Significant work has been dedicated toward the development of new materials and formulations for the delivery of siRNA to hepatocytes (see e.g. refs. 4
). We sought to develop nanoparticles with delivery potential to myeloid cells through the use of lipids and lipid-like materials known as KC2 (an ionizable lipid)4
and C12-200 (a cationic lipid).5
Formulations containing these lipid materials, as well as the excipients: cholesterol, PEG-DMG, and distearoyl phosphatidyl choline were formulated as nanoparticles, encapsulating siRNAs directed to several myeloid-expressed gene targets (including CD45, CD11b, integrin β1, TNFα). The KC2 containing formulations utilized here use a higher molar percent of cationic lipid compared to earlier work,4
further increasing formulation potency. We also optimized the C12-200 containing liposomes increasing the lipid to siRNA ratio in the particle.
Quantitative in vivo whole body imaging and histological localization of LNP-siRNA distribution.
In order to determine LNP-siRNA distribution, we intravenously (i.v.) injected LNP-encapsulated, fluorescently labeled siRNA, and followed whole body fluorescence by fluorescence-mediated tomography/X-ray computed tomography (FMT-CT). FMT-CT is a hybrid imaging approach that quantitates fluorochrome concentration in tissue, while fusion with CT data provides the anatomic localization of the fluorescent signal at high resolution.23
Concentration was sampled 90 minutes after injection of siRNA formulated in KC2 LNP and C12-200 LNP (
), and key organs were then analyzed. Fluorescent signal reporting on siRNA concentration was attributed to anatomical structures using hybrid CT data. Interestingly, we found that the spleen is a major distribution site for both LNP preparations, with high fluorescence per gram of tissue (
). Imaging also provided information about the excretion pathway of LNP siRNA. The signal peaked in the liver, gall bladder and intestine likely reflecting the excretion of the fluorochrome and attached materials. Low signal was observed in the kidneys and the urinary tract (Figure 1
and data not shown). Low siRNA concentration was observed in the lung for both nanoparticles. Ex vivo
fluorescence reflectance imaging corroborated these findings with the spleen showing the brightest signal among major organs (
). Distribution data obtained by imaging are consistent with plasma/tissue siRNA amount derived from a PCR-based method of siRNA quantification,24
which gives us confidence that we are not merely tracking fluorochrome, but fluorescently labeled siRNA. Leuschner et al.
have shown that multiple myeloid cell types in the spleen take up C12-200 LNP-formulated siRNA including macrophages, splenic reservoir monocytes, and dendritic cells.22
Similar analysis carried out for the KC2 formulation did not reveal major difference in the cell populations targeted (data not shown).
Figure 1 In vivo dynamic fluorescence-mediated tomography/X-ray computed tomography (FMT-CT) of lipid nanoparticles (LNP)-small interfering RNA (siRNA) delivery and siRNA localization within reservoir monocyte clusters in the spleen. (a) 3D reconstruction of FMT-CT (more ...) Efficient silencing of gene targets in myeloid cells after i.v. injection.
Encouraged by the distribution of LNP siRNA to sites of immune cell localization, we initiated experiments to determine whether distribution translates into RNAi silencing activity in leukocytes. Macrophage lineage cells specialize in the removal of foreign material, thus most systemically administered particles are taken up by these cells. In fact, cells of the monocyte/macrophage lineage showed the highest fluorescence signal after i.v. injection of fluorescently labeled siRNA (see below). However, uptake often does not translate into siRNA-induced silencing; macrophages specialize in shuttling cargo into lysosomes for degradation, whereas siRNA needs to reach the cytoplasm to induce cleavage of the target mRNA. Indeed, with earlier-generation LNP formulations,6,7
despite good uptake we have not observed siRNA-mediated gene silencing in macrophages after i.v. administration.
To determine which leukocyte populations are subject to silencing in response to i.v. injected LNP-siRNA, we used a CD45 silencing assay. CD45 is a common, highly expressed leukocyte antigen that occupies up to 10% of the cell surface.25
We assayed CD45 protein knockdown by flow cytometric analysis of leukocytes defined by combinations of specific cell surface markers and then measured the decrease of CD45 expression in each of these cell populations (
). We compared leukocytes from animals injected with a formulation containing a CD45-specific siRNA versus animals injected with an identical formulation containing a control siRNA targeting luciferase. We surveyed leukocytes isolated from different organs (spleen, liver, peritoneal cavity, bone marrow, and lymph nodes) 3 days after a single i.v. bolus injection (
and Supplementary Figure S1b
). A 3-day interval between injection and protein level analysis was needed to allow the reduction in mRNA levels to be translated into decreased CD45 protein expression. Using this assay, we found highly effective (~80%) silencing in cells of macrophage lineage (
), good (~40%) silencing in dendritic cells (Supplementary Figure S1b
), some activity (~15%) in B cells (Supplementary Figure S1c
, and no silencing in T cells, NK cells, or GR-1+
granulocytes (Supplementary Figure S1c
). Silencing in all cell types was examined in multiple tissues including spleen, liver, bone marrow, blood, and peritoneal cavity. These findings imply broad applicability of this technology to diseases that involve innate immune activation, infection, and antigen presentation.
