Precision expression compositions for the spleen, vaccine compositions, and uses thereof

Abstract

The present application relates to the technical field of spleen delivery, and specifically discloses a spleen precise expression composition, a vaccine composition and application thereof. The spleen precise expression composition comprises: component (1): at least one nucleic acid comprising a nucleotide sequence encoding a target protein and one or more miRNA targeting binding sequences; component (2): a lipid nanoparticle composition which is easy to accumulate in the spleen. The spleen precise expression composition can be used for preparing a lipid nanoparticle or a drug or vaccine composition, and can be efficiently delivered into the spleen and its tissues and cells, and significantly reduces the expression of target genes in non-spleen organs, avoiding side effects such as inflammatory reactions or other off-target effects caused by undesired expression.

Description

Technical Field

[0001] This application relates to the technical field of drug delivery in immune tissues, organs or cells, and further to the technical field of drug delivery in organs such as the spleen or lymph nodes, and further to spleen-specific expression compositions, vaccine compositions and their applications. Background Technology

[0002] Lipid nanoparticles (LNPs) are a mainstream technology for mRNA vaccine and drug delivery. Ionizable cationic lipids play a crucial role in organ targeting. Most LNP vector systems used for mRNA delivery, including those used in Pfizer and Moderna's marketed COVID-19 vaccines, exhibit liver and spleen enrichment. However, their lack of sufficient expression precision limits the application scenarios of mRNA-LNP technology. For example, target genes or proteins intended for liver expression—such as enzymes or proteins naturally expressed only in the liver, or gene editors or engineered proteins intended to function only in the liver—may simultaneously exhibit significant expression levels in immune organs such as the spleen and lymph nodes, or in immune cells of the liver, leading to side effects such as inflammatory responses. Similarly, antigens or target proteins intended for expression in immune tissues, organs such as the spleen or lymph nodes, or immune cells may be expressed ectopically in the liver due to LNP leakage through blood vessels, resulting in rare hepatitis, such as some rare hepatitis caused by the leakage expression of mRNA COVID-19 vaccines in the liver. Currently, existing mRNA-LNPs are mainly enriched in the liver and spleen after administration, especially the liver. When it is necessary to specifically deliver drugs to the spleen or immune cells, the enrichment expression in the liver is undesirable.

[0003] There are several possible technical approaches to enhance selective targeting. For example, altering the composition, charge, structure, and / or size of ionizable cationic lipids can affect organ and tissue targeting. However, due to the current lack of understanding of the structure-activity relationship of ionizable lipids, LNP delivery systems based solely on chemical design and CADD (Carbon Diet Determination) struggle to achieve the selective delivery and expression of drugs in the liver or immune tissues, organs, and cells (e.g., the spleen). Therefore, technologies that can improve the selective delivery and expression of target proteins in organs, tissues, and cells via LNPs have significant application value.

[0004] Current techniques, building upon organ-enriched LNPs in the spleen and immune cells, further reduce off-target expression caused by ectopic expression in the liver by inserting microRNA-122 or other liver-enriched microRNA (miRNA or miR) target site sequences before and after the coding region of the mRNA, including the UTR region. This can further reduce the expression of target proteins in the liver and improve the spleen / liver ratio of protein expression levels compared to simply using organ-enriched LNPs. For example, inserting miR-122ts between the ORF and UTR can achieve selective shutdown of expression in the liver (Simplified Lipid Nanoparticles for Tissue-And Cell-Targeted mRNA Delivery Facilitate Precision Tumor Therapy in a LungMetastasis Mouse Model. Advanced Materials, 2024). For example, Moderna uses miRts inserted into the 3' UTR to shut down off-target expression in specific organs such as the liver or spleen (Jain et al, MicroRNAs Enable mRNA Therapeutics to Selectively Program Cancer Cells to Self-Destruct[J] Nucleic Acid Therapeutics, 2018). However, inserting miRt sequences into the 5' UTR, protein-coding region ORF, or 3' UTR, which play a crucial role in expression and have stricter sequence structure requirements, may interfere with the expression of target proteins in target organs such as the spleen. Therefore, designing novel, universal switching elements is of great value.

[0005] How to achieve efficient and precise delivery of the spleen remains a hot research topic. Summary of the Invention

[0006] This application provides a spleen-specific expression composition, a vaccine composition, and their applications. The spleen-specific expression composition of this application can be efficiently delivered to the spleen and its tissues and cells, and significantly reduce the expression of target genes in non-splenic organs, avoiding side effects such as inflammatory reactions or other off-target effects caused by undesirable expression of target proteins.

[0007] This application involves the following: 1. A spleen-specific expression composition, the expression composition comprising: Component (1): at least one nucleic acid comprising a nucleotide sequence encoding a target protein and one or more miRNA targeting sequences, wherein the miRNA bound by the miRNA targeting sequence is poorly enriched in the spleen and highly enriched in non-splenic tissues, organs and / or cells; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; In this composition, component (1) is partially or completely encapsulated in the lipid nanoparticle composition of component (2).

[0008] 2. The spleen-specific expression composition according to item 1, wherein the nucleic acid is mRNA.

[0009] 3. The spleen-specific expression composition according to claim 2, wherein the mRNA comprises a polyadenine tail (polyA), and the one or more miRNA targeting sequences are the same or different, wherein the one or more miRNA targeting sequences are inserted outside the nucleotide sequence encoding the target protein, optionally before or within the polyA.

[0010] 4. The spleen-specific expression composition according to item 3, wherein the polyA with one or more miRNA targeting sequences inserted comprises an nA-miRNA targeting sequence-mA structure, wherein: nA represents the n consecutive adenosine nucleotides (A) adjacent to the 5' end of the miRNA targeting sequence. mA represents the m consecutive adenosine nucleotides (A) adjacent to the 3' end of the miRNA targeting sequence. m and n are each independent natural numbers, and n = 0 or n ≥ 1. Choose any of the following: n≤60, n≤30, n≤19, n≤14, or n≤10. Preferably, 14≤n≤30, 19≤n≤30, and 14≤n≤19.

[0011] 5. The spleen-specific expression composition according to item 3 or 4, wherein the polyA containing one or more miRNA targeting sequences comprises an nA-miRNA targeting sequence-mA structure, wherein: m+n≤150, m+n≤120, m+n≤100, m+n≤80, m+n≤60, m+n≤30, m+n≤20, or m+n≤10; Preferably, m+n is any natural number between 150 and 10, including the endpoints.

[0012] 6. The spleen-specific expression composition according to any one of items 3-5, wherein the plurality of miRNA target-binding sequences are directly linked or linked by one or more nucleotides, optionally, the nucleotides being G.

[0013] 7. The spleen-specific expression composition according to any one of items 3-6, wherein the polyA further comprises a GATATC sequence.

[0014] 8. The spleen-specific expression composition according to any one of items 3-7, wherein the polyA comprises the structure of the nA-miRNA targeting binding sequence -mA-GATATC-19A-G-19A-G-17A.

[0015] 9. The spleen-specific expression composition according to any one of items 1-8, wherein the miRNA targeting sequence targets and binds to miR-122 or its seed sequence.

[0016] 10. The spleen-specific expression composition according to any one of items 1-9, wherein the miRNA targeting binding sequence comprises or is a nucleotide sequence as shown in SEQ ID NO.7, or comprises or is a nucleotide sequence having 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 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.7.

[0017] 11. The spleen-specific expression composition according to any one of items 3-10, wherein the polyA or a variant thereof comprises or is a nucleotide sequence shown in any one of SEQ ID NO. 8-39, or comprises or is a nucleotide sequence having 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 100% sequence identity with the sequence shown in any one of SEQ ID NO. 6, 8-39.

[0018] 12. The spleen-specific expression composition according to any one of items 2-11, wherein the mRNA comprises a 5' untranslated region (5'UTR) and / or a 3' untranslated region (3'UTR); Optionally, the 5' uncoding region (5'UTR) contains or is the nucleotide sequence shown in SEQ ID NO.2, or contains or is a nucleotide sequence having 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 100% sequence identity with the sequence shown in SEQ ID NO.2; Optionally, the 3' uncoding region (3'UTR) contains or is the nucleotide sequence shown in SEQ ID NO.4, or contains or is a nucleotide sequence having 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 100% sequence identity with the sequence shown in SEQ ID NO.4.

[0019] 13. The spleen-specific expression composition according to any one of items 2-12, wherein some or all of the uridine in the mRNA molecule is a chemically modified uridine; Preferably, the uridine is a pseudouridine. Optionally, the uridine is N1-methylpseudouridine.

[0020] 14. The spleen-specific expression composition according to any one of items 2-13, wherein the mRNA molecule further comprises a 5' cap structure; Preferably, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

[0021] 15. The spleen-specific expression composition according to any one of items 1-14, wherein the lipid nanoparticle composition comprises ionizable lipids, phospholipids, sterols, and polymer-conjugated lipids; Preferably, the ionizable lipid is selected from compounds of formula IIA, or pharmaceutically acceptable salts thereof, or stereoisomers thereof.

[0022] R 1a and R 1b Independently selected from -CH3; R2 and R3 are independently selected from H; R4 is selected from C8, C9, C10, C11 or C12 straight-chain alkyl groups; R5 and R6 are independently selected from C6, C7 and C8 straight-chain alkyl groups; When X1 and Y1 are different, and X2 and Y2 are different, X1 and X2 are independently selected from C=O or O; Y1 and Y2 are independently selected from C=O or O; o and p are independently selected from 5, 6, 7, and 8; q is selected from 2, 3, or 4; Preferably, R4 is selected from C10 or C11 straight-chain alkyl groups; Preferably, R5 and R6 are independently selected from C6 and C8 straight-chain alkyl groups; more preferably, R5 and R6 are both C8 straight-chain alkyl groups or R5 is a C6 straight-chain alkyl group and R6 is a C8 straight-chain alkyl group. Preferably, q is 3; Preferably, q is 4; Preferably, X1 and X2 are both C=O, and Y1 and Y2 are both O; Preferably, X1 is 0, Y1 is C=0, and X2 is C=0 and Y2 is 0; Preferably, X1 is C=O, Y1 is O, and X2 is O and Y2 is C=O; Preferably, X1 and X2 are both 0, and Y1 and Y2 are both C=0; More preferably, the ionizable lipid is selected from any one or more of the following compounds: , , , , or .

