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：
10833839
负责人：
Tingting Jiang
金额：
$ 24.9万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10833839
关键词：
Address Adenine Affect Asthma Biology C57BL/6 Mouse Cause of Death Cell Line Cell model Cell physiology Cells Chronic Obstructive Pulmonary Disease Classification 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 Genes Genetic Genetic Polymorphism Genome Goals Guide RNA Human Impairment Laboratories 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 VX-770 Variant Work Writing airway epithelium base base editing base editor bronchial epithelium career development clinically relevant cystic fibrosis mouse cystic fibrosis patients delivery vehicle 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

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.

项目摘要肺部疾病是全球死亡的主要原因之一。基因突变是许多肺疾病。例如，囊性纤维化跨膜电导调节剂（CFTR）基因的突变CAS 直接引起囊性纤维化（CF）或增加其他非CF肺部疾病的风险，例如慢性阻塞性肺部疾病和哮喘。迄今为止，在CFTR基因中已经确定了超过2,000个突变。作为CF引起或非CF引起的多态性。纠正引起CF的突变并探索功能非CF引起的多态性可以帮助治疗多种肺部疾病。 CRISPR相关的基础编辑介导了A> g（Adenine Base Editor，Abe）或 C> T（Cytidine Base Editor，CBE）。该提案的目的是使用基础编辑来纠正CF造成CFTR CF主细胞和小鼠模型中的突变，并研究非CF引起的多态性是否影响CFTR活性和肺部细胞功能。初步数据表明，安倍可以纠正引起CF的废话突变（W1282X）以恢复永生的人类肺上皮细胞中的CFTR表达和功能。在 K99阶段，安倍将交付到与临床相关的细胞模型，CF患者衍生的原发支气管将进行上皮细胞系，以纠正W1282X和严格的脱靶分析以研究效率和安倍的特异性（AIM 1）。此外，针对肺气道的体内输送方法将由将Intein-Split ABE系统包装到AAV5中（AIM 2）。交付效率将以野生类型进行测试 C57BL/6鼠。 GFP-CFTR记者细胞系也将使用CRISPR介导的同源性建立修复方法将GFP序列与CFTR基因组基因座相结合（AIM 3）。修订阶段的研究将在尊敬的心理委员会的指导下进行，其专业知识范围从CRISPR 工程和肺部生物学应用。到R00阶段，PI将准备建立一个独立的实验室专注于使用基础编辑技术研究肺部疾病中的点突变。在R00阶段，AAV5-ABE将被输送到W1282X CF小鼠，以确定其作为新颖的潜力基因治疗方法纠正CF中“不可治疗”的CFTR突变（AIM 2）。此外，非CF引起 CBE将在肺气道上皮细胞中引入突变，以了解它们如何影响CFTR 表达，定位和功能，以及它们如何响应已知的CFTR调节剂药物 - 例如ivacaftor （目标3）。这些研究将揭示是否以及非CF引起的突变如何促进肺部疾病，并建议与非CF引起突变有关的肺部疾病的潜在治疗方法。集体，拟议的研究将阐明与CFTR相关的肺部疾病的诊断和治疗多态性。该项目将促进PI的持续科学和专业培训，并促进她过渡到独立。