JP2024523006A - Novel Replicase Cycling Reaction (RCR) - Google Patents

Abstract

The present invention generally relates to a novel method for the production and amplification of RNA/mRNA using viral RNA replicases and/or RNA-dependent RNA polymerase (RdRp) enzymes and their associated mRNAs. The present invention can be used for the production and amplification of any kind of RNA/mRNA sequence having at least one RdRp binding site at the 5'-end or 3'-end or both. The RNA/mRNA thus obtained is useful for producing mRNA vaccines and/or RNA-based medicines as well as for generating mRNA-associated proteins, peptides and/or antibodies under in-vitro and in-cell translation conditions. Mainly, the present invention is a novel RNA replicase-mediated RNA/mRNA amplification method, i.e. replicase cycling reaction (RCR). RNA replicases involved in RCR include, but are not limited to, viral and/or bacteriophage RNA-dependent RNA polymerases (RdRps), in particular coronavirus and Hepatitis C virus (HCV) RdRp enzymes.

Description

CROSS REFERENCE TO RELATED APPLICATIONS:
This invention claims priority to U.S. Provisional Patent Application No. 63/209,969, filed June 12, 2021, entitled "Novel mRNA Composition and Production for Use in Anti-Viral and Anti-Cancer Vaccines." In addition, the present invention claims priority to U.S. Provisional Patent Application No. 63/210,988, filed June 15, 2021, U.S. Provisional Patent Application No. 63/212,657, filed June 19, 2021, and U.S. Provisional Patent Application No. 63/222,398, filed July 15, 2021, all of which are entitled "Novel mRNA Composition and Production Method for Use in Anti-Viral and Anti-Cancer Vaccines." Additionally, the present invention claims priority to U.S. Provisional Patent Application No. 63/270,034, filed October 20, 2021, and U.S. Provisional Patent Application No. 63/280,226, filed November 17, 2021, both of which are entitled "Novel RNA Composition and Production Method for Use in iPS Cell Generation." Additionally, the present invention claims priority to U.S. patent application Ser. No. 17/489,357, filed Sep. 29, 2021, entitled "Novel mRNA Composition and Production Method for Use in Anti-Viral and Anti-Cancer Vaccines." This application is a continuation-in-part of U.S. patent application Ser. No. 17/489,357, filed Sep. 29, 2021, entitled "Novel mRNA Composition and Production Method for Use in Anti-Viral and Anti-Cancer Vaccines," the contents of which are incorporated herein by reference in their entireties.

Field of the invention:
The present invention generally relates to a novel method for the production and amplification of RNA/mRNA using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and their associated mRNAs. The present invention can be used to produce and amplify any kind of RNA/mRNA sequence having at least one RdRp binding site at the 5'-end, 3'-end, or both. The RNA/mRNA thus obtained is useful for producing mRNA vaccines and/or RNA-based medicines, as well as for producing mRNA-associated proteins, peptides, and/or antibodies under translation conditions in-vitro and in-cell. Mainly, the present invention is a novel method for RNA/mRNA amplification via RNA replicase, namely Replicase Cycling Reaction (RCR). RNA replicases involved in RCR include, but are not limited to, viral and/or bacteriophage RNA-dependent RNA polymerases (RdRps), in particular coronavirus and Hepatitis C virus (HCV) RdRp enzymes.

Background:
Conventional polymerase chain reaction (PCR) is a method of amplifying double-stranded DNA sequences from a DNA template using a thermostable DNA polymerase, and does not involve any RNA material. Unlike PCR, RNA replicase-mediated cycling reaction (RCR) is a method of amplifying single-stranded RNA sequences from an RNA template using an RNA-dependent RNA polymerase (RdRp), and does not involve any DNA material. Obviously, PCR and RCR are very different and cannot be compared. Therefore, previous PCR research does not concern RCR.

Lin et al. first reported RCR in 2002 (WO2002/092774 to Lin). Lin discovered that specially designed 5'-capture-molecule-linked primers could be used to trigger viral and/or bacteriophage repliase-mediated RNA amplification from single-stranded RNA templates. This RCR mechanism mimics the replication/amplification mechanisms of some viruses and bacteriophages. However, many RNA species do not have 5'-cap molecules, so the need for special 5'-capture-molecule-linked primers limits its use. In addition, the linked 5'-capture-capture molecules make the resulting RNA product impure. In the production of mRNA vaccines, removing the 5' cap capture molecule from the RNA product is problematic because it is tedious and may cause RNA degradation. Therefore, a new PCR method that does not use a 5' cap capture molecule-linked primer is highly desirable.

This year, in 2021, another PCR method using alphavirus RdRp and its binding/recognition site, the 19-nucleotide (nt) 3'-conserved sequence element (3'-CSE), was proposed by Bloom et al. (Gene Therapy 28:117-129, 2021). Bloom's method does not use a 5'-capped capture primer, but the 3'-CSE used is too long and structural to be efficiently incorporated into the desired RNA template. In routine practice, the traditional method of polymerase chain reaction-in vitro transcription (PCR-IVT) is commonly used to generate RNA templates amenable to RdRp amplification ( FIG. 1 ; U.S. Pat. Nos. 7,662,791, 8,080,652, 8,372,969, and 8,609,831 to Lin; Methods Mol Biol. 221:93-101, 2003, by Lin et al.). However, the 19-nt 3′-CSE is too long and structural to be placed in a PCR primer. In addition, 3'-CSE is a highly structured RNA sequence that interferes with RNA transcription (McDowell et al., Science 266:822-825, 1994), making it impossible to produce efficiently using conventional IVT methods. Most problematic is that 3'-CSE is specifically recognized by alphavirus RdRp, which is not commercially available, further hindering the development of related technologies. Considering the different properties of replicase/RdRp species of different viruses, it is desirable to explore and use another type of replicase/RdRp enzyme with simpler and fewer structural binding/recognition sites to overcome the problems of conventional RCR methods.

In view of the shortcomings of the previous RCR methods, it is highly desirable to develop a novel RCR method that not only uses simpler and less structural binding/recognition sites for replicase and/or RdRp, but also does not use 5'-capped capture primers for highly efficient RNA amplification and production.

