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WO1996037235A1 - Procedes d'inhibition du vih - Google Patents

Procedes d'inhibition du vih Download PDF

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Publication number
WO1996037235A1
WO1996037235A1 PCT/US1996/007518 US9607518W WO9637235A1 WO 1996037235 A1 WO1996037235 A1 WO 1996037235A1 US 9607518 W US9607518 W US 9607518W WO 9637235 A1 WO9637235 A1 WO 9637235A1
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Prior art keywords
hiv
ala
arg
thr
subunit
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PCT/US1996/007518
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English (en)
Inventor
Nahid Mohagheghpour
Daniel Tuse
William J. Estrin
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Panther Scientific, Inc.
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Publication of WO1996037235A1 publication Critical patent/WO1996037235A1/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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/235—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bordetella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus

Definitions

  • the present invention relates to a genetic therapy method for the inhibition of human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • HIV-I human immunodeficiency virus
  • HIV-I HIV type I virus
  • An effective therapy has not been identified to date.
  • a number AIDS-related symptoms and complications are correlated with the viral load imposed on the patient's immune system by HIV (Fauci; Weiss; Verhofstede, et al). Therapeutics which are effective to reduce this viral load may therefore alleviate the severity of some pathological manifestations of HIV.
  • the present invention includes a method of inhibiting the production of infectious retroviral virions in a cell infected by the retrovirus.
  • the method includes providing a retrovirus-infected cell containing a chimeric gene which contains a DNA sequence encoding the SI subunit of pertussis toxin (SI gene) operably linked to a retroviral long terminal repeat (LTR) region, and growing the cell. The growing is carried out under conditions where expression of the chimeric gene is induced. Expression of the SI subunit by the chimeric gene in the cell inhibits the production of infectious retroviral virions by the cell.
  • SI gene pertussis toxin
  • LTR retroviral long terminal repeat
  • the method inhibits the production of infectious Human Immunodeficiency Virus (HIV) virions (e.g., HIV-1 virions), the cell is infected with HIV (e.g., HIV-1) and the LTR region is an HIV LTR region (e.g., an HIV-1 LTR region).
  • HIV Human Immunodeficiency Virus
  • the DNA sequence encoding the SI subunit may code for an amino acid sequence identical or homologous to any of sequences SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, or fragments thereof.
  • the chimeric gene may further include an element effective to reduce, inhibit or eliminate undesired expression of the SI subunit of PT in cells not infected by retrovirus.
  • an element is a DNA fragment containing a head-to-tail trimer (A-trimer) of an SV40 polyadenylation sequence.
  • the element is preferably inserted upstream of the LTR region in the chimeric gene.
  • the infected cell may be any cell susceptible to infection by HIV, such as a monocyte, macrophage or CD4+ T lymphocyte.
  • the present invention provides a chimeric gene, which includes a retroviral LTR region operably linked to a DNA sequence encoding the SI subunit of pertussis toxin.
  • the LTR region is an HIV LTR region (e.g., an HIV-1 LTR region).
  • the chimeric gene may further include an element effective to reduce, inhibit or eliminate undesired expression of the SI subunit of PT in cells not infected by retrovirus, such as the A-trimer described above.
  • the DNA sequence encoding the SI subunit may code for an amino acid sequence identical or homologous to any of sequences SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, or fragments thereof.
  • the present invention includes a retroviral expression vector, containing a chimeric gene as described above.
  • the invention includes a method of reducing the HIV viral load in an HIV-infected subject.
  • the method includes (i) isolating CD4+ lymphocytes from the subject, (ii) transforming the lymphocytes with a chimeric gene comprising an HIV LTR region operably linked to a DNA sequence encoding the SI subunit of pertussis toxin (PT), and (iii) introducing lymphocytes carrying the chimeric gene into the subject.
  • the lymphocytes express the SI subunit of PT, inhibiting production of infectious HIV virions. This inhibition reduces viral load in the HIV-infected subject, and inhibits the spread of infection in the individual.
  • the present invention includes a method of reducing HIV-1 viral load in a subject harboring HIV- 1 -infected cells.
  • the method includes administering to the subject, a retroviral expression vector containing a chimeric gene comprising an HIV-1 LTR region operably linked to a DNA sequence encoding the SI subunit of pertussis toxin (PT), under conditions which promote transfection of the vector into said infected cells.
  • Infected cells carrying the vector express the SI subunit, which inhibits HIV production and results in a reduced viral load in the HIV- 1 -infected subject.
  • the present invention also includes methods of treating HIV infections in HIV- infected subjects.
  • the methods contain the steps outlined above in methods for reduction of viral load in HrV-infected subjects.
  • Also contemplated by the present invention are methods of treatment of an HIV infection and of reducing viral load in an HIV-infected subject which combine any of the methods summarized above with other anti-HIV therapies, such as treatment with AZT, HIV protease inhibitor and the like.
