Gene Therapy (1997) 4, 488–492  1997 Stockton Press All rights reserved 0969-7128/97 $12.00 BRIEF COMMUNICATION Gene transfer and expression of human a-galactosidase from mouse muscle in vitro and in vivo FJ Novo 1,2,3 , DC Go ´recki 1 , G Goldspink 2 and KD MacDermot 1 1 Department of Clinical Genetics and 2 Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK Lysosomal storage disorders are amenable to treatment by rabbit b-myosin heavy chain promoter, or containing the enzyme replacement. Genetic modification of muscle via CMV promoter only. Increased enzymatic activity was direct injection of expression vectors might represent an detectable both in cell extracts and in supernatants. Fur- alternative method of providing the defective enzymes, if thermore, human fibroblasts deficient in a-gal were able to adequate and long-lasting expression levels can be ach- take up the enzyme from medium conditioned by trans- ieved in muscle. We have used the C2C12 mouse myo- fected myoblasts. This did not occur in the presence of genic cell line to study the effect of combination of muscle- mannose-6-phosphate which indicates that the uptake was specific regulatory elements on the expression of the via mannose-6-phosphate receptors. To our knowledge, human lysosomal enzyme a-galactosidase (a-gal). In dif- this is the first report in which a correctly processed form ferentiated myotubes, a construct containing the myosin of human a-gal was expressed and secreted from differen- light chain 1/3 enhancer in combination with the human tiated muscle cells. Direct injection of a plasmid expression cytomegalovirus promoter resulted in higher expression vector into mouse tibialis anterior muscle showed signifi- than constructs combining the same enhancer with the cantly increased levels of a-gal 7 days after injection. Keywords: a-gal; muscle expression vectors; direct muscle injection Anderson–Fabry disease is a lysosomal storage disorder enzyme replacement in the 1970s which demonstrated the feasibility of enzyme therapy for Fabry disease. 1 (LSD) resulting from the deficiency of the lysosomal enzyme a-gal (EC 3.2.1.22). This enzymatic defect leads However, the unavailability of sufficient amounts of the purified human enzyme has prevented a proper evalu- to the deposition of neutral glycosphingolipids in most tissues, the pathological and clinical manifestations of the ation of the efficacy of replacement therapy so far. Alternative ways of providing a source of active disease being the result of progressive accumulation in endothelial cells leading to ischemia and infarction in enzyme for the treatment of LSD have included bone marrow transplantation and, more recently, gene transfer organs like kidney, heart or brain. 1 In addition to the sorting mechanisms operating in the into haematopoietic stem cells or enzyme delivery into the whole organism by genetically modified cells. 4 For trans-Golgi network, lysosomal enzymes can also be recaptured from the extracellular space via mannose-6- instance, it has been recently shown that fibroblasts trans- fected with retroviral vectors and grown on collagen lat- phosphate receptors. 2 In keeping with this, it has been shown that the administration of purified lysosomal tices which were implanted in the peritoneal cavity suc- cessfully secreted b-glucuronidase and corrected the enzymes to the culture medium can correct the enzymatic defect in fibroblasts from patients with various types of storage lesions in the liver and spleen of mucopolysacch- aridosis VII mice. 5 The same approach resulted in long- LSD. 3,4 This ability of cells to take up the enzyme has provided the basis for the use of replacement therapy for term secretion of this enzyme in dogs 6 and similar results were obtained in nude mice transplanted with neo- this group of disorders. In the case of Fabry disease, early studies showed that a-gal partially purified from various organs which were secreting a-l-iduronidase. 7 Since the discovery that skeletal muscle can be trans- sources is taken up by skin fibroblasts from Fabry hemi- zygotes when added to the culture medium and does fected in vivo by intramuscular injection of plasmid DNA, 8–10 this organ system has attracted considerable catabolize the accumulated substrate, globotriaosyl- ceramide (CTH). This prompted several clinical trials of attention as a potential source of secreted therapeutic proteins. Injection of plasmid DNA constructs has been used successfully for the expression of dystrophin, 11 fac- tor VII, 12 apolipoprotein-E 13 and a-1 antitrypsin, 14 Correspondence: FJ Novo whereas intramuscular injection of genetically modified 3 Present address: Departamento de Gene ´tica Universidad de Nav- myoblasts gave encouraging results in the secretion of arro, 31080 Pamploma, Spain Received 5 November 1996; accepted 8 January 1997 human growth hormone, 15 factor IX, 16 b-glucuronidase, 17