Fusion of HIV-1 Tat protein transduction domain to poly-lysine as a new DNA delivery tool

Effective gene therapy depends on the efficient transfer of therapeutic genes to target cells. None of the current technologies, however, satisfy all of the requirements necessary for gene therapy, because the plasma and nuclear membranes of mammalian cells are tight barriers against gene transfer using synthetic delivery systems. The protein transduction domain (PTD) of human immunodeficiency virus type 1 (HIV-1) Tat protein greatly facilitates protein transfer via membrane destabilisation. We synthesised polylysine peptides containing Tat PTD (TAT-pK), or other sequences, and investigated their potential as agents for gene transfer. The synthesised polypeptide TAT-pK retains DNA binding function and mediates delivery of a reporter gene to cultured cells. RGD motif binds with low affinity to alpha integrins which induce cell activation. Two control polypeptides, GGG-pK and RGD-pK, were synthesised and tested, but their gene transfer abilities were weaker than those of TAT-pK. TAT-pK-mediated gene transfer was enhanced in the presence of chloroquine or ammonium chloride, to a greater extent than that of cationic lipid-mediated gene transfer in most cancer cell lines tested. These data suggest that TAT-pK may be a potent candidate delivery vehicle that promotes gene transfer, dependent on the endocytic pathway. We conclude that the TAT-pK/DNA complex is useful as a minimal unit to package therapeutic genes and to transduce them into mammalian cells.