Preclinical development of hammerhead ribozyme gene therapy for autosomal dominant retinitis pigmentosa

Post-transcriptional gene silencing (PTGS) offers therapeutic potential for gene therapy of hereditary retinal degeneration. The human rod opsin (RHO) gene harbors many mutations responsible for autosomal dominant Retinitis Pigmentosa (adRP), making it a candidate disease target for PTGS gene therapy. The major bottleneck in the development of therapeutic PTGS technologies (including ribozymes, antisense, and RNA interference) is the identification of rare sites in target messenger RNAs (mRNAs) that are sensitive to PTGS in live cells. A comprehensive PTGS agent design strategy was developed to systematically evaluate RHO mRNA for potential target sites sensitive to PTGS in human cells in order to develop an efficacious PTGS therapeutic agent.;A major reason for the insensitivity of target mRNA sites arises from the dense secondary and tertiary structure of full-length mRNAs in physiological conditions. Rational design of PTGS agents involved detailed computational analysis to estimate secondary structure accessibility of full-length human RHO mRNA transcripts. A cell-based screening system was developed using engineered secreted human alkaline phosphatase (SEAP) as a reporter of gene expression to experimentally measure cellular efficacy of PTGS agents targeting predicted accessible sites. Combining rational RNA drug design with a cell-based screening platform allowed for rapid identification of a novel target region in RHO mRNA. Optimization of PTGS agents targeting the 725 site resulted in highly efficacious PTGS agents. The hhRz agent developed from the results of this study represents the single most efficacious RHO-targeting hhRz to date.;Intragenic heterogeneity in autosomal dominant disease represents a significant hurdle in the development of therapeutic gene modulation treatments. Over 150 different mutations have been identified in RHO-linked adRP. A mutation independent strategy of decreasing disease gene expression using the optimized PTGS agents combined with the reconstitution of wild-type gene resistant to the PTGS agents was designed and characterized in human cells. The success of this type of mutation independent "knockdown/reconstitute" gene therapy is highly conditional upon the efficacy of the PTGS agent in reducing mutant gene expression, and the systematic analysis of RHO mRNA in this study suggests that the novel 725 target site is the optimal site for PTGS agent targeting.