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

利用高通量连续流合成电荷改变可释放转运蛋白基因递送载体来建立 mRNA 递送的结构功能关系

基本信息

批准号：
9758810
负责人：
Trevor Del Castillo
金额：
$ 6.12万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2021-06-30
项目状态：
已结题

项目摘要

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).

拟议的研究将改善一种有前途的电荷改变的获取和性能可释放的转运蛋白类基因传递载体。这些材料表现出非凡的 mRNA 转染和表达效率高，表观细胞毒性低。这些属性是归因于最初的聚阳离子材料的新型电荷中和降解化学至中性小分子。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、内体方面的功能逃逸和细胞内运输导致活细胞中的货物 mRNA 表达也将被已确立的。如果成功的话，将成为生命科学研究和医学应用的强大工具用于将遗传物质递送至体内和体外细胞。这项技术可以在遗传疾病和癌症的治疗领域具有广泛的促进影响免疫疗法以及生物化学和药物化学的基础实验。该研究策略的一个关键创新是连续流的开发 CART 材料的合成，提供对大型新型 CART 库的高通量访问。这些将利用综合优势快速探索广阔的结构功能空间。这实验方法和研究员将接受培训的技术技能将首先表征 CART 通过NMR和凝胶渗透色谱了解每个CART的分子结构，然后通过动态光散射研究 CART-mRNA 复合物，以记录分子的影响结构对所得纳米粒子的尺寸、zeta 电位和稳定性进行影响，然后最终进行筛选体外新型 CART 与相关细胞培养物的组合文库，以建立功能性结果，特别是关于细胞系特异性和表达（由荧光确定）记者分析）。在本提案未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体内实验。那家伙还将接受负责任的研究、教学、职业行为方面的正式和非正式培训与成为研究教授的未来职业目标相关的发展技能，以及参与外展和指导，以便准备在他们的领域领导成功的外展项目未来。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 Robert Waymouth 教授实验室，与教授。保罗·文德（化学）和罗纳德·利维（医学）。