Development of effective anti-viral agents targeted to the HIV viruses which are specific and yet non-toxic to the host cells has been a difficult task. This proposal seeks to investigate and develop the anti-viral activity of catalytic RNAs (ribozymes) complementary to specific regions of the HIV-1 genome. This new and exciting form of anti-viral agent has great potential in both HIV-1 as well as other viral therapies since it can be designed to specifically destroy only the targeted viral RNAs with no toxicity to the host. The catalytic RNAs proposed in this study have already been demonstrated to effectively, and specifically cleave HIV-1 transcript in vitro. It is the intent of the proposed studies to develop (ribozymes) which are functionally active in vivo for use as anti-HIV therapeutic agents. In order to develop such ribozymes, we propose to take advantage of a powerful quantitative assay in a facile genetic organism, the yeast Saccharomyces cerevisiae. The ribozyme target will be a segment of the HIV-1 genome fused to a yeast promoter and the E. coli lacZ gene. This fusion will allow functional beta-galactosidase expression which can be readily quantitated. Upon expression of the inducible ribozyme targeted to the HIV-1 sequences, the hybrid mRNA will be cleaved and inactivated. The amount of beta-galactosidase activity remaining will provide a quantitative basis for ribozyme activity. A number of modifications in ribozyme structure and modes of expression will be analyzed in this system. Concomitant with the results from yeast, a transient mammalian cell assay system will be utilized to test those ribozymes which are functional in the yeast cells. Ultimately, the most stable and active ribozymes will be cloned into mammalian expression vectors, transfected into human cells capable of infection by HIV-1. Our long range goal is to successfully immunize an animal against HIV-1 infection with a ribozyme.
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