Publication date: Dec 13, 2025
DNA vaccines have garnered considerable attention due to their recent success in humans for SARS-CoV-2 and immunotherapy for cancer. However, conventional methods for creating and manufacturing DNA vaccines at-scale are slow and rate-limiting for timely response. Herein, we introduce a rapid and completely synthetic workflow that harnesses enzymes to create bulk DNA from a sequence text file. This synthetic workflow termed Enzymatic DNA Synthesis & Rolling-Circle Amplification (EDS-RCA) leverages multiple enzymes to print DNA oligos and assemble them into genes prior to cloning into circular constructs for rolling-circle amplification (RCA). We show that the resulting EDS-RCA DNA elicits comparable vaccine immunogenicity as standard plasmid format, despite the DNA being a large concatemeric repeat. The EDS-RCA method generated the hemagglutinin gene of H1N1 at a mean per-base error rate as low as ~1 mutation every 10,000 bases and, upon DNA vaccination, elicited strong antibody and cellular immune responses. Skin delivery of EDS-DNA using gene gun facilitated striking vaccine dose-sparing capabilities in comparison to intramuscular electroporation methods. In total, DNA vaccines produced by EDS-RCA are immunogenic and amenable to numerous delivery-modalities with preclinical mouse models and could offer an alternative for rapid scale-up of DNA vaccines for future human use.
Open Access PDF
| Concepts | Keywords |
|---|---|
| Cancer | Comparable |
| Enzymes | Eds |
| Timely | Enzymatic |
| Vaccines | File |
| Immune | |
| Plasmid | |
| Produced | |
| Rapid | |
| Rate | |
| Rca | |
| Scale | |
| Synthetic | |
| Vaccine | |
| Vaccines | |
| Workflow |