Background One hemophilia treatment concept focuses on rebalancing coagulation and anticoagulation to restore normal blood clotting. Targeting the coagulation regulator, protein S (PS), in hemophilia shows promise to increase the generation of thrombin, a critical enzyme in the clotting process. Objectives This study aimed to: (1) assess whether inhibiting PS increases thrombin generation (TG) in plasma from individuals with hemophilia A (HA) and B (HB); and (2) develop a hepatocyte-targeted PS small interfering RNA (siRNA) therapy using N-acetylgalactosamine conjugation to restore hemostasis in hemophilia without increasing thromboembolic risks. Methods We assessed TG in plasma from patients with HA and HB. To target the liver specifically, we developed a PS-siRNA conjugated with N-acetylgalactosamine. This approach ensures that PS levels remain adequate in other cells, thereby minimizing the risk of thrombosis. Additionally, we evaluated the therapeutic potential of PS-siRNA in preclinical models. Results Inhibiting PS with a polyclonal antibody in plasma resulted in a 3- to 5-fold increase in TG in HA and a 4- to 9-fold increase in HB plasma, with a 70% reduction in plasma PS. In preclinical models, subcutaneous PS-siRNA therapy in HA mice and nonhuman primates successfully lowered PS levels and improved clot formation. It also prevented bleeding in both saphenous vein puncture and knee injury models in HA mice. Notably, it enhanced clotting without triggering widespread clot formation. Conclusion Reducing PS levels enhances TG in hemophilia models, and PS-siRNA therapy shows promise in improving hemostasis. This approach warrants further clinical investigation as a potential treatment for hemophilia.

This study identified advanced pharmacy practice experience (APPE) activities students wish to pursue via a qualitative analysis of goal setting. It also aimed to understand student goal setting alignment with core Entrustable Professional Activities (EPAs).SMART (specific, measurable, achievable, realistic/relevant, time-bound) goal data were reviewed, analyzed, and coded to develop an inductive thematic analysis. Overall frequency of SMART goal coding themes was analyzed using descriptive statistics, along with frequency of themes based on rotation type. Relative percentages of each rotation type were compared. A crosswalk between themes and EPA was created post-coding.Several themes among student SMART APPE goals from one complete academic year were identified (n = 79). A total of 1690 SMART goals were categorized into 22 themes. The top five most frequently utilized themes represent 60.6% of all goals, and included "Assess, Select, Recommend Therapy" (19%); "Patient Communication" (15.4%); "Foundational Knowledge" (12.4%); "Interprofessional Collaboration" (7.9%); and "Workflow, Roles and Responsibilities" (6%). The core EPA Patient Care Provider Domain was most common and accounted for 46.4% of SMART goals. The remainder included Information Master (18.8%); Practice Manager (16.1%); Interprofessional Education Team Member (7.9%); Population Health Promoter (6.6%); and Self-Developer (3.6%).Our analysis revealed that students set goals in alignment with the profession's primary focus of providing direct patient care however some EPAs were infrequently citied by students in their goal setting process which may mean there is less focus on these important skills.

