the potential of rnai to silence any gene has made it an attractive therapeutic modality .
however , the main obstacle to rnai in the clinic is delivery . to be effective
, sirnas must be transported through the body , bind , and be taken up by target cells where they must traverse the plasma membrane and gain access to the cytosolic compartment , where the rnai machinery resides . to be useful ,
ease of formulation and administration , overall cost , and any associated toxicities are essential considerations .
dcs are heterogeneous , with subsets defined phenotypically , functionally and by location ( reviewed in ref .
2 ) . a delivery vehicle that knocks down expression of specific genes in a distinct dc population(s ) would be a valuable tool for targeting diverse diseases including cancers , infectious diseases , autoimmunity and as a vaccine component .
therefore , a platform that delivers sirnas to specific dc subsets in situ would be useful for inhibiting or activating immune responses .
lipid nanoparticles ( lnps ) are one of the most advanced platforms for sirna delivery to hepatocytes , and are under clinical evaluation for conditions that require hepatic gene silencing .
these lnps typically contain ionizable cationic lipids ( pka ~6.5 ) that bind nucleic acids via electrostatic interactions at low ph , but are charge neutral at ph 7.4 . as a well - perfused organ
furthermore , lnp uptake by hepatocytes is mediated by association with serum apoe leading to efficient uptake via low - density lipoprotein receptors in the liver .
following cellular uptake of the lnp , the ionization of the lipid within acidic endosomes is thought to promote sirna escape to the cytosol .
importantly , these lnps are associated with minimal toxicity , including little induction of proinflammatory cytokines following administration of physiologically relevant doses .
in contrast to the liver , sirna delivery to extra - hepatic cells is challenging . in particular , immune cells such as dcs are relatively resistant to sirna uptake in vitro and in vivo .
although designed for hepatic gene silencing , the efficacy of these lnps and their derivatives has been assessed for rnai - mediated gene silencing in macrophages ( mos ) and dcs in vitro and in vivo .
lnp uptake and modest gene silencing was achieved at high doses of lnps ( ~ 5mg / kg ) .
together with a complementary study , this work demonstrates the feasibility of gene silencing in immune cells using lnp technology . clearly
, however , lnp formulations must be modified to achieve optimal gene silencing in immune cells .
enhanced uptake of lnps by primary hepatocytes is mediated by apolipoprotein e binding to the neutral lnps .
this results in recognition by receptors including the low - density lipoprotein receptor and scavenger receptors largely expressed on hepatocytes .
the mechanism of uptake strongly suggests that retargeting of these particles to other receptors or other tissues is possible .
indeed , akinc et al . demonstrated that lnps modified with n - acetylgalactosamine ( galnac ) were retargeted to the asialoglycoprotein receptor ( asgpr ) expressed by hepatocytes in vivo .
for delivery to nonhepatic cells , we have shown in vitro that anti - transferrin receptor aptamers can be used to redirect similar lnps .
more recently , liang et al . described a similar approach using aptamer - coated lnps to target osteoblasts in vivo .
for uptake by immune cells , a recent study coated lnps with a full - length anti - cd4 antibody for sirna delivery to primary cd4 t cells .
lnp uptake and gene silencing was observed in cd4 t cells in various organs including the spleen , lymph nodes , and blood .
these studies demonstrate the potential for attaching exogenous ligands to lnps for delivery to hepatic , and importantly nonhepatic primary cells in vivo . drawing from these works
, we reasoned that this lnp platform could be a useful foundation for the delivery of sirnas to dcs to modulate immune responses . to achieve this
, we modified lnps using a single chain antibody ( scfv ) specific for the dc receptor dec205 , a c - type lectin expressed at high levels on cd8 dcs .
dec205 dcs mediate cross - presentation of antigen resulting in modulation of cd8 t - cell responses . therefore , inhibition of gene expression by dec205 dcs is a potentially powerful approach for regulating cd8 t - cell activation . using this approach , we assessed the ability of dec - lnps ( lnps coated with scfv specific for murine dec205 , containing sirnas specific for costimulatory molecules ) to inhibit immune responses .
injection of dec - lnps resulted in preferential uptake of the dec - lnps by splenic dec205 dcs .
furthermore , when coadministered with adjuvant , lnps containing sirna targeting cd40 , cd80 , and cd86 reduced expression of these costimulatory molecules to levels similar to those observed in immature dcs .
most importantly , dcs isolated from mice injected with a low dose ( ~0.6  mg / kg ) of these dec - lnps , were able to suppress a robust mixed lymphocyte reaction ( mlr ) , demonstrating the functional efficacy of this approach .
