RESUMO
Chronic elevations of plasma apolipoprotein B (apoB) are strongly associated with cardiovascular disease. We have previously demonstrated that inhibition of hepatic apoB mRNA using antisense oligonucleotides (ASO) results in reductions of apoB, VLDL, and LDL in several preclinical animal models and humans. In this study, we evaluated the anti-atherogenic effects of a murine-specific apoB ASO (ISIS 147764) in hypercholesterolemic LDLr deficient (LDLr(-/-)) mice. ISIS 147764 was administered weekly at 25-100 mg/kg for 10-12 weeks and produced dose-dependent reductions of hepatic apoB mRNA and plasma LDL by 60-90%. No effects on these parameters were seen in mice receiving control ASOs. ApoB ASO treatment also produced dose-dependent reductions of aortic en face and sinus atherosclerosis from 50-90%, with high-dose treatment displaying less disease than the saline-treated, chow-fed LDLr(-/-) mice. No changes in intestinal cholesterol absorption were seen with apoB ASO treatment, suggesting that the cholesterol-lowering pharmacology of 147764 was primarily due to inhibition of hepatic apoB synthesis and secretion. In summary, ASO-mediated suppression of apoB mRNA expression profoundly reduced plasma lipids and atherogenesis in LDLr(-/-) mice, leading to the hypothesis that apoB inhibition in humans with impaired LDLr activity may produce similar effects.
Assuntos
Apolipoproteínas B/metabolismo , Aterosclerose/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Receptores de LDL/deficiência , Animais , Apolipoproteínas B/antagonistas & inibidores , Apolipoproteínas B/sangue , Apolipoproteínas B/genética , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/metabolismo , Composição Corporal/efeitos dos fármacos , Colesterol/metabolismo , Feminino , Hipercolesterolemia/prevenção & controle , Immunoblotting , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Knockout , Período Pós-Prandial , Receptores de LDL/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/sangueRESUMO
The potency of second generation antisense oligonucleotides (ASOs) in animals was increased 3- to 5 -fold (ED(50) approximately 2-5 mg/kg) without producing hepatotoxicity, by reducing ASO length (20-mer to 14-mer) and by employing novel nucleoside modifications that combine structural elements of 2'-O-methoxyethyl residues and locked nucleic acid. The ability to achieve this level of potency without any formulation agents is remarkable and likely to have a significant impact on the future design of ASOs as therapeutic agents.
Assuntos
Conformação de Ácido Nucleico , Nucleosídeos/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/toxicidade , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/efeitos dos fármacos , Testes de ToxicidadeRESUMO
We investigated the use of an antisense oligonucleotide (ASO) specific for mRNA of the alpha chain (CD49d) of mouse VLA-4 to down-regulate VLA-4 expression and alter central nervous system (CNS) inflammation. ISIS 17044 potently and specifically reduced CD49d mRNA and protein in cell lines and in ex-vivo-treated primary mouse T cells. When administered prophylactically or therapeutically, ISIS 17044 reduced the incidence and severity of paralytic symptoms in a model of experimental autoimmune encephalomyelitis (EAE). This was accompanied by a significant decrease in the number of VLA-4+ cells, CD4(+) T cells, and macrophages present in spinal cord white matter of EAE mice. ISIS 17044 was found to accumulate in lymphoid tissue of mice, and oligonucleotide was also detected in endothelial cells and macrophage-like cells in the CNS, apparently due to disruption of the blood-brain barrier during EAE. These results demonstrate the potential utility of systemically administered antisense oligonucleotides for the treatment of central nervous system inflammation.
