Retroviral Transfer of Acidα-Glucosidase cDNA to Enzyme-Deficient Myoblasts Results in Phenotypic Spread of the Genotypic Correction by Both Secretion and Fusion

Abstract

Myoblasts have properties that make them suitable vehicles for gene replacement therapy, and lysosomal storage diseases are attractive targets for such therapy. Type II Glycogen Storage Disease, a deficiency of acid α-glucosidase (GAA), results in the abnormal accumulation of glycogen in skeletal and cardiac muscle lysosomes. The varied manifestations of the enzyme deficiency in affected patients are ultimately lethal. We used a retroviral vector carrying the cDNA encoding for GAA to replace the enzyme in deficient myoblasts and fibroblasts and analyzed the properties of the transduced cells. The transferred gene was efficiently expressed, and the de novo-synthesized enzyme reached lysosomes where it digested glycogen. In enzyme-deficient myoblasts after transduction, enzyme activity rose to more than 30-fold higher than in normal myoblasts and increased about five-fold more when the cells were allowed to differentiate into myotubes. The transduced cells secreted GAA that was endocytosed via the mannose-6-phosphate receptor into lysosomes of deficient cells and digested glycogen. Moreover, the transduced myoblasts were able to fuse with and provide enzyme for GAA-deficient fusion partners. Thus, the gene-corrected cells, which appear otherwise normal, may ultimately provide phenotypic correction to neighboring GAA-deficient cells by fusion and to distant cells by secretion and uptake mechanisms. The metabolic disorders affecting muscle cells may be corrected by gene transfer. In this study myoblasts from a patient with acid maltase deficiency (Glycogen Storage Disease Type II) were efficiently transduced by recombinant retrovirus. A high level of expression of acid maltase (GAA) was observed in gene-corrected cells that demonstrated phenotypic normalization when compared to normal control cells. Furthermore, we found evidence that the transduced myoblasts could supply GAA enzymatic activity to GAA-deficient muscle cells by both secretion and fusion mechanisms, providing the basis for a phenotypic spread of the genotypic correction.