An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

Takumi Kishimoto; Ken Nishimura; Kana Morishita; Aya Fukuda; Yusaku Miyamae; Yutaro Kumagai; Kimio Sumaru; Mahito Nakanishi; Koji Hisatake; Masayuki Sano

doi:10.1186/s13036-024-00404-9

An engineered ligand-responsive Csy4 endoribonuclease controls transgene expression from Sendai virus vectors

J Biol Eng. 2024 Jan 16;18(1):9. doi: 10.1186/s13036-024-00404-9.

Authors

Takumi Kishimoto^{1

2}, Ken Nishimura³, Kana Morishita⁴, Aya Fukuda¹, Yusaku Miyamae⁵, Yutaro Kumagai⁴, Kimio Sumaru⁴, Mahito Nakanishi^{4

6}, Koji Hisatake¹, Masayuki Sano⁷

Affiliations

¹ Laboratory of Gene Regulation, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.
² Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
³ Laboratory of Gene Regulation, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan. ken-nishimura@md.tsukuba.ac.jp.
⁴ Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
⁵ Institute of Life and Environment Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.
⁶ TOKIWA-Bio, Inc, 2-1-6 Sengen, Tsukuba, Ibaraki, 305-0047, Japan.
⁷ Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan. m.sano@aist.go.jp.

Abstract

Background: Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer.

Results: We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes.

Conclusion: SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.

Keywords: Csy4; ES cells; Gene regulation; Ligand; Neural differentiation; Sendai virus.

Abstract

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