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WO1996037235A1 - Methods for inhibition of hiv - Google Patents

Methods for inhibition of hiv 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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WIPO (PCT)
Prior art keywords
hiv
ala
arg
thr
subunit
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PCT/US1996/007518
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French (fr)
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/en

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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

Methods of inhibiting HIV-1 using chimeric gene constructs are diclosed. The methods include administering constructs containing a gene encoding the S1 subunit of pertussis toxin (S1 gene) operably linked to an HIV long terminal repeat (LTR) region.

Description

METHODS FOR INHIBITION OF HIV
FIELD OF THE INVENTION
The present invention relates to a genetic therapy method for the inhibition of human immunodeficiency virus (HIV).
REFERENCES
Adachi, A., et al, J. Virol 59:284 (1986).
Aldrovandi, G.M, et al, Nature 363:732 (1993). Anderson, E.L., et al, J. Infect. Dis. 157:731 (1988).
Arya, et al, Science 229:69-73 (1985).
Ausubel, F.M., et al. , CURRENT PROTOCOLS IN MOLECULAR BIOLOGY. John Wiley and Sons, Inc., Media, PA (1988).
Bell, S.D., et al, Clin. Exp. Immunol 87:37-45 (1992). Bergeron, L., et al, J. Virol 66(4):2389-97 (1992).
Bokoch, G.M., et al, J. Biol. Chem. 258:2072-2075 (1983).
Bosma, G., et al, Nature 301:527 (1983).
Braddock, et al, Cell 58:269-279 (1989).
Burnette, W.N., et al, Science 242:72 (1988). Center, D.M., et al, Clin. Res. 37(2):406A (1989).
Civin, C.I., U.S. Patent No. 4,714,680, issued 22 December 1987.
Cone, R.D., and Mulligan, R.C., Proc. Natl. Acad. Sci. USA 81:6349 (1984).
Cruikshank, W., et al, J. Cell Biochem. 0(14-D):166 (1990).
Cullen, Cell 46973-982 (1986). Edery, et al, Cell 56:303-312 (1989).
Eisenberg, D., and Wesson, M., Biopolymers 29(1): 171-177 (1990).
Fauci, A.S., Science 239:618 (1988).
Feinberg, et al, Cell 46:807-817 (1986).
Felber and Pavlaskis, Science 239:184-187 (1988). Felber, et al, Proc. Natl. Acad. Sci. USA 86:1495-1499 (1989).
Graham, F.L., and Van der Eb, A.J., Virology 52:456 (1973).
Green, et al, Cell 58:215-223 (1989).
Grunhaus, A. and Horowitz, M.S., Semin. Virol, 3:237-252 (1992).
Harlow, E., et al, ANTIBODIES: A LABORATORY MANUAL. Cold Spring Harbor Laboratory Press (1988). Hauber, et al, Proc. Natl. Acad. Sci. USA 84:6364-6368 (1987).
Hazeki, O., and Ui, M., J. Biol Chem. 256:2856-2862 (1981).
Hertz, J., and Gerard, R.D., Proc. Natl. Acad. Sci. U.S.A., 90:2812-2816 (1993).
Hsia, J.A., et al, J. Biol. Chem. 259:1086-1090 (1983). Israel, K., and Honigman, A., Gene 104:139 (1991).
Jhappan, G.F., et al, J. Virol. 60:750 (1986).
Jones, et al, Genes & Devel. 2:1101 (1988).
Katada, T., and Ui, M., Biochim. Biophys. Acta 421:57-69 (1976).
Katada, T., and Ui, M., Endocrinol 101:1247-1255 (1977). Katada, T., and Ui, M., J. Biol Chem. 04:1822-1827 (1979).
Katada, T., and Ui, M., J. Biol. Chem. 255:9580-9588 (1980).
Katada, T., and Ui, M., J. Biol Chem. 256:8210-8217 (1981).
Katada, T., and Ui, M., Proc. Natl. Acad. Sci. USA 79:1329-1333 (1982a).
Katada, T., and Ui, M., J. Biol. Chem. 257:7210-7216 (1982b). Katada, T., et al, Arch Biochem. Biophys. 224:290-298 (1983).
Koyanagi, Y., et al, Science 236:819 (1987).
Kure, K., et al, Acta Neurolpathol 80(4):393-400 (1990).
Kurose, H., and Ui, M., J. Cyclic Nuc. & Prot. Phosphoryl Res. 9:305-318 (1983).
Kurose, H., et al, J. Biol. Chem. 258:4870-4875 (1983). Laspia, et al, Cell 59:283-292 (1989).
Lee, W.R., et al J. Virol. 66(3): 1799-803 (1992).
Linder, N.E., and Gilman, A.G., Scientific American July: 56-65 (1992).
Linggood, F.V., et al, Exp. Pathol. 44:177 (1962).
Loussert-Ajaka, I., et al, Res. Virol. 145:337-42 (1994). Mann, R., et al, Cell 33:153 (1983).
Maxwell and Maxwell, DNA 7:557-562 (1988).
Maxwell, I.H., et al, Cancer Res. 46:4660 (1986).
Maxwell, I.H., et al, BioTechniques 7:276 (1989).
McCune, J.M., et al , Cell 64:351 (1991). Miller, A.D., Hum. Gene Ther. \:5 (1990).
Mosier, D.E., et al, Science 260:689 (1993).
Mullis, K.B., U.S. Patent No. 4,683,202, issued 28 July 1987.
Mullis, K.B., et al, U.S. Patent No. 4,683,195, issued 28 July 1987.
Murayama, T., et al, Arch Biochem. Biophys. 221:381-390 (1983). Murayama, T., and Ui, M., J. Biol Chem. 258:3319-3326 (1983).
