WO2023169335A1 - Mrna-fatty acid targeting compound, and preparation method therefor and application thereof - Google Patents

Mrna-fatty acid targeting compound, and preparation method therefor and application thereof Download PDF

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WO2023169335A1
WO2023169335A1 PCT/CN2023/079617 CN2023079617W WO2023169335A1 WO 2023169335 A1 WO2023169335 A1 WO 2023169335A1 CN 2023079617 W CN2023079617 W CN 2023079617W WO 2023169335 A1 WO2023169335 A1 WO 2023169335A1
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mrna
fatty acid
formula
compound
preparation
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胡勇
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武汉瑞佶生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12N9/0048—Uricase (1.7.3.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase

Definitions

  • the present invention relates to the field of genetic engineering technology, and in particular to an mRNA-fatty acid targeting compound and its preparation method and application.
  • Nucleic acid-based therapy is a unique treatment approach.
  • the therapeutic effects of traditional therapies are often short-lived because they tend to target proteins rather than the underlying genetic problem.
  • nucleic acid therapies can achieve long-term effects through gene suppression, addition, replacement or editing.
  • the delivery of mRNA into specific tissue cells can be achieved through different methods, such as lipid nanoparticles, GalNac, etc., but these methods cannot be specifically delivered to muscle cells.
  • Skeletal and cardiac muscle cells rely heavily on the oxidation of long-chain fatty acids for contraction.
  • Fatty acids are transported to muscle tissue through the blood complexed with albumin or covalently bound to triacylglycerols, forming a neutral lipid core of circulating triglyceride-rich lipoproteins (such as chylomicrons or very low-density lipoproteins).
  • the capillary endothelium is the first barrier for fatty acids from the vascular compartment to reach bone and cardiomyocytes.
  • the mechanisms of fatty acid movement across membranes are not fully understood, but recent studies suggest that interaction of albumin-fatty acid complexes with endothelial membranes may promote fatty acid uptake.
  • Serum albumin is an endogenous fatty acid transporter.
  • Albumin the most abundant plasma protein in human blood, is synthesized in the liver and released into the lumen of blood vessels.
  • Albumin interacts with multiple cellular receptors, such as the glycoproteins Gp60, Gp30, and Gp18a, the Megalin/Cubilin complex, and the neonatal Fc receptor (FcRn). Interactions with these receptors enable albumin recycling, pinocytosis, and extended half-life.
  • Albumin contains multiple hydrophobic vesicles that can bind fatty acids and steroids as well as different drugs.
  • cholesterol-binding oligonucleotides can achieve siRNA delivery through low-density lipoprotein receptor-mediated endocytosis.
  • Wolfrum et al. conjugated cholesterol and various lipids to siRNA and demonstrated that long-chain fatty acids and cholesterol can facilitate siRNA entry into cells, resulting in efficient gene silencing in mice.
  • This group of studies also demonstrates that efficient and selective uptake of siRNA conjugates relies on interactions between lipoprotein particles, lipoprotein receptors, and other cell surface receptors.
  • fatty acid-conjugated siRNA has achieved efficient muscle cell-targeted drug delivery.
  • fatty acid-conjugated siRNA has been discovered for many years, its messenger RNA (mRNA) muscle delivery system has not been able to achieve a breakthrough.
  • mRNA messenger RNA
  • the existing technology cannot achieve the coupling of fatty acids and mRNA, and mRNA- Advances in muscle cell-targeted therapies have been hampered by the inefficiency of fatty acid complexes when delivered in vivo.
  • the present invention provides an mRNA-fatty acid targeting compound and its preparation method and application.
  • the targeting compound provided by the invention can achieve efficient coupling of fatty acids and mRNA, and has good muscle targeted delivery effect.
  • R in formula I is -C n H 2n+1 or -C n H 2n-1 , where n represents the number of carbon atoms;
  • the value of n ranges from 15 to 22.
  • the mRNA is an mRNA encoding a functional protein.
  • the invention also provides a method for preparing the mRNA-fatty acid targeting compound described in the above scheme, which includes the following steps:
  • R is -C n H 2n+1 or -C n H 2n-1 , n represents the number of carbon atoms, and A represents adenylate.
  • the preparation method of the oligoadenylate-fatty acid compound having the structure shown in formula b includes the following steps:
  • the compound having the structure shown in formula a and oligoadenylic acid are mixed for a condensation reaction to obtain the oligoadenylic acid-fatty acid compound having the structure shown in formula b; the structural formula of the oligoadenylic acid is as shown in formula c Show;
  • the temperature of the condensation reaction is room temperature and the time is 8 to 15 hours.
  • the preparation method of the compound having the structure shown in formula a includes the following steps:
  • the fatty acid, pentafluorophenyl trifluoroacetate and an alkaline reagent are mixed to perform a transesterification reaction to obtain a compound having the structure shown in formula a; the structural formula of the fatty acid is expressed as R-COOH.
  • the temperature of the transesterification reaction is room temperature and the time is 0.5 to 2 hours.
  • the system components of the RNA ligation reaction include: Tris-HCl buffer, MgCl 2 , PEG 8000, RNA ligase, adenosine triphosphate, target mRNA and oligoadenylate-fatty acid compound.
  • the present invention also provides the application of the mRNA-fatty acid targeting compound described in the above scheme or the mRNA-fatty acid targeting compound prepared by the preparation method described in the above scheme in the preparation of protein replacement drugs and metabolic disease treatment drugs.
  • the dosage forms of the protein replacement drugs and metabolic disease treatment drugs are injections.
  • the invention provides an mRNA-fatty acid targeting compound, the general structural formula of which is shown in Formula I.
  • the present invention provides a brand new mRNA-fatty acid targeting compound, which connects the fatty acid group to the amino group of adenine at the 3' end of the mRNA, solving the problem that fatty acids cannot be efficiently coupled to the mRNA, and the mRNA-fatty acid targeting compound
  • the encoded target protein has high specific expression in muscle tissue and can be directly delivered to muscle cells via intramuscular injection or systemic injection.
  • the present invention also provides a method for preparing the mRNA-fatty acid targeting compound described in the above scheme.
  • the present invention utilizes target mRNA and oligoadenylate-fatty acid compound to perform RNA ligation reaction to obtain the mRNA-fatty acid targeting compound of the present invention, and prepares The steps are simple and easy to operate.
  • Figure 1 is the quantitative results of mRNA in mouse liver in Example 2;
  • Figure 2 is the quantitative results of mRNA in mouse quadriceps muscle in Example 2;
  • Figure 3 shows the expression level test results of the target protein encoded by mRNA-palmitic acid in different tissues in Example 3;
  • Figure 4 shows the expression level test results of the target protein encoded by mRNA-oleic acid in different tissues in Example 5;
  • Figure 5 shows the expression level of the target protein encoded by mRNA-oleic acid in mice and the blood sugar reduction test results in Example 6;
  • Figure 6 shows the uric acid reduction test results in mice of the target protein encoded by mRNA-oleic acid in Example 7.
  • the invention provides an mRNA-fatty acid targeting compound, the general structural formula of which is shown in formula I:
  • R in formula I is -C n H 2n+1 or -C n H 2n-1 , where n represents the number of carbon atoms; represents mRNA.
  • n 15-22, preferably 16-21.
  • the mRNA-fatty acid targeting compound is an mRNA-palmitic acid targeting compound, and its structural formula is as shown in Formula I-1:
  • the mRNA-fatty acid targeting compound is an mRNA-oleic acid targeting compound, and the structural formula is as shown in Formula I-2:
  • the mRNA is an mRNA that can encode a functional protein.
  • the present invention has no special requirements for the specific sequence of the mRNA. It can be selected according to the sequence of the target gene; in specific embodiments of the present invention, the mRNA Preferably it is hEPO mRNA, LUC mRNA, hGLP1 mRNA or hRAS mRNA.
  • the invention also provides a method for preparing the mRNA-fatty acid targeting compound described in the above scheme, which includes the following steps:
  • R is -C n H 2n+1 or -C n H 2n-1 , n represents the number of carbon atoms, and A represents adenylate.
  • the preparation method of the oligoadenylate-fatty acid compound having the structure shown in formula b includes the following steps:
  • the compound having the structure shown in formula a and oligoadenylic acid are mixed for a condensation reaction to obtain the oligoadenylic acid-fatty acid compound having the structure shown in formula b; the structural formula of the oligoadenylic acid is as shown in formula c Show;
  • the preparation method of the compound having the structure shown in formula a includes the following steps:
  • the fatty acid, pentafluorophenyl trifluoroacetate and an alkaline reagent are mixed to perform a transesterification reaction to obtain a compound having the structure shown in formula a; the structural formula of the fatty acid is expressed as R-COOH.
  • fatty acids, pentafluorophenyl trifluoroacetate and alkaline reagents are mixed to perform a transesterification reaction to obtain a compound having a structure represented by formula a.
  • the fatty acid is preferably palmitic acid or oleic acid.
  • the alkaline reagent is preferably triethylamine, which provides a suitable alkaline environment for the transesterification reaction to proceed.
  • the mass ratio of the fatty acid and pentafluorophenyl trifluoroacetate is preferably 0.2:2 to 0.4:1; the dosage ratio of the fatty acid to triethylamine is preferably 0.2 to 0.4g.
  • the pH value of the transesterification reaction at 8 to 10 by controlling the amount of triethylamine; the transesterification reaction is carried out in a solvent; the transesterification reaction is carried out with
  • the solvent is preferably dichloromethane, chloroform or benzene; the present invention has no special requirements on the amount of the solvent, as long as it can dissolve the raw materials and make the reaction proceed smoothly.
  • the temperature of the transesterification reaction is room temperature and the time is 0.5 to 2 hours.
