Title: Therapy and mechanism of Mendelian eye diseases
Description: Retinal degenerative diseases cause varying degrees of irreversible vision loss in millions of people worldwide. Common to all retinal degenerative diseases is the malfunction or demise of photoreceptor cells or its supportive cells, retinal pigment epithelium cell in the retina. A considerable part of these diseases were resulted from the inherited mutations of essential genes expressed in these retinal cells. The understanding of pathologic mechanism as well as developing of therapeutic treatment for these diseases were discussed in this study. A cutting-edge therapeutic genome editing technology is studied in the first part of study. This technology was invented to treat retinitis pigmentosa via engineered nucleases, which has great clinical potential for autosomal dominant genetic disorders that were previously irreparable by conventional gene therapy interventions. Though customizable gene editing tools can be engineered to target specific mutation sites, however it is too daunting for diseases like retinitis pigmentosa, a progressive retinal degenerative condition associated with more than 150 mutations in the rhodopsin gene alone.

Here in this study, we present an “ablate-and-replace” combination strategy that 1) destroys expression of the endogenous gene by CRISPR/Cas9 in a mutation-independent manner, and 2) enables expression of wild-type protein through exogenous cDNA. As proof of concept, we show that our CRISPR-based therapeutic machinery efficiently ablates mRho in vivo, and when combined with gene replacement therapy, ameliorates rod photoreceptor degeneration and improves visual function in two genetically distinct autosomal dominant retinitis pigmentosa animal models. This mutation-independent, ablate-and-replace strategy represents the first electrophysiological recovery by a CRISPR-mediated therapy in an autosomal dominant disorder and it offers a clinically relevant, universal strategy to overcome allelic heterogeneity in debilitating inherited conditions. For the second part of the study, gene editing technology was used to study the pathogenesis of Doyne honey comb dystrophy, another Mendelian disease with extensive similarities to age-related macular degeneration.

This monogenic disorder is caused by a unique point mutation on an extracellular matrix protein EFEMP1, expressed by retinal pigment epithelium cell. To precisely gauge the physiological effect resulted from this mutation, CRISPR-mediated gene correction was used to create isogeneic cell pairs from patient donated tissue-derived stem cells. These stem cells were differentiated into retinal pigment epithelium cell before analysis. We found unfolded protein response and immune response were not involved in the pathogenesis, which contradicts existing theories. Via proteomics analysis, we found expression level of a cholesterol catabolic enzyme was affected by the EFEMP1 mutation while those proteins controlling the cholesterol transport remains constant. This result provides supportive evidence to explain the aberrant intracellular accumulation of cholesterol found in patient retinal pigment epithelium cells. This imbalance in lipid homeostasis also suggests Doyne honey comb dystrophy is a retinal pigment epithelium cell-autonomous disease.