A novel replicase cycling reaction (RCR)

Abstract

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 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

[Technical Field]

[0001] 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." This invention also 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." This invention further 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." This invention further claims priority to U.S. Patent Application No. 17 / 489,357, filed September 29, 2021, both of which are 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 each of which are incorporated herein by reference in their entirety.

[0002] Field of the invention: The present invention generally relates to a novel method for producing and amplifying 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 type of RNA / mRNA sequence that has at least one RdRp binding site at the 5' end, 3' end, or both. The RNA / mRNA thus obtained is useful not only for producing mRNA vaccines and / or RNA-based medicines, but also for producing mRNA-associated proteins, peptides, and / or antibodies under translation conditions in vitro and in cells. Primarily, the present invention is a novel method for amplifying RNA / mRNA via an RNA replicase, i.e., the replicase cycling reaction (RCR). RNA replicases involved in RCR include, but are not limited to, viral and / or bacteriophage RNA-dependent RNA polymerases (RdRps), particularly RdRp enzymes from coronaviruses and hepatitis C virus (HCV). [Background technology]

[0003] Background technology: Conventional polymerase chain reaction (PCR) is a method that uses a thermostable DNA polymerase to amplify double-stranded DNA sequences from a DNA template, without involving any RNA material. Unlike PCR, RNA replicase-mediated cycling reaction (RCR) is a method that uses an RNA-dependent RNA polymerase (RdRp) to amplify single-stranded RNA sequences from an RNA template, without involving any DNA material. Clearly, PCR and RCR are very different and cannot be compared. Therefore, previous PCR research does not pertain to RCR. Summary of the Invention [Problem to be solved by the invention]

[0004] Lin et al. first reported PCR in 2002 (WO2002 / 092774 to Lin). Lin discovered that specially designed 5'-cap-capture molecule-linked primers could be used to induce RNA amplification from single-stranded RNA templates via viral and / or bacteriophage repliases. This PCR mechanism mimics the replication / amplification mechanisms of several viruses and bacteriophages. However, many RNA species lack 5'-cap molecules, limiting its use by requiring special 5'-cap-capture molecule-linked primers. Furthermore, linked 5'-cap-cap molecules contaminate the resulting RNA product. Removal of 5'-cap-capture molecules from RNA products is problematic for mRNA vaccine production, as it is tedious and may lead to RNA degradation. Therefore, novel PCR methods that do not use 5'-cap-capture molecule-linked primers are highly desirable.

[0005] In 2021, Bloom et al. proposed another PCR method using alphavirus RdRp and its binding / recognition site, a 19-nucleotide (nt) 3'-conserved sequence element (3'-CSE) (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 structurally intractable for efficient incorporation into the desired RNA template. In routine practice, the conventional method of polymerase chain reaction-in vitro transcription (PCR-IVT) is commonly used to generate RNA templates amenable to RdRp amplification (Figure 1; U.S. Patent Nos. 7,662,791, 8,080,652, 8,372,969, and 8,609,831 to Lin; Methods Mol Biol. 221:93-101, 2003, Lin et al.). However, the 19-nt 3'-CSE is too long and too structured to be placed in a PCR primer. Furthermore, because the 3'-CSE is a highly structured RNA sequence, it interferes with RNA transcription (McDowell et al., Science 266:822-825, 1994), making it inefficient to produce using conventional IVT methods. Moreover, the most problematic aspect is that the 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 different viral replicase / RdRp species, it is desirable to explore and use other types of replicase / RdRp enzymes with simpler and fewer structural binding / recognition sites to overcome the problems of the conventional RCR method.

[0006] In view of the shortcomings of previous RCR methods, it is highly desirable herein to develop a novel RCR method that not only uses simpler and fewer 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. DETAILED DESCRIPTION OF THE INVENTION

[0007] Summary of the Invention: The principle of the present invention relies on incorporating at least one replicase / RdRp binding (recognition) site for coronavirus and / or hepatitis C virus (HCV) into the 5' and / or 3' ends of a desired RNA template, resulting in cycling amplification of either the sense or antisense strand of the desired RNA sequence, or both. In RCR, the defined replicase / RdRp binding site functions as a promoter and / or enhancer of replicase / RdRp activity. As shown in Figure 2, after incorporating at least one replicase / RdRp binding site into the 5' and / or 3' ends of the desired RNA template, the desired RNA sequence can be amplified approximately 15-fold to over 1000-fold with each cycle of the 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 PCR can provide an RNA amplification rate of approximately 15-fold to over 1000-fold within a specified time, depending on the length and structural complexity of the desired RNA sequence. Therefore, the desired RNA strand can be obtained with a relatively high purity ratio (up to 14 / 15 to >999 / 1000), depending on the termination point of PCR for the sense strand RNA, antisense strand RNA, or both. It is noteworthy that the desired RNA sequence and template in PCR can be one or more types, and the resulting RNA product can be single-stranded or double-stranded.

