Using base editing to investigate CFTR polymorphisms in lung diseases

项目总结 肺部疾病是全球主要的死亡原因之一。基因突变是许多肺的基础 疾病。例如，囊性纤维化跨膜传导调节基因(CFTR)的突变可以 直接导致囊性纤维化(CF)或增加其他非囊性肺疾病的风险，如慢性阻塞性肺疾病 肺部疾病和哮喘。到目前为止，已经发现了2,000多个cftr基因突变，并归类 作为导致或不引起CF的多态。纠正Cf致突变并探索其功能 非致病基因的多态可以帮助治疗多种肺部疾病。 与CRISPR相关的碱基编辑介导A&gT；G(腺嘌呤碱基编辑，ABE)或 C&gt；T(胞苷碱基编辑，CBE)。这项建议的目标是使用基本编辑来纠正导致CF的CFTR Cf原代细胞和小鼠模型中的突变，并调查非Cf引起的多态 影响CFTR活性和肺细胞功能。初步数据显示，安倍可以纠正导致CF的胡言乱语 突变(W1282X)以恢复永生化人肺上皮细胞中CFTR的表达和功能。在 K99期，ABE将被输送到临床相关的细胞模型--CF患者来源的初级支气管 上皮细胞株，以校正W1282X，并进行严格的脱靶分析以研究其效率 和ABE的特异性(目标1)。此外，将通过以下方式开发一种针对肺部呼吸道的体内给药方法 将完整拆分的ABE系统打包成AAV5(目标2)。将在野生型中测试投放效率 C57BL/6小鼠。还将利用CRISPR介导的同源定向建立GFP-CFTR报告细胞系 将GFP序列整合到cftr基因组位点的修复方法(目标3)。指导阶段的研究 将在一个受人尊敬的指导委员会的指导下进行，该委员会的专业知识来自CRISPR 工程及其在肺部生物学中的应用。到R00阶段，PI将准备好建立 独立实验室专注于使用碱基编辑技术来研究肺部疾病的点突变。 在R00阶段，AAV5-ABE将被输送给W1282X CF小鼠，以确定其作为一种新的 基因治疗方法纠正一种“无法治愈”的CFTR突变(目标2)。此外，不会导致CF 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，以及它们对已知的CFTR调节剂药物--如异烟肼--的反应 (目标3)。这些研究将揭示非致病突变是否以及如何导致肺部疾病，并提示 与非致病突变相关的肺部疾病的潜在治疗方法。总体而言， 拟议的研究将阐明与CFTR相关的肺部疾病的诊断和治疗 多态现象。该项目将促进PI的持续科学和专业培训，并为她 向独立过渡。

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.