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Figures
- Fig. 1.
Gene editing to engineer the 521ΔT Sgcg point mutation in mice. (A) Schematic of the human SGCG gene on the left and the 521ΔT mutation on the right. The 521ΔT mutation is the most common LGMD 2C mutation. The single thymine deletion causes a frameshift, the expression of a premature stop codon, and loss of γ-sarcoglycan protein expression. (B) Schematic of the genetic locus and composition of the murine Sgcg gene. Both the human and the mouse genes include eight exons, with exons 2-8 encoding protein. Each coding region is 876 bp with each individual exon having the same number of coding base pairs. Exon 6 is highlighted in red. (C) Both human and murine exon 6 are 73 bp in length, and each has a stretch of five thymines in the same location (highlighted in yellow). Individual base pair mismatches are shown in red. (D) Schematic depicting the CRISPR design strategy to introduce the 521ΔT mutation into the mouse genome. A 20 nt gRNA sequence (blue line) was used to direct Cas9 nuclease to the specific locus, defined by the presence of a -NGG protospacer adjacent motif (PAM, pink line). The nuclease created a double- strand break 3 bp upstream of the PAM (arrowhead). The addition of a 180 nt repair template promoted HDR and contained the single base pair deletion (gray lines depict homology arms).
- Fig. 2.
Reduction of Sgcg transcript and loss of γ-sarcoglycan protein expression in Sgcg 521ΔT muscle. (A) RT-PCR illustrating the presence of Sgcg (715 bp) and Sgcg 521ΔT (714 bp) transcripts. (B) Sequence chromatograms of the 5T region in the Sgcg and Sgcg 521ΔT transcripts illustrating the deletion of one thymine in exon 6 (red box) of the Sgcg 521ΔT transcript. (C) γ-Sarcoglycan protein (red) was readily detected in WT muscle, but not in 521ΔT muscle. Hoechst (blue) marked nuclei. (D) Immunoblot analysis revealed the presence of γ-sarcoglycan protein in WT muscle lysates, whereas γ-sarcoglycan was not detected in 521ΔT muscle lysates. Data are mean±s.e.m. *P<0.05 (t-test). Scale bars: 50 µm.
- Fig. 3.
Reduced mass and myofiber area in Sgcg 521ΔT mice. (A) Body mass was not significantly different among genotypes. (B) Gluteus/hamstring (GH) muscle mass normalized to tibia length was reduced in 521ΔT mice compared with controls. (C) The total mass from muscle groups normalized to tibia length was significantly reduced in 521ΔT mice compared with WT controls (quadriceps, gluteus/hamstring, triceps, diaphragm, heart, tibialis anterior muscle groups combined). (D) Average myofiber cross-sectional area (CSA) was significantly smaller in 521ΔT mice than in WT controls. (E) Percentage of myofibers with >1 internal nuclei was increased in 521ΔT muscle, with 4-month-old 521ΔT myofibers having the most internal nuclei. (F) Representative images of 521ΔT and WT muscle. Anti-dystrophin (green) outlined myofibers. Hoechst (blue) demonstrated nuclei. Data are mean±s.e.m. *P<0.05 (one-way ANOVA with Tukey; n≥5 mice per group). Scale bars: 50 µm.
- Fig. 4.
Marked dystrophic histopathology in Sgcg 521ΔT mice. Tibialis anterior muscle from 521ΔT mice displays hallmark signs of muscle disease including increased immune infiltrate, fibrosis, necrosis, calcification and internal myonuclei visualized through H&E and Masson's Trichrome staining. Older 521ΔT mice (4 months) have more severe muscle pathology than younger 521ΔT (2 months) mice. These features are absent in healthy WT muscle. Scale bars: 100 µm.
- Fig. 5.
