mRNA pharmaceutical compositions, methods of making and using the same

Abstract

The present application relates to the technical field of mRNA therapy, and particularly relates to an mRNA drug composition for improving mRNA delivery efficiency by endosome escape, a C1 type niemann-pick protein (NPC1) inhibitor and mRNA drug combination, and a preparation method and application thereof. The present application relates to an NPC1 inhibitor for improving mRNA delivery system efficiency. The mRNA drug composition disclosed by the present application significantly improves the in vitro and in vivo 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, makes the mRNA delivery system escape from endosomes and lysosomes to the cytoplasm, and expresses more effective proteins. The combination of the NPC1 inhibitor and the mRNA delivery system obtained by the present application has important application prospects in enhancing the preventive and therapeutic effects of mRNA drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mRNA therapy, in particular to an mRNA drug composition for improving mRNA delivery efficiency by endosome escape, an NPC1 inhibitor and mRNA drug combination, and a preparation method and application thereof. BACKGROUND

[0002] Based on the advancement, innovation, safety, personalized potential and multifunctionality of mRNA therapy, mRNA therapy has become a new star in the field of biomedicine. mRNA therapy has made significant progress in some fields including new crown, cancer treatment, genetic disease, immunotherapy, etc., and has shown potential in clinical application (1-4). Intracellular delivery of mRNA therapy has great potential for vaccine and therapy development. Delivery vectors for mRNA therapy include lipid nanoparticles, polymeric nanoparticles, inorganic nanoparticles, protein-based virosome nanoparticles, electroporation, etc. However, the successful application of mRNA therapy depends largely on the engineering design of mRNA delivery system and intracellular delivery and intracellular processing of mRNA to avoid their degradation by enzymes in the circulatory system and promote endosome escape.

[0003] A key determinant for mRNA delivery vehicles to be efficacious is the ability of mRNA to escape the endosomal-lysosomal system after reaching the target tissue, to gain access to the cytoplasm for translation and protein expression. Studies have shown that only less than 2% of mRNA delivered by delivery vehicles can escape the endosomal-lysosomal system to remain in the cytoplasm (5-11). The endosomal-lysosomal escape of mRNA after delivery into cells is limited in time and space. The time for mRNA to enter cells via delivery vehicles and reach the endosome or lysosome is generally between minutes to hours, depending on the cell type and the properties of the vehicle. For example, the process of lipid nanoparticle-mediated delivery of mRNA into cells via endocytosis usually occurs within minutes, and the endocytosed mRNA can be transported to early endosomes within minutes under the encapsulation of vesicles. As the endosome matures, the early endosome will transform into a late endosome, accompanied by a decrease in pH and an increase in enzyme activity in the endosomal environment, which usually takes 30 minutes to several hours. If mRNA fails to escape successfully at the endosomal stage, it will be further transported to the lysosome for degradation or escape. Overall, the transport of mRNA from endocytosis to lysosome can be completed within a few hours. Therefore, endosomal-lysosomal escape usually has to be completed within this time to avoid mRNA degradation (5, 8, 12, 13). In addition, the endosomal-lysosomal escape of mRNA is not only time-constrained, but also limited by the structure and properties of the endosomal membrane (membrane composition, fluidity, stability, pH change, enzyme activity), endosomal-lysosomal transformation (maturation process of endosome and endosomal-lysosomal fusion process), membrane fusion mechanism (membrane rupture and fusion and pH dependence), and spatial barriers inside the endosome (physical structure and local environmental differences inside the endosome) (9, 14-18). The mRNA escape event in the endosomal-lysosomal pathway usually occurs at certain specific regions or stages of the endosomal membrane, and does not occur uniformly throughout the endosome. Therefore, breaking through the endosomal-lysosomal escape of mRNA and prolonging the residence time of mRNA in the cytoplasm is a key step to break the limitation of mRNA delivery.

