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：
10248528
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/10248528
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 Cyclic AMP Cystic Fibrosis Cytidine DNA DNA Double Strand Break Data Deaminase Defect Development Disease Epithelial Epithelial Cells 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 Pharmaceutical Preparations Phenotype Physiological Physiology Point Mutation Process 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 Variant Viscosity Work airway epithelium airway surface liquid base 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 nucleases 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.

项目摘要/摘要 Cystem纤维化（CF）是一种限制生命的单基因疾病，由Cystem纤维化中的突变引起电导常规者（CFTR）基因。被细菌永久殖民，炎症变得突出，阻塞恶化，约95％患者死于呼吸衰竭。恢复某些CFTR突变类功能的疗法从策略中。编辑技术以纠正体细胞中的CFTR突变，重点是响应小分子模块化疗法。将A•T转换为G•C基座，目前有346个良好的引起疾病的变体 CFTR，其中66％是所有单核苷酸突变的点突变。在此提案中使用ABE，我们将使用ABE修改以下4 CFTR突变：1）R553X 2）W1282X是第二个和第三个社区终止密码子突变。剪接突变，代表约12％的CFTR突变。突变，导致蛋白质缺陷和ivacacaftor治疗。证明基本编辑器将在细胞中纠正CFTR并在体外纠正阴离子转运蛋白缺陷，2）定义细胞类型的编辑偏好和细胞分裂在气道细胞中的作用，3）校正阴离子通道原发性气道上皮的缺陷以及猪CF模型气道中的批判性疾病表型。项目1、2和3（以及有价值的核心）紧密合作，直接解决您计划为CF开发分子疗法的目标。实验室在高度协作环境中解决共同的目标。致力于改善CF人民的生活，并使我们对肺部生物学的了解。我们的目标是提供终身基因修复，可以适应许多CF 突变。实验将为其他其他单基因障碍提供指导，从而显着推进基因治疗领域。