Modular Pooled Discovery of Synthetic Knockin Sequences to Program Durable Cell Therapies [ChIP-Seq]

Chronic stimulation can cause T cell dysfunction and limit efficacy of cellular immunotherapies. CRISPR screens have nominated gene targets for engineered T cells, but improved methods are required to compare large numbers of synthetic knockin sequences to reprogram cell functions. Here, we developed Modular Pooled Knockin Screening (ModPoKI), an adaptable platform for modular construction of DNA knockin libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors. Over 30 ModPoKI screens across human TCR and CAR T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically-stimulated CAR T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate a ~10,000-member library of TF combinations. Non-viral knockin of a combined BATF-TFAP4 polycistronic construct further enhanced function. ModPoKI facilitates discovery of complex gene constructs to program cellular functions. Overall design: ChIP-Sequencing with BATF and TFAP4 antibodies using primary human GD2 CAR T cells with BATF, TFAP4, BATF-TFAP4 or control (tNGFR) knockin

慢性刺激可诱发T细胞功能障碍，并限制细胞免疫治疗的疗效。CRISPR筛选已为工程化T细胞筛选出潜在基因靶点，但仍需开发更优方法以大规模比较合成敲入序列，进而重编程细胞功能。
本研究开发了模块化混合敲入筛选技术（Modular Pooled Knockin Screening, ModPoKI）——一种基于条形码多顺反子接头构建DNA敲入文库的灵活适配平台。
我们构建了两个ModPoKI文库，分别包含100种转录因子（transcription factors, TFs）与129种天然及合成表面受体。在人类T细胞受体（T cell receptor, TCR）和嵌合抗原受体T细胞（chimeric antigen receptor T cell, CAR-T）的多种培养条件下开展的逾30次ModPoKI筛选实验，成功鉴定出转录因子AP4（TFAP4）敲入构建体，该构建体可增强慢性刺激后CAR-T细胞的存活适应性，并在体外与体内均提升其抗肿瘤功能。
ModPoKI的模块化特性支持我们构建包含约10000种转录因子组合的文库。通过非病毒方式敲入BATF-TFAP4联合多顺反子构建体，可进一步增强CAR-T细胞功能。ModPoKI可助力发现用于重编程细胞功能的复杂基因构建体。
总体实验设计：使用针对BATF与TFAP4的抗体，对分别敲入BATF、TFAP4、BATF-TFAP4或对照（tNGFR）的原代人GD2嵌合抗原受体T细胞进行染色质免疫共沉淀测序（ChIP-Sequencing）。