mRNA pharmaceutical preparation for treating heart failure and its preparation method and application

Abstract

The present invention relates to the field of biomedicine technology, and specifically to mRNA pharmaceutical preparations for treating heart failure, and preparation methods and applications thereof. The present invention delivers mRNA expressing brain natriuretic peptide (BNP) into the body, and utilizes human cells to autonomously express BNP, promote natriuresis and diuresis, relax blood vessels, reduce systemic vascular resistance and plasma volume, thereby achieving the purpose of treating heart failure. The pharmaceutical preparation provided by the present invention has high expression amount and bioavailability, low immunogenicity, short drug onset time, long duration, and is of great significance in maintaining the body's water and salt balance, blood pressure stability, cardiovascular and kidney organ functions, and has great potential in the diagnosis and treatment of heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an mRNA pharmaceutical preparation for treating heart failure and a preparation method and application thereof. Background Art

[0002] Heart failure (HF), also known as HF, refers to a clinical syndrome caused by myocardial structural or functional disorders due to various reasons, which ultimately leads to poor ventricular pumping or filling function. It is also the terminal stage of the continuous development and deterioration of various cardiovascular diseases. 1-2% of the global population is affected by this disease. According to the location of failure, it can be divided into left heart failure and right heart failure. Left heart failure can lead to pulmonary circulation congestion, manifested as varying degrees of dyspnea and fatigue; right heart failure can lead to systemic circulation congestion, usually manifested as ascites and lower limb edema. Heart failure has a high mortality rate and a poor prognosis. Early detection and early treatment of heart failure are of great clinical significance in delaying the progression of the disease and improving the quality of life of patients.

[0003] Brain natriuretic peptide (BNP) is a vasodilator hormone mainly synthesized by the ventricles. It is secreted in response to ventricular volume expansion and pressure load during heart failure. It can promote sodium excretion and urination, has a strong vasodilator effect, and can inhibit excessive activation of the neuroendocrine system and has a natural anti-cardiac remodeling effect. BNP and NT-proBNP are produced from the same precursor protein proBNP. NT-proBNP is the non-functional N-terminus produced by the cleavage of the protein, while BNP is the C-terminus. Recombinant human brain natriuretic peptide (rhBNP) for injection has the same amino acid sequence, spatial structure and biological activity as endogenous brain natriuretic peptide, and therefore has the same mechanism of action. BNP and NT-proBNP can be used as specific diagnostic markers for heart failure. When the concentration of BNP is less than 100ng / L or NT-proBNP is less than 300ng / L, the negative predictive value of heart failure is 90%; when the concentration of BNP is greater than 300ng / L or NT-proBNP is greater than 1500ng / L, the positive predictive value of heart failure is also 90%. They can also be used as indicators for evaluating the efficacy of heart failure: compared with the baseline, if the BNP or NT-proBNP after treatment decreases by ≥30%, the treatment plan is considered effective. Therefore, BNP has good research prospects and utilization value whether it is used as a diagnostic indicator or a therapeutic drug for heart failure.

[0004] Messenger RNA (mRNA) is a large class of RNA molecules transcribed from a strand of DNA as a template. mRNA carries genetic information and can directly guide protein synthesis. mRNA drugs deliver mRNA molecules into the cytoplasm, where they are transcribed and expressed to generate the desired protein. mRNA has significant advantages as a drug preparation: first, mRNA can theoretically express any protein, so it can treat almost all diseases; second, mRNA is only expressed in the cytoplasm and can start the protein translation process without entering the nucleus, without the risk of integration into the genome, and is more efficient; finally, mRNA is synthesized through an in vitro transcription process, which is relatively cheap and can be quickly applied to various therapies. Therefore, mRNA is a new therapeutic product with great potential and has been widely used in research in the fields of immunology, oncology, innate metabolic diseases, and vaccine development.

