WO2024002269A1 - Protein-mediated mrna targeting molecule, method for preparing same, and use thereof - Google Patents

Protein-mediated mrna targeting molecule, method for preparing same, and use thereof Download PDF

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WO2024002269A1
WO2024002269A1 PCT/CN2023/104036 CN2023104036W WO2024002269A1 WO 2024002269 A1 WO2024002269 A1 WO 2024002269A1 CN 2023104036 W CN2023104036 W CN 2023104036W WO 2024002269 A1 WO2024002269 A1 WO 2024002269A1
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protein
mrna
nucleotide fragment
sequence
guanosine
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French (fr)
Chinese (zh)
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胡勇
张昊
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武汉瑞佶生物科技有限公司
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • 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
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Definitions

  • the invention belongs to the technical field of molecular biology, and in particular relates to a protein-mediated mRNA targeting molecule and its preparation method and application.
  • mRNA The immunogenicity and instability of mRNA are important reasons that limit the research and application of mRNA. Compared with DNA, mRNA can mediate better transfection efficiency and longer protein expression time.
  • the coding information of the mRNA includes the sequence of the protein produced by ribosomes, which must be delivered into the cell to encode the protein. It is not basic until the modified nucleosides are introduced into the sequence of the mRNA and a vector that can deliver the mRNA is developed. Technical problems in the application process were solved.
  • the mRNA chain is a long-chain macromolecule with negative charges, and the surface of the cell membrane also carries negative charges. Electrostatic repulsion makes it difficult for the mRNA molecules to automatically bind to the cell membrane and pass through the cell membrane into the cell.
  • delivery of mRNA into cells can be achieved through different methods, such as electroporation, sonoporation, microinjection or compound transfection.
  • the cytotoxicity and biological safety of these methods are difficult to meet clinical needs, and clinical translation has certain limitations. Difficulty.
  • One object of the present invention is to provide a protein-mediated mRNA targeting molecule.
  • Another object of the present invention is to provide a method for preparing protein-mediated mRNA targeting molecules.
  • Another object of the present invention is to provide an application of protein-mediated mRNA targeting molecules.
  • the invention provides a protein-mediated mRNA targeting molecule and its preparation method and application, which realizes direct and efficient coupling of mRNA and protein molecules, and mediates endocytosis through specific binding to cell surface receptors. Achieve specific targeted delivery of mRNA molecules.
  • the present invention first provides a protein-mediated mRNA targeting molecule.
  • the protein-mediated mRNA targeting molecule is composed of mRNA connected to a targeting protein group through the PolyA at its 3' end, wherein the mRNA
  • the structure contains the 5' cap structure, 5'UTR with Kozak sequence, target gene sequence, 3'UTR and PolyA from the 5' end to the 3' end.
  • the protein-mediated mRNA targeting molecule is also called an mRNA-protein targeting molecule.
  • the targeting protein includes VSV-G, permeabilin, transferrin, GM-SCF, arginine-glycine-aspartate tripeptide, aspartic acid -Glycine-arginine tripeptide, anti-VEGFR antibody, anti-ERBB2 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD25 antibody, anti-tenascin antibody, anti-MUC1 antibody, anti-TAG72 antibody, anti-CEA antibody, etc. .
  • the mRNA in the protein-mediated mRNA targeting molecule of the present invention, includes modified nucleosides and/or unmodified nucleosides, wherein the modified nucleosides are chemical Modified nucleosides.
  • the chemically modified nucleosides include 2-fluoro-2-deoxyadenosine, 2-fluoro-2-deoxyuridine, 2-fluoro-2-deoxycytidine, 2-fluoro-2-deoxyguanosine Glycoside, 2-fluoro-2-deoxy-5-methylcytidine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine, 2-fluoro- 2-Deoxy-N7-methyl-guanosine, 2-fluoro-2-deoxy-5-methoxyuridine, 2-fluoro-2-deoxy-N4-acetylcytidine, 2-fluoro-2-deoxy -N6-methyladenosine, 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetylcytidine and one or more
  • the 5' cap structure of the mRNA includes Cap0, Cap1, Cap2, ARCA, inosine, N1-methyl-guanosine, 2 'Fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, 7-methyl-guanosine -5'-triphosphate-5'-adenosine, guanosine -5'-triphosphate-5'-adenosine, 7-methyl-guanosine -5'-triphosphate-5'-guanosine, guanosine - One of -5'-triphosphate-5'-guanosine and 7-methyl-guanosine-5'-triphosphate-5'-2-methoxyadenine-guanosine.
  • the 3'UTR part of the mRNA includes at least 10 bases, for example, it can be 10-500 bases, preferably 50-50 bases. 200 bases.
  • the length of PolyA of the mRNA is 5-150, preferably 5-120, further preferably 5-100, and even more preferably 5-50.
  • the target gene sequence of the mRNA does not play a role in the delivery of the protein-mediated mRNA targeting molecule in cells.
  • the present invention also provides a method for preparing protein-mediated mRNA targeting molecules, which includes:
  • the protein-mediated mRNA targeting molecule is prepared by connecting the mRNA to the targeting protein group through the PolyA at its 3' end.
  • methods for preparing protein-mediated mRNA targeting molecules of the invention include:
  • the 3' end of the first nucleotide fragment is connected to the 5' end of the second nucleotide fragment with a protein group at the 3' end to prepare a protein-mediated mRNA targeting molecule. ; wherein the first nucleotide fragment and the second nucleotide fragment form the mRNA part in the protein-mediated mRNA targeting molecule.
  • the 3' end of the first nucleotide fragment and the second nucleotide with a protein group at the 3' end are When ligating the 5' ends of the fragments, splint DNA was used to assist.
  • the splint DNA in the method for preparing a protein-mediated mRNA targeting molecule of the present invention, includes a first splint DNA and a second splint DNA; a first splint DNA and a first nucleotide fragment
  • the 3' end sequence of the second splint DNA is complementary to the 5' end sequence of the second nucleotide fragment.
  • the 3' end of the first nucleotide fragment and the second nucleotide with a protein group at the 3' end are When the 5' ends of the fragments are connected, the first splint DNA and the second splint DNA are seamlessly connected (that is, the first splint DNA corresponds to the sequence on the first nucleotide fragment and the second splint DNA corresponds to the sequence on the first nucleotide fragment. There are no intervening bases between sequences on a dinucleotide fragment).
  • the first splint DNA is complementary to the 5-40 base sequences of the 3' end of the first nucleotide fragment
  • the second splint DNA is complementary to the 5-40 base sequences at the 5' end of the second nucleotide fragment.
  • the length of the first splint DNA is 5-40 bases
  • the length of the second splint DNA is 5-40 bases.
  • the 3' end sequence of the first nucleotide fragment complementary to the first splint DNA is a protein-mediated mRNA targeting molecule.
  • the 5' end sequence of the second nucleotide fragment complementary to the second splint DNA includes a protein-mediated mRNA targeting molecule.
  • a part of the sequence in the 3'UTR of the molecule's mRNA may or may not include part or all of PolyA. That is, the second splint DNA is designed for the 3'UTR of the target mRNA, or the 3'UTR and PolyA sequence.
  • the second nucleotide fragment includes a third nucleotide fragment and PolyA in sequence from the 5' end to the 3' end, and the third nucleotide fragment is the 3'UTR of the mRNA of the protein-mediated mRNA targeting molecule. part of the 3' end sequence.
  • the length of the third nucleotide fragment is 5-100 bases, preferably 5-80 bases, further preferably 5-60 bases, even more preferably 5-40 bases.
  • the length of the PolyA is 5-150, preferably 5-120, more preferably 5-100, even more preferably 5-50.
  • the length of the second nucleotide fragment is 10-150 bases, preferably 10-120 bases, further preferably 15-100 bases, even more preferably 15-60 bases.
  • the first splint DNA includes, but is not limited to, what is shown in SEQ ID No. 3, and the second splint DNA includes, but is not limited to, what is shown in SEQ ID No. 4.
  • the preparation method of the protein-mediated mRNA targeting molecule of the present invention includes:
  • a first nucleotide fragment synthesize a plasmid vector with a promoter sequence and a target gene sequence, perform in vitro transcription, and obtain a first nucleotide fragment; the structure of the first nucleotide fragment extends from the 5' end to the 3' end
  • the 'end contains in turn a 5' cap structure, a 5'UTR with a Kozak sequence, a target gene sequence, and a part of the 5' end of the 3'UTR;
  • a second nucleotide fragment with a protein group at the 3' end connect the targeting protein to the 3' end of the second nucleotide fragment to obtain a second nucleotide fragment with a protein group at the 3' end. ; Wherein, the protein group is directly connected to the PolyA of the 3' end sequence of the second nucleotide fragment;
  • the 3' end sequence of the first nucleotide fragment is complementary to the first splint DNA
  • the 5' end sequence of the second nucleotide fragment is complementary to the second splint DNA.
  • ligase such as T4 RNA ligase
  • the 3' end of the first nucleotide fragment is connected to the 5' end of the second nucleotide fragment with a protein group at the 3' end, and further removed by DNase treatment.
  • Splint DNA to obtain protein-mediated mRNA targeting molecules.
  • the promoter may be a T3 or T7 or SP6 promoter.
  • the present invention also provides a pharmaceutical composition, which includes: the protein-mediated mRNA targeting molecule of the present invention, and pharmaceutically acceptable excipients.
  • the present invention also provides the use of the pharmaceutical composition or the protein-mediated mRNA targeting molecule in the preparation of drugs for expressing target polypeptides in mammals or human subjects.
  • the present invention also provides a method for expressing a target polypeptide in a subject, the method comprising: combining the drug
  • the substance or the protein-mediated mRNA targeting molecule is administered to the subject.
  • the subject is a mammal or a human.
  • the target polypeptide may be one or more of protein replacement drugs such as GLP-1, urate oxidase, insulin, collagen, etc.
  • the present invention achieves in vivo delivery independent of traditional liposomes and lipid nanoparticles by using positively charged proteins.
  • these specific protein molecules have specific receptors on the surface of target cells to achieve targeted delivery to tissues and organs.
  • the mRNA-protein targeting molecule of the present invention can be used in a drug delivery system.
  • the 3' end of the mRNA is connected with a specific protein group.
  • the primer is endocytosed, thereby allowing the mRNA to enter the cell.
  • Expression solves the technical problem of targeted delivery of nucleic acid drugs in the drug delivery process.
  • the mRNA with the protein group at the 3' end is synthesized.
  • Targeted delivery in the body is achieved under the action of the invention to achieve the purpose of targeted drug delivery.
  • the method of the present invention does not rely on traditional carriers such as liposomes and lipid nanoparticles.
  • the method is simpler and more reliable, and solves the existing coupling between mRNA and protein. problems, as well as the problems of targeted delivery in vivo.
  • the connection efficiency and accuracy can be greatly improved through the design of splint DNA.
  • Figure 1 is a schematic flow chart of a method for preparing an mRNA-protein targeting molecule in Embodiment 1 of the present invention
  • (a) is a schematic structural diagram of a DNA fragment in plasmid DNA and an mRNA molecule obtained by in vitro transcription
  • (b) is an mRNA molecule
  • (c) is a schematic diagram of the resulting mRNA-protein targeting molecule.
  • Figure 2 is a comparison of cells transfected with mRNA-VSV-G targeting molecules and mRNA molecules.
  • Figure 3 is a schematic flow chart of a method for preparing an mRNA-protein targeting molecule in Embodiment 2 of the present invention
  • (a) is a schematic structural diagram of the DNA fragment in the plasmid DNA and the mRNA molecule obtained by in vitro transcription
  • (b) is the mRNA molecule
  • (c) is a schematic diagram of the resulting mRNA-protein targeting molecule.
  • Figure 4 is a comparison of cells transfected with mRNA-ERBB2 antibody targeting molecules without ERBB2 expression and cells expressing ERBB2 on the cell membrane.
  • Figure 5 is a comparison of cells without CD22 expression and CD22+ cells transfected with mRNA-CD22 antibody targeting molecules.
  • Figure 6 shows the molecular structure and ligation efficiency results of mRNA-VSV-G.
  • This embodiment provides an mRNA-protein targeting molecule, namely, an mRNA-VSV-G protein targeting molecule, which is a structure with VSV-G protein on the 3' end PolyA of the mRNA.
  • the sequence of the mRNA molecule includes a 5' cap, a 5'UTR with a Kozak sequence, a target gene sequence, a 3'UTR and PolyA.
