Development of Novel Micro-dystrophins with Enhanced Functionality.

Gene therapies using adeno-associated viral (AAV) vectors have advanced into clinical trials for several diseases, including Duchenne muscular dystrophy (DMD). A limitation of AAV is the carrying capacity (∼5 kb) available for genes and regulatory cassettes (RCs). These size constraints are problematic for the 2.2-Mb dystrophin gene. We previously designed a variety of miniaturized micro-dystrophins (µDys) that displayed significant, albeit incomplete, function in striated muscles. To develop µDys proteins with improved performance, we explored structural modifications of the dystrophin central rod domain. Eight µDys variants were studied that carried unique combinations of between four and six of the 24 spectrin-like repeats present in the full-length protein, as well as various hinge domains. Expression of µDys was regulated by a strong but compact muscle-restricted RC (CK8e) or by the ubiquitously active cytomegalovirus (CMV) RC. Vectors were evaluated by intramuscular injection and systemic delivery to dystrophic mdx4cv mice, followed by analysis of skeletal muscle pathophysiology. Two µDys designs were identified that led to increased force generation compared with previous µDys while also localizing neuronal nitric oxide synthase to the sarcolemma. An AAV vector expressing the smaller of these (µDys5) from the CK8e RC is currently being evaluated in a DMD clinical trial.

Twelve hours of heat stress induces inflammatory signaling in porcine skeletal muscle.

Heat stress causes morbidity and mortality in humans and animals and threatens food security by limiting livestock productivity. Inflammatory signaling may contribute to heat stress-mediated skeletal muscle dysfunction. Previously, we discovered increased circulating endotoxin and intramuscular oxidative stress and TNF-α protein abundance, but not inflammatory signaling following 24 and 72 h of heat stress. Thus the purpose of this investigation was to clarify the role of inflammatory signaling in heat-stressed skeletal muscle. Crossbred gilts (n = 8/group) were assigned to either thermal neutral (24°C), heat stress (37°C), or pair-fed thermal neutral (24°C) conditions for 12 h. Following treatment, animals were euthanized, and the semitendinosus red (STR) and white (STW) were recovered. Heat stress did not alter inflammatory signaling in STW. In STR, relative heat shock protein abundance was similar between groups, as was nuclear content of heat shock factor 1. In whole homogenate, relative abundance of the NF-κB activator inhibitory κB kinase-α was increased by heat stress, although abundance of NF-κB was similar between groups. Relative abundance of phosphorylated NF-κB was increased by heat stress in nuclear fractions. Activator protein-1 (AP-1) signaling was similar between groups. While there were few differences in transcript expression between thermal neutral and heat stress, 80 and 56% of measured transcripts driven by NF-κB or AP-1, respectively, were increased by heat stress compared with pair-fed thermal neutral. Heat stress also caused a reduction in IL-6 transcript and relative protein abundance. These data demonstrate that short-term heat stress causes inflammatory signaling through NF-κB in oxidative, but not glycolytic, skeletal muscle.

Viral vector-mediated gene therapies.

PURPOSE OF REVIEW: Gene therapy as a treatment for neuromuscular disease has significantly advanced over the past decade. In the present review, the progress of adeno-associated viruses (AAV) vector-mediated gene therapy for Duchenne muscular dystrophy (DMD) during the past year is highlighted. RECENT FINDINGS: Modulating the immune response to AAV vector capsid or the transgene has helped to increase stable transduction efficiency. Full-length dystrophin expression via gene editing with targeted nucleases may ultimately be an ideal treatment option. Also genes with homologues function may ameliorate many aspects of the DMD pathophysiology. SUMMARY: The work during the past year has increased our understanding of AAV vector-mediated therapy and has also validated new approaches to treat DMD. The results will aid in the design of both preclinical and clinical trials.

Dystrophin insufficiency causes selective muscle histopathology and loss of dystrophin-glycoprotein complex assembly in pig skeletal muscle.

