CN110638759A

CN110638759A - A preparation for in vitro transfection and in vivo mRNA delivery

Info

Publication number: CN110638759A
Application number: CN201911041368.1A
Authority: CN
Inventors: 向晟楠; 苏晓晔; 彭育才; 刘隽; 李爽; 刘琪; 雷奕欣
Original assignee: Zhuhai Lifanda Biotechnology Co Ltd
Current assignee: Zhuhai Lifanda Biotechnology Co Ltd
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2020-01-03
Also published as: CN115778904A

Abstract

The invention relates to the field of mRNA delivery, and particularly provides a preparation for in vitro transfection and in vivo delivery of mRNA. The raw materials of the cationic liposome provided by the invention comprise neutral helper lipid, first cationic lipid and second cationic lipid, wherein the second cationic lipid comprises Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA or c 12-200. The cationic liposome is specially obtained by research and development of mRNA, has better transfection effect in-vitro mRNA cell transfection, and has lower toxicity compared with the commercial cationic liposome reagent on the market; has better immune effect in mRNA in-vivo delivery, and can be applied to the field of infectious disease vaccines. When in use, the gene can be directly matched with a specific mRNA sequence to flexibly meet different application requirements.

Description

A preparation for in vitro transfection and in vivo mRNA delivery

Technical Field

The invention relates to the field of mRNA delivery, in particular to a preparation for in vitro transfection and in vivo delivery of mRNA.

Background

Cationic liposomes are widely used for cellular and in vivo delivery of nucleic acids. Compared with other existing delivery technologies such as nano microspheres, lipid nanoparticles and the like, the cationic liposome has the advantages of simple preparation process, deeper research, capability of being matched with different types of nucleic acids at present, and flexible application scene, and is particularly suitable for the development of in-vivo and in-vitro nucleic acid transfection reagents. At present, part of nucleic acid drugs are developed by adopting a cationic liposome technology.

Cationic liposomes are generally composed of a cationic lipid and one or more helper lipids. So-called "liposome complexes" can be formed from cationic liposomes and anionic nucleic acids.

On one hand, compared with other nanoparticle preparations, the cationic liposome is more suitable for in vitro cell transfection, and the in vivo nucleic acid delivery effect is poor, so that the low in vivo nucleic acid delivery efficiency of the cationic liposome limits the application of the cationic liposome in the fields of in vivo transfection reagent development and drug development. The development of a new method for improving the in vivo nucleic acid delivery effect of the cationic liposome can improve the application prospect in the field.

On the other hand, the development of mRNA drugs is one of the current hot areas, however, no cationic liposome reagent specially used for in vivo mRNA delivery is available in the market at present, and the cytotoxicity of mature transfection reagents such as lipofectamine is high in the aspect of in vitro transfection application. There is a lack of low toxicity and high efficacy products. In the research and development of mRNA drugs, the existing cationic liposome technology is only applied to the mRNA immunotherapy of cancer, the cancer immunotherapy mainly relates to a cellular immune pathway, killing immune cells need to be activated to eliminate tumors, and the immunity of infectious diseases needs to generate corresponding antibodies through a humoral immune pathway to generate immune protection to organisms, and the two immune mechanisms are different, and the product effectiveness and requirements are different. Therefore, cationic liposome delivery systems remain blank for infectious disease vaccine applications.

Therefore, a new cationic liposome needs to be developed to meet the requirements of mRNA in vitro cell transfection on toxicity and effectiveness, realize efficient delivery of mRNA in vivo and open up the application of the cationic liposome in mRNA infectious disease vaccines.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a cationic liposome to solve the technical problems of low RNA delivery efficiency, high toxicity and narrow application field of the cationic liposome in the prior art.

The second purpose of the invention is to provide the application of the cationic liposome.

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

The fourth purpose of the present invention is to provide a cationic liposome and mRNA complex preparation, so as to alleviate the technical problems of low in vivo and in vitro transfer efficiency, high toxicity and narrow application range of the cationic liposome and mRNA complex preparation in the prior art.

The fifth object of the present invention is to provide a method for preparing the above cationic liposome/RNA complex preparation.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a cationic liposome comprises neutral helper lipid, first cationic lipid and second cationic lipid;

the second cationic lipid comprises Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA or c 12-200.

Further, the second cationic lipid is Dlin-MC 3-DMA;

preferably, the neutral helper lipid comprises DOPE, DOPC or cholestrol;

preferably, the neutral helper lipid is DOPE.

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

preferably, the first cationic lipid is DOTAP.

Further, 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, the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5): (0.5-3).

Use of cationic liposomes in any one of the following a) to c):

a) mRNA in vitro cell transfection;

b) preparing an mRNA in vivo delivery agent;

c) preparing an mRNA vaccine;

preferably, the mRNA vaccine comprises an mRNA tumor vaccine or an mRNA infectious disease vaccine, preferably an mRNA infectious disease vaccine;

preferably, the in vivo delivery of mRNA is local in vivo delivery of mRNA.

A method for preparing cationic liposome comprises providing a solution containing neutral helper lipid, a first cationic lipid and a second cationic lipid, and sequentially removing solvent, drying, hydrating and ultrasonic treating to obtain cationic liposome.

Further, 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, the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5): (0.5-3);

preferably, the solvent comprises chloroform and/or a mixture of dichloromethane and methanol;

preferably, the drying comprises: vacuum drying for 1.5-2.5 hr;

preferably, the hydrating comprises: shaking ultrapure water without RNase at 35-39 deg.C for 8-12 min, wherein the concentration of the hydrated cationic liposome is 2.5-3.5 mg/ml;

preferably, the ultrasound comprises: ultrasonic treating at 38-42 deg.c for 35-45 min.

