Lipid Nanoparticle Delivery of sirna to Osteocytes Leads to Effective Silencing of SOST and Inhibition of Sclerostin In Vivo

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1 Cittion: Moleculr Therpy Nucleic Acids (216) 5, e363; doi:1.138/mtn Officil journl of the Americn Society of Gene & Cell Therpy Lipid Nnoprticle Delivery of sirna to Osteocytes Leds to Effective Silencing of SOST nd Inhiition of Sclerostin In Vivo Genc Bsh 1, Min Ordodi 1, Wilder R Scott 2, Andrew Cottle 1, Yn Liu 1, Hitng Wng 1 nd Pieter R Cullis 1 Sclerostin is protein secreted y osteocytes tht is encoded y the SOST gene; it decreses one formtion y reducing osteolst differentition through inhiition of the Wnt signling pthwy. Silencing the SOST gene using RNA interference (RNAi) could therefore e n effective wy to tret osteoporosis. Here, we investigte the utility of lipid nnoprticle (LNP) formultions of sirna to silence the SOST gene in vitro nd in vivo. It is shown tht primry mouse emryonic firolsts (MEF) provide useful model system in which the SOST gene cn e induced y incution in osteogenic medi, llowing development of optimized SOST sirna for silencing the SOST gene. Incution of MEF cells with LNP contining optimized SOST sirna produced significnt, prolonged knockdown of the induced SOST gene in vitro, which ws ssocited with n increse in osteogenic mrkers. Intrvenous (i.v.) dministrtion of LNP contining SOST sirna to mice showed significnt ccumultion of LNP in osteocytes in compct one, depletion of SOST mrna nd susequent reduction of circulting sclerostin protein, estlishing the potentil utility for LNP sirna systems to promote one formtion. Moleculr Therpy Nucleic Acids (216) 5, e363; doi:1.138/mtn ; pulished online 13 Septemer 216 Suject Ctegory: sirnas, shrnas nd mirnas Nnoprticles Introduction Tretment of progressive one diseses such s osteoporosis is chllenging prolem nd significnt unmet clinicl need. Osteoporosis is systemic skeletl disorder chrcterized y low one mss nd progressive microrchitecturl deteriortion of one tissue, leding to n increse in one frgility nd ultimtely susceptiility to one frctures. 1 Given the cler link etween modultion of the Wnt/β-ctenin signling pthwy nd certin humn diseses, there hs een n incresing interest in clrifying the role of proteins regulting the Wnt/β-ctenin pthwy nd one mss regultion. 2 Sclerostin, protein secreted lrgely y osteocytes, is negtive regultor of osteolst differentition nd functions y inhiiting the Wnt nd one morphogenetic protein signling pthwys tht re criticl for osteolst prolifertion nd ctivity. 3 5 Humns with inherited sclerostin deficiency (sclerosteosis or vn Buchem disese) hve incresed one mss nd re resistnt to frcture. 5,6 Importntly, in menopusl women with low one mss, dministrtion of ntiodies trgeting sclerostin ws ssocited with incresed one minerl density, one formtion, nd decresed one resorption. 6 This, coupled with the fct tht sclerostin is the only pthwy modultor tht is highly selective for one, with its secretion eing restricted to osteocytes, suggests tht locking the function of the Wnt ntgonist sclerostin could result in new therpeutics to increse one formtion. Recently, RNA interference (RNAi), hs emerged s novel therpeutic modlity, potentilly le to silence ny desired gene. Impressive preclinicl results hve een chieved using RNAi to tret metolic, degenertive, nd genetic disorders, s well s cncer nd inflmmtion. 6,7 Further, new insights into one iology re producing new therpeutic trgets for one pthologies. For exmple, RNAi-sed therpies trgeting genes tht re known to negtively regulte one formtion, such s the SOST gene, could led to tretments for diseses exhiiting impired one formtion. This will require the development nd optimiztion of efficient delivery methods to one tissue. The mjor ostcle for developing sirna for therpeutic pplictions is the need for effective delivery with cliniclly cceptle formultions nd cceptle routes of dministrtion. This is prticulrly true for the delivery to compct one, which exhiits n intrinsiclly poor drug penetrtion nd vsculr perfusion. To dte, formultion of nucleic cid therpeutics in lipid nnoprticles (LNPs) represents one of the most effective strtegies for in vivo delivery, prticulrly for the liver. 7,8 In this study, we investigte first whether expression of the SOST gene cn e effectively inhiited in vitro (using surrogte primry stem cell model) employing n sirna-medited pproch delivered y LNPs. Second, we ssess the functionl effect of SOST knockdown on one osteogenic mrkers in vitro. Finlly, we demonstrte effective delivery of LNP-siRNAs to osteocytes in compct one nd consequent silencing of the SOST gene in vivo following systemic dministrtion of LNP-siRNA-SOST. 1 NnoMedicines Reserch Group, Deprtment of Biochemistry nd Moleculr Biology, Life Sciences Institute, University of British Columi, Vncouver, British Columi, Cnd; 2 Deprtment of Cellulr nd Physiologicl Sciences, Biomedicl Reserch Centre, University of British Columi, Vncouver, British Columi, Cnd. Correspondence: G Bsh, NnoMedicines Reserch Group, Deprtment of Biochemistry nd Moleculr Biology, Life Sciences Institute, University of British Columi, 235 Helth Sciences Mll, Vncouver, British Columi, Cnd V6T 1Z3. E-mil: gsh@mil.uc.c Key words: one formtion; lipid nnoprticles; sclerostin; sirna Received 16 June 216; ccepted 19 July 216; pulished online 13 Septemer 216. doi:1.138/mtn