Murayama, T., and Ui, M., J. Biol Chem. 259:761-769 (1984).
Namikawa, R., et al, Science 242:1684 (1988).
Nardi, G., et al, Bollettino del Istiuto Sieroterapico Nfilanese 68(3):277-283 (1989).
Narvanen, A., et al, AIDS 2(2): 119-123 (1988a).
Narvanen, A., et al, J. Med. Virol. 26(2): 111-118 (1988b).
Nogimori, K., et al, Biochim Biophys Acta 801:232-243 (1984).
Ohta, Y., et al, Immunopharmacol. 19(3): 197-205 (1990). Orloff, G.M., et al, J. Immunol. 146(8) :2578-2587 (1991).
Peault, B. and Uchida, N., Int. Patent Pub. No. WO 95/03693, published 9 Feb., 1995.
Peterlin, et al, Proc. Natl. Acad. Sci. USA 83:9734-9738 (1986).
Piatak, M., et al, Science 259:1749 (1993). Pizza, M., et al. , Science 246:497 (1989).
Robinson, W.E., Jr., et al, J. Virol. 64(H):5301-5305 (1990).
Robinson, W.E., Jr., et al, J. Virol 65(8) :4169-4176 (1991).
Rosen, et al, Cell 41:813-823 (1985).
Rosen, et al, Proc. Natl. Acad. Sci. USA 85:2071-2075 (1988). Rosenfeld, M.A., et al, Science 252:431 (1991).
Rosenfeld, M.A., et al, Cell, 68:143-155 (1992).
Ruegg, C.L., et al, J. Virol 63(8):325-360 (1989).
Sambrook, J., et al., MOLECULAR CLONING: A LABORATORY MANUAL. Second Ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY) (1989). Sato, Y., et al. , Lancet i: 122 (1984).
Sodroski, et al, Science 227:171-173 (1985).
Southern, P.J. and P. Berg, J. Mol Appl Gen. 1:327-341 (1982).
Spiegal, A., N Eng. J. Med. 322(20): 1461-1462 (1990).
Stryer, L., BIOCHEMISTRY. Third Edition, Chapter 38, Freeman, W.H., New York, NY, (1988)
Sumi, T., and Ui, M., Endocrinol. 92:352-358 (1975).
Tamura, M., et al, Biochem. 21:5516-6622 (1982).
Tamura, M., et al, J. Biol. Chem. 258:6756-6761 (1983).
Tas, M., et al, Clin. & Experiment. Immunol. 71(1): 13-18 (1988). Tong-Starksen, et al, Proc. Natl. Acad. Sci. USA 80:6845-6849 (1987). Toyota, T., et al, Diabetologia 14:319-323 (1978). Toyota, T., et al, Tohoku J. Exp. Med. 130:105-116 (1980). Tsukamoto, A., et al, U.S. Patent No. 5,061,620, issued 29 October 1991. Tyler, D.S., et al, J. Immunol 145(10):3276-3282 (1990).
Ui, M., Prot. Nuc. Acid & Enzyme 31:26-49 (1986). Verhofstede, C, et al, AIDS 8:1421-7 (1994). Weiss, R.A., Science 260:1273 (1993). Wright, et al, Science 234:988-992 (1986). Yajima, M., et al, J. Biochem. 83:295-303 (1978a).
Yajima, M., et al, J. Biochem. 83:305-312 (1978b). Yamada, M., et al, J. Virol 65(3): 1370-1376 (1991).
BACKGROUND OF THE INVENTION Acquired Immune Deficiency Syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). It is estimated that 1 to 1.5 million Americans are infected with the HIV type I virus (HIV-I). Of these, approximately 30% will progress to AIDS in 5 or 6 years. There have been approximately 360,000 AIDS patients, of whom approximately 250,000 have died. Health Care for AIDS patients in 1992 was estimated to have cost nearly 10 billion dollars. 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.
SUMMARY OF THE INVENTION
In one aspect, 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. In one embodiment, 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).
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. One example of such 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.
In another aspect, 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. In one embodiment, 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. In a related aspect, the present invention includes a retroviral expression vector, containing a chimeric gene as described above.
In yet another aspect, 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. In 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. In a related aspect, 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. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES 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;α in the region defined by amino acids 597 through 611, and 36 through 50, of the respective proteins.
Figure 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.
BRIEF DESCRIPTION OF THE SEQUENCES
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
I. Definitions
The term "operably linked", as used herein, 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. For example, 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.
"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.
II. Overview of Invention
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. Accordingly, the infection of a cell harboring an HIV LTR/S1 PT construct by HIV results in the production of the SI subunit of PT. 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. One such target is G,α, an endogenous inhibitory mammalian guanine nucleotide binding protein (G-protein). Because the S-1 PT recognition site on GjC. is similar to a region of HIV gp41, 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.
III. Human Immunodeficiency Virus
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 cleavage of the gpl6O glycoprotein into the gpl2O and the gp41 glycoproteins is essential for viral infectivity (Lee, et al, 1992). Based on a number of studies, it is now believed that the transmembrane protein HIV gp41 is the mediator of viral fusion (Yamada, et al, 1991; Kure, et al, 1990; Tas, et al, 1988; Orloff, et al, 1991; Robinson, Jr., et al, 1991; Robinson, Jr., et al, 1990; Tyler, et al, 1990; Nardi, et al, 1989; Ruegg, et al, 1989; Eisenberg and Wesson, 1990; Narvanen, et al, 1988a; Bell, et al, 1992).
IV. G Proteins
The G proteins are heterotrimers, with subunits designated a, β and γ 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 β and γ 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. 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).
Hydrolysis of GTP to GDP initiates deactivation of the G protein. The dissociation of GDP from the G protein is apparently the rate limiting step of this process, and dissociation of GDP is accelerated by interaction between the G protein and the receptor protein. Considerable evidence exists that a cycle of dissociation and association of G protein subunits is superimposed on this regulatory GTPase cycle.