[0004] Niemann-Pick Type C-1 (NPC1), a transmembrane protein located on late endosomes / lysosomes, consists of 1278 amino acid residues, contains 13 transmembrane helices, and has three large luminal domains that mediate the distribution of lipoprotein-derived cholesterol in cells. NPC1 is mainly responsible for cholesterol transport in vivo, and its amino acid mutations can cause "Niemann-Pick disease", which is manifested as delayed neurodevelopment in children, cognitive impairment or deafness, and can even lead to the death of children in severe cases (19-21). NPC1 has been identified as an important regulator of the main circulating pathway for delivering siRNA. NPC1 protein plays a key role in regulating the transport and metabolism of cholesterol, and its defects can lead to abnormal accumulation of cholesterol in lysosomes, triggering lysosomal morphological, transport and functional defects. NPC1 can expel siRNA containing LNP delivery carriers to the extracellular; 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 can enhance the accumulation of intracellular cholesterol by inhibiting the NPC1 target, and inhibit the cytoplasmic transmission of the virus (24). These studies all suggest that the combination of small molecule NPC1 inhibitors with siRNA delivery systems is an attractive strategy to improve the efficacy of siRNA systems by improving the cytoplasmic retention of siRNA. However, the study of NPC1 inhibitors for improving the efficiency of mRNA delivery systems is still blank. The development of mRNA delivery systems that can extend the cytoplasmic retention of mRNA through endosome-lysosome escape to improve the delivery efficiency of mRNA delivery systems is imminent.

[0005] References

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

[0030] To achieve the above invention purposes, the technical solutions provided by the present invention are as follows:

[0031] An mRNA pharmaceutical composition comprising a C1 type Niemann-Pick protein inhibitor (NPC1) and an mRNA pharmaceutical. The NPC1 inhibitor and the mRNA pharmaceutical are combined to improve the delivery efficiency of the mRNA pharmaceutical. The NPC1 inhibitor improves the delivery efficiency by facilitating endo-lysosomal escape of the mRNA pharmaceutical delivery system.

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

[0033] The mRNA pharmaceutical is an mRNA pharmaceutical-containing delivery system, which is a lipid nanoparticle, a polymer nanoparticle, an inorganic nanoparticle, a protein-based virosome nanoparticle, Lipofectamine 3000, or an electroporation delivery system; preferably an mRNA pharmaceutical-containing lipid nanoparticle or an mRNA pharmaceutical-containing Lipofectamine 3000.

[0034] The mRNA pharmaceutical-containing lipid nanoparticle is composed of a carrier and an mRNA pharmaceutical encapsulated in the carrier; the carrier includes a combination of a cationic lipid, an auxiliary phospholipid, cholesterol and a polyethylene glycol lipid; the mRNA pharmaceutical is an mRNA vaccine or an mRNA protein replacement therapy drug, and the mRNA pharmaceutical is an mRNA drug dependent on mRNA translation.

[0035] Preferably, the cationic lipid includes but is not limited to 4(N,N dimethylamino) butanoic acid (dilinoleyl) methyl ester (Dlin-MC3-DMA) or ((4-hydroxybutyl) azabicycloalkyl) bis(hexane-6,1-diyl) bis(2-hexyldecyl ester) (ALC0315); the auxiliary phospholipid includes but is not limited to 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); and the polyethylene glycol lipid includes but is not limited to 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol 2000 (PEG-DMG-2000).

[0036] The molar ratio of the cationic lipid, the helper phospholipid, the cholesterol, the polyethylene glycol lipid and the inhibitor of the C1 type of Niemann-Pick protein 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, the helper phospholipid, the cholesterol, the polyethylene glycol lipid and the inhibitor of the C1 type of Niemann-Pick protein 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 application provides a preparation method of the mRNA drug composition of the first aspect, wherein the inhibitor of the C1 type of Niemann-Pick protein and the mRNA drug are combined by any one of the following methods: direct mixing, encapsulation or modification. Preferably, the inhibitor of the C1 type of Niemann-Pick protein and the mRNA drug are combined by encapsulation. The preparation method comprises the following steps:

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

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

[0040] (3) mixing the mixture organic phase and the mRNA drug aqueous phase by microfluidization to obtain a mixed solution, and then performing ultrafiltration on the mixed solution by using an ultrafiltration tube to obtain the mRNA drug composition.

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

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

[0043] In step (3), the microfluidic method mixing is carried out, the flow rate ratio of the organic phase of the mixture and 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 and 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 application provides the use of the mRNA drug composition of the first aspect in the preparation of an mRNA drug for preventing and / or treating diseases.

