Nucleic acid molecule delivery carrier as well as preparation method and application of freeze-drying preparation of nucleic acid molecule delivery carrier

Abstract

The invention discloses a nucleic acid molecule delivery carrier as well as a preparation method and application of a freeze-drying preparation of the nucleic acid molecule delivery carrier. Wherein the delivery vehicle comprises a nucleic acid molecule encoding interleukin 12 and an ionizable lipid compound. According to the invention, the mRNA of the interleukin 12 with an optimized sequence is used, the mRNA is more stable, the protein expression is increased, and the misfolding rate is reduced. Meanwhile, the interleukin 12 is anchored through a transmembrane sequence, so that the possible immune storm risk is reduced. Besides, on the basis of newly synthesized, degradable and novel ionizable lipid molecules, a high-efficiency and low-toxicity freeze-drying preparation delivery system based on lipid is formed, and the freeze-drying preparation has potential application value in the fields of preventive vaccines and therapeutic drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to a nucleic acid molecule delivery carrier and a preparation method and use of a freeze-dried preparation thereof, and more particularly to an interleukin 12 nucleic acid molecule delivery carrier and a preparation method and use of a freeze-dried preparation thereof. BACKGROUND

[0002] IL-12 (Interleukin 12, IL-12) has a dual role of regulating innate immunity and acquired immunity, and has potential application value in the fields of preventive vaccines and therapeutic drugs. Interleukin 12 was first discovered in the 1990s, and was classified as a type I pro-inflammatory factor in immunology research. It is a heterodimer composed of two subunits p35 (or IL-12α) and p40 (or IL-12β) through two disulfide bonds, and mainly acts as a ligand of IL-12 receptor (Interleukin 12 receptor, IL-12R) to play an important role in innate immunity and adaptive immunity.

[0003] The main physiological producers of interleukin 12 (IL-12) are phagocytes (monocytes / macrophages and neutrophils) and dendritic cells, which respond to pathogens (bacteria, fungi, intracellular parasites and viruses) through Toll-like receptors (TLRs) and other receptors, and deliver membrane-bound signals and soluble signals from activated T cells and natural killer (NK) cells through CD44 and TLRs, as well as components of inflammatory extracellular matrix (such as low molecular weight hyaluronic acid). The most important target cells of IL-12 in physiology are hematopoietic progenitor cells, which synergize with other colony-stimulating factors to induce proliferation and colony formation. IL-12 induces NK cell, NKT cell and T cell proliferation, enhances cytotoxicity and cytotoxic mediator expression and cytokine production, especially interferon-γ (IFN-γ), and facilitates differentiation into type 1 cytokine-producing cells (TH1, TC1 and NK1 cells); for B cells, IL-12 enhances the activation and production of TH1-related immunoglobulin (such as mouse IgG2a) directly or through the action of type 1 cytokines (such as IFN-γ).

[0004] In mice, administration of recombinant IL-12 at the time of RSV attachment protein G vaccination increased IFN-g production and decreased IL-4 and IL-5 in CD4+ and CD8+ T cells, and prevented eosinophilia in a subsequent RSV challenge. IL-12 also plays a key role in host defense against HBV. Impaired IL-12 secretion by dendritic cells is associated with chronicity of HBV infection, and IL-12 can also inhibit HBV replication in HBV-tg mice by inducing IFN-g production, and combination therapy with HBsAg vaccine and IL-12 can effectively eliminate HBV.

[0005] Nonetheless, a number of IL-12 related innate immune modulators and cancer therapeutics have failed to show significant effects in clinical stages. Since the first human clinical trial of IL-12 in 1996, related clinical studies have continued for more than 20 years, but FDA-approved IL-12 products are still not available. Therefore, there is a need in the art to use new technologies to improve the therapeutic effect of IL-12 on tumor diseases.

[0006] Different types of nucleic acid preparations are being developed for the treatment of various major diseases such as infectious diseases, cancer, rare diseases, etc. Such nucleic acid preparations include DNA, antisense nucleic acids (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, ribozyme, etc. Since mRNA vaccines can be deduced, designed and synthesized according to key proteins without consuming a large amount of resources for synthesis by living organisms or cells, they can themselves induce a comprehensive and effective human immune response, and mRNA vaccines do not have the potential reverse danger of attenuated vaccines; there is no problem of recovery mutation of inactivated vaccines. In addition, mRNA is easily degraded after being translated into protein, and its transient expression characteristics not only ensure the safety of mRNA drugs, but also make the dose controllable, avoiding the antigen immune tolerance (a state of no response to a specific antigen) caused by long-term exposure of vaccine drugs. Therefore, mRNA vaccines have revolutionary advantages in safety, rapid preparation and immunogenicity. Based on the continuous updating of mRNA modification and delivery technology, mRNA as a key to the development of new therapeutic drugs has almost unlimited application prospects. In the long run, mRNA technology will gradually mature in the fields of tumor vaccines, gene editing, CAR T cell therapy, protein replacement therapy and other infectious disease preventive vaccines, and various product lines are advancing in clinical development.

[0007] However, on the one hand, nucleic acid preparations are negatively charged and most of them have a large molecular weight and are difficult to directly enter cells, and on the other hand, RNA is unstable and is easily degraded by nucleases and intramolecular hydrolysis during the process of being introduced into the body, thereby losing biological functions. Therefore, developing a safe and efficient and universal nucleic acid delivery system, especially a temperature-stable delivery system such as a freeze-dried preparation technology, and an efficient skin administration route are urgent problems to be solved in the process of nucleic acid drug transformation. SUMMARY

[0008] To solve at least part of the problems in the prior art, the present application provides a nucleic acid molecule delivery carrier and a freeze-dried preparation method and use thereof. Specifically, the present application includes the following contents.

[0009] In a first aspect of the present application, a nucleic acid molecule delivery carrier is provided, which comprises a nucleic acid molecule encoding interleukin 12 and an ionizable lipid compound.

[0010] In certain embodiments, the nucleic acid molecule delivery carrier according to the present application further comprises a phospholipid, a sterol and a PEG-lipid.

