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BACKGROUND: IgA nephropathy is the most common primary GN. Clinical features of IgA nephropathy include proteinuria, which is the strongest known surrogate of progression to kidney failure. Complement pathway activation is a critical driver of inflammation and tissue injury in IgA nephropathy. Cemdisiran is an investigational RNA interference therapeutic that suppresses hepatic production of complement component 5 (C5), thereby potentially reducing proteinuria in IgA nephropathy. We evaluated the efficacy and safety of cemdisiran in adult patients with IgA nephropathy at high risk of kidney disease progression. METHODS: In this phase 2, 36-week, double-blind study, adult patients with IgA nephropathy and urine protein ≥1 g/24 hours were randomized (2:1) to subcutaneous cemdisiran 600 mg or placebo every 4 weeks in combination with the standard of care. The primary end point was percentage change from baseline at week 32 in urine protein-to-creatinine ratio (UPCR) measured by 24-hour urine collection. Additional end points included change from baseline in UPCR measured by spot urine, serum C5 level, and safety assessments. RESULTS: Thirty-one patients were randomized (cemdisiran, N =22; placebo, N =9). Cemdisiran-treated patients had a placebo-adjusted geometric mean change in 24-hour UPCR of -37.4% (cemdisiran-adjusted geometric mean ratio to baseline [SEM], 0.69 [0.10]) at week 32. Spot UPCR was consistent with 24-hour UPCR placebo-adjusted change of -45.8% (cemdisiran-adjusted geometric mean ratio to baseline [SEM], 0.73 [0.11]). Mean (SD) change in serum C5 level from baseline at week 32 was -98.7% (1.2) with cemdisiran and 25.2% (57.7) with placebo. Over 36 weeks, most adverse events were mild or moderate and transient; the most common adverse event after cemdisiran treatment was injection-site reaction (41%). CONCLUSIONS: These findings indicate that treatment with cemdisiran resulted in a reduction of proteinuria at week 32 and was well tolerated.
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Glomerulonefrite por IGA , Adulto , Humanos , Glomerulonefrite por IGA/tratamento farmacológico , Taxa de Filtração Glomerular , Proteinúria/tratamento farmacológico , Proteinúria/etiologia , Testes de Função Renal , Método Duplo-CegoRESUMO
Complement dysregulation underpins the physiopathology of paroxysmal nocturnal hemoglobinuria (PNH). Cemdisiran, an RNA interference investigational treatment, silences complement component 5 (C5) expression in the liver. Previously reported results showed sustained reduction in C5 levels following cemdisiran monotherapy, with >90% reduction in patients with PNH. This phase 1/2 study evaluated single (Part A, n = 32; 50-900 mg) or multiple (Part B, n = 24; 100-600 mg) ascending doses of cemdisiran or placebo (double-blind, randomized 3:1) in healthy adults, or cemdisiran in patients with PNH who were naive to, or receiving, eculizumab (Part C, n = 6; 200 or 400 mg weekly; open-label). The primary objective was to assess the safety and tolerability of cemdisiran. Other assessments included change in complement activity, lactate dehydrogenase levels, and inhibition of hemolysis following cemdisiran treatment. Cemdisiran was generally well tolerated in this study. Overall, 75%, 89%, and 100% of subjects in Parts A, B, and C, respectively, experienced ≥1 non-serious adverse event (AE). Most events were Grade 1 or 2 in severity and the most common AEs included nasopharyngitis and headache. Cemdisiran elicited robust, sustained reductions in the complement activity in healthy adults and patients with PNH. In Part C, exploratory analyses showed that cemdisiran monotherapy was insufficient to prevent hemolysis in patients with PNH as measured by serum lactate dehydrogenase levels. Cemdisiran and eculizumab combination therapy reduced the dose of eculizumab required to provide adequate control of intravascular hemolysis. These results demonstrate a potential benefit of cemdisiran coadministration in patients who are inadequate responders to eculizumab alone.