A composite preparation of cationic liposome and mRNA comprises working solution, mRNA and cationic liposome.

Further, the working solution comprises deionized water, ultrapure water, a sodium chloride solution, a PBS solution or a PBS solution containing 5-40 mug/ml transferrin;

preferably, the sequence of the mRNA is shown as SEQ ID NO. 1;

preferably, the mRNA concentration is 0.01-0.5 mg/ml;

preferably, the mass ratio of the cationic liposome to the mRNA is 1:1-8: 1;

preferably, in the aspect of in vivo delivery of mRNA, the working solution is a PBS solution, and the mass ratio of the cationic liposome to the mRNA is 1:1-2: 1;

preferably, in the aspect of mRNA in vitro cell transfection, the working solution is a PBS solution containing 5-40 μ g/ml transferrin, and the mass ratio of the cationic liposome to the mRNA is 2:1-8: 1.

The preparation method of the cationic liposome and mRNA compound preparation comprises the step of mixing mRNA and cationic liposome in a working solution to obtain the cationic liposome and mRNA compound preparation.

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

the raw materials of the cationic liposome provided by the invention comprise neutral helper lipid, first cationic lipid and second cationic lipid. Traditional cationic liposomes comprise a cationic lipid and a neutral lipid, the cationic lipid can be combined with negatively charged nucleic acid to form nanoparticles and help the nucleic acid enter cells, and part of the neutral lipid can assist fusion between lipid membranes and cell membranes in addition to the function of the constitutive structure to further help the release of the nucleic acid, however, the research finds that the utility is limited. Protonatable lipids have been shown to aid intracellular delivery of nucleic acids and their escape from endosomes. The design of the invention is that cationic lipid is used as a main component, first cationic lipid with positive charge is used as a main chain to combine and agglomerate mRNA to form nano particles, second cationic lipid is added to help the endocytosis of the particles and the escape of endosome, neutral lipid is used to adjust charge and assist the release of nucleic acid, and the three components exert synergistic effect and simultaneously meet the requirements of mRNA encapsulation, endocytosis of cells, escape and release of endosome. For a transfection reagent, effectiveness and safety are two central principles for its value evaluation. The cationic liposome is specially developed and designed aiming at mRNA, has excellent transfection effect in-vitro cell transfection, and has lower toxicity compared with the commercial reagent with complex components on the market because the three used lipid components are all used in the design of other nucleic acid medicaments and the safety of the lipid components is verified in clinical tests; the cationic liposome has excellent delivery and immunization effects in vivo delivery, can be applied to the field of infectious disease vaccines, and fills the technical blank in the field in the prior art. In addition, the prepared cationic liposome can be mixed with specific mRNA before administration according to actual application requirements, and other operations such as preparing nanoparticles by compounding with the mRNA in advance are not needed, so that the application of the cationic liposome is more flexible.

The preparation method of the cationic liposome provided by the invention is simple and easy to operate, has low cost and does not need complex professional equipment.

The composite preparation of cationic liposome and mRNA provided by the invention comprises working solution, mRNA and cationic liposome. The cationic liposome mRNA compound has excellent transfer rate and low cytotoxicity compared with the market products, can achieve the immune effect in a short time without any adjuvant, particularly has good effect on infectious disease treatment, and fills up the technical blank in the field in the prior art.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of the particle size Tf4 on dynamic light scattering in example 8;

FIG. 2 is a graph showing the results of particle size of cationic liposome mRNA complexes by dynamic light scattering in example 9;

FIG. 3 is a graph showing the effect of different mass ratios of Tf4 and mRNA on transfection efficiency of cells in vitro in example 10;

FIG. 4 shows the transfection efficiency of Tf4 into HEK293 cells in vitro in example 11;

FIG. 5 shows the transfection efficiency of Tf4 into DC2.4 cells in vitro in example 11;

FIG. 6 shows in vitro HEK293 cell transfection toxicity of Tf4 in example 12;

FIG. 7 shows the transfection efficiency of HEK293 cells in vitro with different buffer compositions of Tf4 in example 13;

FIG. 8 is an evaluation of the fluorescence in vivo imaging system 2 hours after the intramuscular administration of the legs of the mouse in example 14;

FIG. 9 shows ELISA raw readings of rabies neutralizing antibody in mice 14 days (single dose) after intramuscular injection of different transfection reagents encapsulating rabies G protein mRNA (5 μ G) in mice legs in example 15;

fig. 10 is an immune cycle in vivo of Tf 4-delivered rabies G protein-encoding mRNA vaccine in example 16.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

A cationic liposome comprises neutral helper lipid, first cationic lipid and second cationic lipid, wherein the second cationic lipid comprises Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA or c 12-200.

For a transfection reagent, effectiveness and safety are two central principles for its value evaluation. The cationic liposome is specially developed and designed aiming at mRNA, has excellent transfection effect in-vitro cell transfection, and has lower toxicity compared with the commercial reagent with complex components on the market because the three used lipid components are all used in the design of other nucleic acid medicaments and the safety of the lipid components is verified in clinical tests; the cationic liposome has excellent immune effect in-vivo delivery, can be applied to the field of infectious disease vaccines, and fills up the technical blank in the field in the prior art. In addition, the prepared cationic liposome can be mixed with specific mRNA before administration according to actual application requirements, and other operations such as preparing nanoparticles by compounding with the mRNA in advance are not needed, so that the application of the cationic liposome is more flexible.

