A number of acquired and inherited metabolic diseases are currently treated with repeated subcutaneous or intravenous infusions of purified or recombinant proteins. These include hemophilias A and B, treated with purified factors VIII and IX, pituitary dwarfism, treated with recombinant growth hormone, diabetes mellitus, treated with insulin, and the erythropoietin-responsive anemias, which are treated with recombinant human erythropoietin.(hEpo). Recent evidence strongly suggests that additional inherited metabolic disorders including Fabry's disease, al-antitrypsin deficiency, and mucopolysaccharidoses I and VI could also be successfully treated by delivery of therapeutic proteins to the systemic circulation. Because each of these diseases requires life-long therapy, the development of a cellular transplantation system which could be used to produce stable levels of circulating serum proteins would represent a significant advance in the treatment of such acquired and inherited metabolic diseases. We have recently demonstrated that genetically modified murine myoblasts can be used to stably produce physiological levels of recombinant proteins in the systemic circulation following intramuscular (IM) injection into normal syngeneic mice. In the studies described in this application, we propose to use the erythropoietin-responsive anemias as an animal model system to explore the feasibility of using genetically modified myoblasts for the treatment of acquired and inherited metabolic diseases. Specifically, we will (i) construct both plasmid and retroviral vectors that program high level expression of hEpo in skeletal myoblasts and myotubes in vitro, (ii) produce primary human myoblasts that synthesize high levels of hEpo and bacterial lacZ in vitro and (iii) optimize the protocol for implantation of these genetically modified human myoblasts into the muscle of SCID mice, and (iv) determine the effects on reticulocyte counts and hematocrits of IM implantation of hEpo-secreting primary human myoblasts in SCID mice for up to one year following IM implantation. Following optimization of this approach in the rodent model, we will perform an efficacy and safety study in rhesus monkeys. Briefly, primary rhesus myoblasts will be transfected with an hEpo expression vector and implanted by IM injection into the monkeys from which they were originally isolated. The efficacy of this system will be determined for periods of up to 6 months following implantation using measurements of serum hEpo levels, hematocrits and reticulocyte counts. In addition, the safety of this approach will be carefully assessed. The results of these studies should be directly applicable to the treatment of the erythropoietin-responsive anemias associated with both HIV infection and chronic renal failure, diseases affecting at least 150,000 Americans. In addition, they will have important implications for the treatment of a large number of inherited metabolic diseases.
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