(The) branched form TAT as an effective gene delivery vector into cancer cells

Abstract

The development of the gene delivery vectors for the effective gene therapy have been studied for over 60 years. However, the viral vectors which were commonly used in the gene therapy field caused some safety problems such as immune response, pathogenic reaction and inflammation. To overcome the problems of the viral vectors, the non-viral vectors were de-veloped. As one class of the non-viral vectors, cell penetrating peptides (CPPs) have been used as a delivery tool because of their cell penetrating ability with low cytotoxicity. Although safety problems were solved using the CPPs as a gene delivery vector, the therapeutic effect was not good due to the low transfection efficiency. To maximize the therapeutic effect, we designed branched form CPPs which have high molecular weight and positive charge. In this study, a TAT (47 - 57) peptide which was derived from the HIV transactivator protein is com-posed of an 11-amino acid peptides (YGRKKRRQRRR) were used for gene delivery into HeLa cells. To make the branched form TAT, two TAT peptides were linked to each other by cysteine and then the N-terminus and S-terminus were modified by cysteine which has thiol group. After the synthesis of the modified TAT (mTAT), the branched TAT (BTAT) was syn-thesized by the DMSO oxidation reaction. For the gene delivery, BTAT was complexed with plasmid green fluorescence protein (pGFP) gene with various N/P ratios. The HeLa cells were treated with BTAT/pGFP complexes and the gene expression was observed by CLSM and FACS. As we expected, the transfection efficiency of BTAT/pGFP was about 40 times higher than TAT/pGFP and mTAT/pGFP. Therefore, the branched form TAT may act a powerful tool for cancer therapy and it can be applied to the other type of gene delivery systems.