Differential uptake, kinetics and mechanisms of intracellular trafficking of next-generation antisense oligonucleotides across human cancer cell lines

Emily Linnane,Elisabetta Chiarparin,Mark Edbrooke,Sarah J Ross,Paul Davey,Sanyogitta Puri,A Robert Macleod,Jim C Norman,Pei Zhang,Alexey S Revenko

doi:10.1093/nar/gkz214

Abstract

Antisense oligonucleotides (ASOs) modulate cellular target gene expression through direct binding to complementary RNA. Advances in ASO chemistry have led to the development of phosphorothioate (PS) ASOs with constrained-ethyl modifications (cEt). These next-generation cEt-ASOs can enter cells without transfection reagents. Factors involved in intracellular uptake and trafficking of cEt-ASOs leading to successful target knockdown are highly complex and not yet fully understood. AZD4785 is a potent and selective therapeutic KRAS cEt-ASO currently under clinical development for the treatment of cancer. Therefore, we used this to investigate mechanisms of cEt-ASO trafficking across a panel of cancer cells. We found that the extent of ASO-mediated KRAS mRNA knockdown varied significantly between cells and that this did not correlate with bulk levels of intracellular accumulation. We showed that in cells with good productive uptake, distribution of ASO was perinuclear and in those with poor productive uptake distribution was peripheral. Furthermore, ASO rapidly trafficked to the late endosome/lysosome in poor productive uptake cells compared to those with more robust knockdown. An siRNA screen identified several factors mechanistically involved in productive ASO uptake, including the endosomal GTPase Rab5C. This work provides novel insights into the trafficking of cEt-ASOs and mechanisms that may determine their cellular fate.

Highlights

Antisense oligonucleotides (ASOs) are single-stranded DNA/RNA-like molecules that can be used as biological tools to modulate the expression of specific cellular target RNA
This study focused on cEt-modified ASOs, and is the first to investigate ASO uptake across a panel of clinically relevant cell lines, to characterize differences in productive uptake mechanisms
We have shown a comprehensive analysis of ASO driven target knockdown, kinetics and trafficking

Summary

Introduction

Antisense oligonucleotides (ASOs) are single-stranded DNA/RNA-like molecules that can be used as biological tools to modulate the expression of specific cellular target RNA. ASOs function through Watson–Crick hybridization, directly binding to complementary RNA sequences and modulating function This is done through a number of different mechanisms, including recruitment of the enzyme Ribonuclease H (RNase H) that cleaves the RNA/ASO duplex leading to the down-regulation of target mRNA and protein [1,2]. Recent progress in ASO chemistry has enabled the advancement of therapeutic ASOs with drug-like properties [3,4] These next-generation ASOs have chemical modifications including a phosporothioate (PS) backbone and 2 -4 constrained ethyl chemistry (cEt) at either end of the molecule. These modifications improve the potency of cEt-ASOs compared to the 2 -O-methoxyethyl (2 -MOE) oligonucleotides, and a number of cEt-ASOs are currently being developed in several disease areas including cancer [5,6,7,8]

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Conclusion