Highly potent MyoAAV4A vector reverses GSD III pathology in aged mice and enables long-term muscle disease correction in young adult mice

Figures

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  Figure 1. Biodistribution of AAV vector genomes and pullulanase expression in major affected tissues of aged male and female GSD IIIa mice 3 months after MyoAAV4A-Dual-Pull administration (A) The diagram shows the construct of AAV vector containing the 2.2 kb codon-optimized pullulanase cDNA linked to an HA-tag at the 3′ end under the control of the LSP-CB dual promoter. ITR, inverted terminal repeats; LSP, liver-specific promoter; CB, CMV enhancer/chicken β-actin promoter. (B) Thirteen-month-old GSD IIIa mice were injected with MyoAAV4A-Dual-Pull vector at a dose of 5 × 1012 vg/kg. After 3 months, behavioral tests were performed and tissues were collected. (C) AAV genome copy numbers in the liver, heart, and quadriceps were determined by quantitative PCR. Data are shown as mean ± SD; n = 7 for each MyoAAV4A-treated group. (D) Pullulanase activity was measured in the liver, heart and quadriceps. Data are shown as mean ± SD; n = 5 for untreated (UT) groups, n = 8 for MyoAAV4A-treated groups; Unpaired t test or unpaired Mann-Whitney U test; ∗p < 0.05, ∗∗p < 0.01.

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  Figure 2. MyoAAV4A-Dual-Pull treatment reduced glycogen storage in major affected tissues of aged GSD IIIa mice 3 months after vector administration (A) Glycogen clearance was assessed by measuring the glycogen content in the liver, heart, and quadriceps. Data are shown as the mean ± SD; n = 5 for UT and WT groups, n = 8 for MyoAAV4A-treated groups; one-way ANOVA or Kruskal-Wallis test; ∗p < 0.05, ∗∗p < 0.01. (B) Periodic acid-Schiff (PAS) staining of tissue sections was performed to detect glycogen accumulation in the liver, heart and skeletal muscles. Glycogen is stained in purple. Two mice in each group were analyzed, and representative images for male mice are shown. Scale bars: 100 μm.

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  Figure 3. MyoAAV4A-Dual-Pull treatment prevented liver fibrosis progression in aged male and female GSD IIIa mice 3 months after vector administration (A) Picrosirius red staining of liver sections was performed to detect fibrotic tissues (red). Two mice were examined in each group, and representative images were shown. Scale bars: 100 μm. (B) Relative mRNA expression levels of α-SMA, collagen I, and collagen III were measured in the livers of UT, MyoAAV4A-treated, and WT mice. Data are shown as mean ± SD; n = 5 for UT and WT, n = 8 for MyoAAV4A-treated groups; one-way ANOVA or Kruskal-Wallis test; ∗p < 0.05, ∗∗p < 0.01.

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  Figure 4. MyoAAV4A-Dual-Pull treatment corrected GSD IIIa symptoms and improved muscle function in aged male and female GSD IIIa mice 3 months after vector administration (A and B) Plasma alanine aminotransferase (ALT) and creatine kinase (CK) activities were measured to assess liver and muscle damage, respectively. (C) Urinary concentrations of GSD III biomarker Glc4 (glucose tetrasaccharide) were measured by mass spectrometry and normalized to urinary creatinine levels. For (A)–(C), data are shown as mean ± SD; n = 5 for UT and WT groups, n = 8 for MyoAAV4A-treated groups; one-way ANOVA or Kruskal-Wallis test; ∗p < 0.05, ∗∗p < 0.01. (D) Muscle strength was evaluated by grip strength test and is presented as the maximum of three independent tests (in grams). (E) Wire hang test was performed to evaluate limb muscle strength. Latency to fall was recorded, and the maximum hanging time was used for comparison. (F) Treadmill test was performed to assess exercise intolerance. The graph represents the maximum running distance. For (D)–(F), data are shown as mean ± SD; n = 9 (male) or 10 (female) for UT, n = 8 for MyoAAV4A-treated groups, n = 8 (male) or 7 (female) for WT; one-way ANOVA; ∗p <…

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  Figure 5. Pullulanase activity and glycogen content in major affected tissues of young adult male GSD IIIa mice at 5.5 and 9 months after vector administration (A) Three-month-old GSD IIIa mice were injected with MyoAAV4A-Dual-Pull vector at a dose of 5 × 1012 vg/kg. Behavioral tests were performed and tissues were collected after 5.5 and 9 months. (B) Pullulanase activities were measured in the liver, heart, and quadriceps. Data are shown as mean ± SD; n = 5 for UT groups, n = 4 (5.5 months) or 6 (9 months) for MyoAAV4A-treated groups; Unpaired t test; ∗p < 0.05, ∗∗p < 0.01. (C) Glycogen contents were measured in the liver, heart, and quadriceps. Data are shown as mean ± SD; n = 5 for UT and WT groups, n = 4 (5.5 months) or 6 (9 months) for MyoAAV4A-treated groups; one-way ANOVA or Kruskal-Wallis test; ∗p < 0.05, ∗∗p < 0.01.

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  Figure 6. MyoAAV4A-Dual-Pull treatment improved muscle function in young adult male GSD IIIa mice 5.5 and 9 months after vector administration (A) Plasma creatine kinase (CK) activity was measured to assess the muscle damage. Data are shown as the mean ± SD; n = 5 for UT and WT groups, n = 4 (5.5 months) or 6 (9 months) for MyoAAV4A-treated groups; One-way ANOVA or Kruskal-Wallis test; ∗p < 0.05, ∗∗p < 0.01. (B) Treadmill test was used to evaluate the exercise intolerance. The graph represents the maximum running distance. Data are shown as mean ± SD; n = 10 (5.5 months) or 5 (9 months) for UT groups, n = 11 (5.5 months) or 6 (9 months) for MyoAAV4A-treated groups, n = 7 for WT groups; One-way ANOVA or Kruskal-Wallis test; ∗p < 0.05, ∗∗p < 0.01.

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  Figure 7. MyoAAV4A-Dual-Pull treatment reduced liver fibrosis in young adult male and female GSD IIIa mice 9 months after vector administration (A) Picrosirius red staining of liver sections was performed to detect fibrotic tissues (red). Two mice were examined in each group, and representative images were shown. Scale bars: 100 μm. (B) Relative mRNA expression levels of three fibrotic biomarkers, α-SMA, collagen I, and collagen III, were examined in the livers of UT, MyoAAV4A-Dual-Pull-treated, and WT mice. Data are shown as mean ± SD; n = 5 for UT and WT, n = 6 for AAV-treated groups; one-way ANOVA or Kruskal-Wallis test; ∗p < 0.05, ∗∗p < 0.01.