  • Figure 1A shows an alignment of the amino acid sequences of HIV gp41 and GjO: in the region defined by amino acids 589 through 595, and 12 through 17, of the respective proteins.
  • Figure IB shows an alignment of the amino acid sequences of HIV gp41 and G ; ⁇ in the region defined by amino acids 597 through 611, and 36 through 50, of the respective proteins.
  • FIG. 2 shows a schematic diagram of an expression cassette containing an HIV-1 long terminal repeat (LTR), a sequence encoding the S-1 subunit of pertussis toxin (PT SI) and an SV40 polyadenylation signal.
  • LTR HIV-1 long terminal repeat
  • PT SI pertussis toxin
  • SEQ ID NO: 1 is the nucleotide sequence of the Bordetella pertussis gene for toxin subunit SI (EMBL accession number XI 6347).
  • SEQ ID NO:2 is the predicted amino acid sequence from SEQ ID NO:l.
  • SEQ ID NO:3 is the nucleotide sequence of the B. pertussis gene for toxin subunit SI (GenBank accession number Ml 3223).
  • SEQ ID NO: 4 is the predicted amino acid sequence from SEQ ID NO: 3.
  • SEQ ID NO: 5 is the nucleotide sequence of the B. pertussis gene for toxin subunit SI (EMBL accession number A 13359).
  • SEQ ID NO:6 is the predicted amino acid sequence from SEQ ID NO:5. DETAILED DESCRIPTION OF THE INVENTION
  • operably linked denotes a relationship between a regulatory region (typically a promoter element, but may include an enhancer element) and the coding region of a gene, whereby the transcription of the coding region is under the control of the regulatory region.
  • Chimeric gene refers to a polynucleotide containing heterologous DNA sequences, such as promoter and enhancer elements from one source operably linked to a gene, encoding a desired gene product, from a second source.
  • heterologous DNA sequences such as promoter and enhancer elements from one source operably linked to a gene, encoding a desired gene product, from a second source.
  • a construct containing a human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) region operably linked to a gene encoding the SI subunit of pertussis toxin comprises an exemplary chimeric gene.
  • HSV-1 human immunodeficiency virus 1
  • LTR long terminal repeat
  • Treating" a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.
  • Treating” an infection, such as a viral infection, in an individual refers to administering a therapeutic substance effective to reduce the symptoms of the infection, reduce the viral burden, or reduce the pathogenicity of the infectious agent in the individual.
  • Upstream with respect to location along a polynucleotide refers to a location that is 5' to the reference location.
  • Methods and compositions of the present invention useful in treating infections caused by the human immunodeficiency virus (HIV), employ chimeric gene constructs, such as retroviral expression vectors, containing the gene for the S-1 subunit of pertussis toxin (PT) under the control of the HIV long terminal repeat (LTR).
  • the HIV LTR region serves to initiate transcription of downstream genes in response to the transactivating protein (Tat), which is produced by HIV upon infection of a host cell.
  • Tat transactivating protein
  • the SI subunit may have several effects in the HIV-infected host cell, depending on its level of expression.
  • High levels of expression of SI result in death and lysis of the host cell.
  • the effects are typically mediated through ADP (adenosine diphosphate)-ribosylation of a target protein.
  • a target protein is G, ⁇ , an endogenous inhibitory mammalian guanine nucleotide binding protein (G-protein).
  • G-protein an endogenous inhibitory mammalian guanine nucleotide binding protein
  • the SI subunit of PT may also ADP ribosylate HIV gp41.
  • HIV having ADP ribosylated gp41 has a markedly reduced infectivity and cytopathogenicity.
  • the combined action of killing virus-infected cells, and inhibition of existing virions by ADP ribosylation of HIV gp41 is effective to inhibit HIV and reduce viral burden in infected individuals.
  • HIV belongs to a large, diverse family of viruses known as "retroviruses". It has been identified in and isolated from the CD4+ lymphocytes (T-4 lymphocytes) of AIDS patients.
  • the envelope protein of HIV-1 is composed of a glycoprotein (gpl60) that serves to attach the virus to CD4 receptors on the host cells, as well as facilitating the fusion of the virus with the host cell.
  • the HIV gpl60 glycoprotein is cleaved between the arginine at position 518 (arg518) and the alanine at position 519 (ala519) of the env gene product to produce an N-terminal gpl2O glycoprotein and a smaller transmembrane C-terminal gp41 glycoprotein.
  • the HIV env gp41 glycoprotein consists of 345 amino acids (from ala519 to the leucine at position 863 (leu863)), and has six potential glycosylation sites (Bergeron, et al, 1992).
  • the G proteins are heterotrimers, with subunits designated a, ⁇ and ⁇ in order of decreasing mass.