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Membranous nephropathy (MN) is an antibody-mediated autoimmune disease characterized by glomerular immune complexes containing complement components. However, both the initiation pathways and the pathogenic significance of complement activation in MN are poorly understood. Here, we show that components from all three complement pathways (alternative, classical and lectin) are found in renal biopsies from patients with MN. Proximity ligation assays to directly visualize complement assembly in the tissue reveal dominant activation via the classical pathway, with a close correlation to the degree of glomerular C1q-binding IgG subclasses. In an antigen-specific autoimmune mouse model of MN, glomerular damage and proteinuria are reduced in complement-deficient mice compared with wild-type littermates. Severe disease with progressive ascites, accompanied by extensive loss of the integral podocyte slit diaphragm proteins, nephrin and neph1, only occur in wild-type animals. Finally, targeted silencing of C3 using RNA interference after the onset of proteinuria significantly attenuates disease. Our study shows that, in MN, complement is primarily activated via the classical pathway and targeting complement components such as C3 may represent a promising therapeutic strategy.
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Glomerulonefrite Membranosa , Nefropatias , Camundongos , Animais , Glomerulonefrite Membranosa/genética , Ativação do Complemento , Glomérulos Renais/patologia , Proteínas do Sistema Complemento/metabolismo , Imunoglobulina G , Nefropatias/patologia , Proteinúria/metabolismoRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease caused by uncontrolled complement activation; effective and approved treatments include terminal complement inhibition. This study assessed whether combination cemdisiran (an investigational N-acetylgalactosamine-conjugated RNAi therapeutic that suppresses liver production of complement component C5) and pozelimab (an investigational fully human monoclonal antibody against C5) results in more effective and durable complement activity inhibition than the individual agents alone in non-human primates. Cynomolgus monkeys received a single subcutaneous injection of cemdisiran (5 or 25 mg/kg), pozelimab (5 or 10 mg/kg), or combination cemdisiran and pozelimab (5+5 mg/kg, 5+10 mg/kg, or 25+10 mg/kg, respectively). When given in combination, pozelimab was administered 2 weeks after cemdisiran dosing. Pharmacokinetics and ex vivo pharmacodynamic properties were assessed. The half-life of pozelimab alone was 12.9-13.3 days; this increased to 19.6-21.1 days for pozelimab administered in combination with cemdisiran. In ex vivo classical pathway hemolysis assays (CH50), pozelimab + cemdisiran combinations achieved durable and more complete suppression of complement activity (8-13 weeks) vs monotherapy of either agent. Cemdisiran monotherapy demonstrated dose-dependent suppression of total C5 concentrations, with the higher dose (25 mg/kg) achieving >90% maximum suppression. Total C5 concentrations after administration of pozelimab + cemdisiran combinations were similar compared with administration of cemdisiran alone. The combination of pozelimab + cemdisiran mediates complement activity inhibition more efficiently than either pozelimab or cemdisiran administered alone. The pharmacokinetic/pharmacodynamic profile of combination pozelimab + cemdisiran in non-human primates appears suitable for further clinical investigation as a potential long-acting treatment for PNH and other complement-mediated diseases.
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Hemoglobinúria Paroxística , Animais , Anticorpos Monoclonais/uso terapêutico , Ativação do Complemento , Complemento C5 , Hemoglobinúria Paroxística/tratamento farmacológico , Hemólise , Macaca fascicularisRESUMO
BACKGROUND: Myasthenia gravis (MG) is an autoimmune neuromuscular disorder hallmarked by fluctuating fatigable muscle weakness. Most patients have autoantibodies against acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). These are thought to have three possible pathogenic mode-of-actions: 1) cross-linking and endocytosis of AChRs, 2) direct block of AChRs and 3) complement activation. The relative contributions of these mechanisms to synaptic block and muscle weakness of individual patients cannot be determined. It likely varies between patients and perhaps also with disease course, depending on the nature of the circulating AChR antibodies. NEW METHOD: We developed a new bioassay which specifically enables functional characterization and quantification of complement-mediated synaptic damage at NMJs, without interference of the other pathogenic mechanisms. To this end, we pre-incubated mouse hemi-diaphragm muscle-nerve preparations with mAb35-hG1, a humanized rat AChR monoclonal and subsequently exposed the preparation to normal human serum as a complement source. NMJ-restricted effects were studied. RESULTS: Clearly NMJ-restricted damage occurred. With immunohistology we showed complement deposition at NMJs, and synaptic electrophysiological measurements demonstrated transmission block. In whole-muscle contraction experiments we quantified the effect and characterized its onset and progression during the incubation with normal human serum. COMPARISON WITH EXISTING METHODS: With this new assay the complement-mediated component of myasthenic NMJ pathology can be studied separately. CONCLUSIONS: Our assay will be of importance in detailed mechanistic studies of local complement activation at NMJs, investigations of new complement inhibitors, and laboratory pre-screening of therapeutic efficacy for individual MG patients to optimize care with clinically approved complement inhibitors.
