Therapeutic siRNA silencing in inflammatory monocytes in mice.

Abstract

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes-but not the noninflammatory subset-depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.

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Therapeutic siRNA silencing in inflammatory monocytes
Florian Leuschner*,1, Partha Dutta*,1, Rostic Gorbatov1, Tatiana I. Novobrantseva2, Jessica
Sullivan1, Gabriel Courties1, Kang Mi Lee3, James I. Kim3, James F. Markmann3, Brett
Marinelli1, Peter Panizzi4, Won Woo Lee5, Yoshiko Iwamoto1, Stuart Milstein2, Hila Epstein-
Barash2, William Cantley2, Jamie Wong2, Virna Cortez-Retamozo1, Andita Newton1, Kevin
Love6, Peter Libby7, Mikael J. Pittet1, Filip K. Swirski1, Victor Koteliansky2, Robert
Langer6,8,9, Ralph Weissleder1,10, Daniel G. Anderson6,8,9, and Matthias Nahrendorf1
1Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School,
Simches Research Building, 185 Cambridge St., Boston, MA 02114, USA
2Alnylam Pharmaceuticals, 300 3rd Street, Cambridge, MA 02142, USA
3Department of Surgery, MGH
4Department of Pharmacal Sciences, Harrison School of Pharmacy, Auburn University, Auburn,
AL 36849, USA
5Department of Nuclear Medicine, Seoul National University Bundang Hospital, 166 Gumi-ro,
Seongnam 463-707, Korea
6David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
77 Massachusetts Avenue, Cambridge, MA, 02139, USA
7Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA
8Department of Chemical Engineering, MIT
9Division of Health Science Technology, MIT
10Department of Systems Biology, Harvard Medical School, Boston, MA
Abstract
Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine
receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise
therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential
functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed
siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an
optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically,
Corresponding authors: Matthias Nahrendorf and Ralph Weissleder, Center for Systems Biology, 185 Cambridge Street, Boston, MA
02114, Tel: (617) 643-0500, Fax: (617) 643-6133, mnahrendorf@mgh.harvard.edu, rweissleder@mgh.harvard.edu.
*These authors contributed equally to this work
Contributions: F.L. and P.D. performed experiments, collected and analyzed the data, and contributed to writing the manuscript, R.G.
did surgeries and performed experiments, T.I.N. designed experiments and siRNA screens, analyzed data and contributed to writing
the manuscript; K.M.L. did islet transplantations and analyzed data, J.I.K., J.F.M., B.M., P.P., W.W.L., Y.I., V.C.R., A.N., W.C., J.W.
performed experiments, imaging, collected, analyzed and discussed data, S.M., H.E.B., K.L. formulated siRNA nanoparticles, P.L.,
M.J.P., and F.K.S. conceived experiments and discussed strategy and results; V.K. R.L., R.W., D. A. and M.N. designed experiments,
developed siRNA delivery technology and in vivo imaging strategies and systems, and reviewed, analyzed and discussed data. MN
and RW wrote the manuscript which was edited and approved by all co-authors. M.N. developed and supervised the project.
Competing financial interest: T.I.N., S.M., H.E.B., W.C., J.W. and V.K. are Alnylam Pharmaceuticals employees; K.L., R.L., and
D.G.A. receive funding from Alnylam Pharmaceuticals. R.L. and D.G.A. are consultants with Alnylam. The other authors declare no
competing financial interests.
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Published in final edited form as:
Nat Biotechnol. ; 29(11): 1005–1010. doi:10.1038/nbt.1989.
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had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular
localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2
mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the
treatment attenuated their number in atherosclerotic plaques, reduced infarct size following
coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet
transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated
macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes
selectively modulates functions of innate immune cell subtypes and may allow for the
development of specific anti-inflammatory therapy.
