Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea

Domain: neuroscience
Published: 2025-12-16
Editorial outcome: minor_revision

Abstract
Mutations in the GJB2 gene cause DFNB1, the most common hereditary hearing loss. GJB2 is expressed by cochlear epithelial cells and fibrocytes, but not by sensory hair cells or neurons. Attempts to treat DFNB1 mouse models with gene therapy have not substantially restored function, as inappropriate expression in hair cells and neurons might compromise their electrical activity. Here, we use ATAC-seq to identify candidate gene regulatory elements (GREs) that can drive cell-type-specific expression of GJB2. HA-tagged GJB2, delivered to a conditional knockout mouse with AAV vectors carrying GREs, is expressed by the appropriate cells, prevents degeneration, and rescues hearing by only 10–20 dB. In a Gjb2 partial knockdown model, a vector lacking HA prevents degeneration and completely restores hearing. In cynomolgus monkey cochleas, human GJB2.HA delivered with similar vectors is located in the appropriate cell types and causes little or no compromise of hearing sensitivity. Together, these findings suggest that GRE-mediated expression of GJB2 can prevent hearing loss in DFNB1 patients.

Introduction
Hearing loss is among the most common sensory disorders1,2, affecting 2–3 in 1000 newborn children. Although more than 180 genes have been causally linked to hearing loss3, a single form of recessive, non-syndromic hearing loss, DFNB1, is responsible for up to 50% of congenital deafness worldwide outside of sub-Saharan Africa4,5. In the United States alone, about 3500 children are born each year with mutations in both alleles of the causative gene, GJB24–7. Many are born with profound hearing loss, but two-third have some residual hearing at birth and the majority of those lose hearing over the next few years8, suggesting that a developmental window exists for therapeutic intervention.

GJB2 encodes the gap junction protein GJB2/connexin 26. In the cochlea, GJB2 is expressed in two cell groups: an epithelial system comprising supporting cells of the organ of Corti, epithelial cells of the inner and outer sulcus, and interdental cells; and a connective tissue system comprising fibrocytes of the lateral wall and suprastrial zone, and basal cells of the stria vascularis9–11. GJB2 is absent from the sensory hair cells and the postsynaptic spiral ganglion neurons.

There are several hypotheses for why GJB2 is critical for the cochlea. In one, the K+ that enters hair cells through mechanotransduction channels and leaves through basal K+ channels is shuttled away from the organ of Corti by the epithelial system9. Failure to remove K+ from the extracellular medium would tend to depolarize hair cells and allow toxic Ca2+ influx through voltage-gated calcium channels. Another is that GJB2 in fibrocytes and strial cells is important for transporting K+ into endolymph and producing the endocochlear potential9. A third hypothesis suggests a required developmental role, perhaps involving glucose transport, as mice lacking GJB2 in the inner ear have profound loss of hair cells and supporting cells by P3012–15. If Gjb2 is deleted after P6, the phenotype is much milder16; however, there remains a long-term requirement for GJB2, as hair-cell loss occurs after months even with deletion as late as P1417. A fourth hypothesis involves ATP- and connexin-dependent intracellular Ca2+ signaling in development18. Regardless of etiology, these studies point to a critical role for GJB2 in the early postnatal period in mice, with a sustained but less critical long-term function.

AAV mediated gene addition to neonatal Gjb2 conditional knockout mice might be expected to rescue this pathology. Yet this approach has thus far failed to substantially rescue function12,19,20. There may be various reasons for this failure—whether the capsid transduces all the appropriate cells, whether regulatory elements prevent expression in inappropriate cells, whether the vector dose is adequate, or whether the vector is delivered soon enough to prevent cochlear degeneration—but each study has used different combinations of these elements and their relative importance for functional rescue has not been systematically studied.

Here we describe a successful gene replacement approach for DFNB1-related hearing loss employing ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) identification of gene regulatory elements (GREs) to limit off-target expression of GJB2, which fully restores hearing sensitivity in a Gjb2 mutant mouse model. Furthermore, we demonstrate that a vector employing GREs effectively targets GJB2-expressing cells in a non-human primate (NHP; Macaca fascicularis) model without inducing toxicity. This study demonstrates significant rescue in a Gjb2 mouse model of DFNB1 deafness while also showing safety and tolerability in an NHP model.

Results
Gene regulatory elements restrict viral GJB2 expression
We previously found that several capsids, including AAV9-PHP.B and AAV-S, efficiently transduce the cells in neonatal mouse cochlea that natively express Gjb2, based on expression of a GFP marker gene driven by a ubiquitous promoter21–23. To determine whether we can also express Gjb2 in those cells, we packaged the coding sequence for mouse GJB2 (mmGJB2) in the AAV9-PHP.B vector under the control of the chicken β-actin (CBA) promoter (AAV-CBA-mmGJB2; Fig. 1d) and injected the vector into the cochleas of neonatal (P0 or P1) mice through the round window membrane (RWM). Ubiquitous expression of mmGJB2 was often lethal. Mice injected with AAV-CBA-mmGJB2 exhibited severe shaking and almost all animals died by P5. To determine whether the lethality was a consequence of GJB2 activity, we constructed a similar vector in which the GJB2 sequence contained the common null mutation 35delG, which leads to a frameshift and stop (AAV-CBA-mmGJB2null). Mice injected with this vector survived until at least P30, the last time point evaluated, suggesting that lethality is a consequence of inappropriate gap junction coupling. AAV vectors injected into the mouse cochlea at early postnatal ages can spread to the brain24–27. We reasoned that nonspecific expression of GJB2 in neurons, driven by the CBA promoter, could cause neurons to become electrically coupled to each other and to neighboring glial cells. The high conductance could short-circuit neuronal post-synaptic potentials; if expressed in inhibitory neurons, GJB2 could increase brain excitability and cause lethality.

Indeed, the AAV9-PHP.B capsid was selected for its ability to transduce brain cells in neonatal mouse28. Similarly, in the cochlea, nonspecific expression of GJB2 protein in hair cells could electrically couple them to neighboring supporting cells and attenuate receptor potentials, abolishing the auditory brainstem response (ABR). When we used the promiscuous CBA promoter to drive expression of eGFP (AAV-CBA-eGFP; Fig. 1d) and injected it into the inner ears of P1 mice, we observed high expression of eGFP by P6 in cells of the sensory epithelium, including the inner and outer hair cells (Fig. 2a; Table 1).

We therefore sought to limit virus-mediated expression to those cells that normally express Gjb2, using specific GREs. To identify candidate GREs that control Gjb2 expression in the mouse cochlea, we dissected whole cochleas from neonatal mice (age P2, P5 and P8), purified dissociated nuclei, and employed the Assay for Transposase-Accessible Chromatin with Sequencing (ATAC-seq)29,30 to reveal regions of open chromatin (Fig. 1a), following established protocols31,32. ATAC-seq revealed several dozen such regions, both proximal and distal to the Gjb2 gene (Fig. 1b). To restrict expression to the cochlear fibrocytes and epithelial cells that express Gjb2, we avoided regions that also appeared in a neuronal sample; to select regions more likely to control gene expression, we focused on those conserved among mammalian genomes (Fig. 1b).

We chose two regions containing likely GREs (Fig. 1c, Supplementary Table 1). GRE1 encompassed the noncoding exon 1 and ~200 bp upstream, which includes a putative promoter33; GRE2 constituted the 3’ non-coding segment of exon 2. In order to create a vector that would be more clinically relevant, we used human sequences equivalent to these mouse GREs. The human or mouse GJB2 coding sequence, with or without a C-terminal hemagglutinin epitope (HA) tag, was flanked by the two GREs.

We then constructed AAV vectors that encoded eGFP driven by our selected GREs (Fig. 1d) and injected them into neonatal mouse cochleas. We found that this vector (AAV-GRE-eGFP) resulted in a restricted pattern of eGFP expression that more closely matched endogenous expression of Gjb2 (Figs. 2b and 3a, c). Notably, there was no expression in hair cells (Fig. 2b). Next, we injected mouse cochleas at P1 with vectors encoding either mmGJB2.HA or hsGJB2.HA, adding an HA tag to the C-terminus to track virally expressed GJB2. We analyzed expression in cochleas at P30 using anti-HA staining (Fig. 2c, d). Consistent with normal GJB2 localization, HA-tag expression was detected predominantly in the spiral limbus, inner and outer sulcus epithelia, supporting cells, basal and intermediate cells of the stria vascularis, and fibrocytes of the spiral ligament (Fig. 2c). GJB2 was not observed in inner hair cells (IHCs) or outer hair cells (OHCs). Immunolabeling appeared concentrated in disc-like puncta at cell membranes, indicating that GJB2 was expressed in the appropriate cells and properly trafficked to the cell membrane to form gap junctions (Fig. 2c, d, Table 1). A similar expression pattern was observed for both mmGJB2.HA and hsGJB2.HA.