  • the subunits clearly differ among the members of the G protein family and serve to define the individual G protein. Common ⁇ and ⁇ subunits are probably shared among some subunits to form the specific oligomers.
  • G-protein mediated transmembrane signaling pathways involve the interaction of membrane receptor proteins, G proteins and effector proteins.
  • the receptor proteins typically respond to extracellular stimuli, such as biogenic amines, proteins, polypeptide hormones, autacoids and/or neurotransmitters (Stryer, 1988; Linder and Gilman, 1992).
  • the G proteins couple the activation state of a receptor to the activation or inactivation of an effector protein.
  • G proteins are regulated cyclically by association of (guanosine triphosphate) GTP with the a subunit, hydrolysis of GTP to guanine diphosphate (GDP) and phosphate (Pj), and dissociation of GDP from the G protein.
  • GDP guanine diphosphate
  • Pj phosphate
  • the binding of GTP typically activates the G protein, resulting in corresponding regulation of the activity of the appropriate effector protein (Stryer, 1988; Linder and Gilman, 1992).
  • GjC adenylate cyclase
  • GjC adenylate cyclase
  • the G J Q; oligomers are composed of approximately 354 amino acids and have a calculated molecular weight of about 40,400.
  • Bordetella pertussis produces a substance which activates an insulin-secreting response in mammals (Sumi and Ui, 1975).
  • a protein secreted by B. pertussis was purified and termed Islet-Activating Protein (IAP; Yajima, et al, 1978a; Yajima, et al, 1978b).
  • LPF lymphocytosis promoting factor
  • HSF histamine sensitizing factor
  • IAP ADP-ribosyltransferase and NAD-glycohydrolase
  • IAP is a 77-kD multimeric protein comprised of six subunits which associate through non-covalent interactions (Tamura, et al, 1982).
  • the bioactivity of IAP activity resides on the largest subunit, which has been designated the A (active) protomer, or SI subunit (Katada, et al, 1983).
  • the remaining 5 subunits, which bind specifically to the cell membrane and deliver the A protomer into the cell, have been collectively designated the B
  • the SI subunit is an enzyme which acts as an ADP-ribosyltransferase, transferring the ADP-ribose moiety of NAD to the cysteine residual groups of proteins (Katada and Ui, 1982a; Katada and Ui, 1982b).
  • one substrate (target protein) for this enzyme is the 41-kD guanine nucleotide binding protein Gj discussed above (Katada and Ui, 1982a; Katada and Ui, 1982b; Bokoch, et al, 1983; Hsia, et al, 1983; Ohta, et al, 1990; Center, et al, 1989), which is selectively ADP-ribosylated by IAP (Katada and Ui, 1982a; Katada and Ui, 1982b; Bokoch, et al, 1983; Hsia, et al, 1983; Ohta, et al, 1990; Center, et al, 1989).
  • IAP has also been shown to ADP ribosylate another type of G protein, G hinder ⁇ , which is thought to be involved in the regulation of neuronal potassium and calcium channels, as well as certain types of phospholipase C activation. It is recognized that the active subunits of at least two other toxins, cholera toxin and E. coli heat labile toxin, possesses ADP-ribosyltransferase activity, and accordingly, may be used in place of the SI subunit of pertussis toxin in the practice of the present invention.
  • Pertussis toxin modifies the G s ⁇ protein, effectively blocking the inhibitory effect of GjC. on adenylate cyclase and signal transduction (Stryer, 1988; Linder and Gilman, 1992; Spiegal, 1990; Cruikshank, et al, 1990; Katada and Ui, 1981; Katada and Ui, 1982a; Katada and Ui, 1982b; Bokoch, et al, 1983; Hsia, et al, 1983; Ohta, et al, 1990; Center, et al, 1989; Hazeki and Ui, 1981; Murayama, et al, 1983; Murayama and Ui, 1983; Kurose, et al, 1983; Kurose and Ui, 1983; Murayama and Ui, 1984).
  • IAP activity e.g., activation of the insulin-secreting response (IAP activity; Katada and Ui, 1977; Katada and Ui, 1979; Katada and Ui, 1980), promotion of hydrolysis of neutral lipids (Nogimori, et al, 1984) or suppression of epinephrine-induced hyperglycemia (Katada and Ui, 1976).
  • IAP activity e.g., activation of the insulin-secreting response
  • Katada and Ui, 1977 Katada and Ui, 1979
  • Katada and Ui, 1980 e.g., inhibition of IAP activity
  • IAP activity e.g., activation of the insulin-secreting response
  • Katada and Ui, 1977 Katada and Ui, 1979
  • Katada and Ui, 1980 e.g., depression of the insulin-secreting response
  • hydrolysis of neutral lipids e.gimori, et al, 1984
  • Amino acid sequence alignments performed in support of the present invention and shown in Figures 1A and IB reveal homologies between a region of the HIV env gp41 glycoprotein (the GTPase region) and the human 41kd Gj ⁇ protein.
