siRNA-treated Mice Research Articles

Inhibition of Interferon Regulatory Factor 4 Attenuates Acute Liver Allograft Rejection in Mice.

Acute liver allograft rejection is a serious complication after liver transplantation. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. However, the role of IRF4 in liver transplantation has never been investigated. In our current study, to evaluate the effect of IRF4 inhibition on recipient survival, IRF4 siRNA, or control siRNA, or PBS was injected into the liver allograft recipients through caudal vein. The survival time of mice treated with IRF4 siRNA (MST=31.5days) was prolonged significantly compared with that of mice treated with PBS (MST=6days) or control siRNA (MST=6.5days) (P<0.001). IRF4 siRNA treatment displays lower induction of pro-inflammatory levels, including TNF-α, IL-6 and IFN-γ, and higher induction of anti-inflammatory IL-10 levels. Administration of anti-IL-10 into IRF4 siRNA-treated mice resulted in shortened allograft survival and increased rejection scores. Furthermore, IRF4 inhibition promotes M2 macrophage differentiation invivo and invitro. And inhibition of macrophages with GdCl3 reverses the prolonged liver allograft survival and decreased liver rejection scores induced by IRF4 siRNA. In conclusion, inhibition of IRF4 attenuates acute liver allograft rejection in mice, and this is associated with promoted M2 macrophage differentiation.

Knockdown of NF-κB p65 subunit expression suppresses growth of nude mouse lung tumour cell xenografts by inhibition of Bcl-2 apoptotic pathway.

Nuclear factor-kappaB (NF-κB) is an important transcriptional factor and regulates a variety of pathophysiologic process involved in cell survival and death. This study aimed to assess the effects of NF-κB p65 subunit knockdown in suppression of nude mouse lung tumour cell xenografts and understands the underlying molecular events. A nude mouse Lewis lung carcinoma cell xenograft model was established, and the mice were intraperitoneally injected with NF-κB p65 small interfering RNA (siRNA) and sacrificed after 2weeks of tumour cell injection. Tumour xenografts were harvested for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Western blot and quantitative reverse transcription-polymerase chain reaction analyses. Results found that compared to the phosphate-buffered saline (PBS)-treated or the negative control (NC) siRNA-treated mice tumour xenograft weight and volume were significantly decreased in the NF-κB p65 siRNA-treated mice. The TUNEL positive (apoptosis) cells in xenograft sections were 45 ± 5 in PBS and 38 ± 3 in NC siRNA but increased to 271 ± 11 in p65 siRNA-treated mice. Compared to the PBS or the NC mice, levels of Bcl-2 mRNA and protein in tumour xenografts were significantly downregulated in p65 siRNA-treated mice. Knockdown of NF-κB p65 subunit expression can significantly inhibit the growth of nude mouse Lewis tumour cell xenografts by inducing tumour cell apoptosis and downregulating pro-apoptotic protein Bcl-2 expression. Targeting NF-κB p65 subunit expression as a potential therapeutic strategy needs further evaluation in treating human lung cancer.

Tanshinone IIA exerts protective effects in a LCA-induced cholestatic liver model associated with participation of pregnane X receptor

Tanshinone IIA (Tan IIA) is one of the main natural active ingredients purified from Salvia miltiorrhiza radix, which has long been used in clinical practice in China to treat diseases including liver fibrosis, Alzheimer׳s disease, and cardiovascular diseases. Tan IIA has hepatoprotective properties, and is an efficacious PXR agonist. Our study was designed to observe the function and mechanism of the hepatoprotective properties of Tan IIA. HepG2 cells were used to investigate the vitrol effects of Tan IIA on PXR and CYP3A4. Gut-formed LCA is hepatotoxic, and has been implicated in the pathogenesis of cholestatic diseases. To further investigate the hepatoprotective mechanisms of Tan IIA against LCA-induced cholestasis in vivo, we choose the normal mice and siRNA-treated mice. The in vitro study demonstrated that the effect of Tan IIA on CYP3A4 was mediated by transactivation of PXR in a dose- and time-dependent manner. The in vivo experiments using PXR siRNA revealed that Tan IIA could protect against LCA-induced hepatotoxicity and cholestasis in a dose-dependent manner. These effects were partially caused by the upregulation of PXR, as well as Cyp3a11, Cyp3a13, and Mdr1, which are the enzymes responsible for LCA metabolism. This is the first report showing that the hepatoprotective effects of Tan IIA are partly mediated by PXR.

