Lipid nanoparticles targeting liver sinusoidal endothelial cells and uses thereof

Abstract

This invention discloses lipid nanoparticles (LNPs) targeting hepatic sinusoidal endothelial cells (LSECs) and their applications. The LNPs consist of cationic ionized lipids, helper lipids, ligand-modified PEGylated lipids, and cholesterol. The helper lipids and ligand-modified PEGylated lipids target receptors on the surface of LSECs. The LNPs enhance LSEC-specific uptake through multivalent targeting design and encapsulate RNA drugs for treating allergic or autoimmune diseases. These RNA drugs encode at least one epitope of an antigen that causes allergies or autoimmune diseases. The LNPs can efficiently target LSECs to induce immune tolerance, providing a novel antigen-specific immunomodulatory strategy for the treatment of allergic or autoimmune diseases.

Description

[0001] This application claims priority to a prior Chinese application, application number 2026101924847, filed on February 10, 2026; all of its contents are part of this invention.

[0002] This application claims priority to a prior Chinese application, application number 2025101995778, filed on February 21, 2025; all of its contents are part of this invention.

[0003] This application claims priority to a prior Chinese application, application number 2025102240116, filed on February 27, 2025; all its contents are part of this invention.

[0004] This application claims priority to the earlier Chinese application, application number 2025102826781, filed on March 11, 2025; all its contents are part of this invention.

[0005] This application claims priority to the earlier Chinese application, application number 2025103613072, filed on March 26, 2025; all its contents are part of this invention. Technical Field

[0006] This invention belongs to the field of drug delivery, and more specifically, relates to a lipid nanoparticle that targets hepatic sinusoidal endothelial cells and its application. Background Technology

[0007] Beyond current treatments employing anti-inflammatory, immunosuppressive, targeted monoclonal antibody, or immunomodulatory approaches, the need for developing novel therapies for autoimmune and allergic diseases remains unmet. While most of these therapies offer symptom relief and temporary reduction in disease activity, they do not offer the prospect of long-term suppression of chronic disease activity or a cure. However, there is a growing recognition of the power of regulatory T-cell (Treg) biology and its importance in providing antigen-specific immune tolerance for autoimmune diseases such as rheumatoid arthritis, lupus, and type 1 diabetes, and allergic diseases such as food allergies, anaphylactic reactions, and asthma. Inducing antigen-specific tolerance is one approach that uses biodegradable nanoparticles to initiate and maintain immunomodulatory responses. Based on the ability of these carriers to encapsulate disease-related antigens, these antigens are delivered to antigen-presenting cells (APCs), thereby inducing antigen-specific tolerance.

[0008] The liver's tolerance is well-known because this organ is effective in preventing immune responses to exogenous food antigens from the gastrointestinal and portal venous systems, and in promoting the persistence of tumor metastasis to this organ. Furthermore, the liver enjoys immune privilege during organ transplantation, requiring less immunosuppressive therapy compared to kidney or heart transplants. Studies have also shown that simultaneous kidney or heart and liver transplantation is less likely to result in immune rejection compared to isolated organ transplants.

[0009] The immunosuppressive effect of the liver can be attributed in part to its unique APC system, including naturally tolerant APCs such as Kupffer cells (KCs), dendritic cells (DCs), and hepatic sinusoidal endothelial cells (LSECs). These tolerogenic APCs constitute a component of the hepatic reticuloendothelial system, which plays a crucial role in clearing foreign substances, degradation products, and toxins from sinusoidal blood through phagocytosis and endocytosis. Furthermore, specialized phagocytes (KCs and DCs) preferentially eliminate circulating microscale particulate matter through phagocytosis, while LSECs are more adept at eliminating soluble macromolecules and nanoparticles in the 200 nm range through clathrin-mediated endocytosis. From an immunomodulatory perspective, LSECs induce CD8+ expression by producing antigen-specific Tregs, generating TGF-β, and upregulating the receptor ligand for programmed cell death protein 1 (PD-1) (PD-L1). + and CD4 + They play a crucial role in herd immunity suppression. Therefore, the ability of LSECs to control antigen-specific treg function should be considered when treating autoimmune and allergic diseases.

[0010] Hepatic sinusoidal endothelial cells (LSECs), as core cells regulating hepatic immune tolerance, express a variety of specific receptors on their surface. The biological characteristics and targeting potential of these receptors have been extensively studied in this field, making them key targets for nanocarrier targeted delivery. SR-E family receptors: The SR-E (Scavenger Receptor Class E) family is an important pattern recognition receptor subset on the surface of LSECs. Among them, SR-E1 / LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) and SR-H1 / STABILIN-1 (stabilin-1) can specifically recognize anionic lipid ligands such as phosphatidylserine (PS) and dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), mediating ligand-receptor-dependent endocytosis. Studies have confirmed that these receptors play a crucial role in regulating the uptake of nanoparticles by LSECs. SR-E3 / mannose receptor (CD206) mainly recognizes mannose, trimannose, and L-... Fucose and other carbohydrate ligands promote intracellular delivery via clathrin-mediated endocytosis, making them classic targets for LSECs-based drug delivery. LSECtin / CLEC4G: As a member of the C-type lectin family, LSECtin / CLEC4G is specifically expressed on the surface of LSECs and can bind to carbohydrate ligands such as GlcNAc (N-acetyl-D-glucosamine) and mannose. Its unique intracellular signaling domain can regulate antigen presentation and participate in the induction of immune tolerance; it has been studied as a target for LSECs-based drug delivery. LYVE-1 (lymphatic endothelial hyaluronic acid receptor 1): LYVE-1 is a hyaluronic acid receptor specifically expressed on the surface of LSECs. It can mediate receptor-dependent endocytosis by recognizing hyaluronic acid ligands. Its high expression and ligand binding specificity make it an important candidate target for LSECs-based drug delivery.

[0011] However, efficient delivery of antigens to LSECs remains a challenge. The use of lipid nanoparticles (LNPs) to induce immune tolerance is an active research area, including the surface-decorated peptides / major histocompatibility complex (MHC) as alternative antigen presentation platforms for immune tolerance in the absence of co-stimulation. Recent studies have shown that by adding mannose ligands to the surface of LNPs, LNPs can specifically target LSECs and enhance immune tolerance by increasing cellular uptake, surface epitope presentation, and inducing Tregs. However, this design involves only one ligand and may still suffer from imprecise targeting, leading to uptake by hepatocytes or other non-solid cells.

[0012] Therefore, there is an urgent need for a targeted and specific method of LNPs to efficiently deliver antigens to LSECs. Summary of the Invention

[0013] This invention discloses lipid nanoparticles (LNPs) targeting hepatic sinusoidal endothelial cells (LSECs) and their applications. The LNPs consist of cationic ionized lipids, helper lipids, ligand-modified PEGylated lipids, and cholesterol. The helper lipids and ligand-modified PEGylated lipids target receptors on the surface of LSECs. The LNPs enhance LSEC-specific uptake through multivalent targeting design and encapsulate RNA drugs for treating allergic or autoimmune diseases. These RNA drugs encode at least one epitope of an antigen that causes allergies or autoimmune diseases. The LNPs can efficiently target LSECs to induce immune tolerance, providing a novel antigen-specific immunomodulatory strategy for the treatment of allergic or autoimmune diseases.

[0014] On one hand, the present invention provides a lipid nanoparticle comprising cationic ionized lipids, auxiliary lipids, ligand-modified PEGylated lipids, and cholesterol; wherein the ligand in the ligand-modified PEGylated lipids is capable of targeting receptors on the surface of hepatic sinusoidal endothelial cells, and the auxiliary lipids are selected from lipids capable of targeting receptors on the surface of hepatic sinusoidal endothelial cells or lipids without targeting.

[0015] Existing technologies suffer from two major drawbacks: First, lipid nanoparticles exhibit poor targeting, often resulting in non-specific delivery or targeting with only a single ligand, leading to low LSEC uptake efficiency and a high proportion of uptake by non-targeted cells (such as hepatocytes). Second, the component design is vague, lacking a clear understanding of the synergistic targeting logic between the auxiliary lipids and ligands (e.g., targeting CD206 with a single mannose ligand results in inaccurate targeting). The lipid nanoparticles provided by this invention allow for the selection of auxiliary lipids that target receptors on the surface of LSECs, or auxiliary lipids without targeting function, combined with at least one PEGylated lipid modified with a ligand targeting receptors on the surface of LSECs to achieve monovalent or multivalent targeting. When the auxiliary lipids have targeting function, or when they lack targeting but are combined with at least two PEGylated lipids modified with ligands, the prepared LNPs can multivalently target receptors on the surface of LSECs, rather than targeting the entire liver, thus solving the problem of low LSEC targeting efficiency despite LNPs being enriched in the liver in existing technologies.

[0016] Further, the cationic ionized lipid is SM102; the auxiliary lipid includes any one of PS, DOPS, and DPPS; the ligand-modified PEGylated lipid includes at least any one of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0017] The PS (phosphatidylserine) targets SR-E1 / LOX-1 and SR-H1 / STABILIN-1 on the surface of LSECs.

[0018] The DOPS (1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine) targets the SR-E1 / LOX-1 and SR-H1 / STABILIN-1 receptors of LSECs.

[0019] The DPPS (1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine) targets the SR-E1 / LOX-1 receptor of LSECs.

[0020] The GalNAc-4-sulfate targets the SR-E3 / mannose receptor (CD206) on the surface of LSECs; the mannose, trimannose, L-fucose and GlcNAc target the SR-E3 / mannose receptor (CD206) and LSECtin / CLEC4G on the surface of LSECs; the hyaluronic acid targets LYVE-1 on the surface of LSECs.

[0021] Preferably, the auxiliary lipid is PS.

[0022] In some embodiments, the types of target-specific auxiliary lipids were screened, and when the auxiliary lipid was PS, the prepared LNPs showed the best effect in targeting the liver.

[0023] Furthermore, the ligand-modified PEGylated lipid is any two of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0024] Preferably, the ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

[0025] In some methods, the types and amounts of ligand-modified PEGylated lipids were screened, and the prepared LNPs showed the best liver-targeting effect when the ligand-modified PEGylated lipids were a combination of DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

[0026] Furthermore, the molar ratio of the cationic ionized lipid, auxiliary lipid, ligand-modified PEGylated lipid, and cholesterol is 20~70: 1~15: 1~5: 25~45, and the sum of the molar ratios of each component is 100%; the N / P ratio of the cationic ionized lipid is 2~6.

[0027] Preferably, when the cationic ionized lipid is SM102, the N / P ratio of SM102 is 6.

[0028] Furthermore, the lipid nanoparticles have a particle size of 80-200 nm; the lipid nanoparticles encapsulate RNA drugs for treating allergic or autoimmune diseases.

[0029] The pore diameter in LSECs typically ranges from 50 to 200 nanometers, thus the size of the lipid nanoparticles provided by this invention can be maintained within the sinus space, thereby facilitating the interaction between LSECs and lipid nanoparticles and the opportunity for LSECs to take up lipid nanoparticles.

[0030] Furthermore, the mass ratio of the RNA drug to the lipid nanoparticles is 1~10:20~100.

[0031] Preferably, the mass ratio of the RNA drug to the lipid nanoparticles is 1:40.

[0032] Furthermore, the RNA is mRNA, which includes a 5'-cap structure, a 5'-UTR, a coding region, a 3'-UTR, and a poly A tail.

