CN115010794A - Protein-mediated mRNA (messenger ribonucleic acid) targeting molecule as well as preparation method and application thereof - Google Patents

Protein-mediated mRNA (messenger ribonucleic acid) targeting molecule as well as preparation method and application thereof Download PDF

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CN115010794A
CN115010794A CN202210762863.7A CN202210762863A CN115010794A CN 115010794 A CN115010794 A CN 115010794A CN 202210762863 A CN202210762863 A CN 202210762863A CN 115010794 A CN115010794 A CN 115010794A
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protein
mrna
sequence
nucleotide fragment
targeting molecule
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胡勇
张昊
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Wuhan Ruiji Biotechnology Co ltd
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Wuhan Ruiji Biotechnology Co ltd
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Priority to PCT/CN2023/104036 priority patent/WO2024002269A1/en
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Abstract

The invention provides a protein-mediated mRNA (messenger ribonucleic acid) targeting molecule as well as a preparation method and application thereof. The protein-mediated mRNA targeting molecule is formed by connecting mRNA with a targeting protein group through PolyA at the 3 'end of the mRNA, wherein the structure of the mRNA sequentially comprises a 5' cap structure, a 5'UTR with a Kozak sequence, a target gene sequence, a 3' UTR and the PolyA from the 5 'end to the 3' end. mRNA molecules with protein at the 3' end can be synthesized by directly connecting mRNA expressing a target gene with a polyA sequence coupled with the protein, so that the aim of delivering a targeted drug is fulfilled. The method is simple and reliable, and solves the problem that the existing mRNA is delivered to specific cells in a targeted manner.

Description

Protein-mediated mRNA targeting molecule and preparation method and application thereof
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a protein-mediated mRNA targeting molecule, and a preparation method and application thereof.
Background
The immunogenicity and instability of mRNA are important reasons limiting the research application of mRNA. Compared to DNA, mRNA mediates superior transfection efficiency and longer protein expression times. However, the mRNA coding information covers the sequence of the ribosome-generated protein, and the protein must be encoded by delivering the protein into cells until modified nucleotides are introduced into the sequence of the mRNA, and a carrier capable of delivering the mRNA is developed, so that the technical problem in the application process is basically solved.
mRNA chains are long-chain macromolecules with negative charges, and the surface of a cell membrane also has negative charges, so that the mRNA molecules are difficult to automatically combine with the cell membrane and penetrate through the cell membrane to enter the cell through electrostatic repulsion. Currently, the delivery of mRNA into cells can be achieved by different methods, such as electroporation, sonoporation, microinjection or transfection of compounds, but the cytotoxicity and biosafety of these methods are difficult to meet clinical requirements and clinical transformation is difficult.
The delivery of mRNA targeted to a particular cell type has not been extensively studied in the prior art.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a protein-mediated mRNA targeting molecule, a preparation method and application thereof, which realize the direct and efficient coupling of mRNA and protein molecules and the specific targeting delivery of the mRNA molecules through the specific combination with cell surface receptors and the mediated endocytosis.
In view of the above, the present invention provides a protein-mediated mRNA targeting molecule, which is formed by linking mRNA with a targeting protein group through PolyA at the 3 'end of the mRNA, wherein the mRNA comprises, in order from the 5' end to the 3 'end, a 5' cap structure, a 5'UTR having a Kozak sequence, a target gene sequence, a 3' UTR, and PolyA. In the present invention, the protein-mediated mRNA targeting molecule is also referred to as mRNA-protein targeting molecule.
According to a specific embodiment of the present invention, preferably, the targeting protein includes VSV-G, transmembrane protein, transferrin, GM-SCF, arginine-glycine-aspartic tripeptide, aspartic acid-glycine-arginine tripeptide, anti-VEGFR antibody, anti-ERBB 2 antibody, anti-CD 20 antibody, anti-CD 22 antibody, anti-CD 33 antibody, anti-CD 25 antibody, anti-tenascin antibody, anti-MUC 1 antibody, anti-TAG 72 antibody, anti-CEA antibody, or the like.
According to a particular embodiment of the invention, in the protein-mediated mRNA targeting molecule of the invention, the mRNA comprises modified nucleosides and/or unmodified nucleosides, wherein the modified nucleosides are chemically 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, 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-acetyl cytidine, 2-fluoro-2-deoxy-N6-methyladenosine, 2-fluoro-2-deoxyuridine, 2-fluoro-N-deoxyuridine, and mixtures thereof, One or more of 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetyl cytidine, and N6-methyladenosine.
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, 7-methyl-guanosine-5 '-triphosphate-5' -guanosine, and 7-methyl-guanosine-5 '-triphosphate-5' -2-methoxyadenine-guanosine One kind of (1).
According to a particular embodiment of the invention, in the protein-mediated mRNA targeting molecule of the invention, the 3' UTR part of the mRNA comprises at least 10 bases, which may be, for example, 10-500 bases, preferably 50-200 bases.
According to a particular embodiment of the invention, in the protein-mediated mRNA targeting molecule of the invention, the PolyA of the mRNA is 5 to 150, preferably 5 to 120, more preferably 5 to 100, and even more preferably 5 to 50 in length.
According to a specific embodiment of the present invention, in the protein-mediated mRNA targeting molecule of the present invention, the gene sequence of interest of the mRNA does not contribute to the protein-mediated mRNA delivery of the mRNA targeting molecule in a cell.
