The specific aims of this proposed supplement reflect the original aims of grant DA-10337, funded in June of 1996: (1) Using ribozyme motifs known to function in mammalian cells, we will develop a set of targeted ribozymes and external guide sequences (EGSs) to achieve cleavage of the DOR and NMDA receptor mRNAs. In the supplementary work, we will expand this approach to sites in HIV mRNA sequences which can be cleaved by the ribozyme approach. (2) In both the original and supplementary work, we will test and compare these ribozyme and EGS elements in test tube experiments, using both purified components and subcellular extracts, in order to selected the most promising candidates for introduction into cells. In contrast to the approach with the cellular receptor mRNAs, we will emphasize ribozyme motifs and guide sequence targeted to HIV mRNA which can be synthesized chemically and delivered to cells without the use of vectors. (3) In original proposal for a three-year study, we planned to introduce sequences encoding ribozyme and EGS motifs into the cellular environment, using a retroviral vector system and the cultured neuroblastoma cell line NG108-15, and these experiments remain on track. However, it is unlikely that the research period of the supplement (less than two years) will allow this aim to be pursued in the case of HIV anti-viral ribozymes for the direct uptake of synthetic RNAs by cells and their transport to the nucleus. As in the original research, we will take advantage of the techniques for RNA quantitation which have been worked out over the past several years in the Robertson and Inturrisi laboratories. The long-term goal of these experiments is to reach the point where these ribozymes elements derived from human cells can be introduced into cells and animals, in order to compare their effectiveness in achieving anti-HIV effects, with those obtainable by techniques using DNA antisense oligonucleotides or vector-drive delivery of ribozymes from the plant kingdom.