[0011] In certain embodiments, the nucleic acid molecule delivery carrier according to the present application, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an open reading frame of an IL-12 p40 subunit and an IL-12 p35 subunit and a transmembrane region; preferably, the nucleic acid molecule further comprises a nucleic acid sequence encoding a signal peptide, a 5'UTR, a 3'UTR and a poly-A tail.

[0012] In certain embodiments, the nucleic acid molecule delivery carrier according to the present application, wherein the nucleic acid molecule has a nucleotide sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homologous to the nucleotide sequence set forth in SEQ ID NO: 1, preferably the sequence set forth in SEQ ID NO: 1.

[0013] In certain embodiments, the nucleic acid molecule delivery carrier according to the present application, wherein the ionizable lipid compound has a structure represented by the following formula:

[0014]

[0015] wherein:

[0016] R1is hydrogen, a substituted or unsubstituted straight chain or branched C1-20alkyl, a substituted or unsubstituted straight chain or branched C2-20alkenyl, a substituted or unsubstituted straight chain or branched C2-20alkynyl, said substituted substituent groups are selected from the group consisting of halogen, -OH, =0, straight or branched C1-10alkyl, straight or branched C1-10alkoxy; all alkyl, alkenyl, alkynyl or alkoxy groups above, one or more C atoms in the carbon chain of which can optionally be replaced by a heteroatom selected independently from O, S and N;

[0017] n is a positive integer selected from 1 to 10; m is a positive integer selected from 1 to 10; n and m can be the same or different; preferably, n is a positive integer selected from 1 to 6, and m is a positive integer selected from 1 to 6;

[0018] each R2is independently selected from the group consisting of hydrogen, straight or branched C1-30alkyl, straight or branched C2-30alkenyl, straight or branched C2-30alkynyl; said substituted substituent groups are selected from the group consisting of halogen, -OH, =0, straight or branched C1-10alkyl, straight or branched C1-10alkoxy, straight or branched C2-20alkenyl; all alkyl, alkenyl, alkynyl or alkoxy groups above, one or more C atoms in the carbon chain of which can optionally be replaced by a heteroatom selected independently from O, S and N;

[0019] with the proviso that at least one R2is

[0020] o is independently selected from a positive integer from 1 to 20;

[0021] R' is selected from the group consisting of substituted or unsubstituted straight or branched C1-30alkyl, substituted or unsubstituted straight or branched C2-30alkenyl, substituted or unsubstituted straight or branched C2-30alkynyl; said substituted substituent groups are selected from the group consisting of =0, halogen, -OH, straight or branched C1-20alkyl, straight or branched C1-20alkoxy, straight or branched C2-20alkenyl; all alkyl, alkenyl, alkynyl or alkoxy groups above, one or more C atoms in the carbon chain of which can optionally be replaced by a heteroatom selected independently from O, S and N;

[0022] when there are at least two R2which are they can be the same or different.

[0023] In certain embodiments, the nucleic acid molecule delivery vehicle according to the present application, wherein the ionizable lipid compound has the following formula:

[0024]

[0025] In a second aspect of the present application, there is provided a pharmaceutical vaccine or composition comprising an immunologically effective amount of the nucleic acid molecule delivery vehicle.

[0026] In a third aspect of the present application, a preparation method of the delivery carrier is provided, which comprises:

[0027] i. a step of synthesizing a nucleic acid molecule capable of expressing interleukin 12 and preparing an aqueous phase;

[0028] ii. a step of preparing an organic phase containing an ionizable lipid compound, and mixing the organic phase with the aqueous phase;

[0029] iii. a freeze-drying step of freeze-drying a preparation preservative and a freeze-drying procedure thereof.

[0030] In a fourth aspect of the present application, the use of the delivery carrier in the preparation of vaccines and drugs for preventing, treating or improving diseases is provided.

[0031] In a fifth aspect of the present application, the use of the delivery carrier in combination with other drugs is provided.

[0032] The mRNA used in the present application is an IL-12 optimized sequence, which is more stable, increases protein expression, and reduces the rate of misfolding. At the same time, the present application anchors IL-12 on the lower envelope through a transmembrane sequence, reducing the risk of possible immune storm. In addition, the present application is based on a newly synthesized, degradable, new type of ionizable lipid molecule, forming a lipid-based efficient, low-toxicity freeze-dried preparation delivery system, thereby achieving safe and efficient delivery. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The gel shows the integrity results of in vitro transcription synthesized IL-12 mRNA.

[0034] Figure 2 The nuclear magnetic resonance spectrum of compound 2 is shown.

[0035] Figure 3 The nuclear magnetic resonance spectrum of compound 3 is shown.

[0036] Figure 4 The nuclear magnetic resonance spectrum of compound T7 is shown.

[0037] Figure 5 The mass spectrum of compound T7 is shown.

[0038] Figure 6 The morphology of the freeze-dried preparation is shown as a white loose and full cake.

[0039] Figure 7 The FACS method is shown to detect the expression level of IL-12 protein on the cell membrane of A375, A549, hek293, Hela cells after reconstitution of the freeze-dried preparation and frozen lipid nanoparticles (LNP-mRNA) and positive control mRNA transfection.

[0040] Figure 8 The cell viability of A375, A549, hek293, Hela cells transfected with the freeze-dried preparation reconstituted LNP-mRNA and positive control mRNA after freezing and thawing was detected by CTG method. DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present application will now be described in detail, with reference to the figures. Such description, however, is to be considered in all aspects as illustrative and not restrictive, understood to be a description of certain aspects, features and embodiments of the present application.

[0042] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. The upper and lower limits of these intervening values are also specifically included within the scope of the present application. These smaller ranges are not insubstantial.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0044] A nucleic acid molecule delivery vehicle and a lyophilized formulation thereof

[0045] In one aspect of the present application, there is provided a nucleic acid molecule delivery vehicle, which is a lipid nanoparticle, comprising a nucleic acid molecule encoding interleukin 12 and an ionizable lipid compound.

