WO2021121173A1 - 基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法 - Google Patents

基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法 Download PDF

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WO2021121173A1
WO2021121173A1 PCT/CN2020/136010 CN2020136010W WO2021121173A1 WO 2021121173 A1 WO2021121173 A1 WO 2021121173A1 CN 2020136010 W CN2020136010 W CN 2020136010W WO 2021121173 A1 WO2021121173 A1 WO 2021121173A1
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mrna
sequence
gbd
acetylgalactosamine
puromycin
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PCT/CN2020/136010
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French (fr)
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张苗苗
洪丹
胡迅
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深圳市瑞吉生物科技有限公司
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Priority to KR1020227023976A priority Critical patent/KR102616559B1/ko
Priority to JP2022536655A priority patent/JP2023506635A/ja
Priority to AU2020405482A priority patent/AU2020405482B2/en
Priority to EP20902709.3A priority patent/EP4019632A4/en
Publication of WO2021121173A1 publication Critical patent/WO2021121173A1/zh
Priority to US17/693,173 priority patent/US11759532B2/en

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    • 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
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/54—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 organic compound
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K48/0008—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
    • A61K48/0025—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 wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—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 wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to an mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide and a preparation method thereof.
  • the asialoglycoprotein receptor is an abundant hetero-oligomer endocytic receptor. It mainly exists on the surface of the cell membrane of the liver parenchymal cells facing the sinusoid. The specificity is an endocytic receptor specifically expressed by hepatocytes.
  • ASGPR's high-affinity ligand N-acetylgalactosamine (GalNAc) has been used as a targeting molecule to achieve breakthroughs in the delivery of nucleic acid drugs, such as small interfering RNA (siRNA) in the liver. Sexual progress.
  • siRNA small interfering RNA
  • sexual progress Although the receptor has been discovered for many years, the messenger RNA (mRNA) delivery system based on the receptor and its ligand has failed to achieve a breakthrough because the existing technical means cannot achieve effective coupling of mRNA and GalNAc.
  • the delivery of mRNA into cells can be achieved through different methods, such as electroporation, sonoporation, microinjection, or cell transfection based on polymer compounds, but these methods are relatively toxic to cells and have certain clinical transformations. Difficulty.
  • the present invention discloses a mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide and a preparation method thereof.
  • GalNAc modification of mature mRNA molecules is realized, thereby realizing mRNA liver Targeted drug delivery is of great significance to innovative basic research, new drug design and development.
  • a DNA fragment for constructing an mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide comprising a promoter, a target gene, a specific protease cleavage sequence and a binding N-acetyl half Lactosamine polypeptide GBD (GlaNAc Binding Domain, GBD) sequence.
  • the DNA fragment can be used to construct an mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide.
  • the GBD sequence is one or a combination of SEQ ID Nos. 1 to 5.
  • the sequence of the target gene is shown in SEQ ID No. 6 or 7.
  • the target gene can be replaced with other genes, and the corresponding target gene can be selected according to the treatment of different diseases.
  • the specific protease cleavage sequence is one or more of T2A, P2A, E2A, F2A, TEV, VLP1 and SUMO specific protease cleavage sequences with GBD sequence.
  • the promoter is a T3, T7 or SP6 promoter.
  • the present invention also discloses an mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide, which includes mRNA molecules obtained by in vitro transcription using plasmids containing DNA fragments as described above.
  • the sequence of the molecule includes 5'cap, target gene sequence, specific protease cleavage sequence, and polypeptide GBD protein; the polypeptide GBD protein is obtained by ribosome translation of the GBD sequence; the GBD sequence end of the mRNA molecule is obtained by purine Mycin is linked to GBD protein, which is linked to N-acetylgalactosamine through an enzyme-catalyzed reaction.
  • the above-mentioned DNA fragments are used as templates to synthesize mRNA molecules containing 5'caps, target gene sequences, specific protease cleavage sequences, and GBD sequences in vitro; under the action of T4 ligase, mRNA molecules and DNA-
  • the puromycin linker binds and forms an mRNA-puromycin complex; through the in vitro translation system, puromycin is connected to the tail of the antibody through the ribosome A site to form mRNA-puromycin-GBD-specific protease cleavage Sequence-gene function protein complex; specific protease cleavage is performed on this product to obtain mRNA-puromycin-GBD complex.
  • Puromycin is an analog of transfer RNA (tRNA, transfer RNA), which can bind to the A position of the ribosome during the transcription process and form a peptide bond with the polypeptide fragment being synthesized and prevent the extension of the peptide fragment.
  • tRNA transfer RNA
  • puromycin can also be bound to the 3'end of RNA or DNA. Using these properties, by binding a special peptide with puromycin to the 3'end of the RNA molecule, a peptide-RNA fusion molecule can be formed ( peptide-RNA fusion product).
  • the technical scheme of the present invention based on this principle, designs and synthesizes mRNA-peptide fusion molecules, and realizes the coupling of mRNA molecules and GalNAc through GalNAc modification on special peptides.
  • N-acetylgalactosamine transferase N-acetylgalactosamine specifically binds to the GBD protein sequence to form an mRNA-puromycin-GBD-GalNAc complex, so that mRNA molecules can be targeted to liver cells, thereby achieving specific delivery of mRNA drug molecules.
  • the present invention also discloses a method for preparing an mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide as described above, which comprises the following steps:
  • Step S1 Select specific cell surface receptors according to the delivered tissues, organs or cells, and design a polypeptide GBD sequence that can bind to N-acetylgalactosamine, and cleave the promoter sequence, target gene sequence, and specific protease
  • the sequence and GBD sequence combination are cloned into a plasmid vector to obtain plasmid DNA;
  • Step S2 using the plasmid DNA of step S1 as a template to perform in vitro transcription to obtain an mRNA sequence containing a 5'cap, a target gene sequence, a specific protease cleavage sequence, and a GBD sequence;
  • Step S3 under the action of T4 ligase, the mRNA molecule binds to the DNA-puromycin linker to form an mRNA-puromycin complex;
  • step S4 the mRNA-puromycin complex obtained in step S3 is translated in vitro, and the mRNA-puromycin complex is translated by the ribosome into a gene functional protein-specific protease cleavage sequence-GBD fusion protein sequence;
  • Step S5 at the end of translation, puromycin is connected to the tail of the antibody through the ribosome A position to form an mRNA-puromycin-GBD-specific protease cleavage sequence-gene functional protein complex;
  • step S6 the product obtained in step S5 is cleaved by a specific protease, and the specific protease cleaved sequence-gene function protein part in the mRNA-puromycin-GBD-specific protease cleavage sequence-gene function protein complex is Cut to obtain mRNA-puromycin-GBD complex;
  • Step S7 under the action of N-acetylgalactosamine transferase, N-acetylgalactosamine specifically binds to the GBD protein sequence to form an mRNA-puromycin-GBD-GalNAc complex.
