CN113577258A

CN113577258A - Double-target mRNA vaccine and preparation method thereof

Info

Publication number: CN113577258A
Application number: CN202110876695.XA
Authority: CN
Inventors: 刘明录; 卢永灿; 冯建海; 强邦明; 王立新; 韩庆梅; 金海锋; 王亮; 许淼
Original assignee: Shandong Xinrui Biotechnology Co ltd
Current assignee: Shandong Xinrui Biotechnology Co ltd
Priority date: 2021-07-31
Filing date: 2021-07-31
Publication date: 2021-11-02
Anticipated expiration: 2041-07-31
Also published as: CN113577258B

Abstract

The invention provides a double-target mRNA vaccine, which comprises a liposome mixture and double-target mRNA; the liposome mixture comprises DLin-MC3-DMA, DSPC, cholesterol and PEG200-DMG, and the molar ratio is 50:10:37.5: 2.5; the double-target mRNA is obtained by transcription of linearized recombinant plasmids; the recombinant plasmid contains a target gene segment; the target gene fragment is obtained by sequentially connecting the following modules in series: 5 '-UTR nucleic acid artificial sequence, P53 nucleic acid artificial sequence, Linker nucleic acid artificial sequence, Kras nucleic acid artificial sequence, MITD nucleic acid artificial sequence, and 3' -UTR nucleic acid artificial sequence; the invention also provides a preparation method of the vaccine; the mRNA vaccine prepared by the invention can block the migration of mRNA, protect the mRNA from being degraded and improve the delivery efficiency of the mRNA.

Description

Double-target mRNA vaccine and preparation method thereof

Technical Field

The invention relates to a double-target mRNA vaccine and a preparation method thereof, belonging to the technical field of biological medicines.

Background

The tumor vaccine is a therapeutic tumor treatment method for activating the immune system of an organism to generate specific anti-tumor cell immune response by using tumor cells, tumor cell lysate or tumor antigen, and comprises a whole cell vaccine, a polypeptide vaccine, an RNA/DNA gene vaccine and a dendritic cell vaccine. Among them, the mRNA vaccine, as a novel nucleic acid vaccine, can express any protein and treat almost all protein-based diseases from the gene level. Compared with other traditional vaccines and DNA vaccine technologies, the synthesis of the mRNA vaccine is simple in production process and low in price. Meanwhile, the artificially designed nucleic acid does not induce the immune response of a human body, does not enter cell nucleuses, has no risk of integrating into genomes, and is relatively safer. However, mRNA has poor stability, is easily degraded by nuclease in vivo and in vitro, and has poor ability of penetrating cell membranes, which is a bottleneck for restricting the development of the mRNA. Therefore, mRNA vaccines require a suitable delivery vehicle to deliver them to the body for a better immune response.

At present, the tumor mRNA vaccine uses the nanometer preparation technology which is most widely based on cationic lipid and polymer, can protect nucleic acid drugs from degradation of nuclease, and successfully realizes the stable delivery of the nucleic acid drugs including siRNA, mRNA, Cas9 plasmid and the like in vivo. Cationic Lipid Nanoparticles (LNPs) are composed of cationic lipid, polyethylene glycol (PEG), cholesterol and the like, and encapsulate nucleic acid drugs, and the nucleic acid drugs are endocytosed into cells by cell membranes to form endosome vesicles, and along with a series of structural changes, mRNA is released from endosomes and is combined with ribosomes responsible for protein production to guide the synthesis of viral proteins. LNPs have good biocompatibility and physicochemical properties, exhibit high gene transfection effects both in vitro and in vivo, and are proven to be the safest drug delivery vehicles. RNA drugs (Patisiran injection) have been approved by the FDA for marketing for in vivo delivery of siRNA or other small oligonucleotides, but the materials for mRNA vaccine delivery are very limited. Because mRNA is easily degraded and its physicochemical characteristics are such that the existing nanomaterials have limited efficiency for mRNA delivery, the development and clinical application of mRNA-based tumor vaccines still face many problems. Therefore, there is a need to develop a new therapeutic approach to achieve efficient delivery of mRNA and efficient expression of the encoded antigen.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a double-target mRNA vaccine and a preparation method thereof, which realize the following purposes:

(1) the mRNA vaccine prepared by the invention has high entrapment rate;

(2) the LNP liposome prepared by the invention can block the migration of mRNA and protect the mRNA from being degraded; improving the transfection efficiency of the cells;

(3) the antibody protein produced by the mRNA vaccine is higher than naked mRNA;

(4) the mRNA vaccine can effectively induce T cells to generate immune response;

(5) the mRNA vaccine of the invention has high killing rate to tumor cells.

