Development of a novel RNAi medicine based on shRNA expression mini-plasmid

• Project/Area Number: 24390027
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Drug development chemistry
• Research Institution: The University of Tokushima
• Principal Investigator: MINAKAWA Noriaki 徳島大学, ヘルスバイオサイエンス研究部, 教授 (40209820)
• Co-Investigator(Kenkyū-buntansha): ISHIDA Tstsuhiro 徳島大学, 大学院ヘルスバイオサイエンス研究部, 教授 (50325271)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥18,850,000 (Direct Cost: ¥14,500,000、Indirect Cost: ¥4,350,000); Fiscal Year 2014: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2013: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2012: ¥9,880,000 (Direct Cost: ¥7,600,000、Indirect Cost: ¥2,280,000)
• Keywords: RNAi創薬 / 4'-チオDNA / PCR / 非天然塩基対 / クリックケミストリー / 免疫応答

Outline of Final Research Achievements

In this study, I aim at a development of novel RNAi medicine based on shRNA expression mini-gene. As a result, I succeeded to prepare the mini-gene via PCR amplification using 2’-deoxy-4’-thionucleoside triphosphates (dSNTPs). In addition, I also developed a practical method to cyclize the resulting mini-gene.
The mini-gene showed potent RNAi activity both in vitro and in vivo. In addition, innate immune responses such as type-I INF and inflammatory cytokines were hardly induced by administration of the mini-gene in vivo.

