Cell tropism of adeno-associated viruses within the mouse inner ear in vivo: from embryonic to adult stages

Abstract

Adeno-associated virus (AAV)-based gene therapy is emerging as a promising treatment for deafness and vestibular deficits, due to the variety of available serotypes that offer a large range of cell targeting capabilities. Nevertheless, the tropism of these AAV serotypes for sensory inner ear cells varies greatly as the cochlea matures, presenting a significant burden for successful preclinical trials. Therefore, identifying serotypes with strong tropism for cochlear and vestibular hair cells during key stages of development in mouse inner ear, the most widely used preclinical model, is essential for advancing clinical applications. We conducted a comparative analysis of the cellular tropism and hair-cell transduction rates of four AAV serotypes in the cochlea and vestibular organs during maturation. We used AAV2, AAV8, AAV9-PHP.eB, and Anc80L65 at the embryonic, neonatal, and adult stages. Our results indicate that the cell transduction rate of these four serotypes varies with age. Notably outer hair cells were mostly targeted during the embryonic stage, inner hair cells were primarily transduced principally at the mature stage, and vestibular hair cells were the most permissive at the neonatal stage. These results provide new insights for preclinical gene therapy studies for the inner ear with potential implications for therapeutic outcomes.

Keywords: AAV; Gene therapy; Inner ear; Serotype; Therapeutic time window; Tropism.

Fig. 1

Comparison of the inner ear cell tropism of four AAV serotypes at the embryonic stage. Confocal images of the apical turn of the organ of Corti (left panels) and vestibular organs (right panels) after the injection into the otocyst on E13-E15 of 1 µL of (A) AAV2-CBA-GFP, (B) AAV8-CMV-GFP, (C) AAV-Anc80L65-CMV-GFP or (D) AAV9-PHP.eB-CBA-GFP and immunostaining on P8 for GFP (green) and myosin 7a (red). For the cochlea, the insets are close-up views of the apical (top), medial (middle), and basal (bottom) regions (scale bars: A-D, 100 μm; insets, 5 μm). IHCs, inner hair cells; OHCs, outer hair cells; SGNs, spiral ganglion neurons; DC, Deiters’ cells; NF, nerve fibers; IPhC, inner phalangeal cells, and HC, Hensen’s cells. The right panel shows a low magnification of the macula and ampulla cristae with close-up views shown in the insets (scale bars A-D, 50 μm; insets, 5 μm). GFP-positive vestibular hair cells are outlined by a dashed line and cells other than the sensory hair cells are indicated by arrowheads. (E) Bar graphs showing the percentage of hair cells—IHCs, OHCs and VHCs—transduced with AAV2 (black), AAV8 (pink), Anc80L65 (blue), and AAV9-PHP.eB (purple), (mean percentage ± SEM). Two-way ANOVA, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 2

Comparison of the inner ear cell tropism of four AAV serotypes at the neonatal stage. Confocal images of the apical turn of the organ of Corti (left panels) and vestibular organs (right panels) after the injection on P2, via the RWM, of 2 µL of (A) AAV2-CBA-GFP, (B) AAV8-CMV-GFP, (C) AAV-Anc80L65-CMV-GFP or (D) AAV9-PHP.eB-CBA-GFP, followed by immunostaining on P8 for GFP (green) and myosin 7a (red). For the cochlea (left), insets are high-magnification views of the apical (top), medial (middle), and basal (bottom) regions (scale bars: A–D, 100 μm; insets, 5 μm). IHCs, inner hair cells; OHCs, outer hair cells; IPC, inner pillar cells, DC, Deiters’ cells; IPhC, inner phalangeal cells. For the vestibule (right), insets are close-up views of the macula and ampulla cristae (scale bars A–D, 50 μm; insets, 5 μm). GFP-positive vestibular hair cells are outlined by dashed lines and cells other than sensory hair cells are indicated by arrowheads. (E) Bar graphs showing the percentage of hair cells—IHCs, OHCs and VHCs—transduced with AAV2 (black), AAV8 (pink), Anc80L65 (blue), and AAV9-PHP.eB (purple), (mean percentage ± SEM). Two-way ANOVA, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 3

Comparison of the inner ear cell tropism of four AAV serotypes at early mature stage (P14-P18). Confocal images of the apical turn of the organ of Corti (left panels) and vestibular organs (right panels) after injection through the RWM at P14-18 with 2 µL of (A) AAV2-CBA-GFP, (B) AAV8-CMV-GFP, (C) AAV-Anc80L65-CMV-GFP or (D) AAV9-PHP.eB-CBA-GFP, followed by immunostaining, at P19-25, for GFP (green) and myosin 7a (red). For the cochlea (left), the insets provide higher-magnification views of the apical (top), medial (middle), and basal (bottom) regions (scale bars: A–D, 100 μm; insets, 5 μm). IHCs, inner hair cells; OHCs, outer hair cells; SGNs, spiral ganglion neurons. For the vestibule (right), broad view of the macula and ampulla cristae with corresponding close-up views (scale bars A–D, 50 μm; insets, 5 μm). GFP-positive vestibular hair cells are outlined by dashed lines and cells other than sensory hair cells are indicated by arrowheads. (E) Bar graphs showing the percentage of hair cells—IHCs, OHCs and VHCs—transduced with AAV2 (black), AAV8 (pink), Anc80L65 (blue), or AAV9-PHP.eB (purple), (mean percentage ± SEM). Two-way ANOVA, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 4

Comparison of the inner ear tropism of four AAV serotypes at late mature stage (P30). Confocal images of the apical turn of the organ of Corti (left panels) and vestibular organs (right panels) injected at P30 with 2 µl of (A) AAV2-CBA-GFP, (B) AAV8-CMV-GFP, (C) AAV-Anc80L65-CMV-GFP and (D) AAV9-PHP.eB-CBA-GFP via RWM, immunostained at P37 for GFP (green) and myosin 7a (red). For the cochlea, higher magnification insets of the apical (top), medial (middle), and basal (bottom) regions are inserted (scale bars: A-D, 100 μm; insets, 10 μm). On the right panel, large view of the macula and ampulla cristae with corresponding higher magnification (scale bars A-D, 50 μm; insets, 10 μm). (E) Comparative analyses of the transduction rate of inner hair cells (IHCs), outer hair cells (OHCs) and vestibular hair cells (VHCs) transduced with different serotypes including AAV2 (black), AAV8 (pink), Anc80L65 (blue), and AAV9-PHP.eB (purple), (mean percentage ± SEM). Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.