Transfection of large plasmids in primary human myoblasts

The ex vivo gene therapy approach for Duchenne muscular dystrophy is promis ing since myoblast transplantation in primates is now very efficient. One o bstacle to this treatment is the low transfection efficiency of large DNA c onstructs in human primary myoblasts. Small plasmids can be easily transfec ted with the new phosphonolipid described in this study, However, a dramati c drop in transfection efficiency is observed with plasmids of 12 kb or mor e containing EGFP minidystrophin and EGFP dystrophin fusion genes. The tran sfection of human primary myoblasts with such large plasmids could only be achieved when the DNA was linked to an adenovirus with the use of polyethyl enimine (PEI), with efficiencies ranging between 3 and 5% of transitory tra nsfection. Branched 2 kDa PEI was less toxic in PEI adenofection than branc hed 25 kDa PEI or linear 22 kDa PEI. The adenovirus was an absolute necessi ty for an efficient transfection. An integrin-binding peptide, a nuclear lo calization signal peptide, chloroquine, glycerol or cell cycle synchronizat ion using aphidicolin did not enhance PEI adenofection. Following PEI adeno fection, the adenoviral proteins were detected using a polyclonal antibody. The detected antigens fell below the detectable level after 12 days in cul ture. 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.