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1.
J Lipid Res ; 57(12): 2150-2162, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27707816

RESUMO

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/metabolismo
2.
Antimicrob Agents Chemother ; 60(12): 7077-7085, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27645237

RESUMO

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2'-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 µM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 µM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, ß, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated.


Assuntos
Antivirais/farmacocinética , Antivirais/toxicidade , RNA Polimerases Dirigidas por DNA/metabolismo , Hepacivirus/efeitos dos fármacos , Nucleosídeos/farmacocinética , Animais , Antivirais/administração & dosagem , Linhagem Celular , RNA Polimerases Dirigidas por DNA/genética , Cães , Hepacivirus/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Polifosfatos/metabolismo , Pró-Fármacos/farmacocinética , Pró-Fármacos/toxicidade , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Testes de Toxicidade/métodos , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
3.
J Virol ; 83(8): 3450-62, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19193803

RESUMO

Oncolytic adenoviral vectors that express immunostimulatory transgenes are currently being evaluated in clinic. Preclinical testing of these vectors has thus far been limited to immunodeficient xenograft tumor models since human adenoviruses do not replicate effectively in murine tumor cells. The effect of the immunostimulatory transgene on overall virus potency can therefore not be readily assessed in these models. Here, a model is described that allows the effective testing of mouse armed oncolytic adenovirus (MAV) vectors in immunocompetent syngeneic tumor models. These studies demonstrate that the MAV vectors have a high level of cytotoxicity in a wide range of murine tumor cells. The murine oncolytic viruses were successfully armed with murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) by a novel method which resulted in vectors with a high level of tumor-specific transgene expression. The mGM-CSF-armed MAV vectors showed an improved level of antitumor potency and induced a systemic antitumor immune response that was greater than that induced by unarmed parental vectors in immunocompetent syngeneic tumor models. Thus, the oncolytic MAV-1 system described here provides a murine homolog model for the testing of murine armed oncolytic adenovirus vectors in immunocompetent animals. The model allows evaluation of the impact of virus replication and the host immune response on overall virus potency and enables the generation of translational data that will be important for guiding the clinical development of these viruses.


Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Modelos Animais , Neoplasias/virologia , Vírus Oncolíticos/crescimento & desenvolvimento , Vírus Oncolíticos/imunologia , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Vírus Oncolíticos/genética
4.
Hum Gene Ther ; 17(8): 807-20, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16942441

RESUMO

In this study, recombinant AAV vectors pseudotyped with viral capsids derived from AAV serotypes 7 and 8 were evaluated for gene transfer in the murine striatum relative to vectors pseudotyped with AAV serotypes 2, 5, and 6. In comparison with rAAV serotype 2, pseudotyped vectors derived from AAV-7 and AAV-8 have increased transduction efficiency in the murine CNS, with the rank order rAAV-7 > rAAV-8 > rAAV-5 > rAAV-2 = rAAV-6, with all vectors demonstrating a marked tropism for neuronal transduction. Pseudotyped rAAV vector gene transfer in the brain after preimplantation of a murine 4C8 glioblastoma tumor was also evaluated. Efficiency of gene transfer to the orthotopic tumor was increased when using AAV-6, -7, and -8 capsid proteins in comparison with serotype 2, with the order rAAV-8 = rAAV-7 > rAAV-6 > rAAV-2 > rAAV-5. The increased gene transfer efficiency of rAAV vectors pseudotyped with the rAAV-8 capsid also provided enhanced therapeutic efficacy in a mouse model of glioblastoma multiforme, using vectors encoding an inhibitor of the vascular endothelial growth factor pathway. These studies demonstrate that rAAV vectors pseudotyped with capsids derived from AAV serotypes 7 and 8 provide enhanced gene transfer in the murine CNS and may offer increased therapeutic efficacy in the treatment of neurological disease.


