We propose to conduct research designed to adapt the ribozyme approach to provide a transient or permanent """"""""knockout"""""""" of a target protein (and its associated function) by cleaving the appropriate mRNA with high specificity. This approach is one which is already showing promise in the antiviral field, where a number of advantages for it have emerged. RNA- level approaches in general are succeeding in antiviral therapy for hepatitis B virus, for example; the ribozyme approach is most promising because one ribozyme can attack a number of RNAs because of its catalytic/enzymatic properties. The other major approach-antisense inhibition--works at a 1:1 level. We propose to adapt approaches which are showing promise in the antiviral field to key cellular RNAs, including mRNAs for mouse delta opioid receptors (DORs) and glutamate receptor channel subunits (NMDAs) in test tube experiments using cloned mRNA expression vectors; and in cultured mouse nerve cells, specifically the NGl08-l5 cell line. We plan to test the hypothesis that the ribozyme technique, using either delta hepatitis agent ribozymes retargeted to the relevant mRNAs; or external guide sequences (EGSs) which re-target the cellular tRNA processing enzyme RNase P to exogenous sequences, can provide a transient """"""""knockout"""""""" of a targeted mRNA, and its associated functional protein. The cellular approach is the logical endpoint of the initial research effort, with work in animals to follow. The collaborative efforts of the Robertson Lab (RNA structure/function; ribozyme development) and the Inturrisi Lab (mRNA and cell characterization; RNA detection) have already led to several publications in the field of RNA quantitation. We will collaborate on testing the above mRNAs, comparing the ribozyme approaches mentioned to parallel efforts using antisense deoxyribonucleotides. In this way we should be able to introduce these approaches into the field of drug abuse research, where they should allow an increase in systematic studies of the effects of expression involving various receptor genes.
