A base editor for the long-term restoration of auditory function in mice with recessive profound deafness.

A prevalent recessive mutation (c.2485C>T, p.Q829X) within the OTOF gene leads to profound prelingual hearing loss. Here we show that in Otof mice harbouring a mutation (c.2482C>T, p.Q828X) homozygous to human OTOF that faithfully mimics the hearing-loss phenotype, a base editor (consisting of the deaminase ABE7.10max and the Cas9 variant SpCas9-NG) packaged in adeno-associated viruses and injected into the inner ear of the mice via the round-window membrane effectively corrected the pathogenic mutation, with no apparent off-target effects. The treatment restored the levels of the otoferlin protein in 88% of the inner hair cells and stably rescued the auditory function of the mice to near-wild-type levels for over 1.5 years while improving synaptic exocytosis in the inner hair cells. We also show that an adenine base editor that targets the prevalent human OTOF mutation restored hearing in humanized mice to levels comparable to those of the wild-type counterparts. Base editors may be effective for the treatment of hereditary deafness.

Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation.

Mutations in microRNA-96 (MIR96) cause autosomal dominant deafness-50 (DFNA50), a form of delayed-onset hearing loss. Genome editing has shown efficacy in hearing recovery through intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications, which has not been done. Here, we developed a genome editing therapy for the MIR96 mutation 14C>A by screening different CRISPR systems and optimizing Cas9 expression and the sgRNA scaffold for efficient and specific mutation editing. AAV delivery of the KKH variant of Staphylococcus aureus Cas9 (SaCas9-KKH) and sgRNA to the cochleae of presymptomatic (3-week-old) and symptomatic (6-week-old) adult Mir9614C>A/+ mutant mice improved hearing long term, with efficacy increased by injection at a younger age. Adult inner ear delivery resulted in transient Cas9 expression without evidence of AAV genomic integration, indicating the good safety profile of our in vivo genome editing strategy. We developed a dual-AAV system, including an AAV-sgmiR96-master carrying sgRNAs against all known human MIR96 mutations. Because mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lay the foundation for the development of treatment for patients with DFNA50 caused by MIR96 mutations.

Bilateral gene therapy in children with autosomal recessive deafness 9: single-arm trial results.

Gene therapy is a promising approach for hereditary deafness. We recently showed that unilateral AAV1-hOTOF gene therapy with dual adeno-associated virus (AAV) serotype 1 carrying human OTOF transgene is safe and associated with functional improvements in patients with autosomal recessive deafness 9 (DFNB9). The protocol was subsequently amended and approved to allow bilateral gene therapy administration. Here we report an interim analysis of the single-arm trial investigating the safety and efficacy of binaural therapy in five pediatric patients with DFNB9. The primary endpoint was dose-limiting toxicity at 6 weeks, and the secondary endpoint included safety (adverse events) and efficacy (auditory function and speech perception). No dose-limiting toxicity or serious adverse event occurred. A total of 36 adverse events occurred. The most common adverse events were increased lymphocyte counts (6 out of 36) and increased cholesterol levels (6 out of 36). All patients had bilateral hearing restoration. The average auditory brainstem response threshold in the right (left) ear was >95 dB (>95 dB) in all patients at baseline, and the average auditory brainstem response threshold in the right (left) ear was restored to 58 dB (58 dB) in patient 1, 75 dB (85 dB) in patient 2, 55 dB (50 dB) in patient 3 at 26 weeks, and 75 dB (78 dB) in patient 4 and 63 dB (63 dB) in patient 5 at 13 weeks. The speech perception and the capability of sound source localization were restored in all five patients. These results provide preliminary insights on the safety and efficacy of binaural AAV gene therapy for hereditary deafness. The trial is ongoing with longer follow-up to confirm the safety and efficacy findings. Chinese Clinical Trial Registry registration: ChiCTR2200063181 .

Unveiling a novel GJB2 dominant K22T mutation in a Chinese family with hearing loss.

