MRNA pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention relates to the technical field of mRNA (messenger Ribonucleic Acid) therapy, in particular to an mRNA (messenger Ribonucleic Acid) pharmaceutical composition combined with a C1 type Niemann-Pick protein (NPC1) inhibitor and an mRNA drug for improving the delivery efficiency of mRNA through endosome escape as well as a preparation method and application of the mRNA pharmaceutical composition. The invention relates to an NPC1 inhibitor for improving the efficiency of an mRNA (messenger ribonucleic acid) delivery system, and by utilizing an mRNA pharmaceutical composition, the in-vivo and in-vitro transfection efficiency of mRNA is remarkably improved, the accumulation of the mRNA delivery system in transfection cells is increased, the retention time of the mRNA delivery system in the cells is prolonged, and the mRNA delivery system efficiency is improved. The mRNA delivery system escapes from endosomes and lysosomes to cytoplasm to express more effect proteins. The combination of the NPC1 inhibitor and the mRNA delivery system has an important application prospect in enhancing the prevention and treatment effects of mRNA drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of mRNA therapy, and in particular to an mRNA pharmaceutical composition of a combination of an NPC1 inhibitor and an mRNA drug that improves mRNA delivery efficiency through endosomal escape, and a preparation method and application thereof. Background Art

[0002] Based on its advancedness, innovation, safety, personalization potential, and versatility, mRNA therapy has become an emerging star in the biomedical field. mRNA therapy has made significant progress in a number of fields, including COVID-19, cancer treatment, genetic diseases, and immunotherapy, and has shown potential in clinical applications (1-4). Intracellular delivery of mRNA therapy has great potential for the development of vaccines and treatments. Delivery vectors for mRNA therapy include lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, protein-based virus-like nanoparticles, electroporation, etc. However, the successful application of mRNA therapy depends largely on the engineering design of the mRNA delivery system and the intracellular delivery and intracellular processing of mRNA to avoid their degradation by enzymes in the circulatory system and promote their endosomal escape.

[0003] A key determinant of the effectiveness of mRNA delivery vectors is that after reaching the target tissue, mRNA enters the target cell by endocytosis and escapes through the endosomal-lysosomal system to reach the cytoplasm for translation and protein expression. Studies have shown that less than 2% of delivery vector-mediated mRNA will be retained in the cytoplasm by endosomal-lysosomal escape (5-11). Endosomal-lysosomal escape after mRNA delivery into cells is limited in time and space. The time it takes for mRNA to enter cells through delivery vectors and reach endosomes or lysosomes is generally between minutes and hours, and the specific time varies depending on the cell type and vector characteristics. For example, the process of lipid nanoparticles delivering mRNA into cells by endocytosis usually occurs within a few minutes. The endocytosed mRNA can be transported to the early endosome within a few minutes under the encapsulation of vesicles. As the endosome matures, the early endosome will transform into the late endosome, accompanied by a decrease in pH and an increase in enzyme activity in the endosomal environment. This process usually takes 30 minutes to several hours. If the mRNA fails to successfully escape in the endosomal stage, it will be further transported to the lysosome to be degraded or escape. In general, the arrival of mRNA from endocytosis to lysosomes may be completed within a few hours. Therefore, endosomal-lysosomal escape must usually be completed within this period of time to avoid mRNA degradation (5,8,12,13). In addition, endosomal-lysosomal escape of mRNA is not only constrained by time, but also by the structure and properties of the endosomal membrane (membrane composition, fluidity, stability, pH changes, enzyme activity), endosomal-lysosomal transformation (endosomal maturation process and endosomal-lysosomal fusion process), membrane fusion mechanism (membrane rupture and fusion and pH dependence) and spatial barriers inside endosomes (differences in physical structure and local environment inside endosomes) (9,14-18). mRNA escape events in the endosomal-lysosomal pathway usually occur in certain specific areas or stages of the endosomal membrane, not uniformly throughout the endosome. Therefore, breaking through the endosomal-lysosomal escape of mRNA and prolonging the retention time of mRNA in the cytoplasm are key steps to break the limitations of mRNA delivery.

[0004] Niemann-Pick Type C-1 (NPC1), a transmembrane protein located in late endosomes / lysosomes, consists of 1278 amino acid residues, contains 13 transmembrane helices, and has three large intraluminal domains. It mediates the distribution of lipoprotein-derived cholesterol in cells. NPC1 is mainly responsible for cholesterol transport in the body. Its amino acid mutation may cause "Niemann-Pick disease", which manifests as neurodevelopmental delay, cognitive impairment or deafness in children, and in severe cases, it may lead to premature death in children (19-21). NPC1 has been identified as an important regulator of the main circulation pathway for delivering siRNA. NPC1 protein plays a key role in regulating the transport and metabolism of cholesterol, and its defects may lead to abnormal accumulation of cholesterol in lysosomes, thereby causing lysosomal morphology, transport and functional defects. NPC1 can expel siRNA mediated by LNP delivery vectors to the extracellular space; NPC1-deficient cells show enhanced cellular retention of LNP-mediated siRNA in late endosomes and lysosomes, and increased gene silencing efficiency of target genes (5,11,22,23). Screening of NPC1 inhibitors in the prevention of Ebola virus infection has shown that they can enhance the accumulation of intracellular cholesterol and inhibit the cytoplasmic delivery of the virus by inhibiting NPC1 targets (24). These studies have shown that the combination of small molecule NPC1 inhibitors with siRNA delivery systems to improve the efficacy of siRNA systems by improving the cytoplasmic retention of siRNA is an attractive strategy. However, research on NPC1 inhibitors for improving the efficiency of mRNA delivery systems remains blank. It is urgent to develop applications that can prolong the retention of mRNA in the cytoplasm through endosomal-lysosomal escape to promote the delivery efficiency of mRNA delivery systems.

