Improving siRNA Pharmacokinetics by Chemical

Modification siRNAs are structurally and biochemically very dissimilar to most drugs approved today; they are relatively big with a molecular weight of roughly 14 kDa, highly labile in biological fluids, and highly charged due to their phosphate backbone, which prevent siRNA from penetrating cellular membranes by diffusion. Furthermore, mammalian cells, even phagocytic macrophages and dendritic cells, do not immediately internalize siRNA (117, 169), and in effect, effective delivery into the target cell cytoplasm still poses the major obstacle of siRNA applications in vivo and therapeutics (although inefficient cellular uptake of oligonucleotides and siRNAs have been described in cell culture using high nucleic acids concentrations (170-173)).

8.1. Enhancing Cellular A great number of delivery vehicles have been developed to facili-

Delivery by siRNA tate siRNA delivery across the plasma membrane into the cyto-

Conjugation plasma such as cationic lipids (such as RNAifect, oligoefectamine, lipofectamine, DOTAP, and TransIT TKO), cationic polymer (such as polyethylenimine (PEI), Chitosan, and cyclodextrin), and dendrimers. All these compounds electrostatically adsorb the anionic siRNA onto their surface and subsequently dock on the anionic cell membranes and allow cellular uptake via adsorptive endocytosis. These non-covalently siRNA-binding vehicles and their applications are described in detail elsewhere (171). Chemical modification of the siRNA itself has also been employed to enhance cellular uptake. As the integrity of the guide strand 5' end is required for siRNA function (48, 168), the 3' end of the guide strand and both ends of the passenger strand have been conjugated to various cationic cell-penetrating molecules or liposomes typically via acid-labile or reducible linkages, often thio-linkages. In particular, cell-penetrating peptides (CPP) such as penetratin (174-176), transportan (175), oligoarginine (177), and TAT (176, 178) have been conjugated to siRNA ends. Although not fully understood, these short cationic, hydrophobic, and/or amphipathic CPPs interact electrostatically with proteoglycans on the cell surface and are internalized by endocytosis, thereby bringing conjugated siRNAs to endosomes from which they are subsequently released to the cytoplasm (179). siRNA-TAT conjugates exhibit a dramatic increase in cellular uptake in vitro comparable to commercial transfection reagents and support-efficient RNAi (178), yet no improvement in efficiency of siRNA-TAT conjugates was seen in vivo upon intranasal delivery in mice and TAT alone seemed to induce unspecific side-effects (176). siRNA conjugated to penetratin (derived from the antennapedia protein) and transportin (a fusion peptide between the neuropeptide galanin and mastoparan, a peptide toxin from wasp venom) has been reported to be similarly efficient as cationic liposomes in a variety of cell lines (175), yet penetratin has been shown to trigger innate immune responses in mice upon intratracheal delivery (176). Chemically stabilized siRNA has been modified by cholesterol conjugation to the 3' end of the passenger stand via a pyrrolidine linker to ensure efficient uptake into liver cells upon intravenous injection in mice, thereby resulting in 60% silencing of target apoB mRNA (52). Similarly, another lipophilic conjugate, alfa-tocoph-erol, was conjugated to the 5' end of the guide strand in a dsiRNA design to successfully reduce apoB protein levels in mouse livers upon intravenous injection (120). Finally, siRNA delivery using commercial transfection reagent can be significantly improved, at least in vitro, by siRNA concatamerization using short complementary "sticky" overhangs (180) or end-conjugation of siRNAs via reducible disulphide-bridges (181).

