired ability to support HIV replication when infected with wild type virus.
  • mutated constructs containing a mutated gag gene and expressing the defective protein were individually cotransfected into mammalian cells with wild type (WT) HIV, using known techniques.
  • WT wild type
  • Constructs are represented schematically in Figure 8.
  • RipAn has a HIV 5' LTR and no 3' LTR; a pro-insulin polyadenylation sequence is present at the 3' end. The sequence between the 5' LTR and 3' polyadenylation sequence is the HIV sequence.
  • RipAmen has a 5' LTR, a polyadenylation site in place of the 3' LTR and a frameshift in the env region. Thus, it does not express HIV envelope protein. Mutated constructs designated by CH, followed by a number, have a linker insertion in the HIV sequence at the approximate position indicated by the number. That is, CH10 has a linker insertion at position 1000, CH14 at position 1400, etc.
  • Mutated constructs whose designations begin with ⁇ include a deletion between two sites indicated by the remainder of the designation (e.g., for ⁇ H10X12, a deletion between approximately positions 1000 and 1200).
  • Constructs designated R7. (H10X12, P14S15, H10H17) are the corresponding HIV (i.e., HIV in which the corresponding deletion is present), with a viral 3' LTR.
  • Infected H9 cells were cultured, harvested periodically and assayed, using anti HIV anti-serum and fluorescently labeled antibodies, using known techniques.
  • Example 6 assessed, as described in Example 5, in a transient assay and as described in Example 6, in cells which constitutively express the HIV gag variants.
  • a stable cell line has been constructed which is refractory to HIV replication by virtue of its expression of an altered HIV gene product.
  • Cells which constitutively express the HIV gag variants were created and assayed for their ability to support HIV replication, as described in Example 6.
  • Results demonstrated that H9 cells innoculated with the supernatant from two cells (HT4(III- ⁇ E-dhfr; HT4 (VI - ⁇ E- dhfr) ) constitutively expressing gag mutants released very few infectious particles. They also showed that supernatants of two cells (HT4(I- ⁇ E-dhfr); HT4(III- ⁇ E-dhfr)), which contained grossly comparable amounts of p24
  • HT4(I- ⁇ E-dhfr) is highly infectious and that from HT4(III- ⁇ E-dhfr) appears to contain mostly
  • HT4(III- ⁇ E-dhfr) and HT4 (VI- ⁇ E-dhfr) was also shown not to be due to the loss of CD4 receptor during cloning. Further, the block generated by the gag variants was shown to affect the late stages of the virus cycle.
  • a gag mutant e.g., p ⁇ H10X12, p ⁇ P14A20, p ⁇ 10H17
  • This can be carried out in an uninfected individual (e.g., an uninfected individual at high risk of HIV infection) or in an infected individual, using techniques described above for a recombinant
  • gag proteins have been targeted in the constructs described, it is also possible to use constructs containing an appropriate mutation in a gene encoding any protein which, like the
  • gag-encoded protein is provided in trans.
  • This approach which is a method of inactivating genes by a dominant negative mutation, makes use of an altered gene encoding a mutant product which, when expressed, inhibits the wild-type (HIV) gene in a cell, with the result that the gene product is non- functional.
  • the result in the present invention is the reduction (inhibition or elimination) in production of infectious HIV.
  • methotrexate the methotrexate; the hygro gene, which encodes
  • ⁇ AN7 which is a bacterial plasmid carrying an origin of replication and a suppressor tRNA gene to complement amber mutations in either a ⁇ phage, a bacteria or a bacterial plasmid, was introduced into cells, which then constitutively express the encoded product. Because they contain a marker-encoding gene incorporated within the genome, all cells which express the marker-encoding gene also express HIV and, thus, can be identified by the presence of the encoded substance. Such cells can be used, for example, to titer HIV; to select for viruses which differ from the parental virus; and to carry out HIV expression at high levels in target cells (e.g., in cells used to produce an HIV vaccine).
  • the block may reside at the level of viral assembly or release, of virion stabilization, or of viral entry or uncoating.
  • the degree to which those different steps are affected may vary depending on the nature of the gag mutant: VI, for instance, which has a sixty two amino-acid deletion overlapping the p17/p24 junction, seems to interfere mainly with viral assembly and/or release, because little p24 activity appears in the supernatant of cells expressing this variant whether or not they are infected with wild type HIV.
