Novel inducible cytoplasmic in vitro transcription-like composition and associated vaccine pharmaceutical design

Abstract

To provide an inducible cytoplasmic IVT (ICIVT) composition that is suitable for inducing in vitro transcription (IVT)-like RNA / mRNA amplification in target cells after transfection in vitro, ex vivo, and / or in vivo.SOLUTION: Provided is a composition that comprises a mixture of a promoter-linked RNA / mRNA-coding DNA (PLRcD) template and an mRNA sequence of another DNA-dependent RNA polymerase (DdRP). Preferably, the DdRP mRNA is selected from mRNA of T7, T3, and / or SP6 RNA polymerases or combinations thereof, and the PLRcD template encodes transcripts of antisense RNA oligonucleotides, small interfering RNA, double-stranded RNA, short hairpin RNA, microRNA / microRNA precursors, long non-coding RNA, messenger RNA, and / or self-amplifying RNA / mRNA, or combinations thereof.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority This invention claims priority to U.S. Provisional Patent Application No. 63 / 531,004, entitled "Novel In-Cell Transcription (ICT) Composition and the Related Vaccine Medicine Designs Thereof," filed August 6, 2023. This invention also claims priority to U.S. Provisional Patent Application No. 17 / 489,357, entitled "Novel mRNA Composition and Production Method for Use in Anti-Viral and Anti-Cancer Vaccines," filed September 29, 2021. This application is further a continuation-in-part of U.S. Provisional Patent Application No. 17 / 489,357, entitled "Novel mRNA Composition and Production Method for Use in Anti-Viral and Anti-Cancer Vaccines," filed September 29, 2021.

[0002] The present invention relates to a novel non-vector-based (non-transgenic) induced cytoplasmic in-vitro transcription (ICIVT) composition useful for inducing in-vitro transcription (IVT)-like RNA / mRNA amplification in a desired target cell after in vitro, ex vivo, and in vivo intracellular transfection. The present invention comprises at least a novel mixture composition of a designed promoter-linked RNA / mRNA-coding DNA (PLRcD) template and mRNA sequences of other DNA-dependent RNA polymerases (DdRPs), which is useful for designing and developing various RNA / mRNA-based medicines and / or vaccines. Preferably, the DdRP mRNA is selected from the transcripts of T7, T3 and / or SP6 RNA polymerase, or a combination thereof, while the designed PLRcD template may encode at least one or more transcripts of antisense RNA oligonucleotide (aRNA-ASO), small interfering RNA (siRNA), double-stranded RNA (dsRNA), short hairpin RNA (shRNA), microRNA (miRNA) / microRNA precursor (pre-miRNA), long noncoding RNA (lncRNA), messenger RNA (mRNA), and / or self-amplifying RNA / mRNA (saRNA / samRNA), or a combination thereof.For delivery / transfection into cells of interest, the mixed composition of PLRcD and DdRP may be further mixed, bound, encapsulated, or compounded with at least one reagent selected from, but not limited to, liposomes, nanoparticles, liposomal nanoparticles (LNPs), exosomes, triglycylglycerin (TGG)-derived drugs, sugar / glucosamine / galactosamine-based binding molecules, infusion / transfusion agents, gene gun materials, electroporation agents, and / or transposons / retrotransposons, or combinations thereof. The resulting expression of the encoded RNA / mRNA after transfection into target cells of interest is 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, all types of cancer, and even many types of viral and bacterial infections. [Background technology]

[0003] Conventional in vitro transcription (IVT) technology is a method of synthesizing and amplifying a desired RNA / mRNA sequence from a pre-designed promoter-bound DNA template in a test tube using DNA-dependent RNA polymerase (DdRP) protein, without involving any cells, cytoplasm, or DdRP mRNA. Therefore, based on similar IVT principles but with different compositions and implementation mechanisms, conventional IVT technology is different from the methodology of intracellular-induced cytoplasmic IVT (ICIVT) of the present invention.

[0004] The use of in vitro transcription (IVT) methods to generate sense (+) strand mRNA and / or related mRNA-cDNA hybrids was first reported by Lin et al. (Lin, U.S. Patent Nos. 7,662,791, 8,080,652, 8,372,969, and 8,609,831). As shown in FIG. 1, Lin's method first uses polymerase chain reaction (PCR) and / or reverse transcription (RT) to incorporate specific RNA promoter primers into the resulting DNA product to generate promoter-bound mRNA-encoding DNA templates suitable for IVT-based mRNA amplification (Lin et al, Methods Mol Biol. 221:93-101, 2003). After IVT and further RNase-free DNase digestion, the amplified mRNA can be combined with a delivery / transfection reagent and transfected into target cells of interest to generate the mRNA-encoded protein / peptide. However, although Lin's method succeeded in producing mRNA and related proteins / peptides in vitro, it did not describe IVT-like reactions or intracellular applications. Therefore, Lin's conventional IVT method does not disclose any information regarding the intracellular transcription of the present invention.

[0005] Current mRNA production methods are still mainly based in part or in whole on Lin's PCR-IVT protocol (Lin's U.S. Patents Nos. 7,662,791, 8,080,652, 8,372,969, and 8,609,831). Also, many commercially available semi-automated or fully automated IVT devices have been designed and developed using Lin's IVT concepts and methodologies. However, these conventional IVT-related methods and devices have still not provided an effective solution to overcome the problem of IVT activation in eukaryotic cells, especially mammalian cells, due to the incompatibility of eukaryotic DdRPs to conventional IVT-related bacteriophage promoters (i.e., T7, T3, and SP6 promoters). Because conventional IVT promoters are incompatible with eukaryotic DdRP enzymes, transcription of conventional designed promoter-bound RNA / mRNA-encoding DNA (PLRcD) sequences usually cannot be activated in mammalian cells, resulting in no amplification of RNA / mRNA in the cells. To solve this problem, another method using plasmid / replicon-driven RNA / mRNA expression in prokaryotes (such as bacteria) was also developed by Lin et al. (U.S. Patents 9,637,747 and 9,783,811 to Lin). Nevertheless, this plasmid / replicon-driven RNA / mRNA expression method requires the use of prokaryotic cells and can only be performed in prokaryotes.

