A formulation for in vitro transfection and in vivo delivery of mRNA

Abstract

The present invention relates to the field of mRNA delivery, and specifically, provides a preparation for in vitro transfection and in vivo delivery of mRNA. The cationic liposome provided by the present invention comprises a neutral auxiliary lipid, a first cationic lipid and a second cationic lipid, and the second cationic lipid comprises Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA or c12-200. The cationic liposome is specially designed for mRNA research and development, has a better transfection effect in in vitro mRNA cell transfection, and has lower toxicity than commercial cationic liposome reagents available on the market; has a better immune effect in mRNA in vivo delivery, and can be applied to the field of infectious disease vaccines. When used, it can be directly matched with a specific mRNA sequence to flexibly meet different application requirements.

Description

Technical Field

[0001] The present invention relates to the field of mRNA delivery, and in particular to a preparation for in vitro transfection and in vivo delivery of mRNA. Background Art

[0002] Cationic liposomes are widely used for the delivery of nucleic acids in cells and in vivo. Compared with other existing delivery technologies such as nanospheres and lipid nanoparticles, cationic liposomes have a simple preparation process, are more deeply studied, and can be used with different types of nucleic acids. The application scenarios are flexible, so they are particularly suitable for the development of in vivo and in vitro nucleic acid transfection reagents. At present, some nucleic acid drug research and development also uses cationic liposome technology.

[0003] Cationic liposomes are usually composed of cationic lipids and one or more helper lipids. So-called "liposome complexes" can be formed from cationic liposomes and anionic nucleic acids.

[0004] On the one hand, compared with other nanoparticle preparations, cationic liposomes are more suitable for in vitro cell transfection, and their in vivo nucleic acid delivery effect is poor. Therefore, the low efficiency of cationic liposomes in in vivo nucleic acid delivery limits their application in the development of in vivo transfection reagents and drug development. Developing new methods to improve the in vivo nucleic acid delivery effect of cationic liposomes can improve their application prospects in this field.

[0005] On the other hand, the development of mRNA drugs is one of the current hot areas. However, there are no cationic liposome reagents specifically used for in vivo delivery of mRNA on the market. In terms of in vitro transfection applications, mature transfection reagents such as lipofectamine have high cytotoxicity. There is a lack of low-toxic and high-efficiency products. In the development of mRNA drugs, the existing cationic liposome technology is only used in the mRNA immunotherapy of cancer. Cancer immunotherapy mainly involves cellular immune pathways, which requires the activation of killer immune cells to eliminate tumors, while infectious disease immunity requires the production of corresponding antibodies through humoral immune pathways to provide immune protection to the body. The two immune mechanisms are different, and the product efficacy and requirements are different. Therefore, cationic liposome delivery systems are still blank in the application of infectious disease vaccines.

[0006] Therefore, we need to develop a new type of cationic liposome that meets the toxicity and efficacy requirements of mRNA in vitro cell transfection, and achieve efficient delivery of mRNA in vivo, while opening up its application in mRNA infectious disease vaccines.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The first purpose of the present invention is to provide a cationic liposome to alleviate the technical problems of low RNA delivery efficiency, high toxicity and narrow application field of cationic liposome in the prior art.

[0009] The second object of the present invention is to provide the application of the cationic liposome.

[0010] The third object of the present invention is to provide a method for preparing the cationic liposome.

[0011] The fourth purpose of the present invention is to provide a complex preparation of cationic liposomes and mRNA to alleviate the technical problems of low in vivo and in vitro delivery efficiency, high toxicity and narrow application range of the complex preparation of cationic liposomes and mRNA in the prior art.

[0012] The fifth object of the present invention is to provide a method for preparing the complex preparation of the above-mentioned cationic liposome and RNA.

[0013] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0014] A cationic liposome, the raw materials of which include a neutral auxiliary lipid, a first cationic lipid and a second cationic lipid;

[0015] The second cationic lipid includes Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA or c12-200.

[0016] Further, the second cationic lipid is Dlin-MC3-DMA;

[0017] Preferably, the neutral helper lipid comprises DOPE, DOPC or cholesterol;

[0018] Preferably, the neutral helper lipid is DOPE.

[0019] Further, the first cationic lipid comprises DOTAP, DOTMA or DOSPA;

[0020] Preferably, the first cationic lipid is DOTAP.

[0021] Furthermore, the molar ratio of the sum of the first cationic lipid and the second cationic lipid to the neutral auxiliary lipid is (9-27):9, and the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5):(0.5-3).

[0022] The use of cationic liposomes in any of the following a)-c):

[0023] a) mRNA in vitro cell transfection;

[0024] b) preparing mRNA in vivo delivery reagents;

[0025] c) preparing mRNA vaccines;

[0026] Preferably, the mRNA vaccine comprises an mRNA tumor vaccine or an mRNA infectious disease vaccine, preferably an mRNA infectious disease vaccine;

[0027] Preferably, the mRNA delivery in vivo is local delivery of mRNA in vivo.

[0028] The method for preparing cationic liposomes comprises providing a solution containing a neutral auxiliary lipid, a first cationic lipid and a second cationic lipid, removing the solvent from the solution, drying, hydrating and ultrasonicating the solution in sequence to obtain cationic liposomes.