2 2 sirna-medited SOST Silencing In Vivo Results Chrcteriztion of lipid nnoprticles Prticle size ws nm (PDI =.49) nd nm (PDI =.44) for nd siluciferse prticles, respectively. Encpsultion efficiency mesured using Qunt-iT Riogreen RNA ssy ws greter thn 9% for oth types of LNP (91.87 ±.4976 nd ± 1.45% for nd siluciferse prticles, respectively). Since the pk of the ionizle ctionic lipid component of the prticles, DLin-MC3-DMA, is 6.44, the prticles hve ner neutrl surfce chrge t physiologicl ph of 7.4. In vitro SOST induction is ssocited with upregultion of one osteogenic mrkers Primry mouse emryonic firolsts (MEFs) were isolted nd cultured for 2 weeks in Dulecco s Modified Egle s Medium (DMEM) or in osteogenic medium (OM). During this time, MEFs were hrvested every other dy nd qrt-pcr nlysis ws crried out using SOST-specific primers. MEFs treted with OM generted incresed SOST trnscripts tht were detectle fter 3 dys of tretment nd incresed y up to 1-fold fter 2 weeks incution time (Figure 1). However, cells incuted with DMEM produced only ckground levels of SOST trnscripts fter 2 weeks incution. Sclerostin protein expression ws lso mesured during MEF culture in DMEM or OM using western lot nlysis. It ws oserved tht cells incuted with OM exhiited significntly higher mounts of sclerostin protein fter 1 week incution with further increses fter 2 weeks (Figure 1,c). To confirm the osteogenic chrcter of the MEFs treted with OM, we lso exmined the expression of Alp, n erly mrker of osteogenic differentition. Osteogenic stimultion generted lrge (up to 1,6-fold) increse in Alp trnscripts over 2 weeks of incution s compred to cells treted with DMEM (Figure 1d). This increse ws lso confirmed using n enzymtic ssy for ALP in cells cultured in DMEM or OM over 2 weeks (Figure 1e). After 1 nd 2 weeks, the intensity of lkline phosphtse stining ws significntly incresed s compred to cells treted with DMEM (Figure 1f). To further confirm the osteogenic identity of OM-treted MEFs, we lso exmined the expression of dditionl erly (Sp7 nd Runx2) nd lte-stge (Bsp) osteogenic mrkers. It ws oserved tht ll three genes were differentilly ut significntly upregulted when MEFs were treted with OM, compred to those treted with DMEM (Figure 1g i). Tken together, the dt indicte tht the MEF system incuted in OM provides n in vitro system where SOST is expressed t significnt levels following 1 week of incution. In ddition, the incresed expression of erly nd lte osteogenic mrkers ccompnying incresed SOST expression levels suggest tht MEFs could e utilized s n in vitro system to ssess the impct of SOST downregultion on one osteogenic mrker genes. Mouse emryonic firolsts exhiit sustntil uptke of LNP-siRNA in vitro The second set of experiments were imed t ssessing the ility of primry MEF cells to ccumulte LNP sirna systems. MEFs were incuted with DiI-leled LNP contining Qusr-57-leled sirna. Cellulr uptke of LNPs nd ssocited sirna ws monitored for 15, 3, 6, nd 12 minutes y mesuring the intensity of the DiI nd Qusr-57 fluorescence in MEFs using live confocl microscopy (Figure 2). A high uptke efficiency ws oserved t ll incution times, s indicted y the intrcellulr ccumultion of sirna (red color, Figure 2) or LNP (green color, Figure 2). sirna in the MEFs ws detected s erly s 15 minutes following incution. The intrcellulr delivery of dye leled-sirna nd LNP incresed over time s detected y the mount nd intensity of the fluorescence. Also, sed on the pttern of fluorescence nd its intensity, good correltion ws oserved etween sirna nd LNP uptke y MEFs, indicting tht these LNP systems cn deliver sirna quickly nd efficiently to the cell without ffecting cell viility. Quntifiction of the fluorescence further demonstrted significnt uptke of LNP-siRNAs y MEFs within 15 minutes following incution nd firly good correltion etween LNPs nd sirnas (Figure 2c). To etter quntify the intrcellulr delivery of LNP-siRNA systems in MEFs, flow cytometry-sed ssy ws crried out. As the fluorescence intensity of Alex647-leled sirna cn e sensitive to rekdown y nucleses, the reltive levels of uptke were mesured y hving oth LNPs nd sirna leled with DiO nd Alex647 respectively. Cellulr uptke of sirna nd LNP lipid ws monitored over 48 hours y mesuring the fluorescence intensity of Alex647 nd DiO in MEFs (Figure 2d). For ll incution times tested, significntly more uptke of LNP-siRNA ws oserved reltive to free-sirna, confirming tht delivery with LNPs is solutely necessry to protect sirna from degrdtion nd mximize intrcellulr trnsfer of the pylod. A good correltion of LNP nd sirna ws lso oserved s fluorescence of oth LNPs nd sirnas diminished overtime presumly due to sirna trnsloction in to the cytosol while LNPs were metolized in the cell. As demonstrted y Alex647 fluorescence, free-sirna is degrded within hours in the cell cytoplsm. sirna screening to identify the nti-sost duplex tht medites optimized knockdown of the SOST gene MEFs were trnsfected with the fluorescently-leled trnsfection control duplex TYE 563-red. For convenience, the RNAiMAX trnsfection regent (Life Technologies) ws used in these studies. Cells were exmined t 24 hours fter trnsfection. Microscopy reveled high efficiency of trnsfection of MEFs to out 7 or 8% following incution with 1 or 3 nmol/l TYE 563-leled sirna respectively (Supplementry Figure S1). MEFs pretreted with OM were lso trnsfected with six sirna duplexes (A through F), including the Hprt positive control nd scrmled sirna s negtive control, t doses of 3, 1, nd 3 nmol/l (Supplementry Tle S1). Reltive mrna levels were mesured using qrt-pcr t 48 hours post-trnsfection. Dt were normlized ginst internl Hprt or Tp (for the positive control) housekeeping genes considering the s seline 1%. It ws oserved tht sequence B nd C could significntly reduce the expression of SOST t doses of 3, 1, nd 3 nmol/l (Supplementry Figure S1). Sequence C in prticulr, produced 7 to 75% knockdown of SOST when treted t 1 nd 3 nmol/l, respectively, similr to sirna trgeting Hprt, positive control. Sequence A (not shown) nd the rest of duplexes did not induce significnt downregultion of the trget gene. It ws concluded tht Moleculr Therpy Nucleic Acids

