A monovalent broad-spectrum orthopoxvirus mRNA vaccine and its application

Abstract

The present invention provides an mRNA vaccine for orthopoxviruses that encodes and secretes surface proteins M1R, B6R, C19L, E8L, and H3L derived from the 2022 strain of monkeypox virus, which have high sequence identity with homologous proteins from smallpox virus, vaccinia virus, and mousepox virus. The mRNA vaccine provided by the present invention is immunoprotective against a lethal challenge with mousepox virus, with the mRNA vaccine encoding C19L providing 100% immunoprotection. It can be used as a monovalent, broad-spectrum mRNA vaccine for orthopoxviruses.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to an mRNA vaccine against monkeypox virus. Background Art

[0002] Since early May 2022, the monkeypox virus has been circulating on a large scale outside of Africa for the first time. On July 23, the WHO declared the monkeypox outbreak a global public health emergency of international concern, indicating that the virus poses a significant threat to global human health and requires action to prevent further spread and escalation into a pandemic. As of November 23, 2022, there have been over 80,000 confirmed cases of monkeypox worldwide, affecting 110 countries and regions, 103 of which are new cases. The 2022 monkeypox virus strain belongs to the West African clade and exhibits some sequence variation.

[0003] Monkeypox virus (MPXV) is a double-stranded DNA virus with a genome size of approximately 197 kb, encoding over 200 proteins. It belongs to the genus Orthopoxvirus in the Poxviridae family, a genus that includes viruses such as variola virus, vaccinia virus, mousepox virus, and vaccinia virus. The genomes of VARV and MPV share 90% homology, and smallpox vaccination has an efficacy of 85% in preventing monkeypox. Currently approved monkeypox vaccines for smallpox are viral vector vaccines, including ACAM2000 (Emergent BioSolutions) and MVA-BN (Bavarian Nordic). ACAM2000 is a second-generation, live, attenuated vaccinia virus vaccine approved by the US Food and Drug Administration (FDA) for use before or after exposure to MPV. MVA-BN is a third-generation, attenuated, non-replicating, modified vaccinia Ankara strain vaccine approved for smallpox prevention in the US and Europe and for the prevention of monkeypox infection in 2019.

[0004] Monkeypox virus (MPV) has a brick-shaped, double-membrane structure and can assume two forms: the intracellular mature virion (IMV) and the extracellular enveloped virion (EEV). These forms exhibit distinct surface membrane protein expression profiles, posing significant challenges for the development of recombinant monkeypox vaccines. Currently, candidate vaccines based on DNA, recombinant protein, and viral vector-based vaccines have been reported, including antigens such as vaccinia virus L1R (M1R for the MPV homologous protein), A27L (A29L for the MPV homologous protein), A33R (A35R for the MPV homologous protein), B5R (B6R for the MPV homologous protein), H3L (H3L for the MPV homologous protein), and D8L (E8L for the MPV homologous protein). M1R, A29L, H3L, and D8L are surface proteins of IMV, while A35R and B6R are surface proteins of EEV. The above-mentioned recombinant vaccinia virus vaccines all have a certain immune protection effect, but the antigen components alone are difficult to produce complete protective immunity against lethal challenge. mRNA vaccines have technical advantages such as rapid synthesis and high immunogenicity, and are suitable for screening and identifying protective antigens for complex pathogens such as monkeypox, but no monkeypox mRNA vaccine has yet been approved for marketing. The purpose of the present invention is to provide an mRNA vaccine that can encode the surface protein of the 2022 epidemic strain of monkeypox virus, which has high homology in the genus Orthopoxvirus and has efficient immune protection in a lethal challenge model in mice. Summary of the Invention

[0005] To achieve the above objectives, the present invention first provides an mRNA molecule comprising a promoter region, a 5' UTR region, an antigen protein coding region, a 3' UTR region, and a poly(A) tail region. The antigen protein coding region is an mRNA encoding surface proteins of the 2022 epidemic strain of monkeypox virus, and the antigen proteins include full-length forms of M1R, B6R, C19L, E8L, and H3L. The present invention incorporates a polynucleotide encoding a tPA signal peptide at the 5' end of the polynucleotide encoding the antigen protein. The polynucleotide has been artificially optimized to achieve superior protein expression, immune enhancement, and protective effects.