It is noted that the neutral helper 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 helper lipid may be DOPE, DOPC, etc., and the first cationic lipid may be DOTAP, DOTMA, etc. The CAS number of the Dlin-MC3-DMA is 1224606-06-7, the CAS number of the Dlin-KC2-DMA is 1224606-06-7, the DODMA is 1, 2-Dioleyloxy-3-dimethyllaminopropane is 1, 2-dioloxy-3-dimethylaminopropane, and the C12-200 is 1,1- ((2- (4- (2- ((2-hydroxydocyclyl) amino) ethyl) (2-hydroxydocyclo) amino) ethyl) piperazin-1-yl) ethyl) azanediyl bis (docandiol-2-ol), and the CAS number is 1220890-25-4.

In a preferred embodiment, the second cationic lipid is dilin-MC 3-DMA and the second cationic lipid is a protonatable cationic lipid, which can help mRNA release from the cationic lipid complex in the cell and escape from the endosome into the cytoplasm, so that the addition of the second cationic lipid can significantly improve the delivery efficiency of the cationic liposome, especially the better effect of dilin-MC 3-DMA.

In a preferred embodiment, the neutral helper lipid comprises DOPE, DOPC or cholestrol, further preferably DOPE. Neutral lipids are an important component of cationic liposomes, and some neutral lipids can mediate the fusion process of cationic lipid complexes and cell membranes due to their special structures, thereby helping mRNA enter cells. It should be noted that DOPE is named 1, 2-dioleoyl-sn-glycerol-3-phosphoethanomine as dioleoyl phosphatidylethanolamine, DOPC is named 1, 2-dioleoyl-sn-glycerol-3-phosphocholeline as dioleoyl lecithin, and cholestrol is cholesterol.

In a preferred embodiment, the first cationic lipid comprises DOTAP, DOTMA or DOSPA, further preferably DOTAP. The first cationic lipid is non-protonatable cationic lipid, and usually shows stronger positive charge, which can ensure that the cationic liposome can be effectively combined with mRNA molecules with negative charge under most pH and buffer environments, and the mRNA is wrapped and formed into a nano-complex, so that the delivery of the mRNA in vitro cells and in vivo is mediated. It should be noted that DOTAP is named 1, 2-diolyl-3-trimethylammoniumnium-propane as 2-Dioleoyl hydroxypropyl-3-N, N, N-trimethylammonium, DOTMA is N- [1- (2, 3-Dioleoyl) propyl ] -N, N, N-trimethylammoniumchloride is named N- [1- (2, 3-Dioleoyl) propyl ] -N, N, N-trimethylammonium chloride, and DOSPA is named 2, 3-Dioleoyl-N- [2 (ethylenecarboxylic amide) ethyl ] -N, N-dimethylolnitrile-1-trimethylammoniumacetate.

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, 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, the molar ratio of the first cationic lipid to the second cationic lipid is 8.9: 1.1. the molar ratio of neutral helper lipid to the sum of the first cationic lipid and the second cationic lipid 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 cationic lipid to the second cationic lipid 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.

The cationic liposome provided by the invention can be applied to any one of the following a) to c):

a) mRNA in vitro cell transfection;

b) preparing an mRNA in vivo delivery agent;

c) preparing mRNA vaccine.

In a preferred embodiment, the mRNA vaccine comprises an mRNA tumor vaccine or an mRNA infectious disease vaccine, preferably an mRNA infectious disease vaccine. Experiments show that the cationic liposome provided by the invention has obvious effect in preparing infectious disease vaccines.

In preferred embodiments, the in vivo delivery of mRNA is local in vivo delivery of mRNA. Local delivery of mRNA in vivo can be intramuscular injection or subcutaneous injection, and the like.

The invention provides a preparation method of the cationic liposome, which comprises the steps of providing a solution containing neutral helper lipid, first cationic lipid and second cationic lipid, and sequentially removing a solvent from the solution, drying, hydrating and carrying out ultrasonic treatment to obtain the cationic liposome. The method is simple and easy to operate, has low cost and does not need complex professional equipment.

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, the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5): (0.5-3).

In a preferred embodiment, the solvent comprises chloroform and/or a mixture of dichloromethane and methanol. The solvent may be chloroform methanol solution, dichloromethane methanol solution, or chloroform dichloromethane methanol solution, and the solvent is removed by rotary evaporation.

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

In a preferred embodiment, hydrating comprises: shaking the mixture at 35-39 deg.C for 8-12 min with RNase-free ultrapure water, and hydrating to obtain cationic liposome with concentration of 2.5-3.5 mg/ml. The hydration temperature is typically, but not limited to, 35 ℃, 36 ℃, 37 ℃, 38 ℃ or 39 ℃; hydration times are 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, 2.5mg/ml, 3mg/ml or 3.5 mg/ml.

In a preferred embodiment, the ultrasound comprises: ultrasonic treating at 38-42 deg.c for 35-45 min. The ultrasound temperature is typically, but not limited to, 38 ℃, 39 ℃, 40 ℃, 41 ℃ or 42 ℃; sonication times are typically, but not limited to, 35 minutes, 38 minutes, 40 minutes, 42 minutes, or 45 minutes.

A composite preparation of cationic liposome and mRNA comprises working solution, mRNA and the cationic liposome. The cationic liposome RNA complex has excellent transfer rate and low cytotoxicity compared with the market products, can achieve the immune effect without any adjuvant, particularly has good effect on infectious disease treatment, and fills the technical blank in the field in the prior art. The working solution is a solution used in vivo or in vitro of nucleic acid, and may be ultrapure water, a sodium chloride solution, a PBS solution, or the like.