Hair cells and supporting cells degenerate in conditional knockout mice, resulting in profound deafness
To understand the phenotype of mice lacking GJB2 and to evaluate gene therapies in a model of DFNB1, we used mouse lines with little or no expression of the Gjb2 gene. While humans with two null alleles of GJB2 are viable, mice with constitutive deletion of both alleles are embryonic lethal34. To restrict deletion of Gjb2 to a limited number of tissues, including cochlea, we first used a conditional knockout mouse model that deleted the entire coding sequence (Gjb2tm1Ugds)35; referred to here as Gjb2Pfl, and crossed it to Sox10-Cre mice12,36; referred to here as Cre+. Mice in which Cre recombinase deleted Gjb2 (Gjb2Pfl/Pfl, Cre+) were compared to normal controls without Cre (Gjb2Pfl/Pfl, Cre−) (Fig. 3a–c).

We first characterized normal Gjb2 expression in the cochleas of control mice by immunolabeling cryosections from neonatal (P6) and adult (P30) mice with antibodies to GJB2 and to markers for hair cells and spiral ganglion neurons (SGNs) (Fig. 3). Consistent with previous reports12,36, cochleas at age P6 from Gjb2Pfl/Pfl, Cre− control mice showed prominent Gjb2 expression in the inner and outer sulcus epithelia, supporting cells, the basal and intermediate cells of the stria vascularis, and fibrocytes of spiral ligament and spiral limbus (Fig. 3a). GJB2 was not detected in IHCs or OHCs. By P30, expression levels had increased in these regions (Fig. 3c, Table 1) consistent with earlier findings12,37.

We then evaluated expression in Gjb2Pfl/Pfl, Cre+ knockout mice. At P6, there was no anti-GJB2 labeling in the organ of Corti, or more generally in the epithelial gap junction network that extends from the spiral limbus through the organ of Corti to the spiral prominence (Fig. 3a), validating the GJB2 antibody and confirming Cre-mediated knockout of GJB2 in these cells. Some anti-GJB2 signal was nevertheless present in the fibrocytes of the lateral wall and spiral limbus, suggesting that these cells do not strongly express Sox10 and do not efficiently delete Gjb2, allowing some Gjb2 expression. At P6, despite the absence of a GJB2 network in the organ of Corti, anti-parvalbumin staining showed normal hair cell morphology (Fig. 3a). Furthermore, no ultrastructural abnormalities in IHC and OHC bundle morphology were detected with scanning electron microscopy at P6 (Fig. 3b). By P30, however, Gjb2Pfl/Pfl, Cre+ mice had developed degeneration of both hair cells and supporting cells of the organ of Corti, leading to a flat sensory epithelium (Fig. 3c, Supplementary Fig. 1).

We measured auditory thresholds by ABR and distortion product otoacoustic emissions (DPOAE) recording in both the Gjb2Pfl/Pfl, Cre+ mutants and Gjb2Pfl/Pfl, Cre− control littermates at P30. The Gjb2Pfl/Pfl, Cre+ mice showed profound deafness across all frequencies tested, with ABR thresholds of 120 dB or higher. Gjb2Pfl/Pfl, Cre− animals showed thresholds consistent with wild-type hearing (Supplementary Fig. 2a, b).

Viral delivery of GJB2 with GREs rescues cochlear morphology and hearing in knockout mice
We then injected an AAV9-PHP.B vector encoding mmGJB2.HA under GRE control of expression (AAV-GRE-mmGJB2.HA; Fig. 1d) into cochleas of Gjb2Pfl/Pfl, Cre+ conditional knockout mice at P1 (dose 5×1010 vector genomes [vg]). As with expression of mmGJB2.HA in wild-type cochleas (Fig. 2c), the HA-tagged GJB2 was detected in many cells, including Deiters’ cells, Hensen’s cells, Claudius cells and border cells; fibrocytes type I, II and V of the lateral wall; and fibrocytes of the spiral limbus (Fig. 3d, Table 1)—congruent with the expression of eGFP (Fig. 2b) under the same regulatory elements. Importantly, no GJB2.HA signal was detected in IHCs and OHCs (parvalbumin label, yellow). We observed that restricted expression of GJB2 using AAV-GRE-mmGJB2.HA prevented the degeneration of the organ of Corti typically seen in Gjb2Pfl/Pfl, Cre+ mice at P30. However, these mice still failed to develop the tunnel of the organ of Corti. Cells of the GJB2 network—comprising the epithelium of the inner and outer sulcus and the root cells of the spiral prominence, fibrocytes of the spiral limbus and strial cells—were preserved (Fig. 3d). Hair cells, missing at P30 in untreated Gjb2Pfl/Pfl, Cre+ mice, were present in the vector-treated cochlea (Fig. 3d). Despite the preservation of cochlear structure, expression of GJB2.HA using GREs only slightly mitigated the hearing loss in Gjb2Pfl/Pfl, Cre+ mice (Supplementary Fig. 2a, b): ABR recordings at P30 demonstrated a 10–15 dB SPL improvement. However, no improvement in DPOAE was detected (Supplementary Fig. 2b).

Next, we injected Gjb2Pfl/Pfl, Cre+ mice at P1 with AAV-GRE-hsGJB2 (dose 5×1010 vg), a vector expressing human GJB2 without the HA tag. Thirty days later, the treated Gjb2Pfl/Pfl, Cre+ mice displayed a fully restored GJB2 network (Fig. 3e), matching the levels observed in Gjb2Pfl/Pfl, Cre− control animals, with no signs of histological abnormalities. Additionally, the tunnel of the organ of Corti was rescued (Fig. 3e). When we assessed hearing function in these mice, we observed a significant improvement in ABR thresholds, with an approximate gain of ~50 dB SPL (Supplementary Fig. 2a). However, no improvement in DPOAE was observed (Supplementary Fig. 2b) suggesting failure to rescue OHC function despite a significant improvement in ABR thresholds. When control mice Gjb2Pfl/Pfl, Cre− were injected with AAV-GRE-hsGJB2, their ABR and DPOAE thresholds were similar to those of untreated control mice (Supplementary Fig. 2c, d), indicating lack of vector toxicity.

AAV-mediated GJB2 expression preserves cochlear structure in a milder mouse model of DFNB1
Although incorporation of GREs produced rescue of hearing greater than that previously reported, it did not restore ABR thresholds to wild-type levels. We speculated that GJB2 has a critical developmental role as early as P018, and that vector delivered at P1 might not produce GJB2 protein until P2 or P3. We therefore sought a milder model of DFNB1, in which GJB2 protein expression is not entirely abolished, and turned to a mouse line in which the coding sequence of the upstream Gjb6 gene was replaced with a Neo selection cassette and β-galactosidase reporter38. These substitutions in Gjb6 have the effect of reducing GJB2 protein levels39–41, presumably due to disruption of nearby Gjb2 regulatory sequences, and the mice are referred to here as Gjb2Gjb6–.

Immunofluorescent labeling with antibodies for GJB6 in the normal control Gjb2Gjb6+ cochlea revealed that, similar to Gjb2, GJB6 is widely expressed in the epithelial and connective tissue cells of the cochlea (Fig. 4a; Table 1). The GJB6 signal was detected in Deiters’, Hensen’s, and Claudius cells, as well as in inner and outer pillar cells, in border cells, and in the epithelium of both the inner and outer sulcus. As expected, no GJB6 signal was observed in IHCs or OHCs. Examination of the lateral wall showed that GJB6 expression was high in type I and V fibrocytes, moderate in type II fibrocytes, and absent in type III and IV fibrocytes. In the stria vascularis, GJB6 puncta were found in the basal cells. Additionally, the fibrocytes of the spiral limbus contained GJB6 puncta.

Immunofluorescence analysis of Gjb2Gjb6– cochleas consistently demonstrated complete loss of GJB6 immunoreactivity (Fig. 4a). However, the mutant mice exhibited normal overall gross morphology of the organ of Corti, with normal presence of hair cells.

We observed a significant decrease in Gjb2 expression in Gjb2Gjb6– mice compared to the Gjb2 expression in control Gjb2Gjb6+ mice, particularly in Deiters’, Hensen’s, Claudius cells, and pillar cells, as well as in the epithelium of both the inner and outer sulcus (Fig. 4b, Table 1). However, Gjb2 expression levels in Gjb2Gjb6– mice were similar to normal controls in the fibrocytes of the lateral wall and spiral limbus.

Since there were no observed differences in HA localization following the injection of AAV-GRE-mmGJB2.HA or AAV-GRE-hsGJB2.HA in wild-type control mice (Fig. 2c, d), indicating that mouse and human GJB2 exhibit similar localization and expression patterns in the mouse cochlea, we proceeded to inject Gjb2Gjb6– mice at P1 with AAV-GRE-hsGJB2.HA via the RWM. Thirty days post-injection, we analyzed cochlear morphology and hsGJB2.HA expression (Fig. 5a). Despite strong HA labeling in cells that normally express GJB2, we observed several morphological abnormalities: a reduction in scala media volume, thickening of the tectorial membrane, enlargement of inner sulcus cells attached to the tectorial membrane, and a decrease or absence of the internal spiral sulcus (Fig. 5b). Additionally, there was a reduction or absence of the tunnel of Corti compared to untreated Gjb2Gjb6– and Gjb2Gjb6+ mice. While IHCs appeared largely normal, OHCs were lost. This finding suggests that, despite strong expression and proper trafficking to the cell membrane, the GJB2 carrying the HA tag may not function properly.

