RNAseq analysis of NKG2A-knockout versus wildtype expanded human NK cells. RNAseq analysis of NKG2A-knockout versus wildtype expanded human NK cells

Checkpoint blockage has revolutionized cancer treatment. NKG2A is an inhibitory receptor expressed by cytotoxic lymphocytes, including NK cells. In contrast to other checkpoint inhibitory antibodies, anti-NKG2A antibodies have shown only limited success. Here, we designed a Cas9-based strategy to delete KLRC1 from human NK cells. Electroporation of KLRC1-targeting Cas9-RNP efficiently eliminated NKG2A expression from primary human NK cells. NKG2A-deficient NK cells showed normal proliferation, only minor transcriptional changes related to enhanced NK cell activation and maintained their phenotype and licensing status. Genetic deletion of NKG2A fully bypassed HLA-E inhibition and further enhanced NK cell activity against various tumor cell lines, thereby outperforming anti-NKG2A antibodies. In combination with antibody-coating of tumor cells to induce antibody-dependent cellular cytotoxicity, genetic deletion of NKG2A independently promoted cytotoxicity. Thus, Cas9-mediated targeting of NKG2A is an effective way to target this important inhibitory checkpoint. This technique is easily amenable to adoptive cell therapy in the clinical setting, where NKG2A deletion will promote anti-tumor responses and may help NK cells to better infiltrate and persist in an inhibitory tumor microenvironment. Overall design: 6 paired (wildtype versus NKG2A-knockout) expanded human NK cell samples were collected 7 days after electroporation of KLRC1-targeting RNP. Total RNA was isolated and subjected to RNA re-sequencing on the DNBseq platform.

免疫检查点阻断疗法（Checkpoint blockade）已彻底革新了癌症治疗格局。NKG2A（NKG2A）是一类表达于细胞毒性淋巴细胞（包括NK细胞）的抑制性受体。与其他免疫检查点抑制性抗体相比，抗NKG2A抗体仅取得了有限的临床成效。本研究设计了一种基于Cas9（Cas9）的基因编辑策略，从人NK细胞中敲除KLRC1（KLRC1）基因。将靶向KLRC1的Cas9-RNP复合物经电穿孔转染原代人NK细胞，可有效清除其中的NKG2A表达。NKG2A缺陷型NK细胞的增殖能力未见异常，仅出现与NK细胞活化增强相关的轻微转录组变化，且可维持其固有表型与成熟许可状态。NKG2A基因敲除可完全绕过HLA-E（HLA-E）介导的免疫抑制，并进一步增强NK细胞对多种肿瘤细胞系的杀伤活性，其抗肿瘤效果优于抗NKG2A抗体。若联合肿瘤细胞抗体包被技术以诱导抗体依赖性细胞介导的细胞毒作用（Antibody-dependent cellular cytotoxicity, ADCC），NKG2A基因敲除可独立促进NK细胞的细胞毒活性。因此，基于Cas9的NKG2A靶向编辑技术是靶向这一重要免疫检查点的有效手段。该技术可轻松适配临床环境下的过继性细胞治疗（Adoptive cell therapy, ACT），NKG2A基因敲除可增强抗肿瘤免疫应答，并助力NK细胞更好地浸润并存活于免疫抑制性肿瘤微环境中。实验整体设计：在转染靶向KLRC1的RNP复合物7天后，收集6对扩增后的人NK细胞样本（野生型与NKG2A敲除型）。提取总RNA后，在DNBseq（DNBseq）测序平台上进行RNA重测序。