In order to solve the technical problems, the invention adopts the following technical scheme:

a dual-target mRNA vaccine comprising a liposome mixture and dual-target mRNA; the liposome mixture comprises DLin-MC3-DMA, DSPC, cholesterol and PEG200-DMG, and the molar ratio is 50:10:37.5: 2.5;

the double-target mRNA is obtained by transcription of linearized recombinant plasmids; the recombinant plasmid contains a target gene segment; the target gene fragment is obtained by sequentially connecting the following modules in series: 5 '-UTR nucleic acid artificial sequence, P53 nucleic acid artificial sequence, Linker nucleic acid artificial sequence, Kras nucleic acid artificial sequence, MITD nucleic acid artificial sequence, and 3' -UTR nucleic acid artificial sequence; the artificial sequence of the 5' -UTR nucleic acid is shown as SEQ ID NO.1 in a sequence table; the P53 nucleic acid artificial sequence is shown as SEQ ID NO.2 in the sequence table; the Linker nucleic acid artificial sequence is shown as SEQ ID NO.3 in the sequence table; the artificial sequence of the Kras nucleic acid is shown as SEQ ID NO.4 in a sequence table; the artificial sequence of the MITD nucleic acid is shown as SEQ ID NO.5 in a sequence table; the artificial sequence of the 3' -UTR nucleic acid is shown as SEQ ID NO.6 in the sequence table.

The following is a further improvement of the above technical solution:

the method for preparing the double-target mRNA vaccine comprises the steps of constructing a target gene segment, constructing a recombinant plasmid, linearly digesting and recovering the recombinant plasmid, transcribing to obtain mRNA and packaging the mRNA.

Constructing the recombinant plasmid, namely cloning a target gene fragment to a PVAX1 vector to obtain the recombinant plasmid; the transcription obtains mRNA, and the mRNA with a 5 'cap and a Poly (A) tail at the 3' end is synthesized by transcription.

The packaging mRNA is to adopt liposome mixture to package mRNA.

The packaged mRNA specifically comprises: preparing ethanol solution of liposome mixture, preparing mRNA aqueous solution, synthesizing LNP/mRNA vaccine particles and preparing the vaccine.

Preparation of an ethanol solution of the liposome mixture: dissolving the liposome mixture in ethanol to obtain an ethanol solution of the liposome mixture, wherein the mass-volume ratio of the liposome mixture to the ethanol is 16.1-16.2 mg: 1 mL;

preparation of the aqueous mRNA solution: dissolving the transcribed mRNA in a citric acid buffer solution, blowing, dispersing, adding PBS, and fully and uniformly mixing to obtain an mRNA aqueous solution; the mass-to-volume ratio of the mRNA to the citric acid buffer solution and the PBS is as follows: 0.4-3.2. mu.g: 1mL of: 2 mL;

synthesizing LNP/mRNA vaccine particles by using a nanoparticle synthesis system and synthesizing LNP/mRNA vaccine particles by using an ethanol solution and an mRNA aqueous solution of a liposome mixture in a volume ratio of 1: 3;

and (3) preparing the vaccine, namely dialyzing LNP/mRNA vaccine particles in a PBS solution for 24 hours, concentrating and filtering to obtain the vaccine.

Cancer is a disease caused by somatic gene mutation, and mutations of the P53 gene and the K-ras gene are present in more than half of cancers and overexpressed in tumors. After the P53 gene is inactivated by mutation, the synthesis of the normal P53 protein is influenced, the function of the protein is inhibited, the cell loses the monitoring on the DNA damage, and the mutation is easily accumulated to become a cancer cell; the K-Ras gene is mutated, so that cells can grow continuously, autophagy of the cells is inhibited, intracellular cell transduction is disturbed, cell proliferation is out of control to generate canceration, and a plurality of cancers such as lung cancer, pancreatic cancer, colorectal cancer and the like are related to the K-Ras mutation.

Compared with the prior art, the invention has the following beneficial effects:

(1) the mRNA vaccine prepared by the invention targets P53 and Kras genes expressed by tumor cells, has strong specificity and is not easy to have off-target effect.

(2) The particle size of the mRNA vaccine prepared by the invention is 92-96 nm, the entrapment rate is more than 98%, the specific LNP liposome is selected to wrap the mRNA, the migration of the mRNA can be blocked, the mRNA is protected from being degraded, the delivery efficiency of the mRNA is improved, the mRNA is directly translated and expressed after entering cells, and the protein expression amount of the LNP/mRNA vaccine is 2.3 times that of naked mRNA.

(3) The mRNA vaccine prepared by the invention can effectively induce T cells to generate immune response and improve the secretion of cell factors IL-2 and IFN-gamma.

(4) The mRNA vaccine prepared by the invention has high killing effect on tumor cells, and the killing rate on tumor target cells reaches 98.6%.

(5) The invention modifies the mRNA template transcribed in vitro, adds a cap at the 5 'end and a polyA tail at the 3' end, and protects the mRNA from being degraded.

(6) When the mRNA vaccine constructed by the invention constructs a gene expression vector, the molecular structure of the mRNA vaccine is modified by 5 'capping and 3' ploy (A) tail, so that the mRNA is protected from degradation of nuclease in the transfection process, and the stability of the mRNA is improved.

Drawings

FIG. 1 is a schematic diagram of the structure of PVAX1-P53Kras carrier prepared in example 1.

FIG. 2 is a schematic diagram of the structure of the PVAX1-EGFP vector prepared in example 1.