[0005] Nanoliposome preparation (LNPs) delivery technology has the advantages of simple synthesis process, no immunogenicity, and easy degradation in vivo. It has been successfully used in the preparation of some vaccines and the research of tumor immune targeted drugs. However, mRNA as a drug preparation still has many difficulties. Due to the extremely unstable nature of mRNA, it is easily chemically degraded in vitro and easily degraded and inactivated by nucleases in vivo. Therefore, it is difficult to deliver and effectively express therapeutic amounts of proteins in vivo, and the side effects that may be caused by drugs developed by this technology are not very clear. Safe and efficient delivery systems, optimized mRNA design, and processes that meet commercial production standards are important factors that limit the development and clinical application of this technology. At the same time, mRNA drug preparations are still concentrated in vaccine development and anti-tumor research, and mRNA preparations for the treatment of heart failure are rarely involved. Therefore, providing an mRNA preparation that can be used for treatment other than viral vaccines has positive clinical value, but it still faces many challenges. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an mRNA pharmaceutical preparation for treating heart failure and its preparation method and application. The mRNA pharmaceutical preparation provided by the present invention is to deliver the mRNA nanolipid preparation of rhBNP into the body, express rhBNP by human cells, and further secrete it outside the body cells to inhibit the excessive activation of RAAS, thereby achieving the purpose of vasodilation and improving heart failure.

[0007] The present invention provides an mRNA pharmaceutical preparation, which comprises mRNA and a delivery lipid; wherein:

[0008] The mRNA is the mRNA of recombinant human brain natriuretic peptide rhBNP;

[0009] The delivery lipids include cationic lipids, PEG lipids, phospholipids and cholesterol;

[0010] The molar ratio of the cationic lipid, PEG lipid, phospholipid and cholesterol is (45-53): (0.5-1.8): (9-11): (37-41), preferably (48-52): (0.5-0.99): (9-11): (37-40). In some embodiments, the molar ratio of the cationic lipid, PEG lipid, phospholipid and cholesterol is (49-51): (0.8-1.7): 10.3: (37.2-39.9).

[0011] Furthermore, the nucleotide sequence of the recombinant human brain natriuretic peptide rhBNP of the present invention comprises any of the following:

[0012] 1), the nucleotide sequence shown in SEQ ID No.1;

[0013] II), a nucleotide sequence encoding the amino acid sequence shown in SEQ ID No.2;

[0014] III), a nucleotide sequence having at least 60% homology with the nucleotide sequence shown in I) or II).

[0015] In the present invention, a nucleotide sequence with at least 60% homology refers to a nucleotide sequence with 60%, 70%, 80%, 90%, 95%, 98% or 99% homology to the nucleotide sequence described in I) or II) of the present invention. The protein encoded by the sequence in which one or more nucleotides are replaced, deleted and / or inserted has the same or similar function as the protein encoded by the original sequence.

[0016] Further, the mRNA of the present invention is unmodified or chemically modified, and the chemical modification is selected from pseudouridine, 1-methylpseudouridine, 2-thiouridine, 5-methylcytosine, 4-methoxy-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 4"-thiouridine, 2-thio-1-methyl-pseudouridine, 1-ethylpseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine.

[0017] Specifically, in some embodiments, the 5' end of the mRNA has 5'-CAP and 5'-UTR modifications, and the 3' end has 3'-UTR and Poly A modifications;

[0018] The nucleic acid sequence of the 5'-UTR is shown in SEQ ID No. 3, and the nucleic acid sequence of the 3'-UTR is shown in SEQ ID No. 4;

[0019] The 5'-CAP is selected from Cap0 (m7Gppp), Cap1 (m7GpppmN), Cap2 (m7GpppmNmN) or the anti-reversal cap analog ARCA (3'-O-Me-m7G(5')ppp(5')G);

[0020] The length of the Poly A is 20-300 thymine nucleotides, preferably 110, 150, 180, 200, 250, 280.

[0021] Furthermore, the cationic lipid of the present invention is 3-(docosylamino)-N1,N1,4-triadecyl-1-piperazineethylamine.

[0022] The PEG lipid is selected from one or more of n-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine, PEG2000-dipalmitoylglycerol, and PEG-dilauryloxypropyl;

[0023] Preferably, the PEG lipid is n-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine;

[0024] The phospholipid is selected from one or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoyl-phosphatidylethanolamine, and dimyristoyl-phosphatidylethanolamine;

[0025] Preferably, the phospholipid is dimyristoyl-phosphatidylethanolamine.