Next, we injected Gjb2Gjb6– mice at P1 with AAV-GRE-hsGJB2, a vector encoding hsGJB2 without the HA tag. Thirty days after injection, the treated Gjb2Gjb6– mice receiving AAV-GRE-hsGJB2 showed a GJB2 network completely restored to levels observed in Gjb2Gjb6– control animals, and showed no histological abnormalities (Figs. 4c and 5b, Table 1). Apparently, HA-tagged GJB2 disrupts normal GJB2 function in this mouse model.

Scanning electron microscopy of the apical, middle, and basal turns of the cochlea revealed well-organized hair bundles in both IHCs and OHCs of Gjb2Gjb6+ control mice (Fig. 4d). In untreated Gjb2Gjb6– knockout mice at P30, however, we observed a tonotopic gradient of hair bundle loss from the base to the apex, with hair bundles completely gone in the basal turn and only a few remaining in the middle and apical regions (Fig. 4d). In contrast, Gjb2Gjb6– knockout mice injected with AAV-GRE-hsGJB2 exhibited a robust preservation of hair-bundle morphology across all cochlear turns (Fig. 4e).

AAV-GRE-hsGJB2 rescues hearing in Gjb2Gjb6– mice
We also assessed hearing function in untreated Gjb2Gjb6– and Gjb2Gjb6+ mice, as well as in animals treated with AAV-GRE-hsGJB2.HA or AAV-GRE-hsGJB2, measured with ABR recording in response to broadband clicks and tone bursts, and with DPOAEs. While Gjb2Gjb6+ mice exhibited normal hearing, Gjb2Gjb6– mice were profoundly deaf by P30 (Fig. 6a). Next, we injected Gjb2Gjb6– mice with AAV-GRE-hsGJB2.HA. As expected from previous findings with the HA tag, these mice showed no improvement in hearing, as tested by ABR or DPOAE (Fig. 6a). In contrast, when control mice were injected with AAV-GRE-hsGJB2.HA, their ABR results were similar to those of untreated control mice, and DPOAE levels were only slightly elevated (Fig. 6b). These findings suggest that GJB2.HA is not toxic but is functionally inactive.

Next, we evaluated hearing in mice injected with AAV-GRE-hsGJB2 at high (1.4×1011 vg), medium (7.0×1010 vg) and low (3.0×1010 vg) doses. At P30, Gjb2Gjb6– mice treated with either dose demonstrated complete rescue of click-evoked and tone burst ABR responses, to levels comparable to untreated control mice (Fig. 6c).

We analyzed the average ABR waveform responses to click stimuli at 80 dB SPL in untreated control mice and Gjb2Gjb6– mice treated with high, medium, or low doses of AAV-GRE-hsGJB2. We found that in treated Gjb2Gjb6– mice, ABR waveforms appeared comparable to those of control Gjb2Gjb6+ mice (Fig. 6e). Additionally, we analyzed the amplitude of ABR wave 1 at 16 kHz at P30. The wave 1 amplitudes in mice injected with all doses showed no difference from normal control mice (Fig. 6d). The ABR rescue remained at P60 and P90 (Fig. 6f), with hearing in animals injected with high and medium doses comparable to that of normal hearing mice. However, mice injected with a low dose exhibited an elevation of the threshold at P60 and P90. High amplitudes were preserved up to P90 in mice expressing hsGJB2 (Fig. 6g). At P30, DPOAE measurements demonstrated robust rescue in mice treated with all three doses of AAV-GRE-hsGJB2. This rescue persisted at P60 and P90 with DPOAE levels comparable to those of normal hearing mice in high and medium doses; levels were somewhat decreased with a low dose (Fig. 6h).

Expression of GJB2 with GREs in NHP cochlea is similar to that in mouse
While functional rescue can be tested most easily in mice, larger animals such as NHPs provide a more relevant model for transgene delivery to the human inner ear. We previously reported a successful surgical approach for delivering AAV vectors to the inner ears of cynomolgus monkeys21–23 and showed efficient transgene expression of a GFP reporter in a NHP cochlea following injection of the AAV9 capsid variant PHP.B via the RWM21–23. That vector used a ubiquitous CBA promoter, and we observed expression of the GFP reporter in many cell types, including hair cells. Following our results in mouse, we expected more specific expression of GJB2 in NHP cochlea using GRE-driven viral vectors.

To directly compare cell-specific expression between CBA and GRE-driven constructs, we injected cochleas of three juvenile cynomolgus monkeys (Macaca fascicularis). The study design is indicated in Supplementary Table 2 and Fig. 7a. The first animal received a vector encoding an eGFP marker under control of the CBA promoter by RMW injection in one ear (AAV-CBA-eGFP, 5.8×1011 vg, Fig. 1b). For the two other animals, three ears were injected with a vector encoding hsGJB2.HA under the control of the GREs (AAV-GRE-hsGJB2.HA; 5.0×1011 vg; Fig. 1d); the fourth ear was injected with formulation buffer (Fig. 1c). Five weeks post-injection, animals were tested with ABR recording, then euthanized. Cochleas were extracted from the temporal bones of animals, further trimmed, decalcified and embedded in optimal cutting temperature (OCT) compound, cryosectioned and immunostained (Fig. 7a).

Consistent with our results in mouse and earlier NHP studies21,22, the AAV-CBA-eGFP vector produced wide-ranging expression throughout the NHP organ of Corti, as revealed by anti-eGFP immunostaining of cochlea sections (Fig. 7b, Table 1). Almost all cell types of the cochlea were transduced. In particular, we observed a very high level of eGFP expression in the organ of Corti, outer and inner sulcus cells, spiral limbus, lateral wall, and Reissner’s membrane. We analyzed higher-magnification images of the organ of Corti. In cochleas that received AAV-CBA-eGFP, complete transduction was observed in hair cells, Deiters’ cells, inner phalangeal cells, pillar cells, Hensen’s cells, and Claudius cells. Additionally, the epithelial cells of the inner sulcus and fibrocytes of the basilar membrane showed high transduction. Fibrocytes of the spiral ligament in the lateral wall were significantly transduced. Notably, high-magnification images of the modiolus region revealed high expression of eGFP in satellite glial cells, Schwann cells, and SGNs.

In order to assess AAV-GRE-hsGJB2.HA expression in NHP cochleas, we first evaluated the specificity of the anti-HA antibody on frozen sections of vehicle-injected and AAV-GRE-hsGJB2.HA-injected NHP cochleas. We used antibodies to SOX2 and MYO7A to label supporting cells and hair cells, respectively. In the negative control vehicle-injected cochlea, as expected, we saw anti-SOX2 and MYO7A labeling but no HA signal (Fig. 8a). However, broad anti-HA labeling was detected in AAV-GRE-GJB2.HA-injected NHP cochleas (Fig. 8b). A weak and diffuse signal was detected in SGNs in both vehicle control and vector-injected cochleas, suggesting that it represents SGN autofluorescence.

Next, we investigated the virus-mediated hsGJB2.HA expression in cryosections collected from cochleas injected with AAV-GRE-hsGJB2.HA (Fig. 7d). Because injected cochleas have both endogenous and vector-delivered GJB2, we identified vector-delivered GJB2 with an antibody to the HA tag. With GJB2 expression driven by the GJB2 GREs, IHCs and OHCs did not express hsGJB2.HA, as evidenced by lack of anti-HA labeling (Fig. 7d). Similar to the normal expression of GJB2 (Fig. 7c), the anti-HA signal was detected in SOX2-positive cells, such as Deiters’ cells, Hensen’s cells, Claudius cells and border cells, with intensity similar to that of native GJB2 (Table 1). However, few or no HA-tagged puncta were seen in inner or outer pillar cells. GJB2.HA was also seen in the inner and outer sulcus epithelia and the root cells of spiral prominence. The spiral limbus fibrocytes and the cells lining the scala vestibuli floor expressed GJB2.HA in a pattern similar to that of native GJB2. We detected weak or no HA signal in the spiral limbus interdental cells. Analysis of the lateral wall showed that, like native GJB2, exogenous GJB2.HA was highly expressed in type I, II and V fibrocytes and was not present in type III and IV fibrocytes. As expected, there was also no expression of GJB2.HA in the marginal cells or the epithelium of Reissner’s membrane. There was no GJB2.HA expression in the modiolar region of the cochlea. As with endogenous GJB2, analysis of cochlear turns showed no gradient in GJB2.HA expression between apex, middle and base (Fig. 8b). There were some differences between virus-delivered and endogenous GJB2: the stria vascularis and pillar cells expressed exogenous GJB2.HA at a lower level than the endogenous GJB2.