[0008] To prepare an RCR-compatible RNA template, we first use reverse transcription polymerase chain reaction (RT-PCR) to incorporate at least one coronavirus and / or HCV replicase / RdRp binding site into the 5' 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 PCR primer (called an RCR-compatible PCR primer). Therefore, after RT-PCR, an RCR-compatible RNA template cDNA is generated with the engineered replicase / RdRp binding site incorporated into the 5' or 3' end, or both. The resulting cDNA can then be cloned into a plasmid or viral vector for use in IVT reactions and / or storage. The desired RCR-compatible RNA template is then generated from the cDNA in an IVT reaction. The resulting RCR-compatible RNA template can then be used in PCR to repeatedly amplify and produce the desired RNA sequence. In practice, IVT and RCR can be performed simultaneously under exactly the same buffer conditions, and therefore, in this specification, cDNA incorporating replicase / RdRp binding sites (referred to as RCR-compatible cDNA template) is also preferred as the starting material for amplifying the desired RNA sequence in the combined IVT-RCR reaction.

[0009] 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 following sequences: 5'-AU(G / C)(U / -)G(A / U)-3' (i.e., 5'-AUSUGW-3'; SEQ ID NO: 1) and / or 5'-U(C / -)(U / A)C(U / C)(U / A)A-3' (i.e., 5'-UCWCYWA-3'; SEQ ID NO: 2). Preferably, the 5'-terminal RdRp binding site is selected from sequences containing 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 a combination thereof. Meanwhile, the 3'-terminal RdRp binding site contains 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 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 facilitate the design of PCR primers for RCR, the uridine / uracil (U) content of these RdRp binding sites can be substituted with thymidine (dT) and / or deoxyuridine (dU) in the primers. To enhance RNA stability, the uridine / uracil (U) content of these RdRp binding sites can be further substituted with pseudouridine or other modified nucleotide analogs 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.

[0010] Furthermore, these newly identified 5'- and 3'-terminal RdRp binding sites result in different RNA amplification rates. For example, the amplification rates of SEQ ID NO:1 and SEQ ID NO:7 are estimated to be approximately 25-1400-fold per PCR cycle, depending on the length and structural complexity of the desired RNA sequence, while the amplification rates of SEQ ID NO:2 and SEQ ID NO:8 are estimated to be approximately 10-900-fold per PCR cycle. Because of these different amplification preferences, different combinations of RdRp binding sites can be used to selectively amplify one RNA strand over another, or to selectively amplify certain RNA strands over other RNA strands. By this means, relatively pure single- and / or double-stranded RNA products of desired RNA sequences can be generated and recovered by PCR and further purified by other methods.

[0011] 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 in both its 5' and 3' terminal regions. Because both ends of the desired RNA contain 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 for both the sense and 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 termination point of the PCR. Furthermore, the resulting sense and antisense strand RNAs can further form double-stranded RNAs, promoting the production of siRNA, shRNA, miRNA, and / or piRNA of the desired RNA sequence.

[0012] Alternatively, in another preferred embodiment, the desired RNA sequence contains at least one RdRp binding site in either its 5' or 3' terminal 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. This approach is particularly 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 resulting mRNA vaccines and RNA / antibody-based medicines may be useful for treating 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.

[0013] Our novel RCR method can be used to produce or amplify any type of RNA species containing at least one RdRp-binding site, particularly viral antigen mRNAs and / or known functional RNAs / mRNAs that are useful for the development of antiviral and / or antidisease vaccines and medicines. For example, our two U.S. priority patent applications (U.S. Provisional Patent Applications Nos. 63 / 270,034 and 63 / 280,226 to Lin) demonstrated a novel method for generating iPS cells by cotransfecting an RCR-compatible RNA template with isolated coronavirus RdRp mRNA into human somatic cells. In contrast, those skilled in the art could anticipate 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 (U.S. Patent Application No. 17 / 489,357 to Lin), we have developed novel designs of RdRp-mediated self-amplifying RNA (saRNA) for generating novel mRNA vaccines and pharmaceuticals for treating viral infections and cancer, respectively. Furthermore, mRNA amplified by RCR can be further used in in vitro translation systems to produce encoded proteins, peptides, and / or antibodies. Given the success demonstrated by these prior inventions, we anticipate many developments in the potential applications of the present invention.

