Transduction, Expression, and Secretion of Human Glucocerebrosidase by Murine Myoblasts

However, the treatment is expensive.Enzyme infusion at a dose of 60 units per kilogram of body weight costs $14,700 per infusion for a 70-kg adult.At the recommended frequency of one infusion every 2 weeks, the annual cost per patient for the enzyme alone is $382,200.The alternative treatment of allogeneic bone marrow transplantation is unavailable to most patients, as only 25 to 30% of patients have an =-matched sibling.It is also a high-risk procedure, with associated mortality varying from 5 to 40%.The availability of a cDNA and efficient vectors makes somatic gene therapy an exciting prospect for Gaucher disease.The target cells for gene transfer are hematopoietic stem cells with the potential to provide a long-term source of enzyme-competent macrophages in the patient.An alternative approach is a cellular transport system that can stably produce and deliver the recombinant enzyme into the systemic circulation, where it would be available for uptake by macrophages, the pathobiologically important cell in Gaucher disease.Normally, glucocerebrosidase is synthesized as a single polypeptide (58 kDa) with a signal sequence (2 m a ) at the amino terminal.2Cotranslationally the signal sequence is cleaved off and the enzyme is core glycosylated.This "high mannose" precursor is posttranslationally processed in the Golgi apparatus to a 66-kDa intermediate and further tailored in the lysosome to the mature enzyme (59 m a ) .The mature enzyme is membrane bound, and very little is secreted extracellularly.However, when a cell is producing enzyme at a rate several times normal, as could be expected of a successfidly transduced cell with a high copy number, the posttranslational processes of oligosaccharide side chain modification and membrane incorporation would be overloaded, leading to extracellular secretion of the surplus enzyme, a significant fraction of which may be expected to be in the "high mannose" precursor form.The ideal recombinant protein delivery system would use a cell that can be easily isolated from the recipient, grown in culture, transduced in vi m, and reimplanted in the host.These genetically modified cells should survive for long periods of time and synthesize large quantities of the recombinant protein available to the pathobiologically important sites.Myoblasts are an excellent candidate for such a system.Myoblasts can be readily isolated from muscle biopsies, expanded in culture, and transduced in pitro.3Myoblasts injected intramuscularly survive and fuse into adjacent muscle fibers at the site of injection.*As skeletal muscle is highly vascular, proteins secreted from myoblasts readily enter the circulation.In vim and in vivo expression of the transferred gene have been demonstrated in mouse myoblast cell line C2C12.536To explore the feasibility of developing a "myogenic organoid" for use in Gaucher disease, we transduced murine C2C12 myoblasts with human GC cDNA using a replication-incompetent retrovirus vector, MFG-GC.The transduced myoblasts were transplanted syngeneically into C3H mice for studies of in vivo expression.Here we report the efKciency of the retrovirus vector MFG-GC in transducing murine myoblasts with the human glucocerebrosidase gene, and expression of the transferred gene in vim and in vivo, and in extracellular secretion of the recombinant enzyme.