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Miastenia Gravis , Receptores Colinérgicos , Animais , Autoanticorpos , Bioensaio , Ativação do Complemento , Humanos , Camundongos , Miastenia Gravis/terapia , Junção Neuromuscular/patologia , RatosRESUMO
BACKGROUND: Cemdisiran, an N-acetylgalactosamine (GalNAc) conjugated RNA interference (RNAi) therapeutic, is currently under development for the treatment of complement-mediated diseases by suppressing liver production of complement 5 (C5) protein. This study was designed to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of cemdisiran in healthy subjects and in patients with paroxysmal nocturnal hemoglobinuria (PNH) in order to support dose selection for late-stage clinical trials. METHODS: Healthy volunteers (HVs; n = 32, including 12 Japanese subjects) were randomized (3:1) to receive single doses of subcutaneous cemdisiran (50-900 mg) or placebo, or repeat doses of subcutaneous cemdisiran (100-600 mg) or placebo weekly, biweekly, weekly/biweekly, or weekly/monthly for 5, 8, or 13 weeks (n = 24). Cemdisiran 200 or 400 mg was administered weekly in an open-label manner, for varying durations, as monotherapy in three eculizumab-naïve PNH patients or in combination with eculizumab in three PNH patients who were receiving stable label doses of eculizumab (900 or 1200 mg biweekly) before the start of the study. After the last dose of cemdisiran, patients were followed for safety and ongoing pharmacologic effects with the eculizumab regimen (600 or 900 mg every month). RESULTS: In HVs, cemdisiran was rapidly converted to a major active metabolite, AS(N-2)3'-cemdisiran, both declining below the lower limit of quantification (LLOQ) in plasma within 48 h, and showing minimal renal excretion. AS(N-2)3'-cemdisiran exhibited more than dose-proportional PK. The C5 protein reductions were dose-dependent, with > 90% reduction of C5 protein beginning on days 21-28 and maintained for 10-13 months following single and biweekly doses of 600 mg. The dose-response relationship, described by an inhibitory sigmoid maximum effect (Emax) model, estimated half-maximal effective dose (ED50) of 14.0 mg and maximum C5 reduction of 99% at 600 mg. The PK and PD were similar between Japanese and non-Japanese subjects, and PNH patients and HVs. One of 48 subjects tested transiently positive for antidrug antibody with low titer, with no impact on PK or PD. In PNH patients, C5 suppression by cemdisiran enabled effective inhibition of residual C5 levels with lower dose and/or dosing frequency of eculizumab, which was maintained for 6-10 months after the last dose of cemdisiran. CONCLUSIONS: Consistent with the PK/PD properties of liver targeting GalNac conjugates, cemdisiran and AS(N-2)3'-cemdisiran plasma concentrations declined rapidly while showing rapid and robust C5 suppression maintained up to 13 months following single and multiple doses, which indicates long residence times of cemdisiran within hepatocytes. The long PD duration of action in liver, low immunogenicity and acceptable safety profiles enables low, infrequent SC dosing and support further evaluation of cemdisiran in complement-mediated diseases as monotherapy or in combination with a C5 inhibitor antibody. CLINICAL TRIAL REGISTRATION NO: NCT02352493.
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Complemento C5 , Hemoglobinúria Paroxística , Complemento C5/farmacocinética , Voluntários Saudáveis , Hemoglobinúria Paroxística/tratamento farmacológico , Humanos , Interferência de RNA , Terapêutica com RNAiRESUMO
BACKGROUND: Plasma coagulation Factor XII (FXII) plays a crucial role in contact activation, ultimately regulating both the kinin-kallikrein system and the intrinsic pathway of coagulation. A growing body of evidence suggests that inhibition of FXII can prevent thrombosis. Given FXII does not appear to modulate hemostasis, targeting FXII is a promising strategy for the prevention of pathological thrombus formation without the hemostatic risks typically associated with anticoagulants. To this end, a subcutaneously administered investigational RNAi therapeutic targeting liver F12 mRNA (ALN-F12) was developed. AIM: To investigate the thrombo-protective and hemostatic effects of FXII reduction by ALN-F12 in rodent thrombosis and hemostasis models. METHODS: A single dose of ALN-F12 was subcutaneously administered to C57Bl/6 mice. After reaching steady state FXII reduction, the impact on thrombosis (ferric chloride arterial thrombosis and electrolytic injury induced venous thrombosis models) and hemostasis (saphenous vein injury and tail tip transection bleeding models) was evaluated. RESULT: Administration of ALN-F12 resulted in dose-dependent reductions of both liver F12 mRNA and plasma FXII protein. In mice, ALN-F12 led to dose-dependent reductions in platelet and fibrin accumulation in the venous electrolytic-injury model and in the time to occlusion in the ferric chloride arterial thrombosis model. At 10 mg/kg ALN-F12, the top dose level evaluated, this resulted in >95% reduction of FXII and ~10 fold reduction in fibrin deposition. Finally, hemostasis models showed that >95% reduction of FXII had no impact on bleeding time or blood loss. CONCLUSION: Our findings support that reduction of plasma Factor XII by ALN-F12 provided thrombo-protective effects with no increased bleeding risk in rodent models of thrombosis and hemostasis.
