Fig. 4: In vivo correction of the MPZL2 c.220 C > T mutation by dual AAV-ie-ABE8eWQ-SpRY:sgRNA3. | Nature Communications

Fig. 4: In vivo correction of the MPZL2 c.220 C > T mutation by dual AAV-ie-ABE8eWQ-SpRY:sgRNA3.

From: PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation

Fig. 4: In vivo correction of the MPZL2 c.220 C > T mutation by dual AAV-ie-ABE8eWQ-SpRY:sgRNA3.The alternative text for this image may have been generated using AI.

a Schematic representation of the dual AAV constructs utilizing split-intein for ABE delivery in vivo, resulting in intein-mediated assembly of complete ABE:sgRNA complexes. The dual plasmids were packaged into the AAV serotype AAV-ie. b Assessment of the editing efficiencies of the MPZL2 target adenine and other bystander adenines or cytosines using dual AAV vectors that encoded split-intein ABE8eWQ-SpRY and sgRNA3 (n = 2). c Experimental overview of the in vivo base editing. Dual AAV-ie was used to induce split intein assembly, with the N-terminal and C-terminal components each prepared at 5.0 × 1013 vg/mL. These were mixed at a 1:1 ratio, and 2000 nl was injected into the RWM of P2 hMPZL2Q74X/Q74X mice. BioRender was used to make the Figure. “Created in BioRender. 남, 배. (2025) https://BioRender.com/kz4zbsm”. The images of the syringe, cochlea, and postnatal pup used in the figure were created using BioRender. d In vivo efficiency of A ∙ T to G ∙ C editing at on-target sites (P = 0.001) and bystander effects in DNA extracted from the organ of Corti (n = 8, P < 0.0001). Statistical analyses were performed using a two-tailed unpaired Student’s t-test. In the box-and-whisker plot, the whiskers mark the minimum and maximum, the box includes the 25th to 75th percentiles, and the line within the box indicates the median of the data set. Significance levels are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. e In vivo potential off-target sites relative to the target site. The potential off-target sites were identified using Cas–OFFinder, including those with up to two mismatches and/or up to one DNA/RNA bulge. f RNA off-target test in vivo at 8 weeks after dual AAV-ie-ABE8eWQ-SpRY:sgRNA3 injection. RNA off-target test in the injected mice was performed, and no RNA off-target editing was detected in vivo. n = 2 (Treated) and n = 2 (Control) per group. Source data for all relevant panels are provided within the Source Data file.

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