Search

PDF file - 485K

PDF file - 82K

PDF file - 477K

Purpose: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node metastasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the pharmacologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four different preclinical mouse models.Experimental Design: Atu027 compared with vehicle or control small interfering RNA lipoplexes was tested in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous metastasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules (repeated low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. Primary tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistochemistry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to provide an insight into molecular changes on depletion of PKN3, in support of efficacy results.Results: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formation of spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well as in mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an increase in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change endothelial function, resulting in reduced colonization and micrometastasis formation.Conclusion: Atu027 can be considered as a potent drug for preventing lung metastasis formation, which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy. Clin Cancer Res; 16(22); 5469–80. ©2010 AACR.

PDF file - 174K

Supplementary Figure 4 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Supplementary Figure 1 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Supplementary Figure 3 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Supplementary Table 1 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Supplementary Table 2 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Supplementary Figure 5 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

Supplementary Figure 6 from Atu027, a Liposomal Small Interfering RNA Formulation Targeting Protein Kinase N3, Inhibits Cancer Progression

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density. [Cancer Res 2008;68(23):9788–98]

Summary Beta‐thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co‐receptor for the bone morphogenetic protein (BMP)‐mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in β‐thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in β‐thalassaemia mice (Hbbth3/+). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of β‐thalassaemia and related disorders.

Polycythemia Vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients’ hematocrit and hemoglobin concentration, placing them at risk of life-threatening thrombotic events. Our GWAS of 440 PV cases and 403,351 controls utilising UK Biobank data found that SNPs in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed over-representation of homozygous HFE mutations in PV patients. HFE influences expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of PV mouse models, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Further, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signalling via GP130 coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

Circulating levels of endothelial miR-150 are reduced in pulmonary arterial hypertension (PAH) and act as an independent predictor of patient survival, but links between endothelial miR-150 and vascular dysfunction are not well understood. We studied the effects of endothelial miR-150 supplementation and inhibition in PAH mice and cells from patients with idiopathic PAH. The role of selected mediators of miR-150 identified by RNA sequencing was evaluated in vitro and in vivo. Endothelium-targeted miR-150 delivery prevented the disease in Sugen/hypoxia mice, while endothelial knockdown of miR-150 had adverse effects. miR-150 target genes revealed significant associations with PAH pathways, including proliferation, inflammation, and phospholipid signaling, with PTEN-like mitochondrial phosphatase (PTPMT1) most markedly altered. PTPMT1 reduced inflammation and apoptosis and improved mitochondrial function in human pulmonary endothelial cells and blood-derived endothelial colony-forming cells from idiopathic PAH. Beneficial effects of miR-150 in vitro and in vivo were linked with PTPMT1-dependent biosynthesis of mitochondrial phospholipid cardiolipin and reduced expression of pro-apoptotic, pro-inflammatory, and pro-fibrotic genes, including c-MYB, NOTCH3, transforming growth factor β (TGF-β), and Col1a1. In conclusion, we are the first to show that miR-150 supplementation attenuates pulmonary endothelial damage induced by vascular stresses and may be considered as a potential therapeutic strategy in PAH., Graphical Abstract, Pulmonary arterial hypertension is a severe disease with no cure. Loss of endothelial microRNA-150 is detrimental, while supplementation of microRNA-150 prevents the disease by reducing fibrosis, proliferation, and inflammation and improving mitochondrial energy production in lung and heart via increased production of the mitochondrial phospholipid cardiolipin.

Background & Aims: Perturbations of intracellular magnesium (Mg2+) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH). Methods: Serum Mg2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine. Results: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs. Conclusions: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH. Lay summary: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein., Graphical Abstract

Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNABid was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicleHEPES) and siRNALuci served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNABid caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.

