Gene Editing Strategies to Correct CFTR Mutations

纠正 CFTR 突变的基因编辑策略

基本信息

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
项目类别：
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

项目摘要

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因子（CFTR）基因肺部疾病始于儿童早期，随着时间的推移，气道疾病开始出现。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，而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我们将研究一类新的腺嘌呤碱基编辑器（ABE）。目前，有 346 个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% 是潜在的。可使用 ABE 进行纠正。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 是第二和第三最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，第三种最常见的致病基因。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的努力。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 的疾病这项拟议的研究具有高度创新性。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。