This laboratory has actively pursued the development of ribozymes as potential therapeutic agents for the treatment of HIV-1 infection for the past several years. Recently, ribozyme efficacy was reported against HIV tat and rev targets in retrovirally transduced human T-lymphocytes. Although our pre-clinical studies have demonstrated the feasibility of successfully using ribozymes for the treatment of HIV-1 infection, they also point to the need to continue to improve ribozyme efficacy. The overall goal of the SPIRAT program proposal, of which this project is a part, is the """"""""Transduction Of Hematopoietic Stem Cells Using Ribozymes for Aids."""""""" The goal of this project is the development of a highly effective ribozyme treatment for HIV-1 infection. Project objectives are to improve ribozyme efficacy through (1) basic studies of ribozyme trafficking and (2) information derived from analyses of ribozyme expression and function in a human HIV-1 infected patient population. First, ribozyme trafficking studies will improve ribozyme efficacy by increasing the likelihood that ribozymes and their RNA targets will be present at the same place and time in the complex intracellular environment. The strategy for achieving this co-localization of ribozyme and target will be to use our knowledge of RNA trafficking and localization signals encoded in both cellular and viral RNAs. These signals dictate pathways for RNA travel and final destination in the cell. For this project, several known RNA trafficking and localization signals will be appended to anti-HIV-1 ribozyme transcripts and evaluated for their effect upon anti-HIV-1 ribozyme activity. The most efficacious ribozyme-cis-appended sequences will be incorporated into retroviral and AAV vectors for transduction into human CD34+ cells, and ultimately used in the treatment of HIV-1 infected patients. Second, analyses of anti-HIV-1 ribozyme expression and function in human bone marrow cells, before and after introduction into HIV-1 infected patients, will provide critical insights into ribozyme efficacy in the host environment. The strategy will be to use sensitive, quantitative PCR methodologies to detect ribozyme genes, transcripts and HIV-1 sequences. These methods will not only facilitate the rapid evaluation of ribozyme function in vivo but will also provide valuable information regarding vector transduction frequency.
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