Introduction
Our understanding of the innate immune system's role in homeostasis and disease has
considerably expanded over the last decades. The cellular arm, in particular monocytes and
their lineage descendant macrophages, participate critically in inflammatory activity in
major diseases. The production, recruitment and differentiation of monocytes, their subsets
and their progeny have been studied in detail1-3; however, this knowledge has not yet
resulted in effective clinical therapies. A major hurdle for translation is the multitude of
protective functions of innate immune cells, many of which are too important for survival to
compromise. A possible solution to this problem is a therapy that “surgically” and
temporarily targets cellular subsets with compromising disease activity while leaving other
classes of cells undisturbed, so the latter can maintain homeostasis, foster healing, resolve
inflammation and defend against infection. While such therapeutic precision has remained
elusive, the emerging differential roles of monocyte and macrophage subsets make this goal
more attainable.
Inflammatory monocytes (Ly-6Chigh in the mouse, CD14+CD16- in human)3 give rise to
classical macrophages and promote inflammatory disease activity. While essential early
responders, their excessive or prolonged recruitment hinders resolution of inflammation and
propagates disease progression. The recruitment of inflammatory monocytes depends on the
chemokine/chemokine receptor pair MCP-1/CCR2, distinct from the non-inflammatory
monocyte subset which depends on fractalkine/CX3CR11. CCR2 also mediates egress of
monocytes from the bone marrow4. Accordingly, genetic deletion of either MCP-1 or CCR2
results in profound reduction of inflammation in various disease models5-10. Therapeutic
targeting of CCR2 could thus limit harmful functions of innate immunity while leaving non-
inflammatory monocytes, alternatively activated macrophages, antigen-presenting cells and
other tissue residents unaffected. Small molecules and neutralizing antibodies targeting the
MCP-1/CCR2 axis are being investigated but often lack specificity or in vivo efficacy1.
Small interference RNA (siRNA) technology shows promise to attenuate production of
specific target proteins in vivo by degradation of mRNA (e.g.11,12). While siRNA delivery
has been the focus of considerable work, the development of formulations capable of
efficacious systemic delivery to immune cells with clinically suitable delivery materials has
been challenging13. Recent advances in siRNA design and chemistry now allow
identification of specific, highly potent sequences with nuclease stability and reduced
immunostimulation14, which we here combine with biocompatible nanoparticle delivery
vehicles15. We hypothesized that emerging monocyte-targeting siRNA nanomaterials could
silence CCR2 mRNA in the inflammatory monocyte subset to selectively inhibit migration
(and consequently adverse function) of these cells and their progeny (Supplementary Fig. 1).
Dynamic optical tomography revealed that nanoparticle-encapsulated, fluorescently tagged
siRNA rapidly redistributed from the blood pool to the spleen and bone marrow. Knock
down of CCR2 in monocytes was confirmed at the mRNA, protein and functional levels.
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We then tested this therapeutic approach in mouse atherosclerosis, myocardial infarction,
pancreatic islet transplantation in diabetes, and cancer. Treatment with clinically feasible
doses of siRNA substantially reduced regional recruitment of inflammatory monocytes and
attenuated disease progression in these disease models.
Results
siRNA distribution
The siRNA carrier used in this study was identified in high-throughput in vitro screens of
several hundred compounds, resulting in development of a 70-80 nm lipid-like particle with
efficient cell delivery and knock down15. The nanoparticle was formulated using C12-200
lipid, disteroylphosphatidyl choline, cholesterol, PEG-DMG and siRNA in a spontaneous
vesicle formation procedure. The specific siRNA sequence against CCR2 (siCCR2) was
identified by in vitro screening from 31 candidate sequences (Supplementary Fig. 2), all
chemically modified to minimize off-target effects. The best duplex with the sequence of 5′-
uGcuAAAcGucucuGcAAAdTsdT-3′ (sense), 5′- UUUGcAGAGACGUUuAGcAdTsdT -3′
(anti-sense) was selected for scale up, formulation and subsequent nanoparticle
encapsulation. The final lipid:siRNA weight ratio of the nanoparticle was 12:1 with an
siRNA entrapment efficiency of 95%.