3 sirna-medited SOST Silencing In Vivo 3 Reltive SOST mrna 1,2 2, OM 1,8 OM 1, 1,6 MEFs M 8 MEFs M 1,4 1,2 6 1, Dy Dy 3 Dy 7 Dy 9 Dy 14 Dy Dy 3 Dy 7 Dy 14 e DMEM OM d d 7 d 14 d Reltive Alp mrna D7 D14 D7 D14 Scl β-ctin Reltive Sp7 mrna 7, 6, 5, 4, 3, 2, 1, c Normlized SOST protein Reltive Runx2 mrna % increse of ALP OD 16, 14, 12, 1, 8, 6, 4, 2, g h i OM OM 2, MEFs M MEFs M 1,5 D7 D14 D7 D14 DMEM OM Incution medium Dy Dy 3 Dy 7 1, 5 Dy 14 Dy Dy 3 Dy 7 f Reltive Bsp mrna Dy Dy 7 Dy 14 35,, 3,, OM MEFs M 25,, 2,, 15,, 1,, 5,, Dy 14 Dy Dy 3 Dy 7 Dy 14 Figure 1 In vitro SOST induction nd effect on one osteogenic mrkers. Mouse emryonic firolsts (MEFs) were plted in 12-well pltes t pproprite density to chieve mximum confluence. Cells were incuted in DMEM or OM nd totl RNA ws extrcted to mesure () SOST, (d) Alp, (g) Sp7, (h), RunX2, nd (i) Bsp mrna t time points indicted. Br grphs represent expression of the trget mrna reltive to the Hprt house keeping gene with or without OM. mrna vlues t dy re considered 1%. Dt re shown s men ± SD of triplicte wells nd re representtive of t lest three experiments. () Protein ws extrcted from cell lystes nd the expression of sclerostin ws determined in protein extrcts using SDS-polycrylmide gel electrophoresis nd western lotting followed y nti-sclerostin ntiody stining. Blots re representtive of multiple experiments. (c) Sclerostin expression ws ssessed over time following incution of MEFs with DMEM or OM nd the intensity of the nds were quntified. Br grphs shown s men ± SD represent dt otined from multiple experiments. (e) Alkline ctivity ws ssessed following stining of MEFs incuted in DMEM or OM in triplicte pltes. Dt represent not less thn 3 imges cptured with 2 ojective. (f) Alkline phosphtse expression ws lso quntified. Br grphs shown s men ± SD represent dt otined from t lest three imges nd multiple res cptured from ech imge (P <.1; P <.1). sequence C ws the most effective duplex to e used to ssess SOST knockdown nd its effect in one osteogenic mrkers. As sirna duplexes re susceptile to degrdtion in vivo, sequence C ws chemiclly modified y dding 2 O-methyl ses tht lock exonuclese ctivity nd improves resistnce to nucleses in vivo (stelth sirna). To ssess the efficcy of the modified duplex nd rule out toxicity, MEFs were plted nd treted with the modified (Stelth) nd nonmodified (stndrd) sirna using the sme trnsfection method, nd SOST mrna ws mesured using qrt-pcr. It ws oserved tht Stelth SOST-siRNA showed similr efficcy to unmodified duplex, producing 8% knockdown of SOST without detectle cell toxicity (Supplementry Figure S2). Next, we exmined the effect of nti-sost-sirna () delivered in LNP. MEFs were plted nd treted with LNP siluc s control () or LNP for 3 nd 7 dys. Following tretment, SOST-mRNA nd Tp housekeeping gene trnscripts were mesured using qrt-pcr nlysis. -treted MEFs exhiited significnt downregultion reltive to siluc controls of SOST-mRNA for ll three doses tested, yielding 6, 7, nd 8% knockdown fter 3 dys tretment, while the housekeeping Tp gene expression remined constnt t ll doses tested (Figure 3,). In ddition, 7 dys of tretment reduced SOST expression in dose dependent mnner leding to 9% downregultion following tretment with 1 µg/ml sirna, without ffecting Tp

4 4 sirna-medited SOST Silencing In Vivo minute 15 minutes 3 minutes 1 hour 2 hours c Corrected men fluorescence intensity 4, 35, 3, 25, 2, 15, 1, 5, sirna LNP d MFI 3,5 3, 2,5 2, 1,5 1, 5 Free-siRNA LNP-DiO sirna-647 PBS Incution time (minutes) PBS Chse time (hours) Figure 2 Visuliztion nd quntifiction of effective uptke of LNP-siRNA y MEFs. Mouse emryonic firolsts (MEFs) were seeded in glss ottom culture dishes nd when 8% confluent, () Qusr57-leled LNP-siRNA (red) nd () DiI-leled LNP-siRNA (green) were dded to the cells t 5 µg/ml to monitor the cellulr uptke of sirna nd LNPs. DMEM ws replced with phenol-free medium prior to live cell imging, nd live cells were nlyzed in Leic confocl microscope t time points indicted. A minimum of three imges were exmined for ech time point. Scle r = 5µm. (c) Intrcellulr fluorescence ws lso quntified. Br grphs shown s men ± SD (n = 3) represent dt otined from two different experiments P <.1. (d) MEFs were incuted with 2 μg/ml LNP (DiO green)-sirna (Alex647 fr red) nd fluorescence intensity ws ssessed t time points indicted using flow cytometry. The r grphs depict percent increse (men ± SD, n = 3) of men fluorescence intensity over time reltive to PBS-treted controls nd free sirna. Dt re representtive of two different experiments performed in triplicte (P <.1). (Figure 3c,d). Tken together, the dt indicte tht LNPs cn efficiently deliver sirna to primry MEFs resulting in significnt SOST gene knockdown. sirna-medited SOST knockdown modultes differentition of MEFs To determine the nolic effect of RNAi-medited SOST knockdown, we mesured the expression of erly nd lte mrkers of osteogenic differentition. MEFs were treted with or, hrvested fter 3 nd 7 dys of incution nd one osteogenic mrkers were mesured using qrt-pcr nlysis. It ws oserved tht MEFs treted with generted incresed Alp trnscripts compred with cells treted with nd tht this increse ws dose dependent. The Alp increse ws more evident t 5 nd 1 µg/ml doses (Figure 4). To confirm the osteogenic identity of this trnsformtion of MEFs, we lso exmined the expression of dditionl erly nd lte-stge osteogenic mrkers (Supplementry Tle S2). Except for Pthr1, which remined unchnged, Coll1A1/2, Osteoclcin (Ocn) nd Osterix (OSX) were ll differentilly upregulted following tretment with compred with, nd this upregultion ws generlly dose dependent (Supplementry Figure S3). Seven dys of tretment did not increse the expression of one osteogenic genes. The key regultors of one formtion Alp, Coll1A2, nd Ocn however, remined elevted compred to (Figure 4), wheres other genes exhiited minor downregultion (Supplementry Figure S3). Cthepsin K (involved in one resorption) gene expression incresed fter 3 dys of tretment ut normlized y dy 7. Finlly, Moleculr Therpy Nucleic Acids

5 sirna-medited SOST Silencing In Vivo 5 Reltive SOST mrna Reltive SOST mrna PBS PBS c Reltive Tp mrna d Reltive Tp mrna PBS PBS Figure 3 sirna-medited knockdown of SOST gene in vitro is gene specific. To investigte the sirna-medited knockdown, mouse emryonic firolsts were seeded in 12-well pltes nd, PBS nd encpsulted in LNPs were ll dministered in triplicte wells s indicted. () At dy 3 nd () dy 7 following tretment, SOST mrna expression ws determined y qrt-pcr using the comprtive ΔΔCT method. The SOST mrna ws normlized to housekeeping gene Hprt. Br grphs represent expression of SOST mrna reltive to Hprt where SOST mrna vlues following LNP-siRNA-Ctrl tretment re considered 1%. Dt re shown s men ± SD of triplicte wells nd re representtive of t lest three experiments. Br grphs in (c) nd (d) represent expression of Tp mrna reltive to Hprt. Tp mrna following is considered 1% (P <.1). β-ctenin, gene tht plys mjor role in gene trnscription, remined unchnged throughout the tretment suggesting tht sirna-medited SOST knockdown did not ffect the signling pthwys involved in cell homeostsis. Alp ctivity is