  • the mRNA-protein targeting molecule is prepared using the following steps:
  • Step S1 design and synthesize a plasmid vector with a promoter sequence and target gene sequence.
  • the plasmid vector was purchased from pUC-GW-KANA of Jinweizhi Technology Co., Ltd., the insertion site is EcoRVI, and the linking sequence of each part is 5'UTR- mRNA coding gene sequence-3'UTR;
  • Step S2 use the plasmid vector of Step S1 as a template to perform in vitro transcription to obtain an mRNA molecule whose sequence includes a 5' cap, a 5'UTR including a Kozak sequence, a target gene sequence, and a 3'UTR;
  • Step S3 Under the action of ligase, the mRNA molecule is combined with the VSV-G modified polyadenylate to prepare the mRNA-VSV-G targeting molecule.
  • the nucleotide sequence of the promoter is shown in SEQ ID No. 1. Further, the nucleotide sequence of the target gene is shown in SEQ ID No. 2.
  • SEQ ID No.1 taatacgactcactatagg
  • step S2 the specific synthesis method of the mRNA molecule in step S2 is as follows:
  • the constructed expression plasmid is used to amplify the DNA template according to the following reaction system:
  • Reaction volume 50 ⁇ l (reaction volume of a single tube, multiple tubes can be reacted at the same time);
  • the PCR amplification system (50 ⁇ l): PrimeSTAR Max Premix (2 ⁇ ) 25 ⁇ l, primer F (SEQ ID No. 6: TTGGACCCCTCGTACAGAAGCTAATACG, 10 ⁇ mol/L) 1.2 ⁇ l, primer R (SEQ ID No. 7: TACCGGTTAGCTTCCTACTCAGGCTTTATTCAAAGACCA, 10 ⁇ mol) /L) 1.2 ⁇ l, DNA template (1ng/ ⁇ l) 1 ⁇ l and water 21.6 ⁇ l.
  • the amplification procedure of the PCR is as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; and final extension at 72°C for 10 minutes.
  • Eligibility criteria A single band appears in electrophoresis detection and the size is correct.
  • NanoDrop to detect the concentration of the purified template and the ratios of 260/280 and 260/230. Samples were taken for DNA agarose gel electrophoresis detection (1.5% agarose, 5V/min, 40min).
  • Qualifying standards 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.
  • Millipore 30Kd ultrafiltration tube concentrates the FPLC-purified DNA template, and elutes and dissolves it with RNase-free water. Use NanoDrop to detect the concentration of the template after ultrafiltration and the ratios of 260/280 and 260/230. Finally dilute to 150ng/ ⁇ l with RNase-free water.
  • Reaction volume 1600 ⁇ l (placed in a 2ml RNase-free Tube, it is the reaction volume of a single tube, multiple tubes can be reacted at the same time): RNA-free water 440 ⁇ l, 7.5mM ATP 160 ⁇ l, 7.5mM UTP 160 ⁇ l, 7.5mM CTP 160 ⁇ l, 7.5mM GTP 160 ⁇ l, 7.5mM M7G (2'OMeA)pG 160 ⁇ l, 150ng/ ⁇ l DNA template 40 ⁇ l, 10 ⁇ Buffer 160 ⁇ l and Enzyme Mix 160 ⁇ l.
  • RNA The procedure for in vitro synthesis of RNA is 37°C, 10 h.
  • the concentration of recovered mRNA detected by NanoDrop was 5 ⁇ g/ ⁇ l, A260/A280 was 1.90, and A260/A230 was 2.0.
  • the obtained mRNA sequence is shown in SEQ ID No. 5.
  • the Cap structure is 3'-O-Me-m7G(5')ppp(5')G.
  • step S3 the VSV-G modified polyadenylic acid uses the exposed amino group at the 3' end of the polyadenylic acid (PolyA composed of 10 A's), and the amino acid residues of the protein group.
  • the carboxyl group is reacted.
  • CDI can be added during the reaction and the reaction can be carried out at room temperature for 2 hours.
  • step S3 includes:
  • Lithium chloride solution is added to the reaction product until the final concentration of lithium chloride is 0.5M, and a precipitated product can be obtained, which is the mRNA-VSV-G targeting molecule of the present invention.
  • the mRNA-VSV-G targeting molecule prepared in this example can be used to prepare mRNA drugs for specific drug delivery.
  • the mRNA drug for specific drug delivery may also include one or more pharmaceutically acceptable carriers, such as protective agents, buffers, etc.
  • the 3' end of the mRNA-VSV-G targeting molecule is connected to the VSV-G protein, which mediates endocytosis through VSV-G, allowing the mRNA to enter cells for expression.
  • the specific operation steps are: about 24 hours after passage of 293T cells, observe the cell status in the 6-well plate. Transfection can be carried out when the confluence is about 90%.
  • This embodiment provides an mRNA-protein targeting molecule, that is, an mRNA-anti-ERBB2 antibody targeting molecule, which is a product with an anti-ERBB2 antibody on the 3' end PolyA of the mRNA.
  • the sequence of the mRNA includes a 5' cap, a 5'UTR with a Kozak sequence, a target gene sequence, a 3'UTR, and PolyA (the sequence of the mRNA is the same as in Example 1).
  • the 3’ end of PolyA carries an anti-ERBB2 antibody.
  • the mRNA-anti-ERBB2 antibody targeting molecule is prepared using the following steps:
  • Step S1 design and synthesize a plasmid vector containing a promoter sequence, a complementary sequence of the target gene sequence and the first splint DNA sequence;
  • Step S2 Use the plasmid vector of step S1 as a template to perform in vitro transcription to obtain mRNA molecules.
  • the sequence of the mRNA molecule includes a 5' cap connected in sequence, a 5'UTR with a Kozak sequence, a target gene sequence, and a part of the 3'UTR (the 5' end sequence part of the 3'UTR of the target mRNA, including the sequence with the first
  • the complementary sequence of the splint DNA sequence corresponds to the mRNA sequence).
  • the mRNA synthesis method is the same as in Example 1;
  • Step S3 a PolyA fragment with a protein group at the 3' end, which has 10 A's, and the 5' end of PolyA has an RNA sequence that can be complementary to the second splint DNA sequence (i.e., the 3'UTR of the target mRNA part of the 3' end sequence).
  • the first splint DNA sequence and the second splint DNA sequence are seamlessly connected to form splint DNA.
  • the mRNA molecule with the complementary sequence of the first splint DNA sequence and the PolyA molecule with the complementary pairing sequence with the second splint DNA sequence are complementary to the splint DNA respectively.
  • the DNA sequence of the first splint is shown in SEQ ID No. 3
  • the DNA sequence of the second splint is shown in SEQ ID No. 4.
  • the sequence of the promoter is shown in SEQ ID No. 1.
  • the target gene is shown in SEQ ID No. 2.
  • ligase is T4RNA Ligase I.
  • the mRNA-protein targeting molecules obtained in this example can be used to prepare mRNA drugs for specific drug delivery.
  • An anti-ERBB2 antibody is connected to the 3' end, and the anti-ERBB2 antibody specifically binds to cancer cells expressing ERBB2 on their surface to cause endocytosis, allowing the mRNA to enter the cells for expression.
  • the specific steps are as follows: About 24 hours after passage of 293T (no ERBB2 expression) and H1781 cells (ERBB2 membrane localization), observe the cell status in the 6-well plate. Transfection can be performed when the confluence is about 90%.
  • This embodiment provides an mRNA-protein targeting molecule, that is, an mRNA-anti-CD22 antibody targeting molecule, which is a connection product of an mRNA molecule and an anti-CD22 antibody.
  • the sequence of the mRNA molecule includes a 5' cap, a 5'UTR with a Kozak sequence, a target gene sequence, a 3'UTR, and PolyA (the sequence of the mRNA is the same as in Example 1).
  • the 3' end PolyA of the mRNA molecule carries an anti-CD22 antibody.
  • the mRNA-protein targeting molecule is prepared using the same steps as in Example 2:
  • Step S1 design and synthesize a plasmid vector containing a promoter sequence, a complementary sequence of the target gene sequence and the first splint DNA sequence;
  • Step S2 Use the plasmid vector of Step S1 as a template for in vitro transcription to obtain an mRNA molecule.
  • the sequence of the mRNA molecule includes a 5' cap connected in sequence, a 5'UTR with a Kozak sequence, a target gene sequence, and a 3'UTR.
  • the 5' end partial sequence (including the RNA sequence corresponding to the complementary sequence of the first splint DNA sequence), the in vitro transcription and purification scheme of mRNA are shown in Example 1;
  • Step S3 the PolyA fragment with a protein group at the 3' end has an RNA sequence that can be complementary to the DNA sequence of the second splint at the 5' end; during the annealing reaction, the complementary sequence of the DNA sequence of the first splint is carried
  • the mRNA molecule and the PolyA molecule with the sequence complementary to the second splint DNA sequence are complementary to the splint DNA.
  • T4 RNA ligase the 3' end hydroxyl group of the mRNA molecule and the PolyA molecule with the anti-CD22 antibody
  • the 5' end phosphate group is connected and treated with DNase I to obtain an mRNA targeting molecule with an anti-CD22 antibody.
  • the DNA sequence of the first splint is shown in SEQ ID No. 3
  • the DNA sequence of the second splint is shown in SEQ ID No. 4.
  • the sequence of the promoter is shown in SEQ ID No. 1.
  • the target gene is shown in SEQ ID No. 2.
  • ligase is T4RNA Ligase I.
  • the mRNA-protein targeting molecules obtained in this example can be used to prepare mRNA drugs for specific drug delivery.
  • An anti-CD22 antibody is connected to the 3' end, and the anti-CD22 antibody specifically binds to cancer cells expressing CD22 on their surface to cause endocytosis, allowing the mRNA to enter the cells for expression.
  • the specific steps are as follows: About 24 hours after passage of 293T (no CD22 expression) and CA46 cells (CD22+), observe the cell status in the 6-well plate. Transfection can be carried out when the confluence is about 90%. Take two 200 ⁇ l opti-MEM, add 10 ⁇ g of GFP-encoding mRNA-CD22 antibody complex respectively, mix thoroughly by gently pipetting with the pipe tip, and let stand for 10 minutes.
  • the mRNA-CD22 antibody complex can specifically transfect CA46 cells expressing CD22 on their surface, but cannot transfect ordinary 293T cells.
  • the magnetic beads were washed three times in RIP buffer and then once in PBS;
  • RNA bound to RBP after immunoprecipitation, resuspend the magnetic beads in 1mL TRIzol RNA extraction reagent, and isolate the co-precipitated RNA;
  • the mRNA was reverse transcribed (RT) into cDNA, and RT-PCR analysis was performed.
  • the same quality mRNA sample was used as a positive control to detect the abundance of mRNA bound to VSV-G.
  • the primers used in RT-PCR are F: AAGGAGGACGGCAACATCCTGGGG (SEQ ID No. 8), R: TACAGCTCGTCCATGCCGAGAGTG (SEQ ID No. 9).
  • the present invention can realize that the final product can be used for targeted delivery of mRNA mainly through the connection of poly(A) and protein delivery carriers, and the connection method of poly(A)/protein complex and the end of the mRNA molecule. .

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Abstract

Provide are a protein-mediated mRNA targeting molecule, a method for preparing same, and use thereof. The protein-mediated mRNA targeting molecule is formed by connecting an mRNA to a targeting protein group by means of PolyA at a 3' terminal thereof. The structure of the mRNA sequentially comprises a 5' cap structure, 5'UTR with a Kozak sequence, a target gene sequence, 3'UTR, and PolyA from a 5' terminal to a 3' terminal. The mRNA expressing a target gene can be directly connected to a PolyA sequence coupled with a protein to synthesize an mRNA molecule with the protein at the 3' terminal, thereby achieving the purpose of targeted drug delivery. The method is simple and reliable, and solves the problem of targeted delivery of existing mRNAs to specific cells.

Description

蛋白质介导的mRNA靶向分子及其制备方法和应用Protein-mediated mRNA targeting molecules and preparation methods and applications thereof 技术领域Technical field
本发明属于分子生物学技术领域，尤其涉及一种蛋白质介导的mRNA靶向分子及其制备方法和应用。The invention belongs to the technical field of molecular biology, and in particular relates to a protein-mediated mRNA targeting molecule and its preparation method and application.