[0014] To efficiently generate highly structured RNA templates and RdRp mRNA, our priority patent application (U.S. Patent Application No. 17 / 489,357 to Lin) developed a novel PCR-IVT method to overcome the low efficiency problem of highly structured RNA production. It is well known that the presence of hairpin-like RNA structures significantly inhibits RNA transcription, making it unreasonable to expect that those skilled in the art would be able to 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 preferred method employs a novel IVT system that combines RNA polymerase and helicase activity. The additional helicase activity in IVT (and presumably also in PCR) significantly reduces secondary structure in both the DNA / RNA template and the resulting RNA product, resulting in much more efficient production of highly structured RNA. 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 helicases may be involved in transcription termination in prokaryotes, our study demonstrated that the function of helicases in RNA amplification during IVT is quite different.

[0015] To facilitate intracellular delivery / transfection in vitro, ex vivo, or in vivo, the RCR-compatible cDNA / RNA template and RdRp mRNA can be mixed, complexed, encapsulated, and / or formulated with at least one delivery / transfection agent selected from, but not limited to, glycylglycerol-derived chemicals, liposomes, nanoparticles, liposomal nanoparticles (LNPs), conjugated molecules, infusion / transfusion chemicals, gene gun materials, electroporation agents, transposons / retrotransposons, and combinations thereof.

[0016] The advantages of using RCR-ready cDNA / RNA templates for RNA / mRNA production and amplification include (1) high RNA yield, (2) high RNA purity, (3) easy preparation, as all reaction materials can be combined into a biochemical enzyme kit for RCR and / or combined IVT-RCR reactions, (4) simple reaction procedures compatible with other RT-PCR and IVT reactions, (5) simple equipment requirements that can be easily achieved using a PCR machine or temperature-controlled incubator, and (6) a variety of potential applications. As a result, the RCR-ready cDNA / RNA templates of the present invention are highly useful for producing and amplifying various desired RNA / mRNA sequences and can be used for a variety of pharmaceutical and therapeutic applications, including, but not limited to, the development of mRNA vaccines and RNA / microRNA-related drugs, as well as the generation of proteins, peptides, and antibodies.

[0017] A.Definition To facilitate the understanding of this invention, a number of terms are defined below.

[0018] nucleic acid : A polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that may be single- or double-stranded.

[0019] nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), phosphate, and a nitrogen-containing heterocyclic base. The base is attached to the sugar moiety via the glycosidic carbon (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.

[0020] Deoxyribonucleoside triphosphates (dNTPs) : Building block molecules for DNA synthesis, including dATP, dGTP, dCTP, dTTP, and may also contain modified deoxyribonucleotide analogs.

[0021] Ribonucleoside triphosphates (rNTPs) : Building block molecules for RNA synthesis, containing ATP, GTP, CTP, UTP, and may also contain pseudouridine and other modified ribonucleotide analogs.

[0022] Nucleotide Analogues :A purine or pyrimidine nucleotide that is structurally different from adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U), but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.

[0023] Oligonucleotides : A molecule consisting of two or more, preferably three or more, and 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, including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.

[0024] Nucleic acid compositionA nucleic acid composition refers to a single- or double-stranded molecular structure, an oligonucleotide or polynucleotide, such as a DNA or RNA sequence, or a mixed DNA / RNA sequence.

[0025] gene : A nucleic acid composition whose oligonucleotide or polynucleotide sequence encodes RNA and / or polypeptide (protein). A gene may be RNA or DNA. A gene may encode 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 encode 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 encode a protein-coding RNA that also contains at least one microRNA or shRNA sequence.

[0026] Primary RNA transcript (pre-mRNA) : An RNA sequence directly transcribed from a gene, without any RNA processing or modification.

[0027] 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 includes the 5' untranslated region (UTR), 3' UTR, exons, and introns.

