mRNA expressing tyrosinase and use thereof

Abstract

A mRNA expressing tyrosinase and the use thereof. A coding sequence (CDS sequence) corresponding to the mRNA expressing tyrosinase is one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the half-life period of the mRNA expressing tyrosinase in a cell is 5-15 days. A lipid nanoparticle composition comprises lipid nanoparticles and the mRNA expressing tyrosinase. The cationic lipid in the lipid nanoparticle composition has lipophilicity, and thus can promote the fusion of the lipid nanoparticle composition and cell membranes, so as to deliver the mRNA expressing tyrosinase into cells; and in the cells, the mRNA expressing tyrosinase is released by means of ionization, and the mRNA expressing tyrosinase is translated and expressed by means of the cells of a living body, so as to synthesize tyrosinase and then generate melanin in the cells.

Description

Tyrosinase-expressing mRNA and its uses Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to mRNA expressing tyrosinase and its use. Background Art

[0002] Vitiligo is a relatively common acquired pigmentary skin disease characterized by localized or generalized complete loss of skin and mucous membrane pigmentation. The cause of vitiligo is not fully understood. It is believed that individuals with a genetic predisposition may be stimulated by a variety of internal and external factors, inducing immune dysfunction, neuropsychiatric disorders, and endocrine metabolic abnormalities, leading to inhibition of the tyrosinase system or destruction of melanocytes, ultimately causing skin depigmentation. Treatment options for vitiligo include hormone therapy, phototherapy and photochemotherapy, transplantation, immunosuppressants, and vitamin D3 derivatives.

[0003] The following are the current treatments for vitiligo:

[0004] 1. Hormone therapy

[0005] (1) Systemic treatment is mainly suitable for patients with generalized progressive vitiligo. Oral or intramuscular injection of hormones can quickly stabilize progressive vitiligo.

[0006] (2) Local treatment: Topical glucocorticoids are effective for the treatment of localized vitiligo, but attention should be paid to the adverse reactions caused by long-term use of topical glucocorticoids. Commonly used drugs in clinical practice include halometasone ointment, mometasone furoate cream, etc.

[0007] 2. Phototherapy and photochemotherapy

[0008] (1) Narrowband ultraviolet (NB-UVB) phototherapy has a certain effect in treating localized or generalized vitiligo.

[0009] (2) Photochemotherapy (PUVA) is a good treatment option for localized vitiligo by applying furanocoumarins (8-MOP, psoralea corylifolia tincture) topically and sun exposure. It can be used in adults and children over 5 years old.

[0010] 3. Transplantation therapy

[0011] It is suitable for patients in the stable stage, with a small number of skin lesions and a non-scarring physique. Common methods include autologous epidermal transplantation, microskin grafting, and autologous cultured melanocyte transplantation.

[0012] 4. Immunosuppressants

[0013] For areas that are not suitable for the use of hormones, or to avoid adverse reactions caused by long-term use of hormones, topical calcineurin inhibitors (tacrolimus, pimecrolimus) have a certain effect.

[0014] 5. Vitamin D3 derivatives

[0015] Vitamin D3 derivatives can be combined with NB-UVB, PUVA, etc. They can also be combined with topical hormones and calcineurin inhibitors.

[0016] CN 213374773 U discloses a portable vitiligo Chinese medicine light treatment machine, comprising a device body, a display screen, and a light treatment probe; a groove is provided on the side of the device body, and a clamp is fixedly installed in the groove; a plurality of ultraviolet lamps are arranged inside the light treatment probe, and a handle is connected to the bottom of the light treatment probe, and the handle is accommodated in the clamp inside the groove; a handle is installed at the center of the top of the device body, and a display screen and a plurality of buttons are provided on one side of the handle; a plurality of universal wheels are fixedly installed at the bottom of the device body; an MCU is integrated in the device body, and the MCU is electrically connected to the display screen, a step-down voltage regulator, and a control circuit respectively; a timing circuit is electrically connected to the plurality of ultraviolet lamps in the light treatment probe via wires; and the step-down voltage regulator is connected to a power supply. Although this technical solution solves the problems of existing vitiligo light treatment machines, such as being bulky, complex in structure, and high in cost, it still has the disadvantage of requiring the device to be carried in order to treat vitiligo, which still interferes with normal life activities.

[0017] Based on the above-mentioned existing technology, there are technical problems in the existing technology that need to be solved, such as the limitations of the use of traditional Chinese medicine light machine for vitiligo, the toxicity and side effects of existing drugs, and the inconvenience of carrying vitiligo light machine.

[0018] Summary of the Invention

[0019] In order to solve the above technical problems, the present invention provides an mRNA expressing tyrosinase, wherein the mRNA expressing tyrosinase acts on the site where vitiligo occurs and expresses melanin at the site where vitiligo occurs.

[0020] Furthermore, the coding sequence (CDS sequence) corresponding to the mRNA expressing tyrosinase is one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the half-life of the mRNA expressing tyrosinase in the cell is 5-15 days.

[0021] The present invention also provides a lipid nanoparticle composition, wherein the lipid nanoparticle composition (LNP-mRNA) comprises lipid nanoparticles (LNP) and the above-mentioned tyrosinase-expressing mRNA, wherein the tyrosinase-expressing mRNA is encapsulated inside the lipid nanoparticles, the lipid nanoparticle composition has a particle size of 50-150 nm, a shear plane potential (zeta potential) of -4.2932 to 0.0717 mV, an encapsulation efficiency (EE%) of 87.90-88.13% for the tyrosinase-expressing mRNA by the lipid nanoparticle composition, a polymer dispersibility index (PDI) of 0.088-0.113, and a lipid bilayer structure.

[0022] Among them, the closer the PDI is to 0, the more uniform the particle size distribution is;

[0023] Based on the total mass of the lipid nanoparticle composition, the mass percentage of the mRNA expressing tyrosinase in the lipid nanoparticle composition is 3-25%.