背景技术Background technique
mRNA的免疫原性和不稳定性，是限制mRNA研究应用的重要原因。相比DNA，mRNA能介导更优的转染效率和更长的蛋白表达时间。然而，mRNA编码信息中涵盖了核糖体生成蛋白的序列，必须递送至细胞内才能编码蛋白，直到将被修饰的核苷引入到mRNA的序列中，且研发出可以递送mRNA的载体，才算基本解决了应用过程中的技术难题。The immunogenicity and instability of mRNA are important reasons that limit the research and application of mRNA. Compared with DNA, mRNA can mediate better transfection efficiency and longer protein expression time. However, the coding information of the mRNA includes the sequence of the protein produced by ribosomes, which must be delivered into the cell to encode the protein. It is not basic until the modified nucleosides are introduced into the sequence of the mRNA and a vector that can deliver the mRNA is developed. Technical problems in the application process were solved.
mRNA链是带有负电荷的长链大分子，而细胞膜表面也带有负电荷，静电排斥使得mRNA分子较难自动结合细胞膜并穿过细胞膜进入细胞内。目前，mRNA向细胞内的递送可以通过不同的方法实现，例如电穿孔、声孔效应、显微注射或化合物转染，但这些方法的细胞毒性和生物安全性难以满足临床需求，临床转化具有一定难度。The mRNA chain is a long-chain macromolecule with negative charges, and the surface of the cell membrane also carries negative charges. Electrostatic repulsion makes it difficult for the mRNA molecules to automatically bind to the cell membrane and pass through the cell membrane into the cell. Currently, the delivery of mRNA into cells can be achieved through different methods, such as electroporation, sonoporation, microinjection or compound transfection. However, the cytotoxicity and biological safety of these methods are difficult to meet clinical needs, and clinical translation has certain limitations. Difficulty.
经查，使mRNA靶向递送至特定细胞类型的递送方式在现有技术中尚无深入研究。It has been found that the delivery method for targeted delivery of mRNA to specific cell types has not been thoroughly studied in the existing technology.
发明内容Contents of the invention
本发明的一个目的在于提供一种蛋白质介导的mRNA靶向分子。One object of the present invention is to provide a protein-mediated mRNA targeting molecule.
本发明的另一目的在于提供一种蛋白质介导的mRNA靶向分子的制备方法。Another object of the present invention is to provide a method for preparing protein-mediated mRNA targeting molecules.
本发明的另一目的在于提供一种蛋白质介导的mRNA靶向分子的应用。Another object of the present invention is to provide an application of protein-mediated mRNA targeting molecules.
本发明提供了一种蛋白质介导的mRNA靶向分子及其制备方法和应用，实现了mRNA与蛋白质分子的直接高效偶联，并通过与细胞表面受体的特异性结合，介导胞吞作用实现mRNA分子的特异性靶向递送。The invention provides a protein-mediated mRNA targeting molecule and its preparation method and application, which realizes direct and efficient coupling of mRNA and protein molecules, and mediates endocytosis through specific binding to cell surface receptors. Achieve specific targeted delivery of mRNA molecules.
具体而言，本发明首先提供了一种蛋白质介导的mRNA靶向分子，该蛋白质介导的mRNA靶向分子由mRNA通过其3’端的PolyA与靶向蛋白基团连接而成，其中，mRNA的结构从5’端至3’端依次包含5’帽子结构、带有Kozak序列的5’UTR、目的基因序列、3’UTR以及PolyA。本发明中，所述蛋白质介导的mRNA靶向分子亦称为mRNA-蛋白靶向分子。 Specifically, the present invention first provides a protein-mediated mRNA targeting molecule. The protein-mediated mRNA targeting molecule is composed of mRNA connected to a targeting protein group through the PolyA at its 3' end, wherein the mRNA The structure contains the 5' cap structure, 5'UTR with Kozak sequence, target gene sequence, 3'UTR and PolyA from the 5' end to the 3' end. In the present invention, the protein-mediated mRNA targeting molecule is also called an mRNA-protein targeting molecule.
根据本发明的具体实施方案，优选地，所述靶向蛋白包括VSV-G、透膜蛋白、转铁蛋白、GM-SCF、精氨酸-甘氨酸-天冬氨酸三肽、天冬氨酸-甘氨酸-精氨酸三肽、抗VEGFR抗体、抗ERBB2抗体、抗CD20抗体、抗CD22抗体、抗CD33抗体、抗CD25抗体、抗肌腱蛋白抗体、抗MUC1抗体、抗TAG72抗体、抗CEA抗体等。According to specific embodiments of the present invention, preferably, the targeting protein includes VSV-G, permeabilin, transferrin, GM-SCF, arginine-glycine-aspartate tripeptide, aspartic acid -Glycine-arginine tripeptide, anti-VEGFR antibody, anti-ERBB2 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD25 antibody, anti-tenascin antibody, anti-MUC1 antibody, anti-TAG72 antibody, anti-CEA antibody, etc. .
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子中，所述mRNA包括经修饰的核苷和/或未经修饰的核苷，其中所述经修饰的核苷为化学修饰核苷。更具体地，所述的化学修饰核苷包括2-氟-2-脱氧腺苷、2-氟-2-脱氧尿苷、2-氟-2-脱氧胞苷、2-氟-2-脱氧鸟苷、2-氟-2-脱氧-5-甲基胞苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷、2-氟-2-脱氧-N7-甲基-鸟苷、2-氟-2-脱氧-5-甲氧基尿苷、2-氟-2-脱氧-N4-乙酰基胞苷、2-氟-2-脱氧-N6-甲基腺苷、5-甲基胞苷、假尿苷、N1-甲基-假尿苷、N7-甲基-鸟苷、5-甲氧基尿苷、N4-乙酰基胞苷和N6-甲基腺苷中的一种或多种。According to a specific embodiment of the present invention, in the protein-mediated mRNA targeting molecule of the present invention, the mRNA includes modified nucleosides and/or unmodified nucleosides, wherein the modified nucleosides are chemical Modified nucleosides. More specifically, the chemically modified nucleosides include 2-fluoro-2-deoxyadenosine, 2-fluoro-2-deoxyuridine, 2-fluoro-2-deoxycytidine, 2-fluoro-2-deoxyguanosine Glycoside, 2-fluoro-2-deoxy-5-methylcytidine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine, 2-fluoro- 2-Deoxy-N7-methyl-guanosine, 2-fluoro-2-deoxy-5-methoxyuridine, 2-fluoro-2-deoxy-N4-acetylcytidine, 2-fluoro-2-deoxy -N6-methyladenosine, 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetylcytidine and one or more of N6-methyladenosine.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子中，所述mRNA的5'帽子结构包括Cap0、Cap1、Cap2、ARCA、肌苷、N1-甲基-鸟苷、2'氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷、2-叠氮基-鸟苷、7-甲基-鸟苷-5'-三磷酸-5'-腺苷、鸟苷-5'-三磷酸-5'-腺苷、7-甲基-鸟苷-5'-三磷酸-5'-鸟苷、鸟苷-5'-三磷酸-5'-鸟苷和7-甲基-鸟苷-5'-三磷酸-5'-2-甲氧基腺嘌呤-鸟苷中的一种。According to a specific embodiment of the present invention, in the protein-mediated mRNA targeting molecule of the present invention, the 5' cap structure of the mRNA includes Cap0, Cap1, Cap2, ARCA, inosine, N1-methyl-guanosine, 2 'Fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, 7-methyl-guanosine -5'-triphosphate-5'-adenosine, guanosine -5'-triphosphate-5'-adenosine, 7-methyl-guanosine -5'-triphosphate-5'-guanosine, guanosine - One of -5'-triphosphate-5'-guanosine and 7-methyl-guanosine-5'-triphosphate-5'-2-methoxyadenine-guanosine.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子中，所述mRNA的3’UTR部分至少包括10个碱基，例如可以是10-500个碱基，优选为50-200个碱基。According to a specific embodiment of the present invention, in the protein-mediated mRNA targeting molecule of the present invention, the 3'UTR part of the mRNA includes at least 10 bases, for example, it can be 10-500 bases, preferably 50-50 bases. 200 bases.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子中，所述mRNA的PolyA的长度为5-150、优选为5-120、进一步优选为5-100、更进一步优选为5-50。According to a specific embodiment of the present invention, in the protein-mediated mRNA targeting molecule of the present invention, the length of PolyA of the mRNA is 5-150, preferably 5-120, further preferably 5-100, and even more preferably 5-50.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子中，所述mRNA的目的基因序列不对所述蛋白质介导的mRNA靶向分子在细胞中的递送发挥作用。According to a specific embodiment of the present invention, in the protein-mediated mRNA targeting molecule of the present invention, the target gene sequence of the mRNA does not play a role in the delivery of the protein-mediated mRNA targeting molecule in cells.
另一方面，本发明还提供了一种蛋白质介导的mRNA靶向分子的制备方法，其包括：On the other hand, the present invention also provides a method for preparing protein-mediated mRNA targeting molecules, which includes:
将mRNA通过其3’端的PolyA与靶向蛋白基团连接，制备得到蛋白质介导的mRNA靶向分子。The protein-mediated mRNA targeting molecule is prepared by connecting the mRNA to the targeting protein group through the PolyA at its 3' end.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法 包括：According to specific embodiments of the invention, methods for preparing protein-mediated mRNA targeting molecules of the invention include:
提供第一核苷酸片段、3’端带有蛋白基团的第二核苷酸片段；Provide a first nucleotide fragment and a second nucleotide fragment with a protein group at the 3' end;
在连接酶的作用下，将第一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接，制备得到蛋白质介导的mRNA靶向分子；其中第一核苷酸片段与第二核苷酸片段形成蛋白质介导的mRNA靶向分子中的mRNA部分。Under the action of ligase, the 3' end of the first nucleotide fragment is connected to the 5' end of the second nucleotide fragment with a protein group at the 3' end to prepare a protein-mediated mRNA targeting molecule. ; wherein the first nucleotide fragment and the second nucleotide fragment form the mRNA part in the protein-mediated mRNA targeting molecule.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法中，将第一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接时，采用夹板DNA辅助进行。According to a specific embodiment of the present invention, in the preparation method of the protein-mediated mRNA targeting molecule of the present invention, the 3' end of the first nucleotide fragment and the second nucleotide with a protein group at the 3' end are When ligating the 5' ends of the fragments, splint DNA was used to assist.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法中，所述夹板DNA包括第一夹板DNA与第二夹板DNA；第一夹板DNA与第一核苷酸片段的3’端序列互补，所述第二夹板DNA与第二核苷酸片段的5’端序列互补。According to a specific embodiment of the present invention, in the method for preparing a protein-mediated mRNA targeting molecule of the present invention, the splint DNA includes a first splint DNA and a second splint DNA; a first splint DNA and a first nucleotide fragment The 3' end sequence of the second splint DNA is complementary to the 5' end sequence of the second nucleotide fragment.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法中，将第一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接时，所述第一夹板DNA与第二夹板DNA之间无缝连接(即，第一夹板DNA对应在第一核苷酸片段上的序列与第二夹板DNA对应在第二核苷酸片段上的序列之间，无间隔碱基)。According to a specific embodiment of the present invention, in the preparation method of the protein-mediated mRNA targeting molecule of the present invention, the 3' end of the first nucleotide fragment and the second nucleotide with a protein group at the 3' end are When the 5' ends of the fragments are connected, the first splint DNA and the second splint DNA are seamlessly connected (that is, the first splint DNA corresponds to the sequence on the first nucleotide fragment and the second splint DNA corresponds to the sequence on the first nucleotide fragment. There are no intervening bases between sequences on a dinucleotide fragment).