Hearing loss constitutes one of the most prevalent conditions within the field of otolaryngology. Recent investigations have revealed that mutations in deafness-associated genes, including point mutations and variations in DNA sequences, can cause hearing impairments. With the ethology of deafness remaining unclear for a substantial portion of the affected population, further screenings for pathogenic mutations are imperative to unveil the underlying mechanisms. On this study, by using next-generation sequencing, we examine 129 commonly implicated deafness-related genes in a Chinese family with hearing loss, revealing a novel heterozygous dominant mutation in the GJB2 gene (GJB2: c.65T>G: p. Lys22Thr). This mutation consistently occurs in affected family members but is not detected in unaffected individuals, strongly suggesting its causative role in hearing loss. Structural analysis indicates potential disruption to the Cx26 gap junction channel's hydrogen bond and electrostatic interactions, aligning with predictions from the PolyPhen and SIFT algorithms. In conclusion, our study provides conclusive evidence that the identified heterozygous GJB2 mutation (GJB2: c.65T>G: p. Lys22Thr), specifically the K22T alteration, is the primary determinant of the family's deafness. This contribution enhances our understanding of the interplay between common deafness-associated genes and hearing loss, offering valuable insights for diagnostic guidance and the formulation of therapeutic strategies for this condition.

Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear.

Adeno-associated virus (AAV)-mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a "multiple vectors in one AAV" strategy for promoter construction in vivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof -/- mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.

Hair cell-specific Myo15 promoter-mediated gene therapy rescues hearing in DFNB9 mouse model.

Adeno-associated viral (AAV) vectors are increasingly used as vehicles for gene delivery to treat hearing loss. However, lack of specificity of the transgene expression may lead to overexpression of the transgene in nontarget tissues. In this study, we evaluated the expression efficiency and specificity of transgene delivered by AAV-PHP.eB under the inner ear sensory cell-specific Myo15 promoter. Compared with the ubiquitous CAG promoter, the Myo15 promoter initiates efficient expression of the GFP fluorescence reporter in hair cells, while minimizing non-specific expression in other cell types of the inner ear and CNS. Furthermore, using the Myo15 promoter, we constructed an AAV-mediated therapeutic system with the coding sequence of OTOF gene. After inner ear injection, we observed apparent hearing recovery in Otof-/- mice, highly efficient expression of exogenous otoferlin, and significant improvement in the exocytosis function of inner hair cells. Overall, our results indicate that gene therapy mediated by the hair cell-specific Myo15 promoter has potential clinical application for the treatment of autosomal recessive deafness and yet for other hereditary hearing loss related to dysfunction of hair cells.

[Study on gene therapy for DPOAE and ABR threshold changes in adult Otof-/- mice].

Objective:This study aims to analyze the threshold changes in distortion product otoacoustic emissions（DPOAE） and auditory brainstem response（ABR） in adult Otof-/- mice before and after gene therapy, evaluating its effectiveness and exploring methods for assessing hearing recovery post-treatment. Methods:At the age of 4 weeks, adult Otof-/- mice received an inner ear injection of a therapeutic agent containing intein-mediated recombination of the OTOF gene, delivered via dual AAV vectors through the round window membrane（RWM）. Immunofluorescence staining assessed the proportion of inner ear hair cells with restored otoferlin expression and the number of synapses.Statistical analysis was performed to compare the DPOAE and ABR thresholds before and after the treatment. Results:AAV-PHP. eB demonstrates high transduction efficiency in inner ear hair cells. The therapeutic regimen corrected hearing loss in adult Otof-/- mice without impacting auditory function in wild-type mice. The changes in DPOAE and ABR thresholds after gene therapy are significantly correlated at 16 kHz. Post-treatment,a slight increase in DPOAE was observeds,followed by a recovery trend at 2 months post-treatment. Conclusion:Gene therapy significantly restored hearing in adult Otof-/- mice, though the surgical delivery may cause transient hearing damage. Precise and gentle surgical techniques are essential to maximize gene therapy's efficacy.

AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness.

Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno-associated virus (AAV)-mediated OTOF gene replacement therapy is extensively validated in Otof-deficient mice. However, the clinical safety and efficacy of AAV-OTOF is not reported. Here, AAV-OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non-human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV-OTOF is delivered into one cochlea of a 5-year-old deaf patient and into the bilateral cochleae of an 8-year-old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure-tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5-year-old patient can be restored to the normal range at 1 month after AAV-OTOF injection, while the 8-year-old patient can hear the conversational sounds. Most importantly, the 5-year-old patient can hear and recognize speech only through the AAV-OTOF-injected ear. This study is the first to demonstrate the safety and efficacy of AAV-OTOF in patients, expands and optimizes current OTOF-related gene therapy and provides valuable information for further application of gene therapies for deafness.

AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial.

BACKGROUND: Autosomal recessive deafness 9, caused by mutations of the OTOF gene, is characterised by congenital or prelingual, severe-to-complete, bilateral hearing loss. However, no pharmacological treatment is currently available for congenital deafness. In this Article, we report the safety and efficacy of gene therapy with an adeno-associated virus (AAV) serotype 1 carrying a human OTOF transgene (AAV1-hOTOF) as a treatment for children with autosomal recessive deafness 9. METHODS: This single-arm, single-centre trial enrolled children (aged 1-18 years) with severe-to-complete hearing loss and confirmed mutations in both alleles of OTOF, and without bilateral cochlear implants. A single injection of AAV1-hOTOF was administered into the cochlea through the round window. The primary endpoint was dose-limiting toxicity at 6 weeks after injection. Auditory function and speech were assessed by appropriate auditory perception evaluation tools. All analyses were done according to the intention-to-treat principle. This trial is registered with Chinese Clinical Trial Registry, ChiCTR2200063181, and is ongoing. FINDINGS: Between Oct 19, 2022, and June 9, 2023, we screened 425 participants for eligibility and enrolled six children for AAV1-hOTOF gene therapy (one received a dose of 9 × 1011 vector genomes [vg] and five received 1·5 × 1012 vg). All participants completed follow-up visits up to week 26. No dose-limiting toxicity or serious adverse events occurred. In total, 48 adverse events were observed; 46 (96%) were grade 1-2 and two (4%) were grade 3 (decreased neutrophil count in one participant). Five children had hearing recovery, shown by a 40-57 dB reduction in the average auditory brainstem response (ABR) thresholds at 0·5-4·0 kHz. In the participant who received the 9 × 1011 vg dose, the average ABR threshold was improved from greater than 95 dB at baseline to 68 dB at 4 weeks, 53 dB at 13 weeks, and 45 dB at 26 weeks. In those who received 1·5 × 1012 AAV1-hOTOF, the average ABR thresholds changed from greater than 95 dB at baseline to 48 dB, 38 dB, 40 dB, and 55 dB in four children with hearing recovery at 26 weeks. Speech perception was improved in participants who had hearing recovery. INTERPRETATION: AAV1-hOTOF gene therapy is safe and efficacious as a novel treatment for children with autosomal recessive deafness 9. FUNDING: National Natural Science Foundation of China, National Key R&D Program of China, Science and Technology Commission of Shanghai Municipality, and Shanghai Refreshgene Therapeutics.

Distributional comparison of different AAV vectors after unilateral cochlear administration.

The adeno-associated virus (AAV) gene therapy has been widely applied to mouse models for deafness. But, AAVs could transduce non-targeted organs after inner ear delivery due to their low cell-type specificity. This study compares transgene expression and biodistribution of AAV1, AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB after round window membrane (RWM) injection in neonatal mice. The highest virus concentration was detected in the injected cochlea. AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB transduced both inner hair cells (IHCs) and outer hair cells (OHCs) with high efficiency, while AAV1 transduced IHCs with high efficiency but OHCs with low efficiency. All AAV subtypes finitely transduced contralateral inner ear, brain, heart, and liver compared with the injected cochlea. In most brain regions, the enhanced green fluorescent protein (eGFP) expression of AAV1 and AAV2 was lower than that of other four subtypes. We suggested the cochlear aqueduct might be one of routes for vectors instantaneously infiltrating into the brain from the cochlea through a dye tracking test. In summary, our results provide available data for further investigating the biodistribution of vectors through local inner ear injection and afford a reference for selecting AAV serotypes for gene therapy toward deafness.

RNA base editing therapy cures hearing loss induced by OTOF gene mutation.

