WO2022260718A1 - Nouvelle réaction de cyclage de réplicase (rcr) - Google Patents

Nouvelle réaction de cyclage de réplicase (rcr) Download PDF

Info

Publication number
WO2022260718A1
WO2022260718A1 PCT/US2022/013350 US2022013350W WO2022260718A1 WO 2022260718 A1 WO2022260718 A1 WO 2022260718A1 US 2022013350 W US2022013350 W US 2022013350W WO 2022260718 A1 WO2022260718 A1 WO 2022260718A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
rdrp
seq
mrna
sequence
Prior art date
Application number
PCT/US2022/013350
Other languages
English (en)
Inventor
Shi-Lung Lin
Sam Lin
Chun-Hung Lin
Original Assignee
Lin Shi Lung
Sam Lin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US17/489,357 external-priority patent/US20220396798A1/en
Application filed by Lin Shi Lung, Sam Lin filed Critical Lin Shi Lung
Priority to JP2023575381A priority Critical patent/JP2024523006A/ja
Publication of WO2022260718A1 publication Critical patent/WO2022260718A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07048—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention also claims priority to U.S. Provisional Patent Applications No. 63/210,988 filed on June 15, 2021, No. 63/212,657 filed on June 19, 2021, and No. 63/222,398 filed on July 15, 2021, all of which are entitled “Novel mRNA Composition and Production Method for Use in Anti- Viral and Anti-Cancer Vaccines”.
  • the present invention further claims priority to U.S. Provisional Patent Applications No. 63/270,034 filed on October 20, 2021, and No. 63/280,226 filed on November 17, 2021, both of which are entitled “Novel RNA Composition and Production Method for Use in iPS Cell Generation”.
  • the present application is a continuation-in-part application of the U.S. Patent Application No. 17/489,357 filed on September 29, 2021, which is entitled “Novel mRNA Composition and
  • This invention generally relates to a novel RNA/mRNA production and amplification method using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes as well as the associated mRNAs thereof.
  • the present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least an RdRp-binding site in the 5’- or 3’ -end, or both.
  • the RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but also for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition.
  • the present invention is a novel RNA replicase-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR).
  • RCR Replicase Cycling Reaction
  • the RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA- dependent RNA polymerases (RdRp), particularly coronaviral and hepatitis C viral (HCV) RdRp enzymes.
  • PCR Prior polymerase chain reaction
  • RCR RNA replicase-mediated cycling reaction
  • RdRp RNA-dependent RNA polymerases
  • Lin et al. first reported RCR in year 2002 (W02002/092774 to Lin). Lin had found that using a special design of 5’ -cap-capture-molecule-linked primers can trigger some viral and/or bacteriophage repliase-mediated RNA amplification from single-stranded RNA templates. This RCR mechanism mimics some viral or bacteriophage replication/amplification mechanisms. However, the requirement of specific 5’ -cap-capture- molecule-linked primers limits its use because many RNA species do not carry 5’ -cap molecules. Also, the linked 5’ -cap-capture molecules contaminate the resulting RNA products.
  • the 19-nt 3’-CSE is too long and too structural to be placed into PCR primers. Also, because the 3’-CSE is a highly structured RNA sequence, it hinders RNA transcription (McDowell et al., Science 266:822-825, 1994) and thus can not be efficiently produced by traditional IVT methods. Moreover, most problematically, the 3’-CSE is specifically recognized by Alphavirus RdRp, which is however not commercially available and hence further hinders the development of its related technology.
  • the principle of the present invention is relied on the incorporation of at least a coronaviral and/or hepatitis C viral (HCV) replicase/RdRp-binding (recognition) site into the 5’- or 3’ -ends, or both, of desired RNA templates, leading to the cycling amplification of either the sense strands or antisense strands, or both, of the desired RNA sequences.
  • HCV hepatitis C viral
  • the defined replicase/RdRp-binding sites serve as a promoter and/or enhancer for replicase/RdRp activities. As shown in FIG.
  • the desired RNA sequences can be amplified from about 15 to over 1000 folds in each cycle of replicase/RdRp cycling reaction (RCR).
  • RCR replicase/RdRp cycling reaction
  • the sense-strand RNA sequences are served as templates for amplifying the antisense-strands of the sense-strand RNAs, while the antisense-strand RNA sequences are in turns served as templates for amplifying the sense- strand RNAs.
  • the desired strand(s) of RNA can be obtained in a relatively high purity ratio (maximally 14/15 to >999/1000 purity), depending on the stop point of RCR for the sense-strand or antisense-strand RNAs, or both.
  • the desired RNA sequences and templates in RCR can be more than one kind and the resulting RNA products can be in either single- or double-stranded conformation.
  • RT-PCR reverse transcription-polymerase chain reaction
  • cDNA complementary DNAs
  • RCR-ready PCR primers synthetically embedded in each of the PCR primers
  • the resulting cDNAs can be cloned into a plasmid or viral vector for further IVT reaction and/or storage preservation. Then, an IVT reaction is performed to generate desired RCR-ready RNA templates from the cDNAs. After that, the resulting RCR-ready RNA templates can be used in RCR to repeatedly amplify and produce the desired RNA sequences.
  • the replicase/RdRp-binding site-incorporated cDNAs are herein also preferred to be served as a starting material for amplifying the desired RNA sequences in a combined IVT -RCR reaction.
