Tan Yuto, Kumagai-Takei Naoko, Shimizu Yurika, Yamasaki Akira, Hara-Yamamoto Mari, Mitani Shigeru, Ito Tatsuo
Department of Bone and Joint Surgery, Kawasaki Medical School, Kurashiki, Japan.
Department of Hygiene, Kawasaki Medical School, Kurashiki, Japan.
PLoS One. 2025 Sep 8;20(9):e0331468. doi: 10.1371/journal.pone.0331468. eCollection 2025.
T-cell therapies have proven to be a promising treatment option for cancer patients in recent years, especially in the case of chimeric antigen receptor (CAR)-T cell therapy. However, the therapy is associated with insufficient activation of T cells or poor persistence in the patient's body, which leads to incomplete elimination of cancer cells, recurrence, and genotoxicity. By extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13 in this study, our ultimate goal is to overcome the above-mentioned challenges in the future. PD-1 plays an important role in controlling T cell responses and is expressed at the cell surface of T cells following activation. The receptor PD-1 interferes with T cell receptor (TCR) signaling following interaction with PD-L1. The outcome of stimulation via PD-1 leads to decreases in cytokine secretion and cell proliferation. We extracted the RNA region of PD-1 pre-mRNA using CD8+T cell lines and examined the effect of targeting the Exon2 splice cis-element on the production of cytokines in the present study. In particular, the production of IFN-γ, TNF-α, GM-CSF was lower in RNA-targeted cells than in non-targeted cells, but the cytokine secretion capacity and cell proliferation were maintained in RNA-targeted cells. These results suggested that the use of the RNA editing technology, CRISPR/dCas13 strategy offers a novel approach to mitigate genotoxicity in lymphocytes with cytokine production and cell proliferation.
近年来，T细胞疗法已被证明是癌症患者一种很有前景的治疗选择，尤其是嵌合抗原受体（CAR）-T细胞疗法。然而，该疗法存在T细胞激活不足或在患者体内持久性差的问题，这会导致癌细胞无法被完全清除、复发以及基因毒性。在本研究中，通过基于CRISPR/dCas13的生物学方法提取PD-1前体mRNA的剪接元件，我们的最终目标是在未来克服上述挑战。PD-1在控制T细胞反应中起重要作用，在激活后表达于T细胞的细胞表面。受体PD-1与PD-L1相互作用后会干扰T细胞受体（TCR）信号传导。通过PD-1刺激的结果会导致细胞因子分泌和细胞增殖减少。在本研究中，我们使用CD8+T细胞系提取了PD-1前体mRNA的RNA区域，并研究了靶向Exon2剪接顺式元件对细胞因子产生的影响。特别是，RNA靶向细胞中IFN-γ、TNF-α、GM-CSF的产生低于非靶向细胞，但RNA靶向细胞的细胞因子分泌能力和细胞增殖得以维持。这些结果表明，使用RNA编辑技术CRISPR/dCas13策略为减轻淋巴细胞中基因毒性以及细胞因子产生和细胞增殖提供了一种新方法。
