Developing Gene Editing Therapeutics, Biodegradable Polymeric Delivery Vehicles, and High-throughput Platforms for the Treatment of Cystic Fibrosis

开发用于治疗囊性纤维化的基因编辑疗法、可生物降解的聚合物递送载体和高通量平台

• 批准号: 10477028
• 负责人: Alexandra Sarah Annukka Piotrowski-Daspit
• 金额: $ 10.98万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10477028
• 关键词: 3-Dimensional; Amines; Base Pairing; Biocompatible Materials; Biodistribution; Biology; Biomedical Engineering; Blood Circulation; CRISPR/Cas technology; Cell Line; Cells; Chemical Engineering; Chlorides; Clinical Trials; Communities; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; DNA; DNA Repair; Development; Development Plans; Diagnosis; Disease; Disease model; Doctor of Philosophy; Drug Delivery Systems; Encapsulated; Engineering; Esters; Family; Genes; Genetic; Genetic Diseases; Goals; Guide RNA; Half-Life; Health; Hereditary Disease; Impairment; In Vitro; Interdisciplinary Study; Knowledge; Libraries; Lung; Lung diseases; Lung infections; Mentors; Methods; Modeling; Mutation; Nanotechnology; Nonsense Codon; Oligonucleotides; Organoids; Other Genetics; Patients; Peptide Nucleic Acids; Physiological; Physiology; Polymer Chemistry; Polymers; Postdoctoral Fellow; Proteins; Public Health; RNA-Directed DNA Polymerase; Reagent; Regulator Genes; Reporter; Research; Research Personnel; Reverse engineering; Safety; Scientist; Screening procedure; Site; Structure-Activity Relationship; System; Technology; Testing; Therapeutic; Therapeutic Agents; Tissue Engineering; Tissues; Training; Translations; Treatment Efficacy; United States; Universities; Work; base; base editing; biodegradable polymer; burden of illness; career; career development; cell type; clinical translation; clinically relevant; curative treatments; cystic fibrosis patients; delivery vehicle; design; design and construction; disease-causing mutation; endonuclease; engineered nucleases; experience; experimental study; genome editing; high throughput screening; high throughput technology; improved; in vitro Model; in vivo; innovation; nanomedicine; nanoparticle; novel; nuclease; nucleic acid delivery; nucleic acid-based therapeutics; prime editing; rational design; response; screening; skills; therapeutic development; therapeutic genome editing; three dimensional cell culture; three-dimensional modeling; tool; uptake

Project Summary Cystic fibrosis (CF) is a progressive genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature stop codon mutations including W1282X are among the most severe and there are no curative treatments for patients. Genome editing agents could offer promising therapeutics applicable to all CF patients. Engineered nucleases including CRISPR/Cas9 systems that can catalyze correction of disease-causing mutation(s) have shown promise and entered clinical trials. To mitigate aberrant nuclease activity and reduce off-target effects, prime editing technology combines a catalytically impaired Cas9 endonuclease fused with an engineered reverse transcriptase programmed with a prime editing guide RNA (pegRNA) that also encodes the desired edit. As an alternative technology, triplex-forming peptide nucleic acids (PNAs) have no intrinsic nuclease activity and stimulate endogenous DNA repair with low off-target effects when bound adjacent to the target site and co-delivered with donor DNA oligonucleotides. Despite advances in gene editing technology, in vivo delivery remains a primary barrier to clinical translation. The goal of the proposed research is to develop a genome editing-based therapeutic strategy for treating the W1282X nonsense CF mutation as well as high-throughput technologies for identifying effective vehicles for in vivo therapeutic nucleic acid delivery. In Aim 1, PNA- and CRISPR/Cas9 prime editing-based gene editing reagents will be designed to correct the W1282X mutation, encapsulated into poly(amine-co-ester) (PACE) nanoparticles (NPs), and tested in vitro and in vivo. In Aim 2, novel PACE materials will be developed for in vivo delivery of nucleic acid-based therapeutics to the lungs and assessed using high-throughput in vivo platforms to determine the structure-function relationships guiding physiological fate. In Aim 3, physiologically relevant 3D culture models will be developed as high-throughput screening tools to assess delivery and efficacy of CF therapies. Overall, the proposed interdisciplinary research is highly clinically relevant, furthering the translation of promising gene editing/nucleic acid therapeutics for CF and other genetic diseases. Dr. Piotrowski-Daspit received her Ph.D. in Chemical and Biological Engineering and is currently a postdoctoral fellow in the Department of Biomedical Engineering at Yale University. Thus far, she has been developing polymeric NPs for nucleic acid delivery and high-throughput in vivo tools. The career development plan outlines a comprehensive strategy for acquiring the technical, conceptual, and professional skills required to complete the proposed studies and launch an independent research career. The proposed training would enable her to gain significant experience in therapeutic development for CF and integrate her into the CF research community. The training plan, together with her background in biomedical engineering, biomaterials and drug delivery, will place her among a select group of scientists with the skills and breadth of knowledge necessary to effectively pursue interdisciplinary work on nucleic acid delivery and editing of genetic disorders.

项目摘要 囊性纤维化（CF）是由CF跨膜突变引起的进行性遗传疾病 电导调节剂（CFTR）基因。包括W1282X在内的过早停止密码子突变是最多的 严重，没有治疗治疗的患者。基因组编辑代理可以提供有希望的 适用于所有CF患者的治疗剂。设计的核酸酶，包括CRISPR/CAS9系统 催化引起疾病突变的纠正已显示出希望并进入临床试验。减轻 异常的核酸酶活性并降低脱靶效应，主要编辑技术结合了催化 CAS9核酸内切酶受损，与编程的工程逆转录酶融合在一起 引导RNA（PEGRNA）也编码所需的编辑。作为替代技术，形成三肽 核酸（PNA）没有固有的核酸酶活性，并刺激内源性DNA修复，而低靶向 当与目标部位相邻并与供体DNA寡核苷酸共同交付时，效果。尽管 基因编辑技术的进步，体内递送仍然是临床翻译的主要障碍。目标 拟议的研究是制定基于基因组编辑的治疗策略，以治疗 W1282X废话CF突变以及用于识别有效车辆的高通量技术 用于体内治疗核酸的递送。在AIM 1，PNA和CRISPR/CAS9基于基于编辑的基因 编辑试剂将旨在校正W1282X突变，该突变封装在poly（氨基酯）中 （速度）纳米颗粒（NP），并在体外和体内进行了测试。在AIM 2中，将开发新的速度材料 用于体内基于核酸的疗法到肺部的递送，并使用体内高通量进行评估 确定指导生理命运的结构功能关系的平台。在AIM 3中，生理上 相关的3D文化模型将作为高通量筛查工具开发，以评估交付和功效 CF疗法。总体而言，拟议的跨学科研究在临床上具有很高的相关性，从而进一步 CF和其他遗传疾病的有希望的基因编辑/核酸疗法的翻译。 Piotrowski-Daspit博士获得博士学位。在化学和生物工程中，目前是 耶鲁大学生物医学工程系的博士后研究员。到目前为止，她已经 开发用于核酸输送和体内高通量的聚合物NP。职业发展 计划概述了获取所需技术，概念和专业技能的全面策略 完成拟议的研究并启动独立的研究职业。拟议的培训将 使她能够获得CF的治疗开发经验，并将其整合到CF中 研究社区。培训计划，以及生物材料的生物医学工程背景 和药物输送，她将把她列入具有知识技能和广度的精选科学家组中 有效地从事核酸递送和遗传疾病编辑的跨学科工作所必需的。