EP1029062A1 - Inhibition de la replication du virus de l'immunodeficience humaine (vih-1) - Google Patents

Inhibition de la replication du virus de l'immunodeficience humaine (vih-1)

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EP1029062A1
EP1029062A1 EP98954989A EP98954989A EP1029062A1 EP 1029062 A1 EP1029062 A1 EP 1029062A1 EP 98954989 A EP98954989 A EP 98954989A EP 98954989 A EP98954989 A EP 98954989A EP 1029062 A1 EP1029062 A1 EP 1029062A1
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cell
hiv
vector
cells
pkr
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Robert J. Suhadolnik
Martin E. Adelson
Kathryn T. Iacono
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Temple University of Commonwealth System of Higher Education
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Temple University of Commonwealth System of Higher Education
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P37/04—Immunostimulants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—Special targeting system for viral vectors
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/855—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants

Definitions

  • This invention relates to intracellular immunization methods and compositions for inhibiting the replication of human virus (HIV), and for treating HIV infection and acquired immunodeficiency syndrome (AIDS)
  • HIV human virus
  • AIDS acquired immunodeficiency syndrome
  • HIV Human immunodeficiency virus
  • AIDS acquired immunodeficiencN syndrome
  • HIV intection or AIDS Current strategies against HIV infection and AIDS. including nucleoside analogues and protease inhibitors, have limited effectiveness as evidenced by the evolution of resistant retroviral strains.
  • HIV virions in infected individuals can also aid in the selection of resistant viral strains.
  • HIV protease inhibitors and nonnucleoside reverse transcriptase inhibitors expanded therapeutic options, permitting combination therapies which target multiple stages in the HIV life cycle.
  • these anti-retroviral compounds have diverse side effects which often lead to complications and cessation of drug therapy.
  • long-term studies have not been completed to assess the degree of retroviral mutation associated with combination therapies in vivo (Moyle et al. , Quart. J. Med. 86: 155-63 (1993)).
  • Another difficulty with present methods is cost. Annual costs for the treatment of AIDS with triple combinations of reverse transcriptase and protease inhibitors are staggering, and such therapies may need to be continued indefinitely.
  • Interferons are hormone-like proteins involved in the defense mechanism of cells against viral infection and tumors.
  • Type I interferons have the capacity to induce a series of antiviral gene products, through signal transduction pathways, which can interfere with viral infection. Included within the family of induced genes are three double-stranded RNA dependent enzymes: the 2' ,5'-oligoadenylate synthetase, adenosine deaminase. and the dsRNA-dependent, interferon-inducible protein kinase, PKR (Baglioni. Cell 17:255-264 (1979); Patterson et al . Virology 210:508-511 (1995)).
  • PKR dsRNA-dependent, interferon-inducible protein kinase
  • PTK p68 protein kinase
  • 2-5OAS 2' ,5'-oligoadenylate synthetase
  • RNase L The p68 protein kinase (PKR) and the 2' ,5'-oligoadenylate synthetase (2-5OAS)/RNase L pathways inhibit viral replication through the inhibition of protein synthesis and degradation of single stranded RNA, respectively.
  • PKR serine/threonine kinase
  • the human cDNA for PKR encodes a 2.5 kb RNA, which is translated into a 550 amino acid protein (Meurs et al. , Cell 62:379-390 (1990)).
  • the binding of two molecules of this 68 kDa PKR about a single dsRNA molecule initiates an autophosphorylation event which is followed by phosphorylation of the subunit of eukaryotic initiation factor 2, preventing a GDP-for-GTP recycling reaction and leading to inhibition of protein synthesis initiation (Lee et al. , Virology 193: 1037-1041 ((1993); Matthews et al. , J. Virol. 65:5657-5662 (1991): Pathak et al . Mol.
  • PKR has been demonstrated to effectively prevent infection of eukaryotic cells by encephalomyocarditis and vaccinia viruses (Lee et al.. Virology 193:1037-1041 (1993)).
  • HIV-1 has also devised strategies to interfere with the enzymatic activities of PKR. It has been reported that HIV-1 infection leads to a significant decline in the amount of intracellular PKR protein levels (Roy et al. , Science 247: 1216-1219 (1990)), and that HIV-1 TAR RNA has an optimal PKR activation concentration and conforms with a bell-shaped activation curve (Maitra et al. , Virology 204:823-827 (1994); Mathews. Seminars Virol. 4:247-257 (1993); Mordechai et al , Virology 206:913-922 (1995); Samuel, J. Biol. Chem. 268:7603-7306 (1993)).
  • the 2' , 5' - oligoadenylate synthetase (2-5OAS) is another interferon induced enzyme in the cellular dsRNA-dependent antiviral response.
  • the 2-5OAS converts ATP into 2', 5' - oligoadenylate (2-5A).
  • the 2-5A synthesized by 2-5OAS binds and activates ribonuclease L, which hydrolyzes both cellular and viral mRNA.
  • the level of 2-5 A is inversely correlated with HIV-1 virion production. In the initial stage of infection there is a transient activation of 2-5OAS.
  • Intracellular immunization is the regulated expression of a molecular species designed to interfere with and prevent viral replication.
  • the concept and name "intracellular immunization" were proposed by David Baltimore in 1988 (Nature 335:395-396 (29 September 1988)).
  • Baltimore proposed that cells could be genetically engineered to express a protein which would make them resistant to viral infection.
  • Baltimore mentioned that, in the case of HIV, retroviral vectors could be used to transduce hematopoietic stem cells with the desired recombinant construct.
  • a gene introduced for the purposes of intracellular immunization must be (i) stably expressed in sufficient quantities to interfere with viral replication, (ii) non-toxic to the cell, and (iii) transferred to the target cell population in a highly efficient, non-toxic manner (Wong et al , Curr. Top. Microbiol Immunol. 179: 159-174 (1992)).
  • the desired gene is introduced into cells under control of the HIV long terminal repeat (LTR).
  • the hybrid gene can then be trans-activated by the Tat gene product or by HIV infection.
  • the Tat protein acts on a cis-acting element of the HIV LTR. known as the TAR region, to increase expression from the LTR (Arva et al . Science 229:69-73 (1985)).
  • the Tat protein is believed to regulate gene expression at both the transcriptional and translational levels. Initially, the majority of transcripts which are initiated from within an HIV-1 LTR are incomplete and the resulting RNA is prematurely short, with an average length of sixty to eighty ribonucleotides.
  • the HIV-1 encoded 15 kDa Tat protein has been proposed to interact with this nascent short message and, in conjunction with cellular cofactors. bind to a stable RNA stem-loop structure within the HIV-1 LTR. TAR (Kashanchi et al . Nature (London) 367:295-299 (1994)). This proteimRNA interaction is thought to increase RNA elongation efficiency, leading to high-level production of full-length mRNAs (Foon et al. . J. Biol. Chem. 271:4201-4208 (1996); Mavankal et al , Proc. Natl. Acad. Sci. USA 93:2089-2094 (1996)).
