Human muscle-type phosphofructokinase (M-PFK) deficiency, also known as glycogenosis type VII, is characterized by a metabolic myopathy and erythroenzymopathy. As with other inherited myopathies, there is currently no effective therapy available. We have identified a naturally-occurring disease model in dogs, established an animal colony, characterized the clinicopathologic features and molecular defect (point mutation). and demonstrated the close homology between canine and human M-PFK deficiency. The overall objectives of the proposed research are to develop and evaluate the safety and efficacy of muscle gene transfer as a therapeutic strategy for inherited myopathies in this unique large animal model. The pathogenesis of M-PFK deficiency will be further investigated at the molecular level. The functional and structural effects of the M-PFK mutation will be investigated by expressing normal canine and mutant full length M-PFK cDNAs in cell culture systems. We will also characterize the cis-acting elements of the M-PFK gene in muscle cells in vitro that determine developmental and tissue-specific expression. We will perform myoblast transfer by transplanting genetically normal myogenic cells into discrete muscles of M-PFK deficient dogs. Normal muscle satellite cells will be isolated, cultured, and transplanted into growing or previously injured muscle. We will evaluate the safety of this procedure and determine the ability of donor cells to survive and function in the recipient muscle with biochemical, histochemical, immunologic and molecular studies. We will also employ non-invasive techniques, e.g., magnetic resonance and near infrared spectroscopy to study repeatedly the efficacy of treatments on muscle function in vivo. Bone marrow transplantation will also be performed in M-PFK deficient dogs to determine the effects of correcting the erythroenzymopathy on muscle metabolism. Secondly, we will develop somatic cell gene transfer into M-PFK deficient muscle cells. We will use retroviral vectors to transfer the normal M-PFK gene into cultured myoblasts or hematopoietic stems prior to in vivo transplantation and evaluate the level, duration, and function of the transfected M-PFK expression. Another approach is to inject free or vector-encoded M-PFK DNA with regulatory elements directly into deficient muscle. The results of these therapeutic strategies in this unique well-defined animal model will provide basic knowledge of transferred gene expression and tissue-specific regulation that can be applied to human patients with M-PFK deficiency and a variety of other myopathies.