[0024] Furthermore, the lipid nanoparticles are composed of cationic lipids, auxiliary phospholipids, cholesterol and PEGylated phospholipids, and the cationic lipids promote the accumulation of mRNA expressing tyrosinase inside the lipid nanoparticles.

[0025] Furthermore, the particle size and shear potential (zeta potential) of the lipid nanoparticle composition were measured using a Zetasizer (Malvern, USA), and the encapsulation efficiency (EE%) was measured by RNA fluorescence (RiboGreen) assay (Invitrogen, USA).

[0026] Furthermore, the particle size of the lipid nanoparticle composition is 123.40-126.75 nm.

[0027] Furthermore, the molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEGylated phospholipid is (50-60):(10-15):(35-49):(1-3).

[0028] Furthermore, the molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEGylated phospholipid is 50:10:38.5:1.5.

[0029] Furthermore, the cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl lipid, abbreviated as DLin-MC3-DMA.

[0030] Furthermore, the auxiliary phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and the auxiliary phospholipid cooperates with cationic lipids, cholesterol and PEGylated phospholipids to support the lipid nanoparticle composition to form a lipid bilayer structure.

[0031] Furthermore, the cholesterol acts as a stabilizer to increase the stability of the lipid nanoparticle composition and prolong the half-life of the lipid nanoparticle composition.

[0032] Furthermore, the PEGylated phospholipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000) or polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine (DSPE-MPEG2000), which is used to extend the half-life of the lipid nanoparticle composition.

[0033] The present invention also provides a method for preparing the lipid nanoparticle composition, comprising the following steps:

[0034] S1. Screening of mRNA expressing tyrosinase;

[0035] S2. Encapsulating the tyrosinase-expressing mRNA screened in S1 into lipid nanoparticles to obtain a lipid nanoparticle composition.

[0036] Furthermore, the S1 specifically includes the following steps:

[0037] S1-1. Design coding sequences (CDS sequences, coding sequences) based on the target protein sequence of tyrosinase, wherein the coding sequence is one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;

[0038] S1-2, introducing the coding sequence in S1-1 into a plasmid synthesis device to synthesize a plasmid encoding the tyrosinase sequence;

[0039] S1-3, using T7 RNA polymerase as a medium to transcribe the linearized DNA in the S1-2 synthetic plasmid to produce crude mRNA expressing tyrosinase;

[0040] S1-4, the crude mRNA expressing tyrosinase is purified by phenol / chloroform to remove proteins and free nucleotides, and then subjected to a capping reaction to obtain N1-methylpseudouridine-modified mRNA;

[0041] S1-5, transfecting N1-methylpseudouridine-modified mRNA into human embryonic kidney cells (HEK293T cells) to obtain transfected human embryonic kidney cells;

[0042] S1-6. Perform protein detection on transfected human embryonic kidney cells using Western blot to screen out mRNA expressing tyrosinase with an activity ΔA of 0.4-0.439;

[0043] The screening principle is as follows: Tyrosinase (EC1.14.18.1) is a monophenol monooxygenase and a bifunctional copper-containing glycoprotein. Tyrosinase is a key enzyme in the synthesis of melanin in organisms and can catalyze L-dopa to produce dopachrome, which has a characteristic absorption peak at 475nm. The activity ΔA is determined as follows:

[0044] a. Preheat the spectrophotometer / microplate reader for 30 minutes, adjust the wavelength to 475 nm, and zero the spectrophotometer with distilled water.

[0045] b. Add the sample to the micro-cuvette / 96-well plate and quickly pipette to mix. Record the absorbance value A1 at 10 seconds. Immediately place the plate in a 25-37°C water bath or incubator for 3 minutes. Remove the plate, wipe it dry, and measure the absorbance value A2 at 3 minutes and 10 seconds. Calculate ΔA = A2 - A1.

[0046] S1-7. Perform quality control on the mRNA expressing tyrosinase after screening by S1-6 using electrophoresis test.

[0047] Furthermore, the equipment for synthesizing the plasmid encoding the tyrosinase sequence in S1-2 was purchased from GENEWIZ, named DNA synthesis instrument, model number MerMade 192.

[0048] Furthermore, the T7 RNA polymerase in S1-3 was purchased from Suzhou Nearshore Protein Technology Co., Ltd. and is included in the kit with item number E131. The transcription process is the same as the operating procedure of the kit with item number E131.

[0049] Furthermore, N1-methylpseudouridine in the S1-4 is a modified base, and the modified mRNA is composed of a 5' cap region (Cap1), a 5' untranslated region (5'UTR), a tyrosinase coding region, a 3' untranslated region (3'UTR) and a polyadenine tail (PolyA tail) starting from the 5' end, and the sequence of the tyrosinase coding region is the same as the coding sequence (CDS sequence).

[0050] Furthermore, the operation procedure of the capping reaction in S1-4 is the same as the operation procedure of the kit M082 produced by Suzhou Nearshore Protein Technology Co., Ltd.

[0051] Furthermore, in S1-5, Lipofectamine MessengerMax was used as a transfection reagent, purchased from Thermo Fisher Scientific, and HEK293T cells were transfected according to the transfection operating procedures of Thermo Fisher Scientific to obtain transfected HEK293T cells.

[0052] Furthermore, the electrophoresis test in S1-7 is performed by agarose gel electrophoresis, wherein the agarose gel contains 0.8 wt% agarose, and the electrophoresis time is 45 min. The loading amount of the tyrosinase molecular weight standard material is 3 μL, and the loading amount of the mRNA expressing tyrosinase is 80 ng.

[0053] When the electrophoresis pattern of the mRNA expressing tyrosinase is a single target sequence band, the quality control requirements are met and the mRNA can be used for lipid nanoparticle encapsulation in step S2.

[0054] Furthermore, the S2 lipid nanoparticle composition contains one mRNA expressing tyrosinase.