We covalently labeled siRNA with a near infrared fluorochrome to follow its in vivo
distribution after intravenous injection with dynamic fluorescence imaging. Mice were
positioned inside the Fluorescence Molecular Tomography (FMT) scanner, and data were
acquired serially for up to 24 hours. siRNA concentration was determined every 5 minutes.
Key organs were identified in co-registered X-ray computed tomography (CT). Immediately
after injection, strong fluorescence signal localized in the cardiac region, reflecting the
presence of siRNA in the blood pool (Fig. 1a, arrow). This signal was fitted over time
resulting in an siRNA blood half-life of 8.1 minutes (Fig. 1a). Interestingly, the spleen
showed the highest fluorescence per gram of tissue in the entire body, with a peak
fluorescence of 391±63 pmol/g tissue (Fig. 1a, circle). There was also considerable
accumulation in the bone marrow, while no siRNA was detected in the lung (3±4 pmol/g).
Dynamic imaging identified that the siRNA was excreted via the hepato-biliary route (Fig.
1a). Ex vivo fluorescence reflectance imaging corroborated these findings (Supplementary
Fig. 3).
We next studied the intra-parenchymal siRNA distribution in the spleen by fluorescence
microscopy. Most of the siRNA related fluorescence was found in cells residing in the red
pulp. Spectrally resolved co-staining for CD11b co-localized this myeloid marker with
siRNA in cells that formed the typical splenic reservoir monocyte clusters16 (Fig. 1b). We
also detected uptake in Kupffer cells (Supplementary Fig. 4). Flow cytometric analysis
further refined the uptake profile in cells in the bone marrow and spleen, and in circulating
leukocytes. While lymphocytes showed comparatively low uptake, we detected strong signal
in neutrophils, Ly-6Chigh and Ly-6Clow monocytes, dendritic cells and macrophages (Fig.
1c). The highest uptake was seen in splenic Ly-6Chigh monocytes (Fig. 1d).
In vivo silencing in Ly-6Chigh monocytes
Recent work described a large monocyte reservoir in the spleen which dispatches these cells
to sites of local inflammation16. Systemic treatment with nanoparticle-encapsulated siCCR2
resulted in a significant reduction of CCR2 mRNA in Ly-6Chigh monocytes isolated from
this population (Fig. 2a). A RACE assay in splenic monocytes isolated after intravenous
injection confirmed the RNAi mechanism by detecting specific mRNA cleavage products
(Supplementary Fig. 5). Cleavage of CCR2 mRNA translated into lower protein levels by
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Western blot (Fig. 2b, Supplementary Fig. 6) and flow cytometric staining of CCR2 protein
on Ly-6Chigh monocytes (Fig. 2c). The migratory capacity of Ly-6Chigh monocytes towards
MCP-1 was drastically reduced from 144±31 to 2±2 migrated cells (Fig. 2d).
Silencing of CCR2 reduces myocardial infarct size
Myocardial infarction elicits a strong inflammatory response and profound recruitment of
innate immune cells, including inflammatory splenic reservoir monocytes16. The magnitude
of inflammation influences infarct size and the degree of post-MI heart failure17. Using flow
cytometry analysis of digested myocardium that underwent ischemia followed by
reperfusion, we found a marked reduction of monocytes and macrophages in the hearts of
mice that had been injected with siCCR2 (control siRNA treatment, 16,950±9,470 cells per
mg infarct tissue; siCCR2 treatment, 5,158±2,201; Fig. 3a,b). The treatment lowered the
number of inflammatory Ly-6Chigh monocytes by 71% (Fig. 3a,b). The infarct-to-area-at-
risk ratio, which signifies the benefit of a treatment acutely after myocardial ischemia, was
reduced by 34% in siCCR2 injected mice (Fig. 3c,d). Splenectomy experiments confirmed
that the splenic monocyte reservoir is a major target of siCCR2 treatment in acute
inflammation (Fig. 3e-g). When treatment was started 1 hour after injury, therapeutic effects
were preserved (Supplementary Fig. 7). A head-to-head comparison to small molecule
inhibitors of CCR2 showed superior efficacy of therapeutic silencing (Supplementary Fig.
