In the course of studying inflammatory muscle diseases (polymyositis, dermatomyositis, and related diseases), we have encountered patients with many other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and acid maltase (acid alpha-glucosidase) deficiency (also known as Glycogen Deficiency Type II or GSD II or Pompe Syndrome). For the last several years, we have focused particular attention on GSD II because of its close resemblance to myositis. It is a recessively inherited lysosomal storage disease in which glycogen accumulates in the lysosomes, particularly those in skeletal muscle. When the enzyme, known also as GAA, is completely absent, affected infants are usually sick at birth and die in infancy of heart failure, rarely living longer than a year. Apparently the enzyme is needed in the heart only in infancy since affected individuals with even a small amount of effective enzyme survive without cardiac involvement. Survivors generally develop a progressive proximal myopathy with pulmonary failure secondary to diaphragmatic involvement in later years. The long-term aim of our studies is to prevent and to treat this devastating disease, particularly the adult variety, in which the level of enzyme is only slightly (less than two-fold) below the minimum necessary for a normal life. From quite early in the project, it was driven by the belief that GSDII, being a lysosomal storage disease, should be amenable to enzyme replacement and possibly gene therapy, and since there are well over 30 diseases of abnormal lysosomal storage, our results might have wider application. There are estimated to be several thousand cases of GSD II worldwide, and hundreds in the United States, of which many are fatal in infancy (Pompe Syndrome). Our guiding plan has been to do research directed towards therapy, but without trying to move into areas likely to be covered by pharmaceutical companies.
We aim ed to develop tools that would advance the development of therapy while at the same time learning new biology or developing techniques that might be applicable to other lysosomal or other enzyme deficiency diseases, especially those in muscle. We have followed lines of research designed to provide answers necessary for the development of optimal therapy for GSD II. Starting with the knockout mice, a human GAA expression vector was put in transgenically under the control of either a skeletal muscle or a liver specific promoter controllable by doxicycline [164]. These mice were designed to determine whether the liver, a much easier organ to target, could serve as a suitable depot organ for the synthesis and secretion of GAA that could be taken up by the heart and by skeletal muscle. We discovered that the liver was a superior site to the skeletal muscle for the repair of both heart and skeletal muscle. [164] The achievement of therapeutic levels with skeletal muscle transduction required the entire muscle mass to produce high levels of enzyme of which little found its way to the plasma, whereas liver, comprising <5% of body weight, secreted 100-fold more enzyme, all of which was in the active 110 kDa precursor form. In the past year, we have continued studies on the therapy of the mouse disease. A major step forward was the development of a mouse model that could be used for pre-clinical study of the effects of long term therapy of the disease by repeated injection of the recombinant human enzyme. The barrier to such study has been the immunological response of the mice to the foreign human protein. After a few doses, the animals die of what appears to be anaphylactic shock. In the experiments described above, we developed a strain of mice that produces a minute amount of GAA in the liver ? an amount below the level of biochemical detection, but the mRNA is detectable by PCR. The liver glycogen is very slightly reduced, but the glycogen in other tissues is unaffected so that the mice are phenotypically unchanged. These mice develop no detectable immune response and can receive weekly injections of rhGAA for many months at low or high dose and have no adverse immunological effects. These mice, immunologically speaking, resemble those human infants who have tiny levels of the enzyme and are thereby tolerant to injection of the recombinant protein. The work on this strain has been published, and the mice have been distributed to many investigators and are now used for pre-clinical studies [173]. Our own studies with endogenous expression of the GAA gene in the skeletal muscle {172] and with the intravenous injection of high doses of rhGAA over long periods have shown the remarkable and discouraging finding that unless the enzyme is turned on (controllable transgene) early or the injections are begun early, the stored glycogen in skeletal muscle is only incompletely removed, and clinical recovery of muscle strength is marginal. By contrast, the glycogen stored in cardiac muscle is cleared well. This finding accords with the small clinical experience so far in human infants, in which heart failure is reversed, but skeletal muscle strength has responded little or not at all. This failure of response has now become a major focus of our work. We have discovered that clearance tends to be very good in Type I skeletal fibers and very poor in Type II fibers. We are exploiting the different proportions of these two types of muscle in different fibers to study the possible reasons for this imbalance. The amount of the mannose-6-phosphate receptor is clearly lower in Type II fibers than in Type I fibers or heart muscle, and so are the levels of several other proteins in the lysosomal pathway. We are, with the collaboration of the NIAMS Light Imaging Unit, developing a technique to measure the pH of lysosomes in living, unfixed single muscle fibers in order to determine whether the pH in lysosomes with a large quantity of stored glycogen in Type II fibers is optimal for GAA. We are exploring the difference in gene expression between the two types of muscle fiber in Pompe mice of various ages to look for pathogenetic clues as well as to look for therapeutic avenues. In other studies, we have been working for the past year with the Rapoport lab in NIA to attempt to deliver therapeutic levels of GAA across the blood brain barrier to clear CNS neurons of the large quantities of stored glycogen seen in Pompe mice, and which it is presumed will accumulate in the brains of Pompe infants who survive infancy with the help of exogenous enzyme replacement.
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