There exist several distinct families of a subunits, one of which, termed GjC., inhibits adenylate cyclase and stimulates certain potassium channels. The GJQ; oligomers are composed of approximately 354 amino acids and have a calculated molecular weight of about 40,400.
V. Pertussis Toxin
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).
Previous studies have shown that B. pertussis produces adjuvant active substances including
LPF (lymphocytosis promoting factor), HSF (histamine sensitizing factor),
ADP-ribosyltransferase and NAD-glycohydrolase (Ui, 1986). These activities are now attributed to IAP, and it is generally acknowledged that IAP is the B. pertussis toxin, or pertussis toxin (Ui, 1986).
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
(binding) oligomer (Tamura, et al, 1983).
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). As is described in more detail below, 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„α, 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.
VI. The Action of Pertussis Toxin on Gα and gp41
Pertussis toxin modifies the Gsα 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). This mechanism accounts for both the in vivo and in vitro activity of IAP, 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). The toxicity of IAP in mammalian cells is also due, at least in part, to ADP ribosylation of G;α.
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 Gjα 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 (Εisenberg and Wesson, 1990; Narvanen, et al, 1988a; Bell, et al, 1992; Narvanen, et al, 1988b). The HIV env gp41 retains human Gμ serine-44, deletes Gjα serine-47 and mutates threonine-48 to leucine, a non phosphorylatable amino acid residue. However, the similarity of the HIV gp41 GTP-hydrophobic pocket to that of G,α 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 Gjα. In this regard, according to the teachings of the present invention, a single SI subunit amino acid substitution of lysine (K) for arginine (R) at the 57 position (i.e., K-57 for R-57) can result in specific ADP-ribosylation of cysteine (C-253) on HIV env gp41 without concurrent ADP- ribosylation of Gjα, and G0α. Accordingly, 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.
Due to the relatively high degree of homology between HIV gp41, and the env gp41 of other retroviruses, methods of the present invention may be applied to the inhibition of these other retroviruses as well. 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.
VII. Therapeutic Constructs
According to the present invention, 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). The construction of specific retroviral vectors (pLTR-SlPTl, pLTR-SlPT2 and pLTR-SlPT3) containing the HIV-LTR and pertussis toxin S-1 sequences is described in Example 1. 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. The plasmid constructs may be transfected into cells using any of several methods known to those skilled in the art, including, for example, calcium phosphate precipitation or electroporation as described in Example 1. 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. Other elements may be introduced into the constructs to improve the efficiency and/or selectivity of expression. For example, 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. One example of such an element is the A-trimer (Maxwell, et al, 1989), which is inserted upstream of the LTR in pLTR-SlPT2. 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.
VIII. In Vitro Assays
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. In such assays, 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. 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. In Example 3, control and pLTR-SlPT-transfected CD4+ cells are infected with HIV-1 as described in Example 1. The cultures are then fixed and examined microscopically for syncytia formation (cells with five or more nuclei). Other methods known to those skilled in the art may be used to assess the level of
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.
IX. In Vivo Assays The efficacy of constructs of the present invention to inhibit HIV replication may also be assayed in suitable in vivo models of HIV infection, such as the SCID-Hu mice model (Namikawa, et al, 1988; McCune, et al, 1991; Mosier, et al, 1993; Aldrovandi, et al, 1993) described in Example 4. In this model, mice homozygous for the severe combined immunodeficiency defect (SCID; Bosma, et al, 1983) are transplanted with human fetal thymus and liver (Thy/Liv). The transplantation results in the development of a co-joint human organ which allows normal maturation of human thymocytes.
Five to six months post-implantation, 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. 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.
X. Clinical Results Using 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). Recent studies of recombinant PT SI mutants with reduced cytopathogenicity and conserved protective epitopes suggest that it is possible to separate the toxicity of the molecule (ADP ribosylation of Gjα and/or G0α) from the antigenicity (Burnette, et al; Pizza, et al). Early clinical studies using complete, active IAP multimers to treat diabetes showed some promising therapeutic results (Toyota, et al, 1978, 1980), but were not actively pursued due to problems related to IAP's antigenicity and allergenicity. In order to overcome the neutralization of injected IAP by circulating anti-IAP antibodies, injection of large amounts or repeated administration appeared necessary, thereby increasing the risk of allergic and/or anaphylactic reactions. Methods to reduce the antigenicity of IAP interfere with the target cell-receptor binding that allows SI subunit to enter the cells and ADP- ribosylate the target proteins, decreasing the utility of the toxin as a therapeutic.
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. The expression of 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. One example of such an element is the A trimer (Maxwell, et al, 1989), discussed in Example 1. Further, 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.
XI. Delivery of Constructs to Cells and Tissues Any of a variety of methods known to those skilled in the art may be used to introduce HIV-LTR PT chimeric genes of the present invention into selected target cells. For example, 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. In the method, 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. Other methods for isolating human stem cells have also been described (e.g., Civin, Tsukamoto, et al). Alternatively, 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-defective, such that the virus is not able to replicate in the host cells.
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.).
The following examples illustrate but in no way are intended to limit the present invention. MATERIALS AND METHODS
Unless otherwise indicated, 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).
General recombinant manipulations (Sambrook, et al; Ausubel, et al.) and immunological procedures (Harlow, et al.) are carried out by standard procedures.
EXAMPLE 1 Production of CD4+ T Cells Containing HIV-l LTR/ Pertussis Toxin Vector Constructs Vector pLTR-SlPTl is constructed as follows. 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/Hindϊll 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). 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-Hindϊϊl 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.
