Pathogenic mutations in the OTOF gene cause autosomal recessive hearing loss (DFNB9), one of the most common forms of auditory neuropathy. There is no biological treatment for DFNB9. Here, we designed an OTOF gene therapy agent by dual-adeno-associated virus 1 (AAV1) carrying human OTOF coding sequences with the expression driven by the hair cell–specific promoter Myo15, AAV1-hOTOF. To develop a clinical application of AAV1-hOTOF gene therapy, we evaluated its efficacy and safety in animal models using pharmacodynamics, behavior, and histopathology. AAV1-hOTOF inner ear delivery significantly improved hearing in Otof^−/− mice without affecting normal hearing in wild-type mice. AAV1 was predominately distributed to the cochlea, although it was detected in other organs such as the CNS and the liver, and no obvious toxic effects of AAV1-hOTOF were observed in mice. To further evaluate the safety of Myo15 promoter-driven AAV1-transgene, AAV1-GFP was delivered into the inner ear of Macaca fascicularis via the round window membrane. AAV1-GFP transduced 60%–94% of the inner hair cells along the cochlear turns. AAV1-GFP was detected in isolated organs and no significant adverse effects were detected. These results suggest that AAV1-hOTOF is well tolerated and effective in animals, providing critical support for its clinical translation.

Inner ear gene therapy using adeno-associated viral vectors (AAVs) in neonatal mice can alleviate hearing loss in mouse models of deafness. However, efficient and safe transgene delivery to the adult mouse cochlea is critical for the effectiveness of AAV-mediated therapy. Here, we examined three gene delivery approaches including posterior semicircular canal (PSCC) canalostomy, round window membrane (RWM) injection, and tubing-RWM+PSCC (t-RP) in adult mice. Transduction rates and survival rates of cochlear hair cells were analyzed, hearing function was recorded, AAV distribution in the sagittal brain sections was evaluated, and cochlear histopathologic images were appraised. We found that an injection volume of 1 μL AAV through the PSCC is safe and highly efficient and does not impair hearing function in adult mice, but local injection allows AAV vectors to spread slightly into the brain. We then tested five AAV serotypes (PHP.eB, IE, Anc80L65, AAV2, and PHP.s) in parallel and observed the most robust eGFP expression in inner hair cells, outer hair cells, and spiral ganglion neurons throughout the cochlea after AAV-Anc80L65 injection. Thus, PSCC-injected Anc80L65 provides a foundation for gene therapy in the adult cochlea and will facilitate the development of inner ear gene therapy.

Due to limited spectral resolution, cochlear implants (CIs) do not convey pitch information very well. Pitch cues are important for perception of music and tonal language; it is possible that music training may improve performance in both listening tasks. In this study, we investigated music training outcomes in terms of perception of music, lexical tones, and sentences in 22 young (4.8 to 9.3 years old), prelingually deaf Mandarin-speaking CI users. Music perception was measured using a melodic contour identification (MCI) task. Speech perception was measured for lexical tones and sentences presented in quiet. Subjects received 8 weeks of MCI training using pitch ranges not used for testing. Music and speech perception were measured at 2, 4, and 8 weeks after training was begun; follow-up measures were made 4 weeks after training was stopped. Mean baseline performance was 33.2%, 76.9%, and 45.8% correct for MCI, lexical tone recognition, and sentence recognition, respectively. After 8 weeks of MCI training, mean performance significantly improved by 22.9, 14.4, and 14.5 percentage points for MCI, lexical tone recognition, and sentence recognition, respectively (p < .05 in all cases). Four weeks after training was stopped, there was no significant change in posttraining music and speech performance. The results suggest that music training can significantly improve pediatric Mandarin-speaking CI users’ music and speech perception.

Patients with single-sided deafness (SSD) often experience poor sound localization, reduced speech understanding in noise, reduced quality of life, and tinnitus. The present study aims to evaluate effects of tinnitus and duration of deafness on sound localization and speech recognition in noise by SSD subjects. Sound localization and speech recognition in noise were measured in 26 SSD and 10 normal-hearing (NH) subjects. Speech was always presented directly in front of the listener. Noise was presented to the deaf ear, in front of the listener, or to the better hearing ear. Tinnitus severity was measured using visual analog scale and Tinnitus Handicap Inventory. Relative to NH subjects, SSD subjects had significant deficits in sound localization and speech recognition in all listening conditions (p < .001). For SSD subjects, speech recognition in noise was correlated with mean hearing thresholds in the better hearing ear (p < .001) but not in the deaf ear. SSD subjects with tinnitus performed poorer in sound localization and speech recognition in noise than those without tinnitus. Shorter duration of deafness was associated with greater tinnitus and sound localization difficulty. Tinnitus visual analog scale and Tinnitus Handicap Inventory were highly correlated; the degree of tinnitus was negatively correlated with sound localization and speech recognition in noise. Those experiencing noticeable tinnitus may benefit more from cochlear implantation than those without; subjective tinnitus reduction may be correlated with improved sound localization and speech recognition in noise. Subjects with longer duration of deafness demonstrated better sound localization, suggesting long-term compensation for loss of binaural cues.

Objective. The aim of this study was to investigate the benefits of residual hair cell function for speech and music perception in bimodal pediatric Mandarin-speaking cochlear implant (CI) listeners. Design. Speech and music performance was measured in 35 Mandarin-speaking pediatric CI users for unilateral (CI-only) and bimodal listening. Mandarin speech perception was measured for vowels, consonants, lexical tones, and sentences in quiet. Music perception was measured for melodic contour identification (MCI). Results. Combined electric and acoustic hearing significantly improved MCI and Mandarin tone recognition performance, relative to CI-only performance. For MCI, performance was significantly better with bimodal listening for all semitone spacing conditions (p<0.05 in all cases). For tone recognition, bimodal performance was significantly better only for tone 2 (rising; p<0.05). There were no significant differences between CI-only and CI + HA for vowel, consonant, or sentence recognition. Conclusions. The results suggest that combined electric and acoustic hearing can significantly improve perception of music and Mandarin tones in pediatric Mandarin-speaking CI patients. Music and lexical tone perception depends strongly on pitch perception, and the contralateral acoustic hearing coming from residual hair cell function provided pitch cues that are generally not well preserved in electric hearing.

CasRx, a recently discovered member of the type VI CRISPR system with minimum size, offers a new approach for RNA manipulation with high efficiency and specificity in prokaryotes and eukaryotes. However, in vivo studies of functional recovery using the CasRx system have not been well characterized. Here, we sought to establish an adeno-associated virus (AAV)-CasRx-guide RNA (gRNA) system for the specific knockdown of Htra2 transcript to protect mice from aminoglycosides-induced hearing loss. For the study, we verified an optimized gRNA in vitro, which was packaged into a single AAV with CasRx, and injected the packaged AAV into mice with hearing loss induced by neomycin and auditory functions investigated by auditory brainstem response tests. Upon using the AAV-CasRx-gRNA system, we found the knockdown of Htra2 transcript led to less cochlear hair cell loss and improved auditory function, with low off-target and adverse side effects. Additionally, the decrease in Htra2 significantly inhibits mRNA expression of Casp3 and Casp9. In conclusion, the AAV-CasRx-gRNA-mediated knockdown of Htra2 transcript in mice has been proved effective and safe for preventing hearing loss induced by aminoglycosides and, thus, represents a promising genetic approach for the future clinical applications for treating non-inherited hearing loss.