Generation of a persistent therapeutic transgene expression system by engineered latency associated transcript expression cassette of herpes simplex virus

• Project/Area Number: 19K22505
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 47:Pharmaceutical sciences and related fields
• Research Institution: Nippon Medical School
• Principal Investigator: Yoshitaka Miyagawa 日本医科大学, 医学部, 講師 (90415604)
• Project Period (FY): 2019-06-28 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2021: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2020: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2019: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: ヘルペスウイルス / CRISPRa / インシュレーター / Herpes simplex virus / LAT

Outline of Research at the Start

本研究では、申請者らが近年開発した無毒化したヘルペスウイルス（HSV）ベクターシステムと改変ゲノム編集技術を組み合わせることにより、遺伝子治療において長い間課題点とされてきた治療遺伝子の不活性化の問題解決に挑む。申請者らは、無毒化HSVの特定のゲノム領域LATがインシュレーター配列により周辺のゲノム環境に影響を受けず、一定の転写活性を示すことを見出している。本領域に改変ゲノム編集技術を応用して同領域の転写活性を安定的に高い状態に保つことを実現する。以上により、長期的に治療遺伝子供給可能な新しい遺伝子治療戦略の提案を行う。

Outline of Final Research Achievements

In this study, we generated a persistent therapeutic gene expression system by modifying the latency associated transcript (LAT) locus in a non-toxic herpes virus (HSV) vector that we have recently developed. In this novel expression system, a complex of artificial transcriptional activator dCas9 and single guide RNA (sgRNA) to target the LAT locus were continuously recruited to the LAT locus of non-toxic HSV, which allowed the region to be kept in a transcriptionally active state persistent. Moreover, we found that the transcription in the LAT locus can be further activated by using multiple sgRNAs. Our study showed that the engineered expression system generated in this study can be useful for persistent and stable therapeutic transgene expression from non-toxic HSV vectors.

Academic Significance and Societal Importance of the Research Achievements

従来、sgRNAの発現にはPol IIIプロモーターが用いられ、転写調節因子dCas9とは別々に発現される。この場合、これらの発現系はそれぞれ転写機構が異なるため、人工的に転写調節を同時に行うことは困難である。一方、今回提案する発現系では、miRNA発現系を巧みに利用してPol IIプロモーターによりsgRNAを効率的に発現できる。以上により、本発現系を構成する全ての因子を一括して発現制御することを可能とし、自己プロモーターの恒常的な転写活性化を実現できる。本発現系を用いた遺伝子治療が完成すれば、恒久的な治療効果が期待され、ベクター使用量及びベクター由来免疫応答を最小限に抑えることができる。

