Ribozymes are catalytic RNAs that can be targeted to specifically cleave selected RNAs. Such target RNAs could be viral RNAs or cellular RNAs. One of the goals of this study was to develop ribozymes against HIV-1 as part of a gene therapy in AIDS patients. Our second goal is to develop ribozymes for specific cellular mRNAs in the CNS. A specific combination of ribozyme target sites together with specific promoters for ribozyme expression could be employed in a reverse genetic approach in transgenic mice. Our plan is to generate specific phenotypes in transgenic mice to cause neuronal dysfunction by multitarget-ribozymes in order to study neuronal function. We have previously developed multitarget-ribozymes that cleave HIV-1 RNA at up to nine different sites, and protect cells from HIV-1 pathogenesis in tissue culture. Our data suggest that multitarget-ribozymes are functional in cell nuclei and they cleave unspliced HIV-1 RNA. In addition, we found that the same multitarget-ribozyme transcript can be translated in the cytoplasm. This strongly suggests that a highly effective negative transdominant mutant HIV-1 Rev protein could be expressed from the same mRNA that also functions as a multitarget-ribozyme. We are currently recloning our HIV-1 multitarget-ribozyme for insertion into a retrovirus vector (MMLV). If the combination of multitarget-ribozyme and transdominant negative HIV-1 Rev protein increases antiviral efficacy against HIV-1, these vector may be included in clinical trials with HIV-1 infected and uninfected twins. For our initial studies of multitarget-ribozyme effectiveness in transgenic mice, we have recently synthesized two octaribozymes, one against the ICP4 protein mRNA of herpes simplex virus and the other against the beta-galactosidase protein mRNA. The efficacy of these ribozymes will initially be tested in tissue culture and subsequently in transgenic mice. The purpose of these studies will be to rule out toxicity of multitarget-ribozymes in vivo. Secondly, these transgenic mice may resist herpes virus neuropathogenesis because of the multitarget-ribozyme. The octaribozyme against the beta-galactosidase will be used to inhibit beta-galactosidase expression in select neurons of the hippocampus of already existing transgenic mice. If multitarget- ribozymes are highly effective in vivo, they could complement gene knock- out experiments for the generation of mouse models for neuronal dysfunction.
