Genome editing-based gene therapy for primary immunodeficiency caused by dominant gain-of-function mutations.

• Project/Area Number: 19K08312
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 52050:Embryonic medicine and pediatrics-related
• Research Institution: National Center for Child Health and Development
• Principal Investigator: Uchiyama Toru 国立研究開発法人国立成育医療研究センター, 成育遺伝研究部, 室長 (10436107)
• Co-Investigator(Kenkyū-buntansha): 西増 弘志 東京大学, 先端科学技術研究センター, 教授 (00467044)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2021: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: ゲノム編集 / 原発性免疫不全症 / 機能獲得型変異 / 遺伝子治療 / 変異アレル特異的編集 / Cas9 / 変異アリル特異的

Outline of Research at the Start

原発性免疫不全症の中でも機能獲得型変異による免疫調節異常症に対して、ゲノム編集技術による遺伝子治療法を開発する。活性化PI3KΔ症候群（APDS）は、PI3KのサブユニットであるPIK3CD遺伝子の片アリルの機能獲得型変異によって発症し、PI3K経路の恒常的活性化からリンパ増殖症状とリンパ球の機能不全を呈する疾患である。本開発では新型CRISPR/Cas9システムを用いて、変異アリル特異的にゲノム編集を実施し、機能獲得型変異による免疫不全症への遺伝子治療の可能性を検討する。

Outline of Final Research Achievements

Gain of function mutations in the PIK3CD gene causes the activated PI3K delta syndrome (APDS). The patients show the constitutive activation of PI3K/Akt signaling, which causes the immunologic abnormalities, including lymphadenopathy and immunodeficiency. In a conventional gene-addition strategy using viral vectors, the mutant allele remains and may cause clinical symptoms. Therefore, we developed the genome editing system using Cas9 nuclease to destroy the mutant allele using an indel insertion. Cas9 transfer yielded the frameshift only in mutated allele due to indel at the level of 70% in the patient’s cells. Loss of mutated allele caused the decrease in the phosphorylation of Akt, implying the improvement of constitutive activation in APDS cells. We are now developing the mice model for a long-term evaluation of the efficacy and the safety features.

Academic Significance and Societal Importance of the Research Achievements

レンチウイルスベクターを用いた遺伝子治療は多くの疾患でその有効性が認められ、造血細胞移植ドナーが不在の患者における有力な治療選択肢となりつつある。しかし、その原理は、変異遺伝子はそのままに、染色体上に新たに治療遺伝子を組み込む「付加型遺伝子治療」であり、恒常活性化変異による疾患に対しては、変異遺伝子がそのままとなることから、効果が期待できない。一方で、ゲノム編集技術は、変異遺伝子（変異アレル）のみを直接編集することが可能であり、さらに、活性化変異に対しては、変異アレルの修復ではなく、変異アレルの破壊のみでシグナルの異常活性化が期待でき、よりシンプルな遺伝子治療法の確立が可能である。

