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Using base editing to investigate CFTR polymorphisms in lung diseases

使用碱基编辑研究肺部疾病中的 CFTR 多态性

基本信息

批准号：
10215859
负责人：
Tingting Jiang
金额：
$ 11.71万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10215859
关键词：
Address Adenine Affect Asthma Biology C57BL/6 Mouse Cause of Death Cell Line Cell model Cell physiology Cells Chronic Obstructive Airway Disease Clustered Regularly Interspaced Short Palindromic Repeats Codon Nucleotides Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cytidine DNA DNA Double Strand Break DNA Sequence DNA Sequence Alteration DNA delivery Data Dependovirus Development Diagnosis Disease Engineering Epithelial Cells FDA approved Fostering GTP-Binding Protein alpha Subunits, Gs Genes Genetic Genetic Polymorphism Genome Goals Guide RNA Human Impairment Laboratories Light Liver Liver diseases Lung Lung diseases Measures Mediating Mentors Methods Monitor Mus Mutation Nonsense Mutation Nose Nucleotides Organ Pathogenesis Pharmaceutical Preparations Phase Plasmids Play Point Mutation Predisposition Proteins Public Health Pulmonology Regulator Genes Reporter Research Research Ethics Risk Role Specificity System Techniques Technology Testing Therapeutic Therapeutic Effect Therapeutic Studies Therapeutic Uses Training Variant Work Writing airway epithelium base bronchial epithelium career development clinically relevant cystic fibrosis mouse cystic fibrosis patients design functional restoration gene correction gene therapy genomic locus in vivo intein job market mouse model novel novel therapeutics nuclease protein expression pulmonary function repaired respiratory tool trafficking transcriptome vector

项目摘要

PROJECT SUMMARY Lung diseases are among the leading causes of death worldwide. Genetic mutations underlie many lung diseases. For instance, mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can directly cause cystic fibrosis (CF) or increase the risk of other non-CF lung diseases, like chronic obstructive pulmonary disease and asthma. To date, over 2,000 mutations have been identified in the CFTR gene, classified as CF-causing or non-CF-causing polymorphisms. Correcting CF-causing mutations and exploring the function of non-CF-causing polymorphisms could help treat a wide range of lung diseases. CRISPR-associated base editing mediates the irreversible conversion of A>G (Adenine Base Editor, ABE) or C>T (Cytidine Base Editor, CBE). The goal of this proposal is to use base editing to correct CF-causing CFTR mutations in CF primary cells and mouse models, and to investigate whether non-CF-causing polymorphisms affect CFTR activity and lung cell function. Preliminary data show that ABE can correct a CF-causing nonsense mutation (W1282X) to restore CFTR expression and function in immortalized human lung epithelial cells. In the K99 phase, ABE will be delivered to a clinically-relevant cell model, CF patient-derived primary bronchial epithelial cell line, to correct W1282X, and rigorous off-target analysis will be performed to study the efficiency and specificity of ABE (Aim 1). Moreover, an in vivo delivery method targeting lung airways will be developed by packaging an intein-split ABE system into AAV5 (Aim 2). The delivery efficiency will be tested in wild type C57BL/6 mice. GFP-CFTR reporter cell lines will also be established using CRISPR-mediated homology directed repair method to integrate a GFP sequence to CFTR genomic locus (Aim 3). Research in the mentored phase will be performed under the guidance of an esteemed mentor committee, whose expertise range from CRISPR engineering and application to pulmonary biology. By the R00 phase, the PI will be ready to establish an independent laboratory focused on using base editing techniques to study point mutations in lung disease. During the R00 phase, AAV5-ABE will be delivered to W1282X CF mice to determine its potential as a novel gene therapy method to correct an “untreatable” CFTR mutation in CF (Aim 2). Furthermore, non-CF-causing mutations will be introduced by CBE in lung airway epithelial cells to understand how they affect CFTR expression, localization, and function, and how they respond to known CFTR modulator drugs – e.g. ivacaftor (Aim 3). These studies will reveal if and how non-CF-causing mutations contribute to lung disease, and suggest potential treatment approaches for lung diseases associated with non-CF-causing mutations. Collectively, the proposed studies will shed light on diagnosis and treatment of lung diseases that are associated with CFTR polymorphisms. This project will foster the PI’s continued scientific and professional training and facilitate her transition to independence.

项目总结