Base editing and prime editing for sickle cell disease

项目总结 尽管镰状细胞病(SCD)的医疗护理取得了进展，但大多数患者仍在经历严重的 疼痛、生活质量差、器官进行性退化和过早死亡。异基因造血干细胞 细胞移植(HSCT)可以治愈SCD，但与许多毒性相关，只有20%的患者 有人类白细胞抗原(HLA)匹配的捐赠者。因此，改进和更广泛的访问 需要根治疗法。自体造血干细胞的基因修饰是一种很有前途的实验方法 用于治疗SCD，它绕过了与异基因HSCT相关的一些问题，尽管最佳的 技术战略尚未确立。该提案探讨了腺苷碱基编辑的使用(ABES) 和SCD基因矫正的主要编辑(PES)。与传统的基因组编辑相比，这些新颖的 这些方法独立于双链DNA断裂(DSB)而产生精确的核苷酸改变，这是 可导致结构DNA异常、细胞死亡或恶变。基于腺苷的编辑者转换 目标A·T碱基对到G·C碱基对。主编从引导RNA模板复制编辑过的序列信息 进入一个目标DNA基因座。我们将在3种不同的SCD策略中测试这些潜在的变革性工具 心理治疗。目标1利用ABES创造HSC改变，概括了胎儿的遗传持久性 血红蛋白(HPFH)，一种良性的遗传条件，通过以下方式缓解共遗传SCD的病理生理学 诱导红细胞(RBC)胎儿血红蛋白(HBF)表达，这是一种有效的抗镰刀剂。我们有 使用蛋白质进化策略创造了新的高效ABE，在 在CD34+造血干细胞和祖细胞(HSPC)中高达60%的频率，并诱导HBF 达到抑制红系后代缺氧性镰刀的水平。AIM 2使用ABES来转换突变的SCD密码子 从Valine到Alanine，从而产生“血红蛋白Makassar(HBG)”，一种自然产生的良性非 镰刀形的变种。我们已经开发出一种改变的PAM特异性ABE，在SCD中将HBS等位基因转换为HBG 供者HSPC频率高达80%，并抑制RBC镰刀。Aim 3使用Prime编辑来还原 突变的SCD密码子转变为正常(Val→Glu)，我们已经证明这种突变发生在HEK293T细胞系中 现在的目标是优化受影响个体的HSPC。总体而言，我们的初步研究表明 三种新颖的、独立的编辑方法在不需要丰富编辑的情况下处理SCD的原则 单元格或创建DSB。通过拟议的研究，我们寻求优化这些方法的效率 为进一步确定其安全性和有效性，采用小鼠模型，体外培养 方法采用生化分析方法。同时开发三种方法将使我们能够比较它们的 结果直接，并确定最佳治疗策略，以追求在未来的临床研究。更多 一般来说，我们计划的研究有可能产生新的范例，使用基本编辑器和PE来 通过对造血干细胞进行精确的基因操作来治疗多种遗传性血液疾病。

PROJECT SUMMARY Despite advances in the medical care of sickle cell disease (SCD), most patients continue to experience severe pain, poor quality of life, progressive organ deterioration and premature death. Allogeneic hematopoietic stem cell transplantation (HSCT) can cure SCD but is associated with numerous toxicities and only 20% of patients have Human Leukocyte Antigen (HLA)-matched donors. Therefore, improved and more widely accessible curative therapies are needed. Genetic modification of autologous HSCs is a promising experimental approach for treating SCD that circumvents some of the problems associated with allogeneic HSCT, although the optimal technical strategies are not yet established. This proposal explores the use of adenosine base editors (ABEs) and prime editors (PEs) for genetic correction of SCD. In contrast to conventional genome editing, these novel approaches create precise nucleotide alterations independent of double-stranded DNA breaks (DSBs), which can cause structural DNA abnormalities, cell death or malignant transformation. Adenosine base editors convert targeted A·T base pairs to G·C pairs. Prime editors copy edited sequence information from a guide RNA template into a targeted DNA locus. We will test these potentially transformative tools in 3 different strategies for SCD therapy. Aim 1 employs ABEs to create HSC alterations that recapitulate hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition that alleviates the pathophysiology of co-inherited SCD by inducing the expression of red blood cell (RBC) fetal hemoglobin (HbF), a potent anti-sickling agent. We have used protein evolution strategies to create new high-efficiency ABEs that generate HPFH mutations at frequencies of up to 60% in CD34+ hematopoietic stem and progenitor cells (HSPCs), with HbF being induced to levels that inhibit hypoxic sickling of erythroid progeny. Aim 2 uses ABEs to convert the mutant SCD codon from valine to alanine, thereby generating “Hemoglobin Makassar (HbG)”, a naturally occurring benign non- sickling variant. We have developed an altered PAM-specific ABE that converts HbS alleles to HbG in SCD donor HSPCs at frequencies of up to 80%, with inhibition of RBC sickling. Aim 3 employs prime editing to revert the mutant SCD codon to normal (Val→Glu), which we have shown to occur efficiently in the HEK293T cell line and now aim to optimize in HSPCs from affected individuals. Overall, our preliminary studies have shown proof of principle for three novel, independent editing approaches to treating SCD without the need to enrich for edited cells or to create DSBs. Through the proposed research, we seek to optimize the efficiency of these approaches in primary HSPCs and to further determine their safety and efficacy by using mouse models, in vitro culture methods and biochemical assays. Developing three approaches simultaneously will enable us to compare their outcomes directly and to determine the best therapeutic strategy to pursue in future clinical studies. More generally, our planned studies have the potential to generate new paradigms for using base editors and PEs to treat numerous genetic blood disorders via precise genetic manipulation of HSCs.