Base editing and prime editing for sickle cell disease

项目摘要 尽管镰状细胞病（SCD）的医疗护理取得了进展，但大多数患者仍继续经历严重的贫血。 疼痛、生活质量差、进行性器官退化和过早死亡。异基因造血干 细胞移植（HSCT）可以治愈SCD，但与许多毒性相关，只有20%的患者 人类白细胞抗原（HLA）匹配的捐赠者。因此，改善和更广泛地获得 需要治愈性疗法。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，其避免了与同种异体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器（ABE）的使用 和用于SCD的遗传校正的初级编辑器（PE）。与传统的基因组编辑相比， 这些方法产生了不依赖于双链DNA断裂（DSB）的精确核苷酸改变， 可导致DNA结构异常、细胞死亡或恶性转化。腺苷碱基编辑器转换 靶向A·T碱基对到G·C碱基对。引物编辑器从指导RNA模板复制编辑的序列信息 插入到目标DNA位点我们将在3种不同的SCD策略中测试这些潜在的变革工具 疗法目的1利用ABE产生HSC改变，重现胎儿遗传性持续性 血红蛋白（HPFH）是一种良性遗传性疾病，通过以下方式阐明共同遗传性SCD的病理生理学： 诱导红细胞（RBC）胎儿血红蛋白（HbF）（一种有效的抗镰状化剂）的表达。我们有 使用蛋白质进化策略来创建新的高效ABE，这些ABE在 在CD 34+造血干细胞和祖细胞（HSPC）中的频率高达60%，HbF被诱导 达到抑制红细胞后代缺氧镰状化的水平。目的2使用ABE转换突变的SCD密码子 从缬氨酸到丙氨酸，从而产生“马卡萨尔血红蛋白（HbG）"，一种天然存在的良性非- 镰状变种我们已经开发了一种改变的PAM特异性ABE，其在SCD中将HbS等位基因转化为HbG。 供体HSPC的频率高达80%，抑制RBC镰状化。Aim 3采用Prime编辑来还原 突变的SCD密码子变为正常密码子（瓦尔→Glu），我们已经证明这在HEK 293 T细胞系中有效发生 现在的目标是优化来自受影响个体的HSPC。总的来说，我们的初步研究表明 三个新的原则，独立的编辑方法来治疗SCD，而不需要丰富的编辑 细胞或创建DSB。通过所提出的研究，我们寻求优化这些方法的效率 在原代HSPC中，并通过使用小鼠模型、体外培养 方法和生化测定。同时开发三种方法将使我们能够比较它们的 结果直接，并确定最佳的治疗策略，在未来的临床研究中追求。更 一般来说，我们计划的研究有可能产生新的范例，使用基础编辑器和PE， 通过对HSC的精确遗传操作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.