interestingly , when we performed experiments using 2 ' fluoro ( 2'f ) modified sirna , a formulation reported to be nonimmunostimulatory and nonimmunogenic , significant immune activation of the targeted dcs was detected . this effect was not observed in assays performed with human peripheral blood mononuclear cells ( pbmcs ) or monocyte - derived dcs ( modcs ) and could be mitigated through selective 2-o - methyl ( 2ome ) modification of the sirna .
overall , our study demonstrates the functionality of lnps modified for receptor targeting for sirna delivery to dcs .
additionally our results suggest that specific cell targeting can mediate immunological responses to sirna formulations .
while care needs to be taken in designing and evaluating targeted approaches using lnps , we found that 2ome modification of the sirna was sufficient to negate immune activation .
our approach enhances the efficacy of sirna - mediated gene silencing by ~10-fold when compared to nontargeted delivery .
therefore , this targeted strategy should prove effective for regulating multiple immune - mediated diseases .
lnps containing sirnas specific either for cd80 , cd86 , cd40 , or a control ( nontarget ) sequence were synthesized by extrusion using a mixture of 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc):dlindma : dspe - peg : cholesterol at a ratio of 15:40:5:40 ( based on refs .
murine anti - dec205 scfv with a c - terminal cysteine was attached to the lipid dspe - peg by a maleimide group ( figure 1a , b ) . following synthesis , we subjected lnps to quality control to ensure consistency between batches .
dynamic light scattering was used to determine diameter and polydispersity of lnps , and cryo - electron microscopy confirmed lnp size and their unilamellar structure ( figure 1c , d ) .
the efficiency of sirna incorporation and scfv binding to dspe - peg was also assessed ( figure 1c , e ) . to show specificity of binding to dec205 ,
scfv - lnps containing dy547-labeled sirna were cultured with either parental ( dec205 ) or dec205 cho cells . as seen in figure 2a , at 4 c dec205 cells bind dec - lnps approximately fourfold better compared with nontargeted lnps ( nt - lnps : lnps not coated with any scfv ) , or lnps coated with an isotype scfv ( iso - lnps ; cho - dec205 panel ) . when similar assays were performed at 37 c to allow cell uptake , cell staining increased by at least fivefold .
little uptake was observed when lnps ( targeted or nontargeted ) were incubated with parental cho cells ( cho panel ) .
similar results were obtained when bone marrow - derived dcs ( bmdcs ) , derived from wild - type mice ( b6 ) were incubated with dec - lnps .
we note that as dec205 is expressed at lower levels on bmdcs compared with cho - dec205 , less dec - lnp binding and uptake was observed in bmdcs ( ~2-fold enhancement compared with nt- or iso - lnps ; b6 bmdc panel ) . to further confirm target - specific uptake we performed experiments using bmdcs derived from dec205 mice , and no specific cell binding or uptake
the dec205 antibody is internalized via receptor - mediated endocytosis , and is targeted to late endosomes and lysosomes .
confocal microscopy confirmed that similar to dec205 antibody , dec - lnps were targeted to lamp-1 late endosomes or lysosomes ( figure 2b ) .
having demonstrated dec205-mediated uptake of dec - lnps in vitro , we investigated their localization in vivo .
b6 mice were injected systemically ( intravenous , i.v . ) with fluorescently labeled scfv - lnps .
as the dec205 receptor is endocytosed when it binds its ligand , we could not use this molecule to identify dec205 dcs .
therefore , we used cd8 , a protein that is coexpressed on dec205 dcs , as a surrogate marker for tracking these cells .
more than 50% of cd11ccd8 cells took up dec - lnps , and uptake was largely restricted to the cd8 dc population , with little uptake observed by cd8 dcs ( figure 3a , b ) .
specificity was demonstrated by comparison of uptake of dec - lnps and iso - lnps .
significant uptake was only observed in dec205 dcs following dec - lnp injection , and other splenic immune cells took up little dec- or iso - lnps ( figure 3a , c ) . when dec - lnps were injected into dec205 mice ,
no uptake was detected ( figure 3d ) . to show that dec - lnps were competent for specific gene silencing , we generated dec - lnps containing a sirna specific for cd80 ( having identified the most effective sirna sequence ; see supplementary figure s1 ) and injected into b6 mice .
cd11cdec205 cells took up similar amounts of lnps following injection with dec - lnp containing either cd80- or control - sirna ( lnp uptake panel , mean fluorescence intensity ( mfi ) : dec - lnp - control = 443 versus dec - lnp - sicd80 = 464 ) . in mice injected with dec - lnp encapsulating cd80-specific sirna , cd80 protein expression approached basal levels , and was reduced by ~2-fold when compared with dec - lnp containing control - sirna ( figure 4a ) .