Introduction Replacement factor therapy for hemophilia A (HA) and B (HB), inherited disorders caused by factor VIII (FVIII) or factor IX deficiency, has significant limitations: First, factor prophylaxis is administered intravenously (IV) more than once a week. Second, patients may still experience bleeding and, third, they may develop antibodies against these therapeutic agents. A bispecific antibody, Emicizumab, a functional analog of FVIIIa, has recently been approved in patients with HA, but additional treatment options for hemophilia are still needed. We reported that genetic ablation of anticoagulant protein S (PS) improves hemostasis in HA and HB mice and constitutes a novel therapeutic target (Blood 2018, 131:1360-1371). We also showed that partial reduction of PS by small interfering RNA conjugated to an N-acetylgalactosamine cluster (GalNAc-siRNA), exclusively targeting hepatocyte-expressed PS, protects mice with HA from acute hemarthrosis. (Blood 2020, 136 (Suppl 1): 20-21). Here, we studied hemostatic effects of partial PS reduction in human ex vivo HA models and characterized a novel GalNAc-siRNA drug candidate, SLN140, in a non-human primate (NHP) model of HA. Methods & Results Hemophilia severity correlates with the amount of thrombin generation (TG) in plasma. We therefore assessed the impact of PS lowering on TG in human platelet-free plasma (PFP) with low FVIII activity. Normal and PS-depleted PFP were mixed to obtain PFP samples with free PS levels from 0 to 94%. Addition of anti-FVIII antibody resulted in PFP with FVIII between 0 and 7%. TG assays were run with low tissue factor as a trigger. Reduction of free PS to 56% doubled peak TG in PFP containing 0-7% FVIII. Further PS reduction to 37% restored peak TG in PFP containing 0-7% FVIII, indicating that partial PS depletion is sufficient to normalize TG in HA. Since PS depletion (Stago) may also reduce tissue factor pathway inhibitor level, the next experiments were performed in normal PFP treated with anti-FVIII and anti-PS antibodies to achieve 0% residual FVIII activity and to lower free PS. Anti-FVIII antibodies lowered peak TG (10±0.1 nM vs 78±2 nM in untreated PFP). Concomitant addition of anti-PS antibodies to achieve 0% or 55% residual PS had similar impact of increasing TG peak (21±0.1 and 27±13 nM, respectively), endorsing that partial depletion of PS increases TG in PFP containing 0% FVIII (Fig 1A). These data were confirmed using severe HA patient derived-PFP. Addition of anti-PS antibodies resulted in an increase of peak TG (to 20±5 nM and to 20±8 nM at 0% and 55% free PS respectively, compared with 8±1 nM in untreated HA PFP), indicating that 45% PS reduction increased peak TG in severe HA PFP (Fig 1B). Finally, we assessed the effect of partial PS reduction by SLN140 in a NHP model of acquired HA (AHA). TG assays were run in the presence of 3.5nM soluble thrombomodulin to enable protein C activation and full assessment of all PS anticoagulant functions. AHA was induced by slow bolus IV injection of anti-FVIII antibody (20,000 BU/kg), which resulted in <0.5% FVIII activity and a reduction in peak TG compared to controls (16±3 nM vs 43±2 nM). NHPs then received either SLN140 or 0.9% NaCl subcutaneously (SC). A single dose of 3 or 10 mg/kg SLN140 reduced free PS level from day 14 to day 21 to about 45% and 25% of baseline, respectively (Fig 1C). At day 14, peak TG relative to baseline and endogenous thrombin potential were ~3-fold higher in the SLN140-treated groups than in the 0.9% NaCl treated group. On day 20, anti-FVIII antibody was injected into all NHPs and blood was collected after 4 hours. In this AHA model, SLN140 restored TG to normal range (peak TG relative to baseline: 0.9% NaCl: 18±8%; 3 mg/kg SLN140: 124±55%; 10 mg/kg SLN140: 123±68%, Fig 1D). Treatment with SLN140 was clinically well tolerated. Basic coagulation and safety parameters were not affected by PS reduction and were comparable in the control group and the 3 and 10 mg/kg SLN140 treatment groups (mean±SD): prothrombin time 14.3±1.0, 14.4±1.0, and 14.8±1.0 seconds; fibrinogen 2.6±0.5, 2.8±0.4, and 2.4±0.6 g/L; platelet count 417±65, 436±127, and 454±112 G/L; D-Dimer 134±41, 141±59, and 188±43 ng/mL, respectively. Conclusion PS reduction enhanced TG in human models of HA ex vivo and a single SC administration of SLN140 improved hemostasis in NHPs with severe HA. These results are encouraging for rebalancing hemostasis in hemophilia and support future clinical studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated small interfering RNA (siRNA) with a promising profile for addressing lipoprotein (a)-related cardiovascular risk. Here, we describe the findings from key preclinical safety studies. In vitro, SLN360 specifically reduced LPA expression in primary human hepatocytes with no relevant off-target effects. In rats, 10 mg/kg subcutaneous SLN360 was distributed specifically to the liver and kidney (peak 126 or 246 mg/g tissue at 6 h, respectively), with <1% of peak liver levels observed in all other tested organs. In vitro, no genotoxicity and no effect on human Ether-a-go-go Related Gene currents or proinflammatory cytokine production was observed, whereas in vivo, no SLN360-specific antibodies were detected in rabbit serum. In rat and nonhuman primate 29-day toxicology studies, SLN360 was well tolerated at all doses. In both species, known GalNAc-conjugated siRNA-induced microscopic changes were observed in the kidney and liver, with small increases in alanine aminotransferase and alkaline phosphatase observed in the high dose rats. Findings were in line with previously described siRNA-GalNAc platform-related effects and all observations were reversible and considered nonadverse. In cynomolgus monkeys, liver LPA messenger RNA and serum lipoprotein (a) were significantly reduced at day 30 and after an 8-week recovery period. No dose-related changes in safety assessment endpoints were noted. No SLN360-induced cytokine production, complement activation, or micronucleus formation was observed in vivo. The toxicological profile of SLN360 presented here is restricted to known GalNAc siRNA effects and no other toxicity associated with SLN360 has been noted. The preclinical profile of SLN360 confirmed suitability for entry into clinical studies.

