WO2004061088A2 - Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes - Google Patents

Compositions and methods for inhibiting human immunodeficiency virus infection by down-regulating human cellular genes Download PDF

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WO2004061088A2
WO2004061088A2 PCT/US2003/041790 US0341790W WO2004061088A2 WO 2004061088 A2 WO2004061088 A2 WO 2004061088A2 US 0341790 W US0341790 W US 0341790W WO 2004061088 A2 WO2004061088 A2 WO 2004061088A2
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biological pathway
human host
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human
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WO2004061088A3 (en
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Stephen J. Dunn
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Ppd Development, Lp
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Definitions

  • the invention relates to methods for identifying human cellular genes and their encoded products for use as targets in the design of therapeutic agents for suppressing human immunodeficiency virus (HIV) infection.
  • the invention relates to methods for identifying biochemical pathways, substrates and metabolic products of said pathways, and enzymes that mediate conversion of substrates into metabolic products, wherein said pathways comprise one or a plurality of targets for the design of preventative and therapeutic agents for preventing, inhibiting, suppressing, or immunizing against infection of na ⁇ ve cells with HIV or the production of infectious virus from infected cells.
  • the invention also relates to methods for identifying protective compounds that inhibit HIN infection.
  • the invention further relates to compounds for use in the treatment or prevention of HIN.
  • HIV acquired immunodeficiency syndrome
  • Retroviruses are small, enveloped viruses that contain a diploid, single-stranded R ⁇ A genome, and replicate via a D ⁇ A intermediate produced by a virally encoded reverse transcriptase, an R ⁇ A-dependent D ⁇ A polymerase (Narmus, 1988, Science 240:1427-39).
  • HIN HIN-1
  • HIV-2 Clavel et al, 1986, Science 233:343-46; Guyader et al, 1987, Nature 326:662-69.
  • CD4 + T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al, 1984, Nature 312:763-67; Klatzmann et al, 1984, Nature 312:767-68; Maddon et al, 1986, Cell 47:333-48).
  • Niral entry into cells is dependent upon the binding of viral protein gpl20 to the cellular CD4 receptor molecule (McDougal et al, 1986, Science 231:382-85; Maddon et al, 1986, Cell 47:333-48).
  • HIV infection is pandemic and HIV-associated diseases have become a worldwide health problem.
  • anti-HIV modalities there is, thus far, no successful prophylactic or therapeutic regimen against AIDS.
  • several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al, 1991, FASEB J. 5:2369-81).
  • reverse transcriptase has been a major focus of drug development.
  • a number of reverse transcriptase-targeted drugs including dideoxynucleotide analogs such as AZT, ddl, ddC, and ddT have been shown to be active against HIV (Mitsuya et al, 1990, Science 249:1533-44). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al, 1989, Science 243:1731- 34). h addition, these drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
  • Late-stage processing is dependent on the activity of a virally encoded protease, and drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al, 1993, Persp. Drug Discov. Design 1:69-83).
  • drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al, 1993, Persp. Drug Discov. Design 1:69-83).
  • drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al, 1993, Persp. Drug Discov. Design 1:69-83).
  • the emergence of drug resistant HIV mutants is also a problem; resistance to one inhibitor often confers cross-resistance to other protease inhibitors (Condra et al, 1995, Nature 374:569
  • RNA-based HIV-1 antiviral agents e.g., decoys, antisense, or ribozymes
  • protein-based HIV-1 antiviral agents e.g., transdominant mutants
  • Antisense polynucleotides have been designed to complex with and sequester HIV-1 transcripts (International Pub. ⁇ os. WO 93/11230 and WO 94/10302; European Patent Pub. No. EP 0 594 881; Chatterjee et al, 1992, Science 258:1485). Furthermore, enzymatically active RNAs (i.e., ribozymes) have been used to cleave viral transcripts. The use of a ribozyme to generate resistance to HIV-1 in a hematopoietic cell line has been reported (Ojwang et al,
  • RevMlO a rransdominant Rev protein
  • an intracellular pathogen such as HIV requires the development of interactions of its genes and gene products with multiple cellular components.
  • the interactions of a virus with a host cell involves the binding of the virus to specific cellular receptors, translocation through the cellular membrane, uncoating, replication ofthe viral genome, and transcription of the viral genes.
  • Each of these events occurs in a cell and involves interactions with at least one cellular component.
  • the life cycle of a virus can be completed only if the cell is "permissive" for viral infection.
  • the availability of amino acids and nucleotides for replication of the viral genome and protein synthesis, the energy status of the cell, and the presence of cellular transcription factors and enzymes all contribute to the propagation of the virus in the cell. Consequently, the cellular components, in part, determine host cell susceptibility to infection and can be used as potential targets for the development of new therapeutic interventions, the case of HIN, one cellular component that has been used towards this end is the cell surface molecule for HIV attachment, CD4.
  • chemokine receptors CCR2, CCR3, CCR5, or CXCR4
  • a chemokine receptor normally binds RA ⁇ TES, MLP-l ⁇ , or MlP-l ⁇ as its natural ligand.
  • CD4 first binds to the HIV gpl20 protein on the cell surface followed by binding of this complex to a chemokine receptor, resulting in viral entry into the cell (Cohen, 1997, Science 275:1261). Therefore, chemokine receptors can present an additional cellular target for the design of HIV therapeutic agents. Inhibitors of HlV/chemokine receptor interactions are being tested as anti-HIV agents.
  • the invention relates to methods for identifying human cellular genes that encode products that are necessary for productive HIV infection for use as targets in the design of therapeutic agents for suppressing HIV infection.
  • the invention also relates to methods for identifying biological pathways comprising the products of such cellular genes, as well as substrates and metabolic products of said pathways.
  • the invention further relates to methods for identifying additional human cellular genes that encode products comprising other members of such biological pathways for use as targets in the design of therapeutic agents for suppressing HIV infection.
  • the invention also relates to methods for identifying protective compounds that inhibit HIV infection.
  • the invention further relates to compounds for use in the treatment or prevention of HIV.
  • a first member of a biological pathway in a human host cell is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a population of human host cells by introducing the genetic suppressor element library into the population of human host cells; infecting the population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene that corresponds to the genetic suppressor element
  • an inhibitor of a member of a biological pathway in a human host cell is identified by first identifying the member of the biological pathway.
  • the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated
  • the inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression ofthe human cellular gene.
  • an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection is identified by first identifying the member of the biological pathway.
  • the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element; and determining the polypeptide encoded by the human cellular gene.
  • the inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression of the member of the biological pathway.
  • an inhibitor of a member of a biological pathway in a human host cell is identified by first identifying the member of the biological pathway.
  • the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIN infection to yield D ⁇ A fragments; transferring the D ⁇ A fragments to an expression vector to yield a genetic suppressor element library, wherein each of the D ⁇ A fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the D ⁇ A fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells
  • the inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases biochemical activity of the member of the biological pathway.
  • an inhibitor of a member of a biological pathway in a human host cell wherein the member of the biological pathway is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the member of the biological pathway.
  • the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; and determining the human cellular gene corresponding to the genetic suppressor element.
  • the inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression ofthe human cellular gene.
  • an inhibitor of a member of a biological pathway in a human host cell wherein the member of the biological pathway is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the member of the biological pathway.
  • the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible, to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element; and determining the polypeptide encoded by the human cellular gene.
  • the inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression of the member of the biological pathway.
  • an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection is identified by first identifying the member of the biological pathway.
  • the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIN infection to yield D ⁇ A fragments; transferring the D ⁇ A fragments to an expression vector to yield a genetic suppressor element library, wherein each of the D ⁇ A fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the D ⁇ A fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIN; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element; and determining the polypeptide encoded by the human cellular gene.
  • the inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases biochemical activity of the member of the biological pathway.
  • an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection is identified by first identifying the second member of the biological pathway.
  • the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; determining the first member
  • the inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression ofthe human cellular gene encoding the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases expression of the human cellular gene encoding the first member ofthe biological pathway.
  • an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection is identified by first identifying the second member of the biological pathway.
  • the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first
  • the inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases expression ofthe first member ofthe biological pathway.
  • an inhibitor of a first member of a biological pathway in a human host cell is identified by first identifying the second member of the biological pathway, i this method, the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host
  • the inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases biochemical activity ofthe first member ofthe biological pathway.
  • an inhibitor of a first member of a biological pathway in a human host cell wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the second member of the biological pathway.
  • the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; determining the first member
  • the inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the human cellular gene encoding the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases expression of the human cellular gene encoding the first member ofthe biological pathway.
  • an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection is identified by first identifying the second member of the biological pathway.
  • the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first
  • the inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the first member ofthe biological pathway in the second population of human host cells; and determining whether the test compound decreases expression ofthe first member ofthe biological pathway.
  • an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection is identified by first identifying the second member of the biological pathway.
  • the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first
  • the inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases biochemical activity ofthe first member ofthe biological pathway.
  • the invention includes methods for identifying human cellular genes that encode products that are necessary for productive HIV infection for use as targets in the design of therapeutic agents for suppressing HIV infection.
  • the invention also includes methods for identifying biological pathways comprising the products of such cellular genes.
  • the invention further includes methods for identifying additional human cellular genes that encode products comprising other members of such biological pathways for use as targets in the design of therapeutic agents for suppressing HIV infection.
  • the invention also includes methods for identifying protective compounds that inhibit HIV infection.
  • the invention further includes compounds for use in the treatment or prevention of HIN.
  • Such compounds include chemical compounds and biological compounds.
  • Chemical compounds and biological compounds include any chemical or biological compound that disrupts or inhibits one or more biological functions required for mediation or replication of HIV.
  • Preferred chemical compounds include small molecule inhibitor or substrate compounds, such as products of chemical combinatorial libraries.
  • Preferred biological compounds include peptides, antisense molecules, and antibodies.
  • the invention is based, in part, on the Applicants' discovery that certain nucleic acid molecules - termed genetic suppressor elements (GSEs) - which are isolated from human cells and which correspond to fragments of certain human cellular genes, prevent activation of latent HIN-1 in a CD4 + cell line as well as productive HIN infection in such cells, that regard, any cellular or viral marker associated with HIN infection can be used to select for such nucleic acid molecules.
  • GSEs genetic suppressor elements
  • An example of such a marker is CD4, which is conveniently monitored by using a specific antibody. Additional markers include virus-specific gene products, such as gpl20 and p24.
  • GSEs having the ability to inhibit HIV infection function can be isolated that are functional in either the sense orientation (encoding peptides) or in the antisense orientation (encoding antisense RNAs). Both types of GSEs are believed to down-regulate the corresponding cellular gene from which they were derived - each type of GSE utilizing a different mechanism.
  • the corresponding cellular gene is referred to herein as a "target gene” and its product is referred to as a "target product.”
  • target can also be used generically to describe either the gene or its encoded product.
  • Sense-oriented GSEs exert their effects as transdominant mutants or RNA decoys.
  • Transdominant mutants are expressed proteins or peptides that competitively inhibit the normal function of a wild-type protein in a dominant fashion.
  • RNA decoys are protein binding sites that titrate out the wild-type protein.
  • Antisense-oriented GSEs exert their effects as antisense RNA molecules, i.e., nucleic acid molecules complementary to the mRNA of the target gene. These nucleic acid molecules bind to mRNA and block the translation of the mRNA. In addition, some antisense nucleic acid molecules can act directly at the DNA level to inhibit transcription.
  • the down-regulation of the concentration (expression, level, amount) or activity (function, biological activity) of a target gene or target product by a GSE depletes a cellular component required for progression through the HIV life cycle resulting in an inhibition of HIV infection.
  • the down-regulation of the concentration or activity of a target gene or target product by a GSE depletes a cellular component that interacts with one or more other human cellular genes or gene products that are required for progression through the HIV life cycle, resulting in an inhibition of HIV infection.
  • At least two human cellular genes are members of the same biological pathway and one human cellular gene or gene product regulates the expression or activity of the other human cellular gene or gene product
  • at least two human cellular genes are members of the same biological pathway and the polypeptide encoded by one human cellular gene is a product of a biochemical reaction mediated by the polypeptide encoded by the other human cellular gene.
  • the at least two human cellular genes are members of the same biological pathway and the polypeptide encoded by one human cellular gene is a substrate of a biochemical reaction mediated by the polypeptide encoded by the other human cellular gene.
  • the at least two human cellular genes encode polypeptides that are isozymes of each other.
  • at least one ofthe human cellular genes encodes an enzyme.
  • the target gene encodes a target product that is a member of the glycolysis pathway of the host cell (as shown in BIOCHEMICAL PATHWAYS, (G. Michal, ed.), J. Wiley & Sons, Inc.: New York, 1999, pp. 27-34).
  • Preferred target products include the following members of the glycolysis pathway: enolase 1 (ENO1); enolase 2 (ENO2); enolase 3 (ENO3); fructose- 1,6-bisphosphatase 1 (FBPl); fructose bisphosphate aldolase A (ALDOA); glucosamine-6-phosphate deaminase (GNPDA); glucose transporter-3 (GLUT3); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH); Na + D-glucose cotransport regulator; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K); phosphofructokinase (PFK); phosphoglucomutase (PGM); phosphorylase kinase, alpha-2 (PHKA2); and thyroid hormone binding protein/pyruvate kinase (TCBA).
  • ENO1 enolase 1
  • ENO2
  • Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate.
  • Enolase 1 is one of three enolase isoenzymes found in mammals; it encodes alpha-enolase, a homodimeric soluble enzyme, and also encodes a shorter monomeric structural lens protein, tau-crystallin. The two proteins are made from the same message. The full length protein, the isoenzyme, is found in the cytoplasm. The shorter protein is produced from an alternative translation start, is localized to the nucleus, and has been found to bind to an element in the c-myc promoter.
  • Enolase 2 is a homodimer, is found in mature neurons and cells of neuronal origin.
  • a switch from alpha enolase to gamma enolase occurs in neural tissue during development in rats and primates.
  • Enolase 3 also a homodimer, is found in skeletal muscle cells in the adult.
  • a switch from alpha enolase to beta enolase occurs in muscle tissue during development in rodents. Mutations in this gene can be associated with metabolic myopathies that may result from decreased stability ofthe enzyme.
  • Fructose- 1,6-bisphosphatase 1, a gluconeogenesis regulatory enzyme catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate.
  • Fructose- 1,6-diphosphatase deficiency is associated with hypoglycemia and metabolic acidosis.
  • Fructose-bisphosphate aldolase (aldolase A) is a glycolytic enzyme that catalyzes the reversible conversion of fructose- 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate.
  • Three aldolase isozymes (A, B, and C), encoded by three different genes, are differentially expressed during development. Aldolase A is found in the developing embryo and is produced in even greater amounts in adult muscle. Aldolase A expression is repressed in adult liver, kidney and intestine and similar to aldolase C levels in brain and other nervous tissue. Aldolase A deficiency has been associated with myopathy and hemolytic anemia. Alternative splicing of this gene results in multiple transcript variants which encode the same protein.
  • Glucosamine-6 phosphate deaminase catalyzes a reversible reaction of glucosamine-6-phosphate and water to form fructose 6-phosphate and ammonium.
  • Glucose transporter-3 (GLUT3) is a facilitative glucose transporter responsible for maintaining an adequate glucose supply to neurons, although it is also found in other tissues.
  • Facilitative glucose carriers accelerate the transport of glucose down its concentration gradient by facilitative diffusion, a form of passive transport.
  • Facilitative glucose carriers are expressed by most if not all cells.
  • the facilitative glucose-transporter isoforms have distinct tissue distributions and biochemical properties and contribute to the precise disposal of glucose under varying physiological conditions.
  • Glyceraldehyde-3 -phosphate dehydrogenase catalyzes the conversion of glyceraldehyde-3 -phosphate to 1,3-bisphosphoglycerate.
  • PF2K 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase
  • PF2K is a bifunctional enzyme that catalyses both the synthesis and the degradation of fructose-2,6-bisphosphate [Fru(2,6)R 2 ], a potent stimulator of 6-phosphofructo-l- kinase and inhibitor of fructose 1,6-bisphosphatase.
  • PF2K can be considered to be either a glycolytic or a gluconeogenic enzyme; it acts as a switch between glycolysis and gluconeogenesis in mammalian liver by regulating the level of Fru(2,6)R 2 .
  • the second step in the glycolytic pathway is the conversion of fructose-6- phosphate to fructose- 1,6-diphosphate by phosphofructokinase.
  • Phosphofructokinase utilizes ATP to catalyze the transfer of phosphate to the 1 -position of fructose-6-phosphate.
  • PGM Phosphoglucomutase catalyzes the interconversion of glucosamine-6-phosphate and glucosamine-1 phosphate.
  • PGM a phosphoenzyme, catalyzes an important trafficking point in carbohydrate metabolism.
  • glucosamine-1 - phosphate produced from sugar catabolism is converted to glucoasmine-6- phosphate, the first intermediate in glycolysis.
  • glucosamine-6-phosphate to glucosamine-1 -phosphate provides a substrate for synthesis of UDP-Glucosamine, which is required for synthesis of a variety of cellular constituents, including cell wall polymers and glycoproteins.
  • Phosphorylase kinase participates in a signaling pathway resulting in the breakdown of glycogen to glucose 1 -phosphate and its subsequent metabolism via glycolysis.
  • cAMP generated in response to varied stimuli, activates A-kinase, which in turn phosphorylates phosphorylase kinase.
  • phosphorylase kinase catalyzes the phosphorylation of glycogen phosphorylase, thereby activating it to release glucose molecules from glycogen for use in the glycolysis pathway.
  • Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate to pyruvate. It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, or reagents described herein, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention that will be limited only by the appended claims. All technical and scientific terms used herein have- the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
  • HIV infection refers to the ability of HIV to enter a host cell and/or replicate in the host cell.
  • HIV cell entry refers to the ability of a virus to enter a cell through binding of a virus to a cell and the fusion of the virus to the cell.
  • HIV or virus binding refers to the attachment of a virus to the surface of a cell.
  • HIV or virus fusion refers to the passage of a virus across the cell plasma membrane into the cell.
  • isolated nucleic acid molecule refers to a nucleic acid molecule that has been removed from its natural milieu (i.e., a molecule that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA.
  • An isolated nucleic acid molecule can be isolated from its natural source or can be produced using recombinant DNA technology (e.g., polymerase chain reaction amplification) or chemical synthesis.
  • Isolated nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to promote HIV infection or inhibit HIV infection.
  • nucleic acid molecules can be isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the nucleic acid molecule will be obtained substantially free of other nucleic acid sequences, generally being at least about 50%, and usually at least about 90% pure.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably.
  • an "isolated nucleic acid molecule” refers to a nucleic acid molecule that is the size of or smaller than a gene. Thus, an isolated nucleic acid molecule does not encompass isolated genomic DNA or an isolated chromosome. The term isolated nucleic acid molecule does not connote any specific minimum length unless set forth by reference to a minimum number of nucleotides or by a function of the nucleic acid molecule.
  • the term "gene” has the meaning that is well known in the art, that is, a nucleic acid sequence that includes the translated sequences that code for a protein ("exons") and the untranslated intervening sequences ("introns"), and any regulatory elements ordinarily necessary to transcribe and/or translate the protein. Included in the invention are nucleic acid molecules that are less than a full-length gene or less than a full-length coding sequence, such as fragments of a gene or coding sequence comprising, consisting essentially of, or consisting of a GSE of the present invention.
  • a coding sequence can include genomic DNA without introns, cDNA or RNA that encodes a protein.
  • An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end ofthe sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., are heterologous sequences).
  • an isolated nucleic acid molecule of the present invention is produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • a nucleic acid molecule homologue i.e., a nucleic acid molecule that differs from the reference nucleic acid molecule by one or more insertions, deletions, substitutions, and/or inversions such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a protein or peptide useful in the present invention or to form stable hybrids under stringent conditions with natural gene isolates
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as site- directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid and/or by hybridization with a wild-type gene.
  • the minimum size of a nucleic acid molecule of the present invention is a size sufficient to encode a protein having the desired biological activity, sufficient to have an effect on the expression or biological activity of a cellular gene or protein as discussed herein or to have an effect on HIV inhibitory activity as described herein (e.g., as in a GSE), or sufficient to form a probe or oligonucleotide primer that is capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule.
  • the size of a nucleic acid molecule of the present invention can be dependent on nucleic acid composition and percent homology or identity between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration) and the intended use of the nucleic acid molecule.
  • the minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT-rich.
  • nucleic acid molecule of the present invention can include a fragment of a gene, a portion of a protein encoding sequence, or a nucleic acid sequence encoding a full-length protein (including a complete gene).
  • One embodiment of the present invention includes a recombinant nucleic acid molecule comprising a recombinant vector and a nucleic acid molecule comprising a nucleic acid sequence encoding a cellular gene or fragment thereof as described herein.
  • a recombinant vector is an engineered (i.e., artificially produced) nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice and for introducing such a nucleic acid sequence into a host cell.
  • the recombinant vector is therefore suitable for use in cloning, sequencing, and/or otherwise manipulating the nucleic acid sequence of choice, such as by expressing and/or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell.
  • a vector typically contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid sequence to be cloned or delivered, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid molecules ofthe present invention or which are useful for expression of the nucleic acid molecules of the present invention (discussed in detail below).
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a plasmid.
  • the vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of a recombinant organism (e.g., a microbe or a plant).
  • the entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention.
  • the integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies ofthe nucleic acid molecule can be integrated into the chromosome.
  • a recombinant vector ofthe present invention can contain at least one selectable marker.
  • a recombinant vector used in a recombinant nucleic acid molecule of the present invention is an expression vector.
  • expression vector is used to refer to a vector that is suitable for production of an encoded product (e.g., a protein of interest).
  • a nucleic acid sequence encoding the product to be produced is inserted into the recombinant vector to produce a recombinant nucleic acid molecule.
  • the nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector which enable the transcription and translation of the nucleic acid sequence within the recombinant host cell.
  • a recombinant vector used in a recombinant nucleic acid molecule of the present invention is a targeting vector.