[0033] The mRNA is unstable and carries a negative charge, while the cell membrane surface also carries a negative charge. Electrostatic repulsion makes it difficult for mRNA molecules to pass through the cell membrane and enter the cell. Therefore, the encapsulation of the lipid nanoparticles is required to achieve mRNA delivery and intracellular expression.

[0034] The 5'-UTR is a non-coding polypeptide mRNA region located directly upstream (5') of the start codon (the first codon in the mRNA transcript translated by the ribosome). The 3'-UTR is a non-coding polypeptide mRNA region located directly downstream (3') of the stop codon (the codon in the mRNA transcript that signals the termination of translation). The polyA tail is the 3' end of most eukaryotic mRNAs and helps regulate mRNA stability, transport, and translation. Both the 5'-UTR and 3'-UTR are typically transcribed from genomic DNA and are elements of pre-mature mRNA. The characteristic structural features of mature mRNA (5'-cap structure and polyA tail) are usually added to the transcribed mRNA during mRNA processing.

[0035] Furthermore, the sequences of the 5'UTR and 3'UTR are independently derived from at least one of natural and synthetic proteins.

[0036] Preferably, the natural protein includes any one of α-globulin, β-globulin, and heat shock protein HSP70.

[0037] Furthermore, the 5'-UTR contains a Kozak sequence.

[0038] In this invention, the Kozak sequence is a nucleotide sequence located after the 5'-cap structure of mRNA, which can bind to the promoter and mediate the translation initiation of mRNA containing the 5'-cap structure.

[0039] Further, the nucleotide sequence of the 5'-UTR is shown in SEQ ID NO.5; the nucleotide sequence of the 3'-UTR is shown in SEQ ID NO.6.

[0040] Furthermore, the coding region includes at least one epitope encoding an antigen that causes the treatment of allergic or autoimmune diseases.

[0041] Epitopes are specific chemical groups in antigen molecules that determine antigen specificity, also known as antigenic determinants. They are the basic units for TCR / BCR and antibody-specific binding. Epitopes can be divided into continuous epitopes (linear epitopes) and discontinuous epitopes (conformational epitopes). In the immune response, based on the different TCRs and BCRs recognized by the antigenic epitope, they are divided into T-cell epitopes and B-cell epitopes. Epitopes are generally no more than 20 amino acids in size, can be recognized by the body, and can stimulate the body to produce antibodies. They are the basis of protein antigenicity and the basic structure for inducing the body to produce an immune response. Naturally occurring immune responses cannot recognize all epitopes, but rather concentrate on a relatively small number of epitopes.

[0042] The epitopes mentioned are preferred epitopes. Through AI-assisted design, epitopes with high affinity and hydrophilicity that induce antigens for treating allergic or autoimmune diseases are selected as preferred epitopes.

[0043] Furthermore, when there are multiple tabletops, they are connected in series via connectors.

[0044] The linker links multiple antigenic epitopes together to enable peptide cleavage upon release of the epitopes into the cell.

[0045] Furthermore, the connector is a flexible connector.

[0046] Preferably, the linker is a glycine-serine (Gly-Ser) linker or a GGPPG linker.

[0047] Furthermore, a target sequence is inserted upstream of the coding region.

[0048] The targeting sequence enables the antigen epitope to enter the MHC-II endosome compartment for peptide presentation to Treg precursor cells. In this invention, the targeting sequence is preferably a 1-80 amino acid fragment of the invariant chain (Ii), abbreviated as Ii(1-80). The protein subdomain of this fragment (a molecular chaperone protein of MHC-II, which facilitates peptide loading into MHC-II for antigen presentation) allows the polypeptide epitope to enter MHC-II from the cytoplasm for CD44 expression. + T cell presentation. The target sequence can be Ii (1-80) or transferrin receptor.

[0049] Furthermore, the amino acid sequence of Ii(1-80) is shown in SEQ ID NO. 7, and its corresponding reverse-translated cDNA sequence is shown in SEQ ID NO. 8.

[0050] This invention also optimizes the codons of the template cDNA transcribed into mRNA to achieve optimal gene expression of a non-human cell tRNA library compared to human cells. This was accomplished using the GenScript online codon optimization tool. Furthermore, during transcription, uridine was replaced with N1-methylpseudouridine; after transcription, a 5' cap and a Poly A tail were added to the mRNA, wherein the 5' cap is either CleanCap or ARCA, and the Poly A tail is preferably 100-120 nucleotides in length.

[0051] Furthermore, the present invention provides a method for preparing the mRNA, comprising the following steps: (1) Design and synthesize cDNA containing a 5'-UTR, a coding region and a 3'-UTR; (2) Transcribe the cDNA from step (1) into mRNA; (3) Add a 5'-cap structure and a poly A tail to the mRNA transcribed in step (2).

[0052] Due to the fragility of RNA drugs, using multiple covalent linking steps may lead to instability of LNPs and mRNA. Therefore, this invention employs a "one-pot" synthesis of LNPs and their encapsulated RNA drugs to bind multiple ligands to the same LNP, ensuring the specific targeting of LNPs to LSECs.

[0053] In some methods, microfluidic methods are used to prepare LNPs.

[0054] This invention utilizes an innovative multivalent targeting design to precisely bind to multiple receptors on the surface of LSECs, efficiently delivering RNA drugs encoding disease-associated antigenic epitopes. The core mechanism of action is inducing antigen-specific immune tolerance, thereby fundamentally regulating abnormal immune responses. This mechanism determines that its therapeutic scope is not limited to allergic diseases such as birch pollen allergy and artemisia allergy, or autoimmune diseases such as type 1 diabetes, myasthenia gravis, toxic diffuse goiter, systemic lupus erythematosus, primary cholangitis, multiple sclerosis, and Sjögren's syndrome, but rather covers all allergic and autoimmune diseases that meet the core pathological characteristics of "immune tolerance disruption." For allergic diseases, regardless of the type of allergen (pollen, dust mites, food proteins, pet dander, etc.), the essence is an excessive IgE-mediated immune response to harmless exogenous substances. This invention can induce regulatory T cell (Treg) proliferation and inhibit Th2 cell activation by loading immunodominant epitope RNA corresponding to the allergen, thus blocking the allergic reaction cascade. For autoimmune diseases, regardless of the pathogenic target (pancreatic β cells, myelin sheath, thyroid receptors, bile duct epithelial cells, etc.), the core is the abnormal activation of autoreactive T / B cells attacking the body's own tissues. This invention can specifically induce immune tolerance by loading optimized epitope RNA corresponding to the autoantigen, inhibiting the production of autoantibodies and the release of pro-inflammatory factors, thus alleviating tissue damage. As long as the disease meets the core characteristics of "the presence of a clear pathogenic antigen and an imbalance in immune tolerance," targeted therapy can be achieved by screening for immunodominant epitopes of the corresponding antigen, optimizing the RNA drug coding region sequence, and using the LNPs delivery system of this invention, demonstrating broad applicability and scalability.

[0055] Furthermore, the allergic disease is Artemisia argyi allergy, and the autoimmune disease is myasthenia gravis and toxic diffuse goiter.

[0056] Furthermore, the RNA encapsulated in the lipid nanoparticles for treating Artemisia allergy has a coding region that encodes at least one of the amino acid sequences shown in SEQ ID No. 1-2; the RNA encapsulated in the lipid nanoparticles for treating myasthenia gravis has a coding region that encodes at least one of the amino acid sequences shown in SEQ ID No. 14-19 and 27-29; and the RNA encapsulated in the lipid nanoparticles for treating toxic diffuse goiter has a coding region that encodes at least one of the amino acid sequences shown in SEQ ID No. 36-43.

[0057] Furthermore, the reverse-translated cDNA sequences corresponding to the amino acid sequences shown in SEQ ID No. 1~2 are shown in SEQ ID No. 3~4.

[0058] In some embodiments, the present invention provides a preferred mRNA for treating artemisia allergy, wherein the coding region of the mRNA encodes all preferred epitope amino acid sequences as shown in SEQ ID No. 1-2, and the reverse-translated cDNA sequence corresponding to the coding region amino acid sequence is shown in SEQ ID No. 12.

[0059] Furthermore, the reverse-translated cDNA sequences corresponding to the amino acid sequences shown in SEQ ID No. 14~19, 27~29 are shown in SEQ ID No. 20~25, 30~32.

[0060] In some embodiments, the present invention provides a preferred mRNA for myasthenia gravis (MG), wherein the coding region of the mRNA encodes all preferred epitope amino acid sequences as shown in SEQ ID No. 14-19, 27-29, and the reverse-translated cDNA sequence corresponding to the coding region amino acid sequence is shown in SEQ ID No. 34, with a GC content of 55.08%.

[0061] Furthermore, the reverse-translated cDNA sequences corresponding to the amino acid sequences shown in SEQ ID No. 36~43 are shown in SEQ ID No. 44~51.

[0062] In some embodiments, the present invention provides a preferred mRNA for toxic diffuse goiter (GD disease), wherein the coding region of the mRNA encodes all preferred epitope amino acid sequences as shown in SEQ ID No. 36-43, and the reverse-translated cDNA sequence corresponding to the coding region amino acid sequence is shown in SEQ ID No. 52, with a GC content of 58.63%.

[0063] On the other hand, the present invention provides the use of the lipid nanoparticles as described above in the preparation of formulations that enhance the ability to target hepatic sinusoidal endothelial cells.

[0064] In some embodiments, the multivalent targeted LNPs provided by this invention can improve the ability to target hepatic sinusoidal endothelial cells compared to monovalent targeted LNPs and non-targeted LNPs.

[0065] In another aspect, the present invention provides the use of the lipid nanoparticles described above in the preparation of formulations that enhance the therapeutic effects of allergic or autoimmune diseases.

[0066] In some embodiments, the LNPs provided by this invention are also used to treat artemisia allergy, myasthenia gravis, and toxic diffuse goiter. The LNPs respectively encapsulate mRNAs encoding all preferred epitopes of antigens that cause various allergies or autoimmune diseases. The therapeutic effects of injecting trivalent targeted LNPs (components containing PS+DSPE-PEG2K-trimannose+DSPE-PEG2K-GlcNAc) into various disease mouse models are significantly better than those of monovalent or other multivalent targeted LNPs.

[0067] In another aspect, the present invention provides an mRNA for treating artemisia allergy, the mRNA comprising a coding region, the coding region encoding at least one amino acid sequence as shown in SEQ ID No. 1~2.

[0068] Furthermore, when the coding region encodes multiple amino acids, these amino acids are linked together by flexible linkers.

[0069] Furthermore, the flexible linker is a glycine-serine (Gly-Ser) linker or a GGPPG linker.

[0070] Furthermore, the coding region encodes two amino acid sequences as shown in SEQ ID No. 1~2.

[0071] Furthermore, the mRNA also includes a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly A tail.

[0072] Further, the 5'-cap structure is CleanCap or ARCA; the nucleotide sequence of the 5'-UTR is shown in SEQ ID NO.5; the nucleotide sequence of the 3'-UTR is shown in SEQ ID NO.6; and the length of the poly A tail is 100-120 nucleotides.

[0073] Furthermore, the present invention provides a method for preparing the above-mentioned mRNA for treating Artemisia allergy, comprising the following steps: (1) Design and synthesize cDNA containing a 5'-UTR, a coding region and a 3'-UTR; (2) Transcribe the cDNA from step (1) into mRNA; (3) Add a 5'-cap structure and a poly A tail to the mRNA transcribed in step (2).

