Protease Engineering to Enhance Alphavirus Vector Efficiency: Advancing Vaccine and Therapeutic Strategies

The COVID-19 pandemic has highlighted the need for innovative vaccines and treatments that go beyond traditional methods. One promising approach is self-amplifying RNA (saRNA) vaccines, which use a specialized RNA platform to produce a stronger immune response with smaller doses. For example, the ARCT-154 vaccine, recently approved in Japan, is based on this technology. Unlike standard mRNA vaccines, saRNA includes instructions that help it replicate itself once inside the body, reducing the need for large doses and potentially leading to longer-lasting immunity. This platform uses a modified alphavirus system containing key proteins (called nsPs 1-4) and a viral promoter to enhance RNA production. It also being studied for applications beyond vaccines, such as cancer therapies and targeted delivery of genetic material to specific cells. However, the efficiency of saRNA systems relies heavily on the activity of one protein in particular;nsP2, an alphavirus protease. This protein acts like molecular scissors, processing other proteins to enable RNA amplification. Improving the activity of nsP2 could make saRNA vaccines and therapies more effective. This project aims to enhance nsP2 activity by introducing controlled changes to its structure (structure-based mutagenesis) and generating random variations (random mutagenesis). A library of modified nsP2 proteins will be tested using a fluorescence-based method to identify those with the best activity. By creating more efficient nsP2 variants, this research aims to improve RNA production, reduce side effects, and make vaccines and therapies more accessible. This work could address the limitations of current mRNA technologies and expand the potential of saRNA for a range of medical applications.