FIG. 3 is a structural diagram of a lipid nanoparticle.

FIG. 4 is a graph of the fluorescence expression of 293T cells transfected with different concentrations of LNP/EGFP in example 7.

FIG. 5 is a graph of IL-2 and IFN- γ secretion from different LNP nanoparticle-transfected cells of example 8.

Detailed Description

EXAMPLE 1 acquisition of target Gene and construction of recombinant plasmid

The invention selects P53 and K-ras as targets to design an mRNA vaccine aiming at the tumor.

The nucleic acid sequences of the modules of the PVAX1-P53Kras vector are as follows:

(1) 5' -UTR nucleic acid artificial sequence (SEQ ID NO.1)

(2) P53 nucleic acid artificial sequence (SEQ ID NO.2)

(3) Linker nucleic acid artificial sequence (SEQ ID NO.3)

(4) Kras nucleic acid artificial sequence (SEQ ID NO.4)

(5) MITD nucleic acid artificial sequence (SEQ ID NO.5)

(6) 3' -UTR nucleic acid artificial sequence (SEQ ID NO.6)

The gene sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 are connected in sequence, Nanjing Kingsrey Biotechnology Ltd is entrusted to synthesize the whole expression frame, and the whole expression frame is inserted into a vector pUC57 of a standard vector named as pUC-P53 Kras. The pUC-P53Kras vector and the PVAX1 vector (SEQ ID NO. 7) were subjected to double digestion with Fast Digest BamHI (available from Thermo Fisher) and Fast Digest NotI (available from Thermo Fisher), and the linearized DNA fragment was recovered by cutting the gel and ligated at 16 ℃ overnight to form a PVAX1-P53Kras expression vector. The above PVAX1-P53Kras was transformed into E.coli (DH 5. alpha.), and positive clones were picked up and identified by PCR. The plasmid is extracted and sent to Nanjing Jinslei Biotech limited for sequencing, the sequencing identification is correct, and the construction of PVAX1-P53Kras is successful (the structure of the vector is shown in figure 1). PVAX1-P53Kras plasmid was extracted from the positive clones, diluted to 2. mu.g/. mu.L and stored at-80 ℃ until use.

By adopting the same method, the EGFP gene fragment (SEQ ID NO. 8) is cloned to a PVAX1 vector, named as PVAX1-EGFP (the vector structure is shown in figure 2), the sequencing identification is correct, and the construction of the PVAX1-EGFP is successful. PVAX1-EGFP plasmid was extracted from positive clones, diluted to 2. mu.g/. mu.L and stored at-80 ℃ until use.

Example 2 linearized digestion and recovery of recombinant plasmids

The template for in vitro transcription requires an RNA polymerase promoter, and plasmid DNA must be linearized by restriction enzymes, because circular DNA will generate a long isomeric RNA template, restriction enzyme sites must be downstream of the target gene, and the restriction enzyme must be completely cut, Not I restriction enzymes are respectively selected to singly cut plasmids (PVAX 1-P53Kras and PVAX 1-EGFP), and the optimized cutting system (50 μ L in total) is as follows:

10 xBuffer: 3 mu L of the solution; NotI enzyme: 3 mu L of the solution; PVAX1-P53Kras plasmid: 1 μ L (2 μ g/. mu.L); water: 43 μ L.

10 xBuffer: 3 mu L of the solution; NotI enzyme: 3 mu L of the solution; PVAX1-EGFP plasmid: 1 μ L (2 μ g/. mu.L); water: 43 μ L.

Carrying out single enzyme digestion reaction on the two plasmids respectively;

mixing in 150 μ L centrifuge tube, performing enzyme digestion in 37 deg.C water bath for 1 hr, rapidly transferring into 65 deg.C water bath for 5min, and terminating enzyme digestion reaction. The size of the fragment was determined by electrophoresis on a 1% agarose gel to be as expected. Cutting off the target fragment, and adopting an agarose gel DNA recovery kit to recover the target fragment.

The concentration of the recovered DNA was measured with a spectrophotometer. Measuring the concentration of the fragment after the enzyme digestion of PVAX1-P53Kras to be 1.1 mu g/mu L, wherein A260/280= 1.83; the concentration of the fragment after the PVAX1-EGFP enzyme digestion is 1.2 μ g/μ L, A260/280=1.82, and the result conforms to the DNA determination standard (A260/280 = 1.8-2.1). Storing at-20 deg.C for use.

Example 3 in vitro transcription of mRNA (IVT mRNA)

5' capped mRNA was synthesized by in vitro transcription using the linearized DNA of example 2 as template and a capped mMESSAGE mMACHINE T7 kit (Ambion), as follows:

TABLE 1 transcription reaction System

1. The reaction was prepared on ice (see Table 1) according to the instructions, mixed well, centrifuged to the bottom of the PCR tube and incubated overnight at 37 ℃.