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[0029] The purpose of the present invention is to improve the in vivo and in vitro delivery efficiency of mRNA drugs, thereby achieving the goal of mRNA therapy prevention and treatment development. The present invention provides an application of a small molecule inhibitor drug for improving the efficiency of mRNA delivery. The drug is an NPC1 inhibitor. The present invention proposes a method for combining an NPC1 inhibitor with an mRNA delivery system, further improving the application of mRNA therapy. The present invention particularly relates to an application for improving the in vivo and in vitro expression efficiency of an mRNA delivery system. The present invention relates to improving the transfection of an mRNA delivery system in vitro by using an NPC1 inhibitor, in particular by increasing the accumulation of the mRNA delivery system in cells. The present invention also relates to an NPC1 inhibitor increasing the delivery of an mRNA delivery system to late endosomes and lysosomes. The present invention also relates to an NPC1 inhibitor promoting the escape of an mRNA delivery system in late endosomes and lysosomes, increasing the possibility of mRNA delivery in the cytoplasm. The present invention also includes an application of improving the delivery of mRNA in vivo by combining an NPC1 inhibitor with an mRNA delivery system.

[0030] In order to achieve the above invention objectives, the technical solutions provided by the present invention are as follows:

[0031] An mRNA pharmaceutical composition comprises a C1-type Niemann-Pick protein inhibitor (NPC1) and an mRNA drug. The NPC1 inhibitor and the mRNA drug are used in combination to improve the mRNA drug delivery efficiency. The NPC1 inhibitor improves the delivery efficiency by promoting the endosomal-lysosomal escape of the mRNA drug delivery system.

[0032] Wherein, the NPC1 inhibitor is any one or more combinations of posaconazole, U18666A and itraconazole; preferably posaconazole, which has the effect of inhibiting and reducing the transport of the mRNA delivery system out of the cell after binding to the transmembrane protein NPC1 on the late endosome / lysosome, thereby improving the endosomal-lysosomal escape and delivery expression of mRNA.

[0033] The mRNA drug is a delivery system containing the mRNA drug, and the delivery system is lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, protein-based virus-like nanoparticles, Lipofectamine 3000, or an electroporation delivery system; preferably, lipid nanoparticles containing mRNA drugs or Lipofectamine 3000 containing mRNA drugs.

[0034] Among them, the lipid nanoparticles containing mRNA drugs are composed of a carrier and an mRNA drug encapsulated in the carrier; the carrier includes a combination of cationic lipids, auxiliary phospholipids, cholesterol and polyethylene glycol lipids; the mRNA drug is an mRNA vaccine or an mRNA protein replacement therapy drug, and the mRNA drug is an mRNA drug that depends on mRNA translation.

[0035] Preferably, the cationic lipids include but are not limited to 4(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (Dlin-MC3-DMA) or ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC0315); the auxiliary phospholipids include but are not limited to 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); the polyethylene glycol lipids include but are not limited to 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (PEG-DMG-2000).

[0036] Wherein, the molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol, polyethylene glycol lipid and C1 Niemann-Pick protein inhibitor is 50:9.6-10:38.1-38.5:1.1-1.5:0.01-0.4. Preferably, the molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol, polyethylene glycol lipid and C1 Niemann-Pick protein inhibitor is 50:9.6-10:38.5:1.5:0.01-0.4, and more preferably, the molar ratio is 50:9.6-10:38.5:1.5:0.1-0.4.

[0037] In a second aspect, the present invention provides a method for preparing the mRNA pharmaceutical composition described in the first aspect, wherein the C1-type Niemann-Pick protein inhibitor and the mRNA drug are combined by any of the following methods: direct mixing, encapsulation, or modification. Preferably, the C1-type Niemann-Pick protein inhibitor and the mRNA drug are combined by encapsulation. The method comprises the following steps:

[0038] (1) preparing a mixture organic phase: mixing a C1 type Niemann-Pick protein inhibitor, a cationic lipid, an auxiliary phospholipid, cholesterol and a polyethylene glycol lipid in an organic solvent to obtain a mixture organic phase;

[0039] (2) preparing an mRNA drug aqueous phase: dissolving the mRNA drug in a buffer solution to obtain an mRNA drug aqueous phase;

[0040] (3) The organic phase of the mixture and the aqueous phase of the mRNA drug are mixed by a microfluidic method to obtain a mixed solution; and the mixed solution is ultrafiltered through an ultrafiltration tube to obtain.

[0041] Wherein, in step (1), the organic solvent comprises any one or more of ethanol, isopropanol, chloroform, dichloromethane, methanol, n-hexane and ethyl acetate, preferably ethanol; in the organic phase of the mixture, the total concentration of cationic lipids, auxiliary phospholipids, cholesterol, polyethylene glycol lipids and C1 Niemann-Pick protein inhibitor is 2.85 mg / mL. The mixed molar ratio of the cationic lipids, auxiliary phospholipids, cholesterol, polyethylene glycol lipids and C1 Niemann-Pick protein inhibitor is 50:9.6-10:38.1-38.5:1.1-1.5:0.01-0.4;

[0042] Wherein, in step (2), the buffer solution is a citric acid-sodium citrate buffer solution with an ion concentration of 10 mM and a pH of 4; and the concentration of the mRNA drug in the mRNA drug aqueous phase is 0.05 mg / mL.

[0043] Wherein, in step (3), the flow rate ratio of the organic phase of the mixture to the aqueous phase of the mRNA drug is 1:2-10, and the total flow rate is 8-16 mL / min; the molecular weight cut-off of the ultrafiltration tube is 10 KDa. Preferably, the flow rate ratio of the organic phase of the mixture to the aqueous phase of the mRNA drug is 1:3, and the total flow rate is 12 mL / min.

[0044] In a third aspect, the present invention provides use of the mRNA pharmaceutical composition described in the first aspect in the preparation of mRNA drugs for preventing and / or treating diseases.

[0045] Beneficial effects:

[0046] The present invention combines an NPC1 inhibitor with an mRNA delivery system to prepare an mRNA pharmaceutical composition. The mRNA pharmaceutical composition obtained by the present invention significantly improves the in vivo and in vitro transfection efficiency of mRNA, increases the accumulation of the mRNA delivery system in transfected cells, prolongs the retention time of the mRNA delivery system in cells, and allows the mRNA delivery system to escape from endosomes and lysosomes to the cytoplasm to express more effector proteins. The mRNA pharmaceutical composition obtained by the present invention has important application prospects in enhancing the preventive and therapeutic effects of mRNA drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0048] Figure 1 The fluorescence signal diagram of HEK293T WT cells and NPC1 KO HEK293T cells transfected with Luc-LNPs and eGFP-LNPs. Figure 1 A is the fluorescence signal diagram of Luc-LNPs transfected cells. Figure 1 B is the fluorescence signal diagram of cells transfected with eGFP-LNPs.