[0028] Introns : A portion or part of a gene transcribed sequence that encodes non-protein-reading frames, such as in-frame introns, 5'-UTR, and 3'-UTR.

[0029] 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 / homologues, and derivatives.

[0030] messenger RNA (mRNA) A collection of pre-mRNA exons formed after intron removal by intracellular RNA splicing mechanisms (e.g., spliceosomes) and functioning as 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 / homologues and derivatives.

[0031] complementary DNA (cDNA) : Single- or double-stranded DNA that contains a sequence complementary to an mRNA sequence and does not contain intron sequences.

[0032] Sense : A nucleic acid molecule with the same sequence and composition as a homologous mRNA. The sense form is indicated by the symbol "+", "s" or "sense".

[0033] antisense : A nucleic acid molecule that is complementary to the respective mRNA molecule. Antisense forms are designated by the symbol "-" or by adding the letter "a" or "antisense" before DNA or RNA, as in "aDNA" and "aRNA."

[0034] 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. The relationship is generally achieved by hydrogen bonding. For example, the sense nucleotide sequence "5'-ATCGU-3'" can form a perfect base pair with the antisense sequence "5'-ACGAT-3'."

[0035] 5' end A nucleotide sequence in which the 5'-hydroxyl group of one nucleotide is joined to the 3'-hydroxyl group of the next nucleotide via a phosphodiester bond, lacking the 5' nucleotide. The terminus may also contain one or more other groups, such as phosphates.

[0036] 3' end A nucleotide sequence in which the 5'-hydroxyl group of one nucleotide is linked to the 3'-hydroxyl group of the next nucleotide via a phosphodiester bond, with the 3'-hydroxyl group missing. Other groups, often hydroxyl groups, may also be present at the end.

[0037] Templates : 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 two strands (and necessarily the 3' ends) are at opposite ends of the duplex.

[0038] Nucleic Acid Template It may be 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 single-stranded RNA molecule.

[0039] keep A nucleotide sequence is said to be conserved relative to a preselected (reference) sequence if it hybridizes non-randomly to the exact complement of the preselected sequence.

[0040] Homologous or HomologyHomology: A term that indicates the similarity between a polynucleotide and a gene or mRNA sequence. A nucleic acid sequence may be partially or completely homologous to, for example, a particular gene or mRNA sequence. Homology can be expressed as a percentage determined by the number of similar nucleotides relative to the total number of nucleotides.

[0041] Complementary or Complementarity or Complementation Complementarity: 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'-AGT-3'" is complementary to "5'-ACT-3'" as well as "5'-ACU-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 significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on nucleic acid binding. The percentage of complementarity refers to the ratio of mismatched bases to the total number of bases in a single 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 number of bases differs between the two strands. In this case, complementarity occurs between the bases of the longer strand paired with the bases of the shorter strand.

[0042] complementary bases :These are nucleotides that usually pair when DNA or RNA takes on a double-stranded structure.

[0043] 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 hybridize specifically between the two strands, resulting in hydrogen bonding.

[0044] 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., nonrandom, interactions exist between two complementary polynucleotides and can be competitively inhibited.

[0045] 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 a foreign / viral DNA or RNA transgene or small inhibitory RNAs.

[0046] RNA interference (RNAi) Post-transcriptional gene silencing in eukaryotes can be triggered by small inhibitory RNA molecules, such as microRNAs (miRNAs), small hairpin RNAs (shRNAs), and small interfering RNAs (siRNAs). 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.

[0047] MicroRNA (miRNA)miRNAs are single-stranded RNAs that can bind to the transcripts of target genes that are partially complementary to miRNAs. miRNAs are usually about 17-27 oligonucleotides in length, and depending on the complementarity between the miRNA and its target mRNA, they can either 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, enabling defense against viral infections and the control of gene expression during the development of animals and plants.

[0048] Precursor MicroRNA (Pre-miRNA) Pre-miRNAs are hairpin-shaped single-stranded RNAs with stem-arm and stem-loop regions that interact with intracellular 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 pre-miRNAs can form complete (100%) or partial (mismatched) hybrid duplexes, and the stem loop connects one end of the stem-arm duplex to form a circular or hairpin loop structure. However, in the present invention, precursors of microRNAs can also include pri-miRNAs.

[0049] Small interfering RNA (siRNA) : Short double-stranded RNAs, approximately 18-27 perfectly base-paired ribonucleotide duplexes in size, that can degrade nearly perfectly complementary target gene transcripts.

