WO2023143591A1 - Novel ionizable lipid used for nucleic acid delivery and lnp composition thereof and vaccine - Google Patents

Novel ionizable lipid used for nucleic acid delivery and lnp composition thereof and vaccine Download PDF

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WO2023143591A1
WO2023143591A1 PCT/CN2023/073756 CN2023073756W WO2023143591A1 WO 2023143591 A1 WO2023143591 A1 WO 2023143591A1 CN 2023073756 W CN2023073756 W CN 2023073756W WO 2023143591 A1 WO2023143591 A1 WO 2023143591A1
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lipid
peg
cationic
cationic lipid
mrna
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PCT/CN2023/073756
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French (fr)
Chinese (zh)
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李荩
王浩猛
严志红
原晋波
刘健
宇学峰
邱东旭
朱涛
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康希诺生物股份公司
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • C07C215/00—Compounds containing amino and hydroxy groups bound to the same carbon skeleton
    • C07C215/02—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C215/04—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated
    • C07C215/06—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic
    • C07C215/14—Compounds containing amino and hydroxy groups bound to the same carbon skeleton having hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being saturated and acyclic the nitrogen atom of the amino group being further bound to hydrocarbon groups substituted by amino groups
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    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/08—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom
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    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
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    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/16—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the hydroxy groups esterified by an inorganic acid or a derivative thereof
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/34—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups
    • C07C233/35—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C255/00—Carboxylic acid nitriles
    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/24—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the same saturated acyclic carbon skeleton
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    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a novel ionizable lipid for nucleic acid delivery and its LNP composition and vaccine.
  • the clinically proven system for delivering mRNA is lipid nanoparticle (Lipid Nanoparticle, LNP), which belongs to lipid-formed nanoparticles, and its principle includes cationic lipids.
  • LNP lipid nanoparticle
  • the mRNA expression rate is low.
  • Dlin-MC3-DMA is used as a cationic lipid to construct LNP, and the mRNA expression level is 0.63% (Maugeri, Marco et al. "Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.”Nature Communications, 2019), therefore, the structure of cationic lipids is a key factor affecting the expression of mRNA.
  • Rabies vaccine is the rabies vaccine and anti-rabies serum inoculated after a person is bitten by an animal to prevent infection with rabies.
  • Rabies is a natural foci or zoonotic acute infectious disease caused by rabies virus. It is widespread and has a high fatality rate, posing a serious threat to people's lives and health.
  • rabies The typical clinical manifestation of rabies is hydrophobia, so rabies is also called hydrophobia.
  • rabies In the early stage, it is sensitive to sound, light, wind and other stimuli, and the throat feels tight. When it enters the excited stage, it can manifest as extreme terror, fear of water, and fear of wind. , paroxysmal pharyngeal muscle spasm, dyspnea, etc., and finally the spasm stops and various paralysis occurs, and can quickly die due to respiratory and circulatory failure.
  • Human rabies is mainly caused by biting, scratching or mucous membrane infection of sick animals, and it can also be transmitted through respiratory aerosol under certain conditions.
  • the saliva of infected animals contains rabies virus. Infected animals are mainly dogs (more than 90%), followed by cats.
  • neutral lipid refers to uncharged, non-phosphoglyceride lipid molecules.
  • polyethylene glycol (PEG)-lipid conjugate refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.
  • lipid nanoparticle refers to a particle having at least one nanoscale size, which comprises at least one lipid.
  • vaccine in the present invention refers to a composition suitable for application to animals (including humans), which induces an immune response after administration, and its strength is sufficient to help prevent, ameliorate or cure clinical diseases caused by microbial infection at a minimum.
  • delivery system in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dose in a living body.
  • N/P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.
  • hydrocarbyl in the present invention refers to the remaining group after the corresponding hydrocarbon loses a hydrogen atom, especially refers to aliphatic hydrocarbon groups in the present invention, such as alkyl, alkenyl, alkynyl, especially alkyl.
  • the present invention provides cationic lipids, which have the following formula I structure:
  • G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;
  • G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenene;
  • Ra is H or C 1 -C 12 hydrocarbon group
  • R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R 4 is C 1 -C 12 hydrocarbon group
  • R 5 is H or C 1 -C 6 hydrocarbon group
  • x 0, 1 or 2.
  • the cationic lipid wherein has the following structure (IA):
  • R 6 is independently H, OH, or C 1 -C 24 hydrocarbyl at each occurrence;
  • n is an integer of 1 to 15.
  • the cationic lipid wherein has the following structure (IB):
  • y and z are each independently an integer from 1 to 12.
  • n in the cationic lipid structure is an integer from 2 to 12, preferably, n is 2, 3, 4, 5 or 6; wherein y and z are each independently an integer from 2 to 10, preferably, is an integer from 4 to 9.
  • R 1 and R 2 each independently have the following structure in the cationic lipid structure:
  • R 7a and R 7b are independently H or C 1 -C 12 hydrocarbon groups at each occurrence; and a is an integer from 2 to 12, preferably, a is an integer from 8 to 12;
  • R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms.
  • R 7a in the cationic lipid structure is H, preferably, R 7a is H every time it occurs.
  • R 7b that occurs at least once in the cationic lipid structure is a C 1 -C 8 hydrocarbon group; preferably, wherein the C 1 -C 8 hydrocarbon group is methyl, ethyl, n-propyl, isopropyl, n-propyl Butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.
  • R1 or R2 or both have one of the following structures:
  • the structure of the cationic lipid compound is as follows:
  • the present invention relates to a lipid nanoparticle comprising: (a) the cationic lipid described above; (b) a non-cationic lipid; (c) a polyethylene glycol (PEG)-lipid conjugate.
  • a lipid nanoparticle comprising: (a) the cationic lipid described above; (b) a non-cationic lipid; (c) a polyethylene glycol (PEG)-lipid conjugate.
  • PEG polyethylene glycol
  • cationic lipids, neutral phospholipids, steroidal lipids and/or polyethylene glycol (PEG)-lipid conjugates are included.
  • the polyethylene glycol (PEG)-lipid conjugate wherein is selected from: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159) , 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino( Polyethylene glycol)] (PEG-DSPE), PEG-Disteryl glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE ), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) or one or more combinations of DMG-PEG2000, 1,
  • the neutral lipid wherein is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1,2-dicarnoyl Myristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), 2-Dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), Oleoylphosphatidylcholine (POPC) , 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), one or more combinations, preferably DSPC.
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • the steroidal lipid wherein is selected from the group consisting of avenasterol, ⁇ -sitosterol, brassicasterol, ergocalcidol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergot Calciferol, dihydrocholesterol, dihydroergosterol, nigrosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol and cholesterol modified by peptides; lanosterol, photosterol, seawesterol , sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol .
  • the molar content of cationic lipids is 20-60%, the molar content of neutral phospholipids is about 5%-25%, and the molar content of steroidal lipids is about 25%-55%; polyethylene glycol (PEG) - the molar content of the lipid conjugate is about 0.5% to 15%,
  • the molar ratio of cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate is 30-60:1-20:20-50:0.1-10, preferably wherein the molar ratio of cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate is 43:10:45:2 or 40:10:48:2.
  • the vaccine also contains other adjuvants, wherein the adjuvants are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.
  • the adjuvants are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.
  • the nanoparticles have an average particle size of 50-200 nm or wherein the nanoparticles have a net neutral charge at neutral pH or wherein the nanoparticles have a polydispersity of less than 0.4.
  • the invention relates to a preparation method of lipid nano particle mRNA vaccine. Specifically, cationic lipids, non- Cationic lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved in a solvent and mixed with mRNA.
  • PEG polyethylene glycol
  • cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and mixed with diluted mRNA dilutions, followed by ultrafiltration, dilution, Prepared after filtration; preferably, cationic lipids, neutral phospholipids, steroidal lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and diluted mRNA diluent at a certain flow rate It is prepared by ultrafiltration, dilution and filtration after mixing; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent flow mixing, laminar flow mixing or microfluidic mixing.
  • the diluent may be acetate buffer, citrate buffer, phosphate buffer or tris buffer.
  • the pH of the buffer solution is 3-6, and the concentration is 6.25-200 mM.
  • the solution flow rate ratio of the lipid mixed solution obtained after dissolving cationic lipids, non-cationic lipids, and polyethylene glycol (PEG)-lipid conjugates into a solvent and mRNA after dilution is 1 to 5: 1.
  • the N/P is 2-10, preferably the N/P is 3-8, more preferably, the N/P is 3, 4, 5, 6, 7, 8.
  • the ultrafiltrate is selected from the group consisting of sodium salt and tris(hydroxymethyl)aminomethane (Tris) salt, preferably, the pH of the ultrafiltrate is 6.0-8.0.
  • Tris tris(hydroxymethyl)aminomethane
  • the invention provides a rabies virus lipid nanoparticle mRNA vaccine, specifically, the administration method can be oral administration, intramuscular injection, intravenous injection or inhalation.
  • the dosage form of the rabies virus lipid nanoparticle mRNA vaccine can be an oral preparation, a liquid preparation, a freeze-dried powder, an injection or an inhalation preparation, preferably, an intramuscular injection, an intravenous injection, a dry powder inhalation or an aerosol inhalation.
  • the present invention relates to a rabies virus lipid nanoparticle mRNA vaccine, comprising: (a) mRNA capable of encoding rabies virus G protein; (b) cationic lipid; (c) non-cationic lipid; (d) polyethylene glycol Alcohol (PEG)-lipid conjugates.
  • a rabies virus lipid nanoparticle mRNA vaccine comprising: (a) mRNA capable of encoding rabies virus G protein; (b) cationic lipid; (c) non-cationic lipid; (d) polyethylene glycol Alcohol (PEG)-lipid conjugates.
  • the rabies virus lipid nanoparticle mRNA vaccine wherein the amino acid sequence encoded by the mRNA comprises SEQ ID NO: 1 (G protein of CTN-1 strain (GenBank: ACR39382.1): SEQ ID NO: 1) or The sequence shown in SEQ ID NO: 2 (CTN-1V-T strain G protein).
  • the rabies virus lipid nanoparticle mRNA vaccine comprises: (a) mRNA capable of encoding rabies virus G protein; (b) cationic lipids; (c) neutral phospholipids, steroidal lipids; (d) Polyethylene glycol (PEG)-lipid conjugates.
  • the mRNA encoding rabies virus G protein was encapsulated by lipid nanoparticles prepared by ALC-0315.
  • the rabies virus lipid nanoparticle mRNA vaccine of the present invention can deliver the biologically active substance through oral administration, inhalation or injection.
  • the invention relates to the use of the rabies virus lipid nanoparticle mRNA vaccine in the preparation of medicines for preventing rabies.
  • the present invention relates to novel cationic lipids which differ from the prior art.
  • the mRNA LNP has better stability and transfection efficiency, and can cause a higher specific antibody response in experimental animals.
  • the present invention provides a rabies virus mRNA vaccine of lipid nanoparticles, which uses lipid nanoparticles as a delivery system, and has better physical and chemical properties by constructing a new cationic lipid, and its encapsulation rate is significantly better than that of lipids already on the market.
  • the plasma nanoparticle delivery system obtained a more immunogenic rabies virus lipid nanoparticle mRNA vaccine.
  • Figure 1 shows the LNP formulation stability-particle size data graph
  • Figure 2 shows the LNP preparation stability-encapsulation efficiency data graph
  • Figure 3 shows the LNP formulation stability-mRNA integrity data graph
  • Fig. 4 shows the LNP formulation stability-PDI data figure
  • FIG. 5 shows the immunization program for BALB/c mice
  • Figure 6 shows the serum neutralizing antibody titer on the BALB/c mouse model
  • Figure 7 shows the frequency of CD4+ T cells specifically secreting TNF ⁇ and IFN ⁇ detected by ICS on the BALB/c mouse model
  • Figure 8 shows the frequency of CD8+ T cells specifically secreting TNF ⁇ and IFN ⁇ detected by ICS on the BALB/c mouse model.
  • Heptadecan-9-yl (7-((2-hydroxyethyl)amino)heptyl)carbonate (457mg, 1.0mmol) was dissolved in tetrahydrofuran, acetonitrile was added, 5-bromopentylundecylcarbonate Ester (437mg, 1.2mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h.
  • compound 11b (1.70 g, 2 mmol) was slowly added to a solution of lithium aluminum hydride (379 mg, 10 mmol) in anhydrous THF (10 ml), and the mixture was heated to reflux for 5 hours. After the reaction is complete, lower the temperature and add water to the system to completely decompose the excess reducing agent. After filtration, the filter residue was washed with ethyl acetate, and the obtained filtrate was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 11 (1.45 g, yellow oil) with a yield of 90%.
  • the present invention uses cationic lipids I-II and control lipids III-IV to prepare lipid nanoparticle nucleic acid vaccines, and the structures of the four cationic lipids are shown in the table below.
  • the vaccine stock solution contains the encoded rabies virus G protein, the antigen sequence is as described in SEQ ID NO: 1, and the target antigen is routinely modified,
  • the N-terminal contains a 5'UTR and a cap structure, and the C-terminal contains a 3'UTR and a PolyA tail.
  • the diluted vaccine stock solution is cationic lipid: DSPC: cholesterol: DMG-PEG2000 molar ratio is 43:10:45:2
  • Prepare the lipid mixed solution ; set the total flow rate of the nano-medicine manufacturing equipment to 12ml/min, the flow rate ratio of the mRNA solution and the lipid mixed solution to 3:1 and start encapsulation. sample, and add sucrose to dissolve liquid.
  • N/P ionizable cationic lipid to nucleotide phosphate
  • N/P molar ratios were 3.6, 5.6, 7.6, respectively. Samples were taken to detect encapsulation efficiency, average particle size, PDI and Zeta potential, and the results are shown in the table below.
  • Embodiment 14 mouse immunization and detection
  • mice Six to eight-week-old female BALB/c mice were randomly divided into 14 groups, and immunized by intramuscular injection in the hind legs. Among them, groups 1-14 were immunized with samples 1-14 (prepared in Example 12). As shown in Figure 5, they were immunized on day 0 and day 14 respectively, and the single immunization dose was 5 ⁇ g mRNA-LNP. On the 14th and 28th day of immunization, blood was collected and serum was separated for detection of antibody titer. The detection results are shown in Table 7 and Figure 6.
  • Lipid nanoparticle mRNA vaccine neutralizing antibody titers after table 7 different cationic lipid encapsulation
  • Serum neutralizing antibody titer detection result shows that the rabies virus mRNA vaccine prepared by the present invention can stimulate the body to produce higher protective neutralizing antibody 14 days after immunization, obviously higher than 0.5IU/ml (sufficiently higher than 0.5IU/ml) ml WHO standard functional antibody response); after 14 days of booster immunization, the serum neutralizing antibody titer increased significantly on the 28th day, and cationic lipids 1, 2, 6, 7, 8, 9, 10, 11, 12, 13
  • the titers of the lipid nanoparticle mRNA vaccines prepared by the lipid nanoparticles 3, 4 and 5 were higher than those prepared by the cationic lipids 3, 4 and 5.
  • the vaccine titer of group 3 was higher than that of groups 4 and 5.
  • Samples 1-14 (numbered mRNA-LNP1 to mRNA-LNP14) prepared in the examples were evaluated on the BALB/c mouse model for cellular immune response.
  • Female BALB/c mice aged 6-8 weeks were randomly divided into 14 groups according to 8 mice/group, as shown in Figure 5, BALB/c mice were immunized with 5 ⁇ g of mRNA-LNP on day 0 and day 14, and immunized on day 14.
  • the mice were dissected to separate splenocytes, stimulated with the overlapping peptide library of rabies virus G antigen, and the cytokine-producing cells were detected by intracellular cytokine staining flow cytometry (ICS).
  • ICS intracellular cytokine staining flow cytometry
  • the mRNA vaccine prepared by the present invention not only induces a Th1 biased response, but also significantly activates the CD8+ T cell response, and the cationic lipids 1, 2, 6, 7, 8, 9, 10, 11, 12, 13 and 14
  • the cellular immune response of the lipid nanoparticle mRNA vaccine obtained was better than that of cationic lipids 3, 4 and 5, and group 3 was slightly better than groups 4 and 5.
  • the mRNA vaccine prepared by the present invention shows better potential for preventing rabies virus.

