Death after High-Dose rAAV9 Gene Therapy in a Patient with Duchenne's Muscular Dystrophy.

We treated a 27-year-old patient with Duchenne's muscular dystrophy (DMD) with recombinant adeno-associated virus (rAAV) serotype 9 containing dSaCas9 (i.e., "dead" Staphylococcus aureus Cas9, in which the Cas9 nuclease activity has been inactivated) fused to VP64; this transgene was designed to up-regulate cortical dystrophin as a custom CRISPR-transactivator therapy. The dose of rAAV used was 1×1014 vector genomes per kilogram of body weight. Mild cardiac dysfunction and pericardial effusion developed, followed by acute respiratory distress syndrome (ARDS) and cardiac arrest 6 days after transgene treatment; the patient died 2 days later. A postmortem examination showed severe diffuse alveolar damage. Expression of transgene in the liver was minimal, and there was no evidence of AAV serotype 9 antibodies or effector T-cell reactivity in the organs. These findings indicate that an innate immune reaction caused ARDS in a patient with advanced DMD treated with high-dose rAAV gene therapy. (Funded by Cure Rare Disease.).

PDE10A Inhibition Reduces the Manifestation of Pathology in DMD Zebrafish and Represses the Genetic Modifier PITPNA.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene. Absence of dystrophin protein leads to progressive degradation of skeletal and cardiac function and leads to premature death. Over the years, zebrafish have been increasingly used for studying DMD and are a powerful tool for drug discovery and therapeutic development. In our study, a birefringence screening assay led to identification of phosphodiesterase 10A (PDE10A) inhibitors that reduced the manifestation of dystrophic muscle phenotype in dystrophin-deficient sapje-like zebrafish larvae. PDE10A has been validated as a therapeutic target by pde10a morpholino-mediated reduction in muscle pathology and improvement in locomotion, muscle, and vascular function as well as long-term survival in sapje-like larvae. PDE10A inhibition in zebrafish and DMD patient-derived myoblasts were also associated with reduction of PITPNA expression that has been previously identified as a protective genetic modifier in two exceptional dystrophin-deficient golden retriever muscular dystrophy (GRMD) dogs that escaped the dystrophic phenotype. The combination of a phenotypic assay and relevant functional assessments in the sapje-like zebrafish enhances the potential for the prospective discovery of DMD therapeutics. Indeed, our results suggest a new application for a PDE10A inhibitor as a potential DMD therapeutic to be investigated in a mouse model of DMD.

Repression of phosphatidylinositol transfer protein α ameliorates the pathology of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.

Gamma-sarcoglycan is required for the response of archvillin to mechanical stimulation in skeletal muscle.

Loss of gamma-sarcoglycan (γ-SG) induces muscle degeneration and signaling defects in response to mechanical load, and its absence is common to both Duchenne and limb girdle muscular dystrophies. Growing evidence suggests that aberrant signaling contributes to the disease pathology; however, the mechanisms of γ-SG-mediated mechanical signaling are poorly understood. To uncover γ-SG signaling pathway components, we performed yeast two-hybrid screens and identified the muscle-specific protein archvillin as a γ-SG and dystrophin interacting protein. Archvillin protein and message levels were significantly upregulated at the sarcolemma of murine γ-SG-null (gsg(-/-)) muscle but delocalized in dystrophin-deficient mdx muscle. Similar elevation of archvillin protein was observed in human quadriceps muscle lacking γ-SG. Reintroduction of γ-SG in gsg(-/-) muscle by rAAV injection restored archvillin levels to that of control C57 muscle. In situ eccentric contraction of tibialis anterior (TA) muscles from C57 mice caused ERK1/2 phosphorylation, nuclear activation of P-ERK1/2 and stimulus-dependent archvillin association with P-ERK1/2. In contrast, TA muscles from gsg(-/-) and mdx mice exhibited heightened P-ERK1/2 and increased nuclear P-ERK1/2 localization following eccentric contractions, but the archvillin-P-ERK1/2 association was completely ablated. These results position archvillin as a mechanically sensitive component of the dystrophin complex and demonstrate that signaling defects caused by loss of γ-SG occur both at the sarcolemma and in the nucleus.

Masticatory muscles of mouse do not undergo atrophy in space.

Muscle loading is important for maintaining muscle mass; when load is removed, atrophy is inevitable. However, in clinical situations such as critical care myopathy, masticatory muscles do not lose mass. Thus, their properties may be harnessed to preserve mass. We compared masticatory and appendicular muscles responses to microgravity, using mice aboard the space shuttle Space Transportation System-135. Age- and sex-matched controls remained on the ground. After 13 days of space flight, 1 masseter (MA) and tibialis anterior (TA) were frozen rapidly for biochemical and functional measurements, and the contralateral MA was processed for morphologic measurements. Flight TA muscles exhibited 20 ± 3% decreased muscle mass, 2-fold decreased phosphorylated (P)-Akt, and 4- to 12-fold increased atrogene expression. In contrast, MAs had no significant change in mass but a 3-fold increase in P-focal adhesion kinase, 1.5-fold increase in P-Akt, and 50-90% lower atrogene expression compared with limb muscles, which were unaltered in microgravity. Myofibril force measurements revealed that microgravity caused a 3-fold decrease in specific force and maximal shortening velocity in TA muscles. It is surprising that myofibril-specific force from both control and flight MAs were similar to flight TA muscles, yet power was compromised by 40% following flight. Continued loading in microgravity prevents atrophy, but masticatory muscles have a different set point that mimics disuse atrophy in the appendicular muscle.

Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts.

Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96-microwell plates into 3-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty-one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin-like growth factor-1, creatine, beta-hydroxy-beta-methylbutyrate, trichostatin A, lisinopril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC(50) of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders.

Effect of serotonin modulation on dystrophin-deficient zebrafish.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the dystrophin gene. Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient sapje and sapje-like zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.

Purification of Myogenic Progenitors from Human Muscle Using Fluorescence-Activated Cell Sorting (FACS).

Primary myoblasts derived from human tissue are a valuable tool in research of muscle disease and pathophysiology. However, skeletal muscle biopsies, especially from diseased muscle, contain a plethora of non-myogenic cells, necessitating purification of the myogenic cell population. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study.

Isolation of Primary Human Skeletal Muscle Cells.

Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study.

Exosomal Small Talk Carries Strong Messages from Muscle Stem Cells.

Exosome-dependent intercellular communication is an emerging signaling mechanism involved in tissue repair and regeneration. Now in this issue of Cell Stem Cell, Fry et al. (2016) show that muscle stem cells communicate with fibrogenic cells by exosomal trafficking of microRNA-206 to regulate extracellular matrix deposition and muscle tissue remodeling.

Pharmacological therapeutics targeting the secondary defects and downstream pathology of Duchenne muscular dystrophy.

INTRODUCTION: Since the identification of the dystrophin gene in 1986, a cure for Duchenne muscular dystrophy (DMD) has yet to be discovered. Presently, there are a number of genetic-based therapies in development aimed at restoration and/or repair of the primary defect. However, growing understanding of the pathophysiological consequences of dystrophin absence has revealed several promising downstream targets for the development of therapeutics. AREAS COVERED: In this review, we discuss various strategies for DMD therapy targeting downstream consequences of dystrophin absence including loss of muscle mass, inflammation, fibrosis, calcium overload, oxidative stress, and ischemia. The rationale of each approach and the efficacy of drugs in preclinical and clinical studies are discussed. EXPERT OPINION: For the last 30 years, effective DMD drug therapy has been limited to corticosteroids, which are associated with a number of negative side effects. Our knowledge of the consequences of dystrophin absence that contribute to DMD pathology has revealed several potential therapeutic targets. Some of these approaches may have potential to improve or slow disease progression independently or in combination with genetic-based approaches. The applicability of these pharmacological therapies to DMD patients irrespective of their genetic mutation, as well as the potential benefits even for advanced stage patients warrants their continued investigation.

CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies.

Cell-surface markers for prospective isolation of stem cells from human skeletal muscle have been difficult to identify. Such markers would be powerful tools for studying satellite cell function during homeostasis and in pathogenesis of diseases such as muscular dystrophies. In this study, we show that the tetraspanin KAI/CD82 is an excellent marker for prospectively isolating stem cells from human fetal and adult skeletal muscle. Human CD82+ muscle cells robustly engraft into a mouse model of muscular dystrophy. shRNA knockdown of CD82 in myogenic cells reduces myoblast proliferation, suggesting it is functionally involved in muscle homeostasis. CD82 physically interacts with alpha7beta1 integrin (α7ß1-ITG) and with α-sarcoglycan, a member of the Dystrophin-Associated Glycoprotein Complex (DAPC), both of which have been linked to muscular dystrophies. Consistently, CD82 expression is decreased in Duchenne muscular dystrophy patients. Together, these findings suggest that CD82 function may be important for muscle stem cell function in muscular disorders.

Mature IGF-I excels in promoting functional muscle recovery from disuse atrophy compared with pro-IGF-IA.

Prolonged disuse of skeletal muscle results in atrophy, and once physical activity is resumed, there is increased susceptibility to injury. Insulin-like growth factor-I (IGF-I) is considered a potential therapeutic target to attenuate atrophy during unloading and to enhance rehabilitation upon reloading of skeletal muscles, due to its multipronged actions on satellite cell proliferation, differentiation, and survival, as well as its actions on muscle fibers to boost protein synthesis and inhibit protein degradation. However, the form of IGF-I delivered may alter the success of treatment. Using the hindlimb suspension model of disuse atrophy, we compared the efficacy of two IGF-I forms in protection against atrophy and enhancement of recovery: mature IGF-I (IGF-IS) lacking the COOH-terminal extension, called the E-peptide, and IGF-IA, which is the predominant form retaining the E-peptide. Self-complementary adeno-associated virus harboring the murine Igf1 cDNA constructs were delivered to hindlimbs of adult female C57BL6 mice 3 days prior to hindlimb suspension. Hindlimb muscles were unloaded for 7 days and then reloaded for 3, 7, and 14 days. Loss of muscle mass following suspension was not prevented by either IGF-I construct. However, IGF-IS expression maintained soleus muscle force production. Further, IGF-IS treatment caused rapid recovery of muscle fiber morphology during reloading and maintained muscle strength. Analysis of gene expression revealed that IGF-IS expression accelerated the downregulation of atrophy-related genes compared with untreated or IGF-IA-treated samples. We conclude that mature-IGF-I may be a better option than pro-IGF-IA to promote skeletal muscle recovery following disuse atrophy.

Repression of phosphatidylinositol transfer protein a ameliorates the pathology of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-a (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.