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: 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
• 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/10007583
• 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; mRNA delivery; 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).

拟议的研究将改善有前途的电荷改变的获取和性能