In another aspect, the present invention provides a method for preparing a protein-mediated mRNA targeting molecule, comprising:
and connecting the mRNA with a targeted protein group through the polyA at the 3' end of the mRNA to prepare the protein-mediated mRNA targeted molecule.
According to a specific embodiment of the present invention, the method for preparing the protein-mediated mRNA targeting molecule of the present invention comprises:
providing a first nucleotide fragment, a second nucleotide fragment with a protein group at the 3' end;
under the action of ligase, connecting the 3' end of the first nucleotide fragment with the 5' end of the second nucleotide fragment with a protein group at the 3' end to prepare a protein-mediated mRNA target molecule; wherein the first nucleotide fragment and the second nucleotide fragment form an mRNA portion of a protein-mediated mRNA targeting molecule.
According to a specific embodiment of the present invention, the method for preparing a protein-mediated mRNA targeting molecule of the present invention comprises linking the 3' end of the first nucleotide fragment to the 5' end of the second nucleotide fragment having a protein group at the 3' end, wherein the linking is assisted by splint DNA.
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 comprises a first splint DNA and a second splint DNA; the first splint DNA is complementary to the 3 '-terminal sequence of the first nucleotide fragment, and the second splint DNA is complementary to the 5' -terminal sequence of the second nucleotide fragment.
According to a specific embodiment of the present invention, when the 3' end of the first nucleotide fragment is linked to the 5' end of the second nucleotide fragment having a protein group at the 3' end, the first splint DNA is seamlessly linked to the second splint DNA (i.e., there is no intervening base between the sequence of the first splint DNA corresponding to the first nucleotide fragment and the sequence of the second splint DNA corresponding to the second nucleotide fragment).
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 first splint DNA is complementary to the 5-40 base sequence of the 3 'end of the first nucleotide fragment, and the second splint DNA is complementary to the 5' end of the second nucleotide fragment by the 5-40 base sequence. In other words, the first splint DNA is 5 to 40 bases in length, and the second splint DNA is 5 to 40 bases in length.
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 'terminal sequence of the first nucleotide segment complementary to the first splint DNA is a partial sequence in the 3' UTR of the mRNA of the protein-mediated mRNA targeting molecule. That is, the first splint DNA is designed for the 3' UTR of the target mRNA.
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 'terminal sequence of the second nucleotide segment complementary to the second splint DNA comprises a portion of the sequence in the 3' UTR of the mRNA of the protein-mediated mRNA targeting molecule, and may or may not comprise a portion 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 sequences.
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 second nucleotide segment comprises, in order from 5 'end to 3' end, a third nucleotide segment and PolyA, wherein the third nucleotide segment is a part of a 3 'end sequence in the 3' UTR of mRNA of the protein-mediated mRNA targeting molecule. Preferably, the third nucleotide fragment has a length of 5 to 100 bases, preferably 5 to 80 bases, more preferably 5 to 60 bases, and still more preferably 5 to 40 bases. Specifically, the length of the PolyA is 5 to 150, preferably 5 to 120, more preferably 5 to 100, and still more preferably 5 to 50. Preferably, the length of the second nucleotide fragment is 10 to 150 bases, preferably 10 to 120 bases, more preferably 15 to 100 bases, and still more preferably 15 to 60 bases.
In some embodiments of the invention, the first splint DNA includes, but is not limited to, the DNA set forth in SEQ ID No.3 and the second splint DNA includes, but is not limited to, the DNA set forth in SEQ ID No. 4.
According to a specific embodiment of the present invention, the method for preparing the protein-mediated mRNA targeting molecule of the present invention comprises:
providing a first nucleotide fragment: synthesizing a plasmid vector with a promoter sequence and a target gene sequence, and transcribing in vitro to obtain a first nucleotide fragment; the structure of the first nucleotide fragment comprises a 5 'cap structure, a 5' UTR with a Kozak sequence, a target gene sequence and a partial fragment of the 5 'end of the 3' UTR in sequence from the 5 'end to the 3' end;
providing a second nucleotide fragment bearing a protein group at the 3' end: connecting the target protein to the 3 'end of the second nucleotide fragment to obtain the second nucleotide fragment with a protein group at the 3' end; wherein the protein group is directly linked to the PolyA of the 3' terminal sequence of the second nucleotide fragment;
and (2) preparing a reaction system, enabling the 3' end sequence of the first nucleotide fragment to be complementarily combined with the first splint DNA and the 5' end sequence of the second nucleotide fragment to be complementarily combined with the second splint DNA in an annealing reaction, enabling the 3' end of the first nucleotide fragment to be connected with the 5' end of the second nucleotide fragment with a protein group at the 3' end under the action of RNA ligase (such as T4RNA ligase), and further removing the splint DNA through DNase treatment to obtain the protein-mediated mRNA targeting molecule. In the absence of splint DNA, direct coupling of mRNA molecules to PolyA having a protein group at the 3' end has problems such as a high mismatch rate and low ligation efficiency. The technical scheme of the invention adopts the splint DNA, and can greatly improve the linking efficiency and accuracy.
According to a specific embodiment of the present invention, preferably, the promoter may be a T3 or T7 or SP6 promoter.
In another aspect, the present invention also provides a pharmaceutical composition comprising: the protein-mediated mRNA targeting molecule and pharmaceutically acceptable auxiliary materials.