Transient hepatic steatosis upon liver resection supposes functional relationships between lipid metabolism and liver regeneration. Repin1 has been suggested as candidate gene for obesity and dyslipidemia by regulating key genes of lipid metabolism and lipid storage. Herein, we characterized the regenerative potential of mice with a hepatic deletion of Repin1 (LRep1−/−) after partial hepatectomy (PH) in order to determine the functional significance of Repin1 in liver regeneration. Lipid dynamics and the regenerative response were analyzed at various time points after PH. Hepatic Repin1 deficiency causes a significantly decreased transient hepatic lipid accumulation. Defects in lipid uptake, as analyzed by decreased expression of the fatty acid transporter Cd36 and Fatp5, may contribute to attenuated and shifted lipid accumulation, accompanied by altered extent and chronological sequence of liver cell proliferation in LRep1−/− mice. In vitro steatosis experiments with primary hepatocytes also revealed attenuated lipid accumulation and occurrence of smaller lipid droplets in Repin1-deficient cells, while no direct effect on proliferation in HepG2 cells was observed. Based on these results, we propose that hepatocellular Repin1 might be of functional significance for early accumulation of lipids in hepatocytes after PH, facilitating efficient progression of liver regeneration.

Supplemental Digital Content is available in the text

Background Hepcidin, a peptide hormone consisting of 25-amino-acids, is the central regulator of systemic iron homeostasis. It is synthesized predominantly in hepatocytes, and dysregulation of its production leads to a variety of disorders of iron metabolism, including iron overload as well as congenital or acquired iron-loading anaemias. These conditions are a major source of morbidity and mortality. SLN124 increases hepatic hepcidin synthesis and hence plasma hepcidin by silencing its repressor, TMPRSS6. SLN124 has been shown to lower serum iron levels for at least 6 weeks after single administration in mice and has also been shown to increase haemoglobin in a mouse model of beta-thalassemia. Here we report results from a randomised, double-blind, placebo-controlled phase 1, single ascending dose study to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of subcutaneously (sc) administered SLN124 in healthy volunteers. Objectives The primary objective was to evaluate the safety and tolerability of single ascending doses of SLN124 in healthy subjects. In addition, PK parameters of SLN124 and PD biomarkers of iron metabolism were evaluated. Methods Each subject received a single dose of SLN124 or placebo given by sc injection into their abdomen. Dose levels of 1 mg/kg, 3 mg/kg and 4.5 mg/kg were evaluated. At each dose level, 6 subjects received SLN124 and 2 received matching placebo. Results Three cohorts of 8 subjects (6:2) were included in the study. The mean age of subjects was 31.3 years (SD 7.8) and 71% were male. There were no serious adverse events or severe treatment emergent adverse events (TEAEs) or TEAEs leading to withdrawal. The majority of TEAEs were mild, including transient mild injection site reactions, which resolved without intervention. No dose limiting toxicities were observed. Plasma hepcidin levels at baseline were (mean ± SD) 2.3±1.1, 2.5±1.8 and 2.7±2.0 nM in the three treatment groups, respectively. After a single administration of 1, 3 or 4.5 mg/kg of SLN124, levels were increased by 3.1±2.7, 5.8±2.6 and 7.8±2.9 nM on day 29 and by 2.6±2.6, 2.8±1.4 and 3.5±1.8 nM day 57 post dosing, respectively. Serum iron was reduced by mean (±SD) 32% (26), 40% (14) ,46% (21) on day 8 and by 42% (20), 48% (14) and 47% (26) on day 29 for single doses of 1, 3 and 4.5 mg/kg, respectively. Average percentage changes in all three treatment groups were also still reduced from baseline at day 57. The mean (±SD) percent transferrin saturation (TSAT) was reduced from baseline levels in all single dose administration groups with maximum decreases observed at day 29 but reductions still observed at day 57. (Table 1). The observed PK is consistent with the PK model based on preclinical data. Thus, whereas the effects of SLN124 on plasma hepcidin show clear increments across the dose range tested, the effect on lowering serum iron and transferrin saturation shows something of a plateau effect at the highest dose. In future studies in patients with raised baseline transferrin saturations, it will be of note to determine whether transferrin saturation continues to decrease at the highest SLN124 doses. Conclusion/summary In summary, SLN124, a GalNAc conjugated siRNA targeting TMPRSS6, effectively reduces serum iron levels and has the potential as a therapeutic for patients with alpha and beta-thalassemia, myelodysplastic syndromes, hereditary hemochromatosis and other related disorders. The effect on hepcidin levels at the end of study, day 57, suggest a prolonged duration of action. The encouraging efficacy signal on serum iron and transferrin saturation and the expected benign safety profile of SLN124 supports further development. The GEMINI II trial (NCT04718844) is currently recruiting and wil l evaluate single and multiple doses of SLN124 in patients withthalassemia and myelodysplastic syndromes. Figure 1 Figure 1. Disclosures Porter: bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla Pharmaceuticals: Honoraria; Celgene (BMS): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees. Scrimgeour: Silence Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Martinez: Silence Therapeutics: Current Employment, Current holder of stock options in a privately-held company. James: Silence Therapeutics: Consultancy. Aleku: Silence Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Wilson: Silence Therapeutics: Consultancy. Muckenthaler: Silence Therapeutics: Research Funding. Schaeper: Silence Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Campion: Silence Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company.

