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：
10007583
负责人：
Trevor Del Castillo
金额：
$ 6.53万
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).

拟议的研究将改善有前途的收费的访问和绩效可释放的基因输送载体的转运蛋白类。这些材料表现出了非凡的 mRNA转染和表达的效率低，具有低明显的细胞毒性。这些属性是归因于最初多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理解这些材料及其功能在细胞线特异性，稳定性，转染，内体方面逃生和细胞内贩运导致活细胞中的货物mRNA表达也将是已确立的。如果成功的生命科学研究和药物应用的强大工具将是生产用于将遗传材料递送到体内和体外的细胞。这项技术可以在遗传疾病和癌症的治疗领域具有广泛的启示影响免疫疗法以及生化和药物化学的基本实验。该研究策略的关键创新将是开发连续流程购物车材料的合成，为大型新型购物车提供了高通量的访问。这些合并的优势将被利用，以迅速探索宽阔的结构功能空间。这实验方法和研究员将训练的技术技能将首先描述购物车由NMR和凝胶渗透色谱法了解每个CART的分子结构，然后通过动态光散射研究CART-MRNA复合物，以注意分子的影响在所得纳米颗粒的尺寸，Zeta电位和稳定性上的结构，最后到筛选在体外与相关细胞培养的新型购物车的组合库，以建立功能结果，尤其是关于细胞线的特异性和表达的结果（由荧光确定记者分析）。在未来的未来研究中未涵盖该提议，这是最有前途的候选人将与我们的合作者一起在鼠标模型中进行体内实验。家伙还将在负责任的研究，教学，职业中接受正式和非正式的培训发展技能与他们成为研究教授的未来职业目标有关，参加外展和指导，以准备领导成功的外展计划未来。这些研究将在斯坦福的高度跨学科培训环境中进行化学系罗伯特·韦茅斯（Robert Waymouth）教授实验室的大学，与教授。保罗·温德（化学）和罗纳德·利维（Medicine）。