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CN-121987828-A - MRNA delivery system based on nucleic acid nano structure and preparation method and application thereof

CN121987828ACN 121987828 ACN121987828 ACN 121987828ACN-121987828-A

Abstract

The invention discloses an mRNA delivery system based on a nucleic acid nanostructure, a preparation method and application thereof. The mRNA delivery system comprises an mRNA scaffold chain, an RNA staple chain, a lipid-nucleic acid coupled chain and auxiliary lipid, wherein the mRNA scaffold chain comprises a nucleic acid sequence for encoding target protein mRNA, the mRNA scaffold chain and the RNA staple chain are subjected to base complementary pairing and self-assembly to form a nucleic acid nano structure, and the lipid-nucleic acid coupled chain comprises a lipid molecule and a nucleic acid coupled with the lipid molecule. The invention realizes the purposes of protecting and delivering mRNA by accurately integrating mRNA molecules encoding functional proteins into a nucleic acid nano structure as scaffold chains, and obtains the effect of accurately regulating and controlling the expression of target proteins.

Inventors

  • DING BAOQUAN
  • LIU JIANBING
  • YANG CHANGPING
  • FAN JING
  • Zhu Hanken

Assignees

  • 国家纳米科学中心

Dates

Publication Date
20260508
Application Date
20260202

Claims (10)

