Twelve years into the AIDS epidemic, satisfactory treatment for this disease is still elusive. Our current understanding Of AIDS pathogenesis calls for early intervention with antiviral agents. Although still in its infancy, human gene therapy holds considerable promise for the long term treatment of genetic disorders, cancer and chronic infectious diseases, including AIDS. We have demonstrated previously that a hairpin ribozyme targeting the 5'-leader sequence of HIV-1, when expressed stably in Jurkat cells under the control of the pol Ill tRNA promoter, is able to confer long term resistance to infection by diverse strains of HIV-1,including an uncloned clinical isolate. Furthermore, transduction of primary lymphocytes with the ribozyme gene also confers long term resistance to infection. While successful transduction of T-cells with an HlV inhibitory gene may confer some therapeutic benefits to patients, gene therapy into pluripotent hematopoietic stem (progenitor) cells would be a preferred strategy to achieving sustained immune reconstitution.
The specific aims of this project are: (1) Development of anti-HIV ribozyme gene therapy for CD4 and CD34 cells. We will optimize expression of ribozyme genes delivered in retrovirus vectors in PBL cells and enriched CD34+ cells. To attain greater virus inhibition and to diminish the chance of generating """"""""escape"""""""" mutants, we will construct retrovirus vectors expressing ribozymes against more than one targets. (2) Evaluation of in vivo efficacy of ribozyme gene therapy in a SCID mouse model-Human PBL and CD34+ cells transduced with retrovirus vectors expressing ribozyme will be reconstituted into SCID mouse, which will be challenged with diverse strains of titered HIV-1 and monitored for ribozyme expression, virus burden and CD4 cell count at various time intervals. (3) Development of HIV-2 ribozymes- We will optimize expression and evaluate the efficacy of HlV-2 ribozymes, not only to demonstrate a general applicability of the approach, but also to have the option of testing the ribozyme gene therapy in a primate model, if warranted. We will collaborate with Dr. Anthony Ho (Director, stem cell and bone marrow transplant program, UCSD) and Dr. Don Mosier (Scripps Research Foundation) to conduct these studies.