  • the present invention preferably dilutes, washes and separates the obtained product liquid in sequence, and sequentially dries, filters and concentrates the obtained organic layer to obtain a crude product, which is then purified by silica gel column chromatography. A compound having the structure shown in formula a is obtained.
  • the diluting solvent is preferably the same as the transesterification solvent; the washing is preferably using sodium bicarbonate solution and sodium bisulfate solution in sequence; the concentration of the sodium bicarbonate solution is preferably 5 mmol/L , the concentration of the sodium bisulfate solution is preferably 5mmol/L; the desiccant for drying is preferably anhydrous sodium sulfate, and the desiccant is removed by filtration after drying; the eluent for silica gel column chromatography purification is preferably chloroform ; In the present invention, it is preferred to mix chloroform and silica gel into a homogenate, then pack the column, and then dissolve the crude product and put it on the column for purification; the method for dissolving the crude product is preferably: dissolving the crude product in dimethyl sulfoxide , and then add acetonitrile and triethylamine to obtain a crude product solution; the concentration of acetonitrile in the crude product solution
  • the present invention mixes the compound with the structure shown in formula a and oligoadenylate to perform a condensation reaction to obtain the oligoadenylate-fatty acid compound with the structure shown in formula b ( Denoted as oligo(A)-fatty acid).
  • the temperature of the condensation reaction is preferably room temperature, and the time is preferably 8 to 15 hours;
  • the solvent for the condensation reaction is preferably sodium tetraborate solution, and the concentration of the sodium tetraborate solution is preferably 0.5 to 2 mol/L.
  • the structural formula of the oligoadenylate is as shown in formula c (see above for specific structural formula), and the number of adenylate in the oligoadenylate is preferably 6 to 10; so
  • the molar ratio of the compound having the structure represented by formula a and oligoadenylic acid is preferably 5:1 to 1:1, more preferably 4:1 to 3:1.
  • the present invention has no special requirements on the source of the oligoadenylate, and it can be synthesized by commercially available products or solid-phase synthesis methods well known in the art.
  • the oligoadenylate Acid was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. or Suzhou Jinweizhi Biotechnology Co., Ltd.
  • the present invention preferably dilutes the reaction product obtained with enzyme-free water, and then performs HPLC purification using an ion exchange column to obtain the oligoadenylate-fatty acid compound having the structure represented by formula b.
  • the present invention performs an RNA ligation reaction between the oligoadenylate-fatty acid compound and target mRNA under the catalysis of RNA ligase to obtain formula I.
  • the system composition of the RNA ligation reaction preferably includes: Tris-HCl buffer, MgCl 2 , PEG 8000, T4 RNA Ligase 1, adenosine triphosphate (ATP), target mRNA and oligoadenylate-fatty acid compound;
  • the RNA ligase is preferably T4 RNA Ligase 1.
  • the pH value of the Tris-HCl buffer is preferably 7 to 7.5, more preferably 7.5, and the RNA
  • the concentration of MgCl 2 is preferably 0.1 to 1 mmol/L
  • the concentration of PEG-8000 is preferably 5 to 50 mg/mL
  • the concentration of T4 RNA Ligase 1 is preferably 15 to 25 U, and more preferably 20 U
  • ATP The concentration is preferably 0.5 ⁇ 2.5mmol/L, more preferably 1mmol/L
  • the molar ratio of the target mRNA and oligoadenylate-fatty acid compound is preferably 1:50 ⁇ 50:1, more preferably 1:20 ⁇ 20:1, most preferably 1:2
  • the MgCl 2 and PEG 8000 are used to maintain enzyme activity
  • ATP is used as the enzymatic reaction substrate of T4 RNA Ligase 1 and is added between the two nucleic acid sequences. .
  • all components are preferably 0.1 to 1 mmol/L
  • the temperature of the RNA ligation reaction is preferably 16-25°C, and the reaction time is preferably 30min-32h; in specific embodiments of the invention, when the temperature of the RNA ligation reaction is 16°C, the reaction The time is preferably 2 to 32 hours; when the temperature of the RNA ligation reaction is 25°C, the reaction time is preferably 30 min to 16 hours.
  • the present invention preferably purifies the product liquid to obtain the mRNA-fatty acid targeting compound; the present invention has no special requirements for the purification method, and can adopt methods well known to those skilled in the art, such as layer analysis method, liquid chromatography, lithium chloride precipitation method, phenol chloroform extraction method, ethanol precipitation method or ammonium acetate precipitation method.
  • the lithium chloride precipitation method is preferably used.
  • the lithium precipitation method specifically includes: adding lithium chloride solution into the product liquid until the concentration of lithium chloride in the liquid is 0.5 mol/L, so that the mRNA-fatty acid targeting compound is precipitated.
  • the present invention also provides the application of the mRNA-fatty acid targeting compound described in the above scheme or the mRNA-fatty acid targeting compound prepared by the preparation method described in the above scheme in the preparation of protein replacement drugs and metabolic disease treatment drugs; in the present invention,
  • the dosage forms of the protein replacement drugs and metabolic disease treatment drugs are preferably injections, and the administration route of the injections is preferably intramuscular injection; the target protein encoded by the mRNA-fatty acid targeting compound provided by the invention is specifically expressed in muscle tissue. High, it can be delivered directly to muscle cells through systemic circulation injection, and has broad application prospects in intramuscular injection vaccines.
  • the coding region sequence of hEPO mRNA used in the examples is:
  • the coding region sequence of LUC mRNA used in the examples is:
  • the coding region sequence of hGLP1 mRNA used in the examples is:
  • the coding region sequence of hRAS mRNA used in the examples is:
  • a Dissolve the crude product in DMSO, add acetonitrile and triethylamine to obtain a crude product solution, in which the concentration of the crude product is 40mmol/L, the concentration of acetonitrile is 20mmol/L, and the concentration of triethylamine is 16mmol/L;
  • aqueous phase Dilute LUC mRNA in citric acid buffer at a final concentration of 2 ⁇ g/ ⁇ l to obtain an mRNA buffer; prepare an ethanol phase solution according to Table 1;
  • phase A mRNA buffer
  • phase B ethanol phase solution
  • mice (purchased from Beijing Vitong Lever) aged 6 to 8 weeks were raised under SPF conditions and kept in cages with a 12-hour light and 12-hour dark cycle, and were treated with Luc mRNA-palmitic acid targeting compounds. and LNP-LUC mRNA were injected into the tail vein of mice respectively. The injection doses were 1 mg, 5 mg, 10 mg, and 15 mg respectively. After 24 hours, the liver and quadriceps muscles of the mice were taken, and the mRNA was extracted using a tissue RNA extraction kit for fluorescence quantification. PCR to quantify target mRNA in different tissues. The experimental results are shown in Figures 1-2.
  • Example 2 Other conditions were the same as Example 1, except that LUC mRNA was replaced with hEPO mRNA to obtain hEPO mRNA-palmitic acid targeting compound.
  • Example 4 Other conditions were the same as in Example 4, except that hEPO mRNA was replaced with hGLP1 mRNA to obtain hGLP1 mRNA-oleic acid targeting compound.
  • mRNA-oleic acid encodes exocrine urate oxidase and is specifically expressed in muscle tissue:
  • BKS db/db hyperglycemia model mice (purchased from Beijing Vitong Lever) aged 6-8 weeks were raised under SPF conditions and kept in cages with a 12-hour light and 12-hour dark cycle.
  • hGLP1 mRNA-oil Acid-targeting compounds were injected intramuscularly into mice at a dose of 5 mg. After 24 hours, the serum was taken, the total protein was extracted, and the blood glucose concentration of the mice was quantified by a blood glucose meter. At the same time, the total protein and blood glucose of the mice in the blank control group were measured. Concentration is quantified.
  • the experimental results are shown in Figure 5. The left side of Figure 5 shows the expression of GLP-1, and the right side shows the blood glucose concentration.
  • the experimental group in Figure 5 is the experimental group injected with hGLP1 mRNA-oleic acid targeting compound. According to Figure 5, it can be seen that the experimental group injected with hGLP1 mRNA-oleic acid targeting compound expressed high expression of the target protein product and significantly reduced blood glucose concentration.
  • Example 4 Other conditions were the same as in Example 4, except that hEPO mRNA was replaced with hRAS mRNA to obtain hRAS mRNA-oleic acid targeting compound.
  • mRNA-oleic acid encodes exocrine urate oxidase and is specifically expressed in muscle tissue:

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Abstract

An mRNA-fatty acid targeting compound, and a preparation method therefor and an application thereof, relating to the technical field of genetic engineering. An RNA ligation reaction of an oligoadenylate-fatty acid compound and a target mRNA is carried out to obtain the mRNA-fatty acid targeting compound. Provided is a novel mRNA-fatty acid targeting compound. The problem that a fatty acid cannot be efficiently coupled to an mRNA is solved; moreover, the specific expression amount of an encoded target protein of the mRNA-fatty acid targeting compound in muscle tissue is high, so that direct delivery to muscle cells by means of intramuscular injection or systemic circulation injection can be implemented.

Description

一种mRNA-脂肪酸靶向化合物及其制备方法和应用An mRNA-fatty acid targeting compound and its preparation method and application 技术领域Technical field
本发明涉及基因工程技术领域，尤其涉及一种mRNA-脂肪酸靶向化合物及其制备方法和应用。The present invention relates to the field of genetic engineering technology, and in particular to an mRNA-fatty acid targeting compound and its preparation method and application.
