Gene Editing Strategies to Correct CFTR Mutations

项目概要/摘要 囊性纤维化（CF）是由囊性纤维化基因突变引起的限制生命的单基因疾病， 电导调节因子（CFTR）基因。肺部疾病开始于儿童早期，随着时间的推移， 细菌永久定植，炎症变得突出，梗阻加重，约95% 的病人死于呼吸衰竭。虽然小分子调节剂已经取得了重大进展， 一些CFTR突变类型的功能恢复治疗，约10%的CF患者没有受益 从这些战略。这些拟议研究的目标是应用基础研究的最新进展， 编辑技术，以纠正体细胞中的CFTR突变，重点是不 对小分子调节剂治疗有反应。我们将研究一类新的腺嘌呤碱基编辑器（ABE） 将A·T转化为G·C碱基对的基因目前，有346个很好表征的致病变异体， CFTR，其中66%是点突变。在所有单核苷酸突变中，46%可能是 可使用ABE校正。在本提案中，我们将使用ABE来修饰以下4个CFTR突变：1）R553 X 2）W1282 X是第2和第3位最常见的提前终止密码子突变。3)3849+ 10 kb C>T是一个 剪接突变，占CFTR突变的约12%。4)G551 D，第三大常见致病菌 突变，导致有缺陷的蛋白质门控，并响应于依伐卡托治疗。在此，我们建议：1） 表明碱基编辑器将在CF细胞中校正CFTR并在体外校正阴离子转运蛋白缺陷，2）定义 编辑的细胞类型偏好和细胞分裂在气道细胞中的作用，以及3）校正阴离子通道 原发性气道上皮细胞的缺陷，以及猪CF模型气道中的严重疾病表型。 项目1、2和3（沿着有价值的核心）紧密合作，直接解决 该计划的目标是开发CF的分子疗法。该计划有效地集中了一个团队的人才 实验室在高度协作的环境中实现共同目标。我们的业绩记录支持我们的 致力于改善CF患者的生活，并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，可以适应许多CF引起的疾病。 突变这项研究具有很强的创新性。这些方法产生的试剂、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， 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.