2. Adding 1 μ L of Dnase I, mixing well, incubating at 37 deg.C for 20min, and removing DNA from the product.

3. Purification of mRNA by LiCl precipitation:

(1) adding 25 mu L DEPC water to 50 mu L system;

(2) adding 25 mu L of LiCl solution, and uniformly mixing;

(3) incubating in a refrigerator at-20 deg.C for 30 min;

(4) centrifuging at 4 deg.C and 12000g for 15min, and removing supernatant;

(5) adding 500 μ L of 70% glacial ethanol into the precipitate, centrifuging at 4 deg.C for 15min, and removing supernatant;

(6) repeating the step (5);

(7) the mRNA was resuspended in an appropriate amount of DEPC water and quantified using a microspectrophotometer to determine a concentration of 2. mu.g/. mu.L mRNA.

4. The 3' end of the mRNA was tagged with the Poly (A) tail and the reaction was set up on ice (see Table 2).

TABLE 23' end plus Poly (A) Tail reaction System

5. Mixing well, centrifuging to tube bottom, and incubating at 37 deg.C for 3 h.

6. The mRNA was purified again by LiCl precipitation, which was the same as in step 3. The purified mRNA was resuspended in an appropriate amount of DEPC water to give a mRNA solution, the mRNA was quantitated using a microspectrophotometer to determine the concentration of mRNA as 2. mu.g/. mu.L, 50. mu.L of each tube was dispensed, and the mRNA product was stored in a freezer at-80 ℃.

7. Validation of in vitro transcribed mRNA length:

placing mRNA in a 70 deg.C water bath for 10min, standing on ice for 3min, performing electrophoresis in urea acrylamide gel at constant voltage of 100v for 30min, taking pictures with a gel imager, and verifying the size and integrity of mRNA.

The analysis of the results shows that P53Kras mRNA and EGFP mRNA obtained by in vitro transcription of linearized PVAX1-P53Kras and PVAX1-EGFP are electrophoresed in urea acrylamide gel, the obtained mRNA fragments are 1681bp and 817bp respectively, and the obtained mRNA accords with the expectation.

Example 4 LNP preparation and characterization of packaging mRNA

The LNP composition comprises: ionizable cationic lipid (DLin-MC 3-DMA), which is positively charged, electrostatically interacts with negatively charged mRNA and assembles into virus-sized particles, facilitating intracellular delivery and release of mRNA into the cytoplasm; cholesterol, which can enhance the fluidity of the liposome and stabilize the nanoparticles; phospholipids (DSPC), forming a lipid bilayer structure; PEG-lipid (PEG 200-DMG) improves the stability of the nano structure and prolongs the metabolism time of the nano drug in blood. The cationic liposome is internally coated with mRNA through electrostatic interaction, the hydrophobic end of PEG-lipid is combined with the hydrophobic end of cationic lipid, the hydrophilic end (connected with PEG) of PEG-lipid outwards forms the shell of the nucleic acid lipid nanoparticle, and in order to enhance the stability of the nucleic acid lipid nanoparticle, a proper amount of components such as cholesterol and the like are added, and finally the finished product of the nucleic acid lipid nanoparticle is obtained (see figure 3).

The specific operation steps are as follows:

the preparation method of the P53Kras mRNA vaccine comprises the following steps:

1. preparation of ethanol solution

10.4mg of DLin-MC3-DMA, 2.7mg of DSPC, 4.9mg of cholesterol and 2.2mg of PEG200-DMG as a solid (molar ratio: 50:10:37.5: 2.5) were weighed out and placed in a 15mL RNase-free tube, and 1.25mL of ethanol was added thereto and sufficiently stirred to dissolve it, thereby obtaining an ethanol solution of a liposome mixture.

2. Preparation of aqueous solutions

Mu.l of the mRNA solution prepared in example 3 (the mass of mRNA was 2 μ g) was dissolved in 1.25mL of 10mM citric acid buffer (PH =4), and blown up and down 50 times; adding 2.5mL PBS, blowing and beating for 50 times, and mixing fully to obtain mRNA aqueous solution.

3. Synthesis of LNP/mRNA vaccines

LNP particles were synthesized by mixing an ethanol solution of liposome mixture with an aqueous mRNA solution at a ratio of 1:3 (volume ratio) using a nanoparticle synthesis system (Ignite);

dialyzing the obtained LNP in PBS (PH 7.4) for 24h to obtain a solution containing LNP, concentrating to a volume of 1mL by using an Amicon ultracentrifuge filter, filtering with a 0.22um filter membrane for 2 times, and storing at-20 ℃ for later use to obtain a P53Kras mRNA vaccine expressed by LNP/P53Kras, wherein the concentration of mRNA is 2 mug/mL;

the concentration is the ratio of the mass of the initially added mRNA to the mass of the final vaccine by volume.

Preparation of EGFP mRNA vaccine:

EGFP mRNA vaccine was prepared according to the same method and indicated by LNP/EGFP;

PVAX1-P53Kras (DNA) vaccine:

on the basis of the preparation method of the P53Kras mRNA vaccine, the changes are as follows:

when preparing the aqueous solution, the same amount of PVAX1-P53Kras plasmid of example 1 was used instead of mRNA solution, and the PVAX1-P53Kras (DNA) vaccine was expressed as LNP/PVAX1-P53 Kras;

blank control:

when the aqueous solution is prepared, equal amount of DEPC water is used to replace mRNA solution, the rest preparation method is the same as that for preparing P53Kras mRNA vaccine, and the blank is expressed by LNP/blank.