[0055] Furthermore, in S2, the specific steps of encapsulating the mRNA expressing tyrosinase in the lipid nanoparticles are:

[0056] S2-1, mixing a cationic lipid, an auxiliary phospholipid, cholesterol, and a PEGylated phospholipid in a microfluidic device to prepare lipid nanoparticles, and then dissolving the lipid nanoparticles in an organic solvent to obtain a mixture A having a lipid nanoparticle concentration of 25 mM;

[0057] S2-2, diluting the tyrosinase-expressing mRNA prepared in S1 to 0.35 mg / ml with a 10 mM citric acid buffer solution at pH 3 or a 100 mM sodium acetate solution at pH 4 to obtain a mixture B;

[0058] S2-3. Mixture A and mixture B were mixed using a microfluidic device to prepare a lipid nanoparticle composition (LNP-mRNA), and dialyzed in phosphate buffered saline (PBS).

[0059] Furthermore, the organic solvent in S2-1 is ethanol.

[0060] Furthermore, the parameters of the microfluidic devices in S2-1 and S2-3 are: total flow rate 12 ml / min, water phase / ethanol phase (volume ratio) = 3 / 1, and temperature is room temperature.

[0061] Furthermore, the microfluidic device was purchased from Precision NanoSystems, Vancouver, BC, Canada.

[0062] Furthermore, the dialysis cassette used in S2-3 was purchased from Beijing Novi Biotechnology Co., Ltd. and is named Slide-A-Lyzer TM , Dialysis Cassettes, 3.5K MWCO dialysis card.

[0063] The present invention also provides a vaccine comprising the above-mentioned mRNA expressing tyrosinase.

[0064] The present invention also provides a vaccine, which comprises the above-mentioned lipid nanoparticle composition. The vaccine is injected into a living body. The cationic lipid in the lipid nanoparticle composition has lipophilicity, which promotes the fusion of the lipid nanoparticle composition with the cell membrane, delivers mRNA expressing tyrosinase into the cell, releases the mRNA expressing tyrosinase through ionization in the cell, and utilizes the organism's own cells to translate the mRNA expressing tyrosinase, synthesizes tyrosinase in the cell, and produces melanin.

[0065] The beneficial effects of the present invention are:

[0066] 1. The mRNA expressing tyrosinase designed by the present invention has a half-life of 5-15 days in cells. It expresses the antigen only in the cytoplasm and does not enter the cell nucleus. This allows for more efficient expression of tyrosinase without the risk of DNA insertion causing gene mutations. Furthermore, the selected mRNA expressing tyrosinase can be expressed transiently and is naturally metabolized and eliminated by the organism, further enhancing safety.

[0067] 2. The lipid nanoparticle composition (LNP-mRNA) of the present invention is a lipid nanoparticle (LNP) encapsulating mRNA expressing tyrosinase. By optimizing the process parameters of the overall preparation process and designing the encoding sequence for encapsulation, the mass percentage of the mRNA expressing tyrosinase prepared by the present invention can be 3-25% of the lipid nanoparticle composition, which is higher than the drug loading ratio (5wt%) of the standard LNP encapsulation in the prior art. This allows the lipid nanoparticle composition of the present invention to produce more melanin, significantly improving efficiency.

[0068] 3. Since mRNA cannot enter cells through free diffusion and is easily degraded, and tyrosinase is a key enzyme for the synthesis of melanin in organisms, the present invention encapsulates the mRNA expressing tyrosinase inside lipid nanoparticles (LNPs), protecting the mRNA from degradation. This is safe and effective, and serves as a new delivery system for delivering the lipid nanoparticle composition into the organism. The lipid nanoparticle composition has lipophilicity, which promotes the fusion of the lipid nanoparticle composition with the cell membrane, delivers the encapsulated mRNA expressing tyrosinase into the cell, releases the mRNA expressing tyrosinase through ionization in the cell, and uses the organism's own cells to translate the mRNA, synthesizing tyrosinase in the cell to produce melanin.

[0069] 4. The vaccine provided by the present invention, which comprises the lipid nanoparticle composition, can deliver the mRNA expressing tyrosinase or the lipid nanoparticle composition encapsulating the mRNA expressing tyrosinase into the body by injection, thereby solving the technical problem that vitiligo illumination machines are inconvenient to carry. The production process of the vaccine can be standardized, reducing R&D costs, shortening the production cycle, and making production more flexible. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG1 is a comparison chart of ΔA data of the activity of tyrosinase mRNA expressed in Examples 1-3 of the present invention;

[0071] FIG2 is an electrophoretic pattern of mRNA expressing tyrosinase in Example 1 of the present invention;

[0072] FIG3 is a test diagram of particle size in Example 1 of the present invention;

[0073] FIG4 is a test diagram of potential in Example 1 of the present invention;

[0074] FIG5 is a graph showing changes in melanin production in mice after subcutaneous injection of the lipid nanoparticle composition in Example 4 of the present invention. DETAILED DESCRIPTION

[0075] Examples 1-3 are methods for preparing lipid nanoparticle compositions, and Example 4 is an experiment to verify whether the lipid nanoparticle composition prepared in Example 1 can produce melanin in vivo.

[0076] Example 1

[0077] The method for preparing a lipid nanoparticle composition of this embodiment comprises the following steps:

[0078] S1. Screening for tyrosinase-expressing mRNA:

[0079] S1-1. Design a coding sequence (CDS sequence) based on the target protein sequence of tyrosinase. In this embodiment, the CDS sequence is SEQ ID NO: 1.