8).
Silencing of CCR2 reduces inflammatory atherosclerosis
Increased invasion of Ly-6Chigh monocytes critically promotes progression and
complication of atherosclerosis18,19 while germline deletion of CCR26 or MCP-15
attenuates the disease. A 3 week siCCR2 therapy in apoE-/- mice with established
atherosclerosis reduced the monocyte/macrophage number in atherosclerotic plaques by
82% (Fig. 4a,b). Monocyte subset analysis showed a marked reduction of inflammatory
Ly-6Chigh monocytes (7,188 ± 3,388 versus 2,281 ± 1581). Immunohistochemical staining
for CD11b indicated a 46% reduced presence of myeloid cells, and a 38% reduction of
lesion size in the aortic root (Fig. 4c).
Silencing of CCR2 prolongs islet graft survival
Pancreatic islet transplantation is a treatment option for patients with type 1 diabetes;
however, allograft rejection is a major hurdle to its clinical use20. Ly-6Chigh monocytes are
recruited in transplant rejection21, and monocytes can differentiate into highly active
antigen-presenting cells3. Therefore, we investigated the effect of siCCR2 treatment in
pancreatic islet transplantation in mice with streptozotocin-induced diabetes. Intravenous
injection of nanoparticle-encapsulated siCCR2 significantly prolonged the normoglycemic
period and by association islet graft function (Fig. 5a). Graft-dependent normoglycemia in
siCCR2 treated mice was established by nephrectomy, which removed the grafted islets and
returned mice to a diabetic state. Histology of the grafted islets confirmed that these were
rejected in control groups, but not in euglycemic mice treated with siCCR2 (Fig. 5b,c)
Silencing of CCR2 reduces tumor size and TAM
The host immune system plays an increasingly recognized role in cancer progression,
including tumor vascularisation, growth, metastasis and resistance to chemotherapy22-25.
Inflammatory monocytes contribute substantially to the host response by differentiation into
tumor-associated macrophages (TAM)26, and the number of TAM inversely correlates with
survival in patients with lymphoma27. Accordingly, we investigated the effect of siCCR2
therapy in mice after implantation of lymphoma EL4 cells. Treatment was initiated when a
tumor mass was palpable, which occurred 6 to 10 days after inoculation. Starting on day 14
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after tumor implantation, tumor size was reduced in siCCR2 treated mice (Supplementary
Fig. 9). Computed tomography showed that tumor volume was significantly reduced after
siCCR2 injections (279±144mm3 versus 590±221 mm3, Fig. 6a). Flow cytometric analysis
of tumors showed a 54% reduction of tumor-associated macrophages in mice treated with
siCCR2 (Fig. 6b). Histological analysis confirmed reduced numbers of CD11b+ cells (Fig.
6c) and showed that the expression of VEGF and the number of microvessels was reduced
after treatment with siCCR2 (Fig. 6d,e). In a second cancer model of colorectal CT26
tumors we found a 75% reduction of tumor associated macrophages (Supplementary Fig.
10). Taken together, treatment with siCCR2 reduced the number of TAMs and tumor size,
however; future experiments will have to show impact of this therapy at later disease stages
and in conjunction with chemotherapy.
Cellular specificity and other gene targets
We were interested to better understand the specificity of siCCR2 treatment, and therefore
investigated reduction of other leukocytes in cancerous lesions. As expected, we found that
silencing of CCR2 primarily reduced the number of inflammatory monocytes, which depend
on this receptor for recruitment. Other myeloid cells and lymphocytes were affected less
(Supplementary Fig. 11). However, silencing of the pan-leukocyte marker CD45 was also
possible in splenic macrophages, Ly-6Clow monocytes and dendritic cells (Supplementary
Fig. 12), implying that the approach is broadly applicable for silencing genes in those cells.