[0046] The nucleic acid molecule of the present application comprises a nucleic acid sequence encoding an open reading frame of IL-12 p40 subunit and IL-12 p35 subunit and a transmembrane region. In a preferred embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding a signal peptide, a 5' cap structure, a 5' UTR, a 3' UTR and a poly-A tail.

[0047] According to the present application, the polynucleotide sequence encoding recombinant IL-12 is a codon-optimized sequence. The nucleic acid molecule is mRNA. The codon-optimized sequence comprises at least one substitution of synonymous nucleobases relative to the wild-type nucleotide sequence.

[0048] In the present invention, the IL-12 p40 subunit and the IL-12 p35 subunit are connected via a linker, which is a peptide linker. In some embodiments of the present invention, the peptide linker can be a GS (Gly / Ser) linker, preferably comprising (G n S) m wherein n is an integer from 1 to 20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) and m is an integer from 1 to 20 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). In some embodiments, the GS linker can comprise (G4S)3.

[0049] In the present invention, the 5' cap is typically a modified nucleotide (in particular a guanine nucleotide) added at the 5' end of the mRNA molecule, which also includes atypical cap analogs. Preferably, the 5' cap is added using a 5'-5'-triphosphate linkage (also known as m7GpppN). Additional examples of 5' cap structures include glyceryl, inverted deoxy abasic residue (moieties), 4',5'- methylene nucleotides, 1-(beta-D-erythro furanosyl) nucleotides, 4'-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, alpha- nucleotides, modified base nucleotides, threo pentofuranosyl nucleotides, acyclic 3',4'-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5- dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 1,4- butanediol phosphate, 3'-aminophosphates, hexyl phosphonates, aminohexyl phosphonates, 3'-phosphates, 3' thiophosphates, dithiophosphates or bridged or unbridged methyl phosphonate moieties. These modified 5' cap structures can be used in the context of the present invention to modify the mRNA sequences of the present invention.

[0050] In the present invention, "a nucleotide sequence encoding" refers to the coding sequence of a nucleic acid (e.g. an mRNA or DNA molecule) that encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements, including promoters and polyadenylation signals capable of directing expression in the cells of the individual or mammal to which the nucleic acid is administered.

[0051] The term "homology" as used herein refers to the overall relatedness between polymeric molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" means evolutionary relatedness between two molecules. Thus, two homologous molecules will have a common evolutionary ancestry. In the context of the present disclosure, the term homology includes identity and similarity.

[0052] In some embodiments, a polymeric molecule is considered to be "homologous" to another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the monomers in the molecules are identical (exactly the same monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0053] The term "identity" as used herein refers to the overall monomer conservation between polymeric molecules, for example, between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the percent identity of two polynucleotide sequences can be ascertained using an alignment comparison algorithm with a program that is specifically designed to give the optimal comparison between two sequences (e.g., an empty gap can be introduced in either or both of the first and second nucleic acid sequences for optimal alignment and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0054] Suitable software programs are available from a variety of sources and are used for alignment of both protein and nucleotide sequences. For example, Bl2seq, Needle, Stretcher, Water, or Matcher, among others. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, among others.

[0055] As used herein, the terms "coding region" and "coding regions" refer to the open reading frame (ORF) in a nucleic acid molecule that, when expressed, produces a polypeptide or protein. "Operably linked" refers to functional linkage between two or more molecules, constructs, transcripts, entities, moieties, etc.

[0056] As used herein, the term "domain" when referring to a polypeptide refers to a motif of a polypeptide having one or more identifiable structural or functional features or properties (e.g., binding ability, serving as a site for protein-protein interaction). "Expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template from a DNA sequence (e.g., by transcription); (2) processing of an mRNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of an mRNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0057] In one aspect of the present application, a vector comprising the nucleic acid molecule of the present application is also provided. The vector includes a plasmid vector or a viral vector. In some embodiments, the vector can be used to prepare the mRNA of the present application in vitro by transcription.

[0058] According to the present application, a commonly used plasmid can be used as a vector. In some embodiments of the present application, the plasmid is psp73 or pUC57-kana.

[0059] The nucleic acid molecule (e.g., mRNA) of the present application can be prepared by methods known in the art, including but not limited to chemical synthesis or in vitro transcription, etc. In some embodiments of the present application, the nucleic acid molecule encoding the mRNA can be artificially synthesized. Exemplary methods include: cloning the nucleic acid molecule into a vector, a construct for in vitro transcription plasmid. Transforming the constructed plasmid into a host bacteria for culture amplification, extracting the plasmid. Digesting the extracted plasmid into a linear molecule using a restriction enzyme. Using the prepared linearized plasmid molecule as a template, mRNA is prepared using an in vitro transcription method. An in vitro transcription (IVT) system generally comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. The NTPs can be selected from, but not limited to, natural and unnatural (modified) NTPs. The polymerase can be selected from, but not limited to, T7 RNA polymerase, T3 RNA polymerase, and mutant polymerases. A cap structure analog can be added during the in vitro transcription process to directly obtain mRNA with a cap structure; or a cap structure can be added to the mRNA after the in vitro transcription is completed using a capping enzyme and a dimethyltransferase. The obtained mRNA can be purified using conventional methods in the art, such as chemical precipitation, magnetic bead method, affinity chromatography, etc.

[0060] Sequence list of the present application:

[0061]

[0062]

[0063]

[0064]

[0065] In some embodiments, the nucleic acid molecule is prepared into a lipid nanoparticle together with the ionizable lipid compound, and the lipid nanoparticle is prepared into a therapeutic vaccine. In some embodiments, the ionizable lipid compound is in a mass ratio of (5-20): 1 to the mRNA.

[0066] The ionizable lipid compound of the present application has the following structure: In a preferred embodiment, the ionizable lipid compound has the following structure:

[0067]

[0068] The ionizable lipid compound of the present application can be synthesized by methods known in the art, for example, by reacting one equivalent or more of an amine with one equivalent or more of an epoxy-terminated compound under suitable conditions. The synthesis of the ionizable lipid compound can be carried out with or without a solvent, and the synthesis can be carried out at a relatively high temperature in the range of 50-100 °C. The ionizable lipid compound obtained can be optionally purified. For example, a mixture of ionizable lipid compounds can be purified to obtain a specific ionizable lipid compound. Or a mixture can be purified to obtain a specific stereoisomer or regioisomer. The epoxide can be purchased commercially or synthesized.