[0050] Small or short hairpin RNA (shRNA) : A single-stranded RNA in which a pair of partially or perfectly matched stem-arm nucleotide sequences is separated by a mismatched loop or bubble oligonucleotide, forming a hairpin-like structure. Many natural miRNAs are derived from small hairpin-like RNA precursors, or microRNA precursors (pre-miRNAs).

[0051] vectorA vector is a recombinant nucleic acid composition, such as recombinant DNA (rDNA), that can move and persist in different genetic environments. It generally has another nucleic acid operably linked to it. A vector is capable of autonomous replication within 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 those 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." Particularly important vectors allow for the cloning of cDNA from mRNA produced using reverse transcriptase. The vector can include components consisting of 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- 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.

[0052] promoter A promoter is a nucleic acid that a polymerase molecule recognizes, possibly binds to, and initiates transcription of RNA. For purposes of the present invention, a promoter may be a known polymerase binding site, an enhancer, etc., and may be any sequence that allows the desired polymerase to initiate synthesis of an RNA transcript.

[0053] 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'-end capping, 3'-end poly(A) tailing, and combinations thereof.

[0054] Gene transfer A genetic engineering method selected from the group consisting of: polysomal transfection, liposome transfection, chemical (nanoparticle) transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene-gun penetration, and combinations thereof.

[0055] genetic engineeringThe DNA recombination method is selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene incorporation, transposon insertion, jumping gene integration, retroviral infection, and combinations thereof.

[0056] Transfected cells : A single or multiple eukaryotic cells after artificially inserting 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.

[0057] antibody : A peptide or protein molecule having a preselected conserved domain structure that encodes a receptor capable of binding to a preselected ligand.

[0058] Medicinal and / or therapeutic uses 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.

[0059] B. Composition and Uses (a) providing at least one RNA sequence containing at least one RdRp binding site at the 5' end or the 3' end, or both; (b) providing at least one RNA replicase isolated or modified from a coronavirus or 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; This includes: The buffer conditions include the inclusion of 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.

[0060] 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 RNA sequences 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 combinations thereof. To incorporate these RdRp binding sites into PCR primers, the uridine / uracil (U) content of these RdRp binding sites can be substituted with thymidine (dT) and / or deoxyuridine (dU) in the primers. Additionally, to improve RNA stability, the uridine / uracil (U) content of these RdRp binding sites and the resulting RNA products can be substituted with pseudouridine or other modified nucleotide analogs. [Brief explanation of the drawings]

[0061] Referring to the drawings, which are given for purposes of illustration and not limitation, the following description is given:

[0062] [Figure 1] The step-by-step procedure of the conventional PCR-IVT method is shown. 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.

[0063] [Figure 2] The figure shows a step-by-step procedure for the PCR method of the present invention. To prepare a PCR-ready cDNA / RNA template, conventional RT-PCR techniques are used to incorporate at least one coronavirus and / or HCV replicase / RdRp binding site into the 5' or 3' end of the cDNA of the desired RNA sequence. Then, using some or all of the steps of this novel PCR method, the desired RNA sequence is produced and amplified from the PCR-ready cDNA / RNA template after single or multiple cycles of amplification. Alternatively, because IVT and PCR can be performed simultaneously under the same buffer conditions, the PCR-ready cDNA / RNA template can also be used as starting material to amplify the desired RNA sequence in a combined IVT-RCR reaction.

[0064] [Figure 3] 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 enhance the stability of the RCR-compatible RNA template, the uridine / uracil (U) moieties of the template can be replaced with pseudouridine or other modified nucleotide analogs.

[0065] [Figure 4]Northern blot analysis results showing that expression of miR-302 microRNA (i.e., from top to bottom: b, c, d, a) and RdRp mRNA in transfected human cells after 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 results from cells transfected with only the pre-miR-302 template (middle), providing evidence of RCR in the cells.

[0066] [Figure 5] Northern blot analysis results of RCR-ready cDNA and RNA templates and the resulting amplified RNA products of interest (i.e., mRNA sequences of viral antigen proteins / peptides) are shown, providing evidence of RCR in vitro.

[0067] [Figure 6] 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 Figure 5) and isolated RdRp mRNA (from Figure 4) is shown, demonstrating that the present invention is useful for the development and production of antiviral mRNA vaccines.