In another aspect, the invention also provides the use of said pharmaceutical composition or said protein-mediated mRNA targeting molecule for the preparation of a medicament for the expression of a polypeptide of interest in a mammalian or human subject. The polypeptide of interest may be a protein replacement drug such as GLP-1, urate oxidase, insulin, collagen, and the like.
According to a particular embodiment of the present invention, preferably, the present invention achieves in vivo delivery independent of traditional liposome, lipid nanoparticles, by using positively charged proteins. Meanwhile, specific receptors exist on the surfaces of target cells for the specific protein molecules, so that tissue and organ targeted delivery is realized.
The mRNA-protein targeted molecule can be used in a drug delivery system, the 3' end of the mRNA is connected with a specific protein group, and the mRNA enters cells for expression by specifically binding with a target cell surface protein receptor and performing endocytosis by a primer, so that the technical problem of targeted delivery of a nucleic acid drug in the drug delivery process is solved.
On the whole, by adopting the technical scheme of the invention, the mRNA expressing the target gene is directly connected with the PolyA sequence coupled with the protein group to synthesize the mRNA with the protein group at the 3' end, and the in vivo targeted delivery is realized under the action of the positive charge protein, so that the target administration is realized. Furthermore, the connection efficiency and accuracy can be greatly improved through the design of the splint DNA.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing an mRNA-protein targeting molecule according to example 1 of the present invention; wherein (a) is a structural schematic diagram of DNA fragments in plasmid DNA and mRNA molecules obtained by in vitro transcription; (b) schematic representation of mRNA molecules to be bound to PolyA with VSV-G at the 3' end; (c) schematic representation of the resulting mRNA-protein targeting molecules.
FIG. 2 is a comparison of mRNA-VSV-G targeting molecules and mRNA molecules transfected cells.
FIG. 3 is a schematic flow chart of a method for preparing an mRNA-protein targeting molecule according to example 2 of the present invention; wherein (a) is a schematic structural diagram of DNA fragments in plasmid DNA and mRNA molecules obtained by in vitro transcription; (b) schematic representation of mRNA molecules to be bound to PolyA with anti-ERBB 2 antibody at the 3' end; (c) schematic representation of the resulting mRNA-protein targeting molecule.
FIG. 4 is a comparison of cells expressing ERBB2 on the cell membrane, with cells expressing ERBB2 transfected with mRNA-ERBB2 antibody targeting molecules.
FIG. 5 shows mRNA-CD22 antibody targeting molecules transfected cells without CD22 expression compared to CD22+ cells.
FIG. 6 shows the mRNA-VSV-G molecular structure and ligation efficiency results.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. The method operations not specifically mentioned in the examples were carried out according to the conventional operations of the prior art or the operations suggested by the manufacturer's specifications.
Example 1 preparation of mRNA-VSV-G protein targeting molecule
This example provides an mRNA-protein targeting molecule, mRNA-VSV-G protein targeting molecule, which is a structure with VSV-G protein on PolyA at the 3' end of the mRNA. Wherein the sequence of the mRNA molecule comprises a 5' cap, a 5' UTR with a Kozak sequence, a gene sequence of interest, a 3' UTR, and a PolyA.
As shown in figure 1, the mRNA-protein targeting molecule is prepared by the following steps:
step S1, designing and synthesizing a section of plasmid vector with promoter sequence and target gene sequence, wherein the plasmid vector is purchased from pUC-GW-KANA of Jinwei science and technology Limited, and inserted with the EcoRVI, and the linking sequence of each part is 5 'UTR-mRNA coding gene sequence-3' UTR;
step S2, carrying out in vitro transcription by taking the plasmid vector of step S1 as a template to obtain mRNA molecules, wherein the sequence of the mRNA molecules comprises a 5' cap, a 5' UTR comprising a Kozak sequence, a target gene sequence and a 3' UTR;
step S3, mRNA molecule is combined with VSV-G modified poly A under the action of ligase to prepare mRNA-VSV-G targeting molecule.
In this embodiment, 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. Furthermore, the nucleotide sequence of the target gene is shown as SEQ ID No. 2.
SEQ ID No.1：taatacgactcactatagg；
SEQ ID No.2：
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
In this example, the specific synthesis method of the mRNA molecule of step S2 is as follows:
1. the constructed expression plasmid is used for amplifying the DNA template according to the following reaction system:
reaction volume, 50 μ l (single tube reaction volume, one simultaneous reaction multiple tubes);
the PCR amplified system (50. mu.l): PrimeSTAR Max Premix (2X) 25. mu.l, primer F (SEQ ID No. 6: TTGGACCCTCGTACAGAAGCTAATACG, 10. mu. mol/L) 1.2. mu.l, primer R (SEQ ID No. 7: TACCGGTTAGCTTCCTACTCAGGCTTTATTCAAAGACCA, 10. mu. mol/L) 1.2. mu.l, DNA template (1 ng/. mu.l) 1. mu.l and water 21.6. mu.l.
The amplification procedure for the PCR was as follows: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 deg.C for 10s, annealing at 60 deg.C for 5s, extension at 72 deg.C for 2min, and 34 cycles; finally, the extension is carried out at 72 ℃ for 10 min.
After completion of the reaction, the reaction solutions were combined in a 1.5ml Tube. Mu.l of the DNA was subjected to DNA agarose gel electrophoresis (1.5% agarose, 5V/min, 40 min). The success or failure of the reaction is confirmed according to the size of the band of interest of the electrophoresis.
And (4) qualified standard: the electrophoresis detection shows a single strip and has the correct size.