[0074] In another aspect, the present invention provides an RNA drug delivery system comprising the above-mentioned mRNA for treating artemisia allergy and lipid nanoparticles.

[0075] Furthermore, the lipid nanoparticles include cationic ionized lipids, auxiliary lipids, ligand-modified PEGylated lipids, and cholesterol; the ligands in the ligand-modified PEGylated lipids can target receptors on the surface of hepatic sinusoidal endothelial cells, and the auxiliary lipids are selected from lipids that can target receptors on the surface of hepatic sinusoidal endothelial cells or lipids without targeting.

[0076] Further, the cationic ionized lipid is SM102; the auxiliary lipid includes any one of PS, DOPS, and DPPS; the ligand-modified PEGylated lipid includes at least any one of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0077] Further, the auxiliary lipid is PS; the ligand-modified PEGylated lipid is any two of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0078] Furthermore, the ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

[0079] Furthermore, the molar ratio of the cationic ionized lipid, auxiliary lipid, ligand-modified PEGylated lipid, and cholesterol is 20~70: 1~15: 1~5: 25~45, and the sum of the molar ratios of each component is 100%; the N / P ratio of the cationic ionized lipid is 2~6; and the particle size of the lipid nanoparticles is 80~200 nm.

[0080] In another aspect, the present invention provides the use of the RNA drug delivery system for treating Artemisia allergy as described above in the preparation of a vaccine that improves the therapeutic effect of Artemisia allergy.

[0081] In another aspect, the present invention provides an mRNA for treating myasthenia gravis, the mRNA comprising a coding region, the coding region encoding at least one of the amino acid sequences shown in SEQ ID No. 14-19, 27-29.

[0082] Furthermore, when the coding region encodes multiple amino acids, these amino acids are linked together by flexible linkers.

[0083] Furthermore, the flexible linker is a glycine-serine (Gly-Ser) linker or a GGPPG linker.

[0084] Furthermore, the coding region encodes all amino acid sequences as shown in SEQ ID No. 14~19, 27~29.

[0085] Furthermore, the mRNA also includes a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly A tail.

[0086] Further, the 5'-cap structure is CleanCap or ARCA; the sequence of the 5'-UTR is shown in SEQ ID NO.5; the sequence of the 3'-UTR is shown in SEQ ID NO.6; and the length of the poly A tail is 100-120 nucleotides.

[0087] Furthermore, the present invention provides a method for preparing the above-mentioned mRNA for treating myasthenia gravis, comprising the following steps: (1) Design and synthesize cDNA containing a 5'-UTR, a coding region and a 3'-UTR; (2) Transcribe the cDNA from step (1) into mRNA; (3) Add a 5'-cap structure and a poly A tail to the mRNA transcribed in step (2).

[0088] In another aspect, the present invention provides an RNA drug delivery system comprising the above-mentioned mRNA for treating myasthenia gravis and lipid nanoparticles.

[0089] Furthermore, the lipid nanoparticles include cationic ionized lipids, auxiliary lipids, ligand-modified PEGylated lipids, and cholesterol; the ligands in the ligand-modified PEGylated lipids can target receptors on the surface of hepatic sinusoidal endothelial cells, and the auxiliary lipids are selected from lipids that can target receptors on the surface of hepatic sinusoidal endothelial cells or lipids without targeting.

[0090] Further, the cationic ionized lipid is SM102; the auxiliary lipid includes any one of PS, DOPS, and DPPS; the ligand-modified PEGylated lipid includes at least any one of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0091] Further, the auxiliary lipid is PS; the ligand-modified PEGylated lipid is any two of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0092] Furthermore, the ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

[0093] Furthermore, the molar ratio of the cationic ionized lipid, auxiliary lipid, ligand-modified PEGylated lipid, and cholesterol is 20~70: 1~15: 1~5: 25~45, and the sum of the molar ratios of each component is 100%; the N / P ratio of the cationic ionized lipid is 2~6; and the particle size of the lipid nanoparticles is 80~200 nm.

[0094] In another aspect, the present invention provides the use of the RNA drug delivery system for treating myasthenia gravis as described above in the preparation of a vaccine that improves the therapeutic effect of myasthenia gravis.

[0095] In another aspect, the present invention provides an mRNA for treating toxic diffuse goiter, the mRNA comprising a coding region that encodes at least one amino acid sequence as shown in SEQ ID No. 36-43.

[0096] Furthermore, when the coding region encodes multiple amino acids, these amino acids are linked together by flexible linkers.

[0097] Furthermore, the flexible linker is a glycine-serine (Gly-Ser) linker or a GGPPG linker.

[0098] Furthermore, the coding region encodes all amino acid sequences as shown in SEQ ID No. 36~43.

[0099] Furthermore, the mRNA also includes a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly A tail.

[0100] Further, the 5'-cap structure is CleanCap or ARCA; the sequence of the 5'-UTR is shown in SEQ ID NO.5; the sequence of the 3'-UTR is shown in SEQ ID NO.6; and the length of the poly A tail is 100-120 nucleotides.

[0101] Furthermore, the present invention provides a method for preparing the above-mentioned mRNA for treating toxic diffuse goiter, comprising the following steps: (1) Design and synthesize cDNA containing a 5'-UTR, a coding region and a 3'-UTR; (2) Transcribe the cDNA from step (1) into mRNA; (3) Add a 5'-cap structure and a poly A tail to the mRNA transcribed in step (2).

[0102] In another aspect, the present invention provides an RNA drug delivery system comprising the above-mentioned mRNA and lipid nanoparticles for treating toxic diffuse goiter.

[0103] Furthermore, the lipid nanoparticles include cationic ionized lipids, auxiliary lipids, ligand-modified PEGylated lipids, and cholesterol; the ligands in the ligand-modified PEGylated lipids can target receptors on the surface of hepatic sinusoidal endothelial cells, and the auxiliary lipids are selected from lipids that can target receptors on the surface of hepatic sinusoidal endothelial cells or lipids without targeting.

[0104] Further, the cationic ionized lipid is SM102; the auxiliary lipid includes any one of PS, DOPS, and DPPS; the ligand-modified PEGylated lipid includes at least any one of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0105] Further, the auxiliary lipid is PS; the ligand-modified PEGylated lipid is any two of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0106] Furthermore, the ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

[0107] Furthermore, the molar ratio of the cationic ionized lipid, auxiliary lipid, ligand-modified PEGylated lipid, and cholesterol is 20~70: 1~15: 1~5: 25~45, and the sum of the molar ratios of each component is 100%; the N / P ratio of the cationic ionized lipid is 2~6; and the particle size of the lipid nanoparticles is 80~200 nm.

[0108] In another aspect, the present invention provides the use of the RNA drug delivery system for treating toxic diffuse goiter as described above in the preparation of a vaccine that improves the therapeutic effect of toxic diffuse goiter.

[0109] The present invention has the following beneficial effects: 1. The LNPs provided by this invention have high targeting precision and uptake efficiency: innovative multivalent targeting design (targeting auxiliary lipids + at least one ligand-modified PEGylated lipids) accurately binds to multiple receptors on the surface of LSECs, resulting in a higher proportion of liver-specific uptake; 2. This invention has screened out a better composition of LNPs, further improving targeting and therapeutic efficacy for allergic and autoimmune diseases. 3. The present invention optimizes the mRNA drug loaded with LNPs, wherein the coding region of the mRNA includes at least one preferred antigenic epitope; 4. This invention screens preferred epitopes of antigens that induce Artemisia argyi allergy, myasthenia gravis, and toxic diffuse goiter, and provides the cDNA sequences of the corresponding reverse translation of the epitopes, as well as the most preferred mRNAs for the three diseases. 5. The LNPs provided by this invention can be developed into vaccines and immunomodulators for the prevention or treatment of allergic diseases and autoimmune diseases, and have the characteristics of strong antigen specificity and low side effects. Attached Figure Description

[0110] Figure 1 Fluorescence distribution in mice injected with LNPs prepared from different cationic ionized lipids, as shown in Example 2, as imaged by an in vivo imaging system (IVIS). Figure 2 Fluorescence intensity results of in vivo imaging system (IVIS) for mice injected with LNPs prepared from different cationic ionized lipids in Example 2; Figure 3 The different fluorescence distributions in various organs of mice injected with LNPs prepared by different cationic ionized lipids in Example 2; Figure 4 The different fluorescence percentages in various organs and the different fluorescence intensities in the liver of mice injected with LNPs prepared by different cationic ionized lipids in Example 2. Figure 5 The different fluorescence percentages in various organs of mice injected with SM102-LNPs (trivalent target 1), MC3-LNPs (trivalent target 2), SM102-LNPs (monovalent target 1), SM102-LNPs (monovalent target 2) and SM102-LNPs (monovalent target 3) in Example 3; Figure 6The intensity of different fluorescence in the livers of mice injected with SM102-LNPs (trivalent target 1), MC3-LNPs (trivalent target 2), SM102-LNPs (monovalent target 1), SM102-LNPs (monovalent target 2) and SM102-LNPs (monovalent target 3) in Example 3; Figure 7 This is a schematic diagram of the complete mRNA structure in Example 6; Figure 8 This is a schematic diagram showing the distribution of different epitopes in the α subunit sequence of AChR in Example 7; Figure 9 This is the result of detecting the proliferative response of different epitopes of the α subunit of AChR in four different MG patients (Pt 3 / 7 / 10 / 11) in Example 7; Figure 10 The frequency at which different sequences of the α subunit of AChR were recognized by seven responding MG patients in Example 7 (indicated by light gray bars). Figure 11 The results show the correlation between disease severity scores and MuSK-Ig1 epitope pattern responsiveness in 22 Italian patients with type G in Example 7. Detailed Implementation

[0111] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0112] Example 1: Preparation of a lipid nanoparticle The lipid nanoparticles (LNPs) prepared in this embodiment comprise SM102, phosphatidylserine (PS), DSPE-PEG2K-trimannose, DSPE-PEG2K-GlcNAc, cholesterol, and an encapsulated RNA drug. SM102 is a cationic ionized lipid, PS is an accessory lipid, and DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc are ligand-modified PEGylated lipids. The RNA drug is an RNA drug for treating allergic or autoimmune diseases. The RNA drug is mRNA, comprising a 5'-cap structure, a 5'-UTR, a coding region, a 3'-UTR, and a poly A tail, wherein the coding region encodes at least one epitope of an antigen that can cause allergic or autoimmune diseases.

[0113] LNPs were prepared using a microfluidic method, specifically as follows: the ratio of ethanol phase to aqueous phase was 1:3 (the ethanol phase contained all lipid components, and the aqueous phase contained the RNA drug), the total flow rate was 12 mL / min, and the molar mass ratio of the lipid components SM102:PS:Chol:DSPE-PEG2K-trimannose:DSPE-PEG2K-GlcNAc = 50:10:38.5:0.75:0.75, wherein the N / P ratio of SM102 was 6, and the mass ratio of RNA drug to lipid nanoparticles was 1~10:20~100 (preferably 1:40). The prepared LNPs were dialyzed using PBS.

[0114] In this embodiment, PS, DSPE-PEG2K-trimannose, and DSPE-PEG2K-GlcNAc all have the ability to target receptors on the surface of LSECs, so the prepared LNPs are trivalent targeting LNPs.