  • the Tat protein can induce uninfected quiescent T cells to become highly permissive for productive HIV-1 infection (Li et al.. Proc. Natl. Acad. Sci. USA 94:8116-20 (July 1997)).
  • the Tat protein can also stimulate expression of heterologous genes placed 3' to a TAR region (Tong-Starksen et al. , Proc. Natl. Acad. Sci. USA 80:6845-49 (1987)), a property which can be exploited in intracellular immunization protocols.
  • U.S. Patent No. 5,554,528 is directed to constructs and methods of intracellular immunization in which a chimeric diphtheria toxin gene is placed under the regulatory control of an HIV LTR.
  • the HIV-regulated toxin gene is stably introduced into HIV susceptible cells using a retroviral (or other) vector system, and transformed cells commit suicide in response to HIV infection.
  • U.S. Patent No. 5,554,528 discloses a prophetic example in which SCID mice are reconstituted with peripheral blood monocytic cells or bone marrow cells which have been transfected with the HIV-regulated diphtheria toxin gene.
  • the present invention provides constructs and methods for stably introducing an antiviral enzyme into target cells, using a construct wherein antiviral gene expression is activated in the presence of an HIV trans-acting factor. Upon HIV infection of the target cells, antiviral gene expression is activated and viral replication is inhibited.
  • This intracellular immunization approach can generate a reservoir of self-renewing cells which are unable to support HIV replication.
  • the intracellular immunization approach can also be used in combination with other antiviral agents (single or multiple combinations of drugs such as nucleoside analogs and/or protease inhibitors), resulting in improved methods for prevention of and treatment for HIV infection and AIDS.
  • PKR antiviral enzyme Activation of the antiviral enzyme PKR results in the death of HIV- 1 infected cells, thus preventing further viral replication and the subsequent infection of neighboring cells.
  • the inventors constructed retroviral vectors capable of transferring a PKR coding region, under HIV-1 transcriptional regulation, into target Sup Tl lymphoblastoid cells. The target cells were then challenged with HIV- 1. HIV replication, as measured by syncytia formation, was inhibited up to 93 % in the transduced cells.
  • HIV-1 LTR— PKR cDNA transduced clones showed PKR expression through 96 hours post-infection, concomitant with maintenance of PKR autophosphorylation activity.
  • One of the HIV-1 LTR— PKR cDNA transduced clones when supplemented with 93 % less 3'-azido-3'-deoxythymidine than N2- 20P controls, reduced syncytia formation by 90% .
  • the inventors have also shown that intracellular immunization using a 2-5 OAS gene can be used to control HIV infection.
  • One embodiment of the present invention provides a recombinant nucleic acid comprising: (a) a PKR coding region, and
  • a regulatory element comprising all or part of an HIV LTR.
  • the HIV trans-acting factor is an HIV Tat protein.
  • the invention further provides a vector comprising a recombinant nucleic acid according to the invention.
  • the vector is a viral vector.
  • the vector is a retroviral vector.
  • the vector comprises M-MuLV sequences.
  • the retroviral vector has essentially the characteristics of pMEA105 or pMEAlO ⁇ .
  • the invention also provides a viral vector comprising: (a) a 2-50AS coding region, and (b) a regulatory element; wherein the 2-5OAS coding region and regulatory element are operatively linked such that 2-5OAS expression is activated in the presence of HIV trans-acting factors.
  • the regulatory element comprises all or part of an HIV LTR.
  • the HIV trans-acting factor is an HIV Tat protein.
  • the viral vector comprises M-MuLV sequences .
  • the viral vector comprises 5 ' and 3 ' M-MuLV LTRs and a neomycin resistance gene.
  • the viral vector has essentially the characteristics of pMEA109 or pMEAHO.
  • Another aspect of the invention is a cell comprising a nucleic acid according to the invention, or a cell which has been transduced or transformed with a viral vector according to the invention.
  • the cell is a prokaryotic cell. In another preferred embodiment the cell is a eukaryotic cell. In some preferred embodiments the cell is a stem cell. In a most preferred embodiment the cell is a CD34-expressing cell. In some preferred embodiments the cell is a CD4- expressing cell. In some embodiments the cell is a retroviral packaging cell or a retroviral producer cell. In some embodiments the nucleic acid is stably integrated into the cellular genome in such a way that PKR expression is activated by HIV infection. In some embodiments the 2-50AS coding region and regulatory element are stably integrated into the cellular genome in such a way that the 2-5OAS expression is activated by HIV infection.
  • the invention also provides a method of inhibiting the replication of HIV comprising introducing a nucleic acid or a vector according to the invention into a cell which is susceptible to HIV infection.
  • a PKR coding region operatively linked to a regulatory element is stably integrated into the cellular genome, such that PKR expression is activated by HIV infection.
  • a 2-50AS coding region operatively linked to a regulatory element is stably integrated into the cellular genome, such that 2-5OAS expression is activated by HIV infection.
  • Another aspect of the invention is a method of inhibiting HIV replication in a patient in need of such treatment comprising introducing a nucleic acid or a vector according to the invention into cells from the patient.
  • at least one chemotherapeutic agent is also administered to the patient.
  • the chemotherapeutic agent is selected from the group consisting of nucleoside analogs and protease inhibitors.
  • the chemotherapeutic agent is AZT.
  • the nucleic acid or vector is introduced into the cells ex vivo.
  • the nucleic acid or vector is introduced into the cells in vivo.
  • the nucleic acid or vector specifically targets stem cells.
  • the nucleic acid or vector specifically targets CD34-expressing cells. In some embodiments the nucleic acid or vector specifically targets CD4-expressing cells. In some embodiments of the method a PKR coding region operatively linked to a regulatory element is stably integrated into the cellular genome, such that PKR expression is activated by HIV infection. In some embodiments a 2-5OAS coding region operatively linked to a regulatory element is stably integrated into the cellular genome, such that 2- 5OAS expression is activated by HIV infection.
  • the invention further constitutes a method of preventing HIV replication in a human host comprising introducing a nucleic acid or a vector according to the invention into cells from the host.
  • nucleic acid or vector is introduced into the cells ex vivo. In some embodiments the nucleic acid or vector is introduced into the cells in vivo.
  • the invention also constitutes the use of a nucleic acid or a vector according to the invention for the preparation of a medicament to inhibit or prevent HIV replication in a patient or human host.
  • Figures 1A and IB show the construction and genomic integration of the HIV-1 LTR-PKR cDNA constructs.
  • Figure 1A shows the HIV-I LTR— PKR cDNA— poly(A) sequence cloned in forward (pMEA105) and reverse (pMEA106) orientations into the pN2 retroviral vector.
  • Figure IB shows the genomic integration of these constructs into the GP+envAml2 retroviral producer cell line. The location of Xbal sites in pN2 (N2-20), pMEA105 (105-10), and pMEA106 (106-4). and the size of the fragments which contain sequences complementary to the [ j2 P]-neo probe are shown.