Abstract

The present invention provides a novel cationic lipid, a lipid nanoparticle and a nucleic acid vaccine. It is found that the lipid nanoparticle mRNA vaccine prepared in the present invention by selecting a specific cationic lipid has better in-vitro stability and immunogenicity than LNPs prepared from cationic lipids in the prior art.

Description

一种用于核酸递送的新型可电离脂质及其LNP组合物和疫苗A Novel Ionizable Lipid for Nucleic Acid Delivery and Its LNP Composition and Vaccine 技术领域technical field
本发明涉及生物医药技术领域，具体涉及一种用于核酸递送的新型可电离脂质及其LNP组合物和疫苗。The invention relates to the technical field of biomedicine, in particular to a novel ionizable lipid for nucleic acid delivery and its LNP composition and vaccine.
背景技术Background technique
目前递送mRNA在临床上经过验证的系统为脂质纳米粒(Lipid Nanoparticle，LNP)，属于脂质形成的纳米微粒，其中原理包括阳离子脂质，而现有技术研究表明，mRNA递送至细胞内后，mRNA表达率偏低，例如Dlin-MC3-DMA作为阳离子脂质构建LNP，mRNA的表达量为0.63％(Maugeri,Marco et al.“Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells.”Nature Communications，2019)，因此，阳离子脂质的结构是影响mRNA表达量的关键因素。At present, the clinically proven system for delivering mRNA is lipid nanoparticle (Lipid Nanoparticle, LNP), which belongs to lipid-formed nanoparticles, and its principle includes cationic lipids. , the mRNA expression rate is low. For example, Dlin-MC3-DMA is used as a cationic lipid to construct LNP, and the mRNA expression level is 0.63% (Maugeri, Marco et al. "Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells."Nature Communications, 2019), therefore, the structure of cationic lipids is a key factor affecting the expression of mRNA.
狂犬病疫苗是人被动物咬伤后接种的狂犬疫苗和抗狂犬病血清，预防感染狂犬病。狂犬病是狂犬病毒所致的自然疫源性或动物源性人畜共患急性传染病，流行性广，病死率极高，对人民生命健康造成严重威胁。Rabies vaccine is the rabies vaccine and anti-rabies serum inoculated after a person is bitten by an animal to prevent infection with rabies. Rabies is a natural foci or zoonotic acute infectious disease caused by rabies virus. It is widespread and has a high fatality rate, posing a serious threat to people's lives and health.
狂犬病的典型临床表现为恐水症，故狂犬病又称恐水病，初期对声、光、风等刺激敏感而喉部有发紧感，进入兴奋期可表现为极度恐怖、恐水、怕风、发作性咽肌痉挛、呼吸困难等，最后痉挛发作停止而出现各种瘫痪，可迅速因呼吸和循环衰竭而死亡。人狂犬病主要通过患病动物咬伤、抓伤或由粘膜感染引起，在特定的条件下还可通过呼吸道气溶胶传染。受染动物唾液内含狂犬病毒。传染动物主要是犬(超过90％)，其次是猫。The typical clinical manifestation of rabies is hydrophobia, so rabies is also called hydrophobia. In the early stage, it is sensitive to sound, light, wind and other stimuli, and the throat feels tight. When it enters the excited stage, it can manifest as extreme terror, fear of water, and fear of wind. , paroxysmal pharyngeal muscle spasm, dyspnea, etc., and finally the spasm stops and various paralysis occurs, and can quickly die due to respiratory and circulatory failure. Human rabies is mainly caused by biting, scratching or mucous membrane infection of sick animals, and it can also be transmitted through respiratory aerosol under certain conditions. The saliva of infected animals contains rabies virus. Infected animals are mainly dogs (more than 90%), followed by cats.
人用狂犬病疫苗既往种类较多，现今国内外多使用细胞培养疫苗。我国现在使用的有精制VERO细胞狂犬病疫苗和精制地鼠肾细胞狂犬病疫苗，浓缩地鼠肾细胞狂犬病疫苗已禁用。目前尚无上市mRNA狂犬病疫苗.In the past, there were many types of human rabies vaccines, but cell culture vaccines are mostly used at home and abroad. my country is currently using refined VERO cell rabies vaccine and refined hamster kidney cell rabies vaccine, and the concentrated hamster kidney cell rabies vaccine has been banned. There is currently no marketed mRNA rabies vaccine.
发明内容 Contents of the invention
本发明术语“中性脂质”术语是指不带电荷的、非磷酸甘油酯的脂质分子。The term "neutral lipid" as used herein refers to uncharged, non-phosphoglyceride lipid molecules.
本发明术语“聚乙二醇(PEG)-脂质缀合物”是指包含脂质部分和聚乙二醇部分的分子。The term "polyethylene glycol (PEG)-lipid conjugate" according to the present invention refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.
本发明术语“脂质纳米颗粒”是指具有至少一个纳米量级尺寸的颗粒，其包含至少一种脂质。The term "lipid nanoparticle" according to the present invention refers to a particle having at least one nanoscale size, which comprises at least one lipid.
本发明术语“疫苗”是指适合于应用于动物(包括人)的组合物，在施用后诱导免疫应答，其强度足以最低限度地帮助预防、改善或治愈起因于由微生物感染的临床疾病。The term "vaccine" in the present invention refers to a composition suitable for application to animals (including humans), which induces an immune response after administration, and its strength is sufficient to help prevent, ameliorate or cure clinical diseases caused by microbial infection at a minimum.
本发明术语“递送系统”是指调控生物活性成分在空间、时间及剂量在生物体内分布的制剂或组合物。The term "delivery system" in the present invention refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dose in a living body.
本发明术语，N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。In terms of the present invention, N/P is the molar ratio of N in the cationic lipid to P in the mRNA mononucleotide.
本发明术语“烃基”是指相应的烃失去一个氢原子后剩余的基团，在本发明中特别指脂烃基，例如烷基、烯基、炔基，特别是烷基。The term "hydrocarbyl" in the present invention refers to the remaining group after the corresponding hydrocarbon loses a hydrogen atom, especially refers to aliphatic hydrocarbon groups in the present invention, such as alkyl, alkenyl, alkynyl, especially alkyl.
本发明提供阳离子脂质，具有如下式I结构：
The present invention provides cationic lipids, which have the following formula I structure:
其中：in:
L1和L2至少一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-或-NRa-，At least one of L 1 and L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)- or -NRa-,
并且，and,
L1或L2中的另一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-、-NRa-、-O(C＝O)-、-(C＝O)O-、-C(＝O)-、-S(O)x-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRaC(＝O)NRa-、-OC(＝O)NRa-或-NRaC(＝O)O-；The other of L 1 or L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)-, -NRa-, -O(C =O)-, -(C=O)O-, -C(=O)-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)- , -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-;
G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基；G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;
G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基； G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenene;
Ra为H或C1-C12烃基；Ra is H or C 1 -C 12 hydrocarbon group;
R1和R2各自独立地为C6-C24烷基或C6-C24烯基；R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl;
R3为H、OH、OR4、CN、-C(＝O)OR4、-OC(＝O)R4或–NR5C(＝O)R4；R 3 is H, OH, OR 4 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ;
R4为C1-C12烃基；R 4 is C 1 -C 12 hydrocarbon group;
R5为H或C1-C6烃基；R 5 is H or C 1 -C 6 hydrocarbon group;
x为0、1或2。x is 0, 1 or 2.
具体地，其中的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C＝O)O-、-(C＝O)NH-、-NH(C＝O)-和-NH-。Specifically, L 1 and L 2 in the cationic lipid formula I structure are each independently selected from -O-, -O(C=O)O-, -(C=O)NH-, -NH(C= O)- and -NH-.
具体地，其中的阳离子脂质式I结构中，L1和L2均为-O-，或者，L1和L2均为-O(C＝O)O-，或者，L1和L2均为-NH-，或者，L1为-NH(C＝O)-，L2为-(C＝O)NH-。Specifically, in the cationic lipid formula I structure, L 1 and L 2 are both -O-, or, L 1 and L 2 are both -O(C=O)O-, or, L 1 and L 2 Both are -NH-, or, L 1 is -NH(C=O)-, and L 2 is -(C=O)NH-.
具体地，其中的阳离子脂质其有以下结构(IA)：
Specifically, the cationic lipid wherein has the following structure (IA):
其中：in:
R6在每次出现时独立地为H、OH或C1-C24烃基；R 6 is independently H, OH, or C 1 -C 24 hydrocarbyl at each occurrence;
n为1至15的整数。n is an integer of 1 to 15.
具体地，其中的阳离子脂质其有以下结构(IB)：
Specifically, the cationic lipid wherein has the following structure (IB):
其中y和z各自独立地为1至12的整数。wherein y and z are each independently an integer from 1 to 12.
具体地，其中的阳离子脂质结构中n为2至12的整数，优选的，n为2、3、4、5或6；其中y和z各自独立地为2至10的整数，优选的，为4至9的整数。 Specifically, n in the cationic lipid structure is an integer from 2 to 12, preferably, n is 2, 3, 4, 5 or 6; wherein y and z are each independently an integer from 2 to 10, preferably, is an integer from 4 to 9.
具体地，其中的阳离子脂质结构中R1和R2各自独立地具有以下结构：
Specifically, R 1 and R 2 each independently have the following structure in the cationic lipid structure:
其中：in:
R7a和R7b在每次出现时独立地为H或C1-C12烃基；并且a为2至12的整数，优选的，a为8至12的整数；R 7a and R 7b are independently H or C 1 -C 12 hydrocarbon groups at each occurrence; and a is an integer from 2 to 12, preferably, a is an integer from 8 to 12;
其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms.
具体地，其中的阳离子脂质结构中至少一次出现的R7a为H，优选的，R7a在每次出现时为H。Specifically, at least one occurrence of R 7a in the cationic lipid structure is H, preferably, R 7a is H every time it occurs.
具体地，其中的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基；优选的，其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。Specifically, R 7b that occurs at least once in the cationic lipid structure is a C 1 -C 8 hydrocarbon group; preferably, wherein the C 1 -C 8 hydrocarbon group is methyl, ethyl, n-propyl, isopropyl, n-propyl Butyl, isobutyl, tert-butyl, n-hexyl or n-octyl.
具体地，其中的阳离子脂质结构中R1或R2或两者具有以下结构之一：

