Date of Award

Spring 2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Polymers and High Performance Materials

Committee Chair

Charles L. McCormick

Committee Chair Department

Polymers and High Performance Materials

Committee Member 2

Alex S. Flynt

Committee Member 2 Department

Biological Sciences

Committee Member 3

Sergei I. Nazarenko

Committee Member 3 Department

Polymers and High Performance Materials

Committee Member 4

Derek L. Patton

Committee Member 4 Department

Polymers and High Performance Materials

Committee Member 5

Daniel A. Savin

Abstract

This dissertation focuses on the development and advancement of interpolyelectrolyte complexes (IPECs) and block ionomer complexes (BICs) for the delivery of genetic material, namely RNA, to cells, both human and insect. RNA interference (RNAi) provides a powerful tool for disease treatment and the elimination of crop pests at the genetic level. Therefore, development of successful delivery vehicles for its effector molecules, small interfering and double stranded RNAs (siRNA and dsRNA), is imperative. IPECs and BICs show the most promise as RNAi vectors, and thus this work focuses on ascertaining the structure-property relationships affecting RNA delivery as well as applying such insights toward enabling RNAi in crop pest insects that remain highly resistant to such treatment.

In Section I, BIC-siRNA interactions and effectiveness in cell transfection are reported. Aqueous RAFT polymerization was used to prepare a series of hydrophilic-block-cationic copolymers in which the cationic block statistically incorporates increasing amounts of neutral, hydrophilic monomer such that the number of cationic groups remains unchanged but the cationic charge density is diluted along the polymer backbone. Reduced charge density decreases the electrostatic binding strength between copolymers and siRNA with the goal of improving siRNA release after targeted cellular delivery. However, lower binding strength resulted in decreased transfection and RNA interference pathway activation, leading to reduced gene knockdown. Enzymatic siRNA degradation studies with BICs indicated lowered binding strength increases susceptibility to RNases, which is the likely cause for poor gene knockdown.

Section II discusses how RNAi-based technologies are ideal for pest control as they can provide species specificity and spare non-target organisms. However, in some pests biological barriers prevent use of RNAi, and therefore broad application. In this study we tested the ability of a synthetic cationic polymer, poly-[N-(3-guanidinopropyl)methacrylamide] (pGPMA), that mimics arginine-rich cell penetrating peptides to trigger RNAi in an insensitive animal–Spodoptera frugiperda. Polymer-dsRNA interpolyelectrolyte complexes (IPECs) are efficiently taken up by cells, and can drive highly efficient gene knockdown. These IPECs also trigger target gene knockdown and moderate larval mortality when fed to fall armyworm larva. This effect was sequence specific, which is consistent with the low toxicity we found to be associated with this polymer. A method for oral delivery of dsRNA is critical to development of RNAi-based insecticides. Thus, this technology has the potential to make RNAi-based pest control useful for targeting numerous species and facilitate use of RNAi in pest management practices.

ORCID ID

0000-0002-9847-1884

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