[0080] S1-2, introducing the CDS sequence in S1-1 into a GENEWIZ DNA synthesizer (model: MerMade 192) to synthesize a plasmid encoding the tyrosinase sequence;

[0081] S1-3, using T7 RNA polymerase as a medium to transcribe the linearized DNA in the S1-2 synthetic plasmid to produce crude mRNA expressing tyrosinase. The T7 RNA polymerase was purchased from Suzhou Jinan Protein Technology Co., Ltd. and is included in the kit with catalog number E131. The transcription process is the same as the operating procedure of the kit with catalog number E131;

[0082] S1-4. Purify the crude mRNA expressing tyrosinase by phenol / chloroform to remove proteins and free nucleotides, and then perform a capping reaction to obtain N1-methylpseudouridine-modified mRNA. The capping reaction procedure is the same as the procedure of the kit M082 produced by Suzhou Jinan Protein Technology Co., Ltd. The N1-methylpseudouridine is a modified base, and the modified mRNA consists of a 5' cap region (Cap1), a 5' untranslated region (5'UTR), a tyrosinase coding region, a 3' untranslated region (3'UTR), and a polyadenine tail (PolyA tail) from the 5' end. The sequence of the tyrosinase coding region is the same as the coding sequence (CDS sequence);

[0083] Wherein, the specific steps of phenol / chloroform purification method are:

[0084] a. Dilute 20 μl of crude tyrosinase-expressing mRNA to 180 μl by adding 160 μl of RNase-free water to obtain a diluted mixture a.

[0085] b. Add 20 μl of 3 M sodium acetate (pH = 5.2) to the diluted mixture a and mix thoroughly with a pipette;

[0086] c. Add 200 μl of phenol / chloroform mixture (phenol:chloroform volume ratio is 1:1) for extraction to obtain mixture b. Centrifuge mixture b at room temperature and 10,000 rpm for 5 minutes. Transfer the upper solution (aqueous phase) to a new RNase-free EP tube (plastic centrifuge tube);

[0087] d. Add an equal volume of chloroform to the aqueous phase and extract again twice, collecting the upper aqueous phase after each extraction;

[0088] e. Add 2 volumes of anhydrous ethanol to the upper aqueous phase collected for the last time and mix well. Incubate at -20°C for 30 minutes to obtain a mixture c. Centrifuge the mixture c at 15,000 rpm for 15 minutes.

[0089] f. After discarding the supernatant, add 500 μl of 70% ethanol pre-cooled to below 4°C to wash the precipitate. Centrifuge again at 15,000 rpm at 4°C and discard the supernatant to obtain the crude tyrosinase mRNA product free of proteins and free nucleotides.

[0090] g. The crude mRNA expressing tyrosinase, after removing proteins and free nucleotides, was dried at room temperature for 2 min. After ethanol evaporated, 20 μl of RNase-free water was added to dissolve the precipitate to obtain mixture d.

[0091] h. Store the mixture d at -80°C;

[0092] S1-5, using Lipofectamine MessengerMax as a transfection reagent (the transfection reagent was purchased from Thermo Fisher Scientific), and transfecting HEK293T cells according to the transfection protocol of Thermo Fisher Scientific to obtain transfected HEK293T cells;

[0093] S1-6. Protein detection was performed on the transfected HEK293T cells using Western blot to screen for tyrosinase mRNA expression;

[0094] The screening principle is as follows: tyrosinase (EC1.14.18.1) is a monophenol monooxygenase and a bifunctional copper-containing glycoprotein. Tyrosinase is a key enzyme in the synthesis of melanin in organisms and can catalyze L-dopa to produce dopachrome. It has a characteristic absorption peak at 475 nm. Screening for mRNA expressing tyrosinase, as shown in Figure 1, in this example, the activity ΔA of the mRNA expressing tyrosinase is 0.439;

[0095] The activity ΔA is determined as follows:

[0096] a. Preheat the spectrophotometer / microplate reader for 30 minutes, adjust the wavelength to 475 nm, and zero the spectrophotometer with distilled water.

[0097] b. Add the sample to the micro-glass cuvette / 96-well plate and quickly pipette to mix thoroughly. Record the absorbance value A1 at 10 seconds. Immediately place the plate in a 37°C water bath or incubator for 3 minutes. Remove the plate, wipe it dry, and measure the absorbance value A2 at 3 minutes and 10 seconds. Calculate ΔA = A2 - A1.

[0098] S1-7. Perform quality control on the mRNA expressing tyrosinase after screening in S1-6 by electrophoresis test. The test method is agarose gel electrophoresis. The agarose gel contains 0.8 wt% agarose. The electrophoresis time is 45 min. The loading amount of tyrosinase molecular weight standard substance (numbered: Ladder) is 3 μL, and the loading amount of tyrosinase-expressing mRNA (numbered: M1) is 80 ng.

[0099] As shown in FIG2 , the electrophoresis pattern of the mRNA expressing tyrosinase (numbered: M1) is a single target sequence band, which meets the quality control requirements of the mRNA expressing tyrosinase and can be used for lipid nanoparticle encapsulation in step S2.

[0100] S2. Encapsulate the tyrosinase-expressing mRNA screened in S1 into lipid nanoparticles to obtain a lipid nanoparticle composition, wherein the mass percentage of the tyrosinase-expressing mRNA in the lipid nanoparticle composition is 15% based on the total mass of the lipid nanoparticle composition.

[0101] S2-1. Cationic lipid, auxiliary phospholipid, cholesterol, and PEGylated phospholipid were mixed at a molar ratio of 50:10:38.5:1.5 in a microfluidic device (Precision NanoSystems, Vancouver, BC, Canada) to prepare lipid nanoparticles. The lipid nanoparticles were dissolved in ethanol, an organic solvent, to obtain mixture A with a lipid nanoparticle concentration of 25 mM.

[0102] S2-2, diluting 200 μg of tyrosinase-expressing mRNA prepared in S1 to 0.35 mg / ml with a 100 mM sodium acetate solution at pH 4 to obtain a mixture B;

[0103] S2-3. Mixture A and mixture B were mixed using a microfluidic device (Precision NanoSystems, Vancouver, BC, Canada) to prepare a lipid nanoparticle composition (LNP-mRNA), and dialyzed against phosphate buffered saline (PBS).