  • the present inventors identified several conserved RdRp-binding sites, including 5’- and 3’-end RdRp-binding sites, respectively.
  • the 5’-end RdRp binding site contains at least a sequence of either 5 , -AU(G/C)(U/-)G(A/U)-3’ ⁇ i.e. 5’-AUSUGW-3’; SEQ.ID.NO.1) or 5’-U(C/-)(U/A)C(U/C)(U/A)A-3’ ⁇ i.e. 5’-UCWCYWA-3’; SEQ.ID.NO.2), or both.
  • the 5’-end RdRp binding site is selected from a sequence containing 5’- AUCUGU-3’ (SEQ.ID.NO.3), 5’-UCUCUAA-3’ (SEQ.ID.NO.4), 5’-UCUCCUA-3’
  • the 3’ -end RdRp binding site contains at least a sequence of either 5’-(U/A)C(A/- )(C/G)AU-3’ ⁇ i.e. 5’-WCASAU-3’; SEQ.ID.NO.7) or 5 , -U(A/U)(A/G)G(A/U)(G/-)A-3’ ⁇ i.e.
  • the 3’-end RdRp binding site is selected from a sequence containing 5’-ACAGAU-3’ (SEQ.ID.NO.9), 5’-UUAGAGA-3’ (SEQ.ID.NO.10), 5 ’ -UAGGAGA-3 ’ (SEQ.ID.NO.il), and/or 5’-UUGAA-3’
  • the uridine/uracil (U) contents of these RdRp-binding sites can be replaced by thymidine (dT) and/or deoxyuridine (dU) in the primers.
  • the uridine/uracil (U) contents of these RdRp-binding sites can be further replaced by pseudouridine or other modified nucleotide analogs during IVT and/or RCR.
  • the currently available coronaviral RdRp enzymes can thus be used to efficiently transcribe and amplify either the sense or antisense strands, or both, of desired RNA sequences in vitro, ex vivo as well as in vivo.
  • these newly identified 5’- and 3’ -end RdRp-binding sites provide different RNA amplification rates.
  • the amplification rate of SEQ.ID.NO.l and SEQ.ID.NO.7 is estimated to be ranged from about 25 to 1400 folds per RCR cycle, while that of SEQ.ID.NO.2 and SEQ.ID.NO.8 is ranged from about 10 to 900 folds per RCR cycle, depending on the length and structual complexity of the desired RNA sequences.
  • the desired RNA sequence (i.e, mRNA and/or microRNA, or any other kind of RNA species) contains at least an RdRp-binding site in both of its 5’- and 3’ -end regions. Since both ends of the desired RNA carry at least an RdRp- binding site for RNA amplification with replicase/RdRp activities, the sense-strand RNA sequences can be used to amplify its complementary antisense RNAs (cRNA or aRNA), while the antisense-strand RNA sequences can be used to amplify the sense RNAs as well, so as to form an amplification cycle of both of the sense- and antisense-strand RNAs and thus resulting in a maximal amplification rate of the desired RNAs.
  • cRNA or aRNA complementary antisense RNAs
  • the desired RNAs so obtained can be in either single-stranded or double-stranded conformation, depending on the stop point of RCR.
  • the resulting sense- and antisense-strand RNAs may further form double-stranded RNAs, facilitating the generation of siRNAs, shRNAs, miRNAs, and/or piRNAs of the desired RNA sequences.
  • the desired RNA sequence contains at least an RdRp-binding site in its either 5’-end or 3’-end region.
  • this approach is useful for generating and amplifying either the mRNA or the antisense RNA (aRNA) of a specific functional protein, viral antigen or antibody, facilitating the development of mRNA vaccines and/or RNA/antibody-based medicines.
  • the mRNA vaccines and RNA/antibody-based medicines so obtained may help to treat a variety of human diseases, including but not limited to Alzheimer’s disease, Parkinson’s disease, motor neuron disease, stroke, diabetes, myocardial infraction, hemophilia, anemia, leukemia, and many kinds of cancers as well as many kinds of viral and bacterial infections.
  • human diseases including but not limited to Alzheimer’s disease, Parkinson’s disease, motor neuron disease, stroke, diabetes, myocardial infraction, hemophilia, anemia, leukemia, and many kinds of cancers as well as many kinds of viral and bacterial infections.
  • our new RCR methodology can be used to produce and amplify all varieties of RNA species carrying at least an RdRp binding site, particularly viral antigen mRNAs and/or known functional RNAs/mRNAs, which are useful for developing anti-viral and/or anti-disease vaccines as well as medicines, and likely many more.
  • RdRp binding site particularly viral antigen mRNAs and/or known functional RNAs/mRNAs, which are useful for developing anti-viral and/or anti-disease vaccines as well as medicines, and likely many more.
  • the RCR-amplified mRNAs can be further used in an in-vitro translation system for producing the encoded proteins, peptides and/or antibodies of interest.
  • our priority method adopts a novel IVT system with a mixture of RNA polymerase and helicase activities.
  • the additional helicase activity in IVT markedly reduces the secondary structures of both DNA/RNA templates and the resulting RNA products for far more efficiently producing highly structured RNAs.
  • an improved buffer system is also used to maintain and enhance the efficiency of mixed RNA polymerase/replicase and helicase activities in IVT (and RCR as well).
  • helicase may be involved in prokaryotic transcription termination, our studies however demonstrate a totally different functionality of helicase in RNA amplification during IVT.
  • the RCR- ready cDNA/RNA template(s) and RdRp mRNA can be mixed, conjugated, encapsulated and/or formulated with at least a delivery/transfection agent selected from, but not limited to, glycylglycerin-derived chemicals, liposomes, nanoparticles, liposomal nanoparticles (LNP), conjugating molecules, infusion/transfusion chemicals, gene gun materials, electroporation agents, transposons/retrotransposons, and a combination thereof.