[0045] Advantages:

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

[0047] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

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

[0049] Figure 2 Fig. 2 is the result of in vivo transfection of WT mice and NPC1 KO mice injected with Luc-LNPs, Figure 2 A is an IVIS imaging diagram, Figure 2 B is a quantitative diagram of in vivo transfection of mice (CPS is the quantitative unit).

[0050] Figure 3 Fig. 3 is the transfection result of different NPC1 inhibitors combined with the mRNA delivery system, Figure 3 A is the transfection result of Posaconazole combined with the mRNA delivery system, Figure 3 B is the transfection result of U18666A combined with the mRNA delivery system.Figure 3 C is the transfection result of Itraconazole and mRNA delivery system combination.

[0051] Figure 4 The result of NPC1 inhibitor and LNP delivery system combination on different cell transfection.

[0052] Figure 5 The result of NPC1 inhibitor and Lip3000 delivery system combination transfection.

[0053] Figure 6 The result of NPC1 inhibitor and mRNA delivery system combination on mRNA retention in cells, Figure 6 A is the flow cytometry result graph of Cy5, Figure 6 B is the flow cytometry result graph of eGFP, Figure 6 C is the laser confocal scanning microscope graph of Cy5 and eGFP, Figure 6 D is the laser confocal result graph of Cy5, Figure 6 E is the laser confocal result graph of eGFP, Figure 6 F is the flow cytometry result graph of DIO, Figure 6 G is the laser confocal scanning microscope graph of DIO, Figure 6 H is the laser confocal result graph of DIO.

[0054] Figure 7 The result of NPC1 inhibitor and mRNA delivery system combination on the influence of endosome-lysosome system, Figure 7 A is the influence of NPC1 inhibitor and mRNA delivery system combination on early endosome Rab5, Figure 7 B is the influence of NPC1 inhibitor and mRNA delivery system combination on late endosome Rab7, Figure 7 C is the influence of NPC1 inhibitor and mRNA delivery system combination on lysosome LAMP1.

[0055] Figure 8 The in vitro transfection result of mRNA drug composition prepared by using NPC1 inhibitor and mRNA delivery system combination.

[0056] Figure 9 The in vivo transfection result of mRNA drug composition prepared by using NPC1 inhibitor and mRNA delivery system combination. Figure 10 The result of using RBD-mRNA containing P-LNPs prepared by using NPC1 inhibitor and mRNA delivery system combination to immunize mice. DETAILED DESCRIPTION

[0057] The application will be further described in conjunction with the preferred embodiments thereof with reference to the accompanying drawings. 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, the w / v, unless otherwise specified, represents g / mL; the v / v, unless otherwise specified, represents mL / mL.

[0059] In the following examples, the PBS, unless otherwise specified, represents 1xPBS with pH=7.4; the DPBS, unless otherwise specified, represents 1xDPBS with pH=7.4.

[0060] In the following examples, the complete medium is DMEM medium containing 10% v / v fetal bovine serum (FBS) and 1% v / v double antibiotics, and the double antibiotics are 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: weigh 10 g of NaOH solid and dissolve in 500 mL of sterilized water, filter sterilize with a 0.22 μm filter membrane after complete dissolution, and reserve for use;

[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, dissolve in 500 mL of RNase-free water to prepare a 0.1 M pH=4 citric acid-sodium citrate buffer solution, filter sterilize with a 0.22 μm filter membrane. After diluting the 0.1 M pH=4 citric acid-sodium citrate buffer solution with RNase-free water, prepare a 10 mM citric acid-sodium citrate buffer solution for use.

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

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

[0067] 2. Preparation of ultrafiltration tube:

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

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

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

[0071] (4) Discard the NaOH solution in the ultrafiltration tube and soak it with PBS buffer solution for more than 5 min twice;

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

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

[0074] Take 2.2 mL of mRNA solution with a concentration of 0.05 mg / mL prepared in step 1 and 0.8 mL of mixed organic phase with a concentration of 2.85 mg / mL prepared in step 1 with 2.5 mL of Xinhua syringe, insert into the microfluidic chip (purchased from Maianna, model SHM), set the parameters 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 = 0.35 mL, rear waste = 0.10 mL, obtain about 2.22 mL of LNPs solution for subsequent ultrafiltration and encapsulation rate detection.