[0023] 16. The spleen-specific expression composition according to item 15, wherein the sterol is selected from one or more of the group consisting of: cholesterol, cholesterol hemisuccinate and its derivatives; The preferred sterol is cholesterol; And / or, the phospholipid is selected from one or more of the group consisting of: 1,2-disorcinol-sn-glycerol-3-phosphate ethanolamine (DEPE), 1,2-dicholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (DChemsPC), 1,2-distearyl-sn-glycerol-3-phosphatidylcholine or 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 16-O-monomethyl Ethanolamine phosphate, 16-O-dimethylphosphatidylethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine (DLoPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonium)ethyl hydrogen phosphate (DOCP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonium)ethyl ethyl phosphate (DOCPe), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC) 1,2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS) and its sodium salt, 1,2-distearate-sn-glycerol-3-phosphate-L-serine and its sodium salt, 1,2-diphydanoyl-sn-glycerol-3-phosphate-L-serine and its sodium salt, distearate Acylphosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (DPhyPE), 1,2-diphydanyl-sn-glycerol-3-phosphate choline (DPhyPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-squalene-sn-glycerol-3-phosphate ethanolamine (DSQPE), 1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), distearyl-phosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 18-1-trans-phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (SLPE), 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine and its sodium salt, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (POPS) and its sodium salt, 1-1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine and its sodium salt, 1-O-hexadecyl-2-O-(9Z-octadecenyl)- sn-glycerol-3-phosphate ethanolamine, 1,2-di-O-phytyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (PChemsPC), 1-O-octadecyl-2-O-methyl-sn-glycerol-3-phosphate choline (edifuxin), palmitoyloleylphosphatidylcholine (POPC), and palmitoyloleyl-phosphatidylethanolamine (POPE); Preferably, the phospholipid is DSPC; And / or, the polymer conjugated lipid is a PEG-modified lipid; Preferably, the polymeric conjugated lipid is selected from one or more of the following groups: PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG, Chol-PEG, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), polyethylene glycolated phosphatidylethanolamine (PEG-PE), 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyethylene glycolated ceramide (PEG-cer), and ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate; More preferably, the polymer conjugated lipid is DMG-PEG2000.

[0024] 17. The spleen-specific expression composition according to item 15 or 16, wherein the lipid nanoparticle composition comprises, in which the molar percentage of ionizable lipids is 40-55 mol%, the molar percentage of sterols is 30-50 mol%, the molar percentage of phospholipids is 5-15 mol%, and the molar percentage of polymer-conjugated lipids is 1-3 mol%. Preferably, the molar percentage of the ionizable lipid is 48-50 mol%, the molar percentage of the sterol is 35 mol%-45 mol%, the molar percentage of the phospholipid is 10 mol%-15 mol%, and the molar percentage of the polymer conjugated lipid is 1.5 mol%-2.5 mol.

[0025] 18. The spleen-specific expression composition according to any one of items 1-17, wherein the target protein is a therapeutic protein or antigen; Optionally, the therapeutic protein is selected from one or more of the group consisting of: antibodies, fusion proteins, enzymes, membrane proteins, receptors, ligands, and toxins.

[0026] 19. A method for preventing or treating a disease, comprising administering a therapeutically effective amount of the spleen-precisely expressed composition according to any one of items 1-18 to a subject; Optionally, the disease is selected from one or more of the following groups: spleen diseases, systemic diseases related to immune cell activation or other organ diseases, diseases that require shutting down or reducing the expression level of target proteins in liver tissues and cells to avoid side effects caused by off-target expression (such as diseases with inflammatory responses, such as rare hepatitis caused by small amounts of leakage expression of COVID-19 vaccines in liver tissues). The systemic diseases or other organ diseases related to immune cell activation are selected from one or more of the following groups: cancer (e.g., melanoma, breast cancer, colorectal cancer, lymphoma, leukemia, multiple myeloma, etc.), infectious diseases (e.g., bacterial infections and viral infections, etc.), autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, autoimmune encephalomyelitis, etc.), and inflammatory diseases (e.g., inflammatory bowel disease, atherosclerosis, ischemic stroke, liver fibrosis, kidney fibrosis).

[0027] 20. The spleen disease described in accordance with item 19 is spleen inflammation.

[0028] 21. A pharmaceutical composition comprising the spleen-precise expression composition as described in any one of claims 1-18.

[0029] 22. The pharmaceutical composition according to item 21 is a vaccine composition.

[0030] Invention Effects 1. This application designs a nucleic acid, which can be mRNA, containing one or more miRNA targeting sequences and a polyadenine tail (polyA), with the one or more miRNA targeting sequences inserted before or within the polyA. The miRNA bound by the miRNA targeting sequence is poorly enriched in the spleen but highly enriched in non-splenic organs. This miRNA targeting sequence binds to the miRNA highly enriched in non-splenic organs, cells, or tissues, thereby mediating the degradation of the nucleic acid encoding the target protein in non-splenic organs, cells, or tissues, thus reducing the expression of the target protein in non-splenic organs without affecting the normal expression of the target protein in the spleen. The therapeutic effect is achieved using this method.

[0031] 2. The nucleic acid in this application is partially or completely encapsulated in a lipid nanoparticle composition that is easily enriched in the spleen, so as to facilitate its delivery to the target organ (spleen). This composition can reduce the expression of the target protein in non-splenic organs without affecting the expression of the target protein in the spleen, thereby ultimately resulting in an increased relative expression ratio of the target protein in the spleen and non-splenic organs, achieving precise expression in the spleen, thereby reducing toxic side effects and off-target effects caused by undesirable expression, and increasing drug-likeness.

[0032] 3. This application utilizes the polyA of the inserted miRNAts (miRNA target binding sequence) to selectively shut down the expression of target genes in off-target organs, tissues, and cells. Compared with inserting the miRNA (target site) target site sequence into the UTR region and / or coding region, the insertion site of this application is far away from the UTR region and coding region that may affect the expression level and efficiency, which can reduce the probability of interfering with the expression of target genes.

[0033] 4. This application further screens suitable lipid nanoparticle compositions (especially suitable ionizable lipids) to achieve better precise expression in the spleen. Attached Figure Description

[0034] Figure 1 Expression of luciferase in the spleen of mice after administration of different mRNA-lipid nanoparticles.

[0035] Figure 2 The expression of luciferase in the liver of mice after administration of different mRNA-lipid nanoparticles.

[0036] Figure 3 The relative proportions of luciferase expression in the spleen / liver after administration of different mRNA-lipid nanoparticles in mice. Detailed Implementation

[0037] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in their functions.

[0038] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims. Unless otherwise defined, 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 disclosure pertains.

[0039] As used herein and in the appended claims, the singular forms “a / an” and “the” include the plural objects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as “only” or “merely” in conjunction with the description of the elements of the claim, or for the use of the limitation of “no”.

[0040] As used herein, the term "about XY" has the same meaning as "about X to about Y". References to a value or parameter of "about" herein include (and describe) variations for that value or parameter itself. For example, a description of "about X" includes a description of "X". The term "about" may be used where the parameter or value does not necessarily need to be identical (e.g., 100% identical). Thus, "about" means that a parameter or value may deviate from 0.1% to 20%, preferably from 0.1% to 10%; for example, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. Within this range, "about" has the meaning as used in this application. Furthermore, in some embodiments, for example, if a parameter or value is defined herein as having a length of, for example, "about 5000 nucleotides," then that length may deviate from 0.1% to 20%, preferably from 0.1% to 10%; for example, deviations of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. That is, in some embodiments of this application, the length of the definition may deviate from 5 to 1000 nucleotides, preferably 5 to 500 nucleotides; for example, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 nucleotides.

[0041] As used herein, references to “not” values ​​or parameters generally refer to and describe “except” values ​​or parameters. For example, “The method is not used to treat type X cancer” means that the method is used to treat cancers other than type X.

[0042] As used herein, the term "and / or" in words such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in words such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0043] It should be understood that the embodiments of this application described herein include embodiments “composed of” and / or “substantially composed of”. In this application, the term “selected from the group consisting of” followed by a group of elements (e.g., “A, B, and C”) means that this application is not limited to the specifically listed group. That is, such terminology does not indicate that this disclosure excludes unlisted elements; alternative meanings are also included within the group following the term. For example, “selected from the group consisting of A, B, and C” should be understood as “selected from A, B, and C” or alternatively, “A, B, or C,” and also includes other structurally and functionally relevant and unrelated elements that are not mentioned.

[0044] Nucleic acid The terms “nucleic acid,” “polynucleotide,” and “nucleotide sequence” are used interchangeably and are not particularly limited in terms of the number of bases. They refer to a polymer of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogues. Whenever this document refers to a nucleic acid or nucleic acid sequence encoding a specific protein and / or peptide, the nucleic acid or nucleic acid sequence preferably also includes regulatory sequences to allow its expression in a suitable host (e.g., in humans), i.e., transcription and / or translation of the nucleic acid sequence encoding the specific protein or peptide.

[0045] In some preferred embodiments of this application, "nucleic acid" refers to "artificial mRNA" or "isolated mRNA." Here, "artificial mRNA" (sequence) is generally understood to be a non-naturally occurring mRNA molecule. In other words, an artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such an mRNA molecule may be non-natural due to its unique sequence (which is not naturally occurring) and / or due to other non-natural modifications (e.g., structural modifications of nucleotides, such as uridine being partially or entirely chemically modified uridine). Typically, artificial mRNA molecules can be designed and / or generated using genetic engineering methods to correspond to a desired artificial sequence (heterologous sequence) of nucleotides. In this context, an artificial sequence is generally a sequence that may not be naturally occurring, i.e., it differs from the wild-type sequence by at least one (e.g., one, two, three, four, five, ten, or more) bases. The term "wild-type" can be understood as a sequence that exists in nature. Furthermore, the term "artificial nucleic acid molecule" is not limited to referring merely to "a single molecule" in terms of quantity, but is generally understood to be a collection of molecules containing the same sequence. The term "isolated" refers to nucleic acid molecules (preferably isolated mRNA) or polypeptides that have been exposed to conditions different from their natural environment, such as those separated from blood and / or animal tissue. In some embodiments, the nucleic acid molecules (preferably isolated mRNA) or polypeptides may be in a highly purified form, i.e., greater than 95% purity or greater than 99% purity. The term "isolated" does not exclude the presence of the same nucleic acid molecules or polypeptides in alternative physical forms, such as dimers or alternative phosphorylated or derivatized forms. In some preferred embodiments of this application, the artificial nucleic acid, the nucleic acid is mRNA, and more specifically, isolated mRNA.

[0046] As used herein, the term "wildtype" has the meaning commonly understood by those skilled in the art as referring to the typical form of an organism, strain, gene, or trait that distinguishes it from mutants or variants when it exists in nature. It can be isolated from resources in nature and is not deliberately modified.

