Targeted Inhibition Rabies Virus Replication In Vitro And In Vivo By Single Chain Antibody-Mediated ShRNA Delivery

Rabies is a lethal zoonotic infection disease induced by Rabies virus and its case fatality ratio (CFR) is 100 percent approximately. China is one of the most severe regions of rabies spread. According to the recommendations of WHO rabies experts committee, post-exposure treatment (PET) must be taken as soon as possible after a bite or a scratch occurred. Immunoglobulin is one of the regents in PET, but the zoogenous immunoglobulin is a kind of allergen, and the products made from human blood are infective agents of some body fluid transmitted diseases. Thus, new antiviral approaches that allow efficient and specific destruction of rabies virus are required.RNA interference (RNAi) refers to the small dsRNA-guided gene silencing phenomenon conserved in a wide range of eukaryotic organisms from plants to mammals. It is an important mechanism of cellular defense and differentiation regulation and plays an important role in the regulation of gene expression, the prevention of viral infection and the control of gene transposition. RNAi in target cells could be induced specifically and effectively by 21nt to 23nt short interfering RNA (siRNA) and has become a powerful tool to explore gene functions replacing the knock-out technique. With the progressions of the research about RNAi, some investigations have been performed successfully to inhibit the replication of viruses.RNAi can be induced by the introduction of synthesized double-stranded siRNA or by the intracellular generation of siRNA from the vector-driven expression of precursor small hairpin RNA (shRNA) which is subsequently processed in the cytoplasm by Dicer into siRNA. Synthesized siRNA has a very short half-life of a few minutes in vivo. The gene-silencing effects of the shRNA are long-lasting and reproducible which make the results more reliability, and it can be prepared and produced large-scale at low cost. ShRNA seems to be ideal for delivering of siRNA in vivo. In this study we selected expression plasmids for shRNA to research targeted inhibition rabies virus replication.The field of RNAi is moving forward at a remarkable pace.Because of their ability to induce transient and reversible effects, siRNAs offer a drug-like approach to disease treatment and thus, several clinical trails are being inducted to assess the safety and efficacy of this approach.A major challenge to the development of RNAi-based therapeutics is specific and efficient in vivo delivery to target cells. Naked siRNA has a very short half-life of a few minutes in serum owing to degradation by ribonucleases (RNAase), rapid renal excretion, uptake by the reticuloendothelial system (RES) and aggregation with serum proteins. Recent studies suggest that cell type-specific gene silencing in vivo can be achieved by combining therapeutic RNAi with cell type-specific affinity ligands or monoclonal antibodies. The antibody-directed siRNA complex enters target cells through endocytosis and is subsequently released to the cytosol to specifically silence target gene expression through biologically conserved RNAi pathways. Antibody fragments fused with a small basic nucleic acid-binding protein is an effective delivery vehicle in vivo. The demonstrated specificity of in vivo gene silencing and the lack of nonspecific immune activation and systemic toxicity encourage further delivery of therapeutic RNAi.Based on the multidomain structure of the bacterial Pseudomonas exotoxin A, a recombinant fusion protein was constructed which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis. The protein consists of three functional domains:1)a RV G-specific single chain disulfide-stabilized antibody confers target cell specificity.2) the exotoxin A translocation domain facilitates endosome escape, and 3) a DNA binding domain derived from the yeast GAL4 protein enables sequence-specific high bacterial lysates displayed both RV infected cells specific and DNA sequence-specific binding in vitro and in vivo. We successful obtain the SEG-shRNA complexes, which targeted the RV infected cells. In this thesis, the replication of rabies virus was inhibited by SEG-shRNA complexes in vivo and in vitro experimentally in order to accumulate essential data of single-chain Fv antibody (scFv) mediated shRNA targeted therapy in the research of rabies virus the PET.Targeting N gene of RV, four shRNA were designed and constructed based on the vector pRNATU6.3-Hygro which expresses fusion protein of green fluorescent protein (GFP) as a report gene. Four cell strains (N1, N2, N3, N4) expressing the short hairpin RNAs (shRNA) were obtained