Dissertation Defense: Isom Kelly, Biomedical Engineering
DISSERTATION DEFENSE
Isom Kelly III, Biomedical Engineering
*under the direction of Dr. Craig Duvall
“Developing Endosomolytic Polymer Porous Silicon Nanocomposites For Delivery of Diverse Gene Therapies”
Wednesday, March 23, 2022
1:15PM | 5211 SC
Intracellular-acting biologics hold significant promise as genetic and other modes of human therapy, to address diseases that are otherwise insurmountable. RNA interference (RNAi) and messenger RNA (mRNA) therapies, for example, have started to have tremendous clinical impact in recent years. CRISPR/Cas9 mediated genome modification strategies are advancing at a rapid pace and finding success in some early clinical trials. Nonviral delivery systems are an important part of the safety and efficacy of RNA, protein, and ribonucleoprotein (RNP) complex delivery for therapeutic purposes. While cationic lipid-based nanoparticles are becoming standard for clinical use in RNA (siRNA and mRNA) delivery, there remains a shortage of generalizable technologies that can delivery non-anionic or complex (e.g., RNP) cargoes into the cell and escape from the degradative endolysosomal pathways.
This dissertation project focuses on a pH-responsive polymer and porous silicon-based nanoparticle (PSNP) composite system that is capable of efficient, nonionic loading and intracellular delivery/bioavailability of two different classes of genetic therapies. The first application of PSNPs focuses on delivery of non-ionic peptide nucleic acids (PNA), a class of molecules with advantages to be applied for inhibition of micro-RNAs. Micro-RNAs are small, noncoding, endogenous RNAs regulate gene expression in cells; here, we demonstrate targeting of microRNA 122, a micro-RNA involved in cholesterol processing and metabolism in the liver. The second part of the project explore PSNP surface modification for loading and delivery of Cas9 RNPs. This system is tested for creation of targeted genome double strand breaks both in cells and animals. A reporter system is utilized whereby targeted genomic modification yields fluorescent protein “turn-on”, enabling a robust readout of editing efficiency using flow cytometry and microscopy. Additionally, targeting of the endogenous MMP13 loci to induce gene knockout via non-homologous end joining is demonstrated.