RNA interference as antiviral therapy against Peste des Petits Ruminants: proof of concept of in vivo efficiency using a small animal model

Abstract

Objective: Morbilliviruses are responsible of important diseases in human beings and animals with economical impact in affected countries. “Peste des petits ruminants” is one of these diseases affecting goats and sheep with high mortality and morbidity. Efficient vaccines exist but they are often used in emergency situation in animals and ten days are necessary to induce a sufficient immune protection. Co-administration of an antiviral treatment with the vaccine could limit the disease impact while conferring a long-lasting protection. CIRAD has explored a biological antiviral therapy based on RNA interference. The identification of siRNA against morbilliviruses has been previously published and patented. The objective of this work was to validate the in vivo efficiency of these siRNAs. Methods: The proof of concept for an efficient in vivo delivery of anti-PPRV siRNAs was developed in a mouse model. Briefly, it is based on the siRNA dynamic extinction of a luciferase reporter gene in mice measured by bioimaging. The originality is also based on the use of a double-reporter expression plasmid allowing standardization within and between the trials. The plasmid is made of a firefly gene placed downstream of one of our morbillivirus siRNA target sequence and a renilla gene used as a constant gene-expression system. In the initial phase, mice received a co-injection (double reporter plasmid + relevant or irrelevant siRNA-PPRV) in the tibialis muscle, followed by an electroporation to promote cellular uptake of DNA. The firefly and renilla signals were measured daily using a bio- imager. The firefly expression was normalized using renilla signal. The specificity of RNA interference was checked by comparison with an unrelated siRNA. Once this initial phase validated, a second phase consisted in testing a delivery system for siRNA based on a cell membrane penetrating peptide. Results: The model was validated. In absence of any siRNA treatment, a good correlation was observed between the firefly and renilla luminescence activities. When the irrelevant siRNA was co- administrated, no incidence on these activities was detected. In contrast, mice treated with siRNA-PPRV showed a strong inhibition of about 99% of the firefly signal. This mouse model system is a proof of concept of in vivo siRNA efficiency and a very useful tool to assess in vivo siRNA delivery systems. Several candidates for in vivo delivery systems were investigated in our laboratory. Preliminary results showed that a cell membrane penetrating peptide could efficiently deliver a siRNA and inhibit the expression of firefly when tested in this mouse model. Conclusion: This mouse model system is a very useful tool that can be applied to test siRNA delivery systems in vivo. In this model, a cell penetrating peptide showed encouraging performances for systemic delivery of siRNA but extensive confirmation will be necessary. The model is now available for the screening of alternative delivery systems, including viral expression vectors that might represent a better cost-effective strategy for small ruminant's treatments in emerging countries. (Résumé d'auteur