CN116196406A - mRNA tumor vaccine and carrying system, preparation method and application thereof - Google Patents

Abstract

The present invention describes an mRNA tumor vaccine and its delivery system, preparation method and application, wherein the mRNA tumor vaccine delivery system uses long single-stranded DNA (lssD) as a carrier, and is loaded with: mRNA encoding tumor antigen; and Mn ²⁺ ion. Among them, lssD contains DC-sign receptors that can target DC cells (dendritic cells, DCs), and Mn ²⁺ ions can be used to form insoluble manganese pyrophosphate (Mn2ppi) framework and activate DC cells. This project records the method of rolling circle amplification technology to prepare long single-stranded DNA carrier lssD loaded with Mn ²⁺ ions and mRNA, and finally achieves the use of aptamers with specific sequences to effectively target antigen-presenting cells and activate antigen presentation The STING pathway of cells can improve the ability of antigen presentation, and realize the purpose of more stable in vivo delivery and effective expression of mRNA tumor vaccines.

Description

A kind of mRNA tumor vaccine and its delivery system, preparation method and application

technical field

The invention relates to the field of tumor treatment, in particular to an mRNA tumor vaccine and its delivery system, preparation method and application.

Background technique

Cancer vaccines are a promising strategy in the rapidly developing field of cancer immunotherapeutics. Among them, the use of mRNA cancer vaccines has two major advantages over conventional cancer treatments. First, personalized tumor vaccine design for specific tumor patients can be realized due to the flexibility and programmability of the mRNA preparation process. Second, mRNA tumor vaccines do not face the problem of multidrug resistance (MDR) compared with traditional chemotherapy drugs. However, since mRNA tumor vaccines are difficult to directly enter antigen-presenting cells (APCs), and there is no effective adjuvant to activate APCs, its wider clinical application is hindered.

Therefore, an efficient and clinically transformable mRNA tumor vaccine delivery system is urgently needed for tumor immunotherapy. Antigen-presenting cells (APCs) are indispensable cells in the process of vaccine-induced activation of immune responses, responsible for presenting antigens to T lymphocytes, thereby effectively activating the immune system. Therefore, how to effectively deliver mRNA vaccines to antigen-presenting cells in the body is necessary for the development of mRNA vaccines, but there are certain challenges.

In recent years, a new aptamer has been found to target DC cells (dendritic cells, DCs) through a high affinity with the DC-sign receptor, which provides an opportunity to improve the effectiveness of mRNA cancer vaccines on DCs. Cell targeting offers new strategies. As an important part of innate immunity, STING pathway has been considered as a promising target to enhance tumor immunotherapy in recent years. cGAS plays an important role as a cytoplasmic DNA sensor, supervises the intake of heterologous DNA such as bacteria, viruses and tumors in cells, and catalyzes the production of the second messenger cGAMP. As the natural ligand of STING, cGAMP further activates STING and its downstream signals IRF3 and IKK/NF-κB, and finally promotes the transcription enhancement of type 1 interferon and the activation of the body's innate immunity. Therefore, how to promote the production of cGAMP, enhance the binding affinity of cGAMP/STING, and thus promote the activation of the STING pathway is an important issue in the development of mRNA tumor vaccines.

Contents of the invention

One of the innovative points of the present invention is that the combination of mRNA tumor vaccine and Mn ²⁺ has the potential to obtain a synergistic anti-tumor immunotherapy effect by activating the cGAS/STING pathway. In order to solve the above problems, the present invention provides an mRNA tumor vaccine and its delivery system, preparation method and application,

The aforementioned mRNA tumor vaccine delivery system is characterized in that it uses long single-stranded DNA (lssD) as a carrier and is loaded with:

mRNAs encoding tumor antigens; and

Mn ²⁺ ions are used to form insoluble manganese pyrophosphate (Mn2ppi) framework and activate DC cells (dendritic cells, DCs).

In the above system, the long single-stranded DNA (lssD) also includes a DC-sign receptor that can target DC cells.

In the above system, the long single-stranded DNA (lssD) contains poly(T) as a (special) sequence for linking mRNA.

In the above system, the mRNA contains poly(A), and the poly(T) is cross-linked with the tail of the poly(A).

In the above system, the long single-stranded DNA (lssD) has a flower-like structure.

