This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an RdRp-binding site in the 5- or 3-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.

The present application relates to a replicable RNA molecule and the use thereof. The replicable RNA molecule comprises, from the 5 end to the 3 end, a 5 cap, a 5 UTR, an open reading frame encoding an RNA replicase, a promoter, a sequence of interest, a 3 UTR and a poly(A) tail, wherein the RNA replicase is capable of amplifying the replicable RNA molecule and is capable of amplifying an RNA molecule containing the sequence of interest and 3 UTR, wherein the RNA replicase is a nonstructural protein or a functional variant thereof derived from Mosso das Pedras virus (MDPV), Everglades virus (EVEV), Rio Negro virus (RNV), Mucambo virus (MUCV), Highlands J virus (HJV), Pixuna virus (PIXV), Trocara virus (TROV), Cabassou virus (CABV), Tonate virus (TONV), Bebaru virus (BEBV), Fort Morgan virus (FMV), Getah virus (GETV) or Ndumu virus (NDUV).

This invention generally relates to a novel composition of RNA/mRNA medicines as well as vaccines produced by using replicase- and/or RNA-dependent RNA polymerase (RdRp)-mediated RNA cycling reaction (RCR). The present invention is useful for developing a variety of self-amplifying RNA/mRNA (samRNA) medicines and vaccines containing at least a replicase/RdRp-binding site in the 5- or 3-end, or both, of any desired RNA molecule, including but not limited to antisense RNA (aRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA)/miRNA precursor, long non-coding RNA (lnRNA) and mRNA. These RNA molecules can be either in single-stranded or in double-stranded, or mixed, conformation. The samRNA so obtained is useful not only for producing RNA-based vaccines and/or medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under a proper in-vitro or in-cell translation condition. The replicase/RdRp-binding sites used in samRNA are derived or modified from coronaviral (e.g. COVID-19) and/or hepatitis C viral (HCV) RNA-dependent RNA polymerases (RdRp) in either single-stranded or double-stranded compositions.