Precise repair of the pathogenic mutation in hematopoietic stem cells (HSCs) represents an ideal cure for patients with sickle cell disease (SCD). Here, we demonstrate correction of the SCD phenotype by converting the sickle mutation codon (GTG) into a benign G-Makassar variant (GCG) using in vivo base editing in HSCs. We show successful production of helper-dependent adenoviral vectors expressing an all-in-one base editor mapping to the sickle mutation site. In HSC-enriched cells from SCD patients, transduction with the base editing vector in vitro resulted in 35% GTG > GCG conversion and phenotypic improvements in the derived red blood cells. After ex vivo transduction of HSCs from an SCD mouse model and subsequent transplantation, we achieved an average of 88% editing at the target site in transplanted mice. Importantly, in vivo HSC base editing followed by selection generated 24.5% Makassar variant in long-term repopulating HSCs of SCD mice. The treated animals demonstrated correction of disease hallmarks without any noticeable side effects. Off-target analyses at top-scored genomic sites revealed no off-target editing. This in vivo approach requires a single non-integrating vector, only intravenous/subcutaneous injections, and minimal in vivo selection. This technically simple approach holds potential for scalable applications in resource-limiting regions where SCD is prevalent.

中文翻译：

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通过体内碱基编辑在 HSC 中引入血红蛋白 G-Makassar 变体可治疗小鼠镰状细胞病


精确修复造血干细胞 （HSC） 中的致病性突变是镰状细胞病 （SCD） 患者的理想治疗方法。在这里，我们通过在 HSC 中使用体内 碱基编辑将镰状突变密码子 （GTG） 转化为良性 G-Makassar 变体 （GCG） 来展示对 SCD 表型的校正。我们展示了成功生产辅助依赖性腺病毒载体，表达映射到镰状突变位点的一体化碱基编辑器。在来自 SCD 患者的富含 HSC 的细胞中，用碱基编辑载体体外 转导导致 35% GTG > GCG 转化和衍生红细胞表型改善。 从 SCD 小鼠模型离体转导 HSC 并随后进行移植后，我们在移植小鼠的靶位点实现了平均 88% 的编辑。重要的是，体内 HSC 碱基编辑后选择在 SCD 小鼠的长期再填充 HSC 中产生了 24.5% 的望加锡变体。接受治疗的动物表现出对疾病标志物的纠正，而没有任何明显的副作用。对得分最高的基因组位点的脱靶分析显示没有脱靶编辑。 这种体内方法需要单个非整合载体，只需静脉内/皮下注射，并且需要最少的体内 选择。这种技术简单的方法在 SCD 普遍存在的资源有限地区具有可扩展应用程序的潜力。