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

项目概要 囊性纤维化 (CF) 是一种进行性遗传性疾病，由 CF 跨膜突变引起 电导调节器（CFTR）基因。包括 W1282X 在内的过早终止密码子突变是最常见的突变之一 严重且对患者没有有效的治疗方法。基因组编辑剂可能提供有希望的 适用于所有 CF 患者的治疗方法。工程化核酸酶，包括 CRISPR/Cas9 系统，可以 催化纠正致病突变已显示出希望并进入临床试验。减轻 异常的核酸酶活性并减少脱靶效应，prime 编辑技术结合了催化作用 受损的 Cas9 核酸内切酶与经过 Prime 编辑编程的工程逆转录酶融合 引导 RNA (pegRNA) 也编码所需的编辑。作为替代技术，三链体形成肽 核酸 (PNA) 没有内在的核酸酶活性，可刺激内源 DNA 修复，脱靶率低 当与靶位点相邻结合并与供体 DNA 寡核苷酸共同递送时产生效应。尽管 随着基因编辑技术的进步，体内递送仍然是临床转化的主要障碍。目标 拟议研究的目的是开发一种基于基因组编辑的治疗策略来治疗 W1282X 无义 CF 突变以及识别有效载体的高通量技术 用于体内治疗性核酸递送。在目标 1 中，基于 PNA 和 CRISPR/Cas9 主要编辑的基因 编辑试剂将被设计来纠正 W1282X 突变，封装在聚（胺-酯）中 (PACE) 纳米颗粒 (NP)，并进行了体外和体内测试。在目标 2 中，将开发新型 PACE 材料 用于将基于核酸的治疗药物体内递送至肺部，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。在目标 3 中，生理上 相关的 3D 培养模型将被开发为高通量筛选工具，以评估交付和功效 CF 疗法。总体而言，拟议的跨学科研究具有高度的临床相关性，进一步促进了 转化有前景的基因编辑/核酸疗法治疗 CF 和其他遗传疾病。 Piotrowski-Daspit 博士获得博士学位。化学和生物工程专业，目前是 耶鲁大学生物医学工程系博士后。迄今为止，她已经 开发用于核酸递送的聚合纳米粒子和高通量体内工具。职业发展 计划概述了获取所需技术、概念和专业技能的综合策略 完成拟议的研究并开展独立的研究生涯。拟议的培训将 使她能够获得 CF 治疗开发方面的重要经验，并将她融入 CF 领域 研究社区。培训计划，以及她在生物医学工程、生物材料方面的背景 和药物输送，将使她跻身于一群拥有技能和知识广度的精选科学家之列 对于有效开展核酸传递和遗传疾病编辑方面的跨学科工作是必要的。

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.