[0047] As used herein, the terms “non-naturally occurring,” “engineered,” and “engineered modified” are used interchangeably to refer to artificial intervention. When these terms are used to describe nucleic acid molecules or peptides, they mean that the nucleic acid molecule or peptide is at least substantially free of at least one other component that is naturally associated with or naturally present in it.

[0048] As used herein, the term "miRNA-targeting binding sequence (miRNAts)" refers to a sequence capable of binding to a specific miRNA and mediating the degradation of nucleic acid encoding a target protein in a non-target organ without affecting the expression of the target protein in the target organ. Specifically, the specific miRNA is highly enriched in non-target organs but poorly enriched in target organs. By enabling the "miRNA-targeting binding sequence" to bind to the specific miRNA in non-target organs, the expression of the target protein in non-target organs is significantly reduced, while maintaining normal or increased expression of the target protein in the target organ, thereby achieving the therapeutic objective. In some embodiments, the specific miRNA is miR-122, the target organ is the spleen, and the non-target organ is another organ other than the spleen that highly expresses miR-122, such as the liver. The target protein is any therapeutic protein present in the target organ (e.g., the spleen). miRNAs that are poorly enriched in the spleen or immune tissues, organs, or specific immune cells and highly enriched in non-immune tissues, organs, and / or specific cells can be, for example, any one or more of the following: miR-122, miR-142, miR-126, miR-148a, miR-133, miR-206, miR-208, miR-17-92, miR-16, miR-21, miR-223, miR-24, miR-27, let-7, miR-30c, miR-1d, miR-149, miR-192, miR-194, and miR-204.

[0049] The term "target cell" refers to any one or more target cells. These cells can be found in vitro, in vivo, in situ, or in the tissues or organs of an organism. The term "target organ" refers to any one or more target organs.

[0050] As used herein, the terms "PolyA," "PolyA tail," or "PolyA sequence" refer to a sequence of adenosine residues, either uninterrupted or interrupted, or inserted with non-A bases or foreign sequences, typically located at the 3' end of an RNA molecule. In RNA, the PolyA sequence is attached to the 3' end of the 3' UTR in the presence of the 3' UTR. An uninterrupted PolyA tail is characterized by a continuous sequence of adenosine residues. The PolyA tail can be of any length. In some embodiments, the PolyA tail comprises, or consists of, at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 adenosine (A) residues, particularly about 120 A residues. Typically, the vast majority of nucleotides in the PolyA tail are adenosine, meaning at least 75%, 80%, 85%, or 90% of the nucleotides, etc., but the remaining nucleotides are allowed to be nucleotides other than A (non-A nucleotides), such as U (uridine monophosphate), G (guanine monophosphate), or C (cytidine monophosphate) or their modified nucleotides. In this application, adenine (A), uracil (U), cytosine (C), and guanine (G) are all nitrogenous bases.

[0051] In some embodiments, the mRNA comprises a polyadenine tail (polyA), and the one or more miRNA targeting sequences are inserted before or within the polyA, and the one or more miRNA targeting sequences may be the same or different. For example, in some embodiments, the mRNA comprises a polyadenine tail (polyA), and the one or more miRNA targeting sequences are inserted before the polyA. For example, in some embodiments, the mRNA comprises a polyadenine tail (polyA), and the one or more miRNA targeting sequences are inserted within the polyA. In some embodiments, the plurality of miRNA targeting sequences are the same; in some embodiments, the plurality of miRNA targeting sequences are completely different; in some embodiments, the plurality of miRNA targeting sequences are partially the same. "Partially the same" means that at least two miRNA targeting sequences are the same or at least two miRNA targeting sequences are different.

[0052] It should be noted that, in this application, the miRs bound to polyA refer to miR target-binding sequences (i.e., miRNAs). For example, in the "polyA-miR-122 recombinant plasmid" obtained in the specific embodiment, "miR-122" represents the miR-122 target-binding sequence.

[0053] In some embodiments, the polyA containing one or more miRNA targeting sequences comprises nA-miRNA targeting sequence-mA; wherein: nA represents the n consecutive adenosine nucleotides (A) adjacent to the 5' end of the miRNA targeting sequence. mA represents the m consecutive adenosine nucleotides (A) adjacent to the 3' end of the miRNA targeting sequence. m and n are each independent natural numbers, and n = 0 or n ≥ 1.

[0054] In some embodiments, n is 0, meaning the one or more miRNA targeting sequences are inserted before the polyA. In some embodiments, when n is 0, a miRNA targeting sequence is inserted before the polyA; the miRNA targeting sequence is a miR-122 targeting sequence. In some preferred embodiments, the miR-122 targeting sequence is as shown in SEQ ID NO. 7, and the sequence of the polyA is as shown in SEQ ID NO. 6. In some embodiments, the sequence of the polyA variant obtained after inserting a miRNA targeting sequence before the polyA is as shown in SEQ ID NO. 39. In some embodiments, n ≥ 1, meaning the one or more miRNA targeting sequences are inserted into the polyA. In some embodiments, when n ≥ 1, a miRNA targeting sequence is inserted into the polyA; the miRNA targeting sequence is a miR-122 targeting sequence. In some preferred embodiments, the miR-122 targeting sequence is as shown in SEQ ID NO. 7, and the sequence of the polyA is as shown in SEQ ID NO. 6. In some implementations, the polyA variant obtained by inserting a miRNA targeting binding sequence into the polyA has sequences as shown in any one or more of SEQ ID NO. 8-38.

[0055] In some implementations, n is any range within the range of 1, 5, 8, 10, 14, 19, 25, or n ≥ 1. In some implementations, n can be any range within the optional range of 60, 30, 19, 14, 10, or 60; for example, n can be arbitrarily selected as 80, 75, 70, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some preferred embodiments, 5≤n≤60, 8≤n≤30, 10≤n≤19, 8≤n≤19, 14≤n≤30, 19≤n≤30, or 14≤n≤19, or any range from 1 to 60. In some preferred embodiments, 14≤n≤30, 19≤n≤30, 14≤n≤19, or any range from 14≤n≤30; for example, n can be arbitrarily chosen as 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 14.

[0056] In some embodiments, the polyA containing one or more miRNA targeting sequences comprises an nA-miRNA targeting sequence-mA structure; wherein, optionally, m+n≤150, m+n≤120, m+n≤100, m+n≤80, m+n≤60, m+n≤30, m+n≤19, or m+n≤14, or m+n is any range ≤150. In some implementations, m+n ≤ 150, ≤ 120, ≤ 100, ≤ 80, ≤ 60, ≤ 30, ≤ 19, or ≤ 14; in a specific embodiment, m+n is optionally 150, 147, 145, 142, 140, 138, 135, 133, 130, 128, 125, 124, 122, 120, 118, 116, 115, 113, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 8 1, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 21, 20, 19, 18, 17, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or any natural number less than or equal to 150. In some preferred embodiments, m + n = 60. In some implementations, the 3' and 5' ends of the miRNA binding site do not contain any A other than the A contained in the miRNA targeting binding sequence itself.

[0057] In some embodiments, the plurality of miRNA target-binding sequences are directly linked or linked via one or more nucleotides. Optionally, the nucleotide is G.

[0058] In some implementations, the polyA is further followed by a GATATC sequence.

[0059] In some embodiments, the polyA comprises the structure of the nA-miRNA targeting binding sequence -mA-GATATC-19A-G-19A-G-17A. As used herein, "19A" means 19 consecutive adenosine nucleotides (A); "17A" means 17 consecutive adenosine nucleotides (A).

[0060] As used herein, the two elements connected by "-" (e.g., between nA and the miRNA target-binding sequence, or between the miRNA target-binding sequence and mA) are directly linked. "Directly linked" means that no nucleotides are contained between the two elements, and therefore, the "direct link" can be established by any permissible nucleotide linkage. In some embodiments, the "direct link" refers to a chemical bond. In some embodiments, the "direct link" refers to a phosphate ester bond.

[0061] In some embodiments, the miRNA targeting sequence targets and binds to miR-122 or its seed sequence. miRNA-mediated gene transcriptional degradation and gene silencing are important mechanisms for the epigenetic regulation of biological traits. miRNAs inhibit transcription by complementary pairing with specific sites on mRNA, thereby causing splicing, deadenylation, and degradation of target genes. The term "seed sequence," also known as the "seed region," is defined as the 2nd to 8th nucleotides from the 5' end of the miRNA; the seed sequence is the most evolutionarily conserved region on the miRNA and is typically perfectly complementary to the target site on the 3'UTR of the mRNA. In this application, the seed sequence can be the 2nd to 8th nucleotides from the 5' end of miR-122, which, upon binding to the miRNA targeting sequence, mediates the degradation of target gene transcripts and gene silencing, significantly inhibiting the expression of the target protein.

[0062] In some embodiments, the miRNA-122 targeting sequence comprises or is a nucleotide sequence as shown in SEQ ID NO.7, or comprises or is a nucleotide sequence having 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 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.7; for example, it comprises or is a nucleotide sequence having at least 80%, 82%, 84%, 85%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO.7.

[0063] The term "variant" refers to a nucleic acid sequence variant, that is, a nucleic acid sequence or gene that contains a nucleic acid sequence that differs from the reference (or "parental") nucleic acid sequence due to at least one nucleic acid. Therefore, variant nucleic acids or genes may preferably contain at least one mutation, substitution, insertion, or deletion in their nucleic acid sequences compared to their respective reference sequences. Preferably, the term "variant," as used herein, includes naturally occurring variants and engineered variants of nucleic acid sequences or genes. Thus, a "variant" as defined herein may be derived from a reference nucleic acid sequence, isolated from a reference nucleic acid sequence, associated with a reference nucleic acid sequence, based on a reference nucleic acid sequence, or homologous to a reference nucleic acid sequence. A "variant" may preferably have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with its respective naturally occurring (wild-type) nucleic acid sequence or gene, or its homolog, fragment, or derivative. The term "functional variant" refers to a variant that has a similar or identical function to its respective naturally occurring (wild-type) nucleic acid sequence or gene, or its homolog, fragment, or derivative.

