Rectifying splicing mutations in blood disorders by gene editing

通过基因编辑纠正血液疾病中的剪接突变

• 批准号: 10305646
• 负责人: Daniel Evan Bauer
• 金额: $ 86.17万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-20 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10305646
• 关键词: 5' Splice Site; Adult; Alleles; Autologous Transplantation; Blood; Blood Cells; Bone Marrow Transplantation; CD34 gene; Cell Cycle; Cell physiology; Cells; DNA Repair; Dependence; Development; Disease; Electroporation; Engraftment; Erythrocytes; Erythroid; Gases; Gene Abnormality; Gene Expression; Gene-Modified; Genes; Genetic Diseases; Globin; Goals; Hematological Disease; Hematopoiesis; Hematopoietic stem cells; Hemoglobin; In Vitro; Inherited; Mediating; Messenger RNA; Methods; Modeling; Modification; Mutation; Myelogenous; Nonhomologous DNA End Joining; Outcome; Pathogenicity; Pathway interactions; Patients; Pattern; Persons; Process; Production; Protocols documentation; RNA Splicing; Reagent; Recovery; Regulatory Element; Ribonucleoproteins; Shwachman-Diamond syndrome; Sickle Cell Trait; Site; Site-Directed Mutagenesis; Specificity; System; Therapeutic; Transfusion; Uncertainty; Untranslated RNA; base; base editing; beta Thalassemia; gene repair; gene therapy; genome editing; genome-wide; genotoxicity; homologous recombination; improved; innovation; insertion/deletion mutation; mutant; nuclease; preservation; reconstitution; repaired; restoration; stem cell function; targeted treatment; therapeutic development; therapeutic genome editing

Inherited blood disorders are especially favorable targets for therapeutic genome editing in that ex vivo modification of patient hematopoietic stem cells (HSCs) followed by autologous transplantation can result in lifelong recovery of normal blood cell production. Recently we developed an improved version of SpCas9 (3xNLS-SpCas9) and an efficient electroporation protocol for genome editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) using SpCas9 ribonucleoprotein (RNP) that leads to highly efficient on-target gene modification, preservation of HSC function and undetectable off-target editing. In principle , homologous recombination (HR) or base editing could be harnessed for the precise correction of disease-associated mutations. However, the requirement for co-delivery of donor template sequence, the cell cycle dependence of HR-based gene repair, and the competing nonhomologous end­ joining/microhomology mediated end joining mutagenic repair pathways complicate achieving efficient HR in HSCs. Base editing Is currently limited in its targeting range with uncertainty about potential genotoxicity and HSC efficiency. Nuclease-induced predictable end-joining repair (with indels) is a highly efficient means of gene modification, and could itself be therapeutic depending on the allelic outcome. This strategy may be particularly effective for noncoding mutations that impact regulatory elements, such as those that dictate the pattern of mRNA splicing. We hypothesize that genome editing, by directing efficient non-templated end-joining DNA repair in HSCs, could restore gene expression and provide durable therapy for inherited blood disorders associated with splicing mutations. Two of the most common mutations associated with transfusion-dependent β-thalassemia are HBB IVS1- 11OG>A and IVS2-654C> T which introduce intronic aberrant splice acceptor and donor sites respectively. Using SpCas9 and LbCas12a RNPs, we have successfully disrupted these inappropriate regulatory elements in HSPCs from multiple patient donors. The erythrocytes differentiated in vitro from these nuclease-treated cells display robust increase in normally spliced HBB mRNA and restored adult hemoglobin (HbA) expression, suggesting that this is a potent strategy for therapeutic development. In Aims 1 & 2 we will develop Cas9 and Cas12a editing reagents for these splicing mutations through nuclease optimization, unbiased genome-wide off-target analysis, and assessment of HSC editing rates through xenoengraftment of edited β-thalassemia patient HSPCs. In Aim 3, we will develop efficient strategies for the non-templated gene editing repair of splice junction disrupting mutations for the IVS2+2T>C mutation in SBOS commonly associated with Shwachman­ Diamond syndrome. The successful completion of these studies w/1 define editing approaches for the efficient HSC repair of a range of pathogenic splicing mutations that impact hematopoiesis and enable the development of targeted reagents based on existing nuclease platforms for definitive gene therapy.

遗传性血液疾病是治疗性基因组编辑特别有利的目标 对患者造血干细胞 (HSC) 进行体外修饰，然后进行自体移植 移植可以导致终生恢复正常血细胞生成。最近我们 开发了 SpCas9 的改进版本 (3xNLS-SpCas9) 和高效的电穿孔方案 使用 SpCas9 对 CD34+ 造血干细胞和祖细胞 (HSPC) 进行基因组编辑 核糖核蛋白 (RNP) 可实现高效的靶向基因修饰、保存 HSC 功能和不可检测的脱靶编辑。 原则上，同源重组（HR）或碱基编辑可用于精确的 纠正与疾病相关的突变。然而，共同交付捐赠者的要求 模板序列、基于 HR 的基因修复的细胞周期依赖性以及竞争 非同源末端连接/微同源介导的末端连接诱变修复途径 使 HSC 中实现高效人力资源变得复杂。碱基编辑目前仅限于其目标范围 潜在遗传毒性和 HSC 效率的不确定性。核酸酶诱导的可预测 末端连接修复（使用插入缺失）是一种高效的基因修饰手段，并且可以 其本身是否具有治疗作用取决于等位基因的结果。这个策略可能特别 对于影响调控元件的非编码突变有效，例如那些决定 mRNA 剪接模式。我们假设基因组编辑通过指导有效的非模板化 HSC 中的末端连接 DNA 修复可以恢复基因表达并提供持久的治疗 与剪接突变相关的遗传性血液疾病。 与输血依赖性 β 地中海贫血相关的两种最常见突变是 HBB IVS1- 11OG>A 和 IVS2-654C> T 引入内含子异常剪接受体和供体位点 分别。 使用 SpCas9 和 LbCas12a RNP，我们成功地破坏了这些不适当的监管 来自多个患者捐赠者的 HSPC 中的元素。红细胞在体外分化为 这些核酸酶处理的细胞表现出正常剪接的 HBB mRNA 的强劲增加并恢复 成人血红蛋白 (HbA) 表达，表明这是一种有效的治疗策略 发展。 在目标 1 和 2 中，我们将为这些剪接开发 Cas9 和 Cas12a 编辑试剂 通过核酸酶优化、无偏见的全基因组脱靶分析和评估进行突变 通过异种移植编辑过的 β-地中海贫血患者 HSPC 来提高 HSC 编辑率。在目标 3 中，我们 将开发剪接点非模板基因编辑修复的有效策略 SBOS 中 IVS2+2T>C 突变的破坏性突变通常与 Shwachman 相关 钻石综合症。这些研究的成功完成，定义了编辑方法 有效修复造血干细胞一系列影响造血功能的致病性剪接突变 基于现有核酸酶平台开发靶向试剂，用于明确的基因治疗。