Development of Inducible Anti-influenza Therapies

摘要

Influenza viruses continue to cause significant morbidity and mortality each year despite the development of vaccines and antiviral therapies targeting these viruses. The inherent ability of influenza viruses to accumulate mutations over time has led to the emergence of strains resistant to antiviral therapies. Furthermore, genetic reassortment creates antigenically diverse viruses, making it difficult to develop vaccines that yield broad protection. The objective of the following research studies is to develop two alternative approaches to current methods of antiviral therapeutics.;Six new siRNAs targeting influenza protein expression by RNA interference (RNAi) were characterized. Three siRNAs (M747, M776, M832) knocked down the expression of matrix protein 2 and attenuated influenza infection to a similar degree as MDCK cells treated with a previously published siRNA, M950. The three siRNAs (NS570, NS595, NS615) that target the nonstructural protein 1 and 2 genes promoted the expression of type I interferons, but were unable to attenuate the production of infectious virus. However NS595- and NS615-siRNAs promoted the production of defective interfering viruses. Another siRNA, M331, was able knock down the expression matrix 1 and matrix 2 and attenuate viral replication. Combination siRNA treatment was found to attenuate 20.9% more infectious virus than M950-siRNA treatment alone. Treatment with a single siRNA (M331, NS570, NS595, or NS615) that targets two protein coding sequences was able to knock down the expression of two proteins, thus enhancing the utilities of the siRNAs.;To further take advantage of RNAi as a mechanism to attenuate influenza infection, we developed an inducible anti-influenza therapy containing the influenza conserved promoter that expresses asRNAs only after influenza infection or in the presence of the influenza virus RNA-dependent RNA polymerase (RdRP). asRNA expression was restricted to pM950, pM776, pNS595, or pNA105 treated cells containing the RdRP. The asRNAs expressed from the inducible asRNA expression vectors (pM776 or pNS595) were 84- to 343-fold below the concentration needed to reduce influenza virus infection by RNAi, thus illustrating the need for improved expression kinetics. Limiting expression of asRNAs within influenza infected cells could potentially reduce the adverse effects and limitation of RNAi therapeutics.;In an attempt to reverse antigenic variation and attenuate influenza titer, we developed additional inducible anti-influenza therapies (pUC57 NF-NA and pUC57 F-NA), similar to the inducible asRNA expression vector, which express nonfunctional or functional neuraminidases (NF-NA or F-NA) upon influenza infection. The presence of vector expressed RdRP or influenza infection induced the expression of NF-NA and F-NA. Overexpression of NF-NA was originally hypothesized to attenuate influenza titer; however, NF-NA regained its sialidase activity after RdRP-mediated transcription. pUC57 NF-NA or F-NA transfected cells produced an RNA-intermediate regardless of the presence of the RdRP, whereas the polymerase was required for NF-NA mRNA and protein expression. Interestingly, reinfection of MDCK cells with the supernatant from pUC57 NF-NA or F-NA treated and influenza (N1 subtype) infected cells revealed that the naive MDCK cells generated N2 subtype viruses, indicating the induced N2 viral RNA could be packaged into progeny viruses forcing the N1 virus to become an N2 virus.;These studies demonstrate that RNAi can be an effective means to attenuate influenza infection. Furthermore, incorporation of the influenza conserved promoter into asRNA or neuraminidase expression vectors can be exploited to promote influenza infected cell-specific expression of anti-influenza molecules. This approach may impact the design and advancement of antiviral therapeutics by overcoming the limitations associated with RNAi and allow for current vaccines to protect against influenza infection by forcing influenza viruses to converge into a single subtype.