Figure 2 Efficacious and durable silencing in peritoneal cavity macrophages. (a) Cells were collected at 72 hours after a single intravenous (i.v.) bolus administration of the indicated doses of small interfering RNA (siRNA) in KC2 or C12-200 lipid nanoparticles (more ...)
To address whether ingestion of LNPs might activate leukocytes, we compared mice injected with PBS versus siRNA in LNP. Notably, we used only chemically modified siRNA molecules (2′OMe modified at selected pyrimidine sites) which do not induce cytokine production in the human PBMC assay.18
We investigated leukocyte numbers as well as their activation status in blood, spleen, bone marrow, and peritoneal cavity following an i.v. administration of LNP siRNA. Interestingly, we did not find evidence for overt activation in any of the populations surveyed; there was no increase in costimulatory molecules CD80 and CD86 or in MHCII expression (Supplementary Figure S2
). After injection of Luc LNP-siRNA, we observed a minor influx of CD11b−
low cells in the peritoneal cavity and slight CD45 upregulation on CD11b+
cells (Supplementary Figure S2
To corroborate the CD45 silencing data, we conducted silencing experiments with other gene targets, including green fluorescent protein (GFP), CD11b, integrin β1, and TNFα, where we observed similar or greater silencing efficiencies (Supplementary Figure S3 and data not shown). For instance, silencing of integrin β1 in CD11b+ cells from the bone marrow was more pronounced than that of CD45 in the same cells (Supplementary Figure S3). These observations may reflect both intrinsic differences in the potencies of the specific siRNA (half-maximal concentration required for in vitro silencing of 10 pM for integrin β1 versus 90 pM for CD45) and different characteristics in the target mRNA transcripts (e.g., mRNA half-life).
Interestingly, the anatomical location showing the strongest silencing of CD45 after i.v. injection in mice was the peritoneal cavity with up to 90% reduction in CD45 protein expression, followed by significant silencing in the spleen, and only moderate silencing seen in the bone marrow, lymph nodes, and liver ( and Supplementary Figure S1, data not shown), using shift in mean fluorescent intensity of the total population as the metric. It is noteworthy that within a population with average silencing even at 20–30%, there are subpopulations of cells with silencing as high as >95% at the lower end of distribution. Given the extreme stability and abundance of CD45, it is likely a gene target that is more refractive to silencing.
Next, in dose response experiments, we assessed the potency of in vivo
silencing in peritoneal macrophages and found that C12-200 LNP induced 50% CD45 silencing at doses of ~0.2
mg/kg, and KC2 LNP at doses of ~0.5
). This is a significant improvement compared to similarly formulated LNPs using early generation cationic lipids, where no silencing in leukocytes was seen after i.v. injection of at least tenfold higher doses.7
Even compared to LNPs using the KC2 lipid the formulation used here shows a sixfold improvement in potency with 50% silencing at 0.5
mg/kg versus 3
in peritoneal macrophages. Knowing that peritoneal cells are sessile, we could monitor longevity of silencing in vivo
using cells transferred intraperitoneally from the peritoneum of GFP transgenic mice. To this end, we injected cohorts of animals with CD45 or control LNP-siRNA and sacrificed them at different time points. We observed silencing in peritoneal macrophages for up to 3 weeks after a single injection (
), which is near identical to the duration of silencing observed in hepatocytes.4,5
We observe a longer silencing duration than that seen in peritoneal macrophages put in culture following liposome treatment.21
This may be a reflection of the differences in the assay systems and the gene targets being assayed (CD45 versus GFP).
With such low doses, it is possible to combine several gene targets for silencing in leukocytes, thus, enabling functional genomics studies in the cells central to inflammatory disorders. To validate this utility, we performed an experiment that mixed siRNA to four independent gene targets formulated in C12-200 LNP, each at the 0.2
mg/kg dose, an estimated IC50
dose for the CD45 siRNA. We included siRNA targeting CD45, CD11b, RAB5c, and integrin β1 into this cocktail. Silencing was monitored in total peritoneal cavity cells 24 hours post injection on the mRNA level by reverse transcription-quantitative PCR. We observed silencing ranging from 50 to 80% for each of the targets as compared to the levels seen in animals dosed with control siRNA (
Macrophage uptake of LNP-siRNA relies on phagocytosis.