  • FIG. 1 shows a Southern blot of Xbal digested genomic DNA from the indicated cell sources.
  • Lanes 1-3 GP+envAml2 cells supplemented with 10, 1. and 0.1 ng pMEA106 (2681 bp);
  • lane 4 N2-20 retroviral producing cells (3283 bp),
  • lane 5 N2-20 transduced, G418 selected SupTl cells (3283 bp);
  • lane 6 106-4 retroviral producing cells (2681 bp);
  • lane 7 106-4 transduced, G418 selected SupTI cells (2681 bp).
  • Arrows depict the migration of the expected 3283 and 2681 bp fragments. The migration of fragments from a lambda Hindlll digest is indicated.
  • Figure 2 shows a challenge of HIV- 1 LTR-PKR transduced SupTl cells with HIV-1 IIIB.
  • Clones were infected with HIV-1 IIIB at an m.o.i. of 0.1 and syncytia were scored in quadruplicate at multiple dilutions. A single syncytia score was calculated as described in Example 5.
  • a control in which HIV-1 was incubated with media alone prior to serial dilution and addition of SupTI indicator cells yielded no syncytia 96 hours after infection.
  • the solid and open bars represent 0 and 10 mM 2-aminopurine, respectively. Results represent the mean ⁇ S.E. of two independent experiments .
  • Figures 3A-3F show photographs (magnification x 100) of syncytia generated by HIV-1 IIIB infection. Transduced, selected clones (1 x 10" cells) were infected with HIV-1 IIIB for 2 h at a m.o.i. of 0.1. Cells were washed and replated at 5 x 10 5 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated FBS. Photographs were taken 96 hours p.i. with an Olympus inverted research microscope.
  • Figures 4A-4F show a Western blot analysis of PKR expression during HIV-I IIIB infection. Protein levels of PKR were monitored by Western blot following HIV-1 infection at 24 h intervals. PKR was calculated to be 68 kDa as determined by migration alongside bovine serum albumin (65 kDa, contained within the rainbow molecular weight markers, Amersham). "U” denotes uninfected cells. Results are representative of two independent experiments.
  • Figures 5A and 5B show PKR autophosphorylation levels throughout HIV-1 IIIB infection in HIV-1 LTR-PKR cDNA transduced clones. Cells were collected at the indicated times after infection with HIV-I IIIB. Following protein extraction and immunoprecipitation, autophosphorylation assays were performed. Extracts were supplemented with 0 ( Figure 5A) or 0.1 . t g/ml ( Figure 5B) of poly(rI)-poly(rC) during the enzymatic assay. Results were quantitated by analysis on a Fuji BAS2000 phosphorimager. For each sample, the bars from left to right represent 0. 24, 48. 72, and 96 hours postinfection, respectively.
  • Figure 6 shows NF- ⁇ B activation in HIV-1 LTR-PKR cDNA transduced clones infected with HIV-1 IIIB.
  • Nuclear extracts from HIV-1 IIIB infected ( + ) and uninfected (-) SupTl cells were prepared 72 hours after infection.
  • Five micrograms of nuclear extract protein were incubated with a 0.0375 pmole [ ⁇ - 32 P] ATP end-labeled KB oligonucleotide probe and resolved on a non-denaturing 4% TBE acrylamide gel.
  • Figure 7 shows treatment of HIV-I LTR-PKR cDNA transduced clones with 3' -azido-3' -deoxythymidine.
  • Each clone (2 x 10 s cells) was incubated with the indicated concentration of AZT 1 hour prior to infection with HIV-1 IIIB. Forty-eight hours p.i. , the cells were serially diluted and 2 x 10 5 SupTl indicator cells were added to each well. Syncytia scores were calculated 96 hours
  • the present invention relates to the discovery of constructs and methods for inhibiting the replication of HIV in a cell which is susceptible to HIV infection, and for the control of HIV infection and AIDS.
  • the inventors placed the enzyme PKR under the transcriptional control of an essential HIV-1 transcriptional element and introduced this construct into HIV-1 receptive cells utilizing a Moloney murine leukemia virus (M-MuLV) retroviral-mediated shuttle system.
  • M-MuLV Moloney murine leukemia virus
  • SupTl lymphoblastoid cell lines transduced with the HIV-1 LTR-PKR cDNA constructs exhibited a significant decrease in syncytia formation when challenged with HIV-1 IIIB.
  • the protein and enzymatic levels of PKR in the transduced cell lines remained elevated following retroviral challenge.
  • Intracellular immunization is a gene therapeutic approach to the treatment of viral infection, in which susceptible cells are genetically modified to express a product which interferes with infection or viral replication.
  • a “regulatory element” is any nucleic acid sequence which is capable of regulating gene expression.
  • “Operatively linked regulatory element” means that a polypeptide coding region is connected to a regulatory element in such a way that the regulatory element can permit expression of the polypeptide when appropriate molecules (such as activator proteins and polymerases) are present in a cell or cell free system.
  • Gene expression means the realization of genetic information encoded by a nucleic acid to produce a functional RNA or protein.
  • Proteins are said to have homology, or similarity, if a substantial number of their amino acids are either (1) identical, or (2) characterized by conservative amino acid substitutions. "Conservative amino acid substitutions” are substitutions with an amino acid which has a related side chain R.
  • Amino acids are typically classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
  • side chain R (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
  • Substantial amino acid sequence homology means an amino acid sequence homology greater than about 30 percent, preferably greater than about 60% , more preferably greater than about 80% , and most preferably greater than about 90 percent.
  • a "biologically active fragment" of a protein is a fragment derived from the protein which retains at least one biological activity of the protein.
  • a "biologically active derivative" of a protein is any analogue, variant, derivative, or mutant which is derived from the protein, which has substantial amino acid sequence homology with the protein, and which retains at least one biological property of the protein.
  • nucleic acid is a polymeric compound comprised of covalently linked subunits called nucleotides.
  • Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single- stranded or double-stranded.
  • DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.
  • a "recombinant nucleic acid” is a nucleic acid molecule in which sequences which are not contiguous in a native context are placed next to each other by experimental manipulation.
  • a "vector” is any means for the transfer of a nucleic acid into a host cell.
  • the term vector includes both viral and nonviral means for introducing the nucleic acid into a prokaryotic cell in vitro, ex vivo, or in vivo.
  • Non-viral vectors include but are not limited to plasmids, liposomes, electrically charged lipids (such as cytofectins), DNA-protein complexes, and biopolymers.
  • Viral vectors include but are not limited to vectors derived from retrovirus, adeno- associated virus, pox viruses, baculovirus, vaccinia virus, herpes simplex virus, Epstein-Barr virus, adenovirus and hybrids of two or more viral vector types.
  • a vector may contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
  • cell includes higher eukaryotic cells such as mammalian cells, lower eukaryotic cells such as yeast cells, prokaryotic cells, and archaebacterial cells.