  • III which carries a four amino acids
  • gag variants tested produced a protein with an extensive deletion in the gag
  • mutant allele may be sufficient to repress wild type virus replication. That may constitute a decisive advantage over dominant negative mutants in the tat or rev genes, which are not likely to be as
  • a gag mutant e.g., p ⁇ H10X12, p ⁇ P14A20, p ⁇ 10H17
  • a mutated construct encoding a gag mutant e.g., p ⁇ H10X12, p ⁇ P14A20, p ⁇ 10H17
  • the recombinant construct can be introduced, for example, into haematopoietic stem cells, which are the primary target of HIV infection. Blood cells, T-lymphocytes and monocytes/macrophages derive from haematopoietic stem cells, which can be purified prior to introduction of the construct.
  • bone marrow cells which contain
  • haematopoietic stem cells can be taken from an individual, such as, one thought to be HIV-infected.
  • the total (mixed) cell population or a purified portion of the stem cells can be infected or
  • the bone marrow stem cells containing the construct can be introduced (injected) into the individual.
  • the individual's bone marrow can be partially cleared by irradiation or through use of a cytotoxic drug.
  • the construct- containing composition can be injected into or otherwise administered to the individual to provide a more direct route.
  • the retrovirus will preferably have or be modified to include a surface protein that is stem-cell tropic, thus making it possible for it to be targeted to marrow cells.
  • a mutated construct encoding a gag mutant can be introduced into peripheral blood cells which have been removed from the body and are returned to the individual after introduction of the construct.
  • mutant VI contains a deletion encompassing the cleavage site between p17/p24.
  • This mutant exhibits a strong t:rans-acting inhibitory effect on wild type HIV-1 replication.
  • HIV protease inhibitors might be of tremendous value in limiting viral spread, if they could induce in infected cells the production of a certain amount of uncleaved gag precursor. Those uncleaved precursors would probably interact with the wild type gag monomers, resulting in the formation of potentially non-functional multime rs .
  • produc tion o f infect ious viral particles by infected cells might be dramatically reduced .
  • the recombinant construct i.e., HIV/non-HIV
  • HIV sequences present in the recombinant constructs can be obtained from the HIV or can be DNA having the same nucleic acid sequence as the selected region(s) of the HIV genome which has been obtained using known cloning techniques, by chemical or mechanical synthesis, etc. It is not necessary that the DNA sequence be precisely the same as that of the selected region of the HIV DNA. Rather, it is possible to use DNA which is the functional
  • HIV sequence i.e., a sequence which encodes a product having the same amino acid sequence or function as that encoded by the
  • Non-HIV DNA present in the recombinant constructs (e.g., Herpes simplex virus TK gene, poliovirus protein 2A-encoding DNA) . It can be all or a portion of a selected gene and can be obtained from sources in which they occur in nature or can be synthesized using known cloning, mechanical or chemical methods. They can have the same sequence as that of the selected gene or a sequence which is its functional equivalent.
  • Recombinant constructs of the present invention can be administered to an individual in a
  • compositions whose components are selected according to the method by which it is to be administered.
  • Such compositions can include, for example, a suitable buffer, carrier and/ or adjuvant.
  • HIV-1 long terminal repeat
  • a plasmid designated pHIV/2A, was constructed by fusing a 695-bp BstEII fragment from a poliovirus cDNA (Mahoney strain) in frame with the coding sequence of a bacterial chloramphenical
  • CAT acetyltransferase
  • Plasmid pHIV/2A was deposited (March 1, 1989) under the terms of the Budapest Treaty in the American Type Culture Collection (Rockville, MD) under ATCC #40578. The plasmid will be unconditionally and irrevocably released to the public upon issuance of a U.S. patent to Applicants.
  • pHIV/2A-1 was constructed by replacing the BstEII fragment in pHIV/2A with the BstEII fragment from the 2A-1 mutant cDNA.
  • HIV/2A plasmid was digested with Xbal (nucleotide 3581 in poliovirus sequence), and filled-in the Xbal site.