Specifically, in the cationic lipid structure, R1 or R2 or both have one of the following structures:

具体地，其中的阳离子脂质化合物下结构如下：


Specifically, the structure of the cationic lipid compound is as follows:


本发明涉及一种脂质纳米颗粒，包含：(a)上述阳离子脂质；(b)非-阳离子脂质；(c)聚乙二醇(PEG)-脂质缀合物。优选地，包含：阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。The present invention relates to a lipid nanoparticle comprising: (a) the cationic lipid described above; (b) a non-cationic lipid; (c) a polyethylene glycol (PEG)-lipid conjugate. Preferably, cationic lipids, neutral phospholipids, steroidal lipids and/or polyethylene glycol (PEG)-lipid conjugates are included.
具体地，其中的聚乙二醇(PEG)-脂质缀合物选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油 (PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合，优选的为DMG-PEG2000。Specifically, the polyethylene glycol (PEG)-lipid conjugate wherein is selected from: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159) , 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino( Polyethylene glycol)] (PEG-DSPE), PEG-Disteryl glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE ), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) or one or more combinations of DMG-PEG2000, preferably DMG-PEG2000.
具体地，其中的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合，优选的为DSPC。Specifically, the neutral lipid wherein is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1,2-dicarnoyl Myristoyl-sn-glycerol-3-phosphoethanolamine (DMPE), 2-Dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), Oleoylphosphatidylcholine (POPC) , 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), one or more combinations, preferably DSPC.
具体地，其中的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合，优选的为胆固醇。Specifically, the steroidal lipid wherein is selected from the group consisting of avenasterol, β-sitosterol, brassicasterol, ergocalcidol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergot Calciferol, dihydrocholesterol, dihydroergosterol, nigrosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol and cholesterol modified by peptides; lanosterol, photosterol, seawesterol , sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol .
具体地，其中的阳离子脂质摩尔含量为20～60％、中性磷脂摩尔含量约为5％～25％、甾族脂质摩尔含量约为25％～55％；聚乙二醇(PEG)-脂质缀合物摩尔含量约为0.5％～15％，Specifically, the molar content of cationic lipids is 20-60%, the molar content of neutral phospholipids is about 5%-25%, and the molar content of steroidal lipids is about 25%-55%; polyethylene glycol (PEG) - the molar content of the lipid conjugate is about 0.5% to 15%,
具体地，其中阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10，优选的，其中阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为43:10:45:2或40:10:48:2。Specifically, wherein the molar ratio of cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate is 30-60:1-20:20-50:0.1-10, preferably wherein the molar ratio of cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate is 43:10:45:2 or 40:10:48:2.
具体地，所述疫苗中还包含其他辅料，其中辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种组合。Specifically, the vaccine also contains other adjuvants, wherein the adjuvants are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.
具体地，其中纳米颗粒的平均粒径为50～200nm或其中纳米颗粒在中性pH下具有净中性电荷或其中纳米颗粒具有小于0.4的多分散性。Specifically, wherein the nanoparticles have an average particle size of 50-200 nm or wherein the nanoparticles have a net neutral charge at neutral pH or wherein the nanoparticles have a polydispersity of less than 0.4.
本发明涉及一种脂质纳米颗粒mRNA疫苗的制备方法。具体地，将阳离子脂质、非- 阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合后制得。The invention relates to a preparation method of lipid nano particle mRNA vaccine. Specifically, cationic lipids, non- Cationic lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved in a solvent and mixed with mRNA.
具体地，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得；优选的，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得；优选的，其中的超滤方式为切向流过滤；更优选的，其中的混合方式可为湍流混合、层流混合或微流体混合。Specifically, cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and mixed with diluted mRNA dilutions, followed by ultrafiltration, dilution, Prepared after filtration; preferably, cationic lipids, neutral phospholipids, steroidal lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and diluted mRNA diluent at a certain flow rate It is prepared by ultrafiltration, dilution and filtration after mixing; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent flow mixing, laminar flow mixing or microfluidic mixing.
具体地，稀释液可为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。Specifically, the diluent may be acetate buffer, citrate buffer, phosphate buffer or tris buffer.
具体地，其中缓冲液pH为3～6，浓度为6.25～200mM。Specifically, the pH of the buffer solution is 3-6, and the concentration is 6.25-200 mM.
具体地，将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1～5:1。Specifically, the solution flow rate ratio of the lipid mixed solution obtained after dissolving cationic lipids, non-cationic lipids, and polyethylene glycol (PEG)-lipid conjugates into a solvent and mRNA after dilution is 1 to 5: 1.
具体地，采用脂质包封mRNA时的N/P为2-10，优选的N/P为3-8，更优选的，N/P为3、4、5、6、7、8。Specifically, when the mRNA is encapsulated with lipid, the N/P is 2-10, preferably the N/P is 3-8, more preferably, the N/P is 3, 4, 5, 6, 7, 8.
具体地，其中的超滤液选自由以下组成的组：钠盐和三(羟甲基)氨基甲烷(Tris)盐，优选的，超滤液pH为6.0～8.0。Specifically, the ultrafiltrate is selected from the group consisting of sodium salt and tris(hydroxymethyl)aminomethane (Tris) salt, preferably, the pH of the ultrafiltrate is 6.0-8.0.
本发明提供一种狂犬病毒脂质纳米颗粒mRNA疫苗，具体地，给药方式可为口服、肌肉注射、静脉注射或吸入。The invention provides a rabies virus lipid nanoparticle mRNA vaccine, specifically, the administration method can be oral administration, intramuscular injection, intravenous injection or inhalation.
具体地，狂犬病毒脂质纳米颗粒mRNA疫苗的剂型可为口服制剂、液体制剂、冻干粉剂、注射剂或吸入制剂，优选的，为肌肉注射剂、静脉注射剂、干粉吸入剂或雾化吸入剂。Specifically, the dosage form of the rabies virus lipid nanoparticle mRNA vaccine can be an oral preparation, a liquid preparation, a freeze-dried powder, an injection or an inhalation preparation, preferably, an intramuscular injection, an intravenous injection, a dry powder inhalation or an aerosol inhalation.
本发明涉及一种狂犬病毒脂质纳米颗粒mRNA疫苗，包含：(a)能编码狂犬病毒G蛋白的mRNA；(b)阳离子脂质；(c)非-阳离子脂质；(d)聚乙二醇(PEG)-脂质缀合物。The present invention relates to a rabies virus lipid nanoparticle mRNA vaccine, comprising: (a) mRNA capable of encoding rabies virus G protein; (b) cationic lipid; (c) non-cationic lipid; (d) polyethylene glycol Alcohol (PEG)-lipid conjugates.
具体地，狂犬病毒脂质纳米颗粒mRNA疫苗，其中所述mRNA编码的氨基酸序列包含SEQ ID NO:1(CTN-1毒株的G蛋白(GenBank:ACR39382.1):SEQ ID NO:1)或SEQ ID NO:2所示序列(CTN-1V-T株G蛋白)。 Specifically, the rabies virus lipid nanoparticle mRNA vaccine, wherein the amino acid sequence encoded by the mRNA comprises SEQ ID NO: 1 (G protein of CTN-1 strain (GenBank: ACR39382.1): SEQ ID NO: 1) or The sequence shown in SEQ ID NO: 2 (CTN-1V-T strain G protein).
或者，与SEQ ID NO:1和2所示序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上或100％同一性的氨基酸序列。Alternatively, an amino acid sequence having 80% or more identity to the sequence shown in SEQ ID NO: 1 and 2, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more or 100% identical amino acid sequences.
具体地，其中的狂犬病毒脂质纳米颗粒mRNA疫苗，包含：(a)能编码狂犬病毒G蛋白的mRNA；(b)阳离子脂质；(c)中性磷脂、甾族脂质；(d)聚乙二醇(PEG)-脂质缀合物。Specifically, wherein the rabies virus lipid nanoparticle mRNA vaccine comprises: (a) mRNA capable of encoding rabies virus G protein; (b) cationic lipids; (c) neutral phospholipids, steroidal lipids; (d) Polyethylene glycol (PEG)-lipid conjugates.
具体地，能编码狂犬病毒G蛋白的mRNA被ALC-0315制备的脂质纳米颗粒包裹。Specifically, the mRNA encoding rabies virus G protein was encapsulated by lipid nanoparticles prepared by ALC-0315.
本发明所述的狂犬病毒脂质纳米颗粒mRNA疫苗可以通过口服、吸入或注射的方式递送所述生物活性物质。The rabies virus lipid nanoparticle mRNA vaccine of the present invention can deliver the biologically active substance through oral administration, inhalation or injection.
本发明涉及狂犬病毒脂质纳米颗粒mRNA疫苗在制备用于预防狂犬病的药物中的用途。The invention relates to the use of the rabies virus lipid nanoparticle mRNA vaccine in the preparation of medicines for preventing rabies.
有益的效果：Beneficial effect:
本发明涉及区别于现有技术的新型阳离子脂质。采用本发明所述的脂质化合物制成PEG-脂质/阳离子脂质/中性脂质/甾族脂质-mRNA纳米颗粒(LNP)，显示本发明所述的脂质化合物作为阳离子脂质的mRNA LNP具有较佳的稳定性和转染效率，在实验动物体内可引起较高的特异性抗体应答。The present invention relates to novel cationic lipids which differ from the prior art. Adopt the lipid compound of the present invention to make PEG-lipid/cationic lipid/neutral lipid/steroidal lipid-mRNA nanoparticle (LNP), show the lipid compound of the present invention as cationic lipid The mRNA LNP has better stability and transfection efficiency, and can cause a higher specific antibody response in experimental animals.