In preferred embodiments, the working solution comprises deionized water, ultrapure water, sodium chloride solution, PBS solution, or PBS solution containing 5-40 μ g/ml transferrin. The working solution of the present invention may be any of the above solutions, although the delivery rates of each solution are different, and preferably different solutions for different applications, e.g., for in vivo RNA delivery, the working solution is preferably a PBS solution; for RNA in vitro cell transfection, the working solution is preferably PBS solution containing 5-40. mu.g/ml transferrin.

In a preferred embodiment, the mRNA has the sequence shown in SEQ ID NO. 1. mRNA in rabies vaccines used in the prior art is designed aiming at Pasteur strains, an inventor designs aiming at a special rabies virus strain CTN-1 in China (the homology between the CTN-1 and the Pasteur strains is only 60-70%), RVG-DmRNA shown in SEQ ID NO.1 is obtained through research, and the sequence is optimized by nucleotide codons, so that the sequence is more suitable for expression in mammals, particularly human bodies. The sequence has higher pertinence to Chinese specific strains, and is more suitable for development and use in China. In addition, the complex preparation prepared from the RVG-D mRNA and the cationic liposome provided by the invention has higher neutralizing antibody positive rate and faster and longer immune protection effect when being used as a rabies vaccine.

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

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

The preparation method of the cationic liposome and mRNA compound preparation comprises the step of mixing mRNA and cationic liposome in working solution to obtain the cationic liposome and mRNA compound preparation. The method is simple and easy to operate, can be prepared on site in real time, and avoids complex operation and use of professional equipment.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1

A cationic liposome is prepared from the following raw materials: DOPC, in a molar ratio to the sum of DOTMA and Dlin-KC2-DMA of 9:9, the molar ratio of DOTMA to Dlin-KC2-DMA is 9.5: 0.5.

example 2

A cationic liposome is prepared from the following raw materials: DOPC, in a molar ratio to the sum of DOTMA and Dlin-KC2-DMA of 9:27, the molar ratio of DOTMA to Dlin-KC2-DMA is 7: 3.

example 3

A cationic liposome is prepared from the following raw materials: DOPC, in a molar ratio to the sum of DOTMA and Dlin-KC2-DMA of 9:20, the molar ratio of DOTMA to Dlin-KC2-DMA is 8.9: 1.1.

example 4

A cationic liposome is prepared from the following raw materials: DOPE, in a molar ratio to the sum of DOTAP and Dlin-MC3-DMA of 9:20, the molar ratio of DOTAP and Dlin-MC3-DMA was 8.9: 1.1.

example 5

A preparation method of cationic liposome comprises the following steps:

a) chloroform is used as an organic solvent, and the raw materials are prepared into an organic solution;

b) taking a proper amount of organic solution in a round-bottom flask, removing the organic solvent by using a rotary evaporation method, and preparing a dried lipid membrane;

c. the lipid film in the round-bottom flask was dried for 2 hours using a vacuum drying apparatus.

d. Adding the RNase-free ultrapure water into a round-bottom flask to hydrate the liposome membrane, and oscillating and hydrating in water bath at 37 ℃ for 10 minutes to ensure that the concentration of the hydrated liposome is 3 mg/ml.

e. And (3) placing the hydrated liposome into a water bath ultrasonic instrument, and carrying out ultrasonic treatment for 40 minutes at 40 ℃ to obtain the cationic liposome aqueous suspension product.

Example 6 Complex formulation of cationic liposomes with mRNA-for in vitro cell transfection

A method for preparing a cationic liposome and mRNA compound preparation comprises the following steps:

a. an appropriate amount of mRNA solution was diluted in PBS solution at a concentration of 0.1 mg/ml.

b. And taking a proper amount of cationic liposome suspension to dilute the cationic liposome suspension in a PBS solution containing 10 mu g/ml transferrin, wherein the mass ratio of the cationic liposome suspension to the mRNA is controlled to be 4: 1.

c. Mixing the two solutions by using a pipette gun, lightly blowing for 5 times, and standing for 15 minutes to obtain the cationic liposome and mRNA composite preparation.

Example 7 cationic liposome and mRNA complex formulation-method for preparing a cationic liposome and mRNA complex formulation for RNA delivery in vivo, comprising the steps of:

b. And (3) taking a proper amount of the cationic liposome suspension to dilute the cationic liposome suspension in a PBS solution, and controlling the mass ratio of the cationic liposome suspension to the mRNA to be 2: 1.

Example 8

The cationic liposome, named Tf4, was prepared by following the cationic liposome preparation method of example 5 with the starting material of example 4.

The prepared Tf4 preparation was immediately tested for relevant physicochemical index (Malverzetasizer nanoZS) using a dynamic light scattering nanometer particle size analyzer. During detection, the Tf4 preparation is diluted by 5 times by using ultrapure water and transferred to a sample tank for detection. The sample cell was inserted into the instrument and the sample particle size was measured and the sample average particle size and the monodisperse coefficient (PDI) were recorded. The results show that the Tf4 particle size is shown in FIG. 1, and it can be seen that cationic liposomes with a size of 122nm are obtained, with a monodispersion coefficient of 0.275. The particle size of the sample is continuously detected for multiple times and is about 120-140nm, and the monodispersion coefficient is less than 0.3. The prepared Tf4 was stable in a 4 degree refrigerator for at least 3 months.