Since both KC2 and C12-200 represent potent LNP formulations for myeloid cell silencing in vivo
, we chose to investigate the mechanism of cellular uptake for these two formulations. We performed uptake experiments with primary mouse bone marrow-derived macrophages that were treated with fluorescently labeled siRNA in KC2 or C12-200 LNP. C12-200, relative to KC2 LNP, mediated more efficient uptake of the labeled siRNA by primary macrophages in vitro
consistent with mRNA-silencing results (
). To characterize the mechanism of cellular uptake, we co-exposed primary macrophages to markers of different endocytic pathways. We saw ~60–70% colocalization of labeled siRNA particles with fluorescent latex beads that due to their large 1
µm size enter these cells by phagocytosis (
We saw substantially less colocalization with markers of other pathways: dextran, a marker of macropinocytosis (~40%), and transferrin, a marker of clathrin-mediated endocytosis (10%) (Supplementary Figure S4a–c
). Latex beads and siRNA-containing vesicles colocalized most prominently in the perinuclear region; these likely represent vesicles that have already undergone lysosome fusion (
). Since phagosomes are known to contain the EEA1 marker27
the compartments in which siRNA signal is observed likely correspond to the EEA1 positive structures reported by Basha et al
In addition, LNP-siRNA uptake was inhibited by Cytochalasian D and by Dynasore (inhibitors of actin rearrangement and dynamin, respectively, both previously shown to inhibit different steps of phagocytosis (Supplementary Figure S4d,e
)). We therefore concluded that the primary mechanism of LNP siRNA internalization in macrophages was phagocytosis.
Figure 3 Uptake of KC2 and C12-200 formulations in primary macrophages. (a) Quantitation of cellular uptake of Alexa Fluor-647 labeled small interfering RNA (siRNA) (AF647-si) in KC2 and C12-200 formulations in primary macrophages in vitro. RFU, relative fluorescence (more ...) Silencing of myeloid genes in vivo occurs in both tissue-resident and splenic reservoir cells of monocyte/macrophage lineage.
A key question for therapeutic gene silencing in leukocytes is whether delivery can be achieved to circulating and splenic monocyte/macrophages, including splenic reservoir monocytes that migrate in high numbers to inflammatory sites such as acute myocardial infarcts.28,29,30
One technical challenge in assessing in vivo
gene silencing in leukocytes is their migratory nature. The site of initial LNP-siRNA uptake and the ultimate cell destination when protein downregulation is detectable may not coincide. To address this issue, we injected mice i.v. with LNP-siRNA and isolated monocytes/macrophages from bone marrow, blood, spleen, and peritoneal cavity at 15, 60, and 120 minutes post injection. The cells were then seeded onto plastic to allow time for downregulation of CD45 protein expression. This strategy interrupted the migratory path of the cells, and determined the site of effective uptake independent of subsequent cell relocation. Interestingly, we did not observe any silencing in the bone marrow in this assay, whereas blood monocytes reached maximum silencing at 15 minutes, splenic cells at 1 hour, and peritoneal macrophages at 2 hours after injection with KC2 LNP-formulated CD45 siRNA (
). The maximal levels of CD45 silencing seen in this in vivo/ in vitro
assay (>50%) were comparable to the levels reached 3 days post injection in vivo
). Silencing kinetics were faster following injection of C12-200 LNP-formulated siRNA, with maximal blood silencing as early as 5 minutes post injection (data not shown). These data indicate that LNP siRNA effectively reached the central pool of circulating and splenic monocytes, as well as resident tissue macrophages. Due to the fast migratory kinetics of myeloid cells, many circulating and splenic monocytes relocate to target tissue after ingesting LNP siRNA. Some of these transfected cells appear to migrate to the peritoneal cavity.
Figure 4 Fast and efficient silencing in central monocytes/macrophages coupled with delayed but durable silencing in resident peritoneal cavity myeloid cells. (a) Cells were collected at 15 minutes, 1 hour, or 2 hours after a single intravenous (i.v.) bolus administration (more ...)