背景技术Background technique
基于核酸的疗法是一种独特的治疗方法。传统疗法产生的治疗效果通常比较短暂，因为它们针对的往往是蛋白质，而非根本的基因问题。相比之下，核酸疗法可以通过基因抑制、增添、替换或编辑来达到长期的疗效。目前，mRNA向特异组织细胞内的递送可以通过不同的方法实现，例如脂质纳米颗粒、GalNac等，但这些方法无法特异性递送至肌肉细胞。Nucleic acid-based therapy is a unique treatment approach. The therapeutic effects of traditional therapies are often short-lived because they tend to target proteins rather than the underlying genetic problem. In contrast, nucleic acid therapies can achieve long-term effects through gene suppression, addition, replacement or editing. Currently, the delivery of mRNA into specific tissue cells can be achieved through different methods, such as lipid nanoparticles, GalNac, etc., but these methods cannot be specifically delivered to muscle cells.
骨骼和心肌细胞严重依赖长链脂肪酸的氧化来进行收缩。脂肪酸通过与白蛋白复合或与三酰甘油共价结合的血液运输到肌肉组织，形成循环中富含甘油三酯的脂蛋白(如乳糜微粒或极低密度脂蛋白)中性脂质核心。毛细血管内皮是脂肪酸从血管腔室到达骨骼和心肌细胞的第一道屏障。脂肪酸跨膜运动的机制尚不完全清楚，但最近的研究表明，白蛋白-脂肪酸复合物与内皮膜的相互作用可能促进脂肪酸的摄取。Skeletal and cardiac muscle cells rely heavily on the oxidation of long-chain fatty acids for contraction. Fatty acids are transported to muscle tissue through the blood complexed with albumin or covalently bound to triacylglycerols, forming a neutral lipid core of circulating triglyceride-rich lipoproteins (such as chylomicrons or very low-density lipoproteins). The capillary endothelium is the first barrier for fatty acids from the vascular compartment to reach bone and cardiomyocytes. The mechanisms of fatty acid movement across membranes are not fully understood, but recent studies suggest that interaction of albumin-fatty acid complexes with endothelial membranes may promote fatty acid uptake.
血清白蛋白是内源性脂肪酸的转运蛋白。白蛋白是人血中最丰富的血浆蛋白，它在肝脏中合成并释放到血管腔中。白蛋白与多种细胞受体相互作用，如糖蛋白Gp60、Gp30和Gp18a，Megalin/Cubilin复合物，以及新生儿Fc受体(FcRn)。与这些受体的相互作用实现白蛋白的循环、胞饮和延长半衰期。白蛋白含有多个疏水囊，可以结合脂肪酸和类固醇以及不同的药物。Serum albumin is an endogenous fatty acid transporter. Albumin, the most abundant plasma protein in human blood, is synthesized in the liver and released into the lumen of blood vessels. Albumin interacts with multiple cellular receptors, such as the glycoproteins Gp60, Gp30, and Gp18a, the Megalin/Cubilin complex, and the neonatal Fc receptor (FcRn). Interactions with these receptors enable albumin recycling, pinocytosis, and extended half-life. Albumin contains multiple hydrophobic vesicles that can bind fatty acids and steroids as well as different drugs.
研究表明，胆固醇结合的寡核苷酸通过低密度脂蛋白受体介导的内吞作用可以实现siRNA的递送。Wolfrum等人将胆固醇和各种脂质结合到siRNA上，并证明了长链脂肪酸和胆固醇可以促进siRNA进入细胞，从而在小鼠中实现有效的基因沉默。这组研究还表明siRNA偶联物的有效和选择性吸收依赖于脂蛋白颗粒、脂蛋白受体和其他细胞表面受体之间的相互作用。Studies have shown that cholesterol-binding oligonucleotides can achieve siRNA delivery through low-density lipoprotein receptor-mediated endocytosis. Wolfrum et al. conjugated cholesterol and various lipids to siRNA and demonstrated that long-chain fatty acids and cholesterol can facilitate siRNA entry into cells, resulting in efficient gene silencing in mice. This group of studies also demonstrates that efficient and selective uptake of siRNA conjugates relies on interactions between lipoprotein particles, lipoprotein receptors, and other cell surface receptors.
经过多年持续的研发，脂肪酸缀合的siRNA已实现高效肌肉细胞靶向性药物递送。尽管脂肪酸缀合的siRNA已被发现多年，但是其信使RNA(Messenger RNA，mRNA)肌肉递送系统一直未能取得突破，现有技术不能实现脂肪酸与mRNA的耦合，mRNA- 脂肪酸复合物在体内递送时递送效率低，因而阻碍了肌肉细胞靶向治疗法的进展。After years of continuous research and development, fatty acid-conjugated siRNA has achieved efficient muscle cell-targeted drug delivery. Although fatty acid-conjugated siRNA has been discovered for many years, its messenger RNA (mRNA) muscle delivery system has not been able to achieve a breakthrough. The existing technology cannot achieve the coupling of fatty acids and mRNA, and mRNA- Advances in muscle cell-targeted therapies have been hampered by the inefficiency of fatty acid complexes when delivered in vivo.
发明内容Contents of the invention
有鉴于此，本发明提供了一种mRNA-脂肪酸靶向化合物及其制备方法和应用。本发明提供的靶向化合物能够实现脂肪酸和mRNA的高效耦合，肌肉靶向递送效果好。In view of this, the present invention provides an mRNA-fatty acid targeting compound and its preparation method and application. The targeting compound provided by the invention can achieve efficient coupling of fatty acids and mRNA, and has good muscle targeted delivery effect.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
一种mRNA-脂肪酸靶向化合物，结构如式I所示：
An mRNA-fatty acid targeting compound with a structure shown in Formula I:
式I中的R为-CnH2n+1或-CnH2n-1，其中n表示碳原子数；R in formula I is -C n H 2n+1 or -C n H 2n-1 , where n represents the number of carbon atoms;
表示mRNA。 represents mRNA.
优选地，所述n的取值范围为15～22。Preferably, the value of n ranges from 15 to 22.
优选地，所述R为-(CH2)14CH3或-(CH2)7CH＝CH(CH2)7CH3。Preferably, the R is -(CH 2 ) 14 CH 3 or -(CH 2 ) 7 CH=CH(CH 2 ) 7 CH 3 .
优选地，所述mRNA为可编码功能蛋白的mRNA。Preferably, the mRNA is an mRNA encoding a functional protein.
本发明还提供了上述方案所述mRNA-脂肪酸靶向化合物的制备方法，包括以下步骤：The invention also provides a method for preparing the mRNA-fatty acid targeting compound described in the above scheme, which includes the following steps:
将具有式b所示结构的寡聚腺苷酸-脂肪酸化合物与目标mRNA在RNA连接酶的催化作用下进行RNA连接反应，得到式I所示结构的mRNA-脂肪酸靶向化合物；
Perform an RNA ligation reaction between the oligoadenylate-fatty acid compound having the structure shown in formula b and the target mRNA under the catalysis of RNA ligase to obtain the mRNA-fatty acid targeting compound having the structure shown in formula I;
式b中：R为-CnH2n+1或-CnH2n-1，n表示碳原子数，A表示腺苷酸。In formula b: R is -C n H 2n+1 or -C n H 2n-1 , n represents the number of carbon atoms, and A represents adenylate.
优选地，所述具有式b所示结构的寡聚腺苷酸-脂肪酸化合物的制备方法包括以下步骤：Preferably, the preparation method of the oligoadenylate-fatty acid compound having the structure shown in formula b includes the following steps:
将具有式a所示结构的化合物和寡聚腺苷酸混合进行缩合反应，得到具有式b所示结构的寡聚腺苷酸-脂肪酸化合物；所述寡聚腺苷酸的结构式如式c所示；
The compound having the structure shown in formula a and oligoadenylic acid are mixed for a condensation reaction to obtain the oligoadenylic acid-fatty acid compound having the structure shown in formula b; the structural formula of the oligoadenylic acid is as shown in formula c Show;
优选地，所述缩合反应的温度为室温，时间为8～15h。Preferably, the temperature of the condensation reaction is room temperature and the time is 8 to 15 hours.
优选地，所述具有式a所示结构的化合物的制备方法包括以下步骤：Preferably, the preparation method of the compound having the structure shown in formula a includes the following steps:
将脂肪酸、三氟乙酸五氟苯酯和碱性试剂混合进行酯交换反应，得到具有式a所示结构的化合物；所述脂肪酸的结构式表示为R-COOH。 The fatty acid, pentafluorophenyl trifluoroacetate and an alkaline reagent are mixed to perform a transesterification reaction to obtain a compound having the structure shown in formula a; the structural formula of the fatty acid is expressed as R-COOH.
优选地，所述酯交换反应的温度为室温，时间为0.5～2h。Preferably, the temperature of the transesterification reaction is room temperature and the time is 0.5 to 2 hours.
优选地，所述RNA连接反应的体系组成包括：Tris-HCl缓冲液、MgCl2、PEG 8000、RNA连接酶、三磷酸腺苷、目标mRNA和寡聚腺苷酸-脂肪酸化合物。Preferably, the system components of the RNA ligation reaction include: Tris-HCl buffer, MgCl 2 , PEG 8000, RNA ligase, adenosine triphosphate, target mRNA and oligoadenylate-fatty acid compound.
本发明还提供了上述方案所述mRNA-脂肪酸靶向化合物或上述方案所述制备方法制备的mRNA-脂肪酸靶向化合物在制备蛋白替代药物和代谢疾病治疗药物中的应用。The present invention also provides the application of the mRNA-fatty acid targeting compound described in the above scheme or the mRNA-fatty acid targeting compound prepared by the preparation method described in the above scheme in the preparation of protein replacement drugs and metabolic disease treatment drugs.
优选地，所述蛋白替代药物和代谢疾病治疗药物的剂型为注射剂。Preferably, the dosage forms of the protein replacement drugs and metabolic disease treatment drugs are injections.
