DOI: 10.5061/dryad.x95x69phd
title: Engineering ‘smart’ viral RNA structures for stable and targeted siRNA delivery

description: Innovative delivery strategies are needed in order to realize the
 potential of small interfering RNA (siRNA) in medicine. SiRNAs are short,
 double-stranded RNA molecules that silence genes by co-opting an
 endogenous RNA interference (RNAi) pathway. Because they act on messenger
 RNA (mRNA) sequences via RNAi, siRNAs hold promise as potentially curative
 therapies for genetic defects, autoimmune disorders, cancers, and other
 diseases that cannot be treated with traditional, protein-binding small
 molecule drugs and biologics. However, key physiological barriers largely
 have precluded the translation of siRNA drugs into clinical practice. In
 vivo, naked siRNAs are degraded rapidly by nucleases and cleared by the
 kidneys, resulting in a half-life of less than five minutes. Moreover, by
 comparison to small molecule drugs, siRNA drugs are relatively large,
 hydrophilic molecules that do not distribute widely to tissues or
 passively cross the cell membrane. Therefore, without an effective
 strategy for delivery, the accumulation of siRNA drugs at target sites is
 minimal. Today, there are few prominent siRNA delivery approaches, and
 each has significant limitations. For example, chemical modifications can
 improve nuclease resistance, but with the increase in stability also come
 tradeoffs in potency and safety. Lipid nanoparticles (LNPs) physically
 shield siRNAs from degradation and can be modified to promote
 biodistribution and uptake; but as LNPs are optimized for efficacy, they
 become increasingly complex, posing quality assurance, cost, and
 evaluation problems. Finally, conjugation to the N-Acetylgalactosamine
 (GalNAc) ligand is a promising delivery strategy for the liver, but
 targeted delivery to other tissues is a problem that still remains to be
 solved. Advances in nucleic acid nanotechnology have shown that RNA is an
 emerging platform for drug delivery. In particular, a three-way junction
 (3WJ) derived from bacteriophage prohead RNA (pRNA) has gained prominence
 as a vector for small molecule, microRNA (miRNA), anti-miRNA, and siRNA
 delivery. As a delivery solution for siRNA, RNA-based platforms like the
 pRNA 3WJ have many notable advantages. For example, size and shape can be
 controlled to minimize clearance, and functionalization with aptamers can
 drive cell uptake. Also, RNA is a fundamentally biocompatible molecule
 that is simple, straightforward to produce, and multifunctional. However,
 its metabolic instability is limiting. Exciting new research has uncovered
 ‘smart’ RNA structures that are produced by flaviviruses (e.g., Zika,
 Dengue, West Nile) to thwart nuclease degradation. Compared to other RNA
 structures used for drug delivery, these nuclease-resistant structures
 (NRSs) may be uniquely positioned for in vivo applications. The aim of
 this project is to harness the inherent stability of flaviviral NRSs in
 the creation of a supramolecular platform for siRNA delivery. This project
 has the potential to address a critical need in the field of
 oligonucleotide therapeutics, which has few promising solutions for
 harnessing the power of RNAi in the clinic. Additionally, this project
 will validate new stable structures as building blocks for use in RNA
 nanotechnology and therefore will help researchers design supramolecular
 structures for a variety of applications well beyond those described in
 this proposal.
description: See "Engineering ‘smart’ viral RNA structures for stable and
 targeted siRNA delivery_README" attached.