Example 9

Tf4 in example 8 a cationic liposome-luciferase gene mRNA complex preparation was prepared according to the method of example 7, and the relevant physicochemical index (malverzetasizer nanoZS) was immediately examined using a dynamic light scattering nano-particle size analyzer. During detection, the compound preparation is diluted by ultrapure water and transferred into a sample tank to be detected, and the concentration of the nucleic acid in the sample tank is ensured to be about 1-2 mug/ml. The sample cell was inserted into the instrument and the sample particle size was measured and the sample average particle size and the monodisperse coefficient (PDI) were recorded. The result shows that the Tf4 particle size is shown in figure 2, and the Tf4 forms a monodisperse coefficient with the average particle size of 207nm of 0.197 after being combined with the mRNA molecule of the luciferase gene. The results of several consecutive experiments show that the particle size of the composite is slightly about 200nm and less than 250nm, and the monodispersion coefficient is less than 0.3, usually less than 0.2. The obtained preparation can be stably stored for about 1 month.

Example 10 optimum formulation ratio

The physicochemical differences between different mRNA molecules were minimal, so we used mRNA encoding the luciferase gene (trilink) as a reporter gene to reflect the efficiency of Tf4 in vitro cell and in vivo mRNA delivery. To optimize the mass ratio of Tf4 for optimal complexing with mRNA, Tf4 was mixed with mRNA molecules encoding the luciferase gene at different mass ratios for in vitro HEK293 cell transfection experiments.

HEK293 cells recovered from liquid nitrogen were cultured for one generation and then ready for use. HEK293 cells 24 hours prior to the experiment were seeded into 96-well plates at a cell density of 10000-. The cell state is observed after the culture in the incubator at 37 ℃ for 24 hours, and the experiment can be started when the cell confluence reaches about 80-90%. The preparation of the nano-complexes of Tf4 and mRNA encoding a luciferase gene (trilink) (prepared according to different mass ratios) was performed according to the method provided in example 6, wherein equal amounts of luciferase mRNA (trilink) were used as a negative control group, and the vehicle without mRNA and Tf4 was used as a blank group, which was directly added to the culture solution of cells in a 96-well plate after all reagents were prepared, ensuring that 0.1 μ g of mRNA was added to each well, and the same amount of vehicle was added to the blank group. The cells were then incubated at 37 degrees for 24 hours and luciferase expression was measured.

The luciferase expression level was measured by using a color development kit (see Bright-Glo, available from Promega)^TMThe Luciferase Assay System kit Manual of operation performs the experiments: cell supernatants from 96-well plates were aspirated and hydrated luciferase substrate was added directly to each well for development for 2 minutes, with 100 microliters of substrate per well. The fluorescence value can then be read using a microplate reader. And (4) opening the multifunctional microplate reader, selecting a chemiluminescence mode, reading the plate, recording the reading, and performing comparative analysis. The data analysis adopts a normalization method: and (4) setting the reading of the control group as 1, and dividing the reading of the experimental group by the reading of the control group to obtain the change multiple of the luciferase expression quantity of the experimental group relative to the reading of the control group for result statistics.

The results of the fluorescence readings 24h after transfection of luciferase mRNA are shown in FIG. 3 (Y axis: amount of luciferase gene expression (fold change) in cells), and when the two are combined, the mass ratio is found to have a large influence on the transfection efficiency of cells, and the optimal mass ratio of Tf4 to mRNA is about 4: 1. The ratio is the optimized preparation ratio and is used for subsequent in vitro cell transfection experiments.

Also, the optimal mass ratio of Tf4 to mRNA for in vivo delivery of mRNA was found to be 2:1 by experiment.

Example 11 in vitro cell transfection efficiency

To compare the advantages of Tf4 over the prior art in cell transfection (24 hours), we compared the transfection efficiency of several transfection reagents on HEK293 cells and DC2.4 cells, respectively. The experiment also used encoded luciferase as a reporter gene. It is contemplated herein to select prior art DOTMA/DOPE and DOTAP/DOPE liposomes, as well as two commercial transfection reagents: lipofectamine2000 and Lipofectamine messengermax were used as positive controls.

The specific experimental procedure was in accordance with the procedure provided in example 10. Briefly, the complex of Tf4 and mRNA was prepared in a mass ratio of 4:1 by seeding the KEK293 cells and DC2.4 cells, respectively, into 96-well plates for 24 hours. The components and the proportion of DOTMA/DOPE and DOTAP/DOPE preparations adopt the optimal scheme found in the preliminary experiment. Lipofectamine2000 and Lipofectamine messengermax were both operated with reference to the relevant instructions, and the optimal ratio (v/w) to mRNA was determined using the optimal protocol determined in the preliminary experiments. After all preparations were added to the cells (0.1. mu.g/well of mRNA) and incubated at 37 ℃ for 24 hours, they were examined.

The results for HEK293 cells are shown in FIG. 4, and for DC2.4 cells in FIG. 5. The results show that in 24-hour transfection experiments, compared to prior art DOTAP/DOPE, DOTMA/DOPE cationic liposomes, and commercial transfection reagent lipofectamine2000, Tf4 have higher capacity to transfect cells, and the results were consistent in both HEK293 cells and DC2.4 cells.

Example 12 in vitro cell transfection toxicity

Safety is another key consideration in developing cationic liposome reagents, and we compared Tf4 with the toxicity of each commercial reagent on HEK293 cells.

HEK293 cells were seeded 24 hours in 96-well plates, the number of cells in each well was kept consistent during plating, and 5 duplicate wells were set for statistics per set of experiments. Referring to example 11, each preparation was prepared and added to the cells to continue the culture for 24 hours, after which the cells in the wells were digested with trypsin, the number of living cells remaining after transfection for 24 hours for each group of reagents was counted by the taloflue staining method and compared, and as a result, as shown in fig. 6, no significant effect was observed on the cell activity by the transfection of Tf4, DOTAP/DOPE and DOTMA/DOPE, and a decrease in the number of living cells of 10 to 20% was observed in the cells transfected with lipofectamine series products, relative to the control group and the naked mRNA transfection group. The Tf4 shows that compared with lipofectamine series products, the product has smaller influence on cell viability, and is more suitable for application of in vitro cell transfection and observation and research of long-term gene expression.

