Therapeutic potential for microRNAs

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Abstract

MiRNAs are a conserved class of non-coding RNAs that negatively regulate gene expression post-transcriptionally. Although their biological roles are largely unknown, examples of their importance in cancer, metabolic disease, and viral infection are accumulating, suggesting that they represent a new class of drug targets in these and likely many other therapeutic areas. Antisense oligonucleotide approaches for inhibiting miRNA function and siRNA-like technologies for replacement of miRNAs are currently being explored as tools for uncovering miRNA biology and as potential therapeutic agents. The next few years should see significant progress in our understanding of miRNA biology and the advancement of the technology for therapeutic modulation of miRNA activity.

Introduction

The past five years have seen incredible growth of a new field of research examining the origin and functions of a class of non-coding RNAs called microRNAs (miRNAs). Completely unknown before 1993, more than 400 human miRNAs 19–24 nucleotides in length have now been experimentally identified [1], [2], and bioinformatic predictions suggest there may be over a thousand in total [3]. MiRNAs are expected to regulate more than 30% of all mRNAs post-transcriptionally [4], [5]. Examples of their roles in development and disease are rapidly accumulating, but the biological functions of most of them are completely unknown.

The story began with the mapping of the lin-4 mutation in the nematode worm C. elegans, which caused a disruption of the timing of larval to adult developmental stages [6]. After years of searching for a protein responsible for the phenotype, finally a small RNA was identified from the locus [7]. It bound with partial complementarity to the 3′UTR of lin-14 mRNA, and was shown to negatively regulate lin-14 expression post-transcriptionally [8]. This was an anecdote of worm biology until 2000, when the let-7 mutation, which also resulted in disruption of developmental timing in C. elegans, was mapped to another small RNA [9]. This time, researchers noticed that the let-7 miRNA sequence, along with its expression during development, was conserved in animals from arthropods to humans [10], indicating that miRNAs represent an ancient mechanism of gene regulation. Efforts to clone miRNAs from plants, humans, and a variety of model organisms soon followed and revealed a previously unseen world of hundreds of miRNAs, many of them conserved, and some of which showed specific expression patterns in tissues [11], [12], [13].

The understanding of miRNA biogenesis and action on target mRNAs was significantly advanced the next year with the recognition that the biochemical pathway being dissected for the recently discovered small interfering RNAs (siRNAs), was in fact the pathway used by the endogenously encoded miRNAs [14], [15], [16]. Since then, the pace of miRNA research has grown exponentially, and in just five years our understanding of miRNA biogenesis, mechanisms by which miRNAs regulate gene expression, and the biological roles of miRNAs has expanded considerably. This progress has confirmed that miRNAs are important post-transcriptional regulators of gene expression and identified them as a new class of drug targets across a variety of therapeutic areas. Based on the initial genetic studies in invertebrates, and much work profiling miRNA expression in human cancer cells, miRNAs are believed to be important during development, and for regulation of cell proliferation and apoptosis. However, the widespread significance of miRNA regulation in mammalian systems has yet to be demonstrated. Antisense technologies for inhibition or replacement of miRNA activity will be useful tools for miRNA functionalization, as well as for therapeutic modulation of miRNAs.

Section snippets

Biogenesis of microRNAs

Mature miRNAs are found as one arm of a hairpin structure within longer, capped and polyadenylated RNA polymerase II transcripts called pri-miRNAs [17], [18] (Fig. 1). These transcripts often originate from intergenic regions, where the miRNA sequence can be located in either an exon or an intron of the non-coding transcript. However, half of all miRNAs are found in introns of protein-coding genes, usually in the same orientation as the pre-mRNA. In these cases, the miRNA appears to be

MicroRNA regulation of gene expression

Although endogenously encoded miRNAs and exogenously added siRNAs are both incorporated into RISC, their interactions with target mRNAs diverge. While siRNAs are designed to bind with perfect complementarity to the target mRNA and guide its endonucleolytic cleavage by RISC, miRNAs bind imperfectly to the 3′UTR of target mRNAs and instead cause either translational repression or mRNA degradation without endonucleolytic cleavage. The outcome seems to depend entirely on the degree of

Development

The first miRNAs were identified by virtue of their effects on worm development, and so it is reasonable to expect that they will play similar roles in higher organisms. Mutations in the miRNA pathway components Dicer or Argonaute cause severe developmental phenotypes in several organisms [14], [80], [81], [82], [83], [84], [85], [86]. In mice, a Dicer knockout is lethal at day E7.5, pointing to early and essential roles for miRNAs in development, and the inability of Dicer null ES cells to

Antisense inhibition of mature miRNA

As mature miRNAs are short oligonucleotides, it is difficult to imagine inhibiting a specific miRNA without using Watson–Crick basepairing. ASOs are currently the most readily approachable technology for inhibiting miRNAs therapeutically. Modified ASOs complementary to miRNA, or anti-miRNA ASOs (AMOs) have been used by many groups to inhibit miRNA activity in cell culture. We and others have shown that oligonucleotides with 2′sugar modifications (including 2′-O-methyl (2′-OMe), 2′-O

Future challenges

MiRNAs are an ancient, conserved mechanism of post-transcriptional regulation which has added another layer of complexity to gene regulatory networks. Although their roles in human disease are only beginning to be elucidated, already examples are emerging pointing to miRNAs as a novel class of drug targets for cancer, antivirals, and potentially many other therapeutic areas. However, in a general sense, the significance of miRNA regulation is not yet understood. The examples from C. elegans

Acknowledgements

We would like to thank the members of the microRNA group at Isis Pharmaceuticals for a critical reading of the manuscript, and Tracy Reigle for assistance with the figures.

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    This review is part of the Advanced Drug Delivery Reviews theme issue on "Opportunities and Challenges for Therapeutic Gene Silencing using RNAi and microRNA Technologies".

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