  • targeting vector is used to refer to a vector that is used to deliver a particular nucleic acid molecule into a recombinant host cell, wherein the nucleic acid molecule is used to delete or inactivate an endogenous gene within the host cell or microorganism (i.e., used for targeted gene disruption or knock-out technology).
  • Such a vector may also be known in the art as a "knock-out" vector.
  • a portion of the vector but more typically, the nucleic acid molecule inserted into the vector (i.e., the insert), has a nucleic acid sequence that is homologous to a nucleic acid sequence of a target gene in the host cell (i.e., a gene which is targeted to be deleted or inactivated).
  • the nucleic acid sequence of the vector insert is designed to bind to the target gene such that the target gene and the insert undergo homologous recombination, whereby the endogenous target gene is deleted, inactivated or attenuated (i.e., by at least a portion ofthe endogenous target gene being mutated or deleted).
  • a recombinant nucleic acid molecule includes at least one nucleic acid molecule of the present invention operatively linked to one or more expression control sequences, including transcription control sequences and translation control sequences.
  • the phrase "recombinant molecule” or “recombinant nucleic acid molecule” primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to an expression control sequence, but can be used interchangeably with the phrase “nucleic acid molecule", when such nucleic acid molecule is a recombinant molecule as discussed herein.
  • the phrase "operatively linked” refers to linking a nucleic acid molecule to an expression control sequence (e.g., a transcription control sequence and/or a translation control sequence) in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conducted) into a host cell.
  • Transcription control sequences are sequences which control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.
  • Suitable expression control sequences include any expression control sequence that can function in a host cell or organism into which the recombinant nucleic acid molecule is to be introduced.
  • the term “transfection” is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell.
  • transformation can be used interchangeably with the term “transfection” when such term is used to refer to the introduction of nucleic acid molecules into microbial cells or plants.
  • transformation is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term “transfection.”
  • transformation has acquired a second meaning which can refer to changes in the growth properties of cells in culture (described above) after they become cancerous, for example.
  • transfection is preferably used with regard to the introduction of exogenous nucleic acids into animal cells, including human cells, and is used herein to generally encompass transfection of animal cells and transformation of plant cells and microbial cells, to the extent that the terms pertain to the introduction of exogenous nucleic acids into a cell. Therefore, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, microinjection, hpofection, adsorption, infection and protoplast fusion.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules operatively linked to an expression vector containing one or more expression control sequences.
  • Hybridization has the meaning that is well known in the art, that is, the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain some regions of mismatch.
  • reference to hybridization conditions refers to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62).
  • moderate stringency hybridization and washing conditions can be defined as hybridizations carried out as described above, followed by washing in 0.2X SSC and 0.1% SDS at 42 degrees Celsius (Ausubel et al, 1989, Current Protocols for Molecular Biology, ibid.). More particularly, moderate stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides).
  • High stringency hybridization and washing conditions refer to conditions which permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides).
  • Very high stringency hybridization and washing conditions refer to conditions which permit isolation of nucleic acid molecules having at least about 90%o nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides).
  • stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 20 °C and about 35 °C (low stringency), more preferably, between about 28°C and about 42°C (more stringent), and even more preferably, between about 35 °C and about 45 °C (even more stringent), with appropriate wash conditions.
  • 6X SSC 0.9 M Na +
  • stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 30°C and about 45 °C, more preferably, between about 38 °C and about 50°C, and even more preferably, between about 45 °C and about 55 °C, with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%). Alternatively, T m can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions.
  • hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25 °C below the calculated T m of a particular hybrid
  • wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20°C below the calculated T m of the particular hybrid.
  • hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37°C in about 0.1X-0.5X SSC, followed by at least one wash at about 68°C in about 0.1X-0.5X SSC).
  • any amino acid sequence described herein can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence.
  • the resulting protein or polypeptide can be referred to as "consisting essentially of the specified amino acid sequence.
  • the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived.
  • the phrase "consisting essentially of, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the specified amino acid sequence.
  • the heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.
  • the methods of the present invention can include the steps of identifying cellular targets (genes and their encoded products) that are in a biological pathway that is necessary for productive HIV infection. Such methods include the steps of: (a) synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; (b) transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; (c) genetically modifying a population of human host cells by introducing the genetic suppressor element library into the population of human host cells; (d) infecting the population of human host cells with HIV; (e) isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the population of human host cells; (f) recovering the genetic suppressor element from the isolated genetically modified human host cell
  • a cell-derived library (e.g., an RFE library) can be constructed from nucleic acid molecules of any mammalian cells, and preferably from cDNA of HTV-susceptible cells.
  • Example 1 demonstrates that GSEs can be selected from HL-60 cells that are naturally susceptible to HIV infection, from HeLa cells which are not naturally susceptible to HIV infection due to the lack of CD4 expression, and from peripheral blood mononuclear cells (PBMCs). It has been shown that expression of CD4 on the surface of HeLa cells by means of a retroviral vector renders the cells susceptible to HIV infection. Therefore, cell types not normally susceptible to HIV infection can still be useful as a source of genetic material for the construction of RFE libraries.
  • PBMCs peripheral blood mononuclear cells
  • DNA is first treated with enzymes to produce randomly cleaved fragments. This can be conveniently performed by DNase I cleavage in the presence of Mn ++ (Roninson et al, U.S. Patent No. 5,217,889, column 5, lines 5-20). Thereafter, the randomly-cleaved DNA is size fractionated by gel electrophoresis. Fragments of between 100 and 700 bp are the preferred lengths for constructing RFE libraries. Single strand breaks of the size-selected fragments are repaired by methods well known in the art.
  • the fragments are ligated with 5' and 3' adaptors, which are selected to have non-cohesive restriction sites so that each fragment can be inserted into an expression vector in an oriented fashion. Further, the 5' adaptor contains a start (ATG) codon to allow the translation of the fragments which contain an open reading frame in the correct phase.
  • the fragments are then inserted into appropriate expression vectors. Any expression vector that results in efficient expression of the fragments in host cells can be used.
  • viral-based vectors such as the retroviral vectors LNCX (Miller and Rosman, 1989, BioTechniques 7:980) and LNGFRM are exemplified.
  • adenovirus, adeno-associated virus and herpes virus vectors can also be used for this purpose.
  • the ligated vectors are first transfected into a packaging cell line to produce viral particles.
  • a packaging cell line for retroviral vectors, any amphotropic packaging line such as PA317 (Miller and Buttimore, 1986, Mol. Cell. Biol. 6:2895-2902; ATCC CRL #9078) can be used to efficiently produce virus.
  • the viral vector also contains a selectable gene, such as the neo r gene or a truncated nerve growth factor receptor (NGFR) gene, which allows isolation ofthe cells that contain the vector.
  • NGFR nerve growth factor receptor
  • the number of independent clones present in each RFE expression library can vary.
  • libraries of cell-derived cDNA of about 10° to 10 independent clones can be used.
  • OM10.1 cells are used to select for GSEs, and are maintained in conventional tissue culture as described in Butera, U.S. Patent No. 5,256,534.
  • the purpose of using OM10.1 cells for the selection of GSEs is that they contain a latent HIV-1 provirus which is inducible by TNF- ⁇ .
  • Example 2 also illustrates the use of CEM- ss cells to select for GSEs.
  • Other cell lines can be similarly engineered with an inducible HIV provirus.
  • Examples of cell lines that are infected with latent HIV include, but are not limited to, Ul, U33, 8E5, ACH-2, LL58, THP/HIV and UHC4 (Bednarik and Folks, 1992, AIDS 6:3-16).
  • agents have been shown to be capable of inducing latent HlV-infected cells, and these include TNF- ⁇ , TNF- ⁇ , interleukins-1, -2, -3, -4 and -6, granulocyte-macrophage colony stimulating factors, macrophage-colony stimulating factors, interferon- ⁇ , transforming growth factor- ⁇ , PMA, retinoic acid and vitamin D3 (Poli and Fauci, 1992, AIDS Res. Human Retroviruses 9:191-197).
  • GSEs can be selected on the basis of their ability to directly protect HlV-susceptible cells from HIV infection or inhibit the entry of HIV into cells using methods described herein.
  • the cell-derived RFE library can be introduced into latently HIN-infected cells or HIN-susceptible cells by any technique well known in the art that is appropriate to the vector system employed.
  • the viral vector also contains a selectable marker in addition to a random fragment of cellular D ⁇ A.
  • a suitable marker by way of example only, is the neo r gene, which permits selection of cells containing RFE library members using the drug G-418.
  • the viral vector contains a truncated low affinity nerve growth factor receptor ( ⁇ GFR) that permits selection of the cells using an anti- ⁇ GFR monoclonal antibody
  • ⁇ GFR nerve growth factor receptor
  • the multiplicity of infection of the virions of the library is adjusted so that preselection for cells that are transduced by the vector is not needed.
  • the transduced cell population can be treated with TNF- ⁇ for a period of between about 24-72 hours (preferably about 24 hours) according to the method of Butera.
  • the activation of the latent HIV-1 provirus in OM10.1 can be detected by the suppression of the cell surface CD4.
  • Clones that are resistant to HIV replication continue to express cell surface CD4.
  • Such clones can be selected, for example, by cell sorting using any antibody staining technique for CD4 and a fluorescence activated cell sorter (FACS).
  • FACS fluorescence activated cell sorter
  • the fraction of CD4 + cells that have been transduced with the RFE library can be compared with cells transduced with an expression library consisting ofthe vector only.
  • An increased relative difference between the cell-derived RFE library and the control library can be found with each additional round of TNF- ⁇ induction.
  • nucleic acid molecules corresponding to the GSEs can be recovered from cells that continue to express CD4 following induction of the latent HIV provirus by TNF- ⁇ .
  • the specific GSEs are recovered from genomic DNA isolated from CD4 + cells sorted by FACS after TNF- ⁇ induction.
  • the GSEs in this population are preferably recovered by PCR amplification using primers designed from the sequences ofthe vector.
  • the recovered GSEs can be introduced into an expression vector as discussed in the Examples section herein.
  • the resultant GSEs expression library is known as a secondary library.
  • the secondary library can utilize the same or a different vector from that used for the construction of the primary library.
  • the secondary library can be transduced into another cell population and the resultant population selected, recloned and processed as described herein.
  • each individually recovered GSE can be inserted into cloning vectors for determining its specific nucleotide sequence and its orientation.
  • the sequence of the GSE is then compared with sequences of known genes to determine the portion of the cellular gene with which it corresponds.
  • the PCR products themselves can be directly sequenced to determine their nucleotide sequences.
  • the isolated GSEs can be analyzed to determine their minimal core sequences.
  • a core sequence is a common sequence found by comparison of GSEs with overlapping sequences.
  • the GSEs are further tested for their ability to protect previously uninfected cells from HIN infection.
  • the core sequence of the GSE is determined. This can be done by comparing overlapping sequences of independently derived GSEs. Alternatively, GSEs can be altered by additions, substitutions or deletions and assayed for retention of HIN-suppressive function. Alterations in the GSEs sequences can be generated using a variety of chemical and enzymatic methods which are well known to those skilled in the art. For example, oligonucleotide-directed mutagenesis can be employed to alter the GSE sequence in a defined way and/or to introduce restriction sites in specific regions within the sequence. Additionally, deletion mutants can be generated using D ⁇ A nucleases such as Bal 31 or Exo in and SI nuclease. Progressively larger deletions in the GSE sequences can be generated by incubating the D ⁇ A with nucleases for increased periods of time (see Ausubel, et al, ibid., for a review of mutagenesis techniques).
  • the altered sequences can be evaluated for their ability to suppress expression of HIV proteins such as p24 in appropriate host cells. It is within the scope of the present invention that any altered or shortened GSE nucleic acid molecules that retain their ability to suppress HIN infection can be incorporated into recombinant expression vectors for further use.
  • the GSEs can be transferred into latently HIN infected or into HIN-susceptible host cells followed by HIV infection, hi this connection, GSEs also can be directly selected from a RFE library for their ability to prevent productive infection by HIV. Protection experiments can be performed in any cell type that takes up the potential GSEs and that is otherwise susceptible to HIV infection, h a preferred embodiment by way of example, the CEM-ss cell line is used (Foley et al. 1965, Cancer 18:522-529). The use of CEM-ss cells as targets for quantitative infectivity of HIV-1 has been described by ⁇ ara & Fischinger (1988, Nature 322:469-470).
  • cell lines that are susceptible to HIV infection include, but are not limited to, HUT-78, H9, Jurkat E6-1, A3.01, U-937, AA-2, HeLa CD4 + and C8166.
  • freshly isolated peripheral blood leukocytes can be used.
  • the test of the potential GSEs can be performed using the same expression vector system as that employed in the RFE library trans duction of cells during initial selection steps.
  • the vector system can be modified to achieve higher levels of expression, e.g., the linkers can be employed to introduce a leader sequence that increases the translational efficiency of the message.
  • One such sequence is disclosed by Kozak, 1994, Biochemie 76:815- 821.
  • Another way of testing the effectiveness of a potential GSE against HIV infection is to determine how rapidly HIV-1 variants develop that can negate the effects of that element.
  • Such a test includes infection of a culture of susceptible cells such as CEM-ss cells at a low multiplicity of infection and repeatedly assaying the culture to determine whether and how quickly HIV-1 infection becomes widespread.
  • the range of useful multiplicities of infection is between about 100 to 1000 tissue culture infectious units (TCID 5 0) per 10° CEM-ss cells.
  • the TCID 5 o is determined by an endpoint method and is important for determining the input multiplicity of infection (moi).
  • a parameter that correlates with the development in the test culture of HIN-1 strains that are resistant to the effects of the potential GSEs is the fraction of cells that are infected in the culture. This fraction can be determined by immunofluorescent staining with an antibody specific for the HIV-1 p24 antigen of fixed permeabilized cells. Commercially available reagents are suitable for performing such tests (Lee et al, 1994, J. Virol. 68:8254-8264). Methods to test putative GSEs and similar compounds for the ability to inhibit aspects of HIV infection and replication are also described in detail in U.S. Patent No. 6,613,506, which is incorporated herein by reference in its entirety.
  • Target genes or proteins that are identified using GSEs can be further evaluated using a variety of methods. Such methods include methods that disrupt or “knock out” the expression of a target gene in a cell capable of being infected with HIV. Knockout methods include somatic cell knock-outs and inhibitory RNA molecules such as antisense oligonucleotides, siRNA molecules, and RNA decoys. Other methods for evaluating GSEs include nucleic acid-based experiments such as Northern blots, real time polymerase chain reaction, or high density microarrays.
  • the activity of a member of the glycolysis pathway can be inhibited in a cell capable of being infected with HIV using methods known to those in the art, such as known chemical inhibitors, antibodies, somatic cell gene knock-outs, antisense molecules, or ribozymes.
  • the cell can then be exposed to HIN and HIV infection measured. Inhibition of HIV infection would indicate that a particular glycolysis pathway member is required for progression through the HIN life cycle. Methods for testing HIV infection are described in the Examples herein.
  • Exemplary human cellular targets that have been identified using the methods described herein and which can serve as target genes or products for the assays described herein include any of the genes listed in Table 1 above, a nucleotide sequence for which is provided in SEQ ID ⁇ os:13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39.
  • enolase 1 (ENO1; SEQ TD NO: 14); enolase 2 (ENO2; SEQ ID NO:16); enolase 3 (ENO3; SEQ ID NO:18); fructose- 1,6-bisphosphatase 1 (FBPl; SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA; SEQ ID NO:22); glucosamine-6-phosphate deaminase (GNPDA; SEQ ID NO:24); glucose transporter-3 (GLUT3; SEQ ID NO:26); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH; SEQ ID NO:28); Na + D-glucose cotransport regulator (SEQ ID NO: 30); 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K; SEQ ID NO:32); phosphofructokinase (PF2K; SEQ ID NO:32
  • a full-length gene including a regulatory region of the gene, or a nucleic acid molecule encoding the gene product (protein encoded by the gene) or fragment thereof, or any fragment of such nucleic acid molecules that is suitable for use in an assay, or any encoded product of any such nucleic acid molecules, to identify inhibitors of the cellular gene or its encoded product for the potpose of inhibition of HIV infection or the development of compounds therefore based on a lead compound.
  • an assay can be used for screening and selecting a chemical or biological compound capable of acting as an anti-HIV therapeutic, based on the ability of the chemical or biological compound to down- regulate the expression ofthe target gene or inhibit the activity of its gene product.
  • the methods can include the steps of (a) exposing (contacting) a second population of human host cells to a test compound; (b) measuring the expression of the human cellular gene in the second population of human host cells; and (c) determining whether the test compound regulates (modifies, increases or decreases, changes, modulates) the expression and/or activity of the human cellular gene or ofthe protein product encoded by the human cellular gene.
  • Such a chemical or biological compound can be referred to herein as a therapeutic compound, a test compound, or a lead compound.
  • Such compounds may be identified by first incubating a cell line that naturally expresses the gene of interest, or which has been transfected with the gene or other recombinant nucleic acid molecule encoding a polypeptide of interest as disclosed herein, with various test compounds (putative regulatory compounds, putative therapeutic compounds), such as a chemical or biological compound. A reduction in the expression of the gene or protein of interest and/or an inhibition of the biological activity of the product encoded by the gene of interest would indicate that the chemical or biological compound is a therapeutic compound, or a lead compound to be used for development of drugs. Compounds identified in this manner are then retested in other assays to confirm their activities against HIN infection.
  • the biological activity or biological action of a protein refers to any function(s) exhibited or performed by the protein that is ascribed to the naturally occurring form ofthe protein as measured or observed in vivo (i.e., in the natural physiological environment ofthe protein) or in vitro (i.e., under laboratory conditions).
  • Modifications, activities or interactions which result in a decrease in protein expression or a decrease in the activity of the protein can be referred to as inactivation (complete or partial), down-regulation, reduced action, or decreased action or activity of a protein.
  • modifications, activities or interactions which result in an increase in protein expression or an increase in the activity of the protein can be referred to as amplification, overproduction, activation, enhancement, up-regulation or increased action of a protein.
  • the biological activity of a protein according to the invention can be measured or evaluated using any assay for the biological activity of the protein as known in the art.
  • assays can include, but are not limited to, binding assays, assays to determine internalization of the protein and/or associated proteins, cell signal transduction assays (e.g., phosphorylation assays), enzyme assays and/or assays for determining downstream cellular events that result from activation or binding of the cell surface protein (e.g., expression of downstream genes, production of various biological mediators, etc.).
  • the assay measures the ability of the protein or polypeptide to allow HIV infection.
  • assays are described in detail herein.
  • Reagents suitable for an assay of the invention include any human cellular gene or its gene product.
  • Compounds to be screened include any of those listed herein, such as known organic compounds such as antibodies, products of peptide libraries, and products of chemical combinatorial libraries.
  • Compounds may also be identified using rational drag design relying on the structure of the gene product of a human cellular gene. Such methods are known to those of skill in the art and involve the use of three-dimensional imaging software programs. For example, various methods of drug design, useful to design or select mimetics or other therapeutic compounds useful in the present invention are disclosed in Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is incorporated herein by reference in its entirety.
  • a mimetic refers to any peptide or non-peptide compound that is able to mimic the biological action of a naturally occurring peptide, often because the mimetic has a basic structure that mimics the basic structure of the naturally occurring peptide and/or has the salient biological properties of the naturally occurring peptide.
  • Mimetics can include, but are not limited to: peptides that have substantial modifications from the prototype such as no side chain similarity with the naturally occurring peptide (such modifications, for example, may decrease its susceptibility to degradation); anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous portions of an isolated protein (e.g., carbohydrate structures); or synthetic or natural organic molecules, including nucleic acids and drugs identified through combinatorial chemistry, for example.
  • Such mimetics can be designed, selected and/or otherwise identified using a variety of methods known in the art.
  • a mimetic can be obtained, for example, from molecular diversity strategies (a combination of related strategies allowing the rapid construction of large, chemically diverse molecule libraries), libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the similar building blocks) or by rational, directed or random drug design. See for example, Maulik et al., supra.
  • a molecular diversity strategy large compound libraries are synthesized, for example, from peptides, oligonucleotides, carbohydrates and/or synthetic organic molecules, using biological, enzymatic and/or chemical approaches.
  • the critical parameters in developing a molecular diversity strategy include subunit diversity, molecular size, and library diversity.
  • the general goal of screening such libraries is to utilize sequential application of combinatorial selection to obtain high-affinity ligands for a desired target, and then to optimize the lead molecules by either random or directed design strategies. Methods of molecular diversity are described in detail in Maulik, et al., ibid. Maulik et al.
  • methods of directed design in which the user directs the process of creating novel molecules from a fragment library of appropriately selected fragments
  • random design in which the user uses a genetic or other algorithm to randomly mutate fragments and their combinations while simultaneously applying a selection criterion to evaluate the fitness of candidate ligands
  • grid-based approach in which the user calculates the interaction energy between three dimensional receptor structures and small fragment probes, followed by linking together of favorable probe sites.
  • inhibitors of HIN infection are identified by exposing a mammalian cell to a test compound; measuring the expression of a human cellular gene or an activity of the polypeptide encoded by the human cellular gene in the mammalian cell; and selecting a compound that down-regulates the expression of the human cellular gene or interferes with the activities of its encoded product.
  • a preferred mammalian cell to use in an assay is a mammalian cell that either naturally expresses the human cellular gene or has been transformed with a recombinant form of the human cellular gene. Methods to determine expression levels of a gene are well known in the art.
  • the cellular gene is a gene encoding a protein involved in or associated with the glycolysis pathway, including any of the genes listed in Table 1.
  • test compound or “putative regulatory compound” or “putative inhibitory compound” and the like refers to compounds having an unknown or previously unappreciated regulatory activity in a particular process.
  • identify with regard to methods to identify compounds is intended to include all compounds, the usefulness of which as a regulatory compound for the purposes of inhibiting HIV infection can be determined by a method of the present invention and confirmed with additional assays, if desired or needed.
  • the conditions under which a host cell (or cell lysate, if appropriate for the given assay) of the present invention is contacted with or exposed to a putative regulatory compound, such as by mixing, are any suitable culture or assay conditions and includes an effective medium in which the cell can be cultured or in which the cell lysate can be evaluated in the presence and absence of a putative regulatory compound.
