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

项目摘要 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。 电导调节因子（CFTR）基因。包括W1282 X在内的过早终止密码子突变是最常见的突变之一。 严重，而且没有治愈性的治疗方法。基因组编辑剂可以提供有前途的 适用于所有CF患者的治疗方法。包括CRISPR/Cas9系统的工程化核酸酶， 催化校正致病突变已经显示出前景并进入临床试验。以减轻 异常的核酸酶活性和减少脱靶效应，引物编辑技术结合了催化 受损的Cas9内切核酸酶与用引物编辑编程的工程化逆转录酶融合 指导RNA（pegRNA），其也编码所需的编辑。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，并刺激内源性DNA修复， 当与靶位点相邻结合并与供体DNA寡核苷酸共递送时，尽管 随着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 这项研究的目的是开发一种基于基因组编辑的治疗策略， W1282 X无义CF突变以及用于鉴定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引物编辑的基因 编辑试剂将被设计为校正W1282 X突变，封装到聚（胺-共-酯）中， （PACE）纳米颗粒（NP），并在体外和体内进行测试。在目标2中，将开发新型PACE材料 用于将基于核酸的治疗剂体内递送至肺部，并使用高通量体内评估 平台，以确定指导生理命运的结构-功能关系。目标3：生理学 将开发相关的三维培养模型，作为高通量筛选工具，以评估交付和疗效 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， 翻译有前途的基因编辑/核酸疗法用于CF和其他遗传性疾病。 Piotrowski-Daspit博士获得博士学位。在化学和生物工程，目前是 耶鲁大学生物医学工程系博士后。到目前为止，她一直 开发用于核酸递送和高通量体内工具的聚合物NP。职业发展 该计划概述了获得所需技术、概念和专业技能的综合战略 完成拟议的研究，并开始独立的研究生涯。拟议的培训将 使她能够获得CF治疗开发的重要经验，并将她融入CF 研究社区。培训计划，加上她在生物医学工程，生物材料， 和药物输送，将使她成为一个精选的科学家小组的技能和知识的广度 这是有效开展核酸递送和遗传疾病编辑的跨学科工作所必需的。

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.