肿瘤细胞治疗研究
英文原题：One-step knock-in CAR constructs in human NK cells enable scalable, TGFβ1-resistant immunotherapy for solid tumors.
我们采用无病毒、一步工程化策略，设计了稳健的、抗TGF 1的同种异体CAR-NK细胞，建立了一种通用且临床可规模化的方法，用于工程化改造代谢增强的CAR-NK细胞，使其能够克服实体瘤中TME介导的免疫抑制。
嵌合抗原受体（CAR）工程化的自然杀伤（NK）细胞是肿瘤免疫治疗中一种有前景的方式，但其在实体瘤中的疗效受到肿瘤微环境（TME）免疫抑制信号的限制，尤其是转化生长因子（TGF）。
原代人NK细胞经细胞因子活化（IL-12/15/18），并通过一步电穿孔进行工程化改造，该电穿孔递送Cas9核糖核蛋白和dsDNA供体（1 kb同源臂、SFFV启动子、poly(A)、GRE元件），以敲除TGFBR2并敲入间皮素CAR。我们将其与两步AAV法进行比较，并在生产过程中给予地塞米松（Dex）处理。采用AsPC-1癌细胞杀伤试验、患者来源胰腺癌类器官（caspase-3/7成像、荧光素酶活力、活/死FACS）以及多组学分析（RNA-seq、ATAC-seq、GSEA）评估抗肿瘤功能，以评价代谢和转录修饰。
我们报道了一种精简的一步策略，通过电穿孔同时破坏 TGF 受体 II（TGF RII）并将靶向间皮素的 CAR 转基因整合到原代 NK 细胞中。通过优化单链引导 RNA、掺入糖皮质激素反应元件的供体 DNA 模板以及电穿孔参数，我们实现了敲入效率的显著提高。在基因组编辑期间进行短暂 Dex 处理，在电穿孔和 AAV 介导的平台中均增强了 CAR 表达和细胞毒性功能。Dex 通过促进氧化磷酸化和 ATP 生成，增强了 CAR-NK 细胞的细胞毒性活性。
RATIONALE: Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells represent a promising modality for cancer immunotherapy, yet their efficacy in solid tumors is limited by immunosuppressive cues from the tumor microenvironment (TME), particularly, transforming growth factor (TGF ). METHODS: Primary human NK cells were cytokine-activated (IL-12/15/18) and engineered via a one-step electroporation that delivered Cas9 ribonucleoprotein and a dsDNA donor (1 kb homology arms, SFFV promoter, poly(A), GRE element) to knock out TGFBR2 and knock in a mesothelin CAR. We compared against a two-step AAV method, and treated dexamethasone (Dex) during manufacture. Anti-tumor function was assessed using AsPC-1 cancer killing assays, patient-derived pancreatic cancer organoids (caspase-3/7 imaging, luciferase viability, live/dead FACS), and multi-omics profiling (RNA-seq, ATAC-seq, GSEA) to evaluate metabolic and transcriptional modifications. RESULTS: We report a streamlined, one-step strategy that simultaneously disrupts the TGF receptor II (TGF RII) and integrates a mesothelin-targeting CAR transgene into primary NK cells via electroporation. By optimizing single-guide RNAs, donor DNA templates incorporating glucocorticoid response elements, and electroporation parameters, we achieved markedly improved knock-in efficiency. Transient Dex treatment during genome editing enhanced CAR expression and cytotoxic function in both electroporation- and AAV-mediated platforms. Dex augmented the cytotoxic activity of CAR-NK cells by promoting oxidative phosphorylation and ATP production. CONCLUSIONS: We designed robust, TGF 1-resistant allogeneic CAR-NK cells using a virus-free, one-step engineering strategy, establishing a versatile, clinically scalable method for engineering metabolically fortified CAR-NK cells capable of overcoming TME-mediated suppression in solid tumors.
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