The present invention also relates to a preparation method of an mRNA tumor vaccine delivery system, comprising the following steps:

Step 1: Mix, mix phosphorylated DNA template and DNA primer in NaCl solution;

Step 2: Annealing, heat the mixture obtained in step 1 at a temperature of 95°C for 2 minutes, at a temperature of 65°C for 2 minutes, then gradually cool to 60°C at a rate of 1°C/min, and repeat this process 80 times Gradually cool down to 20°C using a PCR thermal cycler;

Step 3: Incubate, add the annealed product obtained in step 2 to T4 DNA ligase and T4 DNA ligase buffer, and incubate overnight;

Step 4: heating, heating the solution obtained in step 3 at a temperature of 65° C. for 10 minutes to inactivate T4 DNA ligase to obtain a circular DNA template;

DNA聚合酶依次加入含有10mM MnCl₂的RCA反应缓冲液中，进行Mn²⁺的装载，然后进行生物矿化反应，并在30℃的温度下反应2h后，于75℃的温度下灭活15min；Step 5: biomineralization reaction, the circular DNA template obtained in step 4, dNTPs and

DNA polymerase was sequentially added to the RCA reaction buffer containing 10mM MnCl ₂ to carry out the loading of Mn ²⁺ , and then to carry out the biomineralization reaction, and reacted at 30°C for 2h, then inactivated at 75°C for 15min ;

Step 6: Add mRNA to the reaction buffer and incubate at 25°C for 1 hour to load mRNA into lssD.

In the above method, the T4 DNA ligase in step 3 is 2U/μL; the T4 DNA ligase buffer contains 50mM Tris-HCl at pH 7.5, 10mM MgCl ₂ , 10mM DTT and 1mM adenosine triphosphate; the overnight incubation temperature is 16°C.

In the above method, the molar ratio of the phosphorylated DNA template to the DNA primer in the step 1 is 1:2; in the step 4, RCA reaction buffer is added during the heating process.

The present invention also relates to an mRNA tumor vaccine, comprising the above mRNA tumor vaccine delivery system and/or the mRNA tumor vaccine delivery system prepared by the above preparation method.

The present invention also relates to the application of an mRNA tumor vaccine, characterized in that: the mRNA tumor vaccine is used for the treatment of tumors.

Advantage and beneficial effect of the present invention are:

① The present invention provides an mRNA tumor vaccine delivery system, using specially designed long single-stranded DNA (lssD) as the carrier for the first time, which can effectively target antigen-presenting cells by using aptamers of specific sequences;

② In the present invention, long single-stranded DNA (lssD) is used as a carrier, which can effectively activate the STING pathway of antigen-presenting cells through the Mn ²⁺ loaded therein, and improve the antigen-presenting ability;

③ The lssD designed by the present invention contains a poly(T) sequence, thereby effectively binding poly(A) in mRNA

sequence to achieve more stable loading of mRNA tumor vaccines;

④ The present invention used the rolling circle amplification technique for the first time to prepare the long single-stranded DNA carrier lssD loaded with Mn ²⁺ ions and mRNA.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only For some embodiments of the present invention, those skilled in the art can also obtain other drawings based on these drawings without creative work.

Fig. 1 is a schematic structural diagram of lssD in an embodiment of the present invention and the distribution of related elements;

2 is a schematic diagram of the process of delivering mRNA cancer vaccines through long single-stranded DNA (lssD) in the embodiment of the present invention;

Fig. 3 is a diagram of the particle size and potential change of lssD mRNA tumor vaccine when using IssD loaded with different substances in the embodiment of the present invention;

Fig. 4 is an agarose gel electrophoresis diagram for verification of intermediate products produced during the preparation of tumor vaccines in the examples of the present invention;

Fig. 5 is the element and crystal structure characterization analysis diagram of lssD-Mn-DC (mRNA) in the embodiment of the present invention;

Fig. 6 is an experimental result diagram of the effect of lssD targeting antigen-presenting cells in the embodiment of the present invention;

Fig. 7 is a graph showing the experimental results of Mn ²⁺ ions enhancing the expression of STING-related genes to promote DC maturation and antigen presentation in an embodiment of the present invention.

Detailed ways

The specific implementation manners of the present invention will be further described below in conjunction with the drawings and examples. The following examples are only used to illustrate the technical solution of the present invention more clearly, but not to limit the protection scope of the present invention.

mRNA tumor vaccine delivery system

The invention describes an mRNA tumor vaccine delivery system, which uses long single-stranded DNA (lssD) as a carrier, and is loaded with mRNA encoding tumor antigens and Mn ²⁺ ions, wherein the long single-stranded DNA (lssD) has a flower-like structure , as shown in the lssD morphology characterization diagram of Fig. 1. Among them, Figure 1(a) SEM results show that lssD has a uniform structure and a flower-like structure; Figure 1(b) TEM mapping results show that C, N, O, P, Mg and Mn elements are uniformly distributed in lssD.

