{"title":"Building a novel TRUCK by harnessing the endogenous IFN-gamma promoter for cytokine expression.","authors":"Liya Ma, Kaiwen Zhang, Jian Xu, Jian Wang, Ting Jiang, Xiaolong Du, Jiaxin Zhang, Jing Huang, Fengyi Ren, Dong Liu, Weiwei Xue, Dongxu Kan, Mengjiao Yao, Yutian Liang, Hongxing Jason-Sun","doi":"10.1016/j.ymthe.2024.06.017","DOIUrl":null,"url":null,"abstract":"<p><p>Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.</p>","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":null,"pages":null},"PeriodicalIF":12.1000,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular Therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.ymthe.2024.06.017","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/6/15 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Despite the remarkable success of chimeric antigen receptor (CAR) T therapy in hematological malignancies, its efficacy in solid tumors remains limited. Cytokine-engineered CAR T cells offer a promising avenue, yet their clinical translation is hindered by the risks associated with constitutive cytokine expression. In this proof-of-concept study, we leverage the endogenous interferon (IFN)-γ promoter for transgenic interleukin (IL)-15 expression. We demonstrate that IFN-γ expression is tightly regulated by T cell receptor signaling. By introducing an internal ribosome entry site IL15 into the 3' UTR of the IFN-γ gene via homology directed repair-mediated knock-in, we confirm that IL-15 expression can co-express with IFN-γ in an antigen stimulation-dependent manner. Importantly, the insertion of transgenes does not compromise endogenous IFN-γ expression. In vitro and in vivo data demonstrate that IL-15 driven by the IFN-γ promoter dramatically improves CAR T cells' antitumor activity, suggesting the effectiveness of IL-15 expression. Last, as a part of our efforts toward clinical translation, we have developed an innovative two-gene knock-in approach. This approach enables the simultaneous integration of CAR and IL-15 genes into TRAC and IFN-γ gene loci using a single AAV vector. CAR T cells engineered to express IL-15 using this approach demonstrate enhanced antitumor efficacy. Overall, our study underscores the feasibility of utilizing endogenous promoters for transgenic cytokines expression in CAR T cells.
尽管 CAR T 疗法在血液恶性肿瘤中取得了巨大成功,但其在实体瘤中的疗效仍然有限。细胞因子工程化的 CAR T 细胞提供了一条前景广阔的途径,但其临床转化却受到组成型细胞因子表达相关风险的阻碍。在这项概念验证研究中,我们利用内源性 IFN-γ 启动子进行转基因 IL-15 表达。我们证明 IFN-γ 的表达受到 TCR 信号的严格调控。通过 HDR 介导的基因敲入将 IRES-IL15 导入 IFN-γ 基因的 3'-UTR 中,我们证实 IL-15 的表达可与 IFN-γ 以抗原刺激依赖性的方式共同表达。重要的是,转基因的插入不会影响内源性 IFN-γ 的表达。体外和体内数据表明,IFN-γ 启动子驱动的 IL-15 能显著提高 CAR T 细胞的抗肿瘤活性,这表明 IL-15 的表达是有效的。最后,作为临床转化努力的一部分,我们开发了一种创新的双基因敲入方法。这种方法使用单一 AAV 载体将 CAR 和 IL-15 基因同时整合到 TRAC 和 IFN-γ 基因座中。使用这种方法设计的表达 IL-15 的 CAR T 细胞显示出更强的抗肿瘤功效。总之,我们的研究强调了利用内源性启动子在 CAR T 细胞中表达转基因细胞因子的可行性。
期刊介绍:
Molecular Therapy is the leading journal for research in gene transfer, vector development, stem cell manipulation, and therapeutic interventions. It covers a broad spectrum of topics including genetic and acquired disease correction, vaccine development, pre-clinical validation, safety/efficacy studies, and clinical trials. With a focus on advancing genetics, medicine, and biotechnology, Molecular Therapy publishes peer-reviewed research, reviews, and commentaries to showcase the latest advancements in the field. With an impressive impact factor of 12.4 in 2022, it continues to attract top-tier contributions.