Knockdown of NF-κB p65 subunit expression suppresses growth of nude mouse lung tumor cell xenografts by activation of Bax apoptotic pathway.

Nuclear factor-kappaB (NF-κB) is an important transcriptional factor and regulates avariety of pathophysiologic process involved in cell survival and death. The present study assesses the effects of NF-κB p65 subunit knockdown in suppression of nude mouse lung tumor cell xenografts and understands the underlying molecular events.A nude mouse Lewis lung carcinoma cell xenograft model was established and the mice were intraperitoneally injected with NF-κB p65 siRNA and sacrificed after two weeks of tumor cell injection. Tumor xenografts were harvested for TUNEL, Western blot, and qRT-PCR analyses.Compared to the PBS-treated or the negative control (NC) siRNA-treated mice, tumor xenograft weight and volume was significantly decreased in the NF-κB p65 siRNA-treated mice. The TUNEL positive (apoptosis) cells in xenograft sections were 45 ± 5 in PBS and 38 ± 3 in NC siRNA, but increased to 271 ± 11 in p65 siRNA-treated mice. Compared to the PBS or the NC mice, levels of Bax mRNA and protein in tumor xenografts were significantly upregulated in p65 siRNA-treated mice. Knockdown of NF-κB p65 subunit expression significantly inhibited the growth of nude mouse Lewis tumor cell xenografts by induction of tumor cell apoptosis and significantly up-regulation of pro-apoptotic protein Bax expression. Future study will confirm the current data and targeting NF-κB p65 subunit expression as apotential therapeutic strategy in treating human lung cancer. NF-κB, lung cancer, apoptosis, small interfering RNA, xenografts.

Chitosan/siRNA nanoparticles targeting cyclooxygenase type 2 attenuate unilateral ureteral obstruction-induced kidney injury in mice.

Cyclooxygenase type 2 (COX-2) plays a predominant role in the progression of kidney injury in obstructive nephropathy. The aim of this study was to test the efficacy of chitosan/small interfering RNA (siRNA) nanoparticles to knockdown COX-2 specifically in macrophages to prevent kidney injury induced by unilateral ureteral obstruction (UUO). Using optical imaging techniques and confocal microscopy, we demonstrated that chitosan/siRNA nanoparticles accumulated in macrophages in the obstructed kidney. Consistent with the imaging data, the obstructed kidney contained a higher amount of siRNA and macrophages. Chitosan-formulated siRNA against COX-2 was evaluated on RAW macrophages demonstrating reduced COX-2 expression and activity after LPS stimulation. Injection of COX-2 chitosan/siRNA nanoparticles in mice subjected to three-day UUO diminished the UUO-induced COX-2 expression. Likewise, macrophages in the obstructed kidney had reduced COX-2 immunoreactivity, and histological examination showed lesser tubular damage in COX-2 siRNA-treated UUO mice. Parenchymal inflammation, assessed by tumor necrosis factor-alpha (TNF-α) and interleukin 6 mRNA expression, was attenuated by COX-2 siRNA. Furthermore, treatment with COX-2 siRNA reduced heme oxygenase-1 and cleaved caspase-3 in UUO mice, indicating lesser oxidative stress and apoptosis. Our results demonstrate a novel strategy to prevent UUO-induced kidney damage by using chitosan/siRNA nanoparticles to knockdown COX-2 specifically in macrophages.

Abstract 3892: Inhibitory effects and regulatory mechanism of nestin in pancreatic cancer