At the same time, the mRNA tumor vaccine delivery system described in the present invention can also include a DC-sign receptor that can target DC cells, and this receptor can target specific intercellular adhesion molecules (ICAM) on the surface of dendritic cells -3, which captures overexpressed, non-integrated (DC-sign) receptors. In the system disclosed by the present invention, Mn ²⁺ replaces traditional Mg ²⁺ as a cofactor for phi29 DNA polymerase to extend long-chain DNA, and Mn ²⁺ ions can form insoluble manganese pyrophosphate (Mn2ppi) framework and activate DC cells ( dendritic cells, DCs). In the tumor microenvironment, the cGAS-STING pathway in DC cells plays an important role in promoting cross-presentation and priming of tumor-specific CD8-positive T cells. Firstly, the type I interferon produced in DC cells can enhance the cross-presentation of DC cells; then it can improve the lymph node homing ability of DC cells by increasing the expression of CCR7 on DC cells; finally, it can promote the homing of antigen-presenting cells by increasing a variety of Th1 cytokines. Trafficking of nest effector T cells. The Mn ²⁺ described in the present invention can comprehensively promote the activation of cGAS and STING in various aspects, from enhancing the generation of cGAMP to enhancing the binding affinity of cGAMP/STING, thereby enhancing the innate immune ability of DC cells.

The long single-stranded DNA (lssD) described in the present invention contains poly(T) as a special sequence for connecting mRNA, and mRNA contains poly(A), and poly(T) can cross-link with the tail of poly(A).

As shown in Figure 2, a. An aptamer containing DNA-inorganic composite material loaded with Mn ²⁺ ions and caged mRNA cancer vaccine. Figure 2b. Schematic showing that lssD stimulates the activation of defense-presenting cells, promotes the expansion of antigen-specific T cells, and induces a robust antitumor immune response.

Preparation method of mRNA tumor vaccine delivery system

On the other hand, the present invention describes the preparation method of the above-mentioned mRNA tumor vaccine delivery system. For the first time, the present invention constructs a new mRNA delivery system by using long single-stranded DNA (lssD) as a carrier through rolling circle amplification (RCA) reaction platform, this method includes the following steps:

Step 1: mixing, mixing phosphorylated DNA template and DNA primer in NaCl solution, preferably, mixing 100 μM phosphorylated DNA template and 200 μM DNA primer in 1:2 molar ratio in 100 mM NaCl solution;

Step 2: Annealing, heat the mixture obtained in step 1 at a temperature of 95°C for 2 minutes, at a temperature of 65°C for 2 minutes, then gradually cool to 60°C at a rate of 1°C/min, and repeat this process 80 times Gradually cool down to 20°C using a PCR thermal cycler;

Step 3: Incubation, add the annealed product obtained in step 2 to T4 DNA ligase and T4 DNA ligase buffer, and incubate overnight, preferably, the T4 DNA ligase is 2U/μL, and the T4 DNA ligase buffer contains 50mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM DTT and 1 mM ATP, incubated overnight at 16°C.

Step 4: Heating, heat the solution obtained in step 3 at 65°C for 10 minutes to inactivate T4 DNA ligase and obtain a circular DNA template. Preferably, in order to prepare lssD, add RCA reaction buffer during heating Solution, RCA reaction buffer (pH7.5) contains 50mm Tris-HCl, 20mm NH ₄ Cl, 50mm KCl, 5mm MnCl ₂ , 5mM MgCl ₂ , 4mmDTT;

DNA聚合酶依次加入含有10mM MnCl₂的RCA反应缓冲液中，进行Mn²⁺的装载，然后进行生物矿化反应，并在30℃的温度下反应2h后，于75℃的温度下灭活15min，优选地，DNA模板(0.5μM)、dNTPs(1mM)，φ29DNA聚合酶(1U/μL)，且加入含有10mM MnCl₂的RCA反应缓冲液后的最终体积为50μL；Step 5: biomineralization reaction, the circular DNA template obtained in step 4, dNTPs and

