Supplementary Materials and Methods
Oligonucleotide screening
Oligonucleotides to be screened were stored lyophilised at -20 oC until needed, and reconstituted
in nuclease-free water to a starting concentration of 0.5 mM. For screening, oligonucleotides
were diluted to a starting concentration of 5 M in serum-free Optimem (Invitrogen, UK) in a
sterile polypropylene 96-well plate (Matrix, UK) and serialised by one in three dilutions of the
starting material. Fifty microlitre aliquots of the diluted oligonucleotides were transferred into a
sterile black walled, clear bottomed, 96-well tissue culture treated plate (Greiner, UK) and an
equal volume of a 1 in 100 dilution of Lipofectamine 2000 (Invitrogen) in Optimem was added
and left to complex for 30 min at room temperature. A549 cells stably expressing luciferase
(Almedia, CA; passage 26), were dissociated from a growth flask by Accutase (PAA, UK),
washed and resuspended to a final concentration of 2 e 5 cells/ml in Optimem containing 5%
(v/v) fetal calf serum (PAA Gold, UK). Following the incubation period, 0.1 ml/well of cell
suspension was added to the lipid-oligo complex and the plates incubated at 37 oC, 5% CO2, in a
humidified incubator for 48 h. Detection of luciferase (BriteLite Plus, Perkin Elmer, UK), was
performed by addition of 0.05 ml/well of the reconstituted reaction buffer, adhesive opaque
bottoms were attached to each cell plate and luciferase activity detected using the ultrasensitive
Luminescence protocol (EnVision, Perkin Elmer, UK). Data was normalised to controls
containing 2.5 M Luc-2 siRNA (maximal effect) or a non-targetting control (NTC, Dharmacon,
UK). For gymnotic delivery studies 1,000 cells were incubated with the indicated amount of
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ASO and grown in DMEM with high Glucose (6 g/l) without L-Glutamine for 8 days. IC50
values were calculated in GraphPad Prism version 5.0 using the least squares (ordinary) fit
method (variable slope). Results are representative of 2-4 different experiments.
Oligonucleotide modification
Whilst engineering of TLR3 avoidance/antagonism remains elusive, 2’-OMe modifications
facilitate disambiguation of potential TLR-7/8-mediated siRNA off-target effects, as 2’-OMe
nucleosides are well-established TLR-7/8 antagonists. In addition, siRNA stability in lung
bronchoalveolar lavage fluid is substantially longer than that observed in serum preparations
[30]. Therefore, we introduced minimal 2’OMe-only modifications to the sequence on key
nucleosides susceptible to aggressive RNase A degradation (UpA and Un at 3’ and 5’ termini
which are primarily accessed by this endonuclease), with no detectable impact on potency. These
modifications concomitantly increased the stability of the double-stranded region (Figure S7),
which is sufficient to enact RNAi [31].
The delivery success of LNA ASO is largely attributed to the protein-binding capacity of the PS
backbone typically employed yet its’ utility in lung delivery is unknown. We therefore evaluated
all-phosphodiester LNA ASO (PO-LNA) vs all-PS LNA ASO (PS-LNA), reasoning that the
reduced protein binding properties of PO-LNA would lead to rapid elimination of any oligo
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achieving systemic circulation either by RNase activity or excretion, thereby minimising
systemic side effects.
Oligonucleotide labelling
Historically, fluorescent dyes have been used to trace oligo tissue distribution. However, many
dyes are typically quite lipophilic (e.g. Cy5 ΔclogD7.4 = +1.81); although substantially more
lipophillic, cholesterol (ΔclogD7.4 = +12.95) has been shown to increase siRNA duration of
action in the lung [30]. Conceivably, this could be attributed to increased membrane interaction
and partitioning capacity. Thus, to eliminate any potential histological artefacts, we decided to
use sulphonated Cy5 (ΔclogD7.4 = -4.14) as a label (Eurogentec). cLogD values were generated
using ACDlabs software (version 11).
