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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/9758810
• 关键词: Access to Information; Alkenes; Area; Biochemical; Biological Assay; Biological Sciences; Cell Culture Techniques; Cell Line; Cells; Charge; Chemistry; Collaborations; Complex; Data; Development; Disease; Drug Delivery Systems; Educational process of instructing; Environment; Esters; Exhibits; Fluorescence; Formulation; Future; Gel Chromatography; Gene Delivery; Genes; Genetic Diseases; Genetic Materials; Glean; Goals; Immunotherapy; Improve Access; In Vitro; Lead; Length; Libraries; Medicine; Mentors; Messenger RNA; Methodology; Methods; Molecular; Molecular Structure; Nature; Oligonucleotides; Performance; Pharmaceutical Chemistry; Polymers; Positioning Attribute; Property; Reporter; Research; Serum; Specificity; Structure; Structure-Activity Relationship; System; Technical Expertise; Technology; Testing; Training; Transfection; Universities; base; biochemical tools; cancer immunotherapy; career; career development; combinatorial; copolymer; cytotoxicity; design; functional group; functional outcomes; gene therapy; health application; in vivo; innovation; light scattering; lipophilicity; mRNA Expression; mouse model; nanoparticle; novel; outreach; outreach program; professor; responsible research conduct; screening; skill acquisition; small molecule; targeted treatment; tool; trafficking; uptake; vector; zeta potential

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技术的获得和性能。 可释放的转运蛋白类基因递送载体。这些材料显示出显著的 对于mRNA转染和表达的效率，具有低的表观细胞毒性。这些属性 这归因于初始聚阳离子材料的新型电荷中和降解化学 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 逃逸，以及导致活细胞中货物mRNA表达的细胞内运输也将是 确立了习如果成功，将成为生命科学研究和医学应用的有力工具。 产生用于在体内和体外将遗传物质递送至细胞。这项技术可以 在治疗遗传疾病和癌症方面具有广泛的有利影响， 免疫治疗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一种连续流 CART材料的合成，提供对新型CART的大型文库的高通量访问。这些 将利用组合优势快速探索广阔的结构-功能空间。的 实验方法和技术技能的研究员将培训，将首先表征CART 通过NMR和凝胶渗透色谱法来了解每个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， 结构对所得纳米粒子的大小、zeta电位和稳定性的影响，然后最后进行筛选 新CART体外组合文库与相关细胞培养物一起建立功能性 结果，特别是关于细胞系特异性和表达（通过荧光测定 报道基因测定）。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。这家伙 还将接受正式和非正式的培训，负责进行研究，教学，职业生涯 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，以便准备在其 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教授的实验室，与 教授Paul Wender（化学）和罗纳德Levy（医学）。