Verification of gene-edited pig production method from Japan

• Project/Area Number: 21K19527
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 55:Surgery of the organs maintaining homeostasis and related fields
• Research Institution: Osaka University
• Principal Investigator: 宮川 周士 大阪大学, 微生物病研究所, 招へい研究員 (90273648)
• Co-Investigator(Kenkyū-buntansha): 前田 晃 大阪大学, 大学院医学系研究科, 招へい教員 (00319708)
• Project Period (FY): 2021-07-09 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2023: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2022: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2021: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: PERV / GGTA1 / CRISPR/Cas3 / ブタ線維芽細胞 / CMAH / Hanganutziu-Deicher抗原 / ブタ内在性レトロウイルス / 再生医療 / バイオ人工臓器 / ノックアウト / CRISPR

Outline of Research at the Start

バイオ人工臓器（異種移植）の分野では、当初、相同組み替え法は困難を極めたが、新法によるknockout（KO）法が我々も含めブタでも成功している。しかし、CRISPR/Cas9法には特許の問題が絡んでおり、今回研究協力者の真下知士グループが開発したCRISPR/Cas3法を応用してKO-ブタの作出を進める。現時点でブタ細胞での有効性はまだ確立されていない。ブタの繊維芽細胞、等を使って、先ずはこのCas3法で既にCas9法でKO経験のあるブタGalTと4GalNT2のKOを比較しながら行う。次に、SLA ClassI,II、とPERV(内在性レトロウイルス)-CのKOの有効性の検証を行う。

Outline of Annual Research Achievements

異種移植の開発に関して各国のチームが使っているCRISPR/Cas9法のブタへの応用は進んでいるが、“特許”の問題が大きく絡んでいる。我々は、国産のCRISPR/Cas3法を応用して、PERVのKOを検討した。
まず、COSMID:CRISPR Search、susScr3を利用し、mismatch, insertion and/or defictを検討しながら、特にA /Cキメラの場合５’側にPERV-Aが来る事を考慮し、PERV-Cのenvの３‘側にcrRNA のsiteを決定した。そして、U6-crRNAを作成した。
次に、ブタ胎仔線維芽細胞へ、このU6-crRNAを加えたbpNLS-Cascade/ U6-crRNAに加え、bpNLS-Cascade とpCX-EGFP (CAG-EGFP)をco-transfectionして、EGFP陽性の細胞(トランスフェクション効率としては66.7%)をSortingして、そのsorting cellについて変異解析を行った 。
変異解析に使用したPrimerは、1875（CTCTCTAGGCTCAAGGCACTTGAGTGG）と1877(CACATTTGCGCATGTTTCGG)、1813(CCAGGACCACCAAATAATGG)と1864(TCATGCCTAGAGATGTACTCAGGGCCA)でnested PCRを行い、PCR後の泳動ではスメアが無いわけでは無かったが、結局数度にわたり、PCR産物をTOPOクローニングした。出来た大腸菌コロニーをシークエンスしたが、変異は検出できなかった。
また、前年度と同じくブタ血管内皮(SEC)を使い、SEC/GGTA1-KOの作出を試みるも、作出できていない。