a 75% reduction in cd80 mrna was observed in dec205 dcs that had taken up dec - lnps containing cd80-specific sirna ( compared with dec - lnps containing control sirnas ; figure 4b ) .
we confirmed that gene knockdown was rnai - mediated using 5 race to detect sirna - directed mrna cleavage products ( figure 4c ) .
sequencing of the polymerase chain reaction ( pcr ) fragment verified that it was derived from cd80 mrna and that the cut site corresponded with nucleotide positions 1011 of the sirna guide strand ( not shown ) .
interestingly , while the data in figure 4 demonstrate the ability of our dec - lnps to effectively knockdown cd80 expression in dcs in vivo , we observed significant dc activation following injection of dec - lnps containing control sirnas ( figure 4a , right panel ) .
this observation was surprising considering these experiments were performed using sirnas containing 2f modified rna , previously reported to have reduced immunostimulatory activity . to better assess this effect , we synthesized a series of luciferase - specific control sirnas ( luc ) containing 2oh rna ( unmodified ) , 2f pyrimidines or selected 2ome modifications ( see methods for modification strategy ; based on refs .
lnps were generated and their ability to stimulate immune responses using multiple assays was examined .
first , b6 mice were injected with dec - lnps that contained either unmodified , 2f or 2ome modified luc - specific sirnas in the absence of any adjuvant .
one day later , splenic dcs were analyzed for the expression of costimulatory receptors ( supplementary figure s2a ) .
as expected , dcs isolated from mice that had received unmodified sirnas demonstrated significant upregulation of cd40 , cd80 , and cd86 .
consistent with our previous results ( figure 4a ) , animals treated with dec - lnps containing 2f modified sirnas also upregulated these costimulatory molecules . importantly , and similar to work by other groups , we found that 2ome modified sirnas induced minimal immune activation . to attempt to circumvent the need for a time and resource intensive in vivo assay , we wanted to determine whether an in vitro assay could be used to detect immunostimulatory sirnas .
however , when we cultured b6-derived bmdcs with dec - lnps ( containing cd80-specific sirnas ) no difference between 2oh modified sirna , 2f or 2ome modified sirna was detected ( supplementary figure s2b ) .
we also used nt - lnps to assess the effects of various sirna formulations on human pbmcs and modcs using standard preclinical assays ( supplementary figure s2c ) .
pbmcs or modcs were cultured for 24 hours at which time cells were assessed for induction of apoptosis and culture supernatants were tested for the presence of proinflammatory cytokines ( see methods ) .
apoptosis was not observed under any conditions tested ( data not shown ) . as expected , pbmcs cultured with the highest concentration of lnps containing unmodified sirnas elicited the secretion of several cytokines .
2f and 2ome luc sirnas induced production of one cytokine ( il8 ) , also at the highest concentration tested .
no cytokine production was observed following culture of modcs with lnps encapsulating unmodified , 2f or 2ome sirnas .
taken together , these experiments suggest that the assays tested show marked differences in sensitivity . surprisingly , bmdcs and modcs were the least receptive for detecting immunostimulatory sirnas .
conversely , dec - lnp injection was useful for the identification of sirnas with immune - activating potential . from these results
, we used sirnas containing selective 2ome substitutions for all subsequent studies . to target autoimmune diseases , reducing the expression of several costimulatory molecules
therefore , we next determined the ability of dec - lnps that encapsulated 2ome modified cd86 specific sirnas , to reduce gene expression following i.v . administration ( optimal sirna identified as previously ; supplementary figure s1 ) .
as the sirnas were 2ome modified , mice were coinjected with lps to stimulate expression of costimulatory molecules .
analysis of splenic dec205 dcs 1 day following dec - lnp injection showed reduction of cd86 protein to near steady - state levels ( figure 5a ) .
total rna was isolated from dec205 dcs that had taken up dec - lnps encapsulating either cd86 or control sirnas .
an approximately 70% reduction in cd86 mrna levels was observed from dcs isolated from mice that received dec - lnps containing cd86 sirna in comparison to dcs derived from control sirna treated mice . to verify knockdown occurred via the rnai pathway ,
we next tested whether combining sirnas in dec - lnps would induce gene silencing equivalent to that observed with single sirnas .
a combination of 2ome modified sirnas targeting cd80 , cd86 and cd40 were incorporated into dec - lnps and injected into b6 mice along with adjuvant ( lps ) to activate the dcs .