  • a delivery/transfection agent selected from, but not limited to, glycylglycerin-derived chemicals, liposomes, nanoparticles, liposomal nanoparticles (LNP), conjugating molecules, infusion/transfusion chemicals, gene gun materials, electroporation agents, transposons/retrotransposons, and a combination thereof.
  • the advantages of using RCR-ready cDNA/RNA templates for RNA/mRNA production and amplification include (1) high RNA yield rate, (2) high RNA purity, (3) easy preparation in that all reaction materials can be made into a biochemical enzyme kit for performing RCR and/or combined IVT-RCR reactions, (4) simple reaction procedure compatible with other RT-PCR and IVT reactions, (5) simple equipment requirement which can be easily accomplished using a PCR machine or a temperature-controlled incubator, and (6) a variety of potential applications.
  • the RCR-ready cDNA/RNA templates of the present invention are very useful for producing and amplifying a variety of desired RNA/mRNA sequences, which can then be used in all sorts of pharmaceutical and therapeutic applications, including but not limited to the development of mRNA vaccines and RNA/microRNA-associated medicines as well as protein/peptide/antibody generation.
  • Nucleic Acid a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), either single or double stranded.
  • Nucleotide a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (G carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • a nucleoside containing at least one phosphate group bonded to the 3' or 5' position of the pentose is a nucleotide.
  • DNA and RNA are consisted of different types of nucleotide units called deoxyribonucleotide and ribonucleotide, respectively.
  • Deoxyribonucleoside Triphosphates the building block molecules for DNA synthesis, including dATP, dGTP, dCTP, and dTTP and sometimes may further containing some modified deoxyribonucleotide analogs.
  • Ribonucleoside Triphosphates the building block molecules for RNA synthesis, including ATP, GTP, CTP, and UTP and sometimes may further containing pseudouridine and some other modified ribonucleotide analogs.
  • Nucleotide Analog a purine or pyrimidine nucleotide that differs structurally 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.
  • Oligonucleotide a molecule comprised of two or more monomeric units of DNA and/or RNA, preferably more than three, and usually more than ten. An oligonucleotide longer than 13 nucleotide monomers is also called polynucleotiude. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.
  • nucleic Acid Composition refers to an oligonucleotide or polynucleotide such as a DNA or RNA sequence, or a mixed DNA/RNA sequence, in either a single-stranded or a double-stranded molecular structure.
  • Gene a nucleic acid composition whose oligonucleotide or polynucleotide sequence codes for an RNA and/or a polypeptide (protein).
  • a gene can be either RNA or DNA.
  • a gene may encode a non-coding RNA, such as small hairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA, and their RNA precursors as well as derivatives.
  • a gene may encode a protein-coding RNA essential for protein/peptide synthesis, such as messenger RNA (mRNA) and its RNA precursors as well as derivatives.
  • mRNA messenger RNA
  • a gene may encode a protein-coding RNA that also contains at least a microRNA or shRNA sequence.
  • RNA Transcript an RNA sequence that is directly transcribed from a gene without any RNA processing or modification.
  • Precursor messenger RNA primary RNA transcripts of a protein-coding gene, which are produced by eukaryotic type-II RNA polymerase (Ro ⁇ -P) machineries in eukaryotes through an intracellular mechanism termed transcription.
  • a pre-mRNA sequence contains a 5’ -untranslated region (UTR), a 3’-UTR, exons and introns.
  • Intron a part or parts of a gene transcript sequence encoding non-protein-reading frames, such as in-frame intron, 5’ -UTR and 3’ -UTR.
  • cDNA protein-reading frames
  • RNA messenger RNA
  • mRNA messenger RNA
  • RNA splicing machineries e.g. spliceosomes
  • the peptides/proteins encoded by mRNAs include, but not limited, enzymes, growth factors, insulin, antibodies and their analogs/homologs as well as derivatives.
  • cDNA Complementary DNA
  • Sense a nucleic acid molecule in the same sequence order and composition as the homologous mRNA. The sense conformation is indicated with a “+”, “s” or “sense” symbol.
  • Antisense a nucleic acid molecule complementary to the respective mRNA molecule.
  • the antisense conformation is indicated as a symbol or with an "a” or “antisense” in front of the DNA or RNA, e.g., "aDNA” or "aRNA”.
  • Base Pair a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
  • RNA uracil
  • U uracil
  • the partnership is achieved through hydrogen bonding.
  • a sense nucleotide sequence "5’-A-T-C-G-U-3’" can form complete base pairing with its antisense sequence "5’-A-C-G-A-T-3’”.
  • 5 ’-end a terminus lacking a nucleotide at the 5’ position of successive nucleotides in which the 5’-hydroxyl group of one nucleotide is joined to the 3’-hydroyl group of the next nucleotide by a phosphodiester linkage.
  • Other groups, such as one or more phosphates, may be present on the terminus.
  • 3 ’-end a terminus lacking a nucleotide at the 3’ position of successive nucleotides in which the 5’-hydroxyl group of one nucleotide is joined to the 3’-hydroyl group of the next nucleotide by a phosphodiester linkage.
  • Other groups, most often a hydroxyl group, may be present on the terminus.
  • a 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 at opposite ends of the duplex (and, by necessity, so then are the 3' ends).
  • Nucleic Acid Template a double-stranded DNA molecule, double-stranded RNA molecule, hybrid molecules such as DNA-RNA or RNA-DNA hybrid, or single-stranded DNA or RNA molecule.
