Jonathan Kirschman

PhD Defense Presentation

Date: Wednesday, July 12th, 2017

Time: 12-2pm EST

Location: Parker H. Petit Institute for Bioengineering and Bioscience (IBB) room 1128

Committee Members:

Philip Santangelo, PhD (Advisor, GT BME)

Krishnendu Roy, PhD (GT BME)

Garrett Stanley, PhD  (GT BME)

Loren Williams, PhD (GT - Chemistry and Biochemistry)

Thomas Barker, PhD (University of Virginia BME)

Title:

Characterization of in vitro transcribed messenger RNA for gene delivery in therapeutic applications 

Abstract:

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 facilitate mRNA trafficking studies in pre-clinical models. We then demonstrate the effectiveness of this approach through the expression of opsins, light-sensitive ion channels, in primary rat cortical neurons and cardiomyocytes. Overall, we demonstrate the ability to study fundamental mechanisms necessary to optimize delivery and therapeutic strategies, in order to design the next generation of novel mRNA therapeutics and vaccines.