Dual-AAV mediated transfer of full-length otoferlin cDNA into auditory inner hair cells and the effects of different mutations in the OTOF gene on the protein levels and cellular distribution of otoferlin in auditory inner hair cells

Otoferlin is a large multi-C2 domain protein indispensable for hearing and synaptic transmission in auditory inner hair cells (IHCs). Mutations within the OTOF gene, coding for otoferlin, cause non-syndromic recessive hearing loss DFNB9. The severity of the hearing impairment can range from profound deafness to moderate hearing loss depending on the pathogenic OTOF variant the patient is carrying. Three different otoferlin mutant mouse models have been studied during the course of this thesis to understand the effects of these mutations on the expression levels, cellular distribution, ultrastructural subcellular localization, and stability of otoferlin in IHCs and how these factors relate to the impaired IHC physiology and auditory function deficits observed in these mice. Otoferlin knock-out (Otof-/-) mice are deaf and have almost completely abolished IHC exocytosis as a result of absent otoferlin protein. Pachanga (OtofPga/Pga) otoferlin mutant mice, carrying the p.Asp1767Gly missense mutation, show no auditory brain stem responses (ABRs) and have a reduced sustained IHC exocytosis. OtofI515T/I515T otoferlin knock-in mice, carrying the human p.Ile515Thr missense mutation, suffer from a moderate hearing impairment and their sustained IHC exocytosis levels are between the levels of wild-type and OtofPga/Pga IHCs. My analysis revealed that the amount of plasma membrane-bound otoferlin in these IHCs seems to correlate with the sustained exocytosis levels and hearing phenotypes found in all three genotypes. Temperature elevation reduces membrane-bound otoferlin even further in OtofI515T/I515T mutants, thus providing a potential explanation for the temperature-sensitive hearing loss found in individuals carrying this mutation. Additionally, both the p.Asp1767Gly and the p.Ile515Thr mutation appear to interfere with the membrane targeting of otoferlin and its functions in membrane retrieval and synaptic vesicle reformation from endosomal IHC compartments. DFNB9 patients would benefit significantly from gene replacement therapies due to the limitations of cochlear implants, which are the only current available treatment for these individuals. Gene transfer mediated by recombinant adeno-associated viruses (AAVs) is seen as a promising tool to treat inherited deafness forms because of the high safety profile and IHC targeting efficiency of this virus. However, the limited cargo capacity of AAVs (~4.7 kb) hinders the transport of the 6 kb-long full-length otoferlin coding sequence into IHCs. With the goal of overcoming this obstacle, I have designed dual-AAV half vectors, each carrying one half of the full-length otoferlin cDNA. Otof-/- mice co-injected with these dual-AAV half vectors showed full-length otoferlin expression in auditory hair cells and partially recovered sustained IHC exocytosis and auditory function. Broadband click sound-evoked ABR thresholds could be restored to near wild-type thresholds of 40-60 dB SPL. However, trans-splicing and hybrid dual-AAV half vectors were not able to rescue the 40% synaptic ribbon loss in these mice when injected into the cochlea at postnatal day (P) 5-7. Further analysis revealed that the development and maturation of IHC ribbon synapses during the first two postnatal weeks was altered in Otof-/- mice. All of these findings point towards a yet unknown role of otoferlin in IHC ribbon synapse maturation. My data additionally showed that using a different AAV serotype does not improve full-length otoferlin IHC transduction rates or protein levels in transduced Otof-/- IHCs. Optimization of the dual-AAV large transgene reassembly, transcription, and translation is thus crucial to obtain higher protein levels and IHC targeting rates and to subsequently increase ABR wave amplitudes in otoferlin dual-AAV treated animals. Nevertheless, this work shows the first successful gene therapy application using dual-AAV vectors to transfer large genes into the mammalian inner ear in a mouse model for human deafness