Gene Editing Strategies to Correct CFTR Mutations

纠正 CFTR 突变的基因编辑策略

• 批准号: 10677600
• 负责人: PAUL B MCCRAY
• 金额: $ 44.11万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677600
• 关键词: Address; Adenine; Animal Model; Anions; Bacteria; Base Pairing; Bicarbonates; Biogenesis; Biology; CRISPR/Cas technology; Cell Cycle; Cell Differentiation process; Cell Line; Cell division; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Cyclic AMP; Cystic Fibrosis; Cytidine; DNA; DNA Double Strand Break; Data; Deaminase; Defect; Development; Disease; Epithelial Cells; Epithelium; Event; Family suidae; Ferrets; Flow Cytometry; Frequencies; Genes; Goals; Growth; Guide RNA; Height; High-Throughput Nucleotide Sequencing; Host Defense; Human; In Vitro; Inflammation; Interphase Cell; Knowledge; Laboratories; Life; Lung; Lung diseases; Measures; Mediating; Mendelian disorder; Methods; Mismatch Repair; Missense Mutation; Mitotic; Modeling; Molecular; Mucins; Mutation; Nonhomologous DNA End Joining; Nonsense Codon; Nucleotides; Obstruction; Outcome; Pathogenesis; Patients; Peptides; Persons; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Point Mutation; Process; Proliferating; Proteins; Pulmonary Cystic Fibrosis; RNA Splicing; Reagent; Regulator Genes; Research; Respiratory Failure; Ribonucleoproteins; Role; Single Nucleotide Polymorphism; Site; Somatic Cell; Talents; Technology; Testing; Time; Transgenes; Treatment Efficacy; VX-770; Variant; Viscosity; Work; airway epithelium; airway surface liquid; base editing; base editor; cell type; collaborative environment; cystic fibrosis airway; disease phenotype; disease-causing mutation; early childhood; experimental study; functional restoration; gene repair; gene therapy; homologous recombination; improved; in vivo; innovation; insertion/deletion mutation; novel; novel strategies; nuclease; preference; prevent; programs; protein expression; repair strategy; restoration; single-cell RNA sequencing; small molecule; success; targeted treatment; therapeutic evaluation; time interval; tool; vector

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.

项目概要/摘要 囊性纤维化（CF）是一种由囊性纤维化突变引起的限制生命的单基因疾病 电导调节器（CFTR）基因。肺部疾病始于儿童早期，随着时间的推移，气道 细菌永久定植，炎症变得突出，梗阻恶化，约 95% 的患者死于呼吸衰竭。虽然小分子调节剂已取得重大进展 恢复某些 CFTR 突变类别功能的疗法，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应用基础研究的最新进展 编辑技术纠正体细胞中的 CFTR 突变，重点关注那些不影响体细胞的突变类别 对小分子调节剂疗法有反应。我们将研究一类新的腺嘌呤碱基编辑器 (ABE) 将 A•T 碱基对转换为 G•C 碱基对。目前，有 346 种已明确表征的致病变异 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 进行校正。在这个提案中，我们将使用ABE来修改以下4个CFTR突变：1）R553X 2) W1282X 是第二和第三最常见的提前终止密码子突变。 3) 3849+10kb C>T 是 剪接突变，约占 CFTR 突变的 12%。 4) G551D，第三大常见致病菌 突变，导致蛋白质门控缺陷，并对 Ivacaftor 治疗有反应。在此我们建议：1） 表明碱基编辑器将纠正 CF 细胞中的 CFTR 并纠正体外阴离子转运蛋白缺陷，2) 定义 编辑的细胞类型偏好和气道细胞中细胞分裂的作用，以及3）纠正阴离子通道 原发性气道上皮细胞缺陷，以及猪 CF 模型气道的严重疾病表型。 项目 1、2 和 3（以及有价值的核心）紧密合作，直接解决 该计划的目标是开发 CF 分子疗法。该计划有效地集中了一支才华横溢的团队 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 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生基因修复策略，适用于许多导致 CF 的患者 突变。这项拟议的研究具有高度创新性。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，从而显着 推进基因治疗领域。