目的：自然杀伤细胞（Natural Killer cells, NK）作为固有免疫系统的核心效应细胞，在肿瘤免疫治疗领域展现出独特优势。然而，传统NK细胞疗法面临来源受限、体外扩增效率低、基因编辑困难及功能持久性不足等瓶颈。脐带血因其免疫原性低、增殖潜能强、易工程化改造等特性，被视为工程化NK细胞疗法的理想来源。但现有技术体系中滋养层依赖的扩增方案可能引入异源成分干扰，而原代NK细胞的基因编辑效率与功能可塑性尚未系统性优化，制约了其临床应用。本研究旨在构建标准化脐带血NK细胞制备体系，并通过多模态基因编辑技术实现功能强化，为开发通用型NK细胞治疗产品提供技术支撑。本研究聚焦于：（1）建立脐带血NK细胞无滋养层依赖的高效扩增及冻存兼容性体系；（2）优化慢病毒转导与成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）-SpCas9核糖核蛋白（Ribonucleoprotein, RNP）递送技术，突破原代NK细胞基因编辑效率限制；（3）通过高通量CRISPR功能筛选鉴定调控NK细胞杀伤功能的关键基因；（4）验证候选基因编辑的NK细胞在体内外模型中的抗肿瘤效果及安全性。

方法：（1）细胞制备与扩增：采用Ficoll密度梯度离心联合免疫磁珠分选获得高纯度（>99%）脐带血CD56⁺CD3^- NK细胞，开发基于IMDM培养基与IL-2/IL-15/IL-21细胞因子组合的无滋养层扩增体系，实现25天内5000倍扩增。（2）基因递送优化：通过细胞因子预刺激（IL-2/IL-15/IL-21）、助感染剂（Retronectin+Vectofusin-1+硫酸鱼精蛋白+BX795）联用提升慢病毒转导效率至60%；建立BO14缓冲液联合CM137电转程序的CRISPR-SpCas9 RNP递送体系，实现>90%基因编辑效率。（3）高通量功能筛选：构建靶向转录因子（1633基因）的CRISPR文库，通过CD107a表型分选与MAGeCK分析筛选调控NK细胞脱颗粒功能的关键基因。（4）功能验证：采用CRISPR-RNP敲除候选基因，通过流式细胞术、细胞毒性检测及异种移植模型（HL-60血液肿瘤、A549实体瘤）评估功能增益与安全性。

结果：

（1）标准化扩增体系：无滋养层IMDM + IL-2/IL-15/IL-21体系支持NK细胞高效扩增（25天5000倍），冻存复苏后活力>80%，基因编辑适应性无损伤。

（2）基因编辑突破：慢病毒转导效率提升至60%，CRISPR-RNP递送实现单基因编辑效率>90%，双基因敲除（NKG2A/CISH）显著增强肿瘤杀伤（p <0.05）。

（3）功能筛选与机制：高通量筛选鉴定CAMTA2为NK细胞功能负调控因子，敲除后CD107a⁺比例提升1.4倍（p <0.001），K562杀伤率提高50%。

（4）体内疗效验证：sgCAMTA2-NK治疗组在血液瘤模型中使肿瘤负荷降低75%（p <0.001），中位生存期延长28%；实体瘤模型中肿瘤抑制率达55%（p <0.01），中位生存期延长64%。

（5）安全性评估：基因敲除未改变NK细胞成熟表型（CD56/CD16/CD57）、活化/抑制受体谱（NKG2D/KIR/NKG2A）及效应分子（穿孔素/颗粒酶B）表达（p >0.05）。

结论：本研究成功构建脐带血NK细胞无滋养层标准化扩增与基因编辑技术平台，突破原代免疫细胞工程化改造效率瓶颈。通过高通量功能筛选揭示CAMTA2作为调控NK细胞杀伤活性的新型靶点，其敲除可在维持细胞表型完整性的前提下显著增强抗肿瘤效应。临床前模型证实基因编辑NK细胞在血液瘤与实体瘤中均具有显著治疗优势，为开发通用型、强效型NK细胞疗法提供了关键技术路径与理论依据。研究成果为推进NK细胞免疫治疗临床转化奠定了重要基础。