[0006] Transgenic and vector / replicon (e.g., plasmid)-based T7-DdRP-mediated IVT in mammalian cells has also been established (Ghaderi et al, Iran J.Cancer Prev.7:137-141, 2014). In conventional practice, a plasmid vector encoding the T7 DdRP is transfected into the cell nucleus to express T7 DdRP mRNA and protein, and then the T7 DdRP protein drives the expression of a T7 promoter-linked mRNA-encoding plasmid vector co-transfected into the transfected cells. However, since the expressed T7 DdRP may be present in either or both locations in the cell nucleus and cytoplasm, it is unclear whether the second encoded mRNA is expressed in the cell nucleus or cytoplasm. It is also unclear whether the co-transfected T7 promoter-linked mRNA-encoding plasmid is delivered to the cell nucleus or cytoplasm. As this vector-based IVT methodology requires nuclear expression of T7 DdRP mRNA, the risk of transgenic insertion certainly exists. In addition, because T7 DdRP mRNA is expressed by a transcription system in the mammalian cell nucleus, the involvement of another eukaryotic promoter driven by mammalian type II RNA polymerase (Pol-II) is also required. As a result, considering such a complex Pol-II+T7-driven mRNA expression mechanism, this vector (plasmid)-based IVT system in mammalian cells is not a substantial cytoplasmic transcription system.

[0007] Currently, with the rapid development of mRNA vaccines and medicines, it is desirable to induce non-transgenic IVT-like RNA / mRNA amplification in the cytoplasm of transfected target cells. However, none of the conventional IVT methods can achieve this goal. To overcome the problems of conventional IVT methods, the present invention employs a novel induced cytoplasmic IVT (ICIVT) composition and method for inducing specific IVT-like RNA / mRNA amplification in transfected target cells, especially mammalian cells. Summary of the Invention

[0008] The principle of the present invention is based on, but more advanced from, the novel composition design described in the priority application US patent application Ser. No. 17 / 489,357. As shown in Fig. 2, by inserting at least an internal ribosome entry site (IRES)-binding Kozak motif (IRES-kozak) between the promoter and the RNA / mRNA coding sequence, a designed construct of an inducible cytoplasmic IVT (ICIVT)-based promoter-bound RNA / mRNA-encoding DNA (PLRcD) template is generated, which is useful for inducing IVT-like RNA / mRNA amplification in the cytoplasm of transfected target cells and directing the cytoplasmic RNA / mRNA expression system. To activate this cytoplasmic RNA / mRNA expression system, at least the mRNA of DNA-dependent RNA polymerase (DdRP), such as T7, T3, SP6 DdRP mRNA, and the designed PLRcD template must be co-transfected. However, because it is unclear whether exogenous mRNA (such as T7, T3, SP6 DdRP mRNA) can be translated into a functional protein, particularly a properly folded protein, in mammalian cells without undergoing nuclear processing and / or modification, even those skilled in the art will recognize that extensive experimentation and modification must be performed to demonstrate the feasibility of the designed DdRP mRNA to form a functional DdRP protein in the mammalian cytoplasm.

[0009] A Kozak motif may be 5'-RCCRCC-3' (SEQ ID NO:1; R is A or G) or 5'-RCCDCC-3' (SEQ ID NO:2; D is A, G or T / U), an IRES may contain a homologue of at least one of the following hairpin / stem loop containing sequences, in which U (uracil / uridine) and T (thymine / thymidine) are interchangeable, including but not limited to: (1) 5'-GCUCCCUUCA ACUUUAACAU GGAAGUGCUU UCUGUGACUU UAAAAGUAAG UGCUUCCAUG UUUUAGUAGG AGU-3' (73-nt) (SEQ ID NO: 3) (2) 5'-CCUUUGCUUU AACAUGGGGG UACCUGCUGU GUGAAACAAA AGUAAGUGCU UCCAUGUUUC AGUGGAGG-3' (68-nt) (SEQ ID NO: 4) (3) 5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3' (69-nt) (SEQ ID NO: 5) (4) 5'-CCUCUACUUU AACAUGGAGG CACUUGCUGU GACAUGACAA AAAUAAGUGC UUCCAUGUUU GAGUGUGG-3' (68-nt) (SEQ ID NO: 6) (5) 5'-CUGUGUGGCU GUCACUCGGC UGCAUGCUUA GUGCACUCAC GCAG-3' (44-nt) (SEQ ID NO: 7) (6) 5'-CUGUGUGGCU GUCACUCGGC UGCAUGCUUA GUGCACUCAC GCAGUAUAAU UAAUAACUAA UUACU-3' (65-nt) (SEQ ID NO: 8) (7) 5'-GUCGUUGACA GGACACGAGU AACUCGUCUA UCUUCUGCAG GCUGCUUACG GUUUCGUCCG UGUUGCAGCC GAUCAUCAGC ACAUCUAGGU UUCGUCCGGG UGUGACCGAA AGGUAAGAUG GAGAGCCUUG UCCCUGGUUU CAACGAG-3'(147-nt) (SEQ ID NO: 9) (8) 5'-AAUUAUAAAU UACCAGAUGA UUUUACAGGC UGCGUUAUAG CUUGGAAUUC UAACAAUCUU GAUUCUAAGG UUGGUGGUAA UUAUAAUU-3' (88-nt) (SEQ ID NO: 10) (9) 5'-CACAAAUAUU ACCAGAUCCA UCAAAACCAA GCAAGAGGUC AUUUAUUGAA GAUCUACUUU UCAACAAAGU GACACUUGCA GAUGCUGGCU UCAUCAAACA AUAUGGUGAU UGCCUUGGUG AUAUUGCUG-3' (129-nt) (SEQ ID NO: 11) (10) 5'-GCAAAAAUGU GAUCUUGCUU GUAAAUACAA UUUUGAGAGG UUAAUAAAUU ACAAGUAGUG CUAUUUUUGU AUUUAGGUUA GCUAUUUAGC UUUACGUUCC AGGAUGCCUA GUGGCAGCCC CACAAUAUCC AGGAAGCCCU CUCUGCGGUU UUUCAGAUUC GUUAGUCGAA AAACCUAAGA AAUUUAAUG-3' (189-nt) (SEQ ID NO: 12) (11) 5'-CACUCCCCUG UGAGGACUAC UGUCUUCACG CAGAAAGCGU CUAGCCAUGG CGUUAGUAUG AGUGUCGUGC AGCCUCCAGG ACCCCCCCUC CCGGGAGAGC CAUAGUGGUC UGCGGAACCG GUGAGUACAC CGGAAUUGCC AGGACGACCG GGUCCUUUCU UGGAUCAACC CGCUCAAUGC CUGGAGAUUU GGGCGUGCCC CCGCGAGACU GCUAGCCGAG UAGUGUUGGG UCGCGAAAGG CCUUGUGGUA CUGCCUGAUG GGUGCUUGCG AGUGCCCCGG GAGGUCUCGU AGAC-3'(294-nt) (SEQ ID NO: 13) (12) 5'-GGACACGAGU AACUCGUCUA UCUUCUGCAG GCUGCUUACG GUUUCGUCCG UGUUG-3' (55-nt) (SEQ ID NO: 14) (13) 5'-CAGCCGAUCA UCAGCACAUC UAGGUUUUGU CCGGGUGUGA CCGAAAGGUA AG-3' (52-nt) (SEQ ID NO: 15)

