Although many lysosomal disorders are corrected by a small amount of the missing enzyme, it has been generally accepted that 20-30% of normal acid alpha-glucosidase (GAA) activity, provided by gene or enzyme replacement therapy, would be required to reverse the myopathy and cardiomyopathy in Pompe disease. We have addressed the issue of reversibility of the disease in the Gaa(-/-) mouse model. We have made transgenic lines expressing human GAA in skeletal and cardiac muscle of Gaa(-/-) mice, and we turned the transgene on at different stages of disease progression by using a tetracycline-controllable system. We have demonstrated that levels of 20-30% of normal activity are indeed sufficient to clear glycogen in the heart of young Gaa(-/-) mice, but not in older mice with a considerably higher glycogen load. However, in skeletal muscle-a major organ affected in infantile and in milder, late-onset variants in humans-induction of GAA expression in young Gaa(-/-) mice to levels greatly exceeding wildtype values did not result in full phenotypic correction, and some muscle fibers showed little or no glycogen clearance. The results demonstrate that complete reversal of pathology in skeletal muscle or long-affected heart muscle will require much more enzyme than previously expected or a different approach.

Although many lysosomal disorders are corrected by a small amount of the missing enzyme, it has been generally accepted that 20-30% of normal acid alpha-glucosidase (GAA) activity, provided by gene or enzyme replacement therapy, would be required to reverse the myopathy and cardiomyopathy in Pompe disease. We have addressed the issue of reversibility of the disease in the Gaa(-/-) mouse model. We have made transgenic lines expressing human GAA in skeletal and cardiac muscle of Gaa(-/-) mice, and we turned the transgene on at different stages of disease progression by using a tetracycline-controllable system. We have demonstrated that levels of 20-30% of normal activity are indeed sufficient to clear glycogen in the heart of young Gaa(-/-) mice, but not in older mice with a considerably higher glycogen load. However, in skeletal muscle-a major organ affected in infantile and in milder, late-onset variants in humans-induction of GAA expression in young Gaa(-/-) mice to levels greatly exceeding wildtype values did not result in full phenotypic correction, and some muscle fibers showed little or no glycogen clearance. The results demonstrate that complete reversal of pathology in skeletal muscle or long-affected heart muscle will require much more enzyme than previously expected or a different approach.

When knockout mice are used to test the efficacy of recombinant human proteins, the animals often develop antibodies to the enzyme, precluding long-term pre-clinical studies. This has been a problem with a number of models, for example, the evaluation of gene or enzyme replacement therapies in a knockout model of glycogen storage disease type II (GSDII; Pompe syndrome). In this disease, the lack of acid alpha-glucosidase (GAA) results in lysosomal accumulation of glycogen, particularly in skeletal and cardiac muscle. Here, we report that in a GAA-deficient mouse model of GSDII, low levels of transgene-encoded human GAA expressed in skeletal muscle or liver dramatically blunt or abolish the immune response to human recombinant protein. Of two low expression transgenic lines, only the liver-expressing line exhibited a profound GAA deficiency in skeletal muscle and heart indistinguishable from that in the original knockouts. The study suggests that the induction of tolerance in animal models of protein deficiencies could be achieved by restricting the expression of a gene of interest to a particular, carefully chosen tissue.