  • the GBD sequence is shown in SEQ ID Nos. 1 to 5.
  • the DNA sequence of the DNA-puromycin linker is shown in SEQ ID No. 8.
  • step S1 the plasmid vector is modified from pCDNA3.1.
  • the present invention also discloses an application of the above-mentioned mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide, and the N-acetylgalactosamine-based mRNA tissue-specific delivery substance is used for preparing specific drugs Among the delivered mRNA drugs, N-acetylgalactosamine is connected to the 3'end, and the N-acetylgalactosamine binds to the liver cell surface sialoglycoprotein receptors through specific binding primers for endocytosis, thereby allowing the mRNA to enter the cell for expression .
  • the technical solution of the present invention by connecting the 3'end of the mRNA drug molecule to a polypeptide sequence capable of coupling with GalNAc, and connecting GalNAc to the mRNA-polypeptide complex, the GalNAc modification of the mature mRNA molecule is realized; the existing GalNAc is solved. Conjugation technology can only achieve the problem of direct coupling of GalNAc with short RNA.
  • N-acetylgalactosamine on liver cells to form a connection that can specifically bind to specific target cells, thereby increasing the efficacy of mRNA drug molecules, and solving the technical problem of targeted delivery of nucleic acid drugs in the drug delivery process , To achieve the purpose of tissue-specific delivery through GalNAc modification of mRNA without relying on physical methods and chemical transfection reagents.
  • Figure 1 is a schematic flow chart of a method for preparing an mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide according to the present invention; where a) is a schematic diagram of DNA fragments in plasmid DNA; b) is in vitro using plasmid DNA as a template The schematic diagram of the mRNA-puromycin linker to be combined after transcription; c) is the schematic diagram of the mRNA-puromycin complex; d) the schematic diagram of the mRNA-puromycin complex undergoing in vitro translation; e) is the end of translation The mRNA-puromycin-GBD-specific protease cleavage to obtain a schematic diagram of the mRNA-puromycin-GBD complex; f) is a schematic diagram of the coupling of the mRNA-puromycin-GBD complex with GalNAc; g) is A schematic diagram of the finally obtained mRNA-puromycin-GBD-GalNAc complex;
  • FIG. 2 is a schematic diagram of the principle of the GalNAc-mediated mRNA liver cell delivery system of the present invention, in which the GalNAc-mRNA coupler induces endocytosis by binding to the liver cell surface ASGPR, thereby allowing mRNA to enter the cell;
  • Figure 3 is a schematic diagram of the optimization of the GalNAc-mRNA liver cell delivery system of the present invention. Among them, the triple GalNAc-mRNA conjugate has the highest transfection efficiency for liver cells;
  • FIG. 4 is a schematic diagram of the comparison of the GalNAc-mRNA liver cell delivery system of the present invention and the comparative example expressing green fluorescent protein (GFP) in liver cells;
  • GFP green fluorescent protein
  • Fig. 5 is a schematic diagram of the results of the GalNAc-mRNA liver cell delivery system of the present invention and the comparative example delivering luciferase (Luc) to liver tissues in vivo.
  • a DNA fragment for constructing an mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide includes a promoter, a target gene, a specific protease cleavage sequence, and a DNA fragment capable of binding N-acetylgalactosamine
  • the polypeptide GBD sequence is connected in sequence.
  • the GBD sequence is one or a combination of SEQ ID Nos. 1 to 5.
  • the sequence of the target gene is shown in SEQ ID No. 6 or 7.
  • the specific protease cleavage sequence is one or more of T2A, P2A, E2A, F2A, TEV, VLP1 and SUMO specific protease cleavage sequences with GBD sequence.
  • the promoter is a T3, T7 or SP6 promoter.
  • the present invention discloses mRNA targeting molecules based on binding N-acetylgalactosamine polypeptides, which include mRNA molecules that are obtained by in vitro transcription using plasmids containing the DNA fragments described above
  • the sequence of the mRNA molecule in turn includes a 5'cap, a target gene sequence, a specific protease cleavage sequence, and a polypeptide GBD protein; the polypeptide GBD protein is obtained by ribosome translation of the GBD sequence; the GBD of the mRNA molecule
  • the end of the sequence is connected to the GBD protein through puromycin, and the GBD protein is connected to N-acetylgalactosamine through an enzyme-catalyzed reaction.
  • N-acetylgalactosamine-based mRNA tissue-specific delivery substance is prepared by the following steps:
  • Step S1 select specific cell surface receptors according to the delivered tissues, organs or cells, and design a polypeptide sequence (GBD) that can bind to N-acetylgalactosamine (GalNAc), and correlate The combination of cloning elements is cloned into pCDNA3.1 plasmid vector;
  • GBD polypeptide sequence
  • GalNAc N-acetylgalactosamine
  • step S2 in vitro transcription is performed using the plasmid DNA of step S1 as a template, and the mRNA sequence generated after in vitro transcription contains the 5'cap, the target gene sequence, and the specific protease digestion sequence with the GBD sequence;
  • the specific protease digestion sequence is one or more of T2A, P2A, E2A, F2A, TEV, VLP1 and SUMO;
  • Step S3 under the action of T4 ligase, the mRNA molecule is combined with a DNA-puromycin linker (DNA Puromycine Linker) to form an mRNA-puromycin complex;
  • DNA-puromycin linker DNA Puromycine Linker
  • step S4 the mRNA-puromycin complex obtained in step S3 is translated in vitro, and the mRNA-puromycin complex is translated by the ribosome into a gene functional protein-specific protease cleavage sequence-GBD fusion protein sequence;
  • Step S5 at the end of translation, puromycin is connected to the tail of the antibody through the ribosome A position to form an mRNA-puromycin-GBD-specific protease cleavage sequence-gene functional protein complex;
  • Step S6 the product obtained in step S5 is cleaved with a specific protease, and mRNA-puromycin-GBD-specific protease is cleaved under the action of 2A peptide self-cleavage or TEV, VLP1, and SUMO specific protease
  • the specific protease cleavage sequence-gene function protein part is cut to obtain the mRNA-puromycin-GBD complex
  • Step S7 under the action of N-acetylgalactosamine transferase, N-acetylgalactosamine specifically binds to the GBD protein sequence to form an mRNA-puromycin-GBD-GalNAc complex.
