ADENOVIRUS-MEDIATED TRANSFER OF THE ACID ALPHA-GLUCOSIDASE GENE INTO FIBROBLASTS, MYOBLASTS AND MYOTUBES FROM PATIENTS WITH GLYCOGEN-STORAGE-DISEASE TYPE-II LEADS TO HIGH-LEVEL EXPRESSION OF ENZYME AND CORRECTS GLYCOGEN ACCUMULATION

Glycogen storage disease type II (GSD II) is an autosomal recessive di sorder caused by defects in the lysosomal acid alpha-glucosidase (GAA) gene. We investigated the feasibility of using a recombinant adenovir us containing the human GAA gene under the control of the cytomegalovi rus promoter (AdCMV-GAA) to correct the enzyme deficiency in different cultured cells from patients with the infantile form of GSD II. In GA A-deficient fibroblasts infected with AdCMV-GAA, transduction and tran scription of the human transgene resulted in de novo synthesis of GAA protein. The GAA enzyme activity was corrected from the deficient leve l to 12 times the activity of normal cells, The transduced cells overe xpressed the 110 kDa precursor form of GAA, which was secreted into th e culture medium and was taken up by recipient cells. The recombinant GAA protein was correctly processed and was active on both an artifici al substrate 4-methylumbellifer yl-alpha-D-glucopyranoside (4MUG) and glycogen, In GAA-deficient muscle cells, a significant increase in cel lular enzyme level, similar to 20-fold higher than in normal cells, wa s also observed after viral treatment. The transduced muscle cells wer e also able to efficiently secrete the recombinant GAA, Moreover, tran sfer of the human transgene resulted in normalization of cellular glyc ogen content with clearance of glycogen from lysosomes, as assessed by electron microscopy, in differentiated myotubes, These results demons trate phenotypic correction of cultured skeletal muscle from a patient with infantile-onset GSD II using a recombinant adenovirus, We conclu de that adenovirus-mediated gene transfer might be a suitable model sy stem for further in vivo studies on delivering GAA to GSD II muscle, n ot only by direct cell targeting but also by a combination of secretio n and uptake mechanisms.