In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.

Figure 1. CRISPR/Cas9-mediated genomic and transcript deletion of exon 23 through intramuscular AAV-CRISPR administration

(a) The Cas9 nuclease is targeted to introns 22 and 23 by two gRNAs. Simultaneous generation of double stranded breaks (DSBs) by Cas9 leads to excision of the region surrounding the mutated exon 23. The distal ends are repaired through non-homologous end joining (NHEJ). The reading frame of the dystrophin gene is recovered and protein expression is restored. (b) PCR across the genomic deletion region shows the smaller deletion PCR product in treated muscles. Sequencing of the deletion band shows perfect ligation of Cas9 target sites (+, AAV-injected muscles; −, contralateral muscles). (c) ddPCR of deletion products shows 2% genome editing efficiency (n=6, mean+s.e.m.). (d) RT-PCR across exons 22 and 24 of dystrophin cDNA shows a smaller band that does not include exon 23 in treated muscles. Sanger sequencing confirmed exon 23 deletion. (e) ddPCR of intact dystrophin transcripts and Δ23 transcripts shows 59% of transcripts do not have exon 23 (n=6, mean+s.e.m.). bGHpA, bovine growth hormone polyadenylation sequence; ITR, inverted terminal repeat; NLS, nuclear localization signal. Asterisk, significantly different from the sham group (p<0.05).

Figure 2. In vivo genome editing restores dystrophin protein expression

(a) Western blot for dystrophin shows recovery of dystrophin expression (+, AAV-injected muscle; −, contralateral muscle). Comparison to protein from wild-type (WT) mice indicates restored dystrophin is ~8% of normal levels (n=6, mean+s.e.m.). (b) Dystrophin immunofluorescence staining shows abundant (67%) dystrophin-positive fibers in Cas9/gRNA treated groups (scale bar = 100 μm, n=7, mean+s.e.m.). Asterisk, significantly different from the sham group (p<0.05).

Figure 3. CRISPR/Cas9 gene editing restores nNOS activity and improves muscle function

(a) Whole muscle transverse sections show abundant dystrophin expression throughout the tibialis anterior muscle. (b) Staining of serial sections shows recruitment and activity of nNOS in a pattern similar to dystrophin expression. (c) H&E staining shows no obvious adverse response to the AAV/Cas9 treatment. Additionally, there is reduction of regions of necrotic fibers. Scale bars = 600 μm in full-view images and 100 μm in high-power images. (d) Significant improvement in specific twitch force (Pt) and tetanic force (Po) as measured by an in situ contractility assay in Cas9/gRNA-treated muscles. Treated muscles also showed significantly better resistance to damage caused by repeated cycles of eccentric contraction (n=7, mean+s.e.m). Overall treatment effect by ANOVA (p<0.05). Asterisk, significantly different from the sham group (p<0.05).

Figure 4. Systemic delivery of CRISPR/Cas9 by intravenous injection restores dystrophin expression in adult mdx mouse cardiac muscle

(a) PCR across the deletion region in the genomic DNA from cardiac tissues shows the smaller deletion PCR product in all treated mice. (b) RT-PCR across exons 22 and 24 of dystrophin cDNA from cardiac tissue shows a smaller band that does not included exon 23 in treated mice. (c) Western blot for dystrophin in protein lysates from cardiac tissue shows recovery of dystrophin expression (+, AAV injected mice; −, saline injected controls). (d) Dystrophin immunofluorescence staining shows dystrophin recovery in cardiomyocytes. Scale bar = 100μm