Chandraprabha Prathibha Babu, Azhagiri Manoj Kumar K, Venkatesan Vigneshwaran, Magis Wendy, Prasad Kirti, Suresh Sevanthy, Pai Aswin Anand, Marepally Srujan, Srivastava Alok, Mohankumar Kumarasamypet Murugesan, Martin David I K, Thangavel Saravanabhavan
Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, 632002, India.
Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India.
Stem Cell Res Ther. 2024 Dec 31;15(1):504. doi: 10.1186/s13287-024-04117-0.
Sickle cell disease (SCD) and β-thalassemia patients with elevated gamma globin (HBG1/G2) levels exhibit mild or no symptoms. To recapitulate this natural phenomenon, the most coveted gene therapy approach is to edit the regulatory sequences of HBG1/G2 to reactivate them. By editing more than one regulatory sequence in the HBG promoter, the production of fetal hemoglobin (HbF) can be significantly increased. However, achieving this goal requires precise nucleotide conversions in hematopoietic stem and progenitor cells (HSPCs) at therapeutic efficiency, which remains a challenge.
We employed Cas9 RNP-ssODN-mediated homology-directed repair (HDR) gene editing to mimic two naturally occurring HBG promoter point mutations; -175T > C, associated with high HbF levels, and -158 C > T, a common polymorphism in the Indian population that induces HbF under erythropoietic stress, in HSPCs.
Asymmetric, nontarget ssODN induced high rates of complete HDR conversions, with at least 15% of HSPCs exhibiting both the -175T > C and -158 C > T mutations. Optimized conditions and treatment with the small molecule AZD-7648 increased this rate, with up to 57% of long-term engrafting human HSPCs in NBSGW mice containing at least one beneficial mutation. Functionally, in vivo erythroblasts exhibited high levels of HbF, which was sufficient to reverse the cellular phenotype of β-thalassemia. Further support through bone marrow MSC co-culture boosted complete HDR conversion rates to exceed 80%, with minimal InDels, improved cell viability, and induced fetal hemoglobin levels similar to those of Cas9 RNP-mediated indels at BCL11A enhancer and HBG promoter.
Cas9 RNP-ssODN-based nucleotide conversion at the HBG promoter offers a promising gene therapy approach to ameliorate the phenotypes of β-thalassemia and SCD. The developed approach can simplify and broaden applications that require the cointroduction of multiple nucleotide modifications in HSPCs.
镰状细胞病（SCD）和γ珠蛋白（HBG1/G2）水平升高的β地中海贫血患者表现出轻微症状或无症状。为了重现这一自然现象，最令人向往的基因治疗方法是编辑HBG1/G2的调控序列以重新激活它们。通过编辑HBG启动子中的多个调控序列，可以显著增加胎儿血红蛋白（HbF）的产生。然而，要在治疗效率上实现这一目标，需要在造血干细胞和祖细胞（HSPCs）中进行精确的核苷酸转换，这仍然是一个挑战。
我们采用Cas9核糖核蛋白-单链寡脱氧核苷酸（Cas9 RNP-ssODN）介导的同源定向修复（HDR）基因编辑，在HSPCs中模拟两种自然发生的HBG启动子点突变；与高HbF水平相关的-175T＞C，以及印度人群中常见的多态性-158 C＞T，该多态性在红细胞生成应激下诱导HbF。
不对称的非靶向ssODN诱导了高比率的完全HDR转换，至少15%的HSPCs同时表现出-175T＞C和-158 C＞T突变。优化条件并用小分子AZD-7648处理可提高这一比率，在NBSGW小鼠中，高达57%的长期植入的人类HSPCs含有至少一种有益突变。在功能上，体内成红细胞表现出高水平的HbF，这足以逆转β地中海贫血的细胞表型。通过骨髓间充质干细胞共培养进一步支持，将完全HDR转换率提高到超过80%，插入缺失最少，细胞活力提高，并诱导胎儿血红蛋白水平与在BCL11A增强子和HBG启动子处Cas9 RNP介导的插入缺失相似。
基于Cas9 RNP-ssODN的HBG启动子核苷酸转换为改善β地中海贫血和SCD的表型提供了一种有前景的基因治疗方法。所开发的方法可以简化和拓宽在HSPCs中需要同时引入多个核苷酸修饰的应用。
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