  • “Pharmaceutically acceptable carrier” includes diluents and fillers which are pharmaceutically acceptable for method of administration, are sterile, and may be aqueous or oleaginous suspensions formulated using suitable dispersing or wetting agents and suspending agents.
  • the particular pharmaceutically acceptable carrier and the ratio of active compound to carrier are determined by the solubility and chemical properties of the composition, the particular mode of administration, and standard pharmaceutical practice.
  • D10G media composed of 89% DMEM supplemented with 2 mM L-glutamine, 10% calf serum, and 1 % penicillin (10,000 units/ml)/streptomycin (10 mg/ml) antibiotic mixture
  • RNA eIF-2a a subunit of eukaryotic initiation factor 2
  • HIV-2 human immunodeficiency virus type two i.p. intraperitoneal injection
  • the constructs and methods according to the invention make use of nucleic acids encoding PKR or 2-50AS.
  • the native human PKR and 2-5OAS genes have been cloned and sequenced (Mory et al . J. Interferon Research 9:295-304 (1980); Wathelet etal. , FEBS Letters 196: 113-20 (1986); Meurs etal..
  • variants of a given protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification.
  • the skilled artisan can produce derivatives of a protein having single or multiple amino acid substitutions, deletions, additions, or replacements. These derivatives may include, ter alia: (a) derivatives in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) derivatives in which one or more amino acids are added to the protein, (c) derivatives in which one or more of the amino acids includes a substituent group, and (d) derivatives in which the protein is fused with another peptide.
  • the techniques for obtaining these derivatives including genetic (suppressions, deletions, mutations, etc.). chemical, and enzymatic techniques, are known to persons having ordinary skill in the art.
  • PKR and 2-5OAS are intended to be included within the scope of the invention.
  • the PKR coding region or 2-5OAS coding region is operatively linked to a regulatory element such that the expression of PKR or 2-5OAS is activated in the presence of HIV trans-acting factors.
  • the regulatory element comprises all or part of an HIV LTR and the HIV trans-acting factor is an HIV Tat protein.
  • HIV LTR as well as any fragments thereof which retain the ability to respond to trans-activation by the Tat protein are all intended to be encompassed by the invention.
  • the HIV Rev protein can trans-activate the expression of heterologous genes which contain the negative cis-acting repressive sequences (crs) and a correctly oriented Rev responsive element (RRE) (Rosen et al. . Proc. Natl. Acad. Sci. USA 85:2071-75 (1988)). It is further understood that the art may discover other regulatory elements and trans-activating factors which can facilitate the HIV-regulated expression of chimeric constructs. The skilled artisan can also add other known regulatory elements to mediate inducible gene expression, such as regulatory sequences which respond to an environmental signal such as heat, cold, or a chemical compound.
  • F Vectors and Methods of Gene Transfer
  • the protein coding region and regulatory element are introduced into a cell using a vector.
  • the protein coding region and regulatory element become stably integrated into the cellular genome.
  • Non-viral vectors may be transferred into cells using any of the methods known in the art, including calcium phosphate coprecipitation, lipofection (synthetic anionic and cationic liposomes), receptor-mediated gene delivery, naked DNA injection, electroporation and bioballistic or particle acceleration.
  • Viral vectors may be transferred into cells using any method known in the art. including infection and transfection.
  • a retroviral vector is used to introduce the protein coding region and regulatory element into a cell which is susceptible to HIV infection.
  • Retroviruses are integrating viruses which generally infect dividing cells.
  • the retrovirus genome includes two LTRs. an encapsidation sequence and three coding regions (gag, pol and env).
  • the construction of recombinant retroviral vectors is known to those of skill in the art.
  • retroviral vectors the gag, pol, and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.
  • These vectors can be constructed from different types of retrovirus, such as M-MuLV, MSV (murine Moloney sarcoma virus). HaSV (Harvey sarcoma virus), SNV (spleen necrosis virus), RSV (Rous sarcoma virus) and Friend virus.
  • M-MuLV MSV (murine Moloney sarcoma virus).
  • HaSV Hardvey sarcoma virus
  • SNV spleen necrosis virus
  • RSV Ra sarcoma virus
  • Friend virus Friend virus.
  • a plasmid is constructed which contains the LTRs, the encapsidation sequence and the coding sequence.
  • This construct is used to transfect a packaging cell line, which cell line is able to supply in trans the retroviral functions which are deficient in the plasmid.
  • the packaging cell lines are thus able to express the gag, pol and env genes.
  • Such packaging cell lines have been described in the prior art.
  • the cell line PA317 (US 4,861 ,719)
  • the PsiCRIP cell line (WO90/02806)
  • the GP+envAm-12 cell line WO89/07150
  • Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.
  • Retroviral vectors derived from lentiviruses such as HIV-1. HIV-2. and SIV can be used for delivery to nondividing cells. These viruses can be pseudotyped with the surface glycoproteins of other viruses, such as M-MuLV or vesicular stomatitis virus (VSV).
  • M-MuLV vesicular stomatitis virus
  • VSV vesicular stomatitis virus
  • Such lentiviral vectors can infect nondividing cells, have a broad host range, and can be concentrated to high titers by ultracentrifugation.
  • Chimeric adenoviral/retroviral vector systems can also be used to achieve efficient gene delivery and long term gene expression.
  • a chimeric viral system in which adenoviral vectors are used to produce transient retroviral producer cells in vivo, such that progeny retroviral particles infect neighboring cells has been described by Feng et al. (Nature Biotechnology 15:866-70 (September 1997)).
  • M-MuLV can also be pseudotyped with HIV envelope glycoproteins to generate a retroviral vector with specificity for CD4-expressing cells, as disclosed by Schnierle et al. (Proc. Natl. Acad. Sci. USA 94:8640-45 (March 1997)).
  • the recombinant constructs according to the invention may be transferred into hematopoietic progenitor and stem cells or into differentiated cells which are susceptible to HIV infection.
  • Stem cells are a special class of cells which have the capacity to replicate themselves as well as the capacity to generate lineage restricted progenitors which further differentiate and expand into specific lineages.
  • Stem cells may be totipotent (germ line stem cells), pleuripotent (e.g. CD34 + hematopoietic stem cells), or unipotent (e.g. lymphoid progenitor cells).
  • the constructs are introduced into human hematopoietic progenitor and stem cells.
  • These primitive cells which can be isolated from sources including bone marrow, peripheral blood, and cord blood, are capable of giving rise to progeny in all hematopoietic lineages.
  • an HIV-1 regulated antiviral enzyme such as PKR or 2-5 OAS only needs to be performed a single time to provide protection for differentiated cells receptive to macrophage-tropic and T- cell-tropic strains of HIV (Rana et al , J. of Virol. 71:3219-3227 (1997)).
  • Primitive human hematopoietic progenitors and stem cells are characterized, inter alia, by the expression of the CD34 cell surface glycoprotein.
