Date Available

11-23-2018

Year of Publication

2016

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

Advisor

Dr. Peixuan Guo

Co-Director of Graduate Studies

Dr. Eric Munson

Abstract

The emerging field of RNA nanotechnology is developing into a promising platform for therapeutically application. Utilizing the state-of-art RNA nanotechnology, RNA nanoparticles can be designed and constructed with controllable shape, size for both RNA therapeutics and chemical drug delivery. The high homogeneity in particle size and ease for RNA therapeutic module conjugation, made it feasible to explore versatile RNA nanoparticle designs for preclinical studies.

One vital module for therapeutic RNA nanoparticle design is RNA aptamer, which can enable the RNA nanoparticles find its specific target for targeted drug delivery. A system of screening divalent RNA aptamers for cancer cell targeting was developed. The system utilized a highly stable three way junction (3WJ) derived from phi29 bacteriophage packing RNA (pRNA). Instead of using one random loop for aptamer SELEX as traditionally, the divalent RNA nanoparticle library contains two variable loops for substrate binding, similar to protein antibodies. The presence of two binding sites on one aptamer greatly enhanced its affinity, and the thermodynamically stability of pRNA-3WJ motif enables controllable RNA folding of each loop. The selected RNA antibody against epithelial adhesion molecule (EpCAM) A9-8 can deliver therapeutic anti-miR21 to EpCAM positive cancer cells in vitro. The feasibility of using RNA aptamer for targeted chemical drug delivery is explored. A phosphorothioate bond modified DNA (thio-DNA) aptamer targeting annexin A2 was utilized as ligand to build nucleic acid nanoparticles for ovarian cancer targeted drug delivery. A DNA/RNA hybrid nanoparticle was generated by conjugating the thio-DNA aptamer to pRNA-3WJ motif. The DNA/RNA hybrid nanoparticles showed favorable property for delivering doxorubicin to ovarian cancer cells in vitro, also targeted to ovarian cancer xenograft in bio-distribution study in vivo. Utilizing the spatial orientation of pRNA-3WJ, cholesterol modification on the arrow tail of pRNA-3WJ can display RNA nanoparticle on the surface of exosomes/extracellular vesicles (EV) for active targeting. Taking the advantage of RNA ligand for specific targeting; and exosome for efficient membrane fusion, cytosol homing and functional siRNA delivery; the RNA ligand decorated exosomes were constructed for specific delivery of siRNA to cancer cells. PSMA aptamer-displaying exosomes and encapsulated survivin siRNA (PSMAapt/EV/siSurvivin) showed efficient gene silencing both in cell culture and animal trials. After systemically injection of PSMAapt/EV/siSurvivin to prostate cancer xenograft mice, cancer growth was almost completely blocked. These results suggest the advance of RNA nanotechnology can further drive its way towards clinical application as a novel next generation drug delivery system.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2016.432

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