The purpose of this investigation was to determine the extent to which dystrophin insufficiency caused histomorphological changes in a novel pig model of Becker muscular dystrophy. In our procedures, we used a combination of biochemical approaches, including quantitative PCR and Western blots, along with a histological analysis using standard and immunohistological measures. We found that 8-wk-old male affected pigs had a 70% reduction in dystrophin protein abundance in the diaphragm, psoas major, and longissimus lumborum and a 5-fold increase in serum creatine kinase activity compared with healthy male littermates. Dystrophin insufficiency in the diaphragm and the longissimus resulted in muscle histopathology with disorganized fibrosis that often colocalized with fatty infiltration but not the psoas. Affected animals also had an 80-85% reduction in α-sarcoglycan localization in these muscles, indicating compromised assembly of the dystrophin glycoprotein complex. Controls used in this study were 4 healthy male littermates, as they are most closely related to the affected animals. We concluded that pigs with insufficient dystrophin protein expression have a phenotype consistent with human dystrophinopathy patients. Given that and their similarity in body size and physiology to humans, we further conclude that this pig line is an appropriate translational model for dystrophinopathies.

PGC-1α gene transfer improves muscle function in dystrophic muscle following prolonged disease progress.

NEW FINDINGS: What is the central question of this study? Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) gene transfer as a treatment for Duchenne muscular dystrophy is efficacious even with advanced disease. What is the main finding and its importance? PGC-1α pathway activation strategies may be most effective when initiated at the earliest possible time. Duchenne muscular dystrophy is a progressive and fatal muscle wasting disease caused by a dystrophin deficiency. We previously found that gene transfer of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) increased abundance of utrophin and increased mitochondrial biogenesis using prevention and rescue treatment protocols. Our purpose in this investigation was to determine the extent to which PGC-1α gene transfer would rescue dystrophic muscle following prolonged disease progression. One-year-old mdx mice from our colony were injected in one hindlimb with a virus driving expression of PGC-1α, while the contralateral limb was injected with empty capsid. Three months after viral gene transfer, PGC-1α expression was 40-fold greater than in contralateral limbs. Specific tension was increased by ∼ 60% (P < 0.05), and force produced during the final contraction of a fatigue protocol was 60% greater in treated soleus muscles compared with contralateral control muscles (P < 0.05). Histopathology was not improved by PGC-1α overexpression. Also, while there were numerous differences in gene expression between healthy and dystrophic muscle, there were relatively few differences between PGC-1α-treated limbs and contralateral control limbs. These data indicate that PGC-1α pathway activation may interrupt the disease process even if initiated within the context of advanced disease; however, the mechanism that underlies this functional correction is not apparent.

Histological and biochemical outcomes of cardiac pathology in mdx mice with dietary quercetin enrichment.

NEW FINDINGS: What is the central question of this study? Does dietary quercetin enrichment improve biochemical and histological outcomes in hearts from mdx mice? What is the main finding and what is its importance? Biochemical and histological findings suggest that chronic quercetin feeding of mdx mice may improve mitochondrial function and attenuate tissue pathology. Patients with Duchenne muscular dystrophy suffer from cardiac pathology, which causes up to 40% of all deaths because of fibrosis and cardiac complications. Quercetin is a flavonol with anti-inflammatory and antioxidant effects and is also an activator of peroxisome proliferator-activated receptor γ coactivator 1α capable of antioxidant upregulation, mitochondrial biogenesis and prevention of cardiac complications. We sought to determine the extent to which dietary quercetin enrichment prevents (experiment 1) and rescues cardiac pathology (experiment 2) in mdx mice. In experiment 1, 3-week-old mdx mice were fed control chow (C3w6m, n = 10) or chow containing 0.2% quercetin for 6 months (Q3w6m, n = 10). In experiment 2, 3-month-old mdx mice were fed control chow (C3m6m, n = 10) or 0.2% chow containing 0.2% quercetin for 6 months (Q3m6m, n = 10). Hearts were excised for histological and biochemical analyses. In experiment 1, Western blot targets for mitochondrial biogenesis (cytochrome c, P = 0.007) and antioxidant expression (superoxide dismutase 2, P = 0.014) increased in Q3w6m mice compared with C3w6m. Histology revealed increased utrophin (P = 0.025) and decreased matrix metalloproteinase 9 abundance (P = 0.040) in Q3w6m mice compared with C3w6m. In experiment 2, relative (P = 0.023) and absolute heart weights (P = 0.020) decreased in Q3m6m mice compared with C3m6m. Indications of damage (Haematoxylin- and Eosin-stained sections, P = 0.007) and Western blot analysis of transforming growth factor ß1 (P = 0.009) were decreased in Q3m6m mice. Six months of quercetin feeding increased a mitochondrial biomarker, antioxidant protein and utrophin and decreased matrix metalloproteinase 9 in young mice. Given that these adaptations are associated with attenuated cardiac pathology and damage, the present findings may indicate that dietary quercetin enrichment attenuates dystrophic cardiac pathology, but physiological confirmation is needed.

