|Budget Amount *help
¥108,160,000 (Direct Cost: ¥83,200,000、Indirect Cost: ¥24,960,000)
Fiscal Year 2010: ¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000)
Fiscal Year 2009: ¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000)
Fiscal Year 2008: ¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000)
Fiscal Year 2007: ¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000)
Fiscal Year 2006: ¥32,760,000 (Direct Cost: ¥25,200,000、Indirect Cost: ¥7,560,000)
(1) Small interfering RNAs (siRNAs) containing 2'-O-methyl-4'-thioribonucleosides, nuclease-resistant nucleosides, encapsulated in MEND (multifunctional envelope-type nano-device) showed a long-term luciferase gene silencing effect in HeLa cells and lowering-effect of cholesterol concentrations in mouse blood. This modification did not act as triggers of the innate immune response. (2) In order to construct a nuclease-resistant vector, enzymatic incorporation using 2'-deoxy-4'-thioribonucleoside 5'-triphosphates (d^SNTPs) by PCR was tried. Although two dNTPs could be substituted by d^SNTPs to produce 4'-thioDNA with 30-80% efficiency of the unmodified dNTPs, further increased substitution gave unfruitful results. Therefore, chemically synthesized 42mer 4'-thioDNAs (2dNs were substituted by 2d^SNs) were ligated to construct the luciferase vectors by a ligase. When these vectors were transfected into NIH3T3 cells, the modified vectors showed about 20-40% activity of that of the unmodified vector after 24-72 h. (3) siRNAs encapsulated in MENDs which have a shorter length of the pH-sensitive fusogenic peptide (shGALA) or a PEG derivative with a cleavage site of matrix metalloproteinase (PPD) showed improved knockdown ability of the target gene in vitro and in vivo.