Thyroid hormone controls the timing of cochlear ribbon synapse maturation.

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation.

AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness.

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.

Preclinical Efficacy And Safety Evaluation of AAV-OTOF in DFNB9 Mouse Model And Nonhuman Primate.

OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof-related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell-specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual-AAV-OTOF-hybrid strategy to transfer full-length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild-type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.

Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses.

Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8-P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.

Cochlear Sox2+ Glial Cells Are Potent Progenitors for Spiral Ganglion Neuron Reprogramming Induced by Small Molecules.

In the mammalian cochlea, spiral ganglion neurons (SGNs) relay the acoustic information to the central auditory circuits. Degeneration of SGNs is a major cause of sensorineural hearing loss and severely affects the effectiveness of cochlear implant therapy. Cochlear glial cells are able to form spheres and differentiate into neurons in vitro. However, the identity of these progenitor cells is elusive, and it is unclear how to differentiate these cells toward functional SGNs. In this study, we found that Sox2+ subpopulation of cochlear glial cells preserves high potency of neuronal differentiation. Interestingly, Sox2 expression was downregulated during neuronal differentiation and Sox2 overexpression paradoxically inhibited neuronal differentiation. Our data suggest that Sox2+ glial cells are potent SGN progenitor cells, a phenotype independent of Sox2 expression. Furthermore, we identified a combination of small molecules that not only promoted neuronal differentiation of Sox2- glial cells, but also removed glial cell identity and promoted the maturation of the induced neurons (iNs) toward SGN fate. In summary, we identified Sox2+ glial subpopulation with high neuronal potency and small molecules inducing neuronal differentiation toward SGNs.