The efficient silencing seen in mouse peritoneal macrophages 3 days after i.v. injection was surprising, and may be (i) a functional consequence of gene silencing, (ii) due to migration of the cells that had been targeted while circulating in blood or residing in the spleen, and/or (iii) a result of local accumulation of LNP siRNA in the peritoneal cavity and uptake by resident macrophages. Notably, the kinetics of LNP accumulation in peritoneal cavity macrophages was significantly slower than that observed in blood or spleen (). Using GFP-expressing transgenic mice, we found that equally efficient silencing of GFP can also be measured in the peritoneal cavity macrophages, therefore excluding a role for endogenous gene knockdown in localization of these cells to the peritoneal cavity (Supplementary Figure S3a).
To determine whether LNP internalization first occurred in circulation or locally in the peritoneal cavity, we transferred resident GFP+
peritoneal cells into the peritoneum of wild-type mice and injected recipient mice i.v. with LNP-siRNA targeting CD45 30 minutes after cell transfer. CD45 in both, GFP+
and well as GFP−
cells, was silenced to a nearly identical degree in vivo
). This experiment established that gene targets in sessile resident peritoneal cells31
are also silenced by i.v. injected LNP-siRNA.
Silencing mRNA specific for the monocyte/macrophage lineage.
While the CD45 assay allowed us to follow many leukocyte cell types in parallel, it necessitated isolation of leukocytes from different tissues to identify cell types by surface staining. Previous work demonstrated that some tissue resident leukocytes cannot be isolated,32
thereby making it impossible to assess the degree of silencing in such cells by flow cytometry. We therefore decided to focus on all cells of the monocyte/macrophage lineage by devising siRNA against CD11b (Mac-1) and thus enabling total tissue mRNA analysis of silencing in the cells of interest. This siRNA was very active in vitro
with an IC50
of about 4 pM. We identified a 24-hour time point when mRNA for CD11b was significantly silenced, but cell surface protein was still unaffected, to avoid potential effects specific to CD11b function. We performed in vivo
titration of LNP encapsulated CD11b siRNA and found it required ~0.3
mg/kg siRNA in C12-200 and ~1
mg/kg siRNA in KC2 LNP to achieve 50% silencing at the mRNA level in the peritoneal cavity cells (
). These in vivo
potencies were very similar to those found for the CD45 protein and they demonstrate the robustness and reproducibility of silencing across several targets, using mRNA and protein as readouts.
Figure 5 Macrophage-specific CD11b silencing. mRNA ratio of CD11b to GAPDH was measured in peritoneal cavity or total perfused liver lobes 24 hours after bolus intravenous (i.v.) injection of small interfering RNA (siRNA) in lipid nanoparticles (LNP). Bars represent (more ...)
We proceeded to test CD11b silencing in different organs, measuring the ratio of CD11b mRNA level to GAPDH. In spleen and peritoneal cavity, CD11b mRNA was reduced to similar levels when compared with CD45 silencing (
and Supplementary Figure S1
), namely, ~30% in the spleen and 70–90% in peritoneal cavity. Interestingly, we also found efficient CD11b knockdown in the liver (
). We believe that the near absence of CD45 silencing in the macrophages isolated from liver reflects the fact that a true resident population of Kupffer cells is not isolated by the liver digestion protocols employed and was therefore not accessible to flow cytometric analysis.32
CD11b knockdown was also normalized to another macrophage-specific marker, F4/80, and was shown to be very similar to the values obtained by normalization to GAPDH (data not shown): This indicates that observed CD11b knockdown is not due to the loss of macrophages.
CD45 and CD11b silencing is RNAi mediated.
To confirm that the knockdown of myeloid gene mRNA observed in rodents was mediated by an RNAi silencing mechanism, we isolated CD11b+
cells from mice treated with either CD45 or CD11b LNP-siRNA. mRNA from these cells was then subjected to rapid amplification of cDNA ends (5′-RACE), a method previously used to demonstrate siRNA-mediated cleavage.6,33
5′-RACE analysis of peritoneal cavity macrophage-derived mRNA from animals treated with LNP-siRNA revealed products of the expected sizes for both CD45 and CD11b amplicons in their respective cohorts (Supplementary Figure S5
). Sequence analysis of cloned PCR products demonstrated that 46 out of 48 and 24 out of 24 PCR products were derived from the predicted cleavage event at position (CTGGCTGAA/TTTCAGAGCA) for CD45 siRNA in KC2 and C12-200 LNP, respectively; for CD11b, 29 out of 48 and 20 out of 24 PCR products were cleaved at position (TTGTCTCAA/CTGTGATGGA) in KC2 and C12-200 LNP, correspondingly. No specific cleavage site PCR products were derived from the 5′-RACE samples treated with LNP-encapsulated control siRNA (out of 140 sequenced products) except for one likely contaminant clone with CD45 cleavage product derived from CD11b siRNA in C12-200 LNP treated cells. These results clearly demonstrate that the effect of siRNA in LNP treatment on CD45 and CD11b expression levels observed is due to cleavage of the mRNA transcript via an RNAi mechanism.
siRNA-mediated silencing in immune cells substantially inhibits disease progression in a mouse model of RA.