reduction of each of the targeted costimulatory molecules was observed , with cd80 and cd86 knocked down to basal levels ( figure 5c ) .
gene silencing was similar to that achieved following treatment with dec - lnps containing single sirnas ( e.g. , ~50% for cd86 : compare cd86 dec - lnp , figure 5a with mix dec - lnp , figure 5c right panel ) . to determine functional relevance of this knockdown , we used the robust mlr assay .
b6 mice were injected with dec - lnps encapsulated with either a mix of cd80 , cd86 , and cd40 or control sirnas .
after 24 hours , splenic cd8 dcs that had taken up dec - lnps were isolated , irradiated , and cultured with splenic t cells derived from a balb / c mouse .
maximal proliferation was observed when dcs were isolated from mice injected with lps only , or lps plus dec - lnps containing control sirnas . in contrast , proliferation of t cells cultured with dcs derived from mice injected with lps plus dec - lnps containing cd80 , cd86 , and cd40 sirnas was significantly reduced ( figure 5d ) .
currently , the most advanced lnp platforms use the ionizable cationic lipid dlindma , or its dlin - kc2-dma and dlin - mc3-dma derivatives .
following systemic administration , these lnps accumulate in first pass organs , i.e. , liver and spleen , making them attractive vehicles for hepatic gene knockdown .
furthermore , these lnps have been shown to safely and effectively reduce expression of hepatocyte genes in clinical trials .
however , there is only very limited data demonstrating targeted delivery of potentially clinically relevant lnps to nonhepatic cells .
we have shown that conjugation of a scfv , specific for the dc receptor dec205 , is sufficient to direct delivery of dlindma - formulated dec - lnps to splenic dcs .
similar to conventional nt - lnps , dec - lnps incorporated sirnas with high efficiency ( > 80% ) , and were of uniform diameter ( ~100 nmol / l ) .
we found that coating lnps with only ~50 scfv was sufficient for dc delivery ( figures 1 , 2 , and 3 ) .
we showed that dec - lnp uptake correlated well with dec205 receptor density : dcs > b cells , t cells and macrophages ( dec205 expression is 1050-fold less than on dcs ) .
use of dec205 mice further confirmed that uptake occurred via the dec205 receptor ( figures 2a and 3d ) .
we also showed that the intracellular pathway accessed by dec - lnps was consistent with the dec205 pathway ( figure 2b ) .
importantly , we demonstrated that targeted delivery resulted in effective rnai - mediated gene silencing of one , or several genes , to essentially basal expression levels ( figures 4 and 5a  c ) .
this reduction in gene expression was sufficient to inhibit a robust mlr ( figure 5d ) .
coated lnps formulated with the cationic lipid ddab with full - length dec205 antibody for sirna delivery and demonstrated the ability to reduce expression of the costimulatory molecule cd40 in dec205 dcs . while some gene knockdown was achieved , this lipid is known for its adjuvant qualities , and is being developed for applications that require immune response stimulation .
the lnps also proved relatively inefficient at sirna loading ( 10% compared with 85% for dlindma lnps ) .
the sirnas used were also unmodified , which could serve as another possible source of immune stimulation . therefore , the potential for this formulation appears to be limited .
more recently , ramishetti et al . utilized a full - length antibody targeting cd4 to enhance t - cell uptake and induce sirna - mediated gene silencing using a clinically relevant lnp formulation similar to the one we employed in our work .
interestingly , both this study and the work from zheng et al . chose to utilize full - length antibodies for lnp targeting , which may limit their utility when considering the immunogenicity of whole antibodies and their rapid clearance from the circulation by fc - mediated uptake by macrophages .
in fact , in preliminary studies , we coated lnps with full - length dec205 antibody and failed to observe uptake by dec205 dcs . using our dec - lnps
mg / kg ) comparable to that study which used n - acetylgalactosamine ( galnac)-coated lnps to target hepatocytes via the asialoglycoprotein receptor ( asgpr ) in apoe knockout mice .
in contrast , when the ability of nt - lnps to silence antigen presenting cells ( apcs ) , including dcs , was investigated a dose of 5  mg / kg was required to achieve partial silencing : ~10-fold higher when compared with our dec - lnps .
furthermore , the authors used the dlindma derivative , dlin - kc2-dma , which displays improved silencing ability in hepatocytes ( relative ed50 dlin - kc2-dma : 0.1  mg / kg versus dlindma : 1  mg / kg ) . as these lnps were formulated for effective gene silencing in hepatocytes ,
biodistribution studies show that nt - lnps are found in the spleen , although at ~50-fold less than in the liver . while targeting ligands
have been shown to have little effect on the overall biodistribution of nanoparticle formulations , they can enhance cell - specific uptake . therefore , while high doses of nt - lnps can confer partial gene silencing in apcs , at low lnp concentrations , we find that a targeted approach is required to achieve knockdown in a specific dc subset
. injection of low - dose nt - lnps failed to result in detectable uptake / gene silencing .
