Development of human inner ear organoid platforms for human auditory/vestibular disorders

University Of Miami School Of Medicine, Coral Gables FL

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Abstract

Abstract Hearing loss (HL), congenital or acquired, is a major health issue which affects approximately 30 million people in the United States alone. Genetic causes are one of the most common etiologies. So far, >200 genes have been identified as causative for HL in humans. This work is significant because we propose to merge two approaches to develop a platform by generation of human inner ear organoids (hIEOs) from human pluripotent stem cells (hiPSCs) as a model system to test inner ear gene therapies and introduction of advanced genome editing strategies to silence dominant mutations or repair recessive mutations that cause dysfunction in human sensory hair cells. To support the role of identified variants in pathophysiology, we will undertake novel in vitro functional studies of these IEOs. The proposed human studies are complementary with development of a human iPSC-derived model for human audio vestibular organoids and analysis of the effect of HL mutations on IEOs. No single system or experiment is typically conclusive in itself. Therefore, to support the role of identified variants in pathophysiology, we will undertake novel in vitro functional studies of these IEOs including using novel muti-`Omic approaches including scATAC-seq & scRNA-seq, epigenetic, and organoid-on-a-chip' platforms. The short-term impact of this proposal will be to generate a novel in vitro human iPSC-derived model corresponding to the vestibular region affected in hearing and balance and to evaluate the cellular consequences of the CRISPR/Cas9-based exon- skipping or disruption strategies in human cells. The long-term impact will be to establish a new paradigm for personalized genetic models of hIEOs affected in HL for in vitro screening of available therapeutics and multi-omic identification of disease and biomarkers for treatment responses. We hypothesize that human induced pluripotent stem cells (iPSCs) and IEOs provide valuable experimental platforms for testing potential therapeutic strategies and gene targeting for the correction of audiovestibular disorders. We further hypothesize that in vitro CRISPR/Cas9-based gene disruption strategies can rescue HL-associated mutations in human hair cells (HCs) using human IEOs laying the foundation for a human clinical trial in the treatment of HL. Specific aims of the project are: 1 Establishment and optimization of hiPSC-derived 3D inner ear system from patient-with genetic HL. 2.Test in vitro CRISPR/Cas9-based gene disruption and AAV-based gene expression strategies to model HL-associated IEO cell defects and to rescue phenotypes-associated mutations in the human IEOs. These innovative approaches for treatment of genetic HL will pave the way for translation to clinical trials. We are optimistic that by merging these technologies we can successfully translate these innovations for other inner ear disorders and, importantly, broader use in other genetic disorders.