Tile Page Arthritic Joint-Targeting siRNA Encapsulated Liposome:...

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Arthritic Joint-Targeting siRNA Encapsulated Liposome: Implication for Treatment

Strategy for Rheumatoid Arthritis

Yukiko Komano, Nobuhiro Yagi, Ikumi Onoue, Kayoko Kaneko, Nobuyuki Miyasaka and

Toshihiro Nanki

Department of Medicine and Rheumatology, Graduate School of Medical and Dental

Sciences, Tokyo Medical and Dental University, Tokyo, Japan (Y.K., I.O., K.K., N.M.,

T.N.); and Kyowa Hakko Kirin Co., Ltd, Shizuoka, Japan (N.Y.).

JPET Fast Forward. Published on October 12, 2011 as DOI:10.1124/jpet.111.185884

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on October 12, 2011 as DOI: 10.1124/jpet.111.185884

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Running title page

Running title: Treatment of Arthritis using siRNA-Encapsulated Liposome

The number of text pages; 11

The number of figures; 3

The number of references; 18

The number of words; Abstract, 240

Introduction, 334

Discussion, 785.

Abbreviations; siRNA: short interfering RNAs; RA: rheumatoid arthritis; WS: wrapsome;

TNF-α: tumor necrosis factor-α; CIA: collagen induced arthritis; RT-PCR: reverse

transcriptase-polymerase chain reaction; IL: interleukin; GAPDH:

glyceraldehyde-3-phosphate dehydrogenase; MFI: mean fluorescence intensity.

Recommended section assignments; Inflammation, Immunopharmacology, and Asthma.

Corresponding author:

Toshihiro Nanki, MD

Department of Medicine and Rheumatology, Graduate School of Medical and Dental

Sciences, Tokyo Medical and Dental University

1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8519 Japan.

Tel: +81-3-5803-4677

Fax: +81-3-5803-4694

E-mail: [email protected]


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Abstract

RNA interference, mediated by small interfering RNA (siRNA), is effective in silencing

genes with a high degree of specificity. To explore the therapeutic potential of systemically

administered siRNA for rheumatoid arthritis (RA), we tested the complex of siRNA and the

recently developed wrapsome (siRNA/WS), containing siRNA encapsulated liposome, in

mice with collagen-induced arthritis (CIA). Mice with CIA received intravenous injection of

Cy5-labeled siRNA/WS. Fluorescence stereoscopic microscopy and flow cytometry were

used to assess the siRNA/WS tissue distribution. The efficacy of siRNA targeting tumor

necrosis factor (TNF)-α/WS in CIA was evaluated by arthritis score. TNF-α mRNA levels in

the joints were measured by real-time reverse transcriptase-polymerase chain reaction. The

intensity of Cy5-fluorescence was higher in arthritic joints than in non-arthritic sites in

Cy5-siRNA/WS-treated mice, and remained higher up to 48 hrs post-injection, compared

with naked Cy5-siRNA-treated mice. Cy5-fluorescence intensity was higher in synovial cells

than splenocytes, bone marrow cells and peripheral blood leukocytes. The majority of

Cy5-positive synovial cells were CD11b+ with only a few CD3+ cells. Treatment with TNF-α

siRNA/WS resulted in significant decreases in severity of arthritis and TNF-α mRNA level in

the joints compared with control siRNA/WS. In conclusion, the use of our WS allowed

efficient and targeted delivery of siRNAs to arthritic joints. The siRNA/WS was mainly

incorporated into CD11b+ cells, including macrophages and neutrophils, in the inflamed

synovium, suggesting its potential therapeutic effects in RA by silencing the expression of

inflammatory molecules produced by these cells.


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Introduction

RNA interference (RNAi), mediated by small interfering RNA (siRNA), is a powerful

way to silence genes with a high degree of specificity, and potentially become a therapeutic

approach in various diseases including viral infections and proliferative vascular retinopathies

(de Fougerolles et al., 2007; Whitehead et al., 2009). To apply this technology to the

treatment of rheumatoid arthritis (RA), it is important to develop means to deliver siRNA via

systemic injection to multiple affected joints. The delivery tool should be stable, and protect

siRNA from nuclease attack in vivo. Our group used the wrapsome (WS) as a vehicle for

siRNA delivery, which was recently designed with a core composed of a cationic lipid

bilayer and siRNA complex enveloped in a neutral lipid bilayer with polyethylene glycol on

the surface (Yagi et al., 2009). In contrast to the widely used cationic liposomes, the WS is

unique in that it is uncharged. The cationic liposomes are prone to bind in vivo to plasma

proteins due to their cationic nature. Because the WS carries a neutral surface charge, the loss

of WS function by the plasma protein is less likely. For the same reason, the attachment of