[0029] Further, the molar ratio of the sum of the first cationic lipid and the second cationic lipid to the neutral auxiliary lipid is (9-27):9, and the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5):(0.5-3);

[0030] Preferably, the solvent comprises chloroform and / or a mixture of dichloromethane and methanol;

[0031] Preferably, the drying comprises: vacuum drying for 1.5-2.5 hours;

[0032] Preferably, the hydration comprises: using RNAse-free ultrapure water to shake at 35-39° C. for 8-12 minutes, and the concentration of the cationic liposomes after hydration is 2.5-3.5 mg / ml;

[0033] Preferably, the ultrasound comprises: ultrasound at 38-42° C. for 35-45 minutes.

[0034] A cationic liposome and mRNA complex preparation comprises a working solution, mRNA and cationic liposome.

[0035] Furthermore, the working solution includes deionized water, ultrapure water, sodium chloride solution, PBS solution or PBS solution containing 5-40 μg / ml transferrin;

[0036] Preferably, the sequence of the mRNA is as shown in SEQ ID NO.1;

[0037] Preferably, the mRNA concentration is 0.01-0.5 mg / ml;

[0038] Preferably, the mass ratio of cationic liposome to mRNA is 1:1-8:1;

[0039] Preferably, in terms of mRNA in vivo delivery, the working solution is a PBS solution, and the mass ratio of cationic liposomes to mRNA is 1:1-2:1;

[0040] Preferably, in terms of in vitro mRNA cell transfection, the working solution is a PBS solution containing 5-40 μg / ml transferrin, and the mass ratio of cationic liposomes to mRNA is 2:1-8:1.

[0041] The method for preparing a complex preparation of a cationic liposome and mRNA comprises mixing mRNA and a cationic liposome in a working solution to obtain the complex preparation of the cationic liposome and mRNA.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The cationic liposomes provided by the present invention include neutral auxiliary lipids, a first cationic lipid and a second cationic lipid as raw materials. Traditional cationic liposomes include a cationic lipid and a neutral lipid. The cationic lipid can combine with negatively charged nucleic acids to form nanoparticles and help nucleic acids enter cells at the same time. In addition to the function of forming the structure, some neutral lipids can also assist the fusion between the lipid membrane and the cell membrane to further help the release of nucleic acids. However, studies have found that this effect is relatively limited. Protonatable lipids have been shown to help the intracellular delivery of nucleic acids and their escape from endosomes. The design of the present invention is to use cationic lipids as the main component, and to use the first positively charged cationic lipid as the main component to combine and condense mRNA to form nanoparticles, add the second cationic lipid to help the endocytosis of the particles and the escape of the endosomes, and use neutral lipids to adjust the charge and assist the release of nucleic acids. The three components exert a synergistic effect and meet the needs of encapsulating mRNA, endocytosis, and escape and release of endosomes. For a transfection reagent, effectiveness and safety are the two central principles for evaluating its value. The cationic liposome is specially designed for mRNA and has excellent transfection effect in in vitro cell transfection. Since the three lipid components used have been used in the design of other nucleic acid drugs and their safety has been verified in clinical trials, they are less toxic than the commercial reagents with complex components on the market. The cationic liposome has excellent delivery and immune effects in in vivo delivery and can be applied to the field of infectious disease vaccines, filling the technical gap in this field in the existing technology. In addition, after the cationic liposome is prepared, it can be mixed with specific mRNA before administration according to actual application needs, without the need for other operations such as pre-compounding with mRNA to make nanoparticles, making its application more flexible.

[0044] The method for preparing cationic liposomes provided by the invention is simple and easy to operate, has low cost and does not require complicated professional equipment.

[0045] The cationic liposome and mRNA complex preparation provided by the present invention comprises a working solution, mRNA and cationic liposomes. The cationic liposome mRNA complex has an excellent transmission rate, lower cytotoxicity than market products, and can achieve an immune effect in a short time without the use of any adjuvant, especially has a good effect on the treatment of infectious diseases, filling the technical gap in this field in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0047] Figure 1 This is a graph showing the particle size of Tf4 in dynamic light scattering in Example 8;

[0048] Figure 2 The dynamic light scattering result diagram of the particle size of the cationic liposome mRNA complex in Example 9;

[0049] Figure 3 The effect of different mass ratios of Tf4 and mRNA on in vitro cell transfection efficiency in Example 10;

[0050] Figure 4 is the in vitro HEK293 cell transfection efficiency of Tf4 in Example 11;

[0051] Figure 5 is the in vitro DC2.4 cell transfection efficiency of Tf4 in Example 11;

[0052] Figure 6 is the in vitro HEK293 cell transfection toxicity of Tf4 in Example 12;

[0053] Figure 7 is the in vitro HEK293 cell transfection efficiency of different buffer compositions of Tf4 in Example 13;

[0054] Figure 8 This is the evaluation of the fluorescent in vivo imaging system 2 hours after intramuscular injection of the mouse leg in Example 14;

[0055] Fig. 9 The original reading of rabies neutralizing antibody ELISA in mice 14 days after the rabies G protein mRNA (5 μg) was packaged with different transfection reagents in Example 15 and injected into the mouse leg muscle (single dose);

[0056] Fig.10This is the in vivo immunization cycle of the mRNA vaccine encoding rabies virus G protein delivered by Tf4 in Example 16. DETAILED DESCRIPTION

[0057] The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer.

