慢性刺激可能导致T细胞功能障碍并限制细胞免疫疗法的疗效。需要改进的方法来比较大量的合成基因敲入(KI)序列，以重编程细胞功能。在这里，我们开发了模块化合成的KI筛选(ModPoKI)，这是一种适用于使用多标记的多基因表达适配子构建DNA KI文库的可变平台。我们构建了两个ModPoKI文库，其中包括100个转录因子(TFs)和129个天然及合成表面受体(SRs)。在不同条件下进行的30多次ModPoKI筛选中，鉴定出一个转录因子AP4(TFAP4)构建体，能增强长期刺激的CAR-T细胞的适应性和体外及体内的抗癌功能。ModPoKI的模块化设计使我们能够生成一个约10,000个TF组合的文库。对BATF-TFAP4多基因共同表达构建体进行非病毒性敲入(KI)可增强适应性。过度表达的BATF和TFAP4共同调控关键基因靶点以重编程T细胞功能。ModPoKI有助于发现复杂的基因构建体以编程细胞功能。© 2023年作者版权所有。由Elsevier Inc.出版。保留所有权利。

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). 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 an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.