[0064] For nucleic acid sequences, the "sequence identity percentage (%)" is defined as the percentage of nucleotides in a candidate sequence that are identical to nucleotides in a specific nucleic acid sequence after sequence alignment (if necessary) to achieve the maximum sequence identity percentage, allowing gaps (gaps). For peptide, polypeptide, or protein sequences, the "sequence identity percentage (%)" is the percentage of amino acid residues in a candidate sequence that are identically substituted to amino acid residues in a specific peptide or amino acid sequence after sequence alignment (if necessary) to achieve the maximum sequence homology percentage. For the purpose of determining the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0065] In some embodiments, the polyA or its variants comprise or are the nucleotide sequences shown in any one of SEQ ID NO. 6, 8-39, or A nucleotide sequence that contains or is identical to the sequence shown in any one of SEQ ID NO. 6, 8-39 with a sequence identity of 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 100%; for example, a nucleotide sequence that contains or is identical to the sequence shown in any one of SEQ ID NO. 8-39 with a sequence identity of at least 80%, 82%, 84%, 85%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0066] In some embodiments, the length of the polyadenine tail (polyA) is 80 to 240 nt, such as 100 to 200 nt, 101 to 150 nt, 120 to 150 nt, 130 to 140 nt, 123 to 135 nt, 125 to 139 nt, or any range within the range of 80 to 240 nt. In some specific embodiments, the length of the polyadenine tail (polyA) is 80nt, 82nt, 84nt, 85nt, 87nt, 90nt, 91nt, 93nt, 95nt, 97nt, 99nt, 100nt, 102nt, 104nt, 105nt, 108nt, 110nt, 112nt, 115nt, 117nt, 119nt, 121nt, 124nt, 125nt, 126nt, 128nt, 130nt, 133nt, 135nt, 137nt, 139nt, 140nt, 141nt, 143nt, 145nt, 147nt, 149nt, 150nt, 162nt. t, 164nt, 165nt, 168nt, 170nt, 172nt, 175nt, 177nt, 179nt, 181nt, 184nt, 185nt, 186nt, 188nt, 190nt, 193nt, 195nt, 197nt, 199nt, 200nt, 202nt, 204nt, 205nt, 208nt, 210nt, 212nt, 215nt, 217nt, 219nt, 221nt, 224nt, 225nt, 226nt, 228nt, 230nt, 233nt, 235nt, 237nt, 239nt, 240nt, or any length in the range of 80 to 240nt.

[0067] In some embodiments, the mRNA comprises a polyadenine tail (polyA) or a functional variant thereof and a miRNA targeting binding sequence. In some embodiments, the mRNA further comprises a 5' untranslated region (5'UTR) and / or a 3' untranslated region (3'UTR). In some embodiments, the mRNA further comprises an optional Kozak sequence and a coding region ORF. In some embodiments, the mRNA comprises, in sequence, a 5' untranslated region (5'UTR), an optional Kozak sequence, a coding region ORF, a 3' untranslated region (3'UTR), and a polyadenine tail (polyA).

[0068] 3' Uncoding Region (3'UTR): The 3'UTR is typically part of the mRNA, located between the protein-coding region (i.e., open reading frame, ORF) and polyA. The 3'UTR sequence is usually encoded by a gene and transcribed into mRNA, but is not translated. The genome sequence is first transcribed into immature mRNA, which contains optional introns. Then, during maturation, the immature mRNA is further processed into mature mRNA. This maturation process includes: 5' capping, splicing of the immature mRNA to remove optional introns and 3' end modifications, such as polyadenylation of the 3' end of the immature mRNA, and optional endonuclease or exonuclease cleavage. The 3'UTR in this application is located on mature mRNA.

[0069] 5' Uncoding Region (5'UTR): The 5'UTR is generally understood as a specific portion of messenger RNA (mRNA). It is located at the 5' end of the mRNA open reading frame. Typically, the 5'UTR begins at the transcription start site and ends one nucleotide before the start codon of the open reading frame. The 5'UTR may contain elements that control gene expression, also known as regulatory elements. The 5'UTR can be modified posttranscribed, for example by adding a 5' cap structure (e.g., m7G(5')ppp(5')(2'-OMeA)pG).

[0070] In some embodiments, the 5' uncoding region (5'UTR) comprises or is a nucleotide sequence as shown in SEQ ID NO.2, or comprises or is a nucleotide sequence having 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 100% sequence identity with the sequence shown in SEQ ID NO.2; for example, it comprises or is a nucleotide sequence having at least 80%, 82%, 84%, 85%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO.2.

[0071] In some embodiments, the 3' uncoding region (3'UTR) comprises or is a nucleotide sequence as shown in SEQ ID NO.4, or comprises or is a nucleotide sequence having 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 100% sequence identity with the sequence shown in SEQ ID NO.4; for example, it comprises or is a nucleotide sequence having at least 80%, 82%, 84%, 85%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO.4.

[0072] In some embodiments, all uridines in the mRNA molecule are unmodified; in other embodiments, a portion (e.g., one or more uridines) or all of the uridines in the mRNA molecule are chemically modified uridines. In some preferred embodiments, the uridine is a pseudouridine, such as N1-methylpseudouridine.

[0073] In some embodiments, the mRNA molecule further includes a 5' cap structure. As used herein, the common term "5' cap" is located at the 5' end of the mRNA and contains a methylated guanosine monophosphate (GMP) linked to the 5' end of the mRNA via pyrophosphate, forming a 5',5'-triphosphate link with its adjacent nucleotide. There are generally three types of 5' cap structures (m7G5'ppp5'Np, m7G5'ppp5'NmpNp, and m7G5'ppp5'NmpNmpNp), referred to as type O, type I, and type II, respectively. Type O indicates that the ribose of the terminal nucleotide is unmethylated, type I indicates that the ribose of the terminal nucleotide is methylated, and type II indicates that the ribose of both terminal nucleotides are methylated. In some embodiments, the 5' cap may be simultaneously capped on the polynucleotide during in vitro transcription using the following chemical RNA cap analogs, according to the manufacturer's protocol, to produce a 5'-guanosine cap structure: 3′-O-Me-m7G(5')ppp(5')G [ARCA cap], G(5')ppp(5')A, G(5')ppp(5')G, m7G(5')ppp(5')A, m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA), or m7G(5')ppp(5')(2'-OMeA)pG (CleanCap AG). For example, in some embodiments, a vaccinia virus capping enzyme may be used to complete the 5' capping of the modified RNA post-transcriptionally to produce an O-type cap structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Type I cap structures can be generated using both vaccinia virus capping enzyme and 2'-O methyltransferase to produce m7G(5')ppp(5')(2'-OMeA)pG. Type II cap structures can be generated by 2'-O-methylation of the 5'-tertiary nucleotide using 2'-O methyltransferase. Type III cap structures can be generated by 2'-O-methylation of the 5'-quadrupole nucleotide using 2'-O methyltransferase.

[0074] In some preferred embodiments, the 5' cap structure is m7G(5')ppp(5')(2'-OMeA)pG.

[0075] Ionizable lipid compounds The term "ionizable lipid" generally refers to ionizable cationic lipids.

[0076] In some embodiments, the ionizable lipids provided in this application are compounds of formula IIA, or pharmaceutically acceptable salts thereof, or stereoisomers thereof: .

[0077] In some implementation schemes, R 1a and R 1b Independently selected from -CH3. In one specific embodiment, R 1a For -CH3; in a specific embodiment, R 1b It is -CH3.

[0078] In some implementations, R2 and R3 are each independently represented by H.

[0079] In some embodiments, R4 is selected from C8, C9, C10, C11, or C12 straight-chain alkyl groups. In some preferred embodiments, R4 is selected from C10 or C11 straight-chain alkyl groups. For example, R4 is a C8 straight-chain alkyl group, a C9 straight-chain alkyl group, a C10 straight-chain alkyl group, a C11 straight-chain alkyl group, or a C12 straight-chain alkyl group.

[0080] In some embodiments, R5 and R6 are independently selected from C6, C7, and C8 straight-chain alkyl groups; for example, R5 and R6 are independently C6, C7, or C8 straight-chain alkyl groups. In some preferred embodiments, R5 and R6 are independently selected from C6 or C8 straight-chain alkyl groups. For example, R5 and R6 are independently C8 or C6 straight-chain alkyl groups.

[0081] In some embodiments, R5 and R6 are both C8 straight-chain alkyl; in some embodiments, R5 is a C6 straight-chain alkyl and R6 is a C8 straight-chain alkyl; in some embodiments, R5 is a C8 straight-chain alkyl and R6 is a C6 straight-chain alkyl.

[0082] In some implementations, X1 and X2 are both C=0, and Y1 and Y2 are both 0; in some implementations, X1 is 0, Y1 is C=0, X2 is C=0, and Y2 is 0; in some implementations, X1 is C=0, Y1 is 0, X2 is 0, and Y2 is C=0. In some implementations, X1 and X2 are both 0, and Y1 and Y2 are both C=0.

[0083] In some implementation schemes, o and p are each independently selected from 5, 6, 7, and 8.

[0084] In some implementations, q is selected from 2, 3 or 4; in some preferred implementations, q is 3 or 4.

[0085] In some embodiments, the ionizable lipid compound is selected from one of the following structures or a pharmaceutically acceptable salt thereof or a stereoisomer thereof: , , , , or .

[0086] As used herein, “alkyl” means any group derived from a straight-chain or branched saturated hydrocarbon, which may be arbitrarily substituted with one, two, or three substituents. Unless otherwise expressly stated, the term “alkyl” is intended to include saturated, unsaturated, and partially unsaturated aliphatic groups. When specifically referring to an unsaturated group, the terms “alkenyl” or “alkynyl” are used. When referring only to a saturated group, the term “alkyl” is used. Generally, an alkyl group has any number of carbon atoms in the range of 1 to 24, 1 to 12, 1 to 8, 1 to 4, or 1 to 24; in some specific embodiments, an alkyl group has, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms, i.e., represented as C1-C24 alkyl.

[0087] The term "optionally substituted" means that a group may or may not be substituted; for example, optionally substituted alkyl groups include substituted alkyl groups and unsubstituted alkyl groups. When the groups are "substituted," they may be substituted by any suitable one or more substituents. In some embodiments, when the groups are "substituted," it means that they are wholly or partially substituted by one or more of carboxyl, hydroxyl or amino, alkoxy, halogen, alkyl, alkenyl, and cycloalkyl groups.

[0088] The term "pharmaceutically acceptable salt" means an acid addition salt or a base addition salt of a compound. The acid used to obtain the acid addition salt includes organic and inorganic acids. The inorganic acids are, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, carbonic acid, hydrocarbon acid, and thiocyanate. The organic acids include, but are not limited to, acetic acid, aspartic acid, benzenesulfonic acid, benzoic acid, adipic acid, alginic acid, ascorbic acid, 4-acetaminobenzoic acid, camphoric acid, camphor-10-sulfonic acid, methylsulfuric acid, decanoic acid, hexanoic acid, octanoic acid, carbonic acid, cinnamic acid, citric acid, cycloamidoic acid, naphthoic acid, naphthalenesulfonic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactobionic acid, gentian acid, glucohepanoic acid, gluconic acid, glucuronic acid, glutamic acid, glutamate, glutaric acid, and 2... -Oxyglutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucoic acid, naphthalene-1,5-dicarboxylic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, palmitic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sulfosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, trifluoroacetic acid, and undecenoic acid. Base addition salts refer to salts prepared by adding an inorganic or organic base to a free base compound. In some embodiments, the inorganic base includes, but is not limited to, metallic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese salts, and aluminum; the organic base includes, but is not limited to, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dealcohol, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, benethamine, benzathine penicillin, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins.

