Therapeutic siRNA: principles, challenges, and strategies

Abstract

RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

Keywords: RNA interference; chemical modification; delivery; liposome; nanoparticle; siRNA; targeting; therapeutics.

Figure 1

Small interfering RNAs (siRNAs) mediate silencing of target genes by guiding sequence dependent slicing of their target mRNAs. These non-coding, silencing RNAs begin as long double-stranded RNA (dsRNA) molecules, which are processed by endonuclease Dicer into short, active ~21-25 nt constructs. Once generated, a siRNA duplex is loaded by Dicer, with the help of RNA-binding protein TRBP, onto Argonaute (AGO2), the heart of the RNA-induced silencing complex (which here is represented just by AGO2). Upon loading, AGO2 selects the siRNA guide strand, then cleaves and ejects the passenger strand. While tethered to AGO2, the guide strand subsequently pairs with its complementary target mRNAs long enough for AGO2 to slice the target. After slicing, the cleaved target mRNA is released and RISC is recycled, using the same loaded guide strand for another few rounds of slicing [12].

Figure 2

A closer look at the model for siRNA guide-strand tethering by AGO2 and target-mRNA recognition and slicing. The terminal 5’ monophosphate group of the guide strand tucks in between the MID and PIWI domains of AGO2. Meanwhile, AGO2’s PAZ domain has a hydrophobic pocket that specifically recognizes the guide-strands 3’ dinucleotide overhang. This positioning opens up siRNA guide nucleotides 2-8, the “seed region,” for base pairing with complementary target mRNA, and next base pairing at nucleotides 10-11 correctly orients the scissile phosphate between these two for cleavage by AGO2’s PIWI domain, which houses the protein’s “slicer” activity [12].

Figure 3

A) Common chemical modifications to siRNA sugars and backbone. B) Chemical modifications to nucleobases.

Figure 4

Schematic of siRNA nanocarriers. A) Liposomes. B) Polymeric nanoparticles. C) Metallic core nanoparticles. D) Dendrimers. E) Polymeric micelles.