In Vitro Validation of Phosphorodiamidate Morpholino Oligomers.

Steve D Wilton,Sue Fletcher,Craig S Mcintosh,May T Aung-Htut,Kristin A West

doi:10.3390/molecules24162922

Abstract

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.

Highlights

Antisense oligonucleotides (ASOs) are short, single-stranded molecules that can be designed to bind to DNA or RNA via Watson-Crick base pairing, with the aim of modifying specific gene expression
The morphology and viability of fibroblasts and myoblasts treated with phosphorodiamidate morpholino oligomers (PMOs) complexed with the Lipofectamine 3000TM reagent were similar to that of cells treated with the uncomplexed PMO and untreated fibroblasts or myoblasts (Figure 1A,B)
Substantial cell death was observed after fibroblasts were treated with PMO complexed with LipofectinTM (10 μl/mL) and myoblasts treated with PMO: Lipofectamine 2000TM complex (Figure 1C,D)

Summary

Introduction

Antisense oligonucleotides (ASOs) are short, single-stranded molecules that can be designed to bind to DNA or RNA via Watson-Crick base pairing, with the aim of modifying specific gene expression. As natural DNA oligonucleotides with the phosphodiester backbone are susceptible to enzymatic degradation, chemical modifications to bases and backbones were developed to enhance binding affinity, increase resistance to nuclease digestion and allow different modes of action that include specific degradation through RNase H induction or siRNA action, blocking protein translation or redirecting pre-mRNA processing [3–6]. Since these modifications and mechanisms were introduced, research into ASO compounds as therapeutics for many diseases, including those caused by genetic mutations, has grown immensely

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Conclusion