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


Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities1,2. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector3, 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.

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Figure 1: Transduction of organotypic explants of murine cochlea with natural AAV serotypes and Anc80L65.
Figure 2: In vivo cochlear transduction of natural AAV serotypes and Anc80L65.
Figure 3: Extensive inner and outer hair cell transduction in murine cochleas with Anc80L65.
Figure 4: Anc80L65-eGFP transduction in vestibular sensory epithelia.

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This work was supported by the Bertarelli Foundation grants (K.M.S., J.R.H.), the Jeff and Kimberly Barber Gene Therapy Research Fund (J.R.H.), the Patel Gene Therapy Fund (J.R.H.), Department of Defense Grant W81XWH-15-1-0472 (K.M.S.), the National Institutes of Health (NIH) 1R01DC015824 (K.M.S.), Nancy Sayles Day Foundation (K.M.S.), Lauer Tinnitus Research Center (K.M.S.), Giving/Grousbeck (L.H.V.), Foundation Fighting Blindness (L.H.V.), Ush2A Consortium (L.H.V.), and NIH 5DP1EY023177 (L.H.V.). The authors would like to thank H.-C. Lin and S. Narasimhan for help with the brain tissue staining protocol, G. Geleoc and C. Nist-Lund for assistance with vestibular tissue imaging, and R. Xiao, R. Shelke, Y. Lin, and T. Barungi for vector preparation.

Author information




L.D.L., B.P., C.A., S.J.W., S.D.G., and A.G. conducted, analyzed, and reported the mouse gene transfer experiments described, and R.T. and A.F. performed and led the human vestibular transduction experiments. L.H.V. provided vector materials. K.M.S., J.R.H., and L.H.V. conceived the study, led experimental design and oversaw analysis. J.R.H., L.H.V., and L.D.L. drafted the manuscript with topical input from all other authors.

Corresponding authors

Correspondence to Konstantina M Stankovic or Jeffrey R Holt or Luk H Vandenberghe.

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Competing interests

L.H.V. holds founder equity in GenSight Biologics, is a consultant to a number of biotech and pharmaceutical companies, and is an inventor on gene therapy patents, including Anc80L65, which are licensed to various entities. L.H.V. also receives sponsored research from Lonza Houston and Selecta Biosciences, licensees of Anc80L65. L.H.V., K.M.S., and J.R.H. have filed a patent on the use of Anc80L65 in the cochlea.

Integrated supplementary information

Supplementary Figure 1 Representative images of an in vitro comparison of several AAV serotypes regarding eGFP expression in cochlear explants of CBA/CaJ mice.

(A-F): Results after incubation at equal doses of 1010 genome containing (GC) particles for 48 hours. Scale bar = 200 μm.(G-J): Mean indicated by horizontal bar. Each condition had minimally N=3 for 48h, and N=2 for 48h+5d unless otherwise noted.

Supplementary Figure 2 eGFP expression scoring system.

eGFP expression in supporting and limbus cells following AAV exposure of cochlear explants was assessed using a qualitative scoring system ranging from 0 (D, H) (lowest expression) to 3 (A, E) (highest level of expression). Representative images illustrate the range of expression both in terms of intensity and number of transduced cells with “0” representing no noted expression (D, H), “1” select number of cells expressing dimly (C, G), “2” low to moderate levels of expression in significant numbers of cells per microscopic field (B, F), and “3” high percentage of cells expressing eGFP at levels ranging from moderate to high (A, E). Scale bar for all images as in A (for A-D) and E (for E-H) = 20 μm.

Supplementary Figure 3 eGFP expression in limbus, supporting cells, and spiral ganglion neurons (SGNs) in cochlear explants of C57BL/6 mice.

Expression in limbus and supporting cells was assessed using an eGFP scoring system detailed in Supplementary Fig. 2. SGN transduction was evaluated by eGFP-positive cell counts per microscopic field. Mean indicated by horizontal bar. Each condition had minimally N=3 for the 48h, and N=2 for the 48h+5d unless otherwise noted.

Supplementary Figure 4 eGFP expression in limbus, supporting cells, and spiral ganglion neurons (SGNs) in cochlear explants of CBA/CaJ mice.

Expression in limbus and supporting cells was assessed using an eGFP scoring system detailed in Supplementary Fig. 2. SGN transduction was evaluated by eGFP-positive cell counts per microscopic field. Mean indicated by horizontal bar. Each condition had minimally N=3 for the 48h, and N=2 for the 48h+5d unless otherwise noted.

Supplementary Figure 5 Bilateral cochlear transduction from base to apex in mouse cochleas with Anc80.

Mice injected at P1 were sacrificed and cochleas were evaluated for eGFP transgene expression 30 days following injection on histological section stained for TuJ1 (red) and Myo7A (blue). Efficient Anc80 transduction was observed in the injected cochlea extending up to apex (A/F), however also in the contralateral uninjected ear (B-E=from apex to base). (G) Close-up image of eGFP-positive and TuJ1-positive spiral ganglion neurons (SGNs). (H) Reconstructed 3D image for SGN evaluation of Anc80 transduction. Scale bars 100 μm (A-E) and 20 μm (F/G).

Supplementary Figure 6 Expression in the central nervous system and Anti-AAV Neutralizing Antibody (NAB) titers.

(A) Axial section of a mouse brain after unilateral cochlear injection with Anc80. (B) Predominant expression in the cerebellum, in particular Purkinje cells (white arrowheads). Scale bars 1 mm (A) and 300 μm (B). (C) Anti-AAV Neutralizing Antibody (NAB) titers in serum and Cerebrospinal Fluid (CSF) in uninjected (n=4 for CSF, n=2 for serum) and Anc80 RWM-injected animals (n=6 for CSF, n=2 for serum). Titers reflect the dilution of serum or CSF at which 50% inhibition of transduction was observed in the NAB assay. Due to sample volume limitations, the limit of sensitivity for serum NAB was 1/4 and 1/52.5 for CSF.

Supplementary Figure 7 Vestibular function following Anc80 cochlear transduction.

Mice were injected at P1 with Anc80.CMV.eGFP via the RWM and evaluated for expression and balance function on the rotarod device. (A) Expression of eGFP (green) in the vestibular tissue is detected via confocal microscopy with immunofluorescent staining for Myo7A (red). (B) Rotarod data revealed no difference between injected and uninjected controls. The mean time until the mice fell off the device +/- s.d. is plotted. N=3 animals, 5 trials each (injected) and 2 animals 5 trials each (control). Scale bar = 50 μm.

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Landegger, L., Pan, B., Askew, C. et al. A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear. Nat Biotechnol 35, 280–284 (2017).

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