  • sequence of the DNA-puromycin linker is shown in SEQ ID No. 8; and the GBD sequence is shown in SEQ ID No. 1-5.
  • step S1 the plasmid vector is modified from pCDNA3.1.
  • mRNA targeting molecules based on binding N-acetylgalactosamine polypeptide are used in the preparation of mRNA drugs for specific drug delivery to form a GalNAc-mediated mRNA liver cell delivery system, in which the 3'end is connected with N-acetyl Galactosamine, through N-acetylgalactosamine and liver cell surface sialoglycoprotein receptor specific binding primers endocytosis, so that mRNA enters the cell for expression, as shown in Figure 2.
  • GBD GBD-GalNAc sequence
  • it can be combined with only 1 GalNAc GBD, 2 GalNAc GBD, 3 GalNAc GBD, or n GalNAc GBD.
  • the use of triple GalNAc-mRNA conjugate has the highest transfection efficiency for liver cells, and the comparison result is shown in Figure 3.
  • the transfection system takes 200 ⁇ l opti-MEM and add 10 ⁇ g test product (including mRNA-GalNAc1, mRNA-GalNAc2, mRNA-GalNAc3, mRNA/lipo2000, mRNA/lipo3000, mRNA/LNP, mRNA/TransIT, mRNA/lipo RNAiMAX , MRNA/In vivo-jetPEI, concentration 2 ⁇ g/ ⁇ l, 5 ⁇ l) or negative control without carrier GFP-mRNA.
  • the prepared transfection system is directly and evenly dropped into the cultured cells, and then shaken up and down 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).
  • the fluorescence intensity was measured under a fluorescence microscope 36h after transfection.
  • the experimental results are shown in Figure 3.
  • the mRNA expression intensity of the mRNA-GalNAc group is significantly higher than that of other vector groups, and the transfection efficiency of the triple GalNAc-mRNA conjugate is the highest.
  • mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide, which is a new type of mRNA drug with liver cell specific binding ability.
  • the GalNAc modification of the mRNA molecule is bound to the GBD protein sequence of the mRNA-puromycin-GBD molecule by N-acetylgalactosamine transferase to form an mRNA-puromycin-GBD-GalNAc molecule.
  • Puromycin is linked to the GBD polypeptide sequence; the mRNA molecule is obtained by in vitro transcription using a plasmid containing the above-mentioned DNA fragment, and the sequence of the mRNA molecule in turn includes 5'cap, target gene sequence, specific protease cleavage sequence, binding
  • the polypeptide GBD sequence of N-acetylgalactosamine, the GBD polypeptide is obtained by ribosomal translation of the GBD sequence, and is prepared by the following steps:
  • Step S1 according to the delivery tissue is the liver cell, the target gene is selected as the green fluorescent protein mWasabi, and a polypeptide sequence (GBD) that can bind to N-acetylgalactosamine (GalNAc) is designed.
  • GBD polypeptide sequence
  • the promoter sequence, the target gene sequence, the specific protease cleavage sequence, and the GBD sequence are combined and cloned into the pCDNA3.1 plasmid vector to obtain plasmid DNA.
  • the GBD sequence is one or a combination of SEQ ID No. 1-5.
  • This embodiment adopts the GBD shown in the SEQ ID No. 2 sequence.
  • the sequence of the target gene is shown in SEQ ID No. 6.
  • the specific protease cleavage sequence is one or more of T2A, P2A, E2A, F2A, TEV, VLP1 and SUMO specific protease cleavage sequences with GBD sequence.
  • the specific protease cleavage sequence used is Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser), as shown in SEQ ID No.9 and SEQ ID No.10.
  • the promoter is a T3, T7 or SP6 promoter. This embodiment uses the T7 promoter, and the sequence is shown in SEQ ID No. 11:
  • the DNA sequence of the DNA-puromycin linker is shown in SEQ ID No. 8;
  • Step S2 use the plasmid DNA of step S1 as a template for in vitro transcription.
  • the mRNA sequence generated after in vitro transcription contains 5'cap, gene sequence, T2A, P2A, E2A, F2A, TEV, VLP1 and SUMO specificity with GBD sequence Protease digests one or more of the sequences.
  • Step S3 under the action of T4 ligase, the mRNA molecule is combined with a DNA-puromycin linker (DNA Puromycine Linker) to form an mRNA-puromycin complex;
  • DNA-puromycin linker DNA Puromycine Linker
  • Step S4 the mRNA-puromycin complex obtained in step S3 is translated in vitro, and the mRNA-puromycin complex is translated by the ribosome into a fusion protein of gene function protein-specific protease cleavage polypeptide sequence-GBD polypeptide sequence;
  • Step S5 at the end of translation, puromycin is connected to the tail of the antibody through the ribosome A position to form an mRNA-puromycin-GBD-specific protease cleavage sequence-gene functional protein complex;
  • Step S6 the product obtained in step S5 is cleaved with a specific protease, and mRNA-puromycin-GBD-specific protease is cleaved under the action of 2A peptide self-cleavage or TEV, VLP1, and SUMO specific protease
  • the specific protease cleavage sequence-gene function protein part is cut to obtain the mRNA-puromycin-GBD polypeptide complex
  • Step S7 under the action of N-acetylgalactosamine transferase, N-acetylgalactosamine specifically binds to the GBD protein sequence to form an mRNA-puromycin-GBD-GalNAc complex.
  • the mRNA-puromycin-GBD-GalNAc complex can specifically bind to the ASGPR receptor on the surface of liver cells to achieve specific liver delivery of mRNA.
  • the above-mentioned mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide is used to prepare mRNA drugs for specific drug delivery to form a GalNAc-mediated mRNA liver cell delivery system, with N-acetylgalactosamine connected to the 3'end, Through N-acetylgalactosamine and liver cell surface sialoglycoprotein receptor specific binding primers endocytosis, so that mRNA enters the cell for expression.
  • Comparative experiments show that, as shown in Figure 4, the delivery system using the mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide is more effective in liver cells than the existing delivery system of mRNA and mRNA/LNP. Efficient expression of green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • the transfection system takes 200 ⁇ l opti-MEM, add 10 ⁇ g of the test substance (GFP mRNA-GalNAc, GFP mRNA/LNP, concentration 2 ⁇ g/ ⁇ l, 5 ⁇ l) or negative control carrier-free GFP-mRNA (concentration 2 ⁇ g/ ⁇ l, 5 ⁇ l) .
  • the prepared transfection system is directly and evenly dropped into the cultured cells, and then shaken up and down 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).
