A NOVEL DNA-PEPTIDE COMPLEX FOR EFFICIENT GENE-TRANSFER AND EXPRESSION IN MAMMALIAN-CELLS

To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptid es that mimic the functions of viral proteins. We have designed and sy nthesized two peptides which emulate viral functions - a DNA condensin g agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasi ng agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was c onstructed step-wise through a spontaneous self-assembly process invol ving oppositely charged, electrostatic interactions. To assemble DNA-p eptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amin o groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, ne gatively charged DNA was rapidly mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gen e transfer. In the second step and to form the active complex, the cat ionic DNA complex was rapidly mixed with negatively charged GLFEALLELL ESLWELLLEA which spontaneously incorporated through electrostatic inte ractions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three m olecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some application s in gene therapy.