Adenovirus-mediated delivery into myocytes of muscle glycogen phosphorylase, the enzyme deficient in patients with glycogen-storage disease type V.
The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease).
Baqué
S
S
Department de Bioquímica i Fisiologia, Universitat de Barcelona, Spain.
Newgard
C B
CB
Gerard
R D
RD
Guinovart
J J
JJ
Gómez-Foix
A M
AM
D000256:Adenoviridae / Q000235:genetics*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006003:Glycogen / Q000378:metabolism; D006012:Glycogen Storage Disease Type V / Q000201:enzymology*; D006801:Humans; D009132:Muscles / Q000166:cytology / Q000201:enzymology* / Q000502:physiology*; D006005:Phosphorylases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D006003:Glycogen; D006005:Phosphorylases