mrker for osteolst differentition s proliferting osteolsts show Alp ctivity tht is gretly enhnced during their in vitro differentition nd one formtion. To further confirm the osteogenic differentition of sirna-medited SOST knockdown in MEFs, cells were treted with OM to induce expression of sclerostin nd then were treted with phosphte-uffered sline (PBS), nd for 1, 2, nd 3 weeks in n osteogenic stimultory medium. After 1 week of tretment, red stining ws significntly enhnced in -treted cells compred to or PBS, suggesting n increse in ALP ctivity following SOST knockdown (Figure 5,d). The ALP stining intensity incresed t 2 weeks fter tretment nd remined significntly higher compred to PBS- nd -treted cells (Figure 5,d). After 3 weeks, ALP stining of -treted cells exhiited drmtic increse in ALP intensity compred with controls, ppering similr to the OM-treted cells (Figure 5c,d). These results indicte tht sirna-medited knockdown of SOST gene exhiits n osteogenic effect demonstrted y incresed ALP ctivity nd differentition to osteolsts. RNAi-induced prolonged SOST silencing without ffecting cell viility Since development of phenotypic effects my require silencing of SOST for n extended period, it ws importnt to exmine the durtion of RNAi-induced silencing fter single tretment. Following SOST induction, MEFs were treted with or for 3 dys. Tretment ws stopped fter 3 dys y replcing the medium, nd SOST mrna ws mesured every 3 dys for 2 weeks using rel-time qrt-pcr. It ws oserved tht SOST trnscripts were reduced y 6% 3 dys fter single tretment with, compred to (Figure 6). SOST expression reduced progressively demonstrting > 8% knockdown t 12 dys post-tretment ut egn to increse fter 2 weeks, ut remined significntly reduced compred with -treted MEFs. Microscopic monitoring of cells did not revel overt toxicity over 15 dys incution. Importntly, despite significnt downregultion of SOST, the Tp housekeeping gene remined unchnged, confirming trget specificity of RNA interference (Figure 6). To determine the osteogenic effects on MEFs following single tretment, mrna trnscripts of three key osteogenic genes Alp, Coll1A1 nd Ocn ws lso mesured. The dt indicte moderte upregultion of the mrna of ll the tested iochemicl mrkers, which tended to diminish t the

6 6 sirna-medited SOST Silencing In Vivo Reltive Alp mrna PBS PBS Reltive Alp mrna 1 Reltive Coll1A2 mrna Reltive Coll1A2 mrna PBS PBS Reltive Ocn mrna Reltive Ocn mrna PBS PBS Figure 4 sirna-medited SOST knockdown increses expression of one osteogenic mrkers. mrna expression of one osteogenic mrkers ws nlyzed using qrt-pcr following,, or PBS tretment t indicted doses. Reltive expression of trget genes ws quntified nd normlized to the housekeeping gene Hprt. The mrna of -treted smples ws compred to, which ws considered 1%. Grphs depict increses of one osteogenic mrkers reltive to mrna expression () 3 nd () 7 dys following tretment with tht ws considered 1%. Dt re shown s men ± SD of triplicte wells nd re representtive of t lest three experiments (P <.5; P <.1; P <.1). 2 week time point (Figure 6c). Tken together, the dt suggest tht single dose tretment with delivered with LNPs produced prolonged silencing effect on the SOST gene without compromising cell viility nd elicited moderte ut consistent osteogenic effect on MEFs. sirna-lnps exhiit significnt uptke y osteocytes in vivo Next, we investigted the uptke of LNP-siRNAs y osteocytes in vivo. LNP-siRNA leled with DiI or PBS were dministered intrvenously to green fluorescence protein (GFP) expressing mice used s source of uiquitous green cells including osteocytes. Forty-eight hours lter one sections were viewed under confocl microscope. In mice treted with PBS, we could visulize the cellulr component of the compct one, 9% of which consists of osteocytes. Osteocytes were identified sed on the expression of GFP in the cytoplsm nd nuclei, ellipsoid nd stellte shpe, regulr prllel orienttion, nd size. The connecting syncytil network, consisting of smll cytoplsmic dendritic processes in cnliculi, ws lso visile t high power (Figure 7). Sections from mice treted with dye-leled LNP-siRNA (i.v. injection vi til vein) demonstrted similr distriutions of the red nd green stining tht lmost completely overlpped with the osteocyte-like shpes. Of note, there ws unstined re in the center of the red-stined cellulr structure corresponding Moleculr Therpy Nucleic Acids

7 sirna-medited SOST Silencing In Vivo 7 PBS DMEM OM c d 6 % increse of ALP OD PBS PBS PBS DMEM Week 1 Week 3 Tretment nd oservtion time Week 3 OM Figure 5 SOST knockdown enhnces lkline phosphtse ctivity. Mouse emryonic firolsts (MEFs) were seeded in pltes t cells per cm 2 nd incuted with OM. Cells were then treted for 1 week with sirna-sost or sirna-ctrl encpsulted in LNPs, t 1 mg/ml nd plte ws treted with PBS. In one plte, cells were incuted in DMEM nd in nother plte cells were incuted with OM throughout ll the oservtion period. MEFs were sujected to lkline phosphtse stining () 1, () 2, nd (c) 3 weeks following tretment. ALP ctivity ws ssessed sed on the red stining intensity using photomicroscopy. A minimum of 3 imges were exmined for ech tretment nd time point. Dt were nlyzed using the OpenL softwre. (d) Imges were quntified s descried in Mterils nd Methods. The r grphs depict percent increse (men ± SD, n = 3) of ALP ctivity over time reltive to PBS- or -treted cells. Dt re representtive of two different experiments (P <.5). to the nuclei. These results indicte tht sustntil numer of osteocytes hd tken up significnt mounts of dye-leled LNP-siRNA (Figure 7). The intensity nd distriution of the red pttern ws quntified reltive to green osteocytes, s descried. It ws concluded tht out 5% of the osteocytes hd tken up significnt mount of LNP-siRNA, s chrcterized y intrcellulr ccumultion nd cytoplsmic distriution of the dye-leled LNPs. LNP-siRNA nnosystems induce SOST silencing in vivo following systemic dministrtion Since we could chieve knockdown of SOST in vitro nd confirm delivery of LNP-siRNAs in osteocytes in vivo, it ws of ovious interest to determine whether these formultions induce SOST silencing in vivo following systemic dministrtion. In ddition, it ws importnt lso confirm tht SOST expression is restricted to osteocytes in compct one. 3 Totl RNA ws extrcted from mouse one, hert, kidney, liver, lung, nd spleen nd mrna ws determined using qrt-pcr. It ws oserved tht SOST mrna ws highly expressed in mouse ones (Supplementry Figure S4). However, SOST mrna ws minimlly expressed in kidney, lung, nd spleen wheres only trces of SOST mrna were determined in the liver nd hert. Since the cellulr constituents of compct one re 9% osteocytes, we cn conclude tht SOST mrna is expressed lmost exclusively in osteocytes. To determine SOST silencing following LNP-siRNA dministrtion, mice were injected with LNP contining, non-specific or with PBS. Mouse one nd serum were then hrvested t 2, 3, nd 7 dys post-tretment, nd circulting sclerostin nd SOST mrna were mesured using ELISA nd qrt-pcr. The dt showed tht circulting sclerostin ws minimlly