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法中，所述第一夹板DNA与第一核苷酸片段的3’端5-40个碱基序列互补，所述第二夹板DNA与第二核苷酸片段的5’端5-40个碱基序列互补。换而言之，所述第一夹板DNA的长度为5-40个碱基，第二夹板DNA的长度为5-40个碱基。According to a specific embodiment of the present invention, in the preparation method of the protein-mediated mRNA targeting molecule of the present invention, the first splint DNA is complementary to the 5-40 base sequences of the 3' end of the first nucleotide fragment, The second splint DNA is complementary to the 5-40 base sequences at the 5' end of the second nucleotide fragment. In other words, the length of the first splint DNA is 5-40 bases, and the length of the second splint DNA is 5-40 bases.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法中，与第一夹板DNA互补的第一核苷酸片段的3’端序列为蛋白质介导的mRNA靶向分子的mRNA的3’UTR中的一部分序列。即，所述第一夹板DNA是针对目标mRNA的3’UTR而设计。According to a specific embodiment of the present invention, in the method for preparing a protein-mediated mRNA targeting molecule of the present invention, the 3' end sequence of the first nucleotide fragment complementary to the first splint DNA is a protein-mediated mRNA targeting molecule. A portion of the sequence in the 3'UTR of a molecule's mRNA. That is, the first splint DNA is designed for the 3'UTR of the target mRNA.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法中，与第二夹板DNA互补的第二核苷酸片段的5’端序列包括蛋白质介导的mRNA靶向分子的mRNA的3’UTR中的一部分序列，也可以包括或不包括部分或全部的PolyA。即，所述第二夹板DNA是针对目标mRNA的3’UTR、或是3’UTR与PolyA序列而设计。According to a specific embodiment of the present invention, in the method for preparing a protein-mediated mRNA targeting molecule of the present invention, the 5' end sequence of the second nucleotide fragment complementary to the second splint DNA includes a protein-mediated mRNA targeting molecule. A part of the sequence in the 3'UTR of the molecule's mRNA may or may not include part or all of PolyA. That is, the second splint DNA is designed for the 3'UTR of the target mRNA, or the 3'UTR and PolyA sequence.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法 中，第二核苷酸片段从5’端至3’端依次包括第三核苷酸片段与PolyA，所述第三核苷酸片段为蛋白质介导的mRNA靶向分子的mRNA的3’UTR中3’端序列的一部分。优选地，所述第三核苷酸片段的长度为5-100个碱基、优选5-80个碱基、进一步优选5-60个碱基、更进一步优选5-40个碱基。具体地，所述PolyA的长度为5-150、优选为5-120、进一步优选为5-100、更进一步优选为5-50。优选地，所述第二核苷酸片段的长度为10-150个碱基、优选10-120个碱基、进一步优选15-100个碱基、更进一步优选15-60个碱基。According to specific embodiments of the invention, methods for preparing protein-mediated mRNA targeting molecules of the invention , the second nucleotide fragment includes a third nucleotide fragment and PolyA in sequence from the 5' end to the 3' end, and the third nucleotide fragment is the 3'UTR of the mRNA of the protein-mediated mRNA targeting molecule. part of the 3' end sequence. Preferably, the length of the third nucleotide fragment is 5-100 bases, preferably 5-80 bases, further preferably 5-60 bases, even more preferably 5-40 bases. Specifically, the length of the PolyA is 5-150, preferably 5-120, more preferably 5-100, even more preferably 5-50. Preferably, the length of the second nucleotide fragment is 10-150 bases, preferably 10-120 bases, further preferably 15-100 bases, even more preferably 15-60 bases.
在本发明的一些具体实施方案中，所述第一夹板DNA包括但不限于如SEQ ID No.3所示，所述第二夹板DNA包括但不限于如SEQ ID No.4所示。In some embodiments of the present invention, the first splint DNA includes, but is not limited to, what is shown in SEQ ID No. 3, and the second splint DNA includes, but is not limited to, what is shown in SEQ ID No. 4.
根据本发明的具体实施方案，本发明的蛋白质介导的mRNA靶向分子的制备方法包括：According to a specific embodiment of the present invention, the preparation method of the protein-mediated mRNA targeting molecule of the present invention includes:
提供第一核苷酸片段：合成带有启动子序列、目的基因序列的质粒载体，行体外转录，得到第一核苷酸片段；所述第一核苷酸片段的结构从5’端至3’端依次包含5’帽子结构、带有Kozak序列的5’UTR、目的基因序列、3’UTR的5’端的一部分片段；Provide a first nucleotide fragment: synthesize a plasmid vector with a promoter sequence and a target gene sequence, perform in vitro transcription, and obtain a first nucleotide fragment; the structure of the first nucleotide fragment extends from the 5' end to the 3' end The 'end contains in turn a 5' cap structure, a 5'UTR with a Kozak sequence, a target gene sequence, and a part of the 5' end of the 3'UTR;
提供3’端带有蛋白基团的第二核苷酸片段：将靶向蛋白连接至第二核苷酸片段的3’端，得到3’端带有蛋白基团的第二核苷酸片段；其中，蛋白基团直接连接在第二核苷酸片段的3’端序列的PolyA上；Provide a second nucleotide fragment with a protein group at the 3' end: connect the targeting protein to the 3' end of the second nucleotide fragment to obtain a second nucleotide fragment with a protein group at the 3' end. ; Wherein, the protein group is directly connected to the PolyA of the 3' end sequence of the second nucleotide fragment;
配制反应体系，使得在退火反应中，第一核苷酸片段的3’端序列与第一夹板DNA互补结合，第二核苷酸片段的5’端序列与第二夹板DNA互补结合，在RNA连接酶(例如T4RNA连接酶)作用下，使第一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接，进一步经过DNA酶处理去除夹板DNA，得到蛋白质介导的mRNA靶向分子。在没有夹板DNA的情况下，mRNA分子与3’端带有蛋白基团的PolyA若直接进行偶联，会出现错配率高，而且连接效率低等问题。本发明的技术方案采用夹板DNA，能大大提高链接效率和准确率。Prepare the reaction system so that during the annealing reaction, the 3' end sequence of the first nucleotide fragment is complementary to the first splint DNA, and the 5' end sequence of the second nucleotide fragment is complementary to the second splint DNA. In the RNA Under the action of ligase (such as T4 RNA ligase), the 3' end of the first nucleotide fragment is connected to the 5' end of the second nucleotide fragment with a protein group at the 3' end, and further removed by DNase treatment. Splint DNA to obtain protein-mediated mRNA targeting molecules. In the absence of splint DNA, if the mRNA molecule is directly coupled to PolyA with a protein group at the 3' end, problems such as high mismatch rate and low connection efficiency will occur. The technical solution of the present invention uses splint DNA, which can greatly improve the linking efficiency and accuracy.
根据本发明的具体实施方案，优选地，所述启动子可以为T3或T7或SP6启动子。According to specific embodiments of the present invention, preferably, the promoter may be a T3 or T7 or SP6 promoter.
另一方面，本发明还提供了一种药物组合物，其包含：本发明所述的蛋白质介导的mRNA靶向分子，和药学上可接受的辅料。On the other hand, the present invention also provides a pharmaceutical composition, which includes: the protein-mediated mRNA targeting molecule of the present invention, and pharmaceutically acceptable excipients.
另一方面，本发明还提供了所述的药物组合物或所述的蛋白质介导的mRNA靶向分子在制备用于在哺乳动物或人类受试者中表达目标多肽的药物中的应用。换而言之，本发明还提供了一种在受试者中表达目标多肽的方法，该方法包括：将所述的药物组合 物或所述的蛋白质介导的mRNA靶向分子施用与受试者。优选地，所述受试者为哺乳动物或人。优选地，所述目标多肽可以是诸如GLP-1、尿酸氧化酶、胰岛素、胶原蛋白等蛋白替代药物中的一种或多种。On the other hand, the present invention also provides the use of the pharmaceutical composition or the protein-mediated mRNA targeting molecule in the preparation of drugs for expressing target polypeptides in mammals or human subjects. In other words, the present invention also provides a method for expressing a target polypeptide in a subject, the method comprising: combining the drug The substance or the protein-mediated mRNA targeting molecule is administered to the subject. Preferably, the subject is a mammal or a human. Preferably, the target polypeptide may be one or more of protein replacement drugs such as GLP-1, urate oxidase, insulin, collagen, etc.
根据本发明的具体实施方案，优选地，本发明通过使用带正电荷的蛋白实现了不依赖于传统脂质体、脂质纳米颗粒的体内递送。同时这些特定蛋白分子在靶细胞表面存在特异性受体，实现组织器官靶向递送。According to specific embodiments of the present invention, preferably, the present invention achieves in vivo delivery independent of traditional liposomes and lipid nanoparticles by using positively charged proteins. At the same time, these specific protein molecules have specific receptors on the surface of target cells to achieve targeted delivery to tissues and organs.
本发明的mRNA-蛋白靶向分子能够用于药物递送系统中，mRNA的3’端连接有特定蛋白基团，通过与靶细胞表面蛋白受体特异性结合引物细胞内吞，从而使mRNA进入细胞进行表达，解决了核酸药物在药物递送过程中的靶向递送的技术难题。The mRNA-protein targeting molecule of the present invention can be used in a drug delivery system. The 3' end of the mRNA is connected with a specific protein group. By specifically binding to the target cell surface protein receptor, the primer is endocytosed, thereby allowing the mRNA to enter the cell. Expression solves the technical problem of targeted delivery of nucleic acid drugs in the drug delivery process.
整体而言，采用本发明的技术方案，通过将表达目的基因的mRNA直接与偶联有蛋白基团的PolyA序列进行连接，合成出3’端带有蛋白基团的mRNA，在正电荷蛋白的作用下实现体内靶向递送，达到靶向性给药的目的，本发明的方法不依赖脂质体、脂质纳米颗粒等传统载体，方法更加简单可靠，解决了现有的mRNA与蛋白质的耦合难题，以及体内靶向递送的难题。进一步地，通过夹板DNA的设计能大大提高连接效率和准确率。Overall, using the technical solution of the present invention, by directly connecting the mRNA expressing the target gene with the PolyA sequence coupled with the protein group, the mRNA with the protein group at the 3' end is synthesized. Targeted delivery in the body is achieved under the action of the invention to achieve the purpose of targeted drug delivery. The method of the present invention does not rely on traditional carriers such as liposomes and lipid nanoparticles. The method is simpler and more reliable, and solves the existing coupling between mRNA and protein. problems, as well as the problems of targeted delivery in vivo. Furthermore, the connection efficiency and accuracy can be greatly improved through the design of splint DNA.
附图说明Description of drawings
图1是本发明实施例1一种mRNA-蛋白靶向分子的制备方法的流程示意图；其中(a)为质粒DNA中DNA片段以及进行体外转录得到mRNA分子的结构示意图；(b)为mRNA分子与3’端带有VSV-G的PolyA待结合的示意图；(c)为得到的mRNA-蛋白靶向分子的示意图。Figure 1 is a schematic flow chart of a method for preparing an mRNA-protein targeting molecule in Embodiment 1 of the present invention; (a) is a schematic structural diagram of a DNA fragment in plasmid DNA and an mRNA molecule obtained by in vitro transcription; (b) is an mRNA molecule A schematic diagram of PolyA with VSV-G at the 3' end to be combined; (c) is a schematic diagram of the resulting mRNA-protein targeting molecule.
图2是mRNA-VSV-G靶向分子与mRNA分子转染细胞对比。Figure 2 is a comparison of cells transfected with mRNA-VSV-G targeting molecules and mRNA molecules.
图3是本发明实施例2一种mRNA-蛋白靶向分子的制备方法的流程示意图；其中(a)为质粒DNA中DNA片段以及进行体外转录得到mRNA分子的结构示意图；(b)为mRNA分子与3’端带有抗ERBB2抗体的PolyA待结合的示意图；(c)为得到的mRNA-蛋白靶向分子的示意图。Figure 3 is a schematic flow chart of a method for preparing an mRNA-protein targeting molecule in Embodiment 2 of the present invention; (a) is a schematic structural diagram of the DNA fragment in the plasmid DNA and the mRNA molecule obtained by in vitro transcription; (b) is the mRNA molecule A schematic diagram of PolyA to be combined with an anti-ERBB2 antibody at the 3' end; (c) is a schematic diagram of the resulting mRNA-protein targeting molecule.
图4是mRNA-ERBB2抗体靶向分子转染无ERBB2表达的细胞与细胞膜上表达ERBB2的细胞对比。Figure 4 is a comparison of cells transfected with mRNA-ERBB2 antibody targeting molecules without ERBB2 expression and cells expressing ERBB2 on the cell membrane.
图5是mRNA-CD22抗体靶向分子转染无CD22表达细胞与CD22+细胞对比。Figure 5 is a comparison of cells without CD22 expression and CD22+ cells transfected with mRNA-CD22 antibody targeting molecules.
图6显示mRNA-VSV-G分子结构和连接效率结果。 Figure 6 shows the molecular structure and ligation efficiency results of mRNA-VSV-G.
具体实施方式Detailed ways
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but this should not be understood as limiting the implementable scope of the present invention.
除非另外专门定义，本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。实施例中未特别注明的方法操作，按照所属领域现有技术的常规操作或商厂说明书建议的操作进行。Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Method operations not specifically noted in the examples should be performed in accordance with conventional operations in the art or operations recommended by the manufacturer's instructions.