Increased immune infiltrate and fiber type switching in Sgcg 521ΔT muscle. (A) Anti-F4/80 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of F4/80+ macrophages per field in 521ΔT muscle compared with WT controls. Hoechst (blue) labels nuclei. Arrows indicate F4/80+ macrophages. (B) Anti-Ly6 immunofluorescence staining (green) of gastrocnemius muscle from 4-month-old mice revealed an increase in the average number of Ly6+ monocytes/neutrophils in 521ΔT muscle per field. Hoechst (blue) labels nuclei. Arrows indicate Ly6+ monocytes/neutrophils. (C) Representative images and quantification of the fiber type composition of the gastrocnemius muscle from 4-month-old mice, evaluated through immunostaining against the different myosin isoforms. 521ΔT muscle had a significant reduction in the percentage of Type 2B myofibers corresponding with a trend towards an increased number of mixed myofibers (P=0.07). Type 2B fibers (red), Type 2A (green), Type 1 (blue), mixed (composed of Type 2B/X, 2X, 2A/X). Data are mean±s.e.m. *P<0.05 (t-test in A,B; two-way ANOVA with Bonferroni's correction in C; n=3 mice per group). Scale bars: 50 µm (A,B); 1 mm (C).
- Fig. 6.
Increased sarcolemmal membrane leak and fibrosis in Sgcg 521ΔT mice. (A) Mice were injected with Evans Blue dye. Gross imaging shows increased fibrosis (white; arrow) and increased dye uptake (blue streaks; arrowhead) in 521ΔT mice compared with WT controls. (B) Increased dye uptake was seen in 2-month-old 521ΔT muscle by spectrophotometric analysis compared with WT. This increase trended towards a reduction by 4 months of age in 521ΔT muscle (compared with 2-month-old 521ΔT muscle; P=0.07). Combined dye values from abdominal, quadriceps, gluteus/hamstring, triceps, diaphragm, heart and gastroc/soleus muscles. (C) Serum CK was elevated in 521ΔT mice compared with controls, with 4-month-old 521ΔT serum CK exceeding that of 2-month-old mutant animals. (D) Fibrosis was measured as HOP content. HOP content was elevated in 521ΔT muscle, with more seen in 4-month-old 521ΔT muscle than at 2 months of age. Combined values from abdominal, gastroc/soleus, diaphragm and triceps. Data are mean±s.e.m. *P<0.05 (one-way ANOVA with Tukey; n≥4 mice per group). Scale bars: 5 mm.
- Fig. 7.
Reduced muscle function in Sgcg 521ΔT mice. (A) Average forelimb grip strength was decreased in 521ΔT mice. (B) Increased maximum tetanic force was decreased in 521ΔT mice at 4 months of age, measured through in situ force analysis of the tibialis anterior muscle. (C) Specific force was also decreased in 521ΔT muscle compared with WT control (tibialis anterior, 4-month-old mice). (D) Respiratory muscle activity was also impaired. Enhanced pause (Penh) was elevated, consistent with abnormal breathing in 521ΔT mice at 4 months, compared with WT controls. Data are mean±s.e.m. *P<0.05 (one-way ANOVA with Tukey in A; t-test in B,C,D; n≥5 mice per group).
- Fig. 8.
In vivo exon skipping in Sgcg 521ΔT muscle via IM injection of antisense oligonucleotides. (A) Schematic showing the correction of the 521ΔT frameshift mutation (red triangle) with a multi-AON exon-skipping strategy targeting exons 4, 5, 6 and 7 (yellow boxes). This strategy corrects the reading frame by generating a transcript encoding Mini-Gamma, comprising exons 2, 3 and 8. The arrows depict the location of the PCR primers, and the expected amplicon size is designated. (B) Design and results from in vivo study 1, in which analysis was conducted 3 days after IM injections of AONs into the tibialis anterior (TA) and gastrocnemius/soleus (Gas) muscle. Muscles were injected with either the four-AON cocktail or vehicle control (PBS; ctrl). Gel electrophoresis of RT-PCR products from treated muscles demonstrated the 521ΔT transcript in PBS-treated muscle (black arrow), as well as a dose-dependent generation of the predicted transcript encoding Mini-Gamma in muscle treated with the Mini-Gamma vivo-PMO cocktail at a low and mid dose (red arrow). (C) Similar treatment of two additional mice for 7 days demonstrated similar results but suggested the need for higher AON dosing. (D) Design and results from in vivo study 2. RT-PCR results indicate robust generation of the transcript encoding Mini-Gamma after two injections, two weeks apart at both the mid and high doses (red arrow). A concomitant reduction in the unskipped 521ΔT was also seen with both these exposures (black arrow). Mini-Gamma expression was evident 4 weeks after a single mid- or high-dose injection. However, expression was diminished compared with the two-injection treatment, and there were more intermediate products. (E) Gel extraction and subsequent Sanger sequencing confirmed that this product was in-frame Mini-Gamma.
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