[0010] To carry out induced cytoplasmic IVT-like transcription (ICIVT), we have invented a co-transfection method to put at least an artificial DdRP mRNA (T7, T3, and / or SP6 DdRP mRNA) together with the designed PLRcD template into the desired target cells. Also, since the so obtained ICIVT-amplified RNA / mRNA product (present in the cytoplasm / cytosol) may not be effectively capped by the intracellular capping enzyme (in the nucleus), the here invented IRES-Kozak motif in the designed PLRcD template then exerts its function as an internal ribosome entry site (IRES) for ribosome assembly and activates protein / peptide translation, while the Kozak sequence indicates the correct position to start protein / peptide synthesis. Thus, with the applicant's novel design of the IRES-Kozak motif (Figure 2), the ICIVT-based PLRcD template of the present invention can be effectively used not only for intracellular transcription (or called cytoplasmic expression) of the desired RNA / mRNA sequence, but also for inducing translation of the obtained mRNA product into protein / peptide in the transfected cell. In particular, the resulting RNA is either coding mRNA or non-coding RNA, or a combination of both.

[0011] To prevent unwanted interferon-associated immune responses and intracellular Trex1-mediated DNA degradation, the 5'-end of the engineered PLRcD template is dephosphorylated, while 8-hydroxyguanine (8-OHG) and / or its derived analogs, especially 8-hydroxy-2-deoxyguanosine (8-OHdG), are added to the 3'-end. DdRP mRNA is capped at the 5'-end with m7G(5')ppp(5')N (m7G) and / or its related cap (i.e., cap-0 or cap-1 / 2) analogs, while 3'-cap-modified G / U / A molecules such as cap-like modified nucleotide analogs (e.g., 8-oxo-G, 8-oxo-A, or 5' phosphorothioate-U) are added to the 3'-end. For the incorporation of 5'-cap, the capping enzyme includes, but is not limited to, coronavirus NSP12, NSP9 / 14, NSP10 / 16, and / or vaccinia capping enzyme, and 2'-O-methyltransferase, or a combination thereof. For the incorporation of 3'-cap, terminal RNA uridylyltransferase and / or polymerase theta are preferably used. Furthermore, to prevent intracellular immune reaction, U (uridine / uracil) of DdRP mRNA can be further fully or partially replaced by pseudouridine, methyluridine, methoxyuridine, and / or other related or similarly modified nucleotide analogs, or a combination thereof.

[0012] To facilitate delivery / transfection into target cells of interest in vitro, ex vivo and / or in vivo, the mixed composition of the designed PLRcD template and DdRP mRNA (Figure 2) can be mixed, bound, encapsulated and / or formulated with at least one delivery / transfection reagent selected from, but not limited to, liposomes, nanoparticles, liposomal nanoparticles (LNPs), exosomes, sugar / glucosamine / galactosamine based binding molecules, infusion / transfusion agents, glycylglycerin derived molecules, gene gun materials, electroporation agents, and / or transposons / retrotransposons or combinations thereof.

[0013] IRES are highly structured nucleotide sequences that often contain hairpin and / or stem-loop structures. To generate highly structured PLRcD templates, applicants' priority application U.S. Patent Application No. 17 / 489,357 ('357) developed a new PCR-IVT methodology using specific helicase activity to overcome the problem of low efficiency of highly structured RNA amplification in conventional IVT reactions. Because the hairpin-like stem-loop structure is an inherent transcription termination signal for prokaryotic RNA polymerases (McDowell et al, Science 266:822-825, 1994), the priority application '357 method employs a new IVT system using a mixture of RNA polymerase and helicase activity. As a result, this additional helicase activity significantly reduces the secondary structure of the PLRcD template and its RNA / mRNA product in IVT, leading to an improved RNA / mRNA amplification rate. In response to this result, the applicant's research has shown that coronavirus NSP7 and NSP13 proteins are two helicases that have been identified as useful for enhancing the amplification efficiency of IVT and ICIVT.