[0049] Figure 2 The results of in vivo transfection using Luc-LNPs injected intramuscularly into WT mice and NPC1 KO mice. Figure 2 A is the IVIS imaging image. Figure 2 B is the quantification diagram of transfection in mice (CPS is the quantification unit).

[0050] Figure 3 The transfection results of different NPC1 inhibitors combined with mRNA delivery systems are shown in Figure 2. Figure 3 A is the transfection result of Posaconazole combined with mRNA delivery system. Figure 3 B is the transfection result of U18666A combined with mRNA delivery system. Figure 3 C is the transfection result of Itraconazole combined with mRNA delivery system.

[0051] Figure 4 The results of transfection of different cells using NPC1 inhibitor and LNP delivery system.

[0052] Figure 5 This is the result of combined transfection of NPC1 inhibitor and Lip3000 delivery system.

[0053] Figure 6 This is a schematic diagram of the results of the combined use of NPC1 inhibitors and mRNA delivery systems on mRNA retention in cells. Figure 6 A is the flow cytometry result of Cy5. Figure 6 B is the flow cytometry result of eGFP. Figure 6 C is a laser confocal scanning microscope image of Cy5 and eGFP. Figure 6 D is the Cy5 laser confocal result. Figure 6 E is the result of eGFP laser confocal microscopy. Figure 6 F is the flow cytometry result of DIO. Figure 6 G is a DIO laser confocal scanning microscope image. Figure 6 H is the DIO laser confocal image.

[0054] Figure 7 This is a schematic diagram of the effects of the combined use of NPC1 inhibitors and mRNA delivery systems on the endosomal-lysosomal system. Figure 7 A shows the effect of NPC1 inhibitor combined with mRNA delivery system on early endosome Rab5. Figure 7 B shows the effect of NPC1 inhibitor combined with mRNA delivery system on late endosomal Rab7. Figure 7 C shows the effect of NPC1 inhibitor and mRNA delivery system on lysosomal LAMP1.

[0055] Figure 8 The figures are the in vitro transfection results of the mRNA pharmaceutical composition prepared by combining the NPC1 inhibitor and the mRNA delivery system.

[0056] Fig. 9 The in vivo transfection results of the mRNA pharmaceutical composition prepared by combining the NPC1 inhibitor and the mRNA delivery system. Fig.10 Schematic diagram of the results of preparing P-LNPs containing RBD-mRNA for immunization of mice using a combination of NPC1 inhibitor and mRNA delivery system. DETAILED DESCRIPTION

[0057] The specific embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings, but are not intended to be limiting of the present invention. The experimental methods and reagents described in the following examples are all commonly used products in the art unless otherwise specified.

[0058] In the following examples, w / v, unless otherwise specified, represents g / mL; v / v, unless otherwise specified, represents mL / mL.

[0059] In the following examples, the PBS, unless otherwise specified, refers to 1×PBS with a pH of 7.4; the DPBS, unless otherwise specified, refers to 1×DPBS with a pH of 7.4.

[0060] In the following examples, the complete culture medium is a DMEM culture medium containing 10% v / v fetal bovine serum (FBS) and 1% v / v dual antibody, and the dual antibody is a mixture of 100 U / mL penicillin and 0.1 mg / mL streptomycin.

[0061] Example 1 Preparation of lipid nanoparticles (LNPs)

[0062] 1. Solution preparation:

[0063] (1) Preparation of 2% w / v NaOH solution: Dissolve 10 g of solid NaOH in 500 mL of sterile water. After the solution is fully dissolved, filter the solution through a 0.22 μm filter membrane to sterilize it and set aside.

[0064] (2) Preparation of 10 mM citric acid-sodium citrate buffer solution: Weigh 6.88 g of citric acid monohydrate and 5.07 g of sodium citrate dihydrate and dissolve them in 500 mL of RNase-free water to prepare a 0.1 M pH = 4 citric acid-sodium citrate buffer solution, and filter and sterilize with a 0.22 μm filter membrane. After the 0.1 M pH = 4 citric acid-sodium citrate buffer solution is diluted appropriately with RNase-free water, it is prepared into a 10 mM citric acid-sodium citrate buffer solution for use.

[0065] (3) Preparation of the organic phase of the mixture: Using ethanol as the solvent, the lipid organic phase was prepared according to the molar ratio of ALC0315: DSPC: cholesterol: PEG-DMG-2000 = 50:10:38.5:1.5 to prepare a total lipid mixture organic phase with a concentration of 2.85 mg / mL for the preparation of lipid nanoparticles LNPs.

[0066] (4) Preparation of aqueous phase: Dilute the mRNA with 10 mM citric acid-sodium citrate buffer solution to obtain an mRNA solution with a concentration of 0.05 mg / mL for use. The mRNA used here includes mRNA encoding luciferase (Luc) (purchased from Novizan) or mRNA encoding green fluorescent protein (eGFP) (purchased from APExBIO).

[0067] 2. Preparation of ultrafiltration tube:

[0068] (1) Soak and clean the ultrafiltration tube (purchased from Millipore, 10KDa) with pure water;

[0069] (2) Soak the ultrafiltration tube in 75% v / v ethanol for more than 5 minutes;

[0070] (3) Discard the ethanol in the ultrafiltration tube and soak it in the prepared 2% w / v NaOH solution for more than 30 min;

[0071] (4) Discard the NaOH solution in the ultrafiltration tube and wash and soak it in PBS for more than 2 times, each time for more than 5 minutes;

[0072] (5) The prepared ultrafiltration tube can be soaked in 75% v / v ethanol for later use and rinsed with DPBS buffer solution before use.

