A fusion protein and an mRNA delivery system and application based thereon

Abstract

This invention provides a fusion protein and a corresponding mRNA delivery system and applications. The amino acid sequence of the fusion protein is shown in SEQ ID NO.1. The mRNA delivery system comprises a cationic polymer, the aforementioned fusion protein, and mRNA. The cationic polymer can encapsulate and protect the negatively charged mRNA by neutralizing it. The 3'UTR terminus of the mRNA binds to the C-terminus of the L7Ae protein in the fusion protein, and the Listeriolysin O terminus of the fusion protein is linked to the N-terminus of the L7Ae protein. This invention also provides the application of the aforementioned fusion protein in the preparation of mRNA delivery systems, and the application of the aforementioned fusion protein or the aforementioned mRNA delivery system in the preparation of mRNA transfection reagents, mRNA drugs, or mRNA vaccines. The novel fusion protein can be prepared in large quantities with high yield and controllable quality. This novel mRNA delivery system has a simpler composition, a simpler preparation process, and lower cost. Moreover, in this novel mRNA delivery system, the mRNA is encapsulated and protected by a cationic polymer (such as protamine), making it more stable.

Description

Technical Field

[0001] This invention relates to the field of biological delivery, and more specifically to a fusion protein and a mRNA delivery system based thereon and its applications. Background Technology

[0002] mRNA needs to overcome multiple challenges, including the cell's extracellular barrier, endosome escape, and intracellular immunity, to reach its target site and ultimately perform its function. Therefore, delivering mRNA into the cytoplasm of the target cell is a major challenge that mRNA must overcome to function effectively.

[0003] Traditional methods for delivering mRNA vaccines include physical methods and viral vector methods. Electroporation and gene guns are classic physical methods for mRNA delivery, but clinical trials have shown that physical methods are often harmful to cells and unsuitable for in vivo application. Viral vector-based mRNA delivery methods have many problems regarding safety, stability, and efficacy, so more efficient delivery systems urgently need to be developed.

[0004] Lipid nanoparticles (LNPs) have become a promising new mRNA delivery system in recent years due to their low immunogenicity, good biocompatibility, and high mRNA encapsulation efficiency, and are now being used clinically.

[0005] Lipid nanoparticles (LNPs) have the following drawbacks: (1) They have certain stability issues and need to be stored under freezing conditions of -20℃ to -80℃. In addition, LNPs in aqueous solutions will undergo size changes and lose their effectiveness; (2) They have high production costs and require expensive microfluidic equipment for preparation; (3) Their composition is relatively complex and requires the use of four components: ionizable lipids, cholesterol, distearate phosphatidylcholine and PEG. Summary of the Invention

[0006] The purpose of this invention is to provide a novel fusion protein and a novel mRNA delivery system based on the fusion protein. Compared with conventional lipid nanoparticle delivery systems, the novel mRNA delivery system has fewer components, a simpler preparation process, lower cost, and greater stability, and can be used as a transfection reagent and vaccine delivery system.

[0007] The technical solution of this invention is as follows:

[0008] The first objective of this invention is to provide a fusion protein comprising Listeriolysin O (LLO), a GS linker, and an L7Ae protein, wherein Listeriolysin O is linked to the N-terminus of the L7Ae protein via the GS linker.

[0009] Furthermore, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.1:

[0010] SEQ ID NO.1:

[0011] MHHHHHHKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVY

[0012] HGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVE

[0013] NQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPN

[0014] VSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNV

[0015] NEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDA

[0016] AVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVP

[0017] IAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEI

[0018] VQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNR

[0019] NISIWGTTLYPKYSNKVDNPIEGGGGSGGGGSGGGGSMGRSMYVRFEVPEDMQNEALSLLE

[0020] KVRESGKVKKGTNETTKAVERGLAKLVYIAEDVDPPEIVAHLPLLCEEKNVPYIYVKSKNDLGRAVGIEVPCASAAIINEGELRKELGSLVEKIKGLQKGSA*

[0021] A second objective of this invention is to provide an mRNA delivery system based on the aforementioned fusion protein. The mRNA delivery system comprises a cationic polymer, the aforementioned fusion protein, and mRNA. The cationic polymer encapsulates and protects the mRNA. The 3'UTR terminus of the mRNA binds to the C-terminus of the L7Ae protein in the fusion protein, and the Listeneriolysin O in the fusion protein is linked to the N-terminus of the L7Ae protein. The Listeneriolysin O performs endosome escape, while the L7Ae protein binds to the mRNA.