[0006] In one embodiment of the present invention, the amino acid sequence of M1R is shown in SEQ ID NO: 2, and the sequence of the polynucleotide after the fusion of the tPA signal peptide encoding polynucleotide and the optimized M1R encoding polynucleotide is shown in SEQ ID NO: 3. The sequence of the antigen protein coding region in the mRNA molecule is transcribed from the polynucleotide shown in SEQ ID NO: 3.

[0007] In one embodiment of the present invention, the amino acid sequence of B6R is shown in SEQ ID NO: 4, and the sequence of the polynucleotide after the fusion of the polynucleotide encoding the tPA signal peptide and the optimized B6R encoding polynucleotide is shown in SEQ ID NO: 5. The sequence of the antigen protein coding region in the mRNA molecule is transcribed from the polynucleotide shown in SEQ ID NO: 5.

[0008] In one embodiment of the present invention, the amino acid sequence of C19L is shown in SEQ ID NO: 6, and the sequence of the polynucleotide after the fusion of the polynucleotide encoding the tPA signal peptide and the optimized C19L encoding polynucleotide is shown in SEQ ID NO: 7. The sequence of the antigen protein coding region in the mRNA molecule is transcribed from the polynucleotide shown in SEQ ID NO: 7.

[0009] In one embodiment of the present invention, the amino acid sequence of E8L is shown in SEQ ID NO: 8, and the sequence of the polynucleotide after the fusion of the polynucleotide encoding the tPA signal peptide and the optimized E8L encoding polynucleotide is shown in SEQ ID NO: 9. The sequence of the antigen protein coding region in the mRNA molecule is transcribed from the polynucleotide shown in SEQ ID NO: 9.

[0010] In one embodiment of the present invention, the amino acid sequence of H3L is shown in SEQ ID NO: 10, and the sequence of the polynucleotide after the fusion of the polynucleotide encoding the tPA signal peptide and the optimized E8L encoding polynucleotide is shown in SEQ ID NO: 11. The sequence of the antigen protein coding region in the mRNA molecule is transcribed from the polynucleotide shown in SEQ ID NO: 11.

[0011] In a preferred embodiment of the present invention, the 5'UTR region in the mRNA molecule is transcribed from a polynucleotide molecule having a sequence as shown in SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.

[0012] In another preferred embodiment of the present invention, the 3'UTR region in the mRNA molecule is transcribed from a polynucleotide molecule having a sequence as shown in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

[0013] In another preferred embodiment of the present invention, the poly (A) region in the mRNA molecule contains n adenylate nucleotides, wherein n is any integer between 25-200.

[0014] In another preferred embodiment of the present invention, the promoter region in the mRNA molecule is a T7 promoter, and its DNA sequence is shown in SEQ ID NO:18.

[0015] In a specific embodiment of the present invention, the mRNA molecule according to claim 1, the 5'UTR region is transcribed from a polynucleotide molecule with a sequence such as SEQ ID NO: 14, the 3'UTR region is transcribed from a polynucleotide molecule with a sequence such as SEQ ID NO: 17, n is 110, and a BspQI restriction site is further provided at the 3' end of the mRNA molecule.

[0016] In some embodiments of the present invention, modifications to nucleosides in mRNA are included, preferably modifications to uracil, including modifications to pseudouridine and N1-methylpseudouridine. More preferably, the modification is N1-methylpseudouridine, with the modification ratio of uracil being 50%-100%. In a specific embodiment of the present invention, the mRNAs encoding M1R, B6R, C19L, E8L, and H3L all have a 100% N1-methylpseudouridine modification ratio for uracil.

[0017] Secondly, the present invention provides the use of the above-mentioned mRNA molecules in the preparation of drugs for preventing and / or treating monkeypox virus disease.

[0018] Again, the present invention provides a delivery vector containing the above-mentioned mRNA.

[0019] In a preferred embodiment of the present invention, the delivery vehicle is a liposome nanoparticle.