  • Methods of enriching for CD34 " cells using anti-CD34 antibodies including immunosorption, immunomagnetic separation and fluorescence activated cell sorting (FACS), are known in the art.
  • Cells expressing a specific CD34 subtype and/or cells expressing other lineage specific markers may be selected using similar techniques.
  • Cells expressing nonlineage markers may be removed by immunomagnetic depletion, immunoadsorption, or FACS. Differential centrifugation methods may also be used, generally in combination with positive and negative antibody selection, to enrich for a desired cell population.
  • the transfection of progenitor and stem cells with constructs according to the invention can generate a reservoir of immunocompetent HIV resistant cells which can differentiate into more mature components of the hematopoietic system, including CD4 + and macrophage descendants. It is important to select for high titer producers, as described in Example 3. and to transduce a large proportion of the target cells, in order to generate an adequate reservoir of resistant cells.
  • Methods for isolating, identifying, separating, and culturing hematopoietic stem cells are disclosed in U.S. 5,635,387 and U.S. 5,643,741 which are incorporated herein by reference.
  • Hematopoietic stem cells may be expanded in vitro, in the presence or absence of various cytokines, either before or after retroviral transduction. Methods and compositions for retroviral transduction of hematopoietic stem cells are disclosed in WO96/33281 which is incorporated herein by reference.
  • the constructs are introduced into more mature human hematopoietic cells which are already susceptible to infection by HIV. Because these differentiated cells have a finite life span, it may be necessary to repeat the introduction of HIV- 1 regulated antiviral constructs into targeted cells two or more (including many) times. In some embodiments, the antiviral constructs are introduced into both stem cells and differentiated cells.
  • HIV-1 infects T cells through CD4 and chemokine coreceptors.
  • CD4-expressing lymphocytes are the primary targets of HIV, and represent one of the main sources of viral replication.
  • CD4 + cells may be selected from sources including peripheral blood monocytic cells, using anti-CD4 antibodies (for positive selection) and other antibodies (for negative selection) in methods including immunosorption, immunomagnetic separation, and FACS.
  • the transfection of COX lymphocytes with constructs according to the invention can reduce the virus load in the peripheral blood and/or lymph nodes of patients infected with HIV.
  • the methods and constructs according to the invention may be used to treat individuals who are already infected with HIV. This approach generates a reservoir of (otherwise susceptible) cells which are not susceptible to infection, and can prevent or ameliorate the symptoms associated with AIDS.
  • the methods and constructs according to the invention are particularly useful for the treatment of HIV- 1 infected individuals who are resistant to one or more inhibitors of HIV protease and/or reverse transcriptase.
  • the methods and constructs according to the present invention may be used to treat uninfected individuals who are at high risk for HIV infection and AIDS, due to sexual conduct, intravenous drug use, medical condition, employment, or other significant risk. This approach can be used to prevent HIV infection as well as deleterious sequelae.
  • the recombinant constructs according to the invention may be transferred into human cells for clinical applications, using either ex vivo or in vivo approaches.
  • cells are removed from the patient. enriched, cultured, and infected or transfected with the recombinant construct, then reintroduced back into the patient.
  • Methods for ex vivo gene therapy have been disclosed by Blaese et al. (Science 270:475-80 (1995)), Kohn et al (Nature Medicine 1(10): 1017-23 (October 1995)), and Ferrari et al. (Blood 80: 1120-24 (1992)). all of which are incorporated by reference.
  • cells are typically transduced at a multiplicity of infection (m.o.i.) of about 0.1 to about 100.
  • Gene modified cells may be reintroduced into the patient by parenteral methods including intravenous infusion and direct injection into the bone marrow. Gene modified cells are reintroduced into the patient in a saline solution or other pharmaceutically acceptable carrier. The number of cells to be reintroduced depends on the purity of the cell population, but a typical dosage is in the range of about 10 5 to about 10 8 cells per kilogram of patient body weight. For example, when CD34 + cells are selected before ex vivo transduction.
  • recombinant vectors are injected directly into the patient.
  • the vectors are constructed so as to preferentially be incorporated into target (e.g. , CD34 + stem cells or CD4" lymphocytes) cells.
  • target e.g. , CD34 + stem cells or CD4" lymphocytes
  • the pseudotyping of M-MuLV virus with truncated HIV envelope glycoproteins can generate a retroviral vector which specifically infects CD4 " cells (Schnierle et al . Proc. Natl. Acad. Sci. USA 94:8640-45 (August 1997)).
  • about 10 5 to about 10 9 vectors are infused by a parenteral method such as intravenous infusion or direct injection into the bone marrow.
  • the methods and constructs according to the present invention can be used in combination with other antiviral drugs such as AZT, ddl. ddC, protease inhibitors, and combinations thereof.
  • antiviral drugs such as AZT, ddl. ddC, protease inhibitors, and combinations thereof.
  • the intracellular immunization approach allows for a significant reduction in antiviral drugs, the maintenance of HIV-I in a true latent state while maintaining an intact immune system, and decreased side effects.
  • SCID mice which have been reconstituted with human cells are used as an animal model to study HIV infection and AIDS, and to optimize treatment protocols before their use in human subjects. SCID mice are reconstituted with peripheral blood monocytic cells or bone marrow cells transfected, using retroviral vectors, with the HIV-regulated constructs according to the invention. The animals are subsequently exposed to HIV and their susceptibility to infection is monitored. Examples
  • SEQ ID NO:4 sense probe oligonucleotide corresponding to the NF- B binding site 5'-ACAAGGGACTTTCCGCTGGGGACTTTCCA GGGA-3' .
  • NIH/3T3 cells were obtained from the American Type Culture Collection (Rockville, MD). Molt4 cells chronically infected with HIV-1 IIIB (Molt 4 IIIB) and SupTl cells were obtained from the NIH AIDS Research Reference and Reagent Program, and were grown at 37 C C, 5 % C0 2 in RPMI 1640 (GIBCO) containing 10% heat-inactivated donor calf serum supplemented with 100 units/ml penicillin-streptomycin (RIO) (Biofluids, Inc.). The amphotrophic retroviral packaging cell line GP+envAml2 was provided by Dr. Arthur Bank (Columbia University, New York).
  • NIH/3T3 cells and the GP+envAml2 cell line were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated calf serum supplemented with 2 mM glutamine and 100 units/ml penicillin- streptomycin (D10G).
  • DMEM Dulbecco's modified Eagle's medium
  • D10G penicillin- streptomycin
  • Culture supernatants from Molt4 IIIB cells were used as the source of infectious virus. The supernatant was cleared by centrifugation (500 x g. 5 min. 4°C) and stored in 10 ml aliquots at -70°C until use.
  • HIV-1 IIIB titer was calculated by infection of SupTl cells, serial dilution, and scoring of syncytia formation in quadruplicate.
  • HIV-1 LTR-PKR cDNA- ⁇ oly(A) cassette was cloned in forward (pMEA105) and reverse (pMEA106) orientations into the pN2 retroviral vector, as illustrated in Figure 1A.
