Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing

Abstract

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the lack of dystrophin, which maintains muscle membrane integrity. We used an adenine base editor (ABE) to modify splice donor sites of the dystrophin gene, causing skipping of a common DMD deletion mutation of exon 51 (∆Ex51) in cardiomyocytes derived from human induced pluripotent stem cells, restoring dystrophin expression. Prime editing was also capable of reframing the dystrophin open reading frame in these cardiomyocytes. Intramuscular injection of ∆Ex51 mice with adeno-associated virus serotype-9 encoding ABE components as a split-intein trans-splicing system allowed gene editing and disease correction in vivo. Our findings demonstrate the effectiveness of nucleotide editing for the correction of diverse DMD mutations with minimal modification of the genome, although improved delivery methods will be required before these strategies can be used to sufficiently edit the genome in patients with DMD.

Fig. 1. Strategy for in vivo exon skipping mediated by adenine base editing in the ∆Ex51 mouse model.

(A) Schematic showing exon skipping and exon reframing strategies to restore the correct ORF of the Dmd transcript. Shape and color of boxes of Dmd exons indicate reading frame. Deletion of exon 51 (∆Ex51) in the Dmd gene generates a premature stop codon in exon 52 (red). Restoration of the correct ORF can be obtained by skipping of exon 50 or 52 (gray) or reframing by a precise insertion of 3n + 2 nt or deletion of 3n − 1 nt in exon 50 or 52 (green). (B) Illustration of the mEx50 sgRNA-4 binding position in the region of the SDS (green) of mouse Dmd exon 50. Sequence shows sgRNA (blue) and PAM (red). Adenines in the editable window of ABEmax-SpCas9-NG are numbered, starting from the PAM. (C) Representative Sanger sequencing chromatogram of the genomic region of the exon 50 SDS in mouse N2a cells, after transfection with ABEmax-SpCas9-NG and mEx50 sgRNA-4. (D) Percentages of DNA editing in mouse N2a cells after transfection with ABEmax-SpCas9-NG and mEx50 sgRNA-4. On-target edit (A14) is colored green. Dots and bars represent different transfection experiments and are means ± SEM (n = 3).

Fig. 2. Exon skipping by AAV-mediated base editing in the ∆Ex51 mouse model.

(A) Schematic of the dual-AAV9 system for in vivo delivery of ABEmax-SpCas9-NG and two copies of mEx50 sgRNA-4. (B) Overview for the in vivo intramuscular (IM) injection of the dual-AAV9 system in the TA muscle of the left leg of P12 ∆Ex51 mice. Right leg was injected with saline as a control. (C) Percentages of DNA editing of the adenines from TA injected with the dual-AAV9 system. On-target adenine (A14) is colored green. Dots and bars represent biological replicates and are means ± SEM (n = 3). (D) Alignment of the top eight off-target sites in mouse DNA. The target adenine (A14) is colored green. (E) Percentages of DNA editing of A14 in the top eight off-target sites from TA injected with the dual-AAV9 system. Dots and bars represent biological replicates and are means ± SEM (n = 3). (F) RT-PCR analysis of RNA from the TA of wild-type (WT) and ∆Ex51 mice injected with the dual-AAV9 system or saline as control (Ctrl). (G) Sequence of the RT-PCR product of the lower band confirms splicing of exons 49 to 52.

Fig. 3. Dystrophin restoration following AAV-mediated base editing in the ∆Ex51 mouse model.

(A) Western blot analysis of dystrophin protein expression in TA muscles of WT and ∆Ex51 mice 3 weeks after intramuscular injection of saline as control (Ctrl) or the dual-AAV9 system for the expression of ABEmax-SpCas9-NG and mEx50 sgRNA-4. Vinculin is the loading control. (B) Quantification of dystrophin expression from Western blots after normalization to vinculin. Dots and bars represent biological replicates and are means ± SEM (n = 3). (C) Immunohistochemistry of dystrophin in TA muscles 3 weeks after intramuscular injection of the dual-AAV9 system. Indicated is the percentage (means ± SEM) of dystrophin-positive myofibers of TA muscles of ∆Ex51 mice receiving an intramuscular injection of the dual-AAV9 system (n = 3). Dystrophin is indicated in green. Scale bar, 100 μm. (D) H&E staining of TA muscles from WT, ∆Ex51, and corrected ∆Ex51 mice 3 weeks after intramuscular injection. Scale bar, 100 μm.

Fig. 4. Base editing–mediated exon skipping restores dystrophin expression in human ∆Ex51 iPSC–derived cardiomyocytes.

(A) Gene editing strategy to restore the in-frame ORF by exon skipping using base editing. (B) The hEx50 sgRNA-1 binding position in the region of the SDS of human DMD exon 50 (green). Sequence shows sgRNA (blue) and PAM (red). (C) Percentages of DNA editing of adenines in the editing window of ABEmax-SpCas9 with hEx50 sgRNA-1 following nucleofection in human ∆Ex51 iPSCs. On-target edit (A14) is colored green. Dots and bars represent results of different nucleofections and are means ± SEM (n = 3). (D) Representative Sanger sequencing chromatogram of the genomic region of the exon 50 SDS of human iPSCs following nucleofection with ABEmax-SpCas9 and hEx50 sgRNA-1. (E) RT-PCR analysis of RNA from single clones of healthy control (Ctrl), ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes after base editing. (F) Western blot analysis of dystrophin protein expression of Ctrl, ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes. Vinculin is the loading control. (G) Immunocytochemistry of dystrophin in Ctrl, ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes. Dystrophin is indicated in red. Cardiac troponin-I (TnI) is indicated in green. Nuclei are marked by DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bar, 50 μm.

Fig. 5. Prime editing–mediated exon reframing restores dystrophin expression in human ∆Ex51 iPSC–derived cardiomyocytes.

(A) Gene editing strategy to restore the in-frame ORF by exon reframing using prime editing. (B) Illustration of the pegRNA used in the following experiments (red) and the target DNA sequence (blue). PAM is indicated in orange and programmed insertion in green. (C) RT-PCR analysis of RNA from single clones of healthy control (Ctrl), ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes after prime editing with nick-1 or nick-2. (D) Sanger sequencing chromatograms of the RT-PCR product of RNA from ∆Ex51 iPSC–derived cardiomyocytes before and after prime editing. (E) Western blot analysis of dystrophin protein expression of Ctrl, ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes. Vinculin is the loading control. (F) Immunocytochemistry of dystrophin in Ctrl, ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes. Dystrophin is indicated in red. Cardiac troponin-I is indicated in green. Nuclei are marked by DAPI stain in blue. Scale bar, 50 μm. (G) Percentage of arrhythmic calcium traces of Ctrl, ∆Ex51, and corrected ∆Ex51 iPSC–derived cardiomyocytes. Dots and bars represent results of different biological replicates (n = 216 cells across three biological replicate experiments) and are means ± SEM (n = 3). *P < 0.05 and **P < 0.001 using unpaired two-tailed Student’s t test.