[0020] In a more preferred embodiment of the present invention, the liposome nanoparticles include cationic lipids, neutral lipids, PEG-modified lipids and sterols.

[0021] More preferably, the cationic lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanoxy)hexyl)amino)octanoate) (SM-102), the neutral lipid is distearoylphosphatidylcholine (DSPC), the PEG-modified lipid is methoxypolyethylene glycol dimyristoylglycerol (DMG-PEG2000), and the sterol is cholesterol.

[0022] Particularly preferably, the molar ratio of heptadec-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), distearoylphosphatidylcholine, methoxypolyethylene glycol dimyristoylglycerol, and cholesterol is 50:10:1.5:38.5.

[0023] In one embodiment of the present invention, the mRNA-delivering liposome nanoparticles can be prepared by the following steps: SM-102, DSPC, DMG-PEG2000, and cholesterol are dissolved in ethanol at a molar ratio of 50:10:1.5:38.5 to prepare the ethanol phase; the mRNA molecules are dissolved in 50 mM sodium acetate buffer (pH 5.0) to prepare the aqueous phase. During microfluidic packaging, the volume ratio of the ethanol phase to the aqueous phase is 1:3, and the total flow rate is 12 ml / min. After packaging, the solution is concentrated by ultrafiltration and exchanged into PBS buffer. The encapsulation efficiency and effective concentration are determined and the solution is stored at 4°C.

[0024] Finally, the present invention provides an mRNA molecule composition, comprising 2, 3 or 4 different mRNA molecules, wherein the mRNA molecules contain a promoter region, a 5'UTR region, an antigen protein coding region, a 3'UTR region and a poly(A) tail region, wherein the antigen protein coding region of the mRNA molecule is transcribed according to any one of the following polynucleotide molecules:

[0025] A polynucleotide molecule encoding an M1R protein as shown in SEQ ID NO: 3;

[0026] A polynucleotide molecule encoding a B6R protein as shown in SEQ ID NO: 5;

[0027] A polynucleotide molecule encoding the E8L protein as shown in SEQ ID NO: 9;

[0028] The polynucleotide molecule encoding the H3L protein has a sequence as shown in SEQ ID NO: 11.

[0029] Mousepox virus is a natural host of mice, and extremely low challenge doses are lethal to mice. It has been approved by the US FDA as a surrogate model for the evaluation of smallpox virus vaccines and drugs. The mRNA-M1R, mRNA-B6R, mRNA-C19L, mRNA-E8L, and mRNA-H3L vaccines provided herein, encoding surface proteins of the 2022 strain of monkeypox virus, can activate a specific immune response against monkeypox virus in a mouse model. All of the above-mentioned mRNA vaccines provided herein are immunoprotective against lethal challenge with mousepox virus, particularly the mRNA vaccine encoding monkeypox C19L, which exhibits 100% immunoprotection against lethal challenge. C19L is a surface protein of the monkeypox virus (EEV) (the homologous protein in vaccinia virus is F13L), which plays an important role in viral membrane binding, virion encapsulation, and EEV production. The immunoprotective mRNA-C19L vaccine provided herein demonstrates for the first time that C19L can serve as a candidate target for orthopoxvirus vaccines. In addition, the sequence identities of monkeypox virus C19L with the homologous proteins of smallpox virus, vaccinia virus, and mousepox virus are 97.6%, 99.2%, and 98.9%, respectively, indicating that the mRNA-C19L vaccine provided by the present invention is expected to be used as a broad-spectrum mRNA vaccine for orthopoxvirus in a monovalent form, and the mRNA-M1R, mRNA-B6R, mRNA-E8L, and mRNA-H3L vaccines provided by the present invention can provide partial protection against lethal challenge. Sixteen days after mousepox virus challenge, the protection rates reached 50%, 33%, 17%, and 17%, respectively, and are expected to be used as a broad-spectrum mRNA vaccine for orthopoxvirus in a polyvalent form. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the template plasmid structure for preparing mRNA vaccines;

[0031] Figure 2 Capillary electrophoresis analysis of synthetic mRNA molecules encoding monkeypox surface protein;

[0032] Figure 3 Dynamic light scattering analysis of monkeypox mRNA vaccine packaged in liposomal nanoparticles.

