The human rod opsin gene harbors many mutations that cause retinitis pigmentosa (RP). The folded opsin mRNA is a target for ribozyme (Rz) gene therapy for autosomal dominant (ad) RP. Studies have shown that folded mRNAs with hundreds of potential cleavage sites have few accessible regions that permit robust Rz catalysis. The long-range goal is to establish a rapid means of generating active Rz designs as candidate gene therapies for any ad mutant human hereditary retinal degeneration allele. The objective is to build active Rzs that cut human rod opsin adRP mRNAs for potential use in clinical trials. The central hypothesis is that the folded structure of opsin mRNA will shield most potential sites from Rz cleavage. Finding the best cleavage sites requires methods to screen many potential hRz designs against precise models of folded human mRNA. A combination of folding algorithms to predict mRNA structural accessibility, and empirical screens of Rz libraries against naked or protein-coated folded opsin mRNA provide for an optimized research strategy. Active Rz genes so identified can be cloned into expression constructs to determine if the Rz can identify and cleave its target along the normal mRNA processing/trafficking streams when expressed in cultured cells. Designs passing this hierarchial test will be tested for rescue and toxicity in murine retinal degeneration models that express human opsin alleles. The rationale is that in vitro and cell culture systems will permit rapid, high-throughput identification of efficacious Rz designs that function to cleave target human mRNAs transcribed from arbitrary dominant disease alleles in vivo. To test the central hypothesis and accomplish the objective three Specific Aims will be pursued:
Aim 1. Locate the most accessible regions in folded human rod opsin mRNA.
Aim 2. Develop efficacious Rzs to cleave NUH( sites in accessible regions of opsin mRNA.
Aim 3. Conduct a preclinical test of the Knockdown Rz strategy by testing for rescue of retinal degeneration and toxicity in murine models that express human rod opsin transgenes. The proposed work is innovative in that it develops an approach to rapidly accomplish candidate Rz gene therapy agents for new arbitrary human dominant disease alleles, and it provides the first known preclinical test of the knockdown Rz strategy for one known adRP disease allele. The expected results are that the folded structure of rod opsin mRNA will severely constrain the number of accessible Rz cleavage sites, that highly active Rzs can be constructed that have marked impact on opsin expression, and that the knockdown strategy or related modifications will rescue retinal degeneration in an animal model. The significance is that efficacious knockdown Rzs generated by this work, which demonstrate both rescue from degeneration and lack of photoreceptor toxicity, could then be translated into human clinical trials for adRP.