  • This retroviral vector was chosen because it can be used to effect the stable integration of the HIV-1 LTR-PKR cDNA— poly(A) cassette into target cells, using replication-incompetent MMuLV particles.
  • This retro viral-mediated intracellular immunization approach was designed to provide (i) an efficient and well-characterized delivery system, (ii) specificity in that the PKR cDNA constructs are transcribed and activated at high levels only upon HIV-1 infection, and (iii) a system which is tightly regulated and silent in uninfected cells due to its dependency on dsRNA for allosteric activation.
  • PKR cDNA was amplified by standard bacterial culture protocols and digested with Hindlll, PstI, and Hhal to yield 36 fragments (Meurs etal. , Cell 62:379-390 (1990)).
  • An 1826 nucleotide Hindlll/PstI fragment containing the wt PKR cDNA was purified using DEAE-cellulose.
  • the pHIV ⁇ JFN plasmid (provided by Dr. Daniel P. Bednarik, Human Genome Sciences, Rockville, MD) was digested with Xhol and Hindlll, releasing a 727 bp fragment containing portions of the U3 and R regions and an additional 195 nucleotides of viral DNA located in the HIV-1 LTR.
  • plasmids which contain the HIV-1 LTR controlling the expression of wt PKR cDNA. Restriction mapping and DNA sequencing were utilized to identify bacterial colonies carrying the desired plasmid construct.
  • the XhoI-HIV-I LTR- PKR cDNA-poly(A)-XhoI fragment was directly subcloned in two orientations into the pN2 vector. It has been reported that the host can use methylation patterns to inactivate transcription initiation from proviral inserts. The inappropriate expression of an antiproliferative gene by readthrough transcripts originating from the M-MuLV 5 ' LTR in constructs cloned in the forward orientation might precipitate this type of host repression (Bednarik et al . Proc. Natl Acad. Sci. USA 86:4958-4962 (1989)).
  • EXAMPLE 3 Production of Retroviral Producer Cell Lines
  • the plasmids constructed in Example 2 (encoding PKR under the control of an HIV LTR) were transferred into retroviral packaging cell lines in order to produce recombinant virus.
  • the GP+envAml2 cell line is a packaging cell line for the production of amphotropic retroviruses.
  • the cell line was constructed by introducing gag and pol helper functions on one plasmid. and the env helper function on a separate plasmid.
  • the cloned helper functions do not contain packaging signals or 3' -LTR sequences.
  • the structure of the helper functions insures that replication competent viruses (RCRs. a major concern in the area of gene therapy) are not produced. Using this system, three independent recombination events would be required in order to generate replication competent virus.
  • the transfection media was aspirated and replaced with 5 ml fresh D10G Twelve hours later, the D10G was aspirated, the cells rinsed with 2 ml PBS, and aspirated again Trypsin solution (500 pi, 0 25 %) was added to each well followed by a 5 min incubation at 37°C, 5% CO 2
  • the cells were resuspended in 5 ml D10G and diluted to 10 3 in D10G containing 1 mg/ml G418 Fourteen days later, coinciding with a complete loss of viability of mock-transfected controls as measured by Trypan blue exclusion, individual G418 resistant colonies were isolated by mini-trypsinization in cloning wells The isolated colonies were expanded for titenng and further characterization
  • NIH3T3 cells (1 x 10 "1 ) were seeded in individual 60 mm plates and permitted to adhere to the plate surface overnight at 37°C, 5 % C0 Infections were performed at multiple dilutions in duplicate Viral supernatants were harvested from nearly confluent G418-res ⁇ stant amphotrophic retroviral producer cells expanded in 100 mm 2 tissue culture plates and frozen at -70 °C The viral supernatants were thawed by incubation at 37 C with gentle agitation.
  • the isolation of individual clones following plasmid transfection increased the titer of the retroviral producer cell lines
  • the N2-20, 105-10, and 106-4 lines were titered at 1 7 ⁇ 0 4 x 10 3 , 1 1 + O l X 10 4 . and 1 2 ⁇ 0 4 ⁇ 10 3 , respectively
  • Producer cell lines N2-20, 105-10. and 106-4 (2 0 x lO 3 ) were seeded into 100 mm 2 cell culture plate 1; -n a total volume of 10 ml D10G Twelve hours later, the media was aspirated, the cells were washed with 10 ml PBS.
  • transduced cells and control cells were centrifuged (350 x g, 4°C. 15 min).
  • Genomic DNA was isolated according to standard procedures.
  • the pN2 vector was digested with PstI and a 923 bp fragment was isolated and radiolabeled by denaturation and incubation in the presence of Klenow fragment, unlabeled nucleotides (30 ⁇ M each of dCTP, dGTP, and dATP), [ - ?2 P]-dTTP (50 ⁇ Ci, 3000 Ci/mmol), and reaction buffer containing 50 mM Tris-HCl (pH 8.0).
  • the digested genomic DNA was electrophoresed through a 0.8% agarose gel at 20 volts overnight and, following denaturation and neutralization, vacuum transferred to a nylon membrane at 80 lbs/inch 2 pressure for 1 hour
  • the DNA was crosslinked to the nylon membrane with a UV Stratalinker.
  • 2 x 10 7 dpm of the neo probe was incubated with the blot at 68 C C overnight.
  • the membrane was washed with varying concentrations of SSC and SDS, dried, and analyzed with a Fuji BAS2000 phosphor imager.
  • SupTl T lymphoblastoid cell lines were transduced such that PKR would be overexpressed following infection with HIV-I. HIV-1 replication was measured by syncytia formation. Syncytia are fused T lymphocytes, having multiple nuclei and sharing a common membrane, which result from HIV infection.
  • the SupTl parental cell line (CD4+ T lymphocytes) provided a control for cellular PKR expression.
  • the N2-20P clones T cells transduced with neomycin selectable marker but not a PKR cDNA
  • transduced cells and SupTl controls were challenged with HIV-1 IIIB at a m.o.i. of 0.1 for 2 h at 37°C. 5% CO 2 with gentle agitation.
  • the infected cells were washed with 5 volumes of RIO to remove unincorporated virus.
  • Syncytia were scored by microscopic examination 96 hours p.i.
  • a single syncytia score from triplicate assays was calculated by correcting for each dilution factor and averaging of the three values, i.e. a total of nine wells were scored to obtain a single syncytia score.
  • Host-dependent hypermethylation of the CpG sites within the HIV-1 LTR has been reported to inactivate Tat-induced transcription and could have contributed to the differences observed in anti-HIV-1 activity between clonal lines transduced with identical retroviral constructs (Gutebuch et al , J. AIDS 6:541-549 (1993)). Gross rearrangements of integrated DNA, which have been reported with M-MuLV after a single round of replication, were not observed.
  • the inhibition of syncytia formation observed in the HIV-1 LTR-PKR cDNA transduced clones was reversed by treatment of the cells with 2-aminopurine prior to infection (Figure 2, open bars).
