Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair [AmpliconSeq]. Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair [AmpliconSeq]

Homology Directed Repair (HDR) enables precise genome editing and holds great promise in the gene therapy field. However, the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we demonstrate the development of a functional, pooled screening platform utilizing an HDR-based readout to identify protein-based reagents that improve HDR outcomes in human hematopoietic stem and progenitor cells (HSPCs), a clinically relevant cell type for gene therapy. We leveraged this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, and we identified optimized i53 variants that enable new intermolecular bonds and robustly increase HDR. These variants specifically reduce insertion-deletion outcomes and also synergize with a DNAPK inhibitor to increase HDR rates. When applied at manufacturing scale, the incorporation of improved variants results in a significant increase in cells with at least one repaired allele and improved HDR in long-term HSPCs subpopulations, while not increasing off-target editing or gross chromosomal rearrangements. We anticipate the pooled screening platform will enable discovery of future gene editing reagents that improve HDR outcomes, such as the i53 variants reported here. Overall design: we describe the design and implementation of a pooled screen to identify protein variants with optimized HDR boosting capabilities.

同源定向修复（Homology Directed Repair, HDR）可实现精准基因组编辑，在基因治疗领域极具应用前景。然而，相较于利用其他DNA修复途径（如非同源末端连接（Non-Homologous End Joining, NHEJ））的编辑方法，基于HDR的疗法因效率偏低而难以实现临床转化。本研究开发了一种基于HDR读取模式的功能化混合筛选平台，用于筛选可提升人类造血干细胞和祖细胞（hematopoietic stem and progenitor cells, HSPCs）中HDR效果的蛋白质类试剂——此类细胞是基因治疗中具有临床相关性的细胞类型。
我们依托该筛选平台，对NHEJ抑制剂i53与其靶标53BP1的结合界面序列多样性进行了系统探究，并鉴定出优化后的i53变体：这些变体可形成全新的分子间相互作用，且能显著增强HDR效率。上述变体可特异性降低插入缺失事件的发生频率，还可与DNAPK抑制剂协同作用，进一步提升HDR速率。
当以工业化生产规模应用该优化变体时，其引入可显著提高携带至少一个修复等位基因的细胞比例，并在长期造血干细胞和祖细胞亚群中改善HDR效果，且不会增加脱靶编辑或大规模染色体重排的发生风险。
我们预计，该混合筛选平台将助力未来开发更多可提升HDR效果的基因编辑试剂，正如本文所报道的i53变体。
整体实验设计：本研究详细描述了一种混合筛选的设计与实施流程，用于鉴定具备最优HDR增强能力的蛋白质变体。