实施例1、mRNA-VSV-G蛋白靶向分子的制备Example 1. Preparation of mRNA-VSV-G protein targeting molecules
本实施例提供一种mRNA-蛋白靶向分子，即mRNA-VSV-G蛋白靶向分子，其是mRNA的3’端PolyA上带有VSV-G蛋白的结构。其中所述mRNA分子的序列包含5’帽子、带有Kozak序列的5'UTR、目的基因序列、3'UTR和PolyA。This embodiment provides an mRNA-protein targeting molecule, namely, an mRNA-VSV-G protein targeting molecule, which is a structure with VSV-G protein on the 3' end PolyA of the mRNA. The sequence of the mRNA molecule includes a 5' cap, a 5'UTR with a Kozak sequence, a target gene sequence, a 3'UTR and PolyA.
如图1所示，所述mRNA-蛋白靶向分子采用以下步骤制备得到：As shown in Figure 1, the mRNA-protein targeting molecule is prepared using the following steps:
步骤S1，设计并合成一段带有启动子序列、目的基因序列的质粒载体，质粒载体购自金唯智科技有限公司的pUC-GW-KANA，插入位点EcoRVI，各部分链接顺序是5’UTR-mRNA编码基因序列-3’UTR；Step S1, design and synthesize a plasmid vector with a promoter sequence and target gene sequence. The plasmid vector was purchased from pUC-GW-KANA of Jinweizhi Technology Co., Ltd., the insertion site is EcoRVI, and the linking sequence of each part is 5'UTR- mRNA coding gene sequence-3'UTR;
步骤S2，以步骤S1的质粒载体为模板进行体外转录，得到mRNA分子，所述mRNA分子的序列包含5’帽子、包含Kozak序列的5'UTR、目的基因序列、3'UTR；Step S2, use the plasmid vector of Step S1 as a template to perform in vitro transcription to obtain an mRNA molecule whose sequence includes a 5' cap, a 5'UTR including a Kozak sequence, a target gene sequence, and a 3'UTR;
步骤S3，在连接酶的作用下，mRNA分子与VSV-G修饰的多聚腺苷酸结合制备得到mRNA-VSV-G靶向分子。Step S3: Under the action of ligase, the mRNA molecule is combined with the VSV-G modified polyadenylate to prepare the mRNA-VSV-G targeting molecule.
本实施例中，步骤S1所设计的带有启动子序列、目的基因序列的质粒载体中，所述启动子的核苷酸序列如SEQ ID No.1所示。进一步地，所述目的基因的核苷酸序列如SEQ ID No.2所示。In this example, in the plasmid vector with a promoter sequence and a target gene sequence designed in step S1, the nucleotide sequence of the promoter is shown in SEQ ID No. 1. Further, the nucleotide sequence of the target gene is shown in SEQ ID No. 2.
SEQ ID No.1：taatacgactcactatagg；SEQ ID No.1: taatacgactcactatagg;
SEQ ID No.2：

SEQ ID No.2:

本实施例中，步骤S2的mRNA分子的具体合成方法如下：In this example, the specific synthesis method of the mRNA molecule in step S2 is as follows:
1、构建好的表达质粒按如下反应体系进行DNA模板的扩增：1. The constructed expression plasmid is used to amplify the DNA template according to the following reaction system:
反应体积，50μl(为单个管的反应体积，一次同时反应多管)；Reaction volume, 50 μl (reaction volume of a single tube, multiple tubes can be reacted at the same time);
所述PCR扩增的体系(50μl)：PrimeSTAR Max Premix(2×)25μl、引物F(SEQ ID No.6：TTGGACCCTCGTACAGAAGCTAATACG，10μmol/L)1.2μl、引物R(SEQ ID No.7：TACCGGTTAGCTTCCTACTCAGGCTTTATTCAAAGACCA，10μmol/L)1.2μl、DNA模板(1ng/μl)1μl和水21.6μl。The PCR amplification system (50 μl): PrimeSTAR Max Premix (2×) 25 μl, primer F (SEQ ID No. 6: TTGGACCCCTCGTACAGAAGCTAATACG, 10 μmol/L) 1.2 μl, primer R (SEQ ID No. 7: TACCGGTTAGCTTCCTACTCAGGCTTTATTCAAAGACCA, 10 μmol) /L) 1.2μl, DNA template (1ng/μl) 1μl and water 21.6μl.
所述PCR的扩增程序如下：预变性98℃3min；变性98℃10s，退火60℃5s，延伸72℃2min，34个循环；最后延伸72℃，10min。The amplification procedure of the PCR is as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; and final extension at 72°C for 10 minutes.
反应结束后，将反应液合并于1.5ml Tube管中。取10μl进行DNA琼脂糖凝胶电泳(1.5％琼脂糖，5V/min，40min)。根据电泳目的条带的大小对反应成功与否进行确认。After the reaction is completed, combine the reaction solutions into a 1.5ml Tube tube. Take 10 μl for DNA agarose gel electrophoresis (1.5% agarose, 5V/min, 40min). Confirm the success of the reaction based on the size of the electrophoresis target band.
合格标准：电泳检测出现单一的条带，且大小正确。Eligibility criteria: A single band appears in electrophoresis detection and the size is correct.
2、DNA模板超滤2. DNA template ultrafiltration
利用Millipore 30Kd超滤管浓缩上述获得的DNA模板。Concentrate the DNA template obtained above using Millipore 30Kd ultrafiltration tube.
3、DNA模板FPLC纯化3. DNA template FPLC purification
将上述超滤得到的DNA，加入等体积的苯酚/氯仿/异戊醇混合液(苯酚/氯仿/异戊醇＝25/24/1)，充分震荡后，12000g离心15min。Add an equal volume of phenol/chloroform/isoamyl alcohol mixture (phenol/chloroform/isoamyl alcohol = 25/24/1) to the DNA obtained by ultrafiltration above. After shaking thoroughly, centrifuge at 12000g for 15 min.
去掉沉淀，转移上清至新的离心管中，加入上清体积的1/10 3M NaAc(pH5.2)，混匀，然后加入2倍体积的无水乙醇，混匀，至于-20℃静置30min。Remove the precipitate, transfer the supernatant to a new centrifuge tube, add 1/10 of the volume of the supernatant 3M NaAc (pH5.2), mix well, then add 2 times the volume of absolute ethanol, mix well, and stand at -20°C. Leave for 30 minutes.
4℃，12000g离心10min，弃上清。Centrifuge at 12000g for 10 minutes at 4°C and discard the supernatant.
用70％乙醇洗涤沉淀，12000g离心5min，取上清，于超净台晾干5min。Wash the precipitate with 70% ethanol, centrifuge at 12000g for 5 minutes, take the supernatant, and dry it on a clean bench for 5 minutes.
用适当的RNase-free水溶解纯化后的DNA模板。Dissolve the purified DNA template in appropriate RNase-free water.
用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40min)。Use NanoDrop to detect the concentration of the purified template and the ratios of 260/280 and 260/230. Samples were taken for DNA agarose gel electrophoresis detection (1.5% agarose, 5V/min, 40min).
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。Qualifying standards: 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.
4、FPLC纯化后模板超滤4. Template ultrafiltration after FPLC purification
Millipore 30Kd超滤管浓缩FPLC纯化后的DNA模板，用RNase-free水洗脱溶解。 用NanoDrop检测超滤后模板的浓度，以及260/280、260/230的比值。最终用RNase-free水稀释至150ng/μl。Millipore 30Kd ultrafiltration tube concentrates the FPLC-purified DNA template, and elutes and dissolves it with RNase-free water. Use NanoDrop to detect the concentration of the template after ultrafiltration and the ratios of 260/280 and 260/230. Finally dilute to 150ng/μl with RNase-free water.
5、mRNA的体外合成5. In vitro synthesis of mRNA
在恒温反应器中，进行mRNA的体外合成。In a constant-temperature reactor, in vitro synthesis of mRNA is performed.
按照如下合成体系进行(反应试剂按照从上至下添加)：Proceed according to the following synthesis system (reaction reagents are added from top to bottom):
反应体积，1600μl(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)：RNA-free水440μl、7.5mM的ATP 160μl、7.5mM的UTP 160μl、7.5mM的CTP 160μl、7.5mM的GTP 160μl、7.5mM的M7G(2’OMeA)pG 160μl、150ng/μl的DNA模板40μl、10×Buffer 160μl和Enzyme Mix 160μl。Reaction volume, 1600μl (placed in a 2ml RNase-free Tube, it is the reaction volume of a single tube, multiple tubes can be reacted at the same time): RNA-free water 440μl, 7.5mM ATP 160μl, 7.5mM UTP 160μl, 7.5mM CTP 160μl, 7.5mM GTP 160μl, 7.5mM M7G (2'OMeA)pG 160μl, 150ng/μl DNA template 40μl, 10×Buffer 160μl and Enzyme Mix 160μl.
所述RNA体外合成的程序为37℃，10h。The procedure for in vitro synthesis of RNA is 37°C, 10 h.
6、DNase I消化去除DNA模板6. DNase I digestion to remove DNA template
向mRNA体外合成后的每个Tube管中各加入120μl DNase I。Add 120μl DNase I to each Tube tube after in vitro synthesis of mRNA.
上下颠倒10次混匀，1000rpm离心10s。Mix by inverting 10 times and centrifuge at 1000rpm for 10s.
重新置于恒温反应器中，37℃，1h。Place again in the thermostatic reactor at 37°C for 1 hour.
7、mRNA沉淀回收7. mRNA precipitation and recovery
向上一步骤中的每个50ml Tube管中，加入等体积的醋酸铵溶液。Add an equal volume of ammonium acetate solution to each 50ml tube from the previous step.
上下颠倒10次混匀。Mix by inverting 10 times.
置于-20℃2h，沉淀。Place at -20°C for 2h to precipitate.
17000g，4℃离心，30min。Centrifuge at 17000g, 4℃, 30min.
去掉上清，用70％乙醇洗涤沉淀。Remove the supernatant and wash the pellet with 70% ethanol.
17000g，4℃离心，10min。Centrifuge at 17000g, 4℃, 10min.
去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。Remove 70% ethanol, evaporate to dryness in a clean bench, and add 20 ml of RNase-free water to each tube.
静置10min后，用枪头轻吹混匀。After letting it stand for 10 minutes, blow gently with the tip of the pipette to mix.
用NanoDrop检测回收后的mRNA浓度为5μg/μl，A260/A280为1.90、A260/A230为2.0。The concentration of recovered mRNA detected by NanoDrop was 5 μg/μl, A260/A280 was 1.90, and A260/A230 was 2.0.
取1μl，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。Take 1 μl, dilute it 10 times, and perform RNA ScreenTape assay and agarose gel electrophoresis to check the integrity of the fragments.
8、LiCl沉淀纯化mRNA8. LiCl precipitation and purification of mRNA
将上一步骤中回收的mRNA按照其1.5倍体积加入Rnase-free水，混匀。Add 1.5 times the volume of the mRNA recovered in the previous step to RNase-free water and mix well.
加入原mRNA 1.5倍体积-20预冷的LiCl溶液，混匀。 Add 1.5 times the volume of the original mRNA -20 pre-cooled LiCl solution and mix well.
然后于-20℃静至2h。Then stand at -20°C for 2 hours.
16000g离心20min。Centrifuge at 16000g for 20 minutes.
弃上清，用70％乙醇洗涤沉淀，16000g离心15min。Discard the supernatant, wash the precipitate with 70% ethanol, and centrifuge at 16,000 g for 15 min.
取上清，于超净台晾干5min。Take the supernatant and dry it on a clean bench for 5 minutes.
用适当的RNase-free水溶解纯化后的mRNA。Dissolve purified mRNA in appropriate RNase-free water.
本实施例中，所得到的mRNA序列如SEQ ID No.5所示。In this example, the obtained mRNA sequence is shown in SEQ ID No. 5.
SEQ ID No.5：
SEQ ID No.5:
其中，Cap结构为3′-O-Me-m7G(5')ppp(5')G。Among them, the Cap structure is 3'-O-Me-m7G(5')ppp(5')G.
本实施例中，步骤S3中，所述VSV-G修饰的多聚腺苷酸采用多聚腺苷酸(10个A组成的PolyA)的3’端暴露的氨基，与蛋白基团的氨基酸的羧基进行反应得到。通常情况下，反应时可加CDI，室温下反应2个小时。In this example, in step S3, the VSV-G modified polyadenylic acid uses the exposed amino group at the 3' end of the polyadenylic acid (PolyA composed of 10 A's), and the amino acid residues of the protein group. The carboxyl group is reacted. Normally, CDI can be added during the reaction and the reaction can be carried out at room temperature for 2 hours.