2. DNA template ultrafiltration
The DNA template obtained above was concentrated using a Millipore 30Kd ultrafiltration tube.
3. DNA template FPLC purification
The DNA obtained by the above ultrafiltration was added to an equal volume of a phenol/chloroform/isoamyl alcohol mixture (phenol/chloroform/isoamyl alcohol: 25/24/1), and after shaking sufficiently, the mixture was centrifuged at 12000g for 15 min.
Removing precipitate, transferring supernatant to new centrifuge tube, adding 1/103M NaAc (pH5.2) in volume of supernatant, mixing, adding 2 times volume of anhydrous ethanol, mixing, and standing at-20 deg.C for 30 min.
Centrifuge at 12000g for 10min at 4 ℃ and discard the supernatant.
Washing the precipitate with 70% ethanol, centrifuging at 12000g for 5min, collecting supernatant, and air drying on a clean bench for 5 min.
The purified DNA template is dissolved with an appropriate RNase-free water.
The concentration of the purified template was checked by NanoDrop, and the ratio of 260/280 to 260/230. Samples were taken for DNA agarose gel electrophoresis (1.5% agarose, 5V/min, 40 min).
And (4) qualified standard: 260/280 is between 1.8 and 2.1 and 260/230 is between 1.6 and 2.2.
4. FPLC post-purification template ultrafiltration
The FPLC purified DNA template was concentrated in a Millipore 30Kd ultrafiltration tube and eluted and solubilized with RNase-free water. The concentration of the template after ultrafiltration was measured by NanoDrop, and the ratio of 260/280 to 260/230. Finally, the mixture was diluted with RNase-free water to 150 ng/. mu.l.
5. In vitro synthesis of mRNA
In an isothermal reactor, in vitro synthesis of mRNA was performed.
The method is carried out according to the following synthesis system (reaction reagents are added from top to bottom):
reaction volume, 1600. mu.l (single Tube reaction volume, simultaneous reaction multiple tubes in one Tube, in a 2ml RNase-free Tube): RNA-free water 440. mu.l, 7.5mM ATP 160. mu.l, 7.5mM UTP 160. mu.l, 7.5mM CTP 160. mu.l, 7.5mM GTP 160. mu.l, 7.5mM M7G (2' OMeA) pG 160. mu.l, 150 ng/. mu.l DNA template 40. mu.l, 10 XBuffer 160. mu.l and Enzyme Mix 160. mu.l.
The procedure for the in vitro synthesis of RNA was 37 ℃ for 10 h.
6. Removal of DNA template by DNase I digestion
Mu.l of DNase I was added to each Tube after in vitro mRNA synthesis.
The mixture was inverted from the top to the bottom 10 times and centrifuged at 1000rpm for 10 seconds.
The mixture was placed in the constant temperature reactor again at 37 ℃ for 1 hour.
7. mRNA precipitate recovery
To each 50ml Tube in the previous step, an equal volume of ammonium acetate solution was added.
The mixture was inverted up and down 10 times and mixed.
Standing at-20 deg.C for 2h, and precipitating.
17000g, centrifuge at 4 deg.C for 30 min.
The supernatant was removed and the precipitate was washed with 70% ethanol.
17000g, centrifuge at 4 deg.C, 10 min.
70% of ethanol was removed, and the mixture was evaporated to dryness in a clean bench and 20ml of RNase-free water was added to each tube.
Standing for 10min, and slightly blowing with a gun head to mix.
The concentration of the recovered mRNA was 5. mu.g/. mu.l, 1.90 for A260/A280 and 2.0 for A260/A230, as measured by NanoDrop.
Mu.l of the DNA fragment was diluted 10-fold and subjected to RNA ScreenTape assay and agarose gel electrophoresis to check the integrity of the fragment.
8. Purification of mRNA by LiCl precipitation
Adding RNase-free water into the mRNA recovered in the previous step according to the volume of 1.5 times of the mRNA, and mixing uniformly.
Add 1.5 volumes-20 precooled LiCl solution of original mRNA and mix well.
Then, the mixture is stood for 2 hours at the temperature of minus 20 ℃.
16000g and centrifuge for 20 min.
The supernatant was discarded, the precipitate was washed with 70% ethanol and centrifuged at 16000g for 15 min.
And taking the supernatant, and airing for 5min on a super clean bench.
The purified mRNA is dissolved in an appropriate RNase-free water.
In this example, the mRNA sequence obtained is shown in SEQ ID No. 5.
SEQ ID No.5：
Cap-UAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCGCCACCAUGGUGAGCAAGGGCGAGGAGCUGUUCACCGGGGUGGUGCCCAUCCUGGUCGAGCUGGACGGCGACGUAAACGGCCACAAGUUCAGCGUGUCCGGCGAGGGCGAGGGCGAUGCCACCUACGGCAAGCUGACCCUGAAGUUCAUCUGCACCACCGGCAAGCUGCCCGUGCCCUGGCCCACCCUCGUGACCACCCUGACCUACGGCGUGCAGUGCUUCAGCCGCUACCCCGACCACAUGAAGCAGCACGACUUCUUCAAGUCCGCCAUGCCCGAAGGCUACGUCCAGGAGCGCACCAUCUUCUUCAAGGACGACGGCAACUACAAGACCCGCGCCGAGGUGAAGUUCGAGGGCGACACCCUGGUGAACCGCAUCGAGCUGAAGGGCAUCGACUUCAAGGAGGACGGCAACAUCCUGGGGCACAAGCUGGAGUACAACUACAACAGCCACAACGUCUAUAUCAUGGCCGACAAGCAGAAGAACGGCAUCAAGGUGAACUUCAAGAUCCGCCACAACAUCGAGGACGGCAGCGUGCAGCUCGCCGACCACUACCAGCAGAACACCCCCAUCGGCGACGGCCCCGUGCUGCUGCCCGACAACCACUACCUGAGCACCCAGUCCAAGCUGAGCAAAGACCCCAACGAGAAGCGCGAUCACAUGGUCCUGCUGGAGUUCGUGACCGCCGCCGGGAUCACUCUCGGCAUGGACGAGCUGUACAAGUAAGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAGCGAAGGUA
Wherein the Cap structure is 3' -O-Me-m7G (5') ppp (5') G.
