Potent inhibition of influenza virus replication with novel siRNA-chimeric-ribozyme constructs

Abstract

A multitarget approach is needed for effective gene silencing for RNA viruses that combines, more than one antiviral approach. Towards this end, we designed a wild-type (wt) chimeric construct, that consisted of small hairpin siRNA joined by a short intracellular cleavable linker to a known, hammerhead ribozyme (Rz), both targeted against M1 genome segment of influenza A virus. When this, wt chimeric RNA construct was introduced into a mammalian cell line, along with the M1 substrate, encoding DNA, very significant (67%) intracellular down regulation in the levels of target RNA was, observed. When the siRNA portion of this chimeric construct was mutated keeping the Rz region, unchanged, it caused only 33% intracellular reduction. On the contrary, when only the Rz was made, catalytically inactive, keeping the siRNA component unchanged, about 20% reduction in the target M1, specific RNA was observed. This wt chimeric construct showed impressive (>80%) protection against, virus challenge, on the other hand, the selectively disabled mutant constructs were less effective. Thus, in this proof of concept study we show that varying levels of protection against virus challenge was, observed with novel mutant versions of the chimeric constructs.