Chemical Modification

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Whilst polymer-based delivery systems protect siRNAs from serum nucleases, they do not offer the same protection against intracellular nucleases upon cytoplasmic delivery. For this reason, chemical modifications are commonly made to siRNAs to confer resistance to these intracellular nucleases and hence increase silencing persistence [125]. Modifications typically involve the addition of phosphorothioate linkages to the phosphate backbone of siRNA, or substitution of 2′-hydroxyls with 2′-methoxy or 2′-fluoro groups on ribose sugars [126]. Importantly, these modifications do not reduce siRNA activity [127]. In fact recent studies have identified 2′-hydroxyl modifications at defined positions on the guide strand which increase siRNA potency [128].

Chemical modifications can also enhance other pharmacological properties of siRNA. For example, 2′-methoxy groups on the passenger strand, can reduce immunogenicity by preventing siRNA interaction with Toll-like receptors thus averting the type I interferon pathway [129]. Off-target effects, whereby siRNAs trigger silencing of partially complementary mRNA, can also be mitigated by 2′-methoxy substitutions at certain positions on the guide strand [130].

More recently, modifications with locked nucleic acids (LNA) and unlocked nucleic acids (UNA) have shown to improve several aspects of siRNA function [131]. In LNA, a methylene bridge links the 2′-oxygen and 4′-carbon of the ribose, locking the sugar ring in a C3′-endo conformation. Strategic incorporation of LNA into siRNAs increases their thermostability, and can increase nuclease resistance [132, 133], reduce immunogenicity [134], and mitigate off-target effects [135]. Conversely, incorporation of UNA, which lack a C2′-C3′ bond in their ribose groups, at certain positions in siRNA can lead to local destabilisation of the duplex [136]. Such modifications have shown to reduce off-targeting [137] and improve the biostability of siRNAs [138].

One modification which can promote tumour-targeting of siRNAs is the covalent attachment of a nucleic acid aptamer [85]. Similar to antibodies, nucleic acid aptamers can bind to a large number of biomolecules and display a high degree of specificity and affinity to their targets [139]. Aptamers offer several advantages over antibodies. They can be selected against any protein or cell targets via automated in vitro selection. They are amenable to simple chemical modifications as well as relatively cheap mass production. They elicit little immune response and their smaller size may facilitate access to epitopes that are inaccessible to antibodies [140].

The use of aptamer-siRNA chimaeras has been demonstrated by the Giangrande group [141], who used an aptamer targeting the prostate-specific membrane antigen (PSMA), an overexpressed surface marker in prostate cancers, conjugated to siRNAs against BCL2 and polo-like kinase 1. Aptamer-conjugated siRNAs were specifically internalised into prostate cancer cells via PSMA, and induced knockdown of targeted genes as well as cell death. In contrast, few effects were observed with unmodified siRNAs or when aptamer-siRNAs were incubated with non-PSMA expressing cells. These aptamer-siRNA conjugates demonstrated great efficacy when tested in vivo, which led to significant regression of PSMA-expressing tumours upon systemic administration in mice [142].

Six of the fourteen most promising siRNA therapeutics in clinical trials are chemically modified, naked siRNAs [112]. It is notable that despite the advantages of these modifications, such RNA-only formulations may still encounter problems such as rapid renal clearance due to their small size, or undesired electrostatic interactions with serum proteins. Therefore, it seems likely that it will be ultimately more effective to package chemically modified siRNAs inside polymer-based delivery systems.