[0115] Example 2: Screening of cationic ionized lipids and their N / P ratios in lipid nanoparticles In this embodiment, LNPs were prepared according to the method in Example 1, except that the cationic ionized lipid SM102 was replaced with MC3. When the cationic ionized lipid was SM102, its N / P ratio was set to 2, 3, 4, and 6, respectively; when the cationic ionized lipid was MC3, its N / P ratio was set to 4. To facilitate subsequent characterization, experiments, and in vivo tracking of LNPs, the prepared LNPs were all loaded with two mRNAs encoding enhanced green fluorescent protein (EGFP) and luciferase (Luc), respectively. DiR was added to the LNP components for labeling (the molar ratio of DiR was 0.3; since the total molar ratio was 100%, the molar ratio of Chol was reduced from 38.5 to 38.2).

[0116] First, the physicochemical properties of the different LNPs prepared above were characterized, including encapsulation efficiency (EE), particle size (Size), polydispersity index (PDI), zeta potential and acid dissociation constant (pKa). The characterization results are shown in Table 1 below.

[0117] Table 1. Characterization results of the physicochemical properties of LNPs prepared from different cationic ionized lipids. As shown in Table 1, when the cationic ionized lipid SM102-N / P ratio is 2~6, the encapsulation efficiency of the prepared LNPs is higher than that of LNPs prepared when the cationic ionized lipid MC3-N / P ratio is 4. Therefore, the preferred cationic ionized lipid is SM102. Comparing the physicochemical properties of LNPs prepared from SM102 with different N / P ratios, the LNPs prepared with an N / P ratio of 6 have the highest encapsulation efficiency, smallest particle size, and most uniform particle size distribution. Considering all factors, an N / P ratio of 6 is the preferred ratio.

[0118] Furthermore, 20 μg mRNA of LNPs prepared from different cationic ionized lipids was injected subcutaneously into the tail of mice, with an equal volume of PBS injected as a control. Six hours later, the location of LNPs and the expression of mRNA were observed using an in vivo imaging system (IVIS). The results are as follows: Figures 1-2 As shown, the background signal of EGFP is strong and has no reference value. However, by comparing the qualitative and quantitative fluorescence intensity results of DiR and Luc, it was found that when the cationic ionized lipid in LNPs is SM102 and the N / P of SM102 is 6, the fluorescence signals of DiR and Luc are stronger and concentrated in the mouse liver.

[0119] Further, mice in each group were euthanized, and their organs were harvested and imaged to compare the intensity of luminescence or fluorescence. The organs included the kidneys, spleen, lungs, liver, heart, and lymph nodes. The results are as follows: Figures 3-4 As shown, comparing the fluorescence proportion of DiR in different organs and the fluorescence intensity of DiR in the liver of mice in each group, it was found that the fluorescence proportion of DiR in the liver did not differ significantly among the groups. However, mice injected with LNPs containing SM102-N / P6 had the highest DiR fluorescence intensity in the liver. Comparing the fluorescence proportion of Luc and EGFP in different organs and the fluorescence intensity of Luc and EGFP in the liver of mice in each group, it was found that mice injected with LNPs containing SM102-N / P6 had the highest fluorescence proportions of both Luc and EGFP in the liver, as well as the highest fluorescence intensity in the liver. These results indicate that when the cationic ionized lipid in the LNPs is SM102, and the N / P ratio of SM102 is 6, the ability to specifically target the liver and translate mRNA is strongest.

[0120] In summary, the preferred cationic ionized lipid in LNPs is SM102, and the preferred N / P ratio of SM102 is 6.

[0121] Example 3: In vivo targeting validation of LNPs In this embodiment, two types of LNPs prepared in Example 2 when the cationic ionized lipids were SM102-N / P=6 and MC3-N / P=4 were selected as experimental subjects and were respectively denoted as SM102-LNPs (trivalent targeting 1, i.e. PS, DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc targeting LSECs) and MC3-LNPs (trivalent targeting 2, i.e. PS, DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc targeting LSECs). Simultaneously, the combination of DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc in the SM102-LNPs (trivalent targeting 1) is replaced with a single DSPE-PEG2K, denoted as SM102-LNPs (monovalent targeting 1, i.e., only PS targets LSECs); the PS in the SM102-LNPs (trivalent targeting 1) is replaced with a common auxiliary lipid DSPC, and the combination of DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc is replaced with a single DSPE-PEG2K. - Trimannose, denoted as SM102-LNPs (monovalent targeting 2, i.e., only DSPE-PEG2K-trimannose targets LSECs); in the aforementioned SM102-LNPs (trivalent targeting 1), PS is replaced with ordinary auxiliary lipid DSPC, and the combination of DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc is replaced with a single DSPE-PEG2K-GlcNAc, denoted as SM102-LNPs (monovalent targeting 3, i.e., only DSPE-PEG2K-GlcNAc targets LSECs).

[0122] The five LNPs were injected subcutaneously into the tail of mice at a dose of 20 μg mRNA per mouse. Six hours later, the location of the LNPs and the expression of their mRNA were observed using an in vivo imaging system (IVIS). Subsequently, the mice in each group were sacrificed, and their organs were harvested and imaged to compare the luminescence or fluorescence intensity. The organs included the kidney, spleen, lung, liver, heart, and lymph nodes.

[0123] The fluorescence percentages of DiR, Luc, and EGFP in various organs of the five groups of mice, and the fluorescence intensities of DiR, Luc, and EGFP in the liver, are shown below. Figures 5-6 As shown. Comparison Figure 1 The fluorescence ratio of DiR in the middle and Figure 2 The DiR fluorescence intensity in the liver of mice injected with SM102-LNPs (trivalent target 1) and MC3-LNPs (trivalent target 2) was significantly higher than that of mice injected with the other three monovalent target LNPs; (Comparison) Figure 1 The fluorescence ratio of Luc and EGFP and Figure 2The fluorescence intensity of Luc and EGFP in the liver of mice injected with SM102-LNPs (trivalent targeting 1) and MC3-LNPs (trivalent targeting 2), as well as the fluorescence percentage of Luc and EGFP in the liver, were significantly higher than those of mice injected with the other three monovalent targeting LNPs. These results indicate that multivalent targeting LNPs have a stronger ability to specifically target the liver and translate mRNA than monovalent targeting LNPs, and that SM102-LNPs (trivalent targeting 1) have a superior targeting effect compared to MC3-LNPs (multivalent targeting 2).

[0124] Example 4: The effect of assisting lipids on the targeting of lipid nanoparticles In Example 1, the PS, DSPE-PEG2K-trimannose, and DSPE-PEG2K-GlcNAc in the LNPs prepared all have the ability to target receptors on the surface of LSECs, with PS serving as an accessory lipid. In this example, the type of accessory lipid will be replaced. Replaceable accessory lipids include DOPS (1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine), DPPS (1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine), and DSPC (distearylphosphatidylcholine). DOPS and DPPS have the ability to target receptors on the surface of LSECs; DOPS targets the SR-E1 / LOX-1 and SR-H1 / STABILIN-1 receptors of LSECs, while DPPS targets the SR-E1 / LOX-1 receptor of LSECs. Therefore, in this embodiment, when the auxiliary lipids are PS, DOPS and DPPS, the prepared LNPs are trivalent targeting LNPs, that is, the auxiliary lipids and the two ligand-modified PEGylated lipids can target LSECs; when the auxiliary lipid is DSPC, the prepared LNPs are bivalent targeting LNPs, that is, the auxiliary lipids do not target LSECs, and only the two ligand-modified PEGylated lipids can target LSECs.

[0125] Four types of LNPs were prepared by PS and its alternative auxiliary lipids according to the method in Example 1. Each LNP carried two mRNAs encoding EGFP and Luc, respectively. DiR was added to the LNP components for labeling (the molar ratio of DiR was 0.3, and since the total molar ratio was 100%, the molar ratio of Chol was reduced from 38.5 to 38.2).

[0126] The four LNPs were injected subcutaneously into the tail of mice at a dose of 5 μg / mouse. Six hours later, the mice in each group were sacrificed and their organs were harvested. The organs were imaged and their luminescence or fluorescence intensity was compared. The organs included the kidney, spleen, lung, liver, heart, and lymph nodes. The results showed that the fluorescence proportion in the liver of each group of mice was significantly higher than that in the other organs. The fluorescence proportions of DiR, Luc, and EGFP in the liver of each group of mice are shown in Table 2 below.

[0127] Table 2. Fluorescence percentages of DiR, Luc, and EGFP in the livers of mice in each group. According to the data in Table 2, when the auxiliary lipids were targeted PS, DOPS, and DPPS, the fluorescence proportions of DiR, Luc, and EGFP in the liver of the prepared LNPs were all higher than those of LNPs prepared without targeted DSPC. This indicates that the addition of targeted auxiliary lipids improved the liver-specific targeting effect of LNPs. Furthermore, the LNPs prepared with PS showed the highest fluorescence proportions of DiR, Luc, and EGFP, indicating that these LNPs had the best targeting effect on the liver and the strongest ability to translate mRNA in the liver. Therefore, PS is the preferred targeted auxiliary lipid.

[0128] Example 5: Effect of ligand-modified PEGylated lipids on the targeting of lipid nanoparticles This embodiment, based on Example 4, fixes the auxiliary lipid as PS (preferred) and changes the quantity and type of ligand-modified PEGylated lipids. In addition to DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc, the ligand-modified PEGylated lipids also include DSPE-PEG2K-mannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

[0129] LNPs were prepared by combining PEGylated lipids modified with different ligands, as shown in Table 3 below.

[0130] Table 3. Combinations of PEGylated lipids with different ligand modifications Since the auxiliary lipid in this embodiment has been fixed as PS that can target LSECs, the LNPs prepared by the different ligand-modified PEGylated lipid combinations in Table 3 above are at least bivalent LNPs. That is, when there is one type of ligand-modified PEGylated lipid, it is a bivalent LNP; when there are two types of ligand-modified PEGylated lipid, it is a trivalent LNP, and so on. LNPs 1-6 are PEGylated lipids modified with one ligand (molar mass ratio of one lipid is 1.5, preparing divalent LNPs); LNPs 7-21 are PEGylated lipids modified with two ligands (molar mass ratio of two lipids is 0.75:0.75, preparing trivalent LNPs); LNPs 22-25 are PEGylated lipids modified with three ligands (molar mass ratio of three lipids is 0.5:0.5:0.5, preparing tetravalent LNPs); and LNPs 25-31 are PEGylated lipids modified with four ligands (molar mass ratio of four lipids is 0.375:0.375:0.375:0.375, preparing pentavalent LNPs). Each LNP contains two mRNAs encoding EGFP and Luc, respectively. DiR is added to the LNPs for labeling (the molar ratio of DiR is 0.3, and since the total molar ratio is 100%, the molar ratio of Chol is reduced from 38.5 to 38.2).

[0131] The above 31 LNPs were injected subcutaneously into the tail of mice at 5 μg / mouse. After 6 hours, the mice in each group were sacrificed and their organs were harvested. The organs were imaged and their luminescence or fluorescence intensity was compared. The organs included the kidney, spleen, lung, liver, heart and lymph nodes. The results showed that the fluorescence proportion in the liver of each group of mice was significantly higher than that in the other organs. The fluorescence proportions of DiR, Luc and EGFP in the liver of each group of mice are shown in Table 4 below.