Research Products

• [Int'l Joint Research] University of Pittsburgh(米国)
• [Int'l Joint Research] University of Pittsburgh(米国)
• [Journal Article] Evaluation of parameters for efficient purification and long-term storage of herpes simplex virus-based vectors (2022)
  • Author(s): Kuroda Seiji、Miyagawa Yoshitaka、Sukegawa Makoto、Tomono Taro、Yamamoto Motoko、Adachi Kumi、Verlengia Gianluca、Goins William F.、Cohen Justus B.、Glorioso Joseph C.、Okada Takashi
  • Journal Title: Molecular Therapy - Methods & Clinical Development Volume: 26 Pages: 132-143
  • DOI: 10.1016/j.omtm.2022.06.007
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 昆虫細胞を活用したウイルスベクター製造技術開発 (2022)
  • Author(s): 宮川 世志幸
  • Journal Title: PHARMSTAGE Volume: 3月号
• [Journal Article] A PCR-amplified transgene fragment flanked by a single copy of a truncated inverted terminal repeat for recombinant adeno-associated virus production prevents unnecessary plasmid DNA packaging (2021)
  • Author(s): Adachi Kumi、Tomono Taro、Okada Hironori、Shiozawa Yusuke、Yamamoto Motoko、Miyagawa Yoshitaka、Okada Takashi
  • Journal Title: Gene Therapy Volume: - Issue: 7-8 Pages: 449-457
  • DOI: 10.1038/s41434-021-00299-x
  • Peer Reviewed / Open Access
• [Journal Article] Generation of an Oncolytic Herpes Simplex Viral Vector Completely Retargeted to the GDNF Receptor GFRα1 for Specific Infection of Breast Cancer Cells (2020)
  • Author(s): Hall Bonnie L.、Leronni Daniela、Miyagawa Yoshitaka、Goins William F.、Glorioso Joseph C.、Cohen Justus B.
  • Journal Title: International Journal of Molecular Sciences Volume: 21 Issue: 22 Pages: 8815-8815
  • DOI: 10.3390/ijms21228815
  • Peer Reviewed
• [Journal Article] Protocol Optimization for the Production of the Non-Cytotoxic JΔNI5 HSV Vector Deficient in Expression of Immediately Early Genes (2020)
  • Author(s): Kuroda Seiji、Miyagawa Yoshitaka、Sato Yuriko、Yamamoto Motoko、Adachi Kumi、Kinoh Hiromi、Goins William F.、Cohen Justus B.、Glorioso Joseph C.、Taniai Nobuhiko、Yoshida Hiroshi、Okada Takashi
  • Journal Title: Molecular Therapy - Methods & Clinical Development Volume: 17 Pages: 612-621
  • DOI: 10.1016/j.omtm.2020.03.014
• [Presentation] The functional analysis of human mesenchymal stem cells for cancer gene therapy. (2022)
  • Author(s): Makoto Sukegawa, Yoshitaka Miyagawa, Seiji Kuroda, Motoko Yamamoto, Kumi Adachi, Nobuhiko Taniai, Hiroshi Yoshida, Akihiro Umezawa, Mashito Sakai, Takashi Okada.
  • Organizer: The 45th Annual Meeting of the Molecular Biology Society of Japan (MBSJ2022).
• [Presentation] Impacts of purification and storage methods on the yield and functionality of oncolytic herpes simplex virus. (2022)
  • Author(s): Seiji Kuroda, Yoshitaka Miyagawa, Makoto Sukegawa, Yuriko Sato, Yuka Oyama, Motoko Yamamoto, Kumi Adachi, Mashito Sakai, Goins WF, Cohen JB, Glorioso JC, and Takashi Okada.
  • Organizer: The 28th Annual Meeting of Japan Society of Gene and Cell Therapy (JSGCT2022).
• [Presentation] A non-cytotoxic herpes simplex virus vector provides standalone inducible co-expression of four cellular reprogramming factors. (2021)
  • Author(s): Yoshitaka Miyagawa, Gianluca Verlengia, Yukage Kobari, Fang Han, Ryotaro Hashizume, Michele Simonato, Akihiro Umezawa, Justus B. Cohen, and Joseph C. Glorioso.
  • Organizer: The 44th Annual Meeting of the Molecular Biology Society of Japan (MBSJ2021).
• [Presentation] The comparison between human mesenchymal stem cells from different sources for oncolytic viral therapy. (2021)
  • Author(s): Makoto Sukegawa, Yoshitaka Miyagawa, Seiji Kuroda, Motoko Yamamoto, Kumi Adachi, Nobuhiko Taniai, Hiroshi Yoshida, Akihiro Umezawa, Mashito Sakai, Takashi Okada.
  • Organizer: The 44th Annual Meeting of the Molecular Biology Society of Japan (MBSJ2021).
• [Presentation] Functional characterization of vector-producing amniotic mesenchymal stem cells (VP-AMSCs) generated by optimized electroporation protocols. (2021)
  • Author(s): Yoshiyuki Yamazaki, Yuko Nitahara-Kasahara, Kai Miyazaki, Yoshitaka Miyagawa, Takashi Okada.
  • Organizer: The 44th Annual Meeting of the Molecular Biology Society of Japan (MBSJ2021).
• [Presentation] 遺伝子治療目的の高品質無毒化ヘルペスウイルス(HSV)ベクター精製方法および保管条件の検討. (2021)
  • Author(s): 黒田 誠司, 宮川 世志幸, 助川 誠, 酒井 真志人.
  • Organizer: 第 89 回 日本医科大学医学会総会.
• [Presentation] Capsid-dependent restriction of retroviruses by species-specific host factors, including TRIM5a and CPSF6, in bat cell lines. (2021)
  • Author(s): Sadayuki Ohkura, Masumi Shimizu, Masayuki Horie, So Nakagawa, Haruka Osanai, Yoshitaka Miyagawa, Rimpei Morita.
  • Organizer: The 68th Annual Meeting of the Japanese Society for Virology
• [Presentation] Efficacy and safety of non-cytotoxic herpes simplex virus-based vectors in vivo. (2020)
  • Author(s): Yoshitaka Miyagawa, Motoyo Maruyama, Atsushi Sakai, Yuriko Sato, Seiji Kuroda, Hiromi Kinoh, Motoko Yamamoto, Ryotaro Hashizume, Hidenori Suzuki, Justus B. Cohen, Joseph C. Glorioso, Takashi Okada.
  • Organizer: The 43rd Annual Meeting of the Molecular Biology Society of Japan (MBSJ2020)
• [Presentation] Evaluation of Purification and Storage Conditions for Highly Defective, Non-Cytotoxic Herpes Simplex Virus Vectors for Gene Therapy. (2020)
  • Author(s): Seiji Kuroda, Yoshitaka Miyagawa, Makoto Sukegawa, Taro Tomono, Motoko Yamamoto, Kumi Adachi, William F. Goins, Justus B. Cohen, Joseph C. Glorioso, and Takashi Okada.
  • Organizer: The 43rd Annual Meeting of the Molecular Biology Society of Japan (MBSJ2020)
• [Presentation] Improved purification method for herpes simplex virus-based vectors. (2019)
  • Author(s): Seiji Kuroda, Yoshitaka Miyagawa, Gianluca Verlengia, Makoto Sukegawa, Kumi Adachi, Motoko Yamamoto, Justus B. Cohen, Joseph C. Glorioso, Hideyuki Suzuki, Hiroshi Yoshida and Takashi Okada.
  • Organizer: The 43rd Annual Meeting of the Molecular Biology Society of Japan (MBSJ2020)
• [Presentation] Optimization of vector design of non-toxic herpes simplex virus-based vector for efficient in vivo transduction (2019)
  • Author(s): Yoshitaka Miyagawa, Motoyo Maruyama, Seiji Kuroda, Atsushi Sakai, Yuriko Sato, Ryotaro Hashizume, Hiromi Kinoh, Motoko Yamamoto, Justus B. Cohen, Joseph C. Glorioso, Takashi Okada.
  • Organizer: The 43rd Annual Meeting of the Molecular Biology Society of Japan (MBSJ2020)
• [Presentation] In vivo imaging of transgene expression from a non-toxic herpes simplex virus vector (2019)
  • Author(s): Yoshitaka Miyagawa, Motoyo Maruyama, Seiji Kuroda, Atsushi Sakai, Yuriko Sato, Hiromi Kinoh, Motoko Yamamoto, Justus B. Cohen, Joseph C. Glorioso, Takashi Okada.
  • Organizer: The 25th Annual Meeting of Japan Society of Gene and Cell Therapy