  • the fluorescence intensity was measured under a fluorescence microscope 36h after transfection.
  • the experimental results are shown in Figure 4, the mRNA expression intensity of the mRNA-GalNAc group is significantly higher than that of the LNP carrier group.
  • An mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide which is prepared by adopting the following steps:
  • Step S1 select the target gene as luciferase (Luc) according to the delivery tissue in the liver cell, and design a peptide sequence (GBD) that can bind to N-acetylgalactosamine (GalNAc), and combine and clone the relevant cloning elements Into the pCDNA3.1 plasmid vector.
  • the DNA fragments in the plasmid DNA include a promoter, a target gene, a specific protease cleavage sequence, and a polypeptide GBD sequence capable of binding N-acetylgalactosamine, which are connected in sequence.
  • the GBD sequence adopts the GBD shown in SEQ ID No. 2 sequence.
  • the sequence of the target gene is shown in SEQ ID No.7.
  • the specific protease cleavage sequence used is Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser), as shown in SEQ ID No. 9 and SEQ ID No. 10.
  • the promoter is a T3, T7 or SP6 promoter.
  • This embodiment uses the T7 promoter, and the sequence is shown in SEQ ID No. 11:
  • the sequence of the DNA in the DNA-puromycin linker is shown in SEQ ID No. 8;
  • Step S2 use the plasmid DNA of step S1 as a template for in vitro transcription.
  • the mRNA sequence generated after in vitro transcription contains 5'cap, gene sequence, T2A, P2A, E2A, F2A, TEV, VLP1 and SUMO specificity with GBD sequence Protease digests one or more of the sequences.
  • Step S3 under the action of T4 ligase, the mRNA molecule is combined with a DNA-puromycin linker (DNA Puromycine Linker) to form an mRNA-puromycin complex;
  • DNA-puromycin linker DNA Puromycine Linker
  • step S4 the mRNA-puromycin complex obtained in step S3 is translated in vitro, and the mRNA-puromycin complex is translated by the ribosome into a gene functional protein-specific protease cleavage sequence-GBD fusion protein sequence.
  • step S5 when translation is completed, puromycin is connected to the tail of the antibody through the ribosome A position to form an mRNA-puromycin-GBD-specific protease cleavage sequence-gene functional protein complex.
  • Step S6 the product obtained in step S5 is cleaved with a specific protease, and mRNA-puromycin-GBD-specific protease is cleaved under the action of 2A peptide self-cleavage or TEV, VLP1, and SUMO specific protease
  • the specific protease cleavage sequence-gene function protein part is cut to obtain the mRNA-puromycin-GBD complex.
  • Step S7 under the action of N-acetylgalactosamine transferase, N-acetylgalactosamine specifically binds to the GBD protein sequence to form an mRNA-puromycin-GBD-GalNAc complex.
  • the above-mentioned mRNA targeting molecule based on binding N-acetylgalactosamine polypeptide is used to prepare mRNA drugs for specific drug delivery to form a GalNAc-mRNA delivery system.
  • the modified luciferases Luc mRNA-GalNAc, Luc mRNA/LNP and Luc mRNA prepared in the above examples were directly introduced into the mouse systemic circulation through tail vein administration, and the in vivo biofluorescence signal characterizes the expression intensity of the modified mRNA in vivo.
  • mice were fixed on the tail vein injection platform, and 200 ⁇ g of the above three mRNA drugs (1 ⁇ g/ ⁇ g, 200 ⁇ l) were injected respectively. Fluorescence imaging observation was performed after 24 hours.