[0069] In some embodiments of the present application, the cationic lipid compound of the present application can be prepared by the following preparation method,

[0070]

[0071] Step 1: esterification

[0072] A carboxylic acid compound A1 is condensed with an alcohol A2 in the presence of a condensing agent to obtain a compound A3. Examples of the condensing agent include, but are not limited to, DCC, EDCI, and the like. Examples of the solvent used in the reaction include, but are not limited to, ethers (such as diethyl ether, tetrahydrofuran, dioxane, and the like), halogenated hydrocarbons (such as chloroform, dichloromethane, dichloroethane, and the like), hydrocarbons (such as n-pentane, n-hexane, benzene, toluene, and the like), and a mixed solvent formed by two or more of these solvents.

[0073] Step 2: Epoxidation Compound A3 is subjected to epoxidation of the double bond in the presence of a base to obtain epoxide compound A4. Examples of double bond epoxidation reagents include, but are not limited to, meta-chloroperoxybenzoic acid, hydrogen peroxide, and the like. Examples of solvents used in the reaction include, but are not limited to, dichloromethane.

[0074] Step 3: Ring opening reaction Compound A4 is subjected to ring opening reaction with amine A5 (e.g., N,N-di(2-aminoethyl)methylamine) to obtain the final compound. Examples of solvents used in the reaction include, but are not limited to, ethanol, methanol, isopropanol, tetrahydrofuran, chloroform, hexane, toluene, diethyl ether, and the like.

[0075] The ionizable lipid molecules of the present application have degradability, and low toxicity, and have strong extrahepatic targeting. They have high encapsulation efficiency for active substances (e.g., nucleic acids such as mRNA), and better cell transfection efficiency. The lipid nanoparticles prepared therefrom have the characteristic of uniform particle size distribution.

[0076] The delivery vehicle of the present application can be in the form of a particle. In certain embodiments, the particle has a diameter in the range of 1 pm to 1000 pm. In certain embodiments, the particle has a diameter in the range of 1 nm to 1000 nm. For example, the particle has a diameter in the range of 1 pm to 100 pm, or in the range of 1 pm to 10 pm, or in the range of 10 pm to 100 pm, or in the range of 20 nm to 800 nm, or in the range of 50 nm to 500 nm, or in the range of 80 nm to 200 nm, or in the range of 1 nm to 100 nm, or in the range of 1 nm to 10 nm. When the particle has a diameter in the range of 1 nm to 1000 nm. The particle can be prepared using any method known in the art. These methods include, but are not limited to, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, nanoprecipitation, microfluidics, simple and complex coacervation, and other methods well known to those of ordinary skill in the art.

[0077] The delivery vehicle of the present application also includes phospholipids, sterols, and PEG-lipids.

[0078] According to the present application, the phospholipid includes an uncharged lipid molecule or a zwitterionic lipid molecule, such as a phosphatidylcholine compound, or / and a phosphatidylethanolamine compound, examples of the phospholipid include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidylcholine, dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. The proportion of the phospholipid in the delivery carrier is not particularly limited, and can be adjusted as needed.

[0079] According to the present application, the PEG-lipid comprises a lipid moiety and a PEG-based polymer moiety. In some embodiments, the PEG-lipid can be represented as "lipid moiety-PEG-number average molecular weight" or "PEG-lipid moiety" or "PEG-number average molecular weight-lipid moiety", the lipid moiety being a diacylglycerol or diacylglycerol amide selected from the group consisting of dilauric glycerol, dimyristyl glycerol, dipalmitoyl glycerol, distearoyl glycerol, dilauric glycerol amide, dimyristyl glycerol amide, dipalmitoyl glycerol amide, distearoyl glycerol amide, l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; the number average molecular weight of the PEG being from about 130 to about 50,000, for example, from about 150 to about 30,000, from about 150 to about 20,000, from about 150 to about 15,000, from about 150 to about 10,000, from about 150 to about 6,000, from about 150 to about 5,000, from about 150 to about 4,000, from about 150 to about 3,000, from about 300 to about 3,000, from about 1,000 to about 3,000, from about 1,500 to about 2,500, for example, about 2000. In some embodiments, the PEGylated lipid molecule can be selected from the group consisting of PEG-dilauric glycerol, PEG-dimyristyl glycerol (PEG-DMG), PEG-dipalmitoyl glycerol, PEG-distearoyl glycerol (PEG-DSPE), PEG-dilauric glycerol amide, PEG-dimyristyl glycerol amide, PEG-dipalmitoyl glycerol amide, and PEG-distearoyl glycerol amide, PEG-cholesterol (l-[8'-(cholest-5-en-3[beta]-yloxy)formamido-3',6'-dioxa octyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecyloxybenzyl-[omega]-methyl-poly(ethylene glycol) ether), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMG-PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), l,2-distearoyl-sn-glycero-methoxypolyethylene glycol (DSG-PEG2000), poly(ethylene glycol)-2000-dimethacrylate (DMA-PEG2000), and l,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (DSA-PEG2000). The ratio of the PEG-lipid in the delivery vehicle is not particularly limited and can be adjusted as desired.

[0080] According to the present application, examples of sterol include, but are not limited to, cholesterol, 5-heptadecylresorcinol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatin, ursolic acid, alpha-tocopherol and mixtures thereof, cholesterol hemisuccinate. In one embodiment, the sterol is cholesterol (CHOL). In one embodiment, the cholesterol lipid molecule is cholesterol hemisuccinate. The proportion of sterol in the delivery vehicle is not particularly limited and can be adjusted as desired.

[0081] In some embodiments of the present application, the delivery vehicle can further comprise an active substance, which is located in the lipid nanoparticle. The nature of the active substance can be a small molecule compound, a nucleic acid, a protein, a peptide, a metal, an isotopically labeled compound, a vaccine, etc.

