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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 | Cloer C.,McColl Lockwood Laboratory for Muscular Dystrophy Research | And 3 more authors.
Molecular Therapy | Year: 2013

We investigated a series of small-sized polyethylenimine (PEI, 0.8/1.2 k)-conjugated pluronic copolymers (PCMs) for their potential to enhance delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in dystrophic mdx mice. PCM polymers containing pluronics of molecular weight (Mw) ranging 2-6 k, with hydrophilic-lipophilic balance (HLB) 7-23, significantly enhanced PMO-induced exon-skipping in a green fluorescent protein (GFP) reporter-based myoblast culture system. Application of optimized formulations of PCMs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. Consistent with our observations in vitro, optimization of molecular size and the HLB of pluronics are important factors for PCMs to achieve enhanced PMO delivery in vivo. Observed cytotoxicity of the PCMs was lower than Endo-porter and PEI 25 k. Tissue toxicity of PCMs in muscle was not clearly detected with the concentrations used, indicating the potential of the PCMs as effective and safe PMO carriers for treating diseases such as muscular dystrophy. © 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.


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.


Wu B.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Lu P.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Cloer C.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Shaban M.,McColl Lockwood Laboratory for Muscular Dystrophy Research | And 5 more authors.
American Journal of Pathology | Year: 2012

Exon skipping is capable of correcting frameshift and nonsense mutations in Duchenne muscular dystrophy. Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of Duchenne muscular dystrophy patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2′-O-methyl phosphorothioate. Peptide-conjugated phosphorodiamidate morpholino offers significantly higher efficiency than phosphorodiamidate morpholino, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of peptide-conjugated phosphorodiamidate morpholino targeting exon 23 in dystrophic mdx mice. The LD50 of peptide-conjugated phosphorodiamidate morpholino was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg peptide-conjugated phosphorodiamidate morpholino produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg peptide-conjugated phosphorodiamidate morpholino restored dystrophin to >50% normal levels in skeletal muscle, and 15% in cardiac muscle. This was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of peptide-conjugated phosphorodiamidate morpholino can be safely applied to achieve significant therapeutic effects in an animal model. © 2012 American Society for Investigative Pathology.


Hu Y.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Li Z.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Wu X.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Lu Q.,McColl Lockwood Laboratory for Muscular Dystrophy Research
PLoS ONE | Year: 2011

Alpha-dystroglycan (α-DG) is a ubiquitously expressed receptor for extracellular matrix proteins and some viruses, and plays a pivotal role in a number of pathological events, including cancer progression, muscular dystrophies, and viral infection. The O-glycans on α-DG are essential for its ligand binding, but the biosynthesis of the functional O-glycans remains obscure. The fact that transient overexpression of LARGE, a putative glycosyltransferase, up-regulates the functional glycans on α-DG to mediate its ligand binding implied that overexpression of LARGE may be a novel strategy to treat disorders with hypoglycosylation of α-DG. In this study, we focus on the effects of stable overexpression of Large on α-DG glycosylation in Chinese hamster ovary (CHO) cell and its glycosylation deficient mutants. Surprisingly, stable overexpression of Large in an O-mannosylation null deficient Lec15.2 CHO cells failed to induce the functional glycans on α-DG. Introducing the wild-type DPM2 cDNA, the deficient gene in the Lec15.2 cells, fully restored the Large-induced functional glycosylation, suggesting that Large induces the functional glycans in a DPM2/O-mannosylation dependent manner. Furthermore, stable overexpression of Large can effectively induce functional glycans on N-linked glycans in the Lec8 cells and ldlD cells growing in Gal deficient media, in both of which circumstances galactosylation are deficient. In addition, supplement of Gal to the ldlD cell culture media significantly reduces the amount of functional glycans induced by Large, suggested that galactosylation suppresses Large to induce the functional glycans. Thus our results revealed a mechanism by which Large competes with galactosyltransferase to target GlcNAc terminals to induce the functional glycans on α-DG. © 2011 Hu et al.


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.


Wu B.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Benrashid E.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Lu P.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Cloer C.,McColl Lockwood Laboratory for Muscular Dystrophy Research | And 3 more authors.
PLoS ONE | Year: 2011

Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD mouse, a transgenic model carrying the full-length human dystrophin gene, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement. © 2011 Wu et al.


Wang M.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Lu P.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Wu B.,McColl Lockwood Laboratory for Muscular Dystrophy Research | Tucker J.D.,McColl Lockwood Laboratory for Muscular Dystrophy Research | And 3 more authors.
Journal of Materials Chemistry | Year: 2012

A series of low molecular weight polyethyleneimine (LPEI) conjugated Pluronic copolymers (PCMs) were synthesized and evaluated in C2C12 myoblasts and CHO cells in vitro and in dystrophic mdx mice in vivo as gene delivery carriers. Pluronics with different molecular weights (M w) and hydrophilic-lipophilic-balance (HLB), and two LPEIs (M w: 0.8k, 1.2k Da) as composition of PCMs have been synthesized, and the dynamics between the structures and properties were investigated. The conjugation efficiency of PEI ranged from 77.5-95.4% with relatively higher levels of PEI conjugation to the Pluronic size in PCMs, with the Pluronics of smaller size achieving relatively higher levels of PEI conjugation. Almost all of the PCM polymers were able to bind and condense plasmid DNA effectively into particles of approximately 200 nm in solution at the polymer/DNA ratio of 2 and above. The PCMs composed of relatively moderate size (M w: 2000-5000 Da), intermediate HLB (12-23) of Pluronics, and LPEI produce much higher gene delivery efficacy and less cytotoxicity as compared with PEI 25k in C2C12 myoblasts and CHO cells in vitro. The PCMs were also able to enhance gene delivery in mdx mice in vivo, indicating their potential as biocompatible gene delivery carriers. This journal is © The Royal Society of Chemistry 2012.


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.,Levine Childrens Hospital | 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.

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