Several cell lines are transfected with pLTR-SlPT2 using electroporation (Maxwell and Maxwell, 1988; BioRad "GENE PULSER", BioRad Laboratories, Hercules, CA) as follows. 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 2-4X107 cell/ml. Jurkat and EL-4 cells (human and murine T cell lines, respectively) are grown to about 1X106 cells/ml and are suspended at 0.5-1X108 cells/ml. NIH 3T3 cells are grown to about 80% confluence and are suspended at 5X106 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 μ 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 Ψl (Mann, et al, 1983), which contains a packaging-defective murine sarcoma virus (MSV). Forty-eight hours after transfection with pLTR-SlPT3, 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 ΨAM 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 102 to 104 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).
EXAMPLE 2 HIV-l P24 Assay
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 (relative to normal cells) suggest that the pLTR-SlPT construct is effective to inhibit HIV production.
EXAMPLE 3
Syncytium Formation Assay 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 mm2 flat wells of 96-well Nunc Microtest plates (Fisher Scientific, Pittsburgh, PA) for 6 to 24 hours at 37CC. 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.
EXAMPLE 4 SCID-Hu Mouse Assay The efficacy of pLTR-SlPT constructs on HIV-l infection in vivo is assessed using the SCID-hu mouse model for HIV infection (Aldrovandi, et al, 1993). Mice homozygous for the severe combined immunodeficiency defect (SCID; Bosma, et al, 1983) are transplanted with human fetal thymus and liver (Thy/Liv) as described by Aldrovandi, et al, (1993).
Five to six months post-implantation, the implants are injected with —50 μ\ of heat- inactivated HIV-l (control) or 1000 infectious units (IU) of either HIV-1ΓR.CSF strain
(Koyanagi, et al, 1987), HrV-lNL4.3 strain (Adachi, et al, 1986) or a pool of HIV clinical isolates. The group injected with live HIV is split into two sets, one of which is injected with pLTR-SlPT3 virus stock.
Further, uninfected mice can be injected with the pLTR-SlPT3 construct to examine in vivo effects of the construct alone. Also, the pLTR-reporter construct, described above, can be injected into infected and uninfected mice as an in vivo control demonstrating levels of expression. When using the reporter gene, 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. In contrast, 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.
While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Panther Scientific, Inc.
(ii) TITLE OF INVENTION: Methods for Inhibition of HIV (iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Dehlinger & Associates
(B) STREET: 350 Cambridge Avenue, Suite 250
(C) CITY: Palo Alto (D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94306
(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT
(B) FILING DATE: 21-MAY-1996
(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/452,598
(B) FILING DATE: 25-MAY-1995
(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Sholtz, Charles K.
(B) REGISTRATION NUMBER: 38,615
(C) REFERENCE/DOCKET NUMBER: 6215-0001.41
(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 324-0880
(B) TELEFAX: (415) 324-0960
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1316 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Bordetella pertussis gene for toxin subunit SI - X16347
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 507..1316 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GAATTCGTCG CCTCGCCCTG GTTCGCCGTC ATGGCCCCCA AGGGAACCGA CCCCAAGATA 6 ATCGTCCTGC TCAACCGCCA CATCAACGAG GCGCTGCAGT CCAAGGCGGT CGTCGAGGCC 12
TTTGCCGCCC AAGGCGCCAC GCCGGTCATC GCCACGCCGG ATCAGACCCG CGGCTTCATC 18
GCAGACGAGA TCCAGCGCTG GGCCGGCGTC GTGCGCGAAA CCGGCGCCAA GCTGAAGTAG 24
CAGCGCAGCC CTCCAACGCG CCATCCCCGT CCGGCCGGCA CCATCCCGCA TACGTGTTGG 30
CAACCGCCAA CGCGCATGCG TGCAGATTCG TCGTACAAAA CCCTCGATTC TTCCGTACAT 36 CCCGCTACTG CAATCCAACA CGGCATGAAC GCTCCTTCGG CGCAAAGTCG CGCGATGGTA 42