[0068] Working Example: 1. Human Cell Isolation and Culture Starting tissue cells can be obtained from enzymatically dissociated skin cells using Aasen's protocol (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 in a microfuge incubator at 37°C for 1 minute, 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 in the cell culture medium. The cell culture medium and all additives should be refreshed every 2–3 days. Cells were passaged at approximately 50–60% confluence by exposing them to trypsin / EDTA for 1 minute and rinsing twice with HBSS containing trypsin inhibitor. For ASC expansion, cells were replated at a dilution of 1:5 to 1:500 in fresh feeder-free MSC Expansion SFM culture medium supplemented with the formulated pre-miR-302 + RdRp mRNA mixture, LIF, bFGF / FGF2, and / or other optionally defined factors. For keratinocyte culture, cells were 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, 5% CO2.Cultured cells were passaged at 50-60% confluence by exposing them to trypsin / EDTA solution for 1 minute and rinsing once with phenol red-free DMEM medium (Invitrogen). Detached cells were replated at a 1:10 dilution 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 manufacturer's or provider's recommendations. 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).

[0069] 2. In-Vitro RNA Transfection For intracellular transfection, dissolve 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 approximately 20:1 to 1:20) in 0.5 ml of fresh cell culture medium and mix with 1–50 μl of In-VivoJetPEI or other similar transfection reagent. After incubating for 10–30 minutes, add the mixture to cell culture medium containing 50–60% confluence of cultured cells. The medium should be flushed every 12–48 hours, depending on the cell type. This transfection procedure can be repeated to increase transfection efficiency.

[0070] 3. Preparation of PCR-ready cDNA / RNA templates Reverse transcription (RT) of the desired RNA / mRNA is performed by adding approximately 0.01 ng–10 micrograms (µg) of the isolated RNA / mRNA to a 20–50 µL RT reaction mixture (SuperScript III cDNA RT kit, ThermoFisher Scientific, MA, USA) according to the manufacturer's instructions. Depending on the amount of RNA / mRNA, the RT reaction mixture also contains approximately 0.01–20 nmoles of RT primer, an appropriate amount of deoxyribonucleoside triphosphates (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 complexity of the desired RNA / mRNA sequence, to create a complementary DNA (cDNA) template for PCR in the next step. 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.

[0071] Next, polymerase chain reaction (PCR) is performed by adding approximately 0.01 pg to 10 μg of RT-derived cDNA to 20–50 μL of PCR preparation mixture (High-Fidelity PCR master kit, ThermoFisher Scientific, MA, USA) according to the manufacturer's recommendations. The PCR mixture is then incubated for 5–20 cycles, depending on the structure and length of the desired cDNA sequence: denaturation at 94°C for 1 minute, annealing at 30–55°C for 30 seconds to 1 minute, and extension at 72°C for 1–3 minutes. This is followed by another 10–20 cycles of PCR, with the cycling procedure repeating: denaturation at 94°C for 1 minute, annealing at 50–58°C for 30 seconds, and extension at 72°C for 1–3 minutes, 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 PCR. For the preparation of IVT-PCR templates, we designed and used a specific PCR-compatible primer pair containing 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-PCR mRNA product. Meanwhile, we also designed and used another PCR-compatible primer pair containing 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 the human pre-miR-302 familial cluster (pre-miR-302). Primer design can use various sequences and combinations of RNA promoters and RdRp binding sites, such as T7, T3, and / or SP6 promoters, with at least one RdRp binding site incorporated into the 5'- and / or 3'-end primers.

[0072] To generate a PCR-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 PCR-ready cDNA template). The 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), an appropriate amount of ribonucleoside triphosphate molecules (rNTPs), and an 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. Next, incubate the IVT-RCR reaction at 30-40°C for 1-6 hours, depending on the stability and activity of the RdRp and RNA polymerase enzymes used.

[0073] 4. Novel RCR Protocol The starting PCR mixture contains approximately 0.01 ng–10 μg of PCR-ready RNA / mRNA template, approximately 0.1–50 U of isolated coronavirus RdRp / helicase, and an appropriate amount of rNTPs in a 1x transcription buffer. The RdRp / helicase can be 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. The 1x transcription buffer can also 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 h, depending on the stability and activity of the RdRp enzyme used.

