title: Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown.
abstract: A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ~5-15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.
authors.affiliation: 1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Clinical Sciences, Section for Oncology and Pathology, Lund University, Lund, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Wittrup
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Ai
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.affiliation: 1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. [2] Institute of Pathophysiology, Semmelweis University, Budapest, Hungary.
authors.forename: Peter
authors.identifier: 0000000210953564
authors.initials: P
authors.lastname: Hamar
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Radiana
authors.initials: R
authors.lastname: Trifonova
authors.affiliation: Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA.
authors.forename: Klaus
authors.initials: K
authors.lastname: Charisse
authors.affiliation: Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA.
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.affiliation: 1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. [3] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Tomas
authors.initials: T
authors.lastname: Kirchhausen
authors.affiliation: 1] Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
mesh_terms: D011992:Endosomes / Q000378:metabolism*; D055785:Gene Knockdown Techniques / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism / Q000493:pharmacokinetics*; D053758:Nanoparticles / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism / Q000493:pharmacokinetics*
chemical_list: D008055:Lipids; D008164:Luminescent Proteins; D034741:RNA, Small Interfering