Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA; Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
Mol Ther. 2020 Jan 8;28(1):52-63. doi: 10.1016/j.ymthe.2019.10.009. Epub 2019 Oct 15.
Enhancing natural killer (NK) cell cytotoxicity by blocking inhibitory signaling could lead to improved NK-based cancer immunotherapy. Thus, we have developed a highly efficient method for editing the genome of human NK cells using CRISPR/Cas9 to knock out inhibitory signaling molecules. Our method efficiently edits up to 90% of primary peripheral blood NK cells. As a proof-of-principle we demonstrate highly efficient knockout of ADAM17 and PDCD1, genes that have a functional impact on NK cells, and demonstrate that these gene-edited NK cells have significantly improved activity, cytokine production, and cancer cell cytotoxicity. Furthermore, we were able to expand cells to clinically relevant numbers, without loss of activity. We also demonstrate that our CRISPR/Cas9 method can be used for efficient knockin of genes by delivering homologous recombination template DNA using recombinant adeno-associated virus serotype 6 (rAAV6). Our platform represents a feasible method for generating engineered primary NK cells as a universal therapeutic for cancer immunotherapy.
通过阻断抑制性信号来增强自然杀伤 (NK) 细胞的细胞毒性，可能会导致基于 NK 细胞的癌症免疫疗法得到改善。因此，我们开发了一种使用 CRISPR/Cas9 编辑人 NK 细胞基因组的高效方法，以敲除抑制性信号分子。我们的方法可以有效地编辑高达 90%的原代外周血 NK 细胞。作为原理验证，我们证明了 ADAM17 和 PDCD1 的高效敲除，这两个基因对 NK 细胞具有功能影响，并证明这些基因编辑的 NK 细胞具有显著提高的活性、细胞因子产生和癌细胞细胞毒性。此外，我们能够在不丧失活性的情况下将细胞扩增到临床相关数量。我们还证明，我们的 CRISPR/Cas9 方法可以通过使用重组腺相关病毒血清型 6 (rAAV6) 传递同源重组模板 DNA 来高效进行基因敲入。我们的平台代表了一种可行的方法，可用于生成工程化的原代 NK 细胞，作为癌症免疫疗法的通用治疗方法。
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