Development of Chemically-Defined Platform Materials for Localized Delivery of RNA Therapeutics

Abstract

Most nucleic acid therapeutics have been engineered for systemic delivery with accumulation predominantly occurring in the liver. Currently, mRNA and siRNA represent the most clinically advanced nucleic acid modalities which can be used to address underlying under- or over-expression of crucial proteins, respectively. To achieve therapeutic impact, RNA delivery often utilizes lipid nanoparticles (LNP) as non-viral vehicles to encapsulate these cargoes for intracellular uptake and efficacy. However, these drugs demonstrate the broad potential to correct virtually any misregulated protein, and thus should not be confined to efficacy in the liver. A wide array of target organs exist which cannot always be reached through this route of administration. Tissues such as the front of the eye (FotE) or the brain both exhibit biological barriers to systemic drug delivery which could be overcome using localized delivery of RNA.

Significant efforts are required to elucidate the structure-activity relationships capable of developing LNP for localized delivery. In this thesis, we outline several strategies for optimization of localized RNA delivery in specific tissues of interest. Using a considered screen of variables including chemical structure of LNP components and delivery media, we optimize LNP for delivery of mRNA to corneal epithelial cells present in the FotE. These improvements were translatable across LNPs containing different ionizable lipids, generally considered the most important consideration for delivery efficacy. LNP modifications also acted synergistically to combine the improved effects and result in an LNP 26-fold more potent in corneal epithelial cells than benchmark liver-targeting formulations. Another rationally-designed library of LNP identified the most potent LNP (named MG-LNP) for delivery of either mRNA or siRNA to microglia both in vitro and in vivo after localized injection. As microglia are notoriously difficult to transfect, this represents one of the most potent tools available to screen RNA therapeutics in this cell type. Using these MG-LNP we delivered siRNA against the inflammatory transcription factor PU.1 and observed a decrease in neuroinflammation typical of neurodegenerative diseases. Finally, we developed a novel hydrogel system which can be used as a cell culture scaffold through 3D bioprinting. This hydrogel can be used to encapsulate cells of interest for future efforts in high throughput screening to allow for improved selection of RNA therapeutics and LNP design for local delivery.