[0082] The delivery vehicle formed from the ionizable lipid compound of the present application can also modify a targeting molecule, thereby making it a targeting agent capable of targeting a specific cell, tissue or organ. The targeting molecule can be included throughout the delivery vehicle or can be located only on its surface. The targeting molecule can be a protein, a peptide, a glycoprotein, a lipid, a small molecule, a nucleic acid, etc., examples of which include, but are not limited to, an antibody, an antibody fragment, low-density lipoprotein (LDL), transferrin, asialycoprotein, a receptor ligand, sialic acid, an aptamer, etc.

[0083] Vaccine or pharmaceutical composition

[0084] In one aspect of the present application, there is provided a vaccine or pharmaceutical composition comprising an immunologically effective amount of a delivery vehicle for a nucleic acid molecule.

[0085] According to the present application, in the vaccine or pharmaceutical composition, when the lipid nanoparticle is used as a carrier, the mRNA is located in the lipid nanoparticle, which contains 30-60 mol% of ionizable cationic lipid molecules, 5-30 mol% of phospholipids, 30-50 mol% of sterols, and 0.4-10 mol% of PEG-lipids, based on the total lipid molecules; preferably, 30-55 mol% of ionizable cationic lipid molecules, 8-20 mol% of phospholipids, 32-50 mol% of sterols, and 0.5-2.5 mol% of PEG-lipids.

[0086] In some embodiments of the present application, the vaccine or pharmaceutical composition is a lyophilized formulation. In some embodiments of the present application, the vaccine or pharmaceutical composition is a vaccine, in particular an mRNA vaccine. The lyophilized formulation further comprises a lyoprotectant. The lyoprotectant can be any lyoprotectant commonly used in the art, including but not limited to sucrose, trehalose and maltose. The lyophilized formulation can further comprise a buffer salt. The buffer salt can be any buffer salt commonly used in the art, including but not limited to Tris, Hepe, EDTA, citric acid-citrate, acetic acid-acetate, phosphate-biphosphate, carbonate-bicarbonate, etc. The lyophilized formulation can further comprise an osmotic pressure regulator. The osmotic pressure regulator can be any osmotic pressure regulator commonly used in the art, including but not limited to sodium chloride, potassium chloride, calcium chloride, glucose, phosphate, citrate, etc.

[0087] According to the present application, the vaccine or pharmaceutical composition is administered alone or in combination with a second agent. The second agent is another tumor therapeutic agent, such as a checkpoint inhibitor (e.g. anti-PD-1 antibody, anti-PD-L1 antibody and / or anti-CTLA-4 antibody, etc.), a tumor chemotherapeutic agent (including but not limited to: alkylating agents, nitrogen mustards, thiotepa, nitrosoureas, methanesulfonates, platinum compounds, mitomycin, etc., specifically, mechlorethamine, chlorambucil, cyclophosphamide, ifosfamide, thiotepa, carmustine, semustine, busulfan, cisplatin, oxaliplatin, carboplatin, platinum oxalate, mitomycin, etc.; agents affecting nucleic acid synthesis, such as dihydrofolate reductase inhibitors, thymidylate synthetase inhibitors, purine nucleoside synthetase inhibitors, ribonucleotide reductase inhibitors, DNA polymerase inhibitors, specifically, methotrexate, 5-FU, FT-207, capecitabine, 6-mercaptopurine, 6-TG, hydroxyurea, cytarabine, gemcitabine, pemetrexed, etc.; agents acting on nucleic acid transcription, such as actinomycin D, daunorubicin, doxorubicin, epirubicin, aclacinomycin, and mithramycin, etc.; topoisomerase I inhibitors acting on DNA replication, such as irinotecan, topotecan, and hydroxy camptothecin, etc.; agents interfering with microtubulin synthesis in the mitotic M phase, such as paclitaxel, docetaxel, vinblastine, vincristine, vinorelbine, podophyllotoxins, and homoharringtonine, etc.), angiogenesis inhibitors (including but not limited to: those inhibiting the action of vascular endothelial growth factor, such as lenalidomide, thalidomide, anti-vascular endothelial cell growth factor antibodies such as bevacizumab, VEGF receptor tyrosine kinase inhibitors vandetanib, vatalanib, sunitinib, etc.).

[0088] The vaccine or pharmaceutical composition of the present application can be administered in any available route, including but not limited to epicutaneous (applied to the skin), intradermal (into the skin itself), subcutaneous (under the skin), intranasal (through the nose), intratumoral, enteral, gastrointestinal, epidural, oral, transdermal, epidural (epidural), intracerebral (into the brain), intracerebroventricular (into the cerebral ventricles), intravenous (into a vein), intraperitoneal (into the peritoneum), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavernous injection (into the root of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through intact skin for general distribution), transmucosal (diffusion through mucous membranes), insufflation (nasal aspiration), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or ear drops. In some embodiments, the mRNA of the present application is administered parenterally (e.g., including subcutaneous, intravenous, intraperitoneal, intratumoral, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques), intraventricular, oral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or by implanted reservoir.

[0089] In one particular embodiment, the nucleic acid molecule (e.g., mRNA) of the present application is administered subcutaneously by needle-free jet injection; in another particular embodiment, the nucleic acid molecule (e.g., mRNA) of the present application is administered intratumorally.

[0090] According to the present application, the infectious diseases include, but are not limited to, influenza, hepatitis B, bacterial dysentery, meningitis, tuberculosis, acute hemorrhagic conjunctivitis, ascariasis, bacterial dysentery, hepatitis A, hepatitis B, malaria, epidemic encephalitis B, filariasis, schistosomiasis, trachoma, rabies, tetanus, gonorrhea, etc., and the cancers / tumors include, but are not limited to, solid tumors and non-solid tumors, such as: adrenocortical carcinoma, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, cervical cancer, colon / rectum cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, liver cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, adult soft tissue sarcoma, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms' tumor, Hodgkin's disease, Kaposi's sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, etc.

