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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

遗传性血液疾病是热基因组编辑的尤其有利的靶标患者造血干细胞（HSC）的体内修饰，然后自体移植会导致正常血细胞产生的终生恢复。最近我们开发了改进的SPCAS9（3XNLS-SPCAS9）和有效的电穿孔协议的版本用于使用SPCAS9的CD34+造血茎和祖细胞（HSPC）的基因组编辑核糖核蛋白（RNP）可导致高效的靶向基因修饰，保留 HSC功能和无法检测的脱靶编辑。原则上，可以利用同源重组（HR）或基础编辑来精确纠正疾病相关突变。但是，捐赠者共同交付的要求模板序列，基于HR基因修复的细胞周期依赖性以及竞争非本体末端连接/微学介导的末端连接诱变修复途径使HSC中有效的HR复杂化。基础编辑目前在其目标范围内有限对潜在的遗传毒性和HSC效率的不确定性。核酸酶诱导的可预测最终连接维修（带有indels）是一种高效的基因修饰手段，并且可以本身是根据等位基因结果的理论。这种策略可能特别是对于影响监管元件的非编码突变，例如决定了的非编码突变 mRNA剪接的模式。我们通过指导有效的非训练来假设基因组编辑 HSC中的最终结合DNA修复可以恢复基因表达，并提供持久的疗法与剪接突变有关的遗传性血液疾病。与输血依赖性的β-甲性贫血相关的两个最常见的突变是HBB IVS1- 11og> a和ivs2-654c> t引入内含异常的分裂受体和供体站点分别。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助者的HSPC的元素。红细胞在体外与这些核酸酶处理的细胞在正常剪接的HBB mRNA中显示出强大的增加并恢复成人血红蛋白（HBA）表达，这表明这是治疗的潜在策略发展。在AIMS 1和2中，我们将开发CAS9和CAS12A编辑试剂通过核酸酶优化，无偏基因组脱靶分析和评估的突变 HSC编辑速率通过编辑的β-thalassymia患者HSPC的异种汇率。在AIM 3中，我们将制定有效的策略，用于剪接连接的非策略基因编辑修复破坏与Shwachman相关的SBO中IVS2+2T> C突变的突变钻石综合征。这些研究的成功完成了，其中1个定义了编辑方法有效的HSC修复了影响造血的一系列致病剪接突变，并使得能够基于现有的核酸酶平台开发定向基因疗法的试剂。