Hearing loss affects 15-26% of the world's population. Amongst the elderly, it ranks as the most common neurological disability, impacting ~50% of octogenarians. The majority of inherited late-onset deafness is autosomal dominant and non-syndromic. In this new porposal, we will build on our experience and expertise in human genetics and gene therapy to improve genetic deafness for deafness by leveraging the power of single- cell RNA sequencing and to establish whether targeted allele suppression can prevent or potentially reverse hearing loss in mature animal models of human deafness by using RNA interference (RNAi). To achieve these goals, we propose the following specific aims: ? Specific Aim 1. Using single-cell isolation techniques, we propose: o SA1A. To refine our knowledge of individual cell-specific transcription in the organ of Corti and improve comprehensive genetic testing for deafness by identifying and characterizing novel exons and isoforms of genes implicated in both non-syndromic and syndromic forms of hearing loss. o SA1B. To characterize the broader impact of single gene mutations on the transcriptome of individual cells in two murine models of human ADNSHL ? the Kncq4+/- mouse, which is a model of human DFNA2-related hearing loss, and the Tmc1Bth/+ mouse (the Beethoven (Bth) mouse), which is a model of human DFNA36-related hearing loss. ? Specific Aim 2. Using RNAi, we propose: o SA2A. To establish whether targeted allele suppression can prevent or potentially reverse hearing loss in mature animals. o SA2B. To develop a broadly applicable treatment strategy for all types of deafness caused by a single gene using a novel RNAi construct that suppresses both endogenous alleles with concomitant gene replacement by an exogenous wild-type allele engineered to resist RNAi-mediated silencing. o SA2C. To determine whether systemic neutralizing antibodies (NAbs) to AAV impact the transduction efficiency of AAV-mediated cochlear gene therapy. The successful completion of these aims will advance our understanding of the biology of hearing and deafness, enhance the clinical care of persons with hearing loss, and potentially lay the foundation for clinical trials using RNAi as a treatment option for some types of hearing loss.
In Specific Aim 1, full-length single-cell RNA-seq on individual cells isolated from mouse cochleae will be used to identify novel exons in genes implicated in non-syndromic hearing loss and to characterize the effect of single gene mutations on transcriptomes in two mouse models of autosomal dominant non-syndromic hearing loss (ADNSHL). In Specific Aim 2, artificial micro RNAs will be used to establish the feasibility of targeted allele suppression based on RNA interference as a treatment for hearing loss in mature mouse models of ADNSHL. We will also determine whether systemic neutralizing antibodies to AAV impact cochlear AAV transduction efficiency using a passive immunization model in immunocompetent C57BL/6 mice.