Example 5 LNP/mRNA nanoparticle characterization assay

The groups of vaccines prepared in example 4 were subjected to nanoparticle size, charged potential, polydispersity, encapsulation efficiency measurements:

1. measurement of size, charged potential and polydispersity of nanoparticles

The prepared nanoparticles were analyzed for size, charged potential and Polydispersity (PDI) using a Malvern Zetasizer Nano ZS90 nanosize potentiometric analyzer. The specific method comprises the following steps:

respectively preparing 100 mu L of each of three groups of samples of LNP/blank, LNP/P53Kras and LNP/EGFP, supplementing 10mM citric acid buffer solution to 1mL, and dripping into a cuvette; the resulting mixture was placed in an instrument to measure the particle size, potential and PDI.

The results show (see table 3) that LNP/blank, LNP/P53Kras, LNP/EGFP particle diameters are 108nm, 96nm, 92nm, respectively, resulting in a slight reduction in nanoparticle diameter due to electrostatic interaction of LNP with mRNA, and related studies show that small size cationic liposomes are more efficient than large size cationic liposomes; PDI of the three components is less than 0.3, and the dispersion degree is good; the potentials of LNP/blank, LNP/P53Kras, LNP/EGFP were 32.3mV, 20.2mV, and 21.2mV in order, which potential was reduced upon binding to liposomes due to the negative charge of the nucleic acids, and which potential helped the particles cross the cell membrane.

2. Encapsulation efficiency determination

The encapsulation efficiency of the nano particles is measured by an ultracentrifugation method, and the specific operation steps are as follows:

three groups of samples, namely LNP/blank, LNP/P53Kras and LNP/EGFP, were prepared at 1mL, the concentration was designated as C0 (2. mu.g/mL), and the samples were placed in a DEPC-treated ultrafiltration tube, centrifuged at 5000rpm and 4 ℃ for 1 hour to separate non-packaged mRNA and liposomes, and the supernatant after centrifugation was non-packaged mRNA, which was measured as C. The total mRNA amount (C0) was calculated from the sample before the non-centrifugation, and the supernatant after the centrifugation was the amount of free mRNA (C), and the encapsulation efficiency was calculated.

Encapsulation ratio (%) = C0-C/C0 x 100%.

The results show (see Table 3) that the encapsulation rate of the obtained LNP/P53Kras and LNP/EGFP is more than 98 percent, and the mRNA vaccine is basically completely encapsulated.

Table 3 nanoparticle characterization

EXAMPLE 6 gel retardation experiment

To assess the degree of binding of LNP to mRNA, it was verified by gel blocking experiments. The nanoparticle LNP/blank and LNP/P53Kras prepared in example 4 and the mRNA solution (naked mRNA) obtained in step 6 of example 3 were subjected to agarose gel electrophoresis at 150v for 15min and observed under a chemiluminescence gel imager.

The results indicated that the control group LNP/blank did not emit light; the naked mRNA is partially degraded, and the strips are in a dispersed state; LNP/P53Kras is retained in the gel pores and is able to completely block the migration of mRNA. Therefore, the LNP liposome prepared by the invention can block the migration of mRNA and protect the mRNA from degradation.

Example 7 evaluation of transfection Effect of cells in vitro

1. To verify the required dose of LNP/mRNA and the transfection effect on cells, EGFP mRNA was chosen as a model for easy observation and detection.

293T cells were seeded at a density of 5 × 10 on 24-well plates⁵Each cell/well was cultured in 1ml of the ldmmem medium (containing 10% FBS), and when the cell density reached 80%, the cells were transfected with 1ml of LNP/EGFP at different concentrations, for a total of six experiments, 3 replicates per group, grouped as follows:

naked mRNA (2. mu.g/mL), blank (LNP/blank), LNP/EGFP (0.5. mu.g/mL), LNP/EGFP (1. mu.g/mL), LNP/EGFP (2. mu.g/mL), LNP/EGFP (4. mu.g/mL) at 37 ℃ in 5% CO₂Under the conditions, after incubation for 48h, cell suspensions were obtained and the percentage of EGFP positive cells was detected by flow cytometry.

Naked mRNA group: example 3 mRNA solution obtained in step 6.

LNP/EGFP (0.5. mu.g/mL), LNP/EGFP (1. mu.g/mL), and LNP/EGFP (4. mu.g/mL) groups, and the amount of mRNA added was varied according to the method for preparing LNP/EGFP (2. mu.g/mL).

The result shows (see figure 4), the expression rate of EGFP positive cells is increased along with the increase of the LNP/EGFP concentration, and when the LNP/EGFP concentration is 2 mu g/mL, the expression amount of EGFP is the highest and reaches more than 95 percent, which indicates that the mRNA coded by EGFP is translated into fluorescent protein. The green fluorescence expression of the naked mRNA group is low, only 31%, and the naked mRNA is degraded in the transfection process to influence the expression of the protein. Therefore, the liposome prepared by the invention can protect mRNA from being degraded and improve the transfection efficiency of cells.