本实施例中，步骤S3具体过程包括：In this embodiment, the specific process of step S3 includes:
构建如下反应体系：1M Tris-HCl(pH 7.5)，1mM MgCl2，5％(wt/vol)PEG 8000，20units/μl RNase Inhibitor，100units/μl T4RNA Ligase 1，1mM ATP，2.5mM mRNA以及VSV-G修饰的多聚腺苷酸；其中，mRNA和VSV-G修饰的多聚腺苷酸的摩尔比控制在 1：2；Construct the following reaction system: 1M Tris-HCl (pH 7.5), 1mM MgCl 2 , 5% (wt/vol) PEG 8000, 20units/μl RNase Inhibitor, 100units/μl T4RNA Ligase 1, 1mM ATP, 2.5mM mRNA and VSV- G-modified polyadenylate; wherein, the molar ratio of mRNA and VSV-G-modified polyadenylate is controlled at 1:2;
25℃反应30分钟(或者16℃反应2小时至32小时之间)；React at 25°C for 30 minutes (or 16°C for between 2 hours and 32 hours);
在反应产物中加入氯化锂溶液至氯化锂终浓度为0.5M，可得到沉淀产物，即为本发明的mRNA-VSV-G靶向分子。Lithium chloride solution is added to the reaction product until the final concentration of lithium chloride is 0.5M, and a precipitated product can be obtained, which is the mRNA-VSV-G targeting molecule of the present invention.
本实施例制备得到的mRNA-VSV-G靶向分子，可以用于制备特异性药物递送的mRNA药物中。所述特异性药物递送的mRNA药物中除本发明的mRNA-VSV-G靶向分子外，还可包括药学上可接受的载体，例如保护剂、缓冲液等中的一种或多种。mRNA-VSV-G靶向分子的3’端连接有VSV-G蛋白，通过VSV-G介导细胞内吞，从而使mRNA进入细胞进行表达。具体操作步骤为：293T细胞传代后约24小时，观察6孔板内的细胞状态，汇合度在90％左右即可进行转染。取两份200μl opti-MEM，分别加入10μg编码GFP的mRNA(作为对照)和本实施例中制备的mRNA-VSV-G，用枪头轻轻吹打充分混匀，静置10min，得到配制好的转染体系。将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。转染后6h换液，吸掉旧的培养基，每孔换为2ml新鲜培养基(90％DMEM+10％FBS)。转染后24h显微镜下观察。The mRNA-VSV-G targeting molecule prepared in this example can be used to prepare mRNA drugs for specific drug delivery. In addition to the mRNA-VSV-G targeting molecule of the present invention, the mRNA drug for specific drug delivery may also include one or more pharmaceutically acceptable carriers, such as protective agents, buffers, etc. The 3' end of the mRNA-VSV-G targeting molecule is connected to the VSV-G protein, which mediates endocytosis through VSV-G, allowing the mRNA to enter cells for expression. The specific operation steps are: about 24 hours after passage of 293T cells, observe the cell status in the 6-well plate. Transfection can be carried out when the confluence is about 90%. Take two portions of 200 μl opti-MEM, add 10 μg of GFP-encoding mRNA (as a control) and the mRNA-VSV-G prepared in this example, gently pipet with a pipette tip to mix thoroughly, and let stand for 10 minutes to obtain the prepared preparation. transfection system. Directly and evenly drop the prepared transfection system into the cultured cells, and then shake it back and forth to make the transfection system evenly distributed on the cells. The medium was changed 6 hours after transfection, the old medium was aspirated, and each well was replaced with 2 ml of fresh medium (90% DMEM+10% FBS). Observe under a microscope 24 hours after transfection.
如图2所示，编码GFP的mRNA转染体系加入细胞后24小时，荧光显微镜下未能发现GFP的存在，即未能实现GFP的表达；编码GFP的mRNA-VSV-G转染体系加入细胞后24小时，荧光显微镜下明显看到GFP的存在，即实现了GFP的表达。As shown in Figure 2, 24 hours after the mRNA transfection system encoding GFP was added to the cells, the presence of GFP could not be found under a fluorescence microscope, that is, the expression of GFP could not be achieved; the mRNA-VSV-G transfection system encoding GFP was added to the cells. After 24 hours, the presence of GFP was clearly visible under a fluorescence microscope, indicating that the expression of GFP was achieved.
实施例2、mRNA-抗ERBB2抗体靶向分子的制备Example 2. Preparation of mRNA-anti-ERBB2 antibody targeting molecules
本实施例提供一种mRNA-蛋白靶向分子，即mRNA-抗ERBB2抗体靶向分子，其为mRNA的3’端PolyA上带有抗ERBB2抗体的产物。其中，mRNA的序列包含5’帽子、带有Kozak序列的5'UTR、目的基因序列、3'UTR、PolyA(mRNA的序列与实施例1相同)。PolyA的3’端带有抗ERBB2抗体。This embodiment provides an mRNA-protein targeting molecule, that is, an mRNA-anti-ERBB2 antibody targeting molecule, which is a product with an anti-ERBB2 antibody on the 3' end PolyA of the mRNA. Among them, the sequence of the mRNA includes a 5' cap, a 5'UTR with a Kozak sequence, a target gene sequence, a 3'UTR, and PolyA (the sequence of the mRNA is the same as in Example 1). The 3’ end of PolyA carries an anti-ERBB2 antibody.
如图3所示，所述mRNA-抗ERBB2抗体靶向分子采用以下步骤制备得到：As shown in Figure 3, the mRNA-anti-ERBB2 antibody targeting molecule is prepared using the following steps:
步骤S1，设计并合成一段带有启动子序列、目的基因序列和第一夹板DNA序列的互补序列的质粒载体；Step S1, design and synthesize a plasmid vector containing a promoter sequence, a complementary sequence of the target gene sequence and the first splint DNA sequence;
步骤S2，以步骤S1的质粒载体为模板进行体外转录，得到mRNA分子。所述mRNA分子的序列包含依次连接的5’帽子、带有Kozak序列的5'UTR、目的基因序列、3'UTR的一部分(目标mRNA的3’UTR中5’端序列部分，包括与第一夹板DNA序列的互补序列对应的mRNA序列)。mRNA合成方法与实施例1相同； Step S2: Use the plasmid vector of step S1 as a template to perform in vitro transcription to obtain mRNA molecules. The sequence of the mRNA molecule includes a 5' cap connected in sequence, a 5'UTR with a Kozak sequence, a target gene sequence, and a part of the 3'UTR (the 5' end sequence part of the 3'UTR of the target mRNA, including the sequence with the first The complementary sequence of the splint DNA sequence corresponds to the mRNA sequence). The mRNA synthesis method is the same as in Example 1;
步骤S3，3’端带有蛋白基团的PolyA片段，其具有10个A，PolyA的5’端带有一段能与第二夹板DNA序列互补配对的RNA序列(即，目标mRNA的3’UTR中3’端序列的一部分)。所述第一夹板DNA序列和第二夹板DNA序列无缝连接构成夹板DNA。在退火反应中，带有第一夹板DNA序列的互补序列的mRNA分子与带有与第二夹板DNA序列互补配对序列的PolyA分子分别与夹板DNA互补结合，在T4RNA连接酶I作用下，所述mRNA分子的3’端羟基与带有抗ERBB2抗体的PolyA的5’端磷酸基团连接，经过DNase I处理后，得到带有抗ERBB2抗体的mRNA靶向分子。Step S3, a PolyA fragment with a protein group at the 3' end, which has 10 A's, and the 5' end of PolyA has an RNA sequence that can be complementary to the second splint DNA sequence (i.e., the 3'UTR of the target mRNA part of the 3' end sequence). The first splint DNA sequence and the second splint DNA sequence are seamlessly connected to form splint DNA. In the annealing reaction, the mRNA molecule with the complementary sequence of the first splint DNA sequence and the PolyA molecule with the complementary pairing sequence with the second splint DNA sequence are complementary to the splint DNA respectively. Under the action of T4 RNA ligase I, the The 3'-end hydroxyl group of the mRNA molecule is connected to the 5'-end phosphate group of PolyA with anti-ERBB2 antibody. After DNase I treatment, an mRNA targeting molecule with anti-ERBB2 antibody is obtained.
本实施例中，所述第一夹板DNA序列如SEQ ID No.3所示，所述第二夹板DNA序列如SEQ ID No.4所示。In this embodiment, the DNA sequence of the first splint is shown in SEQ ID No. 3, and the DNA sequence of the second splint is shown in SEQ ID No. 4.
SEQ ID No.3：5’-ttcaaagacc-3’；SEQ ID No.3: 5’-ttcaaagacc-3’;
SEQ ID No.4：5’-tcaggcttta-3’。SEQ ID No.4: 5’-tcaggcttta-3’.
所述启动子的序列如SEQ ID No.1所示。The sequence of the promoter is shown in SEQ ID No. 1.
所述目的基因如SEQ ID No.2所示。The target gene is shown in SEQ ID No. 2.
进一步的，所述连接酶为T4RNA Ligase I。Further, the ligase is T4RNA Ligase I.
本实施例得到的mRNA-蛋白靶向分子可以用于制备特异性药物递送的mRNA药物中。3’端连接有抗ERBB2抗体，通过抗ERBB2抗体与表面表达ERBB2的癌细胞特异性结合引起细胞内吞，从而使mRNA进入细胞进行表达。具体操作步骤如下：293T(无ERBB2表达)和H1781细胞(ERBB2膜定位)传代后约24小时，观察6孔板内的细胞状态，汇合度在90％左右即可进行转染。取两份200μl opti-MEM，分别加入10μg编码GFP的mRNA-ERBB2抗体复合物，用枪头轻轻吹打充分混匀，静置10min。将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。转染后6h换液，吸掉旧的培养基，每孔换为2ml新鲜培养基(90％DMEM+10％FBS)。转染后24h显微镜下观察。如图4所示，mRNA-ERBB2抗体复合物能够特异性转染表面表达ERBB2的H1781细胞，不能转染普通293T细胞。The mRNA-protein targeting molecules obtained in this example can be used to prepare mRNA drugs for specific drug delivery. An anti-ERBB2 antibody is connected to the 3' end, and the anti-ERBB2 antibody specifically binds to cancer cells expressing ERBB2 on their surface to cause endocytosis, allowing the mRNA to enter the cells for expression. The specific steps are as follows: About 24 hours after passage of 293T (no ERBB2 expression) and H1781 cells (ERBB2 membrane localization), observe the cell status in the 6-well plate. Transfection can be performed when the confluence is about 90%. Take two 200 μl opti-MEM, add 10 μg of GFP-encoding mRNA-ERBB2 antibody complex respectively, mix thoroughly by gently pipetting with the pipe tip, and let stand for 10 minutes. Directly and evenly drop the prepared transfection system into the cultured cells, and then shake it back and forth to make the transfection system evenly distributed on the cells. The medium was changed 6 hours after transfection, the old medium was aspirated, and each well was replaced with 2 ml of fresh medium (90% DMEM+10% FBS). Observe under a microscope 24 hours after transfection. As shown in Figure 4, the mRNA-ERBB2 antibody complex can specifically transfect H1781 cells expressing ERBB2 on the surface, but cannot transfect ordinary 293T cells.