  • This linear DNA was then partially digested with PstI, and the fragment that was only digested at the PstI site in the poliovirus sequence (nucleotide 3420) was selected using a 1% low-melting point agarose gel. The two ends of this fragment were ligated with a polylinker fragment with a PstI end on one side and a blunt end on the other, isolated from the Bluescript plasmid
  • the coding sequence of the fusion gene contains 73 N-terminal amino acids of CAT; the polioviral sequence including 50 C-terminal amino acids of region P1, the entire 2A and 33 N-terminal amino acids of protein 2B; and 9 random amino acids preceding a stop codon.
  • Similar fusion genes designated pHIV/2A-1 and pHIV/2APX respectively, were also constructed with mutations in the 2A sequence ( Figure 1).
  • pHIV/2A-1 contains a single amino acid insertion in 2A, which results in a mutant poliovirus, 2A-1, which is defective in shutting off host cell protein synthesis upon infection.
  • pHIV/2APX has a 53-amino acid deletion in 2A.
  • Protein 2A has a proteolytic activity and is known to cleave the junction between P1 and itself. Thus, it was expected that the predicted 41 kd
  • Hindlll-Bglll fragments ( Figure 1) containing the coding sequences of the fusion proteins were excised from HIV/2A, 2A-1 and 2APX, and ligated to the Hindlll and BamHI sites of SP64.
  • a Hindlll-Bglll fragment containing the CAT reading frame was also cloned into SP64 to synthesize a control template.
  • the coding strands were synthesized with SP6 polymerase (Promega).
  • RNA was used for the in vitro translation in 50 ul of the nuclease- treated rabbit reticulocyte lysate (Promega) containing
  • the ability of 2A to inhibit cellular mRNA translation was also examined, through the use of cotransfection experiments.
  • the plasmids used were: 1 ) a reporter construct , pRSVCAT ( Gorman et al. ,
  • HeLa cells were plated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum at about 5x10 per 60mm petri dish 12 to 24 hours prior transfection.
  • the low level of expression from the LTR of HIV-1 without tat expression has made it possible to establish HeLa cell lines permanently carrying pHIV/2A.
  • the plasmid pHIV/2A was cotransfected into HeLa cells with the plasmid pSV2neo which expressed the neomycin resistance gene. Southern and Berg, J.Mol. Appl. Genet., 1:327-341 (1982). Several dozen individual neomycin resistant colonies were tested.
  • the expression of the integrated HIV/2A genes could then be activated by transfecting in the tat gene expressed by pSVETA.
  • the inhibitory function of 2A in these cell lines was assayed by cotransfecting pRSVCAT and pSVETA, and measuring the CAT activity of the cotransfected extracts. These cell lines were found to contain variable copies of HIV/2A genes and respond to tat-activation to variable extents.
  • One of the cell lines, 2A-38 carried 10 to 20 copies of HIV/2A gene and
  • Figure 4 demonstrates the cleavage of P220 in 2A-producing HeLa cells.
  • the immunoblot experiment was performed using an antiserum against P220, essentially as described by Bernstein et al ., except that an alkaline phosphatase conjugated anti-rabbit IgG (Promega) was used as the second antibody according to the vendor's instructions. Extracts were prepared 48 hours post- transfection from HeLa cells transfected with or without pSVETA (lanes 1 and 2 of Figure 4), and 2A-38 cells transfected with or without pSVETA (lanes 3 and 4 of Figure 4).
  • Extracts were also prepared from HeLa cells infected with wild type ( Figure 4, lane 6) or 2A-1 mutant virus ( Figure 4, lane 5) at multiplicity of
  • 2A also acts as a protease because 2A mutants are defective in cleavage of 2A from its precursor. 2A is able to induce cleavage of P220, as was also evident from in vitro results, but the mechanism of cleavage, thought to be an indirect effect of 2A, remains obscure.
  • the LTR of HIV can be transactivated several hundred-fold by the tat gene from a very low basal level.
  • the level of expression in HeLa cells after activation is much higher than synthesis from RSV LTR or SV40 early promoter. It has been possible to establish
  • TK Herpes simplex virus thymidine kinase
  • Constructs containing portions of the HIV promoter were produced.
