Gene therapy for hearing loss

• Applicant: Professorin Dr. Ellen Reisinger
• Subject Area: Otolaryngology, Phoniatrics and Audiology ; Molecular Biology and Physiology of Neurons and Glial Cells
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 416116807

Mutations in the gene OTOF, encoding the protein otoferlin, typically cause congenital profound deafness (DFNB9). Otoferlin is required for synaptic transmission from auditory sensory cells to subsequent neurons. To date, patients with this form of deafness receive cochlear implants, which stimulate the auditory pathway directly with electrical signals, thereby enabling people to comprehend speech. However, cochlear implants hardly transmit frequency information and encode only a limited dynamic range of sound intensity. Since in this form of deafness the inner ear is morphologically almost entirely intact, a transfer of otoferlin cDNA into the sensory cells is expected to restore hearing. Such a causal gene therapy could potentially enable hearing with hardly any quality restrictions, yet has not been developed for human patients to date.Recently, my group succeeded to transfer the large otoferlin cDNA into auditory sensory cells of mice by means of a recombinant virus. This was challenging because no viral vector was known that is suitable to transfer large cDNAs into this type of cells. We overcame this challenge by means of a "dual-AAV" method, which served to partially restore hearing in deaf otoferlin knock-out mice. In this method, the cDNA of otoferlin was split to two adeno-associated viruses (AAVs), which re-assemble in the nuclei of target cells to allow transcription of the full length otoferlin mRNA. This proposal focuses on testing variants of these AAVs that shall enhance the correct tail-to-head multimerization, aiming to increase protein expression and thus to further improve hearing. In addition, I will employ a refined method for injection into the inner ear, and a different AAV serotype to test if this is suitable to increase the rate of transduced sensory cells. Next, the synapses and auditory nerve fibers shall be characterized after re-expression of otoferlin. We will then apply this gene therapy approach to two mouse models with human mutations. Moreover, I aim to use this method to test cDNA variants in vivo, e.g. phosphomimetic mutations in otoferlin and two different splice variants, for a potential role in noise-induced synapse loss. Otoferlin expression levels will be compared to wild-type levels with immunofluorescence. The function of re-expressed otoferlin will be assessed by cellular patch-clamp electrophysiology to record Ca2+-triggered fusion of synaptic vesicles. Hearing in mice will be measured by auditory brainstem recordings and behavioral tests.My goal is to refine the causal therapy for DFNB9 to bring it close to clinical application.

• DFG Programme: Research Grants