AAV-GRE-hsGJB2.HA is safe in NHP cochleas
We performed histological analysis on H&E-stained frozen tissue sections from injected cochleas to evaluate pathology (Fig. 9a). Tissue morphology and architecture appeared normal in both AAV-treated and vehicle control groups, with no significant abnormalities observed. Minor changes in the basal turn, likely related to surgery and vector injection, were noted in the left cochlea of NHP #2. To further evaluate immune response activation in the cochlea, we performed staining for ionized calcium-binding protein (IBA1) in frozen tissue sections from injected and control cochleas (Fig. 9b). IBA1 is primarily expressed in microglia cells, which in the cochlea are macrophages. Macrophages are abundant in the cochlea; however, insults such as AAV capsids or surgery can cause an inflammatory response, which includes activation of tissue-resident macrophages as characterized by changes in macrophage morphology, number, and distribution. We observed normal tissue morphology and architecture in all cochleas, with no notable abnormalities. (Fig. 9b). Sensory hair cells and supporting cells appeared normal. We observed no difference in macrophage infiltration in treated cochleas compared to vehicle-injected cochlea, confirming that NHPs injected with AAV-GRE-hsGJB2.HA showed minimal immune response to the vector.

To assess safety physiologically, we also tested the hearing before and after injection with either AAV-GRE-hsGJB2.HA or vehicle (Supplementary Fig. 3). We compared pre-injection thresholds to thresholds at 5 weeks after injection to assess changes that may be due to the vector. In the vehicle-injected cochlea (NHP #1, right ear), no significant difference was seen in the click and the pure tone ABR thresholds after injection. In two ears injected with 5.0×1011 vg (NHP #1 left ear and NHP #2 right ear), we saw no significant change in sensitivity in either click or tone ABRs. However, in one ear injected with 5.0×1011 vg (NHP #2 left ear), hearing threshold was slightly elevated by 15–25 dB nHL at all frequencies. This may be a result of early postoperative changes in the middle ear after mastoid surgery. Because two of three ears showed no elevation of threshold, we believe the injection of vector does not reproducibly cause a loss of sensitivity.

Methods
Study approval
All mouse studies were conducted in compliance with ethical regulations according to protocol IS00003633 approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School and were performed according to the NIH guidelines.

All NHP studies were performed at Biomere Medical Research Models (Worcester, MA) under Testing Facility Study No. NUT22-01, which was reviewed and approved by the facility’s IACUC. The facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) and registered with the United States Department of Agriculture (USDA). All procedures complied with the Animal Welfare Act regulations (9 CFR), the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals.

Mouse models (Mus musculus)
Gjb2 conditional knockout mice (Gjb2tm1Ugds)35, referred to here as Gjb2Pfl/Pfl, have loxP sites flanking the coding sequence in exon 2. Gjb2Pfl/Pfl mice were crossed to Sox10-Cre mice (Tg(Sox10-cre)1Wdr/J, Jackson Laboratory #025807)57, which have a BAC transgene encoding Cre recombinase under the Sox10 promoter, referred to here as Cre+.
Gjb2Pfl/Pfl, Cre+ or Gjb2Pfl/Pfl, Cre− mice were maintained on a C57BL/6J background. Gjb6- constitutive knockout mice (Gjb6tm1Kwi; European Mouse Mutant Archive #EM:00323)38 have the Gjb6 coding region replaced by a Neo cassette and β-galactosidase reporter with a nuclear localization sequence; these substitutions have the effect of reducing GJB2 protein levels39–41, so mice are referred to here as Gjb2Gjb6–. These mice were maintained on mixed C57BL/6J–CBA genetic backgrounds.
Animals of both sexes and ages ranging from P0 to P90 were used in experiments. The mice were housed at the animal facility of the Harvard Medical School in a 12-h-light/12-h-dark cyclic environment. The temperature ranged from 73°F to 76°F, and the humidity from 30% to 40%. Both sexes were used in each experiment, and no differences in genotype distribution between males and females were observed.

AAV vector production and construction
AAV backbone plasmids as initially reported21,31 were used for cloning. Human GREs and coding sequences were synthesized and inserted into AAV backbones using HiFi DNA Assembly Master Mix (NEB, Cat. #E2621). In parallel, mouse coding sequences were amplified from genomic DNA using Q5 Hot Start High-Fidelity DNA Polymerase (NEB, Cat. #M0493) according to the manufacturer’s protocol, and then inserted into AAV backbones with HiFi DNA Assembly Master Mix.
Depending on the construct, candidate GREs identified through ATAC-seq were placed upstream or downstream of the transgene, or the CBA promoter was used directly. All vectors also contained a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a bovine growth hormone polyadenylation signal (BGH).
For all experiments, the capsid was AAV9-PHP.B28. AAV packaging was performed by the Boston Children’s Hospital Viral Core (Boston, MA, USA) or by PackGene Biotech (Houston, TX, USA). All plasmids were sequence-verified prior to packaging. Viral titers were determined by qPCR specific for the inverted terminal repeat of the virus.

ATAC-seq library preparation and sequencing
Neonatal (P2, n=3; P5, n=3; P8, n=4) CD1 mice were sacrificed and cochleas were dissected bilaterally. All tissue from each animal was combined and dounced 10 times in 5 ml of HB buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH pH 7.8, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, DTT 1 mM [Sigma], one-half Roche proteinase inhibitor tablet) in a 7-ml Dounce homogenizer with tight pestle, then supplemented with IGEPAL CA-630 to final concentration of 0.3%, and finally dounced an additional five times with the tight pestle. Nuclei were filtered through a 40-μm strainer and gently combined 1:1 with 50% iodixanol. Nuclei were then underlaid with a gradient of 1 ml 40% iodixanol and 1 ml 30% iodixanol and centrifuged at 10,000×g for 19 min using the no brake setting. Purified nuclei (0.8 ml) were then collected from the 30/40% interface. A 20-μl aliquot was taken for visual inspection and cell counting, with typical yield of ~100,000–200,000 cochlear nuclei per animal.
ATAC-seq (assay for transposase accessible chromatin with sequencing) was performed using the OMNI-ATAC protocol58 immediately following the nuclear isolation steps described above. Nuclei (50,000) were pelleted at 500×g for 10 min at 4°C in a fixed angle centrifuge and resuspended in 50 μl transposition buffer (prepared from Illumina kit components TD buffer and 100 nM transposase, as well as 0.01% digitonin, 0.1% Tween-20) via careful pipetting. Transposition occurred at 37°C in a thermomixer at 1000 rpm for 30 min before resuspension in 250 μl of DNA binding buffer (Zymo DNA Clean and Concentrator Kit) and reaction cleanup per manufacturer’s instructions, with final elution volume of 21 μl in elution buffer. Samples were amplified following NEBNext High-Fidelity 2X PCR Master Mix manufacturer protocols (NEB) in a total reaction volume of 50 μl (1.25 μM each of i5 and i7 sequencing primers) at 72°C for 5 min, 98°C for 30 s, (98°C for 10 s, 63°C for 30 s, and 72°C for 1 min × 10 cycles. PCR samples were re-purified following the Zymo manufacturer protocol and eluted into 21 μl of elution buffer. Size selection was performed via agarose gel (150–1000 bp) followed by Qiagen MinElute gel extraction according to the manufacturer’s instructions, with two serial 10 μl elution steps. Sequencing (75-bp paired-end) was performed on an Illumina NextSeq 500.

ATAC-seq: sequencing, alignment, and genome browser track generation
All ATAC-seq libraries were sequenced on the Illumina NextSeq 500 platform, generating 75-bp single-end reads at a minimum depth of 20 million reads per sample. Raw reads were quality trimmed with Trimmomatic (v0.33)59 using the following command: java -jar trimmomatic-0.33.jar SE -threads 1 -phred33 [FASTQ_FILE] ILLUMINACLIP:[ADAPTER_FILE]:2:30:10 LEADING:5 TRAILING:5 SLIDINGWINDOW:4:20 MINLEN:45. Nextera adapters were removed during trimming. Filtered reads were aligned to the mouse genome (GRCm38/mm10, December 2011) with Subread (v1.4.6-p3)60 using default parameters. PCR duplicates were removed using samtools rmdup. For visualization, BEDtools was used to convert BAM files to BED format (bedtools bamtobed), and mm10 blacklisted regions61 were excluded with command: bedops –not-element-of 1 [BLACKLIST_BED]. Filtered BED files were downsampled to 20 million reads, converted to coverage BED format with BEDtools genomecov, and finally transformed to bigWIG files with UCSC bedGraphToBigWig for genome browser visualization.

