| dc.description.abstracteng | In humans, in whom speech and speech perception is essential for communication and social interaction, deafness is a severe disorder. One of the genetic non-syndromic forms is the autosomal recessive nonsyndromic deafness DFNB 9, which is caused by loss of function mutations in the OTOF gene encoding the protein otoferlin. As otoferlin may serve a dual role in Ca2+-sensing and vesicle replenishment at the IHC ribbon synapse, it is of great interest to establish rescue protocols to (i) obtain a more detailed view on the properties of the protein and its functional domains and (ii) ultimately use the knowledge to develop gene therapy in patients suffering from DFNB9. While other studies have successful performed rescue experiments using adeno-associated viruses, these viruses lack the capability of accommodating the relatively large sequence of otoferlin into their genome.
In the present study, I established and characterized novel in vitro methods to transfer the full-length otoferlin into cochlear IHC in situ. Therefore, two different gene delivery methods, (i) electroporation , as a cheap and flexible screening method with theoretically unlimited insert size and (ii) adenoviruses, which display high transduction efficiency, but currently still evoke immune responses of the host, were evaluated. Alongside these two methods, I further established an in vitro model system of cultured organs of Corti derived from mice of embryonic day E14.5, with which – compared to previous attempts with postnatal gene delivery – higher transfection/transduction rates could be achieved. In a qualitative manner, the development of the embryonic cultures was comparable to cultures of neonatal age with the same cumulative age. After establishing successful gene transfer by EP and Ads, C57Bl6 WT and otoferlin-knockout cultures were genetically manipulated to either express a mutant or wildtype form of otoferlin. | de |