本发明提供了一种mRNA-脂肪酸靶向化合物，结构通式如式I所示。本发明提供了一种全新的mRNA-脂肪酸靶向化合物，将脂肪酸基团连接在mRNA 3’端的腺嘌呤的氨基上，解决了脂肪酸无法与mRNA高效耦合的问题，且该mRNA-脂肪酸靶向化合物的编码目标蛋白在肌肉组织中特异性表达量高，能够通过肌肉注射或体循环注射直接递送至肌肉细胞。The invention provides an mRNA-fatty acid targeting compound, the general structural formula of which is shown in Formula I. The present invention provides a brand new mRNA-fatty acid targeting compound, which connects the fatty acid group to the amino group of adenine at the 3' end of the mRNA, solving the problem that fatty acids cannot be efficiently coupled to the mRNA, and the mRNA-fatty acid targeting compound The encoded target protein has high specific expression in muscle tissue and can be directly delivered to muscle cells via intramuscular injection or systemic injection.
本发明还提供了上述方案所述mRNA-脂肪酸靶向化合物的制备方法，本发明利用目标mRNA与寡聚腺苷酸-脂肪酸化合物进行RNA连接反应，得到本发明的mRNA-脂肪酸靶向化合物，制备步骤简单，容易操作。The present invention also provides a method for preparing the mRNA-fatty acid targeting compound described in the above scheme. The present invention utilizes target mRNA and oligoadenylate-fatty acid compound to perform RNA ligation reaction to obtain the mRNA-fatty acid targeting compound of the present invention, and prepares The steps are simple and easy to operate.
附图说明Description of drawings
图1为实施例2中小鼠肝脏中mRNA的定量结果；Figure 1 is the quantitative results of mRNA in mouse liver in Example 2;
图2为实施例2中小鼠四头肌中mRNA的定量结果；Figure 2 is the quantitative results of mRNA in mouse quadriceps muscle in Example 2;
图3为实施例3中mRNA-棕榈酸编码目标蛋白在不同组织中的表达量测试结果；Figure 3 shows the expression level test results of the target protein encoded by mRNA-palmitic acid in different tissues in Example 3;
图4为实施例5中mRNA-油酸编码目标蛋白在不同组织中的表达量测试结果；Figure 4 shows the expression level test results of the target protein encoded by mRNA-oleic acid in different tissues in Example 5;
图5为实施例6中mRNA-油酸编码目标蛋白在小鼠体内的表达量以及血糖降低测试结果；Figure 5 shows the expression level of the target protein encoded by mRNA-oleic acid in mice and the blood sugar reduction test results in Example 6;
图6为实施例7中mRNA-油酸编码目标蛋白在小鼠体内的尿酸降低测试结果。Figure 6 shows the uric acid reduction test results in mice of the target protein encoded by mRNA-oleic acid in Example 7.
具体实施方式Detailed ways
本发明提供了一种mRNA-脂肪酸靶向化合物，结构通式如式I所示：
The invention provides an mRNA-fatty acid targeting compound, the general structural formula of which is shown in formula I:
式I中的R为-CnH2n+1或-CnH2n-1，其中n表示碳原子数；表示mRNA。R in formula I is -C n H 2n+1 or -C n H 2n-1 , where n represents the number of carbon atoms; represents mRNA.
在本发明中，所述n的取值范围为15～22，优选为16～21。In the present invention, the value range of n is 15-22, preferably 16-21.
在本发明中，所述R为-(CH2)14CH3或-(CH2)7CH＝CH(CH2)7CH3，当所述R为-(CH2)14CH3时，所述mRNA-脂肪酸靶向化合物为mRNA-棕榈酸靶向化合物，结构式如式I-1所示：
In the present invention, the R is -(CH 2 ) 14 CH 3 or -(CH 2 ) 7 CH = CH(CH 2 ) 7 CH 3. When the R is -(CH 2 ) 14 CH 3 , The mRNA-fatty acid targeting compound is an mRNA-palmitic acid targeting compound, and its structural formula is as shown in Formula I-1:
当所述R为-(CH2)7CH＝CH(CH2)7CH3时，所述mRNA-脂肪酸靶向化合物为mRNA-油酸靶向化合物，结构式如式I-2所示：
When the R is -(CH 2 ) 7 CH = CH (CH 2 ) 7 CH 3 , the mRNA-fatty acid targeting compound is an mRNA-oleic acid targeting compound, and the structural formula is as shown in Formula I-2:
在本发明中，所述mRNA为可编码功能蛋白的mRNA，本发明对所述mRNA的具体序列没有特殊要求，根据目的基因序列进行选择即可；在本发明的具体实施例中，所述mRNA优选为hEPO mRNA、LUC mRNA、hGLP1 mRNA或hRAS mRNA。In the present invention, the mRNA is an mRNA that can encode a functional protein. The present invention has no special requirements for the specific sequence of the mRNA. It can be selected according to the sequence of the target gene; in specific embodiments of the present invention, the mRNA Preferably it is hEPO mRNA, LUC mRNA, hGLP1 mRNA or hRAS mRNA.
本发明还提供了上述方案所述mRNA-脂肪酸靶向化合物的制备方法，包括以下步骤：The invention also provides a method for preparing the mRNA-fatty acid targeting compound described in the above scheme, which includes the following steps:
将具有式b所示结构的寡聚腺苷酸-脂肪酸化合物与目标mRNA在RNA连接酶的催化作用下进行RNA连接反应，得到式I所示结构的mRNA-脂肪酸靶向化合物；
Perform an RNA ligation reaction between the oligoadenylate-fatty acid compound having the structure shown in formula b and the target mRNA under the catalysis of RNA ligase to obtain the mRNA-fatty acid targeting compound having the structure shown in formula I;
式b中：R为-CnH2n+1或-CnH2n-1，n表示碳原子数，A表示腺苷酸。In formula b: R is -C n H 2n+1 or -C n H 2n-1 , n represents the number of carbon atoms, and A represents adenylate.
在本发明中，所述具有式b所示结构的寡聚腺苷酸-脂肪酸化合物的制备方法包括以下步骤：In the present invention, the preparation method of the oligoadenylate-fatty acid compound having the structure shown in formula b includes the following steps:
将具有式a所示结构的化合物和寡聚腺苷酸混合进行缩合反应，得到具有式b所示结构的寡聚腺苷酸-脂肪酸化合物；所述寡聚腺苷酸的结构式如式c所示；
The compound having the structure shown in formula a and oligoadenylic acid are mixed for a condensation reaction to obtain the oligoadenylic acid-fatty acid compound having the structure shown in formula b; the structural formula of the oligoadenylic acid is as shown in formula c Show;
在本发明中，所述具有式a所示结构的化合物的制备方法包括以下步骤：In the present invention, the preparation method of the compound having the structure shown in formula a includes the following steps:
将脂肪酸、三氟乙酸五氟苯酯和碱性试剂混合进行酯交换反应，得到具有式a所示结构的化合物；所述脂肪酸的结构式表示为R-COOH。The fatty acid, pentafluorophenyl trifluoroacetate and an alkaline reagent are mixed to perform a transesterification reaction to obtain a compound having the structure shown in formula a; the structural formula of the fatty acid is expressed as R-COOH.
在本发明中，所述式a和R-COOH中的R基团和式b中一致，n的取值范围和式b中一致，在此不再赘述。下面对本发明的制备方法进行详细说明：In the present invention, the R group in formula a and R-COOH is consistent with that in formula b, and the value range of n is consistent with that in formula b, which will not be described again. The preparation method of the present invention is described in detail below:
本发明将脂肪酸、三氟乙酸五氟苯酯和碱性试剂混合进行酯交换反应，得到具有式a所示结构的化合物。在本发明的具体实施例中，所述脂肪酸优选为棕榈酸或油酸。在本发明中，碱性试剂优选为三乙胺，所述碱性试剂为酯交换反应的进行提供合适的碱性环境。In the present invention, fatty acids, pentafluorophenyl trifluoroacetate and alkaline reagents are mixed to perform a transesterification reaction to obtain a compound having a structure represented by formula a. In specific embodiments of the present invention, the fatty acid is preferably palmitic acid or oleic acid. In the present invention, the alkaline reagent is preferably triethylamine, which provides a suitable alkaline environment for the transesterification reaction to proceed.
在本发明中，所述脂肪酸和三氟乙酸五氟苯酯(Pfp-TFA)的质量比优选为0.2:2～0.4:1；所述脂肪酸和三乙胺的用量比优选为0.2～0.4g：2.5mL；在本发明的具体实施例中，优选通过控制三乙胺的用量将酯交换反应的pH值控制在8～10；所述酯交换反应在溶剂中进行；所述酯交换反应用溶剂优选为二氯甲烷、氯仿或苯；本发明对所述溶剂的用量没有特殊要求，能够将原料溶解，使反应顺利进行即可。In the present invention, the mass ratio of the fatty acid and pentafluorophenyl trifluoroacetate (Pfp-TFA) is preferably 0.2:2 to 0.4:1; the dosage ratio of the fatty acid to triethylamine is preferably 0.2 to 0.4g. : 2.5mL; in specific embodiments of the present invention, it is preferred to control the pH value of the transesterification reaction at 8 to 10 by controlling the amount of triethylamine; the transesterification reaction is carried out in a solvent; the transesterification reaction is carried out with The solvent is preferably dichloromethane, chloroform or benzene; the present invention has no special requirements on the amount of the solvent, as long as it can dissolve the raw materials and make the reaction proceed smoothly.
在本发明中，所述酯交换反应的温度为室温，时间为0.5～2h。In the present invention, the temperature of the transesterification reaction is room temperature and the time is 0.5 to 2 hours.
