Gene Silencing and Gene Editing in Phototransduction

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 DESCRIPTION (provided by applicant): Retinal degenerative diseases affect 9 million Americans. Among these conditions, retinitis pigmentosa (RP) is among the most devastating. Mutations in genes encoding subunits of the rod-specific enzyme, cyclic guanosine monophosphate (cGMP) phosphodiesterase 6 (PDE6A and PDE6B), are responsible for approximately 72,000 cases of RP worldwide each year, making therapeutic modeling highly relevant to developing mechanisms based therapies. In both RP and age-related macular degeneration (AMD), progressive atrophy of rod photoreceptors leads to secondary death of cone photoreceptors. Gene therapy could enhance rod viability and prevent secondary cone loss. These FDA trials used wild-type (wt) gene supplementation (i.e., overexpression of a normal version of the gene) in diseased cells to overcome the abnormality of the patients' mutated genes. The first human gene therapy trial for early onset retinal degeneration found visual function recovered initially, but did not retard the rate of photoreceptor degeneration, and the gene therapy treated RP patients now continue their march toward blindness. The failure of FDA trials to attenuate the progression of rod death suggests that there is a "point of no return" for rod viability in retinal disease and presents a major obstacle to the treatment of RP. We hypothesize that this retinal "point of no return" derives from 1) changes in Ca2+ homeostasis and 2) inadequate activation of the mammalian target of rapamycin (mTOR) self-survival pathway. Preliminary data suggest that the "point of no return" could be halted by administering two therapeutic constructs via bipartite vectors in an autosomal recessive PDE6-RP model (arRP) (Aims 1 and 2). In photoreceptors, incoming light activates PDE6, which hydrolyzes free cGMP. Lower free cGMP levels close cGMP-gated Na+/Ca2+ cation (CNG) channels in the plasma membrane, reducing cation influx and propagating nerve impulses. Aims 1 and 2 test therapeutic strategies aimed at remedying PDE6 deficiency. Specifically, shRNA knockdown will be used to identify therapeutic targets using two novel bipartite AAV8 vectors described in detail herein. Finally, wt gene supplementation leaves the patient's mutant genes intact, which could continuously trigger ongoing damage despite the presence of a wt gene in a diseased cell. A gene editing approach could overcome this defect. Thus, Aim 3 explores in vivo AAV mediated CRISPR-gene editing in PDE6-RP models available on campus: 3a) the Pde6a mutant mouse; and 3b) human stem cells from an RP patient bearing the PDE6A mutations (OMIM# 180071), which offers an in vitro model for comparing CRISPR efficacy in human cells vs. Pde6a mouse retina.