AAV-mediated base editing restores cochlear gap junction in GJB2 dominant-negative mutation-associated syndromic hearing loss model
Takao Ukaji, Daisuke Arai, Harumi Tsutsumi, Ryoya Nakagawa, Fumihiko Matsumoto, Katsuhisa Ikeda, Osamu Nureki, Kazusaku Kamiya
Takao Ukaji, Daisuke Arai, Harumi Tsutsumi, Ryoya Nakagawa, Fumihiko Matsumoto, Katsuhisa Ikeda, Osamu Nureki, Kazusaku Kamiya
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Research Article Genetics Therapeutics

AAV-mediated base editing restores cochlear gap junction in GJB2 dominant-negative mutation-associated syndromic hearing loss model

Abstract

Mutations in the gap junction β2 (GJB2) gene, which encodes connexin 26, are the leading cause of genetic deafness. These mutations are characterized by the degeneration and fragmentation of gap junctions and gap junction plaques (GJPs) composed of connexin 26. Dominant-negative mutations of GJB2, such as R75W, cause syndromic hearing loss and palmoplantar keratoderma. We previously reported that the R75W mutation, a single-base substitution where C is replaced by T, causes fragmentation of GJPs. Therefore, an adenine base editor (ABE), which enables A-to-G base conversions, can potentially be useful for the treatment of this genetic disease. Here, we report that an all-in-one adeno-associated virus (AAV) vector, which includes a compact ABE (SaCas9-NNG-ABE8e) with broad targeting range, and a sgRNA targeting the R75W mutation in GJB2 corrected this pathogenic mutation and facilitated the recovery of the gap junction intercellular communication network of GJPs. In a transgenic mouse model with the GJB2 R75W mutation, AAV-mediated base editing also restored the fragmented GJPs to orderly outlines in cochlear supporting cells. Our findings suggest that an ABE-based base-editing strategy could be an optimal treatment for the dominant form of GJB2-related hearing loss, GJB2-related skin diseases, and other deafness-related mutations, especially single-base substitutions.

Authors

Takao Ukaji, Daisuke Arai, Harumi Tsutsumi, Ryoya Nakagawa, Fumihiko Matsumoto, Katsuhisa Ikeda, Osamu Nureki, Kazusaku Kamiya

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Figure 7

All-in-one AAV-mediated base editing in transgenic mice with GJB2 R75W.

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All-in-one AAV-mediated base editing in transgenic mice with GJB2 R75W. ...
(A) Auditory brain stem response (ABR) waveforms of GJB2 R75W–transgenic mice (CX26R75W+) and their nontransgenic littermates (non-TG) at 30 weeks of age (n = 3). The number to the left side of each wave indicates sound pressure level (SPL) expressed in decibels (dB). (B) Strategy for assessing the efficiency of AAV-mediated base editing of CX26R75W+ mice. (C) Representative images of cochlea after all-in-one AAV-mediated base editing. Cochlea organotypic cultures were prepared from P0 CX26R75W+ mice and non-TG littermates (n = 3). Cochlea samples were infected with the all-in-one AAV vector and maintained in DMEM for 72 hours. Samples were then fixed, permeabilized, and incubated with anti-CX26 antibody, followed by incubation with Cy3-conjugated anti-rabbit IgG, and then counterstained with DAPI. Arrowheads indicate distinct GJPs after AAV-mediated base editing. Scale bar: 10 μm. (D) Quantification of data presented in C. Length of GJPs from each group of inner sulcus cells. Box-and-whisker plot shows median, interquartile range, and minimum and maximum values; isolated dots beyond the whiskers correspond to outliers defined as a value that is smaller than the lower quartile −1.5 × the interquartile range or larger than the upper quartile +1.5 times the interquartile range. n = at least 42 per group. Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons test. **P < 0.01, ****P < 0.0001. (E) The all-in-one AAV vector was injected into the cochlear perilymph of left ear via the semicircular canal of adult CX26R75W+ mice (n = 3). After administration of the AAV vector, the length of gap junction in cochlea tissue was evaluated. (F) Representative images of cochlea after AAV-mediated base editing. Cochlea samples were fixed, permeabilized, and incubated with anti-CX26 antibody, followed by incubation with Cy3-conjugated anti-rabbit IgG, and then counterstained with DAPI. Arrowheads indicate distinct GJPs after AAV-mediated base editing. Scale bar: 200 μm (leftmost images), 20 μm (images in other columns). (G) Quantification of data presented in F. Length of GJPs of inner sulcus cells was evaluated. The explanation of the box-and-whisker plot is the same as in D. n = 40 per group. Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons test. ****P < 0.0001.