DNA polymerase was sequentially added to the RCA reaction buffer containing 10mM MnCl ₂ to carry out the loading of Mn ²⁺ , and then to carry out the biomineralization reaction, and reacted at 30°C for 2h, then inactivated at 75°C for 15min , preferably, DNA template (0.5 μM), dNTPs (1mM), φ29 DNA polymerase (1U/μL), and the final volume after adding the RCA reaction buffer containing _10mM MnCl2 is 50 μL;

Step 6: Add mRNA to the reaction buffer and incubate at 25°C for 1 hour to load mRNA into lssD. In order to verify circular DNA templates and single-stranded DNA (ssDNA) templates before cycling, tris-boronic acid edta (TBE, pH 8.0) can be added in a 12% native PAGE gel run at 110V for 2h.

As an option, after the above step 6, a detection step can also be added, namely:

Step 7: detection, the gel in step 6 was stained in ethidium bromide solution, and scanned using a gel imaging analysis system under 302nm UV light. Gel electrophoresis was used to run mRNANPs to check for any free mRNA and/or mRNA leaching from NPs.

mRNA tumor vaccine

The mRNA tumor vaccine described in the present invention is a tumor vaccine comprising the above mRNA tumor vaccine delivery system, and may also be a tumor vaccine comprising the mRNA tumor vaccine delivery system prepared by the above preparation method.

application

The tumor vaccine described in the present invention can be used in the prevention of tumors and in the immunotherapy of tumors.

Specific embodiments of the present invention will be introduced below for illustrative purposes.

Example 1

The particle size and potential of the prepared lssD mRNA tumor vaccine were characterized by nanoparticle size detector (DLS).

The specific experimental steps are as follows:

1. Take 10ul lssD mRNA tumor vaccine and add it to 1ml double distilled water for thorough mixing.

2. Add the mixture into the detection dish, and detect the corresponding particle size and potential change on the machine. As shown in Figure 2, where lssD represents Mg ²⁺ ions instead of Mn ²⁺ ions as co-stimulatory factors and is not loaded with mRNA; lssD-Mn-DC represents Mn ²⁺ ions as co-stimulatory factors and is not loaded with mRNA; lssD-Mn-DC(mRNA) represents the final mRNA tumor vaccine formulation with Mn ²⁺ ions as co-stimulatory factors and loaded with mRNA.

From the graph of particle size and potential change shown in Figure 3, it can be seen that the loading of lssD on mRNA can be achieved by using Mn ²⁺ ions as co-stimulatory factors.

Example 2

Using agarose gel electrophoresis to characterize the process of preparing lssD mRNA tumor vaccines and to verify each expected intermediate product,

The specific experimental steps are as follows:

1. Take 2g of agarose and add it to 100ml of 1xTAE solution, heat it in a microwave oven at high temperature and add it to the mold to cool.

2. Mix the intermediate product in the preparation process with the loading buffer and add it to the sample loading space. Then use 120V voltage to run for 30 minutes.

3. Detect the information of each band in the exposure instrument.

The experimental results are shown in FIG. 4 , and the results of electrophoresis of each gel column are consistent with the expected results of the intermediate product of the preparation method described in the present invention.

Example 3

After the prepared lssD-Mn-DC(mRNA) was lyophilized, its elemental composition and crystal structure were characterized by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD), respectively.

As shown in Figure 5 (a), it is verified that the method described in the present invention can successfully prepare lssD-Mn-DC (mRNA), as shown in Figure 5 (b), the lssD-Mn-DC (mRNA) prepared by the method described in the present invention ( mRNA) has good structural stability.

Example 4

lssD-Mn-DC(mRNA) and lssD-Mn(mRNA) were added to the culture medium of DC2.4 cells, and lssD-Mn-DC(mRNA) were recorded at 0, 0.5, 1, 1.5 and 2 hours respectively ) and lssD-Mn (mRNA) combined with DC2.4 cells. The binding of lssD-Mn-DC(mRNA) and lssD-Mn(mRNA) to DC2.4 cells and the translation of mRNA in cells were detected by confocal and flow cytometry.

Figure 6(a-b) The exposure results of the well plate show that lssD can effectively bind DC cells and successfully translate GFP protein; Figure 6(c) shows various confocal fluorescence images of DC2.4 cells treated with lssD for 1 hour; Figure 6(d-e) The GFP fluorescence intensity in BMDCs after incubation with different formulations of lssD for 1 h was measured together with light exposure. Figure 6 shows that lssD can efficiently target antigen presenting cells (DC cells) and translate related proteins.