2. Detection by ELISA method

293T cells were seeded at a density of 5 × 10 on 24-well plates⁵Cells were cultured in 1mL of DMEM medium (containing 10% FBS) per well, and when the cell density reached 80%, 1mL of LNP/P53Kras (2. mu.g/mL) was added to transfect the cells.

Three sets of experiments were set up, grouped as follows: a naked mRNA group (mRNA solution obtained in step 6 of example 3), a blank group (DMEM medium (containing 10% FBS) with the same volume) and an experimental group LNP/P53Kras (2. mu.g/mL) were incubated for 48 hours, and then 293T cells were centrifuged at 1500rpm for 5min to collect supernatant and the cell pellet was discarded. The cell supernatant was diluted 10-fold and 100-fold with the coating solution directly, and subjected to ELISA detection. The specific operation steps of ELISA detection are as follows:

(1) the diluted solution was added to a 96-well microplate at 100. mu.L/well, and placed in a refrigerator at 4 ℃ overnight.

(2) Washing the plate with PBST prepared in advance for 5 times, each time 200 mu L/hole, staying for at least 1min, and patting to dry by throwing the plate.

(3) 5% BSA blocking solution, 200. mu.L/well, was added and the plates were incubated for 2h at 37 ℃ in an incubator. After incubation, the blocking solution was spun off, and the plate was washed again with PBST for 5 times, as above for plate washing.

(4) Anti-p53 Rabbit Monoclonal Antibody (diluted 1: 600), 100. mu.L/well, was added and the plates were incubated for 2h at 37 ℃ in an incubator. After incubation, the plates were washed again 5 times with PBST, as above for the plates.

(5) Secondary antibodies, Goatanti-MouseIgG, HRP Conjugated (diluted 1: 5000) were added and the plates were incubated for 2h at 37 ℃ in an incubator. After incubation, the plates were washed again 5 times with PBST, as above for the plates.

(6) Adding 100 mu L/hole of TMB color development liquid, reacting for 20min at room temperature in a dark place, and adding 100 mu L/hole stop solution to stop the reaction. And (5) placing the sample in a multifunctional microplate reader to read the light absorption value at 450 nm.

The results are shown in table 4, antigen proteins capable of being specifically combined with the P53 antibody protein are generated by the naked mRNA group and the LNP/P53Kras, and are obviously different from the blank group, and the ELISA detection result shows that the antibody protein generated by the LNP/P53Kras nano vaccine in the experimental group is 2.3 times higher than that of the naked mRNA group. The experimental result further shows that the LNP nano delivery system provided by the invention can protect the degradation of P53Kras mRNA, successfully enter cells and express P53 protein.

TABLE 4293 expression of antigenic proteins in T cells

Example 8 cytokine secretion assay

IL-2 and IFN-gamma are key indicators for assessing whether T cells produce a strong immune response, and cytokine secretion by T cells is detected by the ELSIA method.

1. Cell culture

(1) Effector cell

Activated T cells were seeded at a density of 5 × 10 on 24-well plates⁵Adding 1mL DMEM medium (containing 10% FBS) into each well to culture cells to reach 80% of cell density, adding 1mL LNP/P53Kras (2 μ g/mL), 1mL LNP/PVAX1-P53Kras (2 μ g/mL) and 1mL naked mRNA (2 μ g/mL) to transfect the cells, incubating for 48h, collecting the cells, centrifuging at 1500rpm for 5min, discarding supernatant, collecting the cells as three groups of effector cells,

control groups were not supplemented with vaccine or naked mRNA, and the rest of the procedure was as above.

(2) Target cell

Human hepatoma cells Hep3B (purchased from Shanghai Xinyu) were used as target cells and cultured to logarithmic growth phase using DMEM medium (containing 10% FBS).

(3) Cell co-culture

Regulation of effector cells (1X 10) with DMEM Medium containing 10% FBS⁵Perwell) and target cells (1X 10)⁵Per well) and seeded in 96-well plates, 100 μ L per well, in four groups, as follows:

experimental group a: t cells (LNP/P53 Kras transfected) were co-cultured with Hep3B cells;

experimental group B: t cells (LNP/PVAX 1-P53Kras transfected) were co-cultured with Hep3B cells;

experimental group C: t cells (naked mRNA transfection) were co-cultured with Hep3B cells;

control group: t cells (untransfected) were co-cultured with Hep3B cells;

each group was repeated three times, and the mixture was heated at 37 ℃ with 5% CO₂The culture box is cultured for 72h, and culture supernatant is collected for detecting cytokines IL-2 and IFN-gamma.

2. Detection of IFN-gamma and IL-2 in cell culture supernatants

(1) The IFN-gamma and IL-2 kits were removed and allowed to stand at room temperature, and 5 standard concentration gradients for IFN-gamma and IL-2 were prepared according to the instructions. And adding 100 mu L of standard substance into the antibody pre-coated hole.