[0089] The pharmaceutically acceptable salts of this application can be synthesized from the basic or acidic portions by conventional chemical methods. Typically, these salts can be prepared by reacting the free acidic form of these compounds with a suitable stoichiometric amount of a base (hydroxides, carbonates, bicarbonates, etc. of Na, Ca, Mg, or K), or by reacting the free basic form of these compounds with a suitable stoichiometric amount of an acid. These reactions are typically carried out in water, in an organic solvent, or in a mixture of both. Typically, a non-aqueous medium, such as diethyl ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, is required where appropriate. A list of other suitable salts can be found in Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, Easton, Pa., (1985); and Stahl and Wermuth's Handbook of Pharmaceutical Salts: Properties, Selection, and Use (Wiley-VCH, Weinheim, Germany, 2002).

[0090] This application can also prepare solvates of the compounds of this application, including pharmaceutically acceptable solvates. A “solvate” refers to a complex of variable stoichiometry formed by a solute and a solvent. Such solvents for the purposes of this invention do not affect the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, MeOH, EtOH, and AcOH. A solvate in which water is the solvent molecule generally refers to a hydrate. Hydrates include components containing a stoichiometric amount of water, as well as components containing a variable amount of water.

[0091] As used herein, the term "pharmaceutically acceptable" means a compound suitable for pharmaceutical use. Salts and solvates suitable for pharmaceutical use (e.g., hydrates and salt hydrates) refer to those in which the counterion or binding solvent is a pharmaceutically acceptable alternative. However, salts and solvates having non-pharmaceutically acceptable counterions or binding solvents are also included within the scope of this application, for example, as intermediates in the preparation of other compounds of this application and their pharmaceutically acceptable salts and solvates.

[0092] It should be noted that the compounds (including their salts and solvates) of this application may exist in crystalline, amorphous, or mixtures thereof. The compounds, their salts, or solvates may also exhibit polymorphism, i.e., the ability to appear in different crystalline forms. These different crystalline forms are generally known as "polymorphs." Polymorphs have the same chemical composition but differ in the packing, geometric arrangement, and other descriptive properties of their crystalline solid states. Therefore, polymorphs can have different physical properties, such as shape, density, hardness, deformability, stability, and solubility. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns, all of which can be used for identification. Those skilled in the art will understand that, for example, different polymorphs may be produced by changing or adjusting the conditions used in the crystallization / recrystallization of the compounds of this application.

[0093] This application also includes different isomers of the compounds of this application. The term "isomer" refers to a compound having the same composition and molecular weight but different physical and / or chemical properties. The difference in structure between isomers can be in the structure itself (geometric isomers) or in their ability to rotate plane-polarized light (stereoisomers). Stereoisomers, for example, the compounds of this application may have one or more asymmetric carbon atoms and may appear as racemates, racemic mixtures, and as single enantiomers or diastereomers. Any isomeric forms of the compounds of this application, whether listed above or not, are included within the scope of this application, and mixtures thereof are also included. Further, for example, if the compound contains a double bond, the substituent may be E or Z configuration. If the compound contains a disubstituted cycloalkyl group, the substituent of the cycloalkyl group may have a cis or trans configuration. All of the above are included within the scope of this application. Furthermore, for example, any asymmetric atom (e.g., carbon) in the compounds of this application can exist in racemic or enantiomeric enrichment, such as (R)-, (S)-, or (R,S)- configurations. In some embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)- configuration; for example, having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% enantiomeric excess. Where possible, substituents on atoms having unsaturated double bonds are present in cis-((Z)-) or trans-((E)-) form. Therefore, as used herein, the compounds of the present invention can be in the form of one of the following possible isomers, rotational isomers, rotation-blocked isomers, tautomers, or mixtures thereof, for example as substantially pure geometric isomers (cis or trans), diastereomers, optical isomers (enantiomers), racemates, or mixtures thereof.

[0094] This application also includes both unlabeled and isotopically labeled forms of the compounds of this application. Isotopically labeled compounds have the structure described by the chemical formulas given herein, except that one or more atoms (e.g., two, three, four, five, or more) are replaced by atoms having a selected atomic weight or mass number. Examples of isotopes that can be incorporated into the compounds of this application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine; wherein isotopes of hydrogen are, for example, 2H and 3H; isotopes of carbon are, for example, 11C, 13C, and 14C; isotopes of nitrogen are, for example, 15N; isotopes of fluorine are, for example, 18F; isotopes of phosphorus are, for example, 31P and 32P; isotopes of sulfur are, for example, 35S; isotopes of chlorine are, for example, 36Cl; and isotopes of iodine are, for example, 125I. This application includes compounds labeled with various isotopes as defined herein, such as those containing radioactive isotopes (e.g., 3H and 14C) or those containing non-radioactive isotopes (e.g., 2H and 13C). Those skilled in the art will appreciate that substitution with a heavier isotope, such as, in particular, deuterium (i.e., 2H or D), may result in certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirements or improved therapeutic index. It is understood that, herein, deuterium is considered a substituent for compounds of formula (I). The concentration of this heavier isotope, particularly deuterium, may be determined by an isotope enrichment factor.

[0095] Those skilled in the art will be able to identify the presence of a stereocenter in the compounds of this application. Therefore, this application includes possible stereoisomers, and includes both racemic compounds and single enantiomers. When the desired compound is a single enantiomer, it can be obtained by stereospecific synthesis or by resolution of the end product or any convenient intermediate. Resolution of the end product, intermediate, or starting material can be achieved by any suitable method known in the art. See, for example, "Stereochemistry of Organic Compounds" by E.L. Eliel, S.H. Wilen, and L.N. Mander (Wiley-interscience, 1994).

[0096] Lipid nanoparticle composition As used herein, the term "compound" refers to all isomers and isotopes including the described structure. "Isotope" refers to atoms with the same atomic number but different mass numbers due to differences in the number of neutrons in their nuclei. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, the compounds of this application and their salts can be prepared by conventional methods by combining with solvents or water molecules to form sols and hydrates.

[0097] In some embodiments, the lipid nanoparticle composition includes ionizable lipids, phospholipids, sterols, and polymer-conjugated lipids.

[0098] As used herein, "lipid nanoparticle" or "LNP" refers to particles with a diameter of about 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents. In some embodiments, the lipid nanoparticles contain nucleic acids. In some embodiments, the nucleic acids are partially or completely encapsulated within the lipid nanoparticles.

[0099] In some embodiments, the sterol is selected from any one or any combination of cholesterol, cholesterol hemisuccinate and their derivatives; in some preferred embodiments, the sterol is cholesterol.

[0100] In some embodiments, the phospholipid is selected from 1,2-disorcinol hemisuccinoyl-sn-glycerol-3-phosphate ethanolamine (DEPE), 1,2-dicholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (DChemsPC), 1,2-distearyl-sn-glycerol-3-phosphatidylcholine or 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine (DLPE), 16-O-monomethylphosphoethanolamine, 16 -O-Dimethylphosphatidylethanolamine, 1,2-Dilinoleoyl-sn-glycerol-3-phosphate ethanolamine (DLoPE), 1,2-Dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonium)hydroethyl phosphate (DOCP), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonium)ethylethyl phosphate (DOCPe), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2- Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), phosphatidylethanolamine, dioleoyl-phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS) and its sodium salt, 1,2-distearyl-sn-glycerol-3-phosphate-L-serine and its sodium salt, 1,2-diphydanyl-sn-glycerol-3-phosphate-L-serine and its sodium salt, distearyl... Phosphatidylcholine, dioleoyl-phosphatidylethanolamine (DOPEA), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (DPhyPE), 1,2-diphydanyl-sn-glycerol-3-phosphate choline (DPhyPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-squalene-sn-glycerol-3-phosphate ethanolamine (DSQPE), 1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), distearyl-phosphatidylethanolamine (DSPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), 18-1-trans-phosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate ethanolamine (SLPE), 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate-L-serine and its sodium salt, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (POPS) and its sodium salt, 1 Any one or more combinations thereof selected from 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine and its sodium salt, 1-O-hexadecyl-2-O-(9Z-octadecenyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-di-O-phytyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-cholesterol hemisuccinoyl-sn-glycerol-3-phosphate choline (PChemsPC), 1-O-octadecyl-2-O-methyl-sn-glycerol-3-phosphate choline (edifoxin), palmitoyloleoylphosphatidylcholine (POPC), and palmitoyloleoyl-phosphatidylethanolamine (POPE). In some preferred embodiments, the phospholipid is DSPC; and / or the polymeric conjugated lipid is a PEG-modified lipid.

[0101] Polymer conjugated lipids mainly refer to lipids modified with polyethylene glycol (PEG). Hydrophilic PEG stabilizes LNPs, modulates nanoparticle size by restricting lipid fusion, and increases the half-life of nanoparticles by reducing non-specific interactions with macrophages. In some embodiments, the polymeric conjugated lipid is selected from any one or more of PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG, Chol-PEG, 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG), polyethylene glycolated phosphatidylethanolamine (PEG-PE), 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG), polyethylene glycolated ceramide (PEG-cer), and ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate. In some preferred embodiments, the polymeric conjugated lipid is DMG-PEG2000.

[0102] In some embodiments, the molar percentage of the ionizable lipids in the lipid nanoparticle composition is any range within the range of 40-55 mol%, 45-50 mol%, 48-50 mol%, or 40-55 mol%. In some preferred embodiments, the molar percentage of the ionizable lipids in the lipid nanoparticle composition is any range within the range of 48-50 mol%. In one specific embodiment, the molar percentage of the ionizable lipids in the lipid nanoparticle composition is 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, or within the range of 40-55 mol%.

[0103] In some embodiments, the molar percentage of the sterol in the lipid nanoparticle composition is any range within the range of 30-50 mol%, 35-45 mol%, 30-40 mol%, or 30-50 mol%. In some preferred embodiments, the molar percentage of the sterol in the lipid nanoparticle composition is any range within the range of 35-45 mol%. In a specific embodiment, the molar percentage of the sterol in the lipid nanoparticle composition is any range within the range of 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, or 30-50 mol%.

