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

• 批准号: 10400076
• 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
• 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/10400076
• 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; 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; 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

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 肺部疾病是世界范围内的主要死亡原因之一。基因突变是许多肺部疾病的基础 疾病例如，囊性纤维化跨膜传导调节因子（CFTR）基因的突变可以 直接导致囊性纤维化（CF）或增加其他非CF肺部疾病的风险，如慢性阻塞性肺病 肺病和哮喘。到目前为止，在CFTR基因中已经发现了2,000多个突变， 作为CF引起或非CF引起的多态性。纠正CF引起的突变并探索其功能 非CF引起的多态性可以帮助治疗多种肺部疾病。 CRISPR相关碱基编辑介导A>G（腺嘌呤碱基编辑器，ABE）或A> G（腺嘌呤碱基编辑器，ABE）的不可逆转化。 C>T（胞苷碱基编辑器，CBE）。该提案的目标是使用碱基编辑来纠正导致CF的CFTR CF原代细胞和小鼠模型中的突变，并研究非CF引起的多态性是否 影响CFTR活性和肺细胞功能。初步数据显示，ABE可以纠正导致CF的废话 突变（W1282 X）以恢复CFTR在永生化人肺上皮细胞中的表达和功能。在 K99期，ABE将被递送至临床相关的细胞模型，CF患者来源的原代支气管上皮细胞。 上皮细胞系，以纠正W1282 X，并将进行严格的脱靶分析以研究效率 和ABE的特异性（Aim 1）。此外，靶向肺气道的体内递送方法将由 将内含肽分裂的ABE系统包装到AAV 5中（Aim 2）。将在野生型中测试递送效率 c57 bl/6小鼠还将使用CRISPR介导的同源性指导的GFP-CFTR报告细胞系来建立。 将GFP序列整合到CFTR基因组基因座的修复方法（Aim 3）。指导阶段的研究 将在一个受人尊敬的导师委员会的指导下进行，该委员会的专业知识包括CRISPR 工程和应用于肺生物学。在R 00阶段，PI将准备建立 一个独立的实验室专注于使用碱基编辑技术来研究肺部疾病中的点突变。 在R 00阶段，将AAV 5-ABE递送至W1282 X CF小鼠以确定其作为新型免疫调节剂的潜力。 基因治疗方法，以纠正CF中“无法治疗的”CFTR突变（目的2）。此外，非CF引起的 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-例如依伐卡托 (Aim 3）。这些研究将揭示非CF引起的突变是否以及如何导致肺部疾病，并建议 与非CF引起的突变相关的肺部疾病的潜在治疗方法。统称 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， 过渡到独立。