(2) Sample preparation, 100 μ L of cell culture supernatant was added directly to the well plate, while a blank control was set.

(3) Adding 50 mu L of biotin-labeled primary antibody diluted according to the proportion into each hole, and incubating for 90min at 37 ℃; after the incubation is finished, the supernatant is discarded, and the washing solution is used for washing for 5 times, 1min each time; adding 100 mu L of enzyme-conjugated secondary antibody diluted according to the proportion, and incubating for 30min at 37 ℃; after incubation, the supernatant was discarded and washed with wash solution 5 times for 1min each time.

(4) Adding 100 μ L substrate TMB (3, 3',5,5' -tetramethyl benzidine), incubating at 37 deg.C for developing color for 5-15min, adding 100 μ L stop solution after developing color, and placing in enzyme labeling instrument for OD450 nm reading.

The ELISA results show (see fig. 5 and table 5): the IL-2 and IFN-r concentrations of the LNP/P53Kras group, the DNA group and the naked mRNA group are higher than those of the control group, and the IL-2 and IFN-r concentrations of the LNP/P53Kras group are the highest. Therefore, the LNP/mRNA vaccine constructed by the invention can effectively induce T cells to generate immune response.

TABLE 5 cytokine secretion amounts (pg/mL)

Example 9 in vitro killing experiment of LNP/mRNA vaccine

1. T cell preparation

50mL of peripheral blood was isolated using TBD sample density separation (from an Tianjin tertiary-ocean organism) to obtain PMBC. After induction culture with DMEM (purchased from CORNING corporation, 88-551-CM) medium containing 1000IU/mL of recombinant interferon alpha 2a (purchased from Shenyang Sansheng), 1000IU/mL of recombinant IL-2 (purchased from Shenyang Sansheng), 50ng/mL of OKT-3 and 5% of autologous patient plasma were added for further culture for 24 hours. Adding liquid at a doubling ratio every two days, culturing to the 14 th day, and detecting CD3 in T cells by flow cytometry⁺、CD56⁺Positive expression rate (CD3-FITC, CD16/CD56-PE antibody from BECKMAN, A07735). CD3⁺Positive rate>90%，CD3⁺CD56⁺Double positive rate>20%, the induction of T cells was considered successful.

2. Cell culture

(1) Effector cell

The activated T cells were seeded in 24-well plates at a density of 5 × 10⁵When 1mL of DMEM medium (containing 10% FBS) is added into each well to culture the cells to reach the cell density of 80%, 1mL of LNP/P53Kras (2 mu g/mL), 1mL of LNP/PVAX1-P53Kras (2 mu g/mL) and 1mL of naked mRNA (2 mu g/mL) are respectively added to transfect the cells, the cells are incubated for 48 hours, the cells are collected, the cells are centrifuged at 1500rpm for 5min, the supernatant is discarded, and the obtained cells are three groups of effector cells.

(2) Target cell

Human hepatoma cells Hep3B (purchased from Shanghai Xinyu) were used as target cells and cultured to logarithmic growth phase using DMEM medium and 10% FBS.

(3) Cell co-culture

Regulation of effector cells (1X 10) with DMEM Medium containing 10% FBS⁵/hole) andtarget cell (1X 10)⁵Per well) and seeded in 96-well plates, 100 μ L per well, in three groups, specifically grouped as follows:

LNP/P53Kras group: t cells (LNP/P53 Kras transfected) were co-cultured with Hep3B cells;

LNP/PVAX1-P53Kras group: t cells (LNP/PVAX 1-P53Kras transfected) were co-cultured with Hep3B cells;

naked mRNA group: t cells (naked mRNA transfection) were co-cultured with Hep3B cells;

control group: t cells (untransfected) were co-cultured with Hep3B cells;

culturing in 5% CO2 and 37 deg.C incubator, adding 20mL CCK-8 per well after 24h, incubating for 2h, detecting 450nm wavelength with microplate reader, and reading OD value. Calculating the cell killing rate: killing rate/% ([ 1- (blank OD value-effector OD value)/blank OD value ] × 100%).

The results show that the killing rates of the LNP/P53Kras group, the LNP/PVAX1-P53Kras group and the naked mRNA group are respectively 98.6%, 62.1% and 41.3%, the killing rate of the LNP/P53Kras transfected T cells is the highest and is significantly higher than that of the LNP/PVAX1-P53Kras group and the naked mRNA group, and the killing rates of the LNP/P53Kras transfected cells are respectively 1.6 times and 2.4 times that of the LNP/PVAX1-P53Kras group and the mRNA group. Therefore, the LNP/P53Kras constructed by the invention can protect mRNA from being degraded during transfection, induce cells to generate stronger immune response, and has obvious effect of killing tumor cells.

In conclusion, the invention successfully constructs an effective mRNA vaccine delivery system, which can not only protect mRNA from being degraded in the transfection process, but also enhance the cellular immune response of organisms.