Liver international (2017). doi:10.1111/liv.13595, Published by Wiley-Blackwell, Oxford

Introduction & Aim: Hemophilia A (HA) is an X-linked disorder caused by an absence or a reduction of coagulation factor VIII. Patients with HA often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis. Hemarthrosis is caused by bleeding into joint spaces affecting the synovium, synovial blood vessels as well as cartilage and bone tissues. Current prophylactic treatments are not always effective and hemophilia patients can experience breakthrough bleeds. Recently, we demonstrated that inhibition of protein S (PS), a natural anticoagulant, controls coagulation and constitutes a potential therapeutic target in hemophilia (Blood 2018, 131:1360-1371). Here, we aim to translate our findings using small interfering RNA conjugated to an N-acetylgalactosamine (GalNAc) cluster to target Pros1 gene expression (GalNAc-PS siRNA) in vivo and exclusively in hepatocytes. siRNAs conjugated to a GalNAc cluster bind to asialoglycoprotein receptors expressed predominantly by hepatocytes thereby providing a potentially safe, specific and efficient delivery technology for therapeutic molecules. Methods & Results: Forty-two days after subcutaneous (s.c.) injection of GalNAc-PS siRNA (3mg/kg), wild-type (WT) mice were alive and did not display overt disseminated intravascular coagulation (DIC). In a second study in WT mice, DIC parameters assessed fourteen days after treatment with either 5mg/kg GalNAc-PS siRNA or with vehicle were also comparable between the two groups (platelet count: 578±284 vs 725±186 G/L, p>0.9, n=4-6; prothrombin time: 9.0±0.4 vs 8.9±0.3 seconds, p>0.9, n=4-6; fibrinogen: 1.5±0.5 vs 1.8±0.4 g/L, p>0.85, n=5-6; thrombin anti-thrombin complexes, TAT: 71±63 vs 115±34 μg/L, p>0.9, n=2-4) supporting that GalNAc-PS siRNA treatment can be safe. At the same time, mice treated with GalNAc-PS siRNA displayed lower plasma PS level compared to mice receiving the vehicle (52±12 vs 100±11 %, p To assess if targeting PS using GalNAc-siRNA-PS protects mice from acute hemarthrosis (AH), we applied an AH model to F8-/- mice. Five days after injecting a single dose of 5mg/kg GalNAc-PS s.c., right knees were injured using a 30 gauge-needle and knee diameters were measured 72 hours later. Macroscopically, vehicle treated F8-/- mice developed extensive bleeding in injured knees as compared to GalNAc-siRNA-PS treated mice. Scores for intra-articular bleeding (2.4±0.9 vs 1.0±0.7, p=0.035, n=5-9) and synovial hyperplasia (2.4±0.9 vs 0.6±0.9, p=0.027, n=5-9) were higher in F8-/- mice treated by vehicle than in those treated by GalNAc-PS-siRNA. Moreover, knee joint swelling was reduced in GalNAc-siRNA-PS treated mice compared to those treated by vehicle (0.14±0.15 vs 0.78±0.50 mm, p=0.025, n=7-10). As expected, PS plasma levels were lower in GalNAc-PS siRNA treated mice compared to those which received vehicle (63±9 vs 101±19% of WT PS antigen level, p0.9, n=7-12; prothrombin time: 8.5±0.3 vs 8.4±0.3 seconds, p>0.9, n=4-13; fibrinogen: 2.7±0.6 vs 3.0±0.8 g/L, p=0.73, n=7-12 and TAT: 33±42 vs 45±66 μg/L, p>0.9, n=6-10). Conclusion: These data provide the first evidence that using a GalNAc-siRNA conjugate to modulate Pros1 gene expression is well tolerated and has the ability to reduce plasma PS level and protect F8-/- mice from AH pointing to PS targeting using GalNAc-siRNA-PS as a new valuable therapeutic approach for hemophilia. Further analysis to understand if the inhibition of PS influences also the inflammatory processes causing the hemophilic arthropathy is ongoing. Disclosures Schaeper: Silence Therapeutics GmbH: Current Employment. Dames:Silence Therapeutics: Current Employment. Eisermann:Silence Therapeutics: Current Employment.