ATAC-seq peak calling and quantification
Putative cochlear GREs (regions of ATAC-seq enrichment) were identified using established methods32. Briefly, peaks were determined using MACS2 (v2.1.0; parameters -p 1e-5 –nolambda –keep-dup all –slocal 10000)29. Peaks from individual sample replicates were intersected to find only reproducible regions of enrichment and blacklist regions were removed according to ENCODE guidelines62. To identify sites with enriched ATAC-seq signal (peaks), we applied the IDR pipeline using the MACS2 peak calling algorithm63 with the following parameters: –nomodel –extsize 200 –keep-dup all. An IDR threshold of 0.01 was used for self-consistency, true replicate, and pooled-consistency analyses. The ‘optThresh’ cutoff was then used to obtain a final set of high-confidence, reproducible ATAC-seq peaks for each sample.
To produce a final list of reference coordinates of all genomic regions that were accessible in at least one mouse cochlear ATAC-seq sample, the MACS2-called peaks for each experimental replicate were unioned using the bedops –everything command. Bedtools merge was then used to combine any peaks that overlapped in this unioned bed file; in this way, any region that was significantly called a peak in at least one ATAC-seq dataset was incorporated in the final aggregated peak list. The featureCounts package was then used to obtain ATAC-seq read counts for each of these accessible putative CREs for downstream analyses64, including comparison to previously published mouse neuronal ATAC-seq data65 to identify putative cochlear-restricted sites.

AAV round window membrane injection in neonatal mice
RWM injections were performed under a stereomicroscope (Nikon SMZ1500). P0-P1 pups were anesthetized using cryoanesthesia and kept on an ice pack during the procedure21,23,45. A small incision was made beneath the external ear bud, which was then extended to allow for separation of the soft tissues and exposure of the bulla. The round window niche was identified visually. Using a Nanoliter 2000 Injector (World Precision Instruments), 1 μl of the viral vector solution was injected through a micropipette needle. The incision was closed with a 7-0 Vicryl surgical suture, and standard postoperative care was provided following the injection.

Mouse cochlear histology and imaging
In experiments where transduction efficiency of AAV-eGFP was detected via intrinsic eGFP fluorescence, organ of Corti explants were dissected at P6 in L-15 medium and fixed with 4% formaldehyde in Hank’s balanced salt solution (HBSS) for 1 h, washed three times with HBSS, and then blocked and permeabilized with 10% donkey serum (Jackson ImmunoResearch, #017-000-121) with 0.3% Triton X-100 for 1 h at room temperature. Rabbit anti-GJB2 antibody (1:100, Fisher, #71-0500) was used to label GJB2. Antibody was diluted in 10% donkey serum and incubated overnight at room temperature followed by several washes in HBSS. Next, samples were incubated in blocking solution for 30 min and incubated overnight at room temperature with a donkey anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (1:500, Invitrogen, #A21207) in blocking solution. To label hair bundle actin, we used phalloidin conjugated to Alexa Fluor 405 (1:20; Life Technologies).
In experiments assessed with cryosections, mouse cochleas were dissected in L-15 medium, immediately fixed with 4% formaldehyde in HBSS for 1 h at room temperature, then washed with HBSS and transferred to fresh 10% EDTA for 1-3 days. Next, samples were cryoprotected and embedded in OCT compound and stored at −80°C prior to sectioning. Cryosections were generated using a Leica CM 3050 S cryostat at 20 μm step size.
For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: rabbit anti-HA C29F4 (1:200, Cell Signaling, #3724), rabbit anti-GJB2 (1:50, Fisher, #71-0500), Guinea pig anti-parvalbumin (1:200, Synaptic System, #195004), rabbit anti-GJB6 (1:200, Fisher, #71-2200), rabbit anti-MYO7A (1:200, Proteus Biosciences, #25-6790), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200, Invitrogen, #A21207), donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200, Invitrogen, #A32790), and donkey anti-guinea pig Alexa Fluor 647 (1:200, Invitrogen, #A21450). We used phalloidin conjugated to Alexa Fluor 405 (1:20; Life Technologies) to label hair-bundle actin (1:20), DAPI to label nuclei (1:500, Invitrogen), and BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; Invitrogen, D3835) to label membranes (1:1500). Samples were blocked for 1 h at room temperature. Antibodies were diluted in 10% donkey serum and incubated with samples overnight at room temperature, followed by several rinses in HBSS. Next, samples were incubated in a blocking solution for 30 min and incubated overnight at room temperature with a secondary antibody in the blocking solution. Tissues were mounted on a Colorfrost glass slide (Thermo Fisher Scientific) using Prolong Gold Antifade mounting medium (Thermo Fisher Scientific). Imaging was performed with a Nikon Ti2 inverted spinning disk confocal using a Plan Fluor 40×/1.3 oil objective, Plan Apo λ 60×/1.4 oil objective, or Plan Apo λ 100×/1.45 oil objective.

Scanning electron microscopy
Scanning electron microscopy in neonatal and adult mice was performed using an established method66. Neonatal organ of Corti explants were dissected at P6 in L-15 medium and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) supplemented with 2 mM CaCl2 for 1–2 h at room temperature. The samples were rinsed three times in sodium cacodylate buffer, 0.1 M, pH 7.4 for 10 min, and then briefly rinsed once in distilled water, dehydrated in an ascending series of ethanol concentrations, and critical-point dried from liquid CO2 (Tousimis Autosamdri 815).
For adult mice, cochleas were rapidly extracted from the temporal bones and placed in Leibovitz’s L-15 solution. Under a stereomicroscope, a small hole was created at the cochlear apex using a 27G needle. Immediately after dissection, the cochleas were fixed in 2.5% glutaraldehyde in the same buffer with 2 mM CaCl2 for an hour at room temperature. They were then rinsed in 0.1 M cacodylate buffer (pH 7.2) and subsequently in distilled water. Using a 27G needle, the cochlear bone was carefully removed, allowing for the microdissection of the organ of Corti and the extraction of the tectorial membrane to expose the sensory epithelium. The samples were then incubated in a saturated aqueous solution of 1% osmium tetroxide for 1 h in the dark, followed by a 10-min water rinse and postfixation in 1% tannic acid aqueous solution for another hour in the dark. Finally, the samples were rinsed, dehydrated, and subjected to critical point drying.
Samples were mounted on aluminum stubs with carbon conductive tabs and were sputter-coated (EMS 300T dual-head sputter coater or EMS 150TS Metal Sputter Coater) with platinum to 5 nm and observed in a field-emission scanning electron microscope (Hitachi S-4700).

Mouse ABR and DPOAE testing
ABRs and DPOAEs were recorded using an established protocol45,67, utilizing an acoustic system developed by Massachusetts Eye and Ear, Boston, MA, USA. Briefly, adult mice were administered anesthesia and maintained on a 37°C heating pad throughout the recording session. Tone-pip stimuli (5 ms duration and 0.5 ms rise-fall time) were delivered at frequencies ranging from 4 kHz to 32 kHz. Sound levels were increased in 5-dB increments from 20 to 120 dB sound pressure level (SPL). ABR Peak Analysis software (version 1.1.1.9, Massachusetts Eye and Ear, Boston, MA, USA) was used to measure ABR thresholds and peak amplitudes. DPOAEs were recorded for primary tones with a frequency ratio of f2/f1=1.2, with L1=L2+10 dB. The f2 frequency ranged from 5.6 kHz to 32 kHz in half-octave increments. Primary tone levels were adjusted in 5-dB increments, from 10 to 120 dB SPL for f2.

NHP study design
One female and two male juvenile cynomolgus monkeys (Macaca fascicularis; 2–4 years old, weighing 2.6–3.2 kg) were used in this study. Primate work was performed at Biomere Medical Research Models (Worcester, MA, USA) according to Biomere’s animal use guidelines and approved procedures. Each animal was considered acclimated to the environment at the time of the study. NHPs were healthy and without a history of ear inflammation, ear surgery, signs of balance disorders, or other risk factors for dysfunction of the inner or middle ear. Animals received transmastoid/trans-RWM injections of 10 μl of AAV-GRE-hsGJB2.HA vector with doses of 5.0×1011 vg (n=3 ears) and 20 μl AAV-CBA-eGFP of 5.8×1011 vg (n=1 ear). One animal was injected in one ear with 10 μl formulation buffer control (see Supplementary Table 2). A hearing test was performed before, and 5 weeks after unilateral injection in NHP #1 and NHP #2. Five weeks post-injection, animals were euthanized. Cochleas were extracted from the temporal bones of animals for histological analysis.

NHP surgical procedures
Animals were sedated with ketamine (10 mg/kg) and pre-medicated with atropine (0.04 mg/kg) following overnight food deprivation22. Prior to surgery, dexamethasone (1 mg/kg), buprenorphine (0.03 mg/kg), sustained-release buprenorphine (0.2 mg/kg, SC), and cefazolin (20 mg/kg) were administered. During the surgery, anesthesia was induced with isoflurane (1–4%) and maintained with oxygen (0.5–4 L) and isoflurane (~1–5%). Animals received warmed intravenous fluids, corneal protection, and continuous monitoring of vital signs. The surgical site was prepared aseptically and local anesthesia was provided with bupivacaine. All viral inner-ear administrations were performed via the facial recess approach, with round-window injection22,23. Under direct microscopic visualization, a microinjection pump delivered 20 μl or 10 μl at 2 μl/min using a Hamilton syringe and 29G stainless steel needle. After injection, the surgical site was flushed with warm saline and closed with tension and absorbable sutures. The skin was cleaned with saline and a topical antibiotic ointment was applied. The wounds were monitored for proper healing for 2 weeks. Postoperatively, animals received bupivacaine for incision analgesia, dexamethasone for up to 5 days, and cefazolin on days 0 and 3, and were monitored daily for pain, recovery, and wound healing for 2 weeks.