  • the D-fluorescein substrate was dissolved in physiological saline at a concentration of 15 mg/ml, and 100 ⁇ l of the solution was injected into the mice through the tail vein. After 10 minutes, the IVIS small animal imaging system was used to quantitatively analyze the strength of the lung signal.

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Abstract

一种基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法，含有启动子、目的基因、特异性蛋白酶剪切序列、能结合N-乙酰半乳糖胺的多肽GBD序列依次连接而成的DNA片段的质粒载体通过转录得到mRNA，在T4连接酶的作用下与DNA-嘌呤霉素连接体相连接，通过蛋白翻译，并采用特异性蛋白酶进行剪切，得到mRNA-嘌呤霉素-GBD复合物，在N-乙酰半乳糖胺转移酶作用下，与GBD蛋白序列结合形成mRNA-嘌呤霉素-GBD-GalNAc复合物，从而对mRNA进行GalNAc修饰，在mRNA药物递送过程中，达到精准给药的目的，增加了mRNA药物分子的药效。

Description

基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法
本申请要求于2019年12月17日提交中国专利局、申请号为201911300610.2、发明名称为“基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，尤其涉及基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法。
背景技术
去唾液酸糖蛋白受体(asialoglycoprotein receptor，ASGPR)是数量丰富的一种异源低聚物的内吞型受体，主要存在于肝脏实质细胞朝向窦状隙一侧的细胞膜表面，具有对糖的特异性，是肝细胞特异性表达的一种内吞型受体。近年来，利用ASGPR的高亲和性配体N-乙酰半乳糖胺(GalNAc)作为靶向分子，在核酸药物，如小干扰RNA(Small interfering RNA，siRNA)的肝靶向递送方面取得了突破性进展。尽管该受体已被发现多年，但基于该受体及其配体的信使RNA(Messenger RNA,mRNA)递送***未能取得突破，原因在于，现有技术手段无法实现mRNA与GalNAc的有效耦合。
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，显微注射或基于高分子化合物的细胞转染，但这些方法对细胞是相对有毒的，临床转化具有一定难度。
发明内容
针对以上技术问题，本发明公开了基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法，通过全新的mRNA合成与修饰策略，实现对成熟mRNA分子的GalNAc修饰，从而实现mRNA肝脏靶向性药物递送，对创新基础研究、新型药物设计与开发均具有十分重要的意义。
为了实现上述发明目的，本发明提供了以下技术方案：
一种用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的 DNA片段，所述DNA片段包括依次连接的启动子、目的基因、特异性蛋白酶剪切序列和能结合N-乙酰半乳糖胺的多肽GBD(GlaNAc Binding Domain,GBD)序列。
采用此技术方案，该DNA片段可以用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子。
作为本发明的进一步改进，所述GBD序列为SEQ ID No.1～5中的一种或几种的组合。
作为本发明的进一步改进，所述目的基因的序列如SEQ ID No.6或7所示。其中，所述的目的基因可以替换为其他的基因，可以根据治疗不同的疾病选择相应的目的基因。
作为本发明的进一步改进，所述特异性蛋白酶剪切序列为带有GBD序列的T2A、P2A、E2A、F2A、TEV、VLP1和SUMO特异性蛋白酶酶切序列中的一种或几种。
作为本发明的进一步改进，所述启动子为T3、T7或SP6启动子。
本发明还公开了一种基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子，其包括mRNA分子，所述mRNA分子为采用包含如上所述的DNA片段的质粒通过体外转录得到，所述mRNA分子的序列依次包含5’帽子、目的基因序列、特异性蛋白酶剪切序列、多肽GBD蛋白；所述多肽GBD蛋白为所述GBD序列通过核糖体翻译得到；所述mRNA分子的GBD序列端通过嘌呤霉素连接GBD蛋白，所述GBD蛋白通过酶催化反应连接N-乙酰半乳糖胺。
采用此技术方案，以上述DNA片段为模板在体外转录合成含有5’帽子、目的基因序列、特异性蛋白酶剪切序列、GBD序列的mRNA分子；在T4连接酶的作用下，mRNA分子与DNA-嘌呤霉素连接体结合，并形成mRNA-嘌呤霉素复合体；通过体外翻译***，嘌呤霉素通过核糖体A位连接到抗体的尾部，形成mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物；对此产物进行特异性蛋白酶剪切，得到mRNA-嘌呤霉素-GBD复合物。
嘌呤霉素(Puromycin)是转运RNA(tRNA,transfer RNA)的类似物，在转录过程中能够结合到核糖体的A位并与正在合成的多肽片段形成肽 键并阻止肽段的延伸。此外嘌呤霉素还可以结合到RNA或DNA的3’端，利用这些性质，通过将带有嘌呤霉素的特殊肽段结合到RNA分子3’端，可以形成一个肽段-RNA的融合分子(peptide-RNA fusion product)。本发明的技术方案，基于这一原理，设计并合成mRNA-肽段融合分子，通过在特殊肽段上进行GalNAc修饰实现mRNA分子与GalNAc的耦合，在N-乙酰半乳糖胺转移酶作用下，N-乙酰半乳糖胺特异性地与GBD蛋白序列结合，形成mRNA-嘌呤霉素-GBD-GalNAc复合物，使得mRNA分子能靶向肝脏细胞，从而实现mRNA药物分子的特异性递送。
本发明还公开了一种如上所述的基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的制备方法，包括以下步骤：
步骤S1，根据所递送的组织、器官或细胞选取特异性细胞表面受体，并设计能结合N-乙酰半乳糖胺的多肽GBD序列，并将启动子序列、目的基因序列、特异性蛋白酶剪切序列、GBD序列组合克隆到质粒载体中，得到质粒DNA；
步骤S2，以步骤S1的质粒DNA为模板进行体外转录，得到含有5’帽子、目的基因序列、特异性蛋白酶剪切序列、GBD序列的mRNA序列；
步骤S3，在T4连接酶的作用下，所述mRNA分子与DNA-嘌呤霉素连接体结合，并形成mRNA-嘌呤霉素复合体；
步骤S4，将步骤S3得到的mRNA-嘌呤霉素复合体进行体外翻译，所述mRNA-嘌呤霉素复合体被核糖体翻译出基因功能蛋白-特异性蛋白酶剪切序列-GBD的融合蛋白序列；
步骤S5，翻译结束时，嘌呤霉素通过核糖体A位连接到抗体的尾部，形成mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物；
步骤S6，对步骤S5得到的产物采用特异性蛋白酶进行剪切，mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物中特异性蛋白酶剪切序列-基因功能蛋白部分被剪切，得到mRNA-嘌呤霉素-GBD复合物；