  • the HIV env gp41 glycoprotein is similar to G , but lacks two of the three required phosphorylation sites in a GTP-binding hydrophobic pocket ( ⁇ isenberg and Wesson, 1990; Narvanen, et al, 1988a; Bell, et al, 1992; Narvanen, et al, 1988b).
  • the HIV env gp41 retains human G ⁇ serine-44, deletes G j ⁇ serine-47 and mutates threonine-48 to leucine, a non phosphorylatable amino acid residue.
  • the similarity of the HIV gp41 GTP-hydrophobic pocket to that of G, ⁇ renders HIV gp41 susceptible to ADP ribosylation by pertussis toxin, which disrupts viral production.
  • the specificity of the SI subunit used in methods and compositions of the present invention may be altered by site-directed mutagenesis, such that, for example, a modified SI subunit exhibits a substrate preference for HIV gp41 over Gj ⁇ .
  • a single SI subunit amino acid substitution of lysine (K) for arginine (R) at the 57 position i.e., K-57 for R-57
  • a single SI subunit amino acid substitution of lysine (K) for arginine (R) at the 57 position i.e., K-57 for R-57
  • vectors employing a construct encoding such a modified SI subunit may be particularly useful for inhibiting HIV replication in infected cells with a lower overall toxicity than wild-type SI.
  • the LTR region incorporated into therapeutic constructs directed against a particular virus preferably corresponds to a portion of the LTR region of that virus. Accordingly, such constructs preferably include PT SI operably linked to the appropriate LTR region. Effective constructs express PT SI in cells transformed with the constructs and infected by the selected retrovirus.
  • vector constructs containing HIV-1 long terminal repeat (LTR) control elements and a gene encoding the S-1 subunit of pertussis toxin (PT) may be used to inhibit the production of infectious HIV in target cells carrying the vector and expressing the SI subunit.
  • the HIV LTR has been demonstrated to markedly enhance viral expression in cells already infected with the HIV virus. This enhanced expression is stimulated by trans-acting regulatory factors which act on elements in the long terminal repeat (LTR) sequence of HIV. Heterologous genes placed under control of the HIV LTR sequences are therefore preferentially expressed in HIV-infected cells.
  • the Tat and Rev proteins are two HIV trans-acting factors.
  • the Tat protein acts on a cis-acting element mapped to region + 14 to +44 (referred to as the TAR region) of the HIV LTR to increase viral expression from the LTR (Arya, et al, 1985; Rosen, et al, 1985, 1988; Sodroski, et al, 1985; Green, et al, 1989).
  • the Tat protein appears to exert an effect at both transcriptional (Peterlin, et al, 1986; Hauber, et al, 1987; Laspia, et al, 1989) and post-transcriptional levels (Cullen, 1986; Feinberg, et al, 1986; Wright, et al, 1986; Braddock, et al, 1989; Edery, et al, 1989) and can stimulate expression of heterologous genes placed 3' to the TAR region (Tong-Starksen, et al, 1987; Felber and Pavlaskis, 1988).
  • Vectors pLTR-SlPTl and pLTR-SlPT2 are plasmid constructs, which may be used to transfect mammalian cells, such as peripheral blood mononuclear cells (PBMCs), including monocytes, macrophages and CD4+ T-cells, as well as other cells susceptible to infection by HIV. Transfected cells may then be selected for clones that have stably incorporated the constructs.
  • PBMCs peripheral blood mononuclear cells
  • Transfected cells may then be selected for clones that have stably incorporated the constructs.
  • pLTR-SlPT3 is a retroviral expression vector that may be used to transfect proliferating PBMCs, such as CD4+ T-cells, either in vitro or in vivo. Retroviral vectors typically have a very high efficiency of transfection, with certain vectors being capable of stably transducing close to 100% of the target cells.
  • Target cells are transfected with virions containing pLTR-SlPT3 by co-incubating the cells with viral stocks produced as described in Example 1, or by co-cultivation with the appropriate packaging cell line containing the desired vector.
  • the constructs described in Example 1 have an HIV-1 long terminal repeat (HIV-1 LTR) region placed upstream of a DNA fragment containing the coding sequence for pertussis toxin (PT) SI subunit.
  • HIV-1 LTR HIV-1 long terminal repeat
  • Other elements may be introduced into the constructs to improve the efficiency and/or selectivity of expression.
  • a fragment containing the SV40 small t intron and poly(A) signals is typically inserted downstream of the PT-Sl DNA.
  • the constructs may also contain an element or elements designed to reduce or prevent spurious or undesired expression of the PT-Sl gene in the absence of activation of the HIV-1 LTR by the Tat protein.