  • nucleotide sequence is conserved with respect to a pre-selected (referenced) sequence if it non-randomly hybridizes to an exact complement of the pre selected sequence.
  • Homologous or Homology a term indicating the similarity between a polynucleotide and a gene or mRNA sequence.
  • a nucleic acid sequence may be partially or completely homologous to a particular gene or mRNA sequence, for example.
  • Homology may be expressed as a percentage determined by the number of similar nucleotides over the total number of nucleotides.
  • Complementary or Complementarity or Complementation a term used in reference to matched base pairing between two polynucleotides (i.e. sequences of an mRNA and a cDNA) related by the aforementioned “base pair (bp)” rules.
  • sequence “5’-A-G-T- 3’” is complementary to not only the sequence “5’-A-C-T-3’” but also to "5’-A-C-U-3’”.
  • Complementation can be between two DNA strands, a DNA and an RNA strand, or between two RNA strands.
  • Complementarity may be "partial” or “complete” or “total”. Partial complementarity or complementation occurs when only some of the nucleic acid bases are matched according to the base pairing rules. Complete or total complementarity or complementation occurs when the bases are completely or perfectly matched between the nucleic acid strands.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as in detection methods that depend on binding between nucleic acids.
  • Percent complementarity or complementation refers to the number of mismatch bases over the total bases in one strand of the nucleic acid. Thus, a 50% complementation means that half of the bases were mismatched and half were matched. Two strands of nucleic acid can be complementary even though the two strands differ in the number of bases. In this situation, the complementation occurs between the portion of the longer strand corresponding to the bases on that strand that pair with the bases on the shorter strand.
  • Complementary Bases nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration.
  • Complementary Nucleotide Sequence a sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to that on another single strand to specifically hybridize between the two strands with consequent hydrogen bonding.
  • Hybridize and Hybridization the formation of duplexes between nucleotide sequences which are sufficiently complementary to form complexes via base pairing.
  • a primer or splice template
  • target template
  • Posttranscriptional Gene Silencing a targeted gene knockout or knockdown effect at the level of mRNA degradation or translational suppression, which is usually triggered by either foreign/viral DNA or RNA transgenes or small inhibitory RNAs.
  • RNA Interference a posttranscriptional gene silencing mechanism in eukaryotes, which can be triggered by small inhibitory RNA molecules such as microRNA (miRNA), small hairpin RNA (shRNA) and small interfering RNA (siRNA). These small RNA molecules usually function as gene silencers, interfering with expression of intracellular genes containing either completely or partially complementarity to the small RNAs.
  • miRNA microRNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • MicroRNA single-stranded RNAs capable of binding to targeted gene transcripts that have partial complementarity to the miRNA.
  • MiRNA is usually about 17-27 oligonucleotides in length and is able to either directly degrade its intracellular mRNA target(s) or suppress the protein translation of its targeted mRNA, depending on the complementarity between the miRNA and its target mRNA.
  • Natural miRNAs are found in almost all eukaryotes, functioning as a defense against viral infections and allowing regulation of gene expression during development of plants and animals.
  • Precursor MicroRNA hairpin-like single-stranded RNAs containing stem-arm and stem-loop regions for interacting with intracellular RNaselll endoribonucleases to produce one or multiple microRNAs (miRNAs) capable of silencing a targeted gene or genes complementary to the microRNA sequence(s).
  • the stem-arm of a pre-miRNA can form either a perfectly (100%) or a partially (mis-matched) hybrid duplexes, while the stem- loop connects one end of the stem-arm duplex to form a circle or hairpin-loop conformation.
  • precursor of microRNA may also includes pri-miRNA.
  • siRNA small interfering RNA: short double-stranded RNAs sized about 18-27 perfectly base-paired ribonucleotide duplexes and capable of degrading target gene transcripts with almost perfect complementarity.
  • Small or short hairpin RNA single-stranded RNAs that contain a pair of partially or completely matched stem-arm nucleotide sequences divided by an unmatched loop or bubble oligonucleotide to form a hairpin-like structure.
  • Many natural miRNAs are derived from small hairpin-like RNA precursors, namely precursor microRNA (pre-miRNA).
  • Vector a recombinant nucleic acid composition such as recombinant DNA (rDNA) capable of movement and residence in different genetic environments. Generally, another nucleic acid is operatively linked therein.
  • the vector can be capable of autonomous replication in a cell in which case the vector and the attached segment is 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 for one or more polypeptides and/or non-coding RNAs are referred to herein as “expression vectors” or “expression-competent vectors”.
  • a vector may contain components consisting of a viral or a type-II RNA polymerase (Pol-II or pol-2) promoter, or both, a Kozak consensus translation initiation site, polyadenylation signals, a plurality of restriction/cloning sites, a pUC origin of replication, a 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 structure of a vector can be a linear or circular form of single- or double- stranded DNA selected form the group consisting of plasmid, viral vector, transposon, retrotransposon, DNA transgene, jumping gene, and a combination thereof.