WS to the endothelial cell membrane and its entrapment by the reticuloendothelial systems

are avoidable, which can otherwise lead to adverse effects, including embolism, a

complication reported with the use of cationic liposomes (Litzinger et al., 1996). In addition,

the surface pegylation of WS confers the long half life of WS in systemic circulation (t1/2 of

17.6 hr). Investigator in our group has recently demonstrated that the use of WS results in

accumulation of siRNA into tumors in vivo without any signs of significant toxicity (Yagi, et

al., 2009).

In the present study, we evaluated the accumulation of siRNA/WS complex into the

inflamed joints of mice with experimentally-induced arthritis. Also, the therapeutic potential

of the siRNA/WS complex targeting tumor necrosis factor (TNF)-α, which is produced

primarily by macrophages in the synovia of the affected joints, was assessed in the arthritic


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mice.


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Materials and Methods

Preparation of siRNA Encapsulated Liposome. All siRNAs including fluorescence

labeled randomized-siRNA were synthesized and purified by Hokkaido System Science

(Hokkaido, Japan). The siRNA sequences were as follows: control randomized-siRNA sense

5’- GGU ACA CAU GUG CAC ACA CdTdT -3’ and antisense 5’- GUG UGU GCA CAU

GUG UAC CdTdT -3’; TNF-α-siRNA sense 5’- GUG CCU AUG UCU CAG CCU CdTdT

-3’ and antisense 5’- GAG GCU GAG ACA UAG GCA CdTdT -3’ (Schiffelers et al., 2005).

Preparation of the complex of siRNAs and WS, which was developed as delivery system for

siRNAs, was described previously (Yagi, et al., 2009). The siRNA/WS complex consists of

siRNA and cationic lipids enveloped by a neutral lipid bilayer containing Egg-phosphatidyl

choline, and is ~100 nm in diameter.

Imaging of siRNA/WS Accumulation in Arthritic Joints of Mice with

Collagen-Induced Arthritis (CIA). Murine CIA was induced in 8-week-old male DBA/1J

mice (Charles River Japan, Tokyo, Japan) by twice immunization with bovine type II

collagen (Collagen Research Center, Tokyo) and complete Freund’s adjuvant (Difco

laboratories, Detroit, MI) (Nanki et al., 2005). From the day of the second immunization (day

0), mice were examined for signs of joint inflammation. The severity of arthritis was

evaluated using the following clinical scoring method (Nanki, et al., 2005): score 0, normal; 1,

erythema and mild swelling confined to the mid-foot or ankle joint; 2, erythema and mild

swelling extending from the ankle to the mid-foot; 3, erythema and moderate swelling

extending from the ankle to the metatarsal joint; 4, erythema and severe swelling of the ankle,

foot, and digits. The clinical score was defined as the sum of the scores of all four paws of

each mouse. At day 7, CIA mice showing joint inflammation scored more than 2 in the above

definition at least in a paw, were randomly injected either with a single injection of

Cy5-labeled siRNA/WS, naked Cy5-labeled siRNA, or saline via the tail vein. Considering


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the limited sensitivity for detection of fluorescence in vivo setting, dose of the Cy5-labeled

siRNA were selected to be 50 and 150 μg for whole-body imaging (Yagi, et al., 2009).

Cy5-fluorescence was monitored for up to 96 hrs after administration using fluorescence

stereoscopic microscope (Macro Imaging System, Nippon Roper, Tokyo). The level of

fluorescence labeled-siRNA in the affected joints was quantified by analyzing regions of

interest using image analysis software (Nippon Roper). Approval for the experimental

protocol was obtained from the Institutional Animal Care and Use Committee of Tokyo

Medical and Dental University.

Analysis of siRNA/WS Tissue Distribution in the CIA Mice. At day 7, CIA mice

showing joint inflammation as described above were randomly injected either with 50 μg of

Cy5-labeled siRNA/WS, naked Cy5-labeled siRNA, or saline via the tail vein. Based on the

results of the whole-body imaging, the dosage of Cy5-labeled siRNA/WS for this FACS

analysis was selected. Twenty-four hours later, tissues (synovium, spleen, and bone marrow)

were harvested and single cell suspensions were prepared using standard procedures.