[0075] 4. Ultrafiltration:

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

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

[0078] Quant-iT TM RNA Reagent and Kit were used to detect the mRNA encapsulation rate of mRNA delivery system (LNPs) on mRNA, and the specific steps were as follows:

[0079] (1) Dilute 20xTE buffer with enzyme-free water to 1xTE buffer for standby;

[0080] (2) Dilute QuantiT RiboGreen Reagen with 1xTE buffer by 200 times for standby;

[0081] (3) Dilute Triton-100 with 1xTE buffer to 2% v / v Triton-100 solution for standby;

[0082] (4) Dilute the standard in Quant-iT TM RNA Reagent and Kit with 1xTE buffer to 2 μg / mL. Prepare standard curve solutions with concentrations of 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; and 0 ng / mL.

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

[0084] (6) Take 100 μL of each of the standard, TE buffer sample solution and Triton-100 sample solution, add to a 96-well plate, with 6 repeats for each sample, then add 100 μL of 200-fold diluted QuantiT RiboGreen Reagen for color development. Vibrate for 1 min, and detect the fluorescence value at 480-520 nm.

[0085] (7) Calculate the concentration of mRNA contained in the LNPs according to the obtained fluorescence value, and use DPBS to dilute the ultrafiltrated LNPs to a concentration of 100 μg / mL of RNA contained. The concentration of mRNA in the mRNA delivery system used in the following tests is 100 μg / mL.

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

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

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

[0089] (2) Cell culture: After trypsinizing the WT HEK293T cells or NPC1 KO HEK293T cells in the culture dish for 30 s, add complete culture medium to terminate trypsinization, collect the digested cells into a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. Discard the trypsin-containing culture medium, add fresh complete culture medium, resuspend the cells, and count (1 × 10 5 The cells were inoculated in a 96-well plate at a volume of 100 μL per well, and incubated in a 37°C CO2 incubator to allow the cells to adhere and grow to 70%-80%. Then, 50 ng or 250 ng of mRNA-containing Luc-LNPs or eGFP-LNPs were added to each well for transfection, and incubated for 48 h.

[0090] Transfection result detection: To detect Luc-LNPs, after 48 hours of transfection culture, a Bio-Lite Luciferase Assay System was added, and the luciferase signal value was detected using a microplate reader. For the detection of eGFP-LNPs, the fluorescence intensity at 480-520 nm was detected using a microplate reader after 48 hours of culture.

[0091] The test results are as follows Figure 1 As shown, the detection results of Luc-LNPs transfection show ( Figure 1 A) Transfection efficiency is positively correlated with mRNA concentration; increasing mRNA concentration increases transfection efficiency. Furthermore, the transfection efficiency of NPC1 KO HEK293T cells was significantly higher than that of WT HEK293T cells. At an mRNA concentration of 50 ng, the transfection efficiency of NPC1 KO HEK293T cells was approximately three times that of WT HEK293T cells; and at an mRNA concentration of 250 ng, the transfection efficiency of NPC1 KO HEK293T cells was approximately eight times that of WT HEK293T cells.

[0092] The detection results of eGFP-LNPs transfection showed that ( Figure 1 (B) Transfection efficiency followed the trend of Luc-LNPs, with increasing mRNA concentration leading to increased transfection efficiency. Furthermore, the transfection efficiency of NPC1 KO HEK293T cells was significantly higher than that of WT HEK293T cells. At an mRNA concentration of 50 ng, the transfection efficiency of NPC1 KO HEK293T cells was approximately 6 times that of WT HEK293T cells; at an mRNA concentration of 250 ng, the transfection efficiency of NPC1 KO HEK293T cells was approximately 8 times that of WT HEK293T cells. This indicates that NPC1 is one of the key regulatory factors for improving the transfection efficiency of in vitro mRNA delivery systems.