Summary Increased vascular permeability and leakage are hallmarks of several pathologies and determine disease progression and severity by facilitating inflammatory/metastatic cell infiltration. Using tissue-specific genetic ablation in endothelial cells, we have investigated in vivo the role of Tumor progression locus 2 (Tpl2), a mitogen-activated protein kinase kinase kinase (MAP3K) member with pleiotropic effects in inflammation and cancer. In response to proinflammatory stimuli, endothelial Tpl2 deletion alters tight junction claudin-5 protein expression through inhibition of JNK signaling and lysosomal degradation activation, resulting in reduced vascular permeability and immune cell infiltration. This results in significantly attenuated disease scores in experimental autoimmune encephalomyelitis and fewer tumor nodules in a hematogenic lung cancer metastasis model. Accordingly, pharmacologic inhibition of Tpl2 or small interfering RNA (siRNA)-mediated Tpl2 knockdown recapitulates our findings and reduces lung metastatic tumor invasions. These results establish an endothelial-specific role for Tpl2 and highlight the therapeutic potential of blocking the endothelial-specific Tpl2 pathway in chronic inflammatory and metastatic diseases.

Increased vascular permeability and leakage are hallmarks of several pathologies and determine disease progression and severity by facilitating inflammatory / metastatic cell infiltration. Using tissue-specific genetic ablation in endothelial cells, we have investigated in vivo the role of Tpl2, a MAP3 kinase with pleiotropic effects in inflammation and cancer. In response to pro-inflammatory stimuli, endothelial Tpl2 deletion alters Tight Junction (TJ) claudin-5 protein expression and attenuates the increase in vascular permeability, inflammatory cell infiltration and accumulation of plasma proteins. This results in significantly attenuated disease scores in Experimental Autoimmune Encephalomyelitis and significantly less tumor nodules in a haematogenic lung cancer metastasis model. Accordingly, pharmacologic inhibition of Tpl2 or siRNA-mediated Tpl2 knockdown recapitulates our findings and reduces lung metastatic tumor invasions. These results establish a novel endothelial-specific role for Tpl2 in the regulation of TJ-mediated endothelial permeability and highlight the therapeutic potential of Tpl2 blockade in chronic inflammatory and metastatic diseases.

Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood.To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension.Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition.Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

