protein (Red) and NS3 (green) and a cell surface lipophilic dye (WGA633; Blue), 72 h ... well plates (Nunc, Wiesbaden, Germany) in phenol red-free DMEM.
OMTN, Volume 12
Supplemental Information
Self-Amplifying Replicon RNA Delivery to Dendritic Cells by Cationic Lipids Pavlos C. Englezou, Cedric Sapet, Thomas Démoulins, Panagiota Milona, Thomas Ebensen, Kai Schulze, Carlos-Alberto Guzman, Florent Poulhes, Olivier Zelphati, Nicolas Ruggli, and Kenneth C. McCullough
a Cell viability
CD172low
CD172hi
% viable Cells
DOGTOR NL10
b
RepRNA (FITC) uptake
Lipid-mediated RepRNA delivery to blood Dendritic Cells
Lipid volume + RepRNA (2ug)
c RepRNA alone
DOGTOR 3ul-(RepRNA)
DOGTOR alone
DOGTOR 12ul-(RepRNA)
VRP
DOGTOR 18ul-(RepRNA)
Figure S1: The effect of increasing lipid load on the efficiency of delivery and translation in DC and monocytes. FITC-labelled RepRNA (2 μg/ 106 cells) was complexed with DOGTOR or NL10 and applied to CD172a selected PBMC for 2 h. Data are shown as follows. (a) Cell viability of DC and monocytes 2 h after treatment with the lipoplexes, as assessed with 7-aminoactinomycin-D staining. The x-axis depicts the volume of lipid used to form the lipoplex whilst the y-axis indicates the % of viable cells. (b) Percentage cells positive for RepRNA. The x-axis depicts the volume of lipid encapsulating the RepRNA whilst the y-axis indicates the intensity of RepRNA delivery. (c) RepRNA translation was also assessed using confocal microscopy 72 h postlipofection. Cells were pulsed with 2 μg/ 106 cells of RepRNA complexed with the varying volumes of DOGTOR for 2 h. The lipoplexes were removed and the cells were fixed, permeabilized and labelled for the E2 protein (Red) and NS3 (green) and a cell surface lipophilic dye (WGA633; Blue), 72 h post-lipofection. Analysis was performed using the IMARIS 7.7 software to generate high resolution 3D stacks to assess RepRNA translation. The scale bars indicate 20 μM and data shown only for positive results.
a Cell viability following treatment with Lipids alone (2h) CD172hi
Cell Viability (%)
CD172low
% of 7-AAD+ cells
b Cell viability of RepRNA+ cells following Lipid-mediated transfection (2h)
Figure S2: The effect of lipofection load on the viability of DC and monocytes. FITC-labelled RepRNA (2 μg/ 106 cells) was complexed with the lipids of interest and applied to CD172a selected PBMC for 2 h. Data are shown as follows. (a) Cell viability of DC and monocytes 2 h after treatment with the lipids in the absence of a RepRNA cargo, as assessed with 7-aminoactinomycin-D staining. The x-axis depicts the lipid of interest whilst the y-axis indicates the % of viable cells. (b) The percentage of 7-aminoactinomycin-D positive DCs and monocytes, positive for RepRNA. The x-axis depicts the lipid encapsulating the RepRNA whilst the y-axis indicates the % of 7-aminoactinomycin-D positive DCs and monocytes, positive for RepRNA.
% positives (NS3 expression)
Figure S3: The effect of RepRNA load on lipid-mediated RepRNA translation. RepRNA (0.4-8 μg/ 106 cells) was complexed with DOGTOR of interest and applied to freshly isolated CD172 enriched PBMC for 2 h. RepRNA translation (NS3 expression) was assessed with flow cytometry, 72 h post-transfection and VRP (data not shown) were employed as a positive control. Data are shown as (a) representative histograms and with respect to percentage positives for NS3 translation.
Table S1: Lipid Characteristics. Table providing information on the three main components that form the structure of the cationic lipids; the polar head, the linker molecule and the acyl tail of the lipids.
