Engineering 'smart' viral RNA structures for stable and targeted siRNA delivery

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.See "Engineering 'smart' viral RNA structures for stable and targeted siRNA delivery_README" attached. Funding provided by: Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100001711Award Number: 19086