EFFICIENT GENE DELIVERY AND EXPRESSION IN MAMMALIAN-CELLS USING DNA COUPLED WITH PERFRINGOLYSIN-O

Current non-viral DNA vectors for gene therapy are limited by low cell ular transfection efficiencies and low levels of gene expression due t o inefficient endosomal DNA release. We have used perfringolysin O (PF O), a membrane active bacterial protein, to deliver DNA into cells. PF O belongs to the so-called sulphydryl-activated family of membrane act ive bacterial proteins, which have been used to deliver small molecule s and proteins into cells. PFO was incorporated into DNA complexes thr ough a biotin-streptavidin bridge and the DNA-PFO complexes were used io deliver the Escherichia coli beta-galactosidase gene into cells. Hi gh levels of gene expression were achieved in murine soi 8 myoblast ce lls using these DNA-PFO complexes. The level of gene expression correl ated well with the content of PFO in the complexes. Under optimal cond itions, 15-20% of the cells were stained blue with X-gal. Furthermore, the expression was independent of a receptor ligand. Thus, membrane a ctive bacterial proteins may be an important tool for the future devel opment of nonviral DNA delivery systems for gene therapy.