[0058] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0059] A cationic liposome, the raw materials of which include a neutral auxiliary lipid, a first cationic lipid and a second cationic lipid, wherein the second cationic lipid includes Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA or c12-200.

[0060] For a transfection reagent, effectiveness and safety are the two central principles for evaluating its value. The cationic liposome is specially designed for mRNA and has excellent transfection effect in in vitro cell transfection. Since the three lipid components used have been used in the design of other nucleic acid drugs and their safety has been verified in clinical trials, they are less toxic than the commercial reagents with complex components on the market. The cationic liposome has excellent immune effect in in vivo delivery and can be applied to the field of infectious disease vaccines, filling the technical gap in this field in the existing technology. In addition, after the cationic liposome is prepared, it can be mixed with specific mRNA before administration according to actual application needs, without the need for other operations such as pre-compounding with mRNA to make nanoparticles, making its application more flexible.

[0061] It should be noted that the neutral auxiliary lipid and the first cationic lipid in the present invention are neutral lipids and cationic lipids commonly used in the art for preparing cationic liposomes, for example, the neutral auxiliary lipid can be DOPE, DOPC, etc., and the first cationic lipid can be DOTAP, DOTMA, etc. The CAS number of Dlin-MC3-DMA is 1224606-06-7, the CAS number of Dlin-KC2-DMA is 1224606-06-7, the full name of DODMA is 1,2-Dioleyloxy-3-dimethylaminopropane, the Chinese name is 1,2-dioloxy-3-dimethylaminopropane, the full name of c12-200 is 1,1-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), and the CAS number is 1220890-25-4.

[0062] In a preferred embodiment, the second cationic lipid is Dlin-MC3-DMA, and the second cationic lipid is a protonable cationic lipid. The protonable lipid can help release mRNA from the cationic lipid complex in the cell and can help mRNA escape from the endosome into the cytoplasm. Therefore, the addition of the second cationic lipid can significantly improve the delivery efficiency of cationic liposomes, especially Dlin-MC3-DMA has a better effect.

[0063] In a preferred embodiment, the neutral auxiliary lipid includes DOPE, DOPC or cholesterol, and DOPE is further preferred. Neutral lipids are an important component of cationic liposomes. Due to their special structure, some neutral lipids can mediate the fusion process of cationic lipid complexes with cell membranes, thereby helping mRNA enter cells. It should be noted that the full name of DOPE is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, the Chinese name is dioleoyl phosphatidylethanolamine, the full name of DOPC is 1,2-dioleoyl-sn-glycero-3-phosphocholine, and the Chinese name is dioleoyl phosphatidylethanolamine, and cholesterol is cholesterol.

[0064] In a preferred embodiment, the first cationic lipid includes DOTAP, DOTMA or DOSPA, and more preferably DOTAP. The first cationic lipid is a non-protonable cationic lipid, which usually shows a strong positive charge, which can ensure that the cationic liposome can effectively bind to the negatively charged mRNA molecules under most pH and buffer environments, encapsulate the mRNA and form a nanocomplex, thereby mediating the delivery of mRNA in vitro cells and in vivo. It should be noted that the full name of DOTAP is 1,2-Dioleoyl-3-trimethylammonium-propane, and its Chinese name is 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium; DOTMA is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, and its Chinese name is N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride; and the full name of DOSPA is 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamini umtrifluoroacetate.

[0065] In a preferred embodiment, the molar ratio of the sum of the first cationic lipid and the second cationic lipid to the neutral helper lipid is (9-27):9, and the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5):(0.5-3). Further preferably, the molar ratio of the sum of the first cationic lipid and the second cationic lipid to the neutral helper lipid is 20:9, and the molar ratio of the first cationic lipid to the second cationic lipid is 8.9:1.1. The molar ratio of the neutral helper lipid to the sum of the first and second cationic lipids is typically, but not limited to, 9:9, 9:12, 9:15, 9:17, 9:18, 9:19, 9:20, 9:21, 9:22, 9:23, 9:25, or 9:27; the molar ratio of the first and second cationic lipids is typically, but not limited to, 7:0.5, 7:1, 7:2, 7:3, 8:0.5, 8:1, 8:2, 8:3, 9.5:0.5, 9.5:1, 9.5:2, or 9.5:3.

[0066] The cationic liposome provided by the present invention can be used in any of the following a)-c):

[0067] a) mRNA in vitro cell transfection;

[0068] b) preparing mRNA in vivo delivery reagents;

[0069] c) Preparation of mRNA vaccines.

[0070] In a preferred embodiment, the mRNA vaccine includes an mRNA tumor vaccine or an mRNA infectious disease vaccine, preferably an mRNA infectious disease vaccine. Experiments have found that the cationic liposomes provided by the present invention are effective in preparing infectious disease vaccines.

