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

项目摘要 囊性纤维化是一种进行性遗传性疾病，由囊性纤维化跨膜突变引起。 电导调节因子(CFTR)基因。包括W1282X在内的过早终止密码子突变是最常见的 严重的，而且没有治疗患者的根治方法。基因组编辑代理可以提供有希望的 治疗方法适用于所有慢性萎缩性胃炎患者。工程核酸酶，包括CRISPR/Cas9系统，可以 催化矫正致病突变(S)已显示出希望并进入临床试验。为了减轻 异常的核酸酶活性和减少偏离目标的影响，Prime编辑技术结合了催化 受损的Cas9内切核酸酶与以原始性编辑为程序的工程逆转录酶融合 也编码所需编辑的引导RNA(PegRNA)。作为一种替代技术，三链形成肽 核酸(PNA)没有固有的核酸酶活性，刺激内源性DNA修复，且脱靶能力较低 当结合到靶点附近并与供体DNA寡核苷酸共同传递时的效果。尽管 基因编辑技术的进步，体内传递仍然是临床翻译的主要障碍。目标是 建议的研究之一是开发一种基于基因组编辑的治疗策略，用于治疗 W1282X无意义的CF突变以及用于识别有效车辆的高通量技术 用于体内治疗性核酸输送。在目标1中，以PNA和CRISPR/Cas9为基础的主要编辑基因 编辑试剂将被设计用来纠正W1282X突变，并封装到聚(胺-共酯)中 (PACE)纳米粒(NPs)，并在体外和体内进行测试。在目标2中，将开发新的PACE材料 将基于核酸的治疗药物体内输送到肺部，并使用体内高通量进行评估 决定生理命运的结构-功能关系的平台。在目标3中，生理上 将开发相关的3D培养模型作为高通量筛查工具，以评估交付和疗效 CF疗法的一部分。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 翻译治疗慢性萎缩性胃炎和其他遗传性疾病的有前景的基因编辑/核酸疗法。 Piotrowski-Daspit博士获得化学和生物工程博士学位，目前是一名 耶鲁大学生物医学工程系博士后。到目前为止，她一直是 开发用于核酸输送和高通量体内工具的聚合物纳米颗粒。职业生涯发展 计划概述了获取所需的技术、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事业。拟议的培训将 使她在CF的治疗开发方面获得丰富的经验，并将她融入CF 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，将使她成为一批具有技能和广度知识的科学家之一 有效开展核酸输送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.