Rescue of dystrophic skeletal muscle by PGC-1α involves restored expression of dystrophin-associated protein complex components and satellite cell signaling.

Duchenne muscular dystrophy is typically diagnosed in the preschool years because of locomotor defects, indicative of muscle damage. Thus, effective therapies must be able to rescue muscle from further decline. We have established that peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α) gene transfer will prevent many aspects of dystrophic pathology, likely through upregulation of utrophin and increased oxidative capacity; however, the extent to which it will rescue muscle with disease manifestations has not been determined. Our hypothesis is that gene transfer of Pgc-1α into declining muscle will reduce muscle injury compared with control muscle. To test our hypothesis, adeno-associated virus 6 (AAV6) driving expression of Pgc-1α was injected into single hind limbs of 3-wk-old mdx mice, while the contralateral limb was given a sham injection. At 6 wk of age, treated solei had 37% less muscle injury compared with sham-treated muscles (P < 0.05). Resistance to contraction-induced injury was improved 10% (P < 0.05), likely driven by the five-fold (P < 0.05) increase in utrophin protein expression and increase in dystrophin-associated complex members. Treated muscles were more resistant to fatigue, which was likely caused by the corresponding increase in oxidative markers. Pgc-1α overexpressing limbs also exhibited increased expression of genes related to muscle repair and autophagy. These data indicate that the Pgc-1α pathway remains a good therapeutic target, as it reduced muscle injury and improved function using a rescue paradigm. Further, these data also indicate that the beneficial effects of Pgc-1α gene transfer are more complex than increased utrophin expression and oxidative gene expression.

An antisense microwalk reveals critical role of an intronic position linked to a unique long-distance interaction in pre-mRNA splicing.

Here we report a novel finding of an antisense oligonucleotide (ASO) microwalk in which we examined the position-specific role of intronic residues downstream from the 5' splice site (5' ss) of SMN2 exon 7, skipping of which is associated with spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Our results revealed the inhibitory role of a cytosine residue at the 10th intronic position ((10)C), which is neither conserved nor associated with any known splicing motif. Significance of (10)C emerged from the splicing pattern of SMN2 exon 7 in presence of a 14-mer ASO (L14) that sequestered two adjacent hnRNP A1 motifs downstream from (10)C and yet promoted SMN2 exon 7 skipping. Another 14-mer ASO (F14) that sequestered both, (10)C and adjacent hnRNP A1 motifs, led to a strong stimulation of SMN2 exon 7 inclusion. The inhibitory role of (10)C was found to be tightly linked to its unpaired status and specific positioning immediately upstream of a RNA:RNA helix formed between the targeting ASO and its intronic target. Employing a heterologous context as well as changed contexts of SMN2 intron 7, we show that the inhibitory effect of unpaired (10)C is dependent upon a long-distance interaction involving downstream intronic sequences. Our report furnishes one of the rare examples in which an ASO-based approach could be applied to unravel the critical role of an intronic position that may not belong to a linear motif and yet play significant role through long-distance interactions.

PGC-1α overexpression increases transcription factor EB nuclear localization and lysosome abundance in dystrophin-deficient skeletal muscle.