在本发明的具体实施例中，优选先将脂肪酸和碱性试剂溶解在溶剂中，然后再加入三氟乙酸五氟苯酯进行酯交换反应。 In specific embodiments of the present invention, it is preferred to dissolve the fatty acid and the alkaline reagent in a solvent first, and then add pentafluorophenyl trifluoroacetate to perform transesterification reaction.
酯交换反应完成后，本发明优选将所得产物料液依次进行稀释、洗涤和分层，将所得有机层依次进行干燥、过滤和浓缩，得到粗产物，将所述粗产物进行硅胶柱色谱纯化，得到具有式a所示结构的化合物。在本发明中，所述稀释用溶剂优选和酯交换反应用溶剂相同；所述洗涤优选为依次使用碳酸氢钠溶液和硫酸氢钠溶液洗涤；所述碳酸氢钠溶液的浓度优选为5mmol/L，所述硫酸氢钠溶液的浓度优选为5mmol/L；所述干燥用干燥剂优选为无水硫酸钠，干燥后通过过滤将干燥剂去除；所述硅胶柱色谱纯化用洗脱剂优选为氯仿；本发明优选将氯仿和硅胶混合均浆，之后进行装柱，然后将粗产物溶解后上柱进行纯化；所述粗产物溶解的方法优选为：将所述粗产物溶解于二甲基亚砜中，然后加入乙腈和三乙胺，得到粗产物溶液；所述粗产物溶液中乙腈的浓度优选为20mmol/L，三乙胺的浓度优选为16mmol/L。After the transesterification reaction is completed, the present invention preferably dilutes, washes and separates the obtained product liquid in sequence, and sequentially dries, filters and concentrates the obtained organic layer to obtain a crude product, which is then purified by silica gel column chromatography. A compound having the structure shown in formula a is obtained. In the present invention, the diluting solvent is preferably the same as the transesterification solvent; the washing is preferably using sodium bicarbonate solution and sodium bisulfate solution in sequence; the concentration of the sodium bicarbonate solution is preferably 5 mmol/L , the concentration of the sodium bisulfate solution is preferably 5mmol/L; the desiccant for drying is preferably anhydrous sodium sulfate, and the desiccant is removed by filtration after drying; the eluent for silica gel column chromatography purification is preferably chloroform ; In the present invention, it is preferred to mix chloroform and silica gel into a homogenate, then pack the column, and then dissolve the crude product and put it on the column for purification; the method for dissolving the crude product is preferably: dissolving the crude product in dimethyl sulfoxide , and then add acetonitrile and triethylamine to obtain a crude product solution; the concentration of acetonitrile in the crude product solution is preferably 20 mmol/L, and the concentration of triethylamine is preferably 16 mmol/L.
得到具有式a所示结构的化合物后，本发明将具有式a所示结构的化合物和寡聚腺苷酸混合进行缩合反应，得到具有式b所示结构的寡聚腺苷酸-脂肪酸化合物(记为oligo(A)-脂肪酸)。在本发明中，所述缩合反应的温度优选为室温，时间优选为8～15h；所述缩合反应用溶剂优选为四硼酸钠溶液，所述四硼酸钠溶液的浓度优选为0.5～2mol/L，更优选为1mol/L；所述寡聚腺苷酸的结构式如式c(具体结构式见上文)所示，所述寡聚腺苷酸中腺苷酸的数量优选为6～10；所述具有式a所示结构的化合物和寡聚腺苷酸的摩尔比优选为5:1～1:1，更优选为4:1～3:1。本发明对所述寡聚腺苷酸的来源没有特殊要求，采用市售商品或采用本领域熟知的固相合成法进行合成均可，在本发明的具体实施例中，所述寡聚腺苷酸购自生工生物工程(上海)股份有限公司或苏州金唯智生物科技有限公司。After obtaining the compound with the structure shown in formula a, the present invention mixes the compound with the structure shown in formula a and oligoadenylate to perform a condensation reaction to obtain the oligoadenylate-fatty acid compound with the structure shown in formula b ( Denoted as oligo(A)-fatty acid). In the present invention, the temperature of the condensation reaction is preferably room temperature, and the time is preferably 8 to 15 hours; the solvent for the condensation reaction is preferably sodium tetraborate solution, and the concentration of the sodium tetraborate solution is preferably 0.5 to 2 mol/L. , more preferably 1 mol/L; the structural formula of the oligoadenylate is as shown in formula c (see above for specific structural formula), and the number of adenylate in the oligoadenylate is preferably 6 to 10; so The molar ratio of the compound having the structure represented by formula a and oligoadenylic acid is preferably 5:1 to 1:1, more preferably 4:1 to 3:1. The present invention has no special requirements on the source of the oligoadenylate, and it can be synthesized by commercially available products or solid-phase synthesis methods well known in the art. In specific embodiments of the present invention, the oligoadenylate Acid was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. or Suzhou Jinweizhi Biotechnology Co., Ltd.
缩合反应完成后，本发明优选将所得反应产物用无酶水稀释，之后用离子交换柱进行HPLC纯化，得到具有式b所示结构的寡聚腺苷酸-脂肪酸化合物。在本发明中，所述HPLC纯化用离子交换柱优选为TSKgel IEC色谱柱，所述HPLC纯化用流动相优选为50％乙腈水溶液(pH值＝7.6)。After the condensation reaction is completed, the present invention preferably dilutes the reaction product obtained with enzyme-free water, and then performs HPLC purification using an ion exchange column to obtain the oligoadenylate-fatty acid compound having the structure represented by formula b. In the present invention, the ion exchange column for HPLC purification is preferably a TSKgel IEC chromatographic column, and the mobile phase for HPLC purification is preferably 50% acetonitrile aqueous solution (pH value = 7.6).
得到具有式b所示结构的寡聚腺苷酸-脂肪酸化合物后，本发明将寡聚腺苷酸-脂肪酸化合物与目标mRNA在RNA连接酶的催化作用下进行RNA连接反应，得到式I所示结构的mRNA-脂肪酸靶向化合物。在本发明中，所述RNA连接反应的体系组成优选包括：Tris-HCl缓冲液、MgCl2、PEG 8000、T4 RNA Ligase 1、三磷酸腺苷(ATP)、目标mRNA和寡聚腺苷酸-脂肪酸化合物；所述RNA连接酶优选为T4 RNA Ligase 1。After obtaining the oligoadenylate-fatty acid compound with the structure shown in formula b, the present invention performs an RNA ligation reaction between the oligoadenylate-fatty acid compound and target mRNA under the catalysis of RNA ligase to obtain formula I. Structure of mRNA-fatty acid targeting compounds. In the present invention, the system composition of the RNA ligation reaction preferably includes: Tris-HCl buffer, MgCl 2 , PEG 8000, T4 RNA Ligase 1, adenosine triphosphate (ATP), target mRNA and oligoadenylate-fatty acid compound; The RNA ligase is preferably T4 RNA Ligase 1.
在本发明中，所述Tris-HCl缓冲液的pH值优选为7～7.5，更优选为7.5，所述RNA 连接反应的体系组成中，MgCl2的浓度优选为0.1～1mmol/L，PEG-8000的浓度优选为5～50mg/mL，T4 RNA Ligase 1的浓度优选为15～25U，更优选为20U，ATP的浓度优选为0.5～2.5mmol/L，更优选为1mmol/L；所述目标mRNA和寡聚腺苷酸-脂肪酸化合物的摩尔比优选为1:50～50:1，更优选为1:20～20:1，最优选为1:2；在本发明中，所述MgCl2和PEG 8000用来维持酶活性，ATP作为T4 RNA Ligase 1的酶促反应底物，加在两段核酸序列中间。在本发明的具体实施例中，将上述体系组成中的各组分混合进行RNA连接反应即可。In the present invention, the pH value of the Tris-HCl buffer is preferably 7 to 7.5, more preferably 7.5, and the RNA In the system composition of the ligation reaction, the concentration of MgCl 2 is preferably 0.1 to 1 mmol/L, the concentration of PEG-8000 is preferably 5 to 50 mg/mL, the concentration of T4 RNA Ligase 1 is preferably 15 to 25 U, and more preferably 20 U, ATP The concentration is preferably 0.5~2.5mmol/L, more preferably 1mmol/L; the molar ratio of the target mRNA and oligoadenylate-fatty acid compound is preferably 1:50~50:1, more preferably 1:20 ~20:1, most preferably 1:2; in the present invention, the MgCl 2 and PEG 8000 are used to maintain enzyme activity, and ATP is used as the enzymatic reaction substrate of T4 RNA Ligase 1 and is added between the two nucleic acid sequences. . In specific embodiments of the present invention, all components in the above system composition are mixed to perform RNA ligation reaction.
在本发明中，所述RNA连接反应的温度优选为16～25℃，反应时间优选为30min～32h；在本发明的具体实施例中，当所述RNA连接反应的温度为16℃时，反应时间具体优选为2～32h；当所述RNA连接反应的温度为25℃时，反应时间具体优选为30min～16h。In the present invention, the temperature of the RNA ligation reaction is preferably 16-25°C, and the reaction time is preferably 30min-32h; in specific embodiments of the invention, when the temperature of the RNA ligation reaction is 16°C, the reaction The time is preferably 2 to 32 hours; when the temperature of the RNA ligation reaction is 25°C, the reaction time is preferably 30 min to 16 hours.
