RESUMO
The 37/67 kDa laminin receptor (LRP/LR) acts as a receptor for prions providing a promising target for the treatment of prion diseases. Recently, we selected anti-LRP/LR single-chain antibodies (scFvs) and proved a reduction of the peripheral PrP(Sc) propagation by passive immunotransfer into scrapie-infected mice. Here, we report the development of an in vivo gene delivery system based on adeno-associated virus (AAV) vectors expressing scFvs-S18 and -N3 directed against LRP/LR. Transduction of neuronal and non-neuronal cells with recombinant (r)AAV serotype 2 vectors encoding scFv-S18, -N3 and -C9 verified the efficient secretion of the antibodies. These vectors were administered via stereotactic intracerebral microinjection into the hippocampus of C57BL/6 mice, followed by intracerebral inoculation with 10 % RML at the same site 2 weeks post-injection of rAAV. After 90 days post-infection, scFv-S18 and -N3 expression resulted in the reduction of peripheral PrP(Sc) propagation by approximately 60 and 32 %, respectively, without a significant prolongation of incubation times and survival. Proof of rAAV vector DNA in spleen samples by real-time PCR strongly suggests a transport or trafficking of rAAV from the brain to the spleen, resulting in rAAV-mediated expression of scFv followed by reduced PrP(Sc) levels in the spleen most likely due to the blockage of the prion receptor LRP/LR by scFv.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Doenças Priônicas/terapia , Receptores de Laminina/imunologia , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Especificidade de Anticorpos , Técnicas de Transferência de Genes , Hipocampo/virologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPSc/metabolismo , Recombinação Genética , Baço/metabolismo , Baço/virologia , Transdução GenéticaRESUMO
Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virus-host interactions. Recently, we demonstrated that the IFN-induced 3' --> 5' exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-kappaB factors p50 and p65 bind and activate the kappaB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-kappaB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exonucleases/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , RNA de Cadeia Dupla/farmacologia , Sítios de Ligação , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/genética , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Exonucleases/genética , Exorribonucleases , Células HeLa , Humanos , Fator Regulador 1 de Interferon , Interferon Tipo I/farmacologia , Mutagênese Sítio-Dirigida , NF-kappa B/genética , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Regiões Promotoras Genéticas , RNA de Cadeia Dupla/síntese química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The accumulation of PrP(Sc) in scrapie-infected neuronal cells has been prevented by three approaches: (i) transfection of ScMNB cells with an antisense laminin receptor precursor (LRP) RNA-expression plasmid, (ii) transfection of ScN2a cells and ScGT1 cells with small interfering RNAs (siRNAs) specific for the LRP mRNA, and (iii) incubation of ScN2a cells with an anti-LRP/LR antibody. LRP antisense RNA and LRP siRNAs reduced LRP/LR expression and inhibited the accumulation of PrP(Sc) in these cells. The treatments also reduced PrP(c) levels. The anti-LRP/LR antibody, W3, abolished PrP(Sc) accumulation and reduced PrP(c) levels after seven days of incubation. Cells remained free of PrP(Sc) after being cultured for 14 additional days without the antibody, whereas the PrP(c) level was restored. Our results demonstrate the necessity of the laminin receptor (LRP/LR) for PrP(Sc) propagation in cultured cells and suggest that LRP/LR-specific antibodies could be used as powerful therapeutic tools in the treatment of transmissible spongiform encephalopathies.