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Deficiência do Fator XII , Hemostáticos , Trombose , Animais , Fator XII/genética , Hemostasia , Fígado , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , Trombose/genética , Trombose/prevenção & controleRESUMO
The complement system plays an important role in the pathogenesis of rheumatoid arthritis (RA). Besides driving lectin pathway (LP) activation, the mannan-binding lectin (MBL)-associated serine proteases (MASPs) also play a key role in regulating the alternative pathway (AP). We evaluated the effects of N-acetylgalactosamine (GalNAc)-conjugated MASP-1 and MASP-2 duplexes in vitro and in mice with and without arthritis to examine whether knockdown of MASP-1 and MASP-2 expression affects the development of arthritis. GalNAc-siRNAs for MASP-1 and MASP-2 demonstrated robust silencing of MASP-1 or MASP-2 at pM concentrations in vitro. To evaluate the impact of silencing in arthritic mice, we used the collagen antibody-induced arthritis (CAIA) mouse model of RA. Mice were injected a 10 mg/kg dose of GalNAc-siRNAs 3x s.q. prior to the induction of CAIA. Liver gene expression was examined using qRT-PCR, and protein levels were confirmed in the circulation by sandwich immunoassays and Western blot. At day 10, CAIA mice separately treated with MASP-1 and MASP-2 duplexes had a specific reduction in expression of liver MASP-1 (70-95%, p < 0.05) and MASP-2 (90%, p < 0.05) mRNA, respectively. MASP-1-siRNA treatment resulted in a 95% reduction in levels of MASP-1 protein in circulation with no effect on MASP-2 levels and clinical disease activity (CDA). In mice injected with MASP-2 duplex, there was a significant (p < 0.05) 90% decrease in ex vivo C4b deposition on mannan, with nearly complete elimination of MASP-2 in the circulation. MASP-2 silencing initially significantly decreased CDA by 60% but subsequently changed to a 40% decrease vs. control. Unexpectedly, GalNAc-siRNA-mediated knockdown of MASP-1 and MASP-2 revealed a marked effect of these proteins on the transcription of FD under normal physiological conditions, whereas LPS-induced inflammatory conditions reversed this effect on FD levels. LPS is recognized by Toll-like receptor 4 (TLR4), we found MBL not only binds to TLR4 an interaction with a Kd of 907 nM but also upregulated FD expression in differentiated adipocytes. We show that MASP-2 knockdown impairs the development of RA and that the interrelationship between proteins of the LP and the AP may extend to the transcriptional modulation of the FD gene.
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Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Fator D do Complemento/metabolismo , Via Alternativa do Complemento/genética , Lectina de Ligação a Manose da Via do Complemento/genética , Transcrição Gênica/genética , Animais , Fator D do Complemento/genética , Expressão Gênica , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , TransfecçãoRESUMO
Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.
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In the version of this article originally published, a sentence was erroneously included in the author contributions, and information regarding second shared authorship was missing from the author contributions. The following should not have been included in the author contributions: "C.W. and A.J.R.H. supervised the work presented in Figs. 1, 2, 5, 6; P.Z. and C.S. supervised the work presented in Figs. 3, 4." Additionally, this sentence should have appeared at the beginning of the author contributions: "These authors contributed equally: C.W., P.F.Z., C.S., and A.J.R.H." The errors have been corrected in the print, PDF and HTML versions of the article.