[0033] Figure 4 Analysis of specific antibody levels for monkeypox virus mRNA vaccine candidates in a mouse model;

[0034] Figure 5 . Analytical analysis of the immune protection of monkeypox virus mRNA vaccine candidates against lethal challenge with mousepox virus;

[0035] Figure 6 .Homology analysis diagram of M1R, B6R, C19L, E8L, and H3L proteins. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0037] Example 1. Preparation of mRNA vaccine

[0038] (1) Construction of template plasmid

[0039] The amino acid sequences of the M1R, B6R, C19L, E8L, and H3L proteins are derived from the 2022 monkeypox strain MPXV_USA_2022_MA001 (Genbank ON563414). The tPA signal peptide (sequence shown in SEQ ID NO:1) was fused to the N-terminus. After codon optimization, the resulting target nucleotide sequences were SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11, respectively. The optimization strategy was primarily based on human codon usage frequency and avoided the BspQI cleavage site (SEQ ID NO:19). The codon adaptation indices of the M1R, B6R, C19L, E8L, and H3L target nucleotide sequences reached 0.95, 0.96, 0.96, 0.97, and 0.95, respectively, significantly higher than the wild-type sequences of 0.66, 0.64, 0.66, 0.66, and 0.69. In the 5'->3' direction, the T7 promoter (SEQ ID NO: 18), 5'UTR (SEQ ID NO: 14), target nucleotide, 3'UTR (SEQ ID NO: 17), stop codon, 110 nt poly (A) sequence, and BspQI restriction site were concatenated (SEQ ID NO: 19) and cloned into the PUC57 plasmid to obtain a template plasmid (the template plasmid structure is shown in the figure). Figure 1 shown).

[0040] (2) Linearization of template plasmid

[0041] A 200-μl reaction system contained 20 μg of template plasmid, 10 μl of BspQI enzyme (10 U / μl), 20 μl of 10× BspQI Buffer, and Nuclease-Free H2O. The reaction was incubated at 50°C for 1 hour. The linearized plasmid was purified by phenol-chloroform extraction. An equal volume of phenol-chloroform (Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1) was added to the DNA solution and mixed thoroughly. Centrifuge at 12,000 g for 10 minutes at room temperature. Carefully aspirate the upper aqueous phase, add an equal volume of chloroform solution (chloroform:isoamyl alcohol = 24:1), and mix thoroughly. Centrifuge as above and carefully aspirate the supernatant. As above, the DNA concentration was determined.

[0042] (3) In vitro transcription and purification of mRNA

[0043] For in vitro transcription of mRNAs encoding M1R, B6R, C19L, E8L, and H3L, the modification ratio of uracil to N1-methylpseudouridine was 100%. A 100-μl reaction system contained 5 μg of linearized plasmid, 10 μl of T7 RNA polymerase (50 U / μl), 5 μl of inorganic pyrophosphatase (0.1 U / μl), 5 μl of RNase Inhibitor (40 U / μl), 10 μl of 10× reaction buffer, 10 μl of ATP (100 mM), 10 μl of GTP (100 mM), 10 μl of m1ψ / UTP (100 mM), 10 μl of CTP (100 mM), and Nuclease-Free H2O (all reagents purchased from Novozymes). The mixture was mixed thoroughly and incubated at 37°C for 2 hours. Then, 5 μl of DNase I (1 U / μl) was added to the reaction system and incubated at 37°C for 15 min to remove the transcription DNA template. The mRNA transcript was purified using phenol-chloroform as described above.

[0044] (4) mRNA capping and purification

[0045] A 100-ul reaction system contained 200 μg of transcribed mRNA, 50 μl of 10 × Capping Reaction buffer, 25 μl of GTP (10 mM), 25 μl of SAM (4 mM), 25 μl of Vaccinia Capping Enzyme (10 U / μl), 25 μl of 2'-O-Methyltransferase (50 U / μl), and Nuclease-Free H2O (the above reagents were purchased from Novozymes). After mixing well, the reaction was incubated at 37°C for 1 hour. The mRNA transcript was purified using phenol-chloroform as described above. The molecular integrity of the mRNA transcript was detected by capillary electrophoresis ( Figure 2 ), the results showed that the sizes of the prepared mRNA-M1R, mRNA-B6R, mRNA-C19L, mRNA-E8L, mRNA-H3L, and mRNA-A29L products were in line with expectations, and the purity reached more than 90%.

