Nuclear targeting peptide scaffolds for lipofection of nondividing endothelial cells

Lipofection of nondividing cells is not efficient due to poor endosome escape and nuclear import of transfected DNA. Using a T7 RNA polymerase cytoplasmic transcription assay, the authors observed that nearly 80% of confluent bovine aortic endothelium expressed lipofected plasmid, indicating that cytoplasmic localization of plasmid was widespread in a nondividing cell population, albeit inefficient since most of the lipofected DNA was observed to be in endosomes. Toward overcoming the final rate limiting step of nuclear import during nonviral gene transfer, the authors conjugated a nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1 to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. These levels of nonviral gene transfection are unprecedented in confluent endothelium