Research Products

• [Journal Article] First synthesis of fully modified 4'-selenoRNA and 2'-OMe-4'-selenoRNA based on the mechanistic considerations of an unexpected strand break. (2014)
  • Author(s): Tarashima, N.; Hayashi, K.; Terasaki, M.; Tanike, H.; Inagaki, Y.; Fukuda, Y.; Furukawa, K.; Matsuda, A.; Minakawa,
  • Journal Title: Org. Lett. Volume: 16 Issue: 18 Pages: 4710-4713
  • DOI: 10.1021/ol502077h
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Development of a New Dumbbell-Shaped Decoy DNA Using a Combination of the Unnatural Base Pair ImON:NaNO and a CuAAC Reaction (2014)
  • Author(s): Yosuke Higuchi, Kazuhiro Furukawa, Tadashi Miyazawa, and Noriaki Minakawa
  • Journal Title: Bioconjugate Chem. Volume: 25 Issue: 7 Pages: 1360-1369
  • DOI: 10.1021/bc500225r
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] PCR Amplification of 4'-ThioDNA Using 2'-Deoxy-4'-thionucleoside 5'-Triphosphates. (2013)
  • Author(s): Kojima, T. ; Furukawa, K. ; Maruyama, H. ; Inoue, N. ; Tarashima, N. ; Matsuda, A. ; Minakawa, N.
  • Journal Title: ACS Synth. Biol. Volume: 2 Issue: 9 Pages: 529-536
  • DOI: 10.1021/sb400074w
  • Peer Reviewed
• [Journal Article] Synthesis of a novel 1,2-dithianenucleoside via Pummerer-like reaction, followed by Vorbruggen glycosylation between 1,2-dithiane derivative and uracil. (2013)
  • Author(s): Miyazawa, T. ; Umezaki. K. ; Tarashima, N. ; Furukawa, K. : Ooi, T. ; Minakawa, N.
  • Journal Title: Chem. Commun. Volume: 49 Issue: 71 Pages: 7851-7853
  • DOI: 10.1039/c3cc44848g
  • Peer Reviewed
• [Journal Article] Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2'-modified-4'-thionudeosides. (2013)
  • Author(s): Kukuchi, Y. ; Yamazaki, N. ; Tarashima, N. ; Furukawa, K. : Takiguchi, Y. ; Itoh, K. ; Minakawa, N.
  • Journal Title: Bioorg. Med. Chem. Volume: 21 Issue: 17 Pages: 5292-5296
  • DOI: 10.1016/j.bmc.2013.06.023
  • Peer Reviewed
• [Journal Article] In vitro potimization of 2'-Ome-4'-thioribonucleoside-modified anti-microRNA oligonucleotides and its targeting delivery to mouse liver using a liposomal nanoparticle (2013)
  • Author(s): Mayumi Takahashi, Naoki Yamada, Hiroto Hatakeyama, Manami Murata, Yusuke Sato, Noriaki Minakawa, Hideyoshi Harashima, Akira Matsuda
  • Journal Title: Nucleic Acids Res. Volume: 41 Issue: 22 Pages: 10659-10667
  • DOI: 10.1093/nar/gkt823
  • Peer Reviewed
• [Journal Article] 4'・チオDNAを用いたRNA創薬 (2013)
  • Author(s): 南川典昭
  • Journal Title: YAKUGAKU ZASSHI Volume: 133 Pages: 53-60
  • NAID: 130003361879
  • Peer Reviewed
• [Journal Article] A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleoside (2012)
  • Author(s): Noriko Tarashima, Yosuke Higuchi, Yasuo Komatsu, and Noriaki Minakawa
  • Journal Title: Bioorg. Med. Chem Volume: 20 Issue: 24 Pages: 7095-7100
  • DOI: 10.1016/j.bmc.2012.10.035
  • Peer Reviewed
• [Journal Article] Intracellular stability of 2'-OMe-4'-thioribonucleoside modified siRNA leads to long-term RNAi effect (2012)
  • Author(s): Mayumi Takahashi
  • Journal Title: Nucleic Acids Research Volume: (in press) Issue: 12 Pages: 5787-5793
  • DOI: 10.1093/nar/gks204
  • Peer Reviewed
• [Presentation] Intelligent RNA expression device (iRed) を利用した 新規遺伝子発現抑制法の開発 (2015)
  • Author(s): 田良島 典子, 小島 孝允, 金城 望, 古川 和寛, 安藤 英紀, 石田 竜弘, 南川 典昭
  • Organizer: 第９５回日本化学会
  • Place of Presentation: 日本大学 船橋キャンパス（千葉県船橋市）
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Intelligent RNA expressing device (iRed) を利用した核酸創薬の新手法 (2014)
  • Author(s): 田良島典子，小島孝光，金城望，古川和寛、安藤英紀，石田竜弘，南川典昭
  • Organizer: 第３２回メディシナルケミストリーシンポジウム
  • Place of Presentation: 神戸国際会議場（兵庫県神戸市）
  • Year and Date: 2014-11-26 – 2014-11-28
• [Presentation] A new dumbbell-shaped decoy DNA targeting NF-kB with the unnatural base pair ImON:NaNO (2014)
  • Author(s): Kazuhiro Furukawa, Yosuke Higuchi, Tadashi Miyazawa, Noriaki Minakawa
  • Organizer: 10th Annual meeting of the oligonucleotide therapeutics society
  • Place of Presentation: San Diego (USA)
  • Year and Date: 2014-10-12 – 2014-10-15
• [Presentation] 4’-セレノ核酸の化学 ―4’-セレノ核酸は4’-チオ核酸を超えることが出来るか？― (2014)
  • Author(s): 南川典昭
  • Organizer: アンチセンス・遺伝子・デリバリーシンポジウム２０１４
  • Place of Presentation: 東京医科歯科大学（東京都文京区）
  • Year and Date: 2014-09-08 – 2014-09-09
  • Invited
• [Presentation] Enzymatic synthesis of 4’-selenoDNA (2014)
  • Author(s): Noriko Tarashima, Tatsuya Sumitomo, Kazuhiro Furukawa, Noriaki Minakawa