Otoferlin (OTOF) gene mutations represent the primary cause of hearing impairment and deafness in auditory neuropathy. The c.2485C>T (p. Q829X) mutation variant is responsible for approximately 3% of recessive prelingual deafness cases within the Spanish population. Previous studies have used two recombinant AAV vectors to overexpress OTOF, albeit with limited efficacy. In this study, we introduce an enhanced mini-dCas13X RNA base editor (emxABE) delivered via an AAV9 variant, achieving nearly 100% transfection efficiency in inner hair cells. This approach is aimed at treating OTOFQ829X, resulting in an approximately 80% adenosine-to-inosine conversion efficiency in humanized OtofQ829X/Q829X mice. Following a single scala media injection of emxABE targeting OTOFQ829X (emxABE-T) administered during the postnatal day 0-3 period in OtofQ829X/Q829X mice, we observed OTOF expression restoration in nearly 100% of inner hair cells. Moreover, auditory function was significantly improved, reaching similar levels as in wild-type mice. This enhancement persisted for at least 7 months. We also investigated P5-P7 and P30 OtofQ829X/Q829X mice, achieving auditory function restoration through round window injection of emxABE-T. These findings not only highlight an effective therapeutic strategy for potentially addressing OTOFQ829X-induced hearing loss but also underscore emxABE as a versatile toolkit for treating other monogenic diseases characterized by premature termination codons.

Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation.

Mutations in microRNA-96 ( MIR96 ) cause dominant delayed onset hearing loss DFNA50 without treatment. Genome editing has shown efficacy in hearing recovery by intervention in neonatal mice, yet editing in the adult inner ear is necessary for clinical applications. Here, we developed an editing therapy for a C>A point mutation in the seed region of the Mir96 gene, Mir96 14C>A associated with hearing loss by screening gRNAs for genome editors and optimizing Cas9 and sgRNA scaffold for efficient and specific mutation editing in vitro. By AAV delivery in pre-symptomatic (3-week-old) and symptomatic (6-week-old) adult Mir96 14C>A mutant mice, hair cell on-target editing significantly improved hearing long-term, with an efficacy inversely correlated with injection age. We achieved transient Cas9 expression without the evidence of AAV genomic integration to significantly reduce the safety concerns associated with editing. We developed an AAV-sgmiR96-master system capable of targeting all known human MIR96 mutations. As mouse and human MIR96 sequences share 100% homology, our approach and sgRNA selection for efficient and specific hair cell editing for long-term hearing recovery lays the foundation for future treatment of DFNA50 caused by MIR96 mutations.

Preclinical evaluation of the efficacy and safety of AAV1-hOTOF in mice and nonhuman primates.

Pathogenic mutations in the OTOF gene cause autosomal recessive hearing loss (DFNB9), one of the most common forms of auditory neuropathy. There is no biological treatment for DFNB9. Here, we designed an OTOF gene therapy agent by dual-adeno-associated virus 1 (AAV1) carrying human OTOF coding sequences with the expression driven by the hair cell-specific promoter Myo15, AAV1-hOTOF. To develop a clinical application of AAV1-hOTOF gene therapy, we evaluated its efficacy and safety in animal models using pharmacodynamics, behavior, and histopathology. AAV1-hOTOF inner ear delivery significantly improved hearing in Otof-/- mice without affecting normal hearing in wild-type mice. AAV1 was predominately distributed to the cochlea, although it was detected in other organs such as the CNS and the liver, and no obvious toxic effects of AAV1-hOTOF were observed in mice. To further evaluate the safety of Myo15 promoter-driven AAV1-transgene, AAV1-GFP was delivered into the inner ear of Macaca fascicularis via the round window membrane. AAV1-GFP transduced 60%-94% of the inner hair cells along the cochlear turns. AAV1-GFP was detected in isolated organs and no significant adverse effects were detected. These results suggest that AAV1-hOTOF is well tolerated and effective in animals, providing critical support for its clinical translation.

Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo.

Mutations in Atp2b2, an outer hair cell gene, cause dominant hearing loss in humans. Using a mouse model Atp2b2Obl/+, with a dominant hearing loss mutation (Oblivion), we show that liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Obl allele. Large deletions encompassing the Obl locus and indels were identified as the result of editing. In vivo genome editing promotes outer hair cell survival and restores their function, leading to hearing recovery. We further show that in a double-dominant mutant mouse model, in which the Tmc1 Beethoven mutation and the Atp2b2 Oblivion mutation cause digenic genetic hearing loss, Cas9/sgRNA delivery targeting both mutations leads to partial hearing recovery. These findings suggest that liposome-RNP delivery can be used as a strategy to recover hearing with dominant mutations in OHC genes and with digenic mutations in the auditory hair cells, potentially expanding therapeutics of gene editing to treat hearing loss.

