Leveraging cytoplasmic transcription to develop self-amplifying DNA vaccines

Project Summary The unprecedented speed of COVID-19 vaccine development has demonstrated the value of vaccine platforms. In particular, mRNA vaccines proved surprisingly successful at eliciting a strong immune response while having a remarkable safety profile considering the novelty of this system. Just as important are the remarkable speed and scale of their production. Despite their spectacular success, mRNA vaccines suffer from major limitations. mRNA is an inherently unstable molecule. One consequence of this property is that mRNA vaccines need to be stored in freezers and their shelf-life is measured in hours after they have been thawed. These storage requirements are considered difficult to ensure even in countries with developed healthcare systems and are extremely problematic in many other parts of the world. The second limitation of mRNA vaccines is that their production is unlike any other biomanufacturing process. As a result, it is limited by a critical lack of infrastructure and expertise. The COVID-19 mRNA vaccines provided an incentive to imagine the next generation of nucleic acid vaccines that would be easier to manufacture at scale and distribute to healthcare systems throughout the world. This proposal hypothesizes that a DNA-based vaccine could enable the design and deployment of safe and effective vaccines that would be faster, easier, and cheaper to manufacture at scale. The production of clinical- grade DNA relies on biomanufacturing processes that are some of the simplest, fastest, and most inexpensive processes in the industry. However, DNA vaccines have failed to elicit a protective immune response so far because only a small fraction of the DNA molecules entering a cell are transported to the nucleus where they can be transcribed. In this R21, researchers will test the feasibility of developing a new generation of DNA vaccines by applying methods from synthetic biology to introduce genetic circuits allowing the expression of the antigen to take place in the cytoplasm. Self-amplifying DNA vaccines will include several genes of viral origins that will transcribe the antigenic sequences from plasmids located in the cell cytoplasm. In addition, these vectors will include additional enzymes to modify mRNA molecules to increase their stability and translation efficiency. By introducing several levels of amplification, the expression of the antigen is expected to be several orders of magnitude higher than what can be achieved with traditional DNA vaccines. The project will proceed through eight iterations of the design-build-test-learn cycle to rationally improve vaccine designs using gene expression data in cell culture. If successful, future studies will test the platform compatibility with a broad range of antigens, optimize the delivery of DNA-based vaccines, and analyze the safety and efficacy of candidate vaccines in animal studies.