  1. 1. An mRNA delivery system based on nucleic acid nanostructures, characterized in that the mRNA delivery system comprises an mRNA scaffold strand, an RNA staple strand, a lipid-nucleic acid coupled strand, and a helper lipid; the mRNA scaffold strand includes a nucleic acid sequence encoding a target protein mRNA; the mRNA scaffold strand and the RNA staple strand are self-assembled through base complementary pairing to form a nucleic acid nanostructure; the lipid-nucleic acid coupled strand includes a lipid molecule and a nucleic acid strand coupled thereto.
  2. 2. The nucleic acid nanostructure-based mRNA delivery system of claim 1, wherein the mRNA scaffold strand comprises mRNA encoding EGFP fluorescent protein and the nucleic acid sequence of the RNA staple strand comprises the sequence of SEQ ID NO. 9-SEQ ID NO.24, or, The mRNA scaffold chain comprises mRNA which simultaneously codes two fluorescent proteins of mCherry and EGFP, the nucleic acid sequence of the RNA staple chain comprises sequences shown in SEQ ID NO. 25-SEQ ID NO.45, or, The mRNA scaffold chain comprises mRNA encoding p53 protein, the nucleic acid sequence of the RNA staple chain comprises sequences shown in SEQ ID NO. 46-SEQ ID NO.73, or, The mRNA scaffold chain comprises mRNA for encoding ovalbumin, and the nucleic acid sequence of the RNA staple chain comprises sequences shown in SEQ ID NO. 74-SEQ ID NO. 101; preferably, the shape of the nucleic acid nanostructure comprises any one or a combination of at least two of square, rectangle or triangle.
  3. 3. The nucleic acid nanostructure-based mRNA delivery system of claim 1 or 2, wherein the RNA staple chain contains a nucleic acid capture chain thereon.
  4. 4. The nucleic acid nanostructure-based mRNA delivery system of claim 3, wherein the nucleic acid capture strand is linked to the lipid-nucleic acid conjugate strand by base complementary pairing; preferably, the lipid-nucleic acid coupled chain has a HS-SH C6 modification at the 5 'or 3' end, and the nucleic acid sequence thereof comprises the sequence shown in SEQ ID NO.102 and/or SEQ ID NO. 103; preferably, the lipid in the lipid-nucleic acid coupled chain comprises any one or a combination of at least two of 1, 2-dioleoyl-SN-glycero-3-phosphoethanolamine, distearoyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, or dipalmitoyl phosphatidylethanolamine.
  5. 5. The nucleic acid nanostructure-based mRNA delivery system of any one of claims 1-4, wherein the lipid-nucleic acid conjugate chain is bound to the nucleic acid nanostructure by hydrophobic interactions with the helper lipid; Preferably, the molar ratio of the lipid-nucleic acid coupled chain to the auxiliary lipid is 1 (25-500); Preferably, the helper lipid comprises any one or a combination of at least two of a cationic lipid, a neutral lipid or a pegylated lipid; preferably, the cationic lipid comprises any one or a combination of at least two of N- (2, 3-dioleyloxy) propyl-N, N-triethylammonium chloride, N- (2, 3-dioleyloxy) propyl-N, N-trimethylammonium chloride or N- (1- (2, 3-dioleyloxy) propyl) -N-2- (spermidine carboxamido) ethyl-N, N-dimethyltrifluoroammonium acetate; Preferably, the neutral lipid comprises any one or a combination of at least two of 1, 2-dioleoyl-SN-glycero-3-phosphorylcholine, 1, 2-distearoyl-SN-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-SN-glycero-3-phosphorylcholine, 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine, folic acid modified 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine, or folic acid modified 1, 2-dioleoyl-SN-glycero-3-phosphorylethanolamine; Preferably, the pegylated lipid comprises 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 and/or polyethylene glycol-ditetradecylacetamide.
  6. 6. The nucleic acid nanostructure-based mRNA delivery system of any one of claims 1-5, wherein the helper lipid is modified with a targeting ligand capable of recognizing a cell surface receptor or an immunoadjuvant that activates immune cells, comprising any one or a combination of at least two of folic acid, mannose, RGD peptide, lipopolysaccharide, R848, or CpG.
  7. 7. A method of preparing a nucleic acid nanostructure-based mRNA delivery system according to any one of claims 1 to 6, comprising: preparing a target mRNA scaffold chain, preparing an RNA staple chain, annealing and assembling the mRNA scaffold chain and the corresponding RNA staple chain to form a nucleic acid nanostructure, preparing a lipid-nucleic acid coupled chain, co-assembling the nucleic acid nanostructure and the lipid-nucleic acid coupled chain, and then co-incubating with auxiliary lipid to obtain the mRNA delivery system based on the nucleic acid nanostructure.
  8. 8. The method of preparing a nucleic acid nanostructure-based mRNA delivery system of claim 7, wherein the preparing a nucleic acid nanostructure comprises: obtaining mRNA scaffold chains for encoding target proteins and corresponding RNA staple chains by adopting a PCR amplification and in vitro transcription method; Preferably, the primer nucleic acid sequence for PCR amplification comprises sequences shown in SEQ ID NO. 1-SEQ ID NO. 8; Preferably, the molar ratio of the mRNA scaffold chain to the RNA staple chain is 1 (1-20); Preferably, the annealing assembly condition is that the temperature is 63-67 ℃ and kept for 2-10 min, and the annealing assembly condition is gradually cooled to 10-20 ℃ within 1-4 h.
  9. 9. The method of preparing a nucleic acid nanostructure-based mRNA delivery system according to claim 7 or 8, wherein preparing a lipid-nucleic acid coupled strand comprises: Carrying out click reaction on the nucleic acid chain with the terminal modified sulfhydryl group and the maleimide modified lipid molecule to obtain a lipid-nucleic acid coupled chain; preferably, the molar ratio of the nucleic acid chain with the terminal modified sulfhydryl group to the maleimide modified lipid molecule is 1 (5-40); preferably, the reaction temperature of the click reaction is 20-40 ℃ and the reaction time is 0.5-4 h. Preferably, the preparation method further comprises hybridizing the nucleic acid nanostructure with a lipid-nucleic acid coupled chain and incubating with a helper lipid to obtain a nucleic acid nanostructure-based mRNA delivery system; Preferably, the mole ratio of the nucleic acid capturing chain to the lipid-nucleic acid coupling chain is 1 (1-2); Preferably, the hybridization condition is that the temperature is 20-40 ℃ and kept for 10-90 min, and the temperature is reduced to 2-6 ℃; preferably, the incubation condition is 2-30 ℃ for 10-90 min, and dialysis is performed for 8-24 h.
  10. 10. Use of the nucleic acid nanostructure-based mRNA delivery system of any one of claims 1-6 in the preparation of a tumor therapeutic drug; preferably, the tumor comprises any one or a combination of at least two of cervical cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, non-small cell cancer, skin cancer or lung cancer.