Research Products

• [Journal Article] The hybrid CL-SP-D molecule has the potential to regulate xenogeneic rejection by human neutrophils more efficiently than CD47 (2024)
  • Author(s): K Iemitsu, R Sakai, A Maeda, K Gadomska, S Kogata, D Yasufuku, J Matsui, K Masahata, M Kamiyama, H Eguchi, S Matsumura, Y Kakuta, H Nagashima, H Okuyama, S Miyagawa
  • Journal Title: Transpl Immunol. Volume: 84 Pages: 102020-102020
  • Peer Reviewed
• [Journal Article] A study of the mechanisms responsible for the action of new immunosuppressants and their effects on rat small intestinal transplantation (2022)
  • Author(s): Shuji Miyagawa, Tasuku Kodama, Rei Matsuura, Pei-Chi Lo, Rieko Sakai, Chiyoshi Toyama, Yuichi Takama, Yoshiyuki Ihara, Yoichi Kakuta, Kazuaki Yamanaka, Katsuyoshi Matsunami, Hiroshi Eguchi, Akira Maeda, Hiroomi Okuyama
  • Journal Title: Transpl Immunol . Volume: 70 Pages: 101497-101497
  • DOI: 10.1016/j.trim.2021.101497
  • Peer Reviewed
• [Journal Article] Immunosuppressive effect of a C5a receptor antagonist via macrophage regulation of an intestinal transplant in a rat model (2022)
  • Author(s): Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Koki Takase, Tasuku Kodama, Kazunori Masahata, Masafumi Kamiyama, Takehisa Ueno, Yuko Tazuke, Hiroshi Eguchi, Katsuyoshi Matsunami, Shuji Miyagawa, Hiroomi Okuyama
  • Journal Title: Transpl Immunol. Volume: - Pages: 101559-101559
  • DOI: 10.1016/j.trim.2022.101559
  • Peer Reviewed / Open Access
• [Journal Article] The Innate Cellular Immune Response in Xenotransplantation (2022)
  • Author(s): Akira Maeda, et al.
  • Journal Title: Frontiers in Immunology Volume: in press
  • DOI: 10.3389/fimmu.2022.858604
  • Peer Reviewed
• [Journal Article] Complement regulation in the Triple-KO era, (2022)
  • Author(s): Miyagawa S, Maeda A, Toyama C, Kogata S, Okamatsu C, Yamamoto R, Masahata K, Kamiyama M, Eguchi H, Watanabe M, Nagashima H, Ikawa M, Matsunami K, Okuyama H.
  • Journal Title: Frontiers in Immunol Volume: -
  • DOI: 10.3389/fimmu.2022.860165
  • Peer Reviewed
• [Journal Article] Suppression of macrophage-mediated xenogeneic rejection by the ectopic expression of human CD177. (2022)
  • Author(s): Kogata S, Lo PC, Maeda A, Okamatsu C, Sato K, Yamamoto R, Haneda T, Yoneyama T, Toyama C, Eguchi H, Masahata K, Kamiyama M, Okuyama H, Miyagawa S.
  • Journal Title: Transpl Immunol. Volume: - Pages: 101663-101663
  • DOI: 10.1016/j.trim.2022.101663
  • Peer Reviewed
• [Journal Article] Suppression of xenogeneic innate immune response by a membrane-type human surfactant protein-A. (2022)
  • Author(s): Toyama C, Maeda A, Kogata S, Yamamoto R, Masahata K, Ueno T, Kamiyama M, Tazuke Y, Eguchi H, Okuyama H, Miyagawa S.
  • Journal Title: Exp Ther Med. Volume: - Issue: 3
  • DOI: 10.3892/etm.2022.11527
  • Peer Reviewed
• [Journal Article] Study of the CRISPR/Cas3 System for Xenotransplantation (2022)
  • Author(s): Miyagawa S, Watanabe M, Nagashima H, Sato K, Kogata S, Toyama C, Masahata K, Kamiyama M, Yamamoto R, Eguchi H, Maeda A, Yoshimi K, Mashimo T, Okuyama H.
  • Journal Title: Transplant Proc Volume: 54 Issue: 2 Pages: 522-524
  • DOI: 10.1016/j.transproceed.2021.09.070
  • Peer Reviewed
• [Journal Article] The Regulation of Neutrophil Extracellular Trap-induced Tissue Damage by Human CD177 (2021)
  • Author(s): Yoneyama Tomohisa、Maeda Akira、Kogata Shuhei、Toyama Chiyoshi、Lo Pei-Chi、Masahata Kazunori、Kamiyama Masafumi、Haneda Tomoko、Okamatu Chizu、Eguchi Hiroshi、Tazuke Yuko、Ueno Takehisa、Okuyama Hiroomi、Miyagawa Shuji
  • Journal Title: Transplantation Direct Volume: 7 Issue: 8 Pages: e734-e734
  • DOI: 10.1097/txd.0000000000001175
  • Peer Reviewed
• [Journal Article] Elevation of microRNA-214 is associated with progression of liver fibrosis in patients with biliary atresia (2021)
  • Author(s): Yoneyama Tomohisa、Ueno Takehisa、Masahata Kazunori、Toyama Chiyoshi、Maeda Akira、Tazuke Yuko、Oue Takaharu、Miyagawa Shuji、Okuyama Hiroomi
  • Journal Title: Pediatric Surgery International Volume: 38 Issue: 1 Pages: 115-122
