2010 Fiscal Year Final Research Report
Development of multifunctional envelope-type nano device encapsulating highly nuclease resistant oligonucleotides
| Project/Area Number |
18109001
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| Research Category |
Grant-in-Aid for Scientific Research (S)
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| Allocation Type | Single-year Grants |
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| Research Field |
Drug development chemistry
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| Research Institution | Hokkaido University |
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Principal Investigator |
MATSUDA Akira Hokkaido University, 大学院・薬学研究院, 教授 (90157313)
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| Co-Investigator(Kenkyū-buntansha) |
HARASHIMA Hideyoshi 北海道大学 (00183567)
MINAKAWA Noriaki 北海道大学 (40209820)
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| Project Period (FY) |
2006 – 2010
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| Keywords | ヌクレアーゼ抵抗性 / 4'-チオ核酸 / siRNA / ベクター / 薬物送達システム |
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| Research Abstract |
(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.
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