In this example, in step S3, the VSV-G modified polyadenylic acid is obtained by reacting an amino group exposed at the 3' -end of polyadenylic acid (polyA consisting of 10A) with a carboxyl group of an amino acid of a protein group. In general, CDI is added during the reaction, and the reaction is carried out at room temperature for 2 hours.
In this embodiment, the specific process of step S3 includes:
the following reaction system was constructed: 1M Tris-HCl (pH 7.5), 1mM MgCl 2 5% (wt/vol) PEG 8000, 20units/ul RNase Inhibitor, 100units/ul T4RNA Ligase 1, 1mM ATP, 2.5mM mRNA and VSV-G modified polyadenylic acid; wherein the molar ratio of mRNA to VSV-G modified polyadenylic acid is controlled to be 1: 2;
reaction at 25 ℃ for 30 minutes (or at 16 ℃ for between 2 hours and 32 hours);
adding lithium chloride solution into the reaction product until the final concentration of lithium chloride is 0.5M, and obtaining a precipitation product, namely the mRNA-VSV-G targeting molecule.
The mRNA-VSV-G targeting molecule prepared in the embodiment can be used for preparing mRNA medicaments for specific medicament delivery. The specific drug delivered mRNA drug may include one or more pharmaceutically acceptable carriers, such as protective agents, buffers, and the like, in addition to the mRNA-VSV-G targeting molecule of the invention. The 3' end of the mRNA-VSV-G targeting molecule is connected with VSV-G protein, and the mRNA enters the cell for expression through the mediation of endocytosis of the VSV-G. The specific operation steps are as follows: after 293T cells are passaged for about 24 hours, the state of the cells in a 6-well plate is observed, and transfection can be performed when the degree of confluence is about 90%. Two 200. mu.l parts of opti-MEM were added to 10. mu.g of GFP-encoding mRNA (as a control) and the mRNA-VSV-G prepared in this example, and the mixture was gently blown and stirred with a pipette tip, and left to stand for 10min to obtain a prepared transfection system. The prepared transfection system is directly and evenly dripped into the cultured cells, and then is shaken up front and back and left and right, so that the transfection system is evenly distributed on the cells. The medium was changed 6h after transfection, old medium was aspirated off and 2ml fresh medium (90% DMEM + 10% FBS) was changed per well. Observations were made under the microscope 24h after transfection.
As shown in FIG. 2, 24 hours after the mRNA transfection system encoding GFP was added to the cells, the presence of GFP could not be detected under a fluorescence microscope, i.e., the expression of GFP could not be achieved; the expression of GFP was achieved 24 hours after the addition of the mRNA-VSV-G transfection system encoding GFP to the cells, as evidenced by the presence of GFP under a fluorescent microscope.
Example 2 preparation of mRNA-anti-ERBB 2 antibody targeting molecule
This example provides an mRNA-protein targeting molecule, an mRNA-anti-ERBB 2 antibody targeting molecule, which is the product of PolyA at the 3' end of mRNA with anti-ERBB 2 antibodies. The sequence of the mRNA contained a 5' cap, a 5' UTR with a Kozak sequence, a gene sequence of interest, a 3' UTR, and PolyA (the sequence of the mRNA was the same as in example 1). The 3' end of PolyA carries anti-ERBB 2 antibody.
As shown in fig. 3, the mRNA-anti-ERBB 2 antibody targeting molecule was prepared by the following steps:
step S1, designing and synthesizing a section of plasmid vector with a promoter sequence, a target gene sequence and a complementary sequence of the first splint DNA sequence;
step S2, carrying out in vitro transcription by using the plasmid vector of step S1 as a template to obtain mRNA molecules. The sequence of the mRNA molecule comprises a 5' cap, a 5' UTR with Kozak sequence, the gene sequence of interest, a portion of the 3' UTR (5' terminal sequence portion in the 3' UTR of the target mRNA, including the mRNA sequence corresponding to the complement of the first splint DNA sequence) connected in sequence. The mRNA synthesis method was the same as in example 1;
step S3, a PolyA fragment with a protein group at the 3' end, which has 10A ' S, and the 5' end of the PolyA fragment has an RNA sequence that is complementary to the second splint DNA sequence (i.e., a portion of the 3' end sequence in the 3' UTR of the target mRNA). The first splint DNA sequence and the second splint DNA sequence are connected in a seamless way to form splint DNA. In the annealing reaction, mRNA molecules with complementary sequences of a first splint DNA sequence and PolyA molecules with complementary pairing sequences of a second splint DNA sequence are respectively combined with the splint DNA in a complementary mode, under the action of T4RNA ligase I, 3 'end hydroxyl of the mRNA molecules is connected with 5' end phosphate groups of the PolyA with anti-ERBB 2 antibody, and the mRNA target molecules with the anti-ERBB 2 antibody are obtained after DNase I treatment.