[0073] 3. Preparation of mRNA delivery system-LNPs:

[0074] Use a 2.5mL Xinhua syringe to draw 2.2mL of the mRNA solution with a concentration of 0.05mg / mL prepared in step 1 and use a 1mL Xinhua syringe to draw 0.8mL of the mixed organic phase with a concentration of 2.85mg / mL prepared in step 1, insert them into a microfluidic chip (purchased from Maianna, model SHM), and set the parameters as follows: volume = 2.67mL, flow ratio of mRNA solution to mixed lipid organic phase = 3:1, total flow = 12mL / min, front waste liquid = 0.35mL, rear waste liquid = 0.10mL, and obtain about 2.22mL LNPs solution for subsequent ultrafiltration and encapsulation efficiency detection.

[0075] 4. Ultrafiltration:

[0076] The LNPs solution prepared in step 3 was diluted with 3 times the volume of DPBS and transferred to the ultrafiltration tube prepared in step 2 for centrifugal ultrafiltration, and the ultrafiltration parameters were: 2500rpm, 4°C, 30min. When the sample in the centrifuge tube was 0.5-1.0mL, about 4 times the volume of DPBS was added again for centrifugal ultrafiltration (2500rpm, 4°C, 30min); when the sample in the centrifuge tube was less than 0.5-1mL, about 3 times the volume of DPBS was added, ultrafiltration (2500rpm, 4°C, 30min) was added to about 0.5mL, and the sample was collected and placed in a 4°C refrigerator for mRNA concentration detection and subsequent experiments. When the selected mRNA is mRNA encoding luciferase, Luc-LNPs are prepared; when the selected mRNA is mRNA encoding green fluorescent protein, eGFP-LNPs are prepared.

[0077] 5. mRNA concentration detection and volume determination of mRNA delivery system:

[0078] Using Quant-iT TM RNA Reagent and Kit is used to detect the encapsulation efficiency of mRNA by mRNA delivery system (LNPs). The specific steps are as follows:

[0079] (1) Dilute 20×TE buffer to 1×TE buffer with enzyme-free water and set aside;

[0080] (2) Dilute QuantiT RiboGreen Reagen 200 times with 1×TE buffer and set aside;

[0081] (3) Dilute Triton-100 with 1×TE buffer to a 2% v / v Triton-100 solution for later use;

[0082] (4) Dilute Quant-iT with 1×TE buffer TM The standard in the RNA Reagent and Kit was diluted to 2 μg / mL. The standard curve solution was prepared with the following concentrations: 2 μg / mL; 1 μg / mL; 500 ng / mL; 250 ng / mL; 125 ng / mL; 62.5 ng / mL; 31.25 ng / mL; 15.625 ng / mL; 0 ng / mL.

[0083] (5) Mix 800 μL of 1×TE buffer + 8 μL of the mRNA delivery system sample obtained by ultrafiltration in step 4 to obtain a TEbuffer sample solution; mix 800 μL of 2% v / v Triton-100 solution + 8 μL of the mRNA delivery system sample obtained by ultrafiltration in step 4 to obtain a Triton-100 sample solution, and let it stand for 10 minutes to allow the mRNA delivery system to be fully lysed;

[0084] (6) 100 μL of each standard, TE buffer sample solution, and Triton-100 sample solution were added to a 96-well plate. Six replicates were made for each sample. Then, 100 μL of 200-fold diluted QuantiT RiboGreen Reagen was added for color development. The plate was shaken for 1 min and the fluorescence value was detected at 480-520 nm.

[0085] (7) The concentration of mRNA contained in LNPs was calculated based on the obtained fluorescence value, and the ultrafiltered LNPs were fixed to a RNA concentration of 100 μg / mL using DPBS. The concentration of mRNA in the mRNA delivery used in the following experiments was 100 μg / mL.

[0086] Example 2 Determination of Luc-LNPs and eGFP-LNPs transfected HEK293T WT cells and NPC1 KO HEK293T cells

[0087] In this example, WT and NPC1 knockout (NPC1 KO) HEK293T cells (purchased from Yuanjing Biotechnology) were used as comparisons, and different concentrations of Luc-LNPs or eGFP-LNPs mRNA delivery systems were used to verify whether NPC1 is a key regulatory factor for improving transfection efficiency. The specific steps are briefly described as follows:

[0088] (1) Preparation of Luc-LNPs and eGFP-LNPs: Prepare according to the steps of Example 1.

[0089] (2) Cell culture: WT HEK293T cells or NPC1 KO HEK293T cells in the culture dish were digested with trypsin for 30 seconds, and complete culture medium was added to terminate the trypsin. The digested cells were collected and placed in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The culture medium containing trypsin was discarded, and fresh complete culture medium was added. The cells were resuspended and counted (1×10 5 Cells were inoculated into 96-well plates at 100 μL per well and placed in a CO2 incubator at 37°C to allow the cells to adhere to the wall and grow to 70%-80%. Luc-LNPs or eGFP-LNPs containing 50 ng or 250 ng of mRNA were then added to each well for transfection and cultured for 48 h.

[0090] Transfection result detection: For the detection of Luc-LNPs, after 48h of transfection culture, add Bio-Lite Luciferase Assay System and use a microplate reader to detect the luciferase signal value. For the detection of eGFP-LNPs, use a microplate reader to detect the fluorescence intensity of 480-520nm after 48h of culture.

[0091] The test results are as follows Figure 1 As shown, the detection results of Luc-LNPs transfection showed ( Figure 1 A), transfection efficiency is positively correlated with mRNA concentration, and the transfection efficiency increases with the increase of mRNA concentration. In addition, the transfection efficiency of NPC1 KO HEK293T cells is significantly higher than that of WT HEK293T cells. When the mRNA concentration is 50ng, the transfection efficiency of NPC1 KO HEK293T cells is about 3 times that of WT HEK293T cells; when the mRNA concentration is 250ng, the transfection efficiency of NPC1 KO HEK293T cells is about 8 times that of WT HEK293T cells.

[0092] The results of eGFP-LNPs transfection showed that ( Figure 1 B), the transfection efficiency is consistent with the trend of Luc-LNPs, and the transfection efficiency increases with the increase of mRNA concentration. At the same time, the transfection efficiency of NPC1 KO HEK293T cells is significantly higher than that of WT HEK293T cells. When the mRNA concentration is 50ng, the transfection efficiency of NPC1 KO HEK293T cells is about 6 times that of WT HEK293T cells; when the mRNA concentration is 250ng, the transfection efficiency of NPC1 KO HEK293T cells is about 8 times that of WT HEK293T cells. It can be seen that NPC1 is one of the key regulatory factors to improve the transfection efficiency of in vitro mRNA delivery system.

