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

Abstract

The present invention relates to the identification of a number of human genes as cellular targets for the design of therapeutic agents for suppressing human immunodeficiency virus infection. These genes encode products which appear to be necessary for HIV replication, as evidenced by an inhibition of HIV infection in cells in which the expression of these genes is down-regulated. In addition, the invention also relates to methods for identifying additional cellular genes as therapeutic targets for suppressing HIV infection, and methods of using such cellular genes and their encoded products in screening assays for selecting additional inhibitors of HIV.

What is claimed is:  
     
         1 . An HIV inhibitory composition, comprising an isolated polynucleotide corresponding to a portion of a cellular gene or a complement thereof, said gene encodes an intracellular product which is necessary for a productive HIV infection, said polynucleotide is operably linked to a regulatory sequence, and expression of said polynucleotide in a host cell inhibits infection by HIV.  
     
     
         2 . The composition of  claim 1  in which inhibition of infection by HIV is measured by continued CD4 expression in the cells following HIV infection.  
     
     
         3 . The composition of  claim 1  in which inhibition of infection by HIV is measured by decreased viral p24 expression in the cells following HIV infection.  
     
     
         4 . The composition of  claim 1  in which the cellular gene encodes a subunit of NADH dehydrogenase.  
     
     
         5 . The composition of  claim 4  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO.: 1.  
     
     
         6 . The composition of  claim 4  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO.: 2.  
     
     
         7 . The composition of  claim 4  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO.: 3.  
     
     
         8 . The composition of  claim 4  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 4.  
     
     
         9 . The composition of  claim 4  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 5.  
     
     
         10 . The composition of  claim 4  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 6.  
     
     
         11 . The composition of  claim 1  in which the cellular gene encodes 2-oxoglutarate dehydrogenase.  
     
     
         12 . The composition of  claim 11  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 7.  
     
     
         13 . The composition of  claim 1  in which the cellular gene encodes pyruvate kinase.  
     
     
         14 . The composition of  claim 13  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 8.  
     
     
         15 . The composition of  claim 1  in which the cellular gene encodes calnexin.  
     
     
         16 . The composition of  claim 15  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 9.  
     
     
         17 . The composition of  claim 1  in which the cellular gene encodes an ADP-ribosylation factor 3.  
     
     
         18 . The composition of  claim 17  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 10.  
     
     
         19 . The composition of  claim 1  in which the cellular gene encodes eukaryotic initiation factor 3.  
     
     
         20 . The composition of  claim 19  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 11.  
     
     
         21 . The composition of  claim 1  in which the cellular gene encodes a protein tyrosine phosphatase.  
     
     
         22 . The composition of  claim 21  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 12.  
     
     
         23 . The composition of  claim 21  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 13.  
     
     
         24 . The composition of  claim 1  in which the cellular gene encodes herpesvirus-associated ubiquitin-specific protease.  
     
     
         25 . The composition of  claim 24  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 14.  
     
     
         26 . The composition of  claim 1  in which the cellular gene encodes eukaryotic initiation factor 4B.  
     
     
         27 . The composition of  claim 26  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 15.  
     
     
         28 . The composition of  claim 1  in which the cellular gene encodes phosphatidylinositol 3 kinase.  
     
     
         29 . The composition of  claim 28  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 17.  
     
     
         30 . The composition of  claim 1  in which the cellular gene encodes elongation factor 1 alpha.  
     
     
         31 . The composition of  claim 30  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 18.  
     
     
         32 . The composition of  claim 1  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 19.  
     
     
         33 . The composition of  claim 1  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO: 20.  
     
     
         34 . An HIV inhibitory composition, comprising an isolated polynucleotide corresponding to a portion of a gene encoding human CD44 or a complement thereof, said polynucleotide is operably linked to a regulatory sequence and expression of said polynucleotide in a host cell inhibits infection by HIV.  
     
     
         35 . The composition of  claim 34  in which the polynucleotide comprises the sequence as depicted in SEQ ID NO:16.  
     
     
         36 . An expression vector containing the polynucleotide of  claim 5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  12 ,  14 ,  16 ,  18 ,  20 ,  22 ,  23 ,  25 ,  27 ,  29 ,  31 ,  32 ,  33  or  35 .  
     
     
         37 . A genetically-engineered host cell containing the polynucleotide of  claim 5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  12 ,  14 ,  16 ,  18 ,  20 ,  22 ,  23 ,  25 ,  27 ,  29 ,  31 ,  32 ,  33  or  35 .  
     
     
         38 . A pharmaceutical composition, comprising the polynucleotide of  claim 5 ,  6 ,  7 ,  8 ,  9 ,  10 ,  12 ,  14 ,  16 ,  18 ,  20 ,  22 ,  23 ,  25 ,  27 ,  29 ,  31 ,  32 ,  33  or  35 .  
     