CCGGTCACCG TCCGGACCGT GCTGACCCCC CTGCCATGGT GTGATCCGTA AAATAGGCAC 48
CATCAAAACG CAGAGGGGAA GACGGG ATG CGT TGC ACT CGG GCA ATT CGC CAA 53 Met Arg Cys Thr Arg Ala lie Arg Gin
1 5
ACC GCA AGA ACA GGC TGG CTG ACG TGG CTG GCG ATT CTT GCC GTC ACG 58 Thr Ala Arg Thr Gly Trp Leu Thr Trp Leu Ala lie Leu Ala Val Thr 10 15 20 25
GCG CCC GTG ACT TCG CCG GCA TGG GCC GAC GAT CCT CCC GCC ACC GTA 62 Ala Pro Val Thr Ser Pro Ala Trp Ala Asp Asp Pro Pro Ala Thr Val 30 35 40
TAC CGC TAT GAC TCC CGC CCG CCG GAG GAC GTT TTC CAG AAC GGA TTC 67 Tyr Arg Tyr Asp Ser Arg Pro Pro Glu Asp Val Phe Gin Asn Gly Phe 45 50 55 ACG GCG TGG GGA AAC AAC GAC AAT GTG CTC GAA CAT CTG ACC GGA CGT 72 Thr Ala Trp Gly Asn Asn Asp Asn Val Leu Glu His Leu Thr Gly Arg 60 65 70
TCC TGC CAG GTC GGC AGC AGC AAC AGC GCT TTC GTC TCC ACC AGC AGC 77 Ser Cys Gin Val Gly Ser Ser Asn Ser Ala Phe Val Ser Thr Ser Ser 75 80 85
AGC CGG CGC TAT ACC GAG GTC TAT CTC GAA CAT CGC ATG CAG GAA GCG 82 Ser Arg Arg Tyr Thr Glu Val Tyr Leu Glu His Arg Met Gin Glu Ala 90 95 100 105
GTC GAG GCC GAA CGC GCC GGC AGG GGC ACC GGC CAC TTC ATC GGC TAC 86 Val Glu Ala Glu Arg Ala Gly Arg Gly Thr Gly His Phe lie Gly Tyr 110 115 120
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
GCC GGC GCG CTG GCC ACC TAC CAG AGC GAA TAT CTG GCA CAC CGG CGC 101 Ala Gly Ala Leu Ala Thr Tyr Gin Ser Glu Tyr Leu Ala His Arg Arg 155 160 165
ATT CCG CCC GAA AAC ATC CGC AGG GTA ACG CGG GTC TAT CAC AAC GGC 106 lie Pro Pro Glu Asn lie Arg Arg Val Thr Arg Val Tyr His Asn Gly 170 175 180 185 ATC ACC GGC GAG ACC ACG ACC ACG GAG TAT TCC AAC GCT CGC TAC GTC 110 lie Thr Gly Glu Thr Thr Thr Thr Glu Tyr Ser Asn Ala Arg Tyr Val 190 195 200 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
GTA GCG TCG ATC GTC GGC ACA TTG GTG CGC ATG GCG CCG GTG GTG GGC 120 Val Ala Ser lie Val Gly Thr Leu Val Arg Met Ala Pro Val Val Gly 220 225 230
GCT TGC ATG GCG CGG CAG GCC GAA AGC TCC GAG GCC ATG GCA GCC TGG 125 Ala Cys Met Ala Arg Gin Ala Glu Ser Ser Glu Ala Met Ala Ala Trp 235 240 245
TCC GAA CGC GCC GGC GAG GCG ATG GTT CTC GTG TAC TAC GAA AGC ATC 130 Ser Glu Arg Ala Gly Glu Ala Met Val Leu Val Tyr Tyr Glu Ser lie 250 255 260 265
GCG TAT TCG TTC TA 131
Ala Tyr Ser Phe
270
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :
Met Arg Cys Thr Arg Ala lie Arg Gin Thr Ala Arg Thr Gly Trp Leu 1 5 10 15
Thr Trp Leu Ala lie Leu Ala Val Thr Ala Pro Val Thr Ser Pro Ala 20 25 30
Trp Ala Asp Asp Pro Pro Ala Thr Val Tyr Arg Tyr Asp Ser Arg Pro 35 40 45
Pro Glu Asp Val Phe Gin Asn Gly Phe Thr Ala Trp Gly Asn Asn Asp 50 55 60 Asn Val Leu Glu His Leu Thr Gly Arg Ser Cys Gin Val Gly Ser Ser 65 70 75 80
Asn Ser Ala Phe Val Ser Thr Ser Ser Ser Arg Arg Tyr Thr Glu Val 85 90 95
Tyr Leu Glu His Arg Met Gin Glu Ala Val Glu Ala Glu Arg Ala Gly 100 105 110
Arg Gly Thr Gly His Phe lie Gly Tyr lie Tyr Glu Val Arg Ala Asp 115 120 125
Asn Asn Phe Tyr Gly Ala Ala Ser Ser Tyr Phe Glu Tyr Val Asp Thr 130 135 140 Tyr Gly Asp Asn Ala Gly Arg lie Leu Ala Gly Ala Leu Ala Thr Tyr 145 150 155 160 Gin Ser Glu Tyr Leu Ala His Arg Arg lie Pro Pro Glu Asn lie Arg 165 170 175
Arg Val Thr Arg Val Tyr His Asn Gly lie Thr Gly Glu Thr Thr Thr 180 185 190
Thr Glu Tyr Ser Asn Ala Arg Tyr Val Ser Gin Gin Thr Arg Ala Asn 195 200 205 Pro Asn Pro Tyr Thr Ser Arg Arg Ser Val Ala Ser lie Val Gly Thr 210 215 220
Leu Val Arg Met Ala Pro Val Val Gly Ala Cys Met Ala Arg Gin Ala 225 230 235 240
Glu Ser Ser Glu Ala Met Ala Ala Trp Ser Glu Arg Ala Gly Glu Ala 245 250 255
Met Val Leu Val Tyr Tyr Glu Ser lie Ala Tyr Ser Phe 260 265
(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1500 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Bordetella pertussis gene for toxin subunit SI - M13223
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 507..1316
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAATTCGTCG CCTCGCCCTG GTTCGCCGTC ATGGCCCCCA AGGGAACCGA CCCCAAGATA 6 ATCGTCCTGC TCAACCGCCA CATCAACGAG GCGCTGCAGT CCAAGGCGGT CGTCGAGGCC 12
TTTGCCGCCC AAGGCGCCAC GCCGGTCATC GCCACGCCGG ATCAGACCCG CGGCTTCATC 18
GCAGACGAGA TCCAGCGCTG GGCCGGCGTC GTGCGCGAAA CCGGCGCCAA GCTGAAGTAG 24