DOGTOR DreamFect Lullaby NL10 NL21 NL42
Backbone/Acyl Chains Dioleyl glutamic Distearyl Dioleyl glycerol Dioleyl glutamic Gemini-dioleyl Dioleyl glutamic
Linker cleavable-branched Non cleavable-linear cleavable-linear cleavable-linear cleavable-linear cleavable-linear
Charge/polar head 4+ 4+ 2+ 5+ 4+ 3 + & a mannose group
Supplemental Methods. Synthesis of Lipid-[RepRNA] complexes. Lipoplexes were generated using the different lipidic formulations (kindly provided by OZ Biosciences, Marseille, France) with either different concentrations of RepRNA, but maintaining a ratio of 3:1 (lipid:nucleic acid), or with increasing the volume of lipid (1-21 µL) added to 1 µg of RepRNA. Biodegradable, amino acid based cationic liposomes were synthesized (OZ Biosciences) in RNase free water in the absence or presence of different volumes of the neutral, DOPE helper lipid with final cationic charges ranging between 2-6. For lipoplexes, the appropriate weight of RepRNA (e.g. 10 µg) was diluted into the appropriate volume of lipid aqueous solution (e.g. 250 µL) of sodium chloride (Na2+Cl-, 145 mM). 20 µg of each lipid was diluted into 100 µL Na2+Cl- 145 mM. Complexes were prepared by mixing equal volumes of RepRNA with appropriate volumes of lipid aqueous solution and allowed to stand for 20 minutes. For example, 50 µL of RepRNA (2 µg) solution was mixed with 6 µL of lipid (pre-diluted in 48 µL of RNase-free, PBS solution).
Flow cytometric analysis of Lipoplex-mediated RepRNA uptake and translation. CD172a-selected PBMC were cultured at 1x106 in the presence of 10% porcine serum overnight in 24well plates (Nunc, Wiesbaden, Germany) in phenol red-free DMEM. Cells were pulsed with cationic lipids alone, complexed with fluorescein-labelled RepRNA or RNA alone for 2 h at 39°C. Viral packaged replicons (VRP) were employed as a control. At the end of the incubation period, the cells were assessed for RepRNA delivery or the medium was replaced with fresh, pre-warmed 10% PS-enriched DMEM and the RepRNA was allowed to translate for 72 h at 39°C. Cell preparations were prepared in parallel to assess both RepRNA delivery and translation and the cells received the same Lipid-RepRNA complexes at the same time. In order to assess RepRNA delivery to DC and monocytes, at the end of the incubation period (2 h) with the various lipoplex formulations, the cells were washed with cold PBS/EDTA and incubated with primary antibodies directed against CD172a (74-22-15/a) for 20 minutes on ice. Cells were washed and subsequently incubated with secondary antibodies, (BV421, Phycoerytrhin [BD Biosciences, Switzerland]; PE-Cy7, [Abcam, Switzerland]; Alexa647 [Molecular Probes, Leiden Netherlands]). In order to assess RepRNA translation cells were incubated with the respective primary antibodies (CD172a-FITC) and subsequently fixed with 4% (w/v) paraformaldehyde (PFA, Sigma-Aldrich) for 10 minutes at room temperature. Cells were then stained with antibodies directed against anti-NS3 (C16) and anti-NP (HB65), diluted in 0.3% (w/v) saponin (Serwa; Sigma Aldrich), for 20 minutes on ice. Following a wash with 0.1% (w/v) saponin, the cells were incubated with secondary conjugated antibodies (BV421, [BD Biosciences, Switzerland]; Alexa647 [Molecular Probes, Leiden Netherlands]; PE-Cy7, [Abcam, Switzerland]) for 20 minutes on ice. Following a last wash, cells were re-suspended in sodium azide buffer (0.05%, v/v) and cell acquisition was performed with either a FACS Calibur analytical FCM or a FACS Canto analytical FCM (Becton Dickinson, Basel, Switzerland) and analysis employed both FlowJo versions 9 and 10 (Treestar, San Carlos, CA).
Analysis of Lipoplex-mediated RepRNA translation using confocal microscopy. Primary porcine CD172a-enriched PBMC or MoDCs were cultured at 200,000 in 8-well Lab-Tek II (Nunc) chambers pre-coated with fibronectin. The cells were incubated in phenol red-free DMEM enriched with 10% (v/v) PS and cells were pulsed with the various RepRNA lipoplexes for 2 h at 39°C. At the end of the incubation the medium was replaced with fresh pre-warmed conditioned medium (DMEM enriched with 10% (v/v) PS, 50 U/ml porcine GM-CSF and 100 U/ml IL-4). The cells were allowed 72 h at 39°C before being assessed for RepRNA translation using the procedure described above. Following the incubation with the secondary conjugated antibodies, the cells were washed with PBS and the slides were mounted in Mowiol. Images were acquired using a 63x objectives mounted on a Leica TCS-SL confocal microscope (Leica Microsystems AG, Glattbrugg). The settings and voxel size were adjusted to acquire high-resolution images. Image analysis was performed with Imaris software versions 7.5 and 7.6 (Bitplane AG, Zurich, Switzerland).
Cell viability analysis of Lipoplex-mediated RepRNA. Cell viability of DC and monocytes 2 h after treatment with the lipoplex formulations was assessed with 7-aminoactinomycin-D staining using flow cytometry.