[0093] Example 3 Determination of in vivo transfection of Luc-LNPs injected intramuscularly into WT mice and NPC1 KO mice

[0094] To further determine whether NPC1 is a key gene affecting transfection efficiency, Luc-LNPs were injected intramuscularly into WT mice and NPC1 KO mice (purchased from Saiye Biotechnology Co., Ltd., NPC1 KO mice are mice with NPC1 gene knockout), and the in vivo transfection effect of mice was detected using a small animal in vivo imaging device. The specific steps are as follows:

[0095] Female mice aged 6 weeks were selected and injected intramuscularly with 0.1 mL of Luc-LNPs (mRNA concentration of 100 μg / mL) prepared in Example 1. 12 h after the injection of the above Luc-LNPs, the mice were intraperitoneally injected with 200 μL of D-luciferin substrate (Novizan, 150 mg / kg), and the bioluminescence in the mice was detected using an IVIS imager. IVIS LivingImage software (Perkin Elmer) was used for image acquisition and analysis. The results are shown in Figure 2. Figure 2 The results showed that the in vivo transfection efficiency of NPC1 KO mice was significantly higher than that of WT mice, and its in vivo transfection efficiency was twice that of WT mice. This shows that NPC1 is one of the key regulatory factors for improving the transfection efficiency of the in vivo mRNA delivery system.

[0096] Example 4 Transfection screening assay for optimal NPC1 inhibitors

[0097] According to the above examples, NPC1 is determined to be a key factor affecting transfection efficiency. To this end, this example screened some NPC1 inhibitors (including posaconazole, U18666A or itraconazole) in order to select the small molecules with the greatest impact on the transfection effect for subsequent NPC1 inhibitor and mRNA delivery system combination tests. The specific experimental steps are as follows:

[0098] (1) Preparation of Luc-LNPs: Prepare according to the steps of Example 1.

[0099] (2) Cell culture: WT HEK293T cells were cultured according to the steps of Example 2. After the cells adhered to the wall and grew to 70%-80%, Luc-LNPs containing 50ng or 250ng of mRNA were added to each well, and 100μL of complete culture medium containing Posaconazole (1 or 5μM), U18666A (1 or 5μM) or Itraconazole (1 or 5μM) was added for transfection and culture for 48h. The control group (control) was cultured in the same manner as above, except that the control group did not add NPC1 inhibitor.

[0100] Transfection result detection: After 48 hours of transfection culture, add Bio-Lite Luciferase Assay System and use a microplate reader to detect the luciferase chemiluminescence value.

[0101] The results are as follows Figure 3 As shown, the transfection results of the selected NPC1 inhibitors showed that the transfection effect of posaconazole was significantly better than that of other small molecule inhibitors, and when 5 μM posaconazole was added, the transfection efficiency was about 6 times that of 1 μM posaconazole.

[0102] Example 5 Determination of transfection in different cells using NPC1 inhibitor and LNPs delivery system

[0103] This example uses different cells to test the transfection efficiency of the NPC1 inhibitor and mRNA delivery system. The specific steps are as follows:

[0104] (1) Primary BMDCs cell culture: Cells were obtained from the tibia and femur of mice (6 weeks old, female), and red blood cells were lysed (purchased from Yisheng, 40401ES) and washed. Then, the cells were seeded at 100,000 / well and cultured in a 96-well cell plate. The medium of BMDCs was changed every other day, and transfection was performed on the 7th day.

[0105] (2) Cultivation of passage cells: Culture was performed according to Example 2. When the cells grew to 70%-80%, Luc-LNPs containing 50 ng or 250 ng of Luc-mRNA were added to each well. At the same time, 100 μL of complete culture medium containing posaconazole (1 or 5 μM) was added and the transfection efficiency was detected after continuing the transfection culture for 48 h.

[0106] Transfection result detection: After 48 hours of transfection culture, add Bio-Lite Luciferase Assay System and use a microplate reader to detect the luciferase chemiluminescence value.

[0107] The results are as follows Figure 4 It was shown that the transfection effect of NPC1 inhibitor combined with LNPs delivery system in BMDCs cells was significantly better than that of LNPs transfection alone.

[0108] This example also uses RAW264.7, MH-S, and B16F10 to detect the transfection efficiency of the NPC1 inhibitor and mRNA delivery system. The specific process is the same as the transfection process of BMDCs cells. The difference is that the culture method of each cell is cultured according to its corresponding conventional method. The results are shown in Figure 4 As shown, consistent with the BMDCs transfection results, the transfection effect of the NPC1 inhibitor combined with the mRNA delivery system (Luc-LNPs) in the above cells was significantly better than that of Luc-LNPs alone.

[0109] Example 6 Transfection assay using NPC1 inhibitor and Lip3000 delivery system

[0110] In this example, HEK293T cells were used to detect the transfection efficiency of NPC1 inhibitor and Lipofectamine 3000 (Lip3000) delivery system. The specific steps are as follows:

[0111] (1) The steps of coating Luc-mRNA with Lip3000 (purchased from Thermo Fisher Scientific) were prepared according to the reagent instruction manual.

[0112] (2) Cell culture: After digesting the HEK293T cells in the culture dish with trypsin for 30 seconds, complete culture medium was added to terminate the trypsin. The digested HEK293T cells were collected and placed in a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The culture medium containing trypsin was discarded, and fresh complete culture medium was added. The cells were resuspended and counted (1×10 5 Cells were inoculated into 96-well plates at 100 μL per well and placed in a 37°C CO2 incubator to allow the cells to adhere to the wall and grow to 70%-80%. Subsequently, 50 ng or 250 ng of Lip3000-coated Luc-mRNA was added to each well, and 100 μL of complete culture medium containing Posaconazole (1 or 5 μM) was added to carry out transfection and culture was continued for 48 h.