步骤S7，在N-乙酰半乳糖胺转移酶作用下，N-乙酰半乳糖胺特异性地与GBD蛋白序列结合，形成mRNA-嘌呤霉素-GBD-GalNAc复合物。
作为本发明的进一步改进，所述GBD序列如SEQ ID No.1～5所示。
Figure PCTCN2020136010-appb-000001
作为本发明的进一步改进，所述DNA-嘌呤霉素连接体的DNA的序列如SEQ ID No.8所示；
Figure PCTCN2020136010-appb-000002
作为本发明的进一步改进，步骤S1中，所述质粒载体改造自pCDNA3.1。
本发明还公开了一种如上所述的基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的应用，所述基于N-乙酰半乳糖胺的mRNA组织特异性递送物质用于制备特异性药物递送的mRNA药物中，3’端连接有N-乙酰半乳糖胺，通过N-乙酰半乳糖胺与肝脏细胞表面唾液酸糖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。
与现有技术相比，本发明的有益效果为：
采用本发明的技术方案，通过将mRNA药物分子3’端连接一段能耦合GalNAc的多肽序列，将GalNAc连接到mRNA-多肽复合物中，实现对成熟mRNA分子的GalNAc修饰；解决了现有的GalNAc缀合技术只能实现GalNAc直接与短片段RNA耦合的难题。进一步的，利用肝脏细胞上N-乙酰半乳糖胺形成连接有能特异性结合特定的目标细胞，从而增加mRNA药物分子的药效，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，实现了不依赖于物理方法和化学转染试剂的方式，通过对mRNA进行GalNAc修饰达到组织特异性递送的目的。
附图说明
图1是本发明一种基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的制备方法的流程示意图；其中a)为质粒DNA中DNA片段的示意图；b)为以质粒DNA为模板进行体外转录得到的mRNA与DNA-嘌呤霉素连接体待结合的示意图；c)为mRNA-嘌呤霉素复合体的示意图；d)mRNA-嘌呤霉素复合体进行体外翻译的示意图；e)为翻译结束后得到的mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切得到mRNA-嘌呤霉素-GBD复合物的示意图；f)为mRNA-嘌呤霉素-GBD复合物与GalNAc耦合的示意图；g)为最终得到的mRNA-嘌呤霉素-GBD-GalNAc复合物的示意图；
图2是本发明的GalNAc介导的mRNA肝脏细胞递送***的原理示意图，其中，GalNAc-mRNA耦合物通过结合肝脏细胞表面ASGPR引起细胞内吞，从而使得mRNA进入细胞；
图3是本发明的GalNAc-mRNA肝脏细胞递送***的优化的示意图。其中，三联GalNAc-mRNA耦合物对肝脏细胞的转染效率最高；
图4是本发明的GalNAc-mRNA肝脏细胞递送***与对比例在肝脏细胞中表达绿色荧光蛋白(GFP)的对比示意图；
图5是本发明的GalNAc-mRNA肝脏细胞递送***与对比例在体内递送荧光素酶(Luc)到肝脏组织的结果示意图。
具体实施方式
为了本发明更容易理解，下面对本发明的具体实施方式结合附图作进一步的详细说明。
一种用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的DNA片段，所述DNA片段包括启动子、目的基因、特异性蛋白酶剪切序列、能结合N-乙酰半乳糖胺的多肽GBD序列依次连接而成。
进一步的，所述GBD序列为SEQ ID No.1～5中的一种或几种的组合。
所述目的基因的序列如SEQ ID No.6或7所示。
所述特异性蛋白酶剪切序列为带有GBD序列的T2A、P2A、E2A、F2A、TEV、VLP1和SUMO特异性蛋白酶酶切序列中的一种或几种。
所述启动子为T3、T7或SP6启动子。
基于上述构建的DNA片段，本发明公开了基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子，其包括mRNA分子，所述mRNA分子为采用包含如上所述的DNA片段的质粒通过体外转录得到，所述mRNA分子的序列依次包含5’帽子、目的基因序列、特异性蛋白酶剪切序列和多肽GBD蛋白；所述多肽GBD蛋白为所述GBD序列通过核糖体翻译得到；所述mRNA分子的GBD序列端通过嘌呤霉素连接GBD蛋白，所述GBD蛋白通过酶催化反应连接N-乙酰半乳糖胺。
所述基于N-乙酰半乳糖胺的mRNA组织特异性递送物质采用以下步骤制备得到：
步骤S1，如图1所示，根据所递送的组织、器官或细胞选取特异性细胞表面受体，并设计一段能结合N-乙酰半乳糖胺(GalNAc)的多肽序列(GBD)，并将相关克隆元件组合克隆到pCDNA3.1质粒载体中；
步骤S2，以步骤S1的质粒DNA为模板进行体外转录，体外转录后生成的mRNA序列含有5’帽子、目的基因序列、带有GBD序列的特异性蛋白酶酶切序列；
所述特异性蛋白酶酶切序列为T2A、P2A、E2A、F2A、TEV、VLP1和SUMO中的一种或几种；
步骤S3，在T4连接酶的作用下mRNA分子与DNA-嘌呤霉素连接体(DNA Puromycine Linker)结合，形成mRNA-嘌呤霉素复合体；
步骤S4，将步骤S3得到的mRNA-嘌呤霉素复合体进行体外翻译，所述mRNA-嘌呤霉素复合体被核糖体翻译出基因功能蛋白-特异性蛋白酶剪切序列-GBD的融合蛋白序列；
步骤S5，翻译结束时，嘌呤霉素通过核糖体A位连接到抗体的尾部，形成mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物；
步骤S6，对步骤S5得到的产物采用特异性蛋白酶进行剪切，在2A肽自我剪切作用下或TEV、VLP1、SUMO特异性蛋白酶作用下，mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物中特异性蛋白酶剪切序列-基因功能蛋白部分被剪切，得到mRNA-嘌呤霉素-GBD复 合物；
步骤S7，在N-乙酰半乳糖胺转移酶作用下，N-乙酰半乳糖胺特异性地与GBD蛋白序列结合，形成mRNA-嘌呤霉素-GBD-GalNAc复合物。
其中，所述DNA-嘌呤霉素连接体的序列如SEQ ID No.8所示；所述GBD序列如SEQ ID No.1～5所示。
进一步的，步骤S1中，所述质粒载体改造自pCDNA3.1。
上述基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子用于制备特异性药物递送的mRNA药物中，形成GalNAc介导的mRNA肝脏细胞递送***，该***中，3’端连接有N-乙酰半乳糖胺，通过N-乙酰半乳糖胺与肝脏细胞表面唾液酸糖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，如图2所示。
GBD-GalNAc序列中的GBD有多种设计方案，基于不同的设计，可以根据需要只结合1个GalNAc的GBD，结合2个GalNAc的GBD，结合3个GalNAc的GBD，或结合n个GalNAc的GBD；进一步优选的，采用三联GalNAc-mRNA耦合物对肝脏细胞的转染效率最高，对比结果如图3所示。
具体操作步骤：
细胞转染
接种完293T细胞(购自中国科学院细胞库)后约24h，观察6孔板内的细胞状态，汇合度在88％～92％。在生物安全柜内，配制90％(体积百分含量)DMEM+10％(体积百分含量)FBS培养基。转染前30min弃掉孔板的培养基，每孔加入1ml新鲜培养基，即90％(体积百分含量)DMEM+10％(体积百分含量)FBS培养基。
准备转染体系：取200μl opti-MEM，加入10μg供试品(包括mRNA-GalNAc1、mRNA-GalNAc2、mRNA-GalNAc3、mRNA/lipo2000、mRNA/lipo3000、mRNA/LNP、mRNA/TransIT、mRNA/lipo RNAiMAX、mRNA/In vivo-jetPEI，浓度2μg/μl，5μl)或阴性对照无载体GFP-mRNA。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36h荧光显微镜下 测定荧光强度。实验结果如图3所示，mRNA-GalNAc组的mRNA表达强度明显高于其他载体组，并且三联GalNAc-mRNA耦合物的转染效率最高。