[0104] In some embodiments, the phospholipid molar percentage in the lipid nanoparticle composition is 5-15 mol%, 8-12 mol%, 10-15 mol%, or any range within the range of 5-15 mol%. In some preferred embodiments, the phospholipid molar percentage in the lipid nanoparticle composition is 10-15 mol%, or any range within the range of 10-15 mol%. In a specific embodiment, the phospholipid molar percentage in the lipid nanoparticle composition is 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, 15 mol%, or any molar percentage within the range of 5-15 mol%.

[0105] In some embodiments, the molar percentage of the polymer-conjugated lipid in the lipid nanoparticle composition is any range within the range of 1-3 mol%, 1.2-2.8 mol%, 1.5-2.5 mol%, or 1-3 mol%. In some preferred embodiments, the molar percentage of the polymer-conjugated lipid in the lipid nanoparticle composition is any range within the range of 1.5-2.5 mol%. In a specific embodiment, the molar percentage of the polymer-conjugated lipid in the lipid nanoparticle composition is any molar percentage within the range of 1 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, 3 mol%, or 1-3 mol%.

[0106] In some embodiments of this application, after application of the spleen-precision expression composition lipid nanoparticle composition, the expression level of the target protein in the spleen accounts for 60%, 70%, 80%, 90%, 95%, or 99% or more of the systemic expression level; for example, 60%, 68%, 66%, 63%, 70%, 77%, 75%, 73%, 71%, 80%, 85%, 83%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%.

[0107] The terms "encapsulation" and "encapsulation" are used interchangeably, referring to the incorporation of nucleic acids into or association with lipid nanoparticles. In this application, "encapsulation" refers to complete or partial encapsulation. As used herein, "encapsulation ratio" refers to the amount of a substance with therapeutic and / or preventive and / or modulatory functions that is part of a vaccine composition, relative to the total amount of such substances used in the preparation of the vaccine composition. For example, in this application, "encapsulation ratio" refers to the amount of mRNA that is part of a vaccine composition, relative to the total amount of mRNA used in the preparation of the vaccine composition.

[0108] The term "mol%" refers to the content of a substance expressed as a percentage point, specifically the molar percentage of a particular component in a lipid nanoparticle composition relative to the total molar amount of all lipid components in the composition. For example, "In the lipid nanoparticle composition, the molar percentage of ionizable lipids is 40-55 mol%" means that the molar amount of ionizable lipids relative to the total molar amount of all lipid components in the lipid nanoparticle composition is the molar percentage of ionizable lipids.

[0109] The term "delivery" refers to providing an entity to a destination. For example, delivering a substance with therapeutic and / or preventive and / or modulatory functions to a subject may include administering a lipid nanoparticle composition comprising therapeutic and / or preventive and / or modulatory functions to a subject; further, for example, via intravenous, intramuscular, intradermal, or subcutaneous routes. Administering a lipid nanoparticle composition to a mammal or mammalian organ, tissue, or cell may involve contacting one or more cells with the lipid nanoparticle composition.

[0110] The term "contact" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a lipid nanoparticle composition means that the mammalian cell and the lipid nanoparticle composition share a physical connection.

[0111] The term "therapeutic effective amount" refers to the amount of a therapeutic agent that, when administered to a patient, improves the disease or symptoms. "Prophylactic effective amount" refers to the amount of a preventive agent that, when administered to a subject, prevents the disease or symptoms. The amount of a therapeutic agent constituting a "therapeutic effective amount" or a preventive agent constituting a "prophylactic effective amount" varies depending on the therapeutic / preventive agent, the disease state and its severity, the age and weight of the patient / subject to be treated / prevented, etc. Those skilled in the art can routinely determine the therapeutic and prophylactic effective amounts based on their knowledge and in accordance with this application.

[0112] As used herein, “administration” includes all means of introducing a compound or vaccine composition to a subject in need, including but not limited to oral, intravenous, intramuscular, intraperitoneal, subcutaneous, percutaneous, inhalation, buccal, intraocular, sublingual, vaginal, and rectal administration. The term “subject” (or, alternatively, “patient”) means an animal receiving preventative or therapeutic treatment; preferably, a mammal; most preferably, a human.

[0113] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; In this composition, component (1) is partially or completely encapsulated in the lipid nanoparticle composition of component (2).

[0114] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before or within polyA; The structure of polyA is as follows: nA-miR-122 target binding sequence -mA-GATATC-19A-G-19A-G-17A.

[0115] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before or within polyA; The miR-122 targeting binding sequence is shown in SEQ ID NO.7, and the polyA sequence is shown in SEQ ID NO.6.

[0116] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before the polyA; The miR-122 targeting binding sequence is shown in SEQ ID NO.7, the polyA sequence is shown in SEQ ID NO.6, and the polyA variant obtained by inserting the miR-122 targeting binding sequence before polyA is shown in SEQ ID NO.39.

[0117] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted into the polyA; The miR-122 targeting binding sequence is shown in SEQ ID NO.7, the polyA sequence is shown in SEQ ID NO.6, and the polyA variant obtained by inserting the miR-122 targeting binding sequence into the polyA is shown in any one of SEQ ID NO.8-38.

[0118] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a promoter sequence, a 5' cap structure, a 5' non-coding region, a Kozak sequence, a nucleotide sequence encoding a target protein, a 3' non-coding region, and a polyA variant; wherein the polyA variant comprises polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before the polyA; The miR-122 targeting binding sequence is shown in SEQ ID NO.7, the polyA sequence is shown in SEQ ID NO.6, and the polyA variant obtained by inserting the miR-122 targeting binding sequence before polyA is shown in SEQ ID NO.39.

[0119] In some implementations, the promoter sequence is as shown in SEQ ID NO.1, the 5' uncoded region is as shown in SEQ ID NO.2, the Kozak sequence is as shown in SEQ ID NO.3, and the 3' uncoded region is as shown in SEQ ID NO.4.

[0120] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before the polyA; The structure of polyA is as follows: nA-miR-122 target binding sequence-mA-GATATC-19A-G-19A-G-17A; The lipid nanoparticle composition includes ionizable lipids, phospholipids, sterols, and polymer-conjugated lipids. The molar percentage of the ionizable lipids is 40-55 mol%. The ionizable lipid is a compound of formula (IIA), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0121] Among them, R 1a and R 1b Independently selected from -CH3; R2 and R3 are independently selected from H; R4 is selected from C8, C9, C10, C11 or C12 straight-chain alkyl groups; R5 and R6 are independently selected from C6, C7 and C8 straight-chain alkyl groups; When X1 and Y1 are different, and X2 and Y2 are different, X1 and X2 are independently selected from C=O or O; Y1 and Y2 are independently selected from C=O or O; o and p are independently selected from 5, 6, 7, and 8; q is selected from 2, 3 or 4.

[0122] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before the polyA; The structure of polyA is as follows: nA-miR-122 target binding sequence-mA-GATATC-19A-G-19A-G-17A; The lipid nanoparticle composition includes ionizable lipids, phospholipids, sterols, and polymer-conjugated lipids. The molar percentage of the ionizable lipids is 40-55 mol%. The ionizable lipid is a compound of formula (IIA), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0123] Among them, R 1a and R 1b Independently selected from -CH3; R2 and R3 are independently selected from H; R4 is selected from C9, C10, or C11 straight-chain alkyl groups; R5 is selected from C6 or C8 straight-chain alkyl groups; R6 is a C8 straight-chain alkyl group; When X1 and Y1 are different, and X2 and Y2 are different, X1 and X2 are independently selected from C=O or O; Y1 and Y2 are independently selected from C=O or O; o is selected from 5, 6, 7, or 8; p is selected from 6 or 7; q is selected from 2, 3 or 4.

[0124] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before the polyA; The structure of polyA is as follows: nA-miR-122 target binding sequence-mA-GATATC-19A-G-19A-G-17A; The lipid nanoparticle composition includes ionizable lipids, phospholipids, sterols, and polymer-conjugated lipids. The molar percentage of the ionizable lipids is 40-55 mol%. The ionizable lipid compound is selected from one of the following structures, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: , or .

[0125] In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before the polyA; The structure of polyA is as follows: nA-miR-122 target binding sequence-mA-GATATC-19A-G-19A-G-17A; The lipid nanoparticle composition comprises 40-55 mol% ionizable lipids, 5-15 mol% phospholipids, 30-50 mol% sterols, and 1-3 mol% polymer conjugated lipids. The ionizable lipid is a compound of formula (IIA), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: ; R 1a and R 1b Independently selected from -CH3; R2 and R3 are independently selected from H; R4 is selected from C8, C9, C10, C11 or C12 straight-chain alkyl groups; R5 and R6 are independently selected from C6, C7 and C8 straight-chain alkyl groups; X1 and Y1 are different, X2 and Y2 are different, and X1 and X2 are independently selected from C=O or O; Y1 and Y2 are independently selected from C=O or O; o and p are independently selected from 5, 6, 7, and 8; q is selected from 2, 3, or 4; In some embodiments, the precise expression composition comprises: Component (1): A nucleic acid comprising a nucleotide sequence encoding a target protein and a polyA variant, wherein the polyA variant comprises a polyA and a miR-122 targeting binding sequence; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; Wherein, component (1) is partially or wholly encapsulated in the lipid nanoparticle composition of component (2); The miR-122 targeting binding sequence is inserted before the polyA; The structure of polyA is as follows: nA-miR-122 target binding sequence-mA-GATATC-19A-G-19A-G-17A; The lipid nanoparticle composition comprises 40-55 mol% ionizable lipids, 5-15 mol% phospholipids, 30-50 mol% sterols, and 1-3 mol% polymer conjugated lipids. Wherein, the phospholipid is DSPC, the sterol is cholesterol, and the polymer conjugated lipid is DMG-PEG; The ionizable lipid is a compound of formula (IIA), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0126] Wherein, R1 is selected from H, vinyl, C1 to C3 alkyl, and R2, R3, and R4 are each independently C7-C15 straight-chain alkyl; X1 and Y1 are different, X2 and Y2 are different, X1 and X2 are each independently selected from C=O or O, and Y1 and Y2 are each independently selected from C=O or O; m and n are each independently selected from 5, 6, 7, and 8.

[0127] This application provides the use of the above-described spleen-specific expression composition for regulating the spleen-specific expression of a target gene.

[0128] In some implementations, the regulation can cause the expression level of the target protein in the spleen to be 90%, 95%, or 99% or more of the systemic expression level; for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0129] This application provides a method for preventing or treating a disease, comprising administering a therapeutically effective amount of the above-described spleen-precisely expressed composition to a subject.