as the rnai pathway is resident in the cytoplasm , the intracellular pathway used following receptor ligation is an important consideration .
we chose the well - characterized dec205 receptor for targeting our lnps . following binding ,
the internalized receptor is routed to late endosomes and associated cargo gains access to the cytoplasm .
the dlindma lipid fuses with anionic phospholipids present in the endosomal membrane resulting in release of encapsulated cargo into the cytoplasm .
however , it is likely that different routes of uptake will affect the efficiency of delivery and subsequent gene silencing . thus , a logical extension to our approach is to determine whether we observe improved gene silencing using other dc specific targeting agents and/or more effective dlindma derivatives .
we are currently investigating the efficacy of dec - lnps formulated with the dlin - mc3-dma ( ed50 0.03  mg / kg ) .
having demonstrated that we could efficiently target sirnas to dcs , the lack of toxicity of these lnps and their cargo had to be established .
we observed significant dc activation following injection of targeted lnps containing unmodified and 2f modified sirnas , and incorporation of 2ome modifications at distinct residues was required to maintain costimulatory molecules analyzed at basal levels ( figure 4a and supplementary figure s2a ) .
as injecting mice to determine immune stimulation is time and resource intensive , we assessed immune stimulation in murine and human tissue culture systems .
murine bmdcs were refractory to lnp stimulation suggesting they would not serve as a useful surrogate for gauging immunogenicity of lnps .
taken together , these assays demonstrate a requirement for in vivo analysis to uncover the stimulatory capacity of the lnps .
recently , a human whole blood cytokine assay has been described that showed similar activation profiles elicited by lnps when compared with in vivo injection , potentially circumventing a requirement for cumbersome in vivo analyses . for the purposes of inhibiting a multifactorial immune response , such as that observed in graft rejection or autoimmunity , the simultaneous knockdown of several ( co)stimulatory genes
this approach has been used to demonstrate that combining antibodies targeting cd80 and cd86 can reduce disease severity in experimental autoimmune encephalomyelitis and in antigen - induced arthritis .
transfer of dcs treated ex vivo with cd40 , cd80 , and cd86 antisense oligonucleotides ( as - odn ) into nod mice delayed type 1 diabetes ( t1d ) onset .
a follow - up study demonstrated that multiple injections of microspheres containing cd40 , cd80 , and cd86 as - odns were sufficient to prevent t1d , possibly due to reduced expression of the costimulatory molecules by splenic dcs .
we achieve significant reduction in expression of cd40 , cd80 , and cd86 on splenic dcs using our dec - lnps following systemic delivery , and ongoing studies are addressing the durability of gene knockdown and the potential use of these dec - lnps for treating autoimmune diseases . in summary
, we have shown that a clinically relevant lnp can be modified for cell - specific delivery of sirnas . in vivo administration
did not produce overt inflammatory responses , and we achieved significant rnai - mediated gene - specific knockdown at low sirna doses .
these attributes make this an attractive platform to warrant further investigation as a potential therapeutic agent for various autoimmune diseases .
furthermore , judicious selection of the ligand coating the lnp opens up the potential for targeting multiple cell types .
mice . c57bl/6 ( b6 ) and balb / c mice were purchased from taconic ( hudson , ny ) .
dec205 mice were bred to homozygosity on the b6 background ( dec205 ) , and validated by pcr and flow cytometry .
mice were housed in the barrier facility at the institute for animal studies , albert einstein college of medicine ( bronx , ny ) according to institutional animal care and use committee ( iacuc ) guidelines .
the single - chain fragment variable ( scfv ) antibodies constructed for dec205 ( clone nldc145 ) and isotype ( clone gl117 ) were cloned from antibody constructs , as previously described .
the variable regions of the heavy and light chain sequences were fused with a flexible linker ( g4s)6 separating them and cloned into a pet28a ( emd millipore , billerica , ma ) bacterial expression vector .
further modifications to the constructs included a strep - tag - ii ( wshpqfek ) at the n - terminus and a 10-residue histidine tag ( h10 ) with a short flexible linker ( g4s ) near the c - terminus .
a c - terminal unpaired cysteine residue was introduced for scfv conjugation to the lipid dspe - peg - mal .