[0046] (5) mRNA liposome nanoparticle packaging

[0047] SM-102 (heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), purchased from Synopsys, DSPC (distearoylphosphatidylcholine, purchased from Synopsys), DMG-PEG2000 (methoxypolyethylene glycol dimyristoylglycerol, purchased from Synopsys), and cholesterol (purchased from Avitor) were dissolved in ethanol at a molar ratio of 50:10:1.5:38.5 to prepare the ethanol phase; mRNA molecules were dissolved in 50 mM sodium acetate buffer (pH 5.0) to prepare the aqueous phase. During microfluidic packaging, the volume ratio of the ethanol phase to the aqueous phase was 1:3, and the total flow rate was 12 ml / min. After packaging, the solution was ultrafiltration and concentrated to PBS buffer, and the encapsulation efficiency and effective concentration were determined before storage at 4°C. Dynamic light scattering analysis showed that the mRNA vaccine packaged in liposome nanoparticles had a uniform particle size distribution ( Figure 3 ), the average diameters of LNP-mRNA-M1R, LNP-mRNA-B6R, LNP-mRNA-C19L, LNP-mRNA-E8L, and LNP-mRNA-H3L were 75.8nm, 76.9nm, 76.8nm, 77.3nm, and 79.3nm, respectively, and the dispersion coefficients were all less than 0.05.

[0048] Example 2. Immune Response to mRNA Vaccine

[0049] In the BALB / c mouse model, 5 μg of mRNA-M1R, mRNA-B6R, mRNA-C19L, mRNA-E8L, and mRNA-H3L candidate vaccines were administered intramuscularly on days 0 and 14, respectively (n = 6 in each group). Blood and serum were collected on days 14 and 28 for specific IgG antibody detection.

[0050] (1) Specific IgG antibody response detection

[0051] Recombinant proteins of M1R (Sino Biological, 40904-V07H), B6R (Sino Biological, 40902-V08H), C19L (Sino Biological, 40894-V08B), E8L (AcroBiosystem, E8L-M52H3), and H3L (Sino Biological, 40893-V08H1) were diluted to a concentration of 1 μg / ml and coated overnight in 96-well plates. After blocking, the IgG antibody titer was detected by enzyme-linked immunosorbent assay. The results showed that 14 days after a single immunization, mRNA-M1R, mRNA-B6R, mRNA-C19L, mRNA-E8L, and mRNA-H3L induced specific antibody responses, with titers of 14997, 2042, 2000, 280543, and 25942, respectively; 14 days after the booster immunization (28 days after the first immunization), the IgG antibodies produced further increased, with titers reaching 1458814, 37411, 2404, 3639150, and 2523481, respectively. Figure 4 ).

[0052] (2) Protective effect of lethal attack by mousepox virus

[0053] Mousepox virus (ATCC VR-1374) was amplified and cultured by infection of BS-C-1 cells. 105 days after the first immunization, each mouse was intraperitoneally challenged with 200 PFU of mousepox virus. All mice in the PBS control group died within 6 days after the challenge. At the end of the 16-day observation period, the survival rates of mice in the mRNA-M1R, mRNA-B6R, mRNA-C19L, mRNA-E8L, and mRNA-H3L immunization groups were 50%, 33%, 100%, 17%, and 17%, respectively. Log-rank (Mantel-Cox) test statistical analysis showed that the above-mentioned mRNA vaccine candidates all had a certain degree of immune protection (P value less than 0.05), but only mRNA-C19L had 100% immune protection ( Figure 5 ).