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Apolipoprotein-E (ApoE) has been implicated in Alzheimer's disease, atherosclerosis, and other unresolvable inflammatory conditions but a common mechanism of action remains elusive. We found in ApoE-deficient mice that oxidized lipids activated the classical complement cascade (CCC), resulting in leukocyte infiltration of the choroid plexus (ChP). All human ApoE isoforms attenuated CCC activity via high-affinity binding to the activated CCC-initiating C1q protein (KD~140-580 pM) in vitro, and C1q-ApoE complexes emerged as markers for ongoing complement activity of diseased ChPs, Aß plaques, and atherosclerosis in vivo. C1q-ApoE complexes in human ChPs, Aß plaques, and arteries correlated with cognitive decline and atherosclerosis, respectively. Treatment with small interfering RNA (siRNA) against C5, which is formed by all complement pathways, attenuated murine ChP inflammation, Aß-associated microglia accumulation, and atherosclerosis. Thus, ApoE is a direct checkpoint inhibitor of unresolvable inflammation, and reducing C5 attenuates disease burden.
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Complexo Antígeno-Anticorpo/imunologia , Apolipoproteínas E/imunologia , Doenças das Artérias Carótidas/imunologia , Plexo Corióideo/imunologia , Disfunção Cognitiva/imunologia , Complemento C1q/imunologia , Via Clássica do Complemento/imunologia , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/imunologia , Animais , Aorta/imunologia , Aorta/patologia , Aterosclerose/imunologia , Aterosclerose/patologia , Encéfalo/imunologia , Encéfalo/patologia , Artérias Carótidas/imunologia , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/patologia , Plexo Corióideo/patologia , Disfunção Cognitiva/patologia , Complemento C5 , Feminino , Humanos , Leucócitos , Masculino , Camundongos Knockout para ApoE , Microscopia de Fluorescência , Pessoa de Meia-Idade , Placa Amiloide/imunologia , Placa Amiloide/patologia , Isoformas de Proteínas/imunologia , RNA Interferente PequenoRESUMO
Hereditary angioedema (HAE) is a genetic disorder mostly caused by mutations in the C1 esterase inhibitor gene (C1INH) that results in poor control of contact pathway activation and excess bradykinin generation. Bradykinin increases vascular permeability and is ultimately responsible for the episodes of swelling characteristic of HAE. We hypothesized that the use of RNA interference (RNAi) to reduce plasma Factor XII (FXII), which initiates the contact pathway signaling cascade, would reduce contact pathway activation and prevent excessive bradykinin generation. A subcutaneously administered GalNAc-conjugated small-interfering RNA (siRNA) targeting F12 mRNA (ALN-F12) was developed, and potency was evaluated in mice, rats, and cynomolgus monkeys. The effect of FXII reduction by ALN-F12 administration was evaluated in two different vascular leakage mouse models. An ex vivo assay was developed to evaluate the correlation between human plasma FXII levels and high-molecular weight kininogen (HK) cleavage. A single subcutaneous dose of ALN-F12 led to potent, dose-dependent reduction of plasma FXII in mice, rats, and NHP. In cynomolgus monkeys, a single subcutaneous dose of ALN-F12 at 3 mg/kg resulted in >85% reduction of plasma FXII. Administration of ALN-F12 resulted in dose-dependent reduction of vascular permeability in two different mouse models of bradykinin-driven vascular leakage, demonstrating that RNAi-mediated reduction of FXII can potentially mitigate excess bradykinin stimulation. Lastly, ex vivo human plasma HK cleavage assay indicated FXII-dependent bradykinin generation. Together, these data suggest that RNAi-mediated knockdown of FXII by ALN-F12 is a potentially promising approach for the prophylactic treatment of HAE.
