Schematic representation of expression cassettes using RNA polymerase promoters for generation of small-interfering RNAs (siRNA). (A) Tandem-type promoters express sense and antisense strands individually. After transcription, both strands hybridize forming a duplex siRNA. (B) Short hairpin RNAs (shRNA) are expressed as a single transcript separated by a short loop of 4–10 nt of sequence. The transcripts form a hairpin structure that can be processed by Dicer into functional siRNAs. (C) Expression of imperfect duplex hairpin structures that is based on pre-microRNA (pre-miRNA) structures. pre-miRNAs are processed by Dicer into a mature miRNA, which can direct gene silencing.

Most common chemical modifications introduced in siRNAs. (A) Phosphodiester modifications: unmodified siRNA (phosphodiester RNA); phosphorothioate RNA; boranophosphonate. (B) 2′-Sugar modifications: 2′-O-Methyl RNA; 2′-deoxy-2′-fluoro RNA; locked nucleic acid (LNA).

In vitro evaluation of lipid/siRNA complexes. (A) Chemical structures of lipids used for preparation of cationic liposomes and complex formation with siRNAs. (AtuFECT01) β-L-Arginyl-2,3-L-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochoride; (helper lipid) 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; (PEG-lipid) distearoyl-sn-glycero-3-phospho-ethanolamine sodium salt. (B) Concentration-dependent inhibition of PKN3 protein expression with lipid/siRNA complex, but not with naked siRNA in HeLa cells as assessed by immunoblot. PTEN served as the loading control. (C) Intracellular distribution of lipid/Cy3–siRNA complexes in HeLa cells analyzed by confocal microscopy. (D) Merged high-magnification pictures of a cytoplasmic area from HUVECs showing endosomal/lysosomal localization of siRNAs. Naked siRNAs remain trapped in the endosomal pathway (upper panel), whereas formulated siRNAs escape the late endosomal/lysosomal vesicles (lower panel). Reprinted by permission from Macmillan Publishers Ltd.: [Gene Therapy] (Santel et al.) © (2006).

Mechanisms of RNA interference (RNAi). RNAi is triggered by long double-stranded RNA (dsRNA), small interfering RNA (siRNA), plasmid or virus-based short hairpin RNA (shRNA), or microRNA (miRNA), and siRNA. Long dsRNA, shRNA, and pre-miRNA are processed by Dicer into 21–23-nt siRNA duplexes with symmetric 2-nt 3′ overhangs and 5′-phosphate groups. Processed siRNAs assemble with cellular proteins to form an RNA-induced silencing complex (RISC). During RISC assembly, one strand (passenger) is eliminated, while the other strand (guide) produces an active RISC, which eventually triggers the sequence-specific mRNA degradation (in case of total complementarity) or translational repression (in case of incorporation of partially complementary miRNA).

Activation of immune response by siRNAs. siRNA/shRNA can be delivered by two different ways: (1) Delivery of viral vector followed by endogenous expression of shRNA in the nucleus, which is transported to the cytoplasm and processed by Dicer into siRNAs. The siRNA causes sequence-specific mRNA degradation and does not induce an interferon (IFN) response. (2) Synthetic delivery systems forming an siRNA complex particle, for example, liposome. In this way, the siRNA is taken up by the cell via endocytosis, causing sequence-specific mRNA cleavage through RNAi and activation of the immune response. The immune response can be activated by different pathways: (a) recognition of siRNAs by Toll-like receptors (TLRs); (b) activation of retinoic acid-inducible gene I (RIG-I) by blunt-end siRNAs; (c) activation of dsRNA-dependent protein kinase R (PKR); (d) activation of 2′-5′-oligoadenylate synthetases (2′5′-OAS). Generally, activation of these pathways leads to the induction of several cellular factors, including nuclear factor κB (NF-κB), interferon regulatory factors (IRFs), eukaryotic translation initiation factor 2α (EIF-2α), and RNase L. Induction of interferons (IFNs), inflammatory cytokines, nonspecific mRNA degradation, and general inhibition of protein synthesis are some undesired effects of the immunostimulation caused by siRNAs.

Gene silencing effect of siRNA conjugated with membrane permeant peptides (MPPs). (A) Conjugation of siRNA with two different MPPs: penetratin and transportan. MPPs and siRNAs were synthesized with a thiol group attached. The reaction between the thiol groups on the siRNAs and the MPPs was catalyzed by the oxidant diamide. (B) Silencing effect of siRNA on luciferase activity. CHO-AA8-Luc Tet-Off cells stably expressing luciferase were transfected with 25 nM siRNA conjugated with MPPs (penetratin and transportan) or complexed with Lipofectamine 2000. Luciferase activity was measured at days 1, 2, and 3 post-transfection. Reproduced with permission from Muratovska and Eccles (2004).

Receptor-mediated delivery of lactosylated PEG siRNA conjugate (Lac-PEG–siRNA) complexed with poly-L-lysine (PLL). (A) Chemical structure of Lac-PEG–siRNA. (B) RNAi activities against firefly luciferase gene generated in human hepatoma HuH-7 cells. A significant decrease in the firefly luciferase expression was observed after transfection with Lac-PEG–siRNA (conjugate alone) and with Lac-PEG–siRNA complexed with PLL (polyion complex, PIC micelle) (right panel). Cellular uptake via asialoglycoprotein (ASGP) receptor-mediated endocytosis was confirmed using asialofetuin (ASF), which is the inhibitor for ASGP receptor. The inhibitor of endosomal acidification, nigericin, was used to confirm that the acid-labile linkage in the conjugate contributes to RNAi activity (left panel). Reproduced from Oishi et al. (2005). Copyright (2006) American Chemical Society.