CAGCGCAGCC CTCCAACGCG CCATCCCCGT CCGGCCGGCA CCATCCCGCA TACGTGTTGG 30
CAACCGCCAA CGCGCATGCG TGCAGATTCG TCGTACAAAA CCCTCGATTC TTCCGTACAT 36 CCCGCTACTG CAATCCAACA CGGCATGAAC GCTCCTTCGG CGCAAAGTCG CGCGATGGTA 42
CCGGTCACCG TCCGGACCGT GCTGACCCCC CTGCCATGGT GTGATCCGTA AAATAGGCAC 48
CATCAAAACG CAGAGGGGAA GACGGG ATG CGT TGC ACT CGG GCA ATT CGC CAA 53 Met Arg Cys Thr Arg Ala lie Arg Gin
1 5 ACC GCA AGA ACA GGC TGG CTG ACG TGG CTG GCG ATT CTT GCC GTC ACG 5 Thr Ala Arg Thr Gly Trp Leu Thr Trp Leu Ala He Leu Ala Val Thr 10 15 20 25 GCG CCC GTG ACT TCG CCG GCA TGG GCC GAC GAT CCT CCC GCC ACC GTA 6 Ala Pro Val Thr Ser Pro Ala Trp Ala Asp Asp Pro Pro Ala Thr Val 30 35 40
TAC CGC TAT GAC TCC CGC CCG CCG GAG GAC GTT TTC CAG AAC GGA TTC 6 Tyr Arg Tyr Asp Ser Arg Pro Pro Glu Asp Val Phe Gin Asn Gly Phe 45 50 55
ACG GCG TGG GGA AAC AAC GAC AAT GTG CTC GAC CAT CTG ACC GGA CGT 7 Thr Ala Trp Gly Asn Asn Asp Asn Val Leu Asp His Leu Thr Gly Arg 60 65 70
TCC TGC CAG GTC GGC AGC AGC AAC AGC GCT TTC GTC TCC ACC AGC AGC 7 Ser Cys Gin Val Gly Ser Ser Asn Ser Ala Phe Val Ser Thr Ser Ser 75 80 85
AGC CGG CGC TAT ACC GAG GTC TAT CTC GAA CAT CGC ATG CAG GAA GCG 8 Ser Arg Arg Tyr Thr Glu Val Tyr Leu Glu His Arg Met Gin Glu Ala 90 95 100 105 GTC GAG GCC GAA CGC GCC GGC AGG GGC ACC GGC CAC TTC ATC GGC TAC 8 Val Glu Ala Glu Arg Ala Gly Arg Gly Thr Gly His Phe He Gly Tyr 110 115 120
ATC TAC GAA GTC CGC GCC GAC AAC AAT TTC TAC GGC GCC GCC AGC TCG 9 He 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 9 Tyr Phe Glu Tyr Val Asp Thr Tyr Gly Asp Asn Ala Gly Arg He Leu 140 145 150
GCC GGC GCG CTG GCC ACC TAC CAG AGC GAA TAT CTG GCA CAC CGG CGC 10 Ala Gly Ala Leu Ala Thr Tyr Gin Ser Glu Tyr Leu Ala His Arg Arg 155 160 165
ATT CCG CCC GAA AAC ATC CGC AGG GTA ACG CGG GTC TAT CAC AAC GGC 10 He Pro Pro Glu Asn He Arg Arg Val Thr Arg Val Tyr His Asn Gly 170 175 180 185 ATC ACC GGC GAG ACC ACG ACC ACG GAG TAT TCC AAC GCT CGC TAC GTC 11 He Thr Gly Glu Thr Thr Thr Thr Glu Tyr Ser Asn Ala Arg Tyr Val 190 195 200
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
GTA GCG TCG ATC GTC GGC ACA TTG GTG CGC ATG GCG CCG GTG ATA GGC 12 Val Ala Ser He Val Gly Thr Leu Val Arg Met Ala Pro Val He Gly 220 225 230
GCT TGC ATG GCG CGG CAG GCC GAA AGC TCC GAG GCC ATG GCA GCC TGG 12 Ala Cys Met Ala Arg Gin Ala Glu Ser Ser Glu Ala Met Ala Ala Trp 235 240 245
TCC GAA CGC GCC GGC GAG GCG ATG GTT CTC GTG TAC TAC GAA AGC ATC 13 Ser Glu Arg Ala Gly Glu Ala Met Val Leu Val Tyr Tyr Glu Ser He 250 255 260 265 GCG TAT TCG TTC TAGACCTGGC CCAGCCCCGC CCAACTCCGG TAATTGAACA 13
Ala Tyr Ser Phe
270 GCATGCCGAT CGACCGCAAG ACGCTCTGCC ATCTCCTGTC CGTTCTGCCG TTGGCCCTCC 141
TCGGATCTCA CGTGGCGCGG GCCTCCACGC CAGGCATCGT CATTCCGCCG CAGGAACAGA 147 TTACCCAGCA TGGCAGCCCC TATGGAC 150
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 269 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Arg Cys Thr Arg Ala He Arg Gin Thr Ala Arg Thr Gly Trp Leu 1 5 10 15
Thr Trp Leu Ala He Leu Ala Val Thr Ala Pro Val Thr Ser Pro Ala 20 25 30
Trp Ala Asp Asp Pro Pro Ala Thr Val Tyr Arg Tyr Asp Ser Arg Pro 35 40 45
Pro Glu Asp Val Phe Gin Asn Gly Phe Thr Ala Trp Gly Asn Asn Asp 50 55 60 Asn Val Leu Asp His Leu Thr Gly Arg Ser Cys Gin Val Gly Ser Ser 65 70 75 80
Asn Ser Ala Phe Val Ser Thr Ser Ser Ser Arg Arg Tyr Thr Glu Val 85 90 95
Tyr Leu Glu His Arg Met Gin Glu Ala Val Glu Ala Glu Arg Ala Gly 100 105 110
Arg Gly Thr Gly His Phe He Gly Tyr He Tyr Glu Val Arg Ala Asp 115 120 125
Asn Asn Phe Tyr Gly Ala Ala Ser Ser Tyr Phe Glu Tyr Val Asp Thr 130 135 140 Tyr Gly Asp Asn Ala Gly Arg He Leu Ala Gly Ala Leu Ala Thr Tyr 145 150 155 160
Gin Ser Glu Tyr Leu Ala His Arg Arg He Pro Pro Glu Asn He Arg 165 170 175
Arg Val Thr Arg Val Tyr His Asn Gly He Thr Gly Glu Thr Thr Thr 180 185 190
Thr Glu Tyr Ser Asn Ala Arg Tyr Val Ser Gin Gin Thr Arg Ala Asn 195 200 205
Pro Asn Pro Tyr Thr Ser Arg Arg Ser Val Ala Ser He Val Gly Thr 210 215 220 Leu Val Arg Met Ala Pro Val He Gly Ala Cys Met Ala Arg Gin Ala 225 230 235 240
Glu Ser Ser Glu Ala Met Ala Ala Trp Ser Glu Arg Ala Gly Glu Ala 245 250 255