EXAMPLE 13 in vitro cell transfection working solution optimization

Considering that the vehicle and buffer system of transfection reagents may have a great influence on their cell transfection effect, we optimized the buffer composition specifically for Tf 4. Referring to the prior art, pure water, sodium chloride injection and PBS are selected, and transferrin is added into the PBS to evaluate the influence of several buffer systems and solvents on the Tf4 transfection efficiency. Luciferase was also selected as a reporter in this experiment.

Reference example 11 a nano-complex of Tf4 and mRNA encoding a luciferase gene was prepared. During the preparation process, Tf4 and mRNA were diluted in any of the above buffers and mixed, and the resulting preparation was directly added to HEK293 cells in a 96-well plate, and luciferase expression was measured after 24 hours of culture, with the results shown in fig. 7. It can be seen that the transfection efficiency of mRNA can be significantly improved by PBS in all the solvents and the buffer solution, and in addition, the transfection efficiency of mRNA cells can be further improved by adding 10 mug/ml of human transferrin (sigma) into PBS. Therefore transferrin-containing PBS is the optimal buffer system for Tf4 formulations in vitro cell transfection applications.

Example 14 in vivo delivery efficiency

Luciferase gene was used as a reporter gene. The application prospect of Tf4 in-vivo mRNA delivery is researched by using a small animal fluorescence in-vivo imaging technology.

Referring to the preparation process of example 7, a Tf4+ PBS formulation was used to deliver reporter mRNA to mice, while a formulation containing 5 μ g luciferase mRNA was introduced using tail vein administration and leg intramuscular injection. The administration mice were injected with luciferase substrate 2 hours later, and after 10 minutes, the mice were anesthetized and the expression of luciferase in the mice was observed by a fluorescence live imaging system. It was found that it was difficult to express mRNA in mice by intravenous injection, probably because Tf4 was more granular in complex with mRNA and was thus easily cleared by the immune system. However, in mice administered to the leg muscle, we detected significant gene expression, and the results are shown in FIG. 8. Therefore, the preparation is suitable for the application of in vivo mRNA local delivery, and in addition, the compound of Tf4 and mRNA is presumed to have certain immunogenicity, so that the preparation is suitable for the application and development of vaccine products.

Example 15 in vivo immunization effect of infectious disease vaccine

From the results in example 14, it is conceivable that the Tf4 preparation is suitable for the development of vaccine-based products. In particular infectious disease vaccines, such as rabies vaccines and the like. The existing rabies vaccines are all required to realize a quick and lasting immune effect, and in the early research, the prior art is found to be difficult to meet the above targets, so the rabies virus mRNA vaccine is selected as a practical application to evaluate the difference between the Tf4 provided by the invention and the existing cationic liposome technology.

In the prior art, DOTMA and DOTAP cationic liposome have been applied to mRNA tumor immunotherapy, and in addition, protamine has been used as a drug carrier in the prior art for the development of rabies virus vaccines, so the positive control preparations are considered to be selected to be compared with the delivery effect of the in vivo rabies G protein mRNA of Tf4 provided by the invention.

In the aspect of mRNA sequences, mRNA sequence coding regions are designed aiming at Pasteur strains in the prior art, while the sequence coding regions are designed aiming at rabies virus strain CTN-1 which is specific to China, and the homology between the two is only 60-70%. In addition, the nucleotide encoding factor is optimized to be more suitable for being expressed in mammals, particularly in human bodies. Therefore, compared with the prior art, the sequence used by the Chinese has higher pertinence to Chinese specific strains, and is more suitable for development and use in China.

Furthermore the mRNA sequence additionally comprises:

(a) a 5' -cap structure;

(b) a poly A sequence, preferably 60 to 120A, more preferably 80 to 100A;

(c) a 5 'UTR of 10-200 nucleotides in length, preferably 15-100 nucleotides in length, preferably comprising DNAH 25' UTR, KOZAK sequences;

(d) a 3 'UTR, preferably comprising the 3' UTR sequence of hemoglobin HBA 2;

the above sequence, designated RVG-D mRNA, is of length: 1843bp, the specific sequence is as follows:

GGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGGUACCGAGCUCGGAUCCACUAGUCCAGUGUGGUGGAAUUCGGGAGAAAGCUUACCAUGAUUCCUCAGGCUCUGCUGUUCGUCCCACUGCUGGUCUUCCCACUGUGCUUCGGCAAGUUCCCCAUCUACACUAUUCCUGACAAACUGGGCCCUUGGUCCCCAAUCGACAUCCACCACCUGUCCUGUCCCAACAAUCUGGUGGUGGAGGACGAGGGCUGUACCAAUCUGAGCGGCUUCUCCUACAUGGAGCUGAAGGUGGGCUACAUCUCCGCCAUCAAGGUGAACGGCUUCACAUGUACCGGCGUGGUGACAGAGGCCGAGACAUACACCAACUUCGUGGGCUAUGUGACCACAACCUUCAAGCGGAAGCACUUCAGACCAACCCCUGAUGCCUGUCGGUCCGCCUAUAACUGGAAGAUGGCCGGCGACCCACGGUAUGAGGAGAGCCUGCACAACCCAUACCCCGAUUACCACUGGCUGAGGACAGUGAAGACAACCAAGGAGUCCGUGGUAAUCAUCUCUCCAAGCGUGGCCGAUCUGGACCCAUACGAUAAGUCCCUGCACAGCAGAGUGUUCCCCCGCGGCAAGUGUUCUGGCAUCACAGUGAGCUCCGCCUAUUGCUCUACCAACCACGACUACACCAUCUGGAUGCCUGAGAAUCCAAGACUGGGCACCUCCUGUGACAUCUUUACCAACUCUCGGGGCAAGAGAGCCUCCAAGGGCUCCAAGACAUGUGGCUUCGUGGACGAGAGAGGCCUGUAUAAGUCCCUGAAGGGCGCCUGCAAGCUGAAGCUGUGCGGCGUGCUGGGCCUGAGGCUGAUGGACGGCACCUGGGUGGCCAUCCAGACAUCCAACGAGACCAAGUGGUGCCCUCCUGAUCAGCUGGUGAACCUGCACGACUUUCACAGCGACGAGAUCGAGCACCUGGUGGUGGAGGAGCUGGUGAAGAAGAGAGAGGAGUGCCUGGAUGCCCUGGAGUCCAUCAUGACCACCAAGAGCGUGUCCUUUAGACGGCUGAGCCACCUGAGAAAGCUGGUGCCCGGCUUUGGCAAGGCCUACACCAUCUUCAACAAGACACUGAUGGAGGCCGAUGCCCACUACAAGUCCGUGAGAACCUGGAACGAGAUCAUCCCUAGCAAGGGCUGUCUGAGGGUGGGCGGCAGAUGUCACCCUCACGUGAAUGGCGUGUUCUUUAAUGGCAUCAUCCUGGGCCCAGAUGGCCACGUGCUGAUCCCAGAGAUGCAGUCCUCUCUGCUGCAGCAGCACAUGGAGCUGCUGGAGUCUAGCGUGAUCCCUCUGAUGCACCCACUGGCCGAUCCAAGCACAGUGUUCAAGGACGGCGACGAGGUGGAGGACUUUGUGGAGGUGCACCUGCCAGAUGUGCACAAGCAGGUGUCCGGCGUGGAUCUGGGCCUGCCAAAUUGGGGCAAGGACGUGCUGAUGGGCGCCGGCGUGCUGACCGCCCUGAUGCUGAUGAUCUUCCUGAUGACCUGCUGUAGACGGACAAAUAGAGCCGAGAGCAUCCAGCACUCCCUGGGCGAGACAGGCCGGAAGGUGUCUGUGACCAGCCAGAGUGGACGAGUGAUCUCCUCAUGGGAAUCCUACAAAAGCGGCGGCGAGACCAAACUGUGAGGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCUCCUCCCCUCCUUGCACCGAGAUUAAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAU(SEQ ID NO.1)。

vaccine preparations in which DOTMA, DOTAP, Tf4 and protamine are respectively coated with RVG-D mRNA were prepared for mouse immunization experiments with reference to preliminary experiments and the method of example 14, and the mass ratio of Tf4 to mRNA was 2:1, and an equal volume of PBS was used as a blank. Mouse sera were taken at day 14 after a single administration of 5. mu.g (mRNA) to mouse leg muscles and assayed for rabies virus neutralizing antibodies by ELISA, according to standard veterinary vaccine evaluation. The ELISA kit adopted in the experiment is a whole virus coating kit, and can be used for comparing the immune effect of each group, but the neutralizing antibody titer cannot be accurately quantified. Therefore, the related samples are sent to a qualified third-party organization (southern China university of agriculture) to carry out neutralizing antibody detection, 0.16 is set as a judgment value according to the related results and blank group ELISA actual readings, and if the reading is higher than 0.16, the positive neutralizing antibody is judged, and the mice are immunized successfully. The results of ELISA raw reading (ELISA method) of rabies neutralizing antibody in mice are shown in FIG. 9, and the positive rate of neutralizing antibody in serum is shown in the following table:

DOTMA	DOTAP	Tf4	protamine	Blank group
					Positive rate
0％	30％	80％	0％	0％

The results show that under the same administration conditions, the existing DOTMA and DOTAP liposome delivery effects cannot stimulate the generation of sufficient rabies virus neutralizing antibodies. At present, the human rabies vaccine requires to generate neutralizing antibody after three times of continuous injections for 4 weeks, and the veterinary rabies vaccine generally requires that the neutralizing antibody positive rate can be 70-80% in 14 days after a single injection, so the Tf4 better meets the actual requirement on the current market than the prior art and has larger commercial development prospect.

Example 16 in vivo immune cycle of infectious disease vaccine

A Tf4 formulation encapsulating RVG-D mRNA (w/w 2) was prepared according to the method of example 15 with an equal volume of vehicle as a blank. Sera from mice were taken 2 weeks, 4 weeks, 12 weeks and 28 weeks after immunization of mice with 5 μ g of mRNA and tested for rabies virus neutralizing antibodies by ELISA. The results are shown in FIG. 10.

