Intercellular transfer of the virally derived precursor form of acid alpha-glucosidase corrects the enzyme deficiency in inherited cardioskeletal myopathy Pompe disease

Pompe disease is a lethal cardioskeletal myopathy in infants and results fr om genetic deficiency of the lysosomal enzyme acid alpha -glucosidase (GAA) , Genetic replacement of the cDNA for human GAA (hGAA) is one potential the rapeutic approach, Three months after a single intramuscular injection of 1 0(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level, Direct transduction of a target mu scle, however, may not correct all deficient cells, Therefore, the amount o f enzyme that can be transferred to deficient cells from virally transduced cells was studied, Fibroblasts from an affected patient were transduced wi th Ad-hGAA, washed, and plated on transwell culture dishes to serve as dono rs of recombinant enzyme, Deficient fibroblasts were plated as acceptor cel ls, and were separated from the donor monolayer by a 22-mum pore size filte r, Enzymatic and Western analyses demonstrate secretion of the 110-kDa prec ursor form of hGAA from the donor cells into the culture medium, This recom binant, 110-kDa species reaches the acceptor cells, where it can he taken u p by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks t o lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme, Patient fibroblasts receiving re combinant hGAA by this transfer mechanism reach levels of enzyme activity t hat are comparable to normal human fibroblasts. Skeletal muscle cell cultur es from an affected patient were also transduced with Ad-hGAA, Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunoc ytochemistry, and enzyme activity is transferred to deficient skeletal musc le cells grown in coculture, Transfer of the precursor protein between musc le cells again occurs via mannose 6-phosphate receptors, as evidenced by co mpetitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA- knockout mice demonstrate that hepatic transduction with adenovirus encodin g either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism, Taken to gether, these data show that the mannose 6-phosphate receptor pathway provi des a useful strategy for cell-to-cell distribution of virally derived reco mbinant GAA.