Rectifying splicing mutations in blood disorders by gene editing

遗传性血液疾病是治疗性基因组编辑的特别有利目标 患者造血干细胞(HSCs)的体外修饰和自体移植 移植可以导致终生恢复正常的血细胞生产。最近我们 开发了SpCas9的改进版本(3xNLS-SpCas9)和高效的电穿孔协议 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)，导致高效的靶向基因修饰，保存 HSC功能和不可检测的非目标编辑。 原则上，可以利用同源重组(HR)或碱基编辑来精确地 纠正与疾病相关的突变。然而，对捐赠者联合交付的要求 模板序列、基于HR的基因修复的细胞周期依赖性和竞争 非同源末端连接/微同源介导的末端连接突变修复途径 使HSC中实现高效的人力资源复杂化。基础编辑目前仅限于其目标范围 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性 末端连接修复(与indels)是一种高效的基因修饰手段，可以 根据等位基因的结果，它本身是有治疗作用的。这一策略可能特别 对影响调控元件的非编码突变有效，例如那些规定 信使核糖核酸剪接模式。我们假设基因组编辑，通过引导高效的非模板化 HSCs末端连接DNA修复，可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。 与输血依赖型β-地中海贫血相关的两个最常见的突变是HBBIVS1- 引入内含子异常剪接受体和供体位点的11og&gt；A和IVS2-654C&gt；T 分别进行了分析。 使用SpCas9和LbCas12a RNP，我们已经成功地扰乱了这些不适当的监管 来自多个患者捐献者的HSPC中的元素。小鼠红细胞体外分化的实验研究 这些核酸酶处理的细胞显示出正常剪接的HBB mRNA的强劲增加和恢复 成人血红蛋白(HBA)的表达，表明这是一种有效的治疗策略 发展。在AIMS 1和2中，我们将开发用于这些剪接的Cas9和Cas12a编辑试剂 通过核酸酶优化、无偏见的全基因组非靶标分析和评估的突变 通过异种移植编辑的β-地中海贫血患者的HSC编码率。在目标3中，我们 将为剪接连接的非模板化基因编辑修复开发有效的策略 通常与Shwachman相关的SBO患者IVS2 2T和GT；C突变的破坏性突变 钻石综合症。这些研究的成功完成确定了编辑方法 高效修复一系列影响造血的致病剪接突变并使 开发基于现有核酸酶平台的靶向试剂，用于明确的基因治疗。

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.