after the plasmids were transfected into the BHK-21 cell line and screened under the pressure of Hygromycin B (300μg/ml).These cell strains were infected rabies virus CVS-11 strain respectively, the virus replication were detected and evaluated by directed immunofluorescence assay (DFA) and real-time PCR and 50% tissue culture infective dose (TC1D50). The three detected results were coincidence. We selected effective inhibition RV siRNA targeting N gene, in which N-489 and N-701 were proved that they can specifically inhibit the virus production in BHK-21 cells. To produce a scFv which can specific recognize the RV infected cells, the heavy (VH) and light chain variable region (VL) sequence of monoclonal antibody SO57 which obtained from Genbank were assembled into a scFv gene using a linker sequence. To construct the recombinant human anti-rabies virus single-chain disulfide-stabilized Fv (scdsFv), Cys sites were introduced into framework region (FR) of VH and VL gene using genetic point mutation technology. The scdsFv gene was cloned into expression vector pET-22b (+) and transformed into E.coli BL21(DE3). The target protein was expressed in form of inclusion bodies. The avidity, specificity characteristics and neutralization capacity of scdsFv showed that scdsFv could bind antigen specifically.In order to prepare the chimeric protein which could realize targeting transfer DNA which express shRNA into the RV infected cells, rabies virus scdsFv(G) gene and ETA-GAL4 gene were amplified by PCR from vector scdsFv(G)-T and PE40-GAL4-T respectively. Then, the chimeric gene scdsFv(G)-ETA-GAL4 (SEG) was obtained by lapextension PCR and cloned into the prokaryotic expression vector pET28a(+). Recombinant expression plasmid of pET28a(+)-scdsFv(G)-ETA-GAL4 (pET28a-SEG) was transformed into the competent E.coli BL21(DE3) cells for expression under the induction of IPTG. The SEG proteins mainly expressed to form inclusion bodies. The SEG proteins were purified by Ni-NTA column and renatured to obtain the biological activity. The experiment results showed that the fusion protein SEG could bind affinity to RV and the RV infected cells.We next tested whether SEG could bind and deliver shRNA to RV-infected cells and found that the fusion protein was able to bind shRNA in a dose-dependent manner in a gel-shift assay and 1nM SEG can bind 10nM shRNA molecules. The fusion protein SEG with plasmid pRNATU6.3-shRNA were added to BHK-21 cells culture medium that infected with RV and then green fluorescent protein (GFP) was observed after 24 hours. It was showed that SEG could realize targeting transfer DNA which expressed shRNA into the RV infected cells. Cell viability caused by SEG-shRNA complexes was also determined by MTT assay and the result confirmed that the complexes were almost non-toxic to cell viability. The levels of viral particles from SEG-shRNA-treated infected cells was reduced as demonstrated by RV mRNA and proteins. These results demonstrated that the complex effectively inhibited RV replication in BHK-21 cells.Because there is no good mouse model for rabies virus, we injected mice intramuscular with CVS-24 as a RV infected animal model to test the ability of SEG fusion protein to specifically deliver shRNA into RV-infected cell in vivo.50LD50 RV-infected mice were injected intravenous with complexes SEG-shRNA. Single-cell suspensions from the hind lambs were examined by flow cytometry, which showed that SEG could specific delivery of shRNA into RV-infected hind lamb cells. The results showed SEG protein could deliver shRNA to RV infected cells specifically. To evaluate the therapeutic potential of antibody-mediated shRNA delivery, we injected SEG-shRNA intravenously 8h after the mice infected lethal dose RV CVS-24. On five days when the RV in brain tissue of mice were detected by RT-PCR and western blot shown that mice treated with SEG-shRNA had lower RV RNA and protein levels than untreated controls. Real-time PCR showed that RV was reduced about 4.88 fold compared to the mock cells. The incubation period of the challenged mice were prolonged significantly (P＜0.01). Evaluation of mouse survival of RV-infected mice showed a significant protection from RV infection by SEG-shRNA treatment. The survival was up to 50% while the virus control all died.To rule out the possibility that non-specific interferon (IFN) production mediated the protection, we measured serum IFN levels after administration of SEG-shRNA, and found that IFN was not induced in SEG-shRNA treated animals. Taken together, our results showed that SGE enables the specific delivery of shRNA to silence gene expression and SEG-shRNA can be used for adjutant treatment for rabies.