In vivo luciferase expression and knockdown studies
48 hour after dosing and 20 min prior to bioimaging, animals were dosed intraperitoneally with
firefly luciferin (D-Luciferin K+ salt, 300 mg/kg, Caliper Life Sciences, UK). Animals were
anaesthetized and anti-coagulated by intraperitoneal injection of Pentoject (1mg/g, Animalcare,
UK) and heparin (100 IU, Leo Pharma, UK). Lungs were exposed and the animals were
perfused with Dulbecco’s Phosphate-Buffered Saline (DPBS; Gibco, UK) containing 10 IU/ml
heparin by right ventricular cardiac puncture to remove residual blood. Following perfusion
lungs and livers were removed from the animals, rinsed in DPBS, dissected free of non-lung
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tissue, transferred to 6 well plates, covered with firefly luciferin solution (300 µg/ml in PBS).
Bioimaging of whole animal and whole tissue luciferase activity was performed with an IVISspectrum (Caliper Life Sciences, UK) with no excitation and an open emission filter. Images
were collected for 1 sec, at F2 and light production quantified as photons/s within a ROI drawn
around the lung.
Intratracheal vs intravenous dosing comparison studies
To assess the kinetics of ApoB knockdown following IV administration animals (n=5) were
dosed with the ApoB PS-LNA or control PS-LNA sequence intravenously at 30 mg/kg. Animals
were euthanised designated timepoints, livers and kidneys were harvested during necropsy, and
flash frozen in liquid nitrogen prior to analysis. In the IT-IV comparison study, CD-1 mice (25g
weight) were dosed with oligos or vehicle either intravenously or intratracheally at a dose of 3
mg/kg.
Lung Tissue Digestion Protocol
Isolated lungs were transferred to a tube containing ice-cold digestion buffer (Dublecco’s
Phosphate Buffered Saline (DPBS) supplemented with 2.5% foetal bovine serum (FBS), 0.5 mM
Ca2+, 0.75 mM Mg2+, and 15 µg/ml DNAse I (Sigma, UK) and kept at 4oC until digestion.
Individual lungs were dissociated in 5 ml digestion buffer containing Collagenase 3 (150 U/ml,
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Worthington, UK) using the mouse lung dissociation program 1 on a GentleMACS tissue
dissociator (Miltenyi Biotec Ltd., UK). This was followed by incubation in a shaking incubator
(250 oscillations/min, 37oC, 30 min; Stuart Scientific, UK) and a final GentleMACS dissociation
using the mouse lung dissociation program 2. Cell suspensions were passed through a 40 µm
cell strainer (Falcon, UK) and cells were pelleted by centrifugation at 450g for 10 min at 4ºC.
Pellets were resuspended in 5 ml DPBS- containing 5% FBS and subjected to hypotonic lysis to
remove contaminating red blood cells before cell sorting.
Cell Sorting
The digested mouse lung cell suspension was spun at 400g for 5 minutes to pellet and resuspended in 0.08 ml AutoMACS buffer per 10 e7 cells (Miltenyi Biotec # 130-091-222).
Samples were passed through a filter to ensure a single cell suspension (Miltenyi Biotec # 103041-407). Cells were incubated with 0.020 ml of CD11b microbeads per 10e7 cells (Miltenyi
Biotec # 130-049-601) at 4°C for 15 minutes then washed with 1ml ice cold AutoMACS buffer.
Cells were resuspended in 0.5 ml buffer and run on the AutoMACS Pro using the default
deplete_s programme. Both the unlabelled and labelled fractions were collected into the prechilled sample collection block, spun at 400 g and re-suspended in stain Buffer (BD 554657)
ready for fluorescent labelling. CD11b- fractions were incubated with anti-CD45 (BD
Biosciences, UK) and anti-CD326 (Biolegend, UK) antibodies to remove leukocytes and identify
alveolar epithelial cells, respectively. All antibodies were incubated at 4°C for 30 minutes.
Analysis and sorting was carried out on a BD FACSAria (Becton Dickinson) equipped with a
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488nm and 633nm laser. Sorting was carried out using a 70 µm nozzle and medium pressure
(drop drive frequency 60,000). Laser QC was checked with CS&T beads (BD Biosciences
#345249) and drop delay checked with Accudrop beads (BD Biosciences #642412).