Alternative pathway complement dysregulation with abnormal glomerular C3 deposits and glomerular damage is a key mechanism of pathology in C3 glomerulopathy (C3G). No disease-specific treatments are currently available for C3G. Therapeutics inhibiting complement are emerging as a potential strategy for the treatment of C3G. In this study, we investigated the effects of N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C3 component of complement that inhibits liver C3 expression in the C3G model of mice with heterozygous deficiency of factor H (Cfh +/- mice). We showed a duration of action for GalNAc-conjugated C3 siRNA in reducing the liver C3 gene expression in Cfh +/- mice that were dosed s.c. once a month for up to 7 mo. C3 siRNA limited fluid-phase alternative pathway activation, reducing circulating C3 fragmentation and activation of factor B. Treatment with GalNAc-conjugated C3 siRNA reduced glomerular C3d deposits in Cfh +/- mice to levels similar to those of wild-type mice. Ultrastructural analysis further revealed the efficacy of the C3 siRNA in slowing the formation of mesangial and subendothelial electron-dense deposits. The present data indicate that RNA interference-mediated C3 silencing in the liver may be a relevant therapeutic strategy for treating patients with C3G associated with the haploinsufficiency of complement factor H.

BACKGROUND AND AIMS:The LPA gene encodes apolipoprotein (a), a key component of Lp(a), a potent risk factor for cardiovascular disease with no specific pharmacotherapy. Here we describe the pharmacological data for SLN360, a GalNAc-conjugated siRNA targeting LPA, designed to address this unmet medical need. METHODS:SLN360 was tested in vitro for LPA knockdown in primary hepatocytes. Healthy cynomolgus monkeys received single or multiple subcutaneous doses of the SLN360 sequence ranging from 0.1 to 9.0 mg/kg to determine the pharmacokinetic and pharmacodynamic effects. Liver mRNA and serum biomarker analyses were performed. RESULTS:In vitro, the SLN360 sequence potently reduces LPA mRNA in primary cynomolgus and human hepatocytes, while no effect was observed on the expression of APOB or PLG. In vivo, SLN360 exposure peaks 2 h after subcutaneous injection with near full elimination by 24 h. Specific LPA mRNA reduction (up to 91% 2 weeks after dosing) was observed with only the 3 mg/kg group showing appreciable return to baseline (40%). No consistent dose- or time-dependent effect on the expression of APOB, PLG or a panel of sensitive markers of liver lipid accumulation was observed. Potent (up to 95%) and long lasting (≥9 weeks) serum Lp(a) reduction was observed, peaking in all active groups at day 21. The minimally effective dose was determined to be 0.3 mg/kg with an ED50 of 0.6 mg/kg. CONCLUSIONS:SLN360 induces a sustained reduction in serum Lp(a) levels in cynomolgus monkeys following subcutaneous dosing. SLN360 has potential to address the unmet need of Lp(a) reduction in cardiovascular diseases.

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.

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<0.001, n=6). In the liver, PS mRNA levels were reduced by 69% compared to mice treated just with the vehicle (31±10 vs 100±24 %, p<0.0001, n=6). Importantly, in a murine model for hemophilia A (F8-/- mice) the intrinsically-activated test using ellagic acid (INTEM) assessed by rotative thromboelastometry (ROTEM®) was improved by treatment with GalNAc-PS siRNA (5mg/kg s.c) as compared to F8-/- mice treated with the vehicle (clotting time: 281±193 vs 802±330 seconds, p<0.01, n=6-11; clot formation time: 109±80 vs 657±466 seconds, p<0.05, n=6-11; alpha angle: 70±13 vs 35±24 mm, p<0.1, n=6-11). 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, p<0.0001, n=7-13) with no overt DIC (platelets count: 711±149 vs 681±189 G/L, p>0.9, 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.

