Therapeutic Strategies for Influenza

The influenza A virus (IAV) is the causative organism of acute respiratory infections in humans. IAV poses serious threats to health across the globe. In 2009, the zoonotic swine influenza virus, H1N1, was responsible for the outbreak of pandemic infecting more than 60 million people in the U.S. alone, and a death toll of 120,000 globally. Other Avian influenza strains such as H5N1 and H7N9 are also zoonotic viruses capable of causing serious infections in humans.

Current therapeutic options include vaccination against seasonal influenza, and antiviral therapies such as oseltamivir and zanamivir that belong to neuraminidase inhibitor class of drugs; amantadine, an M2 channel blocker; and, xofluza, an endonuclease inhibitor. The conventional therapeutics have limited efficacy and have the chances of triggering the emergence of viral strains that are resistant to these therapies.

The IAV pathogen has a small genome and depends upon the host cell machinery for its replication, a key process in its life cycle. The IAV genome comprises of 8 negative-sense RNA segments that help in packaging viral RNA into the developing virions. Identification of these dependency factors of IAV that are essential for completion of its life cycle can help in developing therapeutics against IAV infection. Pooled genome-wide CRISPR/Cas9 screens have helped in identifying these factors.

In addition to its role as a genomic screen to identify new targets and inhibitory host factors, CRISPR/Cas technique can be used to develop antiviral strategies to eliminate RNA viruses such as IAV. For example, Class 2, Type VI-D CRISPR/Cas13d system from Ruminococcus flavefaciens XPD3002 is reported to be capable of inhibiting RNA viruses. Cas13d utilises a 22-nt spacer that can be tailored in accordance with the target RNA sequence.

 Recently, Timothy Abbott and his team of researchers from the Standford University developed a prophylactic antiviral CRISPR in human cells (PAC-MAN) strategy to inhibit not only IAV but SARS-CoV-2 and other coronaviruses as well. The team identified the highly conserved regions in the IAV genome and designed crRNAs capable of targeting these conserved viral sequences. By screening the crRNA pools, the most effective crRNA was identified. Cas13d PAC-MAN strategy was found to be able to inhibit viral replication in human lung epithelial cells, by targeting highly conserved RNA regions within the viral genome. Their results also indicated that the Cas13d PAC-MAN strategy could be more effective at inhibiting IAV at lower viral load, implying that the strategy would be more effective in preventing new infections that typically arise from exposure to lower viral loads.

Cas13d PAC-MAN strategy only works for cells that express the Cas13d endonuclease and tailored crRNAs, while other cells remain susceptible to infection. This is the key challenge in translating the potential therapeutic strategy to clinics. To increase the success of this therapy, the CRISPR components need to be expressed in a higher proportion of the human lung epithelial cells.