RNA连接反应完成后，本发明优选对产物料液进行纯化，得到mRNA-脂肪酸靶向化合物；本发明对所述纯化的方法没有特殊要求，采用本领域技术人员熟知的方法即可，具体如层析法、液相色谱法、氯化锂沉淀法、酚氯仿抽提法、乙醇沉淀法或醋酸铵沉淀法，在本发明的具体实施例中，优选采用氯化锂沉淀法，所述氯化锂沉淀法具体包括：将氯化锂溶液加入所述产物料液中，至料液中氯化锂的浓度为0.5mol/L，使mRNA-脂肪酸靶向化合物沉淀出来。After the RNA ligation reaction is completed, the present invention preferably purifies the product liquid to obtain the mRNA-fatty acid targeting compound; the present invention has no special requirements for the purification method, and can adopt methods well known to those skilled in the art, such as layer analysis method, liquid chromatography, lithium chloride precipitation method, phenol chloroform extraction method, ethanol precipitation method or ammonium acetate precipitation method. In specific embodiments of the present invention, the lithium chloride precipitation method is preferably used. The lithium precipitation method specifically includes: adding lithium chloride solution into the product liquid until the concentration of lithium chloride in the liquid is 0.5 mol/L, so that the mRNA-fatty acid targeting compound is precipitated.
本发明还提供了上述方案所述的mRNA-脂肪酸靶向化合物或上述方案所述制备方法制备的mRNA-脂肪酸靶向化合物在蛋白替代药物和代谢疾病治疗药物制备中的应用；在本发明中，所述蛋白替代药物和代谢疾病治疗药物的剂型优选为注射剂，所述注射剂的给药途径优选为肌肉注射；本发明提供的mRNA-脂肪酸靶向化合物的编码目标蛋白在肌肉组织中特异性表达量高，能够通过体循环注射直接递送至肌肉细胞，在肌肉注射疫苗中有广阔的应用前景。The present invention also provides the application of the mRNA-fatty acid targeting compound described in the above scheme or the mRNA-fatty acid targeting compound prepared by the preparation method described in the above scheme in the preparation of protein replacement drugs and metabolic disease treatment drugs; in the present invention, The dosage forms of the protein replacement drugs and metabolic disease treatment drugs are preferably injections, and the administration route of the injections is preferably intramuscular injection; the target protein encoded by the mRNA-fatty acid targeting compound provided by the invention is specifically expressed in muscle tissue. High, it can be delivered directly to muscle cells through systemic circulation injection, and has broad application prospects in intramuscular injection vaccines.
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.
实施例中使用的hEPO mRNA，编码区序列为：

The coding region sequence of hEPO mRNA used in the examples is:

实施例中使用的LUC mRNA，编码区序列为：
The coding region sequence of LUC mRNA used in the examples is:
实施例中使用的hGLP1 mRNA，编码区序列为：
The coding region sequence of hGLP1 mRNA used in the examples is:
实施例中使用的hRAS mRNA，编码区序列为：

The coding region sequence of hRAS mRNA used in the examples is:

实施例1Example 1
mRNA-棕榈酸靶向化合物的制备，步骤如下：Preparation of mRNA-palmitic acid targeting compounds, the steps are as follows:
(1)将0.256g棕榈酸和2.22mL三乙胺溶解在1mL二氯甲烷中，加入1.12g三氟乙酸五氟苯酯(Pfp-TFA)，室温条件下搅拌反应1小时；将所得反应产物用6mL二氯甲烷稀释，然后用3mL 5mmol/L的NaHCO3溶液和3mL 5mmol/L的NaHSO4溶液洗涤；分离以上溶液，得到有机层，将有机层用无水硫酸钠固体干燥，在负压条件下过滤和浓缩，得到粗产物；将所述粗产物进行硅胶柱色谱纯化；纯化步骤如下：(1) Dissolve 0.256g palmitic acid and 2.22mL triethylamine in 1mL methylene chloride, add 1.12g pentafluorophenyl trifluoroacetate (Pfp-TFA), and stir for 1 hour at room temperature; the resulting reaction product Dilute with 6 mL of methylene chloride, and then wash with 3 mL of 5 mmol/L NaHCO 3 solution and 3 mL of 5 mmol/L NaHSO 4 solution; separate the above solution to obtain an organic layer, dry the organic layer with anhydrous sodium sulfate solid, and place under negative pressure Filter and concentrate under the conditions to obtain a crude product; the crude product is purified by silica gel column chromatography; the purification steps are as follows:
a.将粗产物溶解在DMSO中，加入乙腈和三乙胺，得到粗产物溶液，其中粗产物的浓度为40mmol/L，乙腈的浓度为20mmol/L，三乙胺的浓度为16mmol/L；a. Dissolve the crude product in DMSO, add acetonitrile and triethylamine to obtain a crude product solution, in which the concentration of the crude product is 40mmol/L, the concentration of acetonitrile is 20mmol/L, and the concentration of triethylamine is 16mmol/L;
b.选取200-300目硅胶，称取20g，溶解在等体积的氯仿中，搅拌成匀浆；b. Select 200-300 mesh silica gel, weigh 20g, dissolve it in an equal volume of chloroform, and stir to form a homogenate;
c.在柱底用棉花塞紧，加入三分之一体积的氯仿，装上蓄液球，打开柱下活塞，将上述匀浆一次倾入蓄液球内，等待匀浆沉降完毕；c. Plug the bottom of the column with cotton, add one third of the volume of chloroform, install the liquid storage ball, open the piston under the column, pour the above homogenate into the liquid storage ball at once, and wait for the homogenate to settle;
d.沉降完成后，用气泵加压，直至流速稳定；d. After settlement is completed, use an air pump to pressurize until the flow rate is stable;
e.将粗产物溶液上到柱子中，0.5mL收一馏分，将目标产物馏分合并。e. Add the crude product solution to the column, collect a fraction of 0.5 mL, and combine the target product fractions.
(2)将具有式c所示结构的寡聚腺苷酸3.47g溶解在1mol/L四硼酸钠溶液中，浓度为2mM，与上述纯化产物混合，搅拌反应12h；反应产物用无酶水稀释，并用离子交换柱进行HPLC纯化，得到寡聚腺苷酸-脂肪酸化合物(oligo(A)-脂肪酸)。(2) Dissolve 3.47g of oligoadenylate with the structure shown in formula c in 1mol/L sodium tetraborate solution at a concentration of 2mM, mix with the above purified product, and stir for 12 hours; the reaction product is diluted with enzyme-free water , and perform HPLC purification using an ion exchange column to obtain oligoadenylate-fatty acid compound (oligo(A)-fatty acid).
(3)将PH＝7.5的Tris-HCl缓冲液、MgCl2、PEG 8000、T4 RNA Ligase 1、三磷酸腺苷(ATP)、LUC mRNA和oligo(A)-脂肪酸混合进行酶促反应，混合所得反应体系中PEG 8000的浓度为20mg/mL，T4 RNA Ligase 1的浓度为20U，ATP的浓度为1mmol/L，LUC mRNA的浓度为1mmol/L，oligo(A)-脂肪酸的浓度为2mmol/L，所述酶促反应的温度为25℃，反应时间为30min。反应完成后，向反应液中加入氯化锂溶液，至氯化 锂的终浓度为0.5mol/L，在此条件下将产物沉淀出来，得到LUC mRNA-棕榈酸靶向化合物。(3) Mix Tris-HCl buffer with pH=7.5, MgCl 2 , PEG 8000, T4 RNA Ligase 1, adenosine triphosphate (ATP), LUC mRNA and oligo(A)-fatty acid for enzymatic reaction, and mix the resulting reaction system The concentration of PEG 8000 is 20mg/mL, the concentration of T4 RNA Ligase 1 is 20U, the concentration of ATP is 1mmol/L, the concentration of LUC mRNA is 1mmol/L, the concentration of oligo(A)-fatty acid is 2mmol/L, as described The temperature of the enzymatic reaction was 25°C, and the reaction time was 30 minutes. After the reaction is completed, add lithium chloride solution to the reaction solution until chloride The final concentration of lithium was 0.5 mol/L. Under these conditions, the product was precipitated to obtain the LUC mRNA-palmitic acid targeting compound.
实施例2Example 2
mRNA-棕榈酸分子化合物靶向递送验证Validation of targeted delivery of mRNA-palmitic acid molecule compounds
将实施例1制备的LUC mRNA-棕榈酸靶向化合物与纳米脂质颗粒(LNP)递送载体的靶向递送特点进行对比，其中LNP-LUC mRNA的制备方法如下：Compare the targeted delivery characteristics of the LUC mRNA-palmitic acid targeting compound prepared in Example 1 and the lipid nanoparticle (LNP) delivery carrier, where the preparation method of LNP-LUC mRNA is as follows:
配制水相：将LUC mRNA按照2μg/μl的终浓度稀释于柠檬酸缓冲液中，得到mRNA缓冲液；按照表1配制乙醇相溶液；Prepare aqueous phase: Dilute LUC mRNA in citric acid buffer at a final concentration of 2 μg/μl to obtain an mRNA buffer; prepare an ethanol phase solution according to Table 1;
表1乙醇相溶液配方
Table 1 Ethanol phase solution formula
准备好PBS溶液作为LNP稀释液；Prepare PBS solution as LNP diluent;
注射泵仪器操作步骤：Syringe pump instrument operation steps:
(1)将A相(mRNA缓冲液)装入5mL注射器，B相(乙醇相溶液)装入5mL注射器，安装于注射泵，夹紧；(1) Put phase A (mRNA buffer) into a 5mL syringe, and phase B (ethanol phase solution) into a 5mL syringe, install it on the syringe pump, and clamp;
(2)将芯片连接到注射器，设定注射泵流速；(2) Connect the chip to the syringe and set the syringe pump flow rate;
(3)点击注射泵的开始按键，将料液注入芯片；(3) Click the start button of the syringe pump to inject the material liquid into the chip;
(4)观察芯片出口的产品颜色，弃去前5滴乳白色液滴(约为100μl)后，开始收集，将产品收集到60mL PBS溶液中；(4) Observe the color of the product at the chip outlet. After discarding the first 5 milky white droplets (about 100 μl), start collecting and collect the product into 60mL PBS solution;
(5)收集完成的产品，轻柔地上下颠倒混匀，于4℃下保存。(5) Collect the completed product, mix gently by inverting it up and down, and store it at 4°C.