Activation of hepatic stellate cells (HSCs) is a key event in pathogenesis of liver fibrosis and represents an orchestral interplay of inhibiting and activating transcription factors like forkhead box f1 (Foxf1), being described to stimulate pro-fibrogenic genes in HSCs. Here, we evaluated a lipidbased liver-specific delivery system (DBTC) suitable to transfer Foxf1 siRNA specifically to HSCs and examined its antifibrotic potential on primary HSCs and LX-2 cells as well as in a murine model of bile duct ligation (BDL)-induced secondary cholestasis. Foxf1 silencing reduced proliferation capacity and attenuated contractility of HSCs. Systemic administration of DBTC-lipoplexes in mice was sufficient to specifically silence genes expressed in different liver cell types. Using intravital and immunofluorescence microscopy we confirmed the specific delivery of Cy3-labeled DBTC to the liver, and particularly to HSCs. Repeated treatment with DBTC-lipoplexes resulted in siRNA-mediated silencing of Foxf1 early after BDL and finally attenuated progression of the fibrotic process. Decreased HSC activation in-effect ameliorated liver injury as shown by substantial reduction of necrotic area and deposition of extracellular matrix. Our findings suggest that Foxf1 may serve as a target gene to disrupt progression of liver fibrosis and DBTC might provide a potentially feasible and effective tool for HSC-specific delivery of therapeutic RNA.

Development of portosystemic collateral vessels and gastroesophageal varices is responsible for the most serious clinical consequences of portal hypertension, but effective clinical therapies are limited. Here we developed and investigated the therapeutic potential of an innovative liposomally-formulated short-interfering RNA (siRNA) technology based on clinical stage components, capable to attenuate production of the endothelial kinase insert domain receptor (KDR), which controls portosystemic collateralization and contributes to disease progression and aggravation. These siRNAs were first validated in vitro, and then, their therapeutic potential on portosystemic collateralization and pathological angiogenesis was tested in vivo in mouse models of portal hypertension (portal vein-ligation). siRNAKDR-lipoplexes efficiently transported siRNAKDR to vascular endothelial cells in mesenteric microvenules and portal vein of portal hypertensive mice, where collaterogenesis and angiogenesis take place. This systemic treatment significantly downregulated pathological KDR overexpression, without causing complete KDR knockout, preserving homeostatic baseline KDR levels and thus limiting adverse effects. siRNAKDR-lipoplex-induced endothelial-specific KDR knockdown drastically reduced by 73% the portosystemic collateralization, and impaired the pathologic angiogenic potential of vascular endothelial cells at different levels (cell proliferation, sprouting and remodeling). Targeting endothelial KDR with therapeutic siRNAKDR-lipoplexes could be a promising and plausible treatment modality for attenuating the formation of portosystemic collaterals in a clinical setting.

Preeclampsia is a pregnancy-related disorder characterized by hypertension and proteinuria, affecting about 5-8% of pregnancies. It is a major cause of fetal and maternal morbidity/mortality. The disease is most probably multifactorial and caused by several placental dysfunctions. As an important mediator, sFLT1 is induced in the placenta and in the serum of preeclamptic women, acting as a decoy receptor for placenta like growth factors (PLGF) and inducing an antiangiogenic balance. RNAi mediated gene specific silencing could represent a new therapeutic option in preeclampsia management. We tested sFLT1-specific small interfering RNA (siRNA) formulated with the liposomal siRNA delivery system AtuPLEX for the ability to ameliorate symptoms without deteriorating fetal health in an established rat model of preeclampsia. Transgenic rats expressing human angiotensinogen (hAGT) and renin (hREN) were crossed to produce a model of preeclampsia (PE rat) in the dams. Beginning on day 7 of gestation, transgenic hAGT dams were dosed intravenously with 2.8 mg/kg siRNA every third day through gestational day 19. Mean blood pressure was continuously recorded by radiotelemetry and 24 hour urine samples were collected in metabolic cages at day 17/18 of gestation. Rats were euthanized at day 21 of gestation. Biodistribution experiments showed that sFLT1 siRNA formulated with AtuPLEX delivers siRNA to the placenta but not to the embryo. In PE rats, our treatment was able to decrease sFLT1 mRNA expression in placenta, especially in the labyrinth layer. Circulating sFLT1 was also reduced by FLT1 siRNA treatment but did not show statistical significance. Silencing of sFLT1 did not prevent blood pressure increase in the last third of pregnancy in our preeclamptic rat model (159 ± 5 mmHg vehicle vs. 152 ± 2 mmHg sFLT1 siRNA). Proteinuria was not ameliorated (5712 ± 2038 μg/d vehicle vs. 3585 ± 1301 μg/d sFLT1 siRNA). In addition, sFLT1 siRNA had no influence on IUGR, measured by brain to liver weight ratio. AtuPLEX formulated siRNA specifically silenced maternal and placental sFLT1. The mode of delivery appears to be safe for fetal health. However, this sFLT1 knock down showed no protective effect on the preeclamptic phenotype in this Ang-II-based model.