Summary of the invention:
The principle of the present invention relies on incorporating at least a coronavirus and/or hepatitis C virus (HCV) replicase/RdRp binding (recognition) site at the 5'-end or/and 3'-end of a desired RNA template, resulting in cycling amplification of either the sense strand or the antisense strand, or both, of a desired RNA sequence. In RCR, the defined replicase/RdRp binding site functions as a promoter and/or enhancer of replicase/RdRp activity. As shown in FIG. 2, after incorporating at least one replicase/RdRp binding site at the 5'-end and/or 3'-end of a desired RNA template, the desired RNA sequence can be amplified about 15-fold to more than 1000-fold with each cycle of replicase/RdRp cycling reaction (RCR). In RCR, the sense strand RNA sequence serves as a template for amplifying the antisense strand of the sense strand RNA, and the antisense strand RNA sequence serves as a template for amplifying the sense strand RNA. Each cycle of RCR can provide an RNA amplification rate of about 15-fold to more than 1000-fold within a defined time period, depending on the length and structural complexity of the desired RNA sequence, so that the desired RNA strand can be obtained with a relatively high purity ratio (up to 14/15 to >999/1000 purity), depending on the stopping point of the RCR of the sense strand RNA or the antisense strand RNA, or both. It is worth noting that the desired RNA sequence and template in the RCR can be one or more types, and the resulting RNA product can be single-stranded or double-stranded.

To prepare the RCR-compatible RNA template, we first use reverse transcription polymerase chain reaction (RT-PCR) to incorporate at least a coronavirus and/or HCV replicase/RdRp binding site into the 5'-end or 3'-end or both of the complementary DNA (cDNA) of the desired RNA sequence. In our special design, at least one replicase/RdRp binding site is synthetically incorporated into each of the PCR primers (called RCR-compatible PCR primers), so that after RT-PCR, the RCR-compatible RNA template cDNA is formed with the designed replicase/RdRp binding site incorporated into the 5'-end or 3'-end or both. The resulting cDNA can also be cloned into a plasmid or viral vector for IVT reactions and/or storage. Then, an IVT reaction is performed to generate the desired RCR-compatible RNA template from the cDNA. The resulting RCR-compatible RNA template can then be used in RCR to repeatedly amplify and produce the desired RNA sequence. In actual practice, IVT and RCR can be performed simultaneously under exactly the same buffer conditions, so in this specification, cDNA incorporating replicase/RdRp binding sites (called RCR-compatible cDNA template) is also preferably used as the starting material for amplifying the desired RNA sequence in a combined IVT-RCR reaction.

The present inventors performed computer screening of the RNA genomes of more than 17 strains of coronavirus and HCV, and identified several conserved RdRp binding sites, including 5'- and 3'-terminal RdRp binding sites. Specifically, the 5'-terminal RdRp binding site contains at least one of the sequences 5'-AU(G/C)(U/-)G(A/U)-3' (i.e., 5'-AUSUGW-3'; SEQ ID NO: 1) or 5'-U(C/-)(U/A)C(U/C)(U/A)A-3' (i.e., 5'-UCWCYWA-3'; SEQ ID NO: 2), or both. Preferably, the 5'-terminal RdRp binding site is selected from sequences comprising 5'-AUCUGU-3' (SEQ ID NO:3), 5'-UCUCUAA-3' (SEQ ID NO:4), 5'-UCUCCUA-3' (SEQ ID NO:5), and/or 5'-UUCAA-3' (SEQ ID NO:6), or combinations thereof, while the 3'-terminal RdRp binding site comprises at least one of the sequences 5'-(U/A)C(A/-)(C/G)AU-3' (i.e., 5'-WCASAU-3'; SEQ ID NO:7), and/or 5'-U(A/U)(A/G)G(A/U)(G/-)A-3' (i.e., 5'-UWRGWR-3'; SEQ ID NO:8). Preferably, the 3'-terminal RdRp binding site is selected from sequences comprising 5'-ACAGAU-3' (SEQ ID NO: 9), 5'-UUAGAGA-3' (SEQ ID NO: 10), 5'-UAGGAGA-3' (SEQ ID NO: 11), and/or 5'-UUGAA-3' (SEQ ID NO: 12), or combinations thereof. To facilitate the design of RCR-compatible PCR primers, the uridine/uracil (U) contents of these RdRp binding sites can be replaced with thymidine (dT) and/or deoxyuridine (dU) in the primers. To enhance RNA stability, the uridine/uracil (U) contents of these RdRp binding sites can be further replaced with pseudouridine or other modified nucleotide analogues during IVT and/or RCR. With the novel understanding and design of these RdRp binding sites, currently available coronavirus RdRp enzymes can be used to efficiently transcribe and amplify either the sense or antisense strand, or both, of desired RNA sequences in vitro, ex vivo, and in vivo.

Furthermore, these newly identified 5' and 3' end RdRp binding sites result in different RNA amplification rates. For example, SEQ ID NO:1 and SEQ ID NO:7 are estimated to have amplification rates of about 25-1400 fold per RCR cycle, while SEQ ID NO:2 and SEQ ID NO:8 are estimated to have amplification rates of about 10-900 fold per RCR cycle, depending on the length and structural complexity of the desired RNA sequence. These different amplification preferences allow for different combinations of RdRp binding sites to selectively amplify one RNA strand over the other, or to selectively amplify certain RNA strands over other species. By this means, relatively pure single-stranded and/or double-stranded RNA products of the desired RNA sequence can be generated and recovered by RCR and further purified by other methods.

In a preferred embodiment, the desired RNA sequence (i.e., mRNA and/or microRNA, or other RNA species) contains at least one RdRp binding site at both its 5' and 3' terminal regions. Since both ends of the desired RNA have at least one RdRp binding site for RNA amplification with replicase/RdRp activity, the sense strand RNA sequence can be used to amplify its complementary antisense RNA (cRNA or aRNA), while the antisense strand RNA sequence can also be used to amplify the sense RNA, thereby forming an amplification cycle of both the sense strand RNA and the antisense strand RNA, maximizing the amplification rate of the desired RNA. The desired RNA thus obtained can be either single-stranded or double-stranded depending on the stopping point of the RCR. In addition, the obtained sense strand RNA and antisense strand RNA can further form double-stranded RNA to promote the production of siRNA, shRNA, miRNA, and/or piRNA of the desired RNA sequence.

Alternatively, in another preferred embodiment, the desired RNA sequence contains at least one RdRp binding site in either its 5'-end or 3'-end region. In this way, either the sense or antisense strand of the desired RNA can be selectively amplified, allowing for more specific amplification of the desired RNA strand. In particular, this approach is useful for generating and amplifying either mRNA or antisense RNA (aRNA) of specific functional proteins, viral antigens or antibodies, facilitating the development of mRNA vaccines and/or RNA/antibody-based medicines. The mRNA vaccines and RNA/antibody-based medicines thus obtained may be useful for the treatment of various human diseases, including, but not limited to, Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, myocardial infarction, hemophilia, anemia, leukemia, and various cancers, as well as various viral and bacterial infections.