8 8 sirna-medited SOST Silencing In Vivo PBS siluc PBS siluc ns Reltive SOST mrna Reltive Tp mrna c Reltive Alp mrna Dy Dy 3 Dy 6 Dy 9 Dy 12 Dy 15 Dy Dy 3 Dy 6 Dy 9 Dy 12 Dy 15 Dys post-trnsfection PBS 2 2 PBS PBS Dy Dy 9 Dy 12 Dy 15 Reltive Coll1A1 mrna Dy Dy 9 Dy 12 Dy 15 Reltive Ocn mrna Dy Dy 9 Dy 12 Dy 15 Figure 6 Durtion of SOST knockdown in vitro nd its effect on one osteogenic mrkers. Sclerostin expression ws induced in mouse emryonic firolsts followed y tretment with or encpsulted in LNPs. After 3 dys, sirna ws wshed off, the plte ws refilled with DMEM nd totl RNA ws extrcted every 3 dys for over period of 2 weeks. Totl RNA ws extrcted nd mrna of () SOST () Tp housekeeping gene nd (c) one osteogenic mrkers ws determined using qrt-pcr. Br grphs represent mrna expression of SOST, Tp nd one osteogenic mrkers reltive to Hprt housekeeping gene over time. SOST mrna following tretment re considered 1%. Dt re shown s men ± SD of triplicte wells nd re representtive of t lest two experiments (P <.5; P <.1; P <.1). ns, not significnt. downregulted t 2 dys fter tretment (Figure 8), ut significnt downregultion ws oserved t 7 dys post-tretment (P =.1, P <.5). The qrt-pcr nlysis reveled significnt knockdown of the SOST gene t 2 (P =.9, P <.5) nd 3 dys post-tretment (P =.1, P <.5) reching 6% silencing reltive to PBS nd/or (P =.2, P <.5) 7 dys post-tretment (Figure 8). Noticely, following tretment with PBS or, SOST mrna remined t similr levels, indicting tht -medited silencing ws sequence specific. In ddition, neither nor dministrtion ltered the expression of the Tp housekeeping gene, demonstrting tht SOST knockdown ws lso gene specific nd excluding sirna-medited off trget effects. In dditionl experiments, we ttempted to optimize the method nd improve the qulity of RNA extrcted from one. Following further optimiztion of RNA extrction from compct one with further improved RNA Integrity Numer ove 8 (Supplementry Figure S5), new in vivo investigtion ws set to confirm SOST knockdown in vivo. Mice were injected intrvenously with, PBS, or two nonspecific control sirnas trgeting luciferse (siluc) nd humn ndrogen receptors (siarh). RNA ws extrcted 1 week fter tretment nd SOST mrna expression ws mesured y qrt-pcr. An liquot ws used to quntitte RNA. The results demonstrte sustntil knockdown of the SOST mrna reltive to oth negtive controls siluc or siarh, nd no difference of SOST expression ws oserved etween smples treted with PBS or control sirna. In ddition, none of the sirna tretments ltered the expression of Tp housekeeping gene reltive to PBS tretment, confirming the efficcy of LNP-siRNAs to specificlly silence the trget gene. It is importnt to note tht no tretment-relted gross toxic effects were oserved, s determined y monitoring weight loss of the mice (Supplementry Figure S6,). Tken together, these results indicte tht the LNP-siRNAs re highly ctive nd very efficient in silencing SOST gene expression in compct one in vivo. Discussion In this work, we hve shown tht the SOST gene cn e induced in vitro in primry MEFs, surrogte stem cell model for one-mrrow-derived stroml stem cells, nd hve developed n sirna duplex tht effectively silences SOST gene expression. Further, we hve shown tht LNP systems contining SOST sirna cn effectively silence SOST gene expression oth in MEFs in vitro nd in osteocytes in compct one in vivo following systemic (i.v.) dministrtion. There re three spects of these results tht wrrnt further discussion. The first re concerns the utility of the MEF cell line for in vitro studies nd the correltion etween the silencing the SOST gene in vitro with in vivo results. The second topic concerns the potentil dvntges of RNAi therpeutics Moleculr Therpy Nucleic Acids

9 sirna-medited SOST Silencing In Vivo 9 c 14 Reltive numer of LNP +ve cells (%) GFP +ve Osteocytes PBS sirna-lnps sirna-lnps Figure 7 Successful delivery of leled LNP-siRNA to mouse osteocytes following systemic dministrtion. DiI-leled LNP-siRNAs (red) were dministered intrvenously in to GFP mice. Two dys following injection, 5 µm one sections were prepred, processed nd nlyzed under confocl microscope. Imges depict representtive res of compct one 48 hours following systemic dministrtion of () PBS nd () leled LNP-siRNAs. Ellipsoid shpe morphology nd prllel orienttion suggests tht green cell-like structures represent osteocytes wheres the red cell-shped structures indicte LNP-siRNAs. Imges demonstrte high degree of overlp etween osteocytes (green) nd LNP-siRNAs (red) indicting the intrcellulr enrichment with LNP-siRNAs nd their delivery to osteocytes. (c) The numer of osteocytes contining LNP-siRNAs ws quntified s descried. Grphs demonstrte the percentge of the osteocytes contining LNPsiRNAs vs totl numer of osteocytes. Approximtely 1 cells were nlyzed. Dt re representtive of two different experiments. Scle r = 1 µm. nd the limittions of the LNP sirna system employed here, which ws originlly designed for silencing genes in heptocytes following i.v. dministrtion. Finlly, we discuss wys in which the therpeutic utility of the LNP sirna systems could e improved possily leding to novel tretments for osteoporosis. The MEF tissue culture system ws used s surrogte for one-mrrow-derived stroml stem cells. MEFs represent popultion of stem cells tht ehve similrly to one mrrow-derived stem cells in ex vivo nd in vivo ssys, nd hve een used to investigte moleculr mechnisms of stem cell differentition to osteolsts nd chondrocytes. 