下面通过具体的实施例进行进一步的举例说明，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例1
一种基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子，其为具有肝脏细胞特异性结合能力的新型mRNA药物。其中，mRNA分子的GalNAc修饰通过N-乙酰半乳糖胺转移酶结合到mRNA-嘌呤霉素-GBD分子的GBD蛋白序列上，形成mRNA-嘌呤霉素-GBD-GalNAc分子。嘌呤霉素连接GBD多肽序列；所述mRNA分子为采用包含上述DNA片段的质粒通过体外转录得到，所述mRNA分子的序列依次包含5’帽子、目的基因序列、特异性蛋白酶剪切序列、能结合N-乙酰半乳糖胺的多肽GBD序列，所述GBD多肽为所述GBD序列通过核糖体翻译得到，其采用如下步骤制备得到：
步骤S1，根据递送组织为在肝脏细胞，选取目的基因为绿色荧光蛋白mWasabi，并设计一段能结合N-乙酰半乳糖胺(GalNAc)的多肽序列(GBD)。将启动子序列、目的基因序列、特异性蛋白酶剪切序列、GBD序列组合克隆到pCDNA3.1质粒载体中，得到质粒DNA。
本实施例中，所述GBD序列为SEQ ID No.1-5中的一种或几种的组合。
Figure PCTCN2020136010-appb-000003
Figure PCTCN2020136010-appb-000004
本实施例采用如SEQ ID No.2序列所示的GBD。
所述目的基因的序列如SEQ ID No.6所示。
Figure PCTCN2020136010-appb-000005
所述特异性蛋白酶剪切序列为带有GBD序列的T2A、P2A、E2A、F2A、TEV、VLP1和SUMO特异性蛋白酶酶切序列中的一种或几种。本实施例中，采用的特异性蛋白酶剪切序列为Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)，如SEQ ID No.9、SEQ ID No.10所示。
所述启动子为T3、T7或SP6启动子。本实施例采用T7启动子，序列如SEQ ID No.11所示：
Figure PCTCN2020136010-appb-000006
所述DNA-嘌呤霉素连接体的DNA的序列如SEQ ID No.8所示；
Figure PCTCN2020136010-appb-000007
步骤S2，以步骤S1的质粒DNA为模板进行体外转录，体外转录后生成的mRNA序列含有5’帽子、基因序列、带有GBD序列的T2A、P2A、E2A、F2A、TEV、VLP1和SUMO特异性蛋白酶酶切序列中的一种或几种序列。
步骤S3，在T4连接酶的作用下，mRNA分子与DNA-嘌呤霉素连接体(DNAPuromycine Linker)结合，形成mRNA-嘌呤霉素复合体；
步骤S4，将步骤S3得到的mRNA-嘌呤霉素复合体进行体外翻译，所述mRNA-嘌呤霉素复合体被核糖体翻译出基因功能蛋白-特异性蛋白酶剪切多肽序列-GBD多肽的融合蛋白序列；
步骤S5，翻译结束时，嘌呤霉素通过核糖体A位连接到抗体的尾部，形成mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物；
步骤S6，对步骤S5得到的产物采用特异性蛋白酶进行剪切，在2A肽自我剪切作用下或TEV、VLP1、SUMO特异性蛋白酶作用下，mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物中特异性蛋白酶剪切序列-基因功能蛋白部分被剪切，得到mRNA-嘌呤霉素-GBD多肽复合物；
步骤S7，在N-乙酰半乳糖胺转移酶作用下，N-乙酰半乳糖胺特异性的与GBD蛋白序列结合，形成mRNA-嘌呤霉素-GBD-GalNAc复合物。
采用mRNA-嘌呤霉素-GBD-GalNAc复合物，能与肝脏细胞表面ASGPR受体特异性结合，实现mRNA的特异性肝脏递送。
采用上述基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子用于制备特异性药物递送的mRNA药物，形成GalNAc介导的mRNA肝脏细胞递送***，3’端连接有N-乙酰半乳糖胺，通过N-乙酰半乳糖胺与肝脏细胞表面唾液酸糖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。通过对比实验显示，如图4所示，采用包含该基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的递送***与现有mRNA、 mRNA/LNP递送***相比，在肝脏细胞中能更加高效的表达绿色荧光蛋白(GFP)。
具体操作步骤：
细胞转染
接种完293T细胞(购自中国科学院细胞库)后约24h，观察6孔板内的细胞状态，汇合度在88％～92％。在生物安全柜内，配制90％(体积百分含量)DMEM+10％(体积百分含量)FBS培养基。转染前30min弃掉孔板的培养基，每孔加入1ml新鲜培养基，即90％(体积百分含量)DMEM+10％(体积百分含量)FBS培养基。
准备转染体系：取200μl opti-MEM，加入10μg供试品(GFP mRNA-GalNAc，GFP mRNA/LNP，浓度2μg/μl，5μl)或阴性对照无载体GFP-mRNA(浓度2μg/μl，5μl)。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36h荧光显微镜下测定荧光强度。实验结果如图4所示，mRNA-GalNAc组的mRNA表达强度明显高于LNP载体组。
实施例2
一种基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子，其采用如下步骤制备得到：
步骤S1，根据递送组织为在肝脏细胞，选取目的基因为荧光素酶(Luc)，并设计一段能结合N-乙酰半乳糖胺(GalNAc)的多肽序列(GBD)，并将相关克隆元件组合克隆到pCDNA3.1质粒载体中。其中，质粒DNA中DNA片段包括启动子、目的基因、特异性蛋白酶剪切序列、能结合N-乙酰半乳糖胺的多肽GBD序列依次连接而成。
本实施例中，所述GBD序列采用如SEQ ID No.2序列所示的GBD。
所述目的基因的序列如SEQ ID No.7所示。
Figure PCTCN2020136010-appb-000008
Figure PCTCN2020136010-appb-000009
本实施例中，采用的特异性蛋白酶剪切序列为Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)，如SEQ ID No.9和SEQ ID No.10所示。所述启动子为T3、T7或SP6启动子。
本实施例采用T7启动子，序列如SEQ ID No.11所示：
Figure PCTCN2020136010-appb-000010
所述DNA-嘌呤霉素连接体中的DNA的序列如SEQ ID No.8所示；
Figure PCTCN2020136010-appb-000011
步骤S2，以步骤S1的质粒DNA为模板进行体外转录，体外转录后生成的mRNA序列含有5’帽子，基因序列，带有GBD序列的T2A、P2A、E2A、F2A、TEV、VLP1和SUMO特异性蛋白酶酶切序列中的一种或几种序列。
步骤S3，在T4连接酶的作用下mRNA分子与DNA-嘌呤霉素连接体(DNAPuromycine Linker)结合，形成mRNA-嘌呤霉素复合体；
步骤S4，将步骤S3得到的mRNA-嘌呤霉素复合体进行体外翻译，所述mRNA-嘌呤霉素复合体被核糖体翻译出基因功能蛋白-特异性蛋白 酶剪切序列-GBD的融合蛋白序列。
步骤S5，翻译结束时，嘌呤霉素通过核糖体A位连接到抗体的尾部，形成mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物。