Abstract Introduction: Nestin, a progenitor/stem cell marker, is expressed in human pancreatic cancer, and we have previously shown that its expression positively correlates with invasiveness and metastasis of the cancer (Cancer Biol Ther, 2011; Am J Pathol. in press). Here, we examined the inhibitory effects of nestin expression using nestin-targeted small interfering RNA (siRNA) in pancreatic cancer cells. We also investigated the regulatory mechanism of nestin expression in pancreatic cancer. Methods: siRNA targeting nestin was transfected to the pancreatic cancer cell lines PANC-1 and PK-45H, and we analyzed the effect on cell behaviors including proliferation, migration, invasion, sphere-forming ability, and sensitivity to gemcitabine. Next, to clarify the mechanisms of nestin expression, pancreatic cancer cells were injected into the spleen of NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic (NOG) mice, and pancreatic cancer cells were also collected from metastatic foci of NOG mice. We compared the methylation status of the promoter and the 1st and 2nd introns of the nestin gene in metastatic and parental cancer cells using bisulfite sequencing and pyrosequencing. Results: Nestin siRNA inhibited the proliferation, migration, invasion, and sphere-forming ability of the pancreatic cancer cells in vitro. Co-treatment of pancreatic cancer cells with gemcitabine and nestin siRNA decreased cell viability compared to administration of gemcitabine or nestin siRNA alone. Cells derived from the metastatic nodules of NOG mice showed higher expression of nestin compared with parental cells, and methylation of the nestin promoter and the 1st and 2nd introns was lower in the metastatic cells. Compared to control siRNA-treated mice, nestin siRNA significantly inhibited primary and metastatic tumor formation derived from human pancreatic cancer cells in an orthotopic implantation model using NOG mice. In summary, decreasing nestin expression in pancreatic cancer cells using nestin siRNA suppressed cell proliferation, migration, invasion, and sphere formation in vitro and primary and metastatic tumor formation in vivo. Furthermore, nestin siRNA had synergistic effects with gemcitabine in inhibiting pancreatic cancer cell growth. These findings suggest that nestin plays a key role in pancreatic cancer cell metastasis and stemness, and that administration of nestin siRNA is a novel therapeutic strategy for pancreatic cancer. Citation Format: Yoko Matsuda, Toshiyuki Ishiwata, Hisashi Yoshimura, Satoshi Yamashita, Toshikazu Ushijima, Kaiyo Takubo, Tomio Arai. Inhibitory effects and regulatory mechanism of nestin in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3892. doi:10.1158/1538-7445.AM2014-3892

Abstract 252: Renal Rescue of D2R Function in Mice Using an AAV9 Vector Reverses the Renal Injury and High Blood Pressure Induced by D2R Silencing in the Kidney

Lack or downregulation of dopamine D2 receptor (D2R) function in mice increases the vulnerability to renal inflammation. Recent studies from our laboratory showed that long-term (28 days) selective down regulation of D2R in one kidney in mice with two intact kidneys increases systolic blood pressure and the expression of inflammatory and injury markers. We hypothesized that rescuing D2R function by re-expressing the receptor in the kidney of mice with D2R silencing will reduce renal inflammation and injury, and normalize blood pressure. To test our hypothesis we silenced D2R expression by the renal subcapsular infusion of D2RsiRNA and then we rescued the silenced D2R expression using retrograde ureteral infusion of AAV9 vector carrying wild-type D2R (D2RAAV). Mice were treated with non-silencing siRNA (NS siRNA) or D2RsiRNA (3 μg/day, n=6 per group) using 28 day osmotic minipumps. Fourteen days following the initiation of siRNA treatment, each group was treated with control AAV (CAAV) or D2RAAV (n=3 per group, 1e+11 viral genome particles per mouse). Fourteen days following AAV administration, blood pressure was recorded and organs were collected. D2R expression was decreased in D2RsiRNA-treated kidneys compared with NS siRNA-treated kidneys (immunoblotting: 54 ± 0.8 vs 100± 22 %; P&lt;0.05). D2RAAV treatment increased D2R expression (7.5 -11 fold; P&lt;0.01) in both D2R and NS siRNA-treated mice. Mice treated with D2RsiRNA+CAAV had increased systolic blood pressure (121±3 mmHg; P&lt;0.05) in comparison with mice treated with D2RsiRNA+D2RAAV (100±6 mm Hg) (rescued mice), NS siRNA +CAAV (101±4 mm Hg), or NS siRNA +D2RAAV (101±1 mm Hg). The expression of TGF-β was higher in the mice treated with D2RsiRNA+CAAV than in D2R rescued mice (D2RsiRNA+D2RAAV) (100±22 vs 54± 11%; P&lt;0.05). The number of cells positive for the marker of proliferation ki-67 was higher in kidneys of D2RsiRNA+CAAV mice than in D2RsiRNA+D2RAAV (32± 5 vs 15±2 per field; P&lt;0.01). In conclusion, these results demonstrate that increased blood pressure and renal injury due to D2R silencing could be rescued by over expression of D2R using AAV9 vector in the kidney. Furthermore, these data confirms the protective effects of renal D2R and may provide the basis for designing novel therapies for kidney disease.