[0022] Furthermore, in the mRNA delivery system, the molar ratio of the aforementioned fusion protein to mRNA is 0.125 to 1:1; the mass ratio of mRNA to cationic polymer is 6:1.

[0023] Preferably, in the mRNA delivery system, the molar ratio of the aforementioned fusion protein to mRNA is 1:1, 0.5:1, 0.25:1, or 0.125:1;

[0024] More preferably, in the mRNA delivery system, the molar ratio of prefusion protein to mRNA is 1:1.

[0025] Furthermore, the cationic polymer is protamine.

[0026] Furthermore, the mRNA is a negatively charged mRNA, and the cationic polymer neutralizes the negatively charged mRNA and encapsulates and protects it.

[0027] Furthermore, the 3'UTR end of the mRNA is ligated to a Box C / D sequence.

[0028] Preferably, the Box C / D sequence is as shown in SEQ ID NO.2:

[0029] GGGCGUGAUGCGAAAGCUGACCCGGGCGUGAUGCGAAAGCUGACCCGCUCUGACCGA AAGGCGUGAUGAGCGCUCUGACCGAAAGGCGUGAUGAGC.

[0030] The Box C / D sequence shown in SEQ ID NO.2 is artificially synthesized and added to the 3'UTR end of the mRNA sequence. With the Box C / D sequence, the mRNA can bind to the Listeriolysin O+GS linker+L7Ae fusion protein.

[0031] In a particular embodiment, the mRNA is p30-mRNA.

[0032] Furthermore, the protamine encapsulates and protects the mRNA, and the Box C / D sequence at the 3'UTR end of the mRNA binds to the L7Ae protein in the fusion protein, while the Listeriolysin O in the fusion protein is linked to the N-terminus of the L7Ae protein.

[0033] A third objective of this invention is to provide the application of the aforementioned fusion protein in the preparation of mRNA delivery systems.

[0034] A fourth objective of this invention is to provide the use of the aforementioned fusion protein or the aforementioned mRNA delivery system in the preparation of mRNA transfection reagents or in the preparation of mRNA drugs or mRNA vaccines.

[0035] The beneficial effects of this invention are as follows:

[0036] (1) This invention provides a novel fusion protein with a well-defined amino acid sequence, which can be prepared in large quantities with high yield and controllable quality.

[0037] (2) The present invention also provides a novel mRNA delivery system that can replace the liposome mRNA delivery system. Compared with the conventional lipid nanoparticle delivery system, the novel mRNA delivery system has fewer components, a simpler preparation process, lower cost and greater stability. Moreover, in the novel mRNA delivery system, the mRNA is encapsulated and protected by cationic polymers (such as protamine), making it more stable.

[0038] (3) The novel fusion protein and novel mRNA delivery system can be used to prepare mRNA transfection reagents or mRNA drugs or mRNA vaccines. The preparation process is simple, the cost is lower, and it is suitable for industrial application. Attached Figure Description

[0039] Figure 1 Schematic diagram of a novel mRNA delivery system;

[0040] Figure 2 Fluorescence image of 293T cells after transfection with protamine / Listeriolysin O+GS linker+L7Ae / eGFP-mRNA complex solution;

[0041] Figure 3 Immunoblot image of 293T cells after transfection with protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex solution;

[0042] Figure 4 Results of antibody detection in mouse serum after immunization with the protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex. Detailed Implementation

