In vivo gene editing in dystrophic mouse muscle and muscle stem cells

Abstract

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

Figure 1. DYSTROPHIN expression in CRISPR-modified dystrophic satellite cells

(A) Detection of exon23 excision by genomic PCR in myotubes derived from satellite cells transfected with SpCas9 and Ai9 gRNAs (left lanes) or coupled Ai9-Dmd23 gRNAs (right lanes). Unedited genomic product, 1572bp; gene-edited product (red asterisk), 1189bp. M, molecular weight marker. (B) RT-PCR detection of exon23-deleted mRNA. Unedited RT-PCR product: 738bp; exon23-deleted product (blue asterisk): 525bp. (C) Western blot detecting DYSTROPHIN in myotubes derived from gene-edited satellite cells. A.U.: Arbitrary Unit, normalized to GAPDH (loading control). DYS: DYSTROPHIN, WT: wild type (D) DYSTROPHIN immunofluorescence in mdx muscles transplanted with satellite cells transfected ex vivo with SpCas9 + Ai9 gRNAs (top) or SpCas9 + Ai9-Dmd23 coupled gRNAs (bottom). For merge: Green: DYSTROPHIN; Red: tdTomato; Blue: DAPI (nuclei). Scale bar: 200 μm. See also Fig. S1I.

Figure 2. AAV-CRISPR enables in vivo excision of Dmd exon23 and restores DYSTROPHIN expression in adult dystrophic muscle

(A,B) Detection of exon23 excision in TA muscles from mdx;Ai9 mice injected intramuscularly with AAV-Ai9 CRISPR (left lanes) or AAV-Dmd CRISPR (right lanes) by genomic PCR (A; unedited product 1012bp; exon-excised product 470bp) and RT-PCR (B). Asterisks mark gene-edited bands. M, molecular weight marker. (C) Western blot detecting DYSTROPHIN in muscles injected with AAV-Ai9 CRISPR (left) or AAV-Dmd CRISPR (right), with relative signal intensity determined by densitometry at bottom. A.U.: Arbitrary Unit, normalized to GAPDH. (D) Representative immunofluorescence images for DYSTROPHIN (green) and DAPI (blue) in mdx;Ai9 muscles injected with AAV-Ai9 (left) or AAV-Dmd (right) CRISPR. Scale bar: 500 μm. (E,F) Muscle specific force (E) and decrease in force after eccentric damage (F) for wild type mice injected with vehicle (n=9), mdx;Ai9 mice injected with AAV-Dmd CRISPR in the right TA and vehicle in the left TA (n=12), mdx;Ai9 mice injected with AAV-Ai9 CRISPR in the right TA and vehicle in the left TA (n=12). *P<0.05, **P<0.01, n.s., not significant, One-Way ANOVA with Newman-Keuls multiple comparisons test.

Figure 3. Systemic dissemination of AAV-CRISPR targets Dmd exon23 and restores DYSTROPHIN in dystrophic cardiac and skeletal muscles

(A) Exon23-deleted transcripts in muscles quantified by Taqman. Data plotted for individual mice (n=7 receiving AAV-Dmd CRISPR (blue) and n=3 receiving AAV-Ai9 CRISPR (red)) and overlaid with mean ± SEM. (B) Western blots detecting DYSTROPHIN in the indicated muscles of mdx;Ai9 mice receiving systemic AAV-CRISPR. Right lanes correspond to muscles from 7 different mice injected intraperitoneally with AAV-Dmd CRISPR. Relative signal intensity, determined by densitometry, presented as A.U.: Arbitrary Unit normalized to GAPDH. (C) Representative immunofluorescence staining for DYSTROPHIN (green) in mdx;Ai9 mice injected with AAV-Ai9 (top) or AAV-Dmd (bottom) CRISPR. Blue: DAPI (nuclei). Scale bar: 200 μm.

Figure 4. Satellite cells in dystrophic muscles are transduced and targeted with systemically disseminated AAV-CRISPR

(A) Experimental design. (B) Percent of ZsGreen+ satellite cells expressing tdTomato+ after intraperitoneal injection of Pax7-ZsGreen^+/−;mdx;Ai9 mice. Individual data points overlaid with mean ± SD; vehicle (n=3), AAV-Cre (n=4) AAV Ai9 CRISPR (n=5). (C) Representative immunofluorescence of myotubes differentiated from FACSorted satellite cells from mice injected intraperitoneally with vehicle, AAV-Cre, or AAV-Ai9 CRISPR. Green: Myosin heavy chain (MHC); Red: tdTomato. Blue: DAPI (nuclei) Scale bar: 200 μm. (D) Exon23-deleted DMD mRNA in satellite-cell derived myotubes from mice previously injected intraperitoneally with AAV-Dmd CRISPR (right lanes), compared to control AAV-Ai9 CRISPR (left lanes).