[0014] The advantages of using a mixture of designed PLRcD template and DdRP mRNA as the novel composition of the present invention include: (1) intracellular RNA / mRNA amplification similar to the effect of self-amplifying RNA, (2) no transgene issues, (3) no need to add a 5' cap to the resulting RNA / mRNA product, (4) long-lasting effect due to stable DNA construction of the designed PLRcD template, (5) regulatable expression of the encoded RNA / mRNA regulated by the amount of DdRP mRNA used, (6) simple manufacturing procedure, (7) further enhancement by using self-amplifying DdRP mRNA (DdRP-samRNA), and (8) development of various potential applications. The invented novel compositions of PLRcD template and DdRP mRNA mixtures are believed to be useful in the design and development of new pharmaceutical and therapeutic applications, including vaccines and medicines, for the treatment of various human / animal diseases, including but not limited to Alzheimer's disease, Parkinson's disease, motor neuron disease, stroke, diabetes, myocardial infarction, hemophilia, anemia, leukemia and many types of cancer, as well as all types of viral and bacterial infections. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] A.Definition 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.

[0016] 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 (1' carbon of the pentose) and combinations thereof, and the base-sugar combination is a nucleoside. A nucleoside containing 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.

[0017] Deoxyribonucleoside Triphosphates , dNTPs) : The building blocks of DNA synthesis, such as dATP, dGTP, dCTP, dTTP, and may further include modified deoxyribonucleotide analogues.

[0018] Ribonucleoside Triphosphates , rNTP) : The building block molecules of RNA synthesis, such as ATP, GTP, CTP, UTP, and optionally further including pseudouridine, 5' methyluridine, methoxyuridine, and / or other modified ribonucleotide analogues.

[0019] 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 similar enough to substitute for the normal nucleotide in a nucleic acid molecule.

[0020] Oligonucleotides : A molecule composed of two or more monomeric units of DNA and / or RNA, preferably three or more, usually ten or more. Oligonucleotides longer than 13 nucleotide monomers are also called polynucleotides. The actual size depends on many factors, which in turn depend 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.

[0021] 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.

[0022] gene: A nucleic acid composition whose oligonucleotide or polynucleotide sequence codes for RNA and / or polypeptides (proteins). Genes are either RNA or DNA. Genes may code for non-coding RNAs such as small hairpin RNAs (shRNAs), microRNAs (miRNAs), rRNAs, tRNAs, snoRNAs, snRNAs, and their RNA precursors and derivatives. Alternatively, genes may code for protein-coding RNAs essential for protein / peptide synthesis, such as messenger RNAs (mRNAs) and their RNA precursors, derivatives. In some cases, genes may code for protein-coding RNAs that also contain at least a microRNA or shRNA sequence.

[0023] Primary RNA transcripts : An RNA sequence that is transcribed directly from a gene without any RNA processing or modification.

[0024] Precursor messenger RNA (pre-mRNA) : The primary RNA transcript of a protein-coding gene. It is generated by an intracellular mechanism called transcription, by the eukaryotic type II RNA polymerase (Pol-II) machinery in eukaryotes. The pre-mRNA sequence contains a 5'-untranslated region (UTR), a 3'-UTR, exons, and introns.

[0025] Introns : A portion of a gene's transcribed sequence that codes for a non-protein reading frame, such as an in-frame intron, 5'-UTR, or 3'-UTR.

[0026] Exon : A portion of a gene transcribed sequence that codes for a protein reading frame (cDNA), such as the cDNAs of cellular genes, growth factors, insulin, antibodies and their analogs / homologs and derivatives.

[0027] 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. Structurally, an mRNA sequence contains a 5' cap nucleotide [e.g., m7G(5')ppp(5')N-], a 5'-untranslated region (5'-UTR), at least a Kozak consensus translation initiation site (e.g., 5'-GCCACC-3'), at least a protein / peptide coding region, a polyadenylation signal (e.g., 5'-AUAAA-3' or 5'-AUUAAA-3'), and / or a 3'-UTR with or without a polyA tail. Proteins / peptides encoded by mRNA include, but are not limited to, enzymes, growth factors, insulin, antibodies and their analogs / homologs, and derivatives.

[0028] Complementary DNA (cDNA) : Single- or double-stranded DNA that contains a sequence complementary to an mRNA sequence and does not contain intron sequences.

[0029] Sense : A nucleic acid molecule that has the same sequence order and composition as a homologous mRNA. The sense structure is indicated by the symbol "+", "s", or "sense".

[0030] Antisense : A nucleic acid molecule that is complementary to the respective mRNA molecule. Antisense structures are indicated by a "-" symbol, or by adding the letter "a" or "antisense" before the DNA or RNA (e.g., "aDNA" or "aRNA").

[0031] Base Pair (bp) : A partnership between adenine (A) and thymine (T) or cytosine (C) and guanine (G) in a double-stranded DNA molecule. In RNA, thymine is replaced by uracil (U). Generally, the partnership is achieved by hydrogen bonds. For example, the sense nucleotide sequence "5'-ATCGU-3'" can form a perfect base pair with its antisense sequence "5'-ACGAT-3'".

[0032] 5' end A terminus lacking the 5' nucleotide 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, and in which one or more phosphates may be present at the terminus.

[0033] 3' end : an end in which the 5' hydroxyl group of one nucleotide is linked by a phosphodiester bond to the 3' hydroxyl group of the next nucleotide, and in most cases a hydroxyl group is present at the end, with no nucleotide at the 3' position of consecutive nucleotides.

[0034] 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 are opposite each other (and, by necessity, so are the 3' ends).

[0035] 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.

[0036] keep A nucleotide sequence is said to be conserved with respect to a preselected (reference) sequence if it non-randomly hybridizes to the exact complement of the preselected sequence.

[0037] Homologous or homologous: A term indicating the similarity between a polynucleotide and a gene or mRNA sequence. A nucleic acid sequence can be, for example, partially or completely homologous to 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.