[0026] Specifically, in an embodiment of the present invention, the PEG lipid accounts for 0.8% to 1.7% of the total delivered lipid molar percentage; preferably, the PEG lipid molar percentage is less than 1% of the total delivered lipid molar percentage; more preferably, 0.6%, 0.7%, 0.8%;

[0027] The cationic lipid accounts for 48%, 49%, 50%, 51% or 52% of the delivery lipid by mole percentage;

[0028] The phospholipids account for 9.5%, 10%, 10.3% or 10.5% of the delivered lipid molar percentage;

[0029] The cholesterol comprises 37.2%, 37.5%, 38%, 39%, 39.2%, 39.9% or 40% of the delivered lipid mole percentage.

[0030] The mRNA pharmaceutical preparation described in the present invention specifically includes rhBNP mRNA, 3-(docosylamino)-N1,N1,4-triadecyl-1-piperazineethylamine, n-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine, dimyristoyl-phosphatidylethanolamine and cholesterol.

[0031] The mRNA pharmaceutical preparation provided by the present invention, wherein the cationic liposomes contained therein can encapsulate mRNA, cholesterol can stabilize the lipid bilayer, and PEG lipids provide a hydration layer for nanoparticles. Compared with the selection of other excipients, the mRNA pharmaceutical preparation provided by the present invention has stronger colloidal stability, can more effectively reduce protein adsorption, reduce the degradation of mRNA in the body, can efficiently deliver the mRNA encoding rhBNP into the body, enable somatic cells to efficiently express rhBNP, and quickly relieve the symptoms of heart failure in patients.

[0032] The present invention provides a method for preparing the mRNA pharmaceutical preparation, which comprises melting mRNA, a citric acid solution and a delivery lipid subjected to high-temperature film formation, and then subjecting the mixture to ultrasonic treatment, filtration and high-temperature extrusion treatment to obtain the mRNA pharmaceutical preparation.

[0033] Furthermore, the pH value of the citric acid solution is 5.0; the high-temperature film forming includes rotary evaporating the delivery lipid in a 60°C water bath to form a uniform film; and the temperature of the high-temperature extrusion is 50°C.

[0034] Specifically, in some embodiments, the preparation method comprises the following steps:

[0035] I) Dissolve the cationic lipid, PEG-lipid, amphoteric phospholipid and cholesterol in chloroform respectively, and evaporate them in a 60°C water bath to form a uniform film;

[0036] II), uniformly mixing the mRNA and a citric acid solution having a pH of 5.0 with the film-forming liposomes to obtain a liposome solution;

[0037] III), subjecting the liposome solution obtained in step II) to ultrasonic treatment to obtain single-layer liposomes with relatively uniform particle size;

[0038] IV), the single-layer liposome filter membrane obtained in step III) is extruded at 50° C. to obtain a liposome preparation loaded with target mRNA with uniform particle size; after concentration and purification, it can be diluted with a solvent to obtain an mRNA pharmaceutical preparation of corresponding concentration.

[0039] The solvent of the mRNA liposome preparation is selected from physiological saline, PBS solution with pH 7.2 to pH 7.4, PBS solution containing 0.05% to 5% chitosan or sodium alginate, or 5% glucose solution. Preferably, the drug dosage form is a liquid injection preparation, and the solvent is physiological saline.

[0040] The preparation of mRNA in the present invention comprises the following steps:

[0041] S1: synthesize the cDNA fragment encoding rhBNP and construct it downstream of the RNA polymerase promoter to obtain a recombinant plasmid;

[0042] S2: transfecting the recombinant plasmid into E. coli to obtain E. coli expressing the exogenous recombinant plasmid, amplifying the E. coli in large quantities, extracting the recombinant plasmid, and using it as a template to obtain a linearized mRNA fragment template including a T7, T3 or SP6 promoter;

[0043] S3: constructing an mRNA extracellular transcription system reaction to obtain the active ingredient mRNA;

[0044] The present invention also provides the use of the mRNA pharmaceutical preparation in preparing drugs for preventing and / or treating heart failure.

[0045] The present invention also provides a drug for preventing and / or treating heart failure, which comprises the mRNA pharmaceutical preparation as described above.