In this example, the first splint DNA sequence is shown in SEQ ID No.3, and the second splint DNA sequence is shown in SEQ ID No. 4.
SEQ ID No.3：5’-ttcaaagacc-3’；
SEQ ID No.4：5’-tcaggcttta-3’。
The sequence of the promoter is shown as SEQ ID No. 1.
The target gene is shown as SEQ ID No. 2.
Further, the Ligase is T4RNA Ligase I.
The mRNA-protein targeting molecule obtained in the embodiment can be used for preparing specific drug delivery mRNA drugs. The 3' end is connected with an anti-ERBB 2 antibody, and the anti-ERBB 2 antibody is specifically combined with cancer cells with the surface expressing ERBB2 to cause endocytosis of the cells, so that mRNA enters the cells for expression. The specific operation steps are as follows: 293T (without ERBB2 expression) and H1781 cells (with ERBB2 membrane location) were passaged for about 24 hours, the state of the cells in a 6-well plate was observed, and transfection was carried out with a confluency of about 90%. Two 200. mu.l parts of opti-MEM were added to 10. mu.g of mRNA-ERBB2 antibody complex encoding GFP, gently blown with a pipette tip, mixed well, and left to stand for 10 min. The prepared transfection system is directly and evenly dripped into the cultured cells, and then the cells are evenly shaken front and back and left and right, so that the transfection system is evenly distributed on the cells. The medium was changed 6h after transfection, old medium was aspirated off and 2ml fresh medium (90% DMEM + 10% FBS) was changed per well. Observations under the microscope were made 24h after transfection. As shown in fig. 4, the mRNA-ERBB2 antibody complex was able to specifically transfect H1781 cells expressing ERBB2 on their surface, and was unable to transfect normal 293T cells.
Example 3 preparation of mRNA-anti-CD 22 antibody targeting molecule
This example provides an mRNA-protein targeting molecule, an mRNA-anti-CD 22 antibody targeting molecule, which is the ligation product of an mRNA molecule and an anti-CD 22 antibody. Wherein the sequence of the mRNA molecule comprises a 5' cap, a 5' UTR with Kozak sequence, a gene sequence of interest, a 3' UTR, PolyA (mRNA sequence is the same as in example 1). The mRNA molecules bear anti-CD 22 antibodies on the 3' end of PolyA. The mRNA-protein targeting molecule is prepared by adopting the same steps as the example 2:
step S1, designing and synthesizing a section of plasmid vector with a promoter sequence, a target gene sequence and a complementary sequence of the first splint DNA sequence;
step S2, carrying out in vitro transcription by taking the plasmid vector of step S1 as a template to obtain mRNA molecules, wherein the sequence of the mRNA molecules comprises a 5 'cap, a 5' UTR with a Kozak sequence, a target gene sequence and a 5 'end part sequence of a 3' UTR (including an RNA sequence corresponding to a complementary sequence of a first splint DNA sequence) which are connected in sequence, and the scheme of the in vitro transcription and purification of the mRNA is shown in example 1;
step S3, the 3 'end of the poly A fragment with protein group, the 5' end of the poly A fragment with a section of RNA sequence which can be complementarily matched with the second splint DNA sequence; in the annealing reaction, mRNA molecules with complementary sequences of a first splint DNA sequence and PolyA molecules with complementary pairing sequences of a second splint DNA sequence are respectively combined with the splint DNA in a complementary mode, under the action of T4RNA ligase, 3 'end hydroxyl of the mRNA molecules is connected with 5' end phosphate groups of the PolyA with anti-CD 22 antibody, and the mRNA targeting molecules with the anti-CD 22 antibody are obtained after DNase I treatment.
In this example, the first splint DNA sequence is shown in SEQ ID No.3, and the second splint DNA sequence is shown in SEQ ID No. 4.
SEQ ID No.3：5’-ttcaaagacc-3’；
SEQ ID No.4：5’-tcaggcttta-3’。
The sequence of the promoter is shown as SEQ ID No. 1.
The target gene is shown as SEQ ID No. 2.
Further, the Ligase is T4RNA Ligase I.
The mRNA-protein targeting molecule obtained in the embodiment can be used for preparing specific drug delivery mRNA drugs. The 3' end is connected with an anti-CD 22 antibody, and the anti-CD 22 antibody is specifically bound with cancer cells expressing CD22 on the surface to cause endocytosis of the cells, so that mRNA enters the cells for expression. The specific operation steps are as follows: the cell state in the 6-well plate was observed about 24 hours after passage of 293T (no CD22 expression) and CA46 cells (CD22+) and the transfection was carried out with a degree of confluence of about 90%. Two 200. mu.l parts of opti-MEM were added to 10. mu.g of mRNA-CD22 antibody complex encoding GFP, gently blown with a pipette tip, mixed well, and left to stand for 10 min. The prepared transfection system is directly and evenly dripped into the cultured cells, and then the cells are evenly shaken front and back and left and right, so that the transfection system is evenly distributed on the cells. The medium was changed 6h after transfection, old medium was aspirated off and 2ml fresh medium (90% DMEM + 10% FBS) was changed per well. Observations under the microscope were made 24h after transfection. As shown in fig. 5, the mRNA-CD22 antibody complex was able to specifically transfect CA46 cells expressing CD22 on their surface, and was unable to transfect normal 293T cells.
