Micro-dystrophin Gene Therapy Partially Enhances Exercise Capacity in Older Adult mdx Mice

Abstract

Micro-dystrophin (μDys) gene therapeutics can improve striated muscle structure and function in different animal models of Duchenne muscular dystrophy. Most studies, however, used young mdx mice that lack a pronounced dystrophic phenotype, short treatment periods, and limited muscle function tests. We, therefore, determined the relative efficacy of two previously described μDys gene therapeutics (rAAV6:μDysH3 and rAAV6:μDys5) in 6-month-old mdx mice using a 6-month treatment regimen and forced exercise. Forelimb and hindlimb grip strength, metabolic rate (VO₂ max), running efficiency (energy expenditure), and serum creatine kinase levels similarly improved in mdx mice treated with either vector. Both vectors produced nearly identical dose-responses in all assays. They also partially prevented the degenerative effects of repeated high-intensity exercise on muscle histology, although none of the metrics examined was restored to normal wild-type levels. Moreover, neither vector had any consistent effect on respiration while exercising. These data together suggest that, although μDys gene therapy can improve isolated and systemic muscle function, it may be only partially effective when dystrophinopathies are advanced or when muscle structure is significantly challenged, as with high-intensity exercise. This further suggests that restoring muscle function to near-normal levels will likely require ancillary or combinatorial treatments capable of enhancing muscle strength.

Keywords: Duchenne muscular dystrophy; exercise; gene therapy; mdx; microdystrophin.

Figure 1

Micro-dystrophin and Experimental Designs (A) The wild-type dystrophin protein is composed of three general domains: the amino terminus with two calponin homology (CH) domains, the central rod domain composed of spectrin-like repeats (numbered), a cysteine-rich (CR) domain, and a carboxy terminus composed of two coiled-coil repeats. Other more specific domains are aligned to these and include binding domains for actin (ABD); laminin (LBD); intermediate filaments (synemin and plectin); proteins in the DGC complex (β-dystroglycan, syntrophin, and dystrobevin); neuronal nitric oxide synthase (nNOS), which binds to repeats 16 and 17 via α-syntrophin; and four interspersed hinge domains (H1–H4). Horizontal lines indicate relative positions of labeled domains. (B) Structural components of different micro-dystrophins correspond to those labeled in (A). Also indicated are the mass of each protein and the corporate developer. (C) Treatment timeline of experiments. All mice were 6 months old when starting experiments. Forelimb and hindlimb grip strength measures were collected monthly on all mice, for a total of 7 measures over 6 months. Forced treadmill running was used to assess systemic muscle function and to exacerbate the dystrophic phenotype during the last month. This included initial and final VO2 max tests and 6 intervening training sessions (T1–T6) over 4 weeks (W1–W4). Mice were terminated 24 h following the last VO2 max test. Procedures are listed above the timeline, and ages of mice are listed below.

Figure 2

Changes in Forelimb and Hindlimb Grip Strength over Time Mice were injected with 0, 5 × 10¹¹ (low dose, -L), or 5 × 10¹² (high dose, -H) vg of rAAV6:μDys5 (μDys5) or rAAV6:μDysH3 (μDysH3), and grip strength was measured monthly as in Figure 1. The key in (A) applies to the whole figure and each point represents mean ± SEM (n = 6, p ≤ 0.05). (A–D) Absolute forelimb (A), absolute hindlimb (B), normalized forelimb (C), and normalized hindlimb (by body weight) grip strength of wild-type (WT, injected with 0 vg) C57BL/10 and mdx mice injected with all doses (mdx = 0 vg). (E and F) Percent change in absolute forelimb (E) and hindlimb (F) grip strength from the initial measurements (time 0). The only difference between vectors occurred in (B) (μDysH3-H > μDys5-H) at 10 and 11 months. Otherwise, significant differences between dose groups are indicated by letters as follows: a, WT differs from all other groups; b, WT differs from all others, and low doses differ from high and mdx; c, WT differs from others, and both doses differ from mdx; d, WT differs from low doses; e, WT differs from low doses and mdx, while both doses differ from mdx; f, mdx differs from all others; g, WT and high doses differ from others, and low doses differ from mdx; h, the dose groups, WT, and mdx all differ from each other; i, high and low doses both differ from all others; j, high doses and mdx differ from others; k, no differences; l, high doses differ from others.

