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

Biallelic loss-of-function variants in the Otoferlin gene (OTOF) lead to congenital severe-to-profound sensorineural hearing loss in both humans and in mice. We developed DB-OTO, a hair cell-specific adeno-associated virus (AAV)-based dual-vector gene therapy designed to provide hearing to individuals with OTOF-related hearing loss. DB-OTO is composed of two AAV1 vectors that reconstitute a functional hOTOF gene expression cassette to form the full-length human OTOF isoform 5. A promoter based on the murine Myosin 15 (mMyo15) gene promoter 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 ex vivo murine explant models, followed by in vivo studies in wild-type mice and in cynomolgus monkeys. Comparison between the 1.0 kb mMyo15 and a ubiquitous promoter driving expression of hOTOFv5 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 OTOF expression in inner hair cells over a 10-fold dose range sustained for at least 3 months in a mouse model of OTOF-deficiency. These data supported the initiation of a Phase 1/2 clinical trial of DB-OTO in pediatric patients with OTOF-related hearing loss. Overall design: single-cell RNA-seq profiling on the cochleae of WT FVB or Otof+/Q828X mice treated with either AAV1-smCBA-OTOF-v5 or AAV1-1.0kb-mMyo15-OTOF-v5 at a dose of 1e11 vg/ear at mean 7.5 weeks of age