  • the reversibility of the anti-HIV-1 effect observed in the HIV-1 LTR-PKR cDNA clones was demonstrated by treatment with 2-aminopurine prior to HIV-1 infection, as shown in Figure 2, open bars.
  • the compond 2-aminopurine is an ATP analog that inhibits PKR autophosphorylation both in vitro and in vivo, but does not affect the dsRNA binding capacity of PKR (Hu et al , J. Interferon Res. 13:323-328 (1993)).
  • triplicate assays were prepared by infection of 2 x lO "1 cells in the presence or absence of 10 mM 2-aminopurine (added 1 hour prior to infection) with HIV-1 IIIB at a m.o.i. of 0.1. Twenty-four hours after infection, the infected cells were serially diluted up to 1 :27 and 2 x 10 5 SupTl indicator cells were added to each well.
  • Ninety-six hours p.i. , syncytia formation was scored as described above.
  • the blot was rinsed 5 times (twice quickly, 1 x 15 min., 2 x 5 min) in a large volume of TBS-T on a slow shake at room temperature. The blot was then incubated with a 1 : 1000 dilution of rabbit polyclonal anti-PKR antibody in 15 ml of TBS-T for 1 hour.
  • the polyclonal anti-PKR antibody was provided by Dr. Charles E. Samuel (University of California at Santa Barbara).
  • the blot was removed from the antibody solution and washed with TBS-T as described above, then incubated for 1 hour with a 1 :2500 dilution of horseradish peroxidase-conjugated mouse anti-rabbit serum (Pierce) on a slow shake. Following washing with TBS-T, the blot was developed with a chemiluminescent solution (ECL, Amersham), following the instructions of the manufacturer. Exposures on X-OMAT film (Kodak) were developed and quantified utilizing image analysis software (Macintosh NIH Image program, version 1.60).
  • the levels of PKR in the transduced clones were noticeably lower at 72 and 96 hours p.i. , perhaps due to lower levels of available Tat protein to drive the system from a lack of productive infection.
  • the expression of chloramphenicol acetyltransferase under HIV-1 LTR control has been reported to be elevated 322- and 278-fold in HIV-I infected H9 and L8460D Jurkat cells, respectively (Sodroski et al. , Science 225:381-385 (1984); Thomis et al.. Proc. Natl. Acad. Sci. USA 89: 10837-10841 ( 1992)).
  • PKR protein overexpression in eukaryotic systems has proven difficult due to its antiproliferative and autoregulatory properties (London et al. , Proc. Natl Acad. Sci. USA 90:4616- 4620 (1993)).
  • NP-40 extracts (30 ⁇ g protein) prepared at the indicated times p.i. were diluted to a total volume of 300 ⁇ l with buffer A [20 mM Tris-HCl (pH 7.6), 50 mM KC1, 400 mM NaCl. 5 mM 2-mercaptoethanol. 1 % Triton X-100. 1 mM EDTA, lO ⁇ g/ml aprotinin, 0.2 mM PMSF, 20% (vol/vol) glycerol] .
  • buffer A 20 mM Tris-HCl (pH 7.6), 50 mM KC1, 400 mM NaCl. 5 mM 2-mercaptoethanol. 1 % Triton X-100. 1 mM EDTA, lO ⁇ g/ml aprotinin, 0.2 mM PMSF, 20% (vol/vol) glycerol.
  • Ten u ⁇ of anti-human PKR rabbit antiserum was added
  • Protein A Sepharose CL-4B (Phamacia) [50 ⁇ l of a 50% (vol/vol) suspension in buffer A] was added to each tube followed by incubation with continuous rotation for 30 minutes at 4 ' C.
  • the Sepharose was pelleted (420 x g, 4 U C, 5 min) and washed 4 times with 300 ⁇ l buffer B [20 mM Tris-HCl (pH 7.6). 100 mM KC1, 0.1 mM EDTA, 10 ug/ml aprotinin, 20% (vol/vol) glycerol] and twice with buffer C (buffer B with 2 mM MnCl 2 and 2 mM MgCl 2 ).
  • Sepharose was resuspended in 50 ⁇ l of buffer C containing 0 or 0.1 ⁇ g/ml poly(rI)-poly(rC) and 2.5 u ⁇ [ ⁇ 32 P]ATP ( > 7000 Ci/mmol). Samples were incubated for 10 min at 30°C. SDS sample buffer (4X) was added to each sample, and the samples were heated at 95°C for 3 minutes. Proteins were separated by 8.5% SDSPAGE and gels were dried and analyzed by autoradiography.
  • PKR autophosphorylation of the HIV-1 LTR-PKR cDNA transduced clones and controls was examined at 24 hour intervals following HIV-1 IIIB infection without or with 0.1 ⁇ g poly(rl)-poly(rC) ( Figure 5). It has been reported that there is a direct relationship between the levels of PKR autophosphorylation and the quantity of eIF-2 ⁇ phosphorylation (Mordechai et al. , Virology 206:913-22 (1995); Suhadolnik et al , Cancer Res. 43:5462-66 (1983)).
  • PKR autophosphorylation levels in N2-20 extracts of untreated SupTl and N2-20P control lines were lower than that observed for the HIV-1 LTR-PKR cDNA clones.
  • Addition of 0.1 ⁇ g/ml poly(rI)-poly(rC) shifted PKR activation levels to the right on the bell-shaped activation curve.
  • Comparison of the 0.1 ⁇ g/ml poly(rl) poly(rC)-treated SupT 1 and N2-20P extracts with the corresponding untreated extracts revealed that most, if not all, of the PKR is inhibited. Clone 106-4:560 at 24 hours p.i.
  • TPA 12-O-tetradecanoylphorbol 13-acetate
  • Complementary, synthetic oligonucleotides corresponding to the NF- ⁇ B binding site SEQ ID NO:4 were annealed and end-labeled with [ ⁇ - 32 P]ATP utilizing T4 polynucleotide kinase.
  • GEMSA gel electrophoretic mobility shift assay
  • I ⁇ -B ⁇ is a cytoplasmic protein which complexes with NF- ⁇ B and acts to repress its transcriptional enhancer properties. Upon phosphorylation, I ⁇ -B ⁇ undergoes a conformational change which releases NF- ⁇ B to translocate into the nucleus and transactivate promoter elements containing the KB binding site such as I ⁇ -B , NF- ⁇ B, IFN- ⁇ , cytokines. immunomodulators, and viral genes (Tzen et al. Ex. Cell Res. 211: 12-16 (1994)).
  • the intracellular immunization approach was used in combination with a traditional antiviral drug chemotherapeutic approach.
  • Control cells and the transduced clones were treated with the reverse transcriptase inhibitor.
  • 3 '-azido-3 '-deoxythymidine (AZT) prior to HIV-1 infection to investigate a combinatory in vitro effect between the HIV-1 LTR driven PKR cDNA sequences and the AZT.
