Development of an AAV-CRISPR system for inner ear gene therapy

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DESCRIPTION (provided by applicant): Hearing loss in humans has a strong genetic component, and developing gene therapy strategies aimed at repairing deafness causing mutations represents a major focus of auditory research. Here, we propose to apply the Clustered Regularly Interspaced Palindromic Repeat (CRISPR)/Cas technology towards the development of a gene therapy platform for the inner ear. In preliminary studies, we have demonstrated the efficiency of the CRISPR system in modifying several deafness genes in cell lines and mouse models, and are pioneers in applying this technology to deafness research. Its applicability for gene therapy, however, faces several hurdles, such as off-target effects and lack of suitable delivery methods. We expect that the proposed project will make significant inroads into the latter issue: The specific goals of this proposal are 1) to engineer a novel, modified CRISPR system that allows packaging into an adeno-associated vector (AAV), which presently is the only system capable of in vivo delivery of genetic material into adult sensory hai cells, and 2) to apply this tool to repair deafness-causing mutations in the mouse model. An efficient and safe application of the CRISPR technology for gene therapy requires several innovations to the CRISPR system. We are confident that our strategy of size-modifying the Cas9 gene and exploration of CRISPR components from different prokaryotic species will enable the engineering of a CRISPR system adaptable for the AAV system. The successful execution of this project will result in the development of an efficient tool for delivering the CRISPR system into inner ear cells, and in proof-of-principle demonstration that CRISPR-mediated, targeted gene therapy is possible in a living animal. This is a highly significant contribution not only for deafness research, but for biomedical research in general.