NHP cochlear processing, histology, and imaging
NHPs were euthanized at 35 days after vector injection and were perfused with heparinized saline followed by 4% formaldehyde. Collected temporal bones were postfixed for another 48 h in 4% formaldehyde. Then, cochleas were dissected from the temporal bones under a microscope22,23 and decalcified in EDTA. Next, samples were cryoprotected by incubating in gradient concentrations of sucrose and were embedded in OCT compound and stored at −80°C prior to sectioning. Cryosections were generated using a Leica CM 3050 S cryostat at 30-μm step size.
For immunofluorescence labeling, the following primary antibodies and secondary antibodies were used: mouse anti-MYO7A (1:50; Santa Cruz, #sc-74516), rabbit anti-HA C29F4 (1:200, Cell Signaling, #3724), rabbit anti-GJB2 (1:50, Fisher, #71-0500), goat anti-SOX2 (1:50, R&D Systems, #AF2018), rabbit anti-IBA1 (1:200, Wako Chemicals, #019-19741), donkey anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 594 (1:200, Invitrogen, #A21207), donkey anti-goat IgG conjugated to Alexa Fluor 647 (1:200, Invitrogen, #A21447), donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200, Invitrogen, #A32766), donkey anti-mouse IgG conjugated to Alexa Fluor 594 (1:200, Invitrogen, #A21135), and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (1:200, Invitrogen, #A32790). Samples were blocked for 1 h at room temperature. Antibodies were diluted in 10% donkey serum and incubated with samples overnight at room temperature, followed by several rinses in HBSS. Next, samples were incubated in a blocking solution for 30 min and incubated overnight at room temperature with a secondary antibody in the blocking solution. DAPI labeled cell nuclei. Tissues were mounted on Colorfrost glass slides (Thermo Fisher Scientific) using Prolong Gold Antifade mounting medium (Thermo Fisher Scientific). Imaging was performed with a Nikon Ti2 inverted spinning disk confocal using a Plan Fluor 40×/1.3 oil objective and Plan Apo λ 60×/1.4 oil objective.

ABR testing in NHPs
Testing was performed using an established protocol23. A hearing test was performed before, and 5 weeks after the unilateral injection. The animals were anesthetized with a mixture of ketamine and atropine and were masked with isoflurane. During the procedure, an animal was maintained on isoflurane anesthesia. ABRs were acquired using a clinical-grade auditory evoked potential system (GSI Audera v2.7; Grayson-Stadler, Eden Prairie, MN, USA). ABRs were recorded using three subdermal needle electrodes: the reference electrode was positioned on the midline of the scalp between the ears, the recording electrode was placed just posterior to the pinna near the slit, and the ground electrode was situated on the lower back near the tail. The click and tone-pip stimuli were presented at alternate polarity with a tubal insert phone (TIP50 GSI generic; Grayson-Stadler, Eden Prairie, MN, USA). The acoustic click was presented at a repetition rate of 11.1 Hz, and waveforms were averaged across 2004 stimulus repetitions. Clicks were a condensation stimulus of 100-μs duration. Sound levels were incremented in 5-dB steps, from ~15 dB nHL up to 54.5 dB nHL. Tone frequencies of 500, 1000, 2000 and 4000 Hz were used. The tone pips were presented at a repetition rate of 27 Hz and waveforms were averaged across 2016 stimulus repetitions. Sound levels were incremented in 5-dB steps, from ~10 dB nHL up to 59.5 dB nHL. GSI Audera v2.7 software (Grayson-Stadler, Eden Prairie, MN, USA) was used to determine the ABR thresholds.