  • Construct A includes the 700 bases of the HIV promoter from the Xho site present at the 5' end through the Hind III site at the 3' end and the Herpes Simplex virus (HSV) thymidine kinase gene; the HSV TK gene includes the region present between the Bgl site (5' end) and the Bam site (3' end). It, thus, includes the HIV NFKB elements, SP-1 sites, the TATA box and the tat site.
  • Construct B includes the same components a construct A except that there are alterations at the sites indicated in the NFKB elements, which result in inactivation of the elements.
  • Constructs A' and B' are the same, respectively, as constructs A and B except that they begin at a Sea site at the 5' end, rather than the 5' Xho site.
  • Constructs C, D and E are shorter portions of the HIV promoter, as indicated in Figure 5. In each case (Constructs A-E), there are
  • Each construct has been inserted into at least one retroviral vector, both in direct and in reverse orientation. Constructs produced in this manner have been transfected into Psi Crip cells, which were maintained under conditions appropriate for expression of the plasmid DNA.
  • TK- Selection of cells using HAT selection was carried out, resulting in isolation of cells which contained the HIV-TK construct and in which the incorporated TK gene was expressed.
  • the tat gene of HIV was introduced into 143 cells, to produce cells designated 143 tat cells. Subsequent assays were carried out to look for differences in the
  • control cells are those containing control plasmid DNAs.
  • the effects of addition of acyclovir to uninfected 143 cells and 143 tat cells are shown in Figure 7. These controls show that acyclovir has no effect on uninfected control cells.
  • Figure 7 when 10 ⁇ M acyclovir was added to cells expressing a recombinant construct (as shown in Figure 7, 143 tat cells expressing ERH200T or
  • ER165T all cells were killed. The same was evident at 100um acyclovir. In contrast, no control cells were killed. It should be noted that 143 cells (i.e., those not containing the tat gene) were similarly affected by the two concentrations of acyclovir. This indicates that although the desired effect is evident, it is not as selective (specific to cells expressing tat) as desired and that a construct containing less of the HIV promoter (e.g., construct E of Figure 5) would be more useful. In this construct, the minimal HIV promoter consists of only the TATA element and the tat binding sequence. This minimal promoter has been shown by other laboratories to result in the lowest level of transcriptional activity.
  • deletional mutagenesis was performed on a plasmid (W13) carrying a full-length copy of the HIV-HXB2 proviral DNA.
  • This virus is replication competent and confers resistance to methotrexate.
  • the R7-Hyg virus was constructed by insertion of the hygromycin resistance gene, Gritz, L and J. Davies, Gene, 25:179-188 (1983) into the identical region, and is also replication competent.
  • WT- ⁇ E-dhfr and related constructs were made by replacement of the Xho 1 - Xba 1 fragment in WT- ⁇ E and others by the corresponding fragment from R7-dhfr. All cloning manipulations followed standard procedures.
  • COS cells were maintained in Dulbecco modified Eagle Medium (DME) supplemented with 5% fetal calf serum and were transfected using DEAE-dextran and chloroquine. Ausubel, F., et al, Current Protocols in Molecular Biology (Wiley, New York) (1987), or calcium-phosphate precipitation, Chen, C. and H.
  • DME Dulbecco modified Eagle Medium
  • H9 cells were grown in RPM1 supplemented with 10% fetal calf serum.
  • HT4-6C cells Chesebro, B. and Wehrly, K., J. Virol., Vol. 62: pp 3779-3788 (1988) and
  • constructs had further modifications creating large internal deletions in the gag precursor, either overlapping the junction between p17 and p24 (VI), or within the p24 protein itself (VII). Because a functional rev has been shown to be necessary for
  • HIV gag production an additional construct was made to serve as a complete gag-null mutant, by
  • a COS cells were transfected with 5 ⁇ g of DNA; plasmid pBC12/RSV/SEAP (1 ⁇ g) was used as an internal control; at 60 hours post transfection, the supernatant was harvested for measurement of p24 and SEAP activity, and to infect H9 cells. The experiment was repeated three times, and gave consistent results. The level of variability for the internal control "SEAP" was less than 15%.
  • H9 cells were exposed to equal amounts of COS super- natants, and followed by indirect immunofluorescence, using serum from an HIV-1 seropositive individual as detector antibody.
  • a similar result was obtained with the construct carrying a deletion of the p17-p24 cleavage site (VI). For these mutants, it would appear that there was no virus released.
