Development of Genome Editing as Treatment for Genetic Hearing Loss

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Project Summary and Relevance Genetic hearing loss affects one in 1000 newborns and contributes significantly to general populations with hearing loss. Over 130 deafness loci have been mapped and more than 80 have been cloned. Despite the tremendous progress in deafness gene discovery, there is no medical treatment for genetic hearing loss. There is an urgent and unmet medical need to develop treatment for genetic hearing loss. CRISPR/Cas9-mediated genome editing is transforming biomedical research and with a promise of becoming new treatment of disease. It enables the application of nuclease with guide RNA to pair with and modify DNA permanently, which can be developed into new therapies for wide range of diseases. We have successfully used transient and in vivo local delivery of editing agents to treat a mouse model, Beethoven, of human dominant hearing loss DFNA36. In this application, we propose two broad aims to further develop CRISPR technology for inner ear editing and to apply it to treat genetic hearing loss. First, we will characterize CRISPR nuclease variants SaCas9 and Cpf1 for hair cell editing by direct RNP (ribonucleoprotein) delivery. This study will expand deafness mutations to be targeted by genome editing due to additional PAM (protospacer adjacent motif) sequences and nuclease activities, with a possibility of improvement in editing efficiency and specificity. We will apply whole-genome application (WGA) using purified hair cells for high- throughput sequencing (HTS) and to identify insertions and deletions (indels) in hair cells, and will correlate hair cell editing efficiency with the outcome of hearing rescue. We will evaluate editing in mature hair cells by RNP delivery, which is relevant to treatment in patients as human inner ears are fully mature. We will screen new nanoparticle based liposomes to provide the options for efficient inner ear delivery and editing. In second aim, we will perform hearing rescue studies by RNP delivery of editing agents to disrupt mutations in mouse models of human dominant deafness, including 1). Hearing rescue by editing Oblivion (Obl) mutation in the Pmca2 gene that affects outer hair cells; 2). Hearing rescue by editing a mutation in the microRNA 96 (Mir96) that results in delayed onset progressive hearing loss. Hearing rescue in those models will demonstrate general therapeutic application of genome editing targeting dominant mutations of hair cell origin. The study will delineate the relationship between editing efficiency, specificity and off-target effect with the extent of hearing rescue and evaluate long-term rescue effect and the outcome of intervention at late stages. The proposal has potential to be developed as new platform for genome editing based therapy for genetic deafness. Genetic hearing loss affects large number of children and contributes to adult deafness with no treatment available. We are developing CRISPR/Cas9-mediated genome editing as a new treatment platform to target dominant mutations by transient local delivery into inner ear, with the potential to treat diverse forms of genetic hearing loss.