Conceivably, our novel RCR method can be used to produce or amplify any kind of RNA species that has at least one RdRp binding site, especially viral antigen mRNA and/or known functional RNA/mRNA that are useful for developing anti-viral and/or anti-disease vaccines, medicines, etc. For example, by co-transfecting RCR-compatible RNA templates and isolated coronavirus RdRp mRNA into human somatic cells, our two U.S. priority patent applications (U.S. Provisional Patent Applications 63/270,034 and 63/280,226 to Lin) have demonstrated a novel method for generating iPS cells. In response, one skilled in the art can envision using three-/four-Yamanaka-factor mRNAs (i.e. Oct4/3, Sox2, Nanog and/or Lin-28) instead of the claimed miR-302 microRNA precursor (pre-miRNA) for iPS cell generation. Alternatively, as shown in another priority patent application (US patent application Ser. No. 17/489,357 to Lin), we have developed novel designs of RdRp-mediated self-amplifying RNA (saRNA) for generating novel mRNA vaccines and medicines for treating viral infections and cancer, respectively. Furthermore, the RCR-amplified mRNA can be further used in in-vitro translation systems to produce the encoded proteins, peptides and/or antibodies. Considering the achievements demonstrated in these prior inventions, many developments in the potential applications of the present invention are highly anticipated.

To efficiently generate highly structured RNA templates and RdRp mRNAs, in our priority patent application (US Patent Application Serial No. 17/489,357 to Lin), we developed a novel PCR-IVT method to overcome the low efficiency problem of highly structured RNA generation. It is well known that the presence of hairpin-like RNA structures significantly inhibits RNA transcription, so it is not reasonable to expect that those skilled in the art can effectively generate highly structured RNA in vitro. In fact, hairpin-like stem-loop structures are essential transcription termination signals for prokaryotic RNA polymerases (McDowell et al., Science 266:822-825, 1994). To solve this problem, our priority method employs a new IVT system that combines RNA polymerase and helicase activities. The additional helicase activity in IVT (and probably in RCR as well) significantly reduces the secondary structure of both the DNA/RNA template and the resulting RNA product, producing highly structured RNA much more efficiently. Therefore, an improved buffer system is also used to enhance and maintain the efficiency of the mixed RNA polymerase/replicase and helicase activities in IVT (and RCR as well). Interestingly, although some previous studies have reported that helicase may be involved in transcription termination in prokaryotes, our study showed that the function of helicase in RNA amplification during IVT is quite different.

To facilitate intracellular delivery/transfection in vitro, ex vivo or in vivo, the RCR-compatible cDNA/RNA template and the RdRp mRNA can be mixed, complexed, encapsulated and/or formulated with at least one delivery/transfection agent selected from, but not limited to, glycylglycerin-derived chemicals, liposomes, nanoparticles, liposomal nanoparticles (LNPs), conjugated molecules, infusion/transfusion chemicals, gene gun materials, electroporation agents, transposons/retrotransposons, and combinations thereof.

The advantages of using the RCR-enabled cDNA/RNA template for RNA/mRNA production and amplification include: (1) high RNA yield, (2) high RNA purity, (3) easy preparation in that all reaction materials can be made into a biochemical enzyme kit for performing RCR and/or combined IVT-RCR reactions, (4) simple reaction procedure compatible with other RT-PCR and IVT reactions, (5) simple equipment requirements that can be easily achieved using a PCR machine or a temperature-controlled incubator, and (6) various potential applications. As a result, the RCR-enabled cDNA/RNA template of the present invention is very useful for the production and amplification of various desired RNA/mRNA sequences and can be used for all kinds of pharmaceutical and therapeutic applications, including but not limited to the development of mRNA vaccines and RNA/microRNA-related medicines, as well as the generation of proteins/peptides/antibodies.

A. Definitions To facilitate the understanding of this invention, a number of terms are defined below.

Nucleic Acid : A polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that may be single- or double-stranded.

Nucleotide : A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogen-containing heterocyclic base. The base is attached to the sugar moiety via the glycosidic carbon (the 1' carbon of the pentose), and the combination of base and sugar is a nucleoside. A nucleoside with at least one phosphate group attached to the 3' or 5' position of the pentose is a nucleotide. DNA and RNA are composed of different types of nucleotide units called deoxyribonucleotides and ribonucleotides, respectively.

Deoxyribonucleoside triphosphates (dNTPs) : Building block molecules for DNA synthesis, including dATP, dGTP, dCTP, dTTP, and may further include modified deoxyribonucleotide analogs.

Ribonucleoside triphosphates (rNTPs) : Building block molecules for RNA synthesis that include ATP, GTP, CTP, UTP, and may also include pseudouridine and other modified ribonucleotide analogs.

Nucleotide Analog : A purine or pyrimidine nucleotide that is structurally different from adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U), but similar enough to substitute for the normal nucleotides in nucleic acid molecules.

Oligonucleotide : A molecule consisting of two or more, preferably three or more, usually ten or more monomeric units of DNA and/or RNA. Oligonucleotides longer than 13 nucleotide monomers are also called polynucleotides. The exact size depends on many factors, which vary depending on the ultimate function or use of the oligonucleotide. Oligonucleotides may be produced by any method, such as chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.

Nucleic Acid Composition : A nucleic acid composition refers to an oligonucleotide or polynucleotide, such as a DNA or RNA sequence, or a mixed DNA/RNA sequence, in a single- or double-stranded molecular structure.

Gene : A nucleic acid composition whose oligonucleotide or polynucleotide sequence codes for RNA and/or polypeptides (proteins). A gene may be RNA or DNA. A gene may code for non-coding RNA, such as small hairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA, and their RNA precursors and derivatives. Alternatively, a gene may code for protein-coding RNA essential for protein/peptide synthesis, such as messenger RNA (mRNA) and its RNA precursors and derivatives. In some cases, a gene may code for a protein-coding RNA that also contains at least one microRNA or shRNA sequence.

Primary RNA transcript (pre-mRNA) : The RNA sequence directly transcribed from a gene, without any RNA processing or modification.

Pre-messenger RNA (pre-mRNA) : The primary RNA transcript of a protein-coding gene, produced by eukaryotic type-II RNA polymerase (Pol-II) machinery through an intracellular mechanism called transcription. The pre-mRNA sequence contains the 5' untranslated region (UTR), 3' UTR, exons, and introns.

Intron : A portion or part of a gene transcribed sequence that codes for non-protein-reading frames, such as an in-frame intron, 5'-UTR, or 3'-UTR.

Exon : Part or all of a gene transcribed sequence that encodes protein-reading frames (cDNA), such as cDNAs for cellular genes, growth factors, insulin, antibodies and their analogs/homologs and derivatives.

