Chemically Assisted Electrotransfer of DNA

摘要 电转移(Et)或电穿孔已被广泛用于将分子货物运送到细胞中以获得基因组 以及表观基因组编辑、细胞工程和DNA疫苗在疾病治疗和预防中的应用(例如 新冠肺炎大流行)。为了提高ET的效率，不同的化学物质与电脉冲相结合 在细胞治疗方面。然而，这一策略的一个挑战是如何筛选化学物质。在大多数研究中， 筛选过程是在体外经验完成的，但这种方法在体内是不切实际的或令人望而却步的。 此外，对培养细胞中的ET有效的化学处理可能不一定对培养细胞中的ET有效 尸体。为此，拟议的研究将探索一种新的化学筛选方法。由于机械装置 控制不同细胞和组织之间的分子运输是保守的，化学处理可以 辅助DNA转运可在体内和体外普遍改善ET？ 为了回答这个问题，袁实验室将在以往成功的基础上，加深对 传输机制。一个重点领域是研究细胞质运输。具体而言，这项研究将 在核进入之前，确定囊泡运输和DNA从囊泡中逃逸的机制。这个 第二个焦点领域是研究DNA在未分裂细胞中的核进入。这项研究将解决关键问题 这样的问题：DNA是如何穿过核孔复合体的？交通工具依赖于什么？ DNA的大小和结构？核定位量如何传递信号或DNA核靶向 每个DNA分子的序列会影响运输吗？除了机理研究，袁的实验室还将 开发新技术来筛选无毒化合物和纳米颗粒，可用于改善 细胞质运输和核进入。第三个重点领域是探索不同的战略组合 以同时改善DNA在组织内和细胞外的运输。这项研究将证明 联合应用可协同提高ET效率，延长转基因在体内的表达。 袁实验室在分析细胞和组织中的分子运输方面拥有丰富的经验，特别是在 ET研究的背景。这款R35将为实验室提供灵活性和能力，以促进对 运输机制，并为研究和应用开辟新的途径。这些研究的发现 上述将导致制定加强DNA运输的共同战略，这反过来又将 提高体内和体外ET水平。

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.