[0113] (3) Transfection result detection: After 48 h of transfection culture, add the Bio-Lite Luciferase Assay System and use a microplate reader to detect the luciferase chemiluminescence value.

[0114] The results are as follows Figure 5 It was shown that the transfection effect of NPC1 inhibitor combined with Lip3000 delivery system in HEK293T cells was significantly better than that of Lip3000 transfection alone.

[0115] Example 7 Determination of LNP retention in cells using a combination of NPC1 inhibitors and mRNA delivery systems

[0116] HEK293T cells were seeded in 24-well plates at a density of 100,000 / mL, and the cells were allowed to adhere to the wall to grow to a density of about 70%-80%. 1 mL of complete medium containing NPC1 inhibitor posaconazole (0 or 5 μM) was added to the cells, and DIO-labeled Luc-LNPs (containing 500 ng mRNA) or Cy5-labeled eGFP-LNPs (containing 500 ng mRNA) were added for 12 hours. The cells were collected and washed twice with PBS, and the accumulated fluorescence intensity of DIO or Cy5 in the cells and the fluorescence intensity of the expressed eGFP were detected by flow cytometry.

[0117] At the same time, laser confocal microscopy was also used to detect the accumulation of LNPs in cells. Briefly, HEK293T cells were cultured at a density of 100,000 / mL in a confocal dish. After 24 hours of cell attachment, 1 mL of complete medium containing the NPC1 inhibitor posaconazole (0 or 5 μM) was added to the cells, and DIO-labeled Luc-LNPs (containing 500 ng mRNA) or Cy5-labeled eGFP-LNPs (containing 500 ng mRNA) were added for 12 hours. The cells were washed three times with cold PBS, fixed with 4% paraformaldehyde at 4°C for 15 minutes, and then washed three times with cold PBS. DAPI staining was then added for 5 minutes at room temperature, the cells were washed with PBS, and observed using a laser confocal microscope.

[0118] The results are as follows Figure 6 The results showed that the combination of NPC1 inhibitor and mRNA delivery system can increase the retention time of LNP in cells. Figure 6 A and Figure 6 B) shows that the combination of NPC1 inhibitor and mRNA delivery system significantly enhanced the fluorescence intensity of Cy5 and eGFP. Similarly, the laser confocal microscope results ( Figure 6 C, Figure 6 D and Figure 6 E) shows that the combination of NPC1 inhibitor and mRNA delivery system for 12 or 24h significantly increased the intracellular fluorescence intensity of Cy5 and eGFP. Interestingly, compared with 12h, the fluorescence intensity of Cy5 in the control group decreased significantly at 24h, while the fluorescence intensity of Cy5 in the group combined with NPC1 inhibitor and mRNA delivery system showed only a slight downward trend.

[0119] In addition, to prove that the intracellular retention of LNPs induced by the combination of NPC1 inhibitor and mRNA delivery system is not limited to specific mRNA, this example uses DIO-labeled Luc-LNPs to transfect into cells and demonstrates similar results ( Figure 6 F. Figure 6 G and Figure 6 H). These results suggest that the combination of NPC1 inhibitors and mRNA delivery systems can lead to the accumulation of more mRNA delivery systems in cells, which is beneficial for the endosomal-lysosomal escape of mRNA and mRNA translation.

[0120] Example 8 Determination of the effects of NPC1 inhibitor combined with mRNA delivery system on the endosomal-lysosomal system

[0121] HEK293T cells were cultured at a density of 100,000 / mL in a confocal dish. After the cells were grown for 24 hours, 1 mL of complete medium containing NPC1 inhibitor posaconazole (0 or 5 μM) was added to the cells, and DIO-labeled Luc-LNPs (containing 500 ng mRNA) were added to culture the cells for 12 hours.

[0122] Immunofluorescence staining: cells were fixed with 4% paraformaldehyde at 4°C for 15 min, washed three times with pre-cooled PBS, permeabilized with PBS containing 0.3% Triton X-100 for 15 min at room temperature, and blocked with blocking buffer (1×PBS, 5% BSA, 0.3% Triton TM X-100) to block the cells for 60 min. Aspirate the blocking buffer and add primary antibodies (Rab5 for early endosomes, Rab7 for late endosomes, LAMP1 for lysosomes) and buffer (1×PBS, 1% BSA, 0.3% Triton TM X-100) at 4°C overnight. Then rinse with PBS three times and add fluorescently conjugated secondary antibody (1×PBS, 1% BSA, 0.3% Triton TM X-100) and incubated at room temperature in the dark for 2h, rinsed 3 times with PBS. Finally, DAPI was added for staining at room temperature for 5min, and the cells were washed with PBS and observed using a laser confocal microscope. All confocal images were taken using a 60X oil objective lens of an inverted fluorescence microscope (Nikon). A precise 5X magnifying glass was used to take the detailed images of individual cells.

[0123] The results are as follows Figure 7 It can be seen from the laser confocal scanning microscopy that the combination of NPC1 inhibitor and mRNA delivery system can significantly promote the production of Rab7 but does not affect the production of Rab5 and LAMP1. In addition, the combination of NPC1 inhibitor and mRNA delivery system promotes the accumulation of DIO-labeled mRNA delivery system in early endosomes, late endosomes and lysosomes. These results show that the accumulation of mRNA delivery system in cells caused by NPC1 inhibition is mainly due to the increased production of late endosomes, which greatly increases the chance of LNPs escape.

[0124] Example 9 Preparation of mRNA Pharmaceutical Composition P-LNPs

[0125] A 2% w / v NaOH solution, a 10 mM citric acid-sodium citrate buffer solution and an mRNA solution were prepared according to the method described in Example 1; regarding the preparation of the organic phase of the mixture: ethanol was used as the solvent, and each organic phase was prepared according to the molar ratio of each component in Table 1 to prepare a mixture organic phase with a concentration of 2.85 mg / mL for the preparation of an mRNA pharmaceutical composition (NPC1 inhibitor-lipid nanoparticles, P-LNPs).

