Development of a highly specific genome editing technology for human cells using adeno-associated viral vector

• Project/Area Number: 25640107
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Medical genome science
• Research Institution: Aichi Medical University
• Principal Investigator: Hiroyuki Konishi 愛知医科大学, 医学部, 教授 (20344335)
• Co-Investigator(Kenkyū-buntansha): HOSOKAWA YOSHITAKA 愛知医科大学, 医学部, 教授 (60229193)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2013: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: アデノ随伴ウイルスベクター / 遺伝子改変 / CRISPR-Cas9 / ゲノム編集 / 2A

Outline of Final Research Achievements

In this study, we sought to improve the efficiency of gene targeting mediated by adeno-associated viral (AAV) vectors. We found that the efficiency of gene targeting achieved by AAV-based targeting vectors varies depending on the choice of promoters regulating a selection marker gene within the vectors, and that the use of promoters exhibiting strong transcriptional activity, such as the CMV promoter, in AAV targeting vectors does not result in gene targeting with high efficiency. This study also revealed that a higher gene targeting efficiency is achieved by using a 2A peptide, rather than a commonly used internal ribosomal entry site (IRES), within AAV-based targeting vectors which carry no promoter and exploit the promoters of targeted endogenous genes to regulate a selection marker gene within the vectors (i.e., promoter-trap targeting vectors).