[0091] Preparation method and lyophilization process

[0092] In one aspect of the present application, a method for preparing a nucleic acid molecule delivery vehicle is provided, which comprises (1) separately dissolving different lipid compounds in an organic solvent to obtain mother solutions, preferably, the organic solvent is anhydrous ethanol or 90-99% ethanol (e.g., 95% ethanol); (2) mixing the mother solutions of the different lipid compounds obtained in step (1) according to the molar ratio of each lipid compound to obtain a mixed solution of lipids as an organic phase; (3) dissolving mRNA in an aqueous solution to obtain an aqueous phase; and (4) mixing the aqueous phase and the organic phase according to a certain volume ratio to prepare a lipid nanoparticle, preferably, the volume ratio of the organic phase to the aqueous phase is 1:2-4. The present application also provides a method for preparing a freeze-dried preparation of a vaccine or a vaccine composition, which comprises freeze-drying the liquid composition comprising the lipid nanoparticle of the present application after obtaining the liquid composition.

[0093] In some embodiments of the present application, a protective agent is added to the liquid composition of the lipid nanoparticles, which protects the lipid nanoparticles to some extent. The protective agent can be one or more of glucose, a-D-mannopyranose, sucrose, lactose, trehalose, cellobiose, mannose, maltose, muscle sugar, cotton sugar, inulin, dextran, maltodextrin, maltopolysaccharide, octasaccharide sucrose, heparin, 2-hydroxypropyl-B cyclodextrin, etc. The basic freeze-drying process includes pre-freezing, primary drying, and secondary drying, or an annealing procedure. Preferably, the pre-freezing is at -40 to -60°C for 1-12 h; the annealing is at -5 to -30°C, preferably -10 to -20°C, and more preferably at -15°C for 2-4 h; the primary drying is at -30 to -55°C for 10-80 h under a vacuum of 0-10 pa; and the secondary drying is at 4 to 20°C for 10-30 h under a vacuum of 0-10 pa.

[0094] Use

[0095] In one aspect of the present application, the use of the nucleic acid molecule, or the vector, or the lipid nanoparticle comprising the nucleic acid molecule, or the vaccine or pharmaceutical composition in the preparation of an immunopotentiator or vaccine is provided. Preferably, the pharmaceutical is used to activate T cells and / or NK cells. More preferably, the activation of T cells includes inducing T cell proliferation; and more preferably, the activation of T cells includes inducing T cell infiltration in a tumor or increasing the number of tumor infiltrating T cells.

[0096] Example 1

[0097] This example shows the preparation of IL-12 mRNA.

[0098] 1) Artificially synthesize a nucleic acid sequence capable of encoding the mRNA shown in SEQ ID No. 1, and clone the sequence behind the T7 promoter of the pUC57-kana vector to construct a plasmid for in vitro transcription.

[0099] 2) Transform the constructed plasmid into E. coli DH5a, culture and amplify, and extract the plasmid.

[0100] 3) Digest the extracted plasmid with a restriction enzyme immediately behind the polyA tail to linearize the molecule.

[0101] 4) Use the prepared linearized plasmid molecule as a template to prepare mRNA using in vitro transcription (Thermo in vitro transcription kit, Cat#A45975). After in vitro transcription, use a capping enzyme and a dimethyltransferase to add a CAP1 cap structure to the mRNA.

[0102] 5) Purification of mRNA: The obtained mRNA stock solution was purified using affinity chromatography.

[0103] 6) Quality control of mRNA: The prepared mRNA was checked using gel detection, the band of the transcribed mRNA was required to be single and no obvious degradation, Figure 1 as shown.

[0104] Example 2

[0105] This example shows the synthesis of an ionizable lipid compound (compound T7).

[0106]

[0107] Substrate compound 10-undecenoic acid (10.0 g, 54.3 mmol, 1.0 eq) was dissolved in dichloromethane (70 mL), and compound 3-octanol (7.07 g, 54.3 mmol, 1.0 eq) and EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) (12.5 g, 65.1 mmol, 1.2 eq) and 4-dimethylaminopyridine (663 mg, 5.43 mmol, 0.1 eq) were added at room temperature. The resulting reaction solution was stirred at 15 °C for 15 h. TLC (petroleum ether, product Rf value 0.7, iodine coloration) spot plate monitoring showed that the starting material was completely reacted and a main spot with small polarity was generated. The reaction solution was quenched by dropwise addition of 100 mL of water and extracted with dichloromethane (200 mL, 100 mL). The combined organic phase was evaporated and concentrated, and the obtained crude product was purified by column chromatography, eluted with petroleum ether to ethyl acetate 30 / 1 to 20 / 1 to obtain product compound 2 (11.0 g, 37.1 mmol, 68.4% yield) as colorless oil. 1 H NMR: 400 MHz (CDCI3) δ 5.74-5.89 (m, 1H), 4.90-5.07 (m, 2H), 4.82 (quin, J = 6.0 Hz, 1H), 2.29 (t, J = 7.6 Hz, 2H), 2.04 (q, J = 6.8 Hz, 2H), 1.46-1.66 (m, 6H), 1.17-1.40 (m, 16H), 0.81-0.96 (m, 6H).

[0108] Substrate compound 2 (11.0 g, 37.1 mmol, 1.0 eq) was dissolved in dichloromethane (70 mL) and m-chloroperoxybenzoic acid (12.0 g, 55.6 mmol, 1.5 eq) was added at room temperature. The resulting reaction was stirred at 15 °C for 15 h. TLC (petroleum ether / ethyl acetate 10 / 1, product Rf 0.45, iodine visualization) was used to monitor the complete consumption of the starting material and the formation of a major polar spot. The reaction was quenched by the dropwise addition of 200 mL of 10% sodium bisulfite and extracted with dichloromethane (200 mL, 100 mL). The combined organic phases were washed with aqueous sodium bicarbonate solution and evaporated to dryness to give the crude product which was purified by column chromatography eluting with petroleum ether / ethyl acetate 100 / 1 to 10 / 1 to give product compound 3 (10.0 g, 32 mmol, 86.3% yield) as a colorless oil. 1 H NMR: 400 MHz (CDC13) δ 4.82 (quin, J = 6.4 Hz, 1H), 2.86-2.95 (m, 1H), 2.75 (t, J = 4.4 Hz, 1H), 2.47 (dd, J = 4.8, 2.8 Hz, 1H), 2.29 (t, J = 7.2 Hz, 2H), 1.39-1.69 (m, 10H), 1.20-1.39 (m, 14H), 0.84-0.93 (m, 6H).

