Gene-augmentation therapy cannot address autosomal-dominant (ad) disorders, in which a defective protein is expressed. To treat these mutations, such as the dominant form of rhodopsin (RHO)-related retinitis pigmentosa RP (adRP), the only cure is to silence (RNAi) or edit the mutant allele. Precise CRISPR therapeutic editing is predicted to be the best means of treating several dominant disorders, but molecular methods harnessing CRISPR have been slow to develop because homology-directed repair (HDR) occurs only during the S and G2 phases of the cell cycle. To overcome these limitations, we have established a versatile editing technology that uses 2 guide RNAs (gRNAs) to target all 150 dominant RHO mutations at any phase of the cell cycle. To conduct preclinical evaluation of this species-specific technology, we have replaced the nave mouse chromosomal Rho with human RHO mutant sequences (humanized RHO). If successful, the viral vectors used in our therapeutic editing strategy could be directly reused in human trials for adRP without further modification, thus avoiding FDA hurdles and accelerating translation of this research to a clinical setting. Our preclinical adRP model could also be useful for testing of future gene- and drug-based therapies. The proposed research is innovative, as it introduces new methods and model systems as the initial steps of a precision medicine approach toward developing adRP treatment. These methods are also potentially adaptable to therapies based on targeting DNA sequences of genes linked to other dominant disorders.
Autosomal-dominant retinitis pigmentosa (adRP) caused by defects in rhodopsin (RHO) is predicted to blind 10,000 Americans in 2019 and can be caused by 150 dominant RHO mutations. The development of mutation-specific guide RNAs (gRNAs) for CRISPR/Cas9 treatment of adRP is impractical because separate FDA approvals would be required for each gRNA. This proposal seeks to develop a novel and generalizable editing technology that has the potential to restore normal RHO function and could potentially be applied to treatment of other dominantly-inherited conditions in a mutation-independent manner.
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