Sequence listing

<110> Shandong Xingyi Biotechnology Ltd

<120> double-target mRNA vaccine and preparation method thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 89

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 1

tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60

gaagagtaag aagaaatata agagccacc 89

<210> 2

<211> 810

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 2

accaccatcc actacaacta catgtgtaac agttcctgca tgggcggcat gaaccaaagg 60

cccatcctca ccatcatcac actggaagac tccagtggta atctactggg aggatctgga 120

ggtggaggtt caggaggaac caccatccac tacaactaca tgtgtaacag ttcctgcatg 180

ggcggcatga actggaggcc catcctcacc atcatcacac tggaagactc cagtggtaat 240

ctactgggag gatctggagg tggaggttca ggaggatctg actgtaccac catccactac 300

aactacatgt gtaacagttc ctgcatgggc tctatgaacc ggaggcccat cctcaccatc 360

atcacactgg aagactccag tggtaatgga ggatctggag gtggaggttc aggaggaatc 420

acactggaag actccagtgg taatctactg ggacggaaca gctttgaggt gtgtgtttgt 480

gcctgtcctg ggagagaccg gcgcacagag gaagagaatc tccgcaaggg aggatctgga 540

ggtggaggtt caggaggagt ccgcgccatg gccatctaca agcagtcaca gcacatgacg 600

gaggttgtga ggcattgccc ccaccatgag cgctgctcag atagcgatgg tctggcccct 660

cctcagcatg gaggatctgg aggtggaggt tcaggaggac tactgggacg gaacagcttt 720

gaggtgcgtg tttgtgcctg tcctgggaga gactggcgca cagaggaaga gaatctccgc 780

aagaaagggg agcctcacca cgagctgccc 810

<210> 3

<211> 30

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 3

ggaggatctg gaggtggagg ttcaggagga 30

<210> 4

<211> 570

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 4

actgaatata aacttgtggt agttggagct gttggcgtag gcaagagtgc cttgacgata 60

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 120

actgaatata aacttgtggt agttggagct gatggcgtag gcaagagtgc cttgacgata 180

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 240

actgaatata aacttgtggt agttggagct tgtggcgtag gcaagagtgc cttgacgata 300

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 360

actgaatata aacttgtggt agttggagct agaggcgtag gcaagagtgc cttgacgata 420

cagctaattc agaatcactt tgtggatgaa ggaggatctg gaggtggagg ttcaggagga 480

actgaatata aacttgtggt agttggagct gctggcgtag gcaagagtgc cttgacgata 540

cagctaattc agaatcactt tgtggatgaa 570

<210> 5

<211> 171

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 5

gtgggcatca ttgctggcct ggttctcctt ggagctgtga tcactggagc tgtggtcgct 60

gccgtgatgt ggaggaggaa gagctcagat agaaaaggag ggagttacac tcaggctgca 120

agcagtgaca gtgcccaggg ctctgatgtg tccctcacag cttgtaaagt g 171

<210> 6

<211> 119

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 6

tgataatagg ctggagcctc ggtggccatg cttcttgccc cttgggcctc cccccagccc 60

ctcctcccct tcctgcaccc gtacccccgt ggtctttgaa taaagtctga gtgggcggc 119

<210> 7

<211> 2999

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 7

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc 780

ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 840

gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 900

ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 960

attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 1020

gcaggcatgc tggggatgcg gtgggctcta tggcttctac tgggcggttt tatggacagc 1080

aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt 1140

aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca 1200

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 1260

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 1320

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 1380

cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac 1440

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 1500

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 1560

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 1620

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 1680

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 1740

caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 1800

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 1860

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 1920

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 1980

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt 2040

cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacaggtggc 2100

acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 2160

atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca cgtgctaaaa 2220

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 2280

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 2340

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2400

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2460

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2520

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2580

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2640

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2700

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2760

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2820

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2880

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2940

agcaacgcgg cctttttacg gttcctgggc ttttgctggc cttttgctca catgttctt 2999

<210> 8

<211> 717

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 8

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 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

Claims

1. A dual-target mRNA vaccine, characterized by: including liposome mixtures and dual-target mrnas; the liposome mixture comprises DLin-MC3-DMA, DSPC, cholesterol and PEG200-DMG, and the molar ratio is 50:10:37.5: 2.5;

2. A method of making the dual target mRNA vaccine of claim 1, characterized in that: the method comprises the steps of constructing a target gene segment, constructing a recombinant plasmid, linearly digesting and recovering the recombinant plasmid, transcribing to obtain mRNA, and packaging the mRNA.

3. The method of claim 2, wherein: constructing the recombinant plasmid, namely cloning a target gene fragment to a PVAX1 vector to obtain the recombinant plasmid; the transcription obtains mRNA, and the mRNA with a 5 'cap and a Poly (A) tail at the 3' end is synthesized by transcription.

4. The method of claim 2, wherein: the packaging mRNA is to adopt liposome mixture to package mRNA.

5. The method of claim 4, wherein: the packaged mRNA specifically comprises: preparing ethanol solution of liposome mixture, preparing mRNA aqueous solution, synthesizing LNP/mRNA vaccine particles and preparing the vaccine.

6. The method of claim 5, wherein: