日本筑波大学Satoshi Yamazaki等研究人员合作通过单细胞扩增控制基因编辑造血干细胞的遗传异质性。这一研究成果于2023年6月28日在线发表在国际学术期刊《细胞—干细胞》上。

研究人员提出了一种以克隆密度扩大基因编辑造血干细胞（HSC）的方法，允许在移植前对单个克隆进行基因分析。研究人员通过开发一个明确的、基于聚合物的扩增系统，并在预培养的HSC的CD201⁺CD150⁺CD48^-c-Kit⁺Sca-1⁺Lin^-群体中确定长期扩增的克隆。使用Prkdcscid免疫缺陷模型，研究人员证明了其可以扩增和剖析编辑的造血干细胞克隆，从而检查所需的和非预期的修改，包括大的缺失。移植经过Prkdc校正的HSC可以挽救免疫缺陷的表型。这个体外操作平台建立了一个范式来控制造血干细胞基因编辑和治疗的遗传异质性。

据介绍，使用工程核酸酶的基因编辑经常在HSC中产生非预期的遗传病变。所以，经基因编辑的造血干细胞培养物含有异质性的群体，其中大部分不携带所需的编辑内容或隐藏不需要的突变。因此，移植经过编辑的HSC有可能出现效率不理想和移植物中出现不必要的突变。

附：英文原文

Title: Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion

Author: Hans Jiro Becker, Reiko Ishida, Adam C. Wilkinson, Takaharu Kimura, Michelle Sue Jann Lee, Cevayir Coban, Yasunori Ota, Yosuke Tanaka, Meike Roskamp, Tsubasa Sano, Arinobu Tojo, David G. Kent, Satoshi Yamazaki

Issue&Volume: 2023-06-28

Abstract: Gene editing using engineered nucleases frequently produces unintended genetic lesionsin hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneouspopulations, the majority of which either do not carry the desired edit or harborunwanted mutations. In consequence, transplanting edited HSCs carries the risks ofsuboptimal efficiency and of unwanted mutations in the graft. Here, we present anapproach for expanding gene-edited HSCs at clonal density, allowing for genetic profilingof individual clones before transplantation. We achieved this by developing a defined,polymer-based expansion system and identifying long-term expanding clones within theCD201+CD150+CD48c-Kit+Sca-1+Lin population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSCclones to check for desired and unintended modifications, including large deletions.Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity inHSC gene editing and therapy.

DOI: 10.1016/j.stem.2023.06.002

Source: https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(23)00213-8