[0071] In a preferred embodiment, the mRNA in vivo delivery is mRNA local in vivo delivery. The mRNA local in vivo delivery can be intramuscular injection or subcutaneous injection, etc.

[0072] The present invention provides a method for preparing the above cationic liposomes, providing a solution containing a neutral auxiliary lipid, a first cationic lipid and a second cationic lipid, removing the solvent from the solution, drying, hydrating and ultrasonicating the solution in sequence to obtain the cationic liposomes. The method is simple and easy to operate, low in cost and does not require complex professional equipment.

[0073] In a preferred embodiment, the molar ratio of the sum of the first cationic lipid and the second cationic lipid to the neutral auxiliary lipid is (9-27):9, and the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5):(0.5-3).

[0074] In a preferred embodiment, the solvent includes chloroform and / or a mixture of dichloromethane and methanol. The solvent may be a chloroform-methanol solution, a dichloromethane-methanol solution, or a chloroform-dichloromethane-methanol solution, and the method for removing the solvent is preferably a rotary evaporation method.

[0075] In a preferred embodiment, drying comprises: vacuum drying for 1.5-2.5 hours. The vacuum drying time is typically but not limited to 1.5 hours, 2 hours or 2.5 hours.

[0076] In a preferred embodiment, hydration includes: shaking at 35-39°C for 8-12 minutes with RNAse-free ultrapure water, and the concentration of cationic liposomes after hydration is 2.5-3.5 mg / ml. The hydration temperature is typically but not limited to 35°C, 36°C, 37°C, 38°C or 39°C; the hydration time is typically but not limited to 8 minutes, 9 minutes, 10 minutes, 11 minutes or 12 minutes; the concentration of cationic liposomes after hydration is typically but not limited to 2.5 mg / ml, 3 mg / ml or 3.5 mg / ml.

[0077] In a preferred embodiment, the ultrasound comprises: ultrasound at 38-42°C for 35-45 minutes. The ultrasound temperature is typically but not limited to 38°C, 39°C, 40°C, 41°C or 42°C; the ultrasound time is typically but not limited to 35 minutes, 38 minutes, 40 minutes, 42 minutes or 45 minutes.

[0078] A cationic liposome and mRNA complex preparation, comprising a working solution, mRNA and the above-mentioned cationic liposome. The cationic liposome RNA complex has an excellent transfer rate, lower cytotoxicity than market products, and can achieve an immune effect without the use of any adjuvant, especially for the treatment of infectious diseases, filling the technical gap in this field in the prior art. It should be noted that the working solution refers to a solution used until the nucleic acid is used in vivo or in vitro, which can be ultrapure water, sodium chloride solution, PBS solution, etc.

[0079] In a preferred embodiment, the working solution includes deionized water, ultrapure water, sodium chloride solution, PBS solution or PBS solution containing 5-40 μg / ml transferrin. The working solution of the present invention can be any of the above solutions, but the delivery rates of the above solutions are different, and different solutions are preferably used for different applications. For example, for in vivo RNA delivery, the working solution is preferably a PBS solution; for RNA in vitro cell transfection, the working solution is preferably a PBS solution containing 5-40 μg / ml transferrin.

[0080] In a preferred embodiment, the sequence of mRNA is shown in SEQ ID NO.1. The mRNA in the rabies vaccine used in the prior art is designed for the Pasteur strain. The inventor designed it for the rabies virus strain CTN-1 unique to my country (CTN-1 has only 60-70% homology with the Pasteur strain). Through research, the RVG-D mRNA shown in SEQ ID NO.1 was obtained. The sequence has been optimized for nucleotide codes to make it more suitable for expression in mammals, especially humans. This sequence has a higher specificity for strains unique to China and is more suitable for development and use in China. In addition, the complex preparation prepared by RVG-D mRNA and the cationic liposome provided by the present invention has a higher neutralizing antibody positive rate and a faster and longer immune protection effect as a rabies vaccine.

[0081] In a preferred embodiment, the mRNA concentration is 0.01-0.5 mg / ml, preferably 0.05-0.2 mg / ml.

[0082] In a preferred embodiment, the mass ratio of cationic liposomes to mRNA is 1: 1-8: 1. In different application scenarios, the mass ratio of cationic liposomes to mRNA is preferably different ratios. For RNA in vivo delivery, the mass ratio of cationic liposomes to mRNA is preferably 1: 1-2: 1; for RNA in vitro cell transfection, the mass ratio of cationic liposomes to mRNA is 2: 1-8: 1.

[0083] The preparation method of the cationic liposome and mRNA complex preparation is to mix mRNA and cationic liposome in a working solution to obtain the cationic liposome and mRNA complex preparation. The method is simple and easy to operate, and can be prepared in real time on site, avoiding complicated operations and the use of professional equipment.

[0084] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0085] Example 1

[0086] A cationic liposome, the raw materials of which are: DOPC, the molar ratio of the total of DOTMA and Dlin-KC2-DMA is 9:9, and the molar ratio of DOTMA and Dlin-KC2-DMA is 9.5:0.5.

