Methionine Sulfoxide Reductase B3-Targeted In Utero Gene Therapy Rescues Hearing Function in a Mouse Model of Congenital Sensorineural Hearing Loss

Abstract

Aims: Methionine sulfoxide reductase B3 (MsrB3), which stereospecifically repairs methionine-R-sulfoxide, is an important Msr protein that is associated with auditory function in mammals. MsrB3 deficiency leads to profound congenital hearing loss due to the degeneration of stereociliary bundles and the apoptotic death of cochlear hair cells. In this study, we investigated a fundamental treatment strategy in an MsrB3 deficiency mouse model and confirmed the biological significance of MsrB3 in the inner ear using MsrB3 knockout (MsrB3(-/-)) mice.

Results: We delivered a recombinant adeno-associated virus encoding the MsrB3 gene directly into the otocyst at embryonic day 12.5 using a transuterine approach. We observed hearing recovery in the treated ears of MsrB3(-/-) mice at postnatal day 28, and we confirmed MsrB3 mRNA and protein expression in cochlear extracts. Additionally, we demonstrated that the morphology of the stereociliary bundles in the rescued ears of MsrB3(-/-) mice was similar to those in MsrB3(+/+) mice.

Innovation: To our knowledge, this is the first study to demonstrate functional and morphological rescue of the hair cells of the inner ear in the MsrB3 deficiency mouse model of congenital genetic sensorineural hearing loss using an in utero, virus-mediated gene therapy approach.

Conclusion: Our results provide insight into the role of MsrB3 in hearing function and bring us one step closer to hearing restoration as a fundamental therapy.

FIG. 1.

Auditory function following transuterine rAAV-mediated gene delivery in MsrB3^+/+ mice. (A) Click-evoked ABR response of an untreated ear (upper), an operated ear (middle), and a contralateral ear (lower) of an MsrB3^+/+ mouse at P28 after administration of the rAAV2/1-GFP treatment. The amplitude of the response is measured in microvolts (μV). The time is expressed in milliseconds (ms) and is indicated on the x-axis. (B) The average click-evoked ABR thresholds of each group are shown. No significant differences were observed between the groups (n = 6 for each group, p > 0.5). Student's t-tests were conducted for statistical comparisons. The data are shown as the mean ± SD. ABR, auditory brainstem response; GFP, green fluorescent protein; MsrB3, methionine sulfoxide reductase B3; rAAV, recombinant adeno-associated virus.

FIG. 2.

Expression of GFP in MsrB3^+/+ mice following rAAV-mediated gene transfer into the organ of Corti. (A) Immunostaining was performed to detect rAAV-transduced cells. Fluorescence images show the organ of Corti from an MsrB3^+/+ mouse that is immunostained for GFP (green) and phalloidin (red) at P0. (B) Fluorescence images show that the GFP-positive cells consisted primarily of IHCs and OHCs at P28. (C) The graph represents the transduction efficiency of IHCs and OHCs in each of the three turns, the apical, middle, and basal turns, within a 160-μm region of the organ of Corti. No significant differences were observed between the groups (p > 0.1, n = 4 for each group). Student's t-tests were conducted for statistical comparisons. The data are shown as the mean ± SD. IHC, inner hair cell; OHC, outer hair cell. Scale bars: 20 μm.

FIG. 3.

Restoration of auditory function following rAAV2/1-mediated MsrB3 gene transfer into the otocysts of MsrB^−/− mice. (A) Example of click-evoked ABR response of a post-treatment MsrB3^−/− mouse ear compared with that of an MsrB3^+/+ mouse ear at P28. The waveform of the rAAV2/1-MsrB3-GFP-treated ear was changed after treatment. (B) Graphical representation of the ABR data that show the average ABR thresholds for click and tone burst (4, 8, 16, and 32 kHz) stimuli of the five rescued MsrB3^−/− mice. Asterisks indicate statistical differences between the ears of the MsrB3^+/+ mice and the rAAV2/1-MsrB3-GFP-treated ears of the MsrB3^−/− mice (*p < 0.05). p-Values were determined by Student's t-tests. The data are shown as the mean ± SD. n = 5 for each group.

FIG. 4.