采用上述制备得到的产品(LNP-LUC mRNA)进行后续的靶向递送特点对比实验，具体步骤如下：Use the product prepared above (LNP-LUC mRNA) to conduct subsequent comparative experiments on targeted delivery characteristics. The specific steps are as follows:
将6～8周龄的balb/c小鼠(购自北京维通利华)在SPF条件下饲养，保持12小时光照和12小时黑暗循环的同期饲养笼，以Luc mRNA-棕榈酸靶向化合物和LNP-LUC mRNA分别对小鼠进行尾静脉注射，注射剂量分别为1mg，5mg，10mg，15mg，24小时后取小鼠肝脏和四头肌，用组织RNA提取试剂盒提取mRNA，进行荧光定量PCR，对不同组织中的目标mRNA进行定量。实验结果如图1-图2所示。 Balb/c mice (purchased from Beijing Vitong Lever) aged 6 to 8 weeks were raised under SPF conditions and kept in cages with a 12-hour light and 12-hour dark cycle, and were treated with Luc mRNA-palmitic acid targeting compounds. and LNP-LUC mRNA were injected into the tail vein of mice respectively. The injection doses were 1 mg, 5 mg, 10 mg, and 15 mg respectively. After 24 hours, the liver and quadriceps muscles of the mice were taken, and the mRNA was extracted using a tissue RNA extraction kit for fluorescence quantification. PCR to quantify target mRNA in different tissues. The experimental results are shown in Figures 1-2.
根据图1-图2可以看出，在肝脏组织中，LNP-LUC mRNA的含量较高，而在四头肌组织中，Luc mRNA-棕榈酸靶向化合物的含量明显高于LNP-LUC mRNA，说明本发明提供的mRNA-棕榈酸靶向化合物的肌肉靶向递送效果好。According to Figure 1-Figure 2, it can be seen that in liver tissue, the content of LNP-LUC mRNA is higher, while in quadriceps muscle tissue, the content of Luc mRNA-palmitic acid targeting compound is significantly higher than LNP-LUC mRNA. It shows that the mRNA-palmitic acid targeting compound provided by the present invention has good muscle targeting delivery effect.
实施例3Example 3
其他条件和实施例1相同，仅将LUC mRNA替换为hEPO mRNA，得到hEPO mRNA-棕榈酸靶向化合物。Other conditions were the same as Example 1, except that LUC mRNA was replaced with hEPO mRNA to obtain hEPO mRNA-palmitic acid targeting compound.
hEPO mRNA-棕榈酸编码目标蛋白在肌肉组织中特异性表达验证：Verification of specific expression of hEPO mRNA-palmitic acid-encoded target protein in muscle tissue:
将6-8周龄的balb/c小鼠(购自北京维通利华)在SPF条件下饲养，保持12小时光照和12小时黑暗循环的同期饲养笼，将以hEPO mRNA-棕榈酸靶向化合物对小鼠进行尾静脉注射，注射剂量为5mg，24小时后取小鼠心脏、肺、脾脏、肝脏、四头肌和血清，提取总蛋白，通过免疫级联吸附反应(elisa)，对目标蛋白产物在不同组织中的表达进行定量。实验结果如图3所示。6-8-week-old balb/c mice (purchased from Beijing Vitong Lever) were raised under SPF conditions in simultaneous cages with a 12-hour light and 12-hour dark cycle, and hEPO mRNA-palmitic acid was targeted. The compound was injected into the tail vein of mice at a dose of 5 mg. After 24 hours, the heart, lungs, spleen, liver, quadriceps and serum of the mice were taken to extract the total protein. The target was analyzed through immune cascade adsorption reaction (elisa). The expression of protein products in different tissues was quantified. The experimental results are shown in Figure 3.
根据图3可以看出，目标蛋白产物在肌肉中的表达量最高。According to Figure 3, it can be seen that the expression level of the target protein product is the highest in muscle.
实施例4Example 4
mRNA-油酸靶向化合物的制备，步骤如下：Preparation of mRNA-oleic acid targeting compounds, the steps are as follows:
(1)将0.282g油酸和2.22mL三乙胺溶解在1mL二氯甲烷中，加入1.12g三氟乙酸五氟苯酯(Pfp-TFA)，室温条件下搅拌反应1小时；将所得反应产物用6mL二氯甲烷稀释，然后用3mL 5mmol/L的NaHCO3溶液和3mL 5mmol/L的NaHSO4溶液洗涤；分离以上溶液，得到有机层，将有机层用无水硫酸钠固体干燥，在负压条件下过滤和浓缩，得到粗产物；将所述粗产物进行硅胶柱色谱纯化；纯化方法和实施例1一致。(1) Dissolve 0.282g oleic acid and 2.22mL triethylamine in 1mL methylene chloride, add 1.12g pentafluorophenyl trifluoroacetate (Pfp-TFA), stir and react at room temperature for 1 hour; Dilute with 6 mL of methylene chloride, and then wash with 3 mL of 5 mmol/L NaHCO 3 solution and 3 mL of 5 mmol/L NaHSO 4 solution; separate the above solution to obtain an organic layer, dry the organic layer with anhydrous sodium sulfate solid, and place under negative pressure Filter and concentrate under the conditions to obtain a crude product; the crude product is purified by silica gel column chromatography; the purification method is consistent with Example 1.
(2)将具有式c所示结构的寡聚腺苷酸3.47g溶解在1mol/L四硼酸钠溶液中，浓度为2mmol/L，与上述纯化产物混合，搅拌反应12h；反应产物用无酶水稀释，并用离子交换柱进行HPLC纯化，得到寡聚腺苷酸-脂肪酸化合物(oligo(A)-脂肪酸)。(2) Dissolve 3.47g of oligoadenylate with the structure shown in formula c in 1mol/L sodium tetraborate solution at a concentration of 2mmol/L, mix it with the above purified product, and stir for 12 hours; the reaction product is treated with enzyme-free Dilute with water and perform HPLC purification with an ion exchange column to obtain oligoadenylate-fatty acid compound (oligo(A)-fatty acid).
(3)将PH＝7.5的Tris-HCl缓冲液、MgCl2、PEG 8000、T4 RNA Ligase 1、三磷酸腺苷(ATP)、hEPO mRNA和oligo(A)-脂肪酸混合进行酶促反应，混合所得反应体系中PEG 8000的浓度为20mg/mL，T4 RNA Ligase 1的浓度为20U，ATP的浓度为1mmol/L，hEPO mRNA的浓度为1mmol/L，oligo(A)-脂肪酸的浓度为2mmol/L，所述酶促反应的温度为25℃，反应时间为30min。反应完成后，采用氯化锂沉淀法对产物料液进行纯化，纯化方法和实施例1一致，得到hEPO mRNA-油酸靶向化合物。(3) Mix Tris-HCl buffer with pH=7.5, MgCl 2 , PEG 8000, T4 RNA Ligase 1, adenosine triphosphate (ATP), hEPO mRNA and oligo(A)-fatty acid for enzymatic reaction, and mix the resulting reaction system The concentration of PEG 8000 is 20mg/mL, the concentration of T4 RNA Ligase 1 is 20U, the concentration of ATP is 1mmol/L, the concentration of hEPO mRNA is 1mmol/L, the concentration of oligo(A)-fatty acid is 2mmol/L, as described The temperature of the enzymatic reaction was 25°C, and the reaction time was 30 minutes. After the reaction is completed, the product liquid is purified using the lithium chloride precipitation method. The purification method is consistent with Example 1 to obtain the hEPO mRNA-oleic acid targeting compound.
实施例5 Example 5
mRNA-油酸编码目标蛋白在肌肉组织中特异性表达验证：Verification of specific expression of target protein encoded by mRNA-oleic acid in muscle tissue:
将6-8周龄的balb/c小鼠(购自北京维通利华)在SPF条件下饲养，保持12小时光照和12小时黑暗循环的同期饲养笼，将以hEPO mRNA-油酸靶向化合物对小鼠进行尾静脉注射，注射剂量为5mg，24小时后取小鼠心脏、肺、脾脏、肝脏、四头肌和血清，提取总蛋白，通过免疫级联吸附反应(elisa)，对不同组织中的目标蛋白产物在不同组织中的表达进行定量。实验结果如图4所示。根据图4可以看出，目标蛋白产物在肌肉中的表达量最高。6-8 week old balb/c mice (purchased from Beijing Vitong Lever) were raised under SPF conditions in simultaneous cages with a 12-hour light and 12-hour dark cycle, and hEPO mRNA-oleic acid was targeted. The compound was injected into the tail vein of mice at a dose of 5 mg. After 24 hours, the heart, lungs, spleen, liver, quadriceps and serum of the mice were taken to extract the total protein. Through immune cascade adsorption reaction (elisa), different Quantify the expression of target protein products in different tissues. The experimental results are shown in Figure 4. According to Figure 4, it can be seen that the expression level of the target protein product is the highest in muscle.
