Visualization of self-delivering hydrophobically modified siRNA cellular internalization

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.

Diverse lipid conjugates for functional extra-hepatic siRNA deliveryin vivo

“…These results suggest that (i) the amount of siRNAs required to induce silencing is tissue dependent, (ii) in some tissues a large amount of siRNA is not necessary to induce silencing, and (iii) over a certain amount of siRNAs in the tissue, a portion of the siRNAs is trapped in a nonproductive pathway 45,46 .…”

Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles

“…To compare two commercially available strategies to conjugate cholesterol to siRNAs (TEG and C7 linkers), we used a previously developed asymmetric siRNA scaffold, 18,40 characterized by a short duplex region (15 bp) and a fully phosphorothioated tail assisting membrane association 18,41,42 (hsiRNAs). hsiRNAs are either partially modified with 2 0 -fluoro pyrimidines on the antisense strand and 2 0 -Omethyl pyrimidines on the sense strand, or they are fully modified using alternating 2 0 -O-methyl and 2 0 -fluoro pattern providing endonuclease stability and protection from innate immune response.…”

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics

“…In this protocol, we use self-delivering, hydrophobically modified siRNAs (hsiRNAs) for mRNA silencing. Recent progress in the chemistry of oligonucleotides has enabled the design of these stabilized hsiRNAs, which promote cellular internalization, efficient entry into RISC, and potent knockdown of target genes (Byrne et al , 2013; Alterman et al ., 2015; Ly et al ., 2017). These compounds contain 2’-O-methyl and 2’-fluoro modifications on all sugars and phosphorothioate backbone modifications; the oligonucleotides are often conjugated to a hydrophobic moiety, such as cholesterol, to support membrane binding and cellular internalization without toxicity.…”

“…Thus, the self-delivering properties of hsiRNAs represent an effective method to identify new leads in vitro in complex cellular models, such as primary neurons. We have recently demonstrated that hsiRNAs efficiently bind neuronal-cell membranes within seconds after treatment, enter cells, and induce potent gene silencing, both in vitro , in primary neurons, and in vivo, in mouse brain, all without the use of transfection reagents (Alterman et al ., 2015; Ly et al ., 2017). …”