[0104] The parameters of the microfluidic device are as follows: total flow rate 12 ml / min, water phase / ethanol phase (volume ratio) = 3 / 1, and temperature is room temperature;

[0105] The dialysis cassettes were purchased from Beijing Novi Biotechnology Co., Ltd. The dialysis steps and name are Slide-A-Lyzer TM The operating instructions for dialysis cassettes, 3.5K MWCO, are the same.

[0106] The cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl lipid, abbreviated as DLin-MC3-DMA; the auxiliary phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); the cholesterol serves as a stabilizer to increase the stability of the lipid nanoparticle composition; the PEGylated phospholipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000), and both the cholesterol and polyethylene glycol can prolong the half-life of the lipid nanoparticle composition. In this embodiment, the half-life of the lipid nanoparticle composition is 14 days.

[0107] In this example, the particle size and shear surface potential (zeta potential) of the LNP-mRNA were measured using a nanoparticle size potentiometer (Zetasizer (Malvern, USA)). As shown in Figure 3, the particle size detection shows that the particle size range of the LNP-mRNA is 50-150 nm, and the average is 125.45 nm. As shown in Figure 4, the potential detection shows that the shear surface potential (zeta potential) diagram of the present example is obtained. In this example, the potential is -4.2932 mV, and the polymer dispersity index (PDI) of the LNP-mRNA is 0.115. The closer the PDI is to 0, the more uniform the particle size distribution is. Therefore, the particle size distribution of the LNP-mRNA in this example is relatively uniform.

[0108] The encapsulation efficiency of tyrosinase-expressing mRNA was measured using an RNA fluorescence (RiboGreen) assay (Invitrogen, USA). The encapsulation efficiency (EE%) was 87.90%, which was calculated by measuring the concentration of free tyrosinase-expressing mRNA before the lipid nanoparticle composition was demulsified (210.00 ng / μL) and the concentration of free tyrosinase-expressing mRNA after the lipid nanoparticle composition was demulsified (1736.00 ng / μL).

[0109] Example 2

[0110] The method for preparing a lipid nanoparticle composition of this embodiment comprises the following steps:

[0111] S1. Screening for tyrosinase-expressing mRNA:

[0112] S1-1. Design a coding sequence (CDS sequence) based on the target protein sequence of tyrosinase. In this embodiment, the CDS sequence is SEQ ID NO: 2.

[0113] S1-2, introducing the CDS sequence in S1-1 into a GENEWIZ DNA synthesizer (model: MerMade 192) to synthesize a plasmid encoding the tyrosinase sequence;

[0114] S1-3, using T7 RNA polymerase as a medium to transcribe the linearized DNA in the S1-2 synthetic plasmid to produce crude mRNA expressing tyrosinase. The T7 RNA polymerase was purchased from Suzhou Jinan Protein Technology Co., Ltd. and is included in the kit with catalog number E131. The transcription process is the same as the operating procedure of the kit with catalog number E131;

[0115] S1-4. Purify the crude mRNA expressing tyrosinase by phenol / chloroform to remove proteins and free nucleotides, and then perform a capping reaction to obtain N1-methylpseudouridine-modified mRNA. The capping reaction procedure is the same as the procedure of the kit M082 produced by Suzhou Jinan Protein Technology Co., Ltd. The N1-methylpseudouridine is a modified base, and the modified mRNA consists of a 5' cap region (Cap1), a 5' untranslated region (5'UTR), a tyrosinase coding region, a 3' untranslated region (3'UTR), and a polyadenine tail (PolyA tail) from the 5' end. The sequence of the tyrosinase coding region is the same as the coding sequence (CDS sequence);

[0116] Wherein, the specific steps of phenol / chloroform purification method are:

[0117] a. Dilute 20 μl of crude tyrosinase-expressing mRNA to 180 μl by adding 160 μl of RNase-free water to obtain a diluted mixture a.

[0118] b. Add 20 μl of 3 M sodium acetate (pH = 5.2) to the diluted mixture a and mix thoroughly with a pipette;

[0119] c. Add 200 μl of phenol / chloroform mixture (phenol:chloroform volume ratio is 1:1) for extraction to obtain mixture b. Centrifuge mixture b at room temperature and 10,000 rpm for 5 minutes. Transfer the upper solution (aqueous phase) to a new RNase-free EP tube (plastic centrifuge tube);

[0120] d. Add an equal volume of chloroform to the aqueous phase and extract again twice, collecting the upper aqueous phase after each extraction;

[0121] e. Add 2 volumes of anhydrous ethanol to the upper aqueous phase collected for the last time and mix well. Incubate at -20°C for 30 minutes to obtain a mixture c. Centrifuge the mixture c at 15,000 rpm for 15 minutes.

[0122] f. After discarding the supernatant, add 500 μl of 70% ethanol pre-cooled to below 4°C to wash the precipitate. Centrifuge again at 15,000 rpm at 4°C and discard the supernatant to obtain the crude tyrosinase mRNA product free of proteins and free nucleotides.

[0123] g. The crude mRNA expressing tyrosinase, after removing proteins and free nucleotides, was dried at room temperature for 2 min. After ethanol evaporated, 20 μl of RNase-free water was added to dissolve the precipitate to obtain mixture d.