  • Cells of the present invention can be cultured in a variety of containers including, but not limited to, tissue culture flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and carbon dioxide content appropriate for the cell. Such culturing conditions are also within the skill in the art.
  • Cells are contacted with or exposed to a putative regulatory compound under conditions which take into account the number of cells per container contacted, the concentration of putative regulatory compound(s) administered to a cell, the incubation time of the putative regulatory compound with the cell, and the concentration of compound administered to a cell. Determination of effective protocols can be accomplished by those skilled in the art based on variables such as the size of the container, the volume of liquid in the container, conditions known to be suitable for the culture of the particular cell type used in the assay, and the chemical composition of the putative regulatory compound (i.e., size, charge etc.) being tested.
  • a preferred amount of putative regulatory compound(s) comprises between about 1 nM to about 10 mM of putative regulatory compound(s) per well of a 96-well plate.
  • compounds which are selected or identified are compounds for which, after the test or host cell was contacted with or exposed to the test compound, the level of expression or biological activity of the target gene or polypeptide was statistically significantly (i.e., with at least a 95% confidence level, or p ⁇ 0.05) changed (preferably decreased) as compared to the initial level of expression or biological activity of the target gene or polypeptide or the expression or biological activity of the target gene or polypeptide in the absence of the test compound.
  • any statistically significant, detectable or measurable regulation of expression or activity of the target gene or product is indicative that the test compound can be identified as a regulator of the target gene or product and as a putative regulator of HIN infection in the host cell.
  • a 1.5 fold decrease in expression or biological activity of the target gene or protein in the cell as compared to in the absence of the test compound results in selection of the compound as being potentially useful for inhibition of HIN infection.
  • detection of at least about a 3 fold increase, and more preferably at least about a 6 fold increase, and even more preferably, at least about a 12 fold increase, and even more preferably, at least about a 24 fold increase in expression or biological activity of the target gene or protein in the cell as compared to in the absence of the test compound results in selection of the compound as being potentially useful for inhibition of HIN infection.
  • Methods for measuring expression of a human cellular gene include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), in situ hybridization, Northern blot, Southern blot, sequence analysis, polyacrylamide gene analysis, microarray analysis, and detection of a reporter gene.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase PCR
  • in situ hybridization Northern blot
  • Southern blot sequence analysis
  • polyacrylamide gene analysis polyacrylamide gene analysis
  • microarray analysis microarray analysis
  • Protein expression can be measured by any method including, but not limited to, Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, malti- tos, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry.
  • the expression of the human cellular gene is measured by the polymerase chain reaction.
  • the expression of the human cellular gene is measured using an antibody that specifically recognizes the polypeptide encoded by the human cellular gene and is analyzed using methods such as immunoprecipitation, ELISA assays, fluorescence activated cell sorting (FACS), or immunofluorescence microscopy.
  • the expression of the human cellular gene is measured using polyacrylamide gel analysis, chromatography, or spectroscopy.
  • the activity of the polypeptide encoded by the human cellular gene is measured by measuring the amount of product generated in a biochemical reaction mediated by the polypeptide encoded by the human cellular gene
  • the activity of the polypeptide encoded by the human cellular gene is measured by measuring the amount of substrate generated in a biochemical reaction mediated by the polypeptide encoded by the target gene.
  • therapeutic compounds are selected by determining the three-dimensional structure of a human cellular gene product; and then determining the three-dimensional structure of a therapeutic compound based on the three-dimensional structure of a human cellular gene product.
  • the structure of the therapeutic compound is determined using computer software capable of modeling the interaction of the therapeutic compound with the target product.
  • One of skill in the art can select the appropriate three-dimensional stracture, therapeutic compound, and analytical software based on the identity of the target gene.
  • suitable candidate chemical compounds can align to a subset of residues described for a target site.
  • a candidate chemical compound comprises a conformation that promotes the formation of covalent or noncovalent crosslinking between the target site and the candidate chemical compound.
  • a candidate chemical compound binds to a surface adjacent to a target site to provide an additional site of interaction in a complex.
  • the antagonist should bind with sufficient affinity to the binding site or to " substantially prohibit a ligand (i.e., a molecule that specifically binds to the target site) from binding to a target area.
  • the design of a chemical compound possessing stereochemical complementarity can be accomplished by techniques that optimize, chemically or geometrically, the "fit" between a chemical compound and a target site.
  • Such techniques are disclosed by, for example, Sheridan and Nenkataraghavan, Ace. Chem Res., vol. 20, p. 322, 1987: Goodford, J. Med. Chem., vol. 27, p. 557, 1984; Beddell, Chem. Soc. Reviews, vol. 279, 1985; Hoi, Angew. Chem., vol. 25, p. 767, 1986; and Nerlinde and Hoi, Structure, vol. 2, p. 577, 1994, each of which are incorporated by this reference herein in their entirety.
  • a "geometric approach” is used.
  • the number of internal degrees of freedom (and the corresponding local minima in the molecular conformation space) is reduced by considering only the geometric (hard_sphere) interactions of two rigid bodies, where one body (the active site) contains "pockets” or "grooves” that form binding sites for the second body (the complementing molecule, such as a ligand).
  • the geometric approach is described by Kuntz et al., J. Mol. Biol, vol. 161, p. 269, 1982, which is incorporated by this reference herein in its entirety.
  • the algorithm for chemical compound design can be implemented using the software program DOCK Package, Version 1.0 (available from the Regents of the University of California).
  • the shape of the cavity or groove on the surface of a structure at a binding site or interface is defined as a series of overlapping spheres of different radii.
  • One or more extant databases of crystallographic data e.g., the Cambridge Structural Database System maintained by University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 1EW, U.K.
  • the Protein Data Bank maintained by Brookhaven National Laboratory, is then searched for chemical compounds that approximate the shape thus defined.
  • Chemical compounds identified by the geometric approach can be modified to satisfy criteria associated with chemical complementarity, such as hydrogen bonding, ionic interactions or Van der Waals interactions.
  • This method is referred to herein as a "chemical_probe approach.”
  • the chemical_probe approach to the design of a chemical compound useful of the present invention is described by, for example, Goodford, J. Med. Chem., vol. 28, p. 849, 1985, which is incorporated by this reference herein in its entirety, and is implemented using an appropriate software package, including for example, GRID (available from Molecular Discovery Ltd., Oxford OX2 9LL, U.K.).
  • the chemical prerequisites for a site_complementing molecule can be identified at the outset, by probing the active site of a protein with different chemical probes, e.g., water, a methyl group, an amine nitrogen, a carboxyl oxygen and/or a hydroxyl. Preferred sites for interaction between an active site and a probe are determined. Putative complementary chemical compounds can be generated using the resulting three dimensional pattern of such sites.
  • inhibitors of HIV infection are identified by exposing (exposing, contacting, mixing with) a polypeptide encoded by a target gene to a test compound; measuring the binding of the test compound to the polypeptide; and selecting a compound that binds to the polypeptide at a desired concentration, affinity, or avidity.
  • the assay is performed under conditions conducive to promoting the interaction or binding of the compound to the polypeptide.
  • a BIAcore machine can be used to determine the binding constant of a complex between the target protein (a protein encoded by the target gene) and a natural ligand in the presence and absence of the candidate compound.
  • the target protein or a ligand binding fragment thereof can be immobilized on a substrate.
  • a natural or synthetic ligand is contacted with the substrate to form a complex.
  • the dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (O'Shannessy et al. Anal. Biochem. 212:457_468 (1993); Schuster et al., Nature 365:343_347 (1993)).
  • Contacting a test compound at various concentrations with the complex and monitoring the response function e.g., the change in the refractive index with respect to time) allows the complex dissociation constant to be determined in the presence of the test compound and indicates whether the test compound is either an inhibitor or an agonist ofthe complex.
  • test compound can be contacted with the immobilized target protein at the same time as the ligand to see if the test compound inhibits or stabilizes the binding of the ligand to the target protein.
  • suitable assays for measuring the binding of a candidate compound to a target protein or its ligand, and or for measuring the ability of such compound to affect the binding of the target protein to its ligand include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), as well as cell__based assays including, cytokine secretion assays, or infracellular signal fransduction assays that determine, for example, protein or lipid phosphorylation, mediator release or infracellular Ca + mobilization upon binding of a compound to a cell signal fransduction molecule or coreceptor.
  • immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA
  • inhibitors of HIV infection are identified by exposing (exposing, contacting, mixing with) a polypeptide encoded by a target gene to a test compound; measuring the activity of the polypeptide and or the activity of the biological pathway comprising the target polypeptide, and selecting a compound that modulates (regulates, changes, increases or decreases), the activity of the polypeptide or biological pathway.
  • a therapeutic compound can be identified in one embodiment by exposing an enzyme encoded by a target gene to a test compound; measuring the activity of the enzyme encoded by the target gene in the presence and absence of the compound; and selecting a compound that down-regulates the activity of the enzyme encoded by the target gene.
  • Methods to measure enzymatic activity are well known to those skilled in the art and are selected based on the identity of the enzyme being tested. For example, if the enzyme is a kinase, then phosphorylation assays can be used. Other assays for detecting the biological activity and a change in such activity for a target polypeptide will be known to those of skill in the art.
  • the invention also encompasses methods known in the art for down-regulating the expression or function of a target gene of the invention, including any of the target genes set forth in Table 1 herein.
  • an antisense RNA or DNA molecule may be used to directly block translation of mRNA encoded by a target gene by binding to the mRNA and preventing protein translation.
  • Polydeoxyribonucleotides can form sequence- specific triple helices by hydrogen bonding to specific complementary sequences in duplexed DNA to effect specific down-regulation of target gene expression. Formation of specific triple helices may selectively inhibit the replication or expression of a target gene by prohibiting the specific binding of functional transacting factors.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are ribozyme embodiments including engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of cellular RNA sequences. Antisense RNA molecules showing high-affinity binding to target sequences can also be used as ribozymes by addition of enzymatically active sequences known to those skilled in the art.
  • Polynucleotides to be used in triplex helix formation should be single- stranded and composed of deoxynucleotides.
  • the base composition of these polynucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rales, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Polynucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich polynucleotides provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • polynucleotides may be chosen that are purine-rich, for example, containing a stretch of G residues. These polynucleotides will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
  • sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" polynucleotide.
  • Switchback polynucleotides are synthesized in an alternating 5 '-3', 3 '-5' manner, so that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constracts that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into host cells.
  • nucleic acid molecules may be introduced as a means of increasing infracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' or 3' ends ofthe molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
  • methods used to identify therapeutic compounds are customized for each target gene or product.
  • the target product is an enzyme
  • the enzyme will be expressed in cell culture and purified.
  • the enzyme will then be screened in vitro against therapeutic compounds to look for inhibition of that enzymatic activity.
  • the target is a non-catalytic protein, then it will also be expressed and purified.
  • Therapeutic compounds will then be tested for their ability to prevent, for example, the binding of a site-specific antibody or a target- specific ligand to the target product.
  • potential therapeutic compounds that bind to and/or effect the activity of target genes or products are further analyzed in a biological assay that tests for inhibition of HIV infection.
  • HeLa human fibroblast
  • HIV binds to the CD4 receptor on the HeLa cell surface and infects the cell.
  • viral proteins including tat are expressed.
  • Tat then binds to the HIN-1 LTR, promoting the expression of ⁇ -galactosidase, which can be detected and quantified. Inhibition of HIV replication by a compound would prevent or reduce expression of tat and result in reduction of ⁇ -galactosidase expression as compared to control cells.
  • a therapeutic compound is not toxic to a human host cell that is not infected with HIV. In another embodiment, a therapeutic compound promotes apoptosis in a human host cell infected with HIV.
  • a pharmaceutical composition is prepared from a therapeutically-effective amount of a therapeutic compound of the invention and a pharmaceutically-acceptable carrier.
  • Pharmaceutically- acceptable carriers are well known to those with skill in the art.
  • resistance to HIV infection is conferred upon an individual by administering a pharmaceutical composition of the invention.
  • the pharmaceutical compositions of the present invention can be manufactured in a manner that is itself known, e.g., by means of a conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the compounds of the invention can be formulated in appropriate aqueous solutions, such as physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PNP).
  • disintegrating agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions can take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix ofthe compound and a suitable powder base such as lactose or starch.
  • the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water- miscible organic polymer, and an aqueous phase.
  • the cosolvent system can be the NPD co-solvent system.
  • NPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
  • co-solvent system can be varied considerably without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components can be varied: for example, other low-toxicity nonpolar surfactants can be used instead of polysorbate 80; the fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides can substitute for dextrose.
  • hydrophobic pharmaceutical compounds can be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drags.
  • Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity.
  • the compounds can be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained- release capsules can, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • additional strategies for protein and nucleic acid stabilization can be employed.
  • compositions also can comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • the compounds of the invention can be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC50 (effective dose for 50% increase) as determined in cell culture, i.e., the concentration of the test compound which achieves a half- maximal inhibition of HIV replication as assayed by the infected cells to retain CD4 expression, to reduce viral p24 or gpl20, and to prevent syncytia formation.
  • EC50 effective dose for 50% increase
  • Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50.
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • Dosage amount and interval can be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the inhibitory effects.
  • Usual patient dosages for systemic administration range from 100 - 2000 mg/day. Stated in terms of patient body surface areas, usual dosages range from 50 - 910 mg/m 2 /day. Usual average plasma levels should be maintained within 0.1-1000 ⁇ M. In cases of local administration or selective uptake, the effective local concentration ofthe compound can not be related to plasma concentration.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's body surface area, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • Suitable routes of administration can, for example, include oral, rectal, transmucosal, transcutaneous, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • One embodiment of the present invention relates to an isolated nucleic acid molecule comprising, consisting essentially of, or consisting of: a GSE nucleic acid sequence according to the present invention, and to nucleic acid sequence homologues of the GSE that hybridize under stringent conditions to the complement of a GSE of the invention.
  • a nucleic acid molecule comprises, consists essentially of or consists of less than a full-length cellular gene comprising a GSE according to the invention.
  • GSEs are listed in Table 2 of the invention. Any of the GSE sequences described herein can be used, in one embodiment, as a template or stracture on which various other inhibitors of the present invention (e.g., small molecules) can be designed or selected.
  • RFE random fragment expression
  • the HL60 RFE library was prepared by isolating mRNA from uninduced HL60 cells (ATCC Accession No. CCL 240) and then subtracting that mRNA with mRNA isolated from cells induced with TNF- ⁇ . This procedure represents a modification of that described in Coche et al, 199 A, Nucleic Acids Res. 22:1322- 23. Tracer mRNA was isolated from HL-60 cells transduced with the retroviral vector pLNCX (Miller et al, 1989, BioTechniques 7:980-90) at different time points after induction with TNF- ⁇ (Boehringer Mannheim; Indianapolis, IN).
  • RNA isolated from induced and uninduced cells was annealed separately to oligo-dT magnetic beads (Dynal Biotech; Lake Success, NY) and first strand cDNA was synthesized using reverse transcriptase and an oligo-dT primer.
  • the RNA strand was then hydrolyzed and second strand cDNA synthesized from the induced cell first strand cDNA using a primer containing ATG codons in all three reading frames and an additional ten random nucleotides on the 3' end.
  • Single- stranded cDNA fragments were annealed to an excess of driver cDNA attached to the magnetic beads.
  • the HeLa RFE library was prepared using the method described in Gudkov et al, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:3744-48.
  • cDNA was prepared from HeLa cells and then partially digested with DNAse I in the presence of Mn (Sambrook et al, 1989, Molecular Cloning A Laboratory Manual (Cold Spring Harbor Laboratory)). Under these conditions, DNAse I is known to produce mostly double-stranded breaks. The resulting fragments were repaired using both the Klenow fragment of DNA polymerase I and T4 polymerase and then the fragments were ligated to synthetic double-stranded adaptors.
  • the 5' adaptor was prepared from the primers 5'-C-T-C-G-G-A-A-T-T- C-A-A-G-C-T-T-A-T-G-G-A-T-G-G-A-T-G-G-A-T-G-G-3' (SEQ ID NO: 1) and 5'-C-A- T-C-C-A-T-C-C-A-T-A-A-G-C-T-T-G-A-A-T-T-C-C-3' (SEQ ID NO: 2).
  • the 3' adaptor was prepared from the primers 5'-T-G-A-G-T-G-A-G-T-G-A-A-T-C-G- A-T-G-G-A-T-C-C-G-T-C-T-3' (SEQ ID NO: 3) and 5'-T-C-C-T-A-G-A-C-G-G- A-T-C-C-A-T-T-C-A-C-T-C-A-C-T-C-A-3' (SEQ ID NO: 4).
  • This randomly fragmented cDNA was then subjected to a normalization procedure to provide for the uniform abundance of different sequences in the population (Gudkov et al, 1997, Methods in Molecular Biology 69:221 (Humana Press)). This procedure was used to increase the probability of isolating GSEs from rare cDNAs, since total polyA RNA comprises a mixture of unequally represented sequences.
  • the randomly fragmented cDNA population was normalized by first denaturing 20 ⁇ g of cDNA by boiling for 5 minutes in 25 ⁇ l of TE buffer, followed by immediate cooling on ice. Then, 25 ⁇ l of 2X hybridization solution was added, and the mixture was divided equally into four .aliquots in Eppendorf tubes. One to two drops of mineral oil were added to each sample to avoid evaporation, and the tubes were placed into a 68°C water bath for annealing. One tube was frozen every 12 hours. Following the last time-point, each of the annealing mixtures was diluted with water to a final volume of 500 ⁇ l and subjected to hydroxylapatite (HAP) chromatography.
  • HAP hydroxylapatite
  • HAP suspension equilibrated with 0.01 M phosphate-buffered saline (PBS) was placed into Eppendorf tubes so that the volume of HAP pellet was approximately 100 ⁇ l.
  • the tubes with HAP and all the solutions used below were preheated and kept at 65°C. Excess PBS was removed, and diluted annealing solution was added. After mixing by shaking in a 65°C water bath, the tubes were left in the water bath until a HAP pellet was formed (a 15 second centrifugation was used to collect the pellet without exceeding lOOOg in the microcentrifuge to avoid damage of HAP crystals). The supernatant was carefully replaced with 1 ml of preheated 0.01 M PBS, and the process was repeated.
  • PBS phosphate-buffered saline
  • the HAP pellet was suspended in 500 ⁇ l of PBS at the single-strand elution concentration determined (e.g., 0.16 M), the supernatant was collected, and the process was repeated.
  • the supematants were combined and traces of HAP were removed by centrifugation.
  • the ssDNA was concentrated by centrifugation, and washed three times using 1 ml of water on a Centricon-100 column.
  • the isolated ssDNA sequences were amplified by PCR using sense primers from each adapter and a minimal number of cycles to obtain 10 ⁇ g of the product.
  • the size of the PCR product that remained within the desired range (200-500 bp) was ascertained.
  • the normalization quality was tested by Southern or slot-blot hybridization with 32 P-labeled probes for high, moderate- and low- expressing genes using 0.3-1.0 ⁇ g of normalized cDNA/lane.
  • ⁇ -actin and ⁇ - tubulin cDNAs were used as probes for high-expressing genes
  • c-myc and topo II cDNAs were used as probes for moderate-expressing genes
  • c-fos cDNA was used as a probe for low-expressing genes.
  • the cDNAs isolated after different annealing times were compared with the original unnormalized cDNA.
  • the probes were ensured to have a similar size and specific activity.
  • the best- normalized ssDNA fraction i.e., the population which produced the most uniform signal intensity with different probes
  • pLNGFRM differs from pLNCX in that the neo gene has been replaced with a truncated low affinity NGFR gene.
  • Cells transduced with pLNGFRM express a truncated receptor on their surface that can be easily selected by an anti-NGFR antibody during FACS.
  • the ligation mixture was introduced into E.coli, and approximately 100,000 transformants were obtained.
  • the size distribution of the cloned fragments was analyzed by PCR using primers derived from vector sequences adjacent to the adapter sequences.
  • PBMC-1 RFE Library A first PBMC RFE library (PBMC-1) was prepared by isolating buffy coats from four healthy donors, from which PBMCs were purified by Ficoll gradient centrifugation followed by stimulation with PHA (1 ⁇ g/ml). Cells were removed at 5, 10, and 24 hours following the addition of PHA and total RNA was isolated by Trizol extraction. The isolated total RNA collected from the four donors was then pooled, yielding three populations corresponding to the time at which the cells were removed following PHA treatment. Poly-A+ mRNA was purified from the total RNA using the Gibco Superscript Choice system for cDNA synthesis (Gibco BRL; Bethesda, MD) and a random primer.
  • Gibco Superscript Choice system for cDNA synthesis Gibco BRL; Bethesda, MD
  • the cDNA was normalized using the PCR-Select cDNA Subtraction kit (Clontech; Palo Alto, CA), based on the suppression subtractive hybridization methods described in Diatchenko et al, 1996, Proc. Natl. Acad. Sci. U.S.A.
  • the normalized random fragments were digested with Xho I and Sse I, purified on quick spin columns (QIAGEN; Nalencia, CA) and ligated into the Sse I and Xho I sites of a bicistronic retroviral vector, pLXEMCN ⁇ gfr. This vector is based on pLXS ⁇ gfr. Modifications included the replacement of the SV40 promoter with encephalomyocarditis virus (EMVC) internal ribosomal entry site (IRES) isolated from the plasmid pCITE (Amersham Biosciences; Piscataway, ⁇ J). The ligation mixture was introduced into competent cells, and approximately 50 million transformants were obtained.
  • EMVC encephalomyocarditis virus
  • IVS internal ribosomal entry site
  • PBMC-2 peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • the isolated PBMCs consisted of different populations, including total unstimulated PBMCs, CD3/CD28 stimulated PBMCs, purified monocytes and macrophages, and purified memory and na ⁇ ve T cells. Unstimulated total PBMCs were directly harvested following Ficoll purification.
  • CD3/CD28 stimulated PBMCs were generated by adding CD3 and CD28 antibodies (CD3/CD28 stimulation) and harvesting after 6 days in culture.
  • Total RNA was partially purified with oligotex (QIAGEN) to produce polyadenylated mRNA.
