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Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown

Nature Biotechnology volume 33, pages 870–876 (2015)Cite this article

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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.

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Figure 1: Sudden endosomal release and cytosolic dispersion of lipoplexed siRNA trigger gene knockdown.
Figure 2: Lipoplexed siRNA is released from maturing endososomes.
Figure 3: Endosomal release triggers autophagy.
Figure 4: Release of LNP siRNAs from maturing endosomes recruits galectin-8.

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Acknowledgements

The authors thank E. Marino for maintaining the Imaging Resource used in this study and K. Ketman and N. Barteneva for technical assistance. This work was supported by US National Institutes of Health (NIH) grants AI090671 and CA139444 (J.L.), the Swedish Research Council (A.W.), NIH grant GM075252 (T.K.) and New England Regional Center of Excellence in Biodefense and Emerging Infectious Disease, Core Imaging Facility grant AI057159 (T.K.).

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Authors and Affiliations

  1. Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Anders Wittrup, Angela Ai, Xing Liu, Peter Hamar, Radiana Trifonova, Tomas Kirchhausen & Judy Lieberman

  2. Department of Clinical Sciences, Section for Oncology and Pathology, Lund University, Lund, Sweden

    Anders Wittrup

  3. Institute of Pathophysiology, Semmelweis University, Budapest, Hungary

    Peter Hamar

  4. Alnylam Pharmaceuticals, Cambridge, Massachusetts, USA

    Klaus Charisse & Muthiah Manoharan

  5. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

    Tomas Kirchhausen

  6. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA

    Tomas Kirchhausen & Judy Lieberman

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  1. Anders Wittrup
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Contributions

A.W. and J.L. conceived the study, designed the experiments and wrote the manuscript. T.K. provided advice. A.W. performed most of the experiments. A.A., P.H., R.T. and X.L. performed some of the experiments. K.C. and M.M. provided LNPs and advice for their use.

Corresponding authors

Correspondence to Anders Wittrup or Judy Lieberman.

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Competing interests

J.L. is a member of the Scientific Board of Alnylam Pharmaceuticals. K.C. and M.M. are employees of Alnylam Pharmaceuticals.

Integrated supplementary information

Supplementary Figure 1 High dynamic range (HDR) imaging of live cells with spinning disk confocal microscopy.

During image acquisition, z-sections, encompassing most of the cellular volume, are captured with short exposure times adjusted to the brightest structures in the cell. After all short exposures are captured, a single plane with approximately 10 times longer exposure time, is captured from the lower third of the cell. Only a single plane with long exposure time is cap- tured in order to limit phototoxicity.

Supplementary Figure 2 Cytosolic siRNA release coincides with decrease in vesicular siRNA fluorescence.

HeLa cells were incubated with siRNA-AF647 lipoplexes and imaged every 3 s. Blue line: fluorescence intensity traces of releasing vesicle (from maximum projection of short exposure images). Red line: fluorescence intensity traces from cytosolic Region of Interest (ROI) adjacent to the releasing vesicle (from single plane long exposure images). This representative tracing suggests release occurs as sudden discrete events without prior leakage.

Supplementary Figure 3 Cytosolic release of siRNA is associated with cytosolic calcium transients.

HeLa cells pre-loaded with the fluores- cent Ca++ indicator Fluo-4 were incubated with siRNA-AF647 lipoplexes and imaged every 15 s. a, Representative cell exhibiting a small localized calcium release coincident with siRNA release. Scale bar, 10 μm. b, Quantitation of whole-cell cytosolic siRNA fluorescence (red curve, left axis) and Ca++/Fluo-4 fluorescence (blue curve, right axis) in four typical cells (1-4). siRNA release events are indicated with black triangles. Spontaneous Ca++ spikes are seen in cell 4, for comparison. Ca++ transients during release are of similar or smaller magnitude than spontaneous Ca++ fluctuations.

Supplementary Figure 4 Cytosolic release of siRNA in both non-illuminated and continously imaged cells.

HeLa cells were incubated with 100 nM siRNA-AF647 lipoplexes for 1 h and either continously imaged every 10 s (right panels) or left non-illuminated until the end of the incuba- tion (left panels). Red outlines indicate cells that have experienced a release event and white dashed outlines indicate cells without release events. Cells are counterstained to facilitate localization (Fluo-4). Scale bar, 20 μm. Shown is representative fields of >10 imaged. The same observation, i.e., no difference in release probability between illuminated and adjacent fields has been observed throughout the imaging experiments reported herein. In addition, the extent of knockdown measured with flow cytometry in end-point analyses recapitulates the extent of knockdown seen during continous imaging (not shown).

Supplementary Figure 5 Endosomes releasing lipoplex siRNA do not associate with the ER.

HeLa cells expressing an ER-GFP marker were incubated with siRNA-AF647 lipoplexes and imaged every 1.5 min. Whole cell (upper row, scale bar 10 μm) and detail time-lapse images of cell exhibiting a release event. Scale bar, 2 μm. White triangles indicate a representative releasing endosome.

Supplementary Figure 6 LNPs merge with pre-existing EEA1+ struc- tures and rapidly lose EEA1 association around the time of release.

HeLa cells, expressing mCherry-galectin-8 and GFP-EEA1, were incubat- ed with LNP-siRNA-AF647 and imaged every 10 s. Detail time-lapse images of an LNP containing vesicle (indicated with filled white triangles) associating with mCherry-galectin-8, are shown. Open triangles indicate pre-existing EEA1+ vesicle. Scale bar, 2 μm.

Supplementary Figure 7 LC3 is recruited to LNP containing vesicles.

HeLa cells expressing tfLC3 (GFP signal: green) were incubated with LNP-siRNA-AF647 and imaged every 30 s. Detail time-lapse images of an LNP-containing vesicle (white triangles) associating with LC3, are shown. Scale bar, 2 μm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and legends (PDF 3865 kb)

Sudden release and cytosolic dispersion of lipoplex siRNA (MP4 4176 kb)

Correlation of cytosolic release of siRNA and knockdown (MP4 11543 kb)

Endosomal release of lipoplexed siRNA is after the early endosome (MOV 475 kb)

Endosomal release of lipoplexed siRNA is from a Rab5+ compartment (MP4 174 kb)

Endosomal release of lipoplexed siRNA is from a Rab7+ compartment (MOV 811 kb)

Autophagy activation upon endosomal release of lipoplexed siRNA (MOV 402 kb)

Rapid and specific recruitment of galectin-8 to releasing endosomes (MOV 4320 kb)

Rapid and specific recruitment of galectin-8 to releasing endosomes (MOV 758 kb)

Gradual appearance of multiple galectin-8 foci during LNP incubation (MOV 2479 kb)

Specific recruitment of galectin-8 to LNP containing vesicles (MOV 1964 kb)

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Wittrup, A., Ai, A., Liu, X. et al. Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nat Biotechnol 33, 870–876 (2015). https://doi.org/10.1038/nbt.3298

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