Objective:Angiopoietin-2, a protein secreted by stimulated endothelium and an antagonist of the endothelium-stabilizing receptor Tie2, contributes to the pathophysiology of septic multiple organ dysfunction. We tested the therapeutic potential of a pulmonary-endothelium-specific RNA interference–bas

We have previously described a small interfering RNA (siRNA) delivery system (AtuPLEX) for RNA interference (RNAi) in the vasculature of mice. Here we report preclinical data for Atu027, a siRNA-lipoplex directed against protein kinase N3 (PKN3), currently under development for the treatment of advanced solid cancer. In vitro studies revealed that Atu027-mediated inhibition of PKN3 function in primary endothelial cells impaired tube formation on extracellular matrix and cell migration, but is not essential for proliferation. Systemic administration of Atu027 by repeated bolus injections or infusions in mice, rats, and nonhuman primates results in specific, RNAi-mediated silencing of PKN3 expression. We show the efficacy of Atu027 in orthotopic mouse models for prostate and pancreatic cancers with significant inhibition of tumor growth and lymph node metastasis formation. The tumor vasculature of Atu027-treated animals showed a specific reduction in lymph vessel density but no significant changes in microvascular density. [Cancer Res 2008;68(23):9788–98]

Acute liver failure (ALF) is a life threatening disease for which only few treatment options exist. The molecular pathways of disease progression are not well defined, but the death receptor Fas (CD95/Apo-1) appears to play a pivotal role in hepatocyte cell death and the development of ALF. Here, we explored posttranscriptional gene silencing of Fas by RNAi to inhibit pathophysiological gene expression. For targeting Fas expression in mice, Fas siRNA was formulated with the liver-specific siRNA delivery system DBTC. Treatment of mice with DBTC/siRNA(Fas) reduced Fas expression in the liver, but not in the spleen, lung, kidney or heart. Furthermore, silencing of Fas receptor was effective in blocking or reducing several aspects of ALF when it was tested in mice exposed to galactosamine/lipopolysaccharide (G/L), a well-known model of ALF. The application of DBTC/siRNA(Fas) 48 h prior G/L exposure resulted in amelioration of hepatic perfusion, reduction of hepatocellular death and increase of survival rate. The administration of DBTC/siRNA(Fas) formulation further diminished the inflammatory response upon G/L challenge, as indicated by a marked decrease of TNFα mRNA expression. However, IL-6 plasma concentration remained unaffectedly by DBTC/siRNA(Fas) formulation. Since the specific silencing of hepatic Fas expression only partially protected from inflammation, but completely attenuated apoptotic and necrotic cell death as well as microcirculatory dysfunction, the development of therapeutic strategies with DBTC lipoplex formulations to treat ALF should be combined with anti-inflammatory strategies to reach maximal therapeutic efficacy.

Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoiesis. Inhibitors of phosphatidylinositide 3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2. In contrast, Gab1 activation depends on recruitment and phosphorylation by the tyrosine kinase receptor RON, with which it is constitutively associated. RON activation induces the phosphorylation of Gab1, mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but not of signal transducer and activator of transcription 5 (Stat5). RON activation was sufficient to replace EPO in progenitor expansion but not in differentiation. In conclusion, we elucidated a novel mechanism specifically involved in the expansion of erythroblasts involving RON as a downstream target of the EpoR. (Blood. 2004;103:4457-4465)

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met–specific branching morphogenesis. It associates directly with c-Met via the c-Met–binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met–binding site is localized to a 13–amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met–binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1–specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling.

Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in tumorigenesis-restraining activity, we searched for cellular proteins that complex with CtBP. Here, we report the cloning and characterization of a 125-kDa protein, CtIP, that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. Our results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif.

