Gene Editing Strategies to Correct CFTR Mutations

项目摘要/摘要 囊性纤维化是由囊性纤维化基因突变引起的一种限制生命的单基因疾病。 电导调节因子(CFTR)基因。肺病开始于儿童早期，随着时间的推移，呼吸道 被细菌永久定植，炎症突出，梗阻恶化，约95% 的患者死于呼吸衰竭。虽然小分子调制器已经取得了重大进展 恢复某些CFTR突变类别的功能的治疗方法，约10%的CF患者没有受益 从这些策略中。这些拟议研究的目标是将最新的进展应用于BASE 编辑技术以纠正体细胞中的CFTR突变，重点放在不 对小分子调节剂疗法有反应。我们将调查一类新的腺嘌呤碱基编辑(ABE) 将A·T碱基对转换为G·C碱基对。目前，有346种具有良好特征的致病变体 在这些突变中，66%是点突变。在所有单核苷酸突变中，46%是潜在的 可使用ABE纠正。在本提案中，我们将使用ABE来修改以下4个CFTR突变：1)R553X 2)W1282X是第二和第三常见的提前终止密码子突变。3)3849+10kb C&>T是一个 剪接突变，约占CFTR突变的12%。4)G551D，第三大常见致病因素 突变，导致蛋白门控缺陷，并对异烟肼治疗有反应。在此，我们建议：1) 显示碱基编辑将在体外纠正CF细胞中的CFTR和纠正阴离子转运体缺陷，2)定义 编辑的细胞类型偏好和细胞分裂在呼吸道细胞中的作用；3)纠正阴离子通道 猪慢性阻塞性肺疾病模型的初级呼吸道上皮细胞缺陷，以及严重疾病的表型。 项目1、2和3(以及有价值的核心)密切合作，直接解决 该计划的目标是开发治疗慢性萎缩性心脏病的分子疗法。该计划有效地集中了一支有才华的团队 在高度协作的环境中解决共同目标的实验室。我们的过往记录支持我们的 致力于改善CF患者的生活，增加我们对肺部生物学的了解。 我们的目标是提供一种终身的基因修复策略，可以适用于许多引起CF的疾病 突变。这项研究具有很强的创新性。由这些产生的试剂、方法和数据 实验将为其他单基因疾病的碱基编辑提供指导，从而显著 推进基因治疗领域。

Project Summary/Abstract Cystic fibrosis (CF) is a life limiting monogenetic disease caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene. The lung disease begins in early childhood and with time, airways become permanently colonized by bacteria, inflammation becomes prominent, obstruction worsens, and ~95% of patients die of respiratory failure. While significant advances have been made with small molecule modulator therapies to restore function for some CFTR mutation classes, ~10% of people with CF have not benefited from these strategies. The goal of these proposed studies is to apply the recent advancements in base editing technology to correct CFTR mutations in somatic cells, with a focus on mutation classes that do not respond to small molecule modulator therapies. We will investigate a new class of adenine base editors (ABE) that converts A•T to G•C base pairs. Currently, there are 346 well characterized disease-causing variants of CFTR, and of these, 66% are point mutations. Of all single nucleotide mutations, 46% are potentially correctable using ABE. In this proposal, we will use ABE to modify the following 4 CFTR mutations: 1) R553X and 2) W1282X are the 2nd and 3rd most common premature stop codon mutations. 3) 3849+10kb C>T is a splicing mutation and represents ~12% of CFTR mutations. 4) G551D, the 3rd most common disease-causing mutation, results in defective protein gating and is responsive to Ivacaftor treatment. Here we propose to: 1) show that base editors will correct CFTR in CF cells and correct the anion transporter defect in vitro, 2) define the cell type preferences of editing and the role of cell division in airway cells, and 3) correct the anion channel defect in primary airway epithelia, as well as, critical disease phenotypes in the airways of a pig CF model. Projects 1, 2, and 3 (along with the valuable cores) work closely together to directly address the Program’s goal of developing molecular therapies for CF. The Program effectively focuses a team of talented laboratories to address a shared goal in a highly collaborative environment. Our track record supports our commitment to improving the lives of people with CF and increasing our understanding of lung biology. Our goal is to provide a life-long gene repair strategy that could be adapted to for many CF causing mutations. This proposed research is highly innovative. The reagents, methods, and data generated by these experiments will provide guidance for base editing for other monogenic disorders, thereby significantly advancing the gene therapy field.