Duchenne muscular dystrophy (DMD) is caused by the absence of functional dystrophin protein and results in progressive muscle wasting. Dystrophin deficiency leads to a host of dysfunctional cellular processes including impaired autophagy. Autophagic dysfunction appears to be due, at least in part, to decreased lysosomal abundance mediated by decreased nuclear localization of transcription factor EB (TFEB), a transcription factor responsible for lysosomal biogenesis. PGC-1α overexpression decreased disease severity in dystrophin-deficient skeletal muscle and increased PGC-1α has been linked to TFEB activation in healthy muscle. The purpose of this study was to determine the extent to which PGC-1α overexpression increased nuclear TFEB localization, increased lysosome abundance, and increased autophagosome degradation. We hypothesized that overexpression of PGC-1α would drive TFEB nuclear translocation, increase lysosome biogenesis, and improve autophagosome degradation. To address this hypothesis, we delivered PGC-1α via adeno-associated virus (AAV) vector injected into the right limb of 3-week-old mdx mice and the contralateral limbs received a sham injection. At 6 weeks of age, this approach increased PGC-1α transcript by 60-fold and increased TFEB nuclear localization in gastrocnemii from PGC-1α treated limbs by twofold compared to contralateral controls. Furthermore, lamp2, a marker of lysosome abundance, was significantly elevated in muscles from limbs overexpressing PGC-1α. Lastly, increased LC3II and similar p62 in PGC-1α overexpressing-limbs compared to contralateral limbs are supportive of increased degradation of autophagosomes. These data provide mechanistic insight into PGC-1α-mediated benefits to dystrophin-deficient muscle, such that increased TFEB nuclear localization in dystrophin-deficient muscle leads to increased lysosome biogenesis and autophagy.

Porcine models of muscular dystrophy.

Duchenne muscular dystrophy is a progressive, fatal, X-linked disease caused by a failure to accumulate the cytoskeletal protein dystrophin. This disease has been studied using a variety of animal models including fish, mice, rats, and dogs. While these models have contributed substantially to our mechanistic understanding of the disease and disease progression, limitations inherent to each model have slowed the clinical advancement of therapies, which necessitates the development of novel large-animal models. Several porcine dystrophin-deficient models have been identified, although disease severity may be so severe as to limit their potential contributions to the field. We have recently identified and completed the initial characterization of a natural porcine model of dystrophin insufficiency. Muscles from these animals display characteristic focal necrosis concomitant with decreased abundance and localization of dystrophin-glycoprotein complex components. These pigs recapitulate many of the cardinal features of muscular dystrophy, have elevated serum creatine kinase activity, and preliminarily appear to display altered locomotion. They also suffer from sudden death preceded by EKG abnormalities. Pig dystrophinopathy models could allow refinement of dosing strategies in human-sized animals in preparation for clinical trials. From an animal handling perspective, these pigs can generally be treated normally, with the understanding that acute stress can lead to sudden death. In summary, the ability to create genetically modified pig models and the serendipitous discovery of genetic disease in the swine industry has resulted in the emergence of new animal tools to facilitate the critical objective of improving the quality and length of life for boys afflicted with such a devastating disease.

Long-term quercetin dietary enrichment decreases muscle injury in mdx mice.

BACKGROUND & AIMS: Duchenne muscular dystrophy results from a mutation in the dystrophin gene, which leads to a dystrophin-deficiency. Dystrophic muscle is marked by progressive muscle injury and loss of muscle fibers. Activation of the PGC-1α pathway has been previously shown to decrease disease-related muscle damage. Oral administration of the flavonol, quercetin, appears to be an effective and safe method to activate the PGC-1α pathway. The aim of this investigation was to determine the extent to which long term dietary quercetin enrichment would decrease muscle injury in dystrophic skeletal muscle. We hypothesized that a quercetin enriched diet would rescue dystrophic muscle from further decline and increase utrophin abundance. METHODS: Beginning at three-months of age and continuing to nine-months of age mdx mice (n = 10/group) were assigned to either to mdx-control receiving standard chow or to mdx-quercetin receiving a 0.2% quercetin-enriched diet. At nine-months of age mice were sacrificed and costal diaphragms collected. One hemidiaphragm was used for histological analysis and the second hemidiaphragm was used to determine gene expression via RT-qPCR. RESULTS: The diaphragm from the mdx-quercetin group had 24% (p ≤ 0.05) more muscle fibers/area and 34% (p ≤ 0.05) fewer centrally nucleated fibers compared to the mdx-control group. Further, there were 44% (p ≤ 0.05) fewer infiltrating immune cells/area, a corresponding 31% (p ≤ 0.05) reduction in TNF gene expression, and a near 50% reduction in fibrosis. The quercetin-enriched diet increased expression of genes associated with oxidative metabolism but did not increase utrophin protein abundance. CONCLUSIONS: Long-term quercetin supplementation decreased disease-related muscle injury in dystrophic skeletal muscle; however the role of PGC-1α pathway activation as a mediator of this response is unclear.

Development of rabbit monoclonal antibodies for detection of alpha-dystroglycan in normal and dystrophic tissue.

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5-2, 29-5, and 45-3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.