[0124] h. Store the mixture d at -80°C;

[0125] S1-5, using Lipofectamine MessengerMax as a transfection reagent (the transfection reagent was purchased from Thermo Fisher Scientific), and transfecting HEK293T cells according to the transfection protocol of Thermo Fisher Scientific to obtain transfected HEK293T cells;

[0126] S1-6. Protein detection was performed on the transfected HEK293T cells using Western blot to screen for tyrosinase mRNA expression;

[0127] The screening principle is as follows: tyrosinase (EC1.14.18.1) is a monophenol monooxygenase and a bifunctional copper-containing glycoprotein. Tyrosinase is a key enzyme in the synthesis of melanin in organisms and can catalyze L-dopa to produce dopachrome. It has a characteristic absorption peak at 475 nm. Screening for mRNA expressing tyrosinase, as shown in Figure 1, in this example, the activity ΔA of the mRNA expressing tyrosinase is 0.439;

[0128] The activity ΔA is determined as follows:

[0129] a. Preheat the spectrophotometer / microplate reader for 30 minutes, adjust the wavelength to 475 nm, and zero the spectrophotometer with distilled water.

[0130] b. Add the sample to the micro-glass cuvette / 96-well plate and quickly pipette to mix thoroughly. Record the absorbance value A1 at 10 seconds. Immediately place the plate in a 37°C water bath or incubator for 3 minutes. Remove the plate, wipe it dry, and measure the absorbance value A2 at 3 minutes and 10 seconds. Calculate ΔA = A2 - A1.

[0131] S1-7. Quality control was performed on the mRNA expressing tyrosinase after screening by S1-6. The quality control method was electrophoresis test. The test method was agarose gel electrophoresis. The agarose gel contained 0.8 wt% agarose. The electrophoresis time was 45 min.

[0132] S2. Encapsulate the tyrosinase-expressing mRNA screened in S1 into lipid nanoparticles to obtain a lipid nanoparticle composition, wherein the mass percentage of the tyrosinase-expressing mRNA in the lipid nanoparticle composition is 8% based on the total mass of the lipid nanoparticle composition.

[0133] S2-1. Cationic lipid, auxiliary phospholipid, cholesterol, and PEGylated phospholipid were mixed at a molar ratio of 50:10:38.5:1.5 in a microfluidic device (Precision NanoSystems, Vancouver, BC, Canada) to prepare lipid nanoparticles. The lipid nanoparticles were dissolved in ethanol, an organic solvent, to obtain mixture A with a lipid nanoparticle concentration of 25 mM.

[0134] S2-2, diluting 200 μg of tyrosinase-expressing mRNA prepared in S1 to 0.35 mg / ml with a 100 mM sodium acetate solution at pH 4 to obtain a mixture B;

[0135] S2-3. Mixture A and mixture B were mixed using a microfluidic device (Precision NanoSystems, Vancouver, BC, Canada) to prepare a lipid nanoparticle composition (LNP-mRNA), and dialyzed against phosphate buffered saline (PBS).

[0136] The parameters of the microfluidic device are as follows: total flow rate 12 ml / min, water phase / ethanol phase (volume ratio) = 3 / 1, and temperature is room temperature;

[0137] The dialysis cassettes were purchased from Beijing Novi Biotechnology Co., Ltd. The dialysis steps and name are Slide-A-Lyzer TM The operating instructions for dialysis cassettes, 3.5K MWCO, are the same.

[0138] The cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl lipid, abbreviated as DLin-MC3-DMA; the auxiliary phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); the cholesterol acts as a stabilizer to increase the stability of the lipid nanoparticle composition; the PEGylated phospholipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000), and both the cholesterol and polyethylene glycol can prolong the half-life of the lipid nanoparticle composition.

[0139] In this example, the particle size and shear plane potential (zeta potential) of the LNP-mRNA were measured using a nanoparticle size potentiometer (Zetasizer (Malvern, USA)). The particle size range of the LNP-mRNA was 50-150 nm, the average was 123.40 nm, the shear plane potential (zeta potential) was -2.1781 mV, and the polymer dispersity index (PDI) of the LNP-mRNA was 0.088. The closer the PDI was to 0, the more uniform the particle size distribution was. Therefore, the particle size distribution of the LNP-mRNA in this example was relatively uniform.

[0140] The encapsulation efficiency of tyrosinase-expressing mRNA was measured using an RNA fluorescence (RiboGreen) assay (Invitrogen, USA). The encapsulation efficiency (EE%) was 88.05%, which was calculated by measuring the concentration of free tyrosinase-expressing mRNA before and after emulsification of the lipid nanoparticle composition (206.00 ng / μL) and the concentration of free tyrosinase-expressing mRNA after emulsification of the lipid nanoparticle composition (1724.00 ng / μL).

[0141] Example 3

[0142] The method for preparing a lipid nanoparticle composition of this embodiment comprises the following steps:

[0143] S1. Screening for tyrosinase-expressing mRNA:

[0144] S1-1. Design a coding sequence (CDS sequence) based on the target protein sequence of tyrosinase. In this embodiment, the CDS sequence is SEQ ID NO: 3.

[0145] S1-2, introducing the CDS sequence in S1-1 into a GENEWIZ DNA synthesizer (model: MerMade 192) to synthesize a plasmid encoding the tyrosinase sequence;

[0146] S1-3, using T7 RNA polymerase as a medium to transcribe the linearized DNA in the S1-2 synthetic plasmid to produce crude mRNA expressing tyrosinase. The T7 RNA polymerase was purchased from Suzhou Jinan Protein Technology Co., Ltd. and is included in the kit with catalog number E131. The transcription process is the same as the operating procedure of the kit with catalog number E131;

[0147] S1-4. Purify the crude mRNA expressing tyrosinase by phenol / chloroform to remove proteins and free nucleotides, and then perform a capping reaction to obtain N1-methylpseudouridine-modified mRNA. The capping reaction procedure is the same as the procedure of the kit M082 produced by Suzhou Jinan Protein Technology Co., Ltd. The N1-methylpseudouridine is a modified base, and the modified mRNA consists of a 5' cap region (Cap1), a 5' untranslated region (5'UTR), a tyrosinase coding region, a 3' untranslated region (3'UTR), and a polyadenine tail (PolyA tail) from the 5' end. The sequence of the tyrosinase coding region is the same as the coding sequence (CDS sequence);

[0148] Wherein, the specific steps of phenol / chloroform purification method are:

[0149] a. Dilute 20 μl of crude tyrosinase-expressing mRNA to 180 μl by adding 160 μl of RNase-free water to obtain a diluted mixture a.