  • Cells transfected with constructs III, IV and VII produced p24 activities that were approximately 100-fold lower than wild type, also indicating a major defect in viral assembly and/or release.
  • H9 cells were exposed to the supernatant obtained from those cells, and subsequently followed by
  • Construct V the rev mutant, induced p24 activity levels that were less than 1% of wild type; the supernatant of V- transfected cells also appeared to be non-infectious.
  • the linker inserted in this construct in addition to changing a few amino acids in the N-terminal portion of the rev protein, introduces a stop codon near the 3' end of the tat first exon.
  • H9 cells were positive by immunofluorescence (Table 6).
  • a COS cells were cotransfected with W13 and the various mutants, at a ratio of 1:4.
  • Supernatants were harvested after 60 hours and used to infect H9 cells. Those were followed by indirect immunofluorescence, using serum from an HIV-1 seropositive individual as detector antibody.
  • II- ⁇ E expressing a truncated form of gag, did not interfere significantly.
  • a cell line designated
  • HT4(VI- ⁇ E-dhfr) which constitutively expresses a gag mutant has been produced.
  • the cells were further analyzed by
  • HT4(WT- ⁇ E-dhfr) (lane 3) and HT4(III- ⁇ E-dhfr) (lane 4) showed a normal pattern of gag protein, compared to cells infected with a replication competent version of HIV-1, HT4(R7-dhfr) (lane 2); finally,
  • HT4(I- ⁇ E-dhfr); HT4(V-dhfr) and HT4 (III- ⁇ E-dhfr) gave levels three-hundred- fold and twelve-fold lower than HT4(WT- ⁇ E-dhfr), respectively; in the
  • HT4( ⁇ E-dhfr) cells were plated at low density, and exposed to 1 ml. of supernatant from acutely
  • H9 cells were inoculated with equal amounts of supernatant from the HIV-infected HT4( ⁇ E-dhfr) cells, harvested at day 4 post infection. After adsorption, cells were washed, placed in fresh medium, and infection was monitored by measuring p24 activity in the culture medium. H9 cells initially exposed to supernatants from HT4(WT- ⁇ E-dhfr), HT4(I- ⁇ E- dhfr) and HT4(V-dhfr) showed rapidly increasing values of p24 activity in the culture medium, reflecting initial innoculation by highly infectious virus (Figure 13).
  • HT4(III- ⁇ E-dhfr) and HT4(VI- ⁇ E-dhfr) was not due to the loss of the CD4 receptor during the cloning procedure. It was also confirmed that the block generated by the gag variants affected the late stages of the virus life cycle. For this, the experiment was first repeated, not in cloned cell lines, but in pooled populations; results comparable to those shown in Figures 12 and 13 were obtained. The different cell lines were then infected with a replication competent variant of HIV-1 virus,
  • R7-Hyg in which the nef open reading frame has been replaced by the hygromycin resistance gene.
  • This virus confers hygromycin resistance to the cells it infects. Comparable numbers of hygromycin resistant colonies were obtained in all cases. Because the expression of the hygromycin resistance gene was directed by the viral LTR, the finding of resistant colonies depended on the efficient accomplishment of all early steps of the HIV life cycle: entry, uncoating, reverse transcription, integration and gene expression. Therefore, it was concluded that the dominant negative effect exhibited by the gag mutations in III and VI is exerted at a late step of the viral life cycle, concomitant with or subsequent to viral assembly.

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Abstract

Dans le procédé intracellulaire décrit, qui sert à inhiber l'HIV (virus de l'immunodéficience humaine) dans des cellules de mammifères, une structure recombinante est introduite dans les cellules. La structure recombinante comprend: (a) la chaîne de répétition de terminaison longue (LTR) de l'HIV ou une partie de la LTR d'HIV, qui contient un promoteur d'HIV fonctionnel et un ADN d'origine non HIV codant pour un produit qui est toxique aux cellules infectées par l'HIV, lorsqu'il est présent dans ces cellules seul ou en conjonction avec une substance sélectionnée; ou (b) la totalité de la LTR d'HIV ou une partie de la LTR d'HIV, qui contient un promoteur d'HIV fonctionnel et un ADN d'HIV ayant subit une mutation ou une altération. Des compositions servant à inhiber l'HIV, qui contiennent de telles structures recombinantes, sont également décrites.