  • A-trimer Maxwell, et al, 1989
  • the A- trimer is a head-to-tail trimer of an SV40 BcH/BamHI DNA fragment specifying polyadenylation of RNA transcripts. It has been shown to prevent spurious expression of chimeric genes resulting from transcriptional initiation in prokaryotic plasmid sequences in transfected mammalian cells (Maxwell, et al, 1989). Additional elements, such as ones that modify the expression strength of the HIV LTR, may also be introduced. Such elements may be useful for modulating the level of PT SI expression in HIV-infected cells.
  • the anti-HIV efficacy of vector constructs produced in accordance with the present invention may be assessed using any of several in vitro HIV assays known to those skilled in the art.
  • one sample of cells is typically transfected with a vector construct containing a region of the HIV LTR operably linked to a gene encoding the S-1 subunit oi pertussis toxin, and another sample of cells is transfected with a mock construct or left untransfected.
  • the cells are then infected with HIV, and the amount of HIV contained in the samples following a suitable incubation period is assayed using an HIV assay.
  • Effective constructs are those that reduce the amount of HIV in samples of cells transfected with those constructs.
  • Example 2 Two exemplary HIV assays (measurement of HIV p24 protein and syncytium formation) are described herein.
  • Example 2 describes experiments designed to measure the amount of HIV using a p24 antigen assay.
  • Cells transfected with pLTR-SlPT vectors are infected with HIV-1 by co-cultivation with an equal number of virus-infected cells or by incubation with HIV-1 viral stocks. The cells are then assayed for the expression of the HIV-1 p24 antigen using the HIV-1 p24 Antigen Quantitation Panel from Abbott Laboratories (North Chicago, IL).
  • Example 3 describes a syncytium formation assay. Infection of a monolayer of cells by a syncytium-inducing (SI) isolate of HIV typically results in the formation of syncytia (cells with five or more nuclei). Substances which inhibit viral infection and/or replication also typically inhibit the formation of syncytia. Accordingly, the inhibition of syncytia formation can be used as an assay for compounds effective to inhibit HIV.
  • SI syncytium-inducing
  • HIV expression in the cells For example, quantitative or semi-quantitative PCR (Mullis; Mullis, et al.) may be used to asses the relative amounts of proviral HIV DNA or viral HIV RNA in a sample (Piatak, et al , Verhofstede, et al, Loussert-Ajaka, et al).
  • the proviral HIV DNA level in PBMCs has been found to be highly correlated with the viral load in the plasma of HIV-infected individuals (Verhofstede, et al), and a measure of the proviral HIV DNA may therefore be used as a measure of the viral load.
  • mice homozygous for the severe combined immunodeficiency defect SCID; Bosma, et al, 1983
  • Thy/Liv human fetal thymus and liver
  • the implants are infected with HIV, and the effects of therapies designed to inhibit HIV may be assayed, for example, by analyzing thymocyte subset distributions using flow cytometry.
  • Example 4 describes such an analysis using anti-CD4 and anti-CD8 monoclonal antibodies to determine the percentage of
  • CD4/CD8 double positive, CD4+/CD8- and CD4-/CD8+ single positive, and CD4/CD8 double negative cells are untreated animals infected with HPV.
  • Untreated animals infected with HPV typically show significant reductions of CD4+ cells relative to uninfected (control) animals.
  • Infected animals treated with an effective therapy, such as a vector construct of the present invention have CD4+ cell populations more similar to those of the control animals than those of untreated infected animals.
  • pertussis toxin Various forms of pertussis toxin have been utilized in clinical settings for nearly 50 years, beginning with vaccines against pertussis (whooping cough) employing inactivated forms of the bacterium Bordetella pertussis.
  • the success of the early vaccines was due, in large part, to the antigenic nature of PT, which was later shown to confer protection against pertussis (Anderson, et al; Sato, et al).
  • the toxicity of the pertussis toxin molecule however, posed some difficulties, and post-synthesis processes designed to inactivate the toxin often significantly reduced its immunogenicity (Linggood, et al).
  • the present invention reduces or eliminates the antigenicity problems of IAP described above. Because the active subunit of IAP (SI subunit) is expressed inside the target cells, it is not detected by the circulating T-cells. The small amounts of SI PT that may escape following lysis of infected cells are not expected to elevate plasma concentrations of the toxin to the levels that result in clinically significant immune responses. Since the methods of the present invention utilize only the SI subunit of PT, they are considerably safer than applications using the complete protein. While SI is the active subunit, it does not easily penetrate cells in the absence of the B oligomer. This means that any PT SI released by lysed cells would be considerably less toxic than a similar molar amount of the complete multimeric protein.
  • PT SI from constructs of the present invention is preferably minimal to non-existent in the absence of Tat or HIV infection.
  • One way to reduce such undesired expression is by incorporating, into the constructs, elements which act to silence the HIV LTR promoter in the absence of the Tat protein.
  • elements which allow the expression of PT SI to be up- or down- regulated in the presence of HIV infection may also be introduced into vector constructs of the present invention, to provide a finer degree of control over SI expression.