[0150] b. Add 20 μl of 3 M sodium acetate (pH = 5.2) to the diluted mixture a and mix thoroughly with a pipette;

[0151] c. Add 200 μl of phenol / chloroform mixture (phenol:chloroform volume ratio is 1:1) for extraction to obtain mixture b. Centrifuge mixture b at room temperature and 10,000 rpm for 5 minutes. Transfer the upper solution (aqueous phase) to a new RNase-free EP tube (plastic centrifuge tube);

[0152] d. Add an equal volume of chloroform to the aqueous phase and extract again twice, collecting the upper aqueous phase after each extraction;

[0153] e. Add 2 volumes of anhydrous ethanol to the upper aqueous phase collected for the last time and mix well. Incubate at -20°C for 30 minutes to obtain a mixture c. Centrifuge the mixture c at 15,000 rpm for 15 minutes.

[0154] f. After discarding the supernatant, add 500 μl of 70% ethanol pre-cooled to below 4°C to wash the precipitate. Centrifuge again at 15,000 rpm at 4°C and discard the supernatant to obtain the crude tyrosinase mRNA product free of proteins and free nucleotides.

[0155] g. The crude mRNA expressing tyrosinase, after removing proteins and free nucleotides, was dried at room temperature for 2 min. After ethanol evaporated, 20 μl of RNase-free water was added to dissolve the precipitate to obtain mixture d.

[0156] h. Store the mixture d at -80°C;

[0157] S1-5, using Lipofectamine MessengerMax as a transfection reagent (the transfection reagent was purchased from Thermo Fisher Scientific), and transfecting HEK293T cells according to the transfection protocol of Thermo Fisher Scientific to obtain transfected HEK293T cells;

[0158] S1-6. Protein detection was performed on the transfected HEK293T cells using Western blot to screen for tyrosinase mRNA expression;

[0159] The screening principle is as follows: tyrosinase (EC1.14.18.1) is a monophenol monooxygenase and a bifunctional copper-containing glycoprotein. Tyrosinase is a key enzyme in the synthesis of melanin in organisms and can catalyze L-dopa to produce dopachrome. It has a characteristic absorption peak at 475 nm. Screening for mRNA expressing tyrosinase, as shown in Figure 1, in this example, the activity ΔA of the mRNA expressing tyrosinase is 0.4;

[0160] The activity ΔA is determined as follows:

[0161] a. Preheat the spectrophotometer / microplate reader for 30 minutes, adjust the wavelength to 475 nm, and zero the spectrophotometer with distilled water.

[0162] b. Add the sample to the micro-glass cuvette / 96-well plate and quickly pipette to mix thoroughly. Record the absorbance value A1 at 10 seconds. Immediately place the plate in a 37°C water bath or incubator for 3 minutes. Remove the plate, wipe it dry, and measure the absorbance value A2 at 3 minutes and 10 seconds. Calculate ΔA = A2 - A1.

[0163] S1-7. Quality control was performed on the mRNA expressing tyrosinase after screening by S1-6. The quality control method was electrophoresis test. The test method was agarose gel electrophoresis. The agarose gel contained 0.8 wt% agarose. The electrophoresis time was 45 min.

[0164] S2. Encapsulate the tyrosinase-expressing mRNA screened in S1 into lipid nanoparticles to obtain a lipid nanoparticle composition, wherein the mass percentage of the tyrosinase-expressing mRNA in the lipid nanoparticle composition is 25% based on the total mass of the lipid nanoparticle composition.

[0165] S2-1. Cationic lipid, auxiliary phospholipid, cholesterol, and PEGylated phospholipid were mixed at a molar ratio of 50:10:38.5:1.5 in a microfluidic device (Precision NanoSystems, Vancouver, BC, Canada) to prepare lipid nanoparticles. The lipid nanoparticles were dissolved in ethanol, an organic solvent, to obtain mixture A with a lipid nanoparticle concentration of 25 mM.

[0166] S2-2, diluting 200 μg of tyrosinase-expressing mRNA prepared in S1 to 0.35 mg / ml with a 100 mM sodium acetate solution at pH 4 to obtain a mixture B;

[0167] S2-3. Mixture A and mixture B were mixed using a microfluidic device (Precision NanoSystems, Vancouver, BC, Canada) to prepare a lipid nanoparticle composition (LNP-mRNA), and dialyzed against phosphate buffered saline (PBS).

[0168] The parameters of the microfluidic device are as follows: total flow rate 12 ml / min, water phase / ethanol phase (volume ratio) = 3 / 1, and temperature is room temperature;

[0169] The dialysis cassettes were purchased from Beijing Novi Biotechnology Co., Ltd. The dialysis steps and name are Slide-A-Lyzer TM The operating instructions for dialysis cassettes, 3.5K MWCO, are the same.

[0170] The cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl lipid, abbreviated as DLin-MC3-DMA; the auxiliary phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); the cholesterol acts as a stabilizer to increase the stability of the lipid nanoparticle composition; the PEGylated phospholipid is dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG2000), and both the cholesterol and polyethylene glycol can prolong the half-life of the lipid nanoparticle composition.

[0171] In this example, the particle size and shear plane potential (zeta potential) of the LNP-mRNA were measured using a nanoparticle size potentiometer (Zetasizer (Malvern, USA)). The particle size range of the LNP-mRNA was 50-150 nm, the average was 126.75 nm, the shear plane potential (zeta potential) was 0.0717 mV, and the polymer dispersity index (PDI) of the LNP-mRNA was 0.106. The closer the PDI was to 0, the more uniform the particle size distribution was. Therefore, the particle size distribution of the LNP-mRNA in this example was relatively uniform.