Discussion
The controlled recruitment of monocytes is an essential feature of host defense, but
excessive or prolonged recruitment of the inflammatory monocyte subset is harmful as these
cells are mediators of tissue destruction. Here we harnessed RNA interference to silence
expression of the monocytic chemokine receptor CCR2, which is responsible for the
migration of the inflammatory monocyte subset. Migration of monocytes towards MCP-1 is
severely attenuated in heterozygous CCR2 deficient mice28, making CCR2 an efficient
silencing target since even intermediate protein levels translate into impaired migratory
capacity of Ly-6Chigh monocytes.
The motivation for targeting CCR2 in monocytes in this study was based on three major
observations: i) the striking phenotype of CCR2-/- and MCP-1-/- mice1,5-10,29,30; ii) prior
work linking monocyte subsets to distinct functions in disease and healing2,3,17,25,31 and iii)
the ease by which monocytes can be targeted via their phagocytic activity32. Based on recent
advances in siRNA formulation designs, we show by dynamic FMT imaging and flow
cytometry that intravenously injected nanoparticles distributed siRNA to immune cells in the
blood, bone marrow and spleen, resulting in efficient and specific knock down of CCR2.
Consequently, fewer inflammatory monocytes migrated to the site of inflammation and
disease severity was attenuated in multiple models investigated. Because we target a
recruitment mechanism, sessile immune cells remain largely unaffected.
The effects of siCCR2 treatment on splenic reservoir monocytes proved particularly
intriguing. Study of acute myocardial infarction firmly established the dominant role of this
emergency reservoir, as more than half of the monocytes recruited to the infarct originate
from the spleen16. Splenectomy and siCCR2 treatment had no additive effects on
recruitment to the infarct, indicating that the splenic monocyte population is a major target
of siCCR2 therapy, and potentially for any anti-inflammatory strategy focusing on
monocytes. The role of splenic monocytes is less well understood in chronic inflammation,
and future studies will decipher if lesional foam cells in atherosclerosis and tumor-
associated macrophages derive from the splenic monocyte pool. Observed treatment effects
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in atherosclerosis and cancer in our study likely also result from CCR2 silencing in bone
marrow or circulating monocytes.
Our approach is distinct from previous work on gene silencing in leukocytes on several
levels. We targeted a monocyte gene that drives recruitment of a cells. Interruption of cell
migration may be particularly efficient, because it removes a cell population from the
inflammatory site rather than neutralizing a single gene product. To our knowledge, this is
the first demonstration of siRNA delivery to the inflammatory monocyte subset. Silencing
CCR2 provides cellular specificity for this emerging therapeutic target. A previous study
described silencing after oral administration of yeast-based glucan particles. These
micrometer sized particles were reported as taken up by macrophages in the intestine
through a glucan-receptor mediated pathway11. A smaller liposome that is comparable in
size to the nanoparticle used in our study was formed from phospholipids and featured a
monoclonal antibody against integrins as a targeting moiety12. The lipidoid nanoparticle
preparation used in our study consists of chemically defined entities and comes with
significant manufacturing advantages13, which enhance chances of its clinical use for anti-
inflammatory therapy. Structurally related delivery materials have been used successfully
for silencing in the liver by independent research groups in several species, with potent
knock down at doses as low as 0.01mg/kg33.
When compared to conventional anti-inflammatory therapy (e.g. steroids, cyclooxygenase-2
inhibitors, methotrexate), silencing CCR2 is set apart by its cellular specificity. Most
clinically approved drugs may have molecular specificity but broadly target cells in tissues
they distribute to, including cells that should be spared to avoid unwanted side effects. For
example, steroids have been tested in patients with myocardial infarction with disastrous
outcomes34, and their side effects (fluid retention, diabetes) make them an unlikely
candidate for treating cardiovascular disease. Other potent immunosuppressive drugs such as
methotrexate, used for treatment of rheumatoid arthritis, also lack cellular specificity.
