Rectifying splicing mutations in blood disorders by gene editing

遗传性血液病是治疗性基因组编辑的特别有利的靶点， 体外修饰患者造血干细胞（HSC），然后自体移植， 移植可导致正常血细胞产生的终身恢复。最近我们 开发了SpCas9的改进版本（3xNLS-SpCas9）和有效的电穿孔方案， 用于使用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功能和不可检测的脱靶编辑。 原则上，同源重组（HR）或碱基编辑可以用于精确的基因重组。 纠正疾病相关突变。然而，要求捐助者共同提供 模板序列，基于HR的基因修复的细胞周期依赖性，以及 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中实现有效HR复杂化。基础编辑目前在其目标范围内受到限制 对潜在的遗传毒性和HSC效率存在不确定性。核酸酶诱导的可预测 末端连接修复（用indels）是一种高效的基因修饰方法， 其本身是治疗性的，这取决于等位基因的结果。这一战略可能特别 对影响调控元件的非编码突变有效，例如那些决定基因表达的非编码突变。 mRNA剪接的模式。我们假设，基因组编辑，通过指导有效的非模板化 在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病。 与输血依赖性β地中海贫血相关的两种最常见的突变是HBB IVS 1- 11OG> A和IVS2 - 654 C> T，其引入内含子异常剪接受体和供体位点 分别 使用SpCas9和LbCas12a RNP，我们已经成功地破坏了这些不适当的调控。 来自多个患者供体的HSPC中的元素。红细胞在体外分化， 这些核酸酶处理的细胞显示正常剪接的HBB mRNA的稳健增加， 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、无偏全基因组脱靶分析和评估， 通过编辑的β-地中海贫血患者HSPC的异种移植来提高HSC编辑率。在目标3中， 将为剪接点的非模板基因编辑修复开发有效的策略 SBOS中IVS2 + 2T> C突变的破坏性突变通常与Shwachman综合征相关 戴蒙德综合征。这些研究的成功完成，将为 HSC有效修复一系列影响造血的致病性剪接突变， 基于现有核酸酶平台开发靶向试剂用于确定性基因治疗。

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.