步骤S6，对步骤S5得到的产物采用特异性蛋白酶进行剪切，在2A肽自我剪切作用下或TEV、VLP1、SUMO特异性蛋白酶作用下，mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物中特异性蛋白酶剪切序列-基因功能蛋白部分被剪切，得到mRNA-嘌呤霉素-GBD复合物。
步骤S7，在N-乙酰半乳糖胺转移酶作用下，N-乙酰半乳糖胺特异性的与GBD蛋白序列结合，形成mRNA-嘌呤霉素-GBD-GalNAc复合物。
采用上述基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子用于制备特异性药物递送的mRNA药物，形成GalNAc-mRNA递送***。
具体操作步骤：
将上述实施例中制备的修饰荧光素酶Luc mRNA-GalNAc、Luc mRNA/LNP和Luc mRNA，通过尾静脉给药方式直接引入小鼠体循环，体内生物荧光信号表征修饰mRNA在体内的表达强度。
尾静脉注射
将Balb/c小鼠固定在尾静脉注射平台上，分别注射200μg的以上三种mRNA药物(1μg/μg，200μl)。24小时后进行荧光成像观察。
小动物成像
将D-荧光素底物溶解于生理盐水，浓度为15mg/ml，将100μl该溶液经尾静脉注射进入小鼠体内。10min后，使用IVIS小动物成像***定量分析肺部信号强弱。
通过对比实验显示，如图5所示，该GalNAc-mRNA递送***与现有mRNA、mRNA/LNP递送***相比，能更加高效的递送荧光素酶(Luc)到肝脏组织。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (16)

  1. 一种用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的DNA片段，其特征在于：所述DNA片段包括依次连接的启动子、目的基因、特异性蛋白酶剪切序列、能结合N-乙酰半乳糖胺的多肽GBD序列。
  2. 根据权利要求1所述的用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的DNA片段，其特征在于：所述能结合N-乙酰半乳糖胺的多肽GBD序列为SEQ ID No.1～5中的一种或几种的组合。
  3. 根据权利要求2所述的用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的DNA片段，其特征在于：所述目的基因的序列如SEQ ID No.6或SEQ ID No.7所示。
  4. 根据权利要求1～3任意一项所述的用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的DNA片段，其特征在于：所述特异性蛋白酶剪切序列为带有GBD序列的T2A、P2A、E2A、F2A、TEV、VLP1和SUMO特异性蛋白酶酶切序列中的一种或几种。
  5. 根据权利要求4所述的用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的DNA片段，其特征在于：所述特异性蛋白酶剪切序列如SEQ ID No.9或SEQ ID No.10所示。
  6. 根据权利要求1～4任意一项所述的用于构建基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的DNA片段，其特征在于：所述启动子为T3、T7或SP6启动子。
  7. 一种结合N-乙酰半乳糖胺多肽的mRNA靶向分子，其特征在于：其包括mRNA分子，所述mRNA分子采用包含如权利要求1～5任意一项所述的DNA片段的质粒通过体外转录得到；
    所述mRNA分子的序列依次包含5’帽子、目的基因序列、特异性蛋白酶剪切序列和多肽GBD蛋白；
    所述多肽GBD蛋白为所述GBD序列通过核糖体翻译得到；
    所述mRNA分子的GBD序列端通过嘌呤霉素连接GBD蛋白，所述GBD蛋白通过酶催化反应连接N-乙酰半乳糖胺。
  8. 一种如权利要求7所述的基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的制备方法，其特征在于：包括以下步骤：
    步骤S1，根据所递送的组织、器官或细胞选取特异性细胞表面受体，设计能结合N-乙酰半乳糖胺的多肽GBD序列，并将启动子序列、目的基因序列、特异性蛋白酶剪切序列、GBD序列组合克隆到质粒载体中，得到质粒DNA；
    步骤S2，以步骤S1的质粒DNA为模板进行体外转录，得到含有5’帽子、目的基因序列、特异性蛋白酶剪切序列、GBD序列的mRNA序列。
  9. 一种如权利要求7所述的基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子的应用，其特征在于：用于制备特异性药物递送的mRNA药物中，3’端连接有N-乙酰半乳糖胺，通过N-乙酰半乳糖胺与肝脏细胞表面唾液酸糖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。
  10. 一种mRNA-嘌呤霉素-GBD-GalNAc复合物的制备方法，其特征在于：包括以下步骤：
    步骤S1，根据所递送的组织、器官或细胞选取特异性细胞表面受体，设计能结合N-乙酰半乳糖胺的多肽GBD序列，并将启动子序列、目的基因序列、特异性蛋白酶剪切序列、GBD序列组合克隆到质粒载体中，得到质粒DNA；
    步骤S2，以步骤S1的质粒DNA为模板进行体外转录，得到含有5’帽子、目的基因序列、特异性蛋白酶剪切序列、GBD序列的mRNA序列；
    步骤S3，在T4连接酶的作用下，所述mRNA分子与DNA-嘌呤霉素连接体结合，形成mRNA-嘌呤霉素复合体；
    步骤S4，将步骤S3得到的mRNA-嘌呤霉素复合体进行体外翻译，所述mRNA-嘌呤霉素复合体被核糖体翻译出基因功能蛋白-特异性蛋白 酶剪切序列-GBD的融合蛋白序列；
    步骤S5，翻译结束时，嘌呤霉素通过核糖体A位连接到抗体的尾部，形成mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物；
    步骤S6，对步骤S5得到的产物采用特异性蛋白酶进行剪切，mRNA-嘌呤霉素-GBD-特异性蛋白酶剪切序列-基因功能蛋白复合物中特异性蛋白酶剪切序列-基因功能蛋白部分被剪切，得到mRNA-嘌呤霉素-GBD复合物；
    步骤S7，在N-乙酰半乳糖胺转移酶作用下，N-乙酰半乳糖胺特异性地与GBD蛋白序列结合，形成mRNA-嘌呤霉素-GBD-GalNAc复合物。
  11. 根据权利要求10所述的mRNA-嘌呤霉素-GBD-GalNAc复合物的制备方法，其特征在于：所述DNA-嘌呤霉素连接体的序列如SEQ ID No.8所示；所述GBD序列如SEQ IDNo.1～5任一所示。
  12. 根据权利要求10所述的mRNA-嘌呤霉素-GBD-GalNAc复合物的制备方法，其特征在于：步骤S1中，所述质粒载体改造自pCDNA3.1。
  13. 根据权利要求10所述的mRNA-嘌呤霉素-GBD-GalNAc复合物的制备方法，其特征在于，步骤S7中，GBD蛋白序列结合1个N-乙酰半乳糖胺、结合2个N-乙酰半乳糖胺、结合3个N-乙酰半乳糖胺或结合n个N-乙酰半乳糖胺。
  14. 一种GalNAc-mRNA递送***，其特征在于，包括权利要求10～13任意一项所述的制备方法制备获得的mRNA-嘌呤霉素-GBD-GalNAc复合物。
  15. 权利要求14所述的GalNAc-mRNA递送***在靶向治疗肝脏疾病中的应用。
  16. 根据权利要求15所述的应用，其特征在于，将所述GalNAc-mRNA递送***导入体内。
PCT/CN2020/136010 2019-12-17 2020-12-14 基于结合N-乙酰半乳糖胺多肽的mRNA靶向分子及其制备方法 WO2021121173A1 (zh)

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