To determine whether the effective silencing in macrophages translated into disease modifying activity, we tested TNFα-specific siRNA in an antibody-induced arthritis mouse model in which systemic inhibition of soluble TNFα has been previously shown by several groups to be highly effective.34
Using siRNA targeting TNFα, we found inhibition of paw swelling in two independent experiments with C12-200 LNP (
). In fact, the anti-inflammatory activity was comparable to anti-VLA1 i.v. antibody treatment (
), which has been previously demonstrated to be at least as effective as systemic TNFα inhibition.35
In agreement with the near complete absence of redness and swelling in the joints and digits, histological analyses of paw sections showed significantly decreased edema, synovial inflammation, and inflammatory cell infiltration in TNFα siRNA-treated animals (
). To quantitate the degree of inflammation in siRNA-treated animals, we used in vivo
fluorescence tomography of arthritic joints after injection of a pan-cathepsin sensor, which reports on protease activity.36
Using this method, we found that TNFα-specific LNP siRNA reduced joint inflammation by more than half (
). In splenic macrophages from arthritic mice treated with TNFα targeting but not with control siRNA, we found a decrease of intracellular TNFα staining indicating effective silencing (
).Overall, the effect of treatment on mean fluorescent intensity value is highly significant according to an ANOVA (F
= 17.061, P
). Tukey's post-hoc
tests indicate that the pairwise difference between mean MFI in the TNFα siRNA group and the Luc siRNA group is significant (P
= 0.0197; see star in
Figure 6 Inhibition of collagen mAb-induced arthritis by small interfering RNA (siRNA) against tumor necrosis factor-α (TNFα). (a) Preventative treatment of mice with siRNA against TNFα (purple curve) but not against Luc (blue curve) in (more ...) In vivo silencing in circulating monocytes in NHPs.
Finally, in order to determine the translational potential of RNAi-mediated silencing in immune cells, we explored leukocyte gene silencing in NHPs. We first performed a nonterminal study in cynomolgus macaques and examined efficacy of a single dose per formulation and one time point of sampling post dose. To avoid confounding effects due to leukocyte redistribution and trafficking in the days following LNP-siRNA administration, we replicated our mouse protocol and collected blood 1 hour post-i.v. injection followed by 3 days of in vitro
culture and analysis of CD45 protein cell surface expression. We tested CD45 or luciferase siRNA formulated in KC2 (3
mg/kg) or C12-200 (1
mg/kg) and compared pre- and post injection levels of CD45 expression on blood monocytes. In every animal dosed with CD45 siRNA, but not with control siRNA, we found CD45 protein reductions of ~40–50% (
). Encouraged by these results, we next monitored organ resident leukocyte silencing in cynomolgus macaques. The same doses and formulations were used, namely CD45 or luciferase siRNA formulated in KC2 (3
mg/kg) or C12-200 (1
mg/kg). Three days after injection we isolated leukocytes from blood, bone marrow, peritoneal cavity, liver, and spleen to compare surface CD45 expression in animals injected with active versus control siRNA. We observed 30–60% silencing in liver, blood, spleen, and bone marrow-derived cells of monocyte/macrophage lineage (n
= 3 per group,
). Interestingly, peritoneal cavity myeloid cells did not demonstrate any detectable silencing (
). Dot plots from representative animals and overlaid histograms from each animal are shown in Supplementary Figure S6
, demonstrating that there is a significant number of cells with diminished CD45 staining. In this experiment, we could not compare pre and post dose levels; therefore we analyzed group averages with expected and significant variability in nongenetically identical animals. Despite this limitation, robust silencing of CD45 was observed in organ resident and blood circulating myeloid cells. It is worth mentioning that in cynomolgus macaques we were able to detect silencing of CD45 in circulating cells up to 3 days after the injection, which indicates potent and durable silencing in the central compartment of myeloid cells ready for recruitment to inflammatory sites.
Figure 7 Silencing CD45 in vivo in peripheral blood monocytes/macrophages from nonhuman primate (NHP). Blood was drawn from each of the 12 experimental NHPs 1 hours prior and 1 hour after bolus administration of 3mg/kg of small interfering RNA (siRNA) (more ...)