  • Promoter a nucleic acid to which a polymerase molecule recognizes, perhaps binds to, and initiates RNA transcription.
  • a promoter can be a known polymerase binding site, an enhancer and the like, any sequence that can initiate synthesis of RNA transcripts by a desired polymerase.
  • RNA Processing a cellular mechanism responsible for RNA maturation, modification and degradation, including RNA splicing, intron excision, exosome digestion, nonsense- mediated decay (NMD), RNA editing, RNA processing, 5’ -capping, 3’-poly(A) tailing, and a combination thereof.
  • NMD nonsense- mediated decay
  • Gene Delivery a genetic engineering method selected from the group consisting of polysomal transfection, liposomal transfection, chemical (nanoparticle) transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene-gun penetration, and a combination thereof.
  • Transfected Cell a single or a plurality of eukaryotic cells after being artificially inserted with at least a nucleic acid sequence or protien/peptide molecule into the cell(s), selected from the group consisting of a somatic cell, a tissue cell, a stem cell, a germ-line cell, a tumor cell, a cancer cell, a virus-infected cell, and a combination thereof.
  • Antibody a peptide or protein molecule having a pre-selected conserved domain structure coding for a receptor capable of binding a pre-selected ligand.
  • compositions useful for stem cell generation, drug/vaccine development, non-transgenic gene therapy, cancer therapy, disease treatment, wound healing, tissue/organ repair and regeneration, and high-yield production of proteins/peptides/antibodies, drug ingredients, medicines, vaccines and/or food supplies, and a combination thereof.
  • a novel RNA replicase-mediated RNA amplification method comprising: (a) providing at least an RNA sequence, wherein said RNA srquence contains at least a
  • RNA replicase wherein said RNA replicase is isolated or modified from the RNA-dependent RNA polymerases (RdRp) of coronavirus or hepatitis C virus (HCV); and (c) mixing the RNA sequence of (a) and the RNA replicase of (b) under a buffer condition, so as to elicit RNA replicase-mediated production and amplification of said RNA sequence, wherein said buffer condition contains ribonucleoside triphosphate molecules (rNTPs) required for RNA synthesis and is in a pH range from 6.0 to 8.0 as well as in a temperature range from 20°C to 45°C.
  • rNTPs ribonucleoside triphosphate molecules
  • the 5’ -end RdRp binding site contains at least a sequence of either 5’-AUSUGW-3’ (SEQ.ID.NO.l) or 5’-UCWCYWA-3’ (SEQ.ID.NO.2), or both.
  • the 5’ -end RdRp binding site is selected from an RNA sequence 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.
  • the 3’ -end RdRp binding site contains at least a sequence of either 5’-WCASAU-3’ (SEQ.ID.NO.7) or 5’-UWRGWR-3’ (SEQ.ID.NO.8), or both.
  • the 3’ -end RdRp binding site is selected from an RNA sequence containing 5’- ACAGAU-3’ (SEQ.ID.NO.9), 5’-UUAGAGA-3’ (SEQ.ID.NO.10), 5’-UAGGAGA-3’ (SEQ.ID.NO.il), and/or 5’-UUGAA-3’ (SEQ.ID.NO.12), or a combination thereof.
  • the uridine/uracil (U) contents of these RdRp-binding sites can be replaced by thymidine (dT) and/or deoxyuridine (dU) in the primers.
  • the uridine/uracil (U) contents of these RdRp- binding sites as well as the resulting RNA products can be replaced by pseudouridine or other modified nucleotide analogs.
  • FIG. 1 depicts the step-by-step procedure of the prior PCR-IVT methodology.
  • RNA production a part or whole procedure of this PCR-IVT method can be adopted for either single or multiple cycle amplification of desired RNA products.
  • FIG. 2 depicts the step-by-step procedure of the presently invented RCR methodology.
  • RCR-ready cDNA/RNA templates at least a coronaviral and/or HCV replicase/ RdRp-binding site is incorporated into the 5’- or 3’ -ends, or both, of the cDNAs of desired RNA sequences, using conventional RT-PCR methods. Then, a part or whole procedure of this novel RCR method is used to produce and amplify the desired RNA sequences from the RCR-ready cDNA/RNA templates after single or multiple cycle amplification. Alternatively, since IVT and RCR methods can be performed simultaneously under the same buffer condition, the RCR-ready cDNA/RNA templates can also be used as starting materials for amplifying the desired RNA sequences in a combined IVT -RCR reaction.
  • FIG. 3 depicts the designed structures of RCR-ready cDNA/RNA templates. It is noted that the RCR-ready cDNA templates are in double-stranded DNA conformation (useful for IVT and combined IVT -RCR reactions), while the RCR-ready RNA templates are in single-stranded RNA conformation (useful for RCR). For further enhancing the stability of RCR-ready RNA templates, the uridine/uracil (U) contents of the templates can be replaced by pseudouridine or other modified nucleotide analogs.
  • FIG. 4 shows the Northern blot analysis results of markedly increased expressions of miR-302 microRNAs (i.e. from top to bottom: b, c, d, a) and RdRp mRNA in transfected human cells after co-transfection with RCR-ready miR-302 precursor microRNA (pre-miR- 302) and viral RdRp mRNA templates (as shown in most right) compared to the result of cells transfected with only the pre-miR-302 template (in middle), demonstrating the evidence of RCR in cells.
  • FIG. 5 shows Northern blot analysis results of RCR-ready cDNA and RNA templates as well as the resulting amplified RNA products of interest (i.e. mRNA sequences of viral antigen proteins/peptides), demonstrating the evidence of RCR in vitro.
