Gene Therapy for Genetic Hearing Loss: AAV Gene Therapy Shows Promising Results for Autosomal Recessive Deafness 9 (DFNB9)

Hearing loss is a major global health issue, affecting about 466 million people worldwide according to the World Health Organization. In children, it can severely hinder speech, language development, education, and social integration. Over 50% of congenital cases are genetic, with more than 120 causative genes identified. Mutations in the OTOF gene, which encodes otoferlin, cause DFNB9, an autosomal recessive nonsyndromic deafness marked by prelingual, severe-to-profound impairment.

Gene therapy is emerging as a promising alternative for treating genetic hearing loss. Adeno-associated viruses (AAVs) have gained attention as delivery vectors due to their strong safety profile, ability to transduce non-dividing cells, and low immunogenicity. Early clinical trials for OTOF-related deafness have shown encouraging results. In a recently published study in Molecular Therapy – Methods & Clinical Development, researchers from Regeneron Pharmaceuticals & Decibel Therapeutics reported on the development of DB-OTO, a dual-vector, hair cell-specific AAV gene therapy incorporating human OTOF variant 5 (OTOFv5) ⁽¹⁾. This work highlights DB-OTO as a key step in the ongoing advancement of gene therapy for hearing loss.

OTOF Gene and OTOFv5 Variant

The OTOF gene, located on chromosome 2p23.1, spans ~90 kb with 48 exons and encodes otoferlin, a multi-C2 domain protein acting as a calcium sensor in inner hair cells (IHCs). Otoferlin is crucial for synaptic vesicle exocytosis and endocytosis at IHC ribbon synapses, enabling rapid neurotransmitter release in response to sound and ensuring faithful transmission of auditory signals to spiral ganglion neurons and central pathways.

Five human transcript variants of OTOF arise from alternative splicing. Of these, v1 (NM_194248) and v5 (NM_001287489) are most relevant to cochlear function due to their similarity to the predominant mouse cochlear variant. These variants differ mainly in their final exons, which encode distinct C-terminal domains with potentially unique functions.

OTOFv5 is particularly important for therapy. It differs by only one amino acid from the main mouse cochlear variant, suggesting strong functional conservation. In contrast, v1 resembles a brain-enriched mouse variant, not cochlear-specific. This pattern indicates OTOFv5 is optimized for auditory function.

Pathogenic mutations within OTOFv5’s final exon, such as Arg1939Gln, have been linked to hearing loss, underscoring its clinical relevance. Its close homology to the mouse cochlear variant, combined with mutation data, strongly supports OTOFv5 as the optimal therapeutic transgene for OTOF-related hearing loss.

Gene Therapy Studies for Hearing Loss

Gene therapy for hearing loss has developed rapidly in the last twenty years, and third-party studies have confirmed its potential through repeated proof-of-concept research. Early investigations focused on various viral vectors, with adeno-associated virus (AAV) emerging as the preferred delivery system because of superior safety, low immunogenicity, and effectiveness in non-dividing cell types according to previous reports ⁽²⁾.

For hearing loss related to OTOF mutations, independent preclinical assessments have shown gene replacement is feasible. Research groups have demonstrated successful hearing restoration in Otof-null mice after dual-AAV delivery of full-length Otof cDNA, establishing the foundation for gene therapy in DFNB9. Subsequent teams further optimized the approach by investigating alternative promoters and delivery methods, other third-party studies regularly reported improved auditory function after AAV-mediated OTOF gene transfer ⁽³⁾.

Several limitations are noted in the independent literature. Most work treats immature mice, while human cochleae develop fully in utero. Many groups used broadly acting promoters, which resulted in incomplete OTOF expression in certain cochlear cells, particularly outer hair cells. Durability data beyond several months is sparse, and reports indicate that some treated mice lose therapeutic benefit over time ⁽²⁾.

Despite these issues, third-party preclinical research supports translational progress. Ongoing clinical trials testing AAV-based OTOF therapy in humans have been highlighted in recent studies, which show encouraging safety profiles and preliminary hearing restoration in patients, including children and adults ⁽⁴⁾.

DB-OTO: A Novel Gene Therapy Approach

DB-OTO is a hair cell-specific, dual-vector AAV gene therapy designed to restore hearing in individuals with OTOF-related deafness. Its development builds on prior research and addresses key limitations of earlier gene therapy strategies.

A major innovation is the use of a hair cell-specific promoter, avoiding off-target expression that could cause toxicity and reduce long-term safety. Researchers engineered a synthetic promoter from the murine Myosin 15 (Myo15) gene, leveraging regulatory sequences identified from IHC chromatin datasets and conserved vertebrate motifs. The best variant was selected through GFP reporter screening in murine explants, followed by in vivo testing in mice and macaques.

Because the OTOFv5 cDNA (5,979 nt) exceeds the AAV packaging limit (~4.7 kb), DB-OTO employs a dual-vector system. Two AAV1 vectors reconstitute a functional OTOF expression cassette, refining earlier split-gene methods. OTOFv5 was chosen after studies in Otof-deficient mice showed only this variant restored auditory brainstem responses, supporting its role as the optimal therapeutic transgene.

