EXPRESSION OF HUMAN DYSTROPHIN FOLLOWING THE TRANSPLANTATION OF GENETICALLY-MODIFIED MDX MYOBLASTS

Transplantation of genetically modified autologous myoblasts has been proposed as a possible solution to avoid long-term use of immunosuppre ssive drugs. To determine the conditions to be used in this kind of ap proach for possible treatment of dystrophin deficiency, mdx myoblasts were infected at different multiplicities of infection (MOI of 0.01-10 00) with an adenoviral vector containing a CMV promoter/enhancer drive n 6.3 kb human dystrophin cDNA (minigene) and tested in vitro for tran sgene expression, in these cultures, dystrophin mRNA was found to be p roportionate with increasing MOI. Primary myoblast cultures derived fr om transgenic mdx mice expressing p-Gal under a muscle-specific promot er and showing high expression of the human mini-dystrophin transgene introduced by the adenoviral vector were grafted into anterior tibiali s muscles of SCID mice. Ten and 24 days after transplantation, numerou s muscle fibers expressing both human dystrophin and P-Gal were detect ed throughout the mouse muscles by immunohistochemistry using an antib ody specific for human dystrophin. The presence of the human mini-dyst rophin mRNA was also detected by RT-PCR. These results demonstrate tha t three essential conditions in autologous myoblast transplantation ca n be achieved: (1) in vivo survival of at least some of the transduced myoblasts; (2) efficient fusion of these cells with the host muscle f ibers; and (3) the high expression of the dystrophin transgene in situ . Furthermore, this article provides a novel RT-PCR-based technique to quantify the human dystrophin mint gene expression in vitro and in vi vo.