     
         39 . A method for identifying a cellular gene which encodes a product necessary for HIV infection, comprising: 
 (a) constructing an expression library of polynucleotide fragments from cellular DNA;    (b) transferring the expression library into host cells; and    (c) selecting the cells that contain a polynucleotide which inhibits HIV infection by measuring levels of a detectable marker in the cells.    
     
     
         40 . The method of  claim 39 , in which the cells contain an inducible latent HIV provirus.  
     
     
         41 . The method of  claim 40 , in which the host cells are OM10.1.  
     
     
         42 . The method of  claim 40 , in which the cells are induced by TNF-α.  
     
     
         43 . The method of  claim 39 , in which the marker is cellular CD4.  
     
     
         44 . The method of  claim 39 , in which expression of the marker is retained by the host cells.  
     
     
         45 . The method of  claim 39 , in which the marker is viral protein p24.  
     
     
         46 . The method of  claim 39 , in which the marker is viral protein gp120.  
     
     
         47 . The method of  claim 45  or  46  in which expression of the marker is suppressed in the host cells.  
     
     
         48 . A method for protecting host cells from HIV infection, comprising introducing an effective amount of the composition of  claim 1 .  
     
     
         49 . The method of  claim 48 , in which the composition is introduced into the host cells in vitro.  
     
     
         50 . The method of  claim 48 , in which the composition is introduced into the host cells in vivo.  
     
     
         51 . A method for treating HIV infection, comprising administering a therapeutically effective amount of the composition of  claim 1  to an individual to inhibit HIV infection.  
     
     
         52 . A method for inhibiting HIV infection, comprising administering a therapeutically effective amount of an inhibitor of a cellular gene to an individual to inhibit HIV infection.  
     
     
         53 . The method of °  claim 52  in which the inhibitor down-regulates gene expression of a subunit of NADH dehydrogenase.  
     
     
         54 . The method of  claim 53  in which the inhibitor is mofarotene.  
     
     
         55 . The method of  claim 52  in which the inhibitor interferes with enzyme activities.  
     
     
         56 . The method of  claim 55  in which the inhibitor is amytal.  
     
     
         57 . The method of  claim 52  in which the inhibitor is a 2-oxoglutarate dehydrogenase inhibitor.  
     
     
         58 . The method of  claim 52  in which the inhibitor is a pyruvate kinase inhibitor.  
     
     
         59 . The method of  claim 52  in which the inhibitor is a calnexin inhibitor.  
     
     
         60 . The method of  claim 52  in which the inhibitor is an ADP-ribosylation factor 3 inhibitor.  
     
     
         61 . The method of  claim 52  in which the inhibitor is ubiquitin-specific protease inhibitor.  
     
     
         62 . The method of  claim 52  in which the inhibitor is CD44 inhibitor.  
     
     
         63 . The method of  claim 52  in which the inhibitor is tyrosine phosphatase inhibitor.  
     
     
         64 . The method of  claim 52  in which the inhibitor is phosphatidylinositol kinase inhibitor.  
     
     
         65 . The method of  claim 52  in which the inhibitor is an eukaryotic elongation factor inhibitor.  
     
     
         66 . The method of  claim 52  in which the inhibitor is eukaryotic initation factor 3 inhibitor.  
     
     
         67 . The method of  claim 52  in which the inhibitor is eukaryotic initation factor 4B inhibitor.  
     
     
         68 . A method for selecting HIV an inhibitor, comprising: 
 (a) exposing a mammalian cell to a test compound;    (b) measuring the expression of a cellular gene or the activities of its encoded product in said cell; and    (c) selecting the compound which down-regulates expression of said gene or interferes with the activities of its encoded product.    
     
     
         69 . The method of  claim 68  in which the cellular gene encodes a subunit of NADH dehydrogenase.  
     
     
         70 . The method of  claim 68  in which the cellular gene encodes M2-type pyruvate kinase and cytosolic thyroid hormone binding protein.  
     
     
         71 . The method of  claim 68  in which the cellular gene encodes calnexin.  
     
     
         72 . The method of  claim 68  in which the cellular gene encodes ADP-ribosylation factor 3.  
     
     
         73 . The method of  claim 68  in which the cellular gene encodes eukaryotic initiation factor 3.  
     
     
         74 . The method of  claim 68  in which the cellular gene encodes a tyrosine phosphatase.  
     
     
         75 . The method of  claim 68  in which the cellular gene encodes herpes-associated ubiquitin-specific protease.  
     
     
         76 . The method of  claim 68  in which the cellular gene encodes eukaryotic initiation factor 4B.  
     
     
         77 . The method of  claim 68  in which the cellular gene encodes CD44.  
     
     
         78 . The method of  claim 68  in which the cellular gene encodes phosphatidylinositol 3 kinase.  
     
     
         79 . The method of  claim 68  in which the cellular gene encodes elongation factor 1 alpha.