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Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c


Because there are currently no biological treatments for hearing loss, we sought to advance gene therapy approaches to treat genetic deafness. We focused on Usher syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and studied a knock-in mouse model, Ush1c c.216G>A, for Usher syndrome type IC (USH1C). As restoration of complex auditory and balance function is likely to require gene delivery systems that target auditory and vestibular sensory cells with high efficiency, we delivered wild-type Ush1c into the inner ear of Ush1c c.216G>A mice using a synthetic adeno-associated viral vector, Anc80L65, shown to transduce 80–90% of sensory hair cells. We demonstrate recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders.

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Figure 1: SEM of the organ of Corti in Ush1c c.216G>A mice at P8.
Figure 2: Mechanotransduction in hair cells of Ush1c c.216G>A neonatal mice.
Figure 3: Expression and localization of fluorescently labeled harmonin in tissues exposed to adeno-associated viral vectors in vitro and in vivo.
Figure 4: Recovery of mechanotransduction in hair cells of mice injected with Anc80L65 harmonin vectors.
Figure 5: ABR and DPOAE threshold recovery in mice injected at P1 with AAV2/Anc80L65. CMV.harmonin-b1.
Figure 6: Startle response, rotarod performance and open field behavior recovery in mice injected at P1 with AAV2/Anc80L65. CMV.harmonin-a1 and AAV2/Anc80L65.CMV.harmonin-b1.
Figure 7: SEM of the organ of Corti in mice treated with harmonin-b1 and untreated mice.

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This work was supported by the Manton Center for Orphan Disease Pilot Award 2011 to G.S.G. (Boston Children's Hospital), the Bertarelli Foundation, Program in Translational Neuroscience and Neuroengineering, Kidz-b-Kidz Foundation (now Arts for USH), the Jeff and Kimberly Barber Gene Therapy Research Fund and a consortium agreement under a primary award from the Foundation Fighting Blindness, Cost Center #7 5794 (P.I. L. Vandenberghe). A.A.I. received support from R01DC000304 (DP Corey). S.H.-A. was the recipient of the diversity faculty fellowship award from Harvard Medical School. Behavior and Viral Cores at Boston Children's Hospital are supported by the Boston Children's Hospital Intellectual and Developmental Disabilities Research Center (BCH IDDRC), P30 HD18655. M. Hastings' contribution was supported by R01DC012596. We thank M. Valero (EPL, MEEI) for assistance with ABRs, S. Xu (BCH core) for help with the behavior work, C. Wang (BCH viral core) for AAV production, and C. Nist-Lund (BCH) for technical assistance. L. Zheng and J. Bartles45 (Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine, Chicago, IL) graciously provided EGFP-tagged, labeled constructs for harmonin-a1 and harmonin-b1 plasmids.

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Authors and Affiliations



The project was conceived and designed by G.S.G. C.A. performed RWM injections with AAV2/1 vectors, electrophysiological recordings and analysis of OHCs, FM1-43 imaging of the organ of Corti; B.P. carried out RWM injections with AAV2/Anc80L65 vectors, electrophysiological recordings and analysis of IHCs mechanotransduction currents. G.S.G. performed electrophysiological recordings of vestibular hair cells. A.G., S.H.-A. and G.S.G. performed ABR experiments. Semi-quantitative radiolabeled PCR and RT-PCR was performed by F.M.J. and M.L.H. ABR and behavioral data analysis was performed by A.G. and G.S.G. Y.A. designed and prepared the plasmid constructs. SEM was performed by J.J.L. (P18), and A.A.I. (P8 and 6 weeks). DIC, immunostaining and confocal imaging was performed by G.S.G. L.H.V. assisted with production of AAV vectors. J.R.H assisted with the design of the experiments, preparation of figures and manuscript. G.S.G. analyzed, interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to Gwenaëlle S Géléoc.

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Competing interests

A patent #00633-0203P01 on “Materials and methods for delivering nucleic acids to cochlear and vestibular cells” has been deposited by L.H.V., G.S.G. and J.R.H. The Anc80L65 vector is patented by L.H.V., patent #WO/2015/054653 – “Methods of predicting ancestral virus sequence and uses thereof.” L.H.V. is co-founder and SAB member of GenSight Biologics and consultant to various gene therapy companies. L.H.V. receives research support from Selecta Biosciences and Lonza Houston on Anc-AAV development and discovery.

Integrated supplementary information

Supplementary Figure 1 Analysis of hair bundle morphology in Ush1c c.216G>A mice at P18 by SEM.

(a-c) Heterozygous c.216GA mice displayed normal hair bundle morphology at P18. (d-i) Disorganized hair bundles were observed along the organ of P18 Homozygous c.216AA mice. (j-l) IHCs hair bundle were mildly disrupted (arrows) in c.216AA mice. Distance measured from apex tip: Base 3.5-4 mm; Mid 1.8-2.2 mm; Apex 0.6-0.8 mm. Scale bar a-f; j-l: 5 μm; g-i: 1 μm.

Supplementary Figure 2 Mechanotranduction properties in c.216AA mice.

(a-e) Analysis of mechanotransduction in neonatal OHCs from middle and mid-apical turns of the cochlea, P3-P6. Representative current traces from ~Po= 0.5 were fit with a double exponential decay function to assess adaptation in c.216GA and c.216AA mutant (a). Fits were used to generate fast (c) and slow (d) time constants as well as the extent of adaptation (e). The 10-90% operating range was estimated from the second-order Boltzmann fits and was not significantly (NS) altered (b). Extent of adaptation, measured at Popen= 0.5, was significantly less in c.216AA mice than for heterozygous OHCs as shown in this scatter plot (e). (f-j) Analysis of mechanotransduction in neonatal IHCs. 10-90% operating range values were smaller in c.216GA versus c.216AA IHCs (g). Adaptation was always present albeit slightly slower and with a significant lesser extent in c.216AA IHCs (h-j). Statistical analysis is indicated in each plot: *P<0.05, **P<0.01 and ***P<0.001, one-way ANOVA.

