This is the first report describing the effects of long-term, systemic myostatin inhibition in a large-animal model of DMD. scAAV8 designed to express a secreted dnMSTAT peptide under the control of a liver-specific promoter was delivered to GRMD canines via simple IV infusion. AAV serotype 8 was used in this study because it was previously demonstrated to be capable of achieving efficient hepatic gene transfer in canines compared with several other serotypes (Bell et al.
). The dominant-negative construct that was used to inhibit myostatin in this study is a mutated form of myostatin found in hypermuscular Belgian Blue cattle; it contains an 11-bp deletion that causes a premature stop in the C-terminus, leaving only the inhibitory propeptide to be expressed (Kambadur et al.
). In addition, our peptide has been modified to contain a D76A mutation, which makes the inhibitory propeptide resistant to proteolysis (Wolfman et al.
). In effect, this construct is a protease-resistant propeptide that can inhibit endogenous myostatin signaling by preventing receptor binding (Lee, 2004
). We followed GRMD canines for 13 months following injection of the dnMSTAT construct and found that systemic myostatin inhibition resulted in increased muscle mass in GRMD canines as assessed by MRI and confirmed at tissue harvest. We also found that hypertrophy of type IIA fibers was largely responsible for the increased muscle mass and that reductions in serum CK and muscle fibrosis were associated with long-term myostatin inhibition in GRMD.
Although proof-of-principle studies have demonstrated increased muscle mass following gene transfer of myostatin inhibitors in healthy NHPs (Kota et al.
) and canines (Qiao et al.
), this is the first report describing myostatin inhibition in a large-animal model of DMD. Similar to what was observed in the healthy NHP (Kota et al.
), increased muscle mass secondary to myostatin inhibition in the GRMD canine resulted from selective hypertrophy of type II as opposed to type I fibers. Based on data from mice, the mechanism underlying this selectivity is likely higher expression of the activin IIB receptor (myostatin receptor) in type II fibers (Mendias et al.
), although additional investigation will be necessary to confirm this finding in large animals.
We also observed that myostatin inhibition was associated with decreased skeletal muscle fibrosis in GRMD canines at 13 months, a finding that has been previously reported in the mdx
mouse following myostatin inhibition (Qiao et al.
) and genetic ablation of myostatin (Wagner et al.
). Evidence suggests that myostatin can regulate skeletal muscle fibrosis by stimulating proliferation of muscle fibroblasts and expression of extracellular matrix proteins (Li et al.
), although this may not be the case in the heart (Cohn et al.
). Therefore, myostatin inhibition may attenuate the skeletal muscle phenotype of GRMD canines by at least two independent mechanisms: induction of hypertrophy of type II fibers and blockade of proliferation of muscle fibroblasts.
We also observed reduced serum CK in treated versus untreated GRMD canines, which is indicative of reduced muscle damage. We followed treated GRMD canines in this study with serial MRI. MRI enables sensitive measures of muscle size over time, and magnetic resonance proton transverse relaxation time (T2
) of muscle has been proposed as a marker of disease progression and treatment in dystrophic muscle (Pacak et al.
is sensitive to a number of characteristics common in dystrophic disease progression, including muscle damage (Foley et al.
; Mathur et al.
), fatty tissue infiltration (Kan et al.
), and fibrosis (Loganathan et al.
). Reduced damage and associated edema-like fluid accumulation may have contributed to the lower T2
in treated versus untreated GRMD canines. Indeed, we observed that muscle T2
showed a trend to decrease post treatment in all muscles examined, and there was a significant decrease in T2
in the TC of treated versus untreated animals at the conclusion of the study. Overall, whereas the mean T2
value reflects a balance between multiple factors, including fibrosis, edema-like fluid accumulation, and fatty-tissue accumulation, the lower T2
values in the TC of the treated GRMD canines seem most consistent with factors related to reduced susceptibility to injury and the decreased CK levels observed in this study. A rationale for the other muscle groups not demonstrating a significantly lower T2
is difficult to reconcile, but may be due, in part, to the variability of T2
values among the canines, the relatively small changes observed, and the small sample size.
We performed this investigation in the GRMD model because it is the largest available animal model of DMD and is considered the gold standard for evaluation of novel therapeutic interventions for DMD (Wang et al.
). The phenotype of the GRMD model most closely resembles the human disease with progressive muscle wasting, degeneration and fibrosis, and shortened life span secondary to respiratory failure or cardiomyopathy (Wang et al.
). This is in contrast to beagle canine X-linked muscular dystrophy (Shimatsu et al.
) and Cavalier King Charles Spaniel models (Walmsley et al.
), which are smaller and easier to maintain, but which exhibit a less severe phenotype. In addition, because of the larger size of GRMD canines, the AAV dose and route of administration derived in this model should be translatable to patients with DMD without major up-scaling of vector.
In this study, we evaluated systemic myostatin inhibition via liver-directed gene transfer following a single IV infusion, a strategy we previously employed in the mdx
mouse using both dnMSTAT (Morine et al.
