A Status Update of Modified Oligonucleotides for Chemotherapeutics Applications

Yogesh S. Sanghvi1

1 Rasayan Inc., Encinitas, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.1
DOI:  10.1002/0471142700.nc0401s46
Online Posting Date:  September, 2011
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Abstract

This unit presents an update of recent developments and clinical progress in chemically modified oliogonucleotides useful for therapeutic applications. During the last decade, the number of therapeutic oligonucleotides in clinical trials has nearly tripled. This is primarily due to advances in the synthesis protocols, better understanding of the biology, improved delivery, and better formulation technologies. Currently, over 100 clinical trials with oligonucleotide‐based drugs are ongoing in the United States for potential treatment of a variety of life‐threatening diseases. Among various oligonucleotides, antisense technology has been at the forefront, with one product on the market. Antisense technologies represent about half of the active clinical trials. Similarly, siRNA, aptamers, spiegelmers microRNA, shRNA, IMO, and CpG have been other active classes of oligonucleotides that are also undergoing clinical trials. This review attempts to summarize the current status of synthesis, chemical modifications, purification, and analysis in light of the rapid progress with multitude of oligonucleotides pursued as therapeutic modality. Curr. Protoc. Nucleic Acid Chem. 46:4.1.1‐4.1.22. © 2011 by John Wiley & Sons, Inc.

Keywords: antisense; aptamers; microRNA; shRNA; siRNA; RNAi; oligonucleotides

     
 
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Table of Contents

  • Introduction
  • Overview of Manufacturing Process
  • Chemical Modifications in Oligonucleotides
  • Strategies for Optimizing Therapeutic Potential of Oligonucleotide‐Based Drugs
  • Future Outlook
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

  •   FigureFigure 4.1.1 Schematic representation of four key steps during automated solid‐support synthesis of DNA and RNA oligonucleotides using phosphoramidite chemistry.
  •   FigureFigure 4.1.2 Schematic representation of various post‐automated‐synthesis steps leading to the isolation of purified single‐strand oligonucleotide.
  •   FigureFigure 4.1.3 Select list of sulfur transfer reagents.
  •   FigureFigure 4.1.4 Select list of noncritical impurities in phosphoramidites.
  •   FigureFigure 4.1.5 Select list of critical impurities in phosphoramidites.
  •   FigureFigure 4.1.6 Select list of impurities in oligonucleotides.
  •   FigureFigure 4.1.7 Structural representation of modifications currently in clinical use.
  •   FigureFigure 4.1.8 General diagram for “gapmer” design of ONs. Flanks: Generally 3‐5 residues of 2′‐modified nucleosides are placed at the 3′‐ and 5′‐ends of the ONs where R = 2′‐MOE (S.43), 2′‐O‐Me (S.44), 2′‐F (S.45), or LNA (S.47). Both 3′‐ and the 5′‐ends of the ONs carry an unprotected hydroxyl group. The backbone is uniformly modified with PS. Gap: Generally 7‐14 residues of 2′‐deoxynucleosides (S.41) are placed in the middle of the ONs with uniform PS modification in the phosphate backbone. In order to recruit RNase H–mediated cleavage, the minimum size of the gap is 5 residues and ideally it is around 10 residues; expanding the size of the gap to 14 residues improves the RNase H efficiency. The RNase H1 recognizes the heteroduplex (5′‐end of the antisense strand/3′‐RNA strand) and generates a cleavage site around 7 base pairs away from the 5′‐end of antisense sequence. PS backbone: The presence of uniform PS linkage in the ONs improves the stability towards nuclease degradation and increases the protein binding due to lipophilicity of the sulfur atom. A majority of the ONs currently undergoing clinical trials contain the PS modification.

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Literature Cited

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