本发明提供了一种脂质纳米颗粒的狂犬病毒mRNA疫苗，采用脂质纳米颗粒作为递送系统，通过构建全新的阳离子脂质具有更优的理化性质，其包封率显著优于已上市的脂质纳米颗粒递送系统，即获得了免疫原性更强的狂犬病毒脂质纳米颗粒mRNA疫苗。The present invention provides a rabies virus mRNA vaccine of lipid nanoparticles, which uses lipid nanoparticles as a delivery system, and has better physical and chemical properties by constructing a new cationic lipid, and its encapsulation rate is significantly better than that of lipids already on the market. The plasma nanoparticle delivery system obtained a more immunogenic rabies virus lipid nanoparticle mRNA vaccine.
附图说明Description of drawings
图1所示为LNP制剂稳定性-粒径数据图；Figure 1 shows the LNP formulation stability-particle size data graph;
图2所示为LNP制剂稳定性-包封率数据图；Figure 2 shows the LNP preparation stability-encapsulation efficiency data graph;
图3所示为LNP制剂稳定性-mRNA完整性数据图；Figure 3 shows the LNP formulation stability-mRNA integrity data graph;
图4所示为LNP制剂稳定性-PDI数据图； Fig. 4 shows the LNP formulation stability-PDI data figure;
图5所示为BALB/c小鼠免疫程序；Figure 5 shows the immunization program for BALB/c mice;
图6所示为BALB/c小鼠模型上血清中和抗体滴度；Figure 6 shows the serum neutralizing antibody titer on the BALB/c mouse model;
图7所示为BALB/c小鼠模型上ICS法检测特异性分泌TNFα和IFNγ的CD4+T细胞频数；Figure 7 shows the frequency of CD4+ T cells specifically secreting TNFα and IFNγ detected by ICS on the BALB/c mouse model;
图8所示为BALB/c小鼠模型上ICS法检测特异性分泌TNFα和IFNγ的CD8+T细胞频数。Figure 8 shows the frequency of CD8+ T cells specifically secreting TNFα and IFNγ detected by ICS on the BALB/c mouse model.
具体实施方式Detailed ways
下面将结合本发明的附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明的部分实施例，而不是全部。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 solution in the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例1Example 1
化合物1的合成
Synthesis of compound 1
6-溴己基(2-己基癸基)碳酸酯(1a)的合成
Synthesis of 6-bromohexyl(2-hexyldecyl)carbonate (1a)
将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g， 7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入2-己基癸醇(1.36g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基(2-己基癸基)碳酸酯1a(1.53g，淡黄色油状物)，产率68％。Dissolve 6-bromo-n-hexanol (0.91g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.90g, 7.5mmol), then add p-nitrochloroformate phenyl ester (1.20g, 6.0mmol) in batches, and stir at room temperature for 3h, add 2-hexyldecanol (1.36g, 5.6mmol) in this reaction solution, and the mixture is Stir overnight at room temperature, after TLC shows that the reaction is complete, add 20mL of dichloromethane for dilution, then wash with 30mL of saturated brine, the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 6-bromohexyl (2 -hexyldecyl)carbonate 1a (1.53 g, pale yellow oil), yield 68%.
MS m/z(ESI):449.3[M+1]MS m/z(ESI):449.3[M+1]
化合物1的合成
Synthesis of compound 1
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物1(454mg，淡黄色油状物)，产率55％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 1 (454 mg, pale yellow oil) in 55% yield.
MS m/z(ESI):826.9[M+1]MS m/z(ESI):826.9[M+1]
1H NMR(300MHz,CDCl3):δ4.13(t,4H,J＝6.6Hz),4.05(d,4H,J＝5.7Hz),3.56-3.55(m,2H),2.47-2.42(m,6H),1.72-1.67(m,10H),1.53-1.48(m,8H),1.45-1.28(m,52H),0.69(t,12H,J＝6.2Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.13(t, 4H, J=6.6Hz), 4.05(d, 4H, J=5.7Hz), 3.56-3.55(m, 2H), 2.47-2.42(m ,6H),1.72-1.67(m,10H),1.53-1.48(m,8H),1.45-1.28(m,52H),0.69(t,12H,J＝6.2Hz)
实施例2Example 2
化合物2的合成
Synthesis of Compound 2
7-溴庚基十七烷-9-基碳酸酯(2a)的合成
Synthesis of 7-bromoheptylheptadecan-9-yl carbonate (2a)
将7-溴庚醇(0.98g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(1.22g，10mmol)，再分批次加入对硝基氯甲酸苯酯(1.11g，5.5mmol)，反应室温搅拌3h，在此反应液中加入9-羟基十七醇(1.44g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到7-溴庚基十七烷-9-基碳酸酯2a(1.50g，淡黄色油状物)，产率65％。Dissolve 7-bromoheptanol (0.98g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (1.22g, 10mmol), then add phenyl p-nitrochloroformate (1.11g , 5.5mmol), the reaction was stirred at room temperature for 3h, 9-hydroxyheptadecanol (1.44g, 5.6mmol) was added to the reaction solution, and the mixture was stirred at room temperature overnight. After TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, Then washed with 30mL saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 7-bromoheptyl heptadecan-9-yl carbonate 2a (1.50g, light yellow oil) , yield 65%.
MS m/z(ESI):477.3[M+1]MS m/z(ESI):477.3[M+1]
十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(2b)的合成
Synthesis of Heptadecan-9-yl(7-((2-Hydroxyethyl)amino)heptyl)carbonate (2b)
室温条件下，将7-溴庚基十七烷-9-基碳酸酯(2a)(1.38g，3mmol)溶于20mL乙醇中，加入乙醇胺(2.75g，45mmol)，升温至50℃，搅拌8h，监控反应进程，原料消耗完全后降温至45℃旋干除去乙醇，用二氯甲烷溶解粗品，用饱和食盐水洗涤三次，有机相用无水硫酸钠干燥，浓缩得到产品十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯2b(1.35g，淡黄色油状物)。At room temperature, 7-bromoheptylheptadecan-9-yl carbonate (2a) (1.38g, 3mmol) was dissolved in 20mL of ethanol, and ethanolamine (2.75g, 45mmol) was added, heated to 50°C, and stirred for 8h , monitor the reaction process, after the raw materials are completely consumed, cool down to 45°C and spin dry to remove ethanol, dissolve the crude product in dichloromethane, wash with saturated brine three times, dry the organic phase with anhydrous sodium sulfate, and concentrate to obtain the product heptadecane-9- (7-((2-Hydroxyethyl)amino)heptyl)carbonate 2b (1.35 g, pale yellow oil).
MS m/z(ESI):458.4[M+1]MS m/z(ESI):458.4[M+1]
5-溴戊基十一烷基碳酸酯(2c)的合成
Synthesis of 5-bromopentylundecyl carbonate (2c)
将5-溴戊醇(0.84g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(1.22g，10mmol)，再分批次加入对硝基氯甲酸苯酯(1.11g，5.5mmol)，反应室温搅拌3h，在此反应液中加入十一醇(0.97g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到5-溴戊基十一烷基碳酸酯2c(1.20g，淡黄色油状物)，产率66％。Dissolve 5-bromopentanol (0.84g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (1.22g, 10mmol), and then add phenyl p-nitrochloroformate (1.11g , 5.5mmol), the reaction was stirred at room temperature for 3h, undecyl alcohol (0.97g, 5.6mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature. After washing with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 5-bromopentylundecyl carbonate 2c (1.20 g, light yellow oil) with a yield of 66%.
MS m/z(ESI):365.2[M+1]MS m/z(ESI):365.2[M+1]
化合物2的合成
Synthesis of Compound 2
将十七烷-9-基(7-((2-羟乙基)氨基)庚基)碳酸酯(457mg，1.0mmol)溶于四氢呋喃中，加入乙腈，5-溴戊基十一烷基碳酸酯(437mg，1.2mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物2(440mg，淡黄色油状物)，产率57％。Heptadecan-9-yl (7-((2-hydroxyethyl)amino)heptyl)carbonate (457mg, 1.0mmol) was dissolved in tetrahydrofuran, acetonitrile was added, 5-bromopentylundecylcarbonate Ester (437mg, 1.2mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 2 (440 mg, pale yellow oil) in 57% yield.
MS m/z(ESI):742.8[M+1]MS m/z(ESI):742.8[M+1]
1H NMR(300MHz,CDCl3):δ4.71-4.68(m,1H),4.15-4.10(m,6H),3.53(t,2H,J＝5.4Hz),2.94(br,1H),2.58(t,2H,J＝5.4Hz),2.45(t,4H,J＝5.7Hz),1.75-1.34(m,62H),0.90(t,9H,J＝6.3Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.71-4.68 (m, 1H), 4.15-4.10 (m, 6H), 3.53 (t, 2H, J=5.4Hz), 2.94 (br, 1H), 2.58 (t, 2H, J=5.4Hz), 2.45(t, 4H, J=5.7Hz), 1.75-1.34(m, 62H), 0.90(t, 9H, J=6.3Hz)
实施例3Example 3
化合物3的合成
Synthesis of compound 3
6-溴己基十一烷基碳酸酯(3a)的合成
Synthesis of 6-bromohexylundecyl carbonate (3a)
将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此 反应液中加入十一醇(0.97g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基十一烷基碳酸酯3a(1.25g，淡黄色油状物)，产率66％。Dissolve 6-bromo-n-hexanol (0.91g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.90g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.20 g, 6.0mmol), the reaction was stirred at room temperature for 3h, where Undecyl alcohol (0.97g, 5.6mmol) was added to the reaction solution, and the mixture was stirred overnight at room temperature. After TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, then washed with 30mL of saturated brine, and the organic phase was washed with anhydrous sodium sulfate After drying, filtration and concentration, 6-bromohexylundecyl carbonate 3a (1.25 g, light yellow oil) was obtained by separation by column chromatography with a yield of 66%.
MS m/z(ESI):379.2[M+1]MS m/z(ESI):379.2[M+1]
化合物3的合成
Synthesis of Compound 3
将6-溴己基十一烷基碳酸酯(948mg，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物3(412mg，淡黄色油状物)，产率60％。Dissolve 6-bromohexylundecyl carbonate (948mg, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), Potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 3 (412 mg, pale yellow oil) in 60% yield.
MS m/z(ESI):686.8[M+1]MS m/z(ESI):686.8[M+1]
1H NMR(300MHz,CDCl3):δ4.13(t,8H,J＝6.6Hz),3.58(t,2H,J＝5.7Hz),2.52(t,6H,J＝8.4Hz),1.74-1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J＝6.2Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.13(t, 8H, J=6.6Hz), 3.58(t, 2H, J=5.7Hz), 2.52(t, 6H, J=8.4Hz), 1.74- 1.64(m,12H),1.63-1.53(m,5H),1.52-1.39(m,39H),0.86(t,6H,J＝6.2Hz)
实施例4Example 4
化合物4的合成
Synthesis of Compound 4
6-溴己基十七烷-9-基碳酸酯(4a)的合成
Synthesis of 6-bromohexylheptadecan-9-yl carbonate (4a)
