**12. References**

672 Non-Viral Gene Therapy

Fig. 6. Antivascular effects of CD34-TRAIL+ cells. NOD/SCID mice bearing subcutaneous tumor nodules 10 mm in diameter were randomly assigned to receive CD34-TRAIL+ cells, mock-transduced CD34+ cells (3 × 106 cells/mouse, intravenous), recombinant soluble TRAIL (500 µg/mouse, IP), and control vehicle. (**A**) Forty-eight hours after treatment, NOD/SCID mice were intravenously injected with 0.2 mL of sulfo-NHS-LC-biotin (5 mg/mL) to biotinylate tumor vasculature. Tumors were then excised, and biotinylated endothelium was revealed by HRP-streptavidin and 3,3'-diaminobenzidine for light microscopy analysis. Representative histological images of in vivo biotinylated mice receiving the different treatments are shown. (**B**) Sections were analyzed using ImageJ for quantification of vascular parameters. Endothelial area was calculated on whole tissue sections as (streptavidin-HRP stained area)/(total tissue area) × 100. \* P < .0001, compared to controls. # P < .0001, compared to soluble TRAIL . (**C**) Vessel wall thickness was calculated on transversally oriented vessels. \* P < .0001, compared to controls. # P < .0001,

In conclusion, under our experimental conditions the use of transduced CD34+ cells as a vehicle of mTRAIL resulted in an antitumor effect greater than that exerted by soluble TRAIL, likely because of an antivascular action. Our findings appear to be of outstanding interest in the context of the increasing need for therapeutic strategies targeting not only tumor cells but also the tumor microenvironment (De Raeve et al., 2004; Joyce, 2005; Rafii et al., 2002). Finally, the clinical feasibility of such a systemic CD34+ cell-based gene therapy

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**29** 

*Japan* 

**Pyrrole-Imidazole Polyamides for Gene** 

**Therapy: Bioanalytical Methods** 

Tomonori Kamei1, Takahiko Aoyama1, Takahiro Ueno2,

*2Department of Medicine, Division of Nephrology and Endocrinology* 

*4Division of Cancer Genetics, Department of Advanced Medical Science* 

Noboru Fukuda2,3, Hiroki Nagase3,4 and Yoshiaki Matsumoto1

*1Department of Clinical Pharmacokinetics, School of Pharmacy, Nihon University* 

*3Advanced Research Institute for the Science and Humanities, Nihon University* 

Pyrrole(Py)-imidazole(Im)(PI) polyamides are small synthetic molecules composed of aromatic rings of *N*-methylpyrrole and *N*-methylimidazole amino acids (Trauger et al., 1996). Synthetic polyamides recognize and bind to specific nucleotide sequences in the minor groove of double-helical DNA with high affinity (Pilch et al., 1996). Various sequencespecific DNA-binding PI polyamides have been developed to regulate gene expression by targeting the promoter regions of enhancer and transcription factor-binding elements in vitro (Murty et al., 2004). PI polyamides were first identified from duocarmycin A and distamycin A, which bind in the minor groove of DNA (Tao et al., 1999; Trauger et al., 1996). Sequence-specific DNA recognition by PI polyamide depends on the sequence of side-byside amino acid pairs. A pair of Py opposite Im targets the CG base pair, whereas Im opposite Py recognizes the GC base pair, and the Py/Py combination binds to both AT and TA base pairs (White et al., 1997). PI polyamides designed to bind to the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and the TATAbox-binding protein DNA binding site have been shown to inhibit virus replication in isolated human peripheral blood

PI polyamides can easily enter into the nucleus and bind to chromosomal DNA. Many promising observation for gene therapy using PI polyamides have been reported. PI polyamide targeting rat transforming growth factor (TGF)-β1 has been reported to inhibit the expressions of TGF-β1 mRNA and protein in the renal cortex of Dahl-S rats. The targeted PI polyamide also reduced glomerulosclerosis and interstitial fibrosis without side effects. These observations indicate that PI polyamides will be effective for TGF-β1-related diseases, including progressive renal injury (Matsuda et al., 2011; Matsuda et al., 2006). PI polyamides targeting human aurora kinase-A (AURKA) and -B (AURKB) promoters significantly inhibited the promoter activities, and mRNA and protein expression levels of AURKA and

**1. Introduction** 

lymphocytes (Dickinson et al., 1998).

**and Pharmacokinetics** 

*Nihon University School of Medicine* 

*Nihon University School of Medicine* 