[0046] The drug of the present invention also includes pharmaceutically acceptable excipients. The drug is in the form of an oral preparation or an injection preparation, wherein the oral preparation includes tablets, powders, dry suspensions, granules, capsules, suspensions or solutions; and the injection preparation includes water injections, oil injections or powder injections.

[0047] The medicine of the present invention also includes effective doses of other substances that can be used to prevent and treat heart failure, such as furosemide, spironolactone, captopril, enalapril, irbesartan, metoprolol, digoxin, aspirin, clopidogrel or sodium nitroprusside.

[0048] The present invention also provides a method for preventing and / or treating heart failure, comprising administering the mRNA pharmaceutical preparation of the present invention.

[0049] The drug based on the mRNA of rhBNP provided by the present invention can effectively express rhBNP in vivo, quickly correct hemodynamic disorder, and thus relieve heart failure. Compared with the current rhBNP, the mRNA preparation of the present invention has higher bioavailability, rapid in vivo drug response, more stable effect, high expression amount and long expression duration; and the mRNA preparation of the present invention has obvious natriuretic and urinary effects, but has no effect on blood potassium, has reliable biosafety, reduces the toxicity caused by PEG, and greatly improves the patient's medication comfort and compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram showing the structure of rhBNP mRNA after processing and modification;

[0051] Figure 2 It showed that rhBNP mRNA significantly improved hemodynamics (PCWP: pulmonary capillary wedge pressure; PAP: pulmonary artery pressure; PVR: pulmonary circulation vascular resistance; LVEF: left ventricular ejection fraction);

[0052] Figure 3 The results showed that rhBNP mRNA preparation significantly increased urine sodium and urine volume, but had no effect on urine potassium;

[0053] Figure 4 It was shown that the use of rhBNP mRNA preparation significantly reduced NT-proBNP;

[0054] Figure 5 Shows the in vivo half-life of rhBNP mRNA preparations. DETAILED DESCRIPTION

[0055] The present invention provides an mRNA pharmaceutical preparation for treating heart failure and its preparation method and application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0056] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0057] Table 1 Amino acid sequences and nucleic acid sequences involved

[0058]

[0059]

[0060] The present invention will be further described below in conjunction with embodiments:

[0061] Example 1 Construction and amplification of recombinant plasmid

[0062] 1. Synthesize rhBNP sequence.

[0063] 2. Connect the DNA sequence encoding rhBNP mRNA to the pRSF vector plasmid through SfiI digestion and ligation. First, digest the vector and fragment. Then connect the vector and fragment. Recover the reaction product after the previous digestion and dissolve it in ddH2O, and then measure the concentration of the gel recovery product by NanoDrop. After the concentration is determined, add the vector and fragment to the digestion and ligation reaction system and incubate overnight at 15°C.

[0064] 3. Transform the ligated plasmid into E. coli and inoculate it on solid culture medium to continue culturing overnight.

[0065] 4. Perform colony PCR and sequence the E. coli colonies with the correct band size. First, randomly select transformants on the LB culture plate at room temperature, pick up a single colony with a sterilized toothpick or gun tip, and mark it. Then, place the toothpick or gun tip with bacteria in the corresponding PCR tube or 96-well PCR reaction plate. Finally, place the PCR mixture mixed with bacteria in a PCR instrument for amplification.

[0066] 5. Amplify the E. coli with the correct sequence after sequencing and extract a large amount of plasmids. First, add LB culture medium to the colonies with the correct sequence for amplification; then add the amplified bacterial solution to the LB culture medium containing kanamycin resistance and shake the bacteria overnight; finally, extract the endotoxin-free plasmid according to the instructions of the TIANGEN kit, and measure the concentration of the extracted plasmid with NanoDrop and store it.

[0067] Example 2 In vitro transcription (IVT) of target mRNA fragments

[0068] 1. Target fragment PCR: After obtaining a large amount of purified plasmid, the DNA sequence encoding the target mRNA, including the T7 sequence, is amplified using high-fidelity Taq enzyme. The reaction solution after amplification is electrophoresed using 1% agarose gel at a constant voltage of 100V.