  • HIV-LTR PT chimeric genes of the present invention may be used to introduce HIV-LTR PT chimeric genes of the present invention into selected target cells.
  • CD4+ positive cells may be isolated from an HIV-positive individual and transfected in vitro with a HIV-LTR/PT construct of the present invention (e.g., as described in Example 1). The cells may then be infused back into the subject. These cells now provide normal CD4+ immune functions. If the cell becomes infected by HIV, expression of the Tat protein induces expression of pT SI . This blocks the further production of infectious HIV virions in the cell, reducing the viral load in patients treated with the methods and/or compositions of the present invention.
  • Therapeutic protection may also be obtained by isolating and transforming a population of hematopoietic stem cells with vector constructs of the present invention using methods known to those skilled in the art.
  • One such isolation method is described by Peault and Uchida.
  • a mixture of hematopoietic cells is isolated from a hematopoietic source, such as bone marrow or spleen, and is enriched for pluripotent human stem cells using a fluorescence activated cell sorter and monoclonal antibody F84.1, which recognizes a stem cell marker.
  • the cells are then transformed with a construct made according to the guidance herein (e.g., pLTR-SlPTl, pLTR-SlPTl or pLTR-SlPTl), and implanted back into the individual in need of treatment.
  • a construct made according to the guidance herein e.g., pLTR-SlPTl, pLTR-SlPTl or pLTR-SlPTl
  • Other methods for isolating human stem cells have also been described (e.g., Civin, Tsukamoto, et al).
  • replication-defective virions containing hybrid vectors (the chimeric genes along with selected viral sequences) of the present invention may be injected directly into selected organs (e.g., thymus) or into the bloodstream, to infect CD4+ cells or other cell types susceptible HIV infection (e.g., monocytes and macrophages).
  • the virions used to transfect host cells are preferably replication-defect
  • the virions may be produced by co-infection of cultured host cells with a helper virus. Following co-infection, the virions are isolated (e.g., by cesium chloride centrifugation) and any remaining helper virus is inactivated (e.g., by heating). The resulting mature virions contain a chimeric gene of the present invention and may be used to infect host cells in the absence of helper virus.
  • High titers of replication-defective recombinant virus, free of helper virus, may also be produced in packaging cell lines containing those components for which the virus is defective (Miller).
  • Methods for manipulating viral vectors are also known in the art (e.g., Grunhaus and Horowitz; Hertz and Gerard; Rosenfeld, et al, 1991, 1992.).
  • restriction enzymes and DNA modifying enzymes are obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). Other chemicals were purchased from Sigma (St. Louis, MO) or United States Biochemical (Cleveland, OH).
  • HIV-l LTR-SlPTl The HIV-l long terminal repeat (HIV-l LTR) region (nucleotides -1068 to +83, where + 1 is the transcriptional start; Wright, et al, 1986), which includes the Tat-responsive element, TAR, as well as other regulatory sequences, is ligated as a BamHl/Hind ⁇ ll fragment to the 5' end of a DNA fragment containing the coding sequence for pertussis toxin SI subunit (the sequence between nucleotides 507 and 1314 of SEQ ID NO:l).
  • HIV-l LTR HIV-l long terminal repeat
  • the resulting fragment is in turn ligated to the 5' end of a —850 bp Bglll/BamHl fragment containing the SV40 small t intron and poly(A) signals (obtained from pSV2-327-3-globin; Maxwell, et al, 1986).
  • This cassette schematized in Fig. 2, is inserted into the EcoRI site of pBR322 (Ausubel, et al, 1988).
  • the pLTR-SlPTl plasmid is grown in bacteria, purified, and introduced into CD4+ cells using a calcium phosphate precipitation method (Graham and Van der Eb, 1973; Ausubel, et al, 1988; Sambrook, et al, 1989; Israel and Honigman, 1991).
  • Vector pLTR-SlPT2 is constructed as follows.
  • An HIV-l LTR region (nucleotides -167 to +80; Jones, et al, 1988) is isolated as a Xhol-Hind ⁇ l fragment, blunted by filling in with Klenow DNA polymerase and cloned into pBR327.
  • the PT-Sl DNA fragment and SV40 small t intron and poly(A) signals (described above) are cloned immediately downstream of the HIV-l LTR.
  • An A-trimer isolated as a Hindlll fragment from pUC.A.1.5 (Maxwell, et al, 1989), is cloned upstream of the HIV-LTR.
  • the A-trimer is a head-to-tail trimer of an SV40 Bcll/Bami DNA fragment specifying polyadenylation of RNA transcripts.
  • HeLa, Jurkat, EL-4 and NIH 3T3 cells are grown in Opti-MEM medium (Gibco- BRL, Gaithersburg, MD) with 3.8% fetal bovine serum in Falcon T-75 flasks, and are harvested and resuspended in Opti-MEM with 10% fetal bovine serum for the electroporation pulse.