Assuntos
Neoplasias Encefálicas/terapia , Corpo Estriado , Dependovirus , Terapia Genética , Glioblastoma/terapia , Neoplasias Experimentais/terapia , Animais , Capsídeo , Linhagem Celular Tumoral , Terapia Genética/métodos , Vetores Genéticos , Glioblastoma/genética , Humanos , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias/métodos , Neoplasias Experimentais/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Especificidade da Espécie , Transdução Genética/métodos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética
5.
J Cardiovasc Transl Res ; 8(1): 44-53, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25604958

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 , Transfecção
6.
Mol Ther ; 13(5): 956-66, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16580881

RESUMO

The presence of the blood-brain barrier complicates drug delivery in the development of therapeutic agents for the treatment of glioblastoma multiforme (GBM). The use of local gene transfer in the brain has the potential to overcome this delivery barrier by allowing the expression of therapeutic agents directly at the tumor site. In this study, we describe the development of a recombinant adeno-associated (rAAV) serotype 8 vector that encodes an optimized soluble inhibitor, termed sVEGFR1/R2, of vascular endothelial growth factor (VEGF). VEGF is an angiogenic factor highly up-regulated in GBM tumor tissue and correlates with disease progression. In subcutaneous models of GBM, VEGF inhibition following rAAV-mediated gene transfer significantly reduces overall tumor volume and increases median survival time following a single administration of vector. Using orthotopic brain tumor models of GBM, we find that direct intracranial administration of the rAAV-sVEGFR1/R2 vector to the tumor site demonstrates anti-tumor efficacy at doses that are not efficacious following systemic delivery of the vector. We propose that rAAV-mediated gene transfer of a potent soluble VEGF inhibitor in the CNS represents an effective antiangiogenic treatment strategy for GBM.


Assuntos
Sistema Nervoso Central/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Glioblastoma/terapia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Linhagem Celular , Linhagem Celular Tumoral , Dependovirus/classificação , Feminino , Terapia Genética/métodos , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Transplante de Neoplasias , Ratos , Ratos Nus , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sorotipagem , Solubilidade , Transplante Heterólogo
7.
J Mol Cell Cardiol ; 35(7): 739-48, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12818564

RESUMO

Grafting of saphenous vein (SV) conduits into the arterial circulation triggers a number of adaptive pathological changes characterized by progressive medial thickening, neointima formation and accelerated atheroma. Previous studies have shown that modification of vein graft biology is possible by adenovirus (Ad)-mediated gene transfer, although gene expression is transient. Advancement of vascular gene therapy to the clinic is compromised by the lack of safe and efficient vector systems that provide sustained therapeutic gene delivery to the vasculature. Due to inadequacies of both Ad and adeno-associated virus (AAV) serotype-2 (AAV-2) systems, we have evaluated gene delivery to endothelial cells (ECs) and smooth muscle cells (SMCs) using alternate AAV serotypes and a third-generation vesicular stomatis virus glycoprotein-pseudotyped lentiviral system. Transduction of both primary human SV EC and SMC was lower using all alternate AAV serotypes compared to AAV-2. However, transduction of both cell types by lentivirus was efficient even at clinically relevant exposure times (15 min), was without toxicity and was promoter sensitive. Transduction levels at lower doses were further enhanced with the addition of the surfactant Poloxamer-407 (P-407). Direct comparison with Ad and AAV-2 confirmed the unique potential for this system. Moreover, we constructed and overexpressed the therapeutic gene tissue inhibitor of metalloproteinase-3 (TIMP-3) using lentivirus and demonstrated transgene production comparable to Ad with concomitant blockade of SMC migration and induction of cell death. We have demonstrated for the first time the potential for third-generation lentiviral vectors, but not alternate AAV serotypes, as efficient vascular gene delivery vectors.


Assuntos
Sistema Cardiovascular/metabolismo , Terapia Genética , Vetores Genéticos , Lentivirus , Transdução Genética , Dependovirus , Humanos , Vírus da Estomatite Vesicular Indiana
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