Building oligonucleotide therapeutics using non-natural chemistries

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Modified nucleotides are increasingly being utilized in all categories of therapeutic oligonucleotides to increase nuclease-resistance, target affinity and specificity. The extent to which these substitutions are tolerated varies with the different modes of action exploited by various modalities, but fully modified oligonucleotides have now been discovered for most types of therapeutic oligonucleotide. Fully phosphorothioate-substituted antisense oligonucleotides have been used for several years. The first fully modified siRNA was reported in 2006 with a 2′-O-methyl sense strand and a phosphorothioate antisense strand. The first fully modified aptamer (2′-O-methyl) was reported in 2005. It is expected that future candidate therapeutic oligonucleotides will have even more drug-like characteristics as a result of the inclusion of modified nucleotides.

Introduction

Oligonucleotides are becoming increasingly important as candidate therapeutics as different modes of action are discovered and developed. From the beginning of 2004 through mid-2006, several significant milestones were achieved in this field. The first therapeutic aptamer was approved by the FDA for age-related macular degeneration in December 2004, and marketed in 2005. The first human clinical trial of an siRNA was initiated in 2004, and antisense therapeutics have continued to progress through all stages of clinical development. Virtually all therapeutic oligonucleotides contain some fraction of non-natural nucleotides. The driving factors for inclusion of these modifications vary depending upon the mode of action. A common factor is the desire to increase stability by improving resistance to serum nucleases. Nuclease degradation can be blocked by several means including base, sugar and phosphate modifications (which prevent endonuclease and exonuclease attack) as well as caps at the 3′- and 5′-termini (preventing attack by exonucleases). Modifications may also be introduced into candidate therapeutic oligonucleotides to increase target affinity and biological potency, to control biodistribution (including intracellular uptake), and to facilitate synthesis. Appropriate choice of chemistries can simultaneously resolve a range of problems including affinity for undesired targets, the propensity of certain oligonucleotide motifs to self-aggregate, and potential toxicities. In all instances, chemical modifications must be limited to those that do not significantly inhibit activity — depending upon the mode of action of the oligonucleotide therapeutic and the particular context of the modification, different types of chemistries can be tolerated. In the following sections, we review the impact of chemical modifications on therapeutic oligonucleotides described in the period from January 2004 until July 2006. Many of the modified nucleotides discussed in this review are shown in Figure 1.

Section snippets

Antisense

The antisense therapeutic field is experiencing a renaissance with the introduction of successive generations of nucleotide modifications that improve the drug-like characteristics of these molecules. Over the past 15 years, several base, sugar and phosphate changes have been identified and shown to significantly reduce nuclease-degradation rates while simultaneously increasing the efficiency of target mRNA hybridization. Although most antisense approaches have been designed to optimize RNase

Aptamers

Aptamers are short oligonucleotides which fold into well-defined three-dimensional architectures, thereby enabling specific binding to molecular targets such as proteins. These molecules are typically obtained using the SELEX process from combinatorial libraries of transcripts in a manner analogous to phage display. Most aptamers developed for therapeutic applications have relied extensively upon nucleotide modifications to improve their properties. Conceptually these modifications can be

Decoy oligonucleotides

Decoy oligonucleotides are conceptually related to aptamers to the extent that they bind proteins directly, competing with promoter elements for binding by transcription factors, and thus altering the transcription of targeted genes. As with other modalities, nucleotide modifications may be introduced to modify the properties of decoy oligos (e.g. cellular uptake, nuclease resistance) but the benefits must be balanced against potential changes in binding to the targeted transcription factor(s).

Immunostimulatory oligonucleotides

Some oligonucleotide sequences are specifically recognized by pattern recognition receptors of the innate immune system and thereby elicit responses such as up-regulation of IL-6, interferon-α, and, interferon-γ. The archetypal motif in this class is the CpG dinucleotide, which has been shown to activate Toll-like teceptor 9 (TLR9) as part of an ancient eukaryotic immune response mechanism to bacterial DNA. Phosphorothioate-modified oligonucleotides containing CpG motifs are currently being

Small interfering RNA

SiRNAs are short duplexes designed to specifically trigger the enzymatic destruction of specific transcripts via the RNA interference pathway. A typical siRNA construct contains a 21-nucleotide long RNA duplex with two base-pair overhangs at each terminus. Unmodified siRNA constructs are susceptible to degradation by RNases and this is a major limitation to their efficacy. Work with a variety of different modified oligonucleotides shows that not all nuclease-stabilizing modifications are

Delivery

With the exception of aptamers and immunostimulatory molecules that interact directly with extracellular protein targets, therapeutic oligonucleotides must enter cells to be active. Their polyanionic nature intrinsically limits permeation across the cell membrane. Strategies such as reducing or eliminating charge by replacing the phosphodiester linkage between nucleotides with uncharged morpholino [48] or phosphono [49] linkers have been tested. Alternatively, receptor-mediated routes into the

Conclusion

Considerable progress has been recently made in increasing the drug-like characteristics of various oligonucleotide therapeutic modalities by the introduction of modified nucleotides. These characteristics include target affinity, potency, stability, safety and ease of synthesis. Although fully modified antisense (phosphorothioate) molecules have been widely utilized for some time, recent efforts have yielded the first demonstrations of highly activity, fully modified aptamers (2′-O-methyl) and

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

Support from Archemix Corp. is gratefully acknowledged by the authors.

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