Screening Specific Adeno-associated Viruses For Targeting Different Cell Types Of Adult Murine Cochleae

CRISPR/Cas9 gene-editing enables precise prevention hearing loss in neonatal deafness mice,by viral and non-viral delivery.Adeno-associated virus(AAV)-mediated gene delivery systems have been shown to be effective tools for gene replacement in the inner ear.However,application of the AAV-mediated CRISPR/Cas9 gene-editing approach to the inner ear in adult mice has not yet been studied.Based on our previous work,we investigated several AAVs in adult mice by canalostomy injection and found that AAV8 had the top efficiency and specificity to transduce inner hair cells(IHCs).We then tested the ability of Cre-expressing AAV8 to activate Cas9 in floxed-Cas9 knock-in(Cas9 KI)mice.Totally we compared six different promoters(CMV,CAG,h Syn,Ca MKIIa,GFAP,ALB)of AAV8 in inner ear of adult Cas9 KI mice,and found three AAVs(CMV,CAG,h Syn)infected inner ear efficiently with different infection tropism.AAVs in promoters of CMV,CAG,h Syn infected diverse cell types in mature murine cochleae,including IHCs.AAV8-h Syn showed high affinity to IHCs and spiral ganglion neurons(SGNs).Neither the AAV8 virus itself(except AAV8-CAG)nor the surgical procedures caused any damage to HCs or impaired normal hearing.Our studies indicate that inner ear injection of AAV-Cre can induce Cas9 activation to perform safe and efficient gene editing in mature inner ear,and different promoters exhibited diverse infection patterns in cochlea,expanding the repertoire of gene-editing tools for regulating gene expression in target cell type of inner ear as part of efforts to rescue genetic hearing loss,or develop gene therapy techniques.