Compositions and methods for inhibiting human...

Details Compositions and methods for inhibiting human... Compositions and methods for inhibiting human...

2000-06-05

2002-08-20

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S325000, C435S366000, C435S236000

Reexamination Certificate

active

06436634

ABSTRACT:

1. INTRODUCTION
The present invention relates to the identification of several human genes as cellular targets for the design of therapeutic agents for suppressing human immunodeficiency virus (HIV) infection. These genes encode intracellular 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. Therefore, inhibitors of these genes and their encoded products may be used as therapeutic agents for the treatment and/or prevention of HIV infection. 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.
2. BACKGROUND OF THE INVENTION
2.1. The Human Immunodeficiency Virus
The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al., 1983,
Science
220:868-870; Gallo et al., 1984,
Science
224:500-503). HIV causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in their depletion. This, in turn, leads to opportunistic infections, neoplastic growth and death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984,
RNA Tumor Viruses,
Weiss et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, 1988,
Science
240:1427-1439). There are at least two distinct subtypes of HIV: HIV-1 (Barre-Sinoussi et al., 1983,
Science
220:868-870; Gallo et al., 1984,
Science
224:500-503) and HIV-2 (Clavel et al., 1986,
Science
233:343-346; Guyader et al., 1987,
Nature
326:662-669). Genetic heterogeneity exists within each of these HIV 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
312:763-767; Klatzmann et al., 1984,
Nature
312:767-768; Maddon et al., 1986,
Cell
47:333-348). Viral entry into cells is dependent upon viral protein gp120 binding to the cellular CD4 receptor molecule (McDougal et al., 1986
, Science
231:382-385; Maddon et al., 1986,
Cell
47:333-348).
2.2. HIV Treatment
HIV infection is pandemic and HIV-associated diseases have become a world-wide 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.
5:2369-2381). For example, virally-encoded reverse transcriptase has been a major focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2′, 3′-dideoxynucleotide analogs such as AZT, ddI, ddC, and ddT have been shown to be active against HIV (Mitsuya et al., 1990,
Science
249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al., 1989,
Science
243:1731-1734). In 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 the 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
238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990,
Proc Natl. Acad. Sci. USA
87:6574-6579). To date, clinical trials of recombinant, soluble CD4 have produced inconclusive results (Schooley et al., 1990,
Ann. Int. Med.
112:247-253; Kahn et al., 1990,
Ann. Int. Med.
112:254-261; Yarchoan et al., 1989,
Proc Vth Int. Conf. on AIDS,
p. 564, MCP 137).
Additionally, the later stages of HIV replication which involve crucial virus-specific secondary 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,
Persy. 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
374:569-571). These drugs often exhibit toxic side effects such as nausea, altered taste, circumoral parethesias 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.
14:142-160). 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 (decoys, antisense and ribozymes) or protein-based (transdominant mutants) HIV-1 antiviral agents 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
63:601-608; Sullenger et al., 1991,
J. Virol.
65:6811-6816; Lisziewicz et al., 1993,
New Biol.
3:82-89; Lee et al., 1994,
J. Virol.
68:8254-8264), ribozymes (Sarver et al., 1990,
Science
247:1222-1225; Wecrasinghe et al., 1991,
J. Virol.
65:5531-5534; Dropulic et al., 1992,
J. Virol.
66:1432-1441; Ojwang et al., 1992,
Proc. Natl. Acad. Sci. U.S.A.
89:10802-10806; Yu et al., 1993,
Proc Natl. Acad. Sci. U.S.A.
90:6340-6344; 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 gag, tat, rev, env (Sezakiel et al., 1991,
J. Virol.
65:468-472; Chatterjee et al., 1992,
Science
258:1485-1488; Rhodes et al., 1990,
J. Gen. Virol.
71:1965; Rhodes et al., 1991,
AIDS
5:145-151; Sezakiel et al., 1992,
J. Virol.
66:5576-5581; Joshi et al., 1991,
J. Virol.
65:5524-5530) and transdominant mutants including Rev (Bevec et al., 1992,
Proc Natl. Acad. Sci. U.S.A.
89:9870-9874), Tat (Pearson et al., 1990,
Proc Natl. Acad. Sci. U.S.A.
87:5079-5083; Modesti et al., 1991, New Biol. 3:759-768), Gag (Trono et al., 1989,
Cell
59:113-120), Env (Bushschacher et al., 1995,
J. Virol.
69:1344-1348) and protease (Junker et al., 1996,
J. Virol.
70:7765-7772).
Antisense polynucleotides have been designed to complex with and sequester the HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al., WO 94/10302; Kretschmer et al., EP 594,881; and Chatterjee et al., 1992,
Science
258:1485). Furthermore, an enzymatically active RNA, termed ribozyme, has 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. USA
89:10802-06; Yamada et al., 1994,
Gene Therapy
1:38-45; Ho et al., WO 94/26877; and Cech and Sullenger, WO 95/13379). In preclinical studies, RevM10, a transdomi

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