Synovial tissues were harvested from arthritic joints (scored more than 2). Peripheral blood

leukocytes were isolated by depletion of red blood cells with NH4Cl hemolytic solution. The

cells were analyzed for Cy5-fluorescence using a FACSCalibur (BD Bioscience, San Jose,

CA). The following mAbs were used for staining: CD11b-phycoerythrin (eBioscience, San

Diego, CA), F4/80-fluorescein isothiocyanate (eBioscience), CD3-fluorescein isothiocyanate

(BD Pharmingen, Franklin Lakes, NJ), and CD19-phycoerythrin (eBioscience).

Analysis of the Effect of TNF-α-siRNA/WS in CIA Mice. At the day of the second

immunization (day 0), mice were grouped on the basis of equal average clinical scores, and

injected intravenously with TNF-α siRNA/WS, naked TNF-α siRNA, control

randomized-siRNA/WS (1 or 10 μg/day), or buffered saline three times per week until day 10.


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Clinical arthritis score and the incidence of arthritis were evaluated. In addition, thickness of

each paw was measured using calipers.

Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). CIA

mice were sacrificed on day 12, and paws were pulverized using cryopress (Microtec, Chiba,

Japan). Total RNA was prepared from 50 mg of the sample using a RNeasy RNA isolation

kit (Qiagen, Valencia, CA). The levels of TNF-α mRNA were measured by real-time

RT-PCR assay using TaqMan probes [TNF-α (Mm00443258) and

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Mm99999915) (Applied Biosystems,

Foster City, CA)]. Amplification was performed in a thermal cycler (ABI Prism 7000;

Applied Biosystems).

Statistical Analysis. Data are expressed as mean ± SEM. For comparing clinical

scores between groups, the non-paired Student’s t-test was used. For comparing incidence of

arthritis between groups, Fisher's exact test was used. Analysis were made using StatView

program (Abacus Concepts, Berkely, CA). P values <0.05 were considered significant.


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Results

Accumulation of Systemically Administered siRNA/WS in Arthritic Joints of

murine CIA. The potential of WS as a delivery tool of siRNA was investigated in CIA. At

day 7, when the CIA mice developed arthritis (scored more than 2 at least in a paw), they

were injected either with fluorescence (Cy5)-labeled siRNA/WS or naked Cy5-labeled

siRNA (50 or 150 μg). After 12 hrs, observation under stereoscopic microscope showed

accumulation of fluorescence in the arthritic joints but not in non-arthritic sites in mice

treated with Cy5-siRNA /WS (150 μg), and the levels of the accumulation appears higher in

severely swollen joints compared to less swollen or normal joints (Figures 1A and B).

Although the intensity was weaker, fluorescence was detected in arthritic joints in mice

treated with 50 μg of Cy5-siRNA/WS (data not shown). In mice treated with naked

Cy5-siRNA (150 μg), although a low level of fluorescence was observed in arthritic joints

immediately after the injection, the fluorescence disappeared within 12 hrs (Figure 1C). The

intensity of the fluorescence level remained high up to 48 hrs post-injection in the arthritic

joints of the mice treated with Cy5-siRNA/WS (Figure 1D). Although the intensity was

weaker, fluorescence was detected in arthritic joints in mice treated with 50 μg of

Cy5-siRNA/WS (data not shown). Contrary, the intensity of the fluorescence level in the

arthritic joints of the mice treated with naked Cy5-siRNA (150 μg) was much lower

compared to that in the mice treated with Cy5-siRNA/WS (Figure 1D).

Next, the tissue distribution of systemically administered Cy5-siRNA/WS was

examined in the CIA mice. At 24-hr post-injection of 50 μg of Cy5-siRNA/WS, naked

Cy5-siRNA or saline, the Cy5-fluorescence levels in cells from the synovium, spleen, bone

marrow and peripheral blood leukocytes were examined by flow cytometry. Compared to

saline injected mice, the fluorescence intensity of Cy5 was higher in cells harvested from the


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synovium of naked Cy5-siRNA injected CIA mice (Figure 2A). Moreover, the Cy5 intensity

was significantly enhanced in synovial cells from mice injected with Cy5-siRNA/WS

compared to those with naked Cy5-siRNA, with the ratio of mean fluorescence intensity

(MFI) (Cy5-siRNA/WS vs. naked Cy5-siRNA) of 4.4 (Figure 2B). The Cy5 intensity in bone

marrow cells and peripheral blood leukocytes of naked Cy5-siRNA injected CIA mice was

also higher, however, the Cy5 intensity in mice injected with Cy5-siRNA/WS was not

enhanced (MFI ratios: 0.97 and 0.57, respectively). Of note, the MFI of Cy5 was much higher

in synovial cells from Cy5-siRNA/WS injected mice compared to bone marrow cells and

peripheral blood leukocytes (Figure 2B). On the other hand, the fluorescence intensity was

not significantly altered in splenocytes.

