Inherited deficiency of lysosomal acid maltase (GAA) results in glycogen storage disease type II (GSD-II) or acid maltase deficiency. Infantile- onset patients (Pompe's disease) present with massive accumulation of glycogen in cardiac and skeletal muscle, resulting in death in the first year of life. In contrast, adult-onset patients have manifestations limited to skeletal muscle. Currently, there is no treatment or cure for the disease. Gene therapy for diseases that affect cardiac and skeletal muscles or organs (such as GSD-II) may be very difficult because of size, location and transfection efficiency. In ongoing studies in normal mice and in an affected bovine model, we have utilized a vector carrying the cytomegalovirus (CMV) promoter and the human GAA cDNA combined with liposome-complexed DNA in attempts to correct the defect. We demonstrated that the plasmid is taken up by tissues, including skeletal and heart muscle, for three months after a single infusion. However, no control or regulation for over-expression is possible with this system. We therefore propose to develop a muscle- specific regulatory vector system that will control for over-expression and tissue specificity. We will construct a series of vectors containing the cytomegalovirus or creatine kinase or heavy chain myosin promoters-enhancers, plus a prokaryotic regulatory element that """"""""turns off"""""""" transcription in the presence of doxycycline (Dox). We will subclone the human GAA cDNA into each vector. We will also transfect the minigene(s) into human GSD-II fibroblast and muscle cells, plus murine and human tissue culture cells, including muscle, skin and lymphoid cells. Cells will be cultured with and without various levels of Dox. Human GAA transcription, translation and expression will be assessed by GAA enzyme activity, GAA protein by rocket immunoelectrophoresis (RIE) and Western analysis and human GAA mRNA by Northern analysis. The laboratory will be used for oligonucleotide synthesis, DNA sequencing, DNA isolation, RNA isolation, PCR, Northern and Southern analysis, ultracentrifugation, and recombinant DNA techniques.
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