Regulation of N-acetylgalactosamine 4-sulfatase expression in retrovirus-transduced feline mucopolysaccharidosis type VI muscle cells

As a preliminary step toward muscle-mediated gene therapy in the mucopolysa ccharidosis (MPS) type VI cat, we have analyzed the transcriptional regulat ion of feline N-acetylgalactosamine 4-sulfatase (f4S) gene expression from various retroviral constructs in primary cultures of muscle cells. Two retr oviral constructs were made containing the f4S cDNA under the transcription al control of the human polypeptide chain-elongation factor 1 alpha (EF1 al pha) gene promoter or the cytomegalovirus (CMV) immediate-early promoter. T wo further retroviral constructs were made with the murine muscle creatine kinase (mck) enhancer sequence upstream of the internal promoter. Virus mad e from each construct was used to transduce feline MPS VI myoblasts. The mc k enhancer significantly upregulated f4S gene expression from both the EF1 alpha promoter and the CMV promoter in transduced myoblasts and in differen tiated myofibers. The highest level of 4S activity was observed in myoblast s and myofibers transduced with the retroviral construct L<(mck)under bar>c mv4S, in which the f4S gene is under the transcriptional regulation of the mck enhancer and CMV immediate-early promoter. L<(mck)under bar>cmv4S-trans duced myofibers demonstrated correction of glycosaminoglycan storage and co ntained a 58-fold elevated level of 4S activity compared with normal myofib ers. Recombinant f4S secreted from L<(mck)under bar>cmv4S-transduced myofib ers was endocytosed by feline MPS VI myofibers, leading to correction of th e biochemical storage phenotype.