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 1 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 2 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 3 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, 4 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 5 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, 6 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 7 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. 8 References 1. Moschos SA, Spinks K, Williams AE, Lindsay MA (2008). Targeting the lung using siRNA and antisense based oligonucleotides. Curr Pharm Des 14: 3620-3627. 2. Barnes PJ (2008). The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest 118: 3546-3556. 3. Gauvreau GM, et al. (2008). Antisense therapy against CCR3 and the common beta chain attenuates allergen-induced eosinophilic responses. Am J Respir Crit Care Med 177: 952958. 4. Alvarez R, et al. (2009). RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy. Antimicrob Agents Chemother 53: 3952-3962. 5. Vaishnaw AK, et al. (2010). A status report on RNAi therapeutics. Silence 1: 14. 6. Weers JG, et al. (2010). Pulmonary formulations: what remains to be done? J Aerosol Med Pulm Drug Deliv 23 Suppl 2: S5-23. 7. Roy I, Vij N (2010). Nanodelivery in airway diseases: challenges and therapeutic applications. Nanomedicine 6: 237-244. 8. Ponnappa BC (2009). siRNA for inflammatory diseases. Curr Opin Investig Drugs 10: 418-424. 9. Lam JK, Liang W, Chan HK (2011). Pulmonary delivery of therapeutic siRNA. Adv Drug Deliv Rev. 9 10. Straarup EM, et al. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38: 7100-7111. 11. Kurreck J, Wyszko E, Gillen C, Erdmann VA (2002). Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res 30: 1911-1918. 12. Obad S, et al. (2011). Silencing of microRNA families by seed-targeting tiny LNAs. Nat Genet 43: 371-378. 13. Soutschek J, et al. (2004). Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432: 173-178. 14. Stein CA, et al. (2010). Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res 38: e3. 15. Zhang Y, et al. (2011). Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection. Gene Ther 18: 326-333. 16. Zheng BJ, et al. (2004). Prophylactic and therapeutic effects of small interfering RNA targeting SARS-coronavirus. Antivir Ther 9: 365-374. 17. Collinet C, et al. (2010). Systems survey of endocytosis by multiparametric image analysis. Nature 464: 243-249. 18. Donaldson JG, Porat-Shliom N, Cohen LA (2009). Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling. Cell Signal 21: 1-6. 19. Pelkmans L, Burli T, Zerial M, Helenius A (2004). Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell 118: 767-780. 10 20. Sandvig K, Torgersen ML, Raa HA, van Deurs B (2008). Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity. Histochem Cell Biol 129: 267-276. 21. Nicklin PL, et al. (1998). Pulmonary bioavailability of a phosphorothioate oligonucleotide (CGP 64128A): comparison with other delivery routes. Pharm Res 15: 583-591. 22. Griesenbach U, et al. (2006). Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo. Respir Res 7: 26. 23. Glud SZ, et al. (2009). Naked siLNA-mediated gene silencing of lung bronchoepithelium EGFP expression after intravenous administration. Oligonucleotides 19: 163-168. 24. Kleinman ME, et al. (2008). Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 452: 591-597. 25. Robbins M, et al. (2008). Misinterpreting the therapeutic effects of small interfering RNA caused by immune stimulation. Hum Gene Ther 19: 991-999. 26. Ripple MJ, et al. (2010). Immunomodulation with IL-4R alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease. J Immunol 185: 48044811. 27. Tian XR, Tian XL, Bo JP, Li SG, Liu ZL, Niu B (2011). Inhibition of allergic airway inflammation by antisense-induced blockade of STAT6 expression. Chin Med J (Engl) 124: 26-31. 28. Ignowski JM, Schaffer DV (2004). Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells. Biotechnol Bioeng 86: 827-834. 11 29. Brandes C, et al. (1996). Novel features of drosophila period Transcription revealed by real-time luciferase reporting. Neuron 16: 687-692. 30. Moschos SA, et al. (2007). Lung delivery studies using siRNA conjugated to TAT(4860) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjug Chem 18: 1450-1459. 31. Czauderna F, et al. (2003). Functional studies of the PI(3)-kinase signalling pathway employing synthetic and expressed siRNA. Nucleic Acids Res 31: 670-682. 12