Research Products

• [Journal Article] Nonconditioned ADA-SCID gene therapy reveals ADA requirement in the hematopoietic system and clonal dominance of vector-marked clones. (2021)
  • Author(s): Uchiyama T, Sirirat Takahashi S, Nakabayashi K, Okamura K, Edasawa K, Yamada M, Watanabe N, Mochizuki E, Yasuda T, Miura A, Kato K, Tomizawa D, Otsu M, ArigaT , Onodera M.
  • Journal Title: Molecular Therapy: Methods & Clinical Development Volume: 23 Pages: 424-433
  • DOI: 10.1016/j.omtm.2021.10.003
  • Peer Reviewed / Open Access
• [Journal Article] Autoimmune hemolytic anemia associated with Takenouchi-Kosaki syndrome. (2021)
  • Author(s): Ishikawa K, Uchiyama T, Kaname T, Kawai T, Ishiguro A.
  • Journal Title: Pediatr Int. Volume: 63(12) Issue: 12 Pages: 1528-1530
  • DOI: 10.1111/ped.14651
  • Peer Reviewed
• [Journal Article] ETV6-related thrombocytopenia associated with a transient decrease in von Willebrand factor (2021)
  • Author(s): Kanamaru Yuri、Uchiyama Toru、Kaname Tadashi、Yanagi Kumiko、Ohara Osamu、Kunishima Shinji、Ishiguro Akira
  • Journal Title: International Journal of Hematology Volume: - Issue: 2 Pages: 297-300
  • DOI: 10.1007/s12185-021-03136-4
  • Peer Reviewed
• [Journal Article] Severe Liver Disorder Following Liver Transplantation in STING-Associated Vasculopathy with Onset in Infancy (2021)
  • Author(s): Ishikawa Takashi、Tamura Eiichiro、Kasahara Mureo、Uchida Hajime、Higuchi Masataka、Kobayashi Hisato、Shimizu Hirotaka、Ogawa Eiki、Yotani Nobuyuki、Irie Rie、Kosaki Rika、Kosaki Kenjiro、Uchiyama Toru、Onodera Masafumi、Kawai Toshinao
  • Journal Title: Journal of Clinical Immunology Volume: - Issue: 5 Pages: 967-974
  • DOI: 10.1007/s10875-021-00977-w
  • Peer Reviewed
• [Journal Article] Bacillus Calmette-Gu?rin (BCG) Infections at High Frequency in Both AR-CGD and X-CGD Patients Following BCG Vaccination (2020)
  • Author(s): Ishikawa Takashi、Okai Masashi、Mochizuki Emi、Uchiyama Toru、Onodera Masafumi、Kawai Toshinao
  • Journal Title: Clinical Infectious Diseases Volume: - Issue: 9 Pages: e2538-e2544
  • DOI: 10.1093/cid/ciaa1049
  • Peer Reviewed
• [Journal Article] A Distinct Feature of T Cell Subpopulations in a Patient with CHARGE Syndrome and Omenn Syndrome (2020)
  • Author(s): Uchiyama Toru、Kawakami Saori、Masuda Hiroshi、Yoshida Kazue、Niizeki Hironori、Mochizuki Emi、Edasawa Kaori、Ishiguro Akira、Onodera Masafumi
  • Journal Title: Journal of Clinical Immunology Volume: 41 Issue: 1 Pages: 233-237
  • DOI: 10.1007/s10875-020-00875-7
  • Peer Reviewed
• [Journal Article] High prevalence of SMARCB1 constitutional abnormalities including mosaicism in malignant rhabdoid tumors (2020)
  • Author(s): Shirai Ryota、Osumi Tomoo、Terashima Keita、et al
  • Journal Title: European Journal of Human Genetics Volume: 未刊 Issue: 8 Pages: 26-26
  • DOI: 10.1038/s41431-020-0614-z
  • Peer Reviewed / Open Access
• [Journal Article] Low prevalence of maternal microchimerism in peripheral blood of Japanese children with type 1 diabetes (2020)
  • Author(s): Ushijima K.、Okuno M.、Ayabe T.、Kikuchi N.、Kawamura T.、Urakami T.、Yokota I.、Amemiya S.、Uchiyama T.、Kikuchi T.、Ogata T.、Sugihara S.、Fukami M.、The Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes
  • Journal Title: Diabetic Medicine Volume: 37 Issue: 12 Pages: 2131-2135
  • DOI: 10.1111/dme.14221
  • Peer Reviewed
• [Journal Article] Copy-number analysis of Y-linked loci in young men with non-obstructive azoospermia: Implications for the rarity of early onset mosaic loss of chromosome Y. (2020)
  • Author(s): Suzuki E, Kobori Y, Katsumi M, Ushijima K, Uchiyama T, Okada H, Miyado M and Fukami M.
  • Journal Title: Reprod Med Biol. Volume: 19 Issue: 2 Pages: 178-181
  • DOI: 10.1002/rmb2.12321
  • Peer Reviewed / Open Access
• [Journal Article] Efficacy of Antithymocyte Globulin Treatment for Severe Centrilobular Injury Following Pediatric Liver Transplant: Clinical Significance of Monitoring Lymphocyte Subset (2020)
  • Author(s): Uchida Hajime、Sakamoto Seisuke、Shimizu Seiichi、Takeda Masahiro、Yanagi Yusuke、Fukuda Akinari、Uchiyama Toru、Irie Rie、Kasahara Mureo
  • Journal Title: Experimental and Clinical Transplantation Volume: 18 Issue: 3 Pages: 325-333
  • DOI: 10.6002/ect.2019.0387
  • Peer Reviewed
• [Journal Article] Prospective Study of Allogeneic Hematopoietic Stem Cell Transplantation with Post-Transplantation Cyclophosphamide and Antithymocyte Globulin from HLA-Mismatched Related Donors for Nonmalignant Diseases (2020)
  • Author(s): Osumi T、Yoshimura S、Sako M、Uchiyama T、Ishikawa T、Kawai T、Inoue E、Takimoto T、Takeuchi I、Yamada M、Sakamoto K、Yoshida K、Kimura Y、Matsukawa Y、Matsumoto K、Imadome K、Arai K、Deguchi T、Imai K、Yuza Y、Matsumoto K、Onodera M、Kanegane H、Tomizawa D、Kato M
  • Journal Title: Biology of Blood and Marrow Transplantation Volume: 26 Issue: 11 Pages: e286-e291
  • DOI: 10.1016/j.bbmt.2020.08.008
  • Peer Reviewed
• [Journal Article] Comparison of clonazepam and levetiracetam in children for prevention of busulfan-induced seizure in hematopoietic stem cell transplantation. (2020)
  • Author(s): 11.Tsujimoto SI, Shirai R, Utano T, Osumi T, Matsumoto K, Shioda Y, Kiyotani C, Uchiyama T, Deguchi T, Terashima K, Tomizawa D, Matsumoto K, Kato M.
  • Journal Title: Int J Hematol. Volume: 111 Issue: 3 Pages: 463-466
  • DOI: 10.1007/s12185-019-02795-8
  • Peer Reviewed
• [Journal Article] Direct assessment of a single-cell DNA using crudely purified live cells: a proof of concept for noninvasive prenatal definitive diagnosis. (2020)
  • Author(s): Saito T, Ito Y, Samura O, Aoki H, Uchiyama T, Okamoto A, Hata K
  • Journal Title: J Mol Diagn. Volume: 22 Issue: 2 Pages: 132-140
  • DOI: 10.1016/j.jmoldx.2019.10.006
  • Peer Reviewed
• [Journal Article] Haploinsufficiency of A20 caused by a novel nonsense variant or entire deletion of TNFAIP3 is clinically distinct from Beh?et’s disease (2019)
  • Author(s): Tsuchida Naomi、Kirino Yohei、Soejima Yutaro、Onodera Masafumi、Arai Katsuhiro、Tamura Eiichiro、Ishikawa Takashi、Kawai Toshinao、Uchiyama Toru、Nomura Shigeru、Kobayashi Daisuke、Taguri Masataka、Mitsuhashi Satomi、Mizuguchi Takeshi、Takata Atsushi、Miyake Noriko、Nakajima Hideaki、Miyatake Satoko、Matsumoto Naomichi
  • Journal Title: Arthritis Research & Therapy Volume: 21 Issue: 1 Pages: 137-137
  • DOI: 10.1186/s13075-019-1928-5