Statistics and reproducibility
All experiments, except for the ones noted below, were performed in at least three independent replicates using independent biological samples. Only samples displaying notable dissection-related damage were excluded from the analysis. No other data were omitted.
In NHP experiments (Figs. 7–9), an initial cohort of three juvenile cynomolgus monkeys was used, as described in the study design (Supplementary Table 2). Across these animals, a total of five ears were injected (three with AAV-GRE-hsGJB2.HA, one with AAV-CBA-eGFP, and one with formulation buffer control). For each cochlea, at least five slides from different regions were analyzed independently. Histological analyses consistently yielded similar results across animals and slides, confirming reproducibility of the findings.
All figures were assembled using Fiji (version 1.54), Adobe Illustrator 2024 (version 28.7.8), Adobe Illustrator 2025 (version 29.7.1), and Adobe Firefly (web-based; no version number available). ABR data were collected and sorted using ABR Peak Analysis (version 1.3.0) and Microsoft Excel for Microsoft 365 (version 2507). GraphPad Prism (version 10.5.0) was used to generate graphs and perform statistical analyses. SnapGene (version 5.1) was used for plasmid cloning and sequence analysis. Microsoft Word for Microsoft 365 (version 2507) was used to prepare the manuscript. The results are shown as mean ± SEM as indicated in the figure legends. Randomization was used whenever possible.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presents a well-designed and innovative study, demonstrating the use of gene regulatory elements to drive cell-type-specific expression of GJB2 in the cochlea.\nGJB2-associated deafness is the most common form of hereditary hearing loss worldwide. The discovery of the pathogenic variants in this gene in 1997 changed the way diagnostics and genetic counseling is done for deafness, and paving the way for gene therapy. However, given that Gjb2 mutations are often lethal in mice, this commonly used model for human deafness was far from ideal. The lack of appropriate AAV that were compatible with connexin 26 expression, encoded by the GJB2 gene, further complicated attempts to develop gene therapy.\nThe authors of this manuscript chose to identify gene regulatory elements specific for connexin 26, to help circumvent previous hurdles. HA-tagged GJB2 partially rescued hearing and preserved cochlear structure in a conditional mouse mutant. Expression of these GREs was validated in nonhuman primate cochleas.These findings support the potential of GRE-driven GJB2 delivery as a promising strategy for treating DFNB1 in humans.\nComments:\n1. This study used two different gene regulatory elements to drive cell-type-specific expression of Gjb2. Was the effect of each GRE tested separately? If so, what were the differences?\n2. Figure 3 does not include SEM images at P30. Are hair cells absent across all regions of the cochlea?\n3. The authors mentioned that “GJB2 has a critical developmental role as early as P0, and that vector delivered at P1 might not produce GJB2 protein until P2 or P3.” scAAV transduces cells more rapidly and is suitable for small genes like GJB2. Have the authors considered trying scAAV?\n4. How do the authors explain the differing effects of the HA tag in the Cre+ mouse model compared to the Gjb2 partial knockdown mouse?\n5. In figures 4 and 7, how many mice were tested within each group for ABR/DPOAE?\n6. In figure 7, the ABR results are presented in different titer concentrations. Which concentration was used for the cross sections and SEM images in Figure 5?\n7. Do the different vector titers result in variations in expression levels or morphological changes over time, as presented in the ABR and DPOAE measurements at P60 and P90?\nMinor:\n1. The figures are not presented in the same order in which they are mentioned in the text, which makes it difficult to follow the results. I recommend reordering some panels of the figures.\n2. Line 128 refers to Figures 1g and h? These panels do not appear in the figure.", "reviewer_2": "This is an excellent report describing the development of reagents that are successful in preventing deafness resulting from Gjb2 mutations using targeted regulatory elements to express the gene in the correct cell types in mouse and non-human primates. GJB2 mutations are an important cause of deafness in childhood and this report has strong translational relevance. I have a few minor comments to improve clarity.\nLine 81: Please define CBA here.\nFig 1c: Please label GRE1 and GRE2 on the diagram.\nFig 2b: It is difficult to distinguish the colours used for phalloidin and Gjb2 in the sections, particularly the lower middle panel, and confusing that the same colour has been used for phalloidin and Gjb2 in the upper and lower panels respectively. Could the colours be changed to distinguish the two markers more clearly?\nFig 2c,d: Please define PV and BIODIPY in the legend and explain what they are labelling.\nLine 163: Just a matter of interest – why Pfl?\nLine 200: Point out labelling of GJB2 in the basal cell region of the stria vascularis.\nLine 248-249: Was the improvement in ABR thresholds after injection of the human GJB2 consistent with a lack of normal OHC function? Were there any other measures of OHC function?\nLine 301: Should this be “preservation” instead of “restoration”?\nLine 309: Typo bottom\nFig 6: It is not possible to see the thickened tectorial membrane, reduced scala media area, or other features mentioned in these images. A standard H&E or similar image would help.\nFig 7: It may just be my screen, but I found it quite difficult to distinguish the colours in this figure. Would the authors consider more contrasting colours? In particular the dark blue and black were difficult.\nLine 522: Do you mean stereotypes or serotypes?\nDiscussion: This is essentially a summary of the main findings of the project, which is fine but it would be helpful to include discussion of other aspects and put the research into a broader context. For example, why do the human GREs seemed to work better than mouse GREs in the mouse? What do the findings tell us about the role of Gjb6 in hearing – is it redundant? Why do the authors think that the HA tag allows Gjb2 to function in one mutant but not in the other mutant?\nLine 609-615: Could the genetic background of each mouse line be added to the methods? Presumably it is mixed but it would be helpful to know if the two Gjb2 mutants had different backgrounds that may contribute to their phenotypes.\nLine 633: What is meant by “as described above prior to fixation”? Presumably these samples were not fixed?", "reviewer_3": "GJB2 encodes connexin26. Recessive variants of human GJB2 are associated with clinically heterogeneous deafness and are common in many countries. A founder variant of GJB2 accounts for the majority of nonsyndromic deafness in the Ashkenazi Jewish population. Gene therapy for GJB2 deafness is on the horizon and looks more hopeful considering the new data in the manuscript from Ivanchenko and co-authors entitled “Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea”. The authors describe an AAV vector with two cell-specific regulatory cis-acting sequences (GREs) driving the expression connexin26 in the appropriate inner ear cell types. Their new AAV vector can significantly restore hearing in an otherwise deaf mouse and in a partially deaf mouse model. The manuscript is nicely crafted, the figures are of good quality and the data is robust and convincing.\nThere is one concerning issue that hopefully can be resolved so that the data can be reproduced by other investigators. A list below of minor edits are recommended.\nMajor issue: Uncontrolled GJB2 expression using a ubiquitous promoter is lethal, as the authors clearly point out and reference. To spatially control GJB2 expression, the authors performed ATAC-seq and identified two chromosomal regions described in the text and in figure1. The two “GREs” are referred to as GRE1 and GRE2. Unless this reviewer missed the information, the authors did not provide the exact nucleotide sequence of GRE1 and GRE2. On line 112, GRE1 is described as including noncoding exon 1 and ~200 bp of upstream sequence. That’s vague. GRE2 is described as “3’ noncoding segment of exon 2.” That’s entirely inexact. Without the nucleotide sequence included in the manuscript for GRE1 and GRE2, the data and observations by Ivanchenko and co-authors cannot be independently reproducible by other scientists, should they wish to pursue this topic. The Nature Communications editors should decide if this is acceptable secrecy by the authors although it is understandable but not appropriate. Some of the authors have equity in Skylark Bio as indicated on line 1017 (Competing Interests) and likely these authors want to move their observations into the clinical for gene therapy for GJB2-associated human deafness without giving commercial competitors an advantage. That too is understandable from a business point of view. But in a high tier science publication, is it proper to intentionally withhold critical information?\nMinor edits:\nAbstract, lines 24. “partially” and “partial” are vague and not scientific. Please provide some actual data.\nAbstract and Introduction, lines 26 and 70, name the nonhuman primate.\nIntroduction, line 31, Many authors state that hearing loss is the most common sensory deficit. Perhaps. But color blindness is 1 in 12 men and 1 in 200 women.\nIntroduction, line 43, The authors state that “GJB2 [expression] is absent from the sensory hair cells…”. Literally, as stated, the Gjb2 gene is not absent. GJB2 expression, according to data in gEAR, is also not absent from hair cells but is expressed at a low level compared to support cells. Which is it? The data in gEAR about Gjb2 expression or is it a myth about GJB2 not being present in hair cells? Please clarify.\nLine 228, define “vgc”.\nLine 249, provide the genotype of the “control mice”.\nLine 461, Please clarify the statement “A well-developed exogenous GJB2.HA network was also see….” What is meant by an “exogenous … network”? Clarify please."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe manuscript presents a well-designed and innovative study, demonstrating the use of gene\nregulatory elements to drive cell-type-specific expression of GJB2 in the cochlea. GJB2-associated\ndeafness is the most common form of hereditary hearing loss worldwide. The discovery of the\npathogenic variants in this gene in 1997 changed the way diagnostics and genetic counseling is done for\ndeafness, and paving the way for gene therapy. However, given that Gjb2 mutations are often lethal in\nmice, this commonly used model for human deafness was far from ideal. The lack of appropriate AAV\nthat were compatible with connexin 26 expression, encoded by the GJB2 gene, further complicated\nattempts to develop gene therapy.\nThe authors of this manuscript chose to identify gene regulatory elements specific for connexin 26, to\nhelp circumvent previous hurdles. HA-tagged GJB2 partially rescued hearing and preserved cochlear\nstructure in a conditional mouse mutant. Expression of these GREs was validated in nonhuman primate\ncochleas. These findings support the potential of GRE-driven GJB2 delivery as a promising strategy for\ntreating DFNB1 in humans.\nComments:\n1. This study used two different gene regulatory elements to drive cell-type-specific expression of Gjb2.\nWas the effect of each GRE tested separately? If so, what were the differences?\nWe tested about 30 different constructs, encoding GFP, mmGJB2 or hsGJB2. Seven of them did not include\nthe second GRE, and they had lower efficacy, so we did not pursue them. Those experiments were\nexploratory, so we did not have sufficient numbers to make a quantitative comparison to vectors with the\nsecond GRE. We feel it is best not to include partial data in the paper.\n2. Figure 3 does not include SEM images at P30. Are hair cells absent across all regions of the cochlea?\nGjb2Pfl/Pfl, Cre+ cochleas showed degeneration of the sensory epithelium across all cochlear turns, as revealed\nby immunofluorescence on cryosections (new Supplementary Fig. 1), resulting in a flattened epithelium. At\nthis stage, SEM imaging is not informative, as the sensory epithelium is lost and no structures remain for\nvisualization. It is also very difficult, as the thin basilar membrane tends to break.\n3. The authors mentioned that “GJB2 has a critical developmental role as early as P0, and that vector\ndelivered at P1 might not produce GJB2 protein until P2 or P3.” scAAV transduces cells more rapidly\nand is suitable for small genes like GJB2. Have the authors considered trying scAAV?\nWe did, but the numbers don’t work. The CDS plus GREs plus WPRE plus bGH sequences add up to 3741 bp,\ntoo much to allow additional reverse complement sequence for an scAAV.