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 5

Restoration of GJPs by AAV-mediated base editing.

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Restoration of GJPs by AAV-mediated base editing. (A) Schematic diagram ...
(A) Schematic diagram of the genome of the all-in-one AAV vector encoding SaABE_V106W#1. (B) Strategy for assessing the efficiency of AAV-mediated base editing at GJB2 R75W. (C) Representative images of GJPs after all-in-one AAV-mediated base editing. HeLa/CX26 R75W cells were infected with the all-in-one AAV vector. After transfection for 72 hours, cells were fixed, permeabilized, and incubated with anti-CX26 antibody, followed by incubation with Alexa Flour 488–conjugated anti-rabbit IgG and Alexa Flour 555–conjugated cholera toxin subunit B, and then counterstained with DAPI. Scale bar: 10 μm. (D–G) Quantitative length (D), area (E), form factor (F), and LAF (G) data after all-in-one AAV-mediated base editing (MOI of 1 × 105 or 5 × 105 vg/cell). Box-and-whisker plots show 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. Each value was normalized to the value obtained for HeLa/CX26 R75W cells (untreated). n = 44 analyzed per group. Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons test. ****P < 0.0001. (H) Efficiency of correction of the mutation GJB2 R75W in HeLa cells after infection with the all-in-one AAV vector. Data represent the mean ± SEM. n = 4. (I) Pie charts showing the composition of allelic variants by on-target or bystander editing after AAV-mediated base editing. (J) Pie charts showing the composition of allelic variants after AAV-mediated base editing. (K) Representative images of GJPs generated by bystander effect. Scale bar: 10 μm.