GENE-TRANSFER IN REGENERATING MUSCLE

We have compared the efficiency of direct gene transfer in normal and regenerating rat skeletal muscle. Muscle necrosis and regeneration was induced by intramuscular injection of bupivacaine in the soleus muscl e of adult rats. Plasmids containing beta-galactosidase (beta-gal) or chloramphenicol acetyltransferase (CAT) genes driven by viral promoter s were injected 3 days after bupivacaine treatment into the regenerati ng and the contralateral uninjured muscles. Expression of CAT activity was > 80-fold higher in regenerating compared to control muscles at 7 days post-transfection, but decreased at 30 and 60 days. Southern blo t analysis showed that the predominant form of CAT DNA was episomal in transfected muscles; however, CAT activity measurements performed on the same transfected muscles showed no precise correlation between enz ymatic activity and amount of plasmid DNA. Expression of beta-gal was detected in numerous regenerating fibers of the injured soleus muscles at 7 days post-transfection; in contrast, only rare positive fibers w ere found in control muscles. Focal infiltrates of mononuclear cells, which surround and invade selectively beta-gal-positive fiber segments , were observed at 30 days post-transfection, suggesting that immune m echanisms are implicated in the progressive loss of transgenes with ti me. The finding that regenerating muscle fibers display a higher effic iency of transfection may be relevant to gene therapy of Duchenne musc ular dystrophy, because regenerating fibers are numerous in the early stages of the disease.