将6-溴正己醇(0.91g，5.0mmol)溶于30mL二氯甲烷中，加入4-二甲氨基吡啶(0.90g，7.5mmol)，再分批次加入对硝基氯甲酸苯酯(1.20g，6.0mmol)，反应室温搅拌3h，在此反应液中加入9-十七醇(1.44g，5.6mmol)，混合物在室温下搅拌过夜，TLC显示反应完成后，加入20mL二氯甲烷稀释，然后用30mL饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤并浓缩，柱层析分离得到6-溴己基十七烷-9-基碳酸酯4a(1.53g，淡黄色油状物)，产率66％。Dissolve 6-bromo-n-hexanol (0.91g, 5.0mmol) in 30mL of dichloromethane, add 4-dimethylaminopyridine (0.90g, 7.5mmol), and then add phenyl p-nitrochloroformate (1.20 g, 6.0mmol), the reaction was stirred at room temperature for 3h, 9-heptadecanol (1.44g, 5.6mmol) was added to the reaction solution, the mixture was stirred at room temperature overnight, after TLC showed that the reaction was complete, 20mL of dichloromethane was added to dilute, Then washed with 30 mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain 6-bromohexyl heptadecan-9-yl carbonate 4a (1.53 g, pale yellow oil), Yield 66%.
MS m/z(ESI):464.3[M+1]MS m/z(ESI):464.3[M+1]
化合物4的合成
Synthesis of Compound 4
将6-溴己基十七烷-9-基碳酸酯(1.16g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg,2.0mmol)， 在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物4(502mg，淡黄色油状物)，产率59％。Dissolve 6-bromohexylheptadecan-9-yl carbonate (1.16g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4-amino-1-butanol (89.2mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), Stir at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 4 (502 mg, pale yellow oil) in 59% yield.
MS m/z(ESI):855.4[M+1]MS m/z(ESI):855.4[M+1]
1H NMR(300MHz,CDCl3):δ4.71-4.68(m,2H),4.13(t,4H,J＝6.6Hz),3.57(t,2H,J＝5.4Hz),2.49-2.44(m,6H),1.74-1.28(m,76H),0.90(t,12H,J＝6.3Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.71-4.68(m, 2H), 4.13(t, 4H, J=6.6Hz), 3.57(t, 2H, J=5.4Hz), 2.49-2.44(m ,6H),1.74-1.28(m,76H),0.90(t,12H,J＝6.3Hz)
实施例5Example 5
化合物5的合成
Synthesis of compound 5
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，乙醇胺(61.0mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物5(487mg，淡黄色油状物)，产率61％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, ethanolamine (61.0mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg , 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 5 (487 mg, pale yellow oil) in 61% yield.
MS m/z(ESI):798.9[M+1]MS m/z(ESI):798.9[M+1]
1H NMR(300MHz,CDCl3):δ4.14(t,4H,J＝6.6Hz),4.04(d,4H,J＝5.7Hz),3.54(t,2H,J＝5.4Hz),2.58(t,2H,J＝5.4Hz),2.46(t,4H,J＝7.2Hz),1.72-1.65(m,6H),1.49-1.28(m,61H),0.69(t,12H,J＝6.2Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.14(t, 4H, J=6.6Hz), 4.04(d, 4H, J=5.7Hz), 3.54(t, 2H, J=5.4Hz), 2.58( t, 2H, J=5.4Hz), 2.46(t, 4H, J=7.2Hz), 1.72-1.65(m, 6H), 1.49-1.28(m, 61H), 0.69(t, 12H, J=6.2Hz )
实施例6Example 6
化合物6的合成
Synthesis of compound 6
将5-溴戊基十一烷基碳酸酯(910mg，2.5mmol)溶于四氢呋喃中，加入乙腈，乙醇胺(61.0mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物6(410mg，淡黄色油状物)，产率65％。Dissolve 5-bromopentylundecyl carbonate (910mg, 2.5mmol) in tetrahydrofuran, add acetonitrile, ethanolamine (61.0mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol ), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 6 (410 mg, pale yellow oil) in 65% yield.
MS m/z(ESI):630.7[M+1]MS m/z(ESI):630.7[M+1]
1H NMR(300MHz,CDCl3):δ4.10(t,8H,J＝6.6Hz),3.52(d,2H,J＝5.4Hz),2.83(br,1H),2.57(t,2H,J＝5.4Hz),2.45(t,4H,J＝7.2Hz),1.73-1.62(m,8H),1.52-1.39(m,40H),0.69(t,6H,J＝6.2Hz) 1 H NMR (300MHz, CDCl 3 ): δ4.10(t, 8H, J=6.6Hz), 3.52(d, 2H, J=5.4Hz), 2.83(br, 1H), 2.57(t, 2H, J =5.4Hz), 2.45(t, 4H, J=7.2Hz), 1.73-1.62(m, 8H), 1.52-1.39(m, 40H), 0.69(t, 6H, J=6.2Hz)
实施例7Example 7
化合物7的合成
Synthesis of compound 7
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，3-甲氧基丙胺(89mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物7(495mg，淡黄色油状物)，产率60％。 Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 3-methoxypropylamine (89mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol) , potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 7 (495 mg, pale yellow oil) in 60% yield.
MS m/z(ESI):826.7[M+1]MS m/z(ESI):826.7[M+1]
实施例8Example 8
化合物8的合成
Synthesis of Compound 8
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，3-氨基丙腈(70mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物8(469mg，淡黄色油状物)，产率58％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 3-aminopropionitrile (70mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), Potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 8 (469 mg, pale yellow oil) in 58% yield.
MS m/z(ESI):807.7[M+1]MS m/z(ESI):807.7[M+1]
实施例9Example 9
化合物9的合成
Synthesis of compound 9
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，4-氨基丁酸乙酯盐酸盐(167mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物9(546mg，淡黄色油状物)，产率63％。 Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, ethyl 4-aminobutyrate hydrochloride (167mg, 1.0mmol), potassium carbonate (550mg , 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 9 (546 mg, pale yellow oil) in 63% yield.
MS m/z(ESI):868.8[M+1]MS m/z(ESI):868.8[M+1]
实施例10Example 10
化合物10的合成
Synthesis of Compound 10
将6-溴己基(2-己基癸基)碳酸酯(1.12g，2.5mmol)溶于四氢呋喃中，加入乙腈，N-(4-氨基丁基)-乙酰胺盐酸盐(167mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物10(560mg，淡黄色油状物)，产率69％。Dissolve 6-bromohexyl (2-hexyldecyl) carbonate (1.12g, 2.5mmol) in tetrahydrofuran, add acetonitrile, N-(4-aminobutyl)-acetamide hydrochloride (167mg, 1.0mmol) , potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 10 (560 mg, pale yellow oil) in 69% yield.
MS m/z(ESI):867.8[M+1]MS m/z(ESI):867.8[M+1]
实施例11Example 11
化合物11的合成
Synthesis of Compound 11
8-溴-N-(十七烷-9-基)辛酰胺(11a)的合成
Synthesis of 8-bromo-N-(heptadecan-9-yl)octylamide (11a)
将8-溴辛酸(1.12g，5.0mmol)溶于50mL二氯甲烷中，在0℃下分批加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(1.05g，5.5mmol)，搅拌30min后，在反应液中逐滴加入9-氨基十七烷(1.28g，5.0mmol)，滴加完毕后，混合物在室温下搅拌过夜，TLC显示反应完成后，用100ml水洗涤2次，有机相用无水硫酸钠干燥，过滤并浓缩，得到化合物11a(1.95g，黄色油状物)，产率82％。Dissolve 8-bromooctanoic acid (1.12g, 5.0mmol) in 50mL of dichloromethane, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in batches at 0°C (1.05g, 5.5mmol), after stirring for 30min, 9-aminoheptadecane (1.28g, 5.0mmol) was added dropwise to the reaction solution. After the addition was complete, the mixture was stirred overnight at room temperature, and TLC showed that the reaction was complete , washed twice with 100ml of water, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 11a (1.95g, yellow oil), with a yield of 82%.
MS m/z(ESI):461.3[M+1]。MS m/z (ESI): 461.3 [M+1].
化合物11b的合成
Synthesis of Compound 11b
将8-溴-N-(十七烷-9-基)辛酰胺(1.15g，2.5mmol)溶于四氢呋喃中，加入乙腈，4- 氨基-1-丁醇(89.2mg，1.0mmol)，碳酸钾(550mg，4.0mmol)，碘化钾(332mg，2.0mmol)，在83℃下搅拌16-20h。冷却至室温，过滤，滤渣用二氯甲烷洗涤，得到的滤液中加入饱和碳酸氢钠溶液，用二氯甲烷萃取2次，合并有机相，经无水硫酸钠干燥，过滤并浓缩，柱层析分离，得到产物11b(534mg，淡黄色油状物)，产率63％。Dissolve 8-bromo-N-(heptadecan-9-yl) octanamide (1.15g, 2.5mmol) in tetrahydrofuran, add acetonitrile, 4- Amino-1-butanol (89.2mg, 1.0mmol), potassium carbonate (550mg, 4.0mmol), potassium iodide (332mg, 2.0mmol), stirred at 83°C for 16-20h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the obtained filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, column chromatography Isolation afforded product 11b (534 mg, pale yellow oil) in 63% yield.
MS m/z(ESI):848.8[M+1]；MS m/z(ESI):848.8[M+1];
1H NMR(300MHz,CDCl3):δ8.10(s,2H),4.21(s,1H),3.46-3.4(m,4H),3.02(t,6H,J＝6.2Hz),2.14(t,4H,J＝4.8Hz),1.57-1.47(t,14H,J＝6.3Hz),1.36-1.26(m,66H),0.90(t,12H,J＝6.3Hz)。 1 H NMR (300MHz, CDCl 3 ): δ8.10(s, 2H), 4.21(s, 1H), 3.46-3.4(m, 4H), 3.02(t, 6H, J=6.2Hz), 2.14(t , 4H, J=4.8Hz), 1.57-1.47(t, 14H, J=6.3Hz), 1.36-1.26(m, 66H), 0.90(t, 12H, J=6.3Hz).
化合物11的合成
Synthesis of Compound 11
在0℃下，将化合物11b(1.70g，2mmol)缓慢加入四氢铝锂(379mg，10mmol)的无水四氢呋喃(10ml)溶液中，混合物加热回流5小时。反应完全后，降温，在体系中加入水使过量的还原剂完全分解。过滤，滤渣用乙酸乙酯洗涤，得到的滤液用水洗涤，经无水硫酸钠干燥，过滤并浓缩，得到化合物11(1.45g，黄色油状物)，产率90％。At 0°C, compound 11b (1.70 g, 2 mmol) was slowly added to a solution of lithium aluminum hydride (379 mg, 10 mmol) in anhydrous THF (10 ml), and the mixture was heated to reflux for 5 hours. After the reaction is complete, lower the temperature and add water to the system to completely decompose the excess reducing agent. After filtration, the filter residue was washed with ethyl acetate, and the obtained filtrate was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 11 (1.45 g, yellow oil) with a yield of 90%.
MS m/z(ESI):820.8[M+1]；MS m/z(ESI):820.8[M+1];
1H NMR(300MHz,CDCl3):δ4.11(s,1H),3.44(t,2H,J＝4.8Hz),3.32(s,2H),3.00(t,6H,J＝6.3Hz),2.52(t,4H,J＝6.3Hz),2.48-2.43(m,2H),1.61-1.56(m,2H),1.36-1.26(m,82H),0.86(t,12H,J＝4.8Hz)。 1 H NMR (300MHz, CDCl 3 ): δ4.11(s, 1H), 3.44(t, 2H, J=4.8Hz), 3.32(s, 2H), 3.00(t, 6H, J=6.3Hz), 2.52(t, 4H, J=6.3Hz), 2.48-2.43(m, 2H), 1.61-1.56(m, 2H), 1.36-1.26(m, 82H), 0.86(t, 12H, J=4.8Hz) .
实施例12脂质纳米颗粒包裹狂犬病毒G蛋白mRNA抗原Example 12 Lipid Nanoparticle Encapsulated Rabies Virus G Protein mRNA Antigen
本发明分别使用阳离子脂质I～II与对照脂质III～IV制备脂质纳米颗粒核酸疫苗，四种阳离子脂质结构如下表。 The present invention uses cationic lipids I-II and control lipids III-IV to prepare lipid nanoparticle nucleic acid vaccines, and the structures of the four cationic lipids are shown in the table below.
表1：阳离子脂质结构式