The AAV1 serotype was selected for its efficient hair cell transduction and favorable safety record in preclinical and clinical studies. Together, the Myo15 promoter, dual-vector design, and OTOFv5 transgene form a comprehensive strategy for safe and effective restoration of hearing in OTOF-related deafness.

Preclinical Development of DB-OTO

DB-OTO was evaluated for efficacy, safety, and durability in animal models, beginning with a CRISPR-Cas9 knock-in DFNB9 mouse model. A truncating mutation (Q828X), orthologous to a known human OTOF mutation, eliminated otoferlin expression in homozygous mice. These mice lacked ABRs but retained normal DPOAEs, closely mirroring human OTOF-related deafness.

A key comparison was made between ubiquitous (smCBA) and hair cell-specific (Myo15) promoters. Both restored hearing in OtofQ828X/Q828X mice, but only Myo15 maintained long-term responses. In contrast, smCBA-driven expression led to IHC loss, macrophage activation, and declining auditory function, highlighting the importance of restricting expression to hair cells.

DB-OTO, combining AAV1 with the Myo15 promoter driving OTOFv5, produced robust, dose-dependent hearing recovery. A single injection restored hearing in 70–90% of OtofQ828X/Q828X mice across a 10-fold dose range, with ABR improvements of 40–45 dB in the 16–22.6 kHz range. Therapeutic effects were stable through 12 weeks and effective across mice aged 7.6–11.1 weeks, corresponding to mature cochlear development in humans at birth.

Safety assessments showed minimal IHC and OHC loss at all doses, supporting the durability and translational potential of DB-OTO for pediatric congenital hearing loss.

Non-Human Primate Studies

To assess clinical relevance, researchers tested DB-OTO in cynomolgus macaques, focusing on transgene specificity and the impact of pre-existing AAV1 immunity.

Using an AAV1 vector with a 1.0 kb mMyo15 promoter–eGFP construct, they confirmed promoter specificity: vector genomes were widespread in the cochlea, but GFP expression was confined to hair cells of the organ of Corti, validating cell-type specificity in primates. A dual-AAV1 GFP construct further showed robust, frequency-wide IHC transduction after intracochlear delivery.

The effect of pre-existing neutralizing antibodies (NAbs) to AAV1 was also tested. About half of the macaques were NAb-positive before dosing. Following bilateral DB-OTO administration, vector DNA and hOTOF mRNA were detected in temporal bones at 7 and 27 weeks across dose groups, with no correlation between NAb status and gene expression.

These results suggest that the blood-labyrinth barrier, like the blood-retinal barrier, protects against anti-capsid immunity, an important advantage since pre-existing AAV antibodies are common in humans and often limit gene therapy efficacy.

Clinical Implications and Future Directions

Encouraging preclinical results supported the launch of a Phase I/II trial (NCT05788536) in pediatric patients with OTOF-related hearing loss, marking a milestone in gene therapy for genetic deafness. Unlike cochlear implants, which bypass hair cells and provide an artificial sound signal, DB-OTO restores otoferlin in IHCs, aiming for more natural hearing, improved pitch resolution, and better speech perception in noise. Delivered as a single intervention, it may also avoid the long-term rehabilitation and maintenance required with implants.

Key challenges remain. Timing of intervention is critical, early treatment may prevent spiral ganglion degeneration, though safety in very young infants must be established. Delivery currently requires intracochlear injection, an invasive surgical procedure; less invasive methods such as intra-tympanic injection could broaden use.

Long-term durability and safety must also be proven. While stable efficacy has been shown for months in mice and primates, extended human follow-up is needed to confirm years-long benefit and rule out immune or delayed adverse effects.

DB-OTO may also serve as a blueprint for broader genetic hearing loss therapies. Cell type-specific promoters, improved vector design, and novel AAV capsids with enhanced hair cell tropism could expand gene therapy options for diverse forms of deafness, building on DB-OTO’s foundation.

Conclusion

The development of DB-OTO represents a significant maturation of gene therapy for hearing loss. It moves beyond simple proof-of-concept to address the nuanced challenges of safety, specificity, and durability required for clinical application. By rationally selecting the most functional human transgene (OTOFv5), engineering a highly specific synthetic promoter (mMyo15), and demonstrating sustained efficacy without toxicity in mature animal models, the study provides a robust preclinical foundation.

The data convincingly argue that cell-specific expression is non-negotiable for safe and durable inner ear gene therapy. The successful transduction in non-human primates and the lack of impact from pre-existing immunity further de-risk the clinical path. These comprehensive findings have supported the initiation of an ongoing Phase I/II clinical trial (NCT05788536), bringing the promise of biological hearing restoration for children with DFNB9 closer to reality. DB-OTO stands as a testament to how sophisticated vector design and a meticulous focus on biological safety can pave the way for transformative genetic medicines.

References

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4. J, et al.  AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness. Adv Sci (Weinh). 2024 Mar;11(11):e2306788. doi: 10.1002/advs.202306788. Epub 2024 Jan 8. Erratum in: Adv Sci (Weinh). 2025; 12:e2504047. doi: 10.1002/advs.202504047.