title: 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.
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.|RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Socheata
authors.identifier: 0000-0002-7320-4763
authors.initials: S
authors.lastname: Ly
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Deanna M
authors.initials: DM
authors.lastname: Navaroli
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.|RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Marie-Cécile
authors.initials: MC
authors.lastname: Didiot
authors.affiliation: RXi Pharmaceuticals, Marlborough, MA 01752, USA.
authors.forename: James
authors.initials: J
authors.lastname: Cardia
authors.affiliation: RXi Pharmaceuticals, Marlborough, MA 01752, USA.
authors.forename: Lakshmipathi
authors.initials: L
authors.lastname: Pandarinathan
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.|RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Julia F
authors.initials: JF
authors.lastname: Alterman
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Fogarty
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Clive
authors.initials: C
authors.lastname: Standley
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Lifshitz
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Karl D
authors.initials: KD
authors.lastname: Bellve
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Prot
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA.|RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Dimas
authors.initials: D
authors.lastname: Echeverria
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA silvia.corvera@umassmed.edu.
authors.forename: Silvia
authors.initials: S
authors.lastname: Corvera
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01655, USA anastasia.khvorova@umassmed.edu.|RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Khvorova
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002522:Chlorocebus aethiops; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C416362:RBSN protein, human; D034741:RNA, Small Interfering; D014168:Transferrin; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C081786:cyclophilin B; D004815:Epidermal Growth Factor; D002784:Cholesterol; D021983:Cyclophilins