EFFICACY OF A PEPTIDE-BASED GENE DELIVERY SYSTEM DEPENDS ON MITOTIC-ACTIVITY

We have developed and tested a transfection compound based on syntheti c peptides. It consists of a 12 amino acid DNA binding peptide (P2) wi th an alkyl group added to the aminoterminus (P2lip) and a peptide der ived from the hemagglutinin protein (HA). The components aggregate spo ntaneously to particles that proved to be an efficient, easy to use an d chemically stable transfection compound. With this system we found a marked correlation between transfection efficiency and mitotic activi ty. Cells that are allowed to perform a mitosis after exposure to eith er DNA-P2lip/HA or DNA-cationic liposome complexes are transfected muc h more efficiently than cells arrested in the cell cycle. In search of an explanation for this phenomenon we studied transport of plasmid DN A across the nuclear membrane. Plasmid DNA injected into the cytoplasm of quiescent human fibroblasts is not expressed, in contrast to DNA i njected into the nucleus. FISH analysis showed that the plasmid DNA is not transported into the nucleus efficiently. Similarly, DNA-P2lip/HA complexes are readily taken up by both proliferating and nonprolifera ting cells, but do not readily penetrate the nuclear membrane. We conc lude that delivery of plasmid DNA to the cytoplasm is not sufficient f or transfection of eukaryotic cells. The nuclear membrane is apparentl y an important barrier. This explains why a mitotic event is required for efficient transfection with the currently available transfection s ystems. The implications for the further development of transfection c ompounds for use in vivo, where nonproliferating cells are often the t arget, are discussed.