Messenger RNA (mRNA) : A collection of pre-mRNA exons, formed after intron removal by the intracellular RNA splicing machinery (e.g., spliceosome), which functions as a protein-coding RNA for peptide/protein synthesis. Peptides/proteins encoded by mRNA include, but are not limited to, enzymes, growth factors, insulin, antibodies and their analogs/homologs and derivatives.

Complementary DNA (cDNA) : Single- or double-stranded DNA that contains a sequence complementary to an mRNA sequence and does not contain intron sequences.

Sense : A nucleic acid molecule of the same sequence and composition as the homologous mRNA. The sense type is indicated by the symbols "+", "s" or "sense".

Antisense : A nucleic acid molecule complementary to the respective mRNA molecule. Antisense forms are designated by the symbol "-" or by adding the letter "a" or "antisense" before the DNA or RNA, as in "aDNA" and "aRNA."

Base pair (bp) : The relationship between adenine (A) and thymine (T) or between cytosine (C) and guanine (G) in a double-stranded DNA molecule. In RNA, uracil (U) is used instead of thymine. Generally, the relationship is achieved by hydrogen bonding. For example, the sense nucleotide sequence "5'-A-T-C-G-U-3'" can perfectly base pair with the antisense sequence "5'-A-C-G-A-T-3'".

5' end : The end of a sequence of consecutive nucleotides in which the 5'-hydroxyl group of one nucleotide is joined to the 3'-hydroxyl group of the next nucleotide via a phosphodiester bond, omitting the 5' nucleotide. Other groups, such as one or more phosphates, may be present at the end.

3' end : The end of a sequence of consecutive nucleotides in which the 5'-hydroxyl group of one nucleotide is linked to the 3'-hydroxyl group of the next nucleotide by a phosphodiester bond, omitting the 3' nucleotide. Other groups, often hydroxyl groups, may also be present at the end.

Template : A nucleic acid molecule that is copied by a nucleic acid polymerase. The template can be single-stranded, double-stranded, or partially double-stranded, RNA or DNA, depending on the polymerase. The synthesized copy is complementary to the template or to at least one strand of a double-stranded or partially double-stranded template. Both RNA and DNA are synthesized in the 5' to 3' direction. The two strands of a nucleic acid duplex are always aligned so that the 5' ends of the duplex (and necessarily the 3' ends) are at opposite ends of the duplex.

Nucleic acid template : a double-stranded DNA molecule, a double-stranded RNA molecule, a hybrid molecule such as a DNA-RNA or RNA-DNA hybrid, or a single-stranded DNA or RNA molecule.

Conserved : A nucleotide sequence is said to be conserved relative to a preselected (referenced) sequence if it non-randomly hybridizes to the exact complement of the preselected sequence.

Homologous or Homology : A term used to indicate the similarity between a polynucleotide and a gene or mRNA sequence. A nucleic acid sequence may, for example, be partially or completely homologous to a particular gene or mRNA sequence. Homology may be expressed as a percentage determined by the number of similar nucleotides relative to the total number of nucleotides.

Complementary or Complementarity or Complementation : A term referring to matched base pairing between two polynucleotides (i.e., mRNA and cDNA sequences) related by the "base pair (bp)" rules described above. For example, the sequence "5'-A-G-T-3'" is complementary to "5'-A-C-T-3'" as well as "5'-A-C-U-3'". Complementarity can be between two DNA strands, between a DNA strand and an RNA strand, or between two RNA strands. Complementarity can be "partial", "complete", or "total". Partial complementarity or complementation occurs when only a portion of the nucleic acid bases match according to the base pairing rules. Complete or total complementarity or complementation occurs when the bases match completely or perfectly between nucleic acid strands. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on the binding of nucleic acids to each other. The percentage of complementarity refers to the percentage of the number of mismatched bases relative to the total number of bases in one strand of nucleic acid. Thus, 50% complementarity means that half the bases are mismatched and half are matched. Two strands of nucleic acid can be complementary even if the two strands have a different number of bases, where the complementarity occurs between the bases of the longer strand paired with the bases of the shorter strand.

Complementary bases : Nucleotides that normally pair when DNA or RNA forms a double-stranded structure.

Complementary Nucleotide Sequence : A sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to the nucleotides on the other single strand to specifically hybridize between the two strands, resulting in hydrogen bonding.

Hybridize and Hybridization : refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a complex through base pairing. When a primer (or splice template) "hybridizes" with a target (template), such a complex (or hybrid) is stable enough to perform the priming function required for DNA polymerase to initiate DNA synthesis. Specific, i.e., non-random, interactions exist between two complementary polynucleotides and can be competitively inhibited.

Posttranscriptional Gene Silencing : The effect of knocking out or knocking down a target gene at the level of mRNA degradation or translational repression, usually triggered by either an exogenous/viral DNA or RNA transgene, or small inhibitory RNAs.

RNA interference (RNAi) : Post-transcriptional gene silencing in eukaryotes that can be triggered by small inhibitory RNA molecules, such as microRNA (miRNA), small hairpin RNA (shRNA), and small interfering RNA (siRNA). These small RNA molecules typically function as gene silencers, inhibiting the expression of cellular genes that contain segments that are fully or partially complementary to the small RNA.

MicroRNA (miRNA) : Single-stranded RNA that can bind to the transcripts of target genes that are partially complementary to the miRNA. miRNAs are usually about 17-27 oligonucleotides in length, and depending on the complementarity of the miRNA with its target mRNA, they can directly degrade the target mRNA in the cell or suppress the protein translation of the target mRNA. Natural miRNAs are present in almost all eukaryotes, and enable defense against viral infections and control of gene expression during the development of animals and plants.

Precursor MicroRNA (Pre-miRNA) : A hairpin-shaped single-stranded RNA that has stem-arm and stem-loop regions to interact with cellular RNase III endoribonuclease to generate one or more microRNAs (miRNAs) that can silence genes complementary to the microRNA sequence or target genes. The stem arms of the pre-miRNA can form complete (100%) or partial (mismatched) hybrid duplexes, and the stem-loop links one end of the stem-arm duplex to form a circle or hairpin loop structure. However, in the present invention, the precursor of the microRNA can also include a pri-miRNA.

Small interfering RNA (siRNA) : Short double-stranded RNA that is approximately 18-27 perfectly base-paired ribonucleotide duplexes in size and can degrade nearly perfectly complementary target gene transcripts.

Small or short hairpin RNA (shRNA) : A single stranded RNA in which a pair of partially or perfectly matched stem-arm nucleotide sequences are separated by a mismatched loop or bubble oligonucleotide to form a hairpin-like structure. Many natural miRNAs are derived from small hairpin-like RNA precursors, or microRNA precursors (pre-miRNAs).

