Gene Transfer to the Inner Ear

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DESCRIPTION (provided by applicant): The proposed study will determine the efficacy of in vivo gene delivery to the adult mouse cochlea using a modified 2nd generation adenovirus [El-, E2b-, E3-] and test cochlear function, as assessed by distortion product to acoustic emissions (DPOAEs) and auditory brainstem responses (ABRs). The magnitude of DPOAEs are reliable indicators of functioning outer hair cells, while ABR threshold measures are an indicator of functioning inner hair cells. Our initial findings support the ability of adenovirus to deliver transgenes to guinea pig cochlear hair cells in vivo, but it has not yet been determined if such gene-transter techniques can be used to change endogenous levels of genes present in hair cells, and if gene-transfer can be used to affect functional changes in cochlear physiology. The first Specific Aim is to deliver a modified 2nd generation adenovirus to the adult mouse cochlea. We will also determine if there is any toxicity associated with introducing and expressing a foreign transgene by comparing serum levels of transaminase and liver pathology from animals that have had equal titers of modified 2nd generation adenovirus containing a foreign transgene (LacZ) to that of modified 2nd generation adenovirus infection without a foreign transgene, Throughout the experiment, DPOAEs and ABRs will be monitored to determine if there is any loss of cochlear function due to viral infection and possible immune clearance of hair cells. The second Specific Aim will determine if protein expression in the cochlea can be modulated using virally-mediated gene transfer using the KCN04 channel as a model. We will overexpress functional KCN04 channels and dominant-negative mutant (G285S) KCN04 channels. We will then use gene transfer to determine if over-expression of normal and dominant-negative mutant KCN04 channels can be used to change the ability of adult hair cells to transduce auditory signals. In both Aims we will use molecular biological cloning, virus production, immunohistochemical, Western blotting, DPOAE and ABR measurements, and aseptic surgical techniques. KCNQ4 mutations are found in the human autosomal dominant non-syndromic deafness disorder, DFNA2, and families with this mutation exhibit a significant hearing loss. Once we can mimic human disease using gene transfer, future studies can employ gene transfer techniques to rescue mutant phenotypes. Information gained from these studies will aid development of theraupeutic strategies targeted at genes involved in deafness.