  1. 2. Targeted Delivery Targeted delivery of siRNAs to specific tissues or cell types is an by siRNA Conjugation attractive strategy, if not a prerequisite, to develop siRNAs into effective therapeutic drugs; it minimizes the amount of required siRNA and potential side-effects. Several studies have utilized celltargeting ligands such as glycosylated molecules, peptides, antibodies, hormones, vitamines, and aptamers by conjugation to various carrier systems (reviewed in ref. (182)). Direct conjugations of siRNAs to cell-targeting ligands such as peptides, antibodies, aptamers, micelles (183), and nanoparticles (184, 185) have also been reported; this strategy not only confers specificity of targeting, but may also enhance cellular uptake through receptor-mediated endocytosis of the specific ligand. The conjugation of a peptide-mimicking insulin growth factor 1 (IGF1) to the 5' end of the siRNA passenger strand resulted in a 60% KD of target gene expression in MCF7 cells that overexpresses the IGF1 receptor (186). Also, an antibody targeting the transferrin receptor expressed at the blood-brain barrier was conjugated to either passenger strand ends via biotin-streptavidin linkages leading to effective target gene silencing in a rat brain tumour model upon intravenous injection (187). To target siRNA delivery to hepatocytes, Oishi et al. constructed a lactose-PEG-siRNA conjugate with an acid-labile linker between siRNA and lactosylated PEG and delivered high amounts of siRNAs into hepatocytes in a receptor-mediated manner (188). An aptamer targeting prostate-specific membrane antigen (PMSA), a receptor expressed in prostate cancers, has been conjugated to the 5' end of the siRNA passenger strand via a streptavidine linkage. Target gene KD was as efficient as when using a conventional lipid-based reagent in LNCaP cells, a prostate tumour cell lines expressing PMSA (189). A clever chimeric variant of the siRNA-PMSA aptamer has been generated by combining the PMSA aptamer and a dsiRNA design in a single T7 transcript that will be cleaved into effective siRNA by endogenous Dicer upon receptor-mediated endocytosis of the PMSA aptamer (190).
  2. 3. Altering A major obstacle in systemic siRNA delivery in vivo is the rapid

Biodistribution by clearance of siRNAs typically observed upon delivery via passive siRNA Conjugation or hydrodynamic intravenous injections (191). Naked siRNAs are very rapidly cleared from the bloodstream primarily due to their renal excretion (51, 52) and degradation by serum RNases (48, 52, 59, 80, 107). As naked siRNAs are smaller than the size threshold for glomerular filtration, incorporating siRNAs into several types of particles has allowed prolonged siRNA circulation and represents the most efficient strategy to avoid renal excretion (148, 183, 188, 192-197). siRNA bioavailability may be enhanced by using nuclease-resistant siRNAs, albeit this strategy will principally not prevent renal excretion and instead lead to excretion of intact siRNA. Several studies report on the improved efficiency of naked, chemically stabilized siRNAs upon introduction in vivo (198) using LNA (89), UNA (96, 198), PS/2'OMe (52), DNA/2'F/2'OMe/PS (72), inverted abasic moieties, and PS-modified siRNAs (199), whereas other studies did find enhancement of siRNA efficacy by 2'-F substitution upon hydro-dynamic injection in mice (73).

Instead, chemical modification or conjugation of the siRNA itself has also been employed to enhance siRNA biodistribution; cholesterol conjugations have been reported to improve siRNA pharmacokinetics and exhibited a higher binding to blood serum albumin upon intravenous injection in mice resulting in siRNAs being detectable in liver, heart, lung, kidney, and fat tissue after 24 h in contrast to naked siRNAs (52). Also, conjugations to bile acids and various long chain fatty acids have been shown to influence siRNA tissue distribution upon intravenous injection as they bind to various lipoproteins, lipoprotein receptors, and transmembrane proteins in blood facilitating cellular uptake (200). PS modification of the siRNA backbone may be expected to alter siRNA biodistribution as PS oligonucleotide non-specifically binds to cellular proteins (63); however, no alteration in the biodistribution of modified siRNAs is seen in vivo (62).

Delivery strategies, and thereby the chemical modifications needed in siRNA design, may well focus on local rather than systemic delivery and indeed the first clinical trials have relied on intraocular injection of siRNA to treat macular degeneration and inhalation of siRNA to treat respiratory syncytial virus (RSV) infection (201).

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