Characterization of promoters for adeno-associated virus mediated efficient Cas9 activation in adult Cas9 knock-in murine cochleae

CRISPR/Cas9 gene editing enables the treatment of hearing loss in congenitally deaf neonatal mice via both viral and non-viral delivery. While adeno-associated virus (AAV)-mediated gene delivery systems have been shown to be effective tools for gene replacement in the inner ear, application of the AAV-mediated CRISPR/Cas9 gene-editing approach for this purpose is yet to be documented. Based on our previous findings, we focused on the effects of several AAVs delivered via canalostomy injection in adult mice. Among the AAVs examined, AAV8 showed the greatest efficiency and specificity in transducing inner hair cells (IHC). The ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knock-in (Cas9 KI) mice was further evaluated. We compared the effects of six different promoters (CMV, CAG, hSyn, CaMKIIa, GFAP, and ALB) of AAV8 delivered to the inner ear of adult Cas9 KI mice. Our findings showed that three AAV groups (CMV, CAG and hSyn promoters) infected the inner ear efficiently with different tropisms. Notably, AAVs with CMV, CAG, and hSyn promoters infected diverse cell types in mature murine cochleae, including IHCs. In particular, AAV8-hSyn showed high affinity to IHCs and spiral ganglion neurons (SGN). Neither the AAV8 virus itself (except AAV8-CAG) nor the surgical procedures used caused damage to HCs or impaired normal hearing. Our findings indicated that injection of AAV-Cre into mature inner ear efficiently induces Cas9 activation to achieve safe and efficient gene editing and different constituent promoters confer diverse infection patterns in cochlea, expanding the repertoire of gene-editing tools for regulating gene expression in target cells of the inner ear as part of the collective effort to rescue genetic hearing loss and develop effective gene therapy techniques.