[0132] Table 4. Fluorescence percentages of DiR, Luc, and EGFP in the livers of mice in each group. Based on the data in Table 4, comparing the fluorescence proportions of DiR, Luc, and EGFP in the livers of mice injected with LNPs 1-6, 7-21, 22-25, and 26-31, it was found that LNPs 1-6, 22-25, and 26-31 were less effective at targeting the liver and less capable of translating mRNA in the liver than LNPs 7-21. This indicates that LNPs prepared with one, three, or four types of ligand-modified PEGylated lipids were less effective at targeting the liver and less capable of translating mRNA in the liver than LNPs prepared with two types of ligand-modified PEGylated lipids. Further comparison of the experimental data for LNPs 7-21 revealed that the fluorescence proportions of DiR, Luc, and EGFP in the livers of mice injected with LNP 7 were higher than those injected with the remaining LNPs 8-21, indicating that LNP 7 had the best liver-targeting effect and the strongest mRNA translation capacity in the liver. The results above show that more PEGylated lipids modified with ligands do not necessarily lead to better targeting. When the auxiliary lipid is PS, the combination of PEGylated lipids modified with two ligands has a better targeting effect. The optimal choice is DSPE-PEG2K-trimannose + DSPE-PEG2K-GlcNAc. Too many ligands will reduce the targeting efficiency due to steric hindrance and receptor competition.

[0133] Based on the above experimental results, when the auxiliary lipid is PS, the preferred ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

[0134] Example 6: Treatment of Artemisia argyi allergy This embodiment verifies that the LNPs prepared in Example 1 can be used to treat Artemisia argyi allergy.

[0135] 1. Prediction, design, and mRNA preparation of antigenic epitope peptides Bet v1 protein is a major birch allergen identified in the pollen of *Betula pulcherrima* and *Betula pulcherrima*, and it induces immunoglobulin E (IgE) binding in over 95% of patients with birch pollen allergies. IgE can cause type 1 hypersensitivity reactions, manifesting as allergic rhinitis, allergic conjunctivitis, hay fever, allergic asthma, bee venom allergy, and food allergies.

[0136] In this embodiment, the NetMHCIIpan 4.1 eluent (EL) predictor was selected using IEDB to predict the binding of epitopes in Art v1 to MHC-II. While binding affinity can assess the ability of a peptide to bind to an MHC molecule, eluent (EL) prediction further integrates the possibility that the peptide has been naturally processed and presented, making the prediction results more likely to identify true extracellular T cell sites. Table 5 below shows the ranking of epitope prediction results.

[0137] Table 5. Binding of epitopes to MHC-II in Art v1 Next, based on the data in Table 5 above, and taking into account hydrophilicity, hydrophobicity and water solubility, the splicing epitopes in Table 6 below were selected.

[0138] Table 6. Segmentation Table Positions Further, a complete cDNA sequence is constructed, wherein the elements of the cDNA include a 5' UTR (containing the Kozak sequence), a coding region, and a 3' UTR. The cDNA sequence of the 5' UTR is shown in SEQ ID NO. 5, and the cDNA sequence of the 3' UTR is shown in SEQ ID NO. 6.

[0139] The coding region encodes a target sequence and at least one epitope peptide or spliced ​​epitope peptide from Tables 5-6 above. The cDNA sequence of the epitope peptide or spliced ​​epitope peptide is linked as part of the coding region using a flexible linker (in this embodiment, the GGPPG linker is selected, whose reverse-translated cDNA sequence is GGCCCGGGCCCGGGC) to facilitate peptide cleavage upon intracellular epitope release. The target sequence allows the antigen epitope to enter the MHC-II endosome compartment for peptide presentation to Treg precursor cells. In this embodiment, the target sequence is selected as the 1-80 amino acid fragment of the invariant chain (Ii), whose amino acid sequence is shown in SEQ ID NO.7, abbreviated as Ii (1-80), and whose reverse-translated cDNA sequence is shown in SEQ ID NO.8. The protein subdomain of this fragment (a molecular chaperone protein of MHC-II, which helps load the peptide into MHC-II for antigen presentation) allows the peptide epitope to enter MHC-II from the cytoplasm for CD4 cleavage. + T cell presentation. The target sequence can be Ii (1-80) or transferrin receptor.

[0140] Furthermore, codon optimization was performed on the coding region cDNA to achieve optimal gene expression of the tRNA library in non-human cells compared to human cells, using the GenScript online codon optimization tool.

[0141] Further, the optimized cDNA is transcribed into mRNA. During transcription, uridine is replaced with N1-methylpseudouridine; after transcription, a 5' cap and a PolyA tail are added to the mRNA. The 5' cap is either CleanCap or ARCA, and the PolyA tail is 100-120 nucleotides in length. The complete mRNA sequence is as follows: Figure 7 As shown.

[0142] Preferably, mRNA can be transcribed by inserting the coding region cDNA into the pTNT plasmid.

[0143] 2. Preparation of LNPs The LNPs prepared in Example 3, where the auxiliary lipid was DSPC and the ligand-modified PEGylated lipid was a single DSPE-PEG2K-trimannose, are designated as monovalent targeting LNPs. LNPs 1, 7 (same as Example 1), 22, and 26 in Table 4 of Example 5 are designated as bivalent targeting LNPs, trivalent targeting LNPs, tetravalent targeting LNPs, and pentavalent targeting LNPs, respectively. All LNPs contain the mRNA prepared in this example (the cDNA sequence of the coding region encodes the two polypeptides spliced ​​to epitope 1 in Table 6 above, i.e., the coding region cDNA sequence is shown in SEQ ID NO. 12).

[0144] 3. LNPs are used to treat artemisia allergy. A pollen allergy animal model was established using a subcutaneous (sc) sensitization method with Artemisia argyi pollen extract. The specific method was as follows: BALB / c mice were sensitized three times by subcutaneous injection of 25 μg on days 1, 8, and 15. Then, on days 22, 23, and 24, they were challenged by inhaling 1% Art v1 purified protein daily, and blood samples were collected to detect total IgE, antigen-specific IgE, and antigen-specific IgG to verify the sensitization effect. Then, on days 26, 28, 30, and 32, mice were subcutaneously injected with 2 μg of the aforementioned monovalent, bivalent, trivalent, tetravalent, and pentavalent targeted LNPs, respectively. Normal mice (blank control group) and model mice injected with an equal amount of blank LNPs (positive control group) were used for comparison. Then, on days 37, 38, 39, 40, and 41, mice were again inhaled with 1% Art v1 purified protein daily. On day 42, the animals were sacrificed, and blood, BALF, lung, and spleen samples were collected for further analysis. Paraffin sections were prepared from collected lung tissue. H&E staining was used to assess inflammatory cell infiltration in the lungs of mice in each group. Results showed that, compared with the blank control group, the positive control group mice had significant inflammatory cell infiltration in the bronchi; compared with the positive control group, mice treated with different LNPs showed significantly reduced inflammatory cell infiltration and tracheal fibrosis in the bronchi. The inflammatory infiltration of the bronchi in each group of mice was scored, and the scoring results are shown in Table 7 below.

[0145] Table 7. Inflammatory infiltration scores of bronchial pulmonary arteries in mice of each group According to the data in Table 7, compared with the positive control, the bronchial inflammation infiltration scores of mice treated with the five LNPs all decreased, but only the scores of mice injected with trivalent targeted LNPs were closest to those of the blank control (normal mice).

[0146] Cell count, IgE level, IgG level, IgG1 level, IgG2a level, CD4 in BALF + CD25 + FoxP3 + The cell percentage and IL-10 content are shown in Table 8 below.

[0147] Table 8. Cell count, IgE level, IgG level, IgG1 level, IgG2a level, and CD4 count in BALF of mice in each group. + CD25 + FoxP3 + Cell percentage and IL-10 content According to the data in Table 8, compared with the positive control, the total number of cells, IgE level, IgG level, and IgG1 level in the BALF of mice treated with the five LNPs decreased, while the proportion of Tregs and IL-10 content increased, and the IgG2a level remained basically unchanged. However, the total number of cells, IgE level, IgG level, and IgG1 level in the BALF of mice treated with trivalent targeted LNPs were the lowest, and the proportion of Tregs (CD4+) was also highest. + CD25 + FoxP3 + The highest proportion of cells and IL-10 content resulted in the best treatment effect.

[0148] This demonstrates that subcutaneous injection of trivalent targeting LNPs expressing Art v1 Artemisia v1 pollen protein epitopes can significantly induce the production of Tregs and IL-10 cytokines, thereby inhibiting pollen protein-induced allergic lung inflammation.

[0149] 3. Screening of spliced ​​table positions Furthermore, trivalent targeting LNPs were prepared from the mRNAs encoding different splicing epitopes in Table 6 according to the method in Example 1.

[0150] A pollen allergy animal model was established using a subcutaneous injection (sc) of Artemisia argyi pollen extract to induce sensitization, following the same method described above. LNPs containing mRNAs with different splicing epitopes were injected subcutaneously for treatment. Normal mice (blank control group) and model mice injected with the same amount of blank LNPs (positive control group) were compared. The bronchial inflammatory infiltration was scored in each group of mice, and the number of cells, IgE level, IgG level, IgG1 level, IgG2a level, and CD4 count in the bronchoalveolar lavage fluid (BALF) of each group were measured.+ CD25 + FoxP3 + The percentage of cells (regulatory T cells (Tregs)) and the IL-10 content were detected using the same methods as above.

[0151] The results showed that, compared with the blank control group, the positive control group mice had significant inflammatory cell infiltration in the bronchi; compared with the positive control group, mice treated with LNPs encoding mRNAs of different splicing epitopes showed significantly reduced inflammatory cell infiltration and tracheal fibrosis in the bronchi. The inflammatory infiltration of the bronchi in each group of mice was scored, and the scoring results are shown in Table 9 below.

[0152] Table 9. Inflammatory infiltration scores of bronchial pulmonary tracts in mice of each group According to the data in Table 9, compared with the positive control, the bronchial inflammation infiltration scores of mice treated with LNPs decreased, but only the scores of mice injected with LNPs encoding splicing epitope 1 were closest to those of the blank control (normal mice).

[0153] Cell count, IgE level, IgG level, IgG1 level, IgG2a level, CD4 in BALF + CD25 + FoxP3 + The percentage of cells (regulatory T cells (Tregs)) and the IL-10 content are shown in Table 10 below.

[0154] Table 10. Cell count, IgE level, IgG level, IgG1 level, IgG2a level, and CD4 count in BALF of mice in each group. + CD25 + FoxP3 + Cell percentage and IL-10 content According to the data in Table 10, compared with the positive control, the total number of cells, IgE level, IgG level, and IgG1 level in the BALF of mice treated with LNPs encoding spliced ​​epitopes were decreased, while the proportion of Tregs and IL-10 content were increased, and the IgG2a level remained basically unchanged. However, the total number of cells, IgE level, IgG level, and IgG1 level in the BALF of mice treated with LNPs encoding spliced ​​epitope 1 were the lowest, while the proportion of Tregs and IL-10 content were the highest, indicating the best treatment effect.

[0155] Therefore, the preferred combination of epitopes for encoding the mRNA coding region is the epitope combination of spliced ​​epitope 1 in Table 6.

[0156] Furthermore, APPGAAPPPAAGGSP was ultimately selected as preferred epitope 1 (SEQ ID NO. 1) and FCYFDCSKSPPGATP was selected as preferred epitope 2 (SEQ ID NO. 2). The reverse-translated cDNA sequence corresponding to preferred epitope 1 is shown in SEQ ID NO. 3, and the reverse-translated cDNA sequence corresponding to preferred epitope 2 is shown in SEQ ID NO. 4. The optimized coding region cDNA sequences are shown in SEQ ID NO. 9-12, respectively, with GC contents of 63.53%, 57.49%, 60.39%, and 57.79%, respectively. Among them, SEQ ID NO. 9 contains epitope 1, SEQ ID NO. 10 contains epitope 2, SEQ ID NO. 11 contains epitopes 1 and 2, and SEQ ID NO. 12 also contains two preferred epitopes.

