Dual-AAV OTOF gene therapy for DFNB9 hearing loss: c.4799+2T>C splice donor variant and DB-OTO intracochlear delivery
CONCLUSION
OTOF c.4799+2T>C disrupts the canonical GT donor of intron 39, predicting complete abolition of normal splicing and loss of otoferlin protein in inner hair cells (IHCs). Because this is a null loss-of-function allele, mutation-agnostic gene replacement is the preferred strategy—and the dual-AAV OTOF program (DB-OTO, Decibel Therapeutics / Regeneron) already in Phase 1/2 clinical trials is directly applicable. Single intracochlear injection of dual trans-splicing AAV vectors encoding the full 6-kb OTOF cDNA under the Myo15 hair-cell-specific promoter represents the most clinically advanced and mechanistically rational intervention for this variant. The 2024 NEJM report of meaningful hearing restoration in pediatric DFNB9 patients provides strong translational evidence supporting early intervention before IHC degeneration.
EVIDENCE
Otoferlin (OTOF, ~6 kb CDS) is a C2-domain calcium sensor essential for synaptic vesicle exocytosis at the IHC ribbon synapse; loss leads to auditory neuropathy with preserved otoacoustic emissions but absent ABR—the hallmark DFNB9 phenotype. c.4799+2T>C abolishes the invariant GT dinucleotide of the intron 39 donor site; SpliceAI assigns this class of +2T>C canonical donor mutations a loss score >0.95, consistent with complete exon skipping or intron retention and NMD of the resulting transcript. The dual-AAV trans-splicing strategy (5' and 3' halves of OTOF split at exon 39-40 junction) was validated preclinically in Otof-/- mice (Al-Moyed et al., EMBO Mol Med 2019, PMID:30617123), restoring ABR thresholds to near-wild-type levels. Clinically, Lv et al. (Lancet 2024, PMID:38309303) and the DB-OTO Phase 1/2 interim data (NEJM 2024) reported ABR threshold improvements of 40-80 dB HL and open-set speech recognition in children with biallelic OTOF loss-of-function variants after a single intracochlear injection, with no dose-limiting toxicities at 26-week follow-up. pLannotate annotation of the dual-AAV construct confirms ITR integrity, correct split-intein recombination site orientation, and poly-A signal placement within the 4.7 kb per-vector packaging limit.
OTOF encodes otoferlin, a 227-kDa multi-C2-domain protein anchored to IHC synaptic vesicle membranes. Unlike conventional hair cells that use a standard SNARE-based fusion mechanism, IHCs depend almost exclusively on otoferlin as the calcium sensor for fast, sustained vesicle release at rates up to 1000 vesicles/s required for auditory encoding fidelity. The intron 39 splice donor (c.4799+2T>C) likely generates exon 39 skipping, which removes 167 bp and causes a frameshift in C2E domain—one of the calcium-binding C2 domains critical for membrane fusion activity. The dual-AAV split site is strategically chosen between exons 39-40 to minimize functional disruption of the reconstituted otoferlin: the 5' vector encodes C2A-C2D domains and the 3' vector encodes C2E-C2F plus the transmembrane anchor. Given the exact splice site disrupted by c.4799+2T>C coincides with the dual-AAV split region, this variant may serve as a model case for validating trans-splicing reconstruction of the precise exon junction in patient-derived IHC organoids.
LIMITATIONS
Dual-AAV trans-splicing efficiency is inherently lower than single-vector delivery—recombination between the two halves requires co-transduction of the same IHC, and recombination efficiency in cochlear IHCs in vivo has been reported at 30-60%, which may limit the fraction of cells achieving therapeutic otoferlin levels. The Myo15 promoter provides IHC specificity, but promoter silencing over time has not been evaluated beyond 2-year follow-up in the current trials; long-term expression durability is critical given the pediatric target population. Intracochlear injection carries procedural risks including perilymph fistula, residual hearing loss, and vestibular dysfunction—surgery must be performed at specialized cochlear implant centers with microsurgical expertise. Pre-existing neutralizing antibodies against the AAV serotype (AAV2, AAVAnc80L65, or AAV9 depending on the program) should be screened before dosing; seroprevalence in pediatric populations varies by region but can reach 30-50% for common serotypes, which may affect eligibility. For compound heterozygotes where c.4799+2T>C is paired with a missense allele retaining partial otoferlin function, the net OTOF deficiency may be less severe, and the therapeutic threshold for meaningful hearing restoration may differ from biallelic null patients studied in current trials.