嵌合敲除靶向技术在细胞免疫疗法的基因组工程中的应用。
Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.
机构信息
Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; Diabetes Center, University of California, San Francisco, San Francisco, CA, USA; Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA.
出版信息
Cell. 2020 Apr 30;181(3):728-744.e21. doi: 10.1016/j.cell.2020.03.039. Epub 2020 Apr 16.
Adoptive transfer of genetically modified immune cells holds great promise for cancer immunotherapy. CRISPR knockin targeting can improve cell therapies, but more high-throughput methods are needed to test which knockin gene constructs most potently enhance primary cell functions in vivo. We developed a widely adaptable technology to barcode and track targeted integrations of large non-viral DNA templates and applied it to perform pooled knockin screens in primary human T cells. Pooled knockin of dozens of unique barcoded templates into the T cell receptor (TCR)-locus revealed gene constructs that enhanced fitness in vitro and in vivo. We further developed pooled knockin sequencing (PoKI-seq), combining single-cell transcriptome analysis and pooled knockin screening to measure cell abundance and cell state ex vivo and in vivo. This platform nominated a novel transforming growth factor β (TGF-β) R2-41BB chimeric receptor that improved solid tumor clearance. Pooled knockin screening enables parallelized re-writing of endogenous genetic sequences to accelerate discovery of knockin programs for cell therapies.
基因修饰免疫细胞的过继转移为癌症免疫治疗带来了巨大的希望。CRISPR 基因敲入靶向技术可以改善细胞疗法，但需要更多高通量的方法来测试哪种基因敲入构建体最有效地增强原代细胞在体内的功能。我们开发了一种广泛适用的技术来对大型非病毒 DNA 模板的靶向整合进行标记和跟踪，并将其应用于对原代人 T 细胞进行 pooled knockin 筛选。将数十种独特标记的 pooled knockin 模板整合到 T 细胞受体 (TCR)-基因座中，揭示了能够增强体外和体内适应性的基因构建体。我们进一步开发了 pooled knockin 测序 (PoKI-seq)，将单细胞转录组分析与 pooled knockin 筛选相结合，以测量体外和体内的细胞丰度和细胞状态。该平台提名了一种新型转化生长因子 β (TGF-β) R2-41BB 嵌合受体，可改善实体瘤清除率。Pooled knockin 筛选能够并行重写内源性遗传序列，从而加速细胞治疗中 knockin 程序的发现。
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