  • the oligotex-primed polyadenylated mRNA was then used as template to synthesize oligotex-bead linked first-strand cDNA.
  • the oligotex-bead linked first-strand cDNA was used as a driver to subtract abundant gene populations from PBMC total RNA. Normalization was performed with a 1:2 ratio of total RNA to cDNA driver at 37°C for 10 minutes. The abundant genes and rare gene were brought to within 2.5 orders of magnitude as measured by real time PCR.
  • RNA was then purified from the normalized total RNA population and fragmented with weak alkaline-sodium carbonate at 60°C for 10 minutes.
  • the fragmented mRNA served as the template for cDNA synthesis (Ominiscript kit; QIAGEN) using primers that included six random nucleotides linked to a Not I or Fse I site and a sequence for PCR priming (BSF-Fse-N6: 5'- G-C-T-A-T-G-A-C-C-A-T-G-A-T-T-A-C-G-C-C-G-G-C-C-N-N-N-N-N-N-N-N-N-N-N-N-N-3'; SEQ ID NO: 7; BSR-Not-N6: 5'-G-T-A-A-T-A-C-G-A-C-T-C-A-C-T- A-T-A-G-G-C-G-G-
  • the fragments were PCR amplified using the primers: 5'-G-C-T- A-T-G-A-C-C-A-T-G-A-T-T-A-C-G-C-C-A-3' (SEQ ID NO: 9) and 5'-G-T-A- A-T-A-C-G-A-C-T-C-A-C-T-A-T-A-G-G-G-C-3' (SEQ ID NO: 10).
  • the fragments were digested with Not I and Fse I and those in the size range of 100-600 bp were isolated from a 2% agarose gel, eluted, and ligated into the bicistronic retroviral vector, pLXEMCVNgfr.
  • This vector has a multiple cloning site modified to include the Not I and Fse I sites.
  • the ligation mixture was electroporated into DH10B competent cells, generating greater than 100 million transformants.
  • HL-60 RFE libraries prepared as described in Example 1 were introduced into PA317 packaging cells (ATCC Accession No. CRL 9078), and converted into retrovirus for infection of OM10.1 cells (ATCC Accession No. CRL 10850; U.S. Patent No. 5,256,534).
  • OM10.1 cells transduced with a pLNCX-based HL-60 RFE library were co-cultured and selected with G418.
  • OM10.1 cells transduced with a pLNGFRM-based or pLXEMCVNgfr-based HL-60 RFE library were first subjected to spinoculation (centrifugation of target cells at 1200xg for 90 minutes in the presence of filtered retroviral supernatant) and then selected by FACS sorting of the NGFR population. Following selection, OM10.1 cells harboring the entire RFE library were induced with 10 U/ml of TNF- ⁇ at 37°C for 24 hours, stained with antibody, and then sorted for CD4 expression. Genomic DNA from the CD4 + cells was purified and used for PCR amplification of inserts using vector-derived primers. The amplified mixture was digested with EcoR I and BamH I and cloned back into the retroviral vector. This selection was repeated for additional rounds.
  • Genomic DNA was isolated from the selected OM10.1 or CEM-ss cells prepared as described in Example 2 by first centrifuging the selected cells, resuspending the cell pellet in 0.1 % Triton X-100, 20 ⁇ g/ml proteinase K, and IX PCR buffer, incubating at 55°C for 1 hour, and then boiling for 10 minutes. Genomic DNA was used for PCR amplification using vector-derived primers, cloned into the retroviral vector, and introduced into E. coli using standard transformation techniques. Individual plasmids were purified from E. coli clones using QIAGEN plasmid purification kits.
  • Inserts were sequenced by the dideoxy procedure (using the AutoRead Sequencing Kit, Pharmacia Biotech or the Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit, ABI) and analyzed on a Pharmacia LKB A.L.F. or ABI 3700 DNA sequencer. Sequences were analyzed using the DNASTAR program or other proprietary data mining procedure.
  • CD4+ cells were isolated from the challenged cell population (1 x IO 7 cells) by washing the cells twice with Assay Buffer (500 ml PBS, 1 ml of 0.5 mM of EDTA, pH 8, 0.5 ml of 10%) sodium azide, and 10 ml of fetal bovine serum), and then resuspending the cells in 500 ⁇ l PBS containing 50 ⁇ l of anti-CD4 antibody (Q4120 PE; Sigma). Following incubation at 4°C for 30 minutes, 5 ml of Assay Buffer was added and the cells were centrifuged at 1200 rpm for 4 minutes. The cells were then washed twice with Assay Buffer and CD4 + cells sorted from the population by FACS.
  • Assay Buffer 500 ml PBS, 1 ml of 0.5 mM of EDTA, pH 8, 0.5 ml of 10%
  • sodium azide sodium azide
  • 10 ml of fetal bovine serum 500 ⁇ l PBS containing 50 ⁇ l of
  • the aforementioned procedure was performed under sterile conditions.
  • the sorted cell population (1 x . IO 6 cells) was washed twice with Assay Buffer, and then suspended in 100 ⁇ l of Assay Buffer and 2 ml of Ortho PermeaFix Solution (Ortho Diagnostics). The cells were incubated at room temperature for 40 minutes, centrifuged at 1200 rpm and 4°C for 4 minutes, and then resuspended in 2 ml Wash Buffer (500 ml PBS, 25 ml fetal bovine serum, 1.5% bovine serum albumin and 0.0055%) EDTA).
  • the cells were resuspended in 50 ⁇ l Wash Buffer diluted 1:500 with IgG a antibody, and incubated at 4°C for 20 minutes. Following this incubation, 5-10 ⁇ l of anti-p24 antibody (KC57-FITC; Coulter) was added and the cells were incubated at 4°C for 30 minutes. The cells were then washed twice with Wash Buffer and analyzed by flow cytometry.
  • a focused library containing GSEs for a particular target gene is produced using a collection of randomly fragmented full-length cDNA clones or overlapping EST clones obtained from various commercial sources that encode the target gene.
  • cDNA clones are produced from plasmids containing the target gene using the polymerase chain reaction. The cDNA clones are pooled into a mixture and fragmented with DNAse I using the method described in Example 1. Under these conditions, DNAse I is known to produce mostly double-stranded breaks.
  • the resulting fragments are repaired with T4 polymerase and ligated to a synthetic double-stranded adaptor, such as the adaptor prepared from the oligonucleotides Fse-Top-Fse (5'-G-G-C-C-G-G-C-C-T-A-T-A-T-T-T-A-A-G-A-G- G-C-C-G-G-C-C-3'; SEQ ID NO: 11) and Fse-Bot-Fse (5'-G-G-C-C-G-G-C-C— T-C-T-T-A-A-T-A-T-A-T-A-T-A-G-G-C-C-G-G-C-C-3'; SEQ ID NO: 12).
  • Fse-Top-Fse 5'-G-G-C-C-G-G-C-C-T-A-T-A-T-A-T-A-T-A-T-
  • the mixture of fragments with adaptors on both ends is digested with Fse I, column purified, and then ligated into the Fse I site of the bicistronic retroviral vector, pLXEMCVNgfr.
  • the ligation mix is then transfomied into competent cells.
  • the resulting plasmid preparation is used for selection.
  • the orientation of the cDNA inserts is maintained by digestion with Xho I and Sse8387 I and ligation into the Sse8387 URsr H site of pLXEMCVNgfr. Neither Rsr H nor Sse83871 cleaves between the cDNA insert and an Fse I site.

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Abstract

The invention provides methods for identifying human cellular genes and their encoded for use as targets in the design of therapeutic agents for suppressing human immunodeficiency virus (HIV) infection. The invention also provides mehtods for identifying protective compounds that inhibit HIV infection. The invention further provides compounds for use in the treatment or prevention of HIV.

Description

COMPOSITIONS AND METHODS FOR INHIBITING HUMAN IMMUNODEFICIENCY VIRUS INFECTION BY DOWN-REGULATING
HUMAN CELLULAR GENES
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to methods for identifying human cellular genes and their encoded products for use as targets in the design of therapeutic agents for suppressing human immunodeficiency virus (HIV) infection. In particular, the invention relates to methods for identifying biochemical pathways, substrates and metabolic products of said pathways, and enzymes that mediate conversion of substrates into metabolic products, wherein said pathways comprise one or a plurality of targets for the design of preventative and therapeutic agents for preventing, inhibiting, suppressing, or immunizing against infection of naϊve cells with HIV or the production of infectious virus from infected cells. The invention also relates to methods for identifying protective compounds that inhibit HIN infection. The invention further relates to compounds for use in the treatment or prevention of HIN.
2. Background of the Invention
The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al., 1983, Science 220:868-70; Gallo et al., 1984, Science 224:500-03). HIN causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in the depletion of these cells. This, in turn, leads to opportunistic infections, neoplastic growth, and death.
HIN is a member of the lentivirus family of retroviruses (Teich et al, 1984, in SNA Tumor Viruses 949-56 (Weiss et al., eds., CSH-Press)). Retroviruses are small, enveloped viruses that contain a diploid, single-stranded RΝA genome, and replicate via a DΝA intermediate produced by a virally encoded reverse transcriptase, an RΝA-dependent DΝA polymerase (Narmus, 1988, Science 240:1427-39). There are at least two distinct subtypes of HIN: HIV-1 (Barre-Sinoussi et al., 1983; Gallo et al, 1984) and HIV-2 (Clavel et al, 1986, Science 233:343-46; Guyader et al, 1987, Nature 326:662-69). Genetic heterogeneity exists within each of these HIN subtypes. CD4+ T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al, 1984, Nature 312:763-67; Klatzmann et al, 1984, Nature 312:767-68; Maddon et al, 1986, Cell 47:333-48). Niral entry into cells is dependent upon the binding of viral protein gpl20 to the cellular CD4 receptor molecule (McDougal et al, 1986, Science 231:382-85; Maddon et al, 1986, Cell 47:333-48).
HIV infection is pandemic and HIV-associated diseases have become a worldwide health problem. Despite considerable efforts in the design of anti-HIV modalities, there is, thus far, no successful prophylactic or therapeutic regimen against AIDS. However, several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al, 1991, FASEB J. 5:2369-81).
For example, virally encoded reverse transcriptase has been a major focus of drug development. A number of reverse transcriptase-targeted drugs, including dideoxynucleotide analogs such as AZT, ddl, ddC, and ddT have been shown to be active against HIV (Mitsuya et al, 1990, Science 249:1533-44). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al, 1989, Science 243:1731- 34). h addition, these drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
Another stage of the HIV life cycle that has been targeted is viral entry into cells, the earliest stage of HIV infection. This approach has primarily utilized recombinant soluble CD4 protein to inhibit infection of CD4+ T cells by some HIV-1 strains (Smith et al, 1987, Science 238:1704-07). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:6574-79). To date, clinical trials of recombinant, soluble CD4 have produced inconclusive results (Yarchoan et al, 1989, Proc. Vth Int. Conf. on AIDS 564, MCP 137; Schooley et al, 1990, Ann. Int. Med. 112:247-53; Kahn et al, 1990, Ann. Int. Med. 112:254-61; Daar et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:6574; Langner et al, 1993, Arch. Virol. 130:157-70; Schacker et al, 1994, J. Infect Dis. 169:37-40; Shacker et al, 1995, J. Acquir. Immune Defic. Syndr. Hum. Retrovirol 9:145-52).
Yet another stage of the HIV life cycle that has been targeted is the integration ofthe proviral DΝA into the host genome. The viral enzyme integrase catalyzes the process of integration, and inhibitors of integrase have been reported (d'Angelo et al, 2001, Pathol Biol. 49:237-46; Farnet et al, 1996, AIDS 10 Supp. A:S3-11).
Additionally, the later stages of HIV replication (which involve crucial virus-specific processing of certain viral proteins and enzymes) have been targeted for anti-HIV drug development. Late-stage processing is dependent on the activity of a virally encoded protease, and drugs including saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al, 1993, Persp. Drug Discov. Design 1:69-83). With this class of drugs, the emergence of drug resistant HIV mutants is also a problem; resistance to one inhibitor often confers cross-resistance to other protease inhibitors (Condra et al, 1995, Nature 374:569-71). Also, these drugs often exhibit toxic side effects such as nausea, altered taste, circumoral parethesias, development of fat deposits, diarrhea, and nephrolithiasis. Antiviral therapy of HIV using different combinations of nucleoside analogs and protease inhibitors have recently been shown to be more effective than the use of a single drug alone (Torres et al, 1997, Infec. Med. 14:142-60). However, despite the ability to achieve significant decreases in viral burden, there is no evidence to date that combinations of available drugs will afford a curative treatment for AIDS.
Other potential approaches for developing treatment for AIDS include the delivery of exogenous genes into infected cells. One such gene therapy approach involves the use of genetically engineered viral vectors to introduce toxic gene products to kill HIV-infected cells. Another form of gene therapy is designed to protect virally infected cells from cytolysis by specifically disrupting viral replication. Stable expression of RNA-based HIV-1 antiviral agents (e.g., decoys, antisense, or ribozymes) or protein-based HIV-1 antiviral agents (e.g., transdominant mutants) can inhibit certain stages of the viral life cycle. A number of anti-HIV suppressors have been reported, such as decoy RNA of TAR or RRE (Sullenger et al, 1990, Cell 63:601-08; Sullenger et al, 1991, J. Virol. 65:6811- 16; Lisziewicz et al, 1993, New Biol. 3:82-89; Lee et al, 1994, J. Virol. 68:8254- 64), ribozymes (Sarver et al, 1990, Science 247:1222-25; Wecrasinghe et al, 1991, J. Virol. 65:5531-34; Dropulic et al, 1992, J. Virol. 66:1432-41; Ojwang et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10802-06; Yu et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6340-44; Yu et al, 1995, Proc. Natl. Acad. Sci. U.S.A. 92:699-703; Yamada et al, 1994, Gene Therapy 1:38-45), antisense RNA complementary to the mRNA of viral gag, tat, rev, or env genes (Sezakiel et al,
1991, J. Virol. 65:468-72; Chatterjee et al, 1992, Science 258:1485-88; Rhodes et al, 1990, J. Gen. Virol. 71:1965; Rhodes et al, 1991, AIDS 5:145-51; Sezakiel et al, 1992, J. Virol. 66:5576-81; Joshi et al, 1991, J. Virol. 65:5524-30) and rransdominant mutants of Rev (Bevec et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:9870-74), Tat (Pearson et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:5079-83; Modesti et al, 1991, New Biol. 3:759-68), Gag (Trono et al, 1989, Cell 59:113- 20), Env (Bushschacher et al, 1995, J. Virol. 69:1344-48) and protease (Junker et al, 1996, J. Virol. 70:7765-72).
Antisense polynucleotides have been designed to complex with and sequester HIV-1 transcripts (International Pub. Νos. WO 93/11230 and WO 94/10302; European Patent Pub. No. EP 0 594 881; Chatterjee et al, 1992, Science 258:1485). Furthermore, enzymatically active RNAs (i.e., ribozymes) have been used to cleave viral transcripts. The use of a ribozyme to generate resistance to HIV-1 in a hematopoietic cell line has been reported (Ojwang et al,
1992, Proc. Natl. Acad. Sci. U.S.A. 89:10802-06; Yamada et al, 1994, Gene Therapy 1:38-45; International Pub. Nos. WO 94/26877 and WO 95/13379). In preclinical studies, RevMlO, a rransdominant Rev protein, has been transfected ex vivo into CD4+ cells of HIV-infected individuals and shown to confer survival advantage over cells transfected with vector alone (Woffendin et al, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2889-94).
However, despite enormous efforts in the art, reliable, curative anti-HIV therapeutic agents and regimens have not been developed.
In nature, evolution of an intracellular pathogen such as HIV requires the development of interactions of its genes and gene products with multiple cellular components. For instance, the interactions of a virus with a host cell involves the binding of the virus to specific cellular receptors, translocation through the cellular membrane, uncoating, replication ofthe viral genome, and transcription of the viral genes. Each of these events occurs in a cell and involves interactions with at least one cellular component. Thus, the life cycle of a virus can be completed only if the cell is "permissive" for viral infection. The availability of amino acids and nucleotides for replication of the viral genome and protein synthesis, the energy status of the cell, and the presence of cellular transcription factors and enzymes all contribute to the propagation of the virus in the cell. Consequently, the cellular components, in part, determine host cell susceptibility to infection and can be used as potential targets for the development of new therapeutic interventions, the case of HIN, one cellular component that has been used towards this end is the cell surface molecule for HIV attachment, CD4.
Recently, it was reported that HIV entry into a susceptible cell requires the expression of a second type of receptor, the chemokine receptors (CCR2, CCR3, CCR5, or CXCR4), in addition to CD4 (Moore, 1997, Science 276:51-52). A chemokine receptor normally binds RAΝTES, MLP-lα, or MlP-lβ as its natural ligand. hi the case of HIN infection, it has been proposed that CD4 first binds to the HIV gpl20 protein on the cell surface followed by binding of this complex to a chemokine receptor, resulting in viral entry into the cell (Cohen, 1997, Science 275:1261). Therefore, chemokine receptors can present an additional cellular target for the design of HIV therapeutic agents. Inhibitors of HlV/chemokine receptor interactions are being tested as anti-HIV agents.
Thus, there remains a need for the discovery of additional cellular targets for the design of anti-HIV therapeutics, particularly intracellular targets for disrupting viral replication after viral entry into a cell. There also remains a need in the art to isolate and identify human cellular genes that encode products that are necessary for productive HIV infection for use as targets in the design of therapeutic agents for suppressing HIV infection. There further remains a need in the art to identify the biological pathways comprising the products of such cellular genes. There still further remains a need in the art to isolate and identify additional human cellular genes that encode products comprising other members of such biological pathways for use as targets in the design of therapeutic agents for suppressing HIV infection. The identification of human cellular genes that encode products that are necessary for productive HIV infection, biological pathways comprising the products of such cellular genes, and additional human cellular genes that encode products comprising other members of such biological pathways, would allow for the identification of novel protective compounds that inhibit, suppress, or otherwise interfere with HIV infection. SUMMARY OF THE INVENTION
The invention relates to methods for identifying human cellular genes that encode products that are necessary for productive HIV infection for use as targets in the design of therapeutic agents for suppressing HIV infection. The invention also relates to methods for identifying biological pathways comprising the products of such cellular genes, as well as substrates and metabolic products of said pathways. The invention further relates to methods for identifying additional human cellular genes that encode products comprising other members of such biological pathways for use as targets in the design of therapeutic agents for suppressing HIV infection.
The invention also relates to methods for identifying protective compounds that inhibit HIV infection. The invention further relates to compounds for use in the treatment or prevention of HIV.
In one embodiment of the methods of the invention, a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a population of human host cells by introducing the genetic suppressor element library into the population of human host cells; infecting the population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene that corresponds to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first member ofthe biological pathway.
In another embodiment of the methods of the invention, an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, is identified by first identifying the member of the biological pathway. In this method, the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; and determining the human cellular gene corresponding to the genetic suppressor element. The inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression ofthe human cellular gene. h yet another embodiment of the methods of the invention, an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, is identified by first identifying the member of the biological pathway. In this method, the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element; and determining the polypeptide encoded by the human cellular gene. The inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression of the member of the biological pathway.
In yet another embodiment of the methods of the invention, an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, is identified by first identifying the member of the biological pathway. In this method, the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIN infection to yield DΝA fragments; transferring the DΝA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DΝA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DΝA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element; and determining the polypeptide encoded by the human cellular gene. The inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases biochemical activity of the member of the biological pathway. In yet another embodiment of the methods of the invention, an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the member of the biological pathway. In this method, the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; and determining the human cellular gene corresponding to the genetic suppressor element. The inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression ofthe human cellular gene.
In yet another embodiment ofthe methods of the invention, an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the member of the biological pathway. In this method, the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible, to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element; and determining the polypeptide encoded by the human cellular gene. The inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases expression of the member of the biological pathway. In yet another embodiment ofthe methods ofthe invention, an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the member of the biological pathway. In this method, the member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIN infection to yield DΝA fragments; transferring the DΝA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DΝA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DΝA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIN; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element; and determining the polypeptide encoded by the human cellular gene. The inhibitor of the member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the polypeptide encoded by the human cellular gene in the second population of human host cells; and determining whether the test compound decreases biochemical activity of the member of the biological pathway.
In yet another embodiment of the methods of the invention, an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, is identified by first identifying the second member of the biological pathway. In this method, the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; determining the first member of the biological pathway; and determining the human cellular gene that encodes the first member of the biological pathway. The inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression ofthe human cellular gene encoding the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases expression of the human cellular gene encoding the first member ofthe biological pathway. In yet another embodiment ofthe methods of this invention, an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, is identified by first identifying the second member of the biological pathway. In this method, the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first member of the biological pathway. The inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases expression ofthe first member ofthe biological pathway.
In yet another embodiment of the methods of the invention, an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, is identified by first identifying the second member of the biological pathway, i this method, the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first member of the biological pathway. The inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases biochemical activity ofthe first member ofthe biological pathway.
In yet another embodiment of the methods of the invention, an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the second member of the biological pathway. In this method, the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; determining the first member of the biological pathway; and determining the human cellular gene that encodes the first member of the biological pathway. The inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the human cellular gene encoding the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases expression of the human cellular gene encoding the first member ofthe biological pathway. yet another embodiment of the methods of the invention, an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the second member of the biological pathway. In this method, the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first member of the biological pathway. The inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the expression of the first member ofthe biological pathway in the second population of human host cells; and determining whether the test compound decreases expression ofthe first member ofthe biological pathway. h yet another embodiment of the methods of the invention, an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, and wherein the inhibitor confers resistance to HIV infection, is identified by first identifying the second member of the biological pathway. In this method, the second member of the biological pathway is identified by synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; infecting the first population of human host cells with HIV; isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the first population of human host cells; recovering the genetic suppressor element from the isolated genetically modified human host cell; determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway; determining the biological pathway of the second member; and determining the first member of the biological pathway. The inhibitor of the first member of the biological pathway is then identified by exposing a second population of human host cells to a test compound; measuring the activity of the first member of the biological pathway in the second population of human host cells; and determining whether the test compound decreases biochemical activity ofthe first member ofthe biological pathway. Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention includes methods for identifying human cellular genes that encode products that are necessary for productive HIV infection for use as targets in the design of therapeutic agents for suppressing HIV infection. The invention also includes methods for identifying biological pathways comprising the products of such cellular genes. The invention further includes methods for identifying additional human cellular genes that encode products comprising other members of such biological pathways for use as targets in the design of therapeutic agents for suppressing HIV infection.