) and soluble activin IIB receptor (Morine et al.
). Other gene transfer–based approaches to myostatin inhibition previously evaluated in large animals include direct intramuscular injection of vector in healthy NHPs (Kota et al.
) and hydrodynamic limb-vein injection of vector in healthy canines (Qiao et al.
). Although these investigators observed increased muscle mass in the injected muscles, these local delivery techniques would require multiple injections to target every muscle and muscle group in a DMD patient compared with the single IV infusion performed in our strategy. Furthermore, as myostatin inhibition does not prevent turnover of dystrophin-deficient muscle fibers, transgene expression would be lost over time as repair and regeneration occur (Bartoli et al.
; Pacak et al.
). This would not be an issue for liver-directed gene transfer in DMD. In addition, our approach takes advantage of the highly efficient synthetic machinery of the liver to secrete the inhibitory peptide into the systemic circulation.
Other investigators have developed systemic myostatin inhibition strategies based on injection of anti-myostatin antibodies (Bogdanovich et al.
; Wagner et al.
) and soluble activin IIB receptor protein (Pistilli et al.
). However, this type of approach would necessitate serial injections based on protein half-life for the duration of a DMD patient's life compared with a single IV infusion with our strategy. In addition, patients would also be at risk for developing NAbs against these injected foreign proteins. In contrast, our dnMSTAT peptide is identical to the endogenous propeptide with the exception of a single-point mutation, and even if the resulting single amino acid substitution has potential for immune activation, evidence suggests that liver production and secretion may induce tolerance to foreign peptides (Mingozzi et al.
). Although we did not assay for the presence of NAbs against dnMSTAT that could have resulted in submaximal serum levels of dnMSTAT, we did confirm the presence of serum dnMSTAT at levels approximately two- to threefold above endogenous propeptide levels at 13 months post injection in this study. Further analysis will be necessary to determine the extent, if any, of the humoral response against dnMSTAT following liver-directed gene transfer of AAV8-dnMSTAT in this model.
We also performed pathologic analysis of the livers of treated and untreated GRMD canines at 13 months. We did not observe any increase in fibrosis in treated livers following Trichrome staining, nor did we observe any regions of mononuclear cell infiltration in treated livers following H&E staining. Although this analysis at 13 months does not allow us to rule out an earlier immune response, a recent study did not detect a T-cell response directed against AAV8 capsid following liver-directed gene transfer in canines (Bell et al.
). Furthermore, as we confirmed expression of dnMSTAT at 13 months by RT-PCR and western blot, it is unlikely that a significant T-cell response directed against hepatocytes expressing dnMSTAT was mounted at an earlier time point. However, future investigation should be directed at analyzing the T-cell response at an earlier time point following liver-directed gene transfer of AAV8-dnMSTAT in this model to support our preliminary findings.
It should be noted, however, that myostatin inhibition is not a cure for DMD; rather, it is a strategy that can be used to slow the loss of muscle mass characteristic of this disease. Stabilization of DMD would require restoration of dystrophin expression. Several approaches to achieve this are currently being evaluated in clinical trials, including exon skipping (Miyagoe-Suzuki and Takeda, 2010
; Moulton and Moulton, 2010
; Partridge, 2010
), gene transfer of minidystrophin (Miyagoe-Suzuki and Takeda, 2010
), and nonsense suppression (Finkel, 2010
). However, as it is unlikely that any one of these strategies will result in complete dystrophin restoration in every muscle, a combination of myostatin inhibition and dystrophin restoration approaches may provide additional benefit in DMD patients. Furthermore, based on observations in normal mice (Lee et al.
; Lee, 2007a
), a combination of myostatin inhibition strategies that target other TGF-β family members in addition to myostatin [i.e
., follistatin, follistatin-like related gene (FLRG), growth and differentiation factor–associated serum protein-1 (GASP-1), and/or soluble myostatin receptor (Lee, 2004
)] may be more effective than a single-agent approach for increasing muscle mass in DMD. However, our prior experiments suggest that this may not be the case in the mdx
mouse, in which similar benefit was observed with dnMSTAT (Morine et al.
) and the soluble activin IIB receptor (Morine et al.
). Additionally, it is unclear if targeting other TGF-β family members by using the soluble activin IIB receptor or follistatin will result in side effects not observed when targeting myostatin alone with dnMSTAT. Future investigation should be directed at evaluating such combination approaches in GRMD canines.
In summary, this is the first report describing the effects of long-term, systemic myostatin inhibition in a large-animal model of DMD. GRMD canines underwent liver-directed gene transfer of an scAAV8 vector designed to express a secreted dnMSTAT peptide, and we observed increased muscle mass with selective hypertrophy of type IIA fibers in conjunction with reduced serum CK and muscle fibrosis at 13 months. We believe that the safe, simple, and effective nature of our liver-directed gene-transfer strategy makes it an ideal candidate for evaluation as a novel therapeutic approach for DMD patients, perhaps in combination with dystrophin-restoration approaches.