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.