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递送到细胞中用于基因组测序。 以及表观基因组编辑、细胞工程和DNA疫苗接种在疾病的治疗和预防中的应用（例如，的 COVID-19疫情）。为了提高ET效率，已经将不同的化学品与电脉冲结合 在细胞治疗中。然而，这种策略的一个挑战是如何筛选化学品。在大多数研究中， 筛选过程是在体外凭经验完成的，但这种方法在体内是不切实际的或禁止的。 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。 身体为此，拟议的研究将探索一种新的化学筛选方法。由于机制 在不同的细胞和组织中是保守的， 协助DNA转运在体外和体内广泛改善ET？ 为了回答这个问题，Yuan实验室将在以前的成功基础上加深对 运输机制。一个重点领域是研究细胞质转运。具体而言，该研究将 确定囊泡运输机制和DNA在进入核之前从囊泡中逃逸。的 第二个重点领域是研究非分裂细胞中DNA的核进入。该研究将解决关键问题 例如：DNA如何通过核孔复合体？运输如何依赖于 DNA的大小和结构核定位信号或DNA核靶向的量如何 每个DNA分子的序列会影响运输吗？除了机制研究，袁实验室将 开发新技术来筛选无毒化合物和纳米颗粒， 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 该组合可以协同地增强ET效率并延长体内转基因表达。 Yuan实验室在分析细胞和组织中的分子转运方面具有丰富的经验，特别是在 ET研究的背景。该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。研究结果 上面提到的将导致发展共同的战略，以加强DNA运输，反过来将 提高体内外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.