Visualization of self-delivering hydrophobically modified siRNA cellular internalization.

Socheata Ly,Lawrence M Lifshitz,Lakshmipathi Pandarinathan,Matthieu Prot,James Cardia,Deanna M Navaroli,Dimas Echeverria,Anastasia Khvorova,Karl D Bellve,Silvia Corvera,Clive Standley,Julia F Alterman,Marie-Cécile Didiot,Kevin Fogarty

doi:10.1093/nar/gkw1005

Abstract

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.

Highlights

A broad range of human diseases, including cancer, infection and neurodegeneration, can be treated via the silencing of specific genes using small oligonucleotides
We find that sd-rxRNA oligonucleotides internalize selectively through a pathway similar to that taken by the epidermal growth factor (EGF) receptor, which utilizes endosomes enriched in the phosphoinositide-binding protein endosome antigen 1 (EEA1) [37,40,41,42,43]
Significant effort in the siRNA field has focused on determining the mechanisms and pathways of oligonucleotide cellular uptake [58,59]

Summary

INTRODUCTION

A broad range of human diseases, including cancer, infection and neurodegeneration, can be treated via the silencing of specific genes using small oligonucleotides. Oligonucleotides are charged non-biologically stable molecules which need to be modified or formulated to enable cellular delivery Their efficacy is defined by both the ability to be delivered to cells and tissues as well as biological availability inside the cell, with the vast majority of internalized compounds being trapped unproductively in lysosomes and other ‘oligonucleotide sinks’ [13,14,15]. We used a recently developed microscopy platform that combines total internal reflection fluorescence (TIRF) and structured illumination microscopy (SIM) to detect the position of endosomes relative to the plasma membrane during the uptake of ligands [39] Using this methodology, we find that sd-rxRNA oligonucleotides internalize selectively through a pathway similar to that taken by the EGF receptor, which utilizes endosomes enriched in the phosphoinositide-binding protein EEA1 [37,40,41,42,43]. The recognition that hydrophobic oligonucleotide uptake involves distinct intracellular endosomal pathways and is saturable will aid in the development of rational strategies to enhance the potency and utility of RNAi-based therapeutics

MATERIALS AND METHODS

RESULTS

Findings

DISCUSSION