美国波士顿儿童医院Daniel E. Bauer课题组发现，无体外培养的基因编辑可避免人类造血干细胞中的基因毒性。该项研究成果于2024年12月12日在线发表在《细胞—干细胞》杂志上。

研究人员将BCL11A +58和+55增强子的联合CRISPR-Cas9编辑与目前临床研究中的领先基因修饰方法进行比较。使用3xNLS-SpCas9和两条单导向RNA（sgRNA）双重靶向BCL11A +58和+55增强子，能显著提高胎儿血红蛋白（HbF）的诱导水平，包括镰状细胞贫血（SCD）患者异种移植模型。

其原因在于同时破坏了两个增强子上的核心半E-box/GATA基序。由于体外培养刺激细胞增殖，造血干细胞和祖细胞（HSPC）在双链断裂（DSB）修复中的非预期靶向结果，如长片段缺失和着丝粒远端染色体片段丧失，成为副产品。编辑静止期HSPC能够绕过长片段缺失和微核形成，并保持高效的靶向编辑和移植功能。

据介绍，编辑BCL11A红细胞增强子的基因编辑方法已被验证为诱导HbF治疗β-地中海贫血的有效途径，但编辑等位基因分布和HbF反应的异质性可能影响其安全性和疗效。

附：英文原文

Title: Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells

Author: Jing Zeng, My Anh Nguyen, Pengpeng Liu, Lucas Ferreira da Silva, Sébastien Levesque, Linda Y. Lin, David G. Justus, Karl Petri, Kendell Clement, Shaina N. Porter, Archana Verma, Nola R. Neri, Tolulope Rosanwo, Marioara-Felicia Ciuculescu, Daniela Abriss, Esther Mintzer, Stacy A. Maitland, Selami Demirci, Hye Ji Cha, Stuart H. Orkin, John F. Tisdale, David A. Williams, Lihua Julie Zhu, Shondra M. Pruett-Miller, Luca Pinello, J. Keith Joung, Vikram Pattanayak, John P. Manis, Myriam Armant, Danilo Pellin, Christian Brendel, Scot A. Wolfe, Daniel E. Bauer

Issue&Volume: 2024-12-12

Abstract: Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.

DOI: 10.1016/j.stem.2024.11.001

Source: https://www.cell.com/cell-stem-cell/abstract/S1934-5909(24)00400-4