The invention also includes methods for identifying protective compounds that inhibit HIV infection. The invention further includes compounds for use in the treatment or prevention of HIN. Such compounds include chemical compounds and biological compounds. Chemical compounds and biological compounds include any chemical or biological compound that disrupts or inhibits one or more biological functions required for mediation or replication of HIV. Preferred chemical compounds include small molecule inhibitor or substrate compounds, such as products of chemical combinatorial libraries. Preferred biological compounds include peptides, antisense molecules, and antibodies. These embodiments are discussed in more detail below.
The invention is based, in part, on the Applicants' discovery that certain nucleic acid molecules - termed genetic suppressor elements (GSEs) - which are isolated from human cells and which correspond to fragments of certain human cellular genes, prevent activation of latent HIN-1 in a CD4+ cell line as well as productive HIN infection in such cells, that regard, any cellular or viral marker associated with HIN infection can be used to select for such nucleic acid molecules. An example of such a marker is CD4, which is conveniently monitored by using a specific antibody. Additional markers include virus-specific gene products, such as gpl20 and p24. GSEs having the ability to inhibit HIV infection function can be isolated that are functional in either the sense orientation (encoding peptides) or in the antisense orientation (encoding antisense RNAs). Both types of GSEs are believed to down-regulate the corresponding cellular gene from which they were derived - each type of GSE utilizing a different mechanism. The corresponding cellular gene is referred to herein as a "target gene" and its product is referred to as a "target product." The term "target" can also be used generically to describe either the gene or its encoded product. Sense-oriented GSEs exert their effects as transdominant mutants or RNA decoys. Transdominant mutants are expressed proteins or peptides that competitively inhibit the normal function of a wild-type protein in a dominant fashion. RNA decoys are protein binding sites that titrate out the wild-type protein. Antisense-oriented GSEs exert their effects as antisense RNA molecules, i.e., nucleic acid molecules complementary to the mRNA of the target gene. These nucleic acid molecules bind to mRNA and block the translation of the mRNA. In addition, some antisense nucleic acid molecules can act directly at the DNA level to inhibit transcription. h one embodiment of the invention, the down-regulation of the concentration (expression, level, amount) or activity (function, biological activity) of a target gene or target product by a GSE depletes a cellular component required for progression through the HIV life cycle resulting in an inhibition of HIV infection. In another embodiment of the invention, the down-regulation of the concentration or activity of a target gene or target product by a GSE depletes a cellular component that interacts with one or more other human cellular genes or gene products that are required for progression through the HIV life cycle, resulting in an inhibition of HIV infection. In a preferred embodiment of the invention, at least two human cellular genes are members of the same biological pathway and one human cellular gene or gene product regulates the expression or activity of the other human cellular gene or gene product, h another preferred embodiment of the invention, at least two human cellular genes are members of the same biological pathway and the polypeptide encoded by one human cellular gene is a product of a biochemical reaction mediated by the polypeptide encoded by the other human cellular gene. In still another preferred embodiment of the invention, the at least two human cellular genes are members of the same biological pathway and the polypeptide encoded by one human cellular gene is a substrate of a biochemical reaction mediated by the polypeptide encoded by the other human cellular gene. In another embodiment, the at least two human cellular genes encode polypeptides that are isozymes of each other. In a preferred embodiment, at least one ofthe human cellular genes encodes an enzyme.
In one embodiment of the invention, the target gene encodes a target product that is a member of the glycolysis pathway of the host cell (as shown in BIOCHEMICAL PATHWAYS, (G. Michal, ed.), J. Wiley & Sons, Inc.: New York, 1999, pp. 27-34). Preferred target products include the following members of the glycolysis pathway: enolase 1 (ENO1); enolase 2 (ENO2); enolase 3 (ENO3); fructose- 1,6-bisphosphatase 1 (FBPl); fructose bisphosphate aldolase A (ALDOA); glucosamine-6-phosphate deaminase (GNPDA); glucose transporter-3 (GLUT3); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH); Na+ D-glucose cotransport regulator; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K); phosphofructokinase (PFK); phosphoglucomutase (PGM); phosphorylase kinase, alpha-2 (PHKA2); and thyroid hormone binding protein/pyruvate kinase (TCBA). These genes and their public database accession numbers are shown in Table 1.
Table 1 Cellular Gene Targets in the Glycolysis Pathway for HIV
Inhibition
Figure imgf000019_0001
Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. Enolase 1 is one of three enolase isoenzymes found in mammals; it encodes alpha-enolase, a homodimeric soluble enzyme, and also encodes a shorter monomeric structural lens protein, tau-crystallin. The two proteins are made from the same message. The full length protein, the isoenzyme, is found in the cytoplasm. The shorter protein is produced from an alternative translation start, is localized to the nucleus, and has been found to bind to an element in the c-myc promoter. Enolase 2 is a homodimer, is found in mature neurons and cells of neuronal origin. A switch from alpha enolase to gamma enolase occurs in neural tissue during development in rats and primates. Enolase 3, also a homodimer, is found in skeletal muscle cells in the adult. A switch from alpha enolase to beta enolase occurs in muscle tissue during development in rodents. Mutations in this gene can be associated with metabolic myopathies that may result from decreased stability ofthe enzyme. Fructose- 1,6-bisphosphatase 1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. Fructose- 1,6-diphosphatase deficiency is associated with hypoglycemia and metabolic acidosis.
Fructose-bisphosphate aldolase (aldolase A) is a glycolytic enzyme that catalyzes the reversible conversion of fructose- 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Three aldolase isozymes (A, B, and C), encoded by three different genes, are differentially expressed during development. Aldolase A is found in the developing embryo and is produced in even greater amounts in adult muscle. Aldolase A expression is repressed in adult liver, kidney and intestine and similar to aldolase C levels in brain and other nervous tissue. Aldolase A deficiency has been associated with myopathy and hemolytic anemia. Alternative splicing of this gene results in multiple transcript variants which encode the same protein.
Glucosamine-6 phosphate deaminase (GNPDA) catalyzes a reversible reaction of glucosamine-6-phosphate and water to form fructose 6-phosphate and ammonium.
Glucose transporter-3 (GLUT3) is a facilitative glucose transporter responsible for maintaining an adequate glucose supply to neurons, although it is also found in other tissues. Facilitative glucose carriers accelerate the transport of glucose down its concentration gradient by facilitative diffusion, a form of passive transport. Facilitative glucose carriers are expressed by most if not all cells. The facilitative glucose-transporter isoforms have distinct tissue distributions and biochemical properties and contribute to the precise disposal of glucose under varying physiological conditions.
Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3 -phosphate to 1,3-bisphosphoglycerate.
Na+ D-glucose cotransport regulator assists in the provision of the starting product, glucose. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K) is a bifunctional enzyme that catalyses both the synthesis and the degradation of fructose-2,6-bisphosphate [Fru(2,6)R2], a potent stimulator of 6-phosphofructo-l- kinase and inhibitor of fructose 1,6-bisphosphatase. PF2K can be considered to be either a glycolytic or a gluconeogenic enzyme; it acts as a switch between glycolysis and gluconeogenesis in mammalian liver by regulating the level of Fru(2,6)R2.
The second step in the glycolytic pathway is the conversion of fructose-6- phosphate to fructose- 1,6-diphosphate by phosphofructokinase. Phosphofructokinase (PFK) utilizes ATP to catalyze the transfer of phosphate to the 1 -position of fructose-6-phosphate.
Phosphoglucomutase (PGM) catalyzes the interconversion of glucosamine-6-phosphate and glucosamine-1 phosphate. In cells of prokaryotic and eukaryotic organisms, PGM, a phosphoenzyme, catalyzes an important trafficking point in carbohydrate metabolism. In one direction, glucosamine-1 - phosphate produced from sugar catabolism is converted to glucoasmine-6- phosphate, the first intermediate in glycolysis. In the other direction, conversion of glucosamine-6-phosphate to glucosamine-1 -phosphate provides a substrate for synthesis of UDP-Glucosamine, which is required for synthesis of a variety of cellular constituents, including cell wall polymers and glycoproteins. Phosphorylase kinase participates in a signaling pathway resulting in the breakdown of glycogen to glucose 1 -phosphate and its subsequent metabolism via glycolysis. cAMP, generated in response to varied stimuli, activates A-kinase, which in turn phosphorylates phosphorylase kinase. Now in its active, phosphorylated state, phosphorylase kinase catalyzes the phosphorylation of glycogen phosphorylase, thereby activating it to release glucose molecules from glycogen for use in the glycolysis pathway.
Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate to pyruvate. It will be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, or reagents described herein, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention that will be limited only by the appended claims. All technical and scientific terms used herein have- the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
As used herein, the term "HIV infection" refers to the ability of HIV to enter a host cell and/or replicate in the host cell. As used herein, the term "HIV cell entry" refers to the ability of a virus to enter a cell through binding of a virus to a cell and the fusion of the virus to the cell. HIV or virus binding refers to the attachment of a virus to the surface of a cell. HIV or virus fusion refers to the passage of a virus across the cell plasma membrane into the cell.
As used herein, the term "isolated nucleic acid molecule" refers to a nucleic acid molecule that has been removed from its natural milieu (i.e., a molecule that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. An isolated nucleic acid molecule can be isolated from its natural source or can be produced using recombinant DNA technology (e.g., polymerase chain reaction amplification) or chemical synthesis. Isolated nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to promote HIV infection or inhibit HIV infection.
It should also be appreciated that reference to an isolated nucleic acid molecule does not necessarily reflect the extent of purity of the nucleic acid molecule. Nucleic acid molecules can be isolated and obtained in substantial purity, generally as other than an intact chromosome. Usually, the nucleic acid molecule will be obtained substantially free of other nucleic acid sequences, generally being at least about 50%, and usually at least about 90% pure. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably.
According to the invention, reference to an "isolated nucleic acid molecule" refers to a nucleic acid molecule that is the size of or smaller than a gene. Thus, an isolated nucleic acid molecule does not encompass isolated genomic DNA or an isolated chromosome. The term isolated nucleic acid molecule does not connote any specific minimum length unless set forth by reference to a minimum number of nucleotides or by a function of the nucleic acid molecule. As used herein, the term "gene" has the meaning that is well known in the art, that is, a nucleic acid sequence that includes the translated sequences that code for a protein ("exons") and the untranslated intervening sequences ("introns"), and any regulatory elements ordinarily necessary to transcribe and/or translate the protein. Included in the invention are nucleic acid molecules that are less than a full-length gene or less than a full-length coding sequence, such as fragments of a gene or coding sequence comprising, consisting essentially of, or consisting of a GSE of the present invention. A coding sequence can include genomic DNA without introns, cDNA or RNA that encodes a protein. An isolated nucleic acid molecule can also include a specified nucleic acid sequence flanked by (i.e., at the 5' and/or the 3' end ofthe sequence) additional nucleic acids that do not normally flank the specified nucleic acid sequence in nature (i.e., are heterologous sequences).
Preferably, an isolated nucleic acid molecule of the present invention is produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. A nucleic acid molecule homologue (i.e., a nucleic acid molecule that differs from the reference nucleic acid molecule by one or more insertions, deletions, substitutions, and/or inversions such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a protein or peptide useful in the present invention or to form stable hybrids under stringent conditions with natural gene isolates) can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibid.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant DNA techniques, such as site- directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, PCR amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid and/or by hybridization with a wild-type gene.
The minimum size of a nucleic acid molecule of the present invention is a size sufficient to encode a protein having the desired biological activity, sufficient to have an effect on the expression or biological activity of a cellular gene or protein as discussed herein or to have an effect on HIV inhibitory activity as described herein (e.g., as in a GSE), or sufficient to form a probe or oligonucleotide primer that is capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule. As such, the size of a nucleic acid molecule of the present invention can be dependent on nucleic acid composition and percent homology or identity between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration) and the intended use of the nucleic acid molecule. The minimal size of a nucleic acid molecule that is used as an oligonucleotide primer or as a probe is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 18 bases in length if they are AT-rich. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule of the present invention, in that the nucleic acid molecule can include a fragment of a gene, a portion of a protein encoding sequence, or a nucleic acid sequence encoding a full-length protein (including a complete gene).
One embodiment of the present invention includes a recombinant nucleic acid molecule comprising a recombinant vector and a nucleic acid molecule comprising a nucleic acid sequence encoding a cellular gene or fragment thereof as described herein. According to the present invention, a recombinant vector is an engineered (i.e., artificially produced) nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice and for introducing such a nucleic acid sequence into a host cell. The recombinant vector is therefore suitable for use in cloning, sequencing, and/or otherwise manipulating the nucleic acid sequence of choice, such as by expressing and/or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid sequence to be cloned or delivered, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid molecules ofthe present invention or which are useful for expression of the nucleic acid molecules of the present invention (discussed in detail below). The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome of a recombinant organism (e.g., a microbe or a plant). The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention. The integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies ofthe nucleic acid molecule can be integrated into the chromosome. A recombinant vector ofthe present invention can contain at least one selectable marker.
In one embodiment, a recombinant vector used in a recombinant nucleic acid molecule of the present invention is an expression vector. As used herein, the phrase "expression vector" is used to refer to a vector that is suitable for production of an encoded product (e.g., a protein of interest). In this embodiment, a nucleic acid sequence encoding the product to be produced is inserted into the recombinant vector to produce a recombinant nucleic acid molecule. The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector which enable the transcription and translation of the nucleic acid sequence within the recombinant host cell. In another embodiment, a recombinant vector used in a recombinant nucleic acid molecule of the present invention is a targeting vector. As used herein, the phrase "targeting vector" is used to refer to a vector that is used to deliver a particular nucleic acid molecule into a recombinant host cell, wherein the nucleic acid molecule is used to delete or inactivate an endogenous gene within the host cell or microorganism (i.e., used for targeted gene disruption or knock-out technology). Such a vector may also be known in the art as a "knock-out" vector. In one aspect of this embodiment, a portion of the vector, but more typically, the nucleic acid molecule inserted into the vector (i.e., the insert), has a nucleic acid sequence that is homologous to a nucleic acid sequence of a target gene in the host cell (i.e., a gene which is targeted to be deleted or inactivated). The nucleic acid sequence of the vector insert is designed to bind to the target gene such that the target gene and the insert undergo homologous recombination, whereby the endogenous target gene is deleted, inactivated or attenuated (i.e., by at least a portion ofthe endogenous target gene being mutated or deleted).
Typically, a recombinant nucleic acid molecule includes at least one nucleic acid molecule of the present invention operatively linked to one or more expression control sequences, including transcription control sequences and translation control sequences. As used herein, the phrase "recombinant molecule" or "recombinant nucleic acid molecule" primarily refers to a nucleic acid molecule or nucleic acid sequence operatively linked to an expression control sequence, but can be used interchangeably with the phrase "nucleic acid molecule", when such nucleic acid molecule is a recombinant molecule as discussed herein. According to the present invention, the phrase "operatively linked" refers to linking a nucleic acid molecule to an expression control sequence (e.g., a transcription control sequence and/or a translation control sequence) in a manner such that the molecule is able to be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell. Transcription control sequences are sequences which control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable expression control sequences include any expression control sequence that can function in a host cell or organism into which the recombinant nucleic acid molecule is to be introduced.
According to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells or plants. In microbial systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection." However, in animal cells, transformation has acquired a second meaning which can refer to changes in the growth properties of cells in culture (described above) after they become cancerous, for example. Therefore, to avoid confusion, the term "transfection" is preferably used with regard to the introduction of exogenous nucleic acids into animal cells, including human cells, and is used herein to generally encompass transfection of animal cells and transformation of plant cells and microbial cells, to the extent that the terms pertain to the introduction of exogenous nucleic acids into a cell. Therefore, transfection techniques include, but are not limited to, transformation, chemical treatment of cells, particle bombardment, electroporation, microinjection, hpofection, adsorption, infection and protoplast fusion.
A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules operatively linked to an expression vector containing one or more expression control sequences.
"Hybridization" has the meaning that is well known in the art, that is, the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain some regions of mismatch. As used herein, reference to hybridization conditions refers to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety. "Stringent hybridization" has a meaning well-established in the art, that is, hybridization performed at a salt concentration of no more than 1M and a temperature of at least 25 degrees Celsius. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM Sodium Phosphate, 5 mM EDTA, pH 7.4) and a temperature of 55 degrees to 60 degrees Celsius are suitable. For example, in one embodiment, "moderately stringent conditions" can be defined as hybridizations carried out as described above, followed by washing in 0.2X SSC and 0.1% SDS at 42 degrees Celsius (Ausubel et al, 1989, Current Protocols for Molecular Biology, ibid.). More particularly, moderate stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides). High stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides). Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 90%o nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). As discussed above, one of skill in the art can use the formulae in Meinkoth et al., ibid, to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10 °C less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature of between about 20 °C and about 35 °C (low stringency), more preferably, between about 28°C and about 42°C (more stringent), and even more preferably, between about 35 °C and about 45 °C (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na+) at a temperature of between about 30°C and about 45 °C, more preferably, between about 38 °C and about 50°C, and even more preferably, between about 45 °C and about 55 °C, with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%). Alternatively, Tm can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions can include a combination of salt and temperature conditions that are approximately 20-25 °C below the calculated Tm of a particular hybrid, and wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20°C below the calculated Tm of the particular hybrid. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37°C in about 0.1X-0.5X SSC, followed by at least one wash at about 68°C in about 0.1X-0.5X SSC). h one embodiment of the present invention, any amino acid sequence described herein can be produced with from at least one, and up to about 20, additional heterologous amino acids flanking each of the C- and/or N-terminal ends of the specified amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of the specified amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the specified amino acid sequence, or that are not related to the function of the specified amino acid sequence, or that would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the specified amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase "consisting essentially of, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a specified amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the specified amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the specified amino acid sequence as it occurs in the natural gene or do not encode a protein that imparts any additional function to the protein or changes the function of the protein having the specified amino acid sequence.
The methods of the present invention can include the steps of identifying cellular targets (genes and their encoded products) that are in a biological pathway that is necessary for productive HIV infection. Such methods include the steps of: (a) synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; (b) transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; (c) genetically modifying a population of human host cells by introducing the genetic suppressor element library into the population of human host cells; (d) infecting the population of human host cells with HIV; (e) isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIV infection from the population of human host cells; (f) recovering the genetic suppressor element from the isolated genetically modified human host cell; and (g) determining the human cellular gene that corresponds to the genetic suppressor element, wherein the human cellular gene encodes the second member of the biological pathway. A cell-derived library (e.g., an RFE library) can be constructed from nucleic acid molecules of any mammalian cells, and preferably from cDNA of HTV-susceptible cells. In that regard, Example 1 demonstrates that GSEs can be selected from HL-60 cells that are naturally susceptible to HIV infection, from HeLa cells which are not naturally susceptible to HIV infection due to the lack of CD4 expression, and from peripheral blood mononuclear cells (PBMCs). It has been shown that expression of CD4 on the surface of HeLa cells by means of a retroviral vector renders the cells susceptible to HIV infection. Therefore, cell types not normally susceptible to HIV infection can still be useful as a source of genetic material for the construction of RFE libraries. It is also preferred that a normalized cDNA library is prepared (Gudkov and Roninson, 1996, Methods in Molecular Biology 69:229-231). Briefly, in one embodiment, DNA is first treated with enzymes to produce randomly cleaved fragments. This can be conveniently performed by DNase I cleavage in the presence of Mn++ (Roninson et al, U.S. Patent No. 5,217,889, column 5, lines 5-20). Thereafter, the randomly-cleaved DNA is size fractionated by gel electrophoresis. Fragments of between 100 and 700 bp are the preferred lengths for constructing RFE libraries. Single strand breaks of the size-selected fragments are repaired by methods well known in the art. The fragments are ligated with 5' and 3' adaptors, which are selected to have non-cohesive restriction sites so that each fragment can be inserted into an expression vector in an oriented fashion. Further, the 5' adaptor contains a start (ATG) codon to allow the translation of the fragments which contain an open reading frame in the correct phase. The fragments are then inserted into appropriate expression vectors. Any expression vector that results in efficient expression of the fragments in host cells can be used. In a preferred embodiment viral-based vectors such as the retroviral vectors LNCX (Miller and Rosman, 1989, BioTechniques 7:980) and LNGFRM are exemplified. Alternatively, adenovirus, adeno-associated virus and herpes virus vectors can also be used for this purpose.
When viral-based vectors are used, the ligated vectors are first transfected into a packaging cell line to produce viral particles. For retroviral vectors, any amphotropic packaging line such as PA317 (Miller and Buttimore, 1986, Mol. Cell. Biol. 6:2895-2902; ATCC CRL #9078) can be used to efficiently produce virus. In a preferred embodiment of the invention, the viral vector also contains a selectable gene, such as the neo r gene or a truncated nerve growth factor receptor (NGFR) gene, which allows isolation ofthe cells that contain the vector.
The number of independent clones present in each RFE expression library can vary. In a preferred embodiment, libraries of cell-derived cDNA of about 10° to 10 independent clones can be used. hi a specific embodiment illustrated by way of example in Example 2, OM10.1 cells are used to select for GSEs, and are maintained in conventional tissue culture as described in Butera, U.S. Patent No. 5,256,534. The purpose of using OM10.1 cells for the selection of GSEs is that they contain a latent HIV-1 provirus which is inducible by TNF-α. Example 2 also illustrates the use of CEM- ss cells to select for GSEs. Other cell lines can be similarly engineered with an inducible HIV provirus. Examples of cell lines that are infected with latent HIV include, but are not limited to, Ul, U33, 8E5, ACH-2, LL58, THP/HIV and UHC4 (Bednarik and Folks, 1992, AIDS 6:3-16). A variety of agents have been shown to be capable of inducing latent HlV-infected cells, and these include TNF-α, TNF- β, interleukins-1, -2, -3, -4 and -6, granulocyte-macrophage colony stimulating factors, macrophage-colony stimulating factors, interferon-α, transforming growth factor-β, PMA, retinoic acid and vitamin D3 (Poli and Fauci, 1992, AIDS Res. Human Retroviruses 9:191-197). Alternatively, GSEs can be selected on the basis of their ability to directly protect HlV-susceptible cells from HIV infection or inhibit the entry of HIV into cells using methods described herein.
