Compositions and methods for the abrogation of cellular...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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Reexamination Certificate

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06667157

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to compounds which modulate glucocorticoid receptor-complex transactivation activity. The present invention relates to a method of identifying compounds which stimulate GR complex transactivation. The present invention relates to a method of identifying compounds which inhibit GR-complex transactivation. The present invention relates to methods of evaluating the level of infection of individuals infected with human immunodeficiency virus (HIV). The present invention relates to transfection agent, particularly for the delivery of nucleic acid molecules or derivatives thereof into the nucleus of a cell.
BACKGROUND OF THE INVENTION
This application is related to U.S. patent application Ser. No. 08/309,644 filed Sep. 21, 1994, which issued as U.S. Pat. No. 5,763,190 on Jun. 9, 1998, the disclosure of which is incorporated herein by reference in their entirety. This application is related to U.S. patent application Ser. No. 08/167,519 filed Dec. 15, 1993, pending, and U.S. patent application Ser. No. 08/246,177 filed May 19, 1994, which issued as U.S. Pat. No. 5,639,598 on Jun. 17, 1997, the disclosures of each of which are incorporated herein by reference in their entirety. In addition, U.S. Ser. No. 08/019,601 filed Feb. 19, 1993, which issued as U.S. Pat. No. 5,874,225 on Feb. 23, 1999, U.S. Ser. No. 08/167,608 filed Dec. 15, 1993, and PCT Patent Application No. PCT/US94/02191 filed Feb. 22, 1994 are each incorporated herein by reference.
Primate lentiviruses, HIV-1, HIV-2 and-SIV, contain, in addition to the canonical gag/pol/env genes, six additional small open reading frames encoding gene products which assist in the viral life cycle. Among these the function of the 96 amino acid vpr protein remains poorly defined.
The vpr gene of HIV-1 encodes a 15 kD virion-associated polypeptide. Probably all the primate lentiviruses contain a vpr gene whose amino acid sequence is highly conserved within these groups. The conservation of the vpr open reading frame in evolution suggests that vpr carries a function which is nondispensable for the viral lifecycle in vivo, though potentially dispensable for replication in certain cell lines in vitro. The incorporation of vpr into virions (Cohen, et al., 1990a, Human immunodeficiency virus vpr, product is a virion-associated regulatory protein. J. Virol. 64, 3097-3099; Yuan, et al., 1990, Human immunodeficiency virus vpr gene encodes a virion-associated protein. AIDS Res. Hum. Retrovir. 6, 1265-1271) as well as its cellular co-localization with gag (Lu, et al., 1993, Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J. Virol. 67, 6542-6550; Paxton, et al., 1993, Incorporation of vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J. Virol. 67, 7229-7237; Lavallee, et al., 1994, Requirement of the Pr55gag precursor for incorporation of the Vpr product into Human Immunodeficiency Virus type 1 Viral Particles. J. Virol. 68, 1926-1934), has led to speculation that vpr performs a structural role in the virus particle. However, vpr deletion mutant viruses produce virions which appear normal in electron micrographs (Terwilliger, 1992, The accessory gene functions of the primate immunity viruses. in: AIDS Research Reviews, Vol. 2. Koff, W. C., Wong-Staal, F. and Kennedy, R. C. (New York, N.Y.: Marcel Dekker, Inc.) and vpr deletion mutant viruses remain infectious with somewhat lower replication kinetics in the majority of CD4+ T cell lines analyzed in vitro (Dedera, et al., 1989, Viral protein R of human immunodeficiency virus types 1 and 2 is dispensable for replication and cytopathogenicity in lymphoid cells. J. Virol. 63, 3205-3208; Shibata, et al., 1990, Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV
AGM
. J. Virol. 64, 742-747; Ogawa, et al., 1989, Mutational analysis of the human immunodeficiency virus vpr open reading frame. J. Virol. 63, 4110-4114; Cohen, et al., 1990b, Identification of HIV-1 vpr product and function. J. AIDS. 3, 11-18). The presence of vpr in the viral particle is consistent with its possible role early in infection. It has been suggested that delivery of vpr into cells by virus could increase cellular permissiveness to early events in virus replication.
The vpr gene of HIV-1 is sufficient to induce growth arrest and cellular differentiation in rhabdomyosarcoma and osteosarcoma cells which express it, either following infection with HIV-1 virus or by transfection of the vpr gene alone, indicating that vpr is a regulator of cellular events linked to virus production (Levy, et al., 1993, Induction of cell differentiation by human immunodeficiency virus 1 vpr. Cell. 72, 541-550). It has been reported that viral replication in macrophages can be inhibited by vpr antisense ribonucleotides (Balotta, et al., 1993, Antisense phosphorothioate oligodeoxynucleotides targeted to the vpr gene inhibit human immunodeficiency virus type 1 replication in primary human macrophages. J. Virol. 67, 4409-4414). In contrast to the kinetics observed in T cell lines in vitro, vpr deletion mutants are poorly infectious in myeloid lines in vitro (Westervelt, et al., 1992, Dual regulation of silent and productive infection in monocytes by distinct human immunodeficiency virus type 1 determinants. J. Virol. 66, 3925-3931; Nattori, et al., 1990, The human immunodeficiency virus type 2 vpr gene is essential for productive infection of human macrophages. Proc. Natl. Acad. Sci. USA. 87, 8080-8084) indicating an important function for vpr in infection of this lineage. In trans, the vpr protein increases virus replication in T lymphocytes and monocyte/macrophages in vitro (Levy, et al., 1994b, Extracellular vpr as a positive regulator of HIV-1 expression. (submitted)), although the effects on monocytes/macrophages was clearly greater. Importantly, nef-, vpr-mutant SIV replicated poorly in nonhuman primates (Lang, et al., 1993, Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus. J. Virol. 67, 902-912). The presence of vpr protein in serum of HIV+ individuals has been reported, and it has been demonstrated that purified serum-derived vpr reactivates HIV-1 replication in latently infected cell lines and in primary hematopoietic cells (Levy, et al., 1994a, Serum vpr regulates HIV-1 latency. Proc. Nat. Acad. Sci. USA. In press). The molecular mechanism by which vpr exerts its effects is not known. Transfection studies have shown that vpr is a weak transactivator of the HIV LTR and several other heterologous viral promoters, including HTLV-1, EBV, and CMV (Cohen, et al., 1990b, supra). These observations, and the fact that vpr is packaged into virions, have lead some to propose that vpr may function as an activator of viral mRNA transcription in the pre-transcriptional tat independent stage (Haseltine, 1991, Molecular biology of the human immunodeficiency virus type 1. FASEB J. 5, 2349-2360).
Glucocorticoid receptors are members of a superfamily of receptor molecules which are involved in the development, differentiation and general maintenance of homeostasis in lieu of a host of stimuli. These ligand-dependant transcription factors have been shown to upregulate the expression of reporter genes superseded by the LTR sequences of HIV as well as those from other retroviruses. In addition, glucocorticosteroids have been studied for their ability to reactivate HIV gene expression in nonproductively infected, or latent cell lines. This reactivation from latency could be prevented by the exposure of those latent cells to agents such as DHEA, a steroid with many functions, among which is the competitive blocking of the action of glucocorticosteroids. In addition, glucocorticoid response element (GRE) sequences have been shown to exist within the HIV LTR, by footprinting as well as by DNAse protection and gel retardation assays. However no direct link between the glucocorticoid bio

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