Wednesday, December 11, 2024
12/11/2024
Project Summary/Abstract The proposed research will improve the understanding of structure-property-function relationships in formulations of charge-altering releasable transporter (CART) gene delivery vectors. These materials demonstrate remarkable efficiency for mRNA transfection with low cytotoxicity due to charge-neutralizing degradative chemistry and have shown remarkable selectivity towards different organs in vivo depending on the chemical structure of the CART. A predictive understanding of how chemical variation affects nanostructure and biological activity will be established. If successful, a set of design rules for CARTs will guide the optimization of mRNA/CART complexes for improved cellular transfection and catered cellular/organ selectivity enabling the development of site-specific CART / RNA therapeutics. The fellow will be the first to perform detailed structural and dynamic studies using advanced cryogenic transmission electron microscopy (cryoEM) and in situ X-ray scattering, illuminating the morphological properties of mRNA/CART nanoparticles. Additionally, this proposal will establish if nanostructure variation arises from changes in formulation technique and CART chemical structures (cation, lipid) and relate these nano-structural variation to differences in biological function. The fellow will develop microfluidic mixing protocols (t-mixer) and screen mRNA/CARTs with differing sizes/morphologies in vitro with cell cultures to establish correlations between functional outcome and nanostructure. In addition to cryoEM and scattering, supplementary measurements of zeta potential, size, phase, thermal transitions, supramolecular interactions, etc. will be carried out to determine the effect physico-chemical properties has on nanostructure in different CART formulations. In separate future research not covered by this proposal, the most distinct mRNA/CARTs will be further studied with 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 applicable to their future career goals of becoming a research professor and participate in outreach and mentoring. These studies will take place in a highly interdisciplinary training environment at Stanford University in the lab of Prof. Robert Waymouth, in close collaboration with Dr. Christopher Tassone at SSRL (SLAC national laboratory).
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