Vector : A recombinant nucleic acid composition, such as recombinant DNA (rDNA), capable of movement and residence in different genetic environments. Typically, another nucleic acid is operably linked to it. A vector is capable of autonomous replication in a cell, where the vector and associated segments are replicated. One type of preferred vector is an episome, i.e., a nucleic acid molecule capable of extrachromosomal replication. Preferred vectors are vectors capable of autonomous replication and expression of nucleic acids. Vectors capable of directing the expression of genes encoding one or more polypeptides and/or non-coding RNAs are referred to herein as "expression vectors" or "expression-competent vectors." Vectors of particular interest allow for the cloning of cDNA from mRNA produced using reverse transcriptase. The vectors may contain a viral or type II RNA polymerase (Pol-II or pol-2) promoter, or both, a Kozak consensus translation initiation site, a polyadenylation signal, multiple restriction/cloning sites, a pUC origin of replication, an SV40 early promoter for expressing at least an antibiotic resistance gene in replication-competent prokaryotic cells, an optional SV40 origin for replication in mammalian cells, and/or a tetracycline responsive element. The vector structure may be a linear or circular form of single-stranded or double-stranded DNA selected from the group consisting of a plasmid, a viral vector, a transposon, a retrotransposon, a DNA transgene, a jumping gene, and combinations thereof.

Promoter : A nucleic acid that a polymerase molecule recognizes, possibly binds to, and initiates transcription of RNA. For purposes of this invention, a promoter may be a known polymerase binding site, an enhancer, etc., and may be any sequence that can initiate synthesis of an RNA transcript by a desired polymerase.

RNA processing : The cellular machinery responsible for the maturation, modification, and degradation of RNA, including RNA splicing, intron excision, exosome digestion, nonsense-mediated decay (NMD), RNA editing, RNA processing, 5'-capping, 3'-poly(A) tailing, and combinations thereof.

Gene transfer : a genetic engineering method selected from the group consisting of polysomal transfection, liposomal transfection, chemical (nanoparticle) transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene-gun penetration, and combinations thereof.

Genetic engineering : A method of DNA recombination selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene incorporation, transposon insertion, jumping gene integration, retroviral infection, and combinations thereof.

Transfected cell : A single or multiple eukaryotic cells after the artificial insertion of at least one nucleic acid sequence or protein/peptide molecule into a cell selected from the group consisting of somatic cells, tissue cells, stem cells, germ cells, tumor cells, cancer cells, virus-infected cells, and combinations thereof.

Antibody : A peptide or protein molecule having a preselected conserved domain structure that encodes a receptor capable of binding to a preselected ligand.

Pharmaceutical and/or Therapeutic Applications : Biomedical applications and/or devices useful for stem cell generation, drug/vaccine development, non-transgenic gene therapy, cancer treatment, disease treatment, wound healing, tissue/organ repair and regeneration, high yield production of proteins/peptides/antibodies, pharmaceutical ingredients, medicines, vaccines and/or foods, and combinations thereof.

B. Compositions and Uses (a) providing at least one RNA sequence that contains at least one RdRp binding site at the 5' end or at the 3' end, or both;
(b) providing at least one RNA replicase isolated or modified from a coronavirus or a Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp);
(c) mixing the RNA sequence of (a) with the RNA replicase of (b) under buffer conditions to cause RNA replicase-mediated production and amplification of said RNA sequence;
Including,
The buffer conditions include ribonucleoside triphosphate molecules (rNTPs) necessary for RNA synthesis, a pH in the range of 6.0 to 8.0, and a temperature in the range of 20° C. to 45° C.
A novel method for amplifying RNA via RNA replicase.

In the case of coronavirus and/or HCV RdRp enzymes, the 5'-terminal RdRp binding site comprises at least one of the sequences 5'-AUSUGW-3' (SEQ ID NO:1) and/or 5'-UCWCYWA-3' (SEQ ID NO:2), or both. Preferably, the 5'-terminal RdRp binding site is selected from RNA sequences comprising 5'-AUCUGU-3' (SEQ ID NO:3), 5'-UCUCUAA-3' (SEQ ID NO:4), 5'-UCUCCUA-3' (SEQ ID NO:5), and/or 5'-UUCAA-3' (SEQ ID NO:6), or combinations thereof. Meanwhile, the 3'-terminal RdRp binding site comprises at least one of the sequences 5'-WCASAU-3' (SEQ ID NO:7) and/or 5'-UWRGWR-3' (SEQ ID NO:8), or both. Preferably, the 3' terminal RdRp binding site is selected from an RNA sequence containing 5'-ACAGAU-3' (SEQ ID NO: 9), 5'-UUAGAGA-3' (SEQ ID NO: 10), 5'-UAGGAGA-3' (SEQ ID NO: 11), and/or 5'-UUGAA-3' (SEQ ID NO: 12), or a combination thereof. To incorporate these RdRp binding sites into PCR primers, the uridine/uracil (U) contents of these RdRp binding sites can be substituted with thymidine (dT) and/or deoxyuridine (dU) in the primers. Also, to improve RNA stability, the uridine/uracil (U) contents of these RdRp binding sites and the resulting RNA product can be substituted with pseudouridine or other modified nucleotide analogs.

Referring to the drawings, which are for purposes of illustration and not limitation, therein are shown:

1 shows the step-by-step procedure of a conventional PCR-IVT method, for RNA production, some or all of the steps of this PCR-IVT method can be adopted for single or multiple cycle amplification of the desired RNA product.

The step-by-step procedure of the RCR method of the present invention is shown. To prepare the RCR-ready cDNA/RNA template, at least one coronavirus and/or HCV replicase/RdRp binding site is incorporated into the 5'-end or 3'-end or both of the cDNA of the desired RNA sequence using conventional RT-PCR methods. The desired RNA sequence is then produced and amplified from the RCR-ready cDNA/RNA template after single or multiple cycles of amplification using some or all of the steps of this novel RCR method. Alternatively, the RCR-ready cDNA/RNA template can also be used as starting material to amplify the desired RNA sequence in a combined IVT-RCR reaction, since the IVT and RCR methods can be performed simultaneously under the same buffer conditions.

The designed structures of RCR-compatible cDNA/RNA templates are shown. Note that the RCR-compatible cDNA template is a double-stranded DNA structure (useful for IVT and combined IVT-RCR reactions), while the RCR-compatible RNA template is a single-stranded RNA structure (useful for RCR). To further increase the stability of the RCR-compatible RNA template, the uridine/uracil (U) components of the template can be replaced with pseudouridine or other modified nucleotide analogs.

Northern blot analysis shows that expression of miR-302 microRNA (i.e., from top to bottom: b, c, d, a) and RdRp mRNA in transfected human cells following co-transfection with RCR-enabled miR-302 precursor microRNA (pre-miR-302) and viral RdRp mRNA template (shown on the far right) was significantly increased compared to the results in cells transfected with only the pre-miR-302 template (middle), providing evidence of RCR in the cells.