[0172] The encapsulation efficiency of tyrosinase-expressing mRNA was measured using an RNA fluorescence (RiboGreen) assay (Invitrogen, USA). The encapsulation efficiency (EE%) was 88.13%, which was calculated by measuring the concentration of free tyrosinase-expressing mRNA before the lipid nanoparticle composition was demulsified (216.00 ng / μL) and the concentration of free tyrosinase-expressing mRNA after the lipid nanoparticle composition was demulsified (1820.00 ng / μL).

[0173] Example 4

[0174] In this example, the lipid nanoparticle composition (LNP-mRNA) prepared in Example 1 was subcutaneously injected into mice to verify whether it could produce melanin.

[0175] The lipid nanoparticle composition can deliver mRNA expressing tyrosinase into cells, release the mRNA expressing tyrosinase through ionization in the cells, utilize the organism's own cells to translate the mRNA expressing tyrosinase, and synthesize tyrosinase in the cells to generate melanin.

[0176] In this example, mice were divided into 5 groups, numbered G01, G02, G03, G04, and G05, with 8 mice in each group;

[0177] G01 is the blank control group, and G02, G03, G04, and G05 are the experimental groups, i.e., G01 is the blank control group injected with phosphate buffered saline (PBS), and G02, G03, G04, and G05 are the experimental groups injected with a PBS mixed solution containing the lipid nanoparticle composition;

[0178] The injection volume of G01 was 200ul PBS, without lipid nanoparticle composition.

[0179] The injection volume of G02 is 200ul PBS mixed solution containing 1ug of lipid nanoparticle composition.

[0180] The injection volume of G03 is 200ul PBS mixed solution containing 5ug lipid nanoparticle composition.

[0181] The injection volume of G04 is 200ul PBS mixed solution containing 10ug lipid nanoparticle composition.

[0182] The injection volume of G05 is 200ul PBS mixed solution containing 25ug lipid nanoparticle composition.

[0183] Safety can be preliminarily assessed by local injection into the skin.

[0184] Safety Evaluation: In this example, the incidence of events occurring 1 to 21 days after a single injection of a lipid nanoparticle composition was statistically analyzed. For the mouse experiment, the primary focus was on observing post-injection mortality and severe allergic reactions. In this example, no mice died or became ill, indicating that the lipid nanoparticle composition of the present invention exhibits no toxic side effects.

[0185] Taking one mouse from the G04 group as an example, as shown in Figure 5, it can be seen that the color of the mouse darkened significantly on day 6 (a) after subcutaneous injection of the lipid nanoparticle composition. This remained stable on day 12 (b) and gradually faded on day 14 (c). This demonstrates that the lipid nanoparticle composition prepared by the present invention can be delivered into the body by injection and produces melanin after tyrosinase is synthesized in the cells.

[0186] Therefore, the lipid nanoparticle composition prepared by the present invention can be injected into the body by subcutaneous injection, such as a vaccine, and stably produces melanin. In addition, the mRNA expressing tyrosinase selected by the present invention only expresses the antigen in the cytoplasm and does not enter the cell nucleus. It can more effectively express tyrosinase without causing the risk of gene mutation caused by DNA insertion. In addition, the selected mRNA expressing tyrosinase can be transiently expressed and is naturally metabolized and eliminated by the organism, further enhancing safety.

[0187] It should be understood that the present invention is not limited to what has been described above and shown in the accompanying drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. An mRNA expressing tyrosinase, characterized in that The mRNA expressing tyrosinase acts on the site where vitiligo occurs, and expresses melanin at the site where vitiligo occurs.

2. An mRNA expressing tyrosinase, characterized in that The coding sequence corresponding to the mRNA expressing tyrosinase is one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and the half-life of the mRNA expressing tyrosinase in the cell is 5-15 days.

3. A lipid nanoparticle composition, characterized in that: The lipid nanoparticle composition comprises lipid nanoparticles and the mRNA expressing tyrosinase according to any one of claims 1 to 2, wherein the mRNA expressing tyrosinase is encapsulated inside the lipid nanoparticles, the particle size of the lipid nanoparticle composition is 50-150 nm, the potential of the shear surface is -4.2932 to 0.0717 mV, the encapsulation rate of the mRNA expressing tyrosinase by the lipid nanoparticle composition is 87.90-88.13%, the polymer dispersibility index is 0.088-0.113, and the lipid nanoparticle composition has a lipid bilayer structure; Based on the total mass of the lipid nanoparticle composition, the mass percentage of the mRNA expressing tyrosinase in the lipid nanoparticle composition is 3-25%.

4. The lipid nanoparticle composition according to claim 3, characterized in that The lipid nanoparticles consist of cationic lipids, auxiliary phospholipids, cholesterol and polyethylene glycolated phospholipids.

5. The lipid nanoparticle composition according to claim 4, characterized in that The molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol and PEGylated phospholipid is (50-60):(10-15):(35-49):(1-3).

6. The lipid nanoparticle composition according to claim 5, characterized in that The cationic lipid is 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl lipid; The auxiliary phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The polyethylene glycol phospholipid is dimyristoylglycerol-polyethylene glycol 2000 or polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine.

7. A method for preparing the lipid nanoparticle composition according to any one of claims 3 to 6, characterized in that: The steps include: S1. Screening of mRNA expressing tyrosinase; S2. Encapsulating the mRNA expressing tyrosinase screened in S1 into lipid nanoparticles to obtain a lipid nanoparticle composition.

8. The method for preparing the lipid nanoparticle composition according to claim 7, characterized in that: The S2 lipid nanoparticle composition contains an mRNA expressing tyrosinase.

9. A vaccine, characterized in that The vaccine comprises the mRNA expressing tyrosinase according to claim 1.

10. A vaccine, characterized in that The vaccine comprises the lipid nanoparticle composition according to any one of claims 3 to 6.

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