  • AZT was added 1 hour prior to infection at a final concentration of 0, 5, 10. 50, 100. or 1000 nM.
  • ACH-2 is a lymphocytic cell line containing one copy of the HIV provirus in the genome (Pomerantz et al. , Cell 61: 1271-76 (1990)). Ul is a promonocytic cell line containing two integrated copies of the HIV provirus in the genome (Folks et al . Science 238:800-02 (1987)).
  • the chronically infected cell lines, ACH-2 and Ul were transduced with the HIV-1 LTR-PKR cDNA constructs using the retroviral supernatants of the retroviral producer cell lines as described in Example 4. HIV expression was induced by treatment with tumor necrosis factor alpha (TNF- . 50 ng/ml) (Folks et al , Proc. Natl. Acad. Sci. USA 86:2365-68 (1989)). Cells were maintained in culture. The HIV-1 LTR-PKR cDNA-transduced Ul and ACH-2 inhibited HIV-1-induced syncytia formation 99% and 99%, respectively. Western analysis as described by Kon et al. ⁇ J. Biol. Chem. 271: 19983-90 (1996)) showed an increase in PKR expression through 96 hour post-induction in the transduced Ul cells.
  • PBMC peripheral blood monocytic cells
  • CB17 scid/scid mice between 4 and 6 weeks of age are maintained under specific-pathogen-free conditions, with all food, water, and bedding being autoclaved before use.
  • PBMC are transduced by cocultivation with recombinant virus producer lines, as described in Example 4, or by direct infection with a retroviral supernatant or a more concentrated retroviral preparation.
  • Cells are selected for neomycin (G418) resistance, as in Example 3A.
  • SCID mice are reconstituted by intraperitoneal (i.p.) injection of 2xl0 7 transduced PBMC.
  • Reconstituted mice are infected with HIV-1 strain IIIB at two hours, at eight days, or at two weeks after reconstitution.
  • serial tenfold dilutions of 10 5 to 10 2 tissue culture infective doses of HIV-1 are injected i.p. into the reconstituted mice. HIV infection is monitored over time by virus-specific PCR, by p24 assays, and by cocultivation assays using cells obtained by peritoneal lavage.
  • the pMEA002 plasmid was used as a backbone vector upon which to build pMEA003, in which the cDNA encoding PKR was removed and replaced with a cDNA encoding 2-5OAS.
  • the pMEA002 plasmid was digested with Hindlll then blunt ended by treatment with Klenow DNA polymerase in the presence of deoxynucleoside triphosphates. A subsequent digestion with EcoRl released the PKR cDNA from the 3127 bp blunt ended EcoRlpSP72-HIV-l LTR fragment, and this fragment was purified using DEAE cellulose.
  • the 2-50AS cDNA was obtained from pNK04, which has been used for the expression of milligram quantities of 2-5OAS (Kon and Suhadolnik. J. Biol. Chem.271: 19983- 90 (1996)).
  • the pNK04 DNA was digested with Ndel. filled in with Klenow fragment, and digested with EcoRl to release a 1242 bp fragment containing the entire 2-5OAS cDNA. This fragment was purified using DEAE-cellulose.
  • the purified cDNA and vector fragments were ligated together (via the EcoRl cohesive ends and blunt ends) to produce a 4374 bp fragment in which the Hindlll restriction site was regenerated
  • the gation mixture was used to transform E coli DH5 ⁇ cells DNA from 20 colonies was isolated using a plasmid DNA boiling min prep method Plasmid structure was analyzed by restriction analysis and by Sanger dideoxynucleotide sequencing, using T7, HIV- 1 LTR. and SP6 primers
  • pMEA003 was digested with Hpal and Clal and the synthetic oligonucleotide containing the polyadenylation sequences utilized in the construction of the pMEAlOl was inserted downstream of the 2-50AS cDNA sequence
  • the XhoI-HIV-1 LTR-2- 50AS-poly(A)-XhoI sequence was excised, purified, and inserted in the forward and reverse orientation into the Xhol site of pN2 to create pMEA109 and pMEAHO. respectively
  • the plasmids encoding 2-50AS under the control of an HIV LTR were transferred into retroviral packaging cell lines in order to generate retroviral producer cell lines and recombinant virus, using the methods described in Example 3
  • Recombinant retrovirus was transduced into T lymphocytic cells as described in Example 4. and the transduced cells were challenged with HIV-1 strain IIIB as described in Example 5
  • 50AS gene alone or in combination with other chemotherapeutic agents, is a potential long term treatment for the control of HIV infection

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  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • AIDS & HIV (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Cette invention se rapporte à un système antiviral avec régulation du VIH-1. Cette invention propose des produits de synthèse et des procédés permettant de transférer une enzyme antivirale dans des cellules cibles, où cette enzyme se trouve soumise à une régulation du VIH-1. L'infection par VIH-1 de ces cellules cibles entraîne l'activation de l'enzyme antivirale et aboutit à l'inhibition de la réplication virale. Cette approche antivirale contre le VIH-1 peut être combinée avec des approches chimiothérapeutiques traditionnelles, ce qui permet de réduire considérablement l'utilisation des médicaments antiviraux, avec une baisse des effets secondaires qui les accompagnent, et de maintenir le VIH-1 dans un état latent réel.
EP98954989A 1997-10-16 1998-10-15 Inhibition de la replication du virus de l'immunodeficience humaine (vih-1) Withdrawn EP1029062A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US6198497P 1997-10-16 1997-10-16
US61984P 1997-10-16
PCT/US1998/021880 WO1999019496A1 (fr) 1997-10-16 1998-10-15 Inhibition de la replication du virus de l'immunodeficience humaine (vih-1)

Publications (1)

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EP1029062A1 true EP1029062A1 (fr) 2000-08-23

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EP98954989A Withdrawn EP1029062A1 (fr) 1997-10-16 1998-10-15 Inhibition de la replication du virus de l'immunodeficience humaine (vih-1)

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EP (1) EP1029062A1 (fr)
JP (1) JP2001520017A (fr)
AU (1) AU1189899A (fr)
CA (1) CA2306444A1 (fr)
WO (1) WO1999019496A1 (fr)

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Publication number Priority date Publication date Assignee Title
CA2484251C (fr) 2002-04-30 2015-06-23 University Of South Florida Matieres et procedes visant a prevenir et a traiter des maladies provoquees par des virus a arn
US7595303B1 (en) 2002-09-05 2009-09-29 University Of South Florida Genetic adjuvants for immunotherapy

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Publication number Priority date Publication date Assignee Title
JP3901726B2 (ja) * 1993-04-02 2007-04-04 リジェル・ファーマシューティカルズ・インコーポレーテッド ウイルス複製の選択的不活性化のための方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9919496A1 *

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CA2306444A1 (fr) 1999-04-22
JP2001520017A (ja) 2001-10-30
AU1189899A (en) 1999-05-03
WO1999019496A1 (fr) 1999-04-22

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