实施例3、mRNA-抗CD22抗体靶向分子的制备Example 3. Preparation of mRNA-anti-CD22 antibody targeting molecules
本实施例提供一种mRNA-蛋白靶向分子，即mRNA-抗CD22抗体靶向分子，其为mRNA分子与抗CD22抗体的连接产物。其中，所述mRNA分子的序列包含5’帽子、带有Kozak序列的5'UTR、目的基因序列、3'UTR、PolyA(mRNA的序列与实施例1相同)。所述mRNA分子的3’端PolyA上带有抗CD22抗体。所述mRNA-蛋白靶向分子采用与实施例2相同步骤制备得到： This embodiment provides an mRNA-protein targeting molecule, that is, an mRNA-anti-CD22 antibody targeting molecule, which is a connection product of an mRNA molecule and an anti-CD22 antibody. Wherein, the sequence of the mRNA molecule includes a 5' cap, a 5'UTR with a Kozak sequence, a target gene sequence, a 3'UTR, and PolyA (the sequence of the mRNA is the same as in Example 1). The 3' end PolyA of the mRNA molecule carries an anti-CD22 antibody. The mRNA-protein targeting molecule is prepared using the same steps as in Example 2:
步骤S1，设计并合成一段带有启动子序列、目的基因序列和第一夹板DNA序列的互补序列的质粒载体；Step S1, design and synthesize a plasmid vector containing a promoter sequence, a complementary sequence of the target gene sequence and the first splint DNA sequence;
步骤S2，以步骤S1的质粒载体为模板进行体外转录，得到mRNA分子，所述mRNA分子的序列包含依次连接的5’帽子、带有Kozak序列的5'UTR、目的基因序列、3'UTR的5’端部分序列(包括第一夹板DNA序列的互补序列对应的RNA序列)，mRNA体外转录及纯化方案见实施例1；Step S2: Use the plasmid vector of Step S1 as a template for in vitro transcription to obtain an mRNA molecule. The sequence of the mRNA molecule includes a 5' cap connected in sequence, a 5'UTR with a Kozak sequence, a target gene sequence, and a 3'UTR. The 5' end partial sequence (including the RNA sequence corresponding to the complementary sequence of the first splint DNA sequence), the in vitro transcription and purification scheme of mRNA are shown in Example 1;
步骤S3，3’端带有蛋白基团的PolyA片段，其5’端带有一段能与第二夹板DNA序列互补配对的RNA序列；在退火反应中，带有第一夹板DNA序列的互补序列的mRNA分子与带有与第二夹板DNA序列互补配对序列的PolyA分子分别与夹板DNA互补结合，在T4RNA连接酶作用下，所述mRNA分子的3’端羟基与带有抗CD22抗体的PolyA的5’端磷酸基团连接，经过DNase I处理后，得到带有抗CD22抗体的mRNA靶向分子。Step S3, the PolyA fragment with a protein group at the 3' end has an RNA sequence that can be complementary to the DNA sequence of the second splint at the 5' end; during the annealing reaction, the complementary sequence of the DNA sequence of the first splint is carried The mRNA molecule and the PolyA molecule with the sequence complementary to the second splint DNA sequence are complementary to the splint DNA. Under the action of T4 RNA ligase, the 3' end hydroxyl group of the mRNA molecule and the PolyA molecule with the anti-CD22 antibody The 5' end phosphate group is connected and treated with DNase I to obtain an mRNA targeting molecule with an anti-CD22 antibody.
本实施例中，所述第一夹板DNA序列如SEQ ID No.3所示，所述第二夹板DNA序列如SEQ ID No.4所示。In this embodiment, the DNA sequence of the first splint is shown in SEQ ID No. 3, and the DNA sequence of the second splint is shown in SEQ ID No. 4.
SEQ ID No.3：5’-ttcaaagacc-3’；SEQ ID No.3: 5’-ttcaaagacc-3’;
SEQ ID No.4：5’-tcaggcttta-3’。SEQ ID No.4: 5’-tcaggcttta-3’.
所述启动子的序列如SEQ ID No.1所示。The sequence of the promoter is shown in SEQ ID No. 1.
所述目的基因为如SEQ ID No.2所示。The target gene is shown in SEQ ID No. 2.
进一步的，所述连接酶为T4RNA Ligase I。Further, the ligase is T4RNA Ligase I.
本实施例得到的mRNA-蛋白靶向分子可以用于制备特异性药物递送的mRNA药物中。3’端连接有抗CD22抗体，通过抗CD22抗体与表面表达CD22的癌细胞特异性结合引起细胞内吞，从而使mRNA进入细胞进行表达。具体操作步骤如下：293T(无CD22表达)和CA46细胞(CD22+)传代后约24小时，观察6孔板内的细胞状态，汇合度在90％左右即可进行转染。取两份200μl opti-MEM，分别加入10μg编码GFP的mRNA-CD22抗体复合物，用枪头轻轻吹打充分混匀，静置10min。将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。转染后6h换液，吸掉旧的培养基，每孔换为2ml新鲜培养基(90％DMEM+10％FBS)。转染后24h显微镜下观察。如图5所示，mRNA-CD22抗体复合物能够特异性转染表面表达CD22的CA46细胞，不能转染普通293T细胞。The mRNA-protein targeting molecules obtained in this example can be used to prepare mRNA drugs for specific drug delivery. An anti-CD22 antibody is connected to the 3' end, and the anti-CD22 antibody specifically binds to cancer cells expressing CD22 on their surface to cause endocytosis, allowing the mRNA to enter the cells for expression. The specific steps are as follows: About 24 hours after passage of 293T (no CD22 expression) and CA46 cells (CD22+), observe the cell status in the 6-well plate. Transfection can be carried out when the confluence is about 90%. Take two 200 μl opti-MEM, add 10 μg of GFP-encoding mRNA-CD22 antibody complex respectively, mix thoroughly by gently pipetting with the pipe tip, and let stand for 10 minutes. Directly and evenly drop the prepared transfection system into the cultured cells, and then shake it back and forth to make the transfection system evenly distributed on the cells. The medium was changed 6 hours after transfection, the old medium was aspirated, and each well was replaced with 2 ml of fresh medium (90% DMEM+10% FBS). Observe under a microscope 24 hours after transfection. As shown in Figure 5, the mRNA-CD22 antibody complex can specifically transfect CA46 cells expressing CD22 on their surface, but cannot transfect ordinary 293T cells.
实施例4、mRNA-VSV-G分子结构检测Example 4. Detection of mRNA-VSV-G molecular structure
采用以下方法对mRNA-VSV-G分子结构进行检测： The following methods were used to detect the molecular structure of mRNA-VSV-G:
将抗VSV-G抗体(5μg)加入实施例1制备的mRNA-VSV-G产物中(1mg)，VSV-G蛋白作为阴性对照，4℃轻柔搅动孵育过夜；Add anti-VSV-G antibody (5 μg) to the mRNA-VSV-G product prepared in Example 1 (1 mg), and VSV-G protein is used as a negative control, and incubate overnight at 4°C with gentle stirring;
加入protein A/G磁珠(40μL)，4℃轻柔搅动孵育1h；Add protein A/G magnetic beads (40 μL), and incubate for 1 hour at 4°C with gentle stirring;
2,500rpm离心30s沉淀磁珠，移去上清液，在500μl RIP缓冲液中重悬磁珠；Centrifuge at 2,500 rpm for 30 seconds to precipitate the magnetic beads, remove the supernatant, and resuspend the magnetic beads in 500 μl RIP buffer;
磁珠在RIP缓冲液中重复清洗共三次，随后在PBS中清洗一次；The magnetic beads were washed three times in RIP buffer and then once in PBS;
对免疫沉淀后RBP上结合的RNA进行纯化，在1mL TRIzol RNA提取试剂中重悬磁珠，分离共沉淀的RNA；Purify the RNA bound to RBP after immunoprecipitation, resuspend the magnetic beads in 1mL TRIzol RNA extraction reagent, and isolate the co-precipitated RNA;
使用不含核酸酶的水(20μL)洗脱RNA。将约20μL经DEPC处理的水加入mRNA沉淀中；Elute RNA using nuclease-free water (20 μL). Add approximately 20 μL of DEPC-treated water to the mRNA precipitation;
将mRNA逆转录(RT)为cDNA，并进行RT-PCR分析，同质量的mRNA样品作为阳性对照，检测结合在VSV-G上的mRNA的丰度。其中RT-PCR中使用的引物为F:AAGGAGGACGGCAACATCCTGGGG(SEQ ID No.8)，R:TACAGCTCGTCCATGCCGAGAGTG(SEQ ID No.9)。The mRNA was reverse transcribed (RT) into cDNA, and RT-PCR analysis was performed. The same quality mRNA sample was used as a positive control to detect the abundance of mRNA bound to VSV-G. The primers used in RT-PCR are F: AAGGAGGACGGCAACATCCTGGGG (SEQ ID No. 8), R: TACAGCTCGTCCATGCCGAGAGTG (SEQ ID No. 9).
检测结果如图6所示。The detection results are shown in Figure 6.
综上所述，本发明主要通过多聚腺苷酸和蛋白递送载体的连接，以及多聚腺苷酸/蛋白复合物与mRNA分子末端的连接方法，可实现最终产物可以用来靶向递送mRNA。 To sum up, the present invention can realize that the final product can be used for targeted delivery of mRNA mainly through the connection of poly(A) and protein delivery carriers, and the connection method of poly(A)/protein complex and the end of the mRNA molecule. .

Claims (13)

  1. 一种蛋白质介导的mRNA靶向分子，该蛋白质介导的mRNA靶向分子由mRNA通过其3’端的PolyA与靶向蛋白基团连接而成，其中，mRNA的结构从5’端至3’端依次包含5’帽子结构、带有Kozak序列的5’UTR、目的基因序列、3’UTR以及PolyA。A protein-mediated mRNA targeting molecule. The protein-mediated mRNA targeting molecule is composed of mRNA connected to a targeting protein group through the PolyA at its 3' end. The structure of the mRNA is from the 5' end to the 3' end. The end contains the 5' cap structure, the 5'UTR with the Kozak sequence, the target gene sequence, the 3'UTR and PolyA.
  2. 根据权利要求1所述的蛋白质介导的mRNA靶向分子，其中，所述靶向蛋白选自VSV-G、透膜蛋白、转铁蛋白、GM-SCF、精氨酸-甘氨酸-天冬氨酸三肽、天冬氨酸-甘氨酸-精氨酸三肽、抗VEGFR抗体、抗ERBB2抗体、抗CD20抗体、抗CD22抗体、抗CD33抗体、抗CD25抗体、抗肌腱蛋白抗体、抗MUC1抗体、抗TAG72抗体、抗CEA抗体中的一种。The protein-mediated mRNA targeting molecule according to claim 1, wherein the targeting protein is selected from the group consisting of VSV-G, permeabilin, transferrin, GM-SCF, arginine-glycine-aspartate Acid tripeptide, aspartic acid-glycine-arginine tripeptide, anti-VEGFR antibody, anti-ERBB2 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD25 antibody, anti-tenascin antibody, anti-MUC1 antibody, One of anti-TAG72 antibodies and anti-CEA antibodies.
  3. 根据权利要求1所述的蛋白质介导的mRNA靶向分子，其中，所述mRNA包括经修饰的核苷和/或未经修饰的核苷，其中所述经修饰的核苷为化学修饰核苷；The protein-mediated mRNA targeting molecule according to claim 1, wherein the mRNA includes modified nucleosides and/or unmodified nucleosides, wherein the modified nucleosides are chemically modified nucleosides ;
    优选地，所述化学修饰核苷包括2-氟-2-脱氧腺苷、2-氟-2-脱氧尿苷、2-氟-2-脱氧胞苷、2-氟-2-脱氧鸟苷、2-氟-2-脱氧-5-甲基胞苷、2-氟-2-脱氧-假尿苷、2-氟-2-脱氧-N1-甲基-假尿苷、2-氟-2-脱氧-N7-甲基-鸟苷、2-氟-2-脱氧-5-甲氧基尿苷、2-氟-2-脱氧-N4-乙酰基胞苷、2-氟-2-脱氧-N6-甲基腺苷、5-甲基胞苷、假尿苷、N1-甲基-假尿苷、N7-甲基-鸟苷、5-甲氧基尿苷、N4-乙酰基胞苷和N6-甲基腺苷中的一种或多种。Preferably, the chemically modified nucleosides include 2-fluoro-2-deoxyadenosine, 2-fluoro-2-deoxyuridine, 2-fluoro-2-deoxycytidine, 2-fluoro-2-deoxyguanosine, 2-fluoro-2-deoxy-5-methylcytidine, 2-fluoro-2-deoxy-pseudouridine, 2-fluoro-2-deoxy-N1-methyl-pseudouridine, 2-fluoro-2- Deoxy-N7-methyl-guanosine, 2-fluoro-2-deoxy-5-methoxyuridine, 2-fluoro-2-deoxy-N4-acetylcytidine, 2-fluoro-2-deoxy-N6 -Methyladenosine, 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetylcytidine and N6 - one or more of methyladenosine.
  4. 根据权利要求1所述的蛋白质介导的mRNA靶向分子，其中，所述mRNA的5’帽子结构包括Cap0、Cap1、Cap2、ARCA、肌苷、N1-甲基-鸟苷、2'氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷、2-叠氮基-鸟苷、7-甲基-鸟苷-5'-三磷酸-5'-腺苷、鸟苷-5'-三磷酸-5'-腺苷、7-甲基-鸟苷-5'-三磷酸-5'-鸟苷、鸟苷-5'-三磷酸-5'-鸟苷和7-甲基-鸟苷-5'-三磷酸-5'-2-甲氧基腺嘌呤-鸟苷中的一种。The protein-mediated mRNA targeting molecule according to claim 1, wherein the 5' cap structure of the mRNA includes Cap0, Cap1, Cap2, ARCA, inosine, N1-methyl-guanosine, 2'fluoro- Guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, 7-methyl-guanosine-5' -Adenosine triphosphate-5'-, Guanosine-5'-triphosphate-5'-adenosine, 7-Methyl-guanosine-5'-triphosphate-5'-guanosine, Guanosine-5' - One of -5'-guanosine triphosphate and 7-methyl-guanosine-5'-triphosphate-5'-2-methoxyadenine-guanosine.