[0054] Example 3. Analysis of coding sequence homology

[0055] The bioinformatics software (Geneious) was used to analyze the sequence homology of monkeypox M1R, B6R, C19L, E8L, and H3L proteins in different orthopoxvirus genera for the complete genome sequences of monkeypox virus (Genbank ON563414), smallpox virus (Genbank L22579.1), vaccinia virus (Genbank NC_006998.1), and mousepox virus (Genbank AF012825.2). The sequence identities of monkeypox virus M1R and the homologous proteins of smallpox virus, vaccinia virus, and mousepox virus were 99.2%, 98.4%, and 97.6%, respectively. The sequence identities of monkeypox virus B6R and smallpox virus were 99.2%, 98.4%, and 97.6%, respectively. The sequence identities of the homologous proteins of monkeypox virus, vaccinia virus and mousepox virus are 92.4%, 96.5% and 96.5% respectively; the sequence identities of monkeypox virus C19L and the homologous proteins of smallpox virus, vaccinia virus and mousepox virus are 97.6%, 99.2% and 98.9% respectively; the sequence identities of monkeypox virus E8L and the homologous proteins of smallpox virus, vaccinia virus and mousepox virus are 93.1%, 94.4% and 95.7% respectively; the sequence identities of monkeypox virus H3L and the homologous proteins of smallpox virus, vaccinia virus and mousepox virus are 94.2%, 93.8% and 93.5% respectively. Figure 6 The results showed that M1R, B6R, C19L, E8L, and H3L all shared high sequence identity (greater than 90%) among the aforementioned poxviruses, with C19L and M1R sharing the highest identity (greater than 95%). Considering that only mRNA-C19L demonstrated 100% protection against lethal challenge in Example 2, it is promising as a monovalent broad-spectrum mRNA vaccine for orthopoxviruses. mRNA-M1R, mRNA-B6R, mRNA-E8L, and mRNA-H3L, on the other hand, were able to provide partial protection against lethal challenge and are promising as a multivalent broad-spectrum mRNA vaccine for orthopoxviruses.

Claims

1. An mRNA molecule comprising a promoter region, a 5'UTR region, an antigen protein coding region, a 3'UTR region and a poly(A) tail region, wherein: The antigen protein coding region is transcribed from the following polynucleotide molecule: the polynucleotide molecule whose sequence is shown in SEQ ID NO:

7.

2. The mRNA molecule according to claim 1, wherein The 5'UTR region is transcribed from a polynucleotide molecule with a sequence as shown in SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO:

14.

3. The mRNA molecule according to claim 1, wherein The 3'UTR region is transcribed from a polynucleotide molecule with a sequence as shown in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO:

17.

4. The mRNA molecule according to claim 1, characterized in that The poly (A) region contains n adenylate nucleotides, wherein n is any integer between 25-200.

5. The mRNA molecule according to claim 1, wherein The promoter region is a T7 promoter, and its DNA sequence is shown in SEQ ID NO:

18.

6. The mRNA molecule according to claim 1, wherein The 5'UTR region is transcribed from a polynucleotide molecule with a sequence such as SEQ ID NO: 14, the 3'UTR region is transcribed from a polynucleotide molecule with a sequence such as SEQ ID NO: 17, n is 110, and a BspQI enzyme cleavage site is provided at the 3' end of the mRNA molecule.

7. Use of the mRNA molecule according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating monkeypox virus disease.

8. A delivery vector comprising the mRNA molecule according to any one of claims 1-6.

9. The delivery vector according to claim 8, characterized in that The delivery vehicle is a liposome nanoparticle.

10. The delivery vector according to claim 9, characterized in that The liposome nanoparticles include cationic lipids, neutral lipids, PEG-modified lipids and sterols.

11. The delivery vector according to claim 10, characterized in that The cationic lipid is heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanoxy)hexyl)amino)octanoate, the neutral lipid is distearoylphosphatidylcholine, the PEG-modified lipid is methoxypolyethylene glycol dimyristoylglycerol, and the sterol is cholesterol.

12. The delivery vector according to claim 11, characterized in that The molar ratio of the heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanoxy)hexyl)amino)octanoate), distearoylphosphatidylcholine, methoxypolyethylene glycol dimyristoylglycerol, and cholesterol is 50:10:1.5:38.5.

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