Figure 3

Metrics of Exercise Capacity VO₂ max tests were performed on wild-type (WT) and mdx mice, 5 (initial) and 6 (final) months after injecting with 0, 5 × 10¹¹ (low dose, -L), or 5 × 10¹² (high dose, -H) vg of rAAV6:μDys5 (μDys5) or rAAV6:μDysH3 (μDysH3) (see Figure 1). WT and mdx mice were injected with 0 vg, the key in (A) applies to the whole figure, and each histogram bar represents mean ± SEM (n = 4–6). (A and B) VO₂ max (A) and the change from initial to final (B). (C and D) Distance traveled before mice fatigued (C) and the change from initial to final (D). (E and F) Indirect calorimetry was used to calculate the total energy expended (E) and the change from initial to final (F). (G and H) The energy consumption rate was then calculated by normalizing energy expenditure values to total distance traveled (G) and the change in the energy consumption rate (H). Significant differences between any two groups (p ≤ 0.05) are indicated by different letters, whereas shared letters indicate no difference.

Figure 4

Metrics of Exercise Training Wild-type (WT) and mdx mice were injected with 0, 5 × 10¹¹ (low dose, -L), or 5 × 10¹² (high dose, -H) vg of rAAV6:μDys5 (μDys5) or rAAV6:μDysH3 (μDysH3). WT and “mdx” mice were injected with 0 vg, the key in (A) applies to the whole figure, and each histogram bar represents mean ± SEM (n = 6). Between the two VO₂ max tests, mice trained on respiratory treadmills twice a week for 6 sessions, with sub-maximal effort, at the same speed and time while respiratory gas exchange was continuously monitored. (A) Electrical shocks are used to encourage mice to remain on the treadmill and their quantification is a metric of motivation. Differences between groups are as follows: a, none; b, WT < high doses < low dose = mdx. (B and C) The fluctuating respiratory pattern common to dystrophic mice was quantified by calculating the mean VO₂ coefficient of variation for (B) the entire training period or (C) each session. (D and E) This fluctuating pattern often results in lower (D) minimum VO₂ and higher (E) maximum respiratory exchange ratio (RER). (F) Total energy expended during each session was calculated using indirect calorimetry. In (B)–(F), significant differences (p ≤ 0.05) between any two groups are indicated by different letters; shared letters indicate no difference.

Figure 5

Histopathological Metrics (A–D) Fiber and nuclei counts per field in different skeletal muscles (TA, tibialis anterior; GAST, gastrocnemius; QUAD quadriceps; and DIA, diaphragm) of wild-type (WT) and mdx mice injected with 0, 5 × 10¹¹ (low dose, -L), or 5 × 10¹² (high dose, -H) vg of rAAV6:μDys5 (μDys5) or rAAV6:μDysH3 (μDysH3). (A) Total nuclei. (B) Fibers with central nuclei. (C) Central nuclei. (D) Fiber count. WT and “mdx” mice were injected with 0 vg, the key in (A) applies to the whole figure, and each histogram bar represents mean ± SEM (n = 4–6). (E–G) Fiber cross-sectional area (CSA) of (E) TA, (F) GAST, and (G) QUAD muscles parsed by fiber size groups: <3,000 μm, small; 3,001–7,000 μm, medium; and >7,000 μm, large. (H) Mice were sacrificed, and serum was collected 24 h after the final VO₂ max test to quantify serum CK levels. Significant differences (p ≤ 0.05) between any two groups are indicated by different letters, whereas shared letters indicate no difference.