Posttranscriptional gene silencing by RNA interference can be therapeutically exploited to inhibit pathophysiological gene expression. However, in contrast to the established effectiveness of RNAi in vitro, safe and effective delivery of siRNAs to specific organs and cell types in vivo remains the major hurdle. Here, we report the development and in vivo characterization of a novel siRNA delivery system (DACC lipoplex) suitable for modulating target gene expression specifically in the lung vasculature. Systemic administration of DACC in mice delivered siRNA cargo functionally to the lung pulmonary endothelium. A single dose of DACC lipoplexes administered by bolus injection or by infusion was sufficient to specifically silence genes expressed in pulmonary endothelial cells such as CD31, Tie-2, VE-cadherin, or BMP-R2. When tested in a mouse model for lung cancer, repeated treatment with DACC/siRNA(CD31) reduced formation of lung metastases and increased life span in a mouse model of experimental lung metastasis.

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Adenovirus E1B 19 kDa protein protects against cell death induced by viral infection and certain external stimuli. The Bcl-2 protein can functionally substitute for the E1B 19 kDa protein. To identify cellular targets for the 19 kDa protein, we used the two-hybrid screen in yeast. We have isolated cDNAs for three different proteins, designated Nip1, Nip2, and Nip3, that interact with the 19 kDa protein. Mutational analysis indicates that these proteins do not associate with 19 kDa mutants defective in suppression of cell death, suggesting a correlation between interaction of these proteins and suppression of cell death. These proteins also associate with discrete sequence motifs in the Bcl-2 protein that are homologous to motifs of the 19 kDa protein. Our results suggest that two diverse proteins, the E1B 19 kDa and the Bcl-2 proteins, promote cell survival through interaction with a common set of cellular proteins.

We have examined a series of small deletion mutants within exon 2 of the adenovirus 2/5 E1A oncogene product, the 243R protein, for immortalization, ras cooperative transformation, tumorigenesis and metastasis. Compared with wild-type 243R, various deletion mutants located between residues 193 and 243 cooperated more efficiently with ras to induce large transformed foci of less adherent cells that were tumorigenic and metastatic. However, the greatest enhancement of transformation (comparable to that obtained with a deletion of the C-terminal 67 amino acids) was observed with a mutant carrying a deletion of residues 225-238. This mutant was also more defective in immortalization. These results suggest that this 14 amino acid region may contain a function that is important for immortalization and negative modulation of tumorigenesis and metastasis. To identify cellular proteins that may associate with the exon 2-coded region of E1A (C-terminal half) and modulate its transformation potential, we constructed a chimeric gene coding for the C-terminal 68 amino acids of E1a fused to bacterial glutathione-S-transferase (GST). This fusion protein was used to purify cellular proteins that bind to the C-terminal region of E1a. A 48 kDa cellular protein doublet (designated CtBP) was found to bind specifically to the GST-E1a C-terminal fusion protein as well as to bacterially expressed full-length E1a (243R) protein. It also co-immunoprecipitated specifically with E1a. Analysis of a panel of GST-E1a C-terminal mutant proteins indicates that residues 225-238 are required for the association of E1a and CtBP, suggesting a correlation between the association of CtBP and the immortalization and transformation modulating activities of exon 2. CtBP is a phosphoprotein and the level of phosphorylation of CtBP appears to be regulated during the cell cycle, suggesting that it may play an important role during cellular proliferation.

Purpose: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node metastasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the pharmacologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four different preclinical mouse models. Experimental Design: Atu027 compared with vehicle or control small interfering RNA lipoplexes was tested in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous metastasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules (repeated low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. Primary tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistochemistry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to provide an insight into molecular changes on depletion of PKN3, in support of efficacy results. Results: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formation of spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well as in mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an increase in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change endothelial function, resulting in reduced colonization and micrometastasis formation. Conclusion: Atu027 can be considered as a potent drug for preventing lung metastasis formation, which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy. Clin Cancer Res; 16(22); 5469–80. ©2010 AACR.