RESUMO
BACKGROUND: Coxsackievirus B3 (CVB3) is a major heart pathogen against which no therapy exists to date. The potential of a combination treatment consisting of a proteinaceous virus receptor trap and an RNA interference-based component to prevent CVB3-induced myocarditis was investigated. METHODS AND RESULTS: A soluble variant of the extracellular domain of the coxsackievirus-adenovirus receptor (sCAR-Fc) was expressed from an adenoviral vector and 2 short hairpin RNAs (shRdRp2.4) directed against CVB3 were delivered by an adeno-associated virus (AAV) vector. Cell culture experiments revealed additive antiviral activity of the combined application. In a CVB3-induced mouse myocarditis model, both components applied individually significantly reduced inflammation and viral load in the heart. The combination exerted an additive antiviral effect and reduced heart pathology. Hemodynamic measurement revealed that infection with CVB3 resulted in impaired heart function, as illustrated by a drastically reduced cardiac output and impaired contractility and relaxation. Treatment with either sCAR-Fc or shRdRp2.4 significantly improved these parameters. Importantly, the combination of both components led to a further significant improvement of heart function. CONCLUSIONS: Combination of sCAR-Fc and shRdRp2.4 exerted additive effects and was significantly more effective than either of the single treatments in inhibiting CVB3-induced myocarditis and preventing cardiac dysfunction.
Assuntos
Antivirais/farmacologia , Infecções por Coxsackievirus/tratamento farmacológico , Enterovirus Humano B/efeitos dos fármacos , Terapia Genética/métodos , Miocardite/tratamento farmacológico , Interferência de RNA , Animais , Antivirais/metabolismo , Infecções por Coxsackievirus/virologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocardite/virologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Carga Viral/efeitos dos fármacosRESUMO
Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.
Assuntos
Dependovirus/fisiologia , Regulação da Expressão Gênica , Inativação Gênica , Vetores Genéticos/genética , MicroRNAs/genética , Músculo Esquelético/metabolismo , Transgenes , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Ordem dos Genes , Técnicas de Transferência de Genes , Genes Reporter , Vetores Genéticos/metabolismo , Humanos , MicroRNAs/metabolismo , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Especificidade de Órgãos/genética , Tropismo ViralRESUMO
A conformationally biased, agonist of human C5a(65-74) (EP67) was assessed for its adjuvant activities in vitro and in vivo. EP67 induced the release of the inflammatory (Th1) type cytokines from C5a receptor (CD88)-bearing antigen presenting cells (APC). EP67 did not induce the release of these cytokines from splenic APCs obtained from C5a receptor knockouts (CD88(-/-)). Serum from mice immunized with EP67-ovalbumin (OVA) contained high OVA-specific antibody (Ab) titers [IgG1, IgG2a (IGg2c), IgG2b]. Mice receiving OVA alone produced only IgG1 Abs, indicating the ability of EP67 to induce a Th1-like Ab class switch. Spleen cell cultures from wild type mice but not CD88(-/-) mice showed an enhanced OVA-specific proliferative response in vitro. These results indicate the ability of EP67 to drive a Th1-mediated immune response and its potential use as a unique adjuvant.