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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要尽管自 CRISPR-Cas9 出现以来，基因编辑技术取得了许多令人兴奋的进展，但该领域因无法催化大DNA的可编程和可预测插入而受到阻碍 DSB 生成编辑工具的使用已经发现了重大的安全问题，包括存在大量的目标基因组缺失和脱靶此外，基于 Cas9 的方法可用于编辑由大分子引起的疾病。基因突变的多样性，如囊性纤维化 (CF)，需要针对每个等位基因定制单独的引导 RNA，这使得这项技术无法作为一种广泛使用的 CF 临床工具，优化基因编辑。能够执行独立于 DSB 的、可编程的、有针对性地插入大型货物的技术通过插入功能性拷贝，无论患者的突变如何，都可以实现通用 CF 治愈方法内源基因座的 CFTR cDNA。 CRISPR 相关转座子的最新发现和发展提供了一个令人兴奋的新机会插入大型遗传货物（> 10kb）的策略具有高整合效率并且几乎没有脱靶事件。该提案旨在系统地优化人类中的 RNA 引导转座酶（称为 INTEGRATE）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 基因突变的目标 1 将重点关注通过提高 DNA 插入效率的严格优化。整合效应复合物的蛋白质递送和共定位是这一目标的关键组成部分。目标 2 全面评估脱靶编辑事件将指导全长的定向插入。人支气管上皮细胞内源基因位点的 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CFTR的成熟铺平道路。持续开发 RNA 引导的转座酶基因编辑技术，以实现不依赖于 DSB 的基因编辑 CF 的通用校正，对其他遗传疾病具有广泛的适用性。