The cell-derived RFE library can be introduced into latently HIN-infected cells or HIN-susceptible cells by any technique well known in the art that is appropriate to the vector system employed. In one embodiment of the invention, the viral vector also contains a selectable marker in addition to a random fragment of cellular DΝA. A suitable marker, by way of example only, is the neo r gene, which permits selection of cells containing RFE library members using the drug G-418. hi a preferred embodiment, the viral vector contains a truncated low affinity nerve growth factor receptor (ΝGFR) that permits selection of the cells using an anti-ΝGFR monoclonal antibody, hi alternative embodiments, the multiplicity of infection of the virions of the library is adjusted so that preselection for cells that are transduced by the vector is not needed. In the case of OM10.1 cells, by way of example, the transduced cell population can be treated with TNF-α for a period of between about 24-72 hours (preferably about 24 hours) according to the method of Butera. The activation of the latent HIV-1 provirus in OM10.1 can be detected by the suppression of the cell surface CD4. (Without being bound by theory, it is believed that viral protein gpl20 binds to CD4 in the cytoplasm, which prevents subsequent expression of CD4 on the cell surface.) Clones that are resistant to HIV replication continue to express cell surface CD4. Such clones can be selected, for example, by cell sorting using any antibody staining technique for CD4 and a fluorescence activated cell sorter (FACS).
The fraction of CD4+ cells that have been transduced with the RFE library can be compared with cells transduced with an expression library consisting ofthe vector only. An increased relative difference between the cell-derived RFE library and the control library can be found with each additional round of TNF-α induction. Thus, in the preferred embodiment of the invention there are at least two cycles of induction, selection and recloning before the GSEs are recovered from the cells for further characterization.
After selection, specific nucleic acid molecules corresponding to the GSEs can be recovered from cells that continue to express CD4 following induction of the latent HIV provirus by TNF-α. The specific GSEs are recovered from genomic DNA isolated from CD4+ cells sorted by FACS after TNF-α induction. The GSEs in this population are preferably recovered by PCR amplification using primers designed from the sequences ofthe vector.
The recovered GSEs can be introduced into an expression vector as discussed in the Examples section herein. The resultant GSEs expression library is known as a secondary library. The secondary library can utilize the same or a different vector from that used for the construction of the primary library. The secondary library can be transduced into another cell population and the resultant population selected, recloned and processed as described herein. Additionally, each individually recovered GSE can be inserted into cloning vectors for determining its specific nucleotide sequence and its orientation. The sequence of the GSE is then compared with sequences of known genes to determine the portion of the cellular gene with which it corresponds. Alternatively, the PCR products themselves can be directly sequenced to determine their nucleotide sequences. Concurrently, the isolated GSEs can be analyzed to determine their minimal core sequences. A core sequence is a common sequence found by comparison of GSEs with overlapping sequences. The GSEs are further tested for their ability to protect previously uninfected cells from HIN infection.
Once GSEs have been identified, the core sequence of the GSE is determined. This can be done by comparing overlapping sequences of independently derived GSEs. Alternatively, GSEs can be altered by additions, substitutions or deletions and assayed for retention of HIN-suppressive function. Alterations in the GSEs sequences can be generated using a variety of chemical and enzymatic methods which are well known to those skilled in the art. For example, oligonucleotide-directed mutagenesis can be employed to alter the GSE sequence in a defined way and/or to introduce restriction sites in specific regions within the sequence. Additionally, deletion mutants can be generated using DΝA nucleases such as Bal 31 or Exo in and SI nuclease. Progressively larger deletions in the GSE sequences can be generated by incubating the DΝA with nucleases for increased periods of time (see Ausubel, et al, ibid., for a review of mutagenesis techniques).
The altered sequences can be evaluated for their ability to suppress expression of HIV proteins such as p24 in appropriate host cells. It is within the scope of the present invention that any altered or shortened GSE nucleic acid molecules that retain their ability to suppress HIN infection can be incorporated into recombinant expression vectors for further use.
In order to confirm that the selected GSEs can protect uninfected cells from HIN infection, the GSEs can be transferred into latently HIN infected or into HIN-susceptible host cells followed by HIV infection, hi this connection, GSEs also can be directly selected from a RFE library for their ability to prevent productive infection by HIV. Protection experiments can be performed in any cell type that takes up the potential GSEs and that is otherwise susceptible to HIV infection, h a preferred embodiment by way of example, the CEM-ss cell line is used (Foley et al. 1965, Cancer 18:522-529). The use of CEM-ss cells as targets for quantitative infectivity of HIV-1 has been described by Νara & Fischinger (1988, Nature 322:469-470). Other cell lines that are susceptible to HIV infection include, but are not limited to, HUT-78, H9, Jurkat E6-1, A3.01, U-937, AA-2, HeLa CD4+ and C8166. In addition, freshly isolated peripheral blood leukocytes can be used.
The test of the potential GSEs can be performed using the same expression vector system as that employed in the RFE library trans duction of cells during initial selection steps. In other embodiments, the vector system can be modified to achieve higher levels of expression, e.g., the linkers can be employed to introduce a leader sequence that increases the translational efficiency of the message. One such sequence is disclosed by Kozak, 1994, Biochemie 76:815- 821. Another way of testing the effectiveness of a potential GSE against HIV infection is to determine how rapidly HIV-1 variants develop that can negate the effects of that element. Such a test includes infection of a culture of susceptible cells such as CEM-ss cells at a low multiplicity of infection and repeatedly assaying the culture to determine whether and how quickly HIV-1 infection becomes widespread. The range of useful multiplicities of infection is between about 100 to 1000 tissue culture infectious units (TCID50) per 10° CEM-ss cells. The TCID5o is determined by an endpoint method and is important for determining the input multiplicity of infection (moi).
A parameter that correlates with the development in the test culture of HIN-1 strains that are resistant to the effects of the potential GSEs is the fraction of cells that are infected in the culture. This fraction can be determined by immunofluorescent staining with an antibody specific for the HIV-1 p24 antigen of fixed permeabilized cells. Commercially available reagents are suitable for performing such tests (Lee et al, 1994, J. Virol. 68:8254-8264). Methods to test putative GSEs and similar compounds for the ability to inhibit aspects of HIV infection and replication are also described in detail in U.S. Patent No. 6,613,506, which is incorporated herein by reference in its entirety.
Target genes or proteins that are identified using GSEs according to any of the methods of the present invention can be further evaluated using a variety of methods. Such methods include methods that disrupt or "knock out" the expression of a target gene in a cell capable of being infected with HIV. Knockout methods include somatic cell knock-outs and inhibitory RNA molecules such as antisense oligonucleotides, siRNA molecules, and RNA decoys. Other methods for evaluating GSEs include nucleic acid-based experiments such as Northern blots, real time polymerase chain reaction, or high density microarrays.
Once one or more members of a biological pathway are identified as being required for progression through the HIV life cycle, and it is within the skill of one in the art to identify additional members of a biological pathway that are also required for progression through the HIV life cycle. For example, the activity of a member of the glycolysis pathway can be inhibited in a cell capable of being infected with HIV using methods known to those in the art, such as known chemical inhibitors, antibodies, somatic cell gene knock-outs, antisense molecules, or ribozymes. The cell can then be exposed to HIN and HIV infection measured. Inhibition of HIV infection would indicate that a particular glycolysis pathway member is required for progression through the HIN life cycle. Methods for testing HIV infection are described in the Examples herein.
Exemplary human cellular targets that have been identified using the methods described herein and which can serve as target genes or products for the assays described herein include any of the genes listed in Table 1 above, a nucleotide sequence for which is provided in SEQ ID Νos:13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39. These genes encode the following cellular proteins, respectively: enolase 1 (ENO1; SEQ TD NO: 14); enolase 2 (ENO2; SEQ ID NO:16); enolase 3 (ENO3; SEQ ID NO:18); fructose- 1,6-bisphosphatase 1 (FBPl; SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA; SEQ ID NO:22); glucosamine-6-phosphate deaminase (GNPDA; SEQ ID NO:24); glucose transporter-3 (GLUT3; SEQ ID NO:26); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH; SEQ ID NO:28); Na+ D-glucose cotransport regulator (SEQ ID NO: 30); 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K; SEQ ID NO:32); phosphofructokinase (PFK; SEQ TD NO:34); phosphoglucomutase (PGM; SEQ ID NO: 36); phosphorylase kinase, alpha-2 (PHKA2; SEQ ID NO:38); and thyroid hormone binding protein/pyruvate kinase (TCBA; SEQ ID NO:40). In any of the assays described herein, one can use a full-length gene, including a regulatory region of the gene, or a nucleic acid molecule encoding the gene product (protein encoded by the gene) or fragment thereof, or any fragment of such nucleic acid molecules that is suitable for use in an assay, or any encoded product of any such nucleic acid molecules, to identify inhibitors of the cellular gene or its encoded product for the puipose of inhibition of HIV infection or the development of compounds therefore based on a lead compound.
Accordingly, once a human cellular gene has been identified as a potential target for supporting the HIV life cycle, an assay can be used for screening and selecting a chemical or biological compound capable of acting as an anti-HIV therapeutic, based on the ability of the chemical or biological compound to down- regulate the expression ofthe target gene or inhibit the activity of its gene product. The methods can include the steps of (a) exposing (contacting) a second population of human host cells to a test compound; (b) measuring the expression of the human cellular gene in the second population of human host cells; and (c) determining whether the test compound regulates (modifies, increases or decreases, changes, modulates) the expression and/or activity of the human cellular gene or ofthe protein product encoded by the human cellular gene.
Such a chemical or biological compound can be referred to herein as a therapeutic compound, a test compound, or a lead compound. Such compounds may be identified by first incubating a cell line that naturally expresses the gene of interest, or which has been transfected with the gene or other recombinant nucleic acid molecule encoding a polypeptide of interest as disclosed herein, with various test compounds (putative regulatory compounds, putative therapeutic compounds), such as a chemical or biological compound. A reduction in the expression of the gene or protein of interest and/or an inhibition of the biological activity of the product encoded by the gene of interest would indicate that the chemical or biological compound is a therapeutic compound, or a lead compound to be used for development of drugs. Compounds identified in this manner are then retested in other assays to confirm their activities against HIN infection.
In general, the biological activity or biological action of a protein refers to any function(s) exhibited or performed by the protein that is ascribed to the naturally occurring form ofthe protein as measured or observed in vivo (i.e., in the natural physiological environment ofthe protein) or in vitro (i.e., under laboratory conditions). Modifications, activities or interactions which result in a decrease in protein expression or a decrease in the activity of the protein, can be referred to as inactivation (complete or partial), down-regulation, reduced action, or decreased action or activity of a protein. Similarly, modifications, activities or interactions which result in an increase in protein expression or an increase in the activity of the protein, can be referred to as amplification, overproduction, activation, enhancement, up-regulation or increased action of a protein. The biological activity of a protein according to the invention can be measured or evaluated using any assay for the biological activity of the protein as known in the art. Such assays can include, but are not limited to, binding assays, assays to determine internalization of the protein and/or associated proteins, cell signal transduction assays (e.g., phosphorylation assays), enzyme assays and/or assays for determining downstream cellular events that result from activation or binding of the cell surface protein (e.g., expression of downstream genes, production of various biological mediators, etc.). Preferably, the assay measures the ability of the protein or polypeptide to allow HIV infection. Such assays are described in detail herein.
Reagents suitable for an assay of the invention include any human cellular gene or its gene product. Compounds to be screened include any of those listed herein, such as known organic compounds such as antibodies, products of peptide libraries, and products of chemical combinatorial libraries. Compounds may also be identified using rational drag design relying on the structure of the gene product of a human cellular gene. Such methods are known to those of skill in the art and involve the use of three-dimensional imaging software programs. For example, various methods of drug design, useful to design or select mimetics or other therapeutic compounds useful in the present invention are disclosed in Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is incorporated herein by reference in its entirety. A mimetic refers to any peptide or non-peptide compound that is able to mimic the biological action of a naturally occurring peptide, often because the mimetic has a basic structure that mimics the basic structure of the naturally occurring peptide and/or has the salient biological properties of the naturally occurring peptide. Mimetics can include, but are not limited to: peptides that have substantial modifications from the prototype such as no side chain similarity with the naturally occurring peptide (such modifications, for example, may decrease its susceptibility to degradation); anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous portions of an isolated protein (e.g., carbohydrate structures); or synthetic or natural organic molecules, including nucleic acids and drugs identified through combinatorial chemistry, for example. Such mimetics can be designed, selected and/or otherwise identified using a variety of methods known in the art.
A mimetic can be obtained, for example, from molecular diversity strategies (a combination of related strategies allowing the rapid construction of large, chemically diverse molecule libraries), libraries of natural or synthetic compounds, in particular from chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the similar building blocks) or by rational, directed or random drug design. See for example, Maulik et al., supra.
In a molecular diversity strategy, large compound libraries are synthesized, for example, from peptides, oligonucleotides, carbohydrates and/or synthetic organic molecules, using biological, enzymatic and/or chemical approaches. The critical parameters in developing a molecular diversity strategy include subunit diversity, molecular size, and library diversity. The general goal of screening such libraries is to utilize sequential application of combinatorial selection to obtain high-affinity ligands for a desired target, and then to optimize the lead molecules by either random or directed design strategies. Methods of molecular diversity are described in detail in Maulik, et al., ibid. Maulik et al. also disclose, for example, methods of directed design, in which the user directs the process of creating novel molecules from a fragment library of appropriately selected fragments; random design, in which the user uses a genetic or other algorithm to randomly mutate fragments and their combinations while simultaneously applying a selection criterion to evaluate the fitness of candidate ligands; and a grid-based approach in which the user calculates the interaction energy between three dimensional receptor structures and small fragment probes, followed by linking together of favorable probe sites.
In one embodiment of the invention, inhibitors of HIN infection are identified by exposing a mammalian cell to a test compound; measuring the expression of a human cellular gene or an activity of the polypeptide encoded by the human cellular gene in the mammalian cell; and selecting a compound that down-regulates the expression of the human cellular gene or interferes with the activities of its encoded product. A preferred mammalian cell to use in an assay is a mammalian cell that either naturally expresses the human cellular gene or has been transformed with a recombinant form of the human cellular gene. Methods to determine expression levels of a gene are well known in the art. In a preferred embodiment, the cellular gene is a gene encoding a protein involved in or associated with the glycolysis pathway, including any of the genes listed in Table 1.
As used herein, the term "test compound" or "putative regulatory compound" or "putative inhibitory compound" and the like refers to compounds having an unknown or previously unappreciated regulatory activity in a particular process. As such, the term "identify" with regard to methods to identify compounds is intended to include all compounds, the usefulness of which as a regulatory compound for the purposes of inhibiting HIV infection can be determined by a method of the present invention and confirmed with additional assays, if desired or needed.
The conditions under which a host cell (or cell lysate, if appropriate for the given assay) of the present invention is contacted with or exposed to a putative regulatory compound, such as by mixing, are any suitable culture or assay conditions and includes an effective medium in which the cell can be cultured or in which the cell lysate can be evaluated in the presence and absence of a putative regulatory compound. Cells of the present invention can be cultured in a variety of containers including, but not limited to, tissue culture flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and carbon dioxide content appropriate for the cell. Such culturing conditions are also within the skill in the art. Cells are contacted with or exposed to a putative regulatory compound under conditions which take into account the number of cells per container contacted, the concentration of putative regulatory compound(s) administered to a cell, the incubation time of the putative regulatory compound with the cell, and the concentration of compound administered to a cell. Determination of effective protocols can be accomplished by those skilled in the art based on variables such as the size of the container, the volume of liquid in the container, conditions known to be suitable for the culture of the particular cell type used in the assay, and the chemical composition of the putative regulatory compound (i.e., size, charge etc.) being tested. By way of example, a preferred amount of putative regulatory compound(s) comprises between about 1 nM to about 10 mM of putative regulatory compound(s) per well of a 96-well plate. Preferably, compounds which are selected or identified are compounds for which, after the test or host cell was contacted with or exposed to the test compound, the level of expression or biological activity of the target gene or polypeptide was statistically significantly (i.e., with at least a 95% confidence level, or p<0.05) changed (preferably decreased) as compared to the initial level of expression or biological activity of the target gene or polypeptide or the expression or biological activity of the target gene or polypeptide in the absence of the test compound. Any statistically significant, detectable or measurable regulation of expression or activity of the target gene or product is indicative that the test compound can be identified as a regulator of the target gene or product and as a putative regulator of HIN infection in the host cell. In one embodiment, a 1.5 fold decrease in expression or biological activity of the target gene or protein in the cell as compared to in the absence of the test compound results in selection of the compound as being potentially useful for inhibition of HIN infection. More preferably, detection of at least about a 3 fold increase, and more preferably at least about a 6 fold increase, and even more preferably, at least about a 12 fold increase, and even more preferably, at least about a 24 fold increase in expression or biological activity of the target gene or protein in the cell as compared to in the absence of the test compound, results in selection of the compound as being potentially useful for inhibition of HIN infection.
Methods for measuring expression of a human cellular gene include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), in situ hybridization, Northern blot, Southern blot, sequence analysis, polyacrylamide gene analysis, microarray analysis, and detection of a reporter gene. Methods for detection of gene and gene transcription levels are well known in the art, and many of such methods are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989 and/or in Glick et al., Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, 1998; Sambrook et al., ibid., and Glick et al., ibid, are incorporated by reference herein in their entireties. Protein expression can be measured by any method including, but not limited to, Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, malti- tos, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry. In a preferred embodiment, the expression of the human cellular gene is measured by the polymerase chain reaction. In another preferred embodiment, the expression of the human cellular gene is measured using an antibody that specifically recognizes the polypeptide encoded by the human cellular gene and is analyzed using methods such as immunoprecipitation, ELISA assays, fluorescence activated cell sorting (FACS), or immunofluorescence microscopy. In another embodiment, the expression of the human cellular gene is measured using polyacrylamide gel analysis, chromatography, or spectroscopy. In still another preferred embodiment, the activity of the polypeptide encoded by the human cellular gene is measured by measuring the amount of product generated in a biochemical reaction mediated by the polypeptide encoded by the human cellular gene, h still another preferred embodiment, the activity of the polypeptide encoded by the human cellular gene is measured by measuring the amount of substrate generated in a biochemical reaction mediated by the polypeptide encoded by the target gene. h another embodiment of the invention, therapeutic compounds are selected by determining the three-dimensional structure of a human cellular gene product; and then determining the three-dimensional structure of a therapeutic compound based on the three-dimensional structure of a human cellular gene product. Preferably, the structure of the therapeutic compound is determined using computer software capable of modeling the interaction of the therapeutic compound with the target product. One of skill in the art can select the appropriate three-dimensional stracture, therapeutic compound, and analytical software based on the identity of the target gene.
For example, suitable candidate chemical compounds can align to a subset of residues described for a target site. Preferably, a candidate chemical compound comprises a conformation that promotes the formation of covalent or noncovalent crosslinking between the target site and the candidate chemical compound. Preferably, a candidate chemical compound binds to a surface adjacent to a target site to provide an additional site of interaction in a complex. When designing an antagonist, for example, the antagonist should bind with sufficient affinity to the binding site or to "substantially prohibit a ligand (i.e., a molecule that specifically binds to the target site) from binding to a target area. It will be appreciated by one of skill in the art that it is not necessary that the complementarity between a candidate chemical compound and a target site extend over all residues specified here in order to inhibit or promote binding of a ligand. hi general, the design of a chemical compound possessing stereochemical complementarity can be accomplished by techniques that optimize, chemically or geometrically, the "fit" between a chemical compound and a target site. Such techniques are disclosed by, for example, Sheridan and Nenkataraghavan, Ace. Chem Res., vol. 20, p. 322, 1987: Goodford, J. Med. Chem., vol. 27, p. 557, 1984; Beddell, Chem. Soc. Reviews, vol. 279, 1985; Hoi, Angew. Chem., vol. 25, p. 767, 1986; and Nerlinde and Hoi, Structure, vol. 2, p. 577, 1994, each of which are incorporated by this reference herein in their entirety.
As another example, a "geometric approach" is used. In a geometric approach, the number of internal degrees of freedom (and the corresponding local minima in the molecular conformation space) is reduced by considering only the geometric (hard_sphere) interactions of two rigid bodies, where one body (the active site) contains "pockets" or "grooves" that form binding sites for the second body (the complementing molecule, such as a ligand). The geometric approach is described by Kuntz et al., J. Mol. Biol, vol. 161, p. 269, 1982, which is incorporated by this reference herein in its entirety. The algorithm for chemical compound design can be implemented using the software program DOCK Package, Version 1.0 (available from the Regents of the University of California). Pursuant to the Kuntz algorithm, the shape of the cavity or groove on the surface of a structure at a binding site or interface is defined as a series of overlapping spheres of different radii. One or more extant databases of crystallographic data (e.g., the Cambridge Structural Database System maintained by University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 1EW, U.K.) or the Protein Data Bank maintained by Brookhaven National Laboratory, is then searched for chemical compounds that approximate the shape thus defined. Chemical compounds identified by the geometric approach can be modified to satisfy criteria associated with chemical complementarity, such as hydrogen bonding, ionic interactions or Van der Waals interactions.
