Usher syndrome type 3A (USH3A), an autosomal recessive disorder, is characterized by progressiveloss of hearing and vision due to a clarin-1 (CLRN1) gene mutation. A person withe 3 Usher syndrome willusually require cochlea implants by mid- to late adulthood and classified as being legally blind. Themanagement of the chronic, progressive, severe vision loss (retinitis pigmentosa, RP) in USH3A remains achallenge. CLRN1 expression in retina is finely regulated. AAV delivered gene augmentation therapy hasshown efficacy in the inner ear of USH3A mice but shown detrimental effects on retinal function in wildtypemice, indicating the right combination of AAV vector dose, promoter, and delivery method needs to be welloptimized to develop a safe gene therapy for USH3A RP. However, the expression level of AAV deliveredgene expression is hard to control and is subject to gene silencing over time. The alternative approach of geneediting therapy can directly repair a defective gene in the genome, result in expression under endogenousregulatory element control, and may provide a promising therapeutic strategy for treatment of USH3A RP andother genetic eye disorders. Current gene editing tools suffer from off-target safety concerns and low repairefficiency. 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-targetinsertion/deletion events (indels). We believe the use of miCas9 strategy will provide the safety and efficacy toenable the CLRN1 gene correction in the retina. The lack of clinically relevant animal models is the bottleneck to developing effective therapeutics forRP in USH3A. The rabbit is a classic model to study eye diseases. We recently developed rabbit modelscarrying the CLRN1 frameshift (CLRN1â/â) and the CLRN1N48K/N48K point mutation. Preliminary characterizationof 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 theprogressive vision and hearing degeneration phenotype. In this STTR FAST TRACK application, taking advantage of the novel miCas9 tools and the novelrabbit models we have established, we propose to develop a gene editing therapy to treat vision loss inUSH3A. In Phase1, we will characterize the eye phenotypes of the CLRN1N48K/N48K rabbits(aim1) and develop amiCas9 mediated hCLRN1 cDNA targeted integration strategy that fit for all CLRN1 mutations(aim2). Throughthis 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 throughsubretinal injection, to provide the preclinical evidence of miCas9 mediated USH3A gene editing therapy. Thisnew knowledge has the potential to advance a first-in-class clinical treatment for Usher Syndrome Type 3A.
Public Health Relevance Statement: PROJECT NARRATIVE Lack of a clinically relevant animal model restricted the development of therapeutics for Usher Syndrome Type 3A (USH3A). To address this challenge, we created a CLRN1 gene mutant rabbit model showing early onset, and progressive retina degeneration mimicking the Retinitis Pigmentosa symptoms in USH3A patients. Using our novel Cas9 variant (miCas9), known to show lower off-target effects and higher KI efficiency, in this rabbit model, we propose to evaluate a gene editing therapy in the eye for USH3A by insertion of the coding region of the clarin1 gene after its natural promoter to treat any causative clarin1 mutation, potentially altering the progressive vision loss that occurs with this disease.
Project Terms: <21+ years old>
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