[0087] Example 2

[0088] A cationic liposome, the raw materials of which are: DOPC, the molar ratio of the total of DOTMA and Dlin-KC2-DMA is 9:27, and the molar ratio of DOTMA and Dlin-KC2-DMA is 7:3.

[0089] Example 3

[0090] A cationic liposome, the raw materials of which are: DOPC, the molar ratio of the total of DOTMA and Dlin-KC2-DMA is 9:20, and the molar ratio of DOTMA and Dlin-KC2-DMA is 8.9:1.1.

[0091] Example 4

[0092] A cationic liposome, the raw materials are: DOPE, the molar ratio of the total of DOTAP and Dlin-MC3-DMA is 9:20, and the molar ratio of DOTAP and Dlin-MC3-DMA is 8.9:1.1.

[0093] Example 5

[0094] A method for preparing cationic liposomes, comprising the following steps:

[0095] a) using chloroform as an organic solvent, preparing the raw materials into an organic solution;

[0096] b) taking an appropriate amount of organic solution into a round-bottom flask, and removing the organic solvent by rotary evaporation to obtain a dried lipid film;

[0097] c. Dry the lipid film in the round-bottom flask using a vacuum drying device for 2 hours.

[0098] d. Take RNase-free ultrapure water and add it to the round-bottom flask to hydrate the lipid film. Then shake and hydrate it in a 37°C water bath for 10 minutes to ensure that the liposome concentration after hydration is 3 mg / ml.

[0099] e. Place the hydrated liposomes in a water bath sonicator and sonicate at 40°C for 40 minutes to obtain a cationic liposome aqueous suspension product.

[0100] Example 6 Cationic liposome and mRNA complex preparation - for in vitro cell transfection

[0101] A method for preparing a complex preparation of a cationic liposome and mRNA, comprising the following steps:

[0102] a. Take an appropriate amount of mRNA solution and dilute it in PBS solution to a concentration of 0.1 mg / ml.

[0103] b. Take an appropriate amount of cationic liposome suspension and dilute it in a PBS solution containing 10 μg / ml transferrin, controlling the mass ratio of cationic liposome to mRNA to be 4:1.

[0104] c. Use a pipette to mix the two, blow gently 5 times, and let it stand for 15 minutes to obtain a complex preparation of cationic liposomes and mRNA.

[0105] Example 7 Cationic liposome-mRNA complex preparation - for in vivo RNA delivery

[0106] A method for preparing a complex preparation of a cationic liposome and mRNA, comprising the following steps:

[0107] a. Take an appropriate amount of mRNA solution and dilute it in PBS solution to a concentration of 0.1 mg / ml.

[0108] b. Take an appropriate amount of cationic liposome suspension and dilute it in PBS solution to control the mass ratio of cationic liposome to mRNA to be 2:1.

[0109] c. Use a pipette to mix the two, blow gently 5 times, and let it stand for 15 minutes to obtain a complex preparation of cationic liposomes and mRNA.

[0110] Example 8

[0111] The raw materials of Example 4 were used according to the method for preparing cationic liposomes of Example 5 to prepare cationic liposomes, which were named Tf4.

[0112] The prepared Tf4 preparation was immediately tested for relevant physical and chemical indicators using a dynamic light scattering nanoparticle size analyzer (Malvern zetasizer nanoZS). During the test, the Tf4 preparation was diluted 5 times with ultrapure water and transferred to the sample tank for testing. The sample tank was inserted into the instrument and the sample particle size was tested, and the average particle size and monodispersity index (PDI) of the sample were recorded. The results showed that the particle size of Tf4 was Figure 1 As shown, cationic liposomes with a size of 122 nm were obtained, and the monodispersity coefficient was 0.275. The particle size of the sample was detected repeatedly, and the monodispersity coefficient was about 120-140 nm, and the monodispersity coefficient was less than 0.3. The prepared Tf4 can be stably stored in a refrigerator at 4 degrees for at least 3 months.

[0113] Example 9

[0114] The Tf4 in Example 8 was prepared according to the method in Example 7 to obtain a complex preparation of cationic liposomes and luciferase gene mRNA, and the relevant physical and chemical indicators were immediately detected using a dynamic light scattering nanoparticle size analyzer (Malvern zetasizer nanoZS). During the detection, the complex preparation was diluted with ultrapure water and transferred to the sample tank for detection, ensuring that the nucleic acid concentration in the sample tank was about 1-2μg / ml. The sample tank was inserted into the instrument and the sample particle size was detected, and the average particle size and monodispersity index (PDI) of the sample were recorded. The results showed that the particle size of Tf4 was as follows Figure 2 As shown, it can be seen that after Tf4 combines with the luciferase gene mRNA molecule, the average particle size is 207nm and the monodispersity coefficient is 0.197. The results of multiple consecutive experiments show that the particle size of the complex is slightly about 200nm less than 250nm, the monodispersity coefficient is less than 0.3, and usually less than 0.2. The obtained preparation can be stably stored for about 1 month.