As yet another example, one can determine the interaction of chemical groups ("probes") with an active site at sample positions within and around a binding site or interface, resulting in an array of energy values from which three dimensional contour surfaces at selected energy levels can be generated. This method is referred to herein as a "chemical_probe approach." The chemical_probe approach to the design of a chemical compound useful of the present invention is described by, for example, Goodford, J. Med. Chem., vol. 28, p. 849, 1985, which is incorporated by this reference herein in its entirety, and is implemented using an appropriate software package, including for example, GRID (available from Molecular Discovery Ltd., Oxford OX2 9LL, U.K.). The chemical prerequisites for a site_complementing molecule can be identified at the outset, by probing the active site of a protein with different chemical probes, e.g., water, a methyl group, an amine nitrogen, a carboxyl oxygen and/or a hydroxyl. Preferred sites for interaction between an active site and a probe are determined. Putative complementary chemical compounds can be generated using the resulting three dimensional pattern of such sites. hi still another embodiment of the invention, inhibitors of HIV infection are identified by exposing (exposing, contacting, mixing with) a polypeptide encoded by a target gene to a test compound; measuring the binding of the test compound to the polypeptide; and selecting a compound that binds to the polypeptide at a desired concentration, affinity, or avidity. In a preferred embodiment, the assay is performed under conditions conducive to promoting the interaction or binding of the compound to the polypeptide. One of skill in the art can determine such conditions based on the polypeptide and the compound being used in the assay, hi one embodiment, a BIAcore machine can be used to determine the binding constant of a complex between the target protein (a protein encoded by the target gene) and a natural ligand in the presence and absence of the candidate compound. For example, the target protein or a ligand binding fragment thereof can be immobilized on a substrate. A natural or synthetic ligand is contacted with the substrate to form a complex. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip (O'Shannessy et al. Anal. Biochem. 212:457_468 (1993); Schuster et al., Nature 365:343_347 (1993)). Contacting a test compound at various concentrations with the complex and monitoring the response function (e.g., the change in the refractive index with respect to time) allows the complex dissociation constant to be determined in the presence of the test compound and indicates whether the test compound is either an inhibitor or an agonist ofthe complex. Alternatively, the test compound can be contacted with the immobilized target protein at the same time as the ligand to see if the test compound inhibits or stabilizes the binding of the ligand to the target protein. Other suitable assays for measuring the binding of a candidate compound to a target protein or its ligand, and or for measuring the ability of such compound to affect the binding of the target protein to its ligand include, for example, immunoassays such as enzyme linked immunoabsorbent assays (ELISA) and radioimmunoassays (RIA), as well as cell__based assays including, cytokine secretion assays, or infracellular signal fransduction assays that determine, for example, protein or lipid phosphorylation, mediator release or infracellular Ca+ mobilization upon binding of a compound to a cell signal fransduction molecule or coreceptor.
In still another embodiment of the invention, inhibitors of HIV infection are identified by exposing (exposing, contacting, mixing with) a polypeptide encoded by a target gene to a test compound; measuring the activity of the polypeptide and or the activity of the biological pathway comprising the target polypeptide, and selecting a compound that modulates (regulates, changes, increases or decreases), the activity of the polypeptide or biological pathway. For example, a therapeutic compound can be identified in one embodiment by exposing an enzyme encoded by a target gene to a test compound; measuring the activity of the enzyme encoded by the target gene in the presence and absence of the compound; and selecting a compound that down-regulates the activity of the enzyme encoded by the target gene. Methods to measure enzymatic activity are well known to those skilled in the art and are selected based on the identity of the enzyme being tested. For example, if the enzyme is a kinase, then phosphorylation assays can be used. Other assays for detecting the biological activity and a change in such activity for a target polypeptide will be known to those of skill in the art. In addition to methods for identifying and producing a biological compound that inhibits HTV infection, the invention also encompasses methods known in the art for down-regulating the expression or function of a target gene of the invention, including any of the target genes set forth in Table 1 herein. For example, an antisense RNA or DNA molecule may be used to directly block translation of mRNA encoded by a target gene by binding to the mRNA and preventing protein translation. Polydeoxyribonucleotides can form sequence- specific triple helices by hydrogen bonding to specific complementary sequences in duplexed DNA to effect specific down-regulation of target gene expression. Formation of specific triple helices may selectively inhibit the replication or expression of a target gene by prohibiting the specific binding of functional transacting factors.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are ribozyme embodiments including engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of cellular RNA sequences. Antisense RNA molecules showing high-affinity binding to target sequences can also be used as ribozymes by addition of enzymatically active sequences known to those skilled in the art.
Polynucleotides to be used in triplex helix formation should be single- stranded and composed of deoxynucleotides. The base composition of these polynucleotides must be designed to promote triple helix formation via Hoogsteen base pairing rales, which generally require sizeable stretches of either purines or pyrimidines to be present on one strand of a duplex. Polynucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich polynucleotides provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, polynucleotides may be chosen that are purine-rich, for example, containing a stretch of G residues. These polynucleotides will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in GGC triplets across the three strands in the triplex.
Alternatively, sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" polynucleotide. Switchback polynucleotides are synthesized in an alternating 5 '-3', 3 '-5' manner, so that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
Both antisense RNA and DNA molecules, and ribozymes of the invention may be prepared by any method known in the art. These include techniques for chemically synthesizing polynucleotides well known in the art such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constracts that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into host cells.
Various modifications to the nucleic acid molecules may be introduced as a means of increasing infracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' or 3' ends ofthe molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone.
Preferably, methods used to identify therapeutic compounds are customized for each target gene or product. If the target product is an enzyme, then the enzyme will be expressed in cell culture and purified. The enzyme will then be screened in vitro against therapeutic compounds to look for inhibition of that enzymatic activity. If the target is a non-catalytic protein, then it will also be expressed and purified. Therapeutic compounds will then be tested for their ability to prevent, for example, the binding of a site-specific antibody or a target- specific ligand to the target product.
In a preferred embodiment, potential therapeutic compounds that bind to and/or effect the activity of target genes or products are further analyzed in a biological assay that tests for inhibition of HIV infection. In a preferred assay, HeLa (human fibroblast) cells that have been modified to express the β- galactosidase gene under the control of the HIV-1 LTR as well as the HIV CD4 receptor are plated onto 96-well plates. HIV binds to the CD4 receptor on the HeLa cell surface and infects the cell. Upon infection with HIV, viral proteins including tat are expressed. Tat then binds to the HIN-1 LTR, promoting the expression of β-galactosidase, which can be detected and quantified. Inhibition of HIV replication by a compound would prevent or reduce expression of tat and result in reduction of β-galactosidase expression as compared to control cells.
In one embodiment of the invention, a therapeutic compound is not toxic to a human host cell that is not infected with HIV. In another embodiment, a therapeutic compound promotes apoptosis in a human host cell infected with HIV.
In one embodiment of the invention, a pharmaceutical composition is prepared from a therapeutically-effective amount of a therapeutic compound of the invention and a pharmaceutically-acceptable carrier. Pharmaceutically- acceptable carriers are well known to those with skill in the art. In another embodiment, resistance to HIV infection is conferred upon an individual by administering a pharmaceutical composition of the invention. The pharmaceutical compositions of the present invention can be manufactured in a manner that is itself known, e.g., by means of a conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
For injection, the compounds of the invention can be formulated in appropriate aqueous solutions, such as physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal and transcutaneous administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PNP). If desired, disintegrating agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Tn the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix ofthe compound and a suitable powder base such as lactose or starch.
The compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
A pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water- miscible organic polymer, and an aqueous phase. The cosolvent system can be the NPD co-solvent system. NPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system can be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components can be varied: for example, other low-toxicity nonpolar surfactants can be used instead of polysorbate 80; the fraction size of polyethylene glycol can be varied; other biocompatible polymers can replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides can substitute for dextrose.
Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drags. Certain organic solvents such as dimethylsulfoxide also can be employed, although usually at the cost of greater toxicity. Additionally, the compounds can be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained- release capsules can, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein and nucleic acid stabilization can be employed.
The pharmaceutical compositions also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
The compounds of the invention can be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the EC50 (effective dose for 50% increase) as determined in cell culture, i.e., the concentration of the test compound which achieves a half- maximal inhibition of HIV replication as assayed by the infected cells to retain CD4 expression, to reduce viral p24 or gpl20, and to prevent syncytia formation. Such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g. Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch.l, p.l). Dosage amount and interval can be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the inhibitory effects. Usual patient dosages for systemic administration range from 100 - 2000 mg/day. Stated in terms of patient body surface areas, usual dosages range from 50 - 910 mg/m2/day. Usual average plasma levels should be maintained within 0.1-1000 μM. In cases of local administration or selective uptake, the effective local concentration ofthe compound can not be related to plasma concentration.
The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's body surface area, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
Suitable routes of administration can, for example, include oral, rectal, transmucosal, transcutaneous, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternatively, one can administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a specific tissue, often in a depot or sustained release formulation. Furthermore, one can administer the compound in a targeted drag delivery system, for example, in a liposome and/or conjugated with a cell-specific antibody. The liposomes and cell-specific antibody will be targeted to and taken up selectively by HlV-infected cells.
One embodiment of the present invention relates to an isolated nucleic acid molecule comprising, consisting essentially of, or consisting of: a GSE nucleic acid sequence according to the present invention, and to nucleic acid sequence homologues of the GSE that hybridize under stringent conditions to the complement of a GSE of the invention. In one embodiment, such a nucleic acid molecule comprises, consists essentially of or consists of less than a full-length cellular gene comprising a GSE according to the invention. Such GSEs are listed in Table 2 of the invention. Any of the GSE sequences described herein can be used, in one embodiment, as a template or stracture on which various other inhibitors of the present invention (e.g., small molecules) can be designed or selected.
Preferred embodiments of the invention and advantages over previously investigated detection methods are best understood by referring to Examples 1-5.
The Examples, which follow, are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
All publications cited herein are incorporated by reference in their entirety. EXAMPLE 1
Preparation of Random Fragment Libraries for the Isolation and Identification of Human Cell-Derived GSEs Exhibiting HIV Suppressive
Activity
Three random fragment expression (RFE) libraries were constracted from mRNA isolated from HL-60 and HeLa cells, and from phytohemaglutinin (PHA) stimulated peripheral blood mononuclear cells (PBMCs).
A. HL60 RFE Library
The HL60 RFE library was prepared by isolating mRNA from uninduced HL60 cells (ATCC Accession No. CCL 240) and then subtracting that mRNA with mRNA isolated from cells induced with TNF-α. This procedure represents a modification of that described in Coche et al, 199 A, Nucleic Acids Res. 22:1322- 23. Tracer mRNA was isolated from HL-60 cells transduced with the retroviral vector pLNCX (Miller et al, 1989, BioTechniques 7:980-90) at different time points after induction with TNF-α (Boehringer Mannheim; Indianapolis, IN). The pLNCX sequences were used as an internal standard to monitor the enrichment of the sequences present in the tracer after subtraction. RNA isolated from induced and uninduced cells was annealed separately to oligo-dT magnetic beads (Dynal Biotech; Lake Success, NY) and first strand cDNA was synthesized using reverse transcriptase and an oligo-dT primer. The RNA strand was then hydrolyzed and second strand cDNA synthesized from the induced cell first strand cDNA using a primer containing ATG codons in all three reading frames and an additional ten random nucleotides on the 3' end. Single- stranded cDNA fragments were annealed to an excess of driver cDNA attached to the magnetic beads. This procedure was repeated several times until substantial enrichment in the pLNCX sequences were seen. The final population of single- stranded DNA (ssDNA) molecules was amplified using a primer containing TGA codons in all three reading frames and an additional ten random nucleotides on the 3' end. The resulting population of cDNA fragments was then cloned into pLNCX. This step was taken to enrich for cellular sequences encoding products that might be important in supporting certain stages of the HIV life cycle in order to compensate for the low efficiency of retroviral transfer into OM10.1 cells. The HL60 library was found to comprise approximately 1 million transformants. B. HeLa RFE Library
The HeLa RFE library was prepared using the method described in Gudkov et al, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:3744-48. First, cDNA was prepared from HeLa cells and then partially digested with DNAse I in the presence of Mn (Sambrook et al, 1989, Molecular Cloning A Laboratory Manual (Cold Spring Harbor Laboratory)). Under these conditions, DNAse I is known to produce mostly double-stranded breaks. The resulting fragments were repaired using both the Klenow fragment of DNA polymerase I and T4 polymerase and then the fragments were ligated to synthetic double-stranded adaptors. The 5' adaptor was prepared from the primers 5'-C-T-C-G-G-A-A-T-T- C-A-A-G-C-T-T-A-T-G-G-A-T-G-G-A-T-G-G-3' (SEQ ID NO: 1) and 5'-C-A- T-C-C-A-T-C-C-A-T-A-A-G-C-T-T-G-A-A-T-T-C-C-3' (SEQ ID NO: 2). The 3' adaptor was prepared from the primers 5'-T-G-A-G-T-G-A-G-T-G-A-A-T-C-G- A-T-G-G-A-T-C-C-G-T-C-T-3' (SEQ ID NO: 3) and 5'-T-C-C-T-A-G-A-C-G-G- A-T-C-C-A-T-C-G-A-T-T-C-A-C-T-C-A-C-T-C-A-3' (SEQ ID NO: 4).
This randomly fragmented cDNA was then subjected to a normalization procedure to provide for the uniform abundance of different sequences in the population (Gudkov et al, 1997, Methods in Molecular Biology 69:221 (Humana Press)). This procedure was used to increase the probability of isolating GSEs from rare cDNAs, since total polyA RNA comprises a mixture of unequally represented sequences.
The randomly fragmented cDNA population was normalized by first denaturing 20 μg of cDNA by boiling for 5 minutes in 25 μl of TE buffer, followed by immediate cooling on ice. Then, 25 μl of 2X hybridization solution was added, and the mixture was divided equally into four .aliquots in Eppendorf tubes. One to two drops of mineral oil were added to each sample to avoid evaporation, and the tubes were placed into a 68°C water bath for annealing. One tube was frozen every 12 hours. Following the last time-point, each of the annealing mixtures was diluted with water to a final volume of 500 μl and subjected to hydroxylapatite (HAP) chromatography. HAP suspension equilibrated with 0.01 M phosphate-buffered saline (PBS) was placed into Eppendorf tubes so that the volume of HAP pellet was approximately 100 μl. The tubes with HAP and all the solutions used below were preheated and kept at 65°C. Excess PBS was removed, and diluted annealing solution was added. After mixing by shaking in a 65°C water bath, the tubes were left in the water bath until a HAP pellet was formed (a 15 second centrifugation was used to collect the pellet without exceeding lOOOg in the microcentrifuge to avoid damage of HAP crystals). The supernatant was carefully replaced with 1 ml of preheated 0.01 M PBS, and the process was repeated. To elute the ssDNA, the HAP pellet was suspended in 500 μl of PBS at the single-strand elution concentration determined (e.g., 0.16 M), the supernatant was collected, and the process was repeated. The supematants were combined and traces of HAP were removed by centrifugation. The ssDNA was concentrated by centrifugation, and washed three times using 1 ml of water on a Centricon-100 column.
The isolated ssDNA sequences were amplified by PCR using sense primers from each adapter and a minimal number of cycles to obtain 10 μg of the product. The size of the PCR product that remained within the desired range (200-500 bp) was ascertained. The normalization quality was tested by Southern or slot-blot hybridization with 32P-labeled probes for high, moderate- and low- expressing genes using 0.3-1.0 μg of normalized cDNA/lane. β-actin and β- tubulin cDNAs were used as probes for high-expressing genes, c-myc and topo II cDNAs were used as probes for moderate-expressing genes, and c-fos cDNA was used as a probe for low-expressing genes. The cDNAs isolated after different annealing times were compared with the original unnormalized cDNA. The probes were ensured to have a similar size and specific activity. The best- normalized ssDNA fraction (i.e., the population which produced the most uniform signal intensity with different probes) was used for large-scale PCR amplification to synthesize at least 20 μg of the product for cloning. More ssDNA template was used to obtain the desired amount by scaling up the number of PCR cycles or the reaction volume.
Following normalization, the mixture of randomly fragmented cDNA was digested with BamH I and EcoR I, column purified, and then ligated into either pLNCX or pLNGFRM (pLNGFRM differs from pLNCX in that the neo gene has been replaced with a truncated low affinity NGFR gene). Cells transduced with pLNGFRM express a truncated receptor on their surface that can be easily selected by an anti-NGFR antibody during FACS. The ligation mixture was introduced into E.coli, and approximately 100,000 transformants were obtained. The size distribution of the cloned fragments was analyzed by PCR using primers derived from vector sequences adjacent to the adapter sequences.
C. PBMC-1 RFE Library A first PBMC RFE library (PBMC-1) was prepared by isolating buffy coats from four healthy donors, from which PBMCs were purified by Ficoll gradient centrifugation followed by stimulation with PHA (1 μg/ml). Cells were removed at 5, 10, and 24 hours following the addition of PHA and total RNA was isolated by Trizol extraction. The isolated total RNA collected from the four donors was then pooled, yielding three populations corresponding to the time at which the cells were removed following PHA treatment. Poly-A+ mRNA was purified from the total RNA using the Gibco Superscript Choice system for cDNA synthesis (Gibco BRL; Bethesda, MD) and a random primer. The cDNA was normalized using the PCR-Select cDNA Subtraction kit (Clontech; Palo Alto, CA), based on the suppression subtractive hybridization methods described in Diatchenko et al, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:6025-30, and the primers 5'-T-A-G-G-G-C-T-C-G-A-G-C-C-G-C-C-A-C-C-A-T-G-3' (SEQ ID NO: 5) and 5'-A-T-C-C-C-T-G-C-A-G-G-T-C-A-C-T-C-A-C-T-C-A-3' (SEQ ID NO: 6). The normalized random fragments were digested with Xho I and Sse I, purified on quick spin columns (QIAGEN; Nalencia, CA) and ligated into the Sse I and Xho I sites of a bicistronic retroviral vector, pLXEMCNΝgfr. This vector is based on pLXSΝgfr. Modifications included the replacement of the SV40 promoter with encephalomyocarditis virus (EMVC) internal ribosomal entry site (IRES) isolated from the plasmid pCITE (Amersham Biosciences; Piscataway, ΝJ). The ligation mixture was introduced into competent cells, and approximately 50 million transformants were obtained.
P. PBMC-2 RFE Library
A second PBMC library (PBMC-2) was prepared by isolating peripheral blood mononuclear cells (PBMC) from 60 healthy donors over a Ficoll gradient. The isolated PBMCs consisted of different populations, including total unstimulated PBMCs, CD3/CD28 stimulated PBMCs, purified monocytes and macrophages, and purified memory and naϊve T cells. Unstimulated total PBMCs were directly harvested following Ficoll purification. CD3/CD28 stimulated PBMCs were generated by adding CD3 and CD28 antibodies (CD3/CD28 stimulation) and harvesting after 6 days in culture. Purified monocytes and macrophages were generated by sorting PBMCs for CD 14, culturing the sorted cells on glass plates, and harvesting adherent cells at days 2, 4-5, and 10-12. Memory and naϊve cells were generated by sorting PBMCs using CD4, CD45RA, and CD62L antibodies into memory (CD4+, CD45RA-, and CD62L+) and naϊve (CD4+, CD45RA+, and CD62L+) T cell populations followed by CD3/CD28 stimulation for 5-7 days. Total RNA was extracted from all harvested populations using the RNeasy Maxi kit (QIAGEN) and pooled. The normalization strategy was based on self-subtraction at the RNA level.
Total RNA was partially purified with oligotex (QIAGEN) to produce polyadenylated mRNA. The oligotex-primed polyadenylated mRNA was then used as template to synthesize oligotex-bead linked first-strand cDNA. The oligotex-bead linked first-strand cDNA was used as a driver to subtract abundant gene populations from PBMC total RNA. Normalization was performed with a 1:2 ratio of total RNA to cDNA driver at 37°C for 10 minutes. The abundant genes and rare gene were brought to within 2.5 orders of magnitude as measured by real time PCR. Messenger RNA was then purified from the normalized total RNA population and fragmented with weak alkaline-sodium carbonate at 60°C for 10 minutes. The fragmented mRNA served as the template for cDNA synthesis (Ominiscript kit; QIAGEN) using primers that included six random nucleotides linked to a Not I or Fse I site and a sequence for PCR priming (BSF-Fse-N6: 5'- G-C-T-A-T-G-A-C-C-A-T-G-A-T-T-A-C-G-C-C-A-G-G-C-C-G-G-C-C-N-N-N- N-N-N-3'; SEQ ID NO: 7; BSR-Not-N6: 5'-G-T-A-A-T-A-C-G-A-C-T-C-A-C-T- A-T-A-G-G-G-C-G-G-C-C-G-C-N-N-N-N-N-N-3'; SEQ ID NO: 8). Following cDNA synthesis, the fragments were PCR amplified using the primers: 5'-G-C-T- A-T-G-A-C-C-A-T-G-A-T-T-A-C-G-C-C-A-3' (SEQ ID NO: 9) and 5'-G-T-A- A-T-A-C-G-A-C-T-C-A-C-T-A-T-A-G-G-G-C-3' (SEQ ID NO: 10).
The fragments were digested with Not I and Fse I and those in the size range of 100-600 bp were isolated from a 2% agarose gel, eluted, and ligated into the bicistronic retroviral vector, pLXEMCVNgfr. This vector has a multiple cloning site modified to include the Not I and Fse I sites. The ligation mixture was electroporated into DH10B competent cells, generating greater than 100 million transformants. EXAMPLE 2
Transduction and Selection of Human Cell-Derived GSEs
HL-60 RFE libraries prepared as described in Example 1 were introduced into PA317 packaging cells (ATCC Accession No. CRL 9078), and converted into retrovirus for infection of OM10.1 cells (ATCC Accession No. CRL 10850; U.S. Patent No. 5,256,534). OM10.1 cells transduced with a pLNCX-based HL-60 RFE library were co-cultured and selected with G418. OM10.1 cells transduced with a pLNGFRM-based or pLXEMCVNgfr-based HL-60 RFE library were first subjected to spinoculation (centrifugation of target cells at 1200xg for 90 minutes in the presence of filtered retroviral supernatant) and then selected by FACS sorting of the NGFR population. Following selection, OM10.1 cells harboring the entire RFE library were induced with 10 U/ml of TNF-α at 37°C for 24 hours, stained with antibody, and then sorted for CD4 expression. Genomic DNA from the CD4+ cells was purified and used for PCR amplification of inserts using vector-derived primers. The amplified mixture was digested with EcoR I and BamH I and cloned back into the retroviral vector. This selection was repeated for additional rounds.
