Recombinant proteins are used to treat a number of human disorders including diabetes, neutropenia, anemia, and musculoskeletal disorders of the elderly. Muscle atrophy and bone wasting associated with aging can be attenuated using growth hormone (GH), insulin-like growth factor-1 (IGF-1), and parathyroid hormone (PTH) but delivery by daily injection is problematic since the proteins degrade rapidly in vivo, are expensive to manufacture, and have detrimental side effects when delivered at pharmacological doses. Cell based delivery of proteins from genetically-modified implanted cells may provide a more effective and cost-saving alternative. The long-term objective of this project is to develop and optimize the surgical techniques for reversible delivery of proteins from bioartificial muscle platforms. Muscle stem cells (myoblasts) can be isolated by simple needle biopsy and genetically modified to express foreign proteins. When tissue engineered in vitro into skeletal muscle-like bioartificial muscles (BAMs) and implanted in vivo, they serve as a long-term delivery system for biologically active proteins. Advantages of this technology over currently used injected myoblasts or plasmid DNA gene therapy techniques include efficient in vitro fusion of myoblasts into BAMs, preimplantation monitoring of growth factor secretion levels, and reversibility. BAMs secreting recombinant human GH (rhGH) and engineered from a murine C2C12 muscle cell line successfully attenuate skeletal muscle disuse atrophy when implanted subcutaneously under tension in mice. In the current project, primary myoblasts from inbred Fisher 344 rats will be transduced to constitutively express rhGH or IGF-1 using retroviral expression constructs under the control of the LTR viral promoter. New replication defective retrovirus expression vectors with the GH, IGF-1, and PTH genes under control of the regulatable human skeletal alpha -actin (HSA) promoter will also be constructed. BAMs from transduced primary rat myoblasts will be engineered using previously developed protocols and their morphology and protein secretion rates evaluated in vitro and in vivo. Rat BAM (R-BAM) myofiber survival, differentiation, innervation, and vascularization in subcutaneous and muscular sites will be studied by quantitative histological, immunocytochemical and biochemical techniques. The ability of GH, IGF-1 and PTH secreted from R-BAMs to attenuate muscle atrophy and bone wasting will be assessed in hindlimb unloaded adult Fisher rats. New therapeutic treatments with recombinant proteins for chronic musculoskeletal wasting disorders will lead to enhanced quality of life and reduced costs in this 150 billion dollar annual U.S. healthcare market.
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