Northern blot analysis of the RCR-prepared cDNA and RNA templates and the resulting amplified RNA products of interest (ie, viral antigen protein/peptide mRNA sequences) are shown, providing evidence of RCR in vitro.

Immunohistochemical staining of coronavirus (e.g., COVID-19) S2 protein produced in mouse muscle cells in vivo after co-transfection with RCR-amplified S protein mRNA (from FIG. 5) and isolated RdRp mRNA (from FIG. 4) is shown, demonstrating that the present invention is useful for the development and production of anti-viral mRNA vaccines.

Example:
1. Isolation and culture of human cells
Starting tissue cells can be obtained from enzymatically dissociated skin cells using the protocol of Aasen (Nat. Protocols 5, 371-382, 2010) or simply from the buffy coat fraction of heparinized peripheral blood cells. Isolated tissue samples should be kept fresh and used immediately by mixing 4 mg/mL collagenase I and 0.25% TrypLE for 15-45 minutes depending on cell density, rinsing twice with HBSS containing trypsin inhibitor, and then transferring to a sterile microtube containing 0.3 mL of feeder-free SFM medium (Irvine Scientific, CA). The cells were then further dissociated by shaking for 1 minute at 37°C in a microtube incubator, and 0.3 mL of the entire cell suspension was transferred to a 35 mm Matrigel-coated culture dish containing 1 mL of feeder-free SFM medium supplemented with the formulated pre-miR-302+RdRp mRNA mixture, LIF, and bFGF/FGF2, or other optional defined factors. The concentrations of the pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and other optional defined factors ranged from 0.1 to 500 micrograms (μg)/mL, respectively, in the cell culture medium. Cell culture medium and all supplements need to be refreshed every 2-3 days, and cells are passaged at approximately 50-60% confluence by exposure to trypsin/EDTA for 1 min and rinsing twice with HBSS containing trypsin inhibitor. For ASC expansion, cells were replated at 1:5 to 1:500 dilution in fresh feeder-free MSC Expansion SFM culture medium supplemented with formulated pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and/or any other defined factors. For keratinocyte culture, cells are isolated from skin tissue and cultured in EpiLife serum-free cell culture medium supplemented with human keratinocyte growth supplement (HKGS, Invitrogen, Carlsbad, CA) in the presence of appropriate antibiotics at 37°C and 5% _CO2 . Cultured cells were passaged at 50-60% confluence by exposure to trypsin/EDTA solution for 1 min and rinsing once with phenol red-free DMEM medium (Invitrogen), and detached cells were replated at a dilution of 1:10 in fresh EpiLife medium supplemented with HKGS supplement. Human cancer and normal cell lines A549, MCF7, PC3, HepG2, Colo-829, and BEAS-2B were obtained from the American Type Culture Collection (ATCC, Rockville, MD) or collaborators and maintained according to the manufacturers' or suppliers' suggestions. After reprogramming, the resulting iPSCs were cultured and maintained according to Lin's feeder-free or Takahashi's feeder-based iPSC culture protocols (Lin et al., RNA 14:2115-2124, 2008; Lin et al., Nucleic Acids Res. 39:1054-1065, 2011; Takahashi K and Yamanaka S, Cell 126:663-676, 2006).

2. In-Vitro RNA Transfection For intracellular introduction/transfection, 0.5-200 μg of a mixture of RCR-amplified RNA/mRNA (i.e., pre-miR-302 or coronavirus S protein mRNA) and RdRp mRNA (ratios ranging from about 20:1 to 1:20) is dissolved in 0.5 ml of fresh cell culture medium and mixed with 1-50 μl of In-VivoJetPEI or other similar transfection reagent. After incubation for 10-30 minutes, the mixture is added to cell medium containing 50-60% confluent cultured cells. The medium is flushed every 12-48 hours, depending on the cell type. This transfection procedure can be repeated to increase the transfection efficiency.

3. Preparation of PCR-Ready cDNA/RNA Template Reverse transcription (RT) of the desired RNA/mRNA is performed by adding approximately 0.01 ng to 10 micrograms (μg) of the isolated RNA/mRNA to 20-50 μL of RT reaction solution (SuperScript III cDNA RT kit, ThermoFisher Scientific, MA, USA) according to the manufacturer's suggestions. Depending on the amount of RNA/mRNA, the RT reaction mixture further contains approximately 0.01-20 nmoles of RT primer, appropriate amounts of deoxyribonucleoside triphosphate molecules (dNTPs), and reverse transcriptase in 1x RT buffer. The RT reaction is then incubated at 37-65°C for 1-3 hours, depending on the length and structural complexity of the desired RNA/mRNA sequence, to generate a complementary DNA (cDNA) template for the next step, PCR. For isolation of viral RdRp mRNA, we designed the RT-reverse primer 5'-GACAACAGGT GCGCTCAGGT CCT-3' (SEQ ID NO: 13) and used it to generate coronavirus RdRp cDNA.

Next, polymerase chain reaction (PCR) is performed by adding approximately 0.01 pg to 10 μg of RT-derived cDNA to 20 to 50 μL of PCR preparation mixture (High-Fidelity PCR master kit, ThermoFisher Scientific, MA, USA) according to the manufacturer's suggestions. The PCR mixture is then first incubated for 5 to 20 cycles of denaturation at 94°C for 1 min, annealing at 30 to 55°C for 30 s to 1 min, and extension at 72°C for 1 to 3 min, depending on the structure and length of the desired cDNA sequence. Then, another 10 to 20 cycles of PCR are performed, repeating the cycle of denaturation at 94°C for 1 min, annealing at 50 to 58°C for 30 s, and extension at 72°C for 1 to 3 min, depending on the structure and length of the resulting PCR product. Finally, the resulting PCR product is used as a cDNA template for IVT and RCR. For the preparation of IVT-RCR template, we design and use a specific RCR-compatible PCR primer pair including SEQ ID NO:13 and 5'-GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT-3' (SEQ ID NO:14) to incorporate the identified RdRp binding site into the PCR-derived RdRp cDNA template. A 5' cap molecule can then be further incorporated into the resulting IVT-RCR mRNA product. Meanwhile, we also design and use another PCR-compatible PCR primer pair, including 5'-GATATCTAAT ACGACTCACT ATAGGGAGAT CTGTGGGAAC TAGTTCAGGA AGGTAA-3' (SEQ ID NO: 15) and 5'-GTTCTCCTAA GCCTGTAGCC AAGAACTGCA CA-3' (SEQ ID NO: 16), to incorporate the identified RdRp binding site into the PCR-derived cDNA template of human pre-miR-302 familial cluster (pre-miR-302). In the primer design, various sequences and combinations of RNA promoters and RdRp binding sites can be used, such as T7, T3, and/or SP6 promoters, and at least one RdRp binding site is incorporated into the 5'-end and/or 3'-end primer.