  5. 一种蛋白质介导的mRNA靶向分子的制备方法，其包括：A method for preparing protein-mediated mRNA targeting molecules, which includes:
    将mRNA通过其3’端的PolyA与靶向蛋白基团连接，制备得到蛋白质介导的mRNA靶向分子。The protein-mediated mRNA targeting molecule is prepared by connecting the mRNA to the targeting protein group through the PolyA at its 3' end.
  6. 根据权利要求5所述的蛋白质介导的mRNA靶向分子的制备方法，该方法包括：The method for preparing protein-mediated mRNA targeting molecules according to claim 5, which method includes:
    提供第一核苷酸片段、3’端带有蛋白基团的第二核苷酸片段；Provide a first nucleotide fragment and a second nucleotide fragment with a protein group at the 3' end;
    在连接酶的作用下，将第一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接，制备得到蛋白质介导的mRNA靶向分子；其中第一核苷酸片段与第二核苷酸片段形成蛋白质介导的mRNA靶向分子中的mRNA部分。Under the action of ligase, the 3' end of the first nucleotide fragment is connected to the 5' end of the second nucleotide fragment with a protein group at the 3' end to prepare a protein-mediated mRNA targeting molecule. ; wherein the first nucleotide fragment and the second nucleotide fragment form the mRNA part in the protein-mediated mRNA targeting molecule.
  7. 根据权利要求6所述的蛋白质介导的mRNA靶向分子的制备方法，其中，将第 一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接时，采用夹板DNA辅助进行。The method for preparing protein-mediated mRNA targeting molecules according to claim 6, wherein the first When the 3' end of one nucleotide fragment is connected to the 5' end of a second nucleotide fragment with a protein group at the 3' end, splint DNA is used to assist.
  8. 根据权利要求7所述的蛋白质介导的mRNA靶向分子的制备方法，其中，所述夹板DNA包括第一夹板DNA与第二夹板DNA；第一夹板DNA与第一核苷酸片段的3’端序列互补，所述第二夹板DNA与第二核苷酸片段的5’端序列互补。The method for preparing protein-mediated mRNA targeting molecules according to claim 7, wherein the splint DNA includes a first splint DNA and a second splint DNA; 3' of the first splint DNA and the first nucleotide fragment The second splint DNA is complementary to the 5' end sequence of the second nucleotide fragment.
  9. 根据权利要求8所述的蛋白质介导的mRNA靶向分子的制备方法，其中，将第一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接时，所述第一夹板DNA与第二夹板DNA之间无缝连接；The method for preparing protein-mediated mRNA targeting molecules according to claim 8, wherein the 3' end of the first nucleotide fragment is combined with the 5' end of the second nucleotide fragment having a protein group at the 3' end. When the 'end is connected, the first splint DNA and the second splint DNA are seamlessly connected;
    优选地，所述第一夹板DNA与第一核苷酸片段的3’端5-40个碱基序列互补，所述第二夹板DNA与第二核苷酸片段的5’端5-40个碱基序列互补；Preferably, the first splint DNA is complementary to the 5-40 base sequences at the 3' end of the first nucleotide fragment, and the second splint DNA is complementary to the 5-40 base sequences at the 5' end of the second nucleotide fragment. The base sequences are complementary;
    优选地，与第一夹板DNA互补的第一核苷酸片段的3’端序列、与第二夹板DNA互补的第二核苷酸片段的5’端序列均为蛋白质介导的mRNA靶向分子的mRNA的3’UTR中的一部分序列；Preferably, the 3' end sequence of the first nucleotide fragment complementary to the first splint DNA and the 5' end sequence of the second nucleotide fragment complementary to the second splint DNA are both protein-mediated mRNA targeting molecules. A part of the sequence in the 3'UTR of the mRNA;
    优选地，第二核苷酸片段从5’端至3’端依次包括第三核苷酸片段与PolyA，所述第三核苷酸片段为蛋白质介导的mRNA靶向分子的mRNA的3’UTR中3’端序列的一部分；更优选地，所述第三核苷酸片段的长度为5-100个碱基；所述PolyA的长度为5-150；所述第二核苷酸片段的长度为10-150个碱基。Preferably, the second nucleotide fragment includes a third nucleotide fragment and PolyA in sequence from the 5' end to the 3' end, and the third nucleotide fragment is 3' of the mRNA of the protein-mediated mRNA targeting molecule. A part of the 3' end sequence in the UTR; more preferably, the length of the third nucleotide fragment is 5-100 bases; the length of the PolyA is 5-150 bases; the length of the second nucleotide fragment Length is 10-150 bases.
  10. 根据权利要求5-9任一项所述的蛋白质介导的mRNA靶向分子的制备方法，该方法包括：The method for preparing a protein-mediated mRNA targeting molecule according to any one of claims 5-9, which method includes:
    提供第一核苷酸片段：合成带有启动子序列、目的基因序列的质粒载体，行体外转录，得到第一核苷酸片段；所述第一核苷酸片段的结构从5’端至3’端依次包含5’帽子结构、带有Kozak序列的5’UTR、目的基因序列、3’UTR的5’端的一部分片段；Provide a first nucleotide fragment: synthesize a plasmid vector with a promoter sequence and a target gene sequence, perform in vitro transcription, and obtain a first nucleotide fragment; the structure of the first nucleotide fragment extends from the 5' end to the 3' end The 'end contains in turn a 5' cap structure, a 5'UTR with a Kozak sequence, a target gene sequence, and a part of the 5' end of the 3'UTR;
    提供3’端带有蛋白基团的第二核苷酸片段：将靶向蛋白连接至第二核苷酸片段的3’端，得到3’端带有蛋白基团的第二核苷酸片段；其中，蛋白基团直接连接在第二核苷酸片段的3’端序列的PolyA上；Provide a second nucleotide fragment with a protein group at the 3' end: connect the targeting protein to the 3' end of the second nucleotide fragment to obtain a second nucleotide fragment with a protein group at the 3' end. ; Wherein, the protein group is directly connected to the PolyA of the 3' end sequence of the second nucleotide fragment;
    配制反应体系，使得在退火反应中，第一核苷酸片段的3’端序列与第一夹板DNA互补结合，第二核苷酸片段的5’端序列与第二夹板DNA互补结合，在RNA连接酶作用下，使第一核苷酸片段的3’端与3’端带有蛋白基团的第二核苷酸片段的5’端连接，进一步经过DNA酶处理去除夹板DNA，得到蛋白质介导的mRNA靶向分子。Prepare the reaction system so that during the annealing reaction, the 3' end sequence of the first nucleotide fragment is complementary to the first splint DNA, and the 5' end sequence of the second nucleotide fragment is complementary to the second splint DNA. In the RNA Under the action of ligase, the 3' end of the first nucleotide fragment is connected to the 5' end of the second nucleotide fragment with a protein group at the 3' end, and the splint DNA is further treated with DNase to obtain the protein mediator. guided mRNA targeting molecules.
  11. 一种药物组合物，其包含：如权利要求1-4任意一项所述的蛋白质介导的mRNA 靶向分子，和药学上可接受的辅料。A pharmaceutical composition comprising: the protein-mediated mRNA according to any one of claims 1-4 Targeting molecules, and pharmaceutically acceptable excipients.
  12. 权利要求11所述的药物组合物或权利要求1-4任一项所述的蛋白质介导的mRNA靶向分子在制备用于在受试者中表达目标多肽的药物中的应用；Application of the pharmaceutical composition of claim 11 or the protein-mediated mRNA targeting molecule of any one of claims 1 to 4 in the preparation of drugs for expressing target polypeptides in a subject;
    优选地，所述受试者为哺乳动物或人；Preferably, the subject is a mammal or a human;
    优选地，所述目标多肽选自GLP-1、尿酸氧化酶、胰岛素、胶原蛋白中的一种或多种。Preferably, the target polypeptide is selected from one or more of GLP-1, urate oxidase, insulin, and collagen.
  13. 一种在受试者中表达目标多肽的方法，该方法包括：A method for expressing a target polypeptide in a subject, the method comprising:
    将权利要求11所述的药物组合物或权利要求1-4任一项所述的蛋白质介导的mRNA靶向分子施用与受试者；Administering the pharmaceutical composition of claim 11 or the protein-mediated mRNA targeting molecule of any one of claims 1 to 4 to a subject;
    优选地，所述受试者为哺乳动物或人；Preferably, the subject is a mammal or a human;
    优选地，所述目标多肽选自GLP-1、尿酸氧化酶、胰岛素、胶原蛋白中的一种或多种。 Preferably, the target polypeptide is selected from one or more of GLP-1, urate oxidase, insulin, and collagen.
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CN115010794A (en) * 2022-06-30 2022-09-06 武汉瑞佶生物科技有限公司 Protein-mediated mRNA (messenger ribonucleic acid) targeting molecule as well as preparation method and application thereof

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030083272A1 (en) * 1997-09-19 2003-05-01 Lahive & Cockfield, Llp Sense mrna therapy
WO2019063843A1 (en) * 2017-09-29 2019-04-04 Nlife Therapeutics, S.L. COMPOSITIONS AND METHODS FOR THE DELIVERY OF mRNA TO HEPATIC CELLS
CN111744019A (en) * 2020-07-01 2020-10-09 深圳市瑞吉生物科技有限公司 Mannose-based mRNA (messenger ribonucleic acid) targeted delivery system and application thereof
US20200362382A1 (en) * 2017-08-18 2020-11-19 Modernatx, Inc. Methods of preparing modified rna
CN112111524A (en) * 2020-01-10 2020-12-22 深圳市瑞吉生物科技有限公司 Preparation method of mRNA-GalNAc targeting molecule, in-vivo delivery system and application thereof
CN112673106A (en) * 2017-12-21 2021-04-16 贝瑟克里科有限公司 Click-modified mRNA
WO2021186028A1 (en) * 2020-03-19 2021-09-23 Baseclick Gmbh Modified mrnas for vaccine development
CN115010794A (en) * 2022-06-30 2022-09-06 武汉瑞佶生物科技有限公司 Protein-mediated mRNA (messenger ribonucleic acid) targeting molecule as well as preparation method and application thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10258698B2 (en) * 2013-03-14 2019-04-16 Modernatx, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
CN108026527B (en) * 2015-06-15 2022-05-10 Mpeg La有限责任公司 Defined multiconjugated oligonucleotides
AU2017320901B2 (en) * 2016-09-01 2023-11-09 Proqr Therapeutics Ii B.V. Chemically modified single-stranded RNA-editing oligonucleotides
SG11201908579PA (en) * 2017-03-17 2019-10-30 Cubicstars Inc Method for producing complex of rna molecule and peptide, and utilization thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030083272A1 (en) * 1997-09-19 2003-05-01 Lahive & Cockfield, Llp Sense mrna therapy
US20200362382A1 (en) * 2017-08-18 2020-11-19 Modernatx, Inc. Methods of preparing modified rna
WO2019063843A1 (en) * 2017-09-29 2019-04-04 Nlife Therapeutics, S.L. COMPOSITIONS AND METHODS FOR THE DELIVERY OF mRNA TO HEPATIC CELLS
CN112673106A (en) * 2017-12-21 2021-04-16 贝瑟克里科有限公司 Click-modified mRNA
CN112111524A (en) * 2020-01-10 2020-12-22 深圳市瑞吉生物科技有限公司 Preparation method of mRNA-GalNAc targeting molecule, in-vivo delivery system and application thereof
WO2021186028A1 (en) * 2020-03-19 2021-09-23 Baseclick Gmbh Modified mrnas for vaccine development
CN111744019A (en) * 2020-07-01 2020-10-09 深圳市瑞吉生物科技有限公司 Mannose-based mRNA (messenger ribonucleic acid) targeted delivery system and application thereof
CN115010794A (en) * 2022-06-30 2022-09-06 武汉瑞佶生物科技有限公司 Protein-mediated mRNA (messenger ribonucleic acid) targeting molecule as well as preparation method and application thereof

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