Introduction: SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated siRNA with a promising profile for addressing Lp(a)-related cardiovascular risk. We previously reported the pharmacodynamic effects of SLN360 in female (F) cynomolgus monkeys (cynos) 1 . Here we describe the findings from key pre-clinical safety studies. Methods: SLN360 was tested in vitro in primary human hepatocytes for LPA knockdown and effects on hypothetical off targets identified through in silico screening. An in vivo biodistribution study was performed in rats over 21 days (4 males [M]/4 F per timepoint; single 10 mg/kg s.c. injection). In a GLP safety study, healthy cynos received 5 once weekly s.c. injections of saline control or SLN360 (3 M / 3 F per group, up to 200 mg/kg) followed by an 8-week recovery period (2 M / 2 F in saline and top dose). A standard battery of safety assessments was performed. Results: In vitro, SLN360 reduced LPA expression in primary human hepatocytes with no effects on any hypothetical liver-expressed human off-target genes at concentrations >300-fold the human LPA IC 50 . SLN360 showed a typical rodent GalNAc distribution pattern, with significant levels in liver and kidney (peak 126 or 246 mg/g tissue at 6h respectively; estimated liver half-life of 56 hours). Levels in other organs (including reproductive organs) were <1% of peak liver levels. In cynos, no clinical observations were observed following 5 s.c. doses of SLN360. Liver LPA mRNA levels were significantly reduced by 98% at day 30 and 95% after the 8-week recovery period, while serum Lp(a) was undetectable at both timepoints. Findings were restricted to the liver (increased weight and diffuse hepatocyte hypertrophy) and lymph nodes (vacuolated macrophages) at day 30. These were considered non-adverse due to reversibility after recovery. No dose-related changes in clinical chemistry, hematology, circulatory and ECG parameters, respiratory rate, neurobehavior, plasma cytokines, complement activation or CRP were noted. The NOAEL was defined as 200 mg/kg, >60-fold the active dose in cynos. Conclusions: Overall, the off-target profile, biodistribution and NOAEL of SLN360 indicate suitability for entry into clinical studies. 1 Aleku et al., 2019. Circulation. 2019, 140:A9538.

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Introduction: SLN360 is a liver targeted GalNAc-conjugated siRNA developed for the treatment of cardiovascular disorders associated with elevated Lp(a). Lp(a) is a key independent, genetically dete...

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.

Abstract 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.

wnloaded pose: Atu027, a novel RNA interference therapeutic, has been shown to inhibit lymph node tasis in orthotopic prostate cancer mouse models. The aim of this study is to elucidate the acologic activity of Atu027 in inhibiting hematogenous metastasis to the target organ lung in four nt preclinical mouse models. erimental Design: Atu027 compared with vehicle or control small interfering RNA lipoplexes was in two experimental lung metastasis models (Lewis lung carcinoma, B16V) and spontaneous tasis mouse models (MDA-MB-435, MDA-MB-231, mammary fat pad). Different dosing schedules ted low volume tail vein injections) were applied to obtain insight into effective Atu027 treatment. ry tumor growth and lung metastasis were measured, and tissues were analyzed by immunohistostry and histology. In vitro studies in human umbilical vein endothelial cells were carried out to e an insight into molecular changes on depletion of PKN3, in support of efficacy results. ults: Intravenous administration of Atu027 prevents pulmonary metastasis. In particular, formaf spontaneous lung metastasis was significantly inhibited in animals with large tumor grafts as well mice with resected primary mammary fat pad tumors. In addition, we provide evidence that an se in VE-cadherin protein levels as a downstream result of PKN3 target gene inhibition may change helial function, resulting in reduced colonization and micrometastasis formation. clusion: Atu027 can be considered as a potent drug for preventing lung metastasis formation, Con which might be suitable for preventing hematogenous metastasis in addition to standard cancer therapy.