Abstract
We have previously shown that in vitro transduction with bovine adeno–associated viral (BAAV) vectors restores connexin expression and rescues gap junction coupling in cochlear organotypic cultures from connexin–deficient mice that are models DFNB1 nonsyndromic hearing loss and deafness. The aims of this study were to manipulate inner ear connexin expression in vivo using BAAV vectors, and to identify the optimal route of vector delivery. Injection of a BAAV vector encoding a bacterial Cre recombinase via canalostomy in adult mice with floxed connexin 26 (Cx26) alleles promoted Cre/LoxP recombination, resulting in decreased Cx26 expression, decreased endocochlear potential, increased hearing thresholds, and extensive loss of outer hair cells. Injection of a BAAV vector encoding GFP-tagged Cx30 via canalostomy in P4 mice lacking connexin 30 (Cx30) promoted formation of Cx30 gap junctions at points of contacts between adjacent non-sensory cells of the cochlear sensory epithelium. Levels of exogenous Cx30 decayed over time, but were still detectable four weeks after canalostomy. Our results suggest that persistence of BAAV-mediated gene replacement in the cochlea is limited by the extensive remodeling of the organ of Corti throughout postnatal development and associated loss of non-sensory cells.
Highlights
Up to 50% of prelingual hearing impairment is linked to the DFNB1 locus on chromosome 13q11–q121, which comprises the genes encoding two structurally and functionally related gap junction proteins, connexin 26 (Cx26) (GJB2) and connexin 30 (Cx30) (GJB6)[2, 3]
We measured the IV wave thresholds of the auditory brainstem responses (ABRs) for click and tone burst stimuli of 8, 14, 20, 26, 32 kHz and found no differences in injected mice compared with non injected controls (Fig. 1c). These results indicate that delivery of DMEM/F12 to the inner ear via canalostomy causes no adverse effects on hearing and could function as an effective route of vector delivery
Viral transduction performed in adult Gjb[2] conditional knock out mice failed to correct hearing function[72, 73], whereas an early intervention in newborn mice produced limited Cx26 reinstatement and only partial rescue of hearing[73]
Summary
Up to 50% of prelingual hearing impairment is linked to the DFNB1 locus on chromosome 13q11–q121, which comprises the genes encoding two structurally and functionally related gap junction proteins, Cx26 (GJB2) and Cx30 (GJB6)[2, 3]. The indispensable endocochlear potential, which in mice exceeds 100 mV17, is generated by the activity of gap-junction coupled cells in the lateral wall (spiral ligament and stria vascularis)[18, 19] and provides the driving force required for K+ influx from endolymph to hair cell cytosol via mechanically activated channels in the hair bundle[20, 21]. Another essential prerequisite for generating receptor potentials is electrical insulation of the hair cells from the rest of sensory epithelium[22]. An important component of a future therapeutic intervention plan is the optimal route of vector delivery to the inner, and this has not been uniquely identified yet[42,43,44,45,46,47]
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