Title:  Methods for the identification of compounds capable of inhibiting HIV-1 viral replication employing murine cell lines expressing human topoisomerase I

United States Patent:  6,395,541

Assignee:  The Rockefeller University (New York, NY)

Filed:  March 11, 1997

The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target topo I for the treatment of HIV infection. The invention relates to therapeutic modalities and pharmaceutical compositions for the treatment of HIV-infection using human topo I and its interaction with HIV gag and RT as a target for intervention. The invention further relates to the use of human topo I to enhance the activity of RT. The present invention also relates to the expression of human topo I in transgenic animals, in particular mice, as a system to study the HIV life cycle and to screen agents for their ability to interfere with the HIV life cycle.

SUMMARY OF THE INVENTION

The present invention relates to therapeutic protocols and pharmaceutical compositions designed to target topo I for the treatment of HIV infection. The invention relates to therapeutic modalities and pharmaceutical compositions for the treatment of HIV-infection using human topo I and its interaction with HIV gag and RT as a target for intervention.

The present invention relates to animal cell lines expressing human topo I, in particular mouse cell lines, and their use as a system to study the HIV life cycle and screen agents for their ability to interfere with the HIV life cycle. The present invention also relates to human topo I transgenic animals, in particular mice, and their use as a system to study the HIV life cycle and to screen agents for their ability to interfere with the HIV life cycle.

The invention is based, in part, on the Applicants' surprising discoveries that (1) human topo I interacts with and is activated by HIV gag in a species specific manner; (2) the interaction between human topo I and gag is required to enhance HIV RT activity; and (3) the interactions between human topo I and HIV gag and RT are required to support HIV replication. This model is based on the Applicants' observation that murine cells expressing human CD4 are not able to support HIV replication. However, murine cells support HIV replication. The expression of human topo I was also shown to enhance the activity of HIV RT in murine cells.

That human topo I interacts with and is activated by HIV gag, and that this complex is required for activation of HIV RT is further supported by the working examples described infra which demonstrate (1) that gag proteins activate cellular topo I and immunoprecipitated-gag proteins induce topo I activity in a species specific manner; (2) mouse cells expressing both human CD4 and topo I infected with HIV effectively reverse transcribe the HIV RNA genome; and (3) the topo I inhibitor, TAN134A, which attacks the topo I site directly, inhibits HIV RT activity in murine cells.

The invention further relates to a murine model for HIV replication, in which transgenic mice expressing both the human CD4 cell surface protein and human topo I are able to support HIV-1 replication. The present invention also encompasses a murine model for HIV replication, in which transgenic mice express human topo I and a HIV pseudovirus is used to infect the animals. The HIV pseudovirus may contain an envelope protein from a virus with a natural tropism for murine cells, such as the murine leukemia virus, which bypasses internalization of the HIV virus by the murine cells. These transgenic mice have utility to screen for other host cellular components required to support the HIV life cycle (i.e., entry, replication and assembly), in addition to screen for drugs and compounds which may have anti-HIV activity.

The invention relates to various modalities of treatment for HIV infection based on the proposed model. The invention further relates to the use of the murine HIV model system for screening test compounds, such as drugs, ligands (natural or synthetic), proteins, peptides and small organic molecules for their ability to interfere with the interaction between human topo I and HIV gag and RT.

The present invention further relates to the use of such identified inhibitors in pharmaceutical compositions designed to inhibit human topo I and/or the interaction between human topo I and HIV gag and/or HIV RT for the treatment and/or prevention of HIV infection. The present invention further encompasses the preparation of such pharmaceutical compositions for the treatment and/or prevention of HIV infection.

The invention also encompasses combinations of a topoisomerase I inhibitor with a least one other antiviral having a different site of action than the viral replication inhibitor. Such a combination provides an improved therapy based on the dual action of these therapeutics whether the combination is synergistic or additive.

Claim 1 of 4 Claims

What is claimed is:

1. A mouse cell line which expresses a heterologous gene encoding human topoisomerase I, wherein expression of said gene facilitates the replication of an HIV pseudovirion containing a heterologous glycoprotein, wherein said glycoprotein serves to facilitate the entry of psuedovirions into said cells.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.