RESUMO
Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD.
Assuntos
Autofagossomos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Canais de Potássio/genética , alfa-Sinucleína/química , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/patologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Regulação da Expressão Gênica , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Lisossomos/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Modelos Biológicos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Canais de Potássio/deficiência , Cultura Primária de Células , Agregados Proteicos/efeitos dos fármacos , Ratos , alfa-Sinucleína/farmacologiaRESUMO
Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole-exome sequencing. We identified a single base pair substitution in the 5' donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles. The homozygous recessive disruption of ABCA5 leads to reduced lysosome function, which results in an accumulation of autophagosomes, autophagosomal cargos as well as increased endolysosomal cholesterol in CGHT keratinocytes. In an unrelated sporadic case of CGHT, we identified a 1.3 Mb cryptic deletion of chr17q24.2-q24.3 encompassing ABCA5 and found that ABCA5 levels are dramatically reduced throughout patient hair follicles. Collectively, our findings support ABCA5 as a gene underlying the CGHT phenotype and suggest a novel, previously unrecognized role for this gene in regulating hair growth.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Colesterol/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Cabelo/crescimento & desenvolvimento , Hipertricose/congênito , Pré-Escolar , Colesterol/genética , Deleção Cromossômica , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Cabelo/patologia , Humanos , Hipertricose/genética , Hipertricose/patologia , Lactente , Queratinócitos/metabolismo , Queratinócitos/patologia , Mutação , Linhagem , Fenótipo , Splicing de RNA/genética , Deleção de SequênciaRESUMO
SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipídeos/sangue , Proteínas de Membrana/genética , Pró-Proteína Convertase 9/genética , RNA Interferente Pequeno/genética , Receptores de LDL/genética , Animais , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hipolipemiantes/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Fígado/enzimologia , Macaca mulatta , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/metabolismo , Transdução de Sinais , Sinvastatina/farmacologia , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismoRESUMO
X-linked congenital generalized hypertrichosis (Online Mendelian Inheritance in Man 307150) is an extremely rare condition of hair overgrowth on different body sites. We previously reported linkage in a large Mexican family with X-linked congenital generalized hypertrichosis cosegregating with deafness and with dental and palate anomalies to Xq24-27. Using SNP oligonucleotide microarray analysis and whole-genome sequencing, we identified a 389-kb interchromosomal insertion at an extragenic palindrome site at Xq27.1 that completely cosegregates with the disease. Among the genes surrounding the insertion, we found that Fibroblast Growth Factor 13 (FGF13) mRNA levels were significantly reduced in affected individuals, and immunofluorescence staining revealed a striking decrease in FGF13 localization throughout the outer root sheath of affected hair follicles. Taken together, our findings suggest a role for FGF13 in hair follicle growth and in the hair cycle.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica , Hipertricose/congênito , Processamento Alternativo , Animais , Mapeamento Cromossômico , Feminino , Ligação Genética , Genoma Humano , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/fisiologia , Heterozigoto , Humanos , Hipertricose/genética , Queratinócitos/metabolismo , Masculino , Camundongos , Mutagênese Insercional , Linhagem , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diacilglicerol O-Aciltransferase/deficiência , Diacilglicerol O-Aciltransferase/genética , Fígado Gorduroso/genética , Inativação Gênica , RNA Interferente Pequeno/genética , Animais , Apolipoproteínas B/deficiência , Apolipoproteínas B/genética , Colesterol/sangue , Fígado Gorduroso/sangue , Fígado Gorduroso/enzimologia , Fígado Gorduroso/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Triglicerídeos/metabolismoRESUMO
Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.
Assuntos
Anticolesterolemiantes/uso terapêutico , Azetidinas/uso terapêutico , Fluorbenzenos/uso terapêutico , Pirimidinas/uso terapêutico , Serina Endopeptidases/metabolismo , Sulfonamidas/uso terapêutico , Animais , Apolipoproteínas B/sangue , Colesterol/sangue , LDL-Colesterol/sangue , Ensaio de Imunoadsorção Enzimática , Ezetimiba , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Hipercolesterolemia/terapia , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rosuvastatina Cálcica , Serina Endopeptidases/genética , Triglicerídeos/sangueRESUMO
Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoEâ»/â» and low density lipoprotein receptor (LDLr)â»/â» mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETPâº/â» hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETPâº/â» mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.