[0038] Complementary or Complementary or Complementary : A term used in reference to matching base pairs between two polynucleotides (e.g., sequences of mRNA and cDNA) related by the aforementioned "base pair (bp)" rules. For example, the sequence "5'-AGT-3'" is complementary to the sequence "5'-ACT-3'" as well as "5'-ACU-3'". Complementarity can occur 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 or partial complementarity occurs when only a portion of the nucleic acid bases match according to the base pairing rules. Complete or complete complementarity occurs when the bases are perfectly matched between the nucleic acid strands. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids. Percent complementarity or percent complementarity refers to the number of mismatched bases relative to the total bases of one strand of nucleic acid. Thus, 50% complementarity means that half the bases are mismatched and half are matched. Two nucleic acid strands can be complementary even if they differ in the number of bases. In this case, complementation occurs between bases on the short strand and the portion of the longer strand that corresponds to the bases on the paired strand.

[0039] Complementary base : The nucleotides that normally pair together when DNA or RNA adopts a double-stranded structure.

[0040] Complementary nucleotide sequence : A sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to the nucleotides on another single strand to result in specific hybridization between the two strands by hydrogen bonding.

[0041] Hybridization and Hybridization : The formation of a duplex between nucleotide sequences that have sufficient complementarity 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 by DNA polymerase to initiate DNA synthesis. There is a specific, i.e., nonrandom, interaction between two complementary polynucleotides that is competitively controlled.

[0042] Post-transcriptional gene silencing : A targeted gene knockout or knockdown effect at the level of mRNA degradation or translational repression, usually caused by foreign / viral DNA or RNA transgenes, or small inhibitory RNAs.

[0043] RNA interference (RNAi) : A post-transcriptional gene silencing mechanism in eukaryotes, 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, disrupting the expression of cellular genes that are fully or partially complementary to the small RNA.

[0044] MicroRNA (miRNA) miRNAs are single-stranded RNAs that can bind to target gene transcripts that are partially complementary to the miRNA. miRNAs are usually oligonucleotides about 17-27 in length and, depending on the complementarity between the miRNA and its target mRNA, can either directly degrade intracellular mRNA targets or inhibit protein translation of the target mRNA. Natural miRNAs are present in almost all eukaryotes, where they function as a defense against viral infections and allow for the control of gene expression during plant and animal development.

[0045] Precursor microRNA (Pre-miRNA): A hairpin-shaped single-stranded RNA containing a stem arm and a stem loop region that interacts with intracellular RNase III endoribonuclease to generate one or more microRNAs (miRNAs) that can silence target genes or genes complementary thereto. The stem arm of pre-miRNA can form a complete (100%) or partial (mismatch) hybrid duplex, while the stem loop connects one end of the stem arm duplex to form a circle or hairpin loop structure. However, in the present invention, the precursor of microRNA also includes pre-miRNA.

[0046] Small interfering RNA (siRNA) : A short double-stranded RNA with a size of approximately 18-27 perfectly base-paired ribonucleotide duplexes that can degrade target gene transcripts with near perfect complementarity.

[0047] Small or short hairpin RNA (shRNA) : A single-stranded RNA that contains a pair of partially or perfectly matching stem-arm nucleotide sequences and is 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 precursor microRNAs (pre-miRNAs).

[0048] vector: A recombinant nucleic acid composition, such as recombinant DNA (rDNA), that can migrate and exist in different genetic environments. Generally, another nucleic acid is operably linked thereto. A vector may be capable of autonomous replication in a cell, where the segment attached to the vector is 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." Particularly important vectors allow for the cloning of cDNA from mRNA generated using reverse transcriptase. The vector may include components consisting of a viral promoter or a type II RNA polymerase (Pol-II or pol-2) promoter, or both, a Kozak consensus translation initiation site (such as 5'-GCCACC-3'), a polyadenylation signal (such as 5'-AUAAA-3' or 5'-AUUAAA-3'), multiple restriction / cloning sites, a pUC origin of replication, an SV40 early promoter for expressing an antibiotic resistance gene in a replicable gene at least in prokaryotic cells, an optional SV40 origin of replication in mammalian cells, and / or a tetracycline response element. The structure of the vector is 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 a combination thereof.

[0049] promoter A promoter is a nucleic acid that a polymerase molecule recognizes and possibly binds to, initiating RNA transcription. For purposes of the present invention, a promoter is any sequence capable of initiating synthesis of an RNA transcript by a desired polymerase, such as a known polymerase binding site, an enhancer, etc.

[0050] RNA processing: The cellular machinery responsible for RNA maturation, modification, and degradation. RNA splicing, intron excision, exosome digestion, nonsense-mediated decay (NMD), RNA editing, RNA processing, 5'-capping, 3'-poly(A) tailing, and combinations thereof.

[0051] Gene delivery A genetic engineering technique selected from the group consisting of: polysome transfection, liposome transfection, chemical (nanoparticle) transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene gun penetration, and combinations thereof.

[0052] Genetic Engineering A DNA recombination method selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene integration, transposon insertion, jumping gene integration, retroviral infection, and combinations thereof.

[0053] Transfected cells : A single or multiple eukaryotic cells selected from the group consisting of somatic cells, tissue cells, stem cells, germline cells, tumor cells, cancer cells, virus-infected cells, and combinations thereof, after artificially inserting at least a nucleic acid sequence or a protein / peptide molecule into the cells.

[0054] antibody : A peptide or protein molecule having a preselected conserved domain structure that encodes a receptor capable of binding to a preselected ligand.

[0055] Pharmaceutical 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, and high yield production of proteins / peptides / antibodies, drug components, pharmaceuticals, vaccines and / or food products, and combinations thereof.

[0056] B. Composition and Uses A novel mixed composition of at least a promoter-bound RNA / mRNA / samRNA-encoding DNA (PLRcD) template and at least a DNA-dependent RNA polymerase (DdRP) mRNA, wherein the PLRcD template contains an internal ribosome entry site (IRES)-binding Kozak motif (IRES-kozak) located between at least the promoter and the RNA / mRNA / samRNA coding sequence.