[0043] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0044] Example 1: Screening of the ratio of Listeriolysin O+GS linker+L7Ae to mRNA

[0045] 1. Preparation of complexes of Listeneriolysin O+GS linker+L7Ae with mRNA in different proportions

[0046] Listeriolysin O+GS linker+L7Ae was mixed with eGFP-mRNA containing Box C / D sequences at ratios of 1:1, 0.5:1, 0.25:1, and 0.125:1 (mol / mol), i.e., 1.30:3, 0.65:3, 0.32:3, and 0.16:3 (μg / μg), respectively, in 50 μL of PBS solution to obtain solutions A1, A2, A3, and A4. These solutions were then mixed with solution B (protamine solution, obtained by mixing 18 μg of protamine with 50 μL of PBS) and incubated to obtain protamine / Listeriolysin O+GS linker+

[0047] L7Ae / mRNA complex solutions A1B, A2B, A3B, and A4B.

[0048] 2. Transfection of 293T cells

[0049] Stable 293T cells were seeded in six-well plates and transfected when the cells reached 70-80% confluency. The culture medium in the culture dish was aspirated and replaced with 1 mL of Opti-MEM medium. After the mixture of solutions A and B was thoroughly mixed and incubated, complex solutions A1B, A2B, A3B, and A4B were added to the 293T cells, respectively. After 6 hours, the medium was replaced with complete medium, and the cells were cultured for another 24 hours.

[0050] 3. Observe and photograph under a fluorescence microscope.

[0051] The 293T cells in 6-well plates containing the above-mentioned complex solutions A1B, A2B, A3B, and A4B were observed and photographed under a fluorescence microscope. The results are shown below. Figure 2 The Listeriolysin O+GS linker+L7Ae fusion protein and mRNA can be delivered in all four of the above ratios. The delivery effect is best when the mass ratio is 1.30:3, that is, the molar ratio is 1:1, which is no worse than Lip2000:eGFP-mRNA (5μl:3μg).

[0052] Example 2: Preparation of the protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex

[0053] Solution A was prepared by incubating 13 μg of Listeniolysin O+GS linker+L7Ae and 30 μg of p30-mRNA containing Box C / D sequences in 500 μL of PBS. Solution B was prepared by incubating 180 μg of protamine in 500 μL of PBS. Solution A and Solution B were then mixed and incubated to obtain the protamine / Listeriolysin O+GS linker+

[0054] L7Ae / p30-mRNA complex ( Figure 1 ) solution.

[0055] Example 3: Transfection of 293T cells with protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex 1. Transfection of 293T cells

[0056] Stable 293T cells were seeded in 6-well plates and transfected when the cells reached 70-80% confluency. The culture medium in the culture dish was aspirated and replaced with 1 mL of Opti-MEM medium. The protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex solution (containing 3 μg p30-mRNA) prepared in Example 2 was added to the 293T cells, mixed well, and incubated in a CO2 incubator at 37°C for 6 h. After that, the medium was replaced with complete medium, and the cells were cultured for another 24 h.

[0057] 2. Detection of ASFV p30 protein by Western blotting

[0058] Total protein was extracted from transfected cells 48 h after transfection and culture. Polyacrylamide gel electrophoresis was then performed after adding 6× loading buffer and boiling for 5 min. Protein bands separated in the gel were transferred to a cellulose acetate membrane via electrotransfer. The membrane was blocked with 5% milk at room temperature for 60 min and washed three times with TBST. The membrane was then incubated overnight at 4°C with p30 antibody diluted 1000-fold with 1% BSA-TBST solution and washed three times with TBST. HRP-labeled mouse anti-rabbit IgG was added, and the membrane was incubated at room temperature for 120 min and washed three times with TBST. The membrane was then developed using an enhanced chemiluminescence (ECL) kit and scanned for imaging. Results are as follows: Figure 3 As shown, this indicates that the delivery system can successfully deliver p30-mRNA into eukaryotic cells for expression.

