Leveraging CRISPR RNA-guided DNA Transposases for Gene Insertion at the CFTR Locus

利用 CRISPR RNA 引导的 DNA 转座酶在 CFTR 基因座插入基因

• 批准号: 10606698
• 负责人: Rebeca Teresa King
• 金额: $ 4.87万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606698
• 关键词: Affect; Age; Alleles; Biological Assay; CRISPR-associated transposons; CRISPR/Cas technology; Candidate Disease Gene; Caucasians; Cell Separation; Cells; Cessation of life; Chloride Channels; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complementary DNA; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Integration; DNA Transposable Elements; Development; Disease; Doctor of Philosophy; Epithelial Cells; Escherichia coli; Event; Exhibits; Gene Expression Regulation; Gene Mutation; Genes; Genetic; Genetic Diseases; Genome; Genomics; Guide RNA; Human; Human Genome; Individual; Introns; Ion Channel; Length; Messenger RNA; Methods; Modification; Molecular Target; Mutation; Nonhomologous DNA End Joining; Other Genetics; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Phenotype; Physiological; Plasmids; Population; Positioning Attribute; Production; Proteins; RNA; RNA Splicing; Regulator Genes; Respiratory Failure; Ribonucleoproteins; Safety; Specificity; System; Technology; Therapeutic; Transcript; Transfection; Transposase; United States; Western Blotting; autosome; bronchial epithelium; cystic fibrosis patients; functional restoration; genetic payload; genome-wide; genomic locus; improved; insertion/deletion mutation; lipofection; novel therapeutics; reconstitution; repaired; restoration; survival prediction; tool; virtual

PROJECT SUMMARY Despite the many exciting advances in gene editing technologies since the advent of CRISPR-Cas9, the field has been hampered by an inability to catalyze programmable and predictable insertion of large DNA payloads without generating double-strand breaks (DSBs). The use of DSB-generating editing tools has unearthed substantial safety concerns, including the presence of large on-target genomic deletions and off-target insertions and deletions. Additionally, Cas9-based approaches for editing diseases that are caused by a large diversity of mutations in a gene, like cystic fibrosis (CF), require individual guide RNAs tailored to each allele, which makes this technology prohibitive as a broadly accessible clinical tool. For CF, optimizing a gene editing technology with the ability to perform DSB-independent, programmable, targeted insertion of large cargos would allow the advent of a universal CF cure regardless of a patient’s mutation(s) by inserting a functional copy of CFTR cDNA at the endogenous locus. The recent discovery and development of CRISPR-associated transposons offers an exciting new strategy to insert large genetic cargos (>10kb) with high integration efficiencies and virtually no off-target events. This proposal aims to systematically optimize RNA-guided transposases, referred to as INTEGRATE, in human cells to achieve therapeutically relevant editing efficiencies, and to apply them for the universal correction of CFTR gene mutations. Aim 1 will focus on a rigorous optimization of DNA insertion efficiencies by improving protein delivery and colocalization of the INTEGRATE effector complex. A critical component of this aim will be comprehensively assessing on- and off-target editing events. Aim 2 will direct the targeted insertion of full-length CFTR cDNA at the endogenous gene locus in human bronchial epithelial cells, and quantify the production of mature CFTR and the restoration of physiologic CFTR ion channel activity. This study will pave the way for the continued development of RNA-guided transposase gene editing technologies for the DSB-independent universal correction of CF, which carries broad applicability to other genetic diseases.

项目摘要 尽管自CRISPR-CAS9冒险以来，基因编辑技术取得了许多令人兴奋的进步，但 无法催化大型DNA的可编程且可预测的插入来阻碍场 有效载荷而无需产生双链断裂（DSB）。 DSB生成编辑工具的使用具有 出土的实质性安全问题，包括存在大型靶向基因组缺失和脱离目标 插入和删除。此外，基于CAS9的编辑方法是由大型疾病引起的 基因中突变的多样性，例如囊性纤维化（CF），需要针对每个等位基因量身定制的单个引导RNA， 这使得该技术被禁止作为广泛可用的临床工具。对于CF，优化基因编辑 具有执行与DSB无关的，可编程，有针对性的大型货物的技术将 通过插入功能副本，不管患者的突变如何 CFTR cDNA处于内源基因座。 CRISPR相关的转座子的最新发现和开发提供了令人兴奋的新的 插入具有较高整合效率的大遗传兑（> 10KB）的策略，几乎没有靶向事件。 该建议旨在系统地优化RNA引导的转座酶，称为集成，在人类中 细胞以实现治疗相关的编辑效率，并将其应用于普遍校正 CFTR基因突变。 AIM 1将通过提高DNA插入效率的严格优化。 蛋白质递送和集成效应子复合物的共定位。这个目标的关键组成部分将是 全面评估目标和脱离目标编辑事件。 AIM 2将指导全长的目标插入 在人支气管上皮细胞中内源基因基因座的CFTR cDNA，并量化 成熟的CFTR和生理CFTR离子通道活性的恢复。这项研究将为 RNA引导的转座酶基因编辑技术的持续开发用于DSB独立的 CF的普遍校正，该CF对其他遗传疾病具有广泛的适用性。