美国国立卫生研究院Cynthia E. Dunbar和Byung-Chul Lee共同合作，近期取得重要工作进展。他们研究提出，CRISPR/HDR编辑与慢病毒转导对恒河猴HSPC长期移植和克隆动力学的影响。相关研究成果2024年3月19日在线发表于《细胞—干细胞》杂志上。

据介绍，为了通过CRISPR/同源性定向修复（HDR）进行精确的基因组编辑，需要对长期移植的造血干细胞（LT-HSC）进行有效和安全的编辑。目前人们还不清楚HDR对相关大型动物模型中真正的LT-HSC克隆动力学的影响。

为了追踪HDR编辑细胞的输出和克隆性，并提供与体内慢病毒转导的HSC的比较，研究人员开发了一种竞争性恒河猴（RM）自体造血干细胞移植模型，将用表达条形码GFP的慢病毒载体（LV）和CD33基因座编辑的HDR转导的HSC共转染。移植后2个月，CRISPR/HDR编辑的细胞显示出两个对数的下降，与LV转导细胞的长期最小损失相比，通过p53抑制几乎没有改善。与高多克隆LV转导的HSC相比，HDR的长期克隆性是寡克隆的。

总之，这些结果表明，当前基因修饰方法对HSC的影响存在显著的临床相关差异。

附：英文原文

Title: Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

Author: Byung-Chul Lee, Ashley Gin, Chuanfeng Wu, Komudi Singh, Max Grice, Ryland Mortlock, Diana Abraham, Xing Fan, Yifan Zhou, Aisha AlJanahi, Uimook Choi, Suk See DeRavin, Taehoon Shin, Sogun Hong, Cynthia E. Dunbar

Issue&Volume: 2024-03-19

Abstract: For precise genome editing via CRISPR/homology-directed repair (HDR), effective andsafe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required.The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal modelhas not been studied. To track the output and clonality of HDR-edited cells and toprovide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model,co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV)and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decreaseby 2 months following transplantation, with little improvement via p53 inhibition,in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonalitywas oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modificationapproaches on HSCs.

DOI: 10.1016/j.stem.2024.02.010

Source: https://www.cell.com/cell-stem-cell/abstract/S1934-5909(24)00053-5