[0115] Example 10 Exploration of the optimal formulation ratio

[0116] The physicochemical differences between different mRNA molecules are minimal, so we used mRNA encoding the luciferase gene (Trilink) as a reporter gene to reflect the efficiency of Tf4 in vitro and in vivo delivery of mRNA. In order to optimize the mass ratio of Tf4 and mRNA for optimal complexation, Tf4 and mRNA molecules encoding the luciferase gene were mixed at different mass ratios and used in in vitro HEK293 cell transfection experiments.

[0117] HEK293 cells revived from liquid nitrogen were cultured for one generation and then used. HEK293 cells 24 hours before the experiment were planted in a 96-well plate with a cell density of 10,000-20,000 cells per well. The cells were cultured in a 37°C incubator for 24 hours and the cell state was observed. The experiment can be started when the cell confluence reaches about 80-90%. According to the method provided in Example 6, a nanocomplex of Tf4 and mRNA (Trilink) encoding the luciferase gene was prepared (prepared according to different mass ratios), and an equal amount of luciferase mRNA (Trilink) was used as a negative control group, and a solvent without mRNA and Tf4 was used as a blank group. After all reagents were prepared, they were directly added to the 96-well plate cell culture medium to ensure that 0.1 μg of mRNA was added to each well, and an equal amount of solvent was added to the blank group. The cells were then incubated in a 37-degree incubator for 24 hours to detect the expression of luciferase.

[0118] The detection of luciferase expression requires the use of a colorimetric kit, refer to the Bright-Glo purchased from Promega TM The experiment was performed according to the Luciferase Assay System Kit Operation Manual: aspirate the cell supernatant in the 96-well plate, and directly add the hydrated luciferase substrate to each well for 2 minutes, adding 100 microliters of substrate to each well. Then the fluorescence value can be read using an ELISA reader. Turn on the multifunctional ELISA reader, select the chemiluminescence mode, and record the readings after reading the plate for comparative analysis. The normalization method is adopted for data analysis: the reading of the control group is set to 1, and the reading of the experimental group is divided by the reading of the control group to obtain the change multiple of the luciferase expression in the experimental group relative to the control group, which is used for result statistics.

[0119] The fluorescence reading results after 24 hours of luciferase mRNA transfection are as follows Figure 3 As shown in the figure (Y axis: cellular luciferase gene expression (fold change)), it was found that the mass ratio of the two combined had a great influence on the cell transfection efficiency, and the optimal mass ratio of Tf4 to mRNA was about 4:1. This ratio is the optimized formulation ratio for subsequent in vitro cell transfection experiments.

[0120] At the same time, experiments showed that the optimal mass ratio of Tf4 to mRNA for in vivo delivery of mRNA is 2:1.

[0121] Example 11 In vitro cell transfection efficiency

[0122] To compare the advantages of Tf4 and existing technologies in cell transfection (24 hours), we compared the transfection efficiency of several transfection reagents on HEK293 cells and DC2.4 cells. This experiment also used luciferase as a reporter gene. Here, we considered selecting existing technologies DOTMA / DOPE and DOTAP / DOPE liposomes, as well as two commercial transfection reagents: lipofectamine2000 and lipofectamine messengermax as positive controls.

[0123] The specific experimental method is in accordance with the method provided in Example 10. In short, KEK293 cells and DC2.4 cells were seeded into 96-well plates and cultured for 24 hours for standby use, and a complex of Tf4 and mRNA was prepared at a mass ratio of 4:1. The components and proportions of DOTMA / DOPE and DOTAP / DOPE preparations all adopted the optimal solution explored in the preliminary experiment. Lipofectamine2000 and lipofectamine messengermax were operated according to the relevant instructions, and the optimal ratio (v / w) with mRNA adopted the optimal solution determined in the preliminary experiment. After all preparations were added to the cells (0.1 μg / well mRNA), they were cultured at 37°C for 24 hours and then tested.

[0124] HEK293 cell results Figure 4 As shown, the results of DC2.4 cells are as follows Figure 5 The results showed that in a 24-hour transfection experiment, compared with the existing DOTAP / DOPE, DOTMA / DOPE cationic liposomes, and the commercial transfection reagent lipofectamine2000, Tf4 has a higher ability to transfect cells, and the results in HEK293 cells and DC2.4 cells are consistent.

[0125] Example 12 In vitro cell transfection toxicity

[0126] Safety is another key consideration in the development of cationic liposome reagents, and we compared the toxicity of Tf4 with that of various commercial reagents on HEK293 cells.

[0127] HEK293 cells were seeded into 96-well plates 24 hours in advance. The number of cells in each well was ensured to be consistent. Five replicate wells were set for each group of experiments for statistics. Various preparations were prepared with reference to Example 11 and added to the cells for further culturing for 24 hours. Thereafter, the cells in the wells were digested with trypsin, and the number of remaining viable cells in each group of reagents after transfection for 24 hours was counted and compared using the trypan blue staining method. The results are shown in FIG. Figure 6As shown, compared with the control group and naked mRNA transfection group, Tf4, DOTAP / DOPE and DOTMA / DOPE transfection had no significant effect on cell activity, and a 10-20% decrease in the number of live cells was found in cells transfected with lipofectamine series products. This shows that Tf4 has less effect on cell viability than lipofectamine series products and is more suitable for in vitro cell transfection applications and long-term gene expression observation and research.