9 Furthermore, it is estlished tht MEFs re cple of entering nd completing the progrm of chondrogenic differentition ex vivo, from undifferentited progenitor cells to mture chondrocytes, therey providing tool tht enles the study of mesenchyml progenitor cell s journey through the chondrogenic pthwy. 1 As visulized y confocl microscopy, the LNP formultions utilized here demonstrted remrkle ility to deliver sirna into MEFs, nd, following induction of the SOST gene (which is usully only expressed in osteocytes), to silence this gene. It my e noted tht relted LNP sirna systems hve demonstrted ilities to silence genes in erythroid nd myeloid progenitors in one mrrow following i.v. dministrtion (Tm Y. et l., unpulished dt) suggesting high potency in nominlly hrd to trnsfect cell lines. This could e due to the mechnism wherey these LNP sirna systems re ccumulted into cells following ssocition with endogenous ApoE. 11,12 ApoE receptors re expressed t high levels in dult hemtopoietic stem cells nd in neurl stem cells 13,14 In ny event, the MEF cell line proved convenient system to optimize sirna dimers nd demonstrte functionl effect of SOST knockdown, s illustrted y the upregultion of one osteogenic mrkers such s Alp to level similr to tht chieved y incution in osteotrophic medi. The MEF tissue culture system llowed the development of potent sirna duplex for silencing the SOST gene nd showed tht LNP systems contining this duplex could result in gene silencing, leding to in vivo studies to see whether

10 1 sirna-medited SOST Silencing In Vivo Reltive expression of sclerostin PBS Reltive SOST mrna c Reltive Tp mrna Dy 1 Dy 3 Dy 7 14 PBS PBS Dy 1 Dy 3 Dy 7 Dy 1 Dy 2 Dy 3 Dy 7 d e Reltive SOST mrna Reltive Tp mrna PBS siluc siar Dy PBS siluc siar Dy 7 Figure 8 LNP-siRNA medited silencing of SOST in vivo is specific, time dependent nd very effective. () LNP-siRNA trgeting SOST, LNP- or PBS, were dministered intrvenously in to C57Bl6 mice. Blood ws collected nd serum sclerostin ws determined using the enzyme-linked immunosorent ssy Br grphs depict sclerostin expression overtime in mice dministered with reltive to tretment. Sclerostin levels following tretment were considered 1%. P <.5. Following in vivo dministrtion with, or PBS, femurs nd tie were dissected, totl RNA ws extrcted nd SOST nd Tp mrna ws determined t time points indicted. Br grphs represent expression of () SOST nd (c) Tp mrna overtime reltive to Hprt. SOST mrna vlues following tretment re considered 1%. Dt re representtive of 3 different experiments. (c) After in vivo dministrtion of nd (s) s descried, high qulity RNA were otined nd SOST nd Tp mrnas were mesured. Br grphs represent expression of (d) SOST nd (e) Tp mrna reltive to Hprt seven dys fter dministrtion of. mrna expression of SOST following tretment re considered 1%. P <.5 P <.1. similr effects could e oserved in osteocytes in compct one. In this regrd, primry chllenge for the development of sirna therpeutics is the ility to deliver sirna molecules to trget orgns or cells in vivo. RNAi therpeutics were originlly limited to topicl pplictions 15 ut drmtic improvements in LNP systems for heptocyte gene silencing hve enled liver trgets to e exploited. 16,17 However, LNP systems to trget other tissues re less developed. 18 LNP contining permnently chrged ctionic lipids hve een shown to silence trget genes on one-forming surfces following systemic dministrtion, 19,2 ut permnently chrged ctionic lipids cn e highly toxic. 21,22 The studies presented here show effective delivery of LNP-siRNA to osteocytes following i.v. dministrtion s visulized y confocl microscopy, demonstrting the presence of LNP-siRNA in t lest 5% of osteocytes. This delivery ws ccompnied y significnt knockdown of the SOST gene in these osteocytes nd consequent downregultion of serum sclerostin 1 week fter systemic dministrtion of sirna. It is of interest tht levels of Alp did not chnge during the week fter sirna dministrtion, nd other mrkers were only slightly downregulted. A recent study demonstrted tht for menopusl women with low one minerl density, the increse in one-formtion mrkers following dministrtion of nti-sclerostin ntiody were trnsitory nd were only detected 1 week fter the initil dose, nd were lrgest 1 month fter. However tretment with the ntiody ws ssocited with incresed one minerl density nd one formtion. It is likely tht wild-type mice re not the idel model to study the effect of sirna-medited knockdown of SOST, prticulrly with regrd to effects on osteogenic mrkers. With regrd to the potentil dvntges of RNAi-sed therpies for tretment of disorders such s osteoporosis, inhiition of sclerostin using monoclonl ntiodies hs een shown to restore one loss induced y estrogen deficiency, nd increse one mss nd strength in niml models or postmenopusl osteoporosis in humns. 1,23,24 Therefore, inhiition of the Wnt/β-ctenin signling pthwy represents promising therpeutic trget for osteoporosis tretment. 25 However, the sclerostin ntiody cn cuse n immune response leding to rpid clernce nd reduced potency for repet dministrtions. 26 RNAi-sed therpies tht silence the SOST gene in osteocytes could provide more effective pproch tht my not e suject to such limittions. It hs een noted tht trnsthyretin (TTR) gene silencing in heptocytes cn persist for up to 6 dys following i.v. dministrtion of LNP TTR sirna in humns. 16 In ddition, recent work hs shown tht incorportion of hydrophoic corticosteroid pro-drugs in LNP successfully rogtes immune responses to LNP oligonucleotide formultions (S. Chen, unpulished dt). A finl point is tht, regrdless of potentil clinicl pplictions, the studies presented here show tht LNPsiRNA systems re potentilly powerful tool to study loss of function effects for novel gene trgets in niml disese models of one disorders. While LNP sirna systems to silence genes such s SOST my hve therpeutic potentil, it is importnt to note the limittions of the LNP sirna system employed in this work, which contins the ionizle ctionic lipid DLin-MC3-DMA, the neutrl lipids DSPC nd cholesterol together with PEG- DMG coting lipid. This system hs demonstrted remrkle efficcy for gene silencing in heptocytes following i.v. Moleculr Therpy Nucleic Acids

