Chemically modified siRNA: tools and applications

Chemical modification provides solutions to many of the challenges facing siRNA therapeutics. This review examines the various siRNA modifications available, including every aspect of the RNA structure and siRNA duplex architecture. The applications of chemically modified siRNA are then examined, with a focus on specificity (elimination of immune effects and hybridization-dependent off-target effects) and delivery. We also discuss improvement of nuclease stability and potency.

Introduction

It has been ten years since the publication of the seminal paper demonstrating the high potency of long double-stranded RNA in gene knockdown [1]. Shortly thereafter, it was discovered that the same effect could be produced in mammalian cells using synthetic short RNA duplexes [2]. The relatively few years since then have seen an explosion of research into therapeutic applications of RNAi. Several companies have been formed to pursue the technology, and transactions involving these companies have recently been measured in the billions of dollars.

The reason for the excitement is that RNAi allows potent knockdown of virtually any gene. This in turn allows rapid progression from target selection to preclinical trials. siRNA has become the most common tool in functional genomics, and therefore can often also help at the target identification stage. Furthermore, some targets that are not druggable by traditional methods can be targeted by gene knockdown.

In spite of the immense attractiveness of gene knockdown as a therapeutic strategy, siRNA duplexes are not optimal drug-like molecules. RNA is highly vulnerable to serum exo- and endo-nucleases, leading to a short half-life in serum. siRNA duplexes are composed of two strands that can drift apart in a dilute environment-like serum. Because oligonucleotides are polyanions they do not easily cross cell membranes and, because this charge density leads to extensive hydration, they do not easily interact with albumin and other serum proteins, leading to rapid elimination. Unmodified oligonucleotides have limited tissue distribution. And finally, oligonucleotides can have off-target effects, either by stimulating the immune system or by entering other endogenous gene regulation pathways.

A wide variety of chemical modifications have been proposed to address these issues. In this review, we examine the principles of chemical modification of siRNA duplexes. We will briefly look into the toolbox; that is, summarize the possible ways that siRNA duplexes can be modified. Following this, we will review the ways these tools have been applied to move siRNA toward the clinic, including the use of chemical modifications to improve potency, serum stability, specificity and delivery. We will point out the most useful and universal modifications as well as some of the most creative modifications and applications, which stretch our paradigms and open new avenues of research into RNAi-based drugs.

Much excellent work has led to significant growth in understanding the mechanism of RNAi 3, 4, 5 (Fig. 1, also see Glossary of specialist terms). When an exogenous 19–21 bp siRNA is introduced into a mammalian cell the 5′-end is phosphorylated. The duplex is then assembled into the RNA-induced silencing complex (RISC), a multiprotein complex including Argonaute2 (AGO2), Dicer, TRBP (HIV-1 TAR RNA-binding protein) and PACT (a dsRNA-binding protein), as well as other proteins, some of which are yet unknown [3]. The strand with the lower binding affinity at its 5′-end becomes the antisense (guide) strand 6, 7, and the other strand (known as the sense or passenger strand) is cleaved and unwound, to leave a single-stranded RNA associated with Argonaute2 (AGO2), an endonuclease at the heart of RISC that promotes location of complementary mRNA, hybridization and cleavage of the mRNA target. When modifying an siRNA duplex it is important to remember that different modification approaches are required for the sense and antisense strands, because of these very different roles 8, 9.

In most cases it is simply assumed that the RNAi mechanism is unaffected by chemical modification of siRNA duplexes. A few studies using modified siRNA have confirmed this by showing that the cleavage of complementary mRNA occurs between bases 10 and 11, counting from the 5′-end of the guide strand, as is the case for unmodified duplexes 10, 11, 12. However, in principle, this should be verified for each new pattern of modification.