Example 4 detection of mRNA-VSV-G molecular Structure
The following method is adopted to detect the mRNA-VSV-G molecular structure:
anti-VSV-G antibody (5. mu.g) was added to the mRNA-VSV-G product prepared in example 1 (1mg), and the VSV-G protein was used as a negative control and incubated overnight at 4 ℃ with gentle agitation;
adding protein A/G magnetic beads (40 mu L), and incubating for 1h at 4 ℃ under soft agitation;
centrifuging the magnetic beads at 2,500rpm for 30s, removing the supernatant, and resuspending the magnetic beads in 500. mu.l of RIP buffer;
the beads were washed repeatedly three times in RIP buffer followed by one wash in PBS;
purifying RNA combined on RBP after immunoprecipitation, resuspending magnetic beads in 1mL TRIzol RNA extraction reagent, and separating coprecipitated RNA;
RNA was eluted using nuclease-free water (20. mu.L). About 20 μ L of DEPC-treated water was added to the mRNA pellet;
mRNA was Reverse Transcribed (RT) into cDNA and analyzed by RT-PCR, and the abundance of mRNA bound to VSV-G was determined using a homogenous mRNA sample as a positive control. Wherein the primers used in the RT-PCR are F: AAGGAGGACGGCAACATCCTGGGG (SEQ ID No.8) and R: TACAGCTCGTCCATGCCGAGAGTG (SEQ ID No. 9).
The results of the detection are shown in FIG. 6.
In summary, the invention mainly realizes that the final product can be used for targeting delivery of mRNA by the connection of polyadenylic acid and a protein delivery carrier and the connection method of polyadenylic acid/protein complex and the tail end of mRNA molecule.
Sequence listing
<110> Wuhan Rui robust and sturdy Biotechnology Co., Ltd
<120> protein-mediated mRNA targeting molecule and preparation method and application thereof
<130> GAI21CN8019
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> promoter
<400> 1
taatacgact cactatagg 19
<210> 2
<211> 720
<212> DNA
<213> Artificial sequence
<220>
<223> target Gene
<400> 2
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 3
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> first splint DNA sequence
<400> 3
ttcaaagacc 10
<210> 4
<211> 10
<212> DNA
<213> Artificial sequence
<220>
<223> second splint DNA sequence
<400> 4
tcaggcttta 10
<210> 5
<211> 865
<212> RNA
<213> Artificial sequence
<220>
<223> mRNA molecular sequence obtained by transcription
<400> 5
uaagagagaa aagaagagua agaagaaaua uaagagccgc caccauggug agcaagggcg 60
aggagcuguu caccggggug gugcccaucc uggucgagcu ggacggcgac guaaacggcc 120
acaaguucag cguguccggc gagggcgagg gcgaugccac cuacggcaag cugacccuga 180
aguucaucug caccaccggc aagcugcccg ugcccuggcc cacccucgug accacccuga 240
ccuacggcgu gcagugcuuc agccgcuacc ccgaccacau gaagcagcac gacuucuuca 300
aguccgccau gcccgaaggc uacguccagg agcgcaccau cuucuucaag gacgacggca 360
acuacaagac ccgcgccgag gugaaguucg agggcgacac ccuggugaac cgcaucgagc 420
ugaagggcau cgacuucaag gaggacggca acauccuggg gcacaagcug gaguacaacu 480
acaacagcca caacgucuau aucauggccg acaagcagaa gaacggcauc aaggugaacu 540
ucaagauccg ccacaacauc gaggacggca gcgugcagcu cgccgaccac uaccagcaga 600
acacccccau cggcgacggc cccgugcugc ugcccgacaa ccacuaccug agcacccagu 660
ccaagcugag caaagacccc aacgagaagc gcgaucacau gguccugcug gaguucguga 720
ccgccgccgg gaucacucuc ggcauggacg agcuguacaa guaagcugcc uucugcgggg 780
cuugccuucu ggccaugccc uucuucucuc ccuugcaccu guaccucuug gucuuugaau 840
aaagccugag uaggaagcga aggua 865
<210> 6
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<212> DNA
<213> Artificial sequence
<220>
<223> primer F
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ttggaccctc gtacagaagc taatacg 27
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<211> 39
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<213> Artificial sequence
<220>
<223> primer R
<400> 7
taccggttag cttcctactc aggctttatt caaagacca 39
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> RT-RCR primer F
<400> 8
aaggaggacg gcaacatcct gggg 24
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> RT-PCR primer R
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tacagctcgt ccatgccgag agtg 24

Claims (12)

1. A protein-mediated mRNA targeting molecule is formed by connecting mRNA with a targeting protein group through PolyA at the 3 'end of the mRNA, wherein the structure of the mRNA sequentially comprises a 5' cap structure, a 5'UTR with a Kozak sequence, a target gene sequence, a 3' UTR and the PolyA from the 5 'end to the 3' end.
2. The protein-mediated mRNA targeting molecule according to claim 1, wherein the targeting protein is selected from one of VSV-G, transmembrane protein, transferrin, GM-SCF, arginine-glycine-aspartic acid tripeptide, aspartic acid-glycine-arginine tripeptide, anti-VEGFR antibody, anti-ERBB 2 antibody, anti-CD 20 antibody, anti-CD 22 antibody, anti-CD 33 antibody, anti-CD 25 antibody, anti-tenascin antibody, anti-MUC 1 antibody, anti-TAG 72 antibody, anti-CEA antibody.