Table 1: Cationic Lipid Structural Formulas

用100mM醋酸钠缓冲液(pH 4.0)稀释狂犬病毒mRNA疫苗原液至浓度为135μg/ml，该疫苗原液含有编码狂犬病毒G蛋白，抗原序列如SEQ ID NO:1所述，目的抗原经过常规修饰，其中N端含有5’UTR及帽子结构，C端含有3’UTR和PolyA尾等设计；稀释后的疫苗原液按照阳离子脂质：DSPC：胆固醇：DMG-PEG2000摩尔比为43:10:45:2配制脂质混合溶液；设定纳米药物制造设备总流速12ml/min、mRNA溶液与脂质混合溶液流速比3：1并开始包封，包封完成后，切向流过滤系统超滤换液收集样品，并加入蔗糖溶 液。在N/P(可电离的阳离子脂质与核苷酸磷酸盐)摩尔比不同条件下进行试验(N/P摩尔比分别为3.6、5.6、7.6)。取样检测包封率、平均粒径、PDI及Zeta电位，结果如下表。Dilute the rabies virus mRNA vaccine stock solution with 100mM sodium acetate buffer (pH 4.0) to a concentration of 135 μg/ml, the vaccine stock solution contains the encoded rabies virus G protein, the antigen sequence is as described in SEQ ID NO: 1, and the target antigen is routinely modified, The N-terminal contains a 5'UTR and a cap structure, and the C-terminal contains a 3'UTR and a PolyA tail. The diluted vaccine stock solution is cationic lipid: DSPC: cholesterol: DMG-PEG2000 molar ratio is 43:10:45:2 Prepare the lipid mixed solution; set the total flow rate of the nano-medicine manufacturing equipment to 12ml/min, the flow rate ratio of the mRNA solution and the lipid mixed solution to 3:1 and start encapsulation. sample, and add sucrose to dissolve liquid. Experiments were carried out under different conditions of N/P (ionizable cationic lipid to nucleotide phosphate) molar ratio (N/P molar ratios were 3.6, 5.6, 7.6, respectively). Samples were taken to detect encapsulation efficiency, average particle size, PDI and Zeta potential, and the results are shown in the table below.
表2：不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗检测结果

Table 2: Detection results of lipid nanoparticle mRNA vaccine after encapsulation with different cationic lipids

由以上结果可以看出，在相同N/P条件下，阳离子脂质1、2、6、7、8、9、10、11、12、13和14所制得样品的包封率均高于阳离子脂质3和对照阳离子脂质4、5所制得的样品，初步可以得出阳离子脂质1、2、6、7、8、9、10、11、12、13和14对mRNA抗原具有更好的包封效果。3组的包封率略高于4、5组。As can be seen from the above results, under the same N/P conditions, the encapsulation efficiencies of the samples prepared by cationic lipids 1, 2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 were all higher than The samples prepared by cationic lipid 3 and contrast cationic lipid 4, 5 can preliminarily draw that cationic lipid 1, 2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 have Better encapsulation effect. The encapsulation efficiency of group 3 was slightly higher than that of groups 4 and 5.
实施例13不同阳离子制得的LNP-mRNA稳定性考察The LNP-mRNA stability investigation that embodiment 13 different cations make
分别取实施例14种以不同阳离子脂质制得的LNP-mRNA置于25℃恒温培养箱中分别放置1、2、3、4周考察其稳定性，结果如图1、图2、图3、图4、表3、表4、表5和表6所示。Take the 14 kinds of LNP-mRNA prepared with different cationic lipids in Example 14 and place them in a constant temperature incubator at 25°C for 1, 2, 3, and 4 weeks to investigate their stability. The results are shown in Figures 1, 2, and 3. , shown in Figure 4, Table 3, Table 4, Table 5 and Table 6.
表3储存稳定性—平均粒径

Table 3 Storage Stability - Average Particle Size

表4储存稳定性—包封率(％)
Table 4 Storage Stability—Encapsulation Efficiency (%)
表5储存稳定性—mRNA完整性(％)

Table 5 storage stability—mRNA integrity (%)

表6储存稳定性—PDI变化
Table 6 Storage Stability—PDI Change
结果表明，1、2、6、7、8、9、10、11、12、13和14组样品的包封率和mRNA完整性4周内没有明显下降，平均粒径和PDI在4周内没有发生明显增大，并且脂质纳米 颗粒的平均粒径和PDI在4周内基本维持不变，与3、4和5组相比，展现出更好的稳定性。3组的稳定性优于4和5组。The results showed that the encapsulation efficiency and mRNA integrity of samples in groups 1, 2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 did not decrease significantly within 4 weeks, and the average particle size and PDI within 4 weeks No significant enlargement occurred, and lipid nano The average particle size and PDI of the particles remained basically unchanged within 4 weeks, showing better stability compared with groups 3, 4 and 5. The stability of group 3 is better than that of groups 4 and 5.
实施例14小鼠免疫与检测Embodiment 14 mouse immunization and detection
1、狂犬病毒脂质纳米颗粒mRNA疫苗体液免疫评价1. Evaluation of humoral immunity of rabies virus lipid nanoparticle mRNA vaccine
将6-8周龄的雌性BALB/c小鼠按6只/组随机分成14组，采用后腿肌肉注射的免疫途径进行免疫。其中，1-14组免疫样品1-14(实施例12制备)。如图5，分别在第0天和第14天免疫，单次免疫剂量为5μg mRNA-LNP。免疫第14天和28天采血并分离血清，进行抗体滴度检测，检测结果如表7和图6所示。Six to eight-week-old female BALB/c mice were randomly divided into 14 groups, and immunized by intramuscular injection in the hind legs. Among them, groups 1-14 were immunized with samples 1-14 (prepared in Example 12). As shown in Figure 5, they were immunized on day 0 and day 14 respectively, and the single immunization dose was 5 μg mRNA-LNP. On the 14th and 28th day of immunization, blood was collected and serum was separated for detection of antibody titer. The detection results are shown in Table 7 and Figure 6.
表7不同阳离子脂质包封后脂质纳米颗粒mRNA疫苗中和抗体滴度

Lipid nanoparticle mRNA vaccine neutralizing antibody titers after table 7 different cationic lipid encapsulation