PCT/US1990/001266 1989-03-20 1990-03-08 Procede intracellulaire d'inhibition de l'hiv dans des cellules de mammiferes WO1990011359A1 (fr)

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Cited By (21)

* Cited by examiner, † Cited by third party
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WO1991004319A1 (fr) * 1989-09-25 1991-04-04 Innovir Laboratories, Inc. Compositions de ribozymes therapeutiques et vecteurs d'expression
EP0454781A1 (fr) * 1989-01-23 1991-11-06 Chiron Corporation Cellules de recombinaison pour des therapies d'infections et de troubles hyperproliferatifs et la preparation de ces cellules
WO1993008844A1 (fr) * 1991-10-30 1993-05-13 Universite Pierre Et Marie Curie (Paris Vi) Cellules transformees pour la prevention ou le traitement de maladies induites par des virus, notamment retrovirus pathogenes
WO1993011251A1 (fr) * 1991-12-06 1993-06-10 Whitehead Institute For Biomedical Research Virus recombinant a site de clivage proteolytique artificiel
WO1994021806A1 (fr) * 1993-03-19 1994-09-29 Medical Research Council Systeme d'administration commande par des facteurs associes au vih et a la cellule
FR2713657A1 (fr) * 1993-12-13 1995-06-16 Transgene Sa Nouveaux vecteurs pour le traitement du sida.
WO1996014863A1 (fr) * 1994-11-10 1996-05-23 The Regents Of The University Of California Suppression de l'activite proteolytique par formation de protease dysfonctionnelle
US5591625A (en) * 1993-11-24 1997-01-07 Case Western Reserve University Transduced mesenchymal stem cells
EP0816507A1 (fr) * 1995-03-16 1998-01-07 Hisamitsu Pharmaceutical Co., Inc. Nouveau recombinant genique
US5853716A (en) * 1995-07-28 1998-12-29 Yale University Genetically engineered chimeric viruses for the treatment of diseases associated with viral transactivators
US5869306A (en) * 1995-03-17 1999-02-09 Hisamitsu Pharmaceutical Co., Inc. Gene transfer preparation
US5871958A (en) * 1989-05-25 1999-02-16 Duke University Mutant rev genes encoding transdominant repressors of HIV replication
US5965124A (en) * 1991-12-06 1999-10-12 Whitehead Institute For Biomedical Research Replication-competent recombinant viral vaccines and method of producing same
US5989886A (en) * 1991-01-02 1999-11-23 The Johns Hopkins University Method for the inhibition and prevention of viral replication using fusions of a virus protein and a destructive enzyme
US6162898A (en) * 1989-05-25 2000-12-19 Duke University Mutant Rev transdominant repressors of HIV replication
US6602705B1 (en) 1998-12-31 2003-08-05 Chiron Corporation Expression of HIV polypeptides and production of virus-like particles
US6689879B2 (en) 1998-12-31 2004-02-10 Chiron Corporation Modified HIV Env polypeptides
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8263394B2 (en) 1998-12-31 2012-09-11 Novartis Vaccines & Diagnostics Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides, and uses thereof

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EP0454781A1 (fr) * 1989-01-23 1991-11-06 Chiron Corporation Cellules de recombinaison pour des therapies d'infections et de troubles hyperproliferatifs et la preparation de ces cellules
EP0454781A4 (en) * 1989-01-23 1992-07-08 Chiron Corporation Recombinant therapies for infection and hyperproliferative disorders
US5871958A (en) * 1989-05-25 1999-02-16 Duke University Mutant rev genes encoding transdominant repressors of HIV replication
US6162898A (en) * 1989-05-25 2000-12-19 Duke University Mutant Rev transdominant repressors of HIV replication
US6251675B1 (en) 1989-05-25 2001-06-26 Duke University Methods utilizing mutant rev genes encoding transdominant repressors of HIV replication
US6287809B1 (en) 1989-05-25 2001-09-11 Novartis Ag Mutant rex transdominant repressors of HIV replication
US6339150B1 (en) 1989-07-07 2002-01-15 Duke University Mutant rex genes encoding transdominant repressors of HIV/HTLV replication