Objective: Natural killer (NK) cells, as pivotal effector cells of the innate immune system, demonstrate unique advantages in tumor immunotherapy. However, conventional NK cell therapies face limitations such as restricted cell sources, low in vitro expansion efficiency, difficulties in genetic engineering, and insufficient functional persistence. Umbilical cord blood (UCB), with its low immunogenicity, strong proliferative potential, and ease of genetic modification, is considered an ideal source for engineered NK cell therapies. Nevertheless, existing expansion systems relying on feeder cells may introduce xenogeneic contamination, while the gene-editing efficiency and functional plasticity of primary NK cells remain suboptimal, hindering clinical translation. This study aims to establish a standardized UCB-NK cell preparation system and enhance functional efficacy through multimodal gene editing, providing technical support for developing universal NK cell-based therapeutics. This study focuses on: (1) Establishing a feeder-free, high-efficiency expansion and cryopreservation-compatible system for UCB-NK cells. (2) Optimizing lentiviral transduction and CRISPR-SpCas9 ribonucleoprotein (RNP) delivery to overcome the gene-editing limitations in primary NK cells. (3) Identifying key genes regulating NK cell cytotoxicity via high-throughput CRISPR screening. (4) Validating the antitumor efficacy and safety of gene-edited NK cells in in vitro and in vivo models.

Methods: (1) Cell Preparation & Expansion: High-purity (>99%) CD56⁺CD3⁻ UCB-NK cells were isolated via Ficoll density gradient centrifugation and immunomagnetic sorting. A feeder-free expansion system using IMDM medium supplemented with IL-2/IL-15/IL-21 achieved a 5000-fold expansion within 25 days. (2) Gene Delivery Optimization: Pre-stimulation with cytokines (IL-2/IL-15/IL-21) and combinatorial enhancers (Retronectin + Vectofusin-1 + protamine sulfate + BX795) increased lentiviral transduction efficiency to 60%. A CRISPR-SpCas9 RNP delivery system (BO14 buffer + CM137 electroporation) achieved >90% editing efficiency. (3) High-Throughput Functional Screening: A CRISPR library targeting transcription factors (1,633 genes) was constructed. CD107a-based phenotypic sorting and MAGeCK analysis identified key regulators of NK cell degranulation. (4) Functional Validation: CRISPR-RNP-mediated knockout of candidate genes was assessed via flow cytometry, cytotoxicity assays, and xenograft models (HL-60 leukemia, A549 solid tumor).

Results:

(1) Standardized Expansion System: The feeder-free IMDM + IL-2/IL-15/IL-21 system supported robust NK cell expansion (5000-fold in 25 days), with post-thaw viability >80% and preserved gene-editing compatibility.

(2) Gene-Editing Breakthroughs: Lentiviral transduction efficiency reached 60%, while CRISPR-RNP achieved >90% single-gene editing. Dual knockout of NKG2A and CISH significantly enhanced tumor killing (p < 0.05).

(3) Functional Screening & Mechanisms: High-throughput screening identified CAMTA2 as a negative regulator of NK cell function. Their knockout increased CD107a⁺ cells by 1.4-fold (p < 0.001) and K562 cytotoxicity by 50%.

(4) In Vivo Efficacy: In leukemia models, sgCAMTA2-NK reduced tumor burden by 75% (p < 0.001) and extended median survival by 28%. In solid tumors, suppression rates reached 55% (p < 0.01), with a 64% survival benefit.

(5) Safety Profile: Gene editing did not alter NK cell maturation (CD56/CD16/CD57), receptor expression (NKG2D/KIR/NKG2A), or effector molecule production (perforin/granzyme B) (p> 0.05).

Conclusion: This study successfully established a standardized, feeder-free UCB-NK cell expansion and gene-editing platform, overcoming key bottlenecks in primary immune cell engineering. High-throughput screening revealed CAMTA2 as a novel target to enhance NK cell cytotoxicity without compromising phenotypic integrity. Preclinical models demonstrated the therapeutic potential of gene-edited NK cells in both hematologic and solid tumors, providing a critical foundation for universal, high-efficacy NK cell therapies.