Next, we determined the cells that incorporate the Cy5-siRNA/WS in the synovium. At

24-hr post-injection, the synovial cells of mice treated with naked Cy5-siRNA or

Cy5-siRNA/WS were analyzed by flow cytometry after staining with anti-CD11b, -F4/80,

-CD3 or –CD19 mAb. In the Cy5-siRNA/WS injected mice, eighty-one percent of the

synovial cells were Cy5-positive, and most of the Cy5-positive cells (>75%) were CD11b+.

In addition, 92% of CD11b+ and 67% of F4/80+ cells were Cy5-positive, whereas, only 20%

of CD3+ cells were Cy5-positive. Although the majority of CD11b+ cells in the synovium of

naked Cy5-siRNA-treated mice was also Cy5-positive (82%), fewer F4/80+ and CD3+ cells

were Cy5-positive (1% and 10%, respectively) compared with Cy5-siRNA/WS-treated mice

(Figure 2C). CD11b+ synovial cells in CIA mainly consisted of macrophages (70%) and

secondary dominant neutrophils (30%) (data not shown). CD19 positive cells were very few

in the synovial cells (less than 0.1%).

These results suggest that both selective delivery and retention of Cy5-siRNA to

inflamed synovium could be achieved using WS as a carrier of the siRNA, and that


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systemically administered siRNA/WS would efficiently target macrophages and neutrophils

in inflamed joints.

Efficacy of siRNA Targeting TNF-α/WS for CIA. Experimental evidence indicates

that functional blockade of TNF-α effectively inhibits CIA (Kim et al., 2002). Accordingly,

we analyzed the effect of systemic administration of siRNA/WS targeting TNF-α on CIA.

Mice treated with TNF-α siRNA/WS at 1 and 10 μg showed substantially decreased and

delayed incidence of arthritis over those treated with control siRNA/WS (P<0.05. TNF-α

siRNA/WS 10 μg v.s. control 10 μg, at day 7, Figure 3A). Treatment with 10 μg siRNA/WS

targeting TNF-α significantly lowered the disease severity expressed as clinical arthritis score

and paw thickness, compared with control siRNA/WS (Figures 3B and C). On the other hand,

naked TNF-α siRNA had no effect on CIA (data not shown). In addition, TNF-α mRNA

levels in the paws of TNF-α siRNA/WS (10 μg)-treated mice were significantly lower than

those treated with control siRNA/WS (Figure 3D). No signs of toxicity were observed both in

control siRNA/WS and TNF-α siRNA/WS treated mice. These results suggest that

down-regulation of TNF-α expression in the synovial tissue by systemically administered

TNF-α-targeting siRNA/WS inhibited CIA.


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Discussion

In this study, we showed that systemically administered siRNA/WS complex

accumulated in arthritic joint in murine CIA, where the complex was preferably incorporated

into CD11b+ macrophages and neutrophils. Moreover, treatment with siRNA/WS targeting

TNF-α ameliorated CIA.

RA is characterized by chronic synovitis affecting multiple joints, and synovial

macrophages play central roles in the pathogenesis by producing various proinflammatory

cytokines such as TNF-α, interleukin (IL)-1, and IL-6. Treatment with anti-TNF-α mAb was

effective for murine CIA (Williams et al., 1992). Moreover, TNF-α blockade was efficacious

in the treatment of RA (Smolen et al., 2007). However, systemic administration of TNF-α

inhibitor could impair immune system, which increases the development of infectious

diseases (Komano et al., 2011), including reactivation of latent infections such as tuberculosis

(Keane et al., 2001). Therefore, selective delivery of siRNA that targets macrophages in the

affected synovia at multiple anatomical sites is an ideal strategy. Our results demonstrated

that this goal can be achieved using the siRNA/WS formation, and such treatment might be

potentially useful for RA in human by inhibition of cytokine production by synovial

macrophages. Our method could also target other proinflammatory cytokines or intracellular

molecules in the synovial macrophages that are known to play some roles in chronic joint

inflammation (Mosser et al., 2008).