Example 5

lssD-Mg-DC and lssD-Mn-DC were co-incubated with DC2.4 cells for 24 hours, and then the total protein of the cells was analyzed by WB, focusing on the activation of STING-related signaling pathways. In addition to extracting total protein, RNA was also extracted for RT-PCR analysis, focusing on the expression of genes related to the STING pathway. By co-incubating lssD-Mg-DC and lssD-Mn-DC with BMDC cells for 24 hours, the activation of BMDC cells was detected using anti-CD80 ⁺ and CD86 ⁺ antibodies.

Figure 7(a) flow cytometry results show that Mn ²⁺ in lssD can enhance the expression of STING-related proteins and better activate DC cells; Figure 7(b) flow cytometry results show that Mn ²⁺ in lssD can Enhance the expression of LssD on MHCII in DC cells, so as to better achieve the effect of presenting antigens. Figure 7 shows that Mn ²⁺ promotes DC maturation and antigen presentation.

The above description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the application . Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the broadest scope consistent with the principles and novel features disclosed herein, within the spirit and principles of the present invention, Any modifications, equivalent replacements, improvements, etc., should be included within the protection scope of the present invention.

Claims (10)

1. an mRNA tumor vaccine delivery system is characterized in that, take long single-stranded DNA (lssD) as carrier, and is loaded with: mRNAs encoding tumor antigens; and Mn ²⁺ ions are used to form insoluble manganese pyrophosphate (Mn2ppi) framework and activate DC cells (dendritic cells, DCs). 2. The mRNA tumor vaccine delivery system according to claim 1, wherein the long single-stranded DNA (lssD) further comprises a DC-sign receptor capable of targeting DC cells. 3. The mRNA tumor vaccine delivery system according to claim 2, wherein the long single-stranded DNA (lssD) contains poly(T) as a sequence for connecting mRNA. 4. The mRNA tumor vaccine delivery system according to claim 3, wherein the mRNA contains poly(A), and the poly(T) is cross-linked with the tail of the poly(A). 5. The mRNA tumor vaccine delivery system according to claim 1, wherein the long single-stranded DNA (lssD) has a flower-like structure. 6. A method for preparing an mRNA tumor vaccine delivery system, comprising the following steps: Step 1: Mix, mix phosphorylated DNA template and DNA primer in NaCl solution; Step 2: Annealing, heat the mixture obtained in step 1 at a temperature of 95°C for 2 minutes, at a temperature of 65°C for 2 minutes, then gradually cool to 60°C at a rate of 1°C/min, and repeat this process 80 times Gradually cool down to 20°C using a PCR thermal cycler; Step 3: Incubate, add the annealed product obtained in step 2 to T4 DNA ligase and T4 DNA ligase buffer, and incubate overnight; Step 4: heating, heating the solution obtained in step 3 at a temperature of 65° C. for 10 minutes to inactivate T4 DNA ligase to obtain a circular DNA template; Step 5: Biomineralization reaction, the circular DNA template, dNTPs and φ29 DNA polymerase obtained in step 4 were sequentially added to the RCA reaction buffer containing 10 mM MnCl ₂ to carry out Mn ²⁺ loading, followed by biomineralization reaction , and after reacting at a temperature of 30°C for 2h, inactivate at a temperature of 75°C for 15min; Step 6: Add mRNA to the reaction buffer and incubate at 25°C for 1 hour to load mRNA into lssD. 7. The preparation method of a kind of mRNA tumor vaccine delivery system as claimed in claim 6, is characterized in that, the T4 DNA ligase of described step 3 is 2U/μ L; T4 DNA ligase buffer comprises the Tris of 50mM pH 7.5 - HCl, 10 mM MgCl ₂ , 10 mM DTT and 1 mM ATP; overnight incubation at 16°C. 8. The preparation method of a kind of mRNA tumor vaccine delivery system as claimed in claim 6, is characterized in that, in described step 1, the molar ratio of phosphorylated DNA template and DNA primer is 1:2; Described step 4 is heated RCA reaction buffer was added during the process. 9. An mRNA tumor vaccine, characterized in that it contains the mRNA tumor vaccine delivery system according to any one of claims 1-5 and/or the mRNA tumor vaccine delivery system prepared by the preparation method according to claim 6 . 10. An application of the mRNA tumor vaccine according to claim 9, characterized in that: the mRNA tumor vaccine is used for the prevention of tumors.

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