the scfv constructs were transformed into bl21 ( de3 ) plyss competent cells ( promega , fitchburg , wi ) .
the scfv dec205 or isotype were expressed in escherichia coli strain bl21 ( de3 ) plyss as insoluble inclusion bodies . following bacterial growth in luria broth ( lb ) medium and induction of protein expression with isopropyl 1-thio - d - galactopyranoside
cells were lysed , and insoluble protein pelleted by centrifugation . solubilized inclusion bodies were passed over a ni - nta column and the bound protein was eluted with 8  m guanidine hydrochloride .
the purified proteins were refolded by rapid dilution in refolding buffer , and purified by size - exclusion chromatography using superdex 200 ( ge healthcare life sciences , pittsburgh , pa ) .
proteins were buffer - exchanged in phosphate - buffered saline ( pbs ) and concentrated .
scfvs were tested for endotoxin contamination using a kinetic chromogenic limulous amoebocyte lysate ( lal ) assay ( kinetic - qcl lal assay , lonza , walkersville , md ) .
the free cysteine engineered onto the n - terminus of the scfv was reduced using tris-(2-carboxyethyl ) phosphine hydrochloride ( life technologies , carlsbad , ca ) . following buffer exchange into pbs
, the reduced scfv was reacted with a 10-fold molar excess of alexafluor488 ( af488)-c5-maleimide according to manufacturer instructions ( life technologies , carlsbad , ca ) .
polyacrylamide gel electrophoresis ( sds - page ) and coomassie staining and storm molecular phosphorimager scanning of the stained gel ( ge healthcare , piscataway , nj ) . preparation of lnps .
lnps were formed using the following lipids at a 15:40:5:40  mol % : dspc ( avanti polar lipids , alabaster , al ) , cholesterol ( chol ; sigma - aldrich , mo ) , 1,2-distearoyl - sn - glycero-3-phosphatidylethanolamine - n-(maleimide-(polyethylene glycol)-2000 ) ( dspe - peg - mal , avanti polar lipids , alabaster , al ) , and 1,2-dilinoleyloxy-3-dimethylaminopropane ( dlindma ) synthesized by the chemical biology core facility at albert einstein college of medicine as previously described .
a fluorophore conjugated to a lipid , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl ) ( dope - rhodamine b ; avanti polar lipids , alabaster , al ) was incorporated ( 0.1 % ) to monitor lnp delivery .
lnps were formed using a modified version of the spontaneous vesicle formation by ethanol dilution method .
the lipid mixture was extruded ( mini - extruder and 1  ml gas - tight syringes , avanti polar lipids , alabaster , al ) sequentially with polycarbonate 200 and 80  nm pore size membranes .
the extruded lipid mixture was mixed via rapid pipetting and vortexing with sirna , followed by dialysis into pbs .
for attachment of scfv , lnps were mixed with reduced scfv at room temperature for 1 hour , and free maleimide groups were then quenched with -mercaptoethanol ( me ) .
tangential flow filtration ( microkros filter module ) with a 500  kda mwco ( spectrum laboratories , rancho dominguez , ca ) was used for buffer exchange into pbs and concentration of lnps .
lnps were assayed for size and polydispersity by dynamic light scattering ( dynapro plate reader , wyatt technology , santa barbara , ca ) .
cryo - electron microscopy confirmed lnp size , geometry and lamellarity ( analytical imaging facility ; albert einstein college of medicine , bronx , ny ) .
sirna incorporation was determined by quant - it ribogreen rna assay ( life technologies , carlsbad , ca ) . to determine scfv conjugation efficiency , lnps were resolved on sds - page gels .
a 3  kda difference in migration of the scfv was indicative of binding to dspe - peg - mal ( unconjugated scfv ~30  kda , scfv - dspe - peg - mal ~33  kda ) .
a cholesterol quantification kit ( abcam , cambridge , ma ) was use to determine the total lipid concentration . based on lipid concentration , the surface area per lipid head group ( ~0.6  nm ) , lnp diameter and their unilamellar structure , ~5060 scfvs are conjugated to each lnp under the conditions outlined above .
sirna synthesis . modified and unmodified individual sirna sequences were synthesized on an expedite 8909 dna synthesizer ( biolytic , fremont , ca ) and purified by standard protocols , as described .
dylight 547-phosphoramidite was attached to the 5 end of the sense strand for synthesis of fluorescently labeled sirna ( dy547-sirna ; glen research , sterling , va ) .