[0057] The encoded RNA / mRNA / samRNA sequence may further comprise at least a polyA signal and / or a polyA tail. Also, the encoded RNA / mRNA / samRNA may be either an mRNA, a self-amplifying RNA / mRNA (saRNA / samRNA), or a non-coding siRNA, shRNA, lncRNA, and / or a microRNA (miRNA) / pre-miRNA, or a combination thereof, encoding a protein, peptide, and / or antibody. To increase structural stability and prevent intracellular immune responses, the uridine / uracil (U) components in the DdRP mRNA sequence may be fully or partially replaced with pseudouridine, methyluridine, methoxyuridine, or other related modified nucleotide analogs. To prevent unwanted interferon-related immune responses and intracellular Trex1-mediated DNA degradation, the 5' end of the designed PLRcD template is dephosphorylated, while 8-hydroxyguanine (8-OHG) and / or its derived analogs, such as 8-OHdG, are added to the 3' end. Meanwhile, the DdRP mRNA is capped at its 5' end with m7G(5')ppp(5')N (m7G) and / or its related 5' cap analogs (i.e., cap-0, cap-1 and / or cap-2) and at its 3' end with a 3'-cap modified G / U / A molecule such as a cap-like modified nucleotide analog (e.g., 8-oxo-G, 8-oxo-A or 5' phosphorothioate-U). Furthermore, the resulting RNA / mRNA / samRNA product may also contain a 5' cap molecule such as m7G and / or its related cap analogs, preferably added and / or modified by vaccinia capping enzyme, coronavirus NSP12, NSP9 / 14, and / or NSP10 / 16, or a combination thereof. Moreover, the mixed composition of the designed PLRcD template and DdRP mRNA preferably further comprises coronavirus NSP7 and / or NSP13 helicase mRNA to enhance RNA / mRNA / samRNA amplification by ICIVT.In summary, the mixture composition of the designed PLRcD template and DdRP mRNA further comprises the mRNA of NSP7, NSP13, NSP12, NSP9 / 14, and / or NSP10 / 16, or a combination thereof.

[0058] For intracellular delivery, the mixed composition of the designed PLRcD template and DdRP mRNA is further mixed, bound, encapsulated, or formulated with at least one reagent selected from, but not limited to, liposomes, nanoparticles, liposomal nanoparticles (LNPs), exosomes, sugar / glucosamine / galactosamine-based binding molecules, infusion / transfusion agents, triglycylglycerol (TGG)-derived molecules, gene gun materials, electroporation agents, and / or transposons / retrotransposons, or combinations thereof.

[0059] With particular reference to the drawings, which are given for purposes of illustration only and not limitation, the following is shown: [Brief description of the drawings]

[0060] [Figure 1] FIG. 1 shows the step-by-step procedure of Lin's conventional PCR-IVT methodology. For RNA generation, some or all of the steps of this PCR-IVT protocol can be employed for single-cycle or multi-cycle amplification of desired RNA / mRNA sequences. [Diagram 2] 1 shows the structure of the designed promoter-linked RNA / mRNA-encoding DNA (PLRcD) template and the DdRP mRNA. To increase the stability of the DdRP mRNA, all or part of the uridine / uracil (U) components of the DdRP mRNA sequence can be replaced by pseudouridine, methyluridine, methoxyuridine, and / or other related or similarly modified nucleotide analogs. The DdRP mRNA can also be a self-amplifying RNA / mRNA (saRNA / samRNA). [Diagram 3]Figure 1 shows the results of non-denaturing agarose gel electrophoresis of an IVT-like reaction using a mixed composition of a designed PLRcD template (e.g., PLRcD encoding eGFP mRNA) and a T7 DdRP. Notably, the expression level of the encoded mRNA (e.g., eGFP mRNA) is controlled by the co-transfected amount of DdRP used. [Figure 4] FIG. 13 shows the results of microscopy of eGFP protein expression induced by LNP-mediated co-transfection of a mixed composition of the designed PLRcD template (from FIG. 3) and T7 DdRP mRNA into target cells. [Diagram 5] FIG. 13 shows another microscopy result of eGFP protein expression induced by electroporation of a mixed composition of another designed PLRcD template (modified from a commercially available T7 promoter-driven IRES-eGFP plasmid vector) and T7 DdRP into target cells. EXAMPLES

[0061] 1. Human Cell Isolation and Culture Human cancer and normal cell lines MCF7, HepG2, A549, and BEAS-2B were obtained from the American Type Culture Collection (ATCC, Rockville, MD) or the applicant's collaborators and maintained according to the individual manufacturers' recommendations. For intracellular transfection, 0.5–500 μg of the mixed composition of PLRcD template and DdRP mRNA is mixed with 0.5 mL of fresh cell culture medium supplemented with 1–50 μl of In-VivoJetPEI transfection reagent, respectively. After 1–30 min of incubation, the mixture is added to cell cultures containing cultured cells at 50%–60% confluency. The cell culture medium is re-flushed every 12–48 h depending on the type of cells transfected. This intracellular transfection procedure can be performed repeatedly to increase the transfection efficiency. The transfection results are shown in Figure 4.

[0062] 2. In Vivo Delivery / Transfection The composition of the designed PLRcD template and DdRP mRNA mixture (weight ratios ranging from approximately 200:1 to 1:200) is mixed with an appropriate amount of In-VivoJetPEI agent or other similar LNP-based delivery / transfection reagent and injected into an animal's vein or muscle according to the purpose of the relevant application, following the manufacturer's protocol. The delivery / transfection reagent is used to mix, bind, encapsulate and / or formulate the designed mixed composition. This not only protects the DNA / mRNA components from degradation, but also facilitates the delivery / transfection of the mixed composition into specific target cells of interest in vitro, ex vivo and in vivo.

