Characterization of in vitro transcribed MRNA for optimal expression in therapeutic applications

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Kirschman, Jonathan Lee
Santangelo, Philip J.
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The use of synthetic messenger ribonucleic acid (mRNA) to express proteins is a highly promising therapeutic and vaccine approach that avoids many safety issues associated with viral or DNA-based systems. However, in order to optimize mRNA designs and delivery, technology advancements are required to study fundamental mechanisms of mRNA uptake and localization at the single-cell and tissue level. Here, we present a single RNA sensitive fluorescent labeling method which allows us to label and visualize synthetic mRNA without significantly affecting function. This approach enabled single cell characterization of mRNA uptake and release kinetics from endocytic compartments, the measurement of mRNA/protein correlations, and motivated the investigation of mRNA induced cellular stress, all important mechanisms influencing protein production. Using protein expression and cellular stress as metrics, messenger RNA was rationally designed through incorporation of chemically modified nucleotides, variations in UTRs, incorporation of cell-type specific micro RNA sites, and co-delivery with small molecules. In addition, we demonstrated this approach can facilitate near-infrared imaging of mRNA localization in vivo and in ex-vivo tissue sections, which will accelerate mRNA trafficking studies in pre-clinical models. Last, I demonstrate the effectiveness of this labeling approach through the expression of opsins, light-sensitive ion channels, in primary rat cortical neurons and cardiomyocytes. Overall, the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies was demonstrated, in order to design the next generation of novel mRNA therapeutics and vaccines.
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