Silencing of CCR2 in myocarditis.

Myocarditis is characterized by inflammatory cell infiltration of the heart and subsequent deterioration of cardiac function. Monocytes are the most prominent population of accumulating leucocytes. We investigated whether in vivo administration of nanoparticle-encapsulated siRNA targeting chemokine (C-C motif) receptor 2 (CCR2)-a chemokine receptor crucial for leucocyte migration in humans and mice--reduces inflammation in autoimmune myocarditis. In myocardium of patients with myocarditis, CCL2 mRNA levels and CCR2(+) cells increased (P < 0.05), motivating us to pursue CCR2 silencing. Flow cytometric analysis showed that siRNA silencing of CCR2 (siCCR2) reduced the number of Ly6C(high) monocytes in hearts of mice with acute autoimmune myocarditis by 69% (P < 0.05), corroborated by histological assessment. The nanoparticle-delivered siRNA was not only active in monocytes but also in bone marrow haematopoietic progenitor cells. Treatment with siCCR2 reduced the migration of bone marrow granulocyte macrophage progenitors into the blood. Cellular magnetic resonance imaging (MRI) after injection of macrophage-avid magnetic nanoparticles detected myocarditis and therapeutic effects of RNAi non-invasively. Mice with acute myocarditis showed enhanced macrophage MRI contrast, which was prevented by siCCR2 (P < 0.05). Follow-up MRI volumetry revealed that siCCR2 treatment improved ejection fraction (P < 0.05 vs. control siRNA-treated mice). This study highlights the importance of CCR2 in the pathogenesis of myocarditis. In addition, we show that siCCR2 affects leucocyte progenitor trafficking. The data also point to a novel therapeutic strategy for the treatment of myocarditis.

Synergic silencing of costimulatory molecules prevents cardiac allograft rejection

BackgroundWhile substantial progress has been made in blocking acute transplant rejection with the advent of immune suppressive drugs, chronic rejection, mediated primarily by recipient antigen presentation, remains a formidable problem in clinical transplantation. We hypothesized that blocking co-stimulatory pathways in the recipient by induction of RNA interference using small interference RNA (siRNA) expression vectors can prolong allogeneic heart graft survival.MethodVectors expressing siRNA specifically targeting CD40 and CD80 were prepared. Recipients (BALB/c mice) were treated with CD40 and/or CD80 siRNA expression vectors via hydrodynamic injection. Control groups were injected with a scrambled siRNA vector and sham treatment (PBS). After treatment, a fully MHC-mismatched (BALB/c to C57/BL6) heart transplantation was performed.ResultAllogeneic heart graft survival (>100 days) was approximately 70% in the mice treated simultaneously with CD40 and CD80 siRNA expression vectors with overall reduction in lymphocyte interstitium infiltration, vascular obstruction, and edema. Hearts transplanted into CD40 or CD80 siRNA vector-treated recipients had an increased graft survival time compared to negative control groups, but did not survive longer than 40 days. In contrast, allogenic hearts transplanted into recipients treated with scrambled siRNA vector and PBS stopped beating within 10–16 days. Real-time PCR (RT-PCR) and flow cytometric analysis showed an upregulation of FoxP3 expression in spleen lymphocytes and a concurrent downregulation of CD40 and CD80 expression in splenic dendritic cells of siRNA-treated mice. Functional suppressive activity of splenic dendritic cells (DCs) isolated from tolerant recipients was demonstrated in a mixed lymphocyte reaction (MLR). Furthermore, DCs isolated from CD40- and CD80-treated recipients promoted CD4 + CD25 + FoxP3+ regulatory T cell differentiation in vitro.ConclusionThis study demonstrates that the simultaneous silencing of CD40 and CD80 genes has synergistic effects in preventing allograft rejection, and may therefore have therapeutic potential in clinical transplantation.