血清中和抗体滴度检测结果显示，本发明制备的狂犬病毒mRNA疫苗在免疫后14天能够刺激机体产生较高的保护性中和抗体，明显高于0.5IU/ml(充分高于0.5IU/ml WHO标准的功能性抗体应答)；14d加强免疫后，第28d血清中和抗体滴度大幅度提升，并且阳离子脂质1、2、6、7、8、9、10、11、12、13和14所制得的脂质纳米颗粒mRNA疫苗的滴度高于阳离子脂质3、4和5所制得的疫苗滴度。3组的疫苗滴度高于4和5组。Serum neutralizing antibody titer detection result shows that the rabies virus mRNA vaccine prepared by the present invention can stimulate the body to produce higher protective neutralizing antibody 14 days after immunization, obviously higher than 0.5IU/ml (sufficiently higher than 0.5IU/ml) ml WHO standard functional antibody response); after 14 days of booster immunization, the serum neutralizing antibody titer increased significantly on the 28th day, and cationic lipids 1, 2, 6, 7, 8, 9, 10, 11, 12, 13 The titers of the lipid nanoparticle mRNA vaccines prepared by the lipid nanoparticles 3, 4 and 5 were higher than those prepared by the cationic lipids 3, 4 and 5. The vaccine titer of group 3 was higher than that of groups 4 and 5.
2、在BALB/c小鼠模型上的细胞免疫反应评价2. Evaluation of cellular immune response on BALB/c mouse model
将实施例制备的样品1-14(编号为mRNA-LNP1～mRNA-LNP14)分别在BALB/c小鼠模型上进行细胞免疫反应的评价。将6-8周龄的雌性BALB/c小鼠按8只/组随机分成14组，如图5所示，BALB/c小鼠在第0天和14天免疫5μg的mRNA-LNP，免疫第28天采血后解剖小鼠分离脾细胞，并使用狂犬病毒G抗原的重叠肽库进行刺激，通过细胞内细胞因子染色流式细胞术(ICS)方法检测细胞因子产生细胞。结果如表8、表9、表10、表11和图7、图8所示。Samples 1-14 (numbered mRNA-LNP1 to mRNA-LNP14) prepared in the examples were evaluated on the BALB/c mouse model for cellular immune response. Female BALB/c mice aged 6-8 weeks were randomly divided into 14 groups according to 8 mice/group, as shown in Figure 5, BALB/c mice were immunized with 5 μg of mRNA-LNP on day 0 and day 14, and immunized on day 14. After 28 days of blood collection, the mice were dissected to separate splenocytes, stimulated with the overlapping peptide library of rabies virus G antigen, and the cytokine-producing cells were detected by intracellular cytokine staining flow cytometry (ICS). The results are shown in Table 8, Table 9, Table 10, Table 11 and Figure 7 and Figure 8.
表8G特异性CD4+T细胞产生TNF-α的百分比

Table 8G The percentage of TNF-α produced by specific CD4+T cells

表9G特异性CD4+T细胞产生IFN-γ的百分比
Table 9G The percentage of IFN-γ produced by specific CD4+ T cells
表10G特异性CD8+T细胞产生TNF-α的百分比
Table 10G specific CD8+T cells produce the percentage of TNF-α
表11G特异性CD8+T细胞产生IFN-γ的百分比

Table 11G The percentage of IFN-γ produced by specific CD8+T cells

本发明制备的mRNA疫苗不仅诱导了Th1偏向反应，还可以显著激活CD8+T细胞反应，且阳离子脂质1、2、6、7、8、9、10、11、12、13和14所制得的脂质纳米颗粒mRNA疫苗的细胞免疫反应较阳离子脂质3、4和5更好，3组略优于4、5组。The mRNA vaccine prepared by the present invention not only induces a Th1 biased response, but also significantly activates the CD8+ T cell response, and the cationic lipids 1, 2, 6, 7, 8, 9, 10, 11, 12, 13 and 14 The cellular immune response of the lipid nanoparticle mRNA vaccine obtained was better than that of cationic lipids 3, 4 and 5, and group 3 was slightly better than groups 4 and 5.
综上，本发明制备的mRNA疫苗显示出较好的用于预防狂犬病毒的潜力。To sum up, the mRNA vaccine prepared by the present invention shows better potential for preventing rabies virus.
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。 In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

Claims (29)

  1. 一种阳离子脂质，其特征在于，所述的阳离子脂质具有如下式I结构：
    A kind of cationic lipid, is characterized in that, described cationic lipid has following formula I structure:
    其中：in:
    L1和L2至少一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-或-NRa-，At least one of L 1 and L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)- or -NRa-,
    并且，and,
    L1或L2中的另一个为-O-、-O(C＝O)O-、-(C＝O)NRa-、-NRa(C＝O)-、-NRa-、-O(C＝O)-、-(C＝O)O-、-C(＝O)-、-S(O)x-、-S-S-、-C(＝O)S-、-SC(＝O)-、-NRaC(＝O)NRa-、-OC(＝O)NRa-或-NRaC(＝O)O-；The other of L 1 or L 2 is -O-, -O(C=O)O-, -(C=O)NRa-, -NRa(C=O)-, -NRa-, -O(C =O)-, -(C=O)O-, -C(=O)-, -S(O)x-, -SS-, -C(=O)S-, -SC(=O)- , -NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-;
    G1和G2各自独立地为未取代的C1-C12亚烷基或C1-C12亚烯基；G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;
    G3为C1-C24亚烷基、C1-C24亚烯基、C3-C8亚环烷基、C3-C8亚环烯基；G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenene;
    Ra为H或C1-C12烃基；Ra is H or C 1 -C 12 hydrocarbon group;
    R1和R2各自独立地为C6-C24烷基或C6-C24烯基；R 1 and R 2 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl;
    R3为H、OH、OR4、CN、-C(＝O)OR4、-OC(＝O)R4或–NR5C(＝O)R4；R 3 is H, OH, OR 4 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 ;
    R4为C1-C12烃基；R 4 is C 1 -C 12 hydrocarbon group;
    R5为H或C1-C6烃基；R 5 is H or C 1 -C 6 hydrocarbon group;
    x为0、1或2。x is 0, 1 or 2.
  2. 根据权利要求1所述的阳离子脂质，其特征在于，所述的阳离子脂质式I结构中L1和L2各自独立地选自-O-、-O(C＝O)O-、-(C＝O)NH-、-NH(C＝O)-和-NH-。The cationic lipid according to claim 1, characterized in that, in the structure of the cationic lipid formula I, L and L are each independently selected from -O-, -O(C=O)O-, - (C=O)NH-, -NH(C=O)- and -NH-.
  3. 根据权利要求1-2任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质式I结构中所述L1和L2均为-O-，或者，L1和L2均为-O(C＝O)O-，或者，L1和L2均为-NH-，或者，L1为-NH(C＝O)-，L2为-(C＝O)NH-。 The cationic lipid according to any one of claims 1-2, characterized in that, the L and L in the structure of the cationic lipid formula I are -O-, or, L and L Both are -O(C=O)O-, or, both L 1 and L 2 are -NH-, or, L 1 is -NH(C=O)-, and L 2 is -(C=O)NH- .
  4. 根据权利要求1-3任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质具有以下结构(IA)：
    The cationic lipid according to any one of claims 1-3, wherein the cationic lipid has the following structure (IA):
    其中：in:
    R6在每次出现时独立地为H、OH或C1-C24烃基；R 6 is independently H, OH, or C 1 -C 24 hydrocarbyl at each occurrence;
    n为1至15的整数。n is an integer of 1 to 15.
  5. 根据权利要求1-4任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质其有以下结构(IB)：
    According to the cationic lipid described in any one of claims 1-4, it is characterized in that, it has following structure (IB) of described cationic lipid:
    其中y和z各自独立地为1至12的整数。wherein y and z are each independently an integer from 1 to 12.
  6. 根据权利要求1-5任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中n为2至12的整数，优选的，n为2、3、4、5或6；其中y和z各自独立地为2至10的整数，优选的，为4至9的整数。The cationic lipid according to any one of claims 1-5, characterized in that, in the cationic lipid structure, n is an integer from 2 to 12, preferably, n is 2, 3, 4, 5 or 6 wherein y and z are each independently an integer from 2 to 10, preferably an integer from 4 to 9.
  7. 根据权利要求1-6任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中R1和R2各自独立地具有以下结构：
    According to the cationic lipid according to any one of claims 1-6, it is characterized in that, in the described cationic lipid structure, R 1 and R 2 each independently have the following structure:
    其中： in:
    R7a和R7b在每次出现时独立地为H或C1-C12烃基；并且a为2至12的整数，优选的，a为8至12的整数；R 7a and R 7b are independently H or C 1 -C 12 hydrocarbon groups at each occurrence; and a is an integer from 2 to 12, preferably, a is an integer from 8 to 12;
    其中R7a、R7b和a各自被选择为使得R1和R2各自独立地包含6至20个碳原子。wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently contain 6 to 20 carbon atoms.
  8. 根据权利要求1-7任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中至少一次出现的R7a为H，优选的，R7a在每次出现时为H。The cationic lipid according to any one of claims 1-7, characterized in that, at least one occurrence of R 7a in the cationic lipid structure is H, preferably, R 7a is H every time it occurs.
  9. 根据权利要求1-8任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中至少一次出现的R7b为C1-C8烃基；优选的，其中C1-C8烃基为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正己基或正辛基。The cationic lipid according to any one of claims 1-8, characterized in that, at least one occurrence of R 7b in the cationic lipid structure is a C 1 -C 8 hydrocarbon group; preferably, wherein C 1 -C 8 Hydrocarbyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-hexyl or n-octyl.
  10. 根据权利要求1-9任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质结构中R1或R2或两者具有以下结构之一：
    According to the cationic lipid according to any one of claims 1-9, it is characterized in that, in the described cationic lipid structure, R 1 or R 2 or both have one of the following structures:
  11. 根据权利要求1-10任一项所述的阳离子脂质，其特征在于，所述的阳离子脂质化合物 下结构如下：


    The cationic lipid according to any one of claims 1-10, characterized in that, the cationic lipid compound The following structure is as follows:


  12. 一种脂质纳米颗粒，其特征在于，包含：如权利要求1-11任一项所述的阳离子脂质、非-阳离子脂质和/或聚乙二醇(PEG)-脂质缀合物，优选地，包含：阳离子脂质、中性磷脂、甾族脂质和/或聚乙二醇(PEG)-脂质缀合物。A lipid nanoparticle, characterized in that, comprising: cationic lipid, non-cationic lipid and/or polyethylene glycol (PEG)-lipid conjugate as described in any one of claims 1-11 , preferably, comprising: cationic lipids, neutral phospholipids, steroidal lipids and/or polyethylene glycol (PEG)-lipid conjugates.
  13. 根据权利要求12所述的脂质纳米颗粒，其特征在于，所述的聚乙二醇(PEG)-脂质缀合物选自：2-[(聚乙二醇)-2000]-N,N-二十四烷基乙酰胺(ALC-0159)、1,2-二肉豆蔻酰基-sn-甘油甲氧基聚乙二醇(PEG-DMG)、1,2-二硬脂酰基-sn-甘油基-3-磷酸乙醇胺-N-[氨基(聚乙二醇)](PEG-DSPE)、PEG-二甾醇基甘油(PEG-DSG)、PEG-二棕榈油基、PEG-二油基、PEG-二硬脂基、PEG-二酰基甘油酰胺(PEG-DAG)、PEG-二棕榈酰基磷脂酰乙醇胺(PEG-DPPE)、PEG-1,2-二肉豆蔻酰基氧基丙基-3-胺(PEG-c-DMA)或DMG-PEG2000中的一种或多种组合，优选的为DMG-PEG2000。The lipid nanoparticle according to claim 12, wherein the polyethylene glycol (PEG)-lipid conjugate is selected from: 2-[(polyethylene glycol)-2000]-N, N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn -Glyceryl-3-phosphoethanolamine-N-[Amino(polyethylene glycol)](PEG-DSPE), PEG-Disterylglycerol (PEG-DSG), PEG-Dipalmitoleyl, PEG-Dioleyl , PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3 - One or more combinations of amine (PEG-c-DMA) or DMG-PEG2000, preferably DMG-PEG2000.
  14. 根据权利要求12-13任一项所述的脂质纳米颗粒，其特征在于，所述的中性脂质选自1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二油酰-sn-甘油-3-磷酸乙醇胺(DOPE)、1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺(DPPE)、1,2-二肉豆蔻酰-sn-甘油-3-磷酸乙醇胺(DMPE)、2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油)(DOPG)、油酰磷脂酰胆碱(POPC)、1-棕榈酰基-2-油酰基磷脂酰乙醇胺(POPE)中的一种或多种组合，优选的为DSPC。 The lipid nanoparticle according to any one of claims 12-13, wherein the neutral lipid is selected from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC ), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl -sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phosphate-(1 One or more combinations of '-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), preferably DSPC.
  15. 根据权利要求12-14任一项所述的脂质纳米颗粒，其特征在于，所述的甾族脂质选自燕麦甾醇、β-谷甾醇、菜子甾醇、麦角骨化醇、菜油甾醇、胆甾烷醇、胆固醇、粪甾醇、脱氢胆固醇、链甾醇、二氢麦角骨化醇、二氢胆固醇、二氢麦角甾醇、黑海甾醇、表胆甾醇、麦角甾醇、岩藻甾醇、六氢光甾醇、羟基胆固醇以及经多肽修饰后的胆固醇；羊毛甾醇、光甾醇、海藻甾醇、谷甾烷醇、谷甾醇、豆甾烷醇、豆甾醇、胆酸、甘氨胆酸、牛磺胆酸、脱氧胆酸和石胆酸中的一种或多种组合，优选的为胆固醇。The lipid nanoparticle according to any one of claims 12-14, wherein the steroidal lipid is selected from the group consisting of avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholesterol Stanol, Cholesterol, Coprosterol, Dehydrocholesterol, Strepterosterol, Dihydroergocalciferol, Dihydrocholesterol, Dihydroergosterol, Nigasterol, Epicholesterol, Ergosterol, Fucosterol, Hexahydrophotosterol , hydroxycholesterol and cholesterol modified by polypeptide; One or more combinations of cholic acid and lithocholic acid, preferably cholesterol.
  16. 根据权利要求12-15任一项所述的脂质纳米颗粒，其特征在于，所述的阳离子脂质在脂质组分中的摩尔百分含量为20～60％、中性磷脂在脂质组分中的摩尔百分含量为5％～25％、甾族脂质在脂质组分中的摩尔百分含量为25％～55％；聚乙二醇(PEG)-脂质缀合物在脂质组分中的摩尔百分含量为0.5％～15％。The lipid nanoparticle according to any one of claims 12-15, characterized in that, the molar percentage of the cationic lipid in the lipid component is 20% to 60%, and the neutral phospholipid in the lipid The molar percentage of the component is 5% to 25%, and the molar percentage of the steroidal lipid in the lipid component is 25% to 55%; polyethylene glycol (PEG)-lipid conjugate The mole percentage in the lipid component is 0.5%-15%.
  17. 根据权利要求12-16任一项所述的脂质纳米颗粒，其特征在于，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为30-60:1-20:20-50:0.1-10，优选的，所述阳离子脂质：中性磷脂：甾族脂质：聚乙二醇(PEG)-脂质缀合物摩尔比为43:10:45:2或40:10:48:2。The lipid nanoparticle according to any one of claims 12-16, wherein the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate mole The ratio is 30-60:1-20:20-50:0.1-10, preferably, the cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid conjugate molar The ratio is 43:10:45:2 or 40:10:48:2.
  18. 根据权利要求12-17任一项所述的脂质纳米颗粒，其特征在于，所述疫苗中还包含其他辅料，所述辅料为醋酸钠、氨丁三醇、磷酸二氢钾、氯化钠、磷酸氢二钠、蔗糖中的一种或多种的组合。According to the lipid nanoparticle according to any one of claims 12-17, it is characterized in that, other adjuvants are also included in the vaccine, and the adjuvants are sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride , disodium hydrogen phosphate, and a combination of one or more of sucrose.
  19. 根据权利要求12-18任一项所述的脂质纳米颗粒，其特征在于，所述纳米颗粒的平均粒径为50～200nm或所述纳米颗粒在中性pH下具有净中性电荷或所述纳米颗粒具有小于0.4的多分散性。The lipid nanoparticle according to any one of claims 12-18, characterized in that, the average particle diameter of the nanoparticle is 50-200nm or the nanoparticle has a net neutral charge at neutral pH or the The nanoparticles have a polydispersity of less than 0.4.
  20. 一种根据权利要求1-19任一项所述的脂质纳米颗粒的制备方法，其特征在于，包括将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后与mRNA混合的步骤。A method for preparing lipid nanoparticles according to any one of claims 1-19, characterized in that comprising conjugating cationic lipids, non-cationic lipids, polyethylene glycol (PEG)-lipids After the substance is dissolved in the solvent, it is mixed with the mRNA.
  21. 根据权利要求20所述的脂质纳米颗粒的制备方法，其特征在于，将所述阳离子脂质、 中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液混合后经超滤、稀释、过滤后制得；优选的，将阳离子脂质、中性磷脂、甾族脂质、聚乙二醇(PEG)-脂质缀合物溶解至乙醇后与经稀释后的mRNA稀释液按一定流速比混合后经超滤、稀释、过滤后制得；优选的，所述的超滤方式为切向流过滤；更优选的，所述的混合方式可为湍流混合、层流混合或微流体混合。The preparation method of lipid nanoparticle according to claim 20, is characterized in that, described cationic lipid, Neutral phospholipids, steroidal lipids, and polyethylene glycol (PEG)-lipid conjugates are dissolved in ethanol and mixed with diluted mRNA diluents, followed by ultrafiltration, dilution, and filtration; preferably, Dissolve cationic lipids, neutral phospholipids, steroidal lipids, and polyethylene glycol (PEG)-lipid conjugates in ethanol and mix with diluted mRNA diluent at a certain flow rate ratio, then ultrafilter and dilute , obtained after filtration; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent flow mixing, laminar flow mixing or microfluidic mixing.
  22. 根据权利要求20-21任一项所述的脂质纳米颗粒的制备方法，其特征在于，稀释液为乙酸盐缓冲液、柠檬酸盐缓冲液、磷酸盐缓冲液或tris缓冲液。The method for preparing lipid nanoparticles according to any one of claims 20-21, wherein the diluent is acetate buffer, citrate buffer, phosphate buffer or tris buffer.
  23. 根据权利要求20-22任一项所述的脂质纳米颗粒的制备方法，其特征在于，所述缓冲液pH为3～6，浓度为6.25～200mM。The preparation method of lipid nanoparticles according to any one of claims 20-22, characterized in that the buffer solution has a pH of 3-6 and a concentration of 6.25-200 mM.
  24. 根据权利要求20-23任一项所述的脂质纳米颗粒的制备方法，其特征在于，将阳离子脂质、非-阳离子脂质、聚乙二醇(PEG)-脂质缀合物溶解至溶剂后所得的脂质混合溶液与mRNA稀释后的溶液流速比为1～5:1。The preparation method of lipid nanoparticles according to any one of claims 20-23, is characterized in that cationic lipids, non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates are dissolved to The flow rate ratio of the obtained lipid mixed solution after the solvent to the diluted mRNA is 1-5:1.
  25. 根据权利要求20-24任一项所述的脂质纳米颗粒的制备方法，其特征在于，采用脂质包封mRNA时的N/P为2-10，优选的N/P为3-8，更优选的，N/P为3、4、5、6、7、8，所述N/P为阳离子脂质中N与mRNA单核苷酸中P的摩尔比。The preparation method of lipid nanoparticles according to any one of claims 20-24, characterized in that the N/P when using lipid-encapsulated mRNA is 2-10, and the preferred N/P is 3-8, More preferably, N/P is 3, 4, 5, 6, 7, 8, and said N/P is the molar ratio of N in cationic lipid to P in mRNA single nucleotide.
  26. 根据权利要求20-25任一项所述的脂质纳米颗粒的制备方法，其特征在于，所述的超滤液选自由以下组成的组：钠盐和三(羟甲基)氨基甲烷(Tris)盐，优选的，超滤液pH为6.0～8.0。The method for preparing lipid nanoparticles according to any one of claims 20-25, wherein the ultrafiltrate is selected from the group consisting of sodium salt and tris(hydroxymethyl)aminomethane (Tris ) salt, preferably, the pH of the ultrafiltrate is 6.0-8.0.
  27. 根据权利要求20-26任一项所述的脂质纳米颗粒的制备方法，其特征在于，其剂型为口服制剂、液体制剂、冻干粉剂、注射剂或吸入制剂，优选的，为肌肉注射剂、静脉注射剂、干粉吸入剂或雾化吸入剂。 The preparation method of lipid nanoparticles according to any one of claims 20-26, characterized in that its dosage form is oral preparation, liquid preparation, freeze-dried powder, injection or inhalation preparation, preferably, intramuscular injection, intravenous Injection, dry powder inhaler or nebulized inhaler.
  28. 一种狂犬病毒脂质纳米颗粒mRNA疫苗，其特征在于，包含：编码G蛋白、G△TM蛋白、Gtrimer蛋白、M蛋白、Gtrimer蛋白M蛋白中的一种或多种的mRNA，所述mRNA被权利要求12-27任一项所述的脂质纳米颗粒包裹或被ALC-0315制备的脂质纳米颗粒包裹。A rabies virus lipid nanoparticle mRNA vaccine, characterized in that it comprises: one or more mRNAs encoding G protein, GΔTM protein, Gtrimer protein, M protein, Gtrimer protein M protein, and the mRNA is The lipid nanoparticle of any one of claims 12-27 is encapsulated or encapsulated by the lipid nanoparticle prepared by ALC-0315.
  29. 一种权利要求1-19、28任一项所述的狂犬病毒脂质纳米颗粒mRNA疫苗在制备用于预防狂犬病的药物中的用途。 A use of the rabies virus lipid nanoparticle mRNA vaccine according to any one of claims 1-19 and 28 in the preparation of medicaments for preventing rabies.
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