[0093] Example 3: Determination of in vivo transfection of Luc-LNPs by intramuscular injection in WT mice and NPC1 KO mice

[0094] To further determine whether NPC1 is a key gene affecting transfection efficiency, Luc-LNPs were administered intramuscularly to WT mice and NPC1 KO mice (purchased from Cyagen Biosciences Ltd.; NPC1 KO mice are mice with the NPC1 gene knocked out). In vivo transfection efficacy in mice was then assessed using a small animal in vivo imaging system. The specific steps are as follows:

[0095] Female mice of 6 weeks old were selected, and the mice were injected with 0.1 mL of Luc-LNPs (mRNA concentration of 100 μg / mL) prepared in Example 1 by intramuscular injection. At 12 h after the above Luc-LNPs injection, the mice were injected with 200 μL of D-luciferin substrate (Novizen, 150 mg / kg) intraperitoneally, and the bioluminescence in the mice was detected using an IVIS imaging instrument. Image acquisition and analysis were performed using IVIS LivingImage software (Perkin Elmer). The results are shown in Figure 2 It was shown that the in vivo transfection efficiency of NPC1 KO mice was significantly higher than that of WT mice, which was 2 times that of WT mice. It can be seen 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 the best NPC1 inhibitor

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

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

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

[0100] Transfection result detection: after transfection culture for 48 h, Bio-Lite Luciferase Assay System was added, and the luciferase chemiluminescence value was detected using a microplate reader.

[0101] The results are shown in 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 the concentration of posaconazole added was 5 μM, the transfection efficiency was about 6 times that of the addition of 1 μM of posaconazole.

[0102] Example 5 Assay of the combination of NPC1 inhibitor and LNP delivery system in different cells

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

[0104] (1) Primary BMDCs cell culture: Take the cells of the tibia and femur of mice (6 weeks old, female), perform red blood cell lysis (purchased from Yixing, 40401ES) and washing, and inoculate the cells at 100000 per well, culture them in a 96-well cell plate, and change the BMDCs every other day, and perform transfection on the 7th day.

[0105] (2) Culture of passaged cells: culture according to Example 2, when the cells grow to 70%-80%, add Luc-LNPs containing 50ng or 250ng of Luc-mRNA in each well, and at the same time add 100μL of complete culture medium containing Posaconazole (1 or 5μM) to continue transfection culture for 48h before detecting the transfection efficiency.

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

[0107] Results as shown in Figure 4 The combination of NPC1 inhibitor and LNP delivery system in BMDCs cells has a significantly better transfection effect than LNP transfection alone.

[0108] This example also uses RAW264.7, MH-S, B16F10 to detect the transfection efficiency of the combination of NPC1 inhibitor and mRNA delivery system, and the specific process is the same as the transfection process of BMDCs cells, except that each cell is cultured according to its corresponding conventional method. Results as shown in Figure 4 Consistent with the BMDCs transfection results, the combination of NPC1 inhibitor and mRNA delivery system (Luc-LNPs) in the above-mentioned cells has a significantly better transfection effect than Luc-LNPs alone.

[0109] Example 6 Assay of the combination of NPC1 inhibitor and Lip3000 delivery system

[0110] This example uses HEK293T cells to detect the transfection efficiency of the combination of NPC1 inhibitor and Lipofectamine 3000 (Lip3000) delivery system. The specific steps are as follows:

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

[0112] (2) Cell culture: After trypsinizing the HEK293T cells in the culture dish for 30s, add complete medium to terminate trypsin, collect the digested HEK293T cells into a 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes. Discard the trypsin-containing culture medium, add fresh complete medium, resuspend the cells and count (1x10 5 6 / mL). Seed the cells in a 96-well plate at a volume of 100μL per well, and place in a 37°C CO2 incubator to culture the cells to adhere and grow to 70%-80%. Then add 50ng or 250ng of Lip3000-coated Luc-mRNA to each well, and add 100μL of complete medium containing posaconazole (1 or 5μM) for transfection and continue to culture for 48h.

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

[0114] Results are shown in Figure 5 Figure 1, which shows that the transfection effect of the combination of NPC1 inhibitor and Lip3000 delivery system in HEK293T cells is significantly better than that of Lip3000 transfection alone.