  • FIG. 6 shows the immunohistochemical staining of coronaviral (e.g. COVID-19) S 2 proteins produced in the mouse muscle cells in vivo after co-transfection with RCR-amplified S protein mRNA (from FIG. 5) and isolated RdRp mRNA (from FIG. 4), indicating that the present invention is useful for developing and manufacturing anti-viral mRNA vaccines.
  • coronaviral e.g. COVID-19
  • Starting tissue cells can be obtained from either enzymatically dissociated skin cells using Aasen’s protocol ⁇ Nat. Protocols 5, 371-382, 2010) or simply from the buffy coat fraction of heparin-treated peripheral blood cells.
  • the isolated tissue samples must be kept fresh and used immediately by mixing with 4 mg/mL collagenase I and 0.25% TrypLE for 15-45 min, depending on cell density, and rinsed by HBSS containing trypsin inhibitor two times and then transferred to a new sterilized microtube containing 0.3 mL of feeder-free SFM culture medium (IrvineScientific, CA).
  • cells were further dissociated by shaking in a microtube incubator for 1 min at 37°C and then transferred the whole 0.3 mL cell suspension to a 35-mm Matrigel-coated culture dish containing 1 mL of feeder-free SFM culture medium supplemented with formulated pre-miR-302+RdRp mRNA mixture, LIF, and bFGF/FGF2, or other optional defined factors.
  • concentrations of pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and other optional defined factors are ranged from 0.1 to 500 microgram (pg)/mL, respectively, in the cell culture medium.
  • the cell culture medium and all of the supplements must be refreshed every 2 ⁇ 3 days and the cells are passaged at about 50% ⁇ 60% confluence by exposing the cells to trypsin/EDTA for 1 min and then rinsing two times in HBSS containing trypsin inhibitor.
  • the cells were replated at 1:5-1:500 dilution in fresh feeder-free MSC Expansion SFM culture medium supplemented with formulated pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and/or other optional defined factors.
  • keratinocytes For culturing keratinocytes, cells are isolated from skin tissues and cultivated in EpiLife serum-free cell culture medium supplemented with human keratinocyte growth supplements (HKGS, Invitrogen, Carlsbad, CA) in the presence of proper antibiotics at 37°C under 5% CO2. Culture cells are passaged at 50%-60% confluency by exposing cells to trypsin/EDTA solution for 1 min and rinsing once with phenol red-free DMEM medium (Invitrogen), and the detached cells are replated at 1:10 dilution in fresh EpiLife medium with HKGS supplements.
  • HKGS human keratinocyte growth supplements
  • iPSCs Human cancer and normal cell lines A549, MCF7, PC3, HepG2, Colo-829 and BEAS-2B were obtained either from the American Type Culture Collection (ATCC, Rockville, MD) or our collaborators and then maintained according to manufacturer’s or provider’s suggestions. After reprogramming, the resulting iPS cells (iPSCs) were cultivated and maintained following either Lin’s feeder-free or Takahashi’s feeder-based iPSC culture protocols (Lin et ah, RNA 14:2115-2124, 2008; Lin et ah, Nucleic Acids Res. 39:1054-1065, 2011; Takahashi K and Yamanaka S, Cell 126:663- 676, 2006).
  • RNA/mRNA i.e. pre-miR-302 or coronaviral S protein mRNA
  • RdRp mRNA mixture ratio ranged from about 20:1 to 1:20
  • RNA/mRNA i.e. pre-miR-302 or coronaviral S protein mRNA
  • RdRp mRNA mixture ratio ranged from about 20:1 to 1:20
  • the mixture is then added into a cell culture containing 50%-60% confluency of the cultivated cells.
  • the medium is reflashed every 12 to 48 hours, depending on cell types. This transfection procedure may be performed repeatedly to increase transfection efficiency.
  • RT Reverse transcription
  • RNA/mRNA Reverse transcription
  • RT reaction mixture further contains about 0.01-20 nmole RT primer, a proper amount of deoxyribonucleoside triphosphate molecules (dNTPs) and reverse transcriptase in lx RT buffer.
  • dNTPs deoxyribonucleoside triphosphate molecules
  • RT reaction is incubated at 37 ⁇ 65°C for 1-3 hours (hr), depending on the length and structural complexity of the desired RNA/mRNA sequences, so as to make the complementary DNA (cDNA) templates thereof for the next step of PCR.
  • cDNA complementary DNA
  • PCR polymerase chain reaction
  • IVT-RCR template preparation we design and use a specific pair of RCR-ready PCR primers for incorporating the identified RdRp-binding sites into the PCR-derived RdRp cDNA templates, including SEQ.ID.NO.13 and 5 ’ -GATATCTAAT ACGACTCACT AT AGGGAGAG GTATGGTACT TGGTAGTT-3’ (SEQ.ID.NO.14). Later, a 5’-cap molecule may be further incorporated in the resulting mRNA products of IVT-RCR.
  • pre-miR-302 familial cluster pre-miR-302 familial cluster
  • pre-miR-302 familial cluster including 5’-GATATCTAAT ACGACTCACT AT AGGGAGAT CTGTGGGAAC TAGTTCAGGA AGGTAA-3’ (SEQ.ID.N0.15) and 5’- GTTCTCCTAA GCCTGTAGCC AAGAACTGC A CA-3’ (SEQ.ID.NO.16).
  • RNA promoters and RdRp-binding sites can be used, such as T7, T3 and/or SP6 promoter, and at least an RdRp binding site has been incorporated in the 5’- and/or 3’-end primers.
  • an IVT-RCR reaction can then be performed to amplify desired RNA/mRNA sequences from the cDNA templates.
  • the IVT- RCR reaction mixture contains 0.01 ng ⁇ 10 pg of the PCR-derived cDNA product, 0.1-50 U of isolated coronaviral RdRp/helicase (Abeam, MA, USA/Creative Enzymes, NY), a proper amount of ribonucleoside triphosphate molecules (rNTPs) and RNA polymerase (i.e.
  • the transcription buffer is commercially available and may be further adjusted according to the manufacturer’s suggestions.