CARD11 blockade suppresses murine collagen-induced arthritis via inhibiting CARD11/Bcl10 assembly and T helper type 17 response

The scaffold protein caspase recruitment domain-containing protein 11 (CARD11) is implicated in the regulation of inflammation and autoimmunity. The present study aimed to explore the role of CARD11 in the pathogenesis of rheumatoid arthritis (RA). Mice with collagen-induced arthritis (CIA) were treated with either CARD11-targeted interfering RNA (CARD11 siRNA) or control siRNA by intraperitoneal injection every 3 days after CIA establishment. The clinical score of arthritis was recorded every other day. Synovial inflammation and cartilage erosion were evaluated by histology and microcomputed tomography (micro-CT). Serum anti-type II collagen (anti-CII) antibodies and cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The CARD11/Bcl10 formation and nuclear factor-kappa B (NF-κB) activation was assessed by immunoprecipitation and immunoblotting, and the percentage of T helper type 17 (Th17) cells was determined by flow cytometry. Systemic administration of CARD11 siRNA significantly reduced the clinical score of CIA severity. As indicated by the histology, joint inflammation and destruction were attenuated by CARD11 siRNA treatment. Micro-CT demonstrated less severe joint destruction in CARD11 siRNA-treated mice than in control mice. CARD11 siRNA treatment resulted in inhibition of CARD11/Bcl10 formation and the subsequent NF-κB activation. In addition, treatment with CARD11 siRNA resulted in a pronounced decrease in proinflammatory cytokines interleukin (IL)-1β, IL-6 and IL-17. Serum anti-CII antibody and the percentage of Th17 cells were also significantly reduced. CARD11 is involved in the pathogenesis of CIA by formation of the CARD11/Bcl10 complex and enhancement of the Th17 cell response. Targeting CARD11 provides a novel research direction in the development of therapeutic strategies for RA.

Selective inhibition of STAT1 reduces spinal cord injury in mice

The signal transducer and activator of transcription 1 (STAT1) is associated with neuronal cell death after cerebral ischemia. However, the role of STAT1 in the spinal cord injury (SCI) remains unclear. Here, we examined whether STAT1 blockade reduces neural tissue damage and locomotor impairment after SCI in mice. The small interfering RNA against STAT1 (STAT1 siRNA) or control non-targeting siRNA was injected intraperitoneally into SCI mice. Histological damage and locomotor function were evaluated. Inflammatory markers and apoptosis were determined. STAT1 siRNA treatment significantly decreased the histological damage following SCI. STAT1 siRNA-treated mice showed significantly improved locomotor function compared with the controls. Furthermore, TNF-α, IL-1β, and IL-6 levels at the injured site from the STAT1 siRNA-treated group were significantly reduced and IL-10 increased, in comparison with controls. The NF-κB activation and apoptosis in SCI were also inhibited. These results reveal that selective STAT1 inhibition reduced neural tissue damage and locomotor impairment by regulating inflammatory response and possibly apoptosis. STAT1 represents a novel therapeutic target after SCI.

Receptor-Interacting Protein 2 Gene Silencing Attenuates Allergic Airway Inflammation

Persistent activation of NF-κB has been associated with the development of asthma. Receptor-interacting protein 2 (Rip2) is a transcriptional product of NF-κB activation. It is an adaptor protein with serine/threonine kinase activity and has been shown to positively regulate NF-κB activity. We investigated potential protective effects of Rip2 gene silencing using small interfering RNA (siRNA) in an OVA-induced mouse asthma model. Rip2 protein level was found to be upregulated in allergic airway inflammation. A potent and selective Rip2 siRNA given intratracheally knocked down Rip2 expression in OVA-challenged lungs and reduced OVA-induced increases in total and eosinophil counts, and IL-4, IL-5, IL-13, IL-1β, IL-33, and eotaxin levels in bronchoalveolar lavage fluid. Rip2 silencing blocked OVA-induced inflammatory cell infiltration and mucus hypersecretion as observed in lung sections, and mRNA expression of ICAM-1, VCAM-1, E-selectin, RANTES, IL-17, IL-33, thymic stromal lymphopoietin, inducible NO synthase, and MUC5ac in lung tissues. In addition, elevation of serum OVA-specific IgE level in mouse asthma model was markedly suppressed by Rip2 siRNA, together with reduced IL-4, IL-5, and IL-13 production in lymph node cultures. Furthermore, Rip2 siRNA-treated mice produced significantly less airway hyperresponsiveness induced by methacholine. Mechanistically, Rip2 siRNA was found to enhance cytosolic level of IκBα and block p65 nuclear translocation and DNA-binding activity in lung tissues from OVA-challenged mice. Taken together, our findings clearly show that knockdown of Rip2 by gene silencing ameliorates experimental allergic airway inflammation, probably via interruption of NF-κB activity, confirming Rip2 a novel therapeutic target for the treatment of allergic asthma.