[0115] Example 7 Assay of the retention of LNP in cells by the combination of NPC1 inhibitor and mRNA delivery system

[0116] HEK293T cells were seeded in a 24-well plate at a density of 100000 / mL, and the cells were allowed to adhere and grow to a density of about 70%-80%. Add 1mL of complete medium containing NPC1 inhibitor posaconazole (0 or 5μM) to the cells, and add DIO-labeled Luc-LNPs (containing 500ng mRNA) or Cy5-labeled eGFP-LNPs (containing 500ng mRNA) respectively to act for 12h. Collect the cells respectively, and wash the cells twice with PBS, and detect the accumulated DIO or Cy5 fluorescence intensity and the expressed eGFP fluorescence intensity in the cells by flow cytometry.

[0117] Meanwhile, laser confocal was also used to detect the accumulation of LNPs in cells. Briefly, HEK293T were cultured in confocal dishes at a density of 100000 / mL. After 24h of cell adhesion growth, 1 mL of complete medium containing NPC1 inhibitor posaconazole (0 or 5 mM) 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, respectively, for 12h. The cells were washed with cold PBS three times, and the cells were fixed with 4% paraformaldehyde at 4°C for 15 min, followed by washing the cells with cold PBS three times. Then DAPI staining was added at room temperature for 5 min, the cells were washed with PBS, and observed using a laser confocal microscope.

[0118] Results are shown in Figure 6 , which show that the combination of NPC1 inhibitor and mRNA delivery system can increase the residence time of LNPs in cells. Flow cytometry results Figure 6 A and Figure 6 B) show that the combination of NPC1 inhibitor and mRNA delivery system significantly enhances the fluorescence intensity of Cy5 and eGFP. Similarly, laser confocal results Figure 6 C, Figure 6 D and Figure 6 E) show that the combination of NPC1 inhibitor and mRNA delivery system significantly increases the intracellular fluorescence intensity of Cy5 and eGFP for 12 or 24h. Interestingly, compared with 12h, the fluorescence intensity of Cy5 in the control group is significantly reduced at 24h, while the fluorescence intensity of Cy5 in the combination of NPC1 inhibitor and mRNA delivery system group only has a slight downward trend.

[0119] In addition, in order to prove that the accumulation of LNPs in cells induced by the combination of NPC1 inhibitor and mRNA delivery system is not limited to specific mRNA, this embodiment uses DIO-labeled Luc-LNPs to transfect into cells, and proves similar results Figure 6 F, Figure 6 G and Figure 7 H). These results show that the combination of NPC1 inhibitor and mRNA delivery system can accumulate more mRNA delivery systems in cells, which helps the endosome-lysosome escape of mRNA and the translation of mRNA.

[0120] Example 8 Determination of the effect of the combination of NPC1 inhibitor and mRNA delivery system on the endosome-lysosome system

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

[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 at room temperature for 15 min, and then sealed with blocking buffer (1×PBS, 5% BSA, 0.3% Triton X-100). TM Cells were blocked with X-100 for 60 min. The blocking buffer was then removed, and primary antibody (Rab5 labeled with early endosomes, Rab7 labeled with late endosomes, and LAMP1 labeled with lysosomes) buffer (1×PBS, 1% BSA, 0.3% Triton) was added. TM Incubate overnight at 4°C with X-100. Then wash three times with PBS and add fluorescently conjugated secondary antibody (1×PBS, 1% BSA, 0.3% Triton). TM Incubate the cells with X-100 solution at room temperature in the dark for 2 hours, then wash three times with PBS. Finally, stain with DAPI for 5 minutes at room temperature, wash the cells with PBS, and observe them using a laser confocal microscope. All confocal images were taken using a 60X oil objective lens on an inverted fluorescence microscope (Nikon). Detailed images of individual cells were taken using a precise 5X magnifying glass.

[0123] The results are as follows Figure 8 The results, as shown by laser confocal scanning microscopy, indicate that the combination of NPC1 inhibitors and mRNA delivery systems significantly promotes Rab7 production but does not affect Rab5 and LAMP1 production. Furthermore, the combination of NPC1 inhibitors and mRNA delivery systems promotes the accumulation of DIO-labeled mRNA delivery systems in early endosomes, late endosomes, and lysosomes. These results suggest that the accumulation of intracellular mRNA delivery systems induced by NPC1 inhibition is primarily due to increased production of late endosomes, which significantly increases the chance of LNPs escaping.