Supplementary Figure 3 Expression of fluorescently labeled harmonin-a1 and harmonin-b1 at 6 weeks in c.216AA organ of Corti after P1 dual vector injection.

(a-c) Confocal images of the basal turn in 6 weeks old c.216AA mice after P1 co-injection of AAV2/Anc80L65.CMV.tdTomato::harmonin-a1 (0.5 μl; 4.1 × 1012 gc/ml) and AAV2/Anc80L65.CMV.EGFP::harmonin-b1 (0.5 μl; 3.0 × 1012 gc/ml). 69% and 74% of the total number of cells (IHCs and OHCs) expressed EGFP (a) and tdTomato (c), respectively and 65% expressed both markers demonstrating successful co-transduction. Total hair cells were counted based on the phalloidin image (b, blue). EGFP+ (a) and tdTomato+ (c) hair cells were counted based on the confocal images shown. Scale bar: 20 μm.

Supplementary Figure 4 Analysis of ABR response in 6 weeks old control c.216GA and injected rescued c.216AA mice.

(a,d) Example of ABR responses at 8 and 16 kHz for control c.216GA and rescued c.216AA mice, 90dB stimulus. (b,c, e-f) Average wave 1 amplitude (b,e) and latency (c,f) at 8-11.3 and 16 kHz in 6 weeks old mice with comparable thresholds (n=8 c.216GA, n=5 c.216AA + harmonin-b1 RWM P1). Mean ± S.E. While wave 1 amplitude did not differ significantly between each group (t-test: P>0.1 for all sound levels, b and e), peak latency differed significantly for 8-11.3 kHz responses (t-test: P<0.05 for all sound levels, c) and 16 kHz (t-test: P<0.001, f).

Supplementary Figure 5 USH1C expression in the inner ear of mice injected with AAV2/Anc80L65.CMV.harmonin-b1.

Expression was assessed in six-week old c.216GA and AAV2/Anc80L65.CMV.harmonin-b1 (0.8 μl; 1.9 × 1012 gc/ml) injected and non-injected c.216AA mice (a) Semi-quantitative RT-PCR of correctly spliced (450 bp) and truncated (415 bp) mRNA expression revealed vector-derived expression of correctly spliced Ush1c in injected (“I”) and contralateral ears (“C”) of c.216AA rescued mice #1 and #2 (thresholds ≤ 35 dB response at 11.3 kHz from injected ears). Mouse #3 with poor ABR response (thresholds ≥90 dB at 11.3 kHz) showed modest recovery of correct mRNA expression and mouse #4 (thresholds ≥100 dB at 11.3 kHz) showed none. While the correctly spliced form is not detected in uninjected left (“L”) and right (“R”) ears of c.216AA mice (mice #5, 6), both the correct and truncated splice forms were detected in c.216GA mice (mice #7, 8, 9). Corresponding mouse Gapdh shown in the bottom panel was amplified to confirm a consistent amount of material was used. (b) Semi-quantitative radiolabeled PCR analysis confirmed the presence AAV-mUsh1c DNA in injected and contralateral ears of Ush1c.216AA mice. AAV-Ush1c DNA was present but reduced in mice #3 and #4. (c) The relative amount of viral DNA correlates with ABR thresholds. The 11.3 and 16 kHz responses from injected and contralateral cochleas of the four injected mice (#1-4) are illustrated. Linear regressions show high correlation between the amount of viral DNA and ABR threshold responses. Tissues were collected at 6 weeks of age. (d) Immunostaining of harmonin confirmed restored expression of harmonin protein in stereocilia of c.216AA mice that recovered hearing following Anc80L65.CMV.harmonin-b1 P1 RWM injection. Panels show immunofluorescence of harmonin (green) and F-actin (red) in outer hair cell (OHC) bundles in mid apical regions of wild type (WT, 6 weeks old), homozygous c.216AA (4 weeks), injected c.216AA mice with no rescue (4 weeks, no ABR response at 110 dB at any frequency tested), and injected c.216AA mice with rescue (4 weeks, 30 dB at 11 kHz). Scale bar: 10 μm.

Supplementary Figure 6 Long term ABR threshold recovery correlates with OHCs survival in the mid to apical region of the auditory organ.

Hair cell count across the entire Organ of Corti was performed post-mortem in left ears of three uninjected c.216AA and five injected c.216AA (P1 RWM injection, 0.8 μl AAV2/Anc80L65.CMV.harmonin-b1) at 6 months of age. Insert: While two of the mice (#1 and #2) showed poor ABR response (PR) thresholds across the entire range tested (≥95 dB), three (#3-5) responded (R) with thresholds ranging 35 and 55 dB for sound stimuli between 5.6 and 16 kHz. The total number of IHC and OHCs hair cells was increased in injected mice. Comparison of rescued injected mice with those injected that had poor rescue shows that the number of IHCs was not different but a significant number of OHCs were noted in the rescued mice. Analysis across the entire length of the organ showed the difference can be accounted for as an increase in hair cell survival from the mid to apical regions of the organ.

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Pan, B., Askew, C., Galvin, A. et al. Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c. Nat Biotechnol 35, 264–272 (2017).

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