  • the lx transcription buffer may further conatin 0.001-10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), and/or 3-(N-morpholino)propane sulfonic acid (MOPS), and/or a combination thereof.
  • betaine trimethylglycine, TMG
  • DMSO dimethylsulfoxide
  • MOPS 3-(N-morpholino)propane sulfonic acid
  • the IVT-RCR reaction is incubated at 30 ⁇ 40°C for 1-6 hr, depending on the stability and activity of the used RdRp and RNA polymerase enzymes.
  • the starting RCR mixture contains about 0.01 ng ⁇ 10 pg of the RCR-ready RNA/mRNA templates, about 0.1-50 U of isolated coronaviral RdRp/helicase, and a proper amount of rNTPs in lx transcription buffer.
  • RdRp/helicase is either an RdRp enzyme with an additional RNA unwinding activity or a mixture of RdRp and helicase.
  • the transcription buffer is commercially available in the market and may be further adjusted according to the manufacturer’s suggestions.
  • the lx transcription buffer may further conatin 0.001-10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), and/or 3-(N- morpholino)propane sulfonic acid (MOPS), and/or a combination thereof, which facilitates the denaturation of highly structured RNA/DNA sequences, such as hairpins and stem-loop structures.
  • betaine trimethylglycine, TMG
  • DMSO dimethylsulfoxide
  • MOPS 3-(N- morpholino)propane sulfonic acid
  • Desired RNAs (10 pg) are isolated with a mirY anaTM RNA isolation kit (Ambion, Austin, TX) or similar purification filter column, following the manufacturer’s protocol, and then further purified by using either 5%— 10% TBE-urea polyacrylamide or 1%— 3.5% low melting point agarose gel electrophoresis.
  • the gel -fractionated RNAs are electroblotted onto a nylon membrane. Detection of the RNA and its IVT template (the PCR-derived cDNA product) is performed with a labeled [LNAJ-DNA probe complementary to a target sequence of the desired RNA.
  • the probe is further purified by high-performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of either a dye-labeled nucleotide analog or [ 32 P]-dATP (> 3000 Ci/mM, Amersham International, Arlington Heights, IL).
  • Cells (10 6 ) are lysed with a CelLytic-M lysis/extraction reagent (Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF, following the manufacturer’s suggestion. Lysates are centrifuged at 12,000 rpm for 20 min at 4°C and the supernatant is recovered. Protein concentrations are measured using an improved SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, CA). Each 30 pg of cell lysate are added to SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, and boiled for 3 min before loading onto a 6-8% polyacylamide gel.
  • Proteins are resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane and incubated in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hr at room temperature. Then, a primary antibody is applied to the reagent and incubated the mixture at 4°C. After overnight incubation, the membrane is rinsed three times with TBS-T and then exposed to goat anti-mouse IgG conjugated secondary antibody to Alexa Fluor 680 reactive dye (1:2,000; Invitrogen-Molecular Probes), for 1 hr at the room temperature. After three additional TBS-T rinses, fluorescent scanning of the immunoblot and image analysis are conducted using Li-Cor Odyssey Infrared Imager and Odyssey Software v.lO (Li-Cor).
  • Li-Cor Odyssey Infrared Imager and Odyssey Software v.lO
  • the mixture of RCR-amplified RNA/mRNA and RdRp mRNA (ratio ranged from about 20:1 to 1:20) is mixed well with a proper amount of delivery agent, such as an In- VivoJetPEI transfection reagent or other similar LNP -based delivery/transfection agents, following the manufacturer’s protocol, and then injected into blood veins or muscles of an animal, depending the purpose of applications.
  • a proper amount of delivery agent such as an In- VivoJetPEI transfection reagent or other similar LNP -based delivery/transfection agents, following the manufacturer’s protocol, and then injected into blood veins or muscles of an animal, depending the purpose of applications.
  • the delivery/transfection agent is used for mixing, conjugating, encapsulating or formulating the amplified RNA/mRNA and RdRp mRNA mixture, so as to not only protect the RNA contents from degradation but also facilitate the delivery/transfection of the RNA/mRNA and RdRp mRNA mixture into specific target cells of interest in vitro, ex vivo and/or in vivo.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne de manière générale un nouveau procédé de production et d'amplification d'ARN/ARNm utilisant des enzymes ARN réplicase et/ou ARN polymérase ARN-dépendante (RdRp) virales ainsi que les ARNm associés. La présente invention peut être utilisée pour fabriquer et amplifier toutes les variétés de séquences d'ARN/ARNm portant au moins un site de liaison RdRp à l'extrémité 5' ou 3', ou les deux. L'ARN/ARNm ainsi obtenu est utile non seulement pour produire des vaccins à ARNm et/ou des médicaments à base d'ARN mais aussi pour générer les protéines, peptides et/ou anticorps associés à l'ARNm dans des conditions de traduction in-vitro ainsi que dans des cellules. Principalement, la présente invention est un nouveau procédé d'amplification d'ARN/ARNm médié par la réplicase, à savoir la réaction de cyclage de la réplicase (RCR). Les ARN réplicases impliquées dans la RCR comprennent, sans s'y limiter, les ARN polymérases ARN dépendantes (RdRp) virales et/ou bactériophages, en particulier les enzymes RdRp coronavirales et du virus de l'hépatite C (HCV).
PCT/US2022/013350 2021-06-12 2022-01-21 Nouvelle réaction de cyclage de réplicase (rcr) WO2022260718A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023575381A JP2024523006A (ja) 2021-06-12 2022-01-21 新規なレプリカーゼサイクリング反応（ｒｃｒ）

