Base editing and prime editing for sickle cell disease

镰状细胞病的碱基编辑和引物编辑

• 批准号: 10323054
• 负责人: DAVID R LIU
• 金额: $ 71.57万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10323054
• 关键词: Abnormal Cell; Acute Pain; Adenosine; Adult; Affect; Affinity; Alanine; Alleles; Allogenic; American; Amino Acids; Antisickling Agents; Architecture; Autologous; Base Pairing; Benign; Biochemical; Biological Assay; Blood; CD34 gene; Caring; Cell Death; Cell Line; Cell Therapy; Cell physiology; Cells; Cessation of life; Chromosomal Rearrangement; Clinical Research; Codon Nucleotides; Complex; DNA; DNA Double Strand Break; Deterioration; Development; Disease; Elements; Engineering; Engraftment; Enzymes; Erythrocytes; Erythroid Cells; Erythroid Progenitor Cells; Escherichia coli; Evolution; Fetal Hemoglobin; Frequencies; Functional disorder; Future; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Diseases; Genetic Engineering; Genetic Template; Genome; Genomic DNA; Genomics; Globin; Guide RNA; HLA Antigens; Hematological Disease; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hemoglobin; Hemoglobin F Disease; Hemolytic Anemia; Human; Hypoxia; In Vitro; Individual; Inherited; Maintenance; Malignant - descriptor; Mediating; Medical; Messenger RNA; Methods; Missense Mutation; Modification; Morbidity - disease rate; Multiple Organ Failure; Mus; Mutation; Nucleotides; Organ; Outcome; Pain; Patients; Proteins; Quality of life; RNA; Reagent; Recombinants; Regulation; Research; Safety; Sickle Cell; Sickle Cell Anemia; Sickle Cell Trait; Sickle Hemoglobin; Site; Technology; Testing; Therapeutic; Toxic effect; Transcriptional Silencer Elements; Transplantation; Treatment Efficacy; Valine; Variant; Xenograft procedure; base; base editing; base editor; beta Globin; chronic pain; curative treatments; experience; gamma Globin; gene therapy; genetic approach; genetic information; genetic manipulation; genome editing; hemoglobin polymer; improved; in vivo; insight; mortality; mouse model; mutant; new technology; novel; novel strategies; off-target mutation; polymerization; precision genetics; premature; prevent; prime editing; prime editor; promoter; repaired; safety testing; sickle erythroid; sickling; targeted treatment; therapeutic target; tool

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.

项目概要 尽管镰状细胞病 (SCD) 的医疗护理取得了进步，但大多数患者仍然面临严重的症状 疼痛、生活质量差、器官进行性恶化和过早死亡。异体造血干 细胞移植 (HSCT) 可以治愈 SCD，但与多种毒性相关，只有 20% 的患者能够治愈 有人类白细胞抗原 (HLA) 匹配的捐赠者。因此，改进并更广泛地使用 需要进行治疗。自体造血干细胞的基因修饰是一种有前途的实验方法 用于治疗 SCD，可避免与同种异体 HSCT 相关的一些问题，尽管最佳方案 技术战略尚未制定。该提案探讨了腺苷碱基编辑器 (ABE) 的使用 用于 SCD 基因校正的主编辑器 (PE)。与传统的基因组编辑相比，这些新颖的 方法创建独立于双链 DNA 断裂 (DSB) 的精确核苷酸改变，这 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 将 A·T 碱基对定位为 G·C 对。 Prime 编辑器从向导 RNA 模板复制编辑后的序列信息 进入目标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 治疗。目标 1 利用 ABE 来创建 HSC 改变，重现胎儿的遗传持续性 血红蛋白 (HPFH) 是一种良性遗传病，可通过以下方式缓解共同遗传性 SCD 的病理生理学 诱导红细胞 (RBC) 胎儿血红蛋白 (HbF) 的表达，这是一种有效的抗镰状化剂。我们有 使用蛋白质进化策略创建新的高效 ABE，在 CD34+ 造血干细胞和祖细胞 (HSPC) 中的频率高达 60%，并诱导 HbF 达到抑制红系后代缺氧镰状化的水平。目标 2 使用 ABE 转换突变 SCD 密码子 从缬氨酸到丙氨酸，从而产生“望加锡血红蛋白（HbG）”，一种天然存在的良性非 镰状变种。我们开发了一种改变的 PAM 特异性 ABE，可将 SCD 中的 HbS 等位基因转化为 HbG 供体 HSPC 的频率高达 80%，并抑制红细胞镰状化。 Aim 3 使用 Prime 编辑来恢复 将突变的 SCD 密码子转换为正常密码子 (Val→Glu)，我们已证明这种情况在 HEK293T 细胞系中有效发生 现在的目标是优化受影响个体的 HSPC。总的来说，我们的初步研究已经证明 三种新颖的、独立的编辑方法治疗 SCD 的原则，无需丰富编辑 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 并通过使用小鼠模型、体外培养进一步确定其安全性和有效性 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 直接结果并确定未来临床研究中追求的最佳治疗策略。更多的 一般来说，我们计划的研究有可能产生使用碱基编辑器和 PE 的新范例 通过对 HSC 进行精确的基因操作来治疗多种遗传性血液疾病。