Development of Therapeutic-Grade Small Interfering RNAs by Chemical Engineering

Abstract

Recent successes in clinical trials have provided important proof of concept that small interfering RNAs (siRNAs) indeed constitute a new promising class of therapeutics. Although great efforts are still needed to ensure efficient means of delivery in vivo, the siRNA molecule itself has been successfully engineered by chemical modification to meet initial challenges regarding specificity, stability, and immunogenicity. To date, a great wealth of siRNA architectures and types of chemical modification are available for promoting safe siRNA-mediated gene silencing in vivo and, consequently, the choice of design and modification types can be challenging to individual experimenters. Here we review the literature and devise how to improve siRNA performance by structural design and specific chemical modification to ensure potent and specific gene silencing without unwarranted side-effects and hereby complement the ongoing efforts to improve cell targeting and delivery by other carrier molecules.

Keywords: LNA; OMe; RNAi; chemical modification; immunogenicity; off-target effect; siRNA; siRNA therapeutic.

Figure 1

The benefits and limitations of synthetic siRNA application. The most widely used siRNA type is the “canonical” synthetic 21-mer siRNA composed of two 21 nt RNA strands annealed to form a 19-bp dsRNA duplex stem and 2 nt 3′-overhangs at both ends (the passenger strand is shown in black and the guide strand is shown in red). Also synthetic, dicer-substrate 27-mer siRNAs (DsiRNA) has provided a popular alternative. Both design types can be delivered in vivo either unformulated or upon formulation of various types of delivery agents into the cell cytoplasm (light gray circle) where siRNAs are taken up by a RISC loading complex (RLC), which upon a dicer cleavage event (27-mer designs only, 21-mer siRNAs are dicer-independent) is structurally rearranged into a pre-RISC. Here the siRNA passenger strand is cleaved leading to the establishment of an active RISC that assists and ensures efficient degradation of RNA target sharing perfect sequence complementarity to the siRNA guide stands. A number of bottleneck in siRNA applications are currently being resolved by chemical modification strategies (red circles). Unformulated siRNAs are sensitive to nuclease degradation in extracellular environment and, although degradation rates are much lower in the cytoplasm, siRNAs stabilization by modification is suggested to also enhance intracellular availability and silencing persistence . Also, extracellular siRNAs can be rapidly cleared from the body, e.g., by renal filtration and can induce innate immune responses via TLR3 in certain cells . Delivery across the target cell membrane and endosomal release of endocytosed are currently the main bottlenecks in siRNA applications in vivo and siRNAs may induce TLR7/8-mediated immune-responses upon endosome retention in immune cells . Also, all cells can respond to foreign cytoplasmic RNA via the PRRs, PKR, RIG-I, and Mda5 . siRNA may disturb natural miRNA pathways, that processes nuclear pri-miRNA transcripts (dark gray circle) via a pre-miRNA intermediate and miRNA duplex into a single-stranded miRNA in RISC, by direct competition for RISC loading or by clotting the pathway due to slow siRNA processing and turnover . Finally, all siRNA will trigger miRNA-like off-targeting effects on unintended targets upon base-pairing of the guide strand seed region and positions within mRNA 3′ UTRs leading to transcript destabilization and/or translational repression . Please refer to main text for more detail.

Figure 2

Popular siRNA design types. The canonical 21-mer siRNA is the most popular siRNA design today. Dicer-substrate siRNAs such as 27-mer siRNA, shRNA, pre-miRNA mimics, or fork siRNA may enhance siRNA potencies. Asymmetrical siRNAs (aiRNA), asymmetric shorter-duplex siRNA (asiRNA), bulge-siRNAs and sisiRNA may enhance silencing specificity. Blunt-end siRNA are reported to be more nuclease resistant. Single-stranded siRNAs (ss-siRNAs) and 16 mer are functional but may required higher siRNA concentrations. Dumbbell-shaped circular siRNAs may have longer silencing duration. Passenger strands are shown in black and guide strands in red. Please refer to main text for more detail.

Figure 3

Popular chemical modification types in siRNA design. RNA, ribonucleic acid; PS, phosphothioate; PS2, phosphodithioate; EA, 2′-aminoethyl; DNA, deoxyribonucleic acid; 2′-F, 2′-fluoro; 2′-OMe 2′-O-methyl; 2′-MOE, 2′-O-methoxyethyl; F-ANA, 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid; HM, 4′-C-hydroxymethyl-DNA; LNA, locked nucleic acid; carboxylic LNA, 2′,4′-carbocyclic-LNA-locked nucleic acid; OXE, oxetane-LNA; UNA, unlocked nucleic acid; 4′-S, 4′-thioribonucleis acid; F-SRNA, 2′-deoxy-2′-fluoro-4′-thioribonucleic acid; ME-SRNA, 2′-O-Me-4′-thioribonucleic acid; 4′-S-F-ANA, 2′-fluoro-4′-thioarabinonucleic acid; ANA, altritol nucleic acid; HNA, hexitol nucleic acid; B, base.

Figure 4

Suggested siRNA design scheme. Please see text for details.