To generate an RCR-ready RNA/mRNA template, at least one promoter and at least one RdRp binding site are incorporated into the resulting PCR-derived cDNA product (which serves as the RCR-ready cDNA template), so that an IVT-RCR reaction can then be performed to amplify the desired RNA/mRNA sequence from the cDNA template. The IVT-RCR reaction mixture contains 0.01 ng-10 μg of PCR-derived cDNA product, 0.1-50 U of isolated coronavirus RdRp/helicase (Abcam, MA, USA/Creative Enzymes, NY), appropriate amounts of ribonucleoside triphosphate molecules (rNTPs) and RNA polymerase (i.e., T7, T3, or SP6) in 1x transcription buffer. Transcription buffers are commercially available and can be further adjusted according to the manufacturer's suggestions. Preferably, the 1x transcription buffer can further contain 0.001-10 mM betaine (trimethylglycine, TMG), dimethyl sulfoxide (DMSO), and/or 3-(N-morpholino)propanesulfonic acid (MOPS), and/or combinations thereof. The IVT-RCR reaction is then incubated at 30-40°C for 1-6 hours, depending on the stability and activity of the RdRp and RNA polymerase enzymes used.

4. Novel RCR Protocol The starting RCR mixture includes about 0.01 ng-10 μg of RCR-ready RNA/mRNA template, about 0.1-50 U of isolated coronavirus RdRp/helicase, and an appropriate amount of rNTPs in 1x transcription buffer. The RdRp/helicase is either an RdRp enzyme with additional RNA unwinding activity or a mixture of RdRp and helicase. Transcription buffers are commercially available and can be further adjusted according to the manufacturer's suggestions. In addition, the 1x transcription buffer can further contain 0.001-10 mM betaine (trimethylglycine, TMG), dimethyl sulfoxide (DMSO), and/or 3-(N-morpholino)propanesulfonic acid (MOPS), and/or combinations thereof, which facilitate the denaturation of highly structured RNA/DNA sequences such as hairpins and stem-loop structures. The PCR reaction is then incubated at 20-45° C. for 1-6 hours depending on the stability and activity of the RdRp enzyme used.

5. RNA Purification and Northern Blot Analysis The desired RNA (10 μg) is isolated using the mirVana® RNA Isolation Kit (Ambion, Austin, TX) or similar purification filter columns according to the manufacturer's protocol and further purified by 5%-10% TBE-urea polyacrylamide or 1%-3.5% low melting point agarose gel electrophoresis. For Northern blot analysis, the gel fractionated RNA is electroblotted onto a nylon membrane. Detection of the RNA and its IVT template (PCR-derived cDNA product) is performed using a labeled [LNA]-DNA probe complementary to the target sequence of the desired RNA. The probes were further purified by high-performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) in the presence of dye-labeled nucleotide analogs or [ P]-dATP (>3000 ^Ci /mM, Amersham International, Arlington Heights, IL) for 20 min.

6. Protein extraction and Western blot analysis. Cells (10 ⁶ ) are lysed with CelLytic-M lysis/extraction reagent (Sigma) supplemented with protease inhibitors, leupeptin, TLCK, TAME, and PMSF according to the manufacturer's recommendations. Lysates are centrifuged at 12,000 rpm for 20 min at 4°C, and the supernatants are collected. Protein concentrations are measured using the modified SOFTmax protein assay package of an E-max microplate reader (Molecular Devices, CA). Thirty micrograms of each cell lysate are added to SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, boiled for 3 min, and then loaded onto a 6-8% polyacrylamide gel. Proteins are separated by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto nitrocellulose membranes, and incubated with Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hours at room temperature. Primary antibody is then applied to the reagent, and the mixture is incubated at 4°C. After overnight incubation, the membrane is rinsed three times with TBS-T and exposed to goat anti-mouse IgG-conjugated secondary antibody against Alexa Fluor 680 reactive dye (1:2,000; Invitrogen-Molecular Probes) for 1 hour at room temperature. After three further TBS-T rinses, the membranes are imaged using a Li-Cor Odyssey Infrared Imager and Odyssey Software v. Fluorescent scanning and image analysis of the immunoblots is performed using Li-Cor 10 (Li-Cor).

7. Immunostaining assay Cell/tissue samples are fixed in 100% methanol for 30 min at 4°C, followed by 4% paraformaldehyde (in 1x PBS, pH 7.4) for 10 min at 20°C. Samples are then incubated in 1x PBS containing 0.1%-0.25% Triton X-100 for 10 min, followed by washing three times with 1x PBS for 5 min. For immunostaining, primary antibodies were purchased from Invitrogen (CA, USA) and Sigma-Aldrich (MO, USA), respectively. Dye-labeled goat anti-rabbit or horse anti-mouse antibodies are used as secondary antibodies (Invitrogen, CA, USA). Results are examined and analyzed at 100x or 200x magnification under a fluorescent 80i microscopic quantitation system equipped with the Metamorph imaging program (Nikon).

8. In Vivo Transfection Assay The mixture of RCR amplified RNA/mRNA and RdRp mRNA (ratios ranging from about 20:1 to 1:20) is thoroughly mixed with an appropriate amount of a delivery agent, such as In-VivoJetPEI transfection reagent or other similar LNP-based delivery/transfection agent, following the manufacturer's recommended protocol, and then injected into an animal intravenously or intramuscularly depending on the intended application. The delivery/transfection agent is used to mix, bind, encapsulate, or formulate the amplified RNA/mRNA and RdRp mRNA mixture to protect the RNA content from degradation as well as to facilitate delivery/transfection of the mixture of RNA/mRNA and RdRp mRNA to specific target cells of interest in vitro, ex vivo, and/or in vivo.

9. Statistical Analysis All data were presented as mean and standard deviation (SD). The mean value of each test group was calculated by AVERAGE in Microsoft Excel. SD was performed by STDEV. Statistical analysis of the data was performed by One-Way ANOVA. Tukey and Dunnett's t post hoc test was used to determine the significance of data differences in each group. p<0.05 was considered significant (SPSS v12.0, Claritas Inc).