实施例6Example 6
mRNA-脂肪酸靶向化合物蛋白替代试验mRNA-fatty acid targeting compound protein replacement assay
其他条件和实施例4相同，仅将hEPO mRNA替换为hGLP1 mRNA，得到hGLP1 mRNA-油酸靶向化合物。Other conditions were the same as in Example 4, except that hEPO mRNA was replaced with hGLP1 mRNA to obtain hGLP1 mRNA-oleic acid targeting compound.
mRNA-油酸编码外泌型尿酸氧化酶在肌肉组织中特异性表达：mRNA-oleic acid encodes exocrine urate oxidase and is specifically expressed in muscle tissue:
将6-8周龄的BKS db/db高血糖模型小鼠(购自北京维通利华)在SPF条件下饲养，保持12小时光照和12小时黑暗循环的同期饲养笼，以hGLP1 mRNA-油酸靶向化合物对小鼠进行肌肉注射，注射剂量为5mg，24小时后取血清，提取总蛋白，通过血糖仪对小鼠血糖浓度进行定量，同时对空白对照组的小鼠的总蛋白和血糖浓度进行定量。实验结果如图5所示，图5左侧为GLP-1的表达量，右侧为血糖浓度，图5中的实验组为注射hGLP1 mRNA-油酸靶向化合物的实验组。根据图5可以看出，注射hGLP1 mRNA-油酸靶向化合物的实验组目标蛋白产物高表达，血糖浓度显著降低。BKS db/db hyperglycemia model mice (purchased from Beijing Vitong Lever) aged 6-8 weeks were raised under SPF conditions and kept in cages with a 12-hour light and 12-hour dark cycle. hGLP1 mRNA-oil Acid-targeting compounds were injected intramuscularly into mice at a dose of 5 mg. After 24 hours, the serum was taken, the total protein was extracted, and the blood glucose concentration of the mice was quantified by a blood glucose meter. At the same time, the total protein and blood glucose of the mice in the blank control group were measured. Concentration is quantified. The experimental results are shown in Figure 5. The left side of Figure 5 shows the expression of GLP-1, and the right side shows the blood glucose concentration. The experimental group in Figure 5 is the experimental group injected with hGLP1 mRNA-oleic acid targeting compound. According to Figure 5, it can be seen that the experimental group injected with hGLP1 mRNA-oleic acid targeting compound expressed high expression of the target protein product and significantly reduced blood glucose concentration.
实施例7Example 7
mRNA-脂肪酸靶向化合物代谢疾病治疗药物试验mRNA-fatty acid-targeting compound metabolic disease treatment drug trial
其他条件和实施例4相同，仅将hEPO mRNA替换为hRAS mRNA，得到hRAS mRNA-油酸靶向化合物。Other conditions were the same as in Example 4, except that hEPO mRNA was replaced with hRAS mRNA to obtain hRAS mRNA-oleic acid targeting compound.
mRNA-油酸编码外泌型尿酸氧化酶在肌肉组织中特异性表达：mRNA-oleic acid encodes exocrine urate oxidase and is specifically expressed in muscle tissue:
将6-8周龄的balb/c小鼠(购自北京维通利华)在SPF条件下饲养，保持12小时光照和12小时黑暗循环的同期饲养笼，将以hRAS mRNA-油酸靶向化合物对小鼠进行肌肉注射，注射剂量为5mg，24小时后取血清，提取总蛋白，通过免疫级联吸附反应(elisa)，对血尿酸浓度进行定量，同时对空白对照组的小鼠的目标蛋白产物和血尿酸浓度进行定量。实验结果如图6所示，图6中实验组为注射hRAS mRNA-油酸靶向化合物的实验组。根据图6可以看出，血尿酸浓度显著降低。 6-8 week old balb/c mice (purchased from Beijing Vital Lever) were raised under SPF conditions in simultaneous cages with a 12-hour light and 12-hour dark cycle to target hRAS mRNA-oleic acid. The compound was injected intramuscularly into mice at a dose of 5 mg. The serum was taken 24 hours later, and the total protein was extracted. The blood uric acid concentration was quantified through immune cascade adsorption reaction (elisa). At the same time, the target of mice in the blank control group was Protein products and serum uric acid concentrations were quantified. The experimental results are shown in Figure 6. The experimental group in Figure 6 is the experimental group injected with hRAS mRNA-oleic acid targeting compound. According to Figure 6, it can be seen that the blood uric acid concentration was significantly reduced.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。 The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (13)

  1. 一种mRNA-脂肪酸靶向化合物，其中，结构如式I所示：
    An mRNA-fatty acid targeting compound, wherein the structure is shown in formula I:
    式I中的R为-CnH2n+1或-CnH2n-1，其中n表示碳原子数；R in formula I is -C n H 2n+1 or -C n H 2n-1 , where n represents the number of carbon atoms;
    表示mRNA。 represents mRNA.
  2. 根据权利要求1所述的mRNA-脂肪酸靶向化合物，其中，所述n的取值范围为15～22。The mRNA-fatty acid targeting compound according to claim 1, wherein the value of n ranges from 15 to 22.
  3. 根据权利要求1所述的mRNA-脂肪酸靶向化合物，其中，所述R为-(CH2)14CH3或-(CH2)7CH＝CH(CH2)7CH3。The mRNA-fatty acid targeting compound according to claim 1 , wherein the R is - ( CH2 ) 14CH3 or -( CH2 ) 7CH =CH( CH2 ) 7CH3 .
  4. 根据权利要求1～3所述的mRNA-脂肪酸靶向化合物，其中，所述mRNA为可编码功能蛋白的mRNA。The mRNA-fatty acid targeting compound according to claims 1 to 3, wherein the mRNA is an mRNA encoding a functional protein.
  5. 权利要求1～4任意一项所述mRNA-脂肪酸靶向化合物的制备方法，其中，包括以下步骤：The preparation method of the mRNA-fatty acid targeting compound according to any one of claims 1 to 4, which includes the following steps:
    将具有式b所示结构的寡聚腺苷酸-脂肪酸化合物与目标mRNA在RNA连接酶的催化作用下进行RNA连接反应，得到式I所示结构的mRNA-脂肪酸靶向化合物；
    Perform an RNA ligation reaction between the oligoadenylate-fatty acid compound having the structure shown in formula b and the target mRNA under the catalysis of RNA ligase to obtain the mRNA-fatty acid targeting compound having the structure shown in formula I;
    式b中：R为-CnH2n+1或-CnH2n-1，n表示碳原子数，A表示腺苷酸。In formula b: R is -C n H 2n+1 or -C n H 2n-1 , n represents the number of carbon atoms, and A represents adenylate.
  6. 根据权利要求5所述的制备方法，其中，所述具有式b所示结构的寡聚腺苷酸-脂肪酸化合物的制备方法包括以下步骤：The preparation method according to claim 5, wherein the preparation method of the oligoadenylate-fatty acid compound having the structure shown in formula b includes the following steps:
    将具有式a所示结构的化合物和寡聚腺苷酸混合进行缩合反应，得到具有式b所示结构的寡聚腺苷酸-脂肪酸化合物；所述寡聚腺苷酸的结构式如式c所示；
    The compound having the structure shown in formula a and oligoadenylic acid are mixed for a condensation reaction to obtain the oligoadenylic acid-fatty acid compound having the structure shown in formula b; the structural formula of the oligoadenylic acid is as shown in formula c Show;
  7. 根据权利要求6所述的制备方法，其中，所述缩合反应的温度为室温，时间为8～15h。The preparation method according to claim 6, wherein the temperature of the condensation reaction is room temperature and the time is 8 to 15 hours.
  8. 根据权利要求6所述的制备方法，其中，所述具有式a所示结构的化合物的制备方法包括以下步骤： The preparation method according to claim 6, wherein the preparation method of the compound having the structure shown in formula a includes the following steps:
    将脂肪酸、三氟乙酸五氟苯酯和碱性试剂混合进行酯交换反应，得到具有式a所示结构的化合物；所述脂肪酸的结构式表示为R-COOH。The fatty acid, pentafluorophenyl trifluoroacetate and an alkaline reagent are mixed to perform a transesterification reaction to obtain a compound having the structure shown in formula a; the structural formula of the fatty acid is expressed as R-COOH.
  9. 根据权利要求8所述的制备方法，其中，所述酯交换反应的温度为室温，时间为0.5～2h。The preparation method according to claim 8, wherein the temperature of the transesterification reaction is room temperature and the time is 0.5 to 2 hours.
  10. 根据权利要求5所述的制备方法，其中，所述RNA连接反应的体系组成包括：Tris-HCl缓冲液、MgCl2、PEG 8000、RNA连接酶、三磷酸腺苷、目标mRNA和寡聚腺苷酸-脂肪酸化合物。The preparation method according to claim 5, wherein the system composition of the RNA ligation reaction includes: Tris-HCl buffer, MgCl 2 , PEG 8000, RNA ligase, adenosine triphosphate, target mRNA and oligoadenylate-fatty acid compound.
  11. 权利要求1～4任意一项所述mRNA-脂肪酸靶向化合物或权利要求5～10任意一项所述制备方法制备的mRNA-脂肪酸靶向化合物在制备蛋白替代药物和代谢疾病治疗药物中的应用。Application of the mRNA-fatty acid targeting compound according to any one of claims 1 to 4 or the mRNA-fatty acid targeting compound prepared by the preparation method according to any one of claims 5 to 10 in the preparation of protein replacement drugs and metabolic disease treatment drugs .
  12. 根据权利要求11所述的应用，其中，所述蛋白替代药物和代谢疾病治疗药物的剂型为注射剂。The application according to claim 11, wherein the dosage forms of the protein replacement drugs and metabolic disease treatment drugs are injections.
  13. 根据权利要求11所述的应用，其中，所述蛋白替代药物和代谢疾病治疗药物的剂型为肌肉注射剂。 The application according to claim 11, wherein the dosage forms of the protein replacement drugs and metabolic disease treatment drugs are intramuscular injections.
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