[0069] 2. Purification of target PCR fragments: First, observe the gel under ultraviolet light after electrophoresis, cut out the fragments of the correct size, add them to GSB solution, and dissolve them in a 55°C water bath; then, cool the gel solution including the target fragments to room temperature, pour it into a gel recovery column, centrifuge at room temperature, rinse twice with Wash Buffer, and dry the gel recovery column; finally, add ddH2O, let it stand at room temperature, centrifuge at room temperature, and collect the centrifuge liquid.

[0070] 3. In vitro synthesis of target mRNA

[0071] The RNA was transcribed in vitro using the NEB T7 transcription kit. The transcription reaction system is as follows:

[0072] Table 2

[0073] <![CDATA[ddH2O]]> to 20μL 10X buffer 2μL rNTP 2μL template DNA 1 μg RNase inhibitor 1μL DTT 1μL YIPP 0.5μL T7 RNA polymerase 2μL

[0074] After gently mixing the reaction solution described in Table 2, react at 37°C for 3 hours. Subsequently, RNA, anhydrous ethanol and sodium acetate were mixed at a ratio of 10:20:1, and placed at -20°C for 1 to 2 hours for sedimentation. Centrifuge at 12000rpm for 10 minutes and discard the supernatant. Wash the lower precipitate with 1mL of 70% ethanol, centrifuge at 12000rpm for 10 minutes in a 4°C centrifuge, and discard the supernatant. After drying at 55°C for 10 minutes, add 15uL of RNase-Free water to resuspend. Place the above RNA at 65°C for 5 minutes, and then place it on ice for 5 minutes. Prepare the reaction solution according to the following reaction system.

[0075] Table 3

[0076] RNA 15μL 10x capping buffer 2μL GTP 1μL 2mM SAM 1μL Vaccinia capping vazyme 1μL

[0077] The reaction solution in Table 3 was gently mixed and placed at 37°C for 30 min.

[0078] 4. Purification of mRNA: The synthesized mRNA is purified by oligo (dT)-cellulose column chromatography. The target mRNA is prepared by purification, and the 5' end of the target mRNA is sequentially connected to 5'-CAP and 5'-UTR, and the 3' end is sequentially connected to 3'-UTR and poly A. Figure 1 shown.

[0079] Example 3 Preparation of rhBNP mRNA liposome formulation

[0080] 1. According to the prescription ratio in Table 3, the cationic lipid, PEG-lipid, amphoteric phospholipid and cholesterol were dissolved in chloroform in a round-bottom flask respectively, and rotary evaporated in a water bath at 60°C to make the above lipid materials form a uniform film at the bottom of the round-bottom flask;

[0081] 2. Add the mRNA prepared in Example 2 and a citric acid solution with a pH of 5.0 into the round-bottom flask after film formation, and shake the film to disperse it in the mRNA solution, thereby obtaining a liposome solution;

[0082] 3. The liposome solution obtained above is subjected to ultrasonic treatment to obtain single-layer liposomes with relatively uniform particle size;

[0083] 4. The monolayer liposome filter membrane obtained above was subjected to extrusion treatment at 50° C. to obtain a liposome preparation loaded with target mRNA and having a uniform particle size;

[0084] 5. The liposome preparation loaded with the target mRNA obtained above was placed in a microgel centrifuge column and centrifuged at 2500 rpm / min at room temperature for 5 min to concentrate and purify the prepared liposome preparation, and then diluted with 0.9% physiological saline to prepare a liposome preparation of mRNA with the corresponding concentration.

[0085] Table 4

[0086]

[0087]

[0088]

[0089] Example 4 Characterization of liposomal formulation of rhBNP mRNA

[0090] 1. Determine the particle size and particle size distribution index PDI of the liposomes obtained in Example 3. Take the liposomes prepared in the recipes 1-8 of Example 3, dilute them with PBS, first measure the PBS sample background with Mastersizer 3000, and then add the dispersed sample. When the concentration of the dispersed system is stable, start to measure the particle size and PDI of the prepared liposomes loaded with the target mRNA.

[0091] 2. Determine the encapsulation efficiency of the prepared liposomes for mRNA. Add an equal amount of methanol-chloroform (volume ratio 1:2) to the liposome preparations (formulations 1 to 8) prepared in Example 3, mix well, and break the emulsion; at the same time, use RiboGreen to determine the encapsulation efficiency of the liposomes for the target mRNA. The results are shown in the following table.

