Effects of Chronic, Maximal Phosphorodiamidate Morpholino Oligomer (PMO) Dosing on Muscle Function and Dystrophin Restoration in a Mouse Model of Duchenne Muscular Dystrophy.

BACKGROUND: Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is currently used in clinical development to treat Duchenne muscular dystrophy (DMD), with four exon-skipping drugs achieving regulatory approval. Exon skipping elicits a truncated, but semi-functional dystrophin protein, similar to the truncated dystrophin expressed in patients with Becker Muscular dystrophy (BMD) where the disease phenotype is less severe than DMD. Despite promising results in both dystrophic animal models and DMD boys, restoration of dystrophin by exon skipping is highly variable, leading to contradictory functional outcomes in clinical trials. OBJECTIVE: To develop optimal PMO dosing protocols that result in increased dystrophin and improved outcome measures in preclinical models of DMD. METHODS: Tested effectiveness of multiple chronic, high dose PMO regimens using biochemical, histological, molecular, and imaging techniques in mdx mice. RESULTS: A chronic, monthly regimen of high dose PMO increased dystrophin rescue in mdx mice and improved specific force in the extensor digitorum longus (EDL) muscle. However, monthly high dose PMO administration still results in variable dystrophin expression localized throughout various muscles. CONCLUSIONS: High dose monthly PMO administration restores dystrophin expression and increases muscle force; however, the variability of dystrophin expression at both the inter-and intramuscular level remains. Additional strategies to optimize PMO uptake including increased dosing frequencies or combination treatments with other yet-to-be-defined therapies may be necessary to achieve uniform dystrophin restoration and increases in muscle function.

Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia.

Cachexia, progressive loss of fat and muscle mass despite adequate nutrition, is a devastating complication of cancer associated with poor quality of life and increased mortality. Myostatin is a potent tonic muscle growth inhibitor. We tested how myostatin inhibition might influence cancer cachexia using genetic and pharmacological approaches. First, hypermuscular myostatin null mice were injected with Lewis lung carcinoma or B16F10 melanoma cells. Myostatin null mice were more sensitive to tumor-induced cachexia, losing more absolute mass and proportionately more muscle mass than wild-type mice. Because myostatin null mice lack expression from development, however, we also sought to manipulate myostatin acutely. The histone deacetylase inhibitor Trichostatin A has been shown to increase muscle mass in normal and dystrophic mice by inducing the myostatin inhibitor, follistatin. Although Trichostatin A administration induced muscle growth in normal mice, it failed to preserve muscle in colon-26 cancer cachexia. Finally we sought to inhibit myostatin and related ligands by administration of the Activin receptor extracellular domain/Fc fusion protein, ACVR2B-Fc. Systemic administration of ACVR2B-Fc potently inhibited muscle wasting and protected adipose stores in both colon-26 and Lewis lung carcinoma cachexia, without affecting tumor growth. Enhanced cachexia in myostatin knockouts indicates that host-derived myostatin is not the sole mediator of muscle wasting in cancer. More importantly, skeletal muscle preservation with ACVR2B-Fc establishes that targeting myostatin-family ligands using ACVR2B-Fc or related molecules is an important and potent therapeutic avenue in cancer cachexia.

Effect of the IL-1 Receptor Antagonist Kineret® on Disease Phenotype in mdx Mice.

Duchenne muscular dystrophy (DMD) is an X-linked muscle disease caused by mutations in the dystrophin gene. The pathology of DMD manifests in patients with progressive muscle weakness, loss of ambulation and ultimately death. One of the characteristics of DMD is muscle inflammation, and dystrophin-deficient skeletal muscles produce higher levels of the pro-inflammatory cytokine interleukin 1ß (IL-1ß) in response to toll like receptor (TLR) stimulation compared to controls; therefore, blocking the IL-1ß pathway could improve the disease phenotype in mdx mice, a mouse model of DMD. Kineret® or IL-1Ra is a recombinant IL-1 receptor antagonist approved by the FDA for treating rheumatoid arthritis. To determine the efficacy of IL-1Ra in a DMD model, we administered subcutaneous injections of saline control or IL-1Ra (25 mg/kg/day) to mdx mice daily for 45 days beginning at 5 weeks of age. Functional and histological parameters were measured at the conclusion of the study. IL-1Ra only partially inhibited this signaling pathway in this study; however, there were still interesting observations to be noted. For example, although not significantly changed, splenocytes from the IL-1Ra-treated group secreted less IL-1ß after LPS stimulation compared to control mice indicating a blunted response and incomplete inhibition of the pathway (37% decrease). In addition, normalized forelimb grip strength was significantly increased in IL-1Ra-treated mice. There were no changes in EDL muscle-specific force measurements, histological parameters, or motor coordination assessments in the dystrophic mice after IL-1Ra treatment. There was a significant 27% decrease in the movement time and total distance traveled by the IL-1Ra treated mice, correlating with previous studies examining effects of IL-1 on behavior. Our studies indicate partial blocking of IL-1ß with IL-1Ra significantly altered only a few behavioral and strength related disease parameters; however, treatment with inhibitors that completely block IL-1ß, pathways upstream of IL-1ß production or combining various inhibitors may produce more favorable outcomes.

Inhibition of inflammation with celastrol fails to improve muscle function in dysferlin-deficient A/J mice.

The dysferlin-deficient A/J mouse strain represents a homologous model for limb-girdle muscular dystrophy 2B. We evaluated the disease phenotype in 10 month old A/J mice compared to two dysferlin-sufficient, C57BL/6 and A/JOlaHsd, mouse lines to determine which functional end-points are sufficiently sensitive to define the disease phenotype for use in preclinical studies in the A/J strain. A/J mice had significantly lower open field behavioral activity (horizontal activity, total distance, movement time and vertical activity) when compared to C57BL/6 and A/JoIaHsd mice. Both A/J and A/JOIaHsd mice showed decreases in latency to fall with rotarod compared to C57BL/6. No changes were detected in grip strength, force measurements or motor coordination between these three groups. Furthermore, we have found that A/J muscle shows significantly increased levels of the pro-inflammatory cytokine TNF-α when compared to C57BL/6 mice, indicating an activation of NF-κB signaling as part of the inflammatory response in dysferlin-deficient muscle. Therefore, we assessed the effect of celastrol (a potent NF-κB inhibitor) on the disease phenotype in female A/J mice. Celastrol treatment for four months significantly reduced the inflammation in A/J muscle; however, it had no beneficial effect in improving muscle function, as assessed by grip strength, open field activity, and in vitro force contraction. In fact, celastrol treated mice showed a decrease in body mass, hindlimb grip strength and maximal EDL force. These findings suggest that inhibition of inflammation alone may not be sufficient to improve the muscle disease phenotype in dysferlin-deficient mice and may require combination therapies that target membrane stability to achieve a functional improvement in skeletal muscle.