Met Val Leu Val Tyr Tyr Glu Ser He Ala Tyr Ser Phe 260 265 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1500 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Bordetella pertussis gene for toxin subunit SI - A13359 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 507..1316
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GAATTCGTCG CCTCGCCCTG GTTCGCCGTC ATGGCCCCCA AGGGAACCGA CCCCAAGATA 6 ATCGTCCTGC TCAACCGCCA CATCAACGAG GCGCTGCAGT CCAAGGCGGT CGTCGAGGCC 12
TTTGCCGCCC AAGGCGCCAC GCCGGTCATC GCCACGCCGG ATCAGACCCG CGGCTTCATC 18
GCAGACGAGA TCCAGCGCTG GGCCGGCGTC GTGCGCGAAA CCGGCGCCAA GCTGAAGTAG 24 CAGCGCAGCC CTCCAACGCG CCATCCCCGT CCGGCCGGCA CCATCCCGCA TACGTGTTGG 30
CAACCGCCAA CGCGTATGCG TGCAGATTCG TCGTACAAAA CCCTCGATTC TTCCGTACAT 36
CCCGCTACTG CAATCCAACA CGGCATGAAC GCTCCTTCGG CGCAAAGTCG CGCGATGGTA 42
CCGGTCACCG TCCGGACCGT GCTGACCCCC CTGCCATGGT GTGATCCGTA AAATAGGCAC 48
CATCAAAACG CAGAGGGGAA GACGGG ATG CGT TGC ACT CGG GCA ATT CGC CAA 53
Met Arg Cys Thr Arg Ala He Arg Gin 1 5
ACC GCA AGA ACA GGC TGG CTG ACG TGG CTG GCG ATT CTT GCC GTC ACG 58 Thr Ala Arg Thr Gly Trp Leu Thr Trp Leu Ala He Leu Ala Val Thr 10 15 20 25
GCG CCC GTG ACT TCG CCG GCA TGG GCC GAC GAT CCT CCC GCC ACC GTA 62 Ala Pro Val Thr Ser Pro Ala Trp Ala Asp Asp Pro Pro Ala Thr Val 30 35 40 TAC CGC TAT GAC TCC CGC CCG CCG GAG GAC GTT TTC CAG AAC GGA TTC 67 Tyr Arg Tyr Asp Ser Arg Pro Pro Glu Asp Val Phe Gin Asn Gly Phe 45 50 55
ACG GCG TGG GGA AAC AAC GAC AAT GTG CTC GAA CAT CTG ACC GGA CGT 72 Thr Ala Trp Gly Asn Asn Asp Asn Val Leu Glu His Leu Thr Gly Arg 60 65 70
TCC TGC CAG GTC GGC AGC AGC AAC AGC GCT TTC GTC TCC ACC AGC AGC 77 Ser Cys Gin Val Gly Ser Ser Asn Ser Ala Phe Val Ser Thr Ser Ser 75 80 85 AGC CGG CGC TAT ACC GAG GTC TAT CTC GAA CAT CGC ATG CAG GAA GCG 82 Ser Arg Arg Tyr Thr Glu Val Tyr Leu Glu His Arg Met Gin Glu Ala 90 95 100 105 GTC GAG GCC GAA CGC GCC GGC AGG GGC ACC GGC CAC TTC ATC GGC TAC 86 Val Glu Ala Glu Arg Ala Gly Arg Gly Thr Gly His Phe He Gly Tyr 110 115 120
ATC TAC GAA GTC CGC GCC GAC AAC AAT TTC TAC GGC GCC GCC AGC TCG 91 He 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 He Leu 140 145 150
GCC GGC GCG CTG GCC ACC TAC CAG AGC GAA TAT CTG GCA CAC CGG CGC 101 Ala Gly Ala Leu Ala Thr Tyr Gin Ser Glu Tyr Leu Ala His Arg Arg 155 160 165
ATT CCG CCC GAA AAC ATC CGC AGG GTA ACG CGG GTC TAT CAC AAC GGC 106 He Pro Pro Glu Asn He Arg Arg Val Thr Arg Val Tyr His Asn Gly 170 175 180 185 ATC ACC GGC GAG ACC ACG ACC ACG GAG TAT TCC AAC GCT CGC TAC GTC 110 He Thr Gly Glu Thr Thr Thr Thr Glu Tyr Ser Asn Ala Arg Tyr Val 190 195 200
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
GTA GCG TCG ATC GTC GGC ACA TTG GTG CGC ATG GCG CCG GTG GTG GGC 120 Val Ala Ser He Val Gly Thr Leu Val Arg Met Ala Pro Val Val Gly 220 225 230
GCT TGC ATG GCG CGG CAG GCC GAA AGC TCC GAG GCC ATG GCA GCC TGG 125 Ala Cys Met Ala Arg Gin Ala Glu Ser Ser Glu Ala Met Ala Ala Trp 235 240 245
TCC GAA CGC GCC GGC GAG GCG ATG GTT CTC GTG TAC TAC GAA AGC ATC 130 Ser Glu Arg Ala Gly Glu Ala Met Val Leu Val Tyr Tyr Glu Ser He 250 255 260 265 GCG TAT TCG TTC TAGACCTGGC CCAGCCCCGC CCAACTCCGG TAATTGAACA 135
Ala Tyr Ser Phe
270
GCATGCCGAT CGACCGCAAG ACGCTCTGCC ATCTCCTGTC CGTTCTGCCG TTGGCCCTCC 141
TCGGATCTCA CGTGGCGCGG GCCTCCACGC CAGGCATCGT CATTCCGCCG CAGGAACAGA 147
TTACCCAGCA TGGCAGCCCC TATGGAC 150
(2) INFORMATION FOR SEQ ID NO:6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 269 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 6 :
Met Arg Cys Thr Arg Ala He Arg Gin Thr Ala Arg Thr Gly Trp Leu 1 5 10 15
Thr Trp Leu Ala He Leu Ala Val Thr Ala Pro Val Thr Ser Pro Ala 20 25 30
Trp Ala Asp Asp Pro Pro Ala Thr Val Tyr Arg Tyr Asp Ser Arg Pro 35 40 45
Pro Glu Asp Val Phe Gin Asn Gly Phe Thr Ala Trp Gly Asn Asn Asp 50 55 60 Asn Val Leu Glu His Leu Thr Gly Arg Ser Cys Gin Val Gly Ser Ser 65 70 75 80
Asn Ser Ala Phe Val Ser Thr Ser Ser Ser Arg Arg Tyr Thr Glu Val 85 90 95
Tyr Leu Glu His Arg Met Gin Glu Ala Val Glu Ala Glu Arg Ala Gly 100 105 110
Arg Gly Thr Gly His Phe He Gly Tyr He Tyr Glu Val Arg Ala Asp 115 120 125