[0092] Table 5 Average particle size, particle size distribution index and encapsulation efficiency measurement results

[0093] Prescription 1 Prescription 2 Prescription 3 Prescription 4 Prescription 5 Prescription 6 Prescription 7 Prescription 8 Average particle size nm 112 109 94 120 118 106 98 104 PDI 0.212 0.173 0.167 0.181 0.171 0.161 0.183 0.177 Encapsulation rate (%) 90.3％ 91.5％ 77.4％ 81.2％ 88.5％ 92.1％ 89.5％ 90.8％

[0094] The above results show that the use of 3-(dioctylamino)-N1,N1,4-triadecyl-1-piperazineethylamine as the ionic lipid in prescription 2 can effectively reduce the dosage of PEG lipid, especially in the nanolipid prepared with PEG lipid of n-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine, the PEG lipid content can be reduced to below 1%. Since PEG lipid usually causes allergic reactions and thrombosis in nanolipid preparations during injection, resulting in unsafe medication, when 1,2-dilinoleyloxy-N,N-dimethylaminopropane or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane is used as the ionic lipid in prescriptions 3 and 4, the encapsulation efficiency of the nanolipid preparation is significantly reduced.

[0095] Example 5 Identification of Cellular Protein Expression Levels of Liposome Formulations of rhBNP mRNA

[0096] 1. HEK-293T cells were seeded in 6-well plates one day in advance and cultured overnight in high-glucose DMEM containing 10% FBS, so that they adhered to the plate and grew to 70% confluence the next day;

[0097] 2. Transfect the above-prepared liposome preparation loaded with rhBNP mRNA (Formulations 1-2, 6-7 in Example 3) into the above-mentioned adherent cells and incubate them at 37°C;

[0098] 3. Take the supernatant of the cell culture fluid, use the BNP Elisa kit, operate according to the instructions of the Elisa kit, and determine the content of rhBNP secreted by the cells. The results are shown in the table below.

[0099] Table 6

[0100]

[0101] The results showed that formulation 2 could express higher rhBNP in cells compared with other formulations, which may be because the formulation was more easily taken up by cells or more conducive to expression.

[0102] Example 6 Determination of the effect of rhBNP mRNA-based liposome formulation on hemodynamics

[0103] 1. Sprague-Dawley rats were housed in SPF conditions in ventilated cages with a 12-h light and 12-h dark cycle.

[0104] 2. The rats were injected with 2.5 mg / kg of doxorubicin through the tail vein (the control group was given an equal volume of 0.9% sodium chloride solution, once a week for 6 weeks, with a cumulative dose of 15 mg / kg, to induce a heart failure model;

[0105] 3. The SD rats in the heart failure group and the control group underwent cardiac ultrasound examination to measure the pulmonary capillary wedge pressure (PCWP), pulmonary artery pressure (PAP), pulmonary circulation vascular resistance (PVR) and left ventricular ejection fraction (LVEF), which confirmed the successful modeling;

[0106] 4. The rats in the heart failure group were randomly divided into three groups and injected with normal saline injection of rhBNP mRNA liposome (Prescription 2 in Example 3), commercially available rhBNP injection, and normal saline of the same dose through the tail vein respectively;

[0107] 5. 24 hours later, the SD rats underwent cardiac ultrasound examination to measure PCWP, PAP, PVR and LVEF.

[0108] The results of the test are as follows Figure 2 As shown in the results, it can be seen that the prepared rhBNP mRNA liposome pharmaceutical preparation and the commercially available rhBNP injection can significantly reduce PCWP, PAP, PVR, and increase LVEF.

[0109] Example 7 Determination of diuretic and natriuretic effects of rhBNP mRNA-based liposome preparations

[0110] 1. According to the method described in Example 6, a heart failure model of SD rats was prepared;

[0111] 2. The rats in the heart failure group were randomly divided into three groups and injected with normal saline injection of rhBNP mRNA liposome (Prescription 2 in Example 3), commercially available rhBNP injection and normal saline of the same dose through the tail vein respectively;

[0112] 3. Collect 24h urine of three groups of SD rats for accurate dosage. Also measure urine sodium (mEq / hr) and urine potassium (mEq / hr).

