基因组工程技术是基于细胞的免疫疗法中优化或微调细胞功能的强大工具。然而，它们用于多个基因编辑带来了相关的生物学和技术挑战。基于短发夹 RNA (shRNA) 的细胞工程绕过了这些关键问题，是基于 CRISPR 的基因编辑的有效替代方案。在这里，我们描述了一种基于 microRNA (miRNA) 的多重 shRNA 平台，该平台通过将高效的 miRNA 支架组合成嵌合簇而获得，以传递多达四个 shRNA 样序列。由于其尺寸有限，我们的盒可以与所有 CAR 组件一起在一步过程中部署，从而简化工程 CAR T 细胞的生成。 shRNA 平台的即插即用设计使我们能够在不影响系统性能的情况下交换每个 shRNA 衍生的指导序列。通过适当选择目标序列，我们能够实现功能性 KO，或者微调目标基因的表达水平，所有这些都不需要进行基因编辑。通过我们的策略，我们同时实现了多个靶基因的简单、安全、高效和可调的调节。这种方法允许在工程细胞的转录组中有效引入多种功能相关的调整，这可能会导致在具有挑战性的环境（例如实体瘤）中提高性能。© 2023 作者。

Genome engineering technologies are powerful tools in cell-based immunotherapy to optimize or fine-tune cell functionalities. However, their use for multiple gene edits poses relevant biological and technical challenges. Short hairpin RNA (shRNA)-based cell engineering bypasses these criticalities and represents a valid alternative to CRISPR-based gene editing. Here, we describe a microRNA (miRNA)-based multiplex shRNA platform obtained by combining highly efficient miRNA scaffolds into a chimeric cluster, to deliver up to four shRNA-like sequences. Thanks to its limited size, our cassette could be deployed in a one-step process along with all the CAR components, streamlining the generation of engineered CAR T cells. The plug-and-play design of the shRNA platform allowed us to swap each shRNA-derived guide sequence without affecting the system performance. Appropriately choosing the target sequences, we were able to either achieve a functional KO, or fine-tune the expression levels of the target genes, all without the need for gene editing. Through our strategy we achieved easy, safe, efficient, and tunable modulation of multiple target genes simultaneously. This approach allows for the effective introduction of multiple functionally relevant tweaks in the transcriptome of the engineered cells, which may lead to increased performance in challenging environments, e.g., solid tumors.© 2023 The Author(s).