[0063] 3. Preparation of PLRcD Template and DdRP mRNA Reverse transcription (RT) of the RNA / mRNA of interest is performed by adding approximately 0.01ng–10 micrograms (μg) of isolated RNA / mRNA to 20–50 μL of RT reaction according to the manufacturer's protocol (SuperScript III cDNA RT Kit, ThermoFisher Scientific, MA, USA). Depending on the amount of RNA / mRNA, the RT reaction mixture further contains approximately 0.01–20 nanomoles of RT primer, 0.1–10 mM each of deoxyribonucleoside triphosphate molecules (dNTPs, dATP, dTTP, dGTP, and dCTP), and reverse transcriptase in 1xRT buffer. Then, depending on the length and structural complexity of the RNA / mRNA sequence of interest, the RT reaction is incubated at 37–65 °C for 1–3 h to form complementary DNA (cDNA) of the desired RNA / mRNA sequence, which is used for the next step of PCR. For demonstration purposes, but not limited to this design, RT primer design uses 5'-CAGTTCCAATTGTGAAGATTCTC-3' (SEQ ID NO: 16) for RT of the RdRp mRNA sequence of interest, and another 5'-CTTGATGACGTTCTCAGTGC-3' (SEQ ID NO: 17) for RT of a green fluorescent protein (GFP)-encoding mRNA (e.g., eGFP-encoding mRNA) containing another microRNA (miRNA) of interest.

[0064] Next, perform polymerase chain reaction (PCR) by adding approximately 0.01 pg–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. Then, depending on the structure and length of the desired cDNA sequence, incubate the PCR mixture for 20–30 cycles of denaturation at 94 °C for 30 s–1 min, annealing at 50–58 °C for 30 s–1 min, followed by extension at 72 °C for 1–3 min, respectively. For demonstration purposes, but not limited to this example, two PCR primer pairs are used to amplify two COVID-19 virus-related PLRcD templates, including the pair 5'-GATATCTAATACGACTCACTATAGGGAGAGGTGCCACCATGGTACTTGGTAGTT-3' (SEQ ID NO:18) and SEQ ID NO:16 (for amplification of coronavirus RdRp / helicase / S protein coding RNA of approximately 12.5 k nucleotides (nucleotides, nt)) and another pair 5'-GATATCTAAT ACGACTCACT ATAGGGAGAC TAGTGGCCAC CATGTTCTTG TTAACAACT-3' (SEQ ID NO:19) and SEQ ID NO:16 (for amplification of coding mRNA encoding coronavirus S protein of approximately 2.8 k nt). Additionally, Applicants use another distinct PCR primer pair to amplify a GFP-encoding PLRcD template (e.g., eGFP-encoding mRNA) in which a miRNA is included, including 5'-primer 5'-GATATCTAATACGACTCACTATAGGGAGAGGTATGGTACTTGGTAGTT-3' (SEQ ID NO: 20) and SEQ ID NO: 17. In principle, the 5'-forward primer encodes at least a conserved promoter sequence of the IVT, including but not limited to T7, T3, and / or SP6 promoter sequences, in particular 5'-TCTAATACGACTCACTATAGGGAGA-3' (SEQ ID NO: 21).For RNA (e.g., DdRP mRNA) generation, since the resulting PLRcD template has a promoter built into it, prepare a new IVT reaction using 0.01 ng–10 µg of PCR product, 0.1–10 U of helicase (e.g., either coronavirus NSP7 and / or NSP13 proteins), NTPs, and a mixture of RNA polymerase (i.e., T7, T3, and / or SP6) dissolved in 1x transcription buffer. Then, incubate the IVT reaction at 37 °C for 1–6 h, depending on the stability and activity of the RNA polymerase used. The resulting mRNA (e.g., DdRP mRNA) product is preferably further capped by using either vaccine capping enzymes, coronavirus NSP9 / 14, and / or NSP10 / 16 proteins, or a combination thereof. Preferably, NSP7, NSP13, NSP9 / 14 and / or NSP10 / 16 mRNA, or a combination thereof, may also be used in a mixed composition with the designed PLRcD template and DdRP mRNA for intracellular co-transfection and amplification of the 5'-capped mRNA in the transfected cells.

[0065] 4. 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 using 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 probe is further purified by high-performance liquid chromatography (HPLC) and probed with dye-labeled nucleotide analogs or [ 32and tail-labeling with terminal transferase (20 units) in the presence of [ P]-dATP (>3000 Ci / mM, Amersham International, Arlington Heights, IL) for 20 min.

[0066] 5. Protein Extraction and Western Blot Analysis cells (10 6 ) are lysed in 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 at 4 °C for 20 min, and the supernatant is collected. Protein concentration is measured using the improved SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, CA). 30 μg 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 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 in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 h at room temperature. Primary antibodies are then added to the reagent, mixed, and incubated at 4 °C. After overnight incubation, the membrane is rinsed three times with TBS-T and exposed to a goat anti-mouse IgG-conjugated secondary antibody against Alexa Fluor 680-reactive dye (1:2,000; Invitrogen-Molecular Probes) for 1 h at room temperature. After three TBS-T rinses, fluorescent scanning and image analysis of the immunoblots are performed using a Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor).

[0067] 6. Immunostaining assay Cell / tissue samples are first fixed in 100% methanol for 30 min at 4 °C, then in 4% paraformaldehyde (1xPBS, pH 7.4 solution) for 10 min at 20 °C. Afterwards, samples are further incubated in 1xPBS containing 0.1%-0.25% Triton X-100 for 10 min, followed by washing three times for 5 min with 1xPBS. For immunostaining, corresponding primary antibodies are 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 Fluorescence 80i Microscope Quantitative System with Metamorph Imaging Program (Nikon).

[0068] 7.Statistical analysis All data are presented as mean and standard deviation (SD). The mean value of each test group is calculated by AVERAGE in Microsoft Excel. SD is performed by STDEV. Statistical analysis of the data is performed by one-way ANOVA. Tukey and Dunnett's t post-hoc test is used to determine the significance of data differences in each group. p<0.05 is considered significant (SPSS v12.0, Claritas Inc).