[0113] The results of the test are as follows Figure 3 As shown, rhBNP mRNA preparation and commercially available rhBNP injection significantly increased urinary sodium and urine volume, with no statistically significant difference between the two, but had no effect on urinary potassium.

[0114] Example 8: Liposome formulation based on rhBNP mRNA significantly reduces NT-proBNP

[0115] 1. According to the method described in Example 6, a heart failure model of SD rats was prepared;

[0116] 2. The rats in the heart failure group were randomly divided into three groups, and the normal saline injection of rhBNP mRNA liposome (Prescription 2 of Example 3) and the commercially available rhBNP injection (control group) were respectively injected through the tail vein;

[0117] 3. After 24 hours, blood was collected from the orbits of rats in different groups, and the serum was separated and the concentration of NT-proBNP in the serum was determined using an Elisa kit.

[0118] The results of the test are as follows Figure 4 As shown, liposomal formulation of rhBNP mRNA significantly reduced NT-proBNP.

[0119] Example 9 In vivo half-life of liposome formulations encoding rhBNP mRNA

[0120] 1. Sprague-Dawley rats were housed in SPF conditions in ventilated cages with a 12-h light and 12-h dark cycle.

[0121] 2. Injecting different rats with a saline injection of liposomes encoding rhBNP mRNA (Formulations 2 and 8 in Example 2) at a concentration of 10 μg / mL through the tail vein;

[0122] 3. Blood was collected from the rats' orbits every day, and the serum was separated. The rhBNP content in the serum was determined using an Elisa kit according to the instructions of the Elisa kit.

[0123] The results are as follows Figure 5 As shown, the high level of BNP expression can be maintained in prescription 2 for 10 days, but prescription 8 can maintain a certain level of BNP expression within 7 days, and the expression level is significantly reduced on the 10th day. Therefore, the mRNA liposome preparation of prescription 2 of the present invention can continuously express BNP protein for a long time and exert a long-term effect.

[0124] It can be seen from the above examples that the rhBNP mRNA-based drug provided by the present invention has a long duration of action, a short onset time, and low side effects, and can significantly improve the heart failure symptoms of patients with heart failure.

[0125] The above are only preferred embodiments of the present invention. It should be pointed out 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.

Claims

1. An mRNA pharmaceutical preparation, characterized in that: It includes mRNA and delivery lipids; The nucleotide sequence of the mRNA is shown in SEQ ID No. 1, and the 5' end thereof has 5'-CAP and 5'-UTR modifications, and the 3' end thereof has 3'-UTR and PolyA modifications; The delivery lipids include cationic lipids, PEG lipids, phospholipids and cholesterol; The molar ratio of the cationic lipid, PEG lipid, phospholipid and cholesterol is (45-53): (0.5-1.8): (9-11): (37-41); The cationic lipid is 3-(docosylamino)-N1,N1,4-triadecyl-1-piperazineethylamine; The PEG lipid is selected from one or more of n-[(methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine, PEG2000-dipalmitoylglycerol, and PEG-dilauryloxypropyl; The phospholipid is selected from one or more of distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoyl-phosphatidylethanolamine, and dimyristoyl-phosphatidylethanolamine.

2. The mRNA pharmaceutical preparation according to claim 1, characterized in that The PEG lipid is n-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine; The phospholipid is dimyristoyl-phosphatidylethanolamine.

3. The mRNA pharmaceutical preparation according to claim 1, characterized in that It includes rhBNP mRNA, 3-(docosylamino)-N1,N1,4-triadecyl-1-piperazineethylamine, n-[(methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine, dimyristoyl-phosphatidylethanolamine and cholesterol.

4. The mRNA pharmaceutical preparation according to claim 1, characterized in that The PEG lipids accounted for less than 1% of the total delivered lipid mole percentage.

5. The method for preparing the mRNA pharmaceutical preparation according to any one of claims 1 to 4, characterized in that: The method comprises the steps of melting mRNA, citric acid solution and delivery lipid formed by high temperature film, and then subjecting the mixture to ultrasonic treatment, filtration and high temperature extrusion treatment to obtain the mRNA pharmaceutical preparation.

6. Use of the mRNA pharmaceutical preparation according to any one of claims 1 to 4 in the preparation of a drug for preventing and / or treating heart failure.

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