Fig. 1.

The key discoveries and advances in circRNA vaccines. The development of circRNA vaccine can be divided into three stages. Phase 1, natural circRNA is discovered for the first time,⁴³ and the formation mechanism of circRNA is elucidated.⁴⁷ Phase 2 (1981–2022), the development of in vitro synthesis of circRNA. This phase began in 1981. After that, several key advances are developed, including three synthetic method were developed (enzymatic ligation, 1982;⁵⁹ PIE system, 1992;⁶⁰ chemical ligation, 1993⁶¹), foreign sequences circularization (1994),⁶² introduction of IRES to engineer translational circRNA (1995),⁶³ engineering translational circRNA via PIE system (1998),⁶⁴ purification of circRNA by HPLC (2011),⁶⁵ identification of various IRES candidates (2016),⁶⁶ efficient optimization of PIE system (2018),^35,67 reduction of artificial circRNA immunogenicity (2019) ^38,68 and achievement of high translation efficiency (2022).⁶⁹ Phase 3 (2022~), engineering circRNA vaccines are used for the first time in the treatment and prevention of COVID-19 and melanoma.^27–31 Future development will continue to advance