[0157] Example 7: Treatment of myasthenia gravis This embodiment verifies that the LNPs prepared in Example 1 can be used to treat myasthenia gravis (MG).

[0158] 1. Prediction, design, and mRNA preparation of antigenic epitope peptides Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disease caused by autoantibody-mediated disruption of neuromuscular junction (NMJ) signaling, leading to muscle weakness, fatigue, and in severe cases, respiratory failure. The main characteristic of MG is the presence of autoantibodies targeting key NMJ proteins, primarily the acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-associated protein 4 (LRP4). Approximately 85% of MG patients are AChR positive, and their CD4+... + T-cell-dependent autoreactive B-cell activation leads to the production of pathogenic anti-AChR autoantibodies. These antibodies trigger receptor internalization, complement-mediated postsynaptic membrane lysis, and neuromuscular signaling disorders, resulting in progressive muscle weakness, ptosis, and difficulty swallowing or breathing. In contrast, MuSK-positive MG (accounting for 5-10%) involves IgG4 autoantibodies disrupting AChR aggregation, while LRP4-positive MG is rarer and its pathological mechanism is unclear. Immune dysregulation in MG is closely related to HLA inheritance, especially HLA-DR3 and HLA-DQ8, which play key roles in antigen presentation and autoreactive T / B cell responses.

[0159] The main pathogenic targets of MG are AChR and MuSK, so this embodiment will screen the preferred epitopes of these two pathogenic targets.

[0160] (1) Selection of AChR epitopes The results indicate that the α subunit of nicotinic AChR (amino acid sequence shown in SEQ ID NO. 13) is crucial in the pathogenesis of myocardial infarction (MG), an autoimmune paralytic disease, as it contains both epitopes that dominate the anti-AChR antibody response and epitopes recognized by CD4+ AChR-specific helper T (Th) cells. The distribution of different epitopes in the α subunit of AChR is shown in the figure below. Figure 8 As shown.

[0161] like Figure 9 The image shows the results of detecting the proliferative response of different epitopes of the α subunit of AChR in four different MG patients (Pt 3 / 7 / 10 / 11). Among them, Pt 3 / 7 / 10 used CD4+. + Lymphocytes were enriched, and peripheral blood mononuclear cells (PBMCs) were used for Pt11. Results showed that, on the one hand, multiple patients shared common epitopes: α118-137 induced significant proliferation in all four patients with extremely high proliferative intensity, making it the most dominant epitope; α304-322 also significantly proliferated in Pt3 and Pt10, and was another important dominant epitope. On the other hand, there were significant individual differences in the immune response to the α subunit epitopes of AChR among different MG patients: for example, Pt3 induced extremely strong proliferation of α48-67, and Pt10 induced significant proliferation of α387-405, etc.

[0162] Figure 10 The light gray bars with specks represent fresh immune cells (PBMCs and CD4) isolated from the peripheral blood of seven MG patients. + The frequency of recognition of different peptide epitopes of the α subunit of AChR by enriched cells showed that α118-137 had the highest number of responders and was the most widely recognized epitope in the peripheral blood of MG patients. α304-322 was also a frequently recognized epitope, and multiple responses were also observed in regions such as α48-67 and α387-405. The black bars represent those obtained in previous studies by stimulating anti-AChR CD4 cells cultured from four MG patients over a long period. + The frequency of T cell lines recognizing these peptides shows that the black bars for α48-67 and α304-322 are the highest, indicating that these two epitopes are core targets that are widely recognized in four long-term T cell lines. The black bar for α419-437 is found in about three cell lines and is also a high-frequency epitope recognized in cell lines.

[0163] Furthermore, to clarify the epitope repertoire of anti-AChR Th cells, this embodiment tested unsorted blood CD4 cells from 22 MG patients. + The response of cells and / or total lymphocytes to a 20-amino acid overlap synthetic polypeptide covering the full sequence of the human muscle AChR α subunit.

[0164] The test results show: (1) Only the most severely ill patients were identified with α subunit epitopes, and these were mainly young women; (2) In vitro detection of AChR-specific CD4 + The response needs to remove CD8. + Cells: Two patients used CD8 + In cell-depleted samples, a clear response to multiple α-subunit polypeptide sequences was detected, while the total peripheral blood mononuclear cell population showed no response to any α-subunit polypeptide. (3) Patients' peptide recognition patterns are unique to each individual, but long-term AChR-specific CD4 + The four most frequently recognized immunodominant regions in T cell lines or adjacent polypeptide sequences are residues 48-67, 101-137, 293-337, and 408-437; followed by 89-105 and 320-337.

[0165] The key epitope sequences of the AChR α subunit are shown in Table 11 below.

[0166] Table 11. Key epitope sequences of AChR α subunit (2) Selection of Musko site The full-length amino acid sequence of MuSK is shown in SEQ ID NO. 26. MuSK-Ig1 is located at positions 21-125 of the full-length amino acid sequence. This example provides an overview of the correlation between disease severity scores and MuSK-Ig1 epitope pattern responsiveness in 22 Italian patients with genotype G. The correlation results are as follows: Figure 11 As shown, the MuSK-Ig1 epitope is highly correlated with the disease severity score, while other epitopes are less correlated, suggesting that the Ig1 epitope is a major driver of MG disease progression. Therefore, Ig1 was chosen as the target epitope for mRNA immunotherapy.

[0167] The key peptide sequences of the MuSK-Ig1 domain obtained by screening with NetmHciipan_el 4.1 in IEDB are shown in Table 12 below.

[0168] Table 12. Key peptide sequences of the MuSK-Ig1 domain Next, based on hydrophilicity, hydrophobicity, and water solubility, the splicing epitopes in Table 13 below were selected.

[0169] Table 13, Segmentation Table Positions (3) Preparation of mRNA Further, a complete cDNA sequence is constructed, the cDNA comprising a 5' UTR (including the Kozak sequence), a coding region, and a 3' UTR. The nucleotide sequences of the 5' UTR and 3' UTR are the same as in Example 6. The coding region encodes a target sequence and at least one epitope sequence from Tables 11-13, with multiple epitopes linked by linkers as part of the coding region. The leader sequence and linker sequences are the same as in Example 6. The cDNA coding region is further codon-optimized using the same method as in Example 6. The optimized cDNA is then transcribed into mRNA using the same method as in Example 6.

[0170] 2. Preparation of LNPs The LNPs prepared in Example 3, where the auxiliary lipid was DSPC and the ligand-modified PEGylated lipid was a single DSPE-PEG2K-trimannose, are designated as monovalent targeting LNPs. LNPs 1, 7 (same as Example 1), 22, and 26 in Table 4 of Example 5 are designated as bivalent targeting LNPs, trivalent targeting LNPs, tetravalent targeting LNPs, and pentavalent targeting LNPs, respectively. All LNPs contain the mRNA prepared in this example (the cDNA sequence of the coding region encodes the nine polypeptides spliced ​​to epitope 3 in Table 13 above, i.e., the coding region cDNA sequence is shown in SEQ ID NO. 34).

[0171] 3. LNPs are used to treat MG. Using a transgenic mouse model expressing HLA-DR3 and HLA-DQ8, AChR-responsive CD4 was transferred via adoptive transfer. + MG mouse models were constructed by T cell or direct AChR immunization. MG mouse models were intravenously injected with trivalent targeting LNPs containing mRNAs with different splicing epitopes, while the control group was injected with blank LNPs. Foxp3 levels in the spleen of each group of mice were measured. + The efficacy was evaluated by measuring the percentage of Tregs, the levels of pro-inflammatory cytokines (IFN-γ, IL-17), the levels of autoantibodies in the blood, and the clinical score. The test results are shown in Table 14 below.

[0172] Table 14. Foxp3 lymph nodes in each group of mice + The percentage of Tregs, the levels of pro-inflammatory cytokines (IFN-γ, IL-17), the levels of autoantibodies, and clinical scores. Table 14 shows that, compared with the control group, mice in the monovalent or multivalent LNP-targeting groups had Foxp3 levels +Increased Tregs, decreased pro-inflammatory cytokines IFN-γ and IL-17, decreased autoantibody levels, and improved clinical scores; among them, the trivalent LNPs-targeted mice showed the best performance in all test indicators, indicating that it had the strongest ability to induce immune tolerance, the best effect in controlling inflammatory response, effectively blocked autoantibody-mediated neuromuscular junction damage, and basically relieved symptoms such as muscle weakness and ptosis, which were close to normal levels.

[0173] Trivalent targeting of LNPs synergistically targets multiple receptors on the surface of LSECs, with simultaneous binding of multiple receptors significantly enhancing the specific uptake efficiency of LSECs, thereby efficiently presenting antigen epitopes and inducing Foxp3. + Tregs are produced in large quantities, while inhibiting the activation of Th1 or Th17 cells and reducing the production of pro-inflammatory factors such as IFN-γ and IL-17, as well as autoantibodies.

[0174] 4. Screening of spliced ​​table positions Furthermore, trivalent targeting LNPs were prepared from the mRNAs encoding different splicing epitopes in Table 13 according to the method in Example 1.

[0175] The MG mouse model was constructed using the same method as above. MG mice were treated with intravenous injections of LNPs containing different spliced ​​epitopes, while the control group was injected with blank LNPs. Foxp3 levels in the lymph nodes of each group of mice were measured. + The percentage of Tregs, the content of pro-inflammatory cytokines (IFN-γ, IL-17), the level of autoantibodies, and the clinical score were measured using the same methods as above. The results are shown in Table 15 below.

[0176] Table 15. Foxp3 lymph nodes in each group of mice + The percentage of Tregs, the levels of pro-inflammatory cytokines (IFN-γ, IL-17), the levels of autoantibodies, and clinical scores. Table 15 shows that, compared with the control group, mice in the group encoding LNPs mRNA splicing epitopes had Foxp3 levels + Increased Tregs, decreased pro-inflammatory cytokines IFN-γ and IL-17, decreased autoantibody levels, and improved clinical scores; among them, the LNPs group of mice encoding splice epitope 3 showed the best performance in all test indicators, indicating that it had the strongest ability to induce immune tolerance, the best effect in controlling inflammatory response, effectively blocked autoantibody-mediated neuromuscular junction damage, and basically relieved symptoms such as muscle weakness and ptosis, which were close to normal levels.

[0177] Therefore, the preferred combination of epitopes for the mRNA coding region is the epitope combination of spliced ​​epitopes 3 in Table 13.

[0178] Furthermore, the mRNA coding region preferably encodes all the polypeptide sequences shown in SEQ ID NO. 14-19 and 27-29. The cDNA sequence corresponding to the mRNA coding region is shown in SEQ ID NO. 33, and the optimized cDNA sequence of the coding region is shown in SEQ ID NO. 34, with a GC content of 55.08%.

[0179] Example 8: Treatment of toxic diffuse goiter This embodiment verifies that the LNPs prepared in Example 1 can be used to treat toxic diffuse goiter (GD disease).