  • HeLa cells are grown to about 80% confluence and are suspended at 2-4X10 7 cell/ml.
  • Jurkat and EL-4 cells (human and murine T cell lines, respectively) are grown to about 1X10 6 cells/ml and are suspended at 0.5-1X10 8 cells/ml.
  • NIH 3T3 cells are grown to about 80% confluence and are suspended at 5X10 6 cells/ml. Pulses are performed in 0.1 ml volumes in Biorad cuvettes. Gene Pulser settings are between 220 and 290 volts, with a capacitance of 250 ⁇ farads, yielding time constants of 25-30 msec.
  • Stably transfected cells are produced by co-transfecting pLTR-SlPT2 with pSV2neo (Southern and Berg) using electroporation as above and are selected using 400 /zg/ml G418 (Gibco), added fresh every 3-5 days for approximately two weeks. G418-resistant cells are either cloned or maintained as a pooled population. Cells transfected with a vector containing a reporter construct in place of the S1PT gene are assayed for expression of the reporter following treatment with Tat or infection by HIV. Reporter expression is measured 12-24 hours after transfection. Reporter expression may also be used to test HIV LTR promoter modifications which resulted in up- or down-regulated expression upon activation of the LTR promoter.
  • Vector pLTR-SlPT3 is constructed by replacing the structural gene for chloramphenicol acetyltransferase (CAT) in the vector pGVL3CATs (Felber, et al, 1989) with the DNA fragment encoding the SI subunit of pertussis toxin described above.
  • the vector pGVL3CATs which is derived from pGVl (Jhappan, et al, 1986), contains HIV-l LTR (nucleotides -524 to +80), the CAT gene and SV40 sequences including poly(A) signals and the small t splice region.
  • the vector also contains a pBR322 origin of replication (ori), and an SV40 ori and early promoter ligated to the neo gene (which confers neomycin resistance in bacteria and G418 resistance in animal cells).
  • Viral stocks of pLTR-SlPT3 are produced using packaging cell line ⁇ l (Mann, et al, 1983), which contains a packaging-defective murine sarcoma virus (MSV).
  • the medium of the 2 cells containing recombinant retrovirus is collected and used to infect AM cells, which contain a packaging-defective MSV retrovirus carrying an amphotropic env coat to enable infection of human cells (Cone and Mulligan, 1984).
  • the infected ⁇ AM cells are treated with G418, and resistant AM colonies containing integrated recombinant pLTR-SlPT3 proviruses are tested for virus production on HeLa cells as described by Cone and Mulligan (1984).
  • Several independent G418-resistant colonies of AM cells that generate 10 2 to 10 4 infectious viral particles per milliliter are identified. These clones are used for viral production and infection of CD4+ cells and cell lines.
  • CD4+ T-cells and cell lines are transfected with pLTR-SlPT3 virions by co- incubating the cells with viral stocks produced as above or by co-cultivation with the appropriate packaging cell line containing the pLTR-SlPT3 vector using standard methods (Ausubel, et al., 1988).
  • Suitable CD4+ cell lines include H9, U937 and Motl4 (Felber, et al, 1989).
  • CD4+ cells transfected with pLTR-SlPT vectors as described above, as well as corresponding control cells are infected with HIV-l by co-cultivation with an equal number of virus-infected cells or by incubation with HIV-l viral stocks.
  • the infected cultures of transfected and control cells are incubated for 6-48 hours at 37°C, and compared for the expression of the HIV-l p24 antigen using the HIV-l p24 Antigen Quantitation Panel from Abbott Laboratories (North Chicago, IL) according to the manufacturer's instructions.
  • Lower levels of HIV-l p24 antigen in cells carrying a pLTR-SlPT construct suggest that the pLTR-SlPT construct is effective to inhibit HIV production.
  • Syncytium inhibition assays are conducted according to the methods of Verhofstede, et al, (1994).
  • Control and pLTR-SlPT-transfected CD4+ cells such as MT2 cells, are infected with HIV-l as described above and grown in 28 mm 2 flat wells of 96-well Nunc Microtest plates (Fisher Scientific, Pittsburgh, PA) for 6 to 24 hours at 37 C C.
  • the cultures are then fixed, and the cells examined for syncytia (cells with five or more nuclei). Reduction of the number of syncytia observed in cells carrying a pLTR-SlPT vector relative to control cultures suggests that the pLTR-SlPT construct is effective to inhibit HIV production.
  • the implants are injected with —50 ⁇ of heat- inactivated HIV-l (control) or 1000 infectious units (IU) of either HIV-1 ⁇ R .
  • CSF strain Five to six months post-implantation, the implants are injected with —50 ⁇ of heat- inactivated HIV-l (control) or 1000 infectious units (IU) of either HIV-1 ⁇ R .