Silencing TNF-α in macrophages and dendritic cells for arthritis treatment

Objectives: Tumour necrosis factor (TNF)-α secreted by macrophages and dendritic cells (DCs) plays a predominant role in arthritis. Our previous studies suggest that a small peptide, RVG-9R (29-aa peptide derived from the rabies virus glycoprotein, fused to 9R residues), can deliver small interfering RNA (siRNA) to macrophages and DCs. We therefore tested whether knockdown of TNF-α expression in macrophages and DCs by RVG-9R/bound siRNA targeting TNF-α reduces the severity of collagen antibody-induced arthritis (CAIA) in mice.Method: Arthritis was induced in mice by injecting a combination of antibodies to collagen followed by lipopolysaccharide (LPS) treatment. Mice were also injected with TNF-α siRNA complexed with RVG-9R peptide or an irrelevant peptide RVMAT-9R on days 1, 3, 5, and 7. As a positive control, dexamethasone was injected intravenously. Paw thickness was measured every 2 days and the mice were killed on day 10 for testing synovial TNF-α levels and histological analysis of joints.Results: In control mice, arthritis developed on day 4 and reached its peak between day 7 and day 9. Treatment with siTNF-α bound to RVG-9R, but not to RVMAT-9R, resulted in reducing paw thickness scores to the same level as dexamethasone treatment, associated with reduced TNF-α level in synovial fluid. Histological analysis of joints in the control RVMAT-9R/TNF-α siRNA-treated mice showed marked pannus formation and destruction of cartilage and subchondrial bone, as well as severe infiltration of inflammatory cells into the synovium. By contrast, the joint pathology was markedly reduced in RVG-9R/TNF-α siRNA-treated mice resembling the dexamethasone-treated mice.Conclusions: Suppression of TNF-α expression in macrophages and DCs by RVG-9R-mediated siRNA delivery could potentially be a clinically viable strategy for treatment of arthritis.

Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the gastrointestinal tract. The cytokine TNF-alpha (TNF-α) plays a pivotal role in mediating this inflammatory response. RNA interference (RNAi) holds great promise for the specific and selective silencing of aberrantly expressed genes, such as TNF-α in IBD. The aim of this study was to investigate the efficacy of an amphiphilic cationic cyclodextrin (CD) vector for effective TNF-α siRNA delivery to macrophage cells and to mice with induced acute-colitis. The stability of CD.siRNA was examined by gel electrophoresis in biorelevant media reflecting colonic fluids. RAW264.7 cells were transfected with CD.TNF-α siRNA, stimulated with lipopolysaccharide (LPS) and TNF-α and IL-6 responses were measured by PCR and ELISA. Female C57BL/6 mice were exposed to dextran sodium sulphate (DSS) and treated by intrarectal administration with either CD.siRNA TNF-α or a control solution. In vitro, siRNA in CD nanocomplexes remained intact and stable in both fed and fasted simulated colonic fluids. RAW264.7 cells transfected with CD.TNF-α siRNA and stimulated with LPS displayed a significant reduction in both gene and protein levels of TNF-α and IL-6. CD.TNF-α siRNA-treated mice revealed a mild amelioration in clinical signs of colitis, but significant reductions in total colon weight and colonic mRNA expression of TNF-α and IL-6 compared to DSS-control mice were detected. This data indicates the clinical potential of a local CD-based TNF-α siRNA delivery system for the treatment of IBD.

Changes in Innate and Permissive Immune Responses after HBV Transgenic Mouse Vaccination and lLong-Term-siRNA Treatment

