mRNA composition and production method for use in anti-viral and anti-cancer vaccines
This invention relates to a novel mRNA composition and its production method useful for developing and manufacturing RNA-based anti-viral and/or anti-cancer vaccines and medicines. This invention includes two types of mRNA constructs, namely “5′-hairpin messenger RNA (5hmRNA)” and “messenger-hairpin-messenger RNA (mhmRNA)”, respectively. Both of 5hmRNA and mhmRNA contain at least a hairpin-like stem-loop RNA structure. The 5hmRNA contains at least a stem-loop RNA structure in the 5′-UTR of a protein/peptide-coding mRNA, while the mhmRNA contains a middle stem-loop structure flanked with two protein/peptide-coding mRNA sequences on both sides. In mhmRNA, the first 5′-mRNA preferably encodes an RNA replicase, for amplifying the second 3′-mRNA in transfected cells. After transfection into target cells, 5hmRNA and mhmRNA can be further translated into at least a desired protein/peptide. To produce highly structured 5hmRNA and mhmRNA, a novel PCR-IVT methodology has been developed and used with a specially designed RNA polymerase-helicase mixture reaction.
1 . A production method for manufacturing and amplifying self-amplifiable RNA construct exhibiting additional helicase activity, comprising:
(a) providing at least a DNA template encoding at least an RNA sequence, wherein a product of transcription of the RNA sequence is a messenger-hairpin-messenger RNA (mhmRNA) construct the mhmRNA construct contains at least a stem-loop RNA structure flanked with two different mRNA sequences of interest on its both sides comprising a first 5′-mRNA and a second 3′-mRNA, and wherein the first 5′-mRNA encodes COVID-19 virus RdRp, which consists solely of Nsp12, Nsp8, and Nsp7 and exhibits both RNA replicase activity and additional helicase activity, thereby contributing to the self-amplifiability of the mhmRNA construct;
(b) providing an in-vitro transcription (IVT) reaction condition containing NTPs, a preset buffer system, and at least a DNA-dependent RNA polymerase; and said preset buffer system contains chemicals to facilitate the denaturation of highly structured RNA/DNA sequences;
(c) mixing the DNA template of (a) and the IVT reaction condition of (b) to generate and amplify the mhmRNA construct from the encoded RNA sequence,
wherein the stem-loop RNA structure of said mhmRNA construct contains at least a sequence selected from SEQ.ID.NO.3, SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, and a combination thereof.
2 . The production method as defined in claim 1 , wherein said stem-loop RNA structure contains at least a perfectly or imperfectly matched either single or multiple hairpin structure, ranging about 10˜800 nucleotides in length, and said-mhmRNA construct further comprises a short sequence located between the stem-loop RNA structure and a start codon of the following second 3′-mRNA, ranging about 1˜500 nucleotides apart in length.
3 . The production method as defined in claim 2 , wherein said multiple hairpin structures in the stem-loop RNA structure further contains a spacer sequence in between every two hairpin structures, ranging about 2˜500 nucleotides apart in length.
4 . The production method as defined in claim 1 , wherein said second 3′-mRNA not only encodes at least one desired protein or peptide but also contains either SEQ.ID.NO.1 or SEQ.ID.NO.2 in its 3′-end.
5 . The production method as defined in claim 1 , wherein the stem-loop RNA structure further functions as an artificial internal ribosome entry site (IRES) mimic for initiating and enhancing translation of the second 3′-mRNA.
6 . The production method as defined in claim 1 , wherein after transfection into target cells, said mhmRNA construct are capable of being further translated into at least a desired protein/peptide for eliciting a pre-designed, desired biological effect or cellular function.
7 . The production method as defined in claim 1 , wherein said stem-loop RNA structure of said mhmRNA construct contain at least a sequence capable of being further processed in transfected cells after transfection to generate at least a shRNA and/or piRNA useful for silencing at least a specific target gene.
8 . The production method as defined in claim 7 , wherein said specific target genes include a variety of disease-associated cellular and viral genes.
9 . The production method as defined in claim 1 , wherein at least an additional helicase enzyme is added to the IVT reaction condition in order to facilitate the unwinding of RNA/DNA secondary structures.
10 . The production method as defined in claim 1 , wherein, said mhmRNA construct encodes at least an ingredient of an anti-viral vaccine.
11 . The production method as defined in claim 1 , wherein, said mhmRNA construct encodes at least an ingredient of an anti-cancer medicine.
12 . The production method as defined in claim 1 , wherein, said mhmRNA construct is further mixed with at least a delivery agent for cellular transfection in vitro, ex vivo or in vivo.
13 . The production method as defined in claim 12 , wherein said delivery agent includes glycylglycerins, liposomes, nanoparticles, liposomal nanoparticles, conjugating molecules, infusion chemicals, gene gun materials, electroporation particles, transposon, and a combination thereof.
14 . The production method as defined in claim 1 , wherein said preset buffer system contains 1× transcription buffer with additional 0.001˜10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), or 3-(N-morpholino) propane sulfonic acid (MOPS), or a combination thereof.
15 . The production method as defined in claim 1 , wherein said mhmRNA construct further contains at least a modified nucleotide.