[0109] Substrate compound 4 N,N-di(2-aminoethyl)methylamine (1.0 g, 8.53 mmol, 1.0 eq) was dissolved in ethanol (15 mL) and compound 3 (8.0 g, 25.6 mmol, 2.5 eq) was added at room temperature. The resulting reaction was stirred at 90 °C (oil bath temperature) for 10 h under nitrogen atmosphere. The reaction was quenched by the dropwise addition of 200 mL of 10% sodium bisulfite and extracted with dichloromethane (200 mL, 100 mL). The crude product was purified by column chromatography eluting with dichloromethane / methanol 20 / 1 to 5 / 1 using TLC (dichloromethane / methanol = 10 / 1, product Rf 0.4, iodine visualization) to give product compound T7 (6.0 g, 54.1% yield) as a colorless oil. 1 H NMR: 400 MHz (CDC13) δ 4.82 (quin, J = 6.4 Hz, 1H), 2.86-2.95 (m, 1H), 2.75 (t, J = 4.4 Hz, 1H), 2.47 (dd, J = 4.8, 2.8 Hz, 1H), 2.29 (t, J = 7.2 Hz, 2H), 1.39-1.69 (m, 10H), 1.20-1.39 (m, 14H), 0.84-0.93 (m, 6H).

[0110] Example 3

[0111] This example illustrates the preparation of nucleic acid-lipid nanoparticles.

[0112] 1) Method for preparing nucleic acid-lipid nanoparticle compositions

[0113] The compounds T7, DSPC, CHOL, DMG-PEG2000, etc. were accurately weighed, each lipid was dissolved in anhydrous ethanol in a suitable container for use. The lipids were mixed in the molar ratio (45:15:38.5:1.5) in the table below, as the organic phase, and the nucleic acid IL-12 mRNA was dissolved in a pH 4.0 hydrochloric acid solution as the aqueous phase. The organic phase and the aqueous phase were mixed at a volume ratio of 1:3, with a flow rate of 15 mL / min, and the lipid nanoparticle suspension was prepared on a microfluidic platform (e.g., Malvern). The obtained lipid nanoparticle suspension was centrifuged and filtered through a 100KDa ultrafiltration centrifuge tube, purified and concentrated, and the concentrated liquid was aliquoted.

[0114] 2) Preparation of lipid nanoparticle lyophilized samples

[0115] The T7 lipid nanoparticle liquid sample was prepared according to the method for preparing nucleic acid-lipid nanoparticle compositions, loaded with IL-12 mRNA, added with a protective agent, and then lyophilized and stored at -80°C. The lyophilization program included pre-freezing, annealing, primary drying, and secondary drying. Pre-freezing was at -55°C for 10-20 h; annealing was at -15°C for 2-4 h; primary drying was at -50°C for 50 h under a vacuum of 0-10 Pa; and secondary drying was at 10°C for 20 h under a vacuum of 0-10 Pa. The appearance of the lyophilized product was as shown in Figure 5 , which was a white, loose, and full cake.

[0116] Compared with the -80°C stored sample, the lyophilized product maintained good physicochemical properties, good cell activity, and reduced toxicity, and was easy to store and transport when stored at -20°C.

[0117] 3) Physicochemical properties of the prepared LNP-mRNA

[0118] The prepared lipid nanoparticles were measured for particle size, PDI, and potential using a laser nanoparticle size analyzer, and the encapsulation rate (%) was measured using a multifunctional enzyme marker combined with a RiboGreen RNA kit, with the results shown in Table 1 below.

[0119] Table 1

[0120]

[0121] The LNP-mRNA particle size was 85.76 nm, the particle size distribution (PDI) was 0.100, the potential was 27.81 mV, and the encapsulation rate was 97.20. The physicochemical properties showed that the LNP-mRNA particle size was appropriate and uniform. The encapsulation rate reflects the degree of encapsulated substances, and the higher the encapsulation rate, the more difficult it is for the encapsulated substances to be decomposed during in vivo delivery.

[0122] After the freeze-dried lipid nanoparticles were reconstituted with enzyme-free water and the -80°C stored lipid nanoparticles were thawed, the physicochemical quality control data of the liquid samples are shown in Table 2 below:

[0123] Table 2

[0124]

[0125]

[0126] As can be seen from Table 2 above, the physicochemical data of mRNA-LNP after freeze-drying meet the control standards. The particle size of the lipid nanoparticles is 60-300 nm, PDI≤0.3, Zeta potential is -45-50 mV, and the encapsulation rate is≥80%.

[0127] 4) Cell level expression detection

[0128] A375, A549, hek293, Hela cell lines were used as expression systems to transfect LNP-mRNA of different concentrations (2 μg / mL, 1 μg / mL, 0.5 μg / mL). After 24 h of culture, the cells were collected, stained with PE anti-human IL-12 / IL-23p40 (monomer, dimer, heterodimer) Antibody (Biolegend, Cat#501807), and the expression level of IL-12 protein on the cell membrane was detected by flow cytometry to determine whether LNP-mRNA could be translated into protein and anchored on the cell membrane. The IL-12 mRNA transfected with Lipofectamine MessengerMAX Reagent (Invitrogen, Cat#1168-027) was used as a positive control. The results are shown in Figure 7 .

[0129] Figure 7NC: negative control; IgG: flow cytometry negative control; IL-12 mRNA: positive control, Lipofectamine Messenger MAX Reagen transfected IL-12 mRNA; 23081001-C: 230801 formulation cryopreserved sample; 23081001-L: 23081001 formulation lyophilized sample; 2 μg / mL, 1 μg / mL, 0.5 μg / mL: sample amount of transfected mRNA.

