Functional, sustained recovery of hearing in Otoferlin-deficient mice using DB-OTO, a hair-cell-specific AAV-based gene therapy

Biallelic loss-of-function variants in the <i>Otoferlin</i> gene (<i>OTOF</i>) lead to congenital hearing loss in both humans and mice. We developed DB-OTO, a hair-cell-specific adeno-associated virus (AAV)-based dual-vector gene therapy designed to restore hearing in individuals with <i>OTOF</i>-related hearing loss. DB-OTO is composed of two AAV1 vectors that reconstitute a functional <i>OTOF</i> gene expression cassette to form the full-length human <i>OTOF</i> variant 5 (<i>h</i> <i>OTOFv5</i>). A synthetic promoter (<i>mMyo15</i>) based on the murine <i>Myosin 15</i> <i>a</i> gene was engineered to restrict the transgene expression to hair cells. The promoter construct was chosen after comparison of multiple variants using a GFP reporter in <i>ex vivo</i> murine explant models, followed by <i>in vivo</i> studies in wild-type mice and cynomolgus macaques. Comparison between the 1.0 kb <i>mMyo15</i> and a ubiquitous promoter driving expression of <i>h</i> <i>OTOF</i>v5 transgene in mice showed that hair-cell-specific expression is critical for safe expression of otoferlin. DB-OTO induced dose-dependent establishment of auditory brainstem response, and <i>OTOF</i> expression in inner hair cells over a 10-fold dose range sustained for at least 3 months in a mouse model of <i>OTOF</i> deficiency. These data supported the initiation of an ongoing phase I/II clinical trial of DB-OTO in pediatric patients with <i>OTOF</i>-related hearing loss.