Normalized RFE libraries prepared from HeLa cells or PBMCs as described in Example 1 were transferred into CEM-ss cells (Cat. No. 776; NIH AIDS Research and Reference Reagent Program) and neo resistant and NGFR+ populations were isolated. The HeLa and PMBC RFE libraries each comprised 50 x 106 independent recombinant clones. Following introduction of the RFE libraries into CEM-ss cells, the CEM-ss cells were infected with a TCID5o of 3000/106 cells of HlV-lπm (Cat. No. 398; NIH AIDS Research and Reference Reagent Program). Because it has been suggested that syncytia formation can be prevented by blocking the interaction between gpl20 expressed on the surface of an infected cells and CD4 on the surface of an uninfected cells, 3 μg/ml of a purified anti-CD4 monoclonal antibody, L77 (Becton Dickinson), was added at 4 and 7 days following infection. The L77 antibody does not prevent HIV infection of a cell. At 8-10 days after infection, a subpopulation of CD4+/p24" cells corresponding to the uninfected cells was sorted. Genomic DNA from the isolated CD4+/p24" cells was purified and used for PCR amplification of inserts with the vector-derived primers. The amplified mixture was digested with EcoR I and BamH I and then cloned back into the retroviral vector. This selection was repeated for additional rounds.
EXAMPLE 3 Recovery and Sequencing of Human Cell-Derived GSEs
Genomic DNA was isolated from the selected OM10.1 or CEM-ss cells prepared as described in Example 2 by first centrifuging the selected cells, resuspending the cell pellet in 0.1 % Triton X-100, 20 μg/ml proteinase K, and IX PCR buffer, incubating at 55°C for 1 hour, and then boiling for 10 minutes. Genomic DNA was used for PCR amplification using vector-derived primers, cloned into the retroviral vector, and introduced into E. coli using standard transformation techniques. Individual plasmids were purified from E. coli clones using QIAGEN plasmid purification kits. Inserts were sequenced by the dideoxy procedure (using the AutoRead Sequencing Kit, Pharmacia Biotech or the Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit, ABI) and analyzed on a Pharmacia LKB A.L.F. or ABI 3700 DNA sequencer. Sequences were analyzed using the DNASTAR program or other proprietary data mining procedure.
As described in Example 2, two independent selection strategies were performed on two different cell lines (OM10.1 and CEM-ss) into which three independent RFE libraries were introduced. The GSEs identified using these selection strategies, and the human cellular genes from which these GSEs were derived, are indicated in Table 2.
Table 2 GSEs and Human Cellular Genes Involved in Inhibition of HIV Infection
Figure imgf000060_0001
Figure imgf000061_0001
EXAMPLE 4
Cell Population Sorting Based on p24 Expression Using Immunofluorescence and Flow Cytometry
Since infracellular p24 accumulation and surface CD4 down-modulation are associated with HIV-1 replication, successful interference with HTV-1 infection should result in an enrichment in cells displaying a p247CD4+ phenotype. Cells possessing this phenotype (i.e., uninfected cells) were identified by immunofluorescence 8-10 days following challenge with HIV. First, CD4+ cells were isolated from the challenged cell population (1 x IO7 cells) by washing the cells twice with Assay Buffer (500 ml PBS, 1 ml of 0.5 mM of EDTA, pH 8, 0.5 ml of 10%) sodium azide, and 10 ml of fetal bovine serum), and then resuspending the cells in 500 μl PBS containing 50 μl of anti-CD4 antibody (Q4120 PE; Sigma). Following incubation at 4°C for 30 minutes, 5 ml of Assay Buffer was added and the cells were centrifuged at 1200 rpm for 4 minutes. The cells were then washed twice with Assay Buffer and CD4+ cells sorted from the population by FACS. The aforementioned procedure was performed under sterile conditions. To identify cells within the CD4+ population that do not express p24, the sorted cell population (1 x. IO6 cells) was washed twice with Assay Buffer, and then suspended in 100 μl of Assay Buffer and 2 ml of Ortho PermeaFix Solution (Ortho Diagnostics). The cells were incubated at room temperature for 40 minutes, centrifuged at 1200 rpm and 4°C for 4 minutes, and then resuspended in 2 ml Wash Buffer (500 ml PBS, 25 ml fetal bovine serum, 1.5% bovine serum albumin and 0.0055%) EDTA). Following centrifugation at room temperature for 10 minutes, the cells were resuspended in 50 μl Wash Buffer diluted 1:500 with IgG a antibody, and incubated at 4°C for 20 minutes. Following this incubation, 5-10 μl of anti-p24 antibody (KC57-FITC; Coulter) was added and the cells were incubated at 4°C for 30 minutes. The cells were then washed twice with Wash Buffer and analyzed by flow cytometry.
EXAMPLE 5 Construction of Focused Library for a Target Gene
A focused library containing GSEs for a particular target gene is produced using a collection of randomly fragmented full-length cDNA clones or overlapping EST clones obtained from various commercial sources that encode the target gene. cDNA clones are produced from plasmids containing the target gene using the polymerase chain reaction. The cDNA clones are pooled into a mixture and fragmented with DNAse I using the method described in Example 1. Under these conditions, DNAse I is known to produce mostly double-stranded breaks. The resulting fragments are repaired with T4 polymerase and ligated to a synthetic double-stranded adaptor, such as the adaptor prepared from the oligonucleotides Fse-Top-Fse (5'-G-G-C-C-G-G-C-C-T-A-T-A-T-T-A-A-G-A-G- G-C-C-G-G-C-C-3'; SEQ ID NO: 11) and Fse-Bot-Fse (5'-G-G-C-C-G-G-C-C— T-C-T-T-A-A-T-A-T-A-G-G-C-C-G-G-C-C-3'; SEQ ID NO: 12).
The mixture of fragments with adaptors on both ends is digested with Fse I, column purified, and then ligated into the Fse I site of the bicistronic retroviral vector, pLXEMCVNgfr. The ligation mix is then transfomied into competent cells. The resulting plasmid preparation is used for selection.
For subcloning after selection, the orientation of the cDNA inserts is maintained by digestion with Xho I and Sse8387 I and ligation into the Sse8387 URsr H site of pLXEMCVNgfr. Neither Rsr H nor Sse83871 cleaves between the cDNA insert and an Fse I site.
It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.

Claims

WHAT WE CLAIM IS:
1. A method of identifying an inhibitor of a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, comprising identifying an inhibitor of a target cellular protein selected from the group consisting of: enolase 1 (ENOl; SEQ ID NO: 14); enolase 2 (ENO2; SEQ ID NO: 16); enolase 3 (ENO3; SEQ ID NO-18); fructose-l,6-bisphosphatase 1 (FBPl; SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA; SEQ ID NO:22); glucosamine-6-phosphate deaminase (GNPDA; SEQ ID NO:24); glucose transporter-3 (GLUT3; SEQ ID NO:26); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH; SEQ ID NO:28); Na+ D-glucose cotransport regulator (SEQ ID NO: 30); 6-phosphofructo-2- kinase/fractose-2,6-bisphosphatase (PF2K; SEQ ID NO:32); phosphofructokinase (PFK; SEQ ID NO:34); phosphoglucomutase (PGM; SEQ ID NO:36); phosphorylase kinase, alpha-2 (PHKA2; SEQ ID NO:38); and thyroid hormone binding protein/pyruvate kinase (TCBA; SEQ ID NO:40).
2. The method of Claim 1, wherein the step of identifying an inhibitor of a member of a biological pathway comprises the steps of:
(a) contacting a human host cell with a putative inhibitor, wherein the human host cell expresses a target polypeptide selected from the group consisting of: enolase 1 (ENOl; SEQ ID NO:14); enolase 2
(ENO2; SEQ ID NO: 16); enolase 3 (ENO3; SEQ ID NO: 18); fructose-1,6- bisphosphatase 1 (FBPl; SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA; SEQ ID NO:22); glucosamine-6-phosphate deaminase (GNPDA; SEQ ID NO:24); glucose transporter-3 (GLUT3; SEQ ID NO:26); glyceraldehyde-3-ρhosphate dehydrogenase (GAPDH; SEQ ID
NO:28); Na+ D-glucose cotransport regulator (SEQ ID NO:30); 6- phosphofracto-2-kinase/fractose-2,6-bisphosphatase (PF2K; SEQ ID NO:32); phosphofructokinase (PFK; SEQ ID NO:34); phosphoglucomutase (PGM; SEQ ID NO:36); phosphorylase kinase, alpha-2 (PHKA2; SEQ ID NO:38); and thyroid hormone binding protein/pyruvate kinase (TCBA; SEQ ID NO:40); and
(b) assessing inhibition of the expression or activity of the polypeptide, wherein a decrease in the expression or activity of the target polypeptide in the presence of the putative inhibitor as compared to in the absence of the putative inhibitor, indicates that the putative inhibitor inhibits the member ofthe biological pathway.
3. The method of Claim 2, further comprising assessing the ability of the inhibitor identified in (b) to inhibit HIV infection in the host cell.
4. The method of Claim 3, wherein the step of assessing the ability of the inhibitor identified in (b) to inhibit HIV infection in the host cell comprises the steps of: a) contacting a human host cell that is susceptible to HIN- infection with the putative inhibitor, wherein the human host cell expresses the target polypeptide; b) exposing the cell to HIV; and c) assessing inhibition of HIN-infection in the presence of the putative inhibitor as compared to in the absence ofthe inhibitor.
5. A method of identifying a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, the method comprising the steps of: a) synthesizing a randomly fragmented cDΝA population from total mRΝA isolated from a human host cell that is susceptible to HIV infection to yield DΝA fragments; b) transferring the DΝA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DΝA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DΝA fragments in human host cells; c) genetically modifying a population of human host cells by introducing the genetic suppressor element library into the population of human host cells; d) infecting the population of human host cells with HIV; e) isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to HIN infection from the population of human host cells; f) recovering the genetic suppressor element from the isolated genetically modified human host cell; g) determining the human cellular gene that corresponds to the genetic suppressor element, wherein the human cellular gene encodes the second member ofthe biological pathway; h) determining the biological pathway of the second member; and; i) determining the first member of the biological pathway.
6. The method of claim 5, wherein the randomly fragmented cDNA population is a normalized cDNA population.
7. The method of claim 5, wherein the genetic suppressor element is a sense oriented genetic suppressor element encoding a peptide.
8. The method of claim 5, wherein the genetic suppressor element is an antisense-oriented genetic suppressor element encoding an antisense RNA.
9. The method of claim 5, wherein the second member of the biological pathway mediates fusion of HIV with human host cells.
10. The method of claim 9, wherein the second member of the biological pathway inhibits binding of HIV to CD4 receptors on the surface of human host cells.
11. The method of claim 5, wherein the second member of the biological pathway modulates reverse transcription of viral RNA in human host cells.
12. The method of claim 11, wherein the second member of the biological pathway inhibits HIN reverse transcriptase.
13. The method of claim 5, wherein the second member of the biological pathway mediates integration of HIN DΝA in the human host cell genome.
14. The method of claim 13, wherein the second member of the biological pathway inhibits HIN integrase.
15. The method of claim 5, wherein the second member of the biological pathway mediates processing of HIV proteins in human host cells.
16. The method of claim 15, wherein the second member of the biological pathway inhibits HIV protease.
17. The method of claim 5, wherein the first member of the biological pathway regulates the expression or activity of the second member of the biological pathway.
18. The method of claim 17, wherein the second member of the biological pathway regulates the expression or activity of the first member of the biological pathway.
19. The method of claim 5, wherein the first member of the biological pathway is a product of a biochemical reaction mediated by the second member of the biological pathway.
20. The method of claim 5, wherein the second member of the biological pathway is a product of a biochemical reaction mediated by the first member ofthe biological pathway.
21. The method of claim 5, wherein the first member of the biological pathway is a substrate of a biochemical reaction mediated by the second member ofthe biological pathway.
22. The method of claim 21, wherein the second member of the biological pathway is an enzyme.
23. The method of claim 5, wherein the second member of the biological pathway is a substrate of a biochemical reaction mediated by the first member ofthe biological pathway.
24. The method of claim 23, wherein the first member ofthe biological pathway is an enzyme.
25. The method of claim 5, wherein the second member of the biological pathway is a member ofthe glycolysis pathway.
26. The method of claim 25, wherein the member of the glycolysis pathway is selected from the group consisting of: Na+ D-glucose cotransport regulator (SEQ ID NO:30); glucose transporter-3 (GLUT3) (SEQ ID NO:26); phosphorylase kinase, alpha 2 (PHKA2) (SEQ ID NO:38); phosphoglucomutase (PGM) (SEQ ID NO:36); glucosamine-6-phosphate deaminase (GNPDA) (SEQ ID NO:24); 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PF2K) (SEQ ID NO:32); phosphofructokinase (PFK) (SEQ ID NO:33); fractose-1,6- bisphosphatase 1 (FBPl) (SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA) (SEQ ID NO:22); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SEQ ID NO:28); enolase 1 (ENOl) (SEQ ID NO: 14); and thyroid hormone binding protein/pyruvate kinase (TCBA) (SEQ ID NO:40).
27. The method of Claim 5, wherein the human host cells are T cells or macrophages.
28. A method of identifying an inhibitor of a human cellular gene or protein encoded thereby that is a member of a biological pathway in a human host cell, wherein the member of the biological pathway is necessary for productive HIV infection, the method comprising the steps of: a) identifying the member of the biological pathway by: i) synthesizing a randomly fragmented cDNA population from total mRNA isolated from a human host cell that is susceptible to HIV infection to yield DNA fragments; ii) transferring the DNA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the
DNA fragments in human host cells; iii) genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; iv) infecting the first population of human host cells with HIV; v) isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to
HIV infection from the first population of human host cells; vi) recovering the genetic suppressor element from the isolated genetically modified human host cell; and vii) determining the identity of the human cellular gene corresponding to the genetic suppressor element; and b) identifying the inhibitor of the member of the biological pathway by: i) exposing a second population of human host cells to a test compound; ii) measuring the expression or activity of the human cellular gene or protein encoded thereby in the second population of human host cells; and iii) determining whether the test compound decreases the expression or activity of the human cellular gene or protein encoded thereby.
29. The method of Claim 28, wherein step (b) comprises: i) exposing a second population of human host cells to a test compound; ii) measuring the expression of the human cellular gene in the second population of human host cells; and iii) determining whether the test compound decreases the expression ofthe human cellular gene.
30. The method of Claim 28, wherein step (b) comprises: i) exposing a second population of human host cells to a test compound; ii) measuring the expression of the protein encoded by the human cellular gene in the second population of human host cells; and iii) determining whether the test compound decreases the expression ofthe protein encoded by the human cellular gene.
31. The method of Claim 28, wherein step (b) comprises: i) exposing a second population of human host cells to a test compound; ii) measuring the activity of the protein encoded by the human cellular gene in the second population of human host cells; and iii) determining whether the test compound decreases the activity ofthe protein encoded by the human cellular gene.
32. The method of claim 28, further comprising the step of: d) determining whether the test compound inhibits HIN infection.
33. The method of claim 28, wherein the expression of the human cellular gene is measured by polymerase chain reaction.
34. The method of claim 28, wherein the expression of the protein encoded by the cellular gene is measured using an antibody that specifically recognizes the member ofthe biological pathway.
35. The method of claim 28, wherein the activity of the protein encoded by the cellular gene is measured by measuring the amount of a product generated in a biochemical reaction mediated by the member of the biological pathway.
36. The method of claim 28, wherein the activity of the protein encoded by the cellular gene is measured by measuring the amount of a substrate consumed in a biochemical reaction mediated by the member of the biological pathway.
37. The method of claim 28, wherein the test compound is selected by: a) determining the three-dimensional stracture of the member ofthe biological pathway; and b) determining the three-dimensional stracture of a test compound by using computer software capable of modeling the interaction of the member ofthe biological pathway and putative test compounds.
38. The method of claim 28, wherein the member of the biological pathway is a member ofthe glycolysis pathway.
39. The method of claim 38, wherein the member of the glycolysis pathway is selected from the group consisting of: Na+ D-glucose cotransport regulator (SEQ ID NO:30); glucose transporter-3 (GLUT3) (SEQ ID NO:26); phosphorylase kinase, alpha 2 (PHKA2) (SEQ ID NO: 38); phosphoglucomutase (PGM) (SEQ ID NO:36); glucosamine-6-phosphate deaminase (GNPDA) (SEQ ID NO:24); 6-phosphofracto-2-kinase/fractose-2,6-bisphosphatase (PF2K) (SEQ ID NO:32); phosphofructokinase (PFK) (SEQ ID NO:33); fractose-1,6- bisphosphatase 1 (FBPl) (SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA) (SEQ ID NO:22); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) (SEQ ID NO:28); enolase 1 (ENOl) (SEQ ID NO: 14); and thyroid hormone binding protein/pyruvate kinase (TCBA) (SEQ ID NO:40).
40. The method of claim 28, wherein the human host cells are T cells or macrophages.
41. The method of claim 28, wherein the inhibitor confers resistance to
HIN infection.
42. An inhibitor of a member of a biological pathway in a human host cell, wherein the inhibitor is identified by the method of claim 28.
43. The inhibitor of claim 42, wherein the member of the biological pathway is a member ofthe glycolysis pathway.
44. The inhibitor of claim 42, wherein the member of the biological pathway is selected from the group consisting of: Na+ D-glucose cotransport regulator (SEQ ID NO:30); glucose transporter-3 (GLUT3) (SEQ ID NO:26); phosphorylase kinase, alpha 2 (PHKA2) (SEQ ID NO: 38); phosphoglucomutase (PGM) (SEQ ID NO:36); glucosamine-6-phosphate deaminase (GNPDA) (SEQ ID NO:24); 6-phosphofructo-2-kinase/fractose-2,6-bisphosphatase (PF2K) (SEQ ID NO:32); phosphofructokinase (PFK) (SEQ ID NO:33); fructose-1,6- bisphosphatase 1 (FBPl) (SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA) (SEQ ID NO:22); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) (SEQ ID NO:28); enolase 1 (ENOl) (SEQ ID NO: 14); and thyroid hormone binding protein/pyruvate kinase (TCBA) (SEQ ID NO: 40).
45. The inhibitor of claim 42, wherein the inhibitor is not toxic to a human host cell that is not infected with HIV.
46. The inhibitor of claim 42, wherein the inhibitor promotes apoptosis in a human host cell infected with HIV.
47. A pharmaceutical composition comprising a therapeutically- effective amount of the inhibitor of claim 42 and a pharmaceutically-acceptable carrier.
48. A method of conferring resistance to HIN infection in an individual, comprising administering to the individual the pharmaceutical composition of claim 47.
49. A method of identifying an inhibitor of a first member of a biological pathway in a human host cell, wherein the biological pathway comprises a second member of the pathway that is necessary for productive HIV infection, the method comprising the steps of: a) identifying the second member of the biological pathway by: i) synthesizing a randomly fragmented cDΝA population from total mRΝA isolated from a human host cell that is susceptible to HIV infection to yield DΝA fragments; ii) transferring the DΝA fragments to an expression vector to yield a genetic suppressor element library, wherein each of the DNA fragments is operatively linked to a protein translation initiation codon, and wherein the expression vector expresses the DNA fragments in human host cells; iii) genetically modifying a first population of human host cells by introducing the genetic suppressor element library into the first population of human host cells; iv) infecting the first population of human host cells with HIV; v) isolating a genetically modified human host cell containing a genetic suppressor element conferring resistance to
HIN infection from the first population of human host cells; vi) recovering the genetic suppressor element from the isolated genetically modified human host cell; vii) determining the human cellular gene corresponding to the genetic suppressor element, wherein the human cellular gene encodes the second member ofthe biological pathway; viii) determining the biological pathway of the second member; ix) determining the first member of the biological pathway and the human cellular gene that encodes the first member ofthe biological pathway; and b) identifying the inhibitor of the first member of the biological pathway by: i) exposing a second population of human host cells to a test compound; ii) measuring the expression or activity of the human cellular gene encoding the first member of the biological pathway or protein encoded thereby in the second population of human host cells; and iii) determining whether the test compound decreases the expression or activity of the human cellular gene encoding the first member ofthe biological pathway or protein encoded thereby.
50. The method of claim 49, wherein the expression of the human cellular gene encoding the first member of the biological pathway is measured by polymerase chain reaction.
51. The method of claim 49, wherein the expression of the first member of the biological pathway is measured using an antibody that specifically recognizes the first member ofthe biological pathway.
52. The method of claim 49, wherein the activity of the first member of the biological pathway is measured by measuring the amount of a product generated in a biochemical reaction mediated by the first member ofthe biological pathway.
53. The method of claim 49, wherein the activity of the first member of the biological pathway is measured by measuring the amount of a substrate consumed in a biochemical reaction mediated by the first member of the biological pathway.
54. The method of claim 49, wherein the activity of the first member of the biological pathway is measured by measuring the expression or activity of a third member of the biological pathway, wherein the first member mediates the expression or activity ofthe third member ofthe biological pathway.
55. The method of claim 49, wherein the first member ofthe biological pathway is a member of the glycolysis pathway selected from the group consisting of: Na+ D-glucose cotransport regulator (SEQ ID NO:30); glucose transporter-3 (GLUT3) (SEQ ID NO:26); phosphorylase kinase, alpha 2 (PHKA2) (SEQ ID NO:38); phosphoglucomutase (PGM) (SEQ ID NO:36); glucosamine-6-phosphate deaminase (GNPDA) (SEQ ID NO:24); 6-phosphofracto-2-kinase/fructose-2,6- bisphosphatase (PF2K) (SEQ ID NO: 32); phosphofractokinase (PFK) (SEQ ID NO:33); fructose- 1,6-bisphosphatase 1 (FBPl) (SEQ ID NO:20); fructose bisphosphate aldolase A (ALDOA) (SEQ ID NO:22); glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) (SEQ ID NO:28); enolase 1 (ENOl) (SEQ ID NO: 14); and thyroid hormone binding protein/pyruvate kinase (TCBA) (SEQ ID NO:40).
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