[0130] In some embodiments, the disease is a splenic disease or a systemic disease or other organ disease related to immune cell activation; wherein, the systemic disease or other organ disease related to immune cell activation is selected from: cancer (e.g., melanoma, breast cancer, colorectal cancer, lymphoma, leukemia, multiple myeloma, etc.), infectious diseases (e.g., bacterial and viral infections, etc.), autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus, type I diabetes, autoimmune encephalomyelitis, etc.), and inflammatory diseases (e.g., inflammatory bowel disease, atherosclerosis, ischemic stroke, liver fibrosis, kidney fibrosis, etc.). For example, in some embodiments, by activating liver immune cells such as macrophages, macrophages are endowed with the function of killing fibrotic tissue, such as CAR-M targeting fibrotic tissue. It should be noted that the spleen should be regarded as a representative organ of immune organ tissues and cells. The diseases that can be treated by the method of this application should not be limited to splenic diseases, but should also include systemic diseases and other organ diseases after immune cell activation, all of which can be treated by selectively expressing drug targets in immune organs and cells. Those skilled in the art should understand that any disease that can be alleviated by the methods of this application is included within the scope of diseases that this application can treat or alleviate. In some embodiments, the spleen disease is selected from rare spleen diseases, spleen tumors, and spleen inflammation.

[0131] This application also provides a medicament comprising the above-described spleen-precise expression composition. It will be understood that the medicament may also comprise other components, such as any one or more of diluents, lubricants, binders, disintegrants, absorbents, colorants, flavorings, and sweeteners.

[0132] Example Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0133] Example 1: Preparation of the ionizable lipid of the present invention 1. Preparation of Compound 1

[0134] Compound 1 was prepared by referring to the preparation method of Compound 25 in the patent with publication number US 2021 / 0154148.

[0135] 2. Preparation of Compound 2

[0136]

[0137] Specifically, compound B (45.18 g, 242.50 mmol) and DMAP (1.48 g, 12.12 mmol) were added to a dichloromethane (220 ml) solution of compound A (43.91 g, 242.50 mmol), and the mixture was stirred thoroughly at room temperature. DCC (55.04 g, 266.75 mmol) was then added in portions. After the addition was complete, the mixture was stirred at room temperature for 20 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtered, and the filter cake was washed with ethyl acetate (100 ml × 2). The filtrate was concentrated and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0–10%) to give compound C (76.08 g, 89.80%).

[0138] Compound E (126.19 g, 493.03 mmol) and DMAP (3.04 g, 22.41 mmol) were added to a 400 mL solution of dichloromethane containing compound D (100.04 g, 448.21 mmol). The mixture was stirred at room temperature until homogeneous, and then a 100 mL solution of dichloromethane containing DCC (102.63 g, 493.03 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction mixture was filtered, and the filter cake was washed with ethyl acetate (400 mL x 2). The filtrate was concentrated and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0–5%) to give compound F (160.43 g, 77.53%).

[0139] Compound G (25.38 g, 129.99 mmol), NBu4I (3.20 g, 8.67 mmol), and K2CO3 (17.97 g, 129.99 mmol) were added to a DMF (120 mL) solution of compound F (20.00 g, 43.33 mmol). The mixture was stirred at room temperature for 20 hours, and the reaction was monitored by TLC. After the reaction was complete, water (200 mL) and ethyl acetate (200 mL) were added to the reaction solution. The mixture was allowed to stand and separated. The aqueous phase was extracted with ethyl acetate (100 mL). The combined organic phases were washed with saturated brine (200 mL) and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0–5%) to give compound H (22.03 g, yield 88.28%).

[0140] Compound C (9.10 g, 26.05 mmol), NBu4I (12.83 g, 34.73 mmol), and Cs2CO3 (16.97 g, 52.09 mmol) were added to a DMF (50 mL) solution of compound H (10.00 g, 17.36 mmol). The mixture was stirred at room temperature for 16 hours, and the reaction was monitored by TLC. After the reaction was complete, water (100 mL) and ethyl acetate (100 mL) were added to the reaction solution. The mixture was allowed to stand and separated. The aqueous phase was extracted with ethyl acetate (50 mL). The organic phases were combined, washed with saturated brine (100 mL), and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0–5%) to give compound I (13.23 g, yield 85.94%).

[0141] Concentrated hydrochloric acid (60 ml) was added to a solution of compound I (11.23 g, 13.30 mmol) in dichloromethane (100 ml), and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was separated into layers. The organic layer was washed with saturated sodium bicarbonate solution (100 ml), then with saturated brine (100 ml), dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0–5%) to give compound J (9.12 g, 100.00%).

[0142] Compound K (3.75 g, 35.34 mmol) and pyridinium 4-methylbenzenesulfonate (5.92 g, 23.56 mmol) were added to a toluene (150 mL) solution of compound J (8.00 g, 11.48 mmol). The mixture was heated under reflux for 20 hours using a Dean-Stark apparatus, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, washed with saturated brine (100 mL × 2), concentrated, and subjected to silica gel column chromatography (ethyl acetate: petroleum ether = 0–5%) to give compound L (3.14 g).

[0143] A solution of compound L (3.14 g, 4.09 mmol) in dichloromethane (50 mL) was added with methanesulfonic anhydride (1.42 g, 8.19 mmol) and triethylamine (1.1 mL, 8.19 mmol), and stirred for 20 hours. The reaction was monitored by TLC. After the reaction was complete, the sample was washed with water (50 mL), then with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give compound M (3.81 g).

[0144] Compound M (3.81 g) was added to 60 mL of dimethylamine (2 M in THF) solution, and the mixture was stirred at room temperature for 3.5 days under nitrogen protection. The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated and purified by silica gel column chromatography (dichloromethane:methanol = 50:1) to give compound 2 (1.97 g) as an oil. 1H NMR (400 MHz, CDCl3) δ 4.97 – 4.83 (m, 1H), 4.17 – 4.06 (m, 4H), 3.52 (t, J = 8.0Hz, 1H), 2.51 – 2.45 (m, 1H), 2.31 – 2.23 (m, 10H), 1.87 – 1.80 (m, 1H), 1.74 – 1.67 (m, 1H), 1.66 – 1.50 (m, 14H), 1.48 – 1.24 (m, 52H), 0.92 (t, J = 8.0Hz, 9H); MS-ESI (m / z): 795.0 (M+H)+.

[0145] 3. Preparation of Compound 3

[0146] The preparation method is the same as for compound 1, but using 8-bromooctanoic acid and 1-nonanol instead of compounds A and B as raw materials, an oily compound 3 can be obtained. ¹H NMR (400 MHz, CDCl₃) δ 4.97 – 4.82 (m, 1H), 4.17 – 4.06 (m, 4H), 3.52 (t, J = 8.0 Hz , 1H), 2.52 – 2.45 (m, 1H), 2.31 – 2.23 (m, 10H), 1.87 – 1.80 (m, 1H), 1.74 – 1.67 (m, 1H), 1.66 – 1.50 (m, 14H), 1.48 – 1.24 (m, 52H), 0.92 (t, J = 8.0 Hz , 9H); MS-ESI (m / z): 794.6 (M+H)+.

[0147] 4. Preparation of Compound 4

[0148] The preparation method is the same as that for compound 1. Using 6-bromohexanoic acid, undecyl alcohol, and 1,2,5-pentanetriol instead of compounds A, B, and K as raw materials, an oily compound 4 can be prepared. 1H NMR (400 MHz, CDCl3) δ 4.97 – 4.82 (m, 1H), 4.08 (dt, J = 10.0, 6.4 Hz, 4H), 3.52 (t, J = 8.0 Hz , 1H), 2.39 – 2.27 (m, 12H), 1.69 – 1.52 (m, 18H), 1.48 – 1.25 (m, 52H), 0.92 (t, J = 8.0 Hz , 9H).; MS-ESI (m / z): 808.8 (M+H)+.

[0149] 5. Preparation of Compound 5

[0150] The preparation method is the same as that of compound 1, but using 6-bromohexanol, 2-n-hexyldecanoic acid and 1,2,6-hexanetriol instead of compounds D, E and K as raw materials, an oily compound can be prepared. 5,1H NMR (600 MHz, CDCl3) δ 4.03 (dt, J = 22.0, 6.4 Hz, 6H), 3.42 (d, J = 6.2 Hz, 1H), 2.40 – 2.34 (m, 2H), 2.31 (s, 8H), 1.59 (dd, J = 26.0, 7.4 Hz, 14H), 1.44 – 1.38 (m, 5H), 1.36 – 1.30 (m, 11H), 1.24 (s, 44H), 0.86 (t, J = 6.4 Hz, 12H); MS-ESI (m / z): 780.7 (M+H)+.

[0151] 6. Preparation of Compound 6

[0152] The preparation method is the same as that for compound 1. Using 8-bromooctanol, 6-bromohexanol, 2-n-hexyldecanoic acid, and 1,2,6-hexanetriol instead of compounds A, D, E, and K as raw materials, an oily compound 6,1H NMR (400 MHz, CDCl3) was prepared as follows: δ 4.08 (tt, J = 7.8, 3.8 Hz, 6H), 2.43 – 2.28 (m, 11H), 1.69 – 1.60 (m, 10H), 1.59 – 1.41 (m, 8H), 1.32 (dt, J = 17.2, 5.7 Hz, 52H), 0.95 – 0.87 (m, 9H). MS-ESI (m / z): 808.8 (M+H)+.

[0153] 7. Preparation of Compound 7

[0154] The preparation method is the same as that for the synthesis of compound 2 in CN117777089A.

[0155] Example 2. Construction of recombinant plasmids containing polyA or its variants 1) Construct a recombinant plasmid containing the luciferase protein coding region and polyA. The vector uses the E. coli cloning vector pUC57 as its backbone. Between the Xba I restriction site and the SapI restriction site at its multiple cloning site, the following sequences are arranged in order: T7 phage promoter (SEQ ID NO: 1), 5' untranslated region (5'UTR) (SEQ ID NO: 2), Kozak sequence (SEQ ID NO: 3), luciferase protein coding sequence (SEQ ID NO: 5), 3' untranslated region (3'UTR) (SEQ ID NO: 4), and poly(dA:dT) (also known as polyA, polyadenine tail) (SEQ ID NO: 6), resulting in the polyA recombinant plasmid. As a control for the poly(A) variant designed in this application, no regulatory elements were added.

[0156] 2) Construct recombinant plasmids containing the polyA variant. The polyA variant exemplified in this embodiment is a poly(A)-0A followed by a miR-122 targeting binding sequence (SEQ ID NO: 39). The following explanation uses this polyA variant as an example.

