Defining the role of MET components in vestibular hair cell maturation and gene therapy responsiveness

Boston Children'S Hospital, Boston MA

Investigators

Abstract

Abstract Balance and equilibrioception depend on vestibular hair cells that encode motion through mechanoelectrical transduction channels (MET) during movements of the head. Mutations in mechanoelectrical transduction channel genes (Tmc, Cib, Tmie, Lhfpl5) result in issues with hearing and balance in both human patients and mouse models. The long-term goal of this project is to better understand how heterogenous regional expression of MET components contribute to vestibular responses, and whether early expression of MET components define a critical period for gene replacement therapy. TMC1 and TMC2 are components of the MET channel and provide ionic influx of potassium and calcium during mechanical deflection whereas CIB2 and CIB3 localize to the intracellular domain of the MET channel acting as putative calcium sensors. Tmc and Cib genes are heterogeneously expressed in different regions of the mouse utricle and saccule with peak expression levels during neonatal development (P0-P7). However, it is unclear what the functional consequences of heterogenous MET subunit expression are on the development of mature vestibular responses. In Aim 1 I plan to characterize how this heterogenous expression contributes to regional differences in vestibular hair cell activity using calcium (Ca2+) imaging of utricles and saccules. Qualitative comparison of regional hair cell differences in Ca2+ influx will be evaluated with fluorescent Ca2+ indicators, and FM dyes which enter hair cells through functional MET channels. Qualitative differences between striolar versus extrastriolar hair cells will be evaluated in developing (P7) and mature hair cells (P30). Additionally, regional changes in Ca2+ influx will be assessed in Tmc2 KO and Cib2 KO mice where preliminary data shows failure of striolar hair cells to take up FM1-43 dye. In Aim 2 I will investigate whether an early period of regionally heterogenous MET expression precludes gene replacement at later stages. This will be examined by injecting adult (P60) Tmc1 KO, Cib2 KO, and Tmie KO mice with AAVs replacing a functional copy of the gene of interest (GOI). Subsequent testing of Vestiular Evoked Potential (VsEP), vestibulo ocular reflexes (VOR), and FM1-43 dye uptake will determine whether gene replacement is effective in mature hair cells. Preliminary results show that we can restore vestibular function in Tmc1, Cib2, and Tmie mice at neonatal stages, but it is unclear whether the therapeutic window extends to include mature adult vestibular hair cells. RELEVANCE: In the US alone 14.8% of patients seek clinical assistance due to balance issues, and the predominant cause of inner ear dysfunction typically includes a genetic component. Many patients receive diagnoses as adults, but precision treatment may have a limited therapeutic window of efficacy as seen in cochlear hair cells. This proposal will advance our basic understanding of vestibular hair cell function and will assist in the development of gene therapies for MET related balance dysfunction to advance precision treatment.