  • the group injected with live HIV is split into two sets, one of which is injected with pLTR-SlPT3 virus stock.
  • uninfected mice can be injected with the pLTR-SlPT3 construct to examine in vivo effects of the construct alone.
  • the pLTR-reporter construct, described above can be injected into infected and uninfected mice as an in vivo control demonstrating levels of expression.
  • cell samples are typically isolated and directly assayed for the presence of reporter.
  • the thymocyte subset distribution of the implants is analyzed using flow cytometry 10 - 20 days after inoculation with HIV, using anti-CD4 and anti-CD8 monoclonal antibodies (Becton-Dickinson, San Jose, CA) directly conjugated to phycoerythrin or FITC, respectively.
  • Thy/Liv implants from mice injected with heat-inactivated HIV typically have a subset distribution pattern similar to that of normal human thymus (about 80% of cells are CD8+/CD4+ and most of the remaining cells are CD8-/CD4 + .
  • Implants from mice injected with live HIV-l, but not with pLTR-SlPT3, are expected to be depleted of both cell populations.
  • implants from mice injected with both live HIV-l and pLTR- S1PT3 are expected to have a subset distribution more similar to that observed in the control (heat-inactivated HIV) injected animals.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CATCAAAACG CAGAGGGGAA GACGGG ATG CGT TGC ACT CGG GCA ATT CGC CAA 53 Met Arg Cys Thr Arg Ala lie Arg Gin
  • ATC TAC GAA GTC CGC GCC GAC AAC AAT TTC TAC GGC GCC GCC AGC TCG 91 lie Tyr Glu Val Arg Ala Asp Asn Asn Phe Tyr Gly Ala Ala Ser Ser 125 130 135 TAC TTC GAA TAC GTC GAC ACT TAT GGC GAC AAT GCC GGC CGT ATC CTC 96 Tyr Phe Glu Tyr Val Asp Thr Tyr Gly Asp Asn Ala Gly Arg lie Leu 140 145 150
  • Thr Trp Leu Ala lie Leu Ala Val Thr Ala Pro Val Thr Ser Pro Ala 20 25 30
  • MOLECULE TYPE DNA (genomic)
  • CATCAAAACG CAGAGGGGAA GACGGG ATG CGT TGC ACT CGG GCA ATT CGC CAA 53 Met Arg Cys Thr Arg Ala lie Arg Gin
  • AGC CAG CAG ACT CGC GCC AAT CCC AAC CCC TAC ACA TCG CGA AGG TCC 11 Ser Gin Gin Thr Arg Ala Asn Pro Asn Pro Tyr Thr Ser Arg Arg Ser 205 210 215
  • CATCAAAACG CAGAGGGGAA GACGGG ATG CGT TGC ACT CGG GCA ATT CGC CAA 53
  • AGC CAG CAG ACT CGC GCC AAT CCC AAC CCC TAC ACA TCG CGA AGG TCC 115 Ser Gin Gin Thr Arg Ala Asn Pro Asn Pro Tyr Thr Ser Arg Arg Ser 205 210 215
  • MOLECULE TYPE protein
  • xi SEQUENCE DESCRIPTION : SEQ ID NO : 6 :

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Abstract

L'invention concerne des procédés d'inhibition du VIH-1 à l'aide d'un gène chimère reconstruit. Les procédés consistent à administrer des structures contenant un gène codant la sous-unité S1 de la toxine de la coqueluche (gène S1) liée fonctionnellement à une région de longue terminaison répétée (LTR).
PCT/US1996/007518 1995-05-25 1996-05-21 Procedes d'inhibition du vih WO1996037235A1 (fr)

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US45259895A 1995-05-25 1995-05-25
US08/452,598 1995-05-25

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232229A2 (fr) * 1986-01-28 1987-08-12 SCLAVO S.p.A. Clonage et expression d'un ADN codant pour une toxine de Bordetella pertussis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232229A2 (fr) * 1986-01-28 1987-08-12 SCLAVO S.p.A. Clonage et expression d'un ADN codant pour une toxine de Bordetella pertussis

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PROC. NATL. ACAD. SCI. U.S.A., March 1994, Vol. 91, VIEILLARD et al., "Blocking of Retroviral Infection at a Step Prior to Reverse Transcription in Cells Transformed to Constitutively Express Interferon beta", pages 2689-2693. *
PROC. NATL. ACAD. SCI. U.S.A., November 1994, Vol. 91, WOFFENDIN et al., "Nonviral and Viral Delivery of a Human Immunodeficiency Virus Protective Gene Into Primary Human T Cells", pages 11581-11585. *
VIROLOGY, 1994, Vol. 203, CHOWDHURY et al., "Pertussis Toxin Inhibits Induction of Human Immunodeficiency Virus Type 1 in Infected Monocytes", pages 378-383. *

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