Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
Nature. 2021 Jul;595(7866):295-302. doi: 10.1038/s41586-021-03609-w. Epub 2021 Jun 2.
Sickle cell disease (SCD) is caused by a mutation in the β-globin gene HBB. We used a custom adenine base editor (ABE8e-NRCH) to convert the SCD allele (HBB) into Makassar β-globin (HBB), a non-pathogenic variant. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBB to HBB. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBB was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBB base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar β-globin represented 79% of β-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBB-to-HBB editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBB, generates benign HBB, and minimizes the undesired consequences of double-strand DNA breaks.
镰状细胞病 (SCD) 是由β-珠蛋白基因 HBB 中的突变引起的。我们使用定制的腺嘌呤碱基编辑器 (ABE8e-NRCH) 将 SCD 等位基因 (HBB) 转换为非致病变体 Makassar β-珠蛋白 (HBB)。将编码碱基编辑器的 mRNA 与靶向引导 RNA 递送到来自 SCD 患者的造血干细胞和祖细胞 (HSPC) 中，导致 HBB 转化为 HBB 的比例达到 80%。将编辑后的人类 HSPC 移植到免疫缺陷小鼠 16 周后，HBB 的频率为 68%，骨髓网织红细胞的缺氧诱导镰变减少了五倍，表明基因编辑持久。为了评估 HBB 碱基编辑的生理效应，我们将 ABE8e-NRCH 和引导 RNA 递送到来自人类 SCD 小鼠的 HSPC 中，然后将这些细胞移植到辐照小鼠中。16 周后，Makassar β-珠蛋白在血液中的β-珠蛋白蛋白中占 79%，缺氧诱导的镰变减少了三倍。与接受未编辑细胞的小鼠相比，接受碱基编辑 HSPC 的小鼠表现出接近正常的血液学参数和减少的脾脏病理学。编辑后的骨髓的二次移植证实，基因编辑在长期造血干细胞中是持久的，并表明 20%或更多的 HBB 到 HBB 编辑足以实现表型拯救。人类 HSPC 的碱基编辑避免了 Cas9 核酸酶处理后观察到的 p53 激活和更大的缺失。这些发现指向 SCD 的一次性自体治疗，该治疗消除了致病性 HBB，产生良性 HBB，并最大限度地减少双链 DNA 断裂的不良后果。