Applications Claiming Priority (14)

Application Number Priority Date Filing Date Title
US202163209969P 2021-06-12 2021-06-12
US63/209,969 2021-06-12
US202163210988P 2021-06-15 2021-06-15
US63/210,988 2021-06-15
US202163212657P 2021-06-19 2021-06-19
US63/212,657 2021-06-19
US202163222398P 2021-07-15 2021-07-15
US63/222,398 2021-07-15
US17/489,357 US20220396798A1 (en) 2021-06-12 2021-09-29 Novel mrna composition and production method for use in anti-viral and anti-cancer vaccines
US17/489,357 2021-09-29
US202163270034P 2021-10-20 2021-10-20
US63/270,034 2021-10-20
US202163280226P 2021-11-17 2021-11-17
US63/280,226 2021-11-17

Publications (1)

Publication Number Publication Date
WO2022260718A1 true WO2022260718A1 (fr) 2022-12-15

Family

ID=84425225

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2022/013372 WO2022260719A1 (fr) 2021-06-12 2022-01-21 Nouvelle composition d'arn et procédé de production à être utilisés dans la génération de cellules ips
PCT/US2022/013350 WO2022260718A1 (fr) 2021-06-12 2022-01-21 Nouvelle réaction de cyclage de réplicase (rcr)

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2022/013372 WO2022260719A1 (fr) 2021-06-12 2022-01-21 Nouvelle composition d'arn et procédé de production à être utilisés dans la génération de cellules ips

Country Status (2)

Country Link
JP (1) JP2024523006A (fr)
WO (2) WO2022260719A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4219756A1 (fr) 2022-01-24 2023-08-02 Mello Biotech Taiwan Co., Ltd. Fragment d'arn synthétique et ses utilisations pour l'amplification dépendante de l'arn

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6168943B1 (en) * 1995-05-04 2001-01-02 Yale University Methods for making modified recombinant vesiculoviruses
WO2002092774A2 (fr) * 2001-05-14 2002-11-21 Shi-Lung Lin Amplification de la reaction de cyclage de la replicase
US20190330591A1 (en) * 2018-01-10 2019-10-31 Rubius Therapeutics, Inc. Amplifiable rnas for therapeutic cell systems

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009075119A1 (fr) * 2007-12-10 2009-06-18 Kyoto University Procédé efficace d'initialisation de noyaux
EP2742127B1 (fr) * 2011-08-12 2019-10-09 Mello Biotechnology, Inc. Expression inductible dans des procaryotes d'un promoteur eucaryotique pol-2
WO2019056090A1 (fr) * 2017-09-19 2019-03-28 The Governors Of The University Of Alberta Arn polymérase arn-dépendante recombinante de virus à arn
SG11202012786RA (en) * 2018-06-26 2021-01-28 Massachusetts Inst Technology Crispr effector system based amplification methods, systems, and diagnostics

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6168943B1 (en) * 1995-05-04 2001-01-02 Yale University Methods for making modified recombinant vesiculoviruses
WO2002092774A2 (fr) * 2001-05-14 2002-11-21 Shi-Lung Lin Amplification de la reaction de cyclage de la replicase
US20190330591A1 (en) * 2018-01-10 2019-10-31 Rubius Therapeutics, Inc. Amplifiable rnas for therapeutic cell systems

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AHN ET AL.: "Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA- dependent RNA polymerase capable of copying viral RNA templates", ARCHIVES OF VIROLOGY, vol. 157, 13 July 2012 (2012-07-13), pages 2095 - 2104, XP037066910, DOI: 10.1007/s00705-012-1404-x *
CHAN ET AL.: "Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 58, no. 5, 23 April 2020 (2020-04-23), pages 1 - 10, XP055752332, DOI: 10.1128/JCM *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4219756A1 (fr) 2022-01-24 2023-08-02 Mello Biotech Taiwan Co., Ltd. Fragment d'arn synthétique et ses utilisations pour l'amplification dépendante de l'arn
JP2023107748A (ja) * 2022-01-24 2023-08-03 美洛生物科技股▲ふん▼有限公司 合成ｒｎａ断片及びｒｎａ依存性増幅のためのその使用

Also Published As

Publication number Publication date
WO2022260719A1 (fr) 2022-12-15
JP2024523006A (ja) 2024-06-25

Similar Documents

Publication Publication Date Title
AU778474B2 (en) Method and medicament for inhibiting the expression of a defined gene
US7659391B2 (en) Method for the in vitro synthesis of short double stranded RNAs
EP2352744B1 (fr) Arn courts en épingles à cheveux destinés à inhiber l'expression des gènes
US8486910B2 (en) SnoRNAi-small nucleolar RNA degradation by RNA interference in trypanosomatids
WO2008140126A1 (fr) Arn cyclique à simple brin et procédé destiné à produire celui-ci
AU2002338926A1 (en) Method for the in vitro synthesis of short double stranded RNAs
ZA200601368B (en) A self-processing RNA expression cassette
JP4517061B2 (ja) ダンベル型ｄｎａの効率的な製造方法
US20220396798A1 (en) Novel mrna composition and production method for use in anti-viral and anti-cancer vaccines
WO2022260718A1 (fr) Nouvelle réaction de cyclage de réplicase (rcr)
TWI382849B (zh) 產生藉由基因靜默效應以用於皮膚維護之化妝品的方法以及編碼基因靜默效應物之基因重組核苷酸
CA2813163A1 (fr) Agent preventif ou therapeutique destine a la fibrose
CN110295169B (zh) 一种miRNA及其用于杀灭褐飞虱的应用
US20220411848A1 (en) Novel Replicase Cycling Reaction (RCR)
US20230099592A1 (en) Novel replicase cycling reaction (rcr)
US20230295627A1 (en) Novel Replicase Cycling Reaction (RCR) and the Related SamRNA Designs Thereof
EP4269585A1 (fr) Arn guide pour l'édition de séquence signal de polyadénylation d'arn cible
US20240093286A1 (en) Novel Replicase Cycling Reaction (RCR) and the Related RdRP-Binding Site Designs Thereof
JP7364198B2 (ja) ｉＰＳ細胞の生成に使用される新規ＲＮＡ組成物およびその製造方法
CN114457077A (zh) 靶向新型冠状病毒rna及其反义链utr的脱氧核酶及其应用
CN113817729A (zh) 一种抑制新型冠状病毒（CoV19）的siRNA及其组合物和应用
CN113817730A (zh) 一种抑制新型冠状病毒(CoV19)的siRNA及其组合物和应用
AU2017276342A1 (en) Method and medicament for inhibiting the expression of a defined gene
AU2010200327A1 (en) Method and medicament for inhibiting the expression of a defined gene

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22820709

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023575381

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22820709

Country of ref document: EP

Kind code of ref document: A1