Description

MRNA delivery system based on nucleic acid nano structure and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicine, and relates to an mRNA delivery system based on a nucleic acid nanostructure, a preparation method and application thereof. Background Messenger RNA (mRNA) therapy has the advantages of low risk of gene integration, high safety, easy large-scale preparation and the like because the messenger RNA (mRNA) therapy can efficiently express specific proteins in vivo, and has wide application prospects in a plurality of fields such as infectious disease vaccines, tumor immunotherapy, protein substitution therapy, gene editing and the like. However, mRNA is a biomacromolecule with a strong negative charge, and has problems of difficulty in penetrating cell membranes and easiness in degradation, etc., and its in vivo administration is generally dependent on a carrier system, so that its therapeutic effect depends greatly on the delivery efficiency of a delivery carrier. Currently, the development of efficient and safe delivery systems to achieve sustained and controlled protein expression and low side effects remains a significant challenge in the field of mRNA delivery. Thanks to the biological macromolecule property of the nucleic acid, compared with other nano materials, the nucleic acid nano structure has the unique advantages of excellent biological molecule recognition capability, sequence programmability, space addressability, biocompatibility and the like. The preparation method is simple, has universality and high production efficiency, and can integrate a plurality of functional components efficiently through reasonable design. The characteristics lead the sensor to have wide application prospect in the biomedical fields such as biosensing, biological imaging, drug delivery and the like. Drug delivery systems based on nucleic acid nanostructures have been developed for in vivo delivery of small molecule drugs, protein drugs, and portions of nucleic acid drugs, but their use in the field of mRNA delivery is still further explored. In conclusion, by utilizing the advantages of the programmable characteristic, the spatial addressability, the good biocompatibility and the like of the nucleic acid nanostructure, the nucleic acid nanostructure which is based on the accurate encoding of the functional protein of the mRNA molecule is developed and used for safely and efficiently delivering the mRNA, so that the expression and the regulation of the living body level are of great research significance. Disclosure of Invention Aiming at the problems of insufficient protein expression controllability and safety and the like in the mRNA delivery field in the prior art, the invention provides an mRNA delivery system based on a nucleic acid nanostructure, a preparation method and application thereof. The mRNA delivery system is formed by self-assembly of mRNA scaffold strands and RNA staple strands by base-pairing. The mRNA delivery system accurately integrates mRNA molecules encoding functional proteins into a nucleic acid nano structure as a scaffold chain, achieves the purposes of protecting and delivering mRNA, and remarkably improves the protein expression of the mRNA and the tumor treatment effect. In order to achieve the aim of the invention, the invention adopts the following technical scheme: in a first aspect, the present invention provides a nucleic acid nanostructure-based mRNA delivery system comprising an mRNA scaffold strand, an RNA staple strand, a lipid-nucleic acid conjugate strand, and a helper lipid; the mRNA scaffold strand includes a nucleic acid sequence encoding a target protein mRNA; the mRNA scaffold strand and the RNA staple strand are self-assembled through base complementary pairing to form a nucleic acid nanostructure; the lipid-nucleic acid coupled strand includes a lipid molecule and a nucleic acid strand coupled thereto. According to the invention, through reasonable sequence and structural design, the nucleic acid sequence of the functional mRNA molecule is used as a scaffold chain and is self-assembled with an RNA staple chain through base complementation pairing to form a nucleic acid nano structure, and the nucleic acid nano structure not only serves as a drug delivery system to deliver mRNA encoding functional protein into cells, but also serves as a drug to play a role in guiding protein expression. Preferably, the mRNA scaffold chain comprises mRNA encoding EGFP fluorescent protein, and the nucleic acid sequence of the RNA staple chain comprises the sequence shown in SEQ ID NO. 9-SEQ ID NO.24, or, The mRNA scaffold chain comprises mRNA which simultaneously codes two fluorescent proteins of mCherry and EGFP, the nucleic acid sequence of the RNA staple chain comprises sequences shown in SEQ ID NO. 25-SEQ ID NO.45, or, The mRNA scaffold chain comprises mRNA encoding p53 protein, the nucleic acid seq