[0128] Example 13 Optimization of in vitro cell transfection working solution

[0129] Considering that the solvent and buffer system of the transfection reagent may have a great impact on its cell transfection effect, we optimized the buffer composition specifically for Tf4. Referring to the existing technology, we selected pure water, sodium chloride injection, PBS, and added transferrin to PBS to evaluate the effects of several buffer systems and solvents on Tf4 transfection efficiency. This experiment also selected luciferase as the reporter gene.

[0130] The nanocomplex of Tf4 and mRNA encoding the luciferase gene was prepared with reference to Example 11. During the preparation process, Tf4 and mRNA were diluted in any of the above buffers and then mixed. The resulting preparation was directly added to HEK293 cells in a 96-well plate, and the luciferase expression was detected after 24 hours of culture. Figure 7 As shown. It can be seen that PBS can significantly improve the transfection efficiency of mRNA among all solvents and buffers. In addition, adding 10μg / ml human transferrin (sigma) to PBS can further improve the mRNA cell transfection efficiency. Therefore, PBS containing transferrin is the optimal buffer system for Tf4 preparation in the application of in vitro cell transfection.

[0131] Example 14 In vivo delivery efficiency

[0132] Luciferase gene was used as reporter gene. Small animal fluorescence in vivo imaging technology was used to study the application prospects of Tf4 in in vivo mRNA delivery.

[0133] Referring to the preparation process of Example 7, the reporter gene mRNA was delivered into mice using the Tf4+PBS preparation, and a preparation containing 5 μg of luciferase mRNA was introduced into the mice by tail vein administration and leg muscle injection. Luciferase substrate was injected into the mice 2 hours later, and the mice were anesthetized after 10 minutes, and the expression of luciferase in the mice was observed by a fluorescent in vivo imaging system. It was found that it was difficult to express mRNA in mice using intravenous injection, probably because the complex of Tf4 and mRNA had a large particle size and was easily cleared by the immune system. However, we detected obvious gene expression in mice administered with leg muscles, and the results are as follows: Figure 8 Therefore, the preparation is suitable for the application of local delivery of mRNA in vivo. In addition, it is speculated that the complex of Tf4 and mRNA has certain immunogenicity and is suitable for the application and development of vaccine products.

[0134] Example 15 In vivo immune effect of infectious disease vaccines

[0135] According to the results in Example 14, it can be expected that the Tf4 preparation is suitable for the development of vaccine products. In particular, infectious disease vaccines, such as rabies vaccines, etc. Existing rabies vaccines all require rapid and lasting immune effects. In previous studies, we have found that the existing technology is difficult to meet the above goals. Therefore, we selected rabies virus mRNA vaccine as a practical application to evaluate the difference between the Tf4 provided by the present invention and the existing cationic liposome technology.

[0136] In the prior art, DOTMA and DOTAP cationic liposomes have been used in mRNA tumor immunotherapy. In addition, protamine has been used as a drug carrier in the development of rabies virus vaccines in the prior art. Therefore, the above preparations are considered to be selected as positive controls for comparison with the in vivo rabies G protein mRNA delivery effect of Tf4 provided by the present invention.

[0137] In terms of mRNA sequence, the existing technologies all design mRNA sequence coding regions for the Pasteur strain, while our sequence coding regions are designed for the rabies virus strain CTN-1, which is unique to my country. The homology between the two is only 60-70%. In addition, we have optimized the nucleotide codon to make it more suitable for expression in mammals, especially humans. Therefore, compared with the existing technologies, the sequence we use is more targeted to the Chinese-specific strain and is more suitable for development and use in China.

[0138] In addition, the mRNA sequence also contains:

[0139] (a) 5'-cap structure;

[0140] (b) a poly(A) sequence, preferably 60-120 A, more preferably 80-100 A;

[0141] (c) 5'UTR, 10-200 nucleotides in length, preferably 15-100 nucleotides in length, preferably including DNAH2 5'UTR, KOZAK sequences;

[0142] (d) 3'UTR, preferably including the 3'UTR sequence of hemoglobin HBA2;

[0143] The above sequence is named RVG-D mRNA, with a length of 1843 bp. The specific sequence is as follows:

[0144]

[0145] Referring to the methods of the preliminary experiment and Example 14, vaccine preparations in which DOTMA, DOTAP, Tf4 and protamine were respectively encapsulated with RVG-D mRNA were prepared for mouse immunization experiments. The mass ratio of Tf4 to mRNA was 2:1, and an equal volume of PBS was used as a blank group. According to the standard evaluation method for veterinary vaccines, mouse serum was collected on the 14th day after a single administration of 5 μg (mRNA) to the leg muscles of mice, and rabies virus neutralizing antibodies in the mouse serum were detected by ELISA. The ELISA kit used in this experiment is a whole virus coating kit, which can be used to compare the immunization effects of each group but cannot accurately quantify the neutralizing antibody titer. Therefore, we sent the relevant samples to a qualified third-party institution (South China Agricultural University) for neutralizing antibody testing. According to the relevant results and the actual ELISA readings of the blank group, 0.16 was set as the judgment value. A reading higher than 0.16 was judged to be neutralizing antibody positive, and the mouse immunization was successful. The original readings of the ELISA for rabies neutralizing antibodies in mice (ELISA method) are as follows Fig. 9 The positive rate of neutralizing antibodies in serum is shown in the following table:

