Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies
Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies
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DOI：
10.1016/j.cell.2020.03.039
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发表时间：
2020-04-30
期刊：
影响因子：
64.5
通讯作者：
Marson, Alexander
中科院分区：
文献类型：
--
作者：
Roth, Theodore L.;Li, P. Jonathan;Marson, Alexander
基因修饰免疫细胞的过继转移在癌症免疫治疗中具有巨大的前景。CRISPR基因敲入靶向可改善细胞疗法，但需要更多高通量方法来测试哪些基因敲入构建体在体内最有效地增强原代细胞功能。我们开发了一种广泛适用的技术，用于对大型非病毒DNA模板的靶向整合进行条形码标记和追踪，并将其应用于在原代人类T细胞中进行混合基因敲入筛选。将数十种独特的带条形码的模板混合敲入T细胞受体（TCR）位点，揭示了在体外和体内增强适应性的基因构建体。我们进一步开发了混合敲入测序（PoKI - seq），将单细胞转录组分析和混合敲入筛选相结合，以测量体外和体内的细胞丰度和细胞状态。该平台确定了一种新型的转化生长因子β（TGF - β）R2 - 41BB嵌合受体，它可提高实体瘤的清除率。混合敲入筛选能够并行重写内源性基因序列，以加速细胞疗法基因敲入方案的发现。
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 beta (TGF-beta) 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.