Research Products

• [Journal Article] Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide (2016)
  • Author(s): Karnan S, Ota A, Konishi Y, Wahiduzzaman M, Hosokawa Y, and Konishi H
  • Journal Title: Nucleic Acids Research Volume: 44 Issue: 6 Pages: e54-e54
  • DOI: 10.1093/nar/gkv1338
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Inhibition of NADPH oxidase 4 induces apoptosis in malignant mesothelioma: Role of reactive oxygen species (2015)
  • Author(s): Tanaka M, Miura Y, Numanami H, Karnan S, Ota A, Konishi H, Hosokawa Y, and Hanyuda M
  • Journal Title: Oncology Reports Volume: 34 Issue: 4 Pages: 1726-1732
  • DOI: 10.3892/or.2015.4155
  • Peer Reviewed
• [Journal Article] Plumbagin suppresses tumor cell growth in oral squamous cell carcinoma cell lines (2015)
  • Author(s): Ono T, Ota A, Ito K, Nakaoka T, Karnan S, Konishi H, Furuhashi A, Hayashi T, Yamada Y, Hosokawa Y, Kazaoka Y
  • Journal Title: Oral Dis Volume: - Issue: 4 Pages: 501-511
  • DOI: 10.1111/odi.12310
  • Peer Reviewed
• [Journal Article] High-resolution 400K oligonucleotide array comparative genomic hybridization analysis of neurofibromatosis type 1-associated cutaneous neurofibromas (2015)
  • Author(s): Asai A, Karnan S, Ota A, Takahashi M, Damdindorj L, Konishi Y, Hossain E, Konishi H, Nagata A, Yokoo K, Hosokawa Y
  • Journal Title: Gene Volume: 558 Pages: 220-226
  • Peer Reviewed
• [Journal Article] Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages (2015)
  • Author(s): Hossain E, Ota A, Karnan S, Takahashi M, Mannan SB, Konishi H, Hosokawa Y
  • Journal Title: Mol Cell Biochem Volume: 400 Issue: 1-2 Pages: 29-40
  • DOI: 10.1007/s11010-014-2259-0
  • Peer Reviewed
• [Journal Article] A comparative analysis of constitutive promoters located in adeno-associated viral vectors (2014)
  • Author(s): Damdindorj L, Karnan S, Ota A, Hossain E, Konishi Y, Hosokawa Y, Konishi H
  • Journal Title: PLoS ONE Volume: 9 Issue: 8 Pages: e106472-e106472
  • DOI: 10.1371/journal.pone.0106472
  • Peer Reviewed / Open Access
• [Journal Article] Combined arsenic trioxide-cisplatin treatment enhances apoptosis in oral squamous cell carcinoma cells. (2014)
  • Author(s): Nakaoka T, Ota A, Ono T, Karnan S, Konishi H, Furuhashi A, Ohmura Y, Yamada Y, Hosokawa Y, and Kazaoka Y.
  • Journal Title: Cell Oncol Volume: 37 Issue: 2 Pages: 119-129
  • DOI: 10.1007/s13402-014-0167-7
  • Peer Reviewed
• [Journal Article] Arsenic upregulates the expression of angiotensin II Type I receptor in mouse aortic endothelial cells. (2013)
  • Author(s): Hossain E, Ota A, Takahashi M, Karnan S, Damdindorj L, Konishi Y, Konishi H, and Hosokawa Y.
  • Journal Title: Toxicol Lett Volume: 220 Issue: 1 Pages: 70-75
  • DOI: 10.1016/j.toxlet.2013.04.006
  • Peer Reviewed
• [Journal Article] Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation. (2013)
  • Author(s): Wang GM, Wong HY, Konishi H, Blair BG, Abukhdeir AM, Gustin JP, Rosen DM, Denmeade S, Rasheed Z, Matsui W, Garay JP, Mohseni M, Higgins MJ, Cidado J, Jelovac D, Croessmann S, Cochran R, Karnan S, Konishi Y, Ota A, Hosokawa Y, Argani P, Lauring J, and Park BH.
  • Journal Title: Cancer Res Volume: 73 Issue: 11 Pages: 3248-3261
  • DOI: 10.1158/0008-5472.can-12-1578
  • Peer Reviewed
• [Journal Article] Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells. (2013)
  • Author(s): Hossain E, Ota A, Karnan S, Damdindorj L, Takahashi M, Konishi Y, Konishi H, and Hosokawa Y.
  • Journal Title: Toxicol Appl Pharmacol Volume: 273 Issue: 3 Pages: 651-658
  • DOI: 10.1016/j.taap.2013.10.012
  • Peer Reviewed
• [Journal Article] KRAS変異がんの治療法 --- 複数の分子標的薬剤を併用する --- (2013)
  • Author(s): 小西裕之
  • Journal Title: 実験医学 Volume: 31 Pages: 538-539
• [Presentation] 1.400KオリゴアレイCGHを用いたNF1患者の皮膚神経線維腫の解析 (2015)
  • Author(s): Sivasundaram Karnan, 太田明伸, 小西裕之, 細川好孝
  • Organizer: 第38回日本分子生物学会年会第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸国際展示場（兵庫・神戸）
  • Year and Date: 2015-12-01
• [Presentation] 口腔がん細胞株に対するプランバギンのプロオキシダント作用とアポトーシス誘導にはミトコンドリア遺伝子発現系が関与する (2015)
  • Author(s): 太田明伸, Sivasundaram Karnan, 小西裕之, 細川好孝
  • Organizer: 第38回日本分子生物学会年会第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸国際展示場（兵庫・神戸）
  • Year and Date: 2015-12-01
• [Presentation] 癌原性KRAS変異をノックインしたヒト気管支上皮細胞株モデルの樹立と解析 (2015)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 細川好孝
  • Organizer: 第74回日本癌学会学術総会
  • Place of Presentation: 名古屋国際会議場（愛知・名古屋）
  • Year and Date: 2015-10-08
• [Presentation] Delta40p53はHepG2細胞に対して細胞増殖抑制効果を示す (2015)
  • Author(s): 太田明伸, Sivasundaram Karnan, 小西裕之, 細川好孝
  • Organizer: 第74回日本癌学会学術総会
  • Place of Presentation: 名古屋国際会議場（愛知・名古屋）
  • Year and Date: 2015-10-08
• [Presentation] アデノ随伴ウイルスに基づく遺伝子改変ベクターにおいて薬剤耐性遺伝子の発現を制御するプロモーターと改変効率の関係 (2014)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 細川好孝
  • Organizer: 第37回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] リポポリサッカライドはErk1/2シグナルを介したLOX-1の発現増加によってマクロファージの酸化LDLの取り込みを増強する (2014)
  • Author(s): 太田明伸, Ekhtear Hossain, Sivasundaram Karnan, Shahnewaj Mannan, 小西裕之, 細川好孝
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 国立京都国際会館（京都・京都）
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] プランバギンは口腔扁平上皮癌に対して活性酸素産生を介した細胞増殖抑制効果を示す (2014)
  • Author(s): 太田明伸, Sivasundaram Karnan, 小西裕之, 風岡宜暁, 細川好孝
  • Organizer: 第73回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] NF1関連の皮膚神経線維腫における400KオリゴアレイCGH解析 (2014)
  • Author(s): Sivasundaram Karnan, 太田明伸, 小西裕之, 細川好孝
  • Organizer: 第73回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（神奈川・横浜）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] ヒト細胞株のゲノム編集によるKRAS-PI3Kシグナル経路の解析. (2013)
  • Author(s): 小西裕之, Sivasundaram Karnan, 太田明伸, 細川好孝.
  • Organizer: 第72回日本癌学会学術総会
  • Place of Presentation: パシフィコ横浜（横浜）
• [Presentation] ヒ素は酸化LDL受容体LOX-1の発現を増加させることによりマウス血管内皮細胞の酸化LDL取り込み能を増強する. (2013)
  • Author(s): 太田明伸, Ekhtear Hossain, Sivasundaram Karnan, Damdindorj Lkhagvasuren, 小西裕子, 小西裕之, 細川好孝.
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: パシフィコ横浜（横浜）
• [Presentation] KRAS変異およびPIK3CA変異のノックインを施したヒト上皮細胞株の生化学的解析. (2013)
  • Author(s): Sivasundaram Karnan, 太田明伸, 細川好孝, 小西裕之.
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: パシフィコ横浜（横浜）
• [Presentation] Arsenic trioxide inhibits interferon-gamma-mediated nitric oxide production in mouse vascular endothelial cells. (2013)
  • Author(s): Hossain E, Ota A, Karnan S, Damdindorj L, Konishi Y, Konishi H, Hosokawa Y.
  • Organizer: 第86回日本生化学会大会
  • Place of Presentation: パシフィコ横浜（横浜）