Compensation was carried out manually using beads (BD Biosciences #552844) and analysis
carried out in FlowJo software (TreeStar). Live cells were gated on the basis of SSC/FSC
characteristics and doublets gated out using FSC-W. Sorting gates were determined by
fluorescence minus one (FMO) or relevant isotype controls and compensation carried out
manually using compensation beads (BD Biosciences, UK). Cells were sorted directly into 96
well plates delivering 30,000 cells/well (or 60,000 cells for the ‘other’ cells) for luminometry or
MS analysis. Successful enrichment was validated using cytology and automated haematology
analysis. For cytology, cells were smeared on glass slides, air dried and stained with May
Grunwald Giemsa stain (Sigma). Automated cytology was performed using a Sysmex XT2000iV (Sysmex UK, UK) according to the manufacturer’s instructions. Cell type-specific,
single-cell suspensions were also possible to obtain by applying the TDCS method on formalin
fixed tissues stored up to 3 days in formalin pots at room temperature. The entire process
permitted a throughput of 12 animals per day.
MS analyses in cells, tissues and urine
Lung TDCS preparations were centrifuged at 400g for 10 mins at 4oC and the supernatant was
discarded prior to transfer to -80 oC for storage. Snap frozen tissue samples were homogenized in
sterile water in a Precelly’s 24 tissue disruptor (Bertin Technologies, Montigny-le-bretonneux,
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France) and 0.1 ml aliquots collected for analyte extraction. 0.025 ml sample volumes were used
for plasma and urine. Liquid extraction was performed in 5% ammonium hydroxide buffer with
phenol chloroform solution (2:1, v/v) and the oligonucleotides isolated in the aqueous layer were
then further extracted using a HLB solid phase extraction plate (Waters, Milford, US). Analysis
was performed using ion pair reverse phase chromatography gradient on an ACE C18 column at
600C (3 x 50mm, 3μm, HiChrom, UK) with 8 mM triethylamine and 200 mM
hexafluoroisopropanol mobile phase modifiers and methanol elution, with tandem MS/MS
detection (API 4000 triple quad, AB Sciex, Warrington, UK). Individual analytes were measured
using specific multiple reactivity monitoring scans in negative ion mode. Peak integration and
quantitation was performed in AB Sciex Analyst software (version 1.4.2). Plasma stability
studies were performed using CD-1 mouse Li-heparin treated plasma (Charles River
Laboratories). 10 nmol (0.01 ml) of siRNA was added to 0.19 ml plasma, mixed briefly by
vortexing, and incubated for either 15 or 60 minutes at 37 degrees C, with gentle agitation.
Samples were immediately snap frozen in dry ice, and stored until analysed.
b-DNA assays for ApoB
Snap frozen tissue was homogenized in a beat mill (Tissuelyser II, Qiagen, Hilden, Germany)
with grinding jars at temperatures beneath -10°C to yield a pulverized and frozen specimen. 2030 mg of the pulverized sample was subjected to a RNeasy Mini Plus Kit (Qiagen) automated
(QIAcube, Qiagen) mRNA isolation according to manufactures standard protocol. The KD
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studies were performed by using the isolated mRNA in a standard Quanti Gene Assay
(Affymetrix, Santa Clara, CA, USA) detecting indicated target and housekeeper mRNA.
Histology
After formalin fixation lung lobes were paraffin embedded (FFPE) over night (TissueTek VIP,
Sakura Finetek, UK). Blocks were cut into 4.5 µm thick sections using a rotary microtome
(Leica, UK), floated onto water (42oC), collected on SuperfrostPlus slides (VWR, UK) and dried
overnight (incubator, 37oC). For histological analysis, slides were deparaffinised and rehydrated
followed by haemotoxylin and eosin staining. For confocal microscopy, slides were mounted in
Vectashield + DAPI (Vectalabs, UK), covered with a glass coverslip (VWR, UK) and kept in the
dark until visualisation. Frozen tissue blocks were cut into 10 µm thick sections using a rotary
microtome, collected onto SuperfrostPlus slides and kept on dry ice upon confocal imaging.
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