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Kalina Paunovska
BME PhD Thesis Defense
Date: May 6th, 2020
Time: 1:00 PM
Bluejeans: https://bluejeans.com/385189433
Committee Members:
James E. Dahlman, Ph.D. (Advisor)
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
Andres Garcia, Ph.D.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
Philip J. Santangelo, Ph.D.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
Edward Botchwey, Ph.D.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
Julie Champion, Ph.D.
Department of Chemical and Biomolecular Engineering, Georgia Institute of Technology
Title: An investigation of parameters that influence non-hepatocyte RNA delivery in vivo
Abstract: Lipid nanoparticle (LNP)-mediated nucleic acid delivery can regulate the expression of any gene, making it a promising way to treat disease. However, clinically relevant delivery of RNA therapeutics to non-hepatocytes in vivo remains challenging. Most LNPs are created by mixing an ionizable lipid with PEG, a phospholipid, and cholesterol, allowing the possibility for thousands of chemically distinct LNPs. These nanoparticles are typically screened in vitro in easily expandable cell lines, yet these cell culture conditions are not representative of in vivo tissue microenvironments. LNPs that deliver their payload (e.g. DNA, RNA) successfully in vitro are then validated in vivo. However, because LNPs that tend to work in vitro do not necessarily work in vivo, this often leads to a small number of viable candidates. The objective of this thesis is to use high-throughput DNA barcoding to ask fundamental questions about in vivo drug delivery. In particular, this work presents four significant contributions to the field of nucleic acid delivery. First, this work explores in vitro and in vivo LNP delivery in many cell types (e.g. endothelial, macrophage) from many tissues (e.g. heart, lung, bone marrow) and reveals that in vitro LNP delivery is not predictive of in vivo delivery. Second, cholesterol structure – a previously unperturbed LNP component – is found to impact LNP delivery in vivo. Cholesterol variants are naturally trafficked in lipoproteins (e.g. LDL, VLDL) suggesting that LNP targeting can be tuned by using naturally- or synthetically-derived cholesterol variants. Third, LNPs that deliver RNA to non-hepatocytes more efficiently than to hepatocytes are identified. Fourth, manipulating cell metabolism through exogenous administration of a small molecule is found to impact LNP-delivered mRNA translation in vivo. Finally, the potential for related works and new directions worthy of pursuit within the field nucleic acid drug delivery are discussed. Taken together, this work enables understanding and optimization of the factors that influence non-hepatocyte RNA delivery in vivo.