[0069] References: 1. Shi-Lung Lin U.S. Pat. No. 7,662,791 to et al. 2. Shi-Lung Lin U.S. Pat. No. 8,080,652 to et al. 3. Ying SY and Shi-Lung Lin U.S. Patent No. 8,372,969 to 4. Shi-Lung Lin and Ying S.Y., U.S. Pat. No. 8,609,831. 5. Shi-Lung Lin U.S. Pat. No. 9,637,747 to et al. 6. Shi-Lung Lin U.S. Pat. No. 9,783,811 to et al. 7. Shi-Lung Lin No. 60 / 222,479, filed on Oct. 23, 2003. 8. Shi-Lung Lin No. 60 / 290,902, filed on Oct. 13, 2002. 9. WO 2002 / 092774; Shi-Lung Lin and Ji H; Replicase cycling reaction amplification 10. Shi-Lung Lin ; Peptide library construction from RNA-PCR-derived RNAs. Methods Mol Biol. 221:289-293,2003. 11. Shi-Lung Lin and Ji H; cDNA library construction using in-vitro transcriptional amplification. Methods Mol Biol. 221:93-101,2003. 12. McDowell et al.; Determination of intrinsic transcription termination efficiency by RNA polymerase elongation rate. Science 266:822-825,1994. 13. Ghaderi et al.;Construction of an eGFP expression plasmid under control of T7 promoter and IRES sequence for assay of T7 RNA polymerase activity in mammalian cell lines. Iran J. Cancer Prev. 7:137-141,2014.

Claims

1. A novel mixture composition comprising at least a promoter-bound RNA-encoding DNA (PLRcD) template and at least an mRNA of a DNA-dependent RNA polymerase, characterized in that the PLRcD template comprises an internal ribosome entry site-binding Kozak motif linked between the promoter and the encoded RNA sequence.

2. 2. The composition of claim 1, further comprising the mRNA of the proteins NSP7, NSP12, NSP13, NSP9 / 14 and / or NSP10 / 16.

3. The composition of claim 1, wherein the RNA encoded by the PLRcD template is a non-coding RNA.

4. The composition of claim 1, wherein the RNA encoded by the PLRcD template is an mRNA encoding a protein / peptide or an antibody.

5. The composition of claim 1, wherein the RNA encoded by the PLRcD template is a self-amplifying RNA / mRNA.

6. The composition of claim 1, wherein the RNA encoded by the PLRcD template further comprises at least a polyA signal or a polyA tail.

7. The composition of claim 1, wherein the RNA encoded by the PLRcD template is a pharmaceutical compound or composition.

8. The composition of claim 1, wherein the 5' end of the promoter-bound RNA-encoding DNA (PLRcD) template is dephosphorylated.

9. The composition of claim 1, characterized in that the promoter-bound RNA-encoding DNA template is tagged at its 3'-end with 8-hydroxyguanine or a derived analog thereof, in particular 8-hydroxy-2-deoxyguanosine.

10. 2. The composition of claim 1, wherein the promoter of the PLRcD template is a bacteriophage RNA promoter, including T7, T3, and SP6 promoters.

11. 2. The composition of claim 1, wherein the DNA-dependent RNA polymerase (DdRP) mRNA is a bacteriophage RNA polymerase mRNA, including T7, T3, and SP6 RNA polymerase.

12. The composition of claim 1, characterized in that the 5' end of the mRNA of the DNA-dependent RNA polymerase (DdRP) is capped with a 5' cap molecule.

13. The composition of claim 1, characterized in that the 3' end of the mRNA of the DNA-dependent RNA polymerase (DdRP) is further tailed with a cap-like modified nucleotide analogue.

14. 2. The composition of claim 1, wherein the mRNA of the DNA-dependent RNA polymerase (DdRP) further contains modified nucleotide analogues.

15. The composition of claim 1, wherein the U (uridine / uracil) components of the DdRP mRNA are completely or partially replaced by pseudouridine, methyluridine, methoxyuridine, and / or other similar modified nucleotide analogs, or combinations thereof.

16. 2. The composition of claim 1, wherein the promoter-linked RNA-encoding DNA (PLRcD) template is generated using polymerase chain reaction (PCR).

17. 2. The composition of claim 1, wherein the DNA-dependent RNA polymerase (DdRP) mRNA is generated using polymerase chain reaction and in vitro transcription (PCR-IVT).

18. The composition described in claim 1, characterized in that the mixed composition containing at least a PLRcD template and at least a DdRP mRNA is further formulated with at least a delivery agent to promote intracellular transfection in vitro, ex vivo, and in vivo.

19. The composition of claim 18, wherein the delivery agent further comprises a liposome, a nanoparticle, a liposomal nanoparticle, an exosome, a binding molecule, an infusion / transfusion agent, a triglycylglycerin-derived molecule, an electroporation agent, a transposon / retrotransposon, or a combination thereof.

20. The composition of claim 1, characterized in that the internal ribosome entry site-binding Kozak motif (IRES-kozak) comprises at least SEQ ID NO:1 or SEQ ID NO:

2.

21. The composition of claim 1, wherein the internal ribosome entry site-binding Kozak motif comprises at least an internal ribosome entry site sequence at the 5' end of the Kozak motif.

22. The composition of claim 21, wherein the internal ribosome entry site is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:

13.

23. The composition of claim 1, wherein the internal ribosome entry site-binding Kozak motif is engineered into a PLRcD template using PCR.

24. The composition of claim 1, wherein the PLRcD template is a pharmaceutical compound or composition.

25. The composition of claim 1, wherein the DdRP mRNA is a self-amplifying RNA / mRNA.

26. The composition of claim 1, wherein the DdRP mRNA is a pharmaceutical compound or composition.

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