BackgroundCurrently, no licensed therapy can thoroughly eradicate hepatitis B virus (HBV) from the body, including interferon α and inhibitors of HBV reverse-transcription. Small interfering RNA (siRNA) seem to be a promising tool for treating HBV, but had no effect on the pre-existing HBV covalently closed circular DNA. Because it is very difficult to thoroughly eradicate HBV with unique siRNAs, upgrading the immune response is the best method for fighting HBV infection. Here, we aim to explore the immune response of transgenic mice to HBV vaccination after long-term treatment with siRNAs and develop a therapeutic approach that combines siRNAs with immunopotentiators.Methodology/Principal FindingsTo explore the response of transgenic mice to hepatitis B vaccine, innate and acquired immunity were detected after long-term treatment with siRNAs and vaccination. Antiviral cytokines and level of anti-hepatitis B surface antigen antibody (HBsAg-Ab) were measured after three injections of hepatitis B vaccine.ResultsFunctional analyses indicated that toll-like receptor-mediated innate immune responses were reinforced, and antiviral cytokines were significantly increased, especially in the pSilencer4.1/HBV groups. Analysis of CD80+/CD86+ dendritic cells in the mouse liver indicated that dendritic cell antigen presentation was strengthened. Furthermore, the siRNA-treated transgenic mice could produce detectable HBsAg-Ab after vaccination, especially in the CpG oligonucleotide vaccine group.Conclusions/SignificanceFor the first time, our studies demonstrate that siRNAs with CpG HBV vaccine could strengthen the immune response and break the immune tolerance status of transgenic mice to HBV. Thus, siRNAs and HBV vaccine could provide a sharp double-edged sword against chronic HBV infection.

Therapeutic Silencing of Bcl-2 by Systemically Administered siRNA Nanotherapeutics Inhibits Tumor Growth by Autophagy and Apoptosis and Enhances the Efficacy of Chemotherapy in Orthotopic Xenograft Models of ER (−) and ER (+) Breast Cancer

Bcl-2 is overexpressed in about a half of human cancers and 50–70% of breast cancer patients, thereby conferring resistance to conventional therapies and making it an excellent therapeutic target. Small interfering RNA (siRNA) offers novel and powerful tools for specific gene silencing and molecularly targeted therapy. Here, we show that therapeutic silencing of Bcl-2 by systemically administered nanoliposomal (NL)-Bcl-2 siRNA (0.15 mg siRNA/kg, intravenous) twice a week leads to significant antitumor activity and suppression of growth in both estrogen receptor-negative (ER(−)) MDA-MB-231 and ER-positive (+) MCF7 breast tumors in orthotopic xenograft models (P < 0.05). A single intravenous injection of NL-Bcl-2-siRNA provided robust and persistent silencing of the target gene expression in xenograft tumors. NL-Bcl-2-siRNA treatment significantly increased the efficacy of chemotherapy when combined with doxorubicin in both MDA-MB-231 and MCF-7 animal models (P < 0.05). NL-Bcl-2-siRNA treatment-induced apoptosis and autophagic cell death, and inhibited cyclin D1, HIF1α and Src/Fak signaling in tumors. In conclusion, our data provide the first evidence that in vivo therapeutic targeting Bcl-2 by systemically administered nanoliposomal-siRNA significantly inhibits growth of both ER(−) and ER(+) breast tumors and enhances the efficacy of chemotherapy, suggesting that therapeutic silencing of Bcl-2 by siRNA is a viable approach in breast cancers.

Nonviral Delivery of Small Interfering RNA Into Pancreas-associated Immune Cells Prevents Autoimmune Diabetes

The development of small interfering RNA (siRNA) for the treatment of human disorders has been often hampered by their low transfection efficiency in vivo. In order to overcome this major drawback, various in vivo siRNA transfection methods have been developed. However, their capacity to transfect immune or insulin-producing β-cells within the pancreas for the treatment of autoimmune diabetes remains undetermined. We found that lipid- or polyethylenimine-based delivery agents were efficient to address siRNA molecules within pancreas-associated antigen-presenting cells (APCs) (but not β-cells) and particularly a CD11b(+) cell population comprising both CD11b(+)CD11c(neg) macrophages and CD11b(+)CD11c(+) dendritic cells. However, the route of administration and the carrier composition greatly affected the transfection efficacy. Therapeutically, we showed that early (starting at 6-week-old) short-course treatment with lipid/Alox15-specific siRNA complex promoted long-term protection from type 1 diabetes (T1D) in wild-type (WT) nonobese diabetic (NOD) mice. Alox15 downregulation in pancreas-associated CD11b(+) cells significantly upregulated a variety of costimulatory molecules and particularly the programmed death 1 ligand 1 (PD-L1) pathway involved in tolerance induction. Concomitantly, we found that regulatory T cells were increased in the pancreas of lipid/Alox15 siRNA-treated NOD mice. Collectively, our data provide new insights into the development of siRNA-based therapeutics for T1D.