[0180] Prediction, design, and mRNA preparation of antigenic epitope peptides The pathogenesis of Graves' disease (GD) involves a misrecognition by the patient's immune system, which mistakes the thyroid-stimulating hormone receptor (TSHR) on the thyroid follicular cell membrane for a "foreign antigen," thereby inducing B lymphocytes to produce specific autoantibodies against TSHR. Furthermore, patients have an imbalance between the Th1 and Th2 helper somatic cell (Th) subsets. Th2 cells are hyperactive, and their secreted cytokines (such as IL-4 and IL-10) promote B cell activation and the production of large amounts of autoantibodies. Simultaneously, the regulatory T cell (Treg) function is deficient, failing to effectively suppress the abnormal immune response, further exacerbating the autoimmune disorder.

[0181] The amino acid sequence of the TSHR is shown in SEQ ID NO.35. The first to fourth 12 amino acids of the sequence are the extracellular domains of the TSHR, which are the main targets of autoantibodies. The frequency of HLA-DR3 in GD patients is 40-55% (15-30% in the general population), and those who carry HLA-DR3 have a 3-4 times increased risk of developing the disease.

[0182] (1) Investigation of the binding affinity of the extracellular domain of TSHR to HLA-DR3 The predicted affinity of the TSHR extracellular domain for HLA-DR3 based on the NIAID IEDB database is shown in Table 16 below.

[0183] Table 16. Predicted results of TSHR extracellular epitope binding affinity to HLA-DR3 (2) Investigation of the binding affinity of the extracellular domain of TSHR to H2-IAd / H2-IEd Epitope prediction in animal experiments was based on the binding affinity of the TSHR extracellular domain to H2-IAd / H2-IEd, and the results are shown in Table 17 below.

[0184] Table 17. Prediction of binding affinity between TSHR extracellular epitopes and H2-IAd / H2-IEd Based on the results in Tables 16-17 above, and further considering hydrophilicity, hydrophobicity, and water solubility, multiple groups of mRNA splicing epitopes suitable for animal models (human TSHR immunization) and patients were screened, as shown in Table 18 below.

[0185] Table 18. TSHR's preferred epitopes and their reverse-transcribed cDNA sequences (3) Preparation of mRNA Further, a complete cDNA sequence is constructed, the cDNA comprising a 5' UTR (including the Kozak sequence), a coding region, and a 3' UTR. The nucleotide sequences of the 5' UTR and 3' UTR are the same as in Example 6. The coding region encodes a target sequence and at least one epitope sequence from Tables 16-18, with multiple epitopes linked by linkers as part of the coding region. The leader sequence and linker sequences are the same as in Example 6. The cDNA coding region is further codon-optimized using the same method as in Example 6. The optimized cDNA is then transcribed into mRNA using the same method as in Example 6.

[0186] 2. Preparation of LNPs The LNPs prepared in Example 3, where the auxiliary lipid was DSPC and the ligand-modified PEGylated lipid was a single DSPE-PEG2K-trimannose, are designated as monovalent targeting LNPs. LNPs 1, 7 (same as Example 1), 22, and 26 in Table 4 of Example 5 are designated as bivalent targeting LNPs, trivalent targeting LNPs, tetravalent targeting LNPs, and pentavalent targeting LNPs, respectively. All LNPs contain the mRNA prepared in this example (the cDNA sequence of the coding region encodes the eight polypeptides spliced ​​to epitope 1 in Table 18 above, i.e., the coding region cDNA sequence is shown in SEQ ID NO. 52).

[0187] 3. LNPs for the treatment of GD disease The GD mouse model was constructed as follows: Susceptible strain mice (such as BALB / c) were sensitized multiple times by using plasmid DNA encoding the TSHR A subunit (TSHR-289) via muscle electroporation.

[0188] This induction method successfully stimulates the production of biologically active stimulating antibodies (TSAbs). These antibodies bind to TSHR on the surface of thyroid cells and continuously activate downstream signaling pathways, leading to elevated thyroid hormone levels, diffuse thyroid enlargement, and thyroid follicular epithelial hyperplasia.

[0189] GD mouse models were treated with monovalent, bivalent, trivalent, quadrivalent, and pentavalent targeted LNPs, respectively, while the control group of GD mouse models was injected with blank LNPs. The efficacy was evaluated by measuring the levels of thyroid-stimulating hormone receptor antibody (TRAb), the Th1 / Th2 helper somatic cell ratio, and the levels of IL-4 and IL-10 in the blood of each group of mice. The results are shown in Table 19 below.

[0190] Table 19. Levels of thyroid-stimulating hormone receptor antibody (TRAb), the ratio of Th1 to Th2 helper somatic cells, and the levels of IL-4 and IL-10 in the blood of mice in each group. As shown in Table 19, compared with the control group, mice in the monovalent or multivalent LNPs-targeted groups had decreased TRAb levels, increased Th1 / Th2 ratios, decreased IL-4 levels, and increased IL-10 levels. Among them, the trivalent LNPs-targeted group had the lowest TRAb and IL-4 levels, the highest Th1 / Th2 ratios, and the highest IL-10 levels, indicating that it effectively blocked TSHR antibody-mediated thyroid dysfunction. The Th1 / Th2 ratio approached or even returned to the normal range, correcting the characteristic Th2 hyperactivity state of GD disease. The decrease in IL-4 levels significantly inhibited the pro-inflammatory function of Th2 cells, and the increase in IL-10 levels enhanced the anti-inflammatory immune response.

[0191] Trivalent targeting of LNPs synergistically targets multiple receptors on the surface of LSECs, significantly enhancing the specific uptake efficiency of LSECs. After efficient uptake of LNPs, LSECs precisely present the TSHR-encoded preferred epitopes on mRNA, thereby inducing Foxp3. + Tregs proliferate and secrete anti-inflammatory factors such as IL-10; on the other hand, they inhibit Th2 cell activation, reduce IL-4 secretion and TRAb production, and ultimately achieve immune balance restoration.

[0192] 4. Screening of spliced ​​table positions Furthermore, trivalent targeting LNPs were prepared from mRNAs containing different splicing epitopes in Table 18 according to the method in Example 1.

[0193] Construct a GD mouse model using the same method as above.

[0194] GD mouse models were treated with trivalent targeted LNPs containing mRNAs with different splicing epitopes, while control GD mouse models were treated with blank LNPs. The efficacy was evaluated by measuring the levels of thyroid-stimulating hormone receptor antibody (TRAb), the ratio of Th1 to Th2 helper somatic cells, and the levels of IL-4 and IL-10 in the blood of each group of mice. The results are shown in Table 20 below.

[0195] Table 20. Levels of thyroid-stimulating hormone receptor antibody (TRAb), the ratio of Th1 to Th2 helper somatic cells, and the levels of IL-4 and IL-10 in the blood of mice in each group. As shown in Table 20, compared with the control group, mice in the LNPs group (encoding spliced ​​epitopes) had decreased TRAb levels, increased Th1 / Th2 ratios, decreased IL-4 levels, and increased IL-10 levels. Among them, mice in the LNPs group (encoding spliced ​​epitope 1) had the lowest TRAb and IL-4 levels, the highest Th1 / Th2 ratios, and the highest IL-10 levels, indicating that it effectively blocked TSHR antibody-mediated thyroid dysfunction. The Th1 / Th2 ratio approached or even returned to the normal range, correcting the characteristic Th2 hyperactivity state of GD disease. The decrease in IL-4 levels significantly inhibited the pro-inflammatory function of Th2 cells, and the increase in IL-10 levels enhanced the anti-inflammatory immune response.

[0196] Therefore, the preferred combination of epitopes for the mRNA coding region is the one that splices epitopes 1 in Table 18.

[0197] Further, the mRNA coding region preferably encodes all the polypeptide sequences shown in SEQ ID NO. 36-43, and the reverse-transcribed cDNA sequences corresponding to the polypeptide sequences shown in SEQ ID NO. 36-43 are shown in SEQ ID NO. 44-51. When the mRNA coding region encodes all the polypeptide sequences shown in SEQ ID NO. 36-43, the corresponding optimized coding region cDNA sequence is shown in SEQ ID NO. 52, with a GC content of 58.63%.