\n4. How do the authors explain the differing effects of the HA tag in the Cre+ mouse model compared to\nthe Gjb2 partial knockdown mouse?\nThe differing effects of the HA tag in the Gjb2Pfl/Pfl, Cre+ mouse model compared to the Gjb2Gjb6- mouse suggest\nthat, although GJB2.HA is strongly expressed and properly trafficked to the cell membrane in both models,\nit may not be fully functional. In the Gjb2Pfl/Pfl, Cre+ mice, the presence of GJB6 may partially compensate for\nthe impaired function of GJB2.HA, preserving essential ion or metabolite transport. In contrast, when GJB2.HA\nis expressed in the absence of GJB6 (as in Gjb2Gjb6- mice), it leads to detrimental effects in the inner ear, likely\ndue to the loss of GJB2 and GJB6 gap junction function.\n5. In figures 4 and 7, how many mice were tested within each group for ABR/DPOAE?\nWe have now specified the number of mice tested for each condition in the figure legends for Figures 4 and\n7, as well as in the corresponding source data files.\n6. In figure 7, the ABR results are presented in different titer concentrations. Which concentration was\nused for the cross sections and SEM images in Figure 5?\nThe cross-section immunofluorescent images and SEM images in Figure 5 were obtained from ears injected\nwith the medium dose (7.0×10¹⁰ vg/ear). We have now clarified this in the figure legend.\n7. Do the different vector titers result in variations in expression levels or morphological changes over\ntime, as presented in the ABR and DPOAE measurements at P60 and P90?\nWhile we did not assess morphological changes at P60 and P90, we observed sustained functional hearing\nrescue with both high (1.4×1011 vg/ear) and medium (7.0×1010 vg/ear) doses through P90, suggesting stable\nexpression and a lack of progressive degeneration. Given these consistent functional outcomes, we believe\nthat additional morphological analysis at later time points would be unlikely to provide further meaningful\ninsights. In contrast, the low-dose (3.0×1010 vg/ear) group exhibited a gradual elevation in ABR thresholds\nover time, which may reflect suboptimal GJB2 expression and potentially lead to degenerative changes.\nHowever, we agree that this could be an interesting direction for future studies focused on long-term\nstructural effects.\nMinor:\n1. The figures are not presented in the same order in which they are mentioned in the text, which makes\nit difficult to follow the results. I recommend reordering some panels of the figures.\nIn general, the figures are presented in the order mentioned. Some subpanels of a figure are mentioned after\nearlier subpanels of a subsequent figure, but the panels are grouped into figures based on the experimental\nmethods, so some out-of-order discussion is unavoidable.\n2. Line 128 refers to Figures 1g and h? These panels do not appear in the figure.\nThe reference in line 128 should be to Fig. 2c,d (insets), not Fig. 1g,h. We have corrected this in the revised\nmanuscript. Thanks for catching this.\nReviewer #2 (Remarks to the Author):\nThis is an excellent report describing the development of reagents that are successful in preventing\ndeafness resulting from Gjb2 mutations using targeted regulatory elements to express the gene in the\ncorrect cell types in mouse and non-human primates. GJB2 mutations are an important cause of\ndeafness in childhood and this report has strong translational relevance. I have a few minor comments\nto improve clarity.\nLine 81: Please define CBA here.\nDone\nFig 1c: Please label GRE1 and GRE2 on the diagram.\nDone\nFig 2b: It is difficult to distinguish the colours used for phalloidin and Gjb2 in the sections, particularly\nthe lower middle panel, and confusing that the same colour has been used for phalloidin and Gjb2 in\nthe upper and lower panels respectively. Could the colours be changed to distinguish the two markers\nmore clearly?\nWe have updated the figure to use distinct colors for phalloidin and GJB2 to improve clarity and avoid confusion\nbetween panels. The revised version now ensures better visual distinction between the two markers.\nFig 2c,d: Please define PV and BIODIPY in the legend and explain what they are labelling.\nDone\nLine 163: Just a matter of interest – why Pfl?\nThis allele was first used in Paris. We have two other floxed alleles (not described here) and needed to\ndistinguish them.\nLine 200: Point out labelling of GJB2 in the basal cell region of the stria vascularis.\nDone\nLine 248-249: Was the improvement in ABR thresholds after injection of the human GJB2 consistent with\na lack of normal OHC function? Were there any other measures of OHC function?\nWe assessed OHC function using distortion product otoacoustic emissions (DPOAEs). No improvement in\nDPOAE responses was observed following injection of either hsGJB2.HA or untagged hsGJB2 in Gjb2Pfl/Pfl, Cre+\nmice. However, we did observe a significant improvement in ABR thresholds with the untagged hsGJB2,\nsuggesting that the functional rescue occurred despite the absence of detectable OHC function recovery. This\nclarification has now been added to the text.\nLine 301: Should this be “preservation” instead of “restoration”?\nDone\nLine 309: Typo bottom\nDone\nFig 6: It is not possible to see the thickened tectorial membrane, reduced scala media area, or other\nfeatures mentioned in these images. A standard H&E or similar image would help.\nWe have now added standard H&E-stained images to Fig. 6 to better illustrate the structural changes\nmentioned in the text.\nFig 7: It may just be my screen, but I found it quite difficult to distinguish the colours in this figure.\nWould the authors consider more contrasting colours? In particular the dark blue and black were\ndifficult.\nThanks for pointing this out. We have expanded the color range.\nLine 522: Do you mean stereotypes or serotypes?\nThanks; fixed\nDiscussion: This is essentially a summary of the main findings of the project, which is fine but it would\nbe helpful to include discussion of other aspects and put the research into a broader context. For\nexample, why do the human GREs seemed to work better than mouse GREs in the mouse?\nWe did not test the endogenous mouse GREs in our experiments, as the selected human GREs effectively\nrestricted GJB2 expression in the mouse cochlea. We prioritized human GREs from the outset to align with\nfuture translational applications and observed that they yielded a spatial expression pattern consistent with\nendogenous GJB2 expression in mouse cochlear supporting cells and fibrocytes. While we cannot conclude\nthat human GREs 'work better' than mouse GREs in this context, our findings showed that human regulatory\nsequences can function appropriately in the mouse inner ear, supporting their potential use in cross-species\npreclinical studies.\nWe have added a paragraph to the discussion to put the findings into a broader context, commenting on the\ndifferent roles for GJB2 in different cells at different times, and thus the differing ability of our vectors to\ncompensate for GJB2 loss.\nWhat do the findings tell us about the role of Gjb6 in hearing – is it redundant? Why do the authors\nthink that the HA tag allows Gjb2 to function in one mutant but not in the other mutant?\nRegarding the role of GJB6, our findings indicate that it is not redundant with GJB2 but plays a complementary\nrole in supporting normal auditory function. The severe phenotype observed when GJB2.HA was\noverexpressed in Gjb2Gjb6- mice, suggests that, in the absence of GJB6, the precise balance and composition\nof connexin proteins becomes critical, and that altered GJB2 alone cannot compensate.\nAs for the differential effect of the HA tag, we propose that GJB2.HA retains partial function in the Gjb2Pfl/Pfl,\nCre+ model because GJB6 is still present to support gap function. In contrast, in Gjb2Gjb6- mice, the absence\nof GJB6 and the presence of the GJB2.HA together resulting in structural abnormalities and no functional\nrescue.\nLine 609-615: Could the genetic background of each mouse line be added to the methods? Presumably\nit is mixed but it would be helpful to know if the two Gjb2 mutants had different backgrounds that may\ncontribute to their phenotypes.\nGjb2Pfl/Pfl, Cre+ or Gjb2Pfl/Pfl, Cre- mice were maintained on a C57BL/6J background. Gjb2Gjb6-mice were\nmaintained on mixed C57BL/6J–CBA genetic backgrounds. We added this to the text.\nLine 633: What is meant by “as described above prior to fixation”? Presumably these samples were not\nfixed?\nCorrected\nReviewer #3 (Remarks to the Author):\nGJB2 encodes connexin26. Recessive variants of human GJB2 are associated with clinically\nheterogeneous deafness and are common in many countries. A founder variant of GJB2 accounts for the\nmajority of nonsyndromic deafness in the Ashkenazi Jewish population. Gene therapy for GJB2\ndeafness is on the horizon and looks more hopeful considering the new data in the manuscript from\nIvanchenko and co-authors entitled “Cell-specific delivery of GJB2 restores auditory function in mouse\nmodels of DFNB1 deafness and mediates appropriate expression in NHP cochlea”. The authors describe\nan AAV vector with two cell-specific regulatory cis-acting sequences (GREs) driving the expression\nconnexin26 in the appropriate inner ear cell types. Their new AAV vector can significantly restore\nhearing in an otherwise deaf mouse and in a partially deaf mouse model. The manuscript is nicely\ncrafted, the figures are of good quality and the data is robust and convincing. There is one concerning\nissue that hopefully can be resolved so that the data can be reproduced by other investigators. A list\nbelow of minor edits are recommended.\nMajor issue: Uncontrolled GJB2 expression using a ubiquitous promoter is lethal, as the authors clearly\npoint out and reference. To spatially control GJB2 expression, the authors performed ATAC-seq and\nidentified two chromosomal regions described in the text and in figure1. The two “GREs” are referred\nto as GRE1 and GRE2. Unless this reviewer missed the information, the authors did not provide the\nexact nucleotide sequence of GRE1 and GRE2. On line 112, GRE1 is described as including noncoding\nexon 1 and ~200 bp of upstream sequence. That’s vague. GRE2 is described as “3’ noncoding segment\nof exon 2.” That’s entirely inexact. Without the nucleotide sequence included in the manuscript for\nGRE1 and GRE2, the data and observations by Ivanchenko and co-authors cannot be independently\nreproducible by other scientists, should they wish to pursue this topic. The Nature Communications\neditors should decide if this is acceptable secrecy by the authors although it is understandable but not\nappropriate. Some of the authors have equity in Skylark Bio as indicated on line 1017 (Competing\nInterests) and likely these authors want to move their observations into the clinical for gene therapy for\nGJB2-associated human deafness without giving commercial competitors an advantage. That too is\nunderstandable from a business point of view. But in a high tier science publication, is it proper to\nintentionally withhold critical information?\nYes, we withheld the nucleotide sequences of the GREs in the review process but always understood that\nthey would be necessary for final publication. They are now included in the Supplementary Table 1.\n.\nMinor edits:\nAbstract, lines 24. “partially” and “partial” are vague and not scientific. Please provide some actual\ndata.\nWe have added the degree of hearing restoration in decibels.\nAbstract and Introduction, lines 26 and 70, name the nonhuman primate.\nDone.\nIntroduction, line 31, Many authors state that hearing loss is the most common sensory deficit. Perhaps.\nBut color blindness is 1 in 12 men and 1 in 200 women.\nCorrected.\nIntroduction, line 43, The authors state that “GJB2 [expression] is absent from the sensory hair cells…”.\nLiterally, as stated, the Gjb2 gene is not absent. GJB2 expression, according to data in gEAR, is also not\nabsent from hair cells but is expressed at a low level compared to support cells. Which is it? The data in\ngEAR about Gjb2 expression or is it a myth about GJB2 not being present in hair cells? Please clarify.\nBased on our SHIELD database, there is indeed some Gjb2 mRNA in cochlear hair cells in neonatal mice, and\nthe gEAR database shows sparse mRNA in hair cells. However we did not detect GJB2 protein in hair cells\nwith antibodies (Fig. 3a), suggesting post-transcriptional down-regulation.\nLine 228, define “vgc”.\nDone\nLine 249, provide the genotype of the “control mice”.\nDone\nLine 461, Please clarify the statement “A well-developed exogenous GJB2.HA network was also see….”\nWhat is meant by an “exogenous … network”? Clarify please.\nCorrected."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All requests have been adequately addressed.", "reviewer_2": "The authors have dealt with all of my comments on the previous version in a satisfactory way.", "reviewer_3": "The authors have revised the manuscript and am satisfied with their edits."}, "author_response": ""}]

Decision letter