Targeting Gonadotropin-releasing Hormone Receptor Inhibits the Early Step of Ovarian Cancer Metastasis by Modulating Tumor-mesothelial Adhesion

Ovarian cancer has a clear predilection to metastasize to the peritoneum, which represents one of the most important prognostic factors of poor clinical outcome. Gonadotropin-releasing hormone (GnRH) receptor is significantly overexpressed during the malignant progression of human ovarian cancer. Here, using lentiviral-based small interfering RNA (siRNA) technology to downregulate GnRH receptor in metastatic ovarian cancer cells, we show that GnRH receptor is an important mediator of ovarian cancer peritoneal metastasis. GnRH receptor downregulation dramatically attenuated their adhesion to the peritoneal mesothelium. By inhibiting the expression of GnRH receptor, we showed decreased expression of α2β1 and α5β1 integrin and adhesion to specific extracellular matrix (ECM) proteins. This was also associated with a reduction of P-cadherin. Furthermore, adhesion of ovarian cancer cells to different ECMs and the mesothelium were abrogated in response to β1 integrin and P-cadherin reduction, confirming that the effects were β1 integrin- and P-cadherin-specific. Using a mouse model of human ovarian cancer metastasis, we found that the inhibition of GnRH receptor, β1 integrin, and P-cadherin significantly attenuated tumor growth, ascites formation, and the number of metastatic implants. These results define a new role for GnRH receptor in early metastasis and offer the possibility of novel therapeutic targets.

Abstract 5710: Effective siRNA delivery through specific targeting of tumor

Abstract The siRNA has been expected to apply for several diseases such as cancer since siRNA specifically silences the disease-associated genes. However, effective gene carriers should be developed to overcome the low siRNA stability in vivo, form stable complexes and facilitate intracellular uptake of siRNA. Here, we have investigated tumor targetable oligopeptide carrier including tumor-homing peptide, AP-1, and rich positive peptide, nine arginines, for systemic siRNA delivery. Phage display technique is a promising tool for selecting peptides or proteins with specific binding properties from a lot of variants. AP-1 peptide can bind with interleukin-4 receptor which expressed on surface of several human cancer cells like MDA-MB231. Arginine rich peptides have been widely used for forming complex with DNA, which promote efficient transfection in various mammalian cells. Oligopeptides formed ionic complex with siRNA at 150 nm size and these peptide/siRNA complexes were transfected in MDA-MB231. Under in vitro silencing experiments, peptide/siRNA complexes did silencing of Dronpa gene as similar to lipofectamine/siRNA complexes, which were observed by fluorescent microscope, flow cytometry, and western blotting analysis. For in vivo experiments, we prepared tumorbearing mice by inoculation of Dronpa-MDA-MB231 cells (Dronpa-stable cells). Tumor targetable oligopeptide/siRNA complexes were administrated through tale vein when tumor volume reached at 50 mm3 - we observed fluorescent signals of Dronpa in tumor. In tumor targetable oligopeptide/siRNA-treated mice, weak signals of Dronpa were observed compared with free siRNA-treated mice. These results revealed the promising potential of our oligopeptide carrier as a stable and effective siRNA delivery system for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5710. doi:1538-7445.AM2012-5710

Silencing of c-kit with small interference RNA attenuates inflammation in a murine model of allergic asthma

Asthma is a chronic respiratory disease characterized by the inflammation of the airways due to infiltration and activation of several inflammatory cells that produce cytokines. c-kit, a proto-oncogene that encodes a tyrosine kinase receptor, has been found to be associated with allergic inflammation. The aim of the present study was to assess whether silencing of c-kit with small interference RNA (siRNA) would attenuate inflammation in allergic asthma. A mouse model of ovalbumin (OVA)-induced allergic asthma was treated with systemic administration of anti-c-kit siRNA to inhibit the expression of the c-kit gene. siRNAs were injected through the vena caudalis. We measured inflammatory response in both anti-c-kit siRNA-treated and control mice. Systemic administration of siRNA could effectively inhibit the expression of the c-kit gene and reduce the infiltration of inflammatory cells (eosinophils and lymphocytes) into the lung tissue and bronchoalveolar lavage fluid. In addition, we found that c-kit siRNA can decrease the production of the T-helper type 2 (Th2) cytokines, interleukin 4 (IL-4) and IL-5, but has no influence on IFN-γ generation. These results show that inhibition of c-kit expression with siRNA can reduce the inflammatory response in allergic asthma.