[0198] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

[0199] sequence list SEQ ID NO.1 The amino acid sequence of Art v1 preferred epitope 1: APPGAAPPPAAGGSP SEQ ID NO.2 The amino acid sequence of Art v1 preferred epitope 2: FCYFDCSKSPPGATP SEQ ID NO.3 The reverse cDNA sequence of Art v1 preferred epitope 1: GCTCCCCCCGGGGCTGCTCCCCCCCCCGCTGCTGGGGCAGCCCC SEQ ID NO.4 The reverse cDNA sequence of Art v1 preferred epitope 2: TTCTGCTACTTCGATTGCAGCAAAAGCCCCCCCGGCGCGACCCCC SEQ ID NO.5 cDNA sequence of the 5'UTR: GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC SEQ ID NO.6 cDNA sequence of 3'UTR: CTCTTCCTCTATGCTCTTCCTGTGCTCTTCCTCTATGCTCTTCCTCTCAAAAAAAAAAAAAAAGCATAAATAACTAAAATACCCAGTCAAGTTACTATTAGTAGATAG SEQ ID NO. 7 The amino acid sequence of Ii (1-80): MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDK SEQ ID NO. 8 The cDNA sequence of Ii(1-80) reverse translation: ATGCATCGCCGCCGCAGCCGCAGCTGCCGCGAAGATCAGAAACCGGTGATGGATGATCAGCGCGATCTGATTAGCAACAACGAACAGCTGCCGATGCTGGGCCGCCGCCCGGGCGCGCCGGAAAGCAAATGCAGCCGCGGCGCGCTGTATACCGGCTTTAGCATTCTGGTGACCCTGCTGCTGGCGGGCCAGGCGACCACCGCGTATTTTCTGTATCAGCAGCAGGGCCGCCTGGATAAA SEQ ID NO.9 mRNA encoding sequence optimized for the coding region of Art v1 epitope 1: ATGGATGACCAGAGAGACTTGATTTCCAACCACGAGCAACTTCCCATACTGGGCAATAGACCCCGGGAACCAGAACGCTGCAGCCGAGGAGCGCTCTATACTGGTGTTTCTGTCCTGGTGGCCTTACTTTTGGCTGGCCAGGCCACCACAGCCTACTTCCTGTACCAGCAGCAAGGCAGGCTGGATAAACTAACCATCACGTCCCAGAACCTGCAGCTGGAGAGCCTCAGGATGAAGCTCGGGCCTGGGCCCGGAACACCAGCACCACCTGGTGCAGCCCCTCCCCCGGCTGCTGGAGGGTCTCCTTCACCTCCAGCCGGCCCTGGCCCGGGCACTCCTGCGCCCCCGGGAGCTGCCCCACCCCCTGCTGCAGGTGGCAGTCCCAGTCCACCAGCAGGGCCAGGGCCTGGATAA SEQ ID NO.10 mRNA encoding sequence optimized for the coding region of Art v1 epitope 2: ATGGATGACCAGAGGGATCTCATTTCCAACCACGAGCAGCTTCCCATCCTGGGCAACAGACCGCGAGAACCAGAACGCTGCTCGAGAGGAGCCCTGTATACAGGGGTCTCTGTTCTGGTGGCTTTGTTACTAGCAGGTCAAGCCACCACAGCCTACTTCCTCTACCAGCAGCAAGGCCGGCTGGACAAGCTCACCATAACTTCTCAGAATCTGCAGCTGGAGAGCTTGAGGATGAAGCTTGGTCCTGGGCCAGGGTCCTGTTTCTGCTACTTTGACTGTAGTAAAAGTCCCCCGGGGGCGACTCCAGCACCCCCAGGAGGACCTGGACCAGGCAGCTGCTTCTGTTATTTTGATTGCAGCAAATCACCTCCAGGTGCTACGCCTGCTCCTCCCGGAGGCCCCGGCCCTGGCTAA SEQ ID NO.11 mRNA encoding sequence optimized for the coding region of Art v1 epitopes 1 + 2: ATGGATGACCAGAGAGATCTCATTTCCAACCACGAGCAGTTGCCCATCCTGGGAAATAGACCTCGGGAGCCAGAACGCTGTAGCAGGGGTGCTCTCTATACTGGAGTCTCTGTTCTGGTGGCCCTGTTATTGGCTGGCCAGGCCACCACAGCCTACTTCCTCTACCAGCAGCAAGGTCGACTGGACAAGCTTACGATAACCAGCCAGAACCTGCAACTAGAAAGTCTTAGGATGAAGCTGGGCCCCGGGCCAGGCACTCCAGCCCCTCCAGGAGCCGCTCCCCCACCAGCTGCAGGAGGGAGTCCGTCGCCCCCCGCAGGCCCTGGTCCGGGCTCCTGCTTCTGCTATTTTGACTGTTCTAAATCACCCCCTGGTGCGACACCTGCACCTCCCGGAGGCCCAGGGCCTGGGTAA SEQ ID NO.12 [[ID=IO]]Optimized nucleotide sequence of the mRNA coding region for treating Artemisia allergy: ATGGATGACCAGAGGGATCTCATATCCAATCATGAGCAGTTGCCCATCCTGGGGAACAGACCCCGAGAACCTGAACGCTGCAGCCGTGGTGCTCTCTATACTGGAGTTTCAGTCCTGGTGGCCCTTCTACTGGCTGGCCAGGCTACCACAGCCTACTTCCTCTACCAGCAGCAAGGCCGGTTAGACAAACTTACGATCACCTCTCAAAACCTGCAGCTGGAGTCATTGAGGATGAAGCTGGGTCCTGGCCCAGGAGCGGGCAGCAAACTTTGTGAAAAAACCTCGAAAACATATTCTGGGAAGTGTGATAATAAGAAATGTGACAAGAAGTGCATTGAGTGGGAGAAGGCCCAGCACGGAGCGTGCCACAAGAGAGAAGCTGGAAAGGAGAGCTGCTTCTGCTACTTTGACTGTTCAAAATCCCCTCCTGGGGCCACTCCAGCACCCCCAGGTGCAGCCCCGCCACCTGCCGCAGGTGGCAGTCCCAGTCCCCCTGCAGACGGAGGCAGCCCACCGCCGCCAGCTGATGGAGGGTCCCCACCTGTGGATGGGGGCTCTCCACCCCCCCCCAGTACACATGGGCCTGGACCTGGCTAA SEQ ID NO.13 Amino acid sequence of the α subunit of AChR: MEPWPLLLLFSLCSAGLVLGSEHETRLVAKLFKDYSSVVRPVEDHRQVVEVTVGLQLIQLINVDEVNQIVTTNVRLKQQWVDYNLKWNPDDYGGVKKIHIPSEKIWRPDLVLYN NADGDFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDEQNCSMKLGTWTYDGSVVAINPESDQPDLSNFMESGEWVIKESRGWKHSVTYSCCPDTPYLDITYHFVMQ RLPLYFIVNVIIPCLLFSFLTGLVFYLPTDSGEKMTLSISVLLSLTVFLLVIVELIPSTSSAVPLIGKYMLFTMVFVIASIIITVIVINTHHRSPSTHVMPNWVRKVFIDTIPN IMFFSTMKRPSREKQDKKIFTEDIDISDISGKPGPPPMGFHSPLIKHPEVKSAIEGIKYIAETMKSDQESNNAAAEWKYVAMVMDHILLGVFMLVCIIGTLAVFAGRLIELNQQG SEQ ID NO.14 The amino acid sequence of the key epitope of the AChR α subunit: EVNQIVTTNVRLKQQWVDYNLK SEQ ID NO.15 The amino acid sequence of the key epitope of the AChR α subunit: RPDLVLYNNADGDFAIVKFTK SEQ ID NO.16 The amino acid sequence of the key epitope of the AChR α subunit: DFAIVKFTKVLLQYTGHITWTPPAIFKSYCEIIVTHFPFDE SEQ ID NO.17 The amino acid sequence of the key epitope of the AChR α subunit: VIVINTHHRSPSTHVMPNWVRKVFIDTIPNIMFFSTMKRPSREKQDKKI SEQ ID NO.18 The amino acid sequence of the key epitope of the AChR α subunit: DTIPNIMFFSTMKRPSREKQDK SEQ ID NO.19 The amino acid sequence of the key epitope of the AChR α subunit: MDHILLGVFMLVCIIGTLAVFAGRLIELNQQG SEQ ID NO.20 Reverse-translated cDNA sequence of the key epitope of the AChR α subunit: GAAGTGAACCAGATTGTGACCACCAACGTGCGCCTGAAACAGCAGTGGGTGGATTATAACCTGAAA SEQ ID NO.21 Reverse-translated cDNA sequence of the key epitope of the AChR α subunit: CGCCCGGATCTGGTGCTGTATAACAACGCGGATGGCGATTTTGCGATTGTGAAATTTACCAAA SEQ ID NO.22 Reverse-translated cDNA sequence of the key epitope of the AChR α subunit: GATTTTGCGATTGTGAAATTTACCAAAGTGCTGCTGCAGTATACCGGCCATATTACCTGGACCCCGCCGGCGATTTTTAAAAGCTATTGCGAAATTATTGTGACCCATTTTCCGTTTGATGAA SEQ ID NO.23 Reverse-translated cDNA sequence of the key epitope of the AChR α subunit: GTGATTGTGATTAACACCCATCATCGCAGCCCGAGCACCCATGTGATGCCGAACTGGGTGCGCAAAGTGTTTATTGATACCATTCCGAACATTATGTTTTTTAGCACCATGAAACGCCCGAGCCGCGAAAAACAGGATAAAAAAAATT SEQ ID NO.24 Reverse-translated cDNA sequence of the key epitope of the AChR α subunit: GATACATTCCGAACATTATGTTTTTTAGCACCATGAAACGCCCGAGCCGCGAAAAACAGGATAAA SEQ ID NO.25 Reverse-translated cDNA sequence of the key epitope of the AChR α subunit: ATGGATCATATTCTGCTGGGCGTGTTTATGCTGGTGTGCATTATTGGCACCCTGGCGGTGTTTGCGGGCCGCCTGATTGAACTGAACCAGCAGGGC SEQ ID NO.26 The full-length amino acid sequence of MuSK: MRELVNIPLVHILTLVAFSGTEKLPKAPVITTPLETVDALVEEVATFMCAVESYPQPEISWTRNKILIKLFDTRYSIRENGQLLTILSVEDSDDGIYCCTANNGVGGAVESCGALQVKMKPKITRPPINVKIIEGLKAVLPCTTMGNPKPSVSWIKGDSPLRENSRIAVLESGSLRIHNVQKEDAGQYRCVAKNSLGTAYSKVVKLEVEVFARILRAPESHNVTFGSFVTLHCTATGIPVPTITWIENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATISIAEWSKPQKDNKGYCAQYRGEVCNAVLAKDALVFLNTSYADPEEAQELLVHTAWNELKVVSPVCRPAAEALLCNHIFQECSPGVVPTPIPICREYCLAVKELFCAKEWLVMEEKTHRGLYRSEMHLLSVPECSKLPSMHWDPTACARLPHLDYNKENLKTFPPMTSSKPSVDIPNLPSSSSSSFSVSPTYSMTVIISIMSSFAIFVLLTITTLYCCRRRKQWKNKKRESAAVTLTTLPSELLLDRLHPNPMYQRMPLLLNPKLLSLEYPRNNIEYVRDIGEGAFGRVFQARAPGLLPYEPFTMVAVKMLKEEASADMQADFQREAALMAEFDNPNIVKLLGVCAVGKPMCLLFEYMAYGDLNEFLRSMSPHTVCSLSHSDLSMRAQVSSPGPPPLSCAEQLCIARQVAAGMAYLSERKFVHRDLATRNCLVGENMVVKIADFGLSRNIYSADYYKANENDAIPIRWMPPESIFYNRYTTESDVWAYGVVLWEIFSYGLQPYYGMAHEEVIYYVRDGNILSCPENCPVELYNLMRLCWSKLPADRPSFTSIHRILERMCERAEGTVSV SEQ ID NO.27 Amino acid sequence of the key epitope of MuSK-Ig1 domain: TEKLPKAPVITTPLETVDALVEEVATFMCAVESYP SEQ ID NO.28 Amino acid sequence of key epitopes in the MuSK-Ig1 domain: QPEISWTRNKILIKLFDTRYSIRENGQLLTILSVE SEQ ID NO.29 Amino acid sequence of key epitopes in the MuSK-Ig1 domain: DSDDGIYCCTANKVGGAVESCGALQVKMKPKITR SEQ ID NO.30 Reverse-translated cDNA sequence of key epitopes of the MuSK-Ig1 domain: ACCGAAAAACTGCCGAAAGCGCCGGTGATTACCACCCCGCTGGAAACCGTGGATGCGCTGGTGGAAGAAGTGGCGACCTTTATGTGCGCGGTGGAAAGCTATCCG SEQ ID NO.31 Reverse-translated cDNA sequence of key epitopes of the MuSK-Ig1 domain: CAGCCGGAAATTAGCTGGACCCGCAACAAAATTCTGATTAAACTGTTTGATACCCGCTATAGCATTCGCGAAAACGGCCAGCTGCTGACCATTCTGAGCGTGGAA SEQ ID NO.32 Reverse-translated cDNA sequence of key epitopes of the MuSK-Ig1 domain: GATAGCGATGATGGCATTTATTGCTGCACCGCGAACAACGGCGTGGGCGGCGCGTGGAAAGCTGCGGCGCGCTGCAGGTGAAAATGAAACCGAAAATTACCCGC SEQ ID NO.33 The coding region of the mRNA used to treat MG was not optimized in the reverse-translated cDNA sequence. SEQ ID NO.34 The optimized coding region of the mRNA for treating MG was reverse-translated into cDNA sequence. SEQ ID NO.35 Amino acid sequence of TSHR: MRPADLLQLVLLLDLPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMGYKFLRIVVWFVSLLALLGNVFVLLILLTSHYKLNVPRFLMCNLAFADFCMGMYLLLIASVDLYTHSEYYNHAIDWQTGPGCNTAGFFTVFASELSVYTLTVITLERWYAITFAMRLDRKIRLRHACAIMVGGWVCCFLLALLPLVGISSYAKVSICLPMDTETPLALAYIVFVLTLNIVAFVIVCCCYVKIYITVRNPQYNPGDKDTKIAKRMAVLIFTDFICMAPISFYALSAILNKPLITVSNSKILLVLFYPLNSCANPFLYAIFTKAFQRDVFILLSKFGICKRQAQAYRGQRVPPKNSTDIQVQKVTHDMRQGLHNMEDVYELIENSHLTPKKQGQISEEYMQTVL SEQ ID NO.36 Amino acid sequence of preferred epitope of TSHR: NISRIYVSIDVTLQQLESH SEQ