Chemically Assisted Electrotransfer of DNA

DNA 化学辅助电转移

• 批准号: 10406467
• 负责人: FAN YUAN
• 金额: $ 40.25万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-21 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10406467
• 关键词: Address; Area; COVID-19 pandemic; Cells; Chemicals; Cultured Cells; DNA; DNA Structure; Development; Electroporation; In Vitro; Interphase Cell; Mediating; Molecular; Molecular Analysis; Nuclear; Nuclear Localization Signal; Nuclear Pore Complex; Physiologic pulse; Process; Research; Techniques; Tissues; Transport Process; Travel; Vaccination; Vesicle; Work; cellular engineering; disorder prevention; epigenome editing; experience; extracellular; flexibility; genome editing; improved; in vivo; nanoparticle; novel; novel strategies; screening; success; transgene expression; vesicle transport

Abstract Electrotransfer (ET) or electroporation has been widely used to deliver molecular cargo into cells for genome and epigenome editing, cell engineering, and DNA vaccination in treatment and prevention of diseases (e.g., the COVID-19 pandemic). To enhance ET efficiency, different chemicals have been combined with electric pulses in treatment of cells. However, a challenge with this strategy is how to screen for chemicals. In most studies, the screening process is accomplished empirically in vitro, but this approach is impractical or prohibitive in vivo. Furthermore, a chemical treatment that works well for ET in cultured cells may not necessarily work for ET in the body. To this end, the proposed study will explore a new approach to chemical screening. Since mechanisms governing molecular transport are conserved across different cells and tissues, can chemical treatments that assist DNA transport be used to ubiquitously improve ET in vitro and in vivo? To answer this question, the Yuan lab will build upon their previous successes to deepen the understanding of transport mechanisms. One area of focus is to investigate cytoplasmic transport. Specifically, the study will determine mechanisms of vesicular transport and the escape of DNA from vesicles prior to nuclear entry. The second area of focus is to investigate the nuclear entry of DNA in non-dividing cells. The study will address key questions such as: How does DNA travel through the nuclear pore complex? How does the transport depend on the size and structures of DNA? How does the amount of nuclear localization signal or DNA nuclear targeting sequence per DNA molecule influence the transport? In addition to the mechanistic studies, the Yuan lab will develop new techniques to screen for nontoxic compounds and nanoparticles that can be used to improve cytoplasmic transport and nuclear entry. The third area of focus is to explore different combinations of strategies to simultaneously improve intracellular and extracellular transport of DNA in tissues. The study will demonstrate that the combination can synergistically enhance ET efficiency and prolong the transgene expression in vivo. The Yuan lab has extensive experiences in the analysis of molecular transport in cells and tissues, especially in the context of ET research. This R35 will give the lab the flexibility and power to advance the understanding of transport mechanisms, and open new avenues for research and applications. Findings from the studies mentioned above will lead to the development of common strategies to enhance DNA transport that in turn will improve ET in vitro and in vivo.

抽象的 电转移（ET）或电穿孔已被广泛用于将分子货物输送到基因组的细胞中 以及表观基因组编辑，细胞工程和DNA疫苗接种，以治疗和预防疾病（例如， 2019冠状病毒病大流行）。为了提高ET效率，已将不同的化学品与电脉冲结合 在细胞的治疗中。但是，这种策略的挑战是如何筛选化学物质。在大多数研究中， 筛查过程是在体外经验上完成的，但是这种方法在体内是不切实际的或过敏的。 此外，一种适合ET在培养细胞中效果很好的化学处理可能不一定适用于ET 身体。为此，拟议的研究将探索一种新的化学筛查方法。由于机制 治理分子运输是在不同细胞和组织中保守的，可以化学处理 辅助DNA转运可无处不在地改善体外和体内的ET？ 为了回答这个问题，元实验室将基于他们先前的成功，以加深对 运输机制。重点的一个领域是研究细胞质转运。具体而言，该研究将 确定囊泡转运的机制和在进入核进入之前从囊泡中逃脱的机理。这 重点的第二个区域是研究DNA在非分裂细胞中的核进入。该研究将解决关键 诸如：DNA如何通过核孔复合体传播的问题？运输如何依赖 DNA的大小和结构？核定位信号或DNA核靶标的量如何 每个DNA分子的序列会影响转运？除了机械研究之外，元实验室还将 开发新技术来筛选无毒化合物和纳米颗粒，可用于改进 细胞质运输和核进入。重点的第三个领域是探索策略的不同组合 同时改善组织中DNA的细胞内和细胞外转运。该研究将证明 该组合可以协同增强ET效率并延长体内转基因表达。 元实验室在细胞和组织中分子转运的分析中具有广泛的经验，尤其是在 ET研究的背景。该R35将使实验室具有灵活性和力量，以提高对 运输机制，并为研究和应用开放新途径。研究结果 上面提到的将导致发展共同策略以增强DNA运输 改善体外和体内的ET。