Assuntos
Encefalomielite Autoimune Experimental/prevenção & controle , Imunossupressores/uso terapêutico , Integrina alfa4/fisiologia , Oligonucleotídeos Antissenso/uso terapêutico , Paralisia/prevenção & controle , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Imuno-Histoquímica , Imunossupressores/administração & dosagem , Imunossupressores/farmacocinética , Integrina alfa4/biossíntese , Integrina alfa4/genética , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/farmacocinética , Paralisia/imunologia , Paralisia/patologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Medula Espinal/imunologia , Medula Espinal/patologiaRESUMO
Inclusion of C-5 propynyl pyrimidines in phosphorothioate antisense oligonucleotides (ASOs) has been shown to significantly increase their potency for inhibiting gene expression in vitro. This increased potency is believed to be the result of enhanced binding affinity to target RNA. Our results show that C-5 propynyl pyrimidine-modified oligonucleotides caused an increase in the melting temperature (T(m)) of both oligodeoxynucleotides (ODNs) and 2'-O-(2-methoxy)ethyl (2'-MOE)-modified oligonucleotides. The in vitro data show a moderate increase in potency for an antisense oligodeoxynucleotide containing C-5 propynyl pyrimidines targeting the murine PTEN (MMAC1) transcript. Second-generation 2'-MOE chimeric ASOs containing C-5 propynyl pyrimidines showed no improvement in potency in PTEN target reduction in vitro or in vivo compared to their nonpropyne-modified parent. These results suggest that increasing affinity for target RNA beyond that achieved with the 2'-MOE modification does not further increase potency in cell-based assays. To evaluate whether this observation held true for in vivo applications, we evaluated both compounds in mice. We were unable to establish a dose-response relationship with C-5 propynyl pyrimidine-modified ODNs because of severe toxicity. The toxicity was characterized by mortality in animals receiving 50 mg/kg and an increase in infiltrating cells and apoptotic cells in livers of mice receiving 20 mg/kg. C-5 propynyl pyrimidine-modified chimeric oligonucleotides exhibited decreased hepatotoxicity compared with C-5 propynyl-modified ODNs but did not exhibit an increase in potency compared with unmodified chimeric oligonucleotides. The hepatotoxicity could be further limited if incorporation of propynyl pyrimidines was restricted to 2'-MOE nucleosides.
Assuntos
Endotélio Vascular/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Nucleotídeos de Pirimidina/química , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Sequência de Bases , Encéfalo/irrigação sanguínea , Linhagem Celular , Esquema de Medicação , Endotélio Vascular/efeitos dos fármacos , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/toxicidade , Tamanho do Órgão/efeitos dos fármacos , RNA/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine involved in the pathogenesis of inflammatory bowel disease. We have developed a second-generation antisense oligonucleotide (ISIS 25302) specific for murine TNF-alpha and have evaluated this oligonucleotide in two models of gut inflammation of distinct etiology. ISIS 25302 decreased TNF-alpha mRNA in a dose- and sequence-dependent manner in vitro in the mouse macrophage cell line P388D1. It also reduced TNF-alpha mRNA in vivo, in whole adipose tissue and in macrophages isolated from the adipose tissue of db/db mice, a strain with constitutively high expression of TNF-alpha. ISIS 25302 significantly reduced disease activity index scores in mice with both an acute and a chronic form of dextran sodium sulfate (DSS)-induced colitis. It also significantly improved histopathological scores in interleukin (IL)-10-deficient mice. This was accompanied by reductions in both the basal and lipopolysaccharide-stimulated secretion of TNF-alpha and interferon-gamma in colonic organ cultures from IL-10 -/- mice. In this model, efficacy was obtained with both a prophylactic treatment regimen or a therapeutic dosing protocol begun after colitis was already present. In both the DSS and IL-10 -/- models, scrambled and mismatch control oligonucleotides were largely without effect, suggesting that ISIS 25302 was exerting its effects through a sequence-dependent antisense mechanism.
Assuntos
Colite/prevenção & controle , Oligonucleotídeos Antissenso/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Doença Aguda , Animais , Células Cultivadas , Doença Crônica , Colite/induzido quimicamente , Colite/genética , Colo/metabolismo , Colo/patologia , Sulfato de Dextrana , Interleucina-10/deficiência , Interleucina-10/genética , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Fosforotioatos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is crucial for metabolic responses to insulin, and defects in PI3K signaling have been demonstrated in type 2 diabetes. PTEN (MMAC1) is a lipid/protein phosphatase that can negatively regulate the PI3K pathway by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate, but it is unclear whether PTEN is physiologically relevant to insulin signaling in vivo. We employed an antisense oligonucleotide (ASO) strategy in an effort to specifically inhibit the expression of PTEN. Transfection of cells in culture with ASO targeting PTEN reduced PTEN mRNA and protein levels and increased insulin-stimulated Akt phosphorylation in alpha-mouse liver-12 (AML12) cells. Systemic administration of PTEN ASO once a week in mice suppressed PTEN mRNA and protein expression in liver and fat by up to 90 and 75%, respectively, and normalized blood glucose concentrations in db/db and ob/ob mice. Inhibition of PTEN expression also dramatically reduced insulin concentrations in ob/ob mice, improved the performance of db/db mice during insulin tolerance tests, and increased Akt phosphorylation in liver in response to insulin. These results suggest that PTEN plays a significant role in regulating glucose metabolism in vivo by negatively regulating insulin signaling.