11 sirna-medited SOST Silencing In Vivo 11 dministrtion in mice nd nonhumn primtes 27 nd hs recently een shown to e highly effective for treting TTRinduced myloidosis in dvnced clinicl trils. 16,28 However, when pplied to silencing genes in extr-heptic tissues such s one, these LNP systems re much less potent. In mice, 5% gene silencing of trget genes in heptocytes cn e chieved t dose levels s low s.5 mg sirna/kg ody weight. 27 In contrst, the doses required to chieve the SOST gene silencing oserved in this work re 15 mg sirna/kg ody weight, more thn thousnd times higher. It is mesure of the reltive lck of toxicity of the LNP sirna systems tht such doses re not overtly toxic, however in other work, we hve noted toxic effects for repet doses in the rnge of 1 mg sirna/kg ody weight (G. Bsh, unpulished dt). These toxicities rise primrily from the LNP crrier which cn e heptotoxic t high-dose levels. While little toxicity ws oserved here, doses of 1 mg sirna or higher re pproching the mximum tolerted dose nd cnnot e considered s hving potentil therpeutic utility, n increse in potency in the rnge of 1 is required. There re numer of wys in which such lrge improvements in potency my e chieved. The first concerns circultion lifetime; s noted elsewhere, 29,3 LNP systems contining the coting lipid PEG-DMG (which hs C 14 cyl chins) exhiit short (< 1 hour) circultion lifetimes which limits distriution to extr-heptic tissues. Longer circultion lifetimes nd enhnced distriution to fenestrted tissues such s one mrrow cn e chieved y incorporting PEG-lipids with longer cyl chins, such s PEG-DSG (contining C 18 cyl chins) which does not dissocite from the LNP s rpidly s PEG-DMG. 3 LNP size is nother importnt vrile; it hs een noted tht smller nnoprticulte systems exhiit enhnced penetrtion into tumor tissue. 31,32 By nlogy, it would e expected tht smller LNP systems, which cn e redily chieved y incresing PEG content 29,33 will improve delivery to the microvsculture of compct one. An lterntive pproch concerns the oservtion tht s much s 7% of endocytosed LNP systems re recycled to the extrcellulr medium, the presence of gents tht reduce or dely such recycling cn led to sustntil improvements in gene silencing potency. 34 In conclusion, the studies presented here using the MEF primry cell system estlish model in vitro system to study the effects rising from silencing specific genes, such s the SOST gene, on osteogenesis. Further, it is shown tht nonoptimized LNP formultions of sirna cn ccess osteocytes in hrd one following i.v. dministrtion, resulting in SOST gene silencing. This work sets the stge for the development of next-genertion LNP sirna systems tht my provide effective strtegies for tretment of osteoporosis nd other one diseses. Mterils nd methods Cell culture. Primry mouse emryonic firolsts (MEFs) were hrvested, ccording to stndrd protocols. 35 Briefly, uteri were hrvested t the emryonic stge t 13.5 dys post-coitus nd further processed to isolte the emryos nd MEFs. Heds nd viscer were removed nd the remining emryos were wshed in PBS, trnsferred to 35 mm petridishes, minced with pir of scissors nd digested with.25% trypsin/edta 1 mmol/l (Sigm-Aldrich, St. Louis, MO) for 1 minutes t 37 C. Following digestion, out 3 ml Dulecco s modified Egle medium (DMEM), supplemented with 1% fetl ovine serum (FBS), 1% L-glutmine, nd 1% penicillin nd streptomycin (P/S), ws dded nd the tissue pipetted up nd down to get single cell suspension. MEFs were mintined in T 75 flsks in DMEM, supplemented with 1% FBS, 1% L-glutmine, 1% NEAA, 1% sodium pyruvte, nd 1% P/S until 8 85% confluent nd were then pssged t 1:2 rtio. In this study, we used MEFs t pssge 3. SOST induction, sirna trnsfection, nd sequence identifiction. MEFs were seeded in 12-well pltes t density of cells/cm 2 nd incuted with DMEM, 1% FBS, nd 1% P/S. At 8% confluence, cells were incuted with MesenCult Osteogenic Stimultory Kit (STEMCELL Technologies, Vncouver, Cnd) or Osteogenic Medium (OM). The MesenCult Osteogenic Stimultory Kit (Mouse) is specificlly formulted for the in vitro differentition of mouse mesenchyml stem nd progenitor cells from compct one, one mrrow, nd MEFs into osteolsts. This kit induces strong osteogenesis in mouse MSCs nd MEFs. Medi were chnged every second dy for 15 dys. Every 3 dys, cells from triplicte wells were lysed using 35 µl lysis uffer (Invitrogen, Crlsd, CA) nd stored t 8 C. At the end of the experiment, cell lystes were thwed, mixed with equl mounts of 7% ethnol, nd RNA ws extrcted ccording to the mnufcturer s directions (PureLink RNA extrction Kit, Life Technologies). Extricted totl RNA ws used to determine mrna expression of SOST, lkline phosphtse (Alp), n erly one osteogenic mrker, Sp7 (mster regultor of osteolst differentition), RunX2 ( key trnscription fctor ssocited with osteolst differentition), nd one siloprotein (Bsp), component of minerlized tissues. The mrna expression levels of SOST or one osteogenic genes were quntified y rel-time polymerse chin rection (qrt-pcr). PureLink RNA Mini Kits (Life Technologies) were used to extrct totl RNA from cultured cells using the commercil protocol. RNA ws reverse trnscried to complementry DNA (cdna) from 1 μg of totl RNA from ech smple, using High Cpcity cdna Reverse Trnscription kit (Applied Biosystems, Foster City, CA). Rel-time PCR ws performed on StepONE Plus Rel Time PCR system (Applied Biosystems) using TqMn Fst Advnced Mster Mix (Applied Biosystems), which include forwrd nd reverse primers nd the fluorescein midite (FAM)-leled proe for the trget gene respectively purchsed from Integrted DNA Technologies, Iow. In ddition, protein ws extrcted from designted pltes nd determined s previously descried, 28 followed y quntifiction using NIH ImgeJ-win32 softwre in order to mesure sclerostin expression t 7 nd 14 dys following incution in OM. Another set of pltes contining MEFs incuted in DMEM or OM were used to mesure lkline phosphtse ctivity following tretment with DMEM or OM tht ws quntified s descried elow. To test the efficcy of gene-specific sirna on MEFs in vitro, set of three predesigned duplexes (TriFECT, Integrted DNA Technologies) were used, nmely A, B, nd C, including three