[0074] 5. RNA Purification and Northern Blot Analysis Isolate the desired RNA (10 μg) using the mirVana® RNA Isolation Kit (Ambion, Austin, TX) or a similar purification filter column according to the manufacturer's protocol and further purify by 5%–10% TBE-urea polyacrylamide or 1%–3.5% low-melting-point agarose gel electrophoresis. For Northern blot analysis, 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 probe is further purified by high-performance liquid chromatography (HPLC) and labeled with dye-labeled nucleotide analogs or [ 32 The cells were tail-labeled with terminal transferase (20 units) in the presence of [ P]-dATP (>3000 Ci / mM, Amersham International, Arlington Heights, IL) for 20 minutes.

[0075] 6. Protein Extraction and Western Blot Analysis cells (10 6 Cells are lysed in CelLytic-M Lysis / Extraction Reagent (Sigma) supplemented with protease inhibitors, leupeptin, TLCK, TAME, and PMSF, according to the manufacturer's recommendations. The lysates are centrifuged at 12,000 rpm at 4°C for 20 minutes, and the supernatant is collected. Protein concentration is measured using the modified SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, CA). Thirty micrograms of each cell lysate is added to SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, boiled for 3 minutes, 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 antibodies are then applied to the reagent, and the mixture is incubated at 4°C. After overnight incubation, the membranes were rinsed three times with TBS-T and exposed to a goat anti-mouse IgG-conjugated secondary antibody (1:2,000; Invitrogen-Molecular Probes) against an Alexa Fluor 680-reactive dye for 1 hour at room temperature. After three additional TBS-T rinses, fluorescent scanning and image analysis of the immunoblots were performed using a Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor).

[0076] 7. Immunostaining Assay Cell / tissue samples were fixed in 100% methanol for 30 minutes at 4°C, followed by 4% paraformaldehyde (in 1x PBS, pH 7.4) for 10 minutes at 20°C. The samples were then incubated in 1x PBS containing 0.1%–0.25% Triton X-100 for 10 minutes, followed by three 5-minute washes with 1x PBS. For immunostaining, primary antibodies were purchased from Invitrogen (CA, USA) and Sigma-Aldrich (MO, USA), respectively. Dye-conjugated goat anti-rabbit or horse anti-mouse antibodies were used as secondary antibodies (Invitrogen, CA, USA). Results were examined and analyzed at 100x or 200x magnification using a fluorescent 80i microscopic quantitation system equipped with the Metamorph imaging program (Nikon).

[0077] 8. In Vivo Transfection Assay The PCR-amplified RNA / mRNA and RdRp mRNA mixture (ratios ranging from approximately 20:1 to 1:20) is thoroughly mixed with an appropriate amount of a delivery agent, such as In-VivoJet PEI transfection reagent or other similar LNP-based delivery / transfection agents, according to the manufacturer's recommended protocol, and then injected intravenously or intramuscularly into an animal, 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 facilitate delivery / transfection of the RNA / mRNA and RdRp mRNA mixture into specific target cells of interest in vitro, ex vivo, and / or in vivo.

[0078] 9.Statistical analysis All data were expressed as mean and standard deviation (SD). The mean value of each test group was calculated using AVERAGE in Microsoft Excel. SD was calculated using STDEV. Statistical analysis of the data was performed using one-way ANOVA. Tukey and Dunnett's t post hoc test was used to determine the significance of data differences between groups. p<0.05 was considered significant (SPSS v12.0, Claritas Inc).

[0079] References: 1. Shi-Lung Lin WO2002 / 092774 to et al. 2. Shi-Lung Lin U.S. Patent No. 7,662,791 to et al. 3. Shi-Lung Lin U.S. Patent No. 8,080,652 to et al. 4. Ying SY and Shi-Lung Lin U.S. Patent No. 8,372,969 to 5. Shi-Lung Lin and U.S. Patent No. 8,609,831 to Ying SY. 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. GeneTherapy 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 humanskin cancer cells into a pluripotent ES-cell-like state.RNA 14:2115-2124,2008. 11. Shi-Lung Lin etc;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.

