Gene transfer into the inner ear
23 April 2019 (online)
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. 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. In the present study, I established and characterized novel in vitro methods to transfer the full-length otoferlin into cochlear inner hair cells (IHC) in situ. Therefore, two different gene delivery methods, (i) electroporation (EP), as a cheap and flexible screening method with theoretically unlimited insert size and (ii) adenoviruses (Ad), 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 (E) 14.5, 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 (wildtype) and otoferlin-knockout (KO) cultures were genetically manipulated to either express a mutant or wildtype (WT) form of otoferlin.