  • Peer Reviewed / Open Access
• [Journal Article] A prospective study of allogeneic transplantation from unrelated donors for chronic granulomatous disease with target busulfan-based reduced-intensity conditioning. (2019)
  • Author(s): Osumi T, Tomizawa D, Kawai T, Sako M, Inoue E, Takimoto T, Tamura E, Uchiyama T, Imadome K, Taniguchi M, Shirai R, Yoshida M, Ando R, Tsumura Y, Fuji H, Matsumoto K, Shioda Y, Kiyotani C, Terashima K, Onodera M, Matsumoto K, Kato M
  • Journal Title: Bone Marrow Transplant. Volume: 54 Issue: 1 Pages: 168-172
  • DOI: 10.1038/s41409-018-0271-9
  • Peer Reviewed
• [Journal Article] ABO-incompatible liver transplantation for children under 2?years of age: A case report and a single-center review (2018)
  • Author(s): Narumoto Soichi、Sakamoto Seisuke、Sasaki Kengo、Hirata Yoshihiro、Fukuda Akinari、Uchiyama Toru、Irie Rie、Yoshioka Takako、Kasahara Mureo
  • Journal Title: Pediatric Transplantation Volume: 23 Issue: 1
  • DOI: 10.1111/petr.13308
  • Peer Reviewed / Int'l Joint Research
• [Presentation] アデノシン・デアミナーゼ欠損症における酵素補充療法による免疫再構築の評価, (2022)
  • Author(s): 内山徹、山田雅文、宮村能子、小野寺雅史
  • Organizer: 第5回日本免疫不全・自己炎症学会総会・学術集会
• [Presentation] 新生児スクリーニングにて発見され、迅速に酵素補充療法を導入したアデノシン・デアミナーゼ欠損症の一例 (2022)
  • Author(s): 内山徹、岡井真史、坂口大俊、坂本淳、馬場みのり、田中英、入月浩美、長崎啓佑、石川尊士、井口晶裕、河合利尚、今井千速、富澤大輔、小野寺雅史
  • Organizer: 第5回日本免疫不全・自己炎症学会総会・学術集会
• [Presentation] Progress of gene therapy for primary immunodeficiency diseases. (2021)
  • Author(s): 内山徹
  • Organizer: 第27回日本遺伝子細胞治療学会学術集会
• [Presentation] 原発性免疫不全症へのゲノム編集による遺伝子治療法の開発 (2021)
  • Author(s): 内山徹
  • Organizer: 日本ゲノム編集学会第６回大会
• [Presentation] CAR-T細胞の体内動態と有効性、安全性の検討 (2021)
  • Author(s): 内山 徹
  • Organizer: 第11回 国際協力遺伝病遺伝子治療フォーラム
  • Invited
• [Presentation] 再生医療 -遺伝子治療と細胞治療- (2020)
  • Author(s): 内山 徹
  • Organizer: 第20回CRCと臨床試験のあり方を考える会議2020 in長崎
  • Invited
• [Presentation] Progress of gene therapy for primary immunodeficiency diseases. (2020)
  • Author(s): 内山 徹
  • Organizer: 第10回 国際協力遺伝病遺伝子治療フォーラム 第10周年記念大会
  • Invited
• [Presentation] レンチウイルスベクターの品質・安全管理 (2020)
  • Author(s): 内山 徹
  • Organizer: 第20回医薬品等ウイルス安全性シンポジウム
  • Invited
• [Presentation] Progress in the treatment of primary immunodeficiency -enzyme replacement therapy and gene therapy- (2020)
  • Author(s): 内山 徹
  • Organizer: 第3回日本免疫不全・自己炎症学会
  • Invited
• [Presentation] Insertional Oncogenesis in X-CGD Patient after MFGS Retroviral Vector-Mediated Gene Therapy. (2019)
  • Author(s): Toru Uchiyama
  • Organizer: 22nd Annual meeting of American Society of Gene and Cell therapy.
  • Int'l Joint Research
• [Presentation] Single cell-based vector tracing in patients with ADA-SCID treated with stem cell gene therapy. (2019)
  • Author(s): Toru Uchiyama
  • Organizer: 2019 Cell Therapy Seminar
  • Int'l Joint Research / Invited
• [Presentation] Progress of gene therapy for primary immunodeficiency. (2019)
  • Author(s): 内山 徹
  • Organizer: 第25回 日本遺伝子細胞治療学会
  • Invited
• [Presentation] X連鎖慢性肉芽腫症へのレトロウイルスベクターによる遺伝子治療における発がん挿入変異の解析 (2019)
  • Author(s): 内山 徹
  • Organizer: 第81回日本血液学会学術集会
• [Book] いま、本格化する 遺伝子治療 (2020)
  • Author(s): 小澤 敬也
  • Total Pages: 240
  • Publisher: 羊土社
  • ISBN: 9784758103848