Intracellular production of ribozymes to specifically destroy HIV RNA targets represents a potentially exciting form of gene therapy against infection or activation of latent virus. The catalytic properties of several small ribozymes are under intense scrutiny in vitro, but there are a large number of parameters that will require systematic study in vivo if rational design criteria are to be formulated. These include efficient expression, stabilization, correct folding, cellular localization, and minor changes in annealing and catalytic properties under physiological conditions. In this proposal models for optimum ribozyme expression cassettes will be developed using the power of genetic selections in the yeast Saccharomyces cerevisiae. It is possible in yeast to select for efficient ribozyme function by targeting the ribozymes against one of several mRNAs and selecting against the target gene product. Two distinct RNA polymerase III promoters will be used to express mini-libraries of """"""""hammerhead"""""""" and """"""""hairpin"""""""" ribozyme variants directed against multiple target sites in mRNAs from three such genes, LYS2, URA3, and CAN1. Efficient destruction of either low or high copy target mRNA after transformation with the libraries can be demanded by growth on selective media. The properties of constructs producing high levels of bonafide ribozyme activity in vivo will be determined and used to formulate predictive models for appropriate expression cassettes and ribozyme structures. These general models will serve as a basis for constructing expression cassettes and ribozyme inserts for use against HIV-1 targets.
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