SUICIDE OF HIV-INFECTED CELLS BY TAT-INDUCIBLE SHRNA -
DESCRIPTION (provided by applicant): The goal of the proposed project is to generate the pre-clinical data necessary to provide proof-of-principle for future clinical studies of pro-apoptotic lentiviral vector (LV) gene therapy using small hairpin RNA (shRNA) to target "HIV-Associated Life Preserver" (HALP) and ring finger protein 7 (RNF7), 2 of the crucial anti-apoptotic genes in acquired HIV infection. HIV/AIDS is an ideal candidate for a novel gene therapy approach since it is an incurable and terminal disease. Although highly active anti-retroviral therapy has significantly improved the survival of HIV-infected individuals, the duration of response is limited by development of drug-resistant viruses, long-term toxicities and a substantial reservoir of latently infected T-cells. The long half-life of latent resting memory CD4 T-cells renders HIV intrinsically incurable with the treatment regimens currently available. Therefore, there is an urgent need to explore novel molecular and genetic strategies to slow or halt HIV-1 replication and to prevent viral latency. We hypothesize that anti-apoptotic genes like HALP and RNF7 are among the major contributors to survival of HIV-infected cells and latent infection. These cells are resistant to HAART therapy. We predict that by silencing these genes and inducing apoptosis, infected cells will be killed, and viral load and latent infection will decrease. During Phase I of this STTR application we will test our hypothesis through 3 aims. In Aim 1, we will generate LV vectors expressing an inducible short hairpin RNA (shRNA), directed against anti-apoptotic gene products HALP and RNF7, but inducible only in HIV-1 infected cells. These LV vectors will be easily scalable for later clinical use. After performing quality control, vectors will be tested in transduction protocols using primary T-cells and hematopoietic progenitor cells. In Aim 2 we will test whether TAT-inducible shRNAs, directed against HALP and RNF7 anti-apoptotic gene products, inhibit expression of these genes in HIV-1 infected cells, leading to death of infected cells and decreased viral load and latent infection. In Aim 3, we will transduce CD4 T-cells and cord blood CD34+ cells with the lentiviral constructs, adoptively transfer the transduced cells to SCID/NOD mice, and assess engraftment and in vivo vector persistence. The results of the proposed experiments will serve as a basis for future clinical investigation of this approach as a novel therapy focused on killing of infected and latent cells, and induction of decreased viral load during HIV/AIDS infection. This proposal focuses upon developing a novel immunotherapy strategy for HIV/AIDS. Approximately 40 million people worldwide are infected with HIV; 1 million people in the United States alone. The numbers have reached epidemic proportions. Triple cocktail drug therapy (HAART), once believed to have great promise in effectively combating the AIDS virus, has not proven to be a cure for AIDS. Moreover, resistance to this drug therapy is increasing; and thus it has become apparent that drug therapy is not the solution for controlling an AIDS epidemic. Therefore, there is an ultimate need for novel therapies. Here we propose to develop novel therapy that is focused on silencing 2 genes in HIV-infected cells which when overactive contribute to prolongation of HIV infection and inability of drugs to kill infected cells.