Asn Asn Phe Tyr Gly Ala Ala Ser Ser Tyr Phe Glu Tyr Val Asp Thr 130 135 140 Tyr Gly Asp Asn Ala Gly Arg He Leu Ala Gly Ala Leu Ala Thr Tyr 145 150 155 160
Gin Ser Glu Tyr Leu Ala His Arg Arg He Pro Pro Glu Asn He Arg 165 170 175
Arg Val Thr Arg Val Tyr His Asn Gly He Thr Gly Glu Thr Thr Thr 180 185 190
Thr Glu Tyr Ser Asn Ala Arg Tyr Val Ser Gin Gin Thr Arg Ala Asn 195 200 205
Pro Asn Pro Tyr Thr Ser Arg Arg Ser Val Ala Ser He Val Gly Thr 210 215 220 Leu Val Arg Met Ala Pro Val Val Gly Ala Cys Met Ala Arg Gin Ala 225 230 235 240
Glu Ser Ser Glu Ala Met Ala Ala Trp Ser Glu Arg Ala Gly Glu Ala 245 250 255
Met Val Leu Val Tyr Tyr Glu Ser He Ala Tyr Ser Phe 260 265

Claims

IT IS CLAIMED:
1. A method of inhibiting the production of infectious Human Immunodeficiency Virus 1 (HIV-l) virions in an HIV-l -infected cell, comprising providing an HIV-l -infected cell containing a chimeric gene containing a DNA sequence encoding the SI subunit of pertussis toxin (SI gene) operably linked to an HIV-l long terminal repeat (LTR) region, and growing said cell, where said growing is carried out under conditions where expression of said chimeric gene is induced, wherein expression of the SI subunit inhibits production of infectious HIV-l virions.
2. A method of claim 1, where the DNA sequence encodes an SI subunit that contains an amino acid sequence selected from the group consisting of sequences represented as SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
3. A method of claim 1 or 2, where said chimeric gene further includes, upstream of the LTR region, a DNA fragment comprising a head-to-tail trimer of SV40 polyadenylation signal sequences.
4. A method of any of claims 1-3, where said cell is a CD4+ lymphocyte.
5. A method of any of claims 1-3, where said cell is a monocyte cell.
6. A method of any of claims 1-3, where said cell is a macrophage cell.
7. A chimeric gene, comprising an HIV-l LTR region operably linked to a DNA sequence encoding the SI subunit of pertussis toxin.
8. A chimeric gene of claim 7, further including, upstream of the LTR region, a DNA fragment comprising a head-to-tail trimer of SV40 polyadenylation signal sequences.
9. A chimeric gene of claim 7 or 8, where the DNA sequence encodes an SI subunit containing an amino acid sequence selected from the group consisting of sequences represented as SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
10. A retroviral expression vector, comprising a chimeric gene of any of claims 7-9.
11. A method of reducing HIV-l viral load in an HIV-l -infected subject, comprising isolating CD4+ lymphocytes from the subject, transforming the lymphocytes with a chimeric gene comprising an HIV-l LTR region operably linked to a DNA sequence encoding the SI subunit of pertussis toxin (PT), and introducing lymphocytes carrying the chimeric gene into the subject, wherein said lymphocytes express the SI subunit of PT, infectious HIV-l production is inhibited and said inhibition results in a reduced viral load in the HIV-l -infected subject.
12. A method of reducing HIV-l viral load in a subject harboring HIV-l -infected cells, comprising administering to the subject, a retroviral expression vector containing a chimeric gene comprising an HIV-l 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, wherein infected cells carrying the vector express the SI subunit, which inhibits HIV production and results in a reduced viral load in the HIV-l -infected subject.
PCT/US1996/007518 1995-05-25 1996-05-21 Methods for inhibition of hiv WO1996037235A1 (en)

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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 (en) * 1986-01-28 1987-08-12 SCLAVO S.p.A. Cloning and expression of Bordetella pertussis toxin-encoding DNA

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0232229A2 (en) * 1986-01-28 1987-08-12 SCLAVO S.p.A. Cloning and expression of Bordetella pertussis toxin-encoding DNA

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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