In arthritic synovium, incorporation of siRNA/WS was observed in majority of

CD11b+ cells and in more than half of the F4/80+ cells, whereas a few in the CD3+ cells.

CD11b+ synovial cells in our CIA model mainly consisted of macrophages and neutrophils. It

was reported that F4/80+ synovial cells were mature macrophages (Morris et al., 1991). The

mechanism of the selective incorporation of siRNA/WS in macrophages and neutrophils in

the inflamed synovium is not fully understood. It is unlikely that the selective siRNA delivery


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by siRNA/WS complex depends simply on the innate phagocytic capability of the cells.

Investigator in our group reported that the complex was able to transduce therapeutic siRNA

selectively into cancer cells in mice in vivo (Yagi, et al., 2009). It is possible that the

siRNA/WS is preferentially delivered and enter the affected synovium based on increased

angiogenesis and vascular permeability in such inflammatory tissues. Indeed, the size of

siRNA/WS is designed to maximize enhanced permeability and retention effect (Hall et al.,

2007). In addition, we recently found that systemically injected siRNA/WS was accumulated

in 12-O-tetradecanoylphorbol-13-acetate treated skin area in a mouse model of skin

inflammation (Yagi N: unpublished observations). This data also indicates that the

siRNA/WS has nature of accumulating into the inflamed tissue, probably due to increase in

vascular permeability. Alternatively, activated macrophages and neutrophils in the inflamed

synovium may have enhanced siRNA/WS uptake activity. Further studies are needed to

examine these probabilities and to determine the mechanism of action of siRNA/WS. The

siRNA/WS system could be potentially applicable to other systemic diseases in which

macrophages play a central pathogenic role. Understanding the molecular mechanism of

inflammatory tissue-selective siRNA/WS delivery should help us further improve the

therapeutic effect of WS-mediated siRNA delivery.

There are only a few studies that reported the success of siRNA-based therapy for RA

in animal models (Inoue et al., 2005; Khoury et al., 2008; Khoury et al., 2006; Schiffelers, et

al., 2005). Khoury et al. reported that systemic administration of a cationic liposome carrying

siRNA directed against TNF-α or a cocktail of siRNAs directed against proinflammatory

cytokines, including IL-1, -6, and -18, was effective against CIA (Khoury, et al., 2008;

Khoury, et al., 2006). The cationic liposome used in the above studies, however, could

rapidly be distributed to reticuloendotherial systems due to the micron-sized diameter and

positive surface property. In contrast, siRNA/WS was designed to be nano-sized (100 nm in


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diameter) and neutrally charged, resulting in its efficient distribution into peripheral inflamed

tissue (Yagi, et al., 2009). Indeed, we confirmed remarkable accumulation and cellular

incorporation of the siRNA encapsulated WS in inflamed synovium, thus, the relative

contribution of macrophages in the affected joints to the therapeutic effect of siRNA might be

higher in the WS than the previously reported cationic liposome.

Treatment with TNF-α siRNA/WS from day 0 (when clinical arthritis just started

developing) showed therapeutic effect in CIA. However, when we started the treatment from

day 7 (when the arthritis were established), the CIA was not suppressed (data not shown).

Combining siRNA/WS with specific targeting approach (e.g. surface modification of

liposome) may improve therapeutic efficacy of this system (Koning et al., 2006).

In conclusion, our results showed that the siRNA/WS system enabled efficient

delivery of siRNA to arthritic joints. The siRNA/WS were incorporated into CD11b+

macrophages and neutrophils in the inflamed synovium, suggesting that it could be a

therapeutic tool to silence the expression of various molecules associated with the

pathogenesis of RA produced by these cells.


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Acknowledgments

We are grateful to Aya Sato for the excellent technical support.

Authorship Contributions

Participated in research design: Komano, Miyasaka, Nanki.

Conducted experiments: Komano. Yagi, Onoue, Kaneko.

Contributed new reagents or analytic tools: Yagi

Performed data analysis: Komano.

Wrote or contributed to the writing of the manuscript: Komano, Miyasaka, Nanki.


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Footnotes

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry

of Health, Labor and Welfare, the Ministry of Education, Culture, Sports, Science, and

Technology, Japan, and the Japanese Ministry of Education, Global Center of Excellence

(GCOE) Program, International Research Center for Molecular Science in Tooth and Bone

Diseases.