Localization and transduction efficiency of MsrB3 and GFP following gene transfer. (A) An anti-GFP antibody was used to label the organ of Corti of the treated left ears of 28-day-old MsrB3^−/− mice, and F-actin was labeled using Alexa 555-conjugated phalloidin. The tissue samples were then imaged with a fluorescence microscope. The low-magnification images show the distribution of GFP expression following the delivery of rAAV2/1-MsrB3-GFP into the otocysts of MsrB3^−/− mice (upper panel). The high-magnification images show that GFP (green) was detected in both the IHCs and OHCs (lower panel) in the rescued MsrB3^−/− mice. (B) Graphical representation of the percentage of GFP-positive cells that were transfected with rAAV2/1-MsrB3-GFP within a 160-μm region of IHCs and OHCs in each of the three turns, including the apical, middle, and basal turn. No significant differences were observed between the groups (p > 0.05, n = 4 for each group). p-Values were determined by Student's t-tests. The data are shown as the mean ± SD. (C) MsrB3 and GFP were specifically expressed in both types of hair cells within the cochlea (left panel). The higher-resolution images show that MsrB3 and GFP were colocalized in both the IHCs and OHCs in the organ of Corti (right panel). The scale bars represent 25 μm in (A), 100 μm in (C, left panel), and 20 μm in (C, right panel).

FIG. 5.

Quantification of exogenous MsrB3 expression levels using RT-PCR and Western blot analysis. (A) RT-PCR analysis of MsrB3 expression, which confirmed the success of rAAV-mediated gene transfer. Total RNA was extracted from the inner ears of MsrB3^+/+ and rescued MsrB3^−/− mice at P28. MsrB3 mRNA was found in the ears of the MsrB3^+/+ mice and MsrB3^+/− mice and in the rAAV2/1-MsrB3-GFP-treated ears of the MsrB3^−/− mice. Gapdh served as an internal control. (B) Western blot analysis of inner ears extracted from MsrB3^+/+ mice, MsrB3^+/− mice, the rescued ears of MsrB3^−/− mice, and the untreated ears of MsrB3^−/− mice at P28. β-actin was used as a quantitative loading control. MsrB3 protein was also detected in the rescued ears of the MsrB3^−/− mice. (C) Graphical representation of the relative MsrB3 levels, which were normalized to β-actin, in the ears of MsrB3^+/− mice and in the rAAV2/1-MsrB3-GFP-treated and untreated ears of MsrB3^−/− mice compared with those in the ears of MsrB3^+/+ mice. Asterisks denote significant differences between the rAAV2/1-MsrB3-GFP-treated ears of the MsrB3^−/− mice and the untreated ears of MsrB3^+/− or MsrB3^−/− mice (n = 4 for each group, *p < 0.001). p-Values were determined by Student's t-tests. The data are shown as the mean ± SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription–polymerase chain reaction.

FIG. 6.

Immunohistochemical analysis of MsrB3 expression in cochlear sections. Immunohistochemistry was performed on serially sliced inner ear samples from the ears of MsrB3^+/+ mice and from the untreated, rAAV2/1-GFP-treated, and rAAV2/1-MsrB3-GFP-treated ears of MsrB3^−/− mice at P28. The expression of MsrB3 protein (red) and Myo7a (green) was assessed in MsrB3^+/+ mice (A–C). No MsrB3 expression was found in the untreated ears or rAAV2/1-GFP-treated ears of the MsrB3^−/− mice (D–I). In contrast, the rescued ears of the MsrB3^−/− mice exhibited local expression of exogenous MsrB3 in the IHCs and OHCs, and the expression of exogenous MsrB3 overlapped with that of Myo7a (J–L). Scale bars represent 10 μm.

FIG. 7.

Ultrastructural morphology of the stereociliary bundles. MsrB3^+/+, MsrB3^−/−, and rescued MsrB3^−/− mice were sacrificed at P28 for scanning electron microscopy analysis. The images show stereociliary bundles along the three cochlear turns, including the apical, middle, and basal turn. (A) Overview image of a large portion of the organ of Corti that shows the stereociliary bundles of the three rows of OHCs and one row of IHCs in the ears of MsrB3^+/+ (left column) mice, the ears of MsrB3^−/− mice (middle column), and the ears of rescued MsrB3^−/− mice (right column). (B) Representative higher-resolution images of the OHCs (left panel) and IHCs (right panel). IHC, inner hair cell; OHC, outer hair cell. The scale bars represent 10 μm in (A) and 1 μm in (B).