GENE-TRANSFER AND EXPRESSION OF HUMAN ALPHA-GALACTOSIDASE FROM MOUSE MUSCLE IN-VITRO AND IN-VIVO

Lysosomal storage disorders are amenable to treatment by enzyme replac ement. Genetic modification of muscle via direct injection of expressi on vectors might represent an alternative method of providing the defe ctive enzymes, if adequate and long-lasting expression levels can be a chieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase ( alpha-gal). In differentiated myotubes, a construct containing the myo sin light chain 1/3 enhancer in combination with the human cytomegalov irus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter or containing the CMV promoter only. Increased enzymatic activity was det ectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme fro m medium conditioned by transfected myoblasts. This did not occur in t he presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the firs t report in which a correctly processed form of human alpha-gal was ex pressed and secreted from differentiated muscle cells. Direct injectio n of a plasmid expression vector into mouse tibialis anterior muscle s howed significantly increased levels of alpha-gal 7 days after injecti on.