USING THE INFLUENZA POLYMERASE 5'-ENDONUCLEASE ACTIVITY TO DEVELOP NOVEL siRNA THERAPEUTICS
Jesteadt, Eric Matthew Neff
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Influenza is a global public health burden, producing seasonal epidemics with 3-5 million severe cases and 250,000 to 500,000 deaths each year. In addition to seasonal illness, influenza has also been responsible for global pandemics, the most notorious being the 1918 “Spanish Influenza.” While vaccination is the most useful tool for seasonal or pandemic influenza control, such interventions would not help individuals who are already infected. Antiviral drugs such as neuraminidase inhibitors and adamantanes can be effective if taken soon after symptoms appear, but antiviral resistance is developing. An alternative to antiviral drugs is the promising field of therapeutics based on RNA interference (RNAi). RNAi based treatments utilize chemically synthesized short interfering (si)RNAs between 21-24 nt in length to silence genes of interest. A number of research groups have investigated RNAi based antivirals, but the main limitations to their clinical use are risks of off-target effects and difficulty with appropriate dose delivery. A potential solution to both problems would be to use a pro-siRNA which is inert in uninfected cells but activated upon infection, thus limiting off-target effects to infected cells while sparing healthy cells. This approach would also provide dosing flexibility, as the active dose would be modulated by viral activity. In the case of influenza, a pro-siRNA consists of a standard siRNA duplex with a 10-13 nucleotide 7-methyl-guanosine (M7G) capped sequence on the 5’ end of the passenger or positive sense strand. This extension should render the pro-siRNA biologically inert in uninfected cells but be cleaved off in infected cells via PA mediated cap snatching activity, leaving behind a functional siRNA duplex. To test the feasibility of this approach, we designed variants of pro-siRNAs targeting eGFP, which can be expressed in a variety of cell types using transient or stable transfection. Cells were transfected with eGFP producing plasmids and/or siRNAs, infected with influenza 24-26 hours post transfection and fixed and analyzed 24 hours post infection. Through use of flow cytometry, we found that several pro-siRNA designs produced modest reductions in eGFP expression in infected cells, suggesting that the concept is feasible. However, further optimization will be required for capped pro-siRNAs to be a practical antiviral approach against seasonal and pandemic influenza.