[0059] Example 4. Immunization of mice with protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex. 1. Immunization of mice.

[0060] Six female C57 mice weighing 18–20 g were selected; three mice in the immunization group were injected with a protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex solution, and three mice in the control group were injected with PBS solution. Mice in the immunization group were injected intramuscularly with a PBS solution of the protamine / Listeriolysin O+GS linker+L7Ae / p30-mRNA complex (i.e., the mRNA delivery system of the fusion protein of this invention, with 2 μg of p30-mRNA) in the posterior thigh. Mice in the control group were injected intramuscularly with 100 μl of PBS solution in the same manner. Fourteen days after the initial immunization, a booster immunization was performed using the same method and dosage.

[0061] 2. Detection of mouse-specific antibodies by Western blotting

[0062] Fourteen days after booster immunization, blood was collected from the orbital vein of mice, and the serum was separated. The mice were then tested for specific antibodies using the Western blotting method. The specific operating steps are as follows: After culturing the transfected 293T cells from Example 3 for 48 hours, total protein was extracted from the transfected cells. Then, 6× loading buffer was added, followed by boiling in a water bath for 5 minutes, and polyacrylamide gel electrophoresis was performed. The transfected 293T cells from Example 3 were loaded in parallel six times. Gel electrotransfer was used to transfer the protein to a cellulose acetate membrane. The membrane was blocked with 5% milk at room temperature for 60 minutes and washed three times with TBST. The 25kDa–35kDa cellulose acetate membrane corresponding to each sample lane was cut out individually, resulting in six small membranes. Serum from six mice in the vaccine group and blank group was used. Each mouse's serum was diluted 500-fold with 1% BSA-TBST solution as the primary antibody. One mouse's serum corresponded to one small membrane. The membranes were incubated overnight at 4°C and washed three times with TBST. HRP-labeled rabbit anti-mouse IgG was added, and the membranes were incubated at room temperature for 120 minutes and washed three times with TBST. The membranes were developed using an enhanced chemiluminescence immunoassay kit (ECL) and scanned for imaging. The results are as follows: Figure 4 As shown, only mice immunized with the protamine / Listeriolysin O+GSlinker+L7Ae / p30-mRNA complex produced specific antibodies.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein is composed of Listeriolysin O, GS linker, and L7Ae protein, with Listeriolysin O linked to the N-terminus of L7Ae protein via GS linker. The amino acid sequence of the fusion protein is shown in SEQ ID NO.

1.

2. An mRNA delivery system based on the fusion protein of claim 1, characterized in that, The mRNA delivery system comprises a cationic polymer, the fusion protein of claim 1, and mRNA. The cationic polymer encapsulates and protects the mRNA. The 3'UTR end of the mRNA binds to the C-terminus of the L7Ae protein in the fusion protein. The 3'UTR end of the mRNA is linked to a Box C / D sequence. The cationic polymer is protamine. The mRNA is negatively charged mRNA.

3. The mRNA delivery system according to claim 2, characterized in that, In the mRNA delivery system described in claim 1, the molar ratio of the fusion protein to mRNA is 0.125 to 1:1; and the mass ratio of mRNA to cationic polymer is 6:

1.

4. The mRNA delivery system according to claim 3, characterized in that, In the mRNA delivery system described in claim 1, the molar ratio of the fusion protein to the mRNA is 1:1, 0.5:1, 0.25:1, or 0.125:

1.

5. The mRNA delivery system according to claim 4, characterized in that, In the mRNA delivery system described in claim 1, the molar ratio of the fusion protein to the mRNA is 1:

1.

6. The mRNA delivery system according to claim 2, characterized in that, The Box C / D sequence is shown in SEQ ID NO.

2.

7. The use of the fusion protein of claim 1 in the preparation of an mRNA delivery system.

8. The use of the mRNA delivery system according to any one of claims 2 to 6 in the preparation of mRNA transfection reagents or in the preparation of mRNA vaccines.

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