Therefore, while these drugs suppress inflammation, they also diminish protective functions
of the immune system which are involved in the resolution of inflammation, wound healing
and defense against infection. In general, delivery of drugs by nanoparticles can increase
their concentration at the site of action35, which may partially explain the advantage in
efficiency when we compared siCCR2 treatment with small molecule CCR2 inhibitors.
The efficient delivery of siRNA to immune cells provides an avenue to translate the vast
knowledge base gained from germline inactivation of genes involved in immunity. While we
have concentrated on CCR2 in this study, it is logical to extend the approach to other targets
in innate immune cells, for instance proteins involved in proliferation, maturation,
differentiation, antigen presentation, such as transcription factors and cytokines. The ability
to silence CD45 in other myeloid cells suggests that generalization of the approach is
feasible. One particularly appealing aspect of siRNA delivery is the ability to co-silence
multiple targets. While we have shown the ubiquitous (and surprising) efficacy of CCR2
siRNA in cardiovascular disease, cancer and transplant rejection, there are likely even more
diseases that could benefit. In sum, this study describes the merging of in vivo RNA
interference with recent insight into monocyte biology, opening a new translational avenue
to approach the many diseases driven by recruitment of these cells.
Methods
See Supplementary Methods for a detailed description of mouse models, imaging, molecular
biology, immunohistology and statistical analysis. All experiemnts were approved by the
Subcommittee on Animal Research Care at Massachusetts General Hospital.
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Synthesis and in vitro screening of siRNAs
31 siRNAs with the lowest predicted off-target potentials and 100% homology with mouse
CCR2 gene sequence NM_009915.2 were selected for synthesis and screening. Single-
strand RNAs were produced at Alnylam Pharmaceuticals as previously described, annealed
and used as duplexes36. Mouse macrophage line J774A.1 cells were transfected with siRNA
by using Lipofectamine RNAiMAX reagent according to the manufacturer's protocols at
0.1nM and 10nM concentrations. CCR2 mRNA levels were quantified 24h post transfection
by Q-PCR and normalized to GAPDH mRNA. Duplexes showing best knock-down at both
concentrations (marked with arrows in Supplemental Figure 2) were selected for 12 point
dose response ranging from 10nM down to 0.01pM to determine IC50 value for these siRNA
duplexes. The best duplex with the sequence of 5′-uGcuAAAcGucucuGcAAAdTsdT-3′
(sense), 5′- UUUGcAGAGACGUUuAGcAdTsdT -3′ (anti-sense) was selected for scaling
up, formulating and subsequent in vivo work. Small case represents 2'OMe modified
residues. For silencing CD45, we used a duplex with the following sequence: 5′-
cuGGcuGAAuuucAGAGcAdTsdT-3′ (sense), 5′- UGCUCUGAAAUUcAGCcAGdTsdT-3′
(antisense) as published previously37.
siRNA formulation into lipid nanoparticles
Nanoparticles were prepared with the cationic lipid C12-200,15 disteroylphosphatidyl
choline, cholesterol, and PEG–DMG using a spontaneous vesicle formation formulation
procedure. Lipids were dissolved in 90% ethanol solution and mixed with siRNA solution
(25 mM citrate, pH 3 ratio) at fixed speed (1:1 ratio) and diluted immediately with PBS to
final 25% ethanol. The ethanol was then removed and the external buffer replaced with PBS
(155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by dialysis. The particles had a
component molar ratio of ∼50/10/38.5/1.5 (C12-200/disteroylphosphatidyl choline/
cholesterol/PEG–DMG). The final lipid:siRNA weight ratio was ∼7:1. Particle size and zeta
potential were determined using a Malvern Zetasizer NanoZS (Malvern, UK). siRNA
content was determined by ion exchange HPLC (Agilent) assay using DNAPac Pa200
column (Dionex Corporation Dionex, 260nm, 55°C run at 2mL/min). siRNA entrapment
efficiency was determined by the Quant-iT RiboGreen RNA assay (Invitrogen, Carlsbad,
CA)38. Briefly, siRNA entrapment was determined by comparing the signal of the RNA
binding dye RiboGreen in formulation samples in the absence and presence of the detergent
Triton-X100. In the absence of detergent, signal comes from accessible (unentrapped)
siRNA only. In the presence of detergent, signal comes from total siRNA. In all preparations
used the encapsulation was above 82%. The polydispersity index of the final formulation
was 0.036-0.037.
Dynamic FMT-CT imaging of siRNA biodistribution
FMT-CT imaging was used to evaluate the time-resolved biodistribution of lipid
nanoparticle siRNA. This quantitative imaging technique combines non-invasive whole
body fluorescent tomography (determining tissue concentration of AF647-coupled siRNA)
with high resolution CT imaging. To minimize absorbance of photons by fur nude mice
were used for this study, which received a non-fluorescent diet for a week prior to imaging.
After placement of mice into a multimodal imaging cartridge which provides fiducials
detected by both modalities for image fusion, contrast-enhanced microCT (Inveon, Siemens)
was followed by dynamic FMT imaging on an FMT-2500 system (PerkinElmer). Anesthesia
used 1.5 % Isoflurane (O2 2L/min) with a gas delivery system integrated into the
multimodal imaging cartridge. After CT imaging, mice were transferred while in the
multimodal imaging cartridge and positioned in the FMT scanner with a tail vein in place.
Then, a bolus of nanoparticle-encapsulated AF647-coupled siRNA was injected at a dose of
1 mg/kg, and mice were imaged every 5 minutes for 2 hours, and then at 4hrs, 6hrs, 8hrs,
and 24hrs post-injection. FMT imaging used excitation/emission wavelengths of 635 and
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655nm, respectively. 30 frontal slices of 0.5 mm thickness in z-direction, with an in-plane
resolution of at least 1×1 mm were acquired.
Post-processing of FMT data sets was done by using a normalized Born forward equation to
calculate fluorochrome concentration expressed in nM fluorescence per voxel. The 3D
dataset was reconstructed for the entire mouse. Based on CT information, volumes of
interest were defined in respective organs. To co-register FMT and CT images, DICOM-
converted data were imported into OsiriX (The OsiriX foundation, Geneva, Switzerland).
Fiducials on the imaging cartridge were visualized and tagged in FMT and CT images with
point markers to define their location in 3D. This allows point-based registration of FMT
data to CT coordinates as described before39. Fluorescence measurements in volumes of
interest were normalized to weight using average organ weights and expressed as
fluorescence per gram tissue.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors thank Michael Warring and the Ragon Institute (MGH) for cell sorting, the CSB Mouse Imaging
Program (Peter Waterman, Brena Sena), Dr. Brian Bettencourt for designing initial sets of siRNA. We
acknowledge the small, medium and large scale RNA synthesis groups at Alnylam as well as analytical, duplex
annealing and QC groups for synthesizing and characterizing RNAs. This work was funded in part by grants from
the National Institute of Health R01-HL096576, R01-HL095629 (M.N.); R01-EB006432, T32-CA79443, U24-
CA92782, P50-CA86355, HHSN268201000044C (R.W.); R01-CA132091, R01-CA115527, R37-EB000244
(R.L.); Deutsche Herzstiftung (F.L.); the SNUBH Research Fund 02-2007-013 (W.W.L.).
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Figure 1. Nanoparticle-encapsulated siRNA distributes to leukocytes
(a) Dynamic FMT-CT biodistribution imaging of siRNA shows major uptake in the spleen
(circle). The material is rapidly cleared from the blood pool (arrow) and excreted via the
hepato-biliary route into bowel (n = 5). (b) Immunofluorescence microscopy of the splenic
red pulp shows colocalization of siRNA (red), CD11b expression (green). The scale bar
indicates 20μm. (c) Profiling by flow cytometry reveals strong uptake of siRNA into cells of
the mononuclear phagocyte system in the bone marrow, blood and the spleen.
