Genome Editing Therapy for Usher Syndrome Type 3

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PROJECT SUMMARY/ABSTRACT Usher syndrome type 3A (USH3A), an autosomal recessive disorder, is characterized by progressive loss of hearing and vision due to a clarin-1 (CLRN1) gene mutation. A person with type 3 Usher syndrome will usually require cochlea implants by mid- to late adulthood and classified as being legally blind. The management of the chronic, progressive, severe vision loss (retinitis pigmentosa, RP) in USH3A remains a challenge. CLRN1 expression in retina is finely regulated. AAV delivered gene augmentation therapy has shown efficacy in the inner ear of USH3A mice but shown detrimental effects on retinal function in wildtype mice, indicating the right combination of AAV vector dose, promoter, and delivery method needs to be well optimized to develop a safe gene therapy for USH3A RP. However, the expression level of AAV delivered gene expression is hard to control and is subject to gene silencing over time. The alternative approach of gene editing therapy can directly repair a defective gene in the genome, result in expression under endogenous regulatory element control, and may provide a promising therapeutic strategy for treatment of USH3A RP and other genetic eye disorders. Current gene editing tools suffer from off-target safety concerns and low repair efficiency. In 2020, our group reported a novel Cas9 variant, known as meticulous integration Cas9 (miCas9), with improved knock-in (KI) efficiency and dramatically reduced undesirable on- and off-target insertion/deletion events (indels). We believe the use of miCas9 strategy will provide the safety and efficacy to enable the CLRN1 gene correction in the retina. The lack of clinically relevant animal models is the bottleneck to developing effective therapeutics for RP in USH3A. The rabbit is a classic model to study eye diseases. We recently developed rabbit models carrying the CLRN1 frameshift (CLRN1−/−) and the CLRN1N48K/N48K point mutation. Preliminary characterization of these rabbits has shown that CLRN1 mutations lead to severe progressive vision and hearing degeneration. To our knowledge these are the first USH3A translational animal models that convincingly recapitulates the progressive vision and hearing degeneration phenotype. In this STTR FAST TRACK application, taking advantage of the novel miCas9 tools and the novel rabbit models we have established, we propose to develop a gene editing therapy to treat vision loss in USH3A. In Phase1, we will characterize the eye phenotypes of the CLRN1N48K/N48K rabbits(aim1) and develop a miCas9 mediated hCLRN1 cDNA targeted integration strategy that fit for all CLRN1 mutations(aim2). Through this Phase I work, we will establish the feasibility of gene editing in the USH3A rabbit retina. In Phase II work, we will test the safety and efficacy of this USH3A gene editing therapy in our USH3A rabbit model through subretinal injection, to provide the preclinical evidence of miCas9 mediated USH3A gene editing therapy. This new knowledge has the potential to advance a first-in-class clinical treatment for Usher Syndrome Type 3A.