Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin activating protease TMPRSS2

Influenza A viruses constitute a major and ongoing global public health concern. Current antiviral strategies target viral gene products, however, the emergence of drug resistant viruses highlights the need for novel antiviral approaches. Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is crucial for viral infectivity and presents therefore a potential drug target. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA-like antisense agents that readily enter cells and can act as antisense agents by sterically blocking complementary RNA. Here, we evaluated the effect of PPMO targeted to regions of the pre-mRNA or mRNA of the HA-cleaving protease TMPRSS2 on proteolytic activation and spread of influenza viruses in human Calu-3 airway epithelial cells. We found that treatment of cells with a PPMO (T-ex5) designed to interfere with TMPRSS2 pre-mRNA splicing resulted in TMPRSS2 mRNA lacking exon 5 and consequently expression of a truncated and enzymatically inactive form of TMPRSS2. Altered splicing of TMPRSS2 mRNA by T-ex5 PPMO prevented HA cleavage of different human seasonal and pandemic influenza A viruses and suppressed viral titers by 2-3 log10, strongly suggesting that TMPRSS2 is responsible for HA cleavage in Calu-3 airway cells. The data indicate that PPMO provide a useful reagent for investigating HA-activating proteases and may represent a promising strategy for the development of novel therapeutics to address influenza infections.