The results show that Tf4 formulation immunized mice read a significant increase from 2 weeks neutralization positive. Readings peaked by week 12 and at week 28 slightly declined but still well above the positive decision value. The Tf4 vaccine formulation is effective in stimulating at least half a year's immune protective effect in mice. Therefore, the Tf4 has a development prospect in the application aspect of rabies vaccines.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Zhuhai livan der Biotechnology Ltd

<120> a preparation for in vitro transfection and in vivo delivery of mRNA

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1843

<212> RNA

<213> Artificial sequence

<400> 1

gggagaccca agcuggcuag cguuuaaacu uaagcuuggu accgagcucg gauccacuag 60

uccagugugg uggaauucgg gagaaagcuu accaugauuc cucaggcucu gcuguucguc 120

ccacugcugg ucuucccacu gugcuucggc aaguucccca ucuacacuau uccugacaaa 180

cugggcccuu gguccccaau cgacauccac caccuguccu gucccaacaa ucugguggug 240

gaggacgagg gcuguaccaa ucugagcggc uucuccuaca uggagcugaa ggugggcuac 300

aucuccgcca ucaaggugaa cggcuucaca uguaccggcg uggugacaga ggccgagaca 360

uacaccaacu ucgugggcua ugugaccaca accuucaagc ggaagcacuu cagaccaacc 420

ccugaugccu gucgguccgc cuauaacugg aagauggccg gcgacccacg guaugaggag 480

agccugcaca acccauaccc cgauuaccac uggcugagga cagugaagac aaccaaggag 540

uccgugguaa ucaucucucc aagcguggcc gaucuggacc cauacgauaa gucccugcac 600

agcagagugu ucccccgcgg caaguguucu ggcaucacag ugagcuccgc cuauugcucu 660

accaaccacg acuacaccau cuggaugccu gagaauccaa gacugggcac cuccugugac 720

aucuuuacca acucucgggg caagagagcc uccaagggcu ccaagacaug uggcuucgug 780

gacgagagag gccuguauaa gucccugaag ggcgccugca agcugaagcu gugcggcgug 840

cugggccuga ggcugaugga cggcaccugg guggccaucc agacauccaa cgagaccaag 900

uggugcccuc cugaucagcu ggugaaccug cacgacuuuc acagcgacga gaucgagcac 960

cugguggugg aggagcuggu gaagaagaga gaggagugcc uggaugcccu ggaguccauc 1020

augaccacca agagcguguc cuuuagacgg cugagccacc ugagaaagcu ggugcccggc 1080

uuuggcaagg ccuacaccau cuucaacaag acacugaugg aggccgaugc ccacuacaag 1140

uccgugagaa ccuggaacga gaucaucccu agcaagggcu gucugagggu gggcggcaga 1200

ugucacccuc acgugaaugg cguguucuuu aauggcauca uccugggccc agauggccac 1260

gugcugaucc cagagaugca guccucucug cugcagcagc acauggagcu gcuggagucu 1320

agcgugaucc cucugaugca cccacuggcc gauccaagca caguguucaa ggacggcgac 1380

gagguggagg acuuugugga ggugcaccug ccagaugugc acaagcaggu guccggcgug 1440

gaucugggcc ugccaaauug gggcaaggac gugcugaugg gcgccggcgu gcugaccgcc 1500

cugaugcuga ugaucuuccu gaugaccugc uguagacgga caaauagagc cgagagcauc 1560

cagcacuccc ugggcgagac aggccggaag gugucuguga ccagccagag uggacgagug 1620

aucuccucau gggaauccua caaaagcggc ggcgagacca aacugugagg acuaguuaua 1680

agacugacua gcccgauggg ccucccaacg ggcccuccuc cccuccuugc accgagauua 1740

auaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1800

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aau 1843

Claims

1. A cationic liposome is characterized in that the raw material comprises neutral helper lipid, first cationic lipid and second cationic lipid;

2. The cationic liposome of claim 1, wherein the second cationic lipid is Dlin-MC 3-DMA;

preferably, the neutral helper lipid comprises DOPE, DOPC or cholestrol;

preferably, the neutral helper lipid is DOPE.

3. The cationic liposome of claim 1, wherein the first cationic lipid comprises DOTAP, DOTMA or DOSPA;

preferably, the first cationic lipid is DOTAP.

4. The cationic liposome of any one of claims 1-3, wherein 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, the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5): (0.5-3).

5. Use of the cationic liposome of any one of claims 1 to 4 in any one of the following a) to c):

a) mRNA in vitro cell transfection;

b) preparing an mRNA in vivo delivery agent;

c) preparing an mRNA vaccine;

preferably, the in vivo delivery of mRNA is local in vivo delivery of mRNA.

6. The method for producing cationic liposomes according to any one of claims 1 to 4, wherein a solution containing a neutral helper lipid, a first cationic lipid and a second cationic lipid is provided, and the solution is subjected to solvent removal, drying, hydration and sonication in this order to obtain cationic liposomes.

7. The method of claim 6, wherein 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, the molar ratio of the first cationic lipid to the second cationic lipid is (7-9.5): (0.5-3);

preferably, the drying comprises: vacuum drying for 1.5-2.5 hr;

8. A complex preparation of cationic liposome and mRNA, comprising a working solution, mRNA and the cationic liposome of any one of claims 1 to 4.

9. The complex preparation of cationic liposome and mRNA according to claim 8, wherein said working solution comprises deionized water, ultrapure water, sodium chloride solution, PBS solution or PBS solution containing transferrin at 5-40 μ g/ml;

preferably, the sequence of the mRNA is shown as SEQ ID NO. 1;

preferably, the mRNA concentration is 0.01-0.5 mg/ml;

preferably, the mass ratio of the cationic liposome to the mRNA is 1:1-8: 1;

preferably, the working solution in the in vivo delivery application is a PBS solution, and the mass ratio of the cationic liposome to the mRNA is 1:1-2: 1;

preferably, the working solution in the in vitro cell transfection is a PBS solution containing 5-40 mug/ml transferrin, and the mass ratio of the cationic liposome to the mRNA is 2:1-8: 1.

10. The method for producing a cationic liposome/mRNA complex preparation according to claim 8 or 9, wherein the mRNA and the cationic liposome are mixed in a working solution to obtain a cationic liposome/mRNA complex preparation.