Transfection of large plasmids in primary human myoblasts.

The ex vivo gene therapy approach for Duchenne muscular dystrophy is promising since myoblast transplantation in primates is now very efficient. One obstacle to this treatment is the low transfection efficiency of large DNA constructs in human primary myoblasts. Small plasmids can be easily transfected with the new phosphonolipid described in this study. However, a dramatic drop in transfection efficiency is observed with plasmids of 12 kb or more containing EGFP minidystrophin and EGFP dystrophin fusion genes. The transfection of human primary myoblasts with such large plasmids could only be achieved when the DNA was linked to an adenovirus with the use of polyethylenimine (PEI), with efficiencies ranging between 3 and 5% of transitory transfection. Branched 2 kDa PEI was less toxic in PEI adenofection than branched 25 kDa PEI or linear 22 kDa PEI. The adenovirus was an absolute necessity for an efficient transfection. An integrin-binding peptide, a nuclear localization signal peptide, chloroquine, glycerol or cell cycle synchronization using aphidicolin did not enhance PEI adenofection. Following PEI adenofection, the adenoviral proteins were detected using a polyclonal antibody. The detected antigens fell below the detectable level after 12 days in culture. We thus provide in this study an efficient and reproducible method to permit efficient delivery of large plasmids to human primary myoblasts for the ex vivo gene therapy of Duchenne muscular dystrophy.