Cellular reprogramming of cochlear glial cells to regenerate auditory neurons - PROJECT ABSTRACT Sensorineural hearing loss is a major public health issue; by 2050, over 900 million people will have some form of auditory impairment. The spiral ganglion nerve (SGN) plays a crucial role in hearing by transmitting acoustic signals from the inner ear to the brain. Because the auditory nerve lacks intrinsic regenerative capacity, damage to the nerve leads to permanent deafness. Cochlear implants are ineffective when the SGN is damaged or lost, as observed in auditory neuropathy (AN) cases. Therefore, there is a critical need to develop novel treatments for AN, and regenerative medicine holds enormous potential to restore SGN and treat this condition. The spiral ganglion glial cells support, nourish, and protect the SGN and are critical for auditory nerve function, development, and homeostasis. Direct neuronal reprogramming converts somatic cells to induced neurons by overexpression of neuronal transcription factors (NTFs), bypassing the pluripotent state. The spiral ganglion glial cells are considered a promising source for cellular reprogramming in the inner ear because of their plasticity, proliferative capacity, survival post-nerve damage, and proximity to the nerve. Recent work has shown that neonatal spiral ganglion glial cells can be converted into SGNs by overexpression of NTFs in vitro and in vivo. However, despite administering multiple NTFs, reprogramming spiral ganglion glial cells remains inefficient. Recent cellular reprogramming strategies incorporate neuroregulatory microRNAs (miRNAs) to enhance the conversion of fibroblasts into induced neurons in combination with NTFs. The role of these miRNAs in regulating SGN fate is unknown. Our primary goal in this grant proposal is to convert spiral ganglion glial cells into SGNs via gene therapy and to advance cellular reprogramming in the inner ear. We identify three knowledge gaps in advancing cellular reprogramming in the inner ear: 1) We know very little of the glial cell response after SGN damage, 2) driving exogenous transgenes specifically into glial cells is challenging, and 3) reprogramming of spiral ganglion glial cells into neurons is very inefficient, and it is not clear if this generates the correct neuronal subtypes. We hypothesize that spiral ganglion glial cells can be converted to SGNs in normal and deafened mice with a combination of NTFs and miRNA delivered to the glial cells via an AAV vector. The aims of this project are (1) the characterization of age-related glial cell response to acute SGN apoptosis by creating a neonatal mouse model of AN, (2) to optimize exogenous transgene delivery into cochlear glial cells in neonatal mice, and (3) explore direct reprogramming of normal or damaged glial cells in vitro. The expected results of this study will be (1) an improved understanding of age-related changes in reactive gliosis in mice in response to SGN death, (2) improved targeting of cochlear glial cells with a combination of glial cell-specific AAV serotypes and promoters, and (3) providing experimental evidence of the role of NTFs and miRNA in inducing SGNs from normal and post-damaged glial cells.