  • DOI: 10.1007/s00383-021-05009-7
  • Peer Reviewed
• [Presentation] The suppression of xenogeneic NK cell degranulation by human CD31. (2023)
  • Author(s): Akira Maeda, Shuhei Kogata, Pei-Chi Lo, Keigo Iemitsu, Makiko Tani, Fitri Hasnaulia, Koki Takase, Kazunori Masahata, Masafumi Kamiyama, Hiroshi Eguchi, Hiroomi Okuyama, Shuji Miyagawa
  • Organizer: The 17th International Xenotransplantation Association
  • Int'l Joint Research
• [Presentation] he hybrid molecule CL-SP-D has the potential to regulate xenogeneic rejection by human neutrophils (2023)
  • Author(s): Shuji Miyagawa, Keigo Iemitsu, Akira Maeda, Rieko Sakai, Daiki Yasufuku, Makiko Tani, Fitri Hasnaulia, Katarzyna Gadomska, Kazunori Masahata, Masafumi Kamiyama, Hiroshi Eguchi, Hiroshi Nagashima, Hiroomi Okuyama.
  • Organizer: The 17th International Xenotransplantation Association
  • Int'l Joint Research
• [Presentation] The suppression of macrophage-mediated intestinal transplant rejection by C5a receptor antagonist. (2022)
  • Author(s): Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Koki Takase, Tasuku Kodama, Kazunori Masahata, Takehisa Ueno, Masafumi Kamiyama, Yuko Tazuke, Hiroshi Eguchi, Katsuyoshi Matsunami, Shuji Miyagawa, Hiroomi Okuyama
  • Organizer: The Transplantation Society 2022
• [Presentation] Effect of a C5a receptor antagonist on macrophage function in an intestinal transplant rat model (2022)
  • Author(s): Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Koki Takase, Tasuku Kodama, Kazunori Masahata, Takehisa Ueno, Masafumi Kamiyama, Yuko Tazuke, Hiroshi Eguchi, Katsuyoshi Matsunami, Shuji Miyagawa, Hiroomi Okuyama
  • Organizer: The Transplantation Society 2022
• [Presentation] Macrophages in xenotransplantation (2022)
  • Author(s): Shuji Miyagawa, Takuji Kawamura, Hiroomi Okuyama, and Akira Maeda
  • Organizer: ATW 2022
• [Presentation] T Human CD177 suppresses the neutrophil-mediated xenocytotoxicity (2021)
  • Author(s): Tomohisa Yoneyama, Akira Maeda, Shuhei Kogata, Chiyoshi Toyama, Kazunori Masahata, Masafumi Kamiyama, Chizu Okamatu, Yuko Tazuke, Takehisa Ueno, Hiroshi Eguchi, Hiroomi Okuyama, Shuji Miyagawa
  • Organizer: CAST 2021
  • Int'l Joint Research
• [Presentation] T A membrane-type surfactant protein A (SP-A) suppresses macrophage-mediated cytotoxicity in swine endothelial cells (2021)
  • Author(s): Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Kazuki Sato, Chizu Okamatsu, Hiroshi Eguchi, Masafumi Kamiyama, Yuko Tazuke, Takehisa Ueno, Hiroomi Okuyama, Shuji Miyagawa
  • Organizer: CAST 2021
  • Int'l Joint Research
• [Presentation] Testing the CPISPR / Cas3 system for the xenotransplantation field. (2021)
  • Author(s): Shuji Miyagawa, Masahito Watanabe, Kazuki Sato, Shuhei Kogata, Chiyoshi Toyama, Kazunori Masahata, Masahumi Kamiyama, Hiroshi Eguchi, Akira Maeda, Kazuto Yoshimi, Tomoji Mashimo, Hiroomi Okuyama, Hiroshi Nagashima
  • Organizer: CAST 2021
  • Int'l Joint Research
• [Presentation] CPISPR / Cas3 system for pig Gal-T (GGTA1 (2021)
  • Author(s): Shuji Miyagawa, Masahito Watanabe, Kazuki Sato, Shuhei Kogata, Chiyoshi Toyama, Kazunori Masahata, Masafumi Kamiyama, Hiroshi Eguchi, Akira Maeda, Kazuto Yoshimi, Tomoji Mashimo, Hiroomi Okuyama, Hiroshi Nagashima
  • Organizer: The 16th International Xenotransplantation Association
  • Int'l Joint Research
• [Presentation] Human CD177 Suppress Macrophage-Mediated Xenogeneic Rejection (2021)
  • Author(s): Shuhei Kogata, Akira Maeda, Pei-Chi Lo, Chizu Okamatsu, Kazuki Sato, Riho Yamamoto, Tomohisa Yoneyama, Chiyoshi Toyama, Hiroshi Eguchi, Yuko Tazuke, Hiroomi Okuyama, Shuji Miyagawa
  • Organizer: The 16th International Xenotransplantation Association
  • Int'l Joint Research
• [Book] 異種移植研究の臨床への現状。 人工臓器、vol 52 no.3 (2023)
  • Author(s): 宮川周士
  • Total Pages: 37
  • Publisher: 日本人工臓器学会
• [Book] 異種移植の現状。「異種移植の現状と展望」 医学のあゆみ、vol283 no.4 (2022)
  • Author(s): 宮川周士
  • Total Pages: 3
  • Publisher: 医歯薬出版株式会社
• [Book] 移植と補体。「補体と疾患」 (2022)
  • Author(s): 宮川周士
  • Total Pages: 6
  • Publisher: 日本臨床