CO-TRANSPLANTATION OF PLASMID-TRANSFECTED MYOBLASTS AND MYOTUBES INTORAT BRAINS ENABLES HIGH-LEVELS OF GENE-EXPRESSION LONG-TERM

We have previously proposed the use of primary muscle cells as a ''pla tform,'' or ''vehicle'' for intracerebral transgene expression. Brain grafts of minced muscle, or cultured muscle cells persisted in rat bra ins for at least 6 mo without any decrease in graft size, or tumor for mation. Stable, but moderate levels of intracerebral transgene express ion were obtained by transplanting plasmid-transfected myotubes in cul ture. In the present study, high and stable levels of intracerebral tr ansgene expression were achieved by the co-transplantation of plasmid- transfected myoblasts and myotubes in culture. Approximately 5 x 10(5) myoblasts and myotubes were transfected with 10 mug pRSVL plasmid DNA , and 30 mug Lipofectin (BRL), respectively. They were mixed together (total cell number was 1 million), and stereotactically injected into the caudate nucleus of an adult rat brain. The activity of luciferase, the product of transgene expression, was stable for at least 4 mo, an d much higher than the levels in myotube grafts, or co-grafts of myobl asts and minced muscle. Presumably, the myotubes served as a framework on which the myoblasts can form myotubes. The sections of brains tran splanted with co-graft of myoblasts, and myotubes transfected with pRS VLac-Z were stained immunofluorescently for beta-galactosidase activit y. The muscle grafts contained beta-galaclosidase positive myofibers 4 mo after transplantation. Such high and stable levels of in vivo expr ession after postnatal gene transfer have rarely been achieved. Primar y muscle cells are useful vehicle for transgene expression in brains, and potentially valuable for gene therapy of degenerative neurological disorders.