AAV-mediated editing to treat human autosomal dominant hearing loss DFNA41 and DFNA2

Project Summary/Abstract 1 in 500 newborns suffers from genetic hearing loss (HL), and there are no FDA-approved drugs or biological treatments for any type of HL. Genome editing that disrupts or repairs DNA mutations or modulates RNA levels is being developed

into new therapies for many diseases. Despite the unprecedented progress, the translation of genome editing into therapy

in humans has been challenging due to the obstacles in delivery, safety, and efficacy. This is particularly relevant for in

vivo editing therapy due to the added complexity, including damped editing efficiency, inefficiency in reaching the target

cells, and the lack of evidence of translatability from animal (mouse) into humans. In this proposal, we will develop first- in-class CRISPR-Cas-based therapeutics for genetic hearing loss by conducting IND-enabling studies of AAV2-mediated delivery of SaCas9-KKH/gRNA to target two dominant genetic hearing loss, DFNA41 due to a P2RX2 mutation and

DFNA2 due to a KCNQ4 mutation. This project will be conducted by a consortium of five core teams with unique and complementary expertise and experience in gene and editing treatments for genetic hearing loss, from animal models to

human trials, editing technology, and regulatory processes. We have recently conducted the first successful gene therapy

of OTOF clinical trial by dual adeno-associated virus (AAV) that leads to hearing restoration in children with congenital genetic hearing loss DFNB9, which included a safety study of AAV in non-human primates (NHPs) and the development of a minimally invasive surgical procedure for AAV inner ear delivery in humans. We have further developed a large

animal pig model of dominant genetic hearing loss DFNA36 by inserting a mutation into the TMC1 gene and successfully rescued hearing in the pig model by local AAV delivery of editing complex, demonstrating the cross-species translatability of editing therapy for genetic hearing loss. For the current proposal, we have obtained the therapeutic

benefit of hearing rescue results by AAV2 delivery of SaCas9-KKH/gRNA to treat DFNA41 and DFNA2 mouse models, which lays the foundation of an IND-enabling study. Leveraging our unparalleled expertise and experience in human genetic hearing loss, including efficacious editing treatment in mouse and pig models, AAV safety study in NHP, and the

first successful human gene therapy clinical trial, we will conduct the following studies: 1). Establish off-target editing profiles in human cells and human inner ear tissues; 2). Efficacy study by escalating doses of AAV2 in DFNA41 and DFNA2 mouse models; 3). Biodistribution and toxicity study in WT mice by an escalating dose of AAV2; 4).

Biodistribution and toxicity study of GLP grade AAV2 in NHP; 5). Patient natural history study, and 6). Regulatory Interactions & IND completion. We have assembled a team of experts in each area with a project manager to execute the plan, develop a streamlined regulatory path for two types of DFNA deafness, and disseminate this regulatory information

to the scientific community. Successful completion of the IND-enabling studies makes our approach applicable to over 20 dominant genetic hearing loss due to hair cell mutations.