References:
1. WO2002/092774 to Shi-Lung Lin et al.
2. U.S. Patent No. 7,662,791 to Shi-Lung Lin et al.
3. U.S. Patent No. 8,080,652 to Shi-Lung Lin et al.
4. U.S. Patent No. 8,372,969 to Ying S.Y. and Shi-Lung Lin .
5. U.S. Patent No. 8,609,831 to Shi-Lung Lin and Ying S.Y.
6. Shi-Lung Lin and Ji H; cDNA library construction using in-vitro transcriptional amplification. Methods Mol Biol. 221:93-101, 2003.
7. Bloom et al.; Self-amplifying RNA vaccines for infectious diseases. Gene Therapy 28:117-129, 2021.
8. McDowell et al.; Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate. Science 266:822-825,1994.
9. Aasen et al.; Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. Nat. Protocols 5:371-382, 2010.
10. Shi-Lung Lin et al.; Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14:2115-2124,2008.
11. Shi-Lung Lin et al.; Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res. 39:1054-1065, 2011.
12. Takahashi K and Yamanaka S; Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-676, 2006.

Sequence Listing
(1) General information:
(iii) Number of sequences: 16

(2) Information of SEQ ID NO:1:
(i) Sequence characteristics:
(A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence Description: SEQ ID NO:1:
AUSUGW6

(2) Information of SEQ ID NO:2:
(i) Sequence characteristics:
(A) Length: 7 bases (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:2:
U.C.W.Y.W.A. 7

(2) Information of SEQ ID NO:3:
(i) Sequence characteristics:
(A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:3:
AUCUGU 6

(2) Information of SEQ ID NO:4:
(i) Sequence characteristics:
(A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:4:
UCUCUAA7

(2) Information of SEQ ID NO:5:
(i) Sequence characteristics:
(A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:5:
UCUCCCUA 7

(2) Information of SEQ ID NO:6:
(i) Sequence characteristics:
(A) Length: 5 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:6:
UUCAA5

(2) Information of SEQ ID NO:7:
(i) Sequence characteristics:
(A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:7:
WCASAU6

(2) Information of SEQ ID NO:8:
(i) Sequence characteristics:
(A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:8:
UWRGWR 6

(2) Information of SEQ ID NO:9:
(i) Sequence characteristics:
(A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:9:
ACAGAU 6

(2) Information of SEQ ID NO:10:
(i) Sequence characteristics:
(A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:10:
UUAGAGAA 7

(2) Information of SEQ ID NO:11:
(i) Sequence characteristics:
(A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:11:
UAGGAGA 7

(2) Information of SEQ ID NO:12:
(i) Sequence characteristics:
(A) Length: 5 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: RNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:12:
UUGAA5

(2) Information of SEQ ID NO:13:
(i) Sequence characteristics:
(A) Length: 23 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: DNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: YES

(xi) Sequence description: SEQ ID NO:13:
GACAACAGGT GCGCTCAGGT CCT 23

(2) Information of SEQ ID NO:14:
(i) Sequence characteristics:
(A) Length: 48 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: DNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:14:
GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTTACT TGGTAGTTT 48

(2) Information of SEQ ID NO:15:
(i) Sequence characteristics:
(A) Length: 56 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: DNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: NO

(xi) Sequence description: SEQ ID NO:15:
GATATCTAAT ACGACTCACT ATAGGGAGAT CTGTGGGAAC TAGTTCAGGA AGGTAA 56

(2) Information of SEQ ID NO:16:
(i) Sequence characteristics:
(A) Length: 32 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Linear

(ii) Molecule type: DNA
(A) Description: /desc="synthetic"

(iii) HYPOTHETICAL: NO

(iv) Antisense: YES

(xi) Sequence description: SEQ ID NO:16:
GTTCTCCTAA GCCTGTAGCC AAGAACTGCA CA 32

Claims (21)

(a) providing at least one RNA sequence comprising at least one RdRp binding site at the 5' end or at the 3' end, or both;
(b) providing at least one RNA replicase isolated or modified from a coronavirus or Hepatitis C virus RNA-dependent RNA polymerase (RdRp);
(c) mixing the RNA sequence of (a) with the RNA replicase of (b) under buffer conditions such that RNA replicase-mediated production and amplification of the RNA sequence occurs;
The buffer conditions include ribonucleoside triphosphate molecules (rNTPs) necessary for RNA synthesis, a pH in the range of 6.0 to 8.0, and a temperature in the range of 20° C. to 45° C.
A novel method for amplifying RNA via RNA replicase. The method of claim 1, wherein the RNA sequence can include one or more stranded structures or one type of RNA species. The method of claim 1, wherein the 5'-terminal RdRp binding site comprises one of the sequences of SEQ ID NO: 1 or SEQ ID NO: 2. The method of claim 3, wherein the 5'-terminal RdRp binding site is selected from a sequence comprising SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or a combination thereof. The method of claim 1, wherein the 3'-terminal RdRp binding site comprises one of the sequences of SEQ ID NO: 7 or SEQ ID NO: 8. The method of claim 5, wherein the 3'-terminal RdRp binding site is selected from a sequence comprising SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12, or a combination thereof. The method of claim 1, wherein the starting RNA sequence is generated using a novel polymerase chain reaction in vitro transcription (PCR-IVT) method that uses a mixed activity of RNA polymerase and RdRp/helicase. The method of claim 1, wherein the RdRp mRNA is generated using a novel polymerase chain reaction in vitro transcription (PCR-IVT) method that uses mixed RNA polymerase and RdRp/helicase activity. The method of claim 1, wherein the buffer condition is 1x transfer buffer optionally supplemented with 0.001-10 mM betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), or 3-(N-morpholino)propanesulfonic acid (MOPS), or a combination thereof. The method of claim 1, wherein the ribonucleoside triphosphate molecules (rNTPs) include ATP, GTP, CTP, and UTP. The method of claim 1, wherein the ribonucleoside triphosphate molecules (rNTPs) can further include pseudouridine or other modified nucleotide analogs. The method of claim 1, wherein the uridine/uracil (U) components of the RNA sequence can be replaced with pseudouridine or other modified nucleotide analogs. The method of claim 1, wherein the RNA sequence is further formulated with at least one delivery agent to facilitate in vitro, ex vivo and/or in vivo intracellular transfection. The method of claim 1, wherein the delivery agent comprises glycylglycerin, liposomes, nanoparticles, liposomal nanoparticles (LNPs), conjugate molecules, infusion chemicals, gene gun materials, electroporation agents, transposons, and combinations thereof. The method of claim 1, wherein the RNA sequence is mRNA. The method of claim 15, wherein the mRNA is useful for producing and developing mRNA vaccines and medicines. The method of claim 15, wherein the mRNA is useful for producing proteins/peptides and antibodies. The method of claim 1, wherein the RNA sequence is a microRNA precursor (pre-miRNA). The method according to claim 18, wherein the pre-miRNA is useful for producing and developing an anticancer drug. The method of claim 18, wherein the pre-miRNA is useful for generating iPS cells. The method of claim 1 , wherein the RNA sequence is used as a medicine or therapeutic component.