[0146] DOTMA DOTAP Tf4 Protamine Blank Group Positive rate 0％ 30％ 80％ 0％ 0％

[0147] The results showed that under the same dosing conditions, the existing DOTMA and DOTAP liposome delivery effects could not stimulate the production of sufficient rabies virus neutralizing antibodies. Currently, human rabies vaccines require the production of neutralizing antibodies 4 weeks after three consecutive injections, while veterinary rabies vaccines generally require a 70-80% neutralizing antibody positive rate 14 days after a single injection. Therefore, Tf4 is more in line with the actual needs of the current market than existing technologies and has greater commercial development prospects.

[0148] Example 16 In vivo immune cycle of infectious disease vaccines

[0149] According to the method of Example 15, Tf4 preparations (w / w=2) encapsulating RVG-D mRNA were prepared, and an equal volume of solvent was used as a blank control. After immunization of mice with 5 μg mRNA, mouse serum was collected and rabies virus neutralizing antibodies were detected by ELISA at 2, 4, 12 and 28 weeks. The results are shown in Fig.10 .

[0150] The results showed that the readings of mice immunized with Tf4 preparations showed a significant increase from the 2nd week when the neutralizing antibodies were positive. The readings reached a peak at 12 weeks, and the readings at 28 weeks dropped slightly but were still far above the positive judgment value. The Tf4 vaccine preparation can effectively stimulate mice to produce an immune protection effect that lasts at least half a year. Therefore, Tf4 has development prospects in the application of rabies vaccines.

[0151] Although the present invention has been illustrated and described with specific embodiments, it will be appreciated that many other changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, it is intended to include all such changes and modifications within the scope of the present invention in the appended claims.

Claims

1. A cationic liposome, characterized in that: The raw materials are a neutral helper lipid, a first cationic lipid, and a second cationic lipid; The second cationic lipid is Dlin-MC3-DMA; The neutral helper lipid is DOPE; The first cationic lipid is DOTAP; The molar ratio of the sum of the first cationic lipid and the second cationic lipid to the neutral auxiliary lipid is 20:9, and the molar ratio of the first cationic lipid to the second cationic lipid is 8.9:1.

1.

2. The cationic liposome of claim 1 is used in any of the following a)-c): a) mRNA in vitro cell transfection; b) preparing mRNA in vivo delivery reagents; c) Preparation of mRNA vaccines.

3. The use according to claim 2, characterized in that: The mRNA vaccine includes an mRNA tumor vaccine or an mRNA infectious disease vaccine.

4. The use according to claim 2, characterized in that: mRNA delivery in vivo is local delivery of mRNA in vivo.

5. The method for preparing the cationic liposome according to claim 1, characterized in that: A solution containing a neutral auxiliary lipid, a first cationic lipid and a second cationic lipid is provided, and the solution is sequentially subjected to solvent removal, drying, hydration and ultrasonication to obtain cationic liposomes.

6. The preparation method according to claim 5, characterized in that: The solvent includes chloroform and / or a mixture of dichloromethane and methanol.

7. The preparation method according to claim 5, characterized in that: The drying comprises: vacuum drying for 1.5-2.5 hours.

8. The preparation method according to claim 5, characterized in that: The hydration comprises: using RNAse-free ultrapure water to shake at 35-39° C. for 8-12 minutes, and the concentration of the cationic liposomes after hydration is 2.5-3.5 mg / ml.

9. The preparation method according to claim 5, characterized in that: The ultrasound comprises: ultrasound at 38-42° C. for 35-45 minutes.

10. A cationic liposome and mRNA complex preparation, characterized in that: The method comprises a working solution, mRNA and the cationic liposome according to claim 1.

11. The complex preparation according to claim 10, characterized in that The working solution includes deionized water, ultrapure water, sodium chloride solution, PBS solution or PBS solution containing 5-40 μg / ml transferrin.

12. The composite preparation according to claim 10, characterized in that The sequence of the mRNA is shown in SEQ ID NO.

1.

13. The complex preparation according to claim 12, characterized in that The mRNA concentration is 0.01-0.5 mg / ml.

14. The complex preparation according to claim 13, characterized in that The mass ratio of cationic liposomes to mRNA is 1:1-8:

1.

15. The composite preparation according to claim 14, characterized in that In the in vivo delivery application, the working solution is a PBS solution, and the mass ratio of cationic liposomes to mRNA is 1:1-2:

1.

16. The complex preparation according to claim 14, characterized in that In in vitro cell transfection, the working solution is a PBS solution containing 5-40 μg / ml transferrin, and the mass ratio of cationic liposomes to mRNA is 2:1-8:

1.

17. The method for preparing the complex preparation of cationic liposome and mRNA according to any one of claims 10 to 16, characterized in that: The mRNA and cationic liposomes are mixed in a working solution to obtain a complex preparation of cationic liposomes and mRNA.

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