[0080] Sequence Listing (1) General Information: (iii) Number of sequences: 16 (2) Information on SEQ ID NO: 1: (i) Sequence characteristics: (A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO: 1: AUSUGW 6 (2) Information on sequence number 2: (i) Sequence characteristics: (A) Length: 7 bases (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO:2: UCWCYWA 7 (2) Information on SEQ ID NO: 3: (i) Sequence characteristics: (A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (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 on SEQ ID NO: 4: (i) Sequence characteristics: (A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO: 4: UCUCUAA 7 (2) Information on SEQ ID NO: 5: (i) Sequence characteristics: (A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO:5: UCUCCUA 7 (2) Information on SEQ ID NO: 6: (i) Sequence characteristics: (A) Length: 5 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO:6: UUCAA 5 (2) Information on SEQ ID NO: 7: (i) Sequence characteristics: (A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO: 7: WCASAU 6 (2) Information on SEQ ID NO: 8: (i) Sequence characteristics: (A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (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 on SEQ ID NO: 9: (i) Sequence characteristics: (A) Length: 6 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (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 on SEQ ID NO: 10: (i) Sequence characteristics: (A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO: 10: UUAGAGA 7 (2) Information on SEQ ID NO: 11: (i) Sequence characteristics: (A) Length: 7 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (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 on SEQ ID NO: 12: (i) Sequence characteristics: (A) Length: 5 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: RNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO: 12: UUGAA 5 (2) Information on SEQ ID NO: 13: (i) Sequence characteristics: (A) Length: 23 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (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 on SEQ ID NO: 14: (i) Sequence characteristics: (A) Length: 48 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (ii) Molecule type: DNA (A) DESCRIPTION: / desc="synthetic" (iii) HYPOTHETICAL: NO (iv) Antisense: NO (xi) Sequence description: SEQ ID NO: 14: GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT 48 (2) Information on SEQ ID NO: 15: (i) Sequence characteristics: (A) Length: 56 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (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 on SEQ ID NO: 16: (i) Sequence characteristics: (A) Length: 32 base pairs (B) Type: Nucleic acid (C) Strandedness: Single (D) Topology: Straight line (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

1. (a) providing at least one RNA sequence comprising at least a 5'-terminal RdRp binding site and a 3'-terminal RdRp binding site, wherein the 5'-terminal RdRp binding site comprises the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, and the 3'-terminal RdRp binding site comprises the sequence of SEQ ID NO: 10 or SEQ ID NO: 11; (b) providing at least an RNA replicase that is a coronavirus RNA-dependent RNA polymerase (RdRp); (c) mixing the RNA sequence of (a) with the RNA replicase of (b) under buffer conditions to produce and amplify the RNA sequence mediated by the RNA replicase; 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. An RNA replicase-mediated RNA amplification method for in vitro synthesis of self-amplifiable RNA (saRNA).

2. 10. The method of claim 1, wherein the RNA sequence is single-stranded, double-stranded, or both.

3. 2. The method of claim 1, wherein the RdRp binding sites at the 5' end and the 3' end of the RNA sequence are incorporated into the RNA sequence by using a polymerase chain reaction in vitro transcription (PCR-IVT) method that uses a mixed activity of RNA polymerase and RdRp / helicase.

4. 10. The method of claim 1, wherein the buffer conditions are used for a coronavirus RdRp enzyme and an RNA polymerase (T7, T3, or SP6).

5. 2. The method of claim 1, wherein the ribonucleoside triphosphate molecules (rNTPs) include ATP, GTP, CTP, and UTP.

6. 2. The method of claim 1, wherein the ribonucleoside triphosphate molecules (rNTPs) further comprise pseudouridine.

7. 2. The method of claim 1, wherein the uridine / uracil (U) components of the RNA sequence are substituted with pseudouridine.

8. The method of claim 1, wherein the amplified RNA sequence is further formulated with at least one delivery agent to facilitate intracellular transfection.

9. 9. The method of claim 8, wherein the delivery agent comprises glycylglycerin, liposomes, nanoparticles, liposomal nanoparticles (LNPs), conjugated molecules, infusion chemicals, gene gun materials, electroporation agents, transposons, and combinations thereof.

10. The method of claim 1 , wherein the RNA sequence is mRNA.

11. The method of claim 10, wherein the mRNA is used to produce and develop mRNA vaccines and medicines.

12. The method of claim 10, wherein the mRNA is used to produce proteins / peptides and antibodies.

13. 2. The method of claim 1, wherein the RNA sequence is a precursor microRNA (pre-miRNA).

14. The method of claim 13, wherein the pre-miRNA is used to produce and develop anti-cancer drugs.

15. The method of claim 13, wherein the pre-miRNA is used to generate iPS cells.

16. The method of claim 1, wherein the RNA sequence is used as a pharmaceutical or therapeutic component.

Images