Enhancement of Gene Transfer Using YIGSR Analog of Tat-Derived Peptide Amer F. Alhaj Saleh, Harmesh S. Aojula, Alain Pluen Drug Delivery Group, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, M13 9PT, UK Received 26 June 2007; revised 18 September 2007; accepted 18 September 2007 Published online 27 September 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20854 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com INTRODUCTION I n the last decade several cell penetrating peptides (CPPs) with membrane translocation properties have been used to enhance the cellular delivery of a wide variety of cargo molecules. 1–3 Tat-derived peptide is one of the most widely characterized CPPs and has been successfully employed in different biological studies. 4–8 Tat-derived pep- tide is an arginine-rich sequence (comprising amino acids 49–57) derived from the transactivating transcriptional acti- vator protein of human immunodeficiency virus type 1 (HIV-1 TAT protein). 9 The highly cationic nature of Tat sequence arising from multiple arginine residues has already been utilized in gene transfer either by covalent coupling of the peptide to oligonucleotides, 10–12 or by simple mixing of plasmid DNA with the peptide to form peptide/DNA com- plexes via noncovalent electrostatic interactions. 13–16 How- ever, the transfection ability of such complexes remains low. In addition, the cellular selectivity of Tat is generally regarded nonspecific as it relies mainly on electrostatic interaction with negatively charged membrane components. 7,17–21 Non- specific uptake of CPPs in different cell types or tissues repre- sents a major challenge for in vivo biomedical applications. Hence the development of new approaches to improve the Enhancement of Gene Transfer Using YIGSR Analog of Tat-Derived Peptide Correspondence to: A. Pluen; e-mail: alain.pluen@manchester.ac.uk This article contains supplementary material available via the Internet at http:// www.interscience.wiley.com/jpages/0006-3525/suppmat. ABSTRACT: Cell penetrating peptide based gene carriers are notably known for low level of gene transfer. To remedy this, as laminin receptor (LR) has been previously linked to tumor metastasis, the LR-binding domain (YIGSR) as well as a scrambled sequence (SGIYR) were added to Tat- derived peptide sequence (YIGSR-Tat and SGIYR-Tat respectively). Peptides cellular uptake was assessed with high-LR (HT1080) and low-LR (HT29) cell lines by flow cytometry. Their ability to form complexes with DNA was examined using YOPRO-1 fluorescence assay and their transfection efficiencies evaluated using a luciferase reporter gene assay. DNA complexes were formed at (1/2) charge ratios as low as 2:1. While no conclusion could be drawn on the effect of YIGSR sequence on peptides uptake in both cell lines, a significant improvement in gene transfection in HT1080 cells was achieved using YIGSR-Tat compared to Tat and SGIYR- Tat. Additionally this increased efficiency was inhibited by excess free YIGSR. No significant difference in transfection efficiency was observed between Tat, SGIYR- Tat and YIGSR-Tat based complexes in HT29 cells. These studies demonstrate that attachment of receptor-binding ligand (YIGSR) to Tat-derived peptide can improve the efficiency of gene transfer in LR-positive cells (HT1080). # 2007 Wiley Periodicals, Inc. Biopolymers 89: 62–71, 2008. Keywords: DNA delivery; Tat peptide; YIGSR peptide; targeting; laminin receptor V V C 2007 Wiley Periodicals, Inc. 62 Biopolymers Volume 89 / Number 1