3. The protein-mediated mRNA targeting molecule according to claim 1, wherein the mRNA comprises modified nucleosides and/or unmodified nucleosides, wherein the modified nucleosides are chemically modified nucleosides;
preferably, the chemically modified nucleoside comprises 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-acetyl cytidine, 2-fluoro-2-deoxy-N6-methyladenosine, 2-fluoro-2-deoxy-N6-methyl-uridine, or 2-fluoro-2-deoxy-pseudouridine One or more of 5-methylcytidine, pseudouridine, N1-methyl-pseudouridine, N7-methyl-guanosine, 5-methoxyuridine, N4-acetyl cytidine, and N6-methyladenosine.
4. The protein-mediated mRNA targeting molecule of claim 1, wherein the mRNA 5 'Cap structure comprises one of 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, 7-methyl-guanosine-5 '-triphosphate-5' -guanosine, and 7-methyl-guanosine-5 '-triphosphate-5' -2-methoxyadenine-guanosine And (4) seed preparation.
5. A method of making a protein-mediated mRNA targeting molecule, comprising:
and connecting the mRNA with a targeted protein group through the polyA at the 3' end of the mRNA to prepare the protein-mediated mRNA targeted molecule.
6. The method of making a protein-mediated mRNA targeting molecule of claim 5, comprising:
providing a first nucleotide fragment, a second nucleotide fragment with a protein group at the 3' end;
under the action of ligase, connecting the 3' end of the first nucleotide fragment with the 5' end of the second nucleotide fragment with a protein group at the 3' end to prepare a protein-mediated mRNA target molecule; wherein the first nucleotide fragment and the second nucleotide fragment form an mRNA portion of a protein-mediated mRNA targeting molecule.
7. The method of claim 6, wherein the ligation of the 3' end of the first nucleotide fragment to the 5' end of the second nucleotide fragment having a protein group at the 3' end is performed with the aid of splint DNA.
8. The method of claim 7, wherein the splint DNA comprises a first splint DNA and a second splint DNA; the first splint DNA is complementary to the 3 '-terminal sequence of the first nucleotide fragment, and the second splint DNA is complementary to the 5' -terminal sequence of the second nucleotide fragment.
9. The method for preparing a protein-mediated mRNA targeting molecule according to claim 8, wherein when the 3' end of the first nucleotide fragment is linked to the 5' end of the second nucleotide fragment having a protein group at the 3' end, the first splint DNA is seamlessly linked to the second splint DNA;
preferably, the first splint DNA is complementary to the 5-40 base sequence of the 3 'end of the first nucleotide fragment, and the second splint DNA is complementary to the 5' end of the second nucleotide fragment by 5-40 base sequences;
preferably, the 3' terminal sequence of the first nucleotide segment complementary to the first splint DNA and the 5' terminal sequence of the second nucleotide segment complementary to the second splint DNA are both part of the sequence in the 3' UTR of the mRNA of the protein-mediated mRNA targeting molecule;
preferably, the second nucleotide segment comprises, in order from 5 'end to 3' end, a third nucleotide segment and PolyA, the third nucleotide segment being a portion of the 3 'end sequence in the 3' UTR of the mRNA of the protein-mediated mRNA targeting molecule; more preferably, the third nucleotide fragment is 5-100 bases in length; the length of the PolyA is 5-150; the second nucleotide fragment is 10-150 bases in length.
10. A method of making a protein-mediated mRNA targeting molecule according to any one of claims 5-9, the method comprising:
providing a first nucleotide fragment: synthesizing a plasmid vector with a promoter sequence and a target gene sequence, and transcribing in vitro to obtain a first nucleotide fragment; the structure of the first nucleotide fragment comprises a 5 'cap structure, a 5' UTR with a Kozak sequence, a target gene sequence and a partial fragment of the 5 'end of the 3' UTR from the 5 'end to the 3' end in sequence;
providing a second nucleotide fragment bearing a protein group at the 3' end: connecting the target protein to the 3 'end of the second nucleotide fragment to obtain the second nucleotide fragment with a protein group at the 3' end; wherein the protein group is directly linked to the PolyA of the 3' terminal sequence of the second nucleotide fragment;
and preparing a reaction system, wherein in an annealing reaction, the 3' end sequence of the first nucleotide fragment is complementarily combined with the first splint DNA, the 5' end sequence of the second nucleotide fragment is complementarily combined with the second splint DNA, under the action of RNA ligase, the 3' end of the first nucleotide fragment is connected with the 5' end of the second nucleotide fragment with a protein group at the 3' end, and the splint DNA is further removed through DNase treatment to obtain the protein-mediated mRNA targeting molecule.
11. A pharmaceutical composition comprising: the protein-mediated mRNA targeting molecule of any one of claims 1 to 4, and a pharmaceutically acceptable excipient.
12. Use of a pharmaceutical composition according to claim 11 or a protein-mediated mRNA targeting molecule according to any one of claims 1-4 for the preparation of a medicament for expressing a polypeptide of interest in a mammalian subject.
CN202210762863.7A 2022-06-30 2022-06-30 Protein-mediated mRNA (messenger ribonucleic acid) targeting molecule as well as preparation method and application thereof Pending CN115010794A (en)

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