[0126] Table 1 Molar ratio of each component in different organic phases

[0127]

[0128] After the above solution is prepared, 2.2 mL of mRNA solution with a concentration of 0.05 mg / mL (the mRNA used here is mRNA encoding luciferase) is drawn with a 2.5 mL Xinhua syringe and 0.8 mL of a mixed lipid solution (organic phase 0 to 9) with a concentration of 2.85 mg / mL is drawn with a 1 mL Xinhua syringe, and inserted into a microfluidic chip (purchased from Maianna, model SHM), and the parameters are set as follows: volume = 2.67 mL, flow ratio of mRNA solution to mixed lipid organic phase = 3:1, total flow = 12 mL / min, front waste liquid = 0.35 mL, rear waste liquid = 0.10 mL, and about 2.22 mL of P-LNPs solution is obtained. P-LNPs0 to P-LNPs9 solutions are prepared in sequence using organic phases 0 to 9 shown in Table 1.

[0129] The prepared P-LNPs0 to P-LNPs9 solutions were diluted with 3 volumes of DPBS and transferred to the ultrafiltration tube prepared according to the method described in Example 1 for centrifugal ultrafiltration, and the ultrafiltration parameters were: 2500rpm, 4°C, 30min. When the sample in the centrifuge tube was 0.5-1.0mL, about 4 volumes of DPBS were added again for centrifugal ultrafiltration (2500rpm, 4°C, 30min); when the sample in the centrifuge tube was less than 0.5-1mL, about 3 volumes of DPBS were added, and ultrafiltration (2500rpm, 4°C, 30min) was performed until it was less than 0.5mL, and the sample was collected, and the volume was fixed to 0.7mL with DPBS, and stored in a 4°C refrigerator.

[0130] The above steps were used to complete the preparation of the mRNA pharmaceutical composition P-LNPs0 to P-LNPs9, and used for further characterization and encapsulation efficiency determination to screen out the optimal mRNA pharmaceutical composition preparation scheme.

[0131] Example 10 Characterization of mRNA pharmaceutical composition and encapsulation efficiency determination

[0132] After screening out the NPC1 inhibitor posaconazole and the mRNA delivery system for the greatest effect on the delivery effect through the above examples, this example integrates and prepares the optimal P-LNPs by optimizing the molar ratio of posaconazole in the mRNA delivery system, and the preparation steps are as in Example 9.

[0133] 1. Particle size, potential, and polydispersity index (PDI) determination of mRNA delivery system:

[0134] The mRNA pharmaceutical composition prepared in Example 9 was diluted 200 times with DPBS and measured with a Malvern particle size potentiometer.

[0135] 2. Determination of encapsulation efficiency of mRNA delivery system:

[0136] The characterization and encapsulation efficiency were determined by the method described in step 5 "mRNA concentration detection and volume determination of mRNA delivery system" of Example 1. The best preparation method was comprehensively screened out based on the characterization and encapsulation efficiency (as shown in Table 2). The results showed that the use of NPC1 inhibitors to replace cholesterol (organic phase 4, organic phase 5, organic phase 6) and PEG (organic phase 1, organic phase 2, organic phase 3) increased the particle size of the prepared LNPs, and the PDI increased and was greater than 0.3. However, the particle sizes of P-LNPs7, P-LNPs8 and P-LNPs9 prepared by using NPC1 inhibitors to replace a certain molar ratio of DSPC (organic phase 7, organic phase 8, organic phase 9) were approximately between 100 and 120 nm, the PDI was stable and less than 0.3, and the encapsulation efficiency was greater than 95%. PDI reflects the uniformity of the size distribution of P-LNPs. The lower the PDI value, the more uniform the size distribution of P-LNPs. It can be seen that using NPC1 inhibitors to replace a certain molar ratio of DSPC to prepare P-LNPs is an effective method to integrate small NPC1 inhibitors into mRNA delivery systems.

[0137] Table 2 Performance evaluation of mRNA delivery system (P-LNPs)

[0138]

[0139] The results are shown in Table 2. Compared with P-LNPs0 (92 nm), the particle size of P-LNPs1 to 9 prepared after the integration of NPC1 inhibitor posaconazole and mRNA delivery system increased slightly. The comprehensive evaluation of particle size, PDI and encapsulation efficiency showed that formulations P-LNPs7, P-LNPs8 and P-LNPs9 with a particle size of approximately 100 nm, a PDI of less than 0.3 and an encapsulation efficiency of greater than 95% had similar physicochemical properties to LNPs. These results show that P-LNPs7, P-LNPs8 and P-LNPs9 are ideal candidates for mRNA drug compositions containing posaconazole.

[0140] In addition, HEK293T cells were transfected with LNPs (i.e., P-LNPs0), P-LNPs7, P-LNPs8, and P-LNPs9 according to the method described in Example 2. Figure 8 The results showed that the delivery efficiency of P-LNPs7, P-LNPs8, and P-LNPs9 was significantly higher than that of LNPs, and the efficiency increased with the increase of posaconazole content. Therefore, P-LNPs9, as the best LNPs containing posaconazole, was renamed as P-LNPs and used in subsequent experiments.

[0141] Example 11 Determination of in vivo transfection of mRNA pharmaceutical composition prepared by combining NPC1 inhibitor and mRNA delivery system

[0142] To detect the in vivo transfection effect of the mRNA pharmaceutical composition P-LNPs prepared by combining the NPC1 inhibitor and the mRNA delivery system, the organic phase 0 and organic phase 9 in Table 1 of Example 9 were used to prepare LNPs and P-LNPs containing Luc-mRNA, respectively. 6-week-old mice were injected intramuscularly with 0.1 mL of LNPs or P-LNPs, respectively, so that the total mRNA injection concentration in each mouse was 10 μg. 12 hours after the injection, the mice were intraperitoneally injected with 200 μL of D-luciferin substrate (Novizan, 150 mg / kg), and the bioluminescence of mice or organs was detected using an IVIS imager. Image acquisition and analysis were performed using IVIS Living Image software (Perkin Elmer).

[0143] The results are as follows Fig. 9 The results showed that the in vivo transfection efficiency of mice injected with P-LNPs prepared by combining NPC1 inhibitor and mRNA delivery system was significantly higher than that of mice injected with LNPs, and the in vivo delivery efficiency of P-LNPs was about 2 times that of LNPs. This fully proves that the combination of NPC1 inhibitor and mRNA delivery system can significantly improve the delivery efficiency of mRNA delivery system in vivo.