[0157] The polyA recombinant plasmid obtained in step 1) was double-digested with two restriction endonucleases to remove poly(dA:dT) of SEQ ID NO: 6; and ligated with T4 DNA ligase. The polyA containing the miR-122 targeting binding sequence was ligated into the vector that removed poly(dA:dT) to obtain the polyA-miR-122 recombinant plasmid.

[0158] Example 3. Preparation of mRNA 1) Plasmid linearization The recombinant plasmids finally constructed in Example 1 (polyA recombinant plasmid and polyA-miR-122 recombinant plasmid, respectively) have a SapI restriction site after the last A in the polyA or its variant sequence. The plasmids containing the target gene were linearized after digestion with the restriction endonuclease SapI. The reaction system is shown in Table 1. The digestion was carried out at 37℃ for 3 h.

[0159] Table 1. Plasmid linearization enzyme digestion system

[0160] Two μL of the enzyme digestion product was subjected to 1% agarose gel electrophoresis to detect the linearization of the plasmid. The linearized plasmid was purified using a PCR product recovery kit (Comway).

[0161] (2) In vitro transcription and purification The linearized recombinant plasmid obtained in step (1) was used as a template for in vitro transcription using a high-yield T7 RNA transcription kit (product name: High Yield T7 RNA Synthesis Kit, Shanghai Zhaowei Technology Development Co., Ltd., product catalog number: ON-040). The components of the high-yield T7 RNA transcription kit were: 5× Reaction Buffer, 100mM ATP Solution, 100mM CTP Solution, 100mM GTP Solution, Enzyme mix, DNase I, Ammonium Acetate Stop Solution, Lithium Chloride (LiCl) Precipitation Solution, and 100mM ΨUTP Solution (pseudouridine triphosphate, full name: N1-Me-pUTP; 100mM). Each component was added according to the system shown in Table 2 below (taking a 20μL reaction system as an example), mixed well, and reacted at 37℃ for 3h.

[0162] Table 2 In vitro transcription system

[0163] Among them, CleanCap AG is m7G(5')ppp(5')(2'-OMeA)pG.

[0164] After the transcription reaction was complete, 1 μL of DNase I was added, and the mixture was incubated at 37°C for 15 min. Then, 15 μL of Ammonium Acetate Stop Solution was added and mixed well. Next, 1 / 3 volume of 7.5 M Lithium Chloride (LiCl) Precipitation Solution was added (to a final concentration of 2.5 M), and the mixture was incubated at -20°C for 30 min. The mixture was centrifuged at 12000 g for 15 min, and the RNA precipitate was discarded. 1 mL of 70% ethanol was added to wash the RNA, and the mixture was centrifuged at 12000 g for 5 min, discarding the supernatant. After air-drying, 50 μL of RNase-free water was added to dissolve the precipitate, and mRNA quantification was performed using a UV spectrophotometer to obtain capped in vitro transcribed mRNA.

[0165] Using the above method, two mRNAs were prepared from polyA recombinant plasmid and polyA-miR-122 recombinant plasmid, respectively.

[0166] Example 4. Preparation of mRNA-lipid nanoparticles Example 3 ultimately yielded two types of mRNA. Acetic acid solution was added to the two mRNA stock solutions obtained in Example 3 until the final concentration of acetic acid was 20 mmol / L and the final concentration of mRNA was 200 μg / ml, respectively. The mixture was stirred and mixed to obtain two working solutions of mRNA.

[0167] Two mRNA working solutions were separately mixed with a lipid mixture using a T-mixing device at a flow ratio of 3:1 (this can be within the range of 2:1 to 4:1; 3:1 is selected as an example in this embodiment) to prepare LNPs. The molar ratio of each lipid in the lipid mixture was as follows: Any ionizable lipid from compounds 1-4: DSPC:cholesterol:DMG-PEG2000 = 50 mol%: 10 mol%: 38.5 mol%: 1.5 mol%. The LNPs were then diluted 2-5 times with 2 mmol / L acetic acid solution, and then replaced at least 3 times with 2 mmol / L acetic acid solution to concentrate the solution to the target concentration of 200 μg / ml. Sucrose solution was added to adjust the osmotic pressure, and the pH was adjusted to 7.0-8.0 with Tris solution to obtain different mRNA-lipid nanoparticles (LNPs). The concentration and particle size of the mRNA loaded in the LNPs were determined using a Ribogreen RNA quantification kit (Invitrogen, R11490) and a Darwin ZetaSizer particle size analyzer, respectively. The specific results are shown in Table 3.

[0168] Table 3. Particle size and LNP performance of different mRNA-lipid nanoparticles

[0169] Application example: Study on the delivery level of mRNA-lipid nanoparticles in mouse organs. C57BL / 6 mice were randomly divided into groups of six based on body weight and acclimatized for 2-3 days. Mice were administered the drug via tail vein injection, with each mouse receiving 0.05 mg / kg of mRNA-LNPs (1 μg) in a 100 μL injection volume. A blank control group received an equal volume of PBS. Six hours after drug administration, Luciferin (200 μL) was injected intraperitoneally at a dose of 150 mg / kg. Ten minutes later, the mice were anesthetized, and imaging was performed using a small animal imaging system. The liver and spleen were dissected and imaged again. The experiment was repeated once. Fluorescence values ​​from the liver and spleen of each mouse were plotted as bar graphs. (See details below.) Figures 1-3 .

[0170] In the attached figure, "-" indicates that the polyA of the mRNA does not contain the miRNA-122 target binding sequence, and "+" indicates that the polyA of the mRNA contains the miRNA-122 target binding sequence, which can mediate the degradation of mRNA in liver organs enriched with miRNA-122 and reduce the expression level of the target protein in the liver.

[0171] Among them, miRNA-122 itself accumulates less in the spleen and more readily in the liver. When mice were injected with mRNA-lipid nanoparticles encapsulated with either polyA (SEQ ID NO. 6) without the miRNA-122 targeting sequence or a polyA variant (SEQ ID NO. 39) containing the miRNA-122 targeting sequence, the effects of the reaction on the liver were observed. Figure 1 and Figure 2 The results showed that, compared with the injection of mRNA-lipid nanoparticles encapsulated with polyA (SEQ ID NO. 6) which does not contain the miRNA-122 targeting binding sequence, the expression level of fluorescent protein in the liver of mice injected with mRNA-lipid nanoparticles encapsulated with the polyA variant (SEQ ID NO. 39) containing the miRNA-122 targeting binding sequence was significantly reduced, while the expression level of fluorescent protein in the spleen did not change much.

[0172] Depend on Figure 3It was found that different lipid + polyA regulatory elements exhibited different expression regulation effects. Among them, compounds 2-4 in the G2-G4 groups showed better mRNA-LNP superimposed with polyA regulatory elements than compound 1 (SM012) in the G1 group. Compound 4 in the G4 group, with mRNA-LNP superimposed with polyA regulatory elements, showed excellent specific expression regulation function and a better spleen / liver ratio than other compounds, making it more suitable for spleen-based drug delivery.

[0173] This invention forms an organ expression regulatory element by inserting a microRNA targeting binding sequence into polyA and superimposing a spleen-specific lipid-LNP, thereby achieving efficient delivery and precise expression regulation in the spleen. This significantly reduces the expression level of the target protein in the liver and avoids the side effects caused by drug accumulation in the liver.

[0174] sequence list The sequences used in the above embodiments of this application are shown in the following sequence list. It should be understood that the following sequences... The sequences listed below are merely exemplary sequences for embodiments of this application and are not intended to limit the scope of this application. The nucleic acid sequences in the following sequence listing may represent DNA or RNA sequences; when representing an RNA sequence, "T" represents uridine.

[0175]

[0176]

[0177]

[0178]

[0179] .

Claims

1. A spleen-specific expression composition, the expression composition comprising: Component (1): at least one nucleic acid comprising a nucleotide sequence encoding a target protein and one or more miRNA targeting sequences, wherein the miRNA bound by the miRNA targeting sequence is poorly enriched in the spleen and highly enriched in non-splenic tissues, organs and / or cells; and Component (2): A composition of lipid nanoparticles that are easily enriched in the spleen; in, The component (1) is partially or completely encapsulated in the lipid nanoparticle composition of the component (2).

2. The spleen-specific expression composition according to claim 1, wherein the nucleic acid is mRNA.

3. The spleen-specific expression composition according to claim 2, wherein the mRNA comprises a polyadenine tail (polyA), and the one or more miRNAs have the same or different targeting binding sequences, wherein, The one or more miRNA targeting sequences are inserted outside the nucleotide sequence encoding the target protein, optionally before or within the polyA.

4. The spleen-specific expression composition according to claim 3, wherein the polyA with one or more miRNA targeting sequences inserted comprises or is an nA-miRNA targeting sequence-mA structure, wherein: nA represents the n consecutive adenosine nucleotides (A) adjacent to the 5' end of the miRNA targeting sequence. mA represents the m consecutive adenosine nucleotides (A) adjacent to the 3' end of the miRNA targeting sequence. m and n are each independent natural numbers, and n = 0 or n ≥ 1. Choose any of the following: n≤60, n≤30, n≤19, n≤14, or n≤10. Preferably, 14≤n≤30, 19≤n≤30, and 14≤n≤19.

5. The spleen-specific expression composition according to claim 3 or 4, wherein the polyA with one or more miRNA targeting sequences inserted comprises an nA-miRNA targeting sequence-mA structure, wherein: m+n≤150, m+n≤120, m+n≤100, m+n≤80, m+n≤60, m+n≤30, m+n≤20, or m+n≤10; Preferably, m+n is any natural number between 150 and 10, including the endpoints.

6. The spleen-specific expression composition according to any one of claims 3-5, wherein the plurality of miRNA target-binding sequences are directly linked or linked by one or more nucleotides, optionally, the nucleotides being G.

7. The spleen-specific expression composition according to any one of claims 3-6, wherein the polyA further comprises a GATATC sequence.

8. The spleen-specific expression composition according to any one of claims 3-7, wherein the polyA comprises or is the structure of the nA-miRNA targeting binding sequence -mA-GATATC-19A-G-19A-G-17A.

9. The spleen-specific expression composition according to any one of claims 1-8, wherein the miRNA targeting sequence targets and binds to miR-122 or its seed sequence.

10. The spleen-specific expression composition according to any one of claims 1-9, wherein the miRNA targeting binding sequence comprises or is a nucleotide sequence as shown in SEQ ID NO. 7, or comprises or is a nucleotide sequence having 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 100% sequence identity with the nucleotide sequence shown in SEQ ID NO. 7.

Images