A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear

Abstract

Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector, for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

Figure 1. Transduction of organotypic explant of murine cochlea with natural AAV serotypes and Anc80L65

Representative confocal projections of an in vitro comparison of several AAV serotypes for eGFP transgene expression in cochlear explants of C57BL/6 mice. A–G: Expression at the cochlear base for all serotypes, and apex and base for Anc80L65 after incubation with 10¹⁰ genome containing (GC) particles for 48 hours. Scale bar = 100 μm. (Top: Myo7A+TuJ1, bottom: eGFP only, middle: overlay, S. Cells: Supporting Cells, OHC: Outer Hair Cell, IHC: Inner Hair Cell) H–K: Percentage of eGFP-positive hair cells per 100 μm after 48h or 48h+5 days of incubation. Mean indicated by horizontal bar. Each condition had minimally N=3 for the 48h, and N=2 for the 48h+5d unless otherwise noted.

Figure 2. In vivo cochlear transduction of natural AAV serotypes and Anc80L65

(A) Confocal images of mouse organs of Corti, counterstained with Alexa546-phalloidin (red) and imaged for eGFP (green). A total of 5 (AAV1), 4 (AAV2), 2 (AAV8), 1 (AAV6), 3 (Anc80L65) C57BL/6 mice were injected with 1 μL of AAV stock solution in one ear at the titer indicated above each panel. Scale bar = 50 μm. Quantification of eGFP-positive IHCs (B) and OHC (C) in the base and apex of a representative AAV-eGFP injected cochlea for each vector. Quantification of eGFP-positive OHCs in the base and apex of AAV-eGFP injected cochleas. (D) Families of sensory transduction currents recorded at P7 (left) from eGFP-negative OHCs (black) and eGFP-positive OHCs (green). Hair bundles were deflected between −0.1 and 1 μm in 0.1 μm increments. Vertical scale bar indicates 200 pA; horizontal indicates 20 msec. Currents from eGFP-negative (black) and eGFP-positive (green) P35 IHCs are shown on the right. Vertical scale bar indicates 100 pA; horizontal indicates 20 msec. (E) Sensory transduction current amplitudes plotted for 103 IHCs and OHCs at the ages indicated at the bottom. Data from eGFP-negative (black) and eGFP-positive (green) are shown. The numbers of cells in each group are shown on the graph. All mice were injected at P1. (F) Mean ± standard deviation (SD). ABR thresholds plotted for four Anc80L65-injected ears (green) and four uninjected ears (black) together with data from one injected ear that had no eGFP fluorescence due to injection-related damage (red). (G) Mean ± SD. DPOAE thresholds are plotted for four Anc80L65-injected ears (green) and four uninjected ears (black) and one negative control ear with injection damage without eGFP fluorescence (red). Injection titers for data points in B–G are as in A.

Figure 3. Extensive Inner and Outer Hair Cell transduction in murine cochleas with Anc80L65

Low- (A) and high (B) magnification image of the entire apical portion of a mouse cochlea injected at P1 with 1 μL of Anc80L65-eGFP at 1.70 × 10¹² GC/mL. The cochlea was harvested at P10 and stained with Alexa546-phalloidin (red) and imaged for eGFP (green). Scale bar = 100 μm in (A), 20 μm in (B). (C, D) Quantitative comparison of inner and outer hair cell transduction efficiency at an equal dose for all serotypes following round window injection of P1-2 C57BL/6 mice. Mouse cochleas in (C,D) were injected with 1.36 × 10¹² of AAV1, AAV2, AAV8, and Anc80L65 and harvested at 7–9 days for live-cell imaging and quantification by epifluorescence microscopy (n=8 per group unless otherwise noted). Error bars represent SD. (E) Dose-dependency of Anc80L65 hair cell transduction. Cochleas exposed to two different Anc80L65-eGFP titers (1.80 × 10¹² versus 1.36 × 10¹² GC) were fixed and stained with Alexa546-phalloidin (red) and imaged for eGFP (green). Scale bar = 20 μm.

Figure 4. Anc80L65-eGFP transduction in vestibular sensory epithelia

(A) Mouse utricle from a P1 mouse injected with 1 μL Anc80L65-eGFP (1.7 × 10¹² GC/mL). The tissue was harvested at P10 fixed and stained with Alexa546-phalloidin (red) and imaged for eGFP (green). Morphological assessment across multiple focal planes of eGFP-positive cells demonstrated transduction of stereotypical flask-shaped morphology of type I cells and the cylinder morphology of type II cells in every sample examined (not shown). Scale bar = 100 μm. (B) The crista of the posterior semicircular canal from the same mouse described for panel A. Scale bar = 50 μm. (C) The sensory epithelium of a human utricle. The tissue was exposed to 10¹⁰ GC Anc80L65.CMV.eGFP.WPRE for 24 hours, cultured for 10 days, fixed, stained with Alexa546-phalloidin (red) and imaged for eGFP fluorescence (green). Scale bar = 100 μm. (D) High-magnification view of a human epithelium in the utricle stained with Alexa546-phalloidin (red) and Myo7A (blue) and imaged for eGFP (green) transduced in identical conditions as in C. White arrows in the overlay panel indicate selected eGFP-positive/Myo7A-positive cells. Scale bar = 20 μm.