[0144] Example 12 Combination of NPC1 inhibitor and mRNA delivery system to prepare mRNA pharmaceutical composition containing RBD-mRNA for determination of immune effect of immunized mice

[0145] In order to detect the in vivo delivery effect of P-LNPs prepared by combining NPC1 inhibitors and mRNA delivery systems, organic phase 0 and organic phase 9 in Table 1 of Example 9 were used to prepare LNPs and P-LNPs containing RBD-mRNA (purchased from Novazon Biotech Co., Ltd.). Six-week-old mice were injected intramuscularly with 0.1 mL of LNPs or P-LNPs on days 0 and 14, respectively, and the RBD-mRNA concentration was 10 μg / mouse / time. On days 21 and 28, mouse blood was collected to detect the content of specific RBD antibodies in mouse serum. The detection steps are as follows:

[0146] (1) Coating: Coat the ELISA plate with carbonate buffer (pH = 9.6) containing 1 ug / mL RBD, 100 uL per well, incubate at 4°C overnight, discard the supernatant, pat dry, and wash three times with 1x PBST (pH = 7.4);

[0147] (2) Blocking: Block the coated plate with PBS containing 2% BSA, 200uL per well. Incubate at 37°C for 2h, and wash 3 times with 1x PBST;

[0148] (3) Adding primary antibody: Add 100 μL of serum to be tested to each well, repeat two wells for each sample, incubate at 37°C for 1 h, and wash 3 times with 1xPBST;

[0149] (4) Adding secondary antibody: add 100 μL HRP-labeled goat anti-mouse secondary antibody (purchased from Wuhan Tri-Taiwan Biotechnology Co., Ltd.) to each well, incubate at 37°C for 1 h, and wash three times with 1xPBST;

[0150] (5) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37°C in the dark for 7 min.

[0151] (6) Termination: Add 50 μL of H2SO4 (2 M) to each well to terminate the reaction.

[0152] (7) Reading: Measure OD using a microplate reader 450 The value in nm.

[0153] The results are as follows Fig.10 The results showed that the RBD-specific IgG antibody levels of P-LNPs were significantly higher than those of LNPs at both 21 and 28 days, and the RBD-specific IgG antibody level on day 28 was higher than that on day 21. The results showed that the combination of NPC1 inhibitors and mRNA delivery systems can significantly improve the delivery efficiency of the mRNA delivery system in vivo and enhance the specific antibody effect of RBD-mRNA vaccines in vivo.

[0154] The present invention provides an mRNA pharmaceutical composition and a method for preparing and applying the same. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be noted that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. An mRNA pharmaceutical composition, characterized in that: It contains a C1-type Niemann-Pick protein inhibitor and an mRNA drug.

2. The mRNA pharmaceutical composition according to claim 1, characterized in that The C1-type Niemann-Pick protein inhibitor is any one or more combinations of posaconazole, U18666A and itraconazole; The mRNA drug is a delivery system containing mRNA drugs, and the delivery system is lipid nanoparticles, polymer nanoparticles, inorganic nanoparticles, protein-based virus-like nanoparticles, Lipofectamine 3000, or an electroporation delivery system; the mRNA drug is preferably lipid nanoparticles containing mRNA drugs or Lipofectamine 3000 containing mRNA drugs.

3. The mRNA pharmaceutical composition according to claim 2, characterized in that The mRNA drug-containing lipid nanoparticles are composed of a carrier and the mRNA drug encapsulated in the carrier; The carrier comprises a combination of cationic lipids, auxiliary phospholipids, cholesterol and polyethylene glycol lipids; The mRNA drug is an mRNA vaccine or an mRNA protein replacement therapy drug.

4. The mRNA pharmaceutical composition according to claim 3, characterized in that The molar ratio of the cationic lipid, auxiliary phospholipid, cholesterol, polyethylene glycol lipid and C1 type Niemann-Pick protein inhibitor is 50:9.6-10:38.1-38.5:1.1-1.5:0.01-0.

4.

5. The method for preparing the mRNA pharmaceutical composition according to any one of claims 1 to 4, characterized in that: The C1-type Niemann-Pick protein inhibitor and the mRNA drug are combined by any of the following methods: direct mixing, encapsulation, or modification.

6. The preparation method according to claim 5, characterized in that: The C1 type Niemann-Pick protein inhibitor and the mRNA drug are combined by encapsulation.

7. The preparation method according to claim 6, characterized in that: The steps include: (1) preparing a mixture organic phase: mixing a C1 type Niemann-Pick protein inhibitor, a cationic lipid, an auxiliary phospholipid, cholesterol and a polyethylene glycol lipid in an organic solvent to obtain a mixture organic phase; (2) preparing an mRNA drug aqueous phase: dissolving the mRNA drug in a buffer solution to obtain an mRNA drug aqueous phase; (3) mixing the organic phase of the mixture and the aqueous phase of the mRNA drug by microfluidics to obtain a mixed solution; The mixed solution is ultrafiltered through an ultrafiltration tube to obtain the product.

8. The preparation method according to claim 7, characterized in that: In step (1), the organic solvent includes any one or more combinations of ethanol, isopropanol, chloroform, dichloromethane, methanol, n-hexane and ethyl acetate; In the organic phase of the mixture, the total concentration of cationic lipids, auxiliary phospholipids, cholesterol, polyethylene glycol lipids and C1 type Niemann-Pick protein inhibitor is 2.85 mg / mL.

9. The preparation method according to claim 7, characterized in that: In step (2), the buffer solution is a citric acid-sodium citrate buffer solution with an ion concentration of 10 mM; and the concentration of the mRNA drug in the mRNA drug aqueous phase is 0.05 mg / mL.

10. The use according to claim 7, characterized in that: In step (3), the microfluidic mixing method has a flow rate ratio of 1:2 to 10 between the organic phase of the mixture and the mRNA drug aqueous phase, and a total flow rate of 8 to 16 mL / min; the molecular weight cutoff of the ultrafiltration tube is 10 KDa.

11. Use of the mRNA pharmaceutical composition according to any one of claims 1 to 4 in the preparation of mRNA drugs for preventing and / or treating diseases.

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