US5225347A (en) * 1989-09-25 1993-07-06 Innovir Laboratories, Inc. Therapeutic ribozyme compositions and expression vectors
WO1991004319A1 (fr) * 1989-09-25 1991-04-04 Innovir Laboratories, Inc. Compositions de ribozymes therapeutiques et vecteurs d'expression
US5989886A (en) * 1991-01-02 1999-11-23 The Johns Hopkins University Method for the inhibition and prevention of viral replication using fusions of a virus protein and a destructive enzyme
WO1993008844A1 (fr) * 1991-10-30 1993-05-13 Universite Pierre Et Marie Curie (Paris Vi) Cellules transformees pour la prevention ou le traitement de maladies induites par des virus, notamment retrovirus pathogenes
AU674134B2 (en) * 1991-12-06 1996-12-12 American Cyanamid Company Recombinant viruses comprising artificial proteolytic cleavage site
CN1055726C (zh) * 1991-12-06 2000-08-23 怀特黑德生物制剂研究所 有复制能力的重组体病毒的制备方法
WO1993011251A1 (fr) * 1991-12-06 1993-06-10 Whitehead Institute For Biomedical Research Virus recombinant a site de clivage proteolytique artificiel
US5965124A (en) * 1991-12-06 1999-10-12 Whitehead Institute For Biomedical Research Replication-competent recombinant viral vaccines and method of producing same
WO1994021806A1 (fr) * 1993-03-19 1994-09-29 Medical Research Council Systeme d'administration commande par des facteurs associes au vih et a la cellule
AU696399B2 (en) * 1993-03-19 1998-09-10 Medical Research Council Delivery system controlled through factors associated with HIV and cell
US5591625A (en) * 1993-11-24 1997-01-07 Case Western Reserve University Transduced mesenchymal stem cells
WO1995016784A1 (fr) * 1993-12-13 1995-06-22 Transgene S.A. Vecteurs exprimant un interferon humain pour le traitement du sida
FR2713657A1 (fr) * 1993-12-13 1995-06-16 Transgene Sa Nouveaux vecteurs pour le traitement du sida.
WO1996014863A1 (fr) * 1994-11-10 1996-05-23 The Regents Of The University Of California Suppression de l'activite proteolytique par formation de protease dysfonctionnelle
US6165794A (en) * 1994-11-10 2000-12-26 The Regents Of The University Of California Suppression of proteolytic activity by dysfunctional protease formation
EP0816507A4 (fr) * 1995-03-16 1998-07-01 Hisamitsu Pharmaceutical Co Nouveau recombinant genique
EP0816507A1 (fr) * 1995-03-16 1998-01-07 Hisamitsu Pharmaceutical Co., Inc. Nouveau recombinant genique
US5869306A (en) * 1995-03-17 1999-02-09 Hisamitsu Pharmaceutical Co., Inc. Gene transfer preparation
US5853716A (en) * 1995-07-28 1998-12-29 Yale University Genetically engineered chimeric viruses for the treatment of diseases associated with viral transactivators
US6602705B1 (en) 1998-12-31 2003-08-05 Chiron Corporation Expression of HIV polypeptides and production of virus-like particles
US6689879B2 (en) 1998-12-31 2004-02-10 Chiron Corporation Modified HIV Env polypeptides
US8263394B2 (en) 1998-12-31 2012-09-11 Novartis Vaccines & Diagnostics Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides, and uses thereof
US8168418B2 (en) 1998-12-31 2012-05-01 Susan W Barnett Expression of HIV polypeptides and production of virus-like particles
US7348177B2 (en) 1998-12-31 2008-03-25 Novartis Vaccines And Diagnostics, Inc. Expression of HIV polypeptides and production of virus-like particles
US7662916B2 (en) 1998-12-31 2010-02-16 Novartis Vaccines & Diagnostics, Inc Modified HIV Env polypeptides
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8133494B2 (en) 2001-07-05 2012-03-13 Novartis Vaccine & Diagnostics Inc Expression cassettes endcoding HIV-1 south african subtype C modified ENV proteins with deletions in V1 and V2
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US9598469B2 (en) 2001-07-05 2017-03-21 Novartis Vaccines And Diagnostics, Inc. HIV-1 south african subtype C env proteins
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof

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