Optimization of a Rhodopsin Targeting Ribozyme Knockdown-Reconstitute Strategy for Autosomal Dominant Retinitis Pigmentosa
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Autosomal Dominant Retinitis Pigmentosa (adRP) is a progressive retinal degenerative disease that leads to blindness, with around 400,000 cases worldwide. Mutations in the gene coding for Rhodopsin are responsible for around 30% of cases, and to date over 200 unique mutations in Rhodopsin have been identified to cause the disease with varying phenotypes of vision loss. A main way vision is impaired is as misfolded Rhodopsin protein forms aggregates that kill the cells of the retina. This gain of function disease is an excellent candidate for Post-Transcriptional Gene Silencing (PTGS) therapy. PTGS therapy knocks out expression of toxic protein by decreasing the amount of mutated RNA expressed. The Sullivan lab has historically developed ribozymes, RNA enzymes that are capable of cleaving targeted mRNAs, designed against wildtype Rhodopsin mRNA (RHO) in a Mutation Independent (MI) manner. Design of a therapeutic that targets wildtype and mutant RHO allows for a therapeutic that will be effective for a larger amount of the patient pool. To avoid haploinsufficiency in patients, a Knockdown-Reconstitute (KdR) strategy in which a reconstituted RHO vector that creates wildtype protein but is immune to ribozyme cleavage is used. The work described in this manuscript based PhD dissertation utilizes RNA secondary structural modeling techniques to analyze target RHO structure in evolutionary relationships, during natural processing found in vivo, and unique mutations in adRP patients. Data from studying target RHO sequence and structure was then used to both optimize ribozyme binding and cleavage in vitro and to create ribozyme immune reconstituted RHO for use in the KdR strategy.