sirnas were modified by 2f substitutions at every pyrimidine or by 2ome substitutions at the bolded nucleotides : cd40 sense 5-gaagauuaucccggucauauu-3 ; cd40 anti - sense 5-uaugaccg ; ggauaaucuucuu-3 ; cd80 sense 5-gaauuaccuggcaucaauauu-3 ; cd80 anti - sense 5-uauugaugccagguaauucuu-3 ; cd86 sense 5-caacuggacucuacg acuuuu-3 ; cd86 anti - sense 5-aagucguagaguccaguuguu-3 ; control sense 5-uagcgacuaaacacaucaauu-3 ; control anti - sense 5-uugauguguuuaguc gcuauu-3. luciferase sense 5-gauuauguccgguuauguauu-3 ; luciferase anti - sense 5-uacauaaccggacauaaucuu-3. lnp binding , uptake , and intracellular localization .
cho cells , cho cells that stably express dec205 ( cho - dec ) , a dec205 b lymphoma line ( a20 cells ) and bmdcs , derived from b6 and dec205 mice , were used to assess lnp binding and uptake .
cells were grown in complete medium ( dulbecco 's modified eagle 's medium , 5% fbs , 10 mmol / l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 20 mmol / l l - glutamine ) .
bmdcs were generated from bone marrow derived from the femur and tibia as previously described .
adherent cells ( a20 , cho ) were harvested using ethylenediaminetetraacetic acid to preserve receptor expression and were incubated at 4 c , 30 minutes with 500 nmol / l scfv - lnps ( containing either 0.1% dope - rhodamine b or cy3-labeled sirnas ) . for uptake ,
cells were washed with fluorescence - activated cell sorting buffer ( pbs containing 0.5% bsa , 0.1% na n3 , 2 mmol / l edta ) and analyzed by flow cytometry ( lsrii , becton dickinson , franklin lakes , nj ; flowjo , ashland , or ) . to monitor intracellular localization , a20 cells were incubated with scfv - lnps , containing sirnas labeled with dylight 547 ( ge dharmacon , lafayette , co ) , 1 hour , 37  c .
following one wash with pbs , cells were pipetted onto poly - l - lysine coated coverslips and allowed to adhere .
adhered cells were fixed with 4% paraformaldehyde , washed with pbs and stained with anti - lamp1 ( clone 1d4b ; ebioscience , san diego , ca ) and anti - eea1 ( clone c45b10 ; cell signaling technology , danvers , ma ) .
stained cells were mounted with vectashield mounting media containing 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories , burlingame , ca ) and samples were visualized on a leica sp5 aobs confocal microscope ( leica , buffalo grove , il ) .
image processing ( linear adjustments to brightness and contrast ) was performed on image files using nih imagej software ( bethesda , md ) .
lnps ( 0.61  mg / kg ) were injected ( retroorbital ( r.o . ) ) into b6 mice and 25  ng ultrapure lps ( invivogen , san diego , ca ) was injected r.o .
one day later , spleens were harvested , collagenase treated to maximize dc yield , and single cell suspensions subjected to red cells lysis .
cells were stained with cell surface markers and analyzed by flow cytometry to determine lnp uptake and expression of costimulatory molecules .
antibodies were used for detection of the following cell surface markers : cd11c , cd8 , dec205 , cd40 , cd80 , cd86 , cd19 , b220 , cd3 , tcr , f4/80 , cd11b , h2k , dcir2 , cd49b , cd45.1 .
cell viability was monitored using a live / dead exclusion dye , i.e. , live / dead blue ( life technologies , carlsbad , ca ) .
cells were acquired using an lsrii cytometer and data analyzed using flowjo software . to measure gene knockdown , cd11ccd8 splenocytes that had taken up lnps ( i.e. , sirna - dy547 ) were sorted by flow cytometry ( facs aria iii , becton dickinson , franklin lakes , nj ) into rnaprotect ( qiagen , valencia , ca ) to stabilize rna .
total rna was isolated using the rneasy rna isolation kit ( qiagen , valencia , ca ) , reverse transcribed using superscript iii ( life technologies , carlsbad , ca ) and random hexamers ( idt , coralville , ia ) , according to manufacturer 's instructions .
quantitative pcr was performed using platinum taq polymerase ( life technologies , carlsbad , ca ) and sybr green to detect amplified products on an iq5 icycler ( biorad , hercules , ca ) .
