Development of recombinant adeno-associated virus vector delivering short-hairpin RNAs to inhibit the replication of influenza viruses

Abstract

OBJECTIVES: RNA interference is a powerful tool to inhibit viral infection. Recombinant Adeno-associated virus (rAAV) vectors are promising and widely used for gene delivery. This study is to select nucleocapsid protein (NP) and matrix protein (M2) of influenza A virus as targets and to develop rAAV vector to deliver antiviral short hairpin RNAs (shRNAs) to combat influenza A. METHODS: rAAV plasmid (pAAV-H1-GFP) was constructed, which contained H1 promoter and GFP marker gene. shRNAs targeting M2(shM2) and NP(shNP) genes were inserted into the pAAV-H1-GFP. rAAV-shM2 and rAAV-shNP were packaged using a modified AAV Helper-Free system. The physical and biological titers of purified rAAV were quantified by real time PCR and flow cytometry and the shRNAs expression were checked by real-time RT-PCR. rAAV and rAAV-shM2 and rAAV-shNP infected MDCK cells respectively. At 48 hours (h) post-infection, MDCK cells were infected by H1N1 (MOI=1) and then incubated at 37°C for10 h. After aspirating medium, the MDCK cells were washed with PBS and added fresh medium and then incubated at 37°C for 1 h again to collect the supernatants to check the titers of H1N1 by plaque assay. RESULTS: The pAAV-H1-GFP was transfected into Hela, 293FT and MDCK cells and rAAV pseudo-virus infected MDCK cells. At 24 h post-transfection and 48 h post-infection, strong green flourenscence can be observed under fluorensence microscope and at 48 h post-infection, shRNA can be checked. The physical and biological titers of purified rAAV are more than 1013 and 1010/ml respectively. The H1N1 virus titer of the collected supernatant for rAAV and rAAV-shNP and rAAV-shM2 is 2.9±104, 1.1±104 and 6.6±103 PFU/ml, respectively. Therefore, 62.1% and 77.2% H1N1 virus were inhibited by rAAV-shNP and rAAV-shM2 respectively. CONCLUSIONS: pAAV-H1-GFP which can express shRNAs and GFP has been constructed and rAAV pseudo-virus with high titer and infectivity has been packaged and purified successfully. rAAV-shM2 and rAAV-shNP can infect MDCK cells and efficiently knock down M2 and NP specific mRNA of H1N1 to inhibit the replication of the H1N1 in MDCK cells. These preliminary results show that rAAV vector can be developed to deliver antiviral-shRNAs to combat influenza A and encourage us to further evaluate anti-H5N1 effects of rAAV-shM2 and rAAV-shNP in vitro and in vivo.