Leveraging high-throughput continuous-flow synthesis of Charge-Altering Releasable Transporter gene delivery vectors to establish structure-function relationships for mRNA delivery

拟议的研究将改善一种有前景的电荷改变的途径和性能 可释放的转运体类的基因传递载体。这些材料表现出了非凡的魅力 转基因效率高，表达效率高，细胞毒性低。这些属性是 归因于初始聚阳离子材料的新型电荷中和降解化学 到中性小分子。对分子结构之间关系的预测性理解 这些材料及其在细胞系特异性、稳定性、转染性、内涵体等方面的功能 逃逸，而细胞内运输导致活细胞中的Cargo mRNA表达也会 已经成立了。如果成功，生命科学研究和医学应用的一个强大工具将是 生产用于将遗传物质输送到体内和体外的细胞。这项技术可以 在遗传病和癌症的治疗领域产生广泛的有利影响 免疫疗法以及生物化学和药物化学的基础实验。 这一研究战略的一个关键创新将是开发连续流 合成购物车材料，提供对新购物车的大型库的高通量访问。这些 发挥综合优势，快速探索广阔的结构-功能空间。这个 实验方法和技术技能将被训练，将首先确定手推车的特征 通过核磁共振和凝胶渗透层析来了解每辆车的分子结构， 然后用动态光散射法研究CART-mRNA复合体，以注意分子的影响 对得到的纳米颗粒的大小、Zeta电位和稳定性进行结构分析，最后进行筛选 新型手推车体外组合文库与相关细胞培养建立功能性 结果，特别是关于细胞系的特异性和表达(通过荧光来确定 记者分析)。在这项提案没有涵盖的单独的未来研究中，最有希望的 候选人将与我们的合作者一起进行小鼠模型的活体实验。那个家伙 还将接受负责任的研究、教学、职业生涯方面的正式和非正式培训 与他们未来成为研究教授的职业目标相关的发展技能，以及 参与外展和指导，以准备领导成功的外展计划 未来。这些研究将在斯坦福大学高度跨学科的培训环境中进行。 大学化学系罗伯特·韦茅斯教授的实验室，与 教授们。保罗·文德(化学)和罗纳德·利维(医学)。

The proposed research will improve access to and performance of a promising charge-altering releasable transporter class of gene delivery vectors. These materials demonstrate remarkable efficiency for mRNA transfection and expression with low apparent cytotoxicity. These properties are attributed to the novel charge-neutralizing degradation chemistry of the initially polycationic materials to neutral small molecules. A predictive understanding of the relationship between molecular structure of these materials and their function in terms of cell-line specificity, stability, transfection, endosomal escape, and intracellular trafficking leading to cargo mRNA expression in living cells will also be established. If successful a powerful tool for life science research and medicinal applications will be produced for the delivery of genetic materials to cells both in vivo and in vitro. This technology could have wide-ranging enabling impacts, in the areas of treatment of genetic disease and cancer immunotherapy as well as in fundamental experimentation in biochemical and medicinal chemistry. A critical innovation for this research strategy will be the development of a continuous-flow synthesis of CART materials, providing high-throughput access to a large library of novel CARTs. These combined advantages will be leveraged to rapidly explore a wide structure-function space. The experimental approach, and technical skills the fellow will train in, will be to first characterize the CARTs by NMR and gel permeation chromatography to understand the molecular structure of each CART, then to study CART-mRNA complexes by dynamic light scattering to note influence of molecular structure on the size, zeta potential, and stability of the resulting nanoparticles, then finally to screen the combinatorial library of novel CARTs in vitro with relevant cell cultures to establish functional outcomes, especially regarding cell-line specificity and expression (to be determined by fluorescence reporter assays). In separate future research not covered by this proposal, the most promising candidates will advance to in vivo experimentation in mouse models with our collaborators. The fellow will also receive formal and informal training in the responsible conduct of research, teaching, career development skills relevant to their future career goals of becoming a research professor, and participate in outreach and mentoring in order to prepare to lead successful outreach programs in their future. These studies will take place in a highly interdisciplinary training environment at Stanford University in the lab of Prof. Robert Waymouth, Department of Chemistry, in close collaboration with Profs. Paul Wender (Chemistry) and Ronald Levy (Medicine).