The Preparation And Evaluation Research Of RNA Interference Targeting Drug Against Rabies Virus

Infection with the rabies virus (RABV) manifests as a neurodegenerative diseaseknown as rabies, which is fatal in the clinical stage. Worldwide, there are at least55,000human cases of rabies each year. Affecting both animals and humans, rabiescases are reported worldwide but are most prevalent in undeveloped countries,specifically in rural areas of Africa and Asia. No definitive treatment exists to cure thedisease once the clinical stage has been reached, the current strategy to control rabiesis pre-exposure prophylaxis by vaccine injection, but research efforts are focused ondeveloping an effective antiviral strategy against established RABV infection.RNA interference (RNAi) therapy, which silences expression of specific targetgenes, represents a promising approach for treating viral infections in mammalianhosts. The RNAi approach has already been successfully applied to several humanpathogenic viruses. And the siRNA used in age-related macular degeneration (AMD)and respiratory syncytial virus (RSV) treatment has entered clinical trials. However,currently used RNAi are aimed at the local drug delivery, such as the eyes and lungs.And most of the treatment of diseases requires systemic drug delivery, so a efficienttargeted delivery system is needed.Recently, Kumar et al (2007) developed a transvascular method to deliver siRNAacross the BBB to the brain via intravenous injection. This method involvedcomplexing the siRNA to a short peptide (RVG29) derived from the rabies virusglycoprotein that binds specifically to acetylcholine receptors (AchRs) on neuronalcells. Adding nine d-Arginines to the carboxy terminus of this peptide (RVG29-9R)enabled the peptide to electrostatically interact with the siRNA and specificallydeliver it to neurons in the mouse brain, thereby suppressing protein expression.Following the discovery of this RVG peptide, several studies have demonstrated the successful delivery of genes to the mouse brain by overcoming the blood–brainbarrier (BBB) using RVG29. However, as far as we know, no RVG29-based DNAdelivery system has been shown to be effective in rabies treatment. In addition, sincethe RVG29currently used are synthetized by commercial biotech companies, from thepoint of view of clinical application, it may not be economically feasible to produceand use RVG29as a targeted delivery vehicle. With this in mind, we describe aneconomically viable production method for the manufacture of RVG29.In the present study, we aimed to develop a universal siRNA agent that is capableof targeting different RABV strains. The N gene was chosen for this study since it isthe most highly conserved among the five RABV genes. We designed six smallinterfering (si)RNAs (N473, N580, N783, N796, N799and N1227) that target theconserved region of the RABV challenge virus standard (CVS)-11strainnucleoprotein (N) gene. Using a plasmid-based transient expression model, wedemonstrated that N796, N580and N799were capable of significantly inhibiting viralreplication in vitro and in vivo. These three siRNAs effectively suppressed RABVexpression in infected baby hamster kidney-21(BHK-21) cells, as evidenced by directimmunofluorescence assay, viral titer measurements, real-time PCR, and Westernblotting. And the significant inhibitive effect lasted for4days. In vivo, all of the threecould partially protect mice from a lethal dose of CVS-11rabies virus.In this study we constructed, expressed and purified, a novel recombinant plasmidtermed pGEX-4T-1/RVG29-9R-6His. Forty milligrams of a recombinant fusionprotein (GST-RVG29-9R-6His), and6mg of peptide (RVG29-9R-6His) wereobtained from100mL of bacterial culture. We assessed the physiochemical propertiesof RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro2a cells (which express the acetylcholine receptor). Our results revealed that theRVG29-9R-6His peptide recognized Neuro2a cells in a dose-dependent manner and itwas also able to bind plasmid DNA and deliver it into the Neuro2a cells effectively.After intravenous injection into mice, RVG29-9R-6His delivered a rabies virusspecific shRNA expression plasmid into the mouse brain resulting in specific genesilencing, and affording robust protection against the rabies virus, survival was60%. Heart, liver, spleen, kidney, brain tissue in mice was taken with immunohistochemicalanalysis48h after intravenous administration, the results showed that drugs weremainly concentrated in brain tissue, less peripheral tissue distribution usingRVG29-9R-6His delivery system; On the contrary, drugs were mainly focused onperipheral tissue, less brain tissue distribution using liposomes delivery system.In conclusion, we developed three siRNAs (N796, N580and N799) thatsuccessfully targeted the N gene of RABV. All three effectively inhibited RABVreplication and protected mice from challenges with the CVS-11strain. And we havesuccessfully generated a recombinant RVG29-9R-6His and demonstrated that iteffectively recognized and bound to Neuron2a cells, and delivered DNA into thebrain in vivo, suggests a novel, more economical strategy, for brain-targeted DNAdelivery in vivo.