Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B

Abstract

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a^{4626SB/4626SB}) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

Keywords: AAV; MYO7A; USH1B; Usher syndrome; dual-AAV; gene therapy; hearing loss; vestibular dysfunction.

Graphical abstract

Figure 1

Dual-AAV8(Y733F)-MYO7A vectors restore MYO7A expression and prolong the survival of shaker-1 cochlear hair cells (A) Representative confocal images of inner hair cells (IHCs) and outer hair cells (OHCs) of the cochlear middle turn. In an untreated wild-type mouse, MYO7A expression (green) is found in the IHCs and OHCs. The shaker-1 mouse does not express MYO7A. Shaker-1 mice that were treated with dual-AAV8(Y733F)-MYO7A vectors exhibit restoration of MYO7A in transduced IHCs and OHCs. Images were taken at P30–P37. The scale bar represents 20 μm. (B and C) Quantification of IHC (B) and OHC (C) transduction rates in shaker-1 mice that were treated with dual-AAV8(Y733F)-MYO7A vectors. (D and E) Quantification of IHC (D) and OHC (E) survival in shaker-1 mice that were treated with dual-AAV8(Y733F)-MYO7A vectors. Shaker-1 mice that were treated with dual-AAV8(Y733F)-MYO7A vectors showed significant improvement in hair cell survival compared with untreated shaker-1 mice. n = the number of animals used. Error bars represent standard errors. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Figure 2

Dual-AAV8(Y733F)-MYO7A vectors did not improve stereocilium organization of cochlear hair cells or auditory function in shaker-1 mice (A) Representative confocal images showing stereocilium organization in IHCs and OHCs. Shaker-1 mice have highly disorganized stereocilium bundles compared to wild-type animals. Dual-AAV8(Y733F)-MYO7A vectors did not improve stereocilium organization in shaker-1 cochlear hair cells, as revealed by phalloidin staining to visualize F-actin. The scale bar represents 10 μm. (B) Auditory brain stem response (ABR) thresholds at the four measured frequencies (4, 8, 16, and 32 kHz). An ABR threshold of 100 dB SPL represents no response. ABR testing was done at ∼P30. Dual-AAV8(Y733F)-MYO7A vectors did not improve ABR in shaker-1 mice. Error bars represent standard errors.

Figure 3

Dual-AV8(Y733F)-MYO7A vectors restore MYO7A expression in transduced vestibular hair cells (A) Representative confocal images of vestibular hair cells from mouse utricles. Images were taken at P30–P37. The shaker-1 mice that were treated with dual-AAV8(Y733F)-MYO7A showed robust MYO7A expression (green), whereas the untreated shaker-1 mice showed no MYO7A expression. A higher-magnification view of the treated shaker-1 stereocilia revealed MYO7A expression along the stereocilia bundles (inset). Scale bars represent 10 μm. (B) Quantification of vestibular hair cell transduction rates. (C) Quantification of hair cell survival in mouse utricles. In contrast to the shaker-1 cochlear hair cells, the vestibular hair cells do not undergo degeneration in shaker-1 mice. Error bars represent standard errors.

Figure 4

Dual-AAV8(Y733F)-MYO7A vectors improve vestibular stereocilium organization and circling behavior in shaker-1 mice (A) Representative confocal images of stereocilium bundles of vestibular hair cells from mouse utricles stained by phalloidin to visualize F-actin. The shaker-1 vestibular hair cells have highly disorganized stereocilium bundles compared with wild-type hair cells. Shaker-1 mice treated with dual-AAV8(Y733F)-MYO7A vectors showed significant improvement in vestibular stereocilium organization compared with untreated shaker-1 vestibular hair cells. The scale bar represents 10 μm. (B) Representative track plots from a wild-type mouse, an untreated shaker-1 mouse, and a shaker-1 mouse treated with dual-AAV8(Y733F)-MYO7A vectors. Dual-AAV8(Y733F)-MYO7A vectors decreased circling behavior in treated shaker-1 mice. (C) Quantification of circling behavior was performed at ∼P30. Shaker-1 mice that were treated with dual-AAV8(Y733F)-MYO7A showed significant reduction in circling compared with untreated shaker-1 mice. Error bars represent standard errors. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 5

Dual-AAV8(Y733F)-MYO7A vectors improved VsEP thresholds in shaker-1 mice (A) Representative vestibular sensory-evoked potential (VsEP) recordings from a wild-type mouse, an untreated shaker-1 mouse, and a shaker-1 mouse treated with dual-AAV8(Y733F)-MYO7A vectors. The untreated shaker-1 mouse had no measurable VsEP responses, whereas the treated shaker-1 mouse had measurable VsEP responses. (B) VsEP threshold measurements. Shaker-1 mice that were treated with dual-AAV8(Y733F)-MYO7A vectors showed improvement in VsEP thresholds compared with untreated shaker-1 mice. Error bars represent standard errors. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.