  • Organizer: XXI Round Table on Nucleosides, Nucleotides and Nucleic Acids
  • Place of Presentation: Poznan(Poland)
  • Year and Date: 2014-08-24 – 2014-08-28
• [Presentation] HOW TO PREPARE 4’-SELENORNA? (2014)
  • Author(s): Noriaki Minakawa, Noriko Tarashima, Koya Hayashi, Maki Terasaki, Hirotsugu Taniike, Yusuke Inagaki, Shinji Fukuda, Kazuhiro Furukawa and Akira Matsuda
  • Organizer: XXI Round Table on Nucleosides, Nucleotides and Nucleic Acids
  • Place of Presentation: Poznan(Poland)
  • Year and Date: 2014-08-24 – 2014-08-28
• [Presentation] 人工塩基対ImNN:NaOOのPCR増幅とRNAi創薬への応用 (2014)
  • Author(s): 南川典昭
  • Organizer: 日本薬学会第１３４年会
  • Place of Presentation: 熊本大学（熊本県）
• [Presentation] クリックケミストリーを用いたRNA簡便精製法の開発 (2014)
  • Author(s): 南川典昭
  • Organizer: 日本薬学会第１３４年会
  • Place of Presentation: 熊本大学（熊本県）
• [Presentation] 2’-デオキシ-4’-セレノピリミジンヌクレオシドの合成とDNAオリゴマーへの導入 (2014)
  • Author(s): 南川典昭
  • Organizer: 日本薬学会第１３４年会
  • Place of Presentation: 熊本大学（熊本県）
• [Presentation] 新規Im : Na塩基対のDNAポリメラーゼによる基質認識 (2013)
  • Author(s): 南川典昭
  • Organizer: 日本薬学会第133年会
  • Place of Presentation: パシフィコ横浜(神奈川県)
  • Year and Date: 2013-03-28
• [Presentation] Huisgen反応を利用したImO^N : NaN^o塩基対を含む環状DNAの合成 (2013)
  • Author(s): 南川典昭
  • Organizer: 日本薬学会第133年会
  • Place of Presentation: パシフィコ横浜(神奈川県)
  • Year and Date: 2013-03-28
• [Presentation] 新規含硫ヌクレオシド誘導体の合成 (2013)
  • Author(s): 南川典昭
  • Organizer: 日本薬学会第133年会
  • Place of Presentation: パシフィコ横浜(神奈川県)
  • Year and Date: 2013-03-28
• [Presentation] A novel approach of gene suppression using an intelligent shRNA expressing device (iRed) (2013)
  • Author(s): Noriaki Minakawa
  • Organizer: 9th Annual Meeting of the Oligonucleotide Therapeutics Society
  • Place of Presentation: Royal Continental Hotel (Italy)
• [Presentation] 新規1, 2-ジチアンヌクレオシドの合成と性質 (2013)
  • Author(s): 南川典昭
  • Organizer: 第５２回日本薬学会中国四国支部学術大会
  • Place of Presentation: 松山大学（愛媛県）
• [Presentation] 2'-デオキシ-4'-セレノピリミジンヌクレオシドの合成 (2013)
  • Author(s): 南川典昭
  • Organizer: 第５２回日本薬学会中国四国支部学術大会
  • Place of Presentation: 松山大学（愛媛県）
• [Presentation] Enzymatic incorporation of unnatural ImNN:NaOO base pair consisting of four hydrogen bonds (2013)
  • Author(s): 南川典昭
  • Organizer: 第４０回国際核酸化学シンポジウム
  • Place of Presentation: 神奈川大学（神奈川県）
• [Presentation] Synthesis of circular decoy DNA containing unnatural base pair ImON:NaNO by CuAAC reaction (2013)
  • Author(s): 南川典昭
  • Organizer: 第４０回国際核酸化学シンポジウム
  • Place of Presentation: 神奈川大学（神奈川県）
• [Presentation] Consideration of insufficient yield in oligonucleotide synthesis containing 4’-selenonucleosides (2013)
  • Author(s): 南川典昭
  • Organizer: 第４０回国際核酸化学シンポジウム
  • Place of Presentation: 神奈川大学（神奈川県）
• [Presentation] ALLOSTERIC CONTROL OF DNA-HYDROLYZING DEOXYRIBOZYME BY SHORT OLIGONUCLEOTIDES (2013)
  • Author(s): 南川典昭
  • Organizer: 第４０回国際核酸化学シンポジウム
  • Place of Presentation: 神奈川大学（神奈川県）
• [Presentation] 改変U1 snRNAによる遺伝子発現抑制効果の検討 (2013)
  • Author(s): 南川典昭
  • Organizer: 第２３回アンチセンスシンポジウム
  • Place of Presentation: 徳島大学（徳島県）
• [Presentation] 複数の改変U1 snRNAを用いた遺伝子発現抑制 (2013)
  • Author(s): 南川典昭
  • Organizer: 第２３回アンチセンスシンポジウム
  • Place of Presentation: 徳島大学（徳島県）
• [Presentation] Chemistry & Biology of 4'-thioDNA (2012)
  • Author(s): Noriaki Minakawa
  • Organizer: Memorial Seminar of International Academic Exchange between Dongguk University & The University of Tokushima
  • Place of Presentation: Dongguk University (Korea)(招待講演)
  • Year and Date: 2012-12-21
• [Presentation] Gene silencing by 2' -modified-4' -thio oligonucleotides via U1i machinery (2012)
  • Author(s): Noriaki Minakawa
  • Organizer: 第39回国際核酸化学シンポジウム
  • Place of Presentation: 名古屋大学(愛知県)
  • Year and Date: 2012-11-15
• [Presentation] 核酸医薬の現状と将来 (2012)
  • Author(s): 南川典昭
  • Organizer: プロセス化学東四国フォーラムセミナー
  • Place of Presentation: 徳島文理大学(徳島県)(招待講演)
  • Year and Date: 2012-09-29
• [Presentation] Huisgen反応を利用したImO^N : NaN^o塩基対を含む環状DNAの合成研究 (2012)
  • Author(s): 南川典昭
  • Organizer: アンチセンス・遺伝子・デリバリーシンポジウム2012
  • Place of Presentation: 仙台市民会館(宮城県)
  • Year and Date: 2012-09-25
• [Presentation] 4'-チオDNAを利用した新規RNAi法の開発 (2012)
  • Author(s): 南川典昭
  • Organizer: 第6回バイオ関連化学シンポジウム
  • Place of Presentation: 北海道大学(北海道)
  • Year and Date: 2012-09-07
• [Presentation] 化学修飾DNAを利用するRNAi創薬の新展開 (2012)
  • Author(s): 南川典昭
  • Organizer: 第19回ファーマサイエンスフォーラム
  • Place of Presentation: 北海道大学(北海道)(招待講演)
  • Year and Date: 2012-07-25