[0130] From Figure 7 It can be seen from the above table that after T7 nanoparticle encapsulating IL-12 mRNA transfected cells, all cell lines can translate IL-12 protein which can be anchored on the cell membrane. There is still a large amount of IL-12 anchored on the cell membrane after transfection of 0.5 μg / mL LNP. The expression amount of lyophilized sample is similar to that of cryopreserved sample, and is similar to that of positive control.

[0131] 5) Cytotoxicity experiment

[0132] A375, A549, hek293, Hela cell lines were used as expression system to transfect LNP-mRNA of different concentrations (C represents cryopreserved sample, L represents lyophilized sample). After 48 h of culture, cells were collected, and CTG method was used to detect cell viability to evaluate the cytotoxicity of LNP-mRNA. The mRNA transfected by Lipofectamine Messenger MAX Reagent (Invitrogen, Cat# 1168-027) was used as positive control. The results are shown in Figure 8 .

[0133] Figure 8 IL-12 mRNA: positive control sample, Lipofectamine Messenger MAX Reagen transfected IL-12 mRNA; 23081001-C: 230801 formulation cryopreserved sample; 23081001-L: 23081001 formulation lyophilized sample; STS: CTG detection positive control; IC50: half inhibitory concentration.

[0134] From Figure 8 It can be seen from the above table that after T7 nanoparticle encapsulating IL-12 mRNA transfected cells, all cell lines can translate IL-12 protein which can be anchored on the cell membrane. There is still a large amount of IL-12 anchored on the cell membrane after transfection of 0.5 μg / mL LNP. The expression amount of lyophilized sample is similar to that of cryopreserved sample, and is similar to that of positive control.

[0135] Finally, it needs to be explained that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can still be modified, or some technical features can be replaced by equivalent ones. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A nucleic acid molecule delivery vehicle, characterized in that, A nucleic acid molecule encoding interleukin 12 and an ionizable lipid compound.

2. The nucleic acid molecule delivery vehicle of claim 1, wherein, Further comprising a phospholipid, a sterol and a PEG-lipid.

3. The nucleic acid molecule delivery vehicle of claim 1 or 2, wherein, The nucleic acid molecule comprises an open reading frame nucleic acid sequence encoding an IL-12 p40 subunit and an IL-12 p35 subunit; Preferably, the nucleic acid molecule further comprises a nucleic acid sequence encoding a transmembrane region; Preferably, the nucleic acid molecule further comprises a nucleic acid sequence encoding a signal peptide, a 5' UTR, a 3' UTR and a poly-A tail.

4. The nucleic acid molecule delivery vehicle of claim 3, wherein, The nucleic acid molecule has a nucleotide sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or about 100% homologous to the nucleotide sequence shown in SEQ ID NO: 1, preferably the sequence shown in SEQ ID NO:

1.

5. The nucleic acid molecule delivery vehicle of claim 1 or 2, wherein, The ionizable lipid compound has the structure shown in the following formula: wherein: R1is hydrogen, substituted or unsubstituted linear or branched C1-20alkyl, substituted or unsubstituted linear or branched C2-20alkenyl, substituted or unsubstituted linear or branched C2-20alkynyl, said substituted substituent is selected from the group consisting of halogen, -OH, =0, linear or branched C1-10alkyl, linear or branched C1-10alkoxy; all of the above alkyl, alkenyl, alkynyl or alkoxy groups, one or more than one C atom in the carbon chain of which can optionally be replaced by a heteroatom independently selected from the group consisting of O, S and N; n is a positive integer selected from 1-10; m is a positive integer selected from 1-10; n and m can be the same or different; preferably, n is a positive integer selected from 1-6, m is a positive integer selected from 1-6; Each R2 is independently selected from hydrogen. The substituted or unsubstituted straight-chain or branched C1-30 alkyl group, the substituted or unsubstituted straight-chain or branched C2-30 alkenyl group, and the substituted or unsubstituted straight-chain or branched C2-30 alkynyl group are selected from halogens, -OH, =O, straight-chain or branched C1-10 alkyl groups, straight-chain or branched C1-10 alkoxy groups, and straight-chain or branched C2-20 alkenyl groups. In all of the above alkyl, alkenyl, alkynyl, or alkoxy groups, one or more carbon atoms in the carbon chain may optionally be replaced by heteroatoms independently selected from O, S, and N. provided that at least one R2is o is independently selected from a positive integer from 1-20; R' is selected from a substituted or unsubstituted straight chain or branched C1-30 alkyl, a substituted or unsubstituted straight chain or branched C2-30 alkenyl, a substituted or unsubstituted straight chain or branched C2-30 alkynyl; the substituent of the substitution is selected from =O, halogen, -OH, straight chain or branched C1-20 alkyl, straight chain or branched C1-20 alkoxy, straight chain or branched C2-20 alkenyl; one or more C atoms in the carbon chain of all the above alkyl, alkenyl, alkynyl or alkoxy can be optionally replaced by a heteroatom independently selected from O, S and N; When there are at least two R2 values At the same time, they can be the same or different.

6. The nucleic acid molecule delivery vehicle of claim 5, wherein, The ionizable lipid compound has the structure shown in the following formula:

7. Vaccine or pharmaceutical composition, characterized in that, A nucleic acid molecule delivery vehicle comprising an immunologically effective amount of the nucleic acid molecule according to any one of claims 1-6.

8. A method for preparing the nucleic acid molecule delivery carrier and the freeze-dried preparation thereof according to any one of claims 1 to 6, characterized by, Comprising: i. a step of synthesizing a nucleic acid molecule capable of expressing interleukin 12 and preparing an aqueous phase; ii. a step of preparing an organic phase containing an ionizable lipid compound and mixing the organic phase with the aqueous phase; iii. a step of lyophilizing a preparation protective agent and a lyophilization program thereof.

9. Use of the nucleic acid molecule delivery vehicle according to any one of claims 1-6 for the manufacture of a vaccine or a medicament for preventing, treating or ameliorating a disease.

10. Use of the nucleic acid molecule delivery vehicle according to any one of claims 1-6 in combination with other vaccines or medicaments.

Images