Long-term maintenance of dystrophin expression and resistance to injury of skeletal muscle in gene edited DMD mice

Abstract

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, we subjected ΔEx44 DMD mice to systemic delivery of AAV9-expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. We found that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection, we observed a loss of gene edited dystrophin expression. Analysis of on- and off-target editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.

Keywords: AAV; CRISPR/Cas9; Duchenne muscular dystrophy; exon reframing; gene editing.

Graphical abstract

Figure 1

CRISPR/Cas9 genome editing prevents skeletal muscle injury induced by downhill running in ΔEx44 mice (A) ΔEx44 mice were injected intraperitoneally (IP) with ssAAV-SpCas9 and scAAV-sgRNA, each at 8 × 10¹³ vg/kg on postnatal day 4 (P4) (corrected ΔEx44). At 4 weeks of age, mice were subjected to sedentary or downhill running conditions for 4 weeks. (B) H&E staining of the quadriceps muscle from WT, ΔEx44, and corrected ΔEx44 mice that were either sedentary or run downhill. Scale bar, 100 μm. (C) Quantification of centralized nuclei in sedentary or downhill run WT, ΔEx44, and corrected ΔEx44 quadriceps muscle. Data are shown as mean ± SEM. Unpaired Student’s t test was performed. ∗∗∗p < 0.001 (n = 6). (D) Quantification of necrotic area in sedentary or downhill run WT, ΔEx44, and corrected ΔEx44 quadriceps muscle. Data are shown as mean ± SEM. Unpaired Student’s t test was performed. ∗∗∗∗p < 0.0001 (n = 6). (E) Hindlimb grip strengths of WT, ΔEx44, and corrected ΔEx44 mice, measured following 4 weeks of downhill running. Hindlimb grip strength was normalized to body weight. Data are shown as mean ± SEM. A one-way ANOVA and a post hoc Tukey’s multiple comparison test were performed. ∗∗∗p < 0.001 (n ≥ 5). (F) Specific force generated by the extensor digitorum longus (EDL) and soleus muscles of WT, ΔEx44, and corrected ΔEx44 mice following 4 weeks of downhill running. Data are shown as mean ± SEM. A one-way ANOVA and a post hoc Tukey’s multiple comparison test were performed. ∗p < 0.05, ∗∗p < 0.005 (n ≥ 5).

Figure 2

Expression of dystrophin is retained in corrected ΔEx44 mice following chronic injury (A) Immunohistochemistry shows retention of dystrophin-positive fibers in the diaphragm, tibialis anterior, and quadriceps muscles of corrected ΔEx44 mice following 4 weeks of downhill running. Dystrophin is shown in green. Scale bar, 100 μm. (B) Western blot analysis shows retention of dystrophin protein in the diaphragm, tibialis anterior, and quadriceps muscles of corrected ΔEx44 mice following 4 weeks of downhill running. Vinculin was loading control. (C) Quantification of dystrophin protein in diaphragm, tibialis anterior, and quadriceps muscles. Dystrophin protein levels were first normalized to vinculin loading control and then to WT sedentary controls. Data are shown as mean ± SEM. Unpaired Student’s t test was performed (n = 3).

Figure 3

Transcriptional homeostasis is maintained in quadriceps muscle of corrected ΔEx44 mice following downhill running (A) Heatmap of differentially expressed genes in the quadriceps muscles of 4-week-old WT, ΔEx44, and corrected ΔEx44 mice. Gene expression is represented as a transformed Z score (n = 3). (B) Selected GO terms for up- and downregulated genes in corrected ΔEx44 quadriceps muscle relative to ΔEx44 quadriceps muscle. (C) Volcano plot of differentially expressed genes between corrected ΔEx44 and WT quadriceps muscles. (D) Heatmap of differentially expressed genes in the quadriceps of 8-week-old WT, ΔEx44, and corrected ΔEx44 mice following 4 weeks of downhill running. Gene expression is represented as a transformed Z score (n = 2). (E) Selected GO terms for up- and downregulated genes in downhill-run-corrected ΔEx44 quadriceps muscle relative to ΔEx44 quadriceps muscle. (F) Volcano plot of differentially expressed genes between downhill-run-corrected ΔEx44 and WT quadriceps muscles.

Figure 4

BaCl₂-induced acute injury results in the loss of gene edited dystrophin (A) ΔEx44 mice were injected intraperitoneally (IP) with ssAAV-SpCas9 and scAAV-sgRNA, each at 8 × 10¹³ vg/kg on postnatal day 4 (P4) (corrected ΔEx44). At 3 months of age, the corrected ΔEx44 tibialis anterior muscle was injected with BaCl₂. Two months after BaCl₂ injury, tissues were harvested for analysis. (B) TIDE analysis of Dmd exon 45 in saline-injected or BaCl₂ treated WT, ΔEx44, and corrected ΔEx44 tibialis anterior muscle. (C) Immunohistochemistry shows the loss of dystrophin-positive fibers in corrected ΔEx44 tibialis anterior muscle following BaCl₂ injury. Dystrophin is shown in green. Scale bar, 250 μm (n = 4). (D) Western blot analysis shows a loss of dystrophin protein in the tibialis anterior muscle of corrected ΔEx44 mice following BaCl₂ injury. Vinculin was loading control. (E) Quantification of dystrophin protein in saline or BaCl₂ injected, corrected ΔEx44 TA muscle. Dystrophin protein levels were first normalized to vinculin loading control and then to corresponding WT controls. Data are shown as mean ± SEM. Unpaired Student’s t test was performed. ∗∗∗∗p < 0.0001 (n = 3).

Figure 5

Gene edited dystrophin is retained in 18-month-old corrected ΔEx44 mice (A) Immunohistochemistry shows dystrophin-positive muscle fibers in the diaphragm and tibialis anterior of 18-month-old corrected ΔEx44 mice. Dystrophin is shown in green. Scale bar, 100 μm. (B) Western blot analysis shows sustained dystrophin and SpCas9 expression in the diaphragm, tibialis anterior, and quadriceps muscles of 18-month-old corrected ΔEx44 mice. Vinculin was loading control. (C) Quantification of dystrophin protein in the diaphragm, tibialis anterior, and quadriceps muscles. Dystrophin protein levels were first normalized to vinculin loading control and then to WT controls. Data are shown as mean ± SEM. (D) H&E staining of the diaphragm, tibialis anterior, and quadriceps muscles from 18-month-old WT, ΔEx44, and corrected ΔEx44 mice. Scale bar, 100 μm. (E) INDEL analysis of the on-target and top 10 off-target sites in 18-month-old corrected ΔEx44 tibialis anterior muscle. INDELs have been normalized to a WT control (n = 3).

Figure 6

Transcriptional homeostasis is maintained in 18-month-old corrected ΔEx44 quadriceps muscle (A) Heatmap of differentially expressed genes in 18-month-old WT, ΔEx44, and corrected ΔEx44 quadriceps muscles. Gene expression is represented as a transformed Z score (n = 3). (B) Selected GO terms for up- and downregulated genes in 18-month-old corrected ΔEx44 quadriceps muscle relative to ΔEx44 quadriceps muscle. (C) Volcano plot of differentially expressed genes between 18-month-old corrected ΔEx44 and WT quadriceps muscles.