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Mizunoya W.,University of Manitoba | Upadhaya R.,University of Manitoba | Burczynski F.J.,University of Manitoba | Wang G.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Anderson J.E.,University of Manitoba
American Journal of Physiology - Cell Physiology | Year: 2011

In Duchenne muscular dystrophy (DMD), palliative glucocorticoid therapy can produce myopathy or calcification. Since increased nitric oxide synthase activity in dystrophic mice promotes regeneration, the outcome of two nitric oxide (NO) donor drugs, MyoNovin (M) and isosorbide dinitrate (I), on the effectiveness of the anti-inflammatory drug prednisone (P) in alleviating progression of dystrophy was tested. Dystrophic mdx mice were treated (18 days) as controls or with an NO donor ± P. Fiber permeability and DNA synthesis were labeled by Evans blue dye (EBD) and bromodeoxyuridine uptake, respectively. P decreased body weight gain, M increased quadriceps mass, and I increased heart mass. P increased fiber permeability (%EBD+ fibers) and calcification in diaphragm. Treatment with NO donors ± P (M+P, I+P) reduced %EBD+ fibers and calcification vs. P alone. %EBD+ fibers in M+P diaphragm did not differ from control. NO donor treatment reduced proliferation and the population of c-met+ cells and accelerated fiber regeneration. Concurrent with P, NO donor treatment suppressed two important detrimental effects of P in mice, possibly by accelerating regeneration, rebalancing satellite cell quiescence and activation in dystrophy, and/or increasing perfusion. Results suggest that NO donors could improve current therapy for DMD. © 2011 the American Physiological Society.

Lu Q.-L.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Yokota T.,Research Center for Genetic Medicine | Takeda S.,National Institute of Neuroscience | Garcia L.,University Pierre and Marie Curie | And 2 more authors.
Molecular Therapy | Year: 2011

Duchenne muscular dystrophy (DMD) is associated with mutations in the dystrophin gene that disrupt the open reading frame whereas the milder Becker's form is associated with mutations which leave an in-frame mRNA transcript that can be translated into a protein that includes the N- and C- terminal functional domains. It has been shown that by excluding specific exons at, or adjacent to, frame-shifting mutations, open reading frame can be restored to an out-of-frame mRNA, leading to the production of a partially functional Becker-like dystrophin protein. Such targeted exclusion can be achieved by administration of oligonucleotides that are complementary to sequences that are crucial to normal splicing of the exon into the transcript. This principle has been validated in mouse and canine models of DMD with a number of variants of oligonucleotide analogue chemistries and by transduction with adeno-associated virus (AAV)-small nuclear RNA (snRNA) reagents encoding the antisense sequence. Two different oligonucleotide agents are now being investigated in human trials for splicing out of exon 51 with some early indications of success at the biochemical level. © The American Society of Gene & Cell Therapy.

Blaeser A.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Keramaris E.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Chan Y.M.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Sparks S.,Carolinas Medical Center | And 3 more authors.
Human Genetics | Year: 2013

Dystroglycanopathies are characterized by a reduction in the glycosylation of alpha-dystroglycan (α-DG). A common cause for this subset of muscular dystrophies is mutations in the gene of fukutin-related protein (FKRP). FKRP mutations have been associated with a wide spectrum of clinical severity from severe Walker-Warburg syndrome and muscle-eye-brain disease with brain and eye defects to mild limb-girdle muscular dystrophy 2I with myopathy only. To examine the affects of FKRP mutations on the severity of the disease, we have generated homozygous and compound heterozygous mouse models with human mutations in the murine FKRP gene. P448Lneo+ and E310delneo+ mutations result in severe dystrophic and embryonic lethal phenotypes, respectively. P448Lneo+/E310delneo+ compound heterozygotes exhibit brain defects and severe muscular dystrophies with near absence of α-DG glycosylation. Removal of the Neor cassette from the P448Lneo+ homozygous mice eliminates overt brain and eye defects, and reduces severity of dystrophic phenotypes. Furthermore, introduction of the common L276I mutation to generate transgenic L276Ineo+ homozygous and L276Ineo+/P448Lneo+ and L276Ineo+/E310delneo+ compound heterozygotes results in mice displaying milder dystrophies with reduced α-DG glycosylation and no apparent brain defects. Limited sampling and variation in functionally glycosylated α-DG levels between and within muscles may explain the difficulties in correlating FKRP expression levels with phenotype in clinics. The nature of individual mutations, expression levels and status of muscle differentiation all contribute to the phenotypic manifestation. These mutant FKRP mice are useful models for the study of disease mechanism(s) and experimental therapies. © 2013 Springer-Verlag Berlin Heidelberg.

Xu L.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Lu P.J.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Wang C.-H.,University of North Carolina at Chapel Hill | Keramaris E.,McColl Lockwood Laboratory for Muscular Dystrophy Research | And 5 more authors.
Molecular Therapy | Year: 2013

Mutations in the FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker-Warburg syndrome and muscle-eye-brain disease. The characteristic biochemical feature of these diseases is the hypoglycosylation of α-dystroglycan (α-DG). Currently there is no effective treatment available. In this study, we examined the adeno-associated virus serotype 9 vector (AAV9)-mediated gene therapy in the FKRP mutant mouse model with a proline to leucine missense mutation (P448L). Our results showed that intraperitoneal administration of AAV9-FKRP resulted in systemic FKRP expression in all striated muscles examined with the highest levels in cardiac muscle. Consistent with our previous observations, FKRP protein is localized in the Golgi apparatus in myofibers. Expression of FKRP consequently restored functional glycosylation of α-DG in the skeletal and cardiac muscles. Significant improvement in dystrophic pathology, serum creatine kinase levels and muscle function was observed. Only limited FKRP transgene expression was detected in kidney and liver with no detectable toxicity. Our results provided evidence for the utility of AAV-mediated gene replacement therapy for FKRP-related muscular dystrophies. © The American Society of Gene & Cell Therapy.

Wang M.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Wu B.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Lu P.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Tucker J.D.,McColl Lockwood Laboratory for Muscular Dystrophy Research | And 4 more authors.
Gene Therapy | Year: 2014

A series of small-size polyethylenimine (PEI)-conjugated pluronic polycarbamates (PCMs) have been investigated for the ability to modulate the delivery of 2′-O-methyl phosphorothioate RNA (2′-OMePS) in vitro and in dystrophic mdx mice. The PCMs retain strong binding capacity to negatively charged oligomer as demonstrated by agarose gel retardation assay, with the formation of condensed polymer/oligomer complexes at a wide-range weight ratio from 1:1 to 20:1. The condensed polymer/oligomer complexes form 100-300 nm nanoparticles. Exon-skipping effect of 2′-OMePS was dramatically enhanced with the use of the most effective PCMs in comparison with 2′-OMePS alone in both cell culture and in vivo, respectively. More importantly, the effective PCMs, especially those composed of moderate size (2k-5kDa) and intermediate hydrophilic-lipophilic balance (7-23) of pluronics, enhanced exon-skipping of 2′-OMePS with low toxicity as compared with Lipofectamine-2000 in vitro or PEI 25k in vivo. The variability of individual PCM for delivery of antisense oligomer and plasmid DNA indicate the complexity of interaction between polymer and their cargos. Our data demonstrate the potential of PCMs to mediate delivery of modified antisense oligonucleotides to the muscle for treating muscular dystrophy or other appropriate myodegenerative diseases. © 2014 Macmillan Publishers Limited.

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