12 12 sirna-medited SOST Silencing In Vivo dditionl duplexes, D, E, nd F, ll from the RefSeq dtse in GenBnk (Supplementry Tle S1). To optimize the experimentl setting of RNAi for our trget gene SOST, three controls were used: fluorescent dye-leled duplex (Tye 3 DS trnsfection control), universl negtive control duplex (NC1) tht trgets site tht is sent from humn, mouse nd rt genomes, nd positive control duplex (Hprt- S1 DS-positive control) tht trgets site in the hypoxnthine phosphoriosyntrnsferse (Hprt) 1 gene common etween humn, mouse, nd rt. MEFs were seeded in 24-well pltes nd forwrd trnsfection ws performed in triplicte ccording to the mnufcturer s directions, y mixing the RNAiMx (Invitrogen) trnsfection regent with sirna to otin finl concentrtion of 3,1, nd 3 nmol/l, including positive nd negtive controls. Trnsfection progressed for 2 hours efore the first medium renewl. After 24 nd 48 hours in culture, cells were lysed with 35 µl lysis uffer (Life Technologies) nd stored t 8 C. Trnsfection efficiency ws ssessed following intrcellulr visuliztion of Tye 563-lelled sirna using Zeiss Axiovert 2 microscope with QImging Retig EX mono 12-it cmer. Fluorescence nd phsecontrst imges were overlid to visulize the intrcellulr sirna using confocl microscopy. Preprtion of lipid nnoprticles (LNPs) nd sirna encpsultion. LNPs were prepred y mixing one volume of the following lipid composition dilinoleylmethyl-4-dimethylminoutyrte (DLin-MC3-DMA), disteroylphosphtidylcholine (DSPC), cholesterol, nd polyethylene glycol-dimyristol glycerol (PEG-DMG) t 5:1:38.5:1.5 mole rtio) dissolved in ethnol nd three volumes of sirna (1:1 w/w sirna to lipid) suspended in cette uffer. The ctionic lipid ws synthesized y Biofine Interntionl (Vncouver, BC). A detiled description of the ctionic lipid nd its ctivities hs een descried previously. 27 Formultion ws performed in microfluidic mixer, provided y Precision Nnosystems (Vncouver, BC), y pumping oth solutions through the micromixer t comined flow rte of 4 ml/minute (1 ml/minute for ethnol nd 3 ml/minute for queous uffer). The resultnt mixture ws dilyzed ginst phosphte-uffered sline (PBS), ph 7.4 for 16 hours to remove ethnol, nd then filtered. A detiled description of the method hs een descried previously. 29 To prepre fluorescently lelled LNPs,.2 mol% 1,1 -Dioctdecyl-3,3,3,3 -Tetrmethylindodicrocynine- 5,5 -disulfonic cid (DiI-C 18) ) from Invitrogen, ws dded to the lipid mix. Dynmic light scttering ws used to determine LNP size (numer weighting) using the Mlvern Zetsizer NnoZS (Worcestershire, UK). Size mesurements were performed in PBS (ph 7.4). Encpsultion ws mesured using the Qunt-iT Riogreen RNA ssy (Life Technologies, Burlington, ON). Prticles were incuted t 37 C in the presence or sence of 1% Triton X-1 (Sigm-Aldrich, St. Louis, MO) for 1 minutes. Following incution, Qunt-iT Riogreen RNA regent ws dded nd fluorescence intensity mesured (Ex/Em 48/52 nm). Triton X-1 tretment mesurements represented totl sirna while non-triton X-1 tretments represented un-encpsulted sirna. Intrcellulr delivery of sirna, knockdown efficiency in vitro, nd quntittive rel-time PCR (qrt-pcr). Prior to testing knockdown efficiency, it ws importnt to ssess the sirna delivery in primry MEFs following encpsultion in LNPs. 8% confluent MEFs were seeded in 35-mm glss ottom culture dishes (MtTek Corportion, MA) t pproprite concentrtions nd LNPs contining sirna leled with Qusr 57 Ex/Em 548/566 nm, were dded to the cells t concentrtion of 5 µg/ml. In nother seprte, prllel experiment, LNPs leled with DiI (DiI C18 Ex/Em 549/565 nm, Invitrogen) contining sirna were lso incuted with MEFs t the sme sirna concentrtion. The sirna ws encpsulted in LNPs contining DLin-MC3-DMA nd cellulr uptke of sirna nd LNPs ws monitored for 15, 3, 6, nd 12 minutes y mesuring the ccumultion nd the intensity of the fluorescence of DiI nd Qusr 57 in MEFs. The incution ws stopped y removing the medi contining the LNP-siRNAs nd replenishing the cells with FluoroBrite DMEM (Life Technologies) without Phenol red s ph indictor. Next, live cell imging ws conducted using Leic TCS SP8 lser scnning confocl microscope (Leic, Germny) nd ll imges were processed nd recorded using LAS AF 3 softwre. The DiI nd Qusr 57 fluorescent proes were excited with the 561 nm lser nd the intrcellulr presence of LNP-siRNAs ws exmined using NIH ImgeJ-win32 nd Photoshop 9. CS2. Fluorescence nd phse-contrst imges were overlid to determine presence nd intensity of fluorescence. A minimum of three imges were exmined for ech time point. Dt nlysis nd quntifiction of intrcellulr delivery of LNP-siRNA ws performed using the NIH ImgeJ-win32 s descried elow. To etter investigte the intrcellulr delivery of LNPsiRNA pulse-chse experiment ws undertken. MEFs were grown in 24-well pltes, treted with 2 μg/ml sirna- Alex647 (fr red) in free form or encpsulted in LNPs DiO (green) from Invitrogen, including PBS control, nd mintined t 37 C. Incution ws stopped fter 2 hours y wshing off the medi nd refilling the plte with fresh medi. Cells were hrvested fter 2, 4, 6, 8, 24, nd 48 hours. Following tretment, MEFs were trypsinized (.25% Trypsin), trnsferred into microcentrifuge tues, spun t 12, rpm for 4 minutes nd fter three wshes they were resuspended in 3 μl FACS stining uffer. Smples were cquired using n LSRII flow cytometer to ssess the presence of LNP (DiO) nd sirna-alex647 intrcellulrly. The Alex647 fluorophore ws excited using the HeNe 633 lser line nd detected t the Alex647 (FL 5) chnnel wheres the DiO ws excited y the Argon lser nd detected t FL1 chnnel. Dt were cquired using FACSDiv softwre nd nlyzed y FlowJo softwre. Mesurements were collected for 5 events. Fluorescence intensity ws normlized ginst the PBS controls nd ws expressed s percent increse of men fluorescence intensity. To investigte the sirna-medited knockdown, MEFs were seeded in 12-well pltes s descried. The sequence of the mouse SOST gene (GenBnk ccession no. NM_24449(1) ws extrcted from the NCBI Entrez nucleotide dtse. The SOST-siRNA ws composed of two complementry RNA strnds: sense strnd (SOST-S) 5 -mgmcramgrumgrururar ArUmArUmCrGmCrUmUrUrGrUrGrAmAG-3 nd ntisense strnd (SOST-AS): 5 -rcrumurcrarcraramargmcrgmarur ArUrUrArArCrArCmUrGmCmCmU-3. The two strnds of Moleculr Therpy Nucleic Acids