reactions were performed in triplicate with the following primers : u6 forward : 5-ctcgcttcggcagcacatatacta-3 ; u6 reverse : 5-acgaatttgcgtgtcatccttgcg-3 ; cd40 forward : 5-cggctgtgcgcgctatg-3 ; cd40 reverse : 5-ggctgagaattcgcctgagtc-3 ; cd80 forward : 5-ctactctcttatcatcctgggcct-3 ; cd80 reverse : 5-cccggaagcaaagcaggtaatcct-3 ; cd86 forward : 5-gccacccacaggatcaattatcct-3 ; cd86 reverse : 5-aaagagagaggctgttggagatac-3. relative amounts of mrna were normalized to u6 mrna and primer specificity verified by melt curve analysis . to detect rnai - mediated cleavage products ,
total rna was isolated from cd11ccd8 splenocytes that had taken up lnps , i.e. , sirna - dy547 ( as for gene knockdown by qpcr ) , ligated at the 5 end to an adapter rna oligonucleotide and first strand cdna was synthesized using a gene - specific primer ( gsp ) .
pcr amplification using an adapter - specific primer and a nested gsp primer will amplify the cleavage product and identify the cut site which corresponds to position 10/11 of the sirna guide strand if mediated via rnai .
the primers and oligonucleotide sequences used : adapter rna : 5-cgacuggagcacgaggacacugacauggacugaaggaguagaaa-3 ; gsp forward : 5-cgactggagcacgaggacactga-3 ; cd80 gsp reverse : 5-tgcgccgaatcctgccccaa-3 ; cd80 reverse nested : 5-atcaggagggtcttctgggg-3 ; cd86 gsp reverse : 5-agtaactgaagctgtaatctccttccaata-3 ; cd86 reverse nested : 5-ctgtgacattatcttgtgatatctgcatgt-3. analysis of immune stimulation .
cd11c murine bmdcs ( day 6 ; macs purified , miltenyi biotec , san diego , ca ) were cultured with dec - lnps and monitored for activation ( cd40 , cd80 , cd86 ) 1 day later by flow cytometry . in vivo stimulation
was assessed by injection of dec - lnps r.o . into b6 mice , followed by analysis of activation markers ( cd40 , cd80 , cd86 ) on splenic dcs after 24 hours by flow cytometry .
the pbmc fraction was used directly or following purification of cd14 cells ( stemcell technologies , vancouver , british columbia , canada ) , monocytes were cultured for 5 days in the presence of granulocyte - macrophage colony - stimulating factor and il4 to generate modcs .
nt - lnps ( 5 , 1 , 0.2 , 0.04 , 0.008 mol / l ) were cultured with pbmcs or modcs overnight and cell culture supernatants collected for cytokine analysis ( ifn , il1 , il2 , il4 , il5 , il6 , il7 , il8 , il10 , il12 , il13 , tnf , granulocyte - macrophage colony - stimulating factor ) using a luminex multiplex format ( life technologies carlsbad , ca ) .
cd11ccd8 splenic dcs were isolated by flow cytometry from b6 mice injected the previous day with dec - lnps containing either a 1:1:1 mix of cd40 , cd80 , and cd86 sirnas or control sirnas .
splenic t cells from balb / c mice were isolated by macs using a pan t cell isolation kit ii ( miltenyi , san diego , ca ) .
macs sorted balb / c t cells were carboxyfluorescein succinimidyl ester labeled according to published protocols , and carboxyfluorescein succinimidyl ester - labeled t cells were cocultured with the irradiated b6 dcs .
three days later , dilution of carboxyfluorescein succinimidyl ester was monitored by flow cytometry to determine t - cell activation .
 due to their ability to knock down the expression of any gene , sirnas have been heralded as ideal candidates for treating a wide variety of diseases , including those involving  
 undruggable  targets . however , the therapeutic potential of sirnas remains severely limited by a lack of effective delivery vehicles . 
 recently , lipid nanoparticles ( lnps ) containing ionizable cationic lipids have been developed for hepatic sirna delivery . 
 however , their suitability for delivery to other cell types has not been determined . 
 we have modified lnps for preferential targeting to dendritic cells ( dcs ) , central regulators of immune responses . to achieve directed delivery , we coated lnps with a single - chain antibody ( scfv ; dec - lnps ) , specific to murine dec205 , which is highly expressed on distinct dc subsets . 
 here we show that injection of sirnas encapsulated in dec - lnps are preferentially delivered to dec205 + dcs . 
 gene knockdown following uptake of dec - lnps containing sirnas specific for the costimulatory molecules cd40 , cd80 , and cd86 dramatically decreases gene expression levels . 
 we demonstrate the functionality of this knockdown with a mixed lymphocyte response ( mlr ) . 
 overall , we report that injection of lnps modified to restrict their uptake to a distinct cell population can confer profound gene knockdown , sufficient to inhibit powerful immune responses like the mlr . 
Introduction
Results
Discussion
Materials and Methods
Supplementary Material