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Angioedemas Hereditários/tratamento farmacológico , Bradicinina/biossíntese , Fator XII/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêutico , Animais , Permeabilidade Capilar/efeitos dos fármacos , Proteína Inibidora do Complemento C1/genética , Fator XII/análise , Feminino , Humanos , Cininogênios/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RatosRESUMO
Mannan-binding lectin-associated serine protease 3 (MASP-3) regulates the alternative pathway of complement and is predominantly synthesized in the liver. The role of liver-derived MASP-3 in the pathogenesis of rheumatoid arthritis (RA) is unknown. We hypothesized that liver-derived MASP-3 is essential for the development of joint damage and that targeted inhibition of MASP-3 in the liver can attenuate arthritis. We used MASP-3-specific small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) to specifically target the liver via asialoglycoprotein receptors. Active GalNAc-MASP3-siRNA conjugates were identified, and in vivo silencing of liver MASP-3 mRNA was demonstrated in healthy mice. The s.c. treatment with GalNAc-MASP-3-siRNAs specifically decreased the expression of MASP-3 in the liver and the level of MASP-3 protein in circulation of mice without affecting the levels of the other spliced products. In mice with collagen Ab-induced arthritis, s.c. administration of GalNAc-MASP-3-siRNA decreased the clinical disease activity score to 50% of controls, with decrease in histopathology scores and MASP-3 deposition. To confirm the ability to perform MASP-3 gene silencing in human cells, we generated a lentivirus expressing a short hairpin RNA specific for human MASP-3 mRNA. This procedure not only eliminated the short-term (at day 15) expression of MASP-3 in HepG2 and T98G cell lines but also diminished the long-term (at day 60) synthesis of MASP-3 protein in T98G cells. Our study demonstrates that isoform-specific silencing of MASP-3 in vivo modifies disease activity in a mouse model of RA and suggests that liver-directed MASP3 silencing may be a therapeutic approach in human RA.
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BACKGROUND: Inclisiran (ALN-PCSsc) is a long-acting RNA interference (RNAi) therapeutic agent that inhibits the synthesis of proprotein convertase subtilisin-kexin type 9 (PCSK9), a target for the lowering of low-density lipoprotein (LDL) cholesterol. METHODS: In this phase 1 trial, we randomly assigned healthy volunteers with an LDL cholesterol level of at least 100 mg per deciliter in a 3:1 ratio to receive a subcutaneous injection of inclisiran or placebo in either a single-ascending-dose phase (at a dose of 25, 100, 300, 500, or 800 mg) or a multiple-dose phase (125 mg weekly for four doses, 250 mg every other week for two doses, or 300 or 500 mg monthly for two doses, with or without concurrent statin therapy); each dose cohort included four to eight participants. Safety, the side-effect profile, and pharmacodynamic measures (PCSK9 level, LDL cholesterol level, and exploratory lipid variables) were evaluated. RESULTS: The most common adverse events were cough, musculoskeletal pain, nasopharyngitis, headache, back pain, and diarrhea. All the adverse events were mild or moderate in severity. There were no serious adverse events or discontinuations due to adverse events. There was one grade 3 elevation in the γ-glutamyltransferase level, which was considered by the investigator to be related to statin therapy. In the single-dose phase, inclisiran doses of 300 mg or more reduced the PCSK9 level (up to a least-squares mean reduction of 74.5% from baseline to day 84), and doses of 100 mg or more reduced the LDL cholesterol level (up to a least-squares mean reduction of 50.6% from baseline). Reductions in the levels of PCSK9 and LDL cholesterol were maintained at day 180 for doses of 300 mg or more. All multiple-dose regimens reduced the levels of PCSK9 (up to a least-squares mean reduction of 83.8% from baseline to day 84) and LDL cholesterol (up to a least-squares mean reduction of 59.7% from baseline to day 84). CONCLUSIONS: In this phase 1 trial, no serious adverse events were observed with inclisiran. Doses of 300 mg or more (in single or multiple doses) significantly reduced levels of PCSK9 and LDL cholesterol for at least 6 months. (Funded by Alnylam Pharmaceuticals and the Medicines Company; ClinicalTrials.gov number, NCT02314442 .).
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Anticolesterolemiantes/administração & dosagem , Inibidores de PCSK9 , RNA Interferente Pequeno/administração & dosagem , Terapêutica com RNAi , Acetilgalactosamina/administração & dosagem , Adolescente , Adulto , Idoso , Anticolesterolemiantes/efeitos adversos , LDL-Colesterol/sangue , Feminino , Voluntários Saudáveis , Humanos , Injeções Subcutâneas , Masculino , Pessoa de Meia-Idade , Pró-Proteína Convertase 9/biossíntese , Pró-Proteína Convertase 9/sangue , RNA Interferente Pequeno/efeitos adversos , Complexo de Inativação Induzido por RNA/metabolismo , Método Simples-Cego , Adulto JovemRESUMO
RATIONALE: Macrophages reside in the healthy myocardium, participate in ischemic heart disease, and modulate myocardial infarction (MI) healing. Their origin and roles in post-MI remodeling of nonischemic remote myocardium, however, remain unclear. OBJECTIVE: This study investigated the number, origin, phenotype, and function of remote cardiac macrophages residing in the nonischemic myocardium in mice with chronic heart failure after coronary ligation. METHODS AND RESULTS: Eight weeks post MI, fate mapping and flow cytometry revealed that a 2.9-fold increase in remote macrophages results from both increased local macrophage proliferation and monocyte recruitment. Heart failure produced by extensive MI, through activation of the sympathetic nervous system, expanded medullary and extramedullary hematopoiesis. Circulating Ly6C(high) monocytes rose from 64±5 to 108±9 per microliter of blood (P<0.05). Cardiac monocyte recruitment declined in Ccr2(-/-) mice, reducing macrophage numbers in the failing myocardium. Mechanical strain of primary murine and human macrophage cultures promoted cell cycle entry, suggesting that the increased wall tension in post-MI heart failure stimulates local macrophage proliferation. Strained cells activated the mitogen-activated protein kinase pathway, whereas specific inhibitors of this pathway reduced macrophage proliferation in strained cell cultures and in the failing myocardium (P<0.05). Steady-state cardiac macrophages, monocyte-derived macrophages, and locally sourced macrophages isolated from failing myocardium expressed different genes in a pattern distinct from the M1/M2 macrophage polarization paradigm. In vivo silencing of endothelial cell adhesion molecules curbed post-MI monocyte recruitment to the remote myocardium and preserved ejection fraction (27.4±2.4 versus 19.1±2%; P<0.05). CONCLUSIONS: Myocardial failure is influenced by an altered myeloid cell repertoire.
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Fenômenos Biomecânicos/fisiologia , Proliferação de Células/fisiologia , Insuficiência Cardíaca/patologia , Macrófagos/fisiologia , Miocárdio/citologia , Animais , Células Cultivadas , Doença Crônica , Feminino , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Myocardial infarction (MI) leads to a systemic surge of vascular inflammation in mice and humans, resulting in secondary ischemic complications and high mortality. We show that, in ApoE(-/-) mice with coronary ligation, increased sympathetic tone up-regulates not only hematopoietic leukocyte production but also plaque endothelial expression of adhesion molecules. To counteract the resulting arterial leukocyte recruitment, we developed nanoparticle-based RNA interference (RNAi) that effectively silences five key adhesion molecules. Simultaneously encapsulating small interfering RNA (siRNA)-targeting intercellular cell adhesion molecules 1 and 2 (Icam1 and Icam2), vascular cell adhesion molecule 1 (Vcam1), and E- and P-selectins (Sele and Selp) into polymeric endothelial-avid nanoparticles reduced post-MI neutrophil and monocyte recruitment into atherosclerotic lesions and decreased matrix-degrading plaque protease activity. Five-gene combination RNAi also curtailed leukocyte recruitment to ischemic myocardium. Therefore, targeted multigene silencing may prevent complications after acute MI.
Assuntos
Moléculas de Adesão Celular/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/terapia , Infiltração de Neutrófilos/fisiologia , RNA Interferente Pequeno/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Moléculas de Adesão Celular/genética , Selectina E/genética , Selectina E/metabolismo , Feminino , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Infarto do Miocárdio/imunologia , Nanopartículas , Infiltração de Neutrófilos/genética , Selectina-P/genética , Selectina-P/metabolismo , Parabiose , Interferência de RNA , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Macrófagos/fisiologia , Monócitos/fisiologia , Células Mieloides/fisiologia , Infarto do Miocárdio/imunologia , Proteínas Nucleares/metabolismo , Receptores CCR2/metabolismo , Fatores de Transcrição/metabolismo , Animais , Movimento Celular/genética , Células Cultivadas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Modelos Animais , Mielopoese/genética , Infarto do Miocárdio/cirurgia , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Receptores CCR2/genética , Proteínas Repressoras , Fatores de Transcrição/genética , Cicatrização/genéticaRESUMO
Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.
Assuntos
Hematopoese Extramedular/imunologia , Células-Tronco Hematopoéticas/imunologia , Macrófagos/imunologia , Mielopoese/imunologia , Baço/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/imunologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Hematopoese Extramedular/genética , Células-Tronco Hematopoéticas/patologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Mielopoese/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Nanopartículas , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Interferência de RNA , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Baço/patologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.