Representative dot-plots on the left illustrate the gating strategy (lin: lineage markers).
Histograms on the right illustrate the uptake of siRNA (red) in the respective cells (columns)
and organs (rows). Blue indicates control fluorescence in cells harvested from non-injected
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mice. (d) Comparison of siRNA content in Ly-6Chigh monocytes retrieved from different
organs (n = 3 mice per group). Mean ± SD, * P < 0.05.
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Figure 2. Intravenous injection of nanoparticle-encapsulated siRNA results in knock down in
monocytes
(a) PCR analysis of FACS-sorted splenic Ly-6Chigh monocytes after intravenous
administration of nanoparticle-encapsulated siRNA targeting CCR2 (siCCR2). siCON:
control siRNA treatment. n = 3 per group. (b) Western blot of Ly-6Chigh monocytes isolated
from the spleen of mice that were either treated with siCCR2 or control. The reduced band
in the siCCR2 lane confirms a lower expression of CCR2 receptor protein in splenic
monocytes after intravenous treatment (experiment was done in triplicate, representative blot
is cropped. The full length blot is presented in Supplementary Figure 6). (c) FACS analysis
of CCR2 protein on splenic Ly-6Chigh monocytes. Representative histogram shows isotype
antibody staining (black), control treatment (red) and siCCR2 treatment (blue). Bar graph of
mean fluorescent intensity (MFI, y axis starts at mean fluorescence intensity of the isotype
control; n = 6 per group). (d) Migration assay of sorted Ly-6Chigh monocytes using MCP-1
as chemoattractant. Cells were harvested from mice injected with either siCCR2 or control
siRNA (n = 3 per group). Stained membranes on the left, bar graph shows enumeration of
migrated cells. Mean ± SD, * P < 0.05.
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Figure 3. Treatment with siCCR2 reduces ischemia reperfusion injury
(a) FACS of hearts 24 hours after ischemia reperfusion injury (IRI). Dot-plots show a
reduction of monocytes/macrophages (blue gate) in the infarct tissue after siCCR2
treatment. Right column of dot plots displays subset analysis with a drastically reduced
Ly-6Chigh monocyte population in the lower right quadrant. (b) Cell tissue numbers for
monocytes/macrophages (Mo/MΦ, left) and for Ly-6Chigh monocytes (n = 9 per group). (c)
Fluorescence reflectance images display the area at risk, which is void of microspheres
injected during ischemia (left column). TTC staining of the same myocardial short axis slice
(right column). The pale, unstained infarct is outlined by a dotted line. (d) Infarct size
normalized to the non-perfused area at risk (n = 7-9 per group). (e-f) FACS of infarcts 24
hours after permanent coronary ligation. Spleen “minus” indicates removal of the spleen at
time of myocardial infarction. (e) Histogram of Ly-6Chigh monocytes in infarcts. (f) Number
of Ly-6Chigh monocytes in infarcts. (g) Number of monocytes/macrophages in infarcts. No
statistically significant difference was detected between treatment groups (n = 4-8 per
group). Mean ± SD, * P < 0.05.
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Fig. 4. Treatment with siCCR2 reduces inflammation in atherosclerotic lesions in apoE-/- mice
(a) FACS analysis of cell suspensions retrieved from aortas after 3 weeks of treatment with
siRNA targeting CCR2 (siCCR2) or control siRNA treatment (siCON). (b) Number of
monocytes and macrophages (Mo/MΦ) and Ly-6Chigh monocytes in the aortas of apoE-/-
mice (n = 10 per group). (c) Immunohistochemical analysis of aortic roots for CD11b and
lesion size (n = 8-14 per group). ROI: region of interest. AU: arbitrary units. Mean ± SD, *
P < 0.05.
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