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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Cell-Penetrating Peptides as Adenovirus Vector CarrierShinya Kida 1 , Yusuke Eto 2 , Yasuo Yoshioka 2 , Shinsaku Nakagawa 2 ,Keiko Hojo 1 , Mitsuko Maeda 1 , and Koichi Kawasaki 11 Life Science Center, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1,Minatojima, Chuo-ku, Kobe 650-8586, Japan; 2 Graduate School of PharmaceuticalSciences, Osaka University, 1-6, Yamadaoka, Suita, Osaka, 565-0871, JapanIntroductionApplication of gene therapy is expected for the treatment of not only genetic disease butalso other intractable diseases such as cancer. A key aspect of gene therapy lies in thevector used for transgenesis. Adenovirus (Ad) with appropriate gene-transduction andgene-expression properties is widely used in gene therapy studies; however, for routineclinical procedures, more efficient transfer vectors must be developed. We aimed to modifythe viable virus, Ad, with peptide transporters for preparation of a peptide-Ad hybrid asshown in Figure 1.Peptide-GC-NH 2SONOOHNCell PenetratingPeptideBifunctionalCross-LinkerAdenovirusVectorFig. 1. Structure of cell penetrating peptide-Ad hybrid.Results and DiscussionWe reported that Asp-Gly-Arg (RGD)-peptide conjugate of Ad can transfer genesefficiently through integrin-mediated endocytosis. Although Ad also transfers genesthrough its receptor, coxsackie-adenovirus receptor (CAR), the RGD-conjugated Adexhibits efficient gene transfer activity even in a CAR-negative cell line, B16BL6 [1]. Wealso reported that Tat(48-60,GRKKRRQRRRPPQ) conjugated Ad exhibited excellent genetransfer efficiency in B16BL6 cells [2]. Following the Tat-related peptide hybrid of Ad,Pro-rich peptide derivative and octaarginine-related peptide hybrids of Ad were preparedand examined for cell-penetrating ability. A Pro-rich peptide, (Val-Arg-Leu-Pro-Pro-Pro) 3 ,derived from the N-terminal domain of maize -zein, was reported as a new family ofpotential carriers, which have cell internalization property with no toxicity[3,4]. Theoctaarginine peptide contains eight arginine residues optimal for efficient translocation ofArg-rich peptides, as proposed by Futaki, et al. [5]. The Pro-rich peptide derivative[(acetyl-(VRLPPP) 3 -GC-amide, ProrGC] and the octaarginine derivative (acetyl-RRRRRRRR-GCamide,R8GC) were prepared by the solid phase method. Each synthetic peptide wascoupled with the heterobifunctional cross-linking reagent, 6-maleimidohexanoic acidN-hydroxysuccinimide ester (MHSu) and then conjugated to the Ad vector containingluciferase gene. Each peptide-modified Ad was examined for gene transfer activity inB16BL6 cells and A549 cells.The gene transduction efficiency of each novel conjugate was compared with that ofWT-Ad in cells with and without Car, which transports Ad across the plasma membrane:A549 (CAR-positive) and B16BL6 (CAR-negative) cells. The gene transduction efficiency,or luciferase activity, of R8GC-Ad and ProrGC-Ad is given in Tables 2 and 3, respectively.R8GC-Ad and ProrGC-Ad exhibited lower luciferase activity in A549 cells. Modificationof Ad might disturb the binding between Ad and its receptor, CAR. On the other hand, bothR8GC-Ad and ProrGC-Ad exhibited higher luciferase activity than WT-Ad in B16BL6cells: the activity was about 30 and 20 times, respectively, as high as WT-Ad. These resultssuggest that cell-penetrating peptide conjugation to Ad enhances Ad infection in theabsence of CAR, where gene transduction was difficult for Wt-Ad. It suggests that Admodified with cell-penetrating peptides has a wide CAR-independent infection area. The374