Abstract:
Hearing loss can result from a variety of factors, including chronic diseases, ear infections, and genetic mutations. One genetic contributor is the OTOF gene, which encodes the protein otoferlin, essential for synaptic transmission from auditory inner hair cells (IHCs) to spiral ganglion neurons (SGNs), the first neurons in the auditory pathway. Mutations in OTOF cause autosomal recessive prelingual hearing impairment, classified as DFNB9, which is typically severe to profound in degree.
Otoferlin contains multiple C2 domains and a C2-FerA domain which forms a flexible superhelix composed of four α-helices and has been shown to bind calcium ions (Ca²⁺) and phospholipids. However, its specific role in auditory function remains unclear.
In this thesis, a novel mouse line, designated Otof-p. KL>M, was characterized. This line carries a three-base pair deletion resulting in the substitution of lysine 824 and leucine 825 with a methionine, predicted to shorten one α-helical structure within the C2-FerA domain. Auditory brainstem response (ABR) measurements revealed that homozygous Otof-p. KL>M mice exhibit reduced ABR wave amplitudes at a young age and develop significant age-related hearing loss. By twelve months of age, these mice displayed elevated hearing thresholds reaching 90 dB sound pressure level (SPL) at the most sensitive frequency (4 kHz).
As anticipated, the Otof-p. KL>M mutation did not affect distortion product otoacoustic emissions (DPOAEs), indicating preserved outer hair cell (OHC) function. Consistently, the number of OHCs remained unchanged across all ages. In contrast, an age-dependent loss of IHCs was observed throughout all cochlear turns in Otof-p. KL>M mice. Moreover, IHCs in mutant mice demonstrated reduced otoferlin expression with increasing age compared to wild-type controls. Collectively, these findings highlight a critical role for the otoferlin C2-FerA domain in maintaining IHC integrity and function.
Several gene therapy strategies targeting the otoferlin coding gene have been investigated in otoferlin-deficient mouse models and early-stage clinical trials. These studies have shown that dual-adeno-associated virus (AAV) gene therapy can effectively deliver a functional OTOF sequence to the inner ear. Nonetheless, optimization of these approaches remains necessary. One avenue of improvement involves engineering novel AAV capsids with enhanced tropism and transduction efficiency for both IHCs and OHCs. In this thesis, a novel in vitro AAV transduction method was developed using explanted organs of Corti to facilitate pre-screening of AAV serotypes. Initial testing of various GFP-expressing AAV serotypes identified PHP.eB (produced in-house) and AAV2 PT (provided by AG Michalakis, LMU Munich) as having the highest transduction efficiencies and specificity in hair cells. Furthermore, three AAV capsids were evaluated for dual-AAV transduction in in vitro experiments using otoferlin knockout organ of Corti cultures. Among these, the PHP.B serotype yielded the most promising results, demonstrating successful transduction of both IHCs and OHCs. These findings validate the utility of the newly developed in vitro method for assessing dual-AAV strategies. Importantly, this approach offers a time-efficient method to pre-screen AAV serotypes in vitro and thereby reducing the number of animals required.
In addition to OTOF, other genes also play vital roles in auditory function. In five patients from Tübingen and Heidelberg presenting with progressive hearing loss, a dominantly inherited point mutation in the RAB4B gene, encoding the Ras-related protein RAB4B, was identified. These individuals exhibit increasing difficulty with speech comprehension, despite only moderately elevated hearing thresholds. To date, RAB4B has not been associated with auditory disorders. To investigate Rab4b expression in the auditory system, immunohistochemistry and RNA Scope in situ hybridization were performed on wild-type mouse inner ears. Both Rab4b protein and mRNA were detected, with expression localized to IHCs, OHCs, and strongly in SGNs. Subsequently, genetically engineered mice carrying the same point mutation as the human patients were generated to assess the functional consequences of the variant. The results presented in this thesis offer initial insights into the potential role of Rab4b in auditory physiology and pathophysiology.
Inner Ear