[0124] Example 9: Preparation of mRNA drug composition P-LNPs

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

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

[0127]

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

[0129] The prepared P-LNPs0-P-LNPs9 solutions are diluted with 3 volumes of DPBS and transferred to an ultrafiltration tube prepared according to the method described in Example 1 for centrifugal ultrafiltration, with the following ultrafiltration parameters: 2500 rpm, 4°C, 30 min. When the sample in the centrifuge tube is left with 0.5-1.0 mL, add about 4 volumes of DPBS and centrifugal ultrafiltrate (2500 rpm, 4°C, 30 min); when the sample in the centrifuge tube is left with 0.5-1 mL or less, add about 3 volumes of DPBS and ultrafiltrate (2500 rpm, 4°C, 30 min) to 0.5 mL or less, collect the sample, and use DPBS to dilute to 0.7 mL, and store in a 4°C refrigerator.

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

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

[0132] After screening the NPC1 inhibitor posaconazole and mRNA delivery system combination that has the greatest impact on the delivery effect through the above examples, this example optimizes the molar ratio of posaconazole in the mRNA delivery system to integrate the preparation of the optimal P-LNPs, and the preparation steps are as shown in Example 9.

[0133] 1. Particle size, zeta potential, and polydispersity index (PDI) of the mRNA delivery system were determined.

[0134] The mRNA drug composition prepared in Example 9 was diluted 200 times with DPBS, and measured using a Malvern particle potential instrument.

[0135] 2. Encapsulation efficiency of the mRNA delivery system was determined.

[0136] The characterization and encapsulation efficiency were determined using the method described in Example 1, Step 5 “mRNA concentration detection and volume setting of the mRNA delivery system”. The optimal preparation method was screened according to 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) both increased the particle size of the prepared LNP and increased the PDI to greater than 0.3. However, the use of NPC1 inhibitors to replace a certain molar ratio of DSPC (organic phase 7, organic phase 8, organic phase 9) resulted in the preparation of P-LNPs7, P-LNPs8, and P-LNPs9 with particle sizes of about 100-120 nm, stable PDI less than 0.3, and encapsulation efficiency greater than 95%. PDI reflects the uniformity of the size distribution of P-LNPs, and the lower the PDI value, the more uniform the size distribution of P-LNPs. Therefore, the use of NPC1 inhibitors to replace a certain molar ratio of DSPC to prepare P-LNPs is an effective integration method for using small NPC1 inhibitors in mRNA delivery systems.

[0137] Table 2 Performance evaluation table 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-LNPs1-9 prepared by integrating the NPC1 inhibitor posaconazole and the mRNA delivery system increased slightly. The comprehensive evaluation of particle size, PDI, and encapsulation efficiency showed that the formulations P-LNPs7, P-LNPs8, and P-LNPs9 with a particle size of about 100 nm, a PDI less than 0.3, and an encapsulation efficiency greater than 95% had similar physical and chemical properties to LNP. These results showed that P-LNPs7, P-LNPs8, and P-LNPs9 were 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, and the results are shown in Figure 9 It is shown that the delivery efficiency of P-LNPs7, P-LNPs8 and P-LNPs9 is significantly higher than that of LNPs, and the efficiency increases with the increase of posaconazole content. Therefore, P-LNPs9 is re-named as P-LNPs and used for subsequent experiments as the best LNPs containing posaconazole.

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

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

[0143] The results are shown in Figure 10 It is shown that the in vivo transfection efficiency of mice injected with P-LNPs, an mRNA pharmaceutical composition prepared by combination of NPC1 inhibitor and mRNA delivery system, is significantly higher than that of mice injected with LNPs, and the in vivo delivery efficiency of P-LNPs is about twice that of LNPs. It is fully proved 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 Determination of immunization effect of RBD-mRNA-containing mRNA pharmaceutical composition prepared by combination of NPC1 inhibitor and mRNA delivery system for immunizing mice

[0145] To test the delivery effect of P-LNPs prepared by combination of NPC1 inhibitor and mRNA delivery system in vivo, LNPs and P-LNPs containing RBD-mRNA (purchased from Novavax Biotech Co., Ltd.) were prepared using organic phase 0 and organic phase 9 in Table 1 of Example 9, respectively. Six-week-old mice were injected with 0.1 mL of LNPs or P-LNPs in the muscle at day 0 and day 14, respectively, and the concentration of RBD-mRNA was 10 μg per mouse per time. At day 21 and day 28, the blood of mice was collected to detect the content of specific RBD antibodies in the serum of mice. The detection steps are as follows:

[0146] (1) Coating: coat the ELISA plate with 100 uL of carbonate buffer (pH = 9.6) containing 1 ug / mL RBD per well, 4°C overnight, discard the supernatant, pat dry, wash 3 times with 1x PBST (pH = 7.4);

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

[0148] (3) Add primary antibody: add 100 uL of the serum to be tested to each well, repeat two wells for each sample, incubate at 37°C for 1 h, wash 3 times with 1x PBST;

[0149] (4) Add secondary antibody: add 100 uL of HRP-labeled goat anti-mouse secondary antibody (purchased from Wuhan Sanying Biotechnology Co., Ltd.) to each well, incubate at 37°C for 1 h, wash 3 times with 1x PBST;

[0150] (5) Color development: add 100 uL of TMB color developing solution to each well, incubate at 37°C in the dark for 7 min,

[0151] (6) Termination: add 50 uL of H2SO4 (2M) to each well to terminate.

[0152] (7) Reading: measure the value at OD 450 nm using an enzyme marker.

[0153] The results are shown in ​ P-LNPs, and the RBD-specific IgG antibody level at day 28 was higher than that at day 21. The results prove that the combination of NPC1 inhibitor and mRNA delivery system can significantly improve the delivery efficiency of mRNA delivery system in vivo, and enhance the specific antibody effect of RBD-mRNA vaccine in vivo.

[0154] The application provides an mRNA drug composition, a preparation method and application ideas thereof, and many methods and approaches for specifically implementing the technical solutions, and the above description is only preferred embodiments of the application, and it should be pointed out that, for ordinary skilled persons in the technical field, several improvements and refinements can be made without departing from the principles of the application, and these improvements and refinements should also be regarded as the protection scope of the application. The components not explicitly described in the embodiments can be implemented by using the prior art.

Claims

1. An mRNA pharmaceutical composition, characterized in that, It comprises a C1 type Niemann-Pick protein inhibitor and an mRNA drug; the C1 type Niemann-Pick protein inhibitor is posaconazole; The preparation method of the mRNA drug composition comprises the following steps: (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; the molar ratio of the cationic lipid, the auxiliary phospholipid, the cholesterol, the polyethylene glycol lipid and the C1 type Niemann-Pick protein inhibitor is 50:9.6~10:38.5:1.5:0.1~0.4; (2) preparing an mRNA drug aqueous phase: dissolving an mRNA drug in a buffer solution to obtain an mRNA drug aqueous phase; (3) mixing the mixture organic phase and the mRNA drug aqueous phase by a microfluidic method to obtain a mixed solution; The mixed solution is ultrafiltered by an ultrafiltration tube, and the mRNA drug composition is obtained.

2. The mRNA pharmaceutical composition according to claim 1, characterized in that The mRNA drug is an mRNA vaccine or an mRNA protein replacement therapy drug.

3. The mRNA pharmaceutical composition according to claim 1, characterized in that In step (1), the organic solvent comprises any one or a combination of multiple of ethanol, isopropanol, chloroform, dichloromethane, methanol, n-hexane and ethyl acetate. In the mixture organic phase, the total concentration of the cationic lipid, the auxiliary phospholipid, the cholesterol, the polyethylene glycol lipid and the C1 type Niemann-Pick protein inhibitor is 2.85 mg / mL.

4. The mRNA pharmaceutical composition according to claim 1, characterized in that In step (2), the buffer solution is a citric acid-sodium citrate buffer solution, and the ion concentration is 10 mM; in the mRNA drug aqueous phase, the concentration of the mRNA drug is 0.05 mg / mL.

5. The mRNA pharmaceutical composition according to claim 1, characterized in that In step (3), the microfluidic mixing method, the flow rate ratio of the mixture organic phase and the mRNA drug aqueous phase 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.

6. The mRNA drug composition of any one of claims 1-5 for use in the preparation of an mRNA drug for preventing and / or treating a disease.

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