Developing gene editing platforms for retinal degeneration.

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ABSTRACT Inherited retinal dystrophies (IRDs) are a heterogenous group of orphan diseases, inherited in an autosomal dominant, recessive or X-linked pattern in addition to mitochondrial transmission, all leading to the loss of functional vision and often progressing to blindness. As a group, IRDs are due to mutations in over 280 genes. Currently, there is only one FDA approved gene therapy for this large family of retinal degenerations. Prime editing, a new versatile genome editing tool, allows for all 12 base-to-base changes, insertions up to 44 bases long and deletions of up to 80 bases. Prime editors are capable of correcting 89% of known genetic variants associated with human disease, but are still in their infancy for in-vivo use. Our long-term goal is to optimize prime editing platforms for IRDs. Lipid based nanoparticles (LNPs) are a modular platform that can encapsulate and deliver genome editors. Delivering nucleases as mRNA has been an optimal alternative strategy for transient protein expression rather than persistent expression of DNA cutting machinery associated with viral vectors. LNPs are capable of rapid and efficient delivery of mRNA to the retinal pigment epithelium, however, they have limited capacity to transfect photoreceptors, which is necessary to target the many genes associated with IRDs. We hypothesize that by employing phage display techniques, we will isolate promising targeting peptides which will decorate our LNPs and effectively deliver prime editing cargo to the photoreceptors. Our main goal is to generate peptide-targeted LNPs that lead to cell-specific delivery of prime editing components for the treatment of IRDs. To achieve this goal, we propose the following specific aims: 1) Optimize in-vivo phage display biopanning for the identification of targeting peptide moieties that allow for photoreceptor-specific lipid nanoparticle-based gene delivery, 2) Dissect the mechanism of peptide-targeted LNP entry into photoreceptors, and 3) Evaluate the efficacy, and any associated toxicity, of prime editing in two rodent models of IRD. Thus far, we have identified novel peptides that can steer LNPs toward photoreceptor gene delivery and determined that LNPs can package all prime editing components together and lead to efficient prime editing of reporter genes in-vitro and in-vivo. Successful completion of this project will lead to the development of cell-specific gene editing platforms that will advance treatment for IRDs.