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Legends for Figures

Figure 1. Accumulation of systemically administered siRNA/WS in arthritic sites

At day 7 after induction of CIA, mice with arthritis (scored more than 2 at least in a paw)

were injected with saline, naked Cy5-siRNA or Cy5-siRNA/WS. A, mice were examined 12

hrs later for accumulation of the Cy5-fluorescence by stereoscopic microscope. The arrows

indicate the sites where the fluorescence was observed. B, Arabic numerals on each paw

indicate arthritis score (top), and arrows show the accumulation of the fluorescence observed

in the corresponding paws (bottom). C, Fluorescence observed in arthritic hind paws of mice

injected either with naked Cy5-siRNA(150 μg /body) or Cy5-siRNA/WS (150 μg /body) at 0

min (immediately after the injection) and 12 hrs. D, Time course of the fluorescence intensity

in arthritic hind paws (arthritis score =3) of mice injected with naked Cy5-siRNA (150 μg

/body) or Cy5-siRNA/WS (150 μg /body). Data are mean ± SEM, n=3. A-C, Representative

photographs of 8 mice are shown.

Figure 2. Tissue distributions of systemically administered siRNA/WS

At day 7 after induction of CIA, mice with arthritis (scored more than 2 at least in a paw)

were injected with saline, naked Cy5-siRNA or Cy5-siRNA/WS. A, Twelve hrs after the

injection, the levels of Cy5-fluorescence in cells extracted from the synovium, spleen, bone

marrow and peripheral blood leukocytes were analyzed by flow cytometry. B, Mean

fluorescence intensity (MFI) of Cy5 in the tissues. RFU, relative fluorescence unit. C,

Synovial cells from the mice injected with naked Cy5-siRNA or Cy5-siRNA/WS were

stained with anti-CD11b, -F4/80, or -CD3 mAb, and Cy5 expression in CD11b+, F4/80+, or

CD3+ cells was analyzed by flow cytometry. The percentage of Cy5-positive cells was

determined for each cell type. n=2-3 for each condition (A-C). Synovial cells from the three

mice were analyzed altogether (A-C).


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JPET #185884

21

Figure 3. Effects of systemic administration of siRNA targeting TNF-α/WS on CIA

A-C, TNF-α siRNA/WS (1 or 10 μg) or control siRNA/WS (10 μg) was injected

intravenously in CIA mice three times per week starting from the day of the second

immunization (day 0) to day 10. The incidence of arthritis (A), arthritis score (B), and paw

thickness (C) were evaluated continuously until day 10. D, At day 12, TNF-α mRNA

expression in the paw was analyzed by real-time RT-PCR. Data are mean ± SEM of 5-13

mice in each group. *P<0.05 mice treated with TNF-α siRNA/WS 10 μg v.s. control

siRNA/WS. ■; control siRNA/WS 10 μg/body, △; TNF-α siRNA/WS 1 μg/body, ●; TNF-α

siRNA/WS 10 μg/body, ◆; normal


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0

10000

20000

30000

40000

A C

0 3

1 3

2 0

3 1

B

３ 2 ３2

0 hr

12 hrs

Naked Cy5-siRNA150 μg

Cy5-siRNA/WL150 μg

Fluorescenceimage

Photograph

Cy5-siRNA/WL (150 μg) injected mice

Cy5-siRNA/WL (150 μg) injected mice

Fluorescence image of arthritic hind paws

D

Figure 1

12 24 48 72 960relative fluorescence intensity

（hrs）

post-injection

⃟ naked Cy5-siRNA

■ Cy5-siRNA/WS

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spleen

peripheral blood

leukocyte

synovium

bone marrow

Cy5 fluorescence

A

C

0 200 400 600 800

peripheral W BC

bone m arrow

spleen

synovium

B

0 200 400 600 800

MFI (RFU)

spleen

synovium

bone marrow

saline

naked Cy5-siRNA

Cy5-siRNA/WS

peripheral blood

leukocyte

Figure 2

20

40

60

80

0Cy-5 positive cells

%100

□ naked Cy5-siRNA-treated mice

■ Cy5-siRNA/WS-treated mice

CD11ｂ＋ F4/80＋ CD3＋




JPET





incid

ence o

f art

hritis (

%)

0

20

40

60

80

100

0 3 5 7 10

0

2

4

6

8

0 3 5 7 10

art

hritis s

core

A C

days after the second immunization


**

*

B

-2.22E-16

0.2

0.4

0.6

0.8

1

1.2

control siRNA/WS TNF-α siRNA/WSrela

tive tra

nscript le

vel

0

*

Figure 3

1.5

2

2.5

3

1 2 3 4thic

kness o

f paw

s (

mm

)

D

0 5 7 10


**




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