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Svyatoslav Silin
Svyatoslav Silin

Mature Girdle Models

There are more than 40 known muscular dystrophies caused by genetic mutations that affect muscle function. These disorders have variable ages of onset and clinical severity, but most often result in severe physical disabilities and premature death. Over what can be decades, patients with muscular dystrophy experience progressive muscle weakness and decreased mobility, making everyday tasks difficult and often impossible to perform even during early disease. Overall, there are fewer than 200,000 cases of muscular dystrophy diagnosed each year in the United States, but this prolonged disease progression places a significant long-term burden on patients and their families and the health care system.

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The primary benefit of iMyoblasts is their ability to regenerate, or multiply, to create more progenitor cells in addition to differentiating into mature muscle cells. More than 25 years ago, scientists unraveled the biology behind how embryos make mature muscle. Another leap forward came about 10 years ago when methods were developed to produce differentiated muscle from patient iPCSs. But these earlier technologies were limited in their ability to make muscle stem cells that can both differentiate and regenerate muscles. Their utility in the laboratory and clinic, where organisms need to respond to injury and age, were hampered by these constraints.

In contrast, skin cells taken from a patient with any form of muscular dystrophy can be turned into iMyoblast progenitor cells. When iMyoblasts are then engrafted into animal models, they give rise to adult human muscle cells with the muscular-dystrophy-causing mutation. These disease models are key to developing new therapeutics with the potential to treat muscular dystrophies.

Using iMyoblasts, Emerson and his group were able to develop animal models for four distinct forms of muscular dystrophy: facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy types R7 and R9, and Walker-Warburg syndrome. These models successfully replicated the molecular disease pathologies of the disorders and were responsive to small molecule and gene editing therapeutics.

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The Pacific flatheaded borerprefers weak or injured trees. Newly planted or grafted trees are particularlysusceptible to attack because of the stress caused by planting and thepossibility of sunburn on the bark and at the bud union. The larvae feed in thecambium and can completely girdle and kill young or newly grafted trees in ashort period of time. Limbs of older trees (particularly if sunburned) are alsoattacked, but they rarely die from flatheaded borer attack. However, olderlimbs can be weakened to such an extent that other borers such as the shotholeborer can attack successfully.

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Duchenne muscular dystrophies (DMD) and Limb-girdle muscular dystrophies (LGMD) are common inherited degenerative muscle diseases caused by mutations in genes coding for memberance associated proteins in muscle cells. DMD and LGMD often manifest themselves in young age and lead to severe morbidity and fatality, with no currently available effective treatment. In addition, the diseases are usually genotypically recessive, which makes them suitable for gene replacement therapy with vectors. Recombinant adeno-associated virus (rAAV) is one such vector based on defective human parvoviruses. rAAV system has attracted attention due to its non-pathogenicity, genomic integration, transduction of quiescent cells, and apparent lack of cellular immune reactions. In contrast to other viral vectors, rAAV is capable of efficiently bypassing the myofiber basal lamina and tranducing mature muscle cells. We have demonstrated that rAAV vectors harboring a foreign gene can achieve highly efficient and sustained gene transfer in mature muscle of immunocompetent animals for more than 1.5 years without detectable toxicity. Recently, significant improvement in vector production methodology has made it possible to generate high titer and high quality rAAV vectors completely free of helper adenovirus contamination. However, no experiments using rAAV vectors to restore the functional deficits in muscle tissue itself have been reported to date. In this proposal, we will use delta-SG as the target disease gene, the delta-SG deficient hamster as the LGDM animal model, and rAAV as the gene delivery vector to test our general hypothesis that safe, efficient and sustained functional rescue of muscle deficiency can be achieved by genetic complementation of inherited muscular dystrophies with rAAV vectors. Specifically, we would like to achieve the following aims: 1) To study gene transfer efficiency and functional rescue in the LGMD hamster model by local intramuscular delivery of delta-SG-rAAV vectors and examine their short term ability to correlate the genetic defect in both skeletal and cardiac muscle. 2) To evaluate the gene therapy efficacy after systemic delivery of rAAV vectors through intra-artery or intra-ventricle injection. 3) To investigate the molecular kinetics and fate of rAAV vectors, especially after systemic vector delivery. 4) To develop new generation, high titer, helper-virus-free rAAV producer cells, which not only harbor vector and packaging genes, but also contain the necessary helper genes from adenovirus in a highly regulated and inducible manner.

Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies. Here, we developed a dystroglycanopathy mouse model using inducible or muscle-specific promoters to conditionally disrupt fukutin (Fktn), a gene required for dystroglycan processing. In conditional Fktn-KO mice, we observed a near absence of functionally glycosylated dystroglycan within 18 days of gene deletion. Twenty-week-old KO mice showed clear dystrophic histopathology and a defect in glycosylation near the dystroglycan O-mannose phosphate, whether onset of Fktn excision driven by muscle-specific promoters occurred at E8 or E17. However, the earlier gene deletion resulted in more severe phenotypes, with a faster onset of damage and weakness, reduced weight and viability, and regenerating fibers of smaller size. The dependence of phenotype severity on the developmental timing of muscle Fktn deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional Fktn-KO mouse allows the generation of tissue- and timing-specific defects in dystroglycan glycosylation, avoids embryonic lethality, and produces a phenotype resembling patient pathology, it is a promising new model for the study of secondary dystroglycanopathy. 041b061a72


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