MPZL2 variant analysis with whole exome sequencing in a cohort of Chinese hearing loss patients.

BACKGROUND: Hearing loss is a genetically heterogeneous disease with more than 100 genes identified. Pathogenic variants in the MPZL2 gene cause autosomal recessive non-syndromic hearing loss. MPZL2 patients showed mild to moderate progressive hearing loss with onset age around 10 years old. To date, four pathogenic variants have been identified. AIMS: To explore the clinical characteristics and variants of MPZL2-related hearing loss, and summarize the prevalence rate in overall hearing loss patients. MATERIAL AND METHODS: To determine the prevalence of MPZL2-related hearing loss in the Chinese population, we analyzed MPZL2 variants of whole exome sequencing data derived from a cohort of 385 hearing loss patients. RESULTS: Overall, homozygous MPZL2 variants were identified in 5 sporadic cases (diagnostic rate = 1.30%). A novel missense variant c.52C > T;p.Leu18Phe was identified in one other patient with compound heterozygous mutations in MPZL2, but the pathogenicity was uncertain according to the American College of Medical Genetics guidelines (2015). A patient homozygous for the c.220C > T,p.Gln74Ter variant showed congenital profound hearing loss at all frequencies, a phenotype different from previous reports. CONCLUSIONS: Our results enriched the mutation and phenotype spectrum of MPZL2-related hearing loss. Comparisons between allele frequencies of MPZL2:c.220C > T;p.Gln74Ter and other common deafness variants suggested that MPZL2:c.220C > T;p.Gln74Ter should be included in the group of common deafness variants for prescreening.

The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice.

Mutations in GJB2 (Gap junction protein beta 2) are the most common genetic cause of non-syndromic hereditary deafness in humans, especially the 35delG and 235delC mutations. Owing to the homozygous lethality of Gjb2 mutations in mice, there are currently no perfect mouse models carrying Gjb2 mutations derived from patients for mimicking human hereditary deafness and for unveiling the pathogenesis of the disease. Here, we successfully constructed heterozygous Gjb2+/35delG and Gjb2+/235delC mutant mice through advanced androgenic haploid embryonic stem cell (AG-haESC)-mediated semi-cloning technology, and these mice showed normal hearing at postnatal day (P) 28. A homozygous mutant mouse model, Gjb235delG/35delG, was then generated using enhanced tetraploid embryo complementation, demonstrating that GJB2 plays an indispensable role in mouse placenta development. These mice exhibited profound hearing loss similar to human patients at P14, i.e., soon after the onset of hearing. Mechanistic analyses showed that Gjb2 35delG disrupts the function and formation of intercellular gap junction channels of the cochlea rather than affecting the survival and function of hair cells. Collectively, our study provides ideal mouse models for understanding the pathogenic mechanism of DFNB1A-related hereditary deafness and opens up a new avenue for investigating the treatment of this disease.

Asymmetric pendrin homodimer reveals its molecular mechanism as anion exchanger.

Pendrin (SLC26A4) is an anion exchanger expressed in the apical membranes of selected epithelia. Pendrin ablation causes Pendred syndrome, a genetic disorder associated with sensorineural hearing loss, hypothyroid goiter, and reduced blood pressure. However its molecular structure has remained unknown, limiting our understanding of the structural basis of transport. Here, we determine the cryo-electron microscopy structures of mouse pendrin with symmetric and asymmetric homodimer conformations. The asymmetric homodimer consists of one inward-facing protomer and the other outward-facing protomer, representing coincident uptake and secretion- a unique state of pendrin as an electroneutral exchanger. The multiple conformations presented here provide an inverted alternate-access mechanism for anion exchange. The structural and functional data presented here disclose the properties of an anion exchange cleft and help understand the importance of disease-associated variants, which will shed light on the pendrin exchange mechanism.