Assuntos
Apolipoproteínas B/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/genética , LDL-Colesterol/sangue , Modelos Animais de Doenças , Receptores de LDL/genética , Animais , Apolipoproteínas B/sangue , Apolipoproteínas E/sangue , Apolipoproteínas E/genética , Aterosclerose/patologia , Linhagem Celular Tumoral , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Efeito Fundador , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hemizigoto , Humanos , Metabolismo dos Lipídeos/genética , Lipossomos/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nanopartículas/administração & dosagem , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de LDL/metabolismo , Triglicerídeos/sangueRESUMO
The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D(4)-cholic acid (D(4)-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of â¼90% gene expression was achieved followed by reduction in the reconjugation of D(4)-CA to D(4)-taurocholic acid (D(4)-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many D(4) and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.
Assuntos
Ácidos e Sais Biliares/metabolismo , Cromatografia Líquida/métodos , Proteínas de Transporte de Ácido Graxo/metabolismo , Fígado/patologia , Espectrometria de Massas/métodos , Metabolômica/métodos , Animais , Inativação Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Ambras syndrome (AS) is a rare form of congenital hypertrichosis with excessive hair on the shoulders, face and ears. Cytogenetic studies have previously implicated an association with rearrangements of chromosome 8. Here we define an 11.5 Mb candidate interval for AS on chromosome 8q based on cytogenetic breakpoints in three patients. TRPS1, a gene within this interval, was deleted in a patient with an 8q23 chromosomal rearrangement, while its expression was significantly downregulated in another patient with an inversion breakpoint 7.3 Mb downstream of TRPS1. Here, we describe the first potential long-range position effect on the expression of TRPS1. To gain insight into the mechanisms by which Trps1 affects the hair follicle, we performed a detailed analysis of the hair abnormalities in Koa mice, a mouse model of hypertrichosis. We found that the proximal breakpoint of the Koa inversion is located 791 kb upstream of Trps1. Quantitative real-time polymerase chain reaction, in situ hybridization and immunofluorescence analysis revealed that Trps1 expression levels are reduced in Koa mutant mice at the sites of pathology for the phenotype. We determined that the Koa inversion creates a new Sp1 binding site and translocates additional Sp1 binding sites within a highly conserved stretch spanning the proximal breakpoint, providing a potential mechanism for the position effect. Collectively, these results describe a position effect that downregulates TRPS1 expression as the probable cause of hypertrichosis in AS in humans and the Koa phenotype in mice.
Assuntos
Cromossomos Humanos Par 8/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição GATA/genética , Hipertricose/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/genética , Animais , Sítios de Ligação , Quebra Cromossômica , Inversão Cromossômica , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Fatores de Transcrição GATA/metabolismo , Rearranjo Gênico , Folículo Piloso/anormalidades , Humanos , Hipertricose/congênito , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Proteínas Repressoras , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
OBJECTIVE: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation. METHODS: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene. RESULTS: Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions. CONCLUSIONS: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.
Assuntos
Adipócitos/citologia , Adipogenia , Diabetes Mellitus/genética , Redes Reguladoras de Genes , Mapas de Interação de Proteínas , Aciltransferases/genética , Aciltransferases/metabolismo , Adipócitos/metabolismo , Adipócitos/patologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Diabetes Mellitus/patologia , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Resistência à Insulina , Perilipina-1/genética , Perilipina-1/metabolismo , Fenótipo , TranscriptomaRESUMO
Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.
Assuntos
Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Dislipidemias/metabolismo , Hepatócitos/metabolismo , Obesidade/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteínas B/metabolismo , Células Cultivadas , Diacilglicerol O-Aciltransferase/genética , Modelos Animais de Doenças , Inativação Gênica , Humanos , Lipoproteínas VLDL/metabolismo , Macaca fascicularis , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: SREBP cleavage-activating protein (SCAP) is a cholesterol binding endoplasmic reticulum (ER) membrane protein that is required to activate SREBP transcription factors. SREBPs regulate genes involved in lipid biosynthesis. They also influence lipid clearance by modulating the expression of LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Inhibiting SCAP decreases circulating PCSK9, triglycerides (TG), and LDL-cholesterol (LDL-C), both in vitro and in vivo. Type 2 diabetics with dyslipidemia are at high risk for cardiovascular diseases. These patients present a unique pathophysiological lipid profile characterized by moderately elevated LDL-C, elevated TG and reduced HDL-cholesterol (HDL-C). The spontaneous dysmetabolic rhesus monkey model (DysMet RhM) recapitulates this human dyslipidemia and therefore is an attractive preclinical model to evaluate SCAP inhibition as a therapy for this disease population. The objective to of this study was to assess the effect of SCAP inhibition on the lipid profile of DysMet RhM. METHOD: We assessed the effect of inhibiting hepatic SCAP on the lipid profile of DysMet RhM using an siRNA encapsulated lipid nanoparticle (siRNA-LNP). RESULTS: The SCAP siRNA-LNP significantly reduced LDL-C, PCSK9 and TG in DysMet RhM; LDL-C was reduced by ≥20%, circulating PCSK9 by 30-40% and TG by >25%. These changes by the SCAP siRNA-LNP agree with the predicted effect of SCAP inhibition and reduced SREBP tone on these endpoints. CONCLUSION: These data demonstrate that a SCAP siRNA-LNP improved the lipid profile in a clinically relevant preclinical disease model and provide evidence for SCAP inhibition as a therapy for diabetic dyslipidemic patients.
Assuntos
Dislipidemias/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , RNA Interferente Pequeno/uso terapêutico , Animais , Biomarcadores/sangue , Colesterol/sangue , Feminino , Lipídeos/sangue , Fígado/metabolismo , Macaca mulatta , Masculino , Nanopartículas , Ácido Palmítico/sangue , Pró-Proteína Convertase 9/sangue , RNA Interferente Pequeno/administração & dosagem , Triglicerídeos/metabolismoRESUMO
Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85-95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes.
RESUMO
The benefits of novel oral anticoagulants are hampered by bleeding. Since coagulation factor IX (fIX) lies upstream of fX in the coagulation cascade, and intermediate levels have been associated with reduced incidence of thrombotic events, we evaluated the viability of fIXa as an antithrombotic target. We applied translational pharmacokinetics/pharmacodynamics (PK/PD) principles to predict the therapeutic window (TW) associated with a selective small molecule inhibitor (SMi) of fIXa, compound 1 (CPD1, rat fIXa inhibition constant (Ki, 21 nmol/L) relative to clinically relevant exposures of apixaban (rat fXa Ki 4.3 nmol/L). Concentrations encompassing the minimal clinical plasma concentration (C min) of the 5 mg twice daily (BID) dose of apixaban were tested in rat arteriovenous shunt (AVS/thrombosis) and cuticle bleeding time (CBT) models. An I max and a linear model were used to fit clot weight (CW) and CBT. The following differences in biology were observed: (1) antithrombotic activity and bleeding increased in parallel for apixaban, but to a lesser extent for CPD1 and (2) antithrombotic activity occurred at high (>99%) enzyme occupancy (EO) for fXa or moderate (>65% EO) for fIXa. translational PK/PD analysis indicated that noninferiority was observed for concentrations of CPD1 that provided between 86% and 96% EO and that superior TW existed between 86% and 90% EO. These findings were confirmed in a study comparing short interfering (si)RNA-mediated knockdown (KD) modulation of fIX and fX mRNA. In summary, using principles of translational biology to relate preclinical markers of efficacy and safety to clinical doses of apixaban, we found that modulation of fIXa can be superior to apixaban.
RESUMO
Lipoprotein(a) [Lp(a)] has recently been recognized as an independent risk factor for coronary heart disease. While plasma Lp(a) levels are correlated with cardiovascular risk, the mechanism by which this particle contributes to atherosclerosis is largely unknown. Although humanized transgenic mouse model has recently been described to study Lp(a) biology, non-human primates (NHP) are the only preclinical model available that allow study of the role of Lp(a) in atherosclerosis in an innate setting. We describe targeting of LPA using lipid nanoparticle formulated short interfering RNAs (siRNAs) in lean rhesus macaque monkeys. We show >90 % LPA mRNA lowering in the liver and >95 % Lp(a) plasma reduction for over 3 weeks after a single siRNA dose. Given the potency of LPA siRNAs, siRNA approach may enable chronic reduction of Lp(a) in atherosclerotic NHP and help to unmask the role for Lp(a) in the genesis and progression of atherosclerosis in man.
Assuntos
Aterosclerose/terapia , Lipoproteína(a)/genética , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Animais , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Humanos , Lipídeos/química , Lipoproteína(a)/sangue , Fígado/metabolismo , Macaca mulatta , Masculino , Nanomedicina , Nanopartículas , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
The greater genomic conservation between humans and non-human primates (NHP) enables target validation studies for developing of therapeutic strategies for human diseases. Together with predicting activity and potential adverse clinical signs, the inclusion of NHP testing bequeaths to efficacy models for dose titration and pharmacodynamic effects. We have used lipid nanoparticle encapsulated siRNA to silence ApoB in the liver and assessed the phenotypic effects on serum lipids with various levels of hepatic ApoB mRNA knockdown in healthy lean cynomolgus monkeys. ApoB siRNA dosed animals demonstrated significant reductions of hepatic ApoB mRNA and serum APOB protein, with a substantial lowering of plasma lipid levels without obvious signs of toxicity. Microarray based assessment of ApoB siRNA mediated effects revealed a number of differentially expressed genes which mapped onto biological pathways and processes related to lipid and cholesterol metabolism. Furthermore, we identified potential targets and cellular effects that could be studied for therapeutic benchmarking of APOB mediated effects. The network of ApoB regulated genes should be of significance for the understanding and development of novel hypercholesterolemia therapies.
Assuntos
Apolipoproteínas B/genética , Regulação da Expressão Gênica , Fígado/metabolismo , Interferência de RNA , Animais , Biópsia , Colesterol/metabolismo , Genoma , Genômica , Humanos , Metabolismo dos Lipídeos , Lipídeos/sangue , Lipídeos/química , Fígado/patologia , Macaca fascicularis , Masculino , Nanopartículas/química , Análise de Sequência com Séries de Oligonucleotídeos , Biossíntese de Proteínas , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
This report aims at exploring quantitatively the relationship between FXII inhibition and thromboprotection. FXII full and partial null in rats were established via zinc finger nuclease-mediated knockout and siRNA-mediated knockdown, respectively. The rats were subsequently characterized in thrombosis and hemostasis models. Knockout rats exhibited complete thromboprotection in both the arteriovenous shunt model (â¼100% clot weight reduction) and the FeCl3-induced arterial thrombosis model (no reduction in blood flow), without any increase in cuticle bleeding time compared with wild-type control rats. Ex-vivo aPTT and the ellagic acid-triggered thrombin generation assay (TGA) exhibited anticoagulant changes. In contrast, ex-vivo PT or high tissue factor-triggered TGA was indistinguishable from control. Rats receiving single doses (0, 0.01, 0.03, 0.1, 0.3, 1âmg/kg) of FXII siRNA exhibited dose-dependent knockdown in liver FXII mRNA and plasma FXII protein (95 and 99%, respectively, at 1âmg/kg) at day 7 post dosing. FXII knockdown was associated with dose-dependent thromboprotection (maximal efficacy achieved with 1âmg/kg in both models) and negligible change in cuticle bleeding times. Ex-vivo TGA triggered with low-level (0.5âµmol/l) ellagic acid tracked best with the knockdown levels and efficacy. Our findings confirm and extend literature reports of an attractive benefit-to-risk profile of targeting FXII for antithrombotic therapies. Titrating of FXII is instructive for its pharmacological inhibition. The knockout rat is valuable for evaluating both mechanism-based safety concerns and off-target effects of FXII(a) inhibitors. Detailed TGA analyses will inform on optimal trigger conditions in studying pharmacodynamic effects of FXII(a) inhibition.
Assuntos
Endodesoxirribonucleases/genética , Fator XII/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Terapia Trombolítica/métodos , Trombose/terapia , Animais , Derivação Arteriovenosa Cirúrgica , Cloretos/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ácido Elágico/farmacologia , Endodesoxirribonucleases/metabolismo , Fator XII/genética , Fator XII/metabolismo , Compostos Férricos/farmacologia , Técnicas de Inativação de Genes , Hemorragia/prevenção & controle , Humanos , Fígado/metabolismo , Masculino , Tempo de Tromboplastina Parcial , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Trombina/metabolismo , Trombose/induzido quimicamente , Trombose/genética , Trombose/patologia , Dedos de Zinco/genéticaRESUMO
The present study aimed at establishing feasibility of delivering short interfering RNA (siRNA) to target the coagulation cascade in rat and rabbit, two commonly used species for studying thrombosis and hemostasis. siRNAs that produced over 90% mRNA knockdown of rat plasma prekallikrein and rabbit Factor X (FX) were identified from in vitro screens. An ionizable amino lipid based lipid nanoparticle (LNP) formulation for siRNA in vivo delivery was characterized as tolerable and exerting no appreciable effect on coagulability at day 7 postdosing in both species. Both prekallikrein siRNA-LNP and FX siRNA-LNP resulted in dose-dependent and selective knockdown of target gene mRNA in the liver with maximum reduction of over 90% on day 7 following a single dose of siRNA-LNP. Knockdown of plasma prekallikrein was associated with modest clot weight reduction in the rat arteriovenous shunt thrombosis model and no increase in the cuticle bleeding time. Knockdown of FX in the rabbit was accompanied with prolongation in ex vivo clotting times. Results fit the expectations with both targets and demonstrate for the first time, the feasibility of targeting coagulation factors in rat, and, more broadly, targeting a gene of interest in rabbit, via systemic delivery of ionizable LNP formulated siRNA.
RESUMO
Haemophilia A and B are characterised by a life-long bleeding predisposition, and several lines of evidence suggest that risks of atherothrombotic events may also be reduced. Establishing a direct correlation between coagulation factor levels, thrombotic risks and bleeding propensity has long been hampered by an inability to selectively and specifically inhibit coagulation factor levels. Here, the exquisite selectivity of gene silencing combined with a gene knockout (KO) approach was used to define the relative contribution of factor IX (fIX) to thrombosis and primary haemostasis in the rat. Using a lipid nanoparticle (LNP) formulation, we successfully delivered fIX siRNAs to the liver by intravenous administration. The knockdown (KD) of target gene mRNA was achieved rapidly (within 24 hour post-siRNA dosing), sustained (maintained for at least 7 days post dosing) and not associated with changes in mRNA expression levels of other coagulation factors. We found that intermediate levels of liver fIX mRNA silencing (60-95 %) translating into a 50-99 % reduction of plasma fIX activity provided protection from thrombosis without prolonging the cuticle bleeding time. Over 99 % inhibition of fIX activity was required to observe increase in bleeding, a phenotype confirmed in fIX KO rats. These data provide substantial evidence of a participation of fIX in the mechanisms regulating thrombosis prior to those regulating primary haemostasis, therefore highlighting the potential of fIX as a therapeutic target. In addition, hepatic mRNA silencing using LNP-encapsulated siRNAs may represent a promising novel approach for the chronic treatment and prevention of coagulation-dependent thrombotic disorders in humans.
Assuntos
Fator IX/genética , Hemofilia B/genética , Hemorragia/genética , Fígado/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Trombose/prevenção & controle , Animais , Linhagem Celular , Cloretos , Modelos Animais de Doenças , Fator IX/metabolismo , Compostos Férricos , Regulação da Expressão Gênica , Genótipo , Hemofilia B/sangue , Hemorragia/sangue , Hemostasia/genética , Masculino , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Trombose/sangue , Trombose/induzido quimicamente , Trombose/genética , Fatores de Tempo , TransfecçãoRESUMO
Angiotensinogen (AGT) is the precursor of active vasoconstrictive octapeptide angiotensin II (Ang II) in the renin-angiotensin-aldosterone system. Blocking the AGT-converting enzymes in the pathway and the Ang II receptor through pharmacological agents has been proven to be effective in lowering blood pressure (BP) in hypertensive patients. In this study, we developed chemically modified small interfering RNAs (siRNA) to target hepatic AGT mRNA in rats. Lipid nanoparticle encapsulated siRNAs were efficiently delivered to rat liver and resulted in significant reduction in hepatic Agt mRNA levels and plasma AGT concentration without impairing liver function. Single intravenous injection of Agt siRNA led to significant and sustained BP lowering in spontaneous hypertensive rats and in Sprague-Dawley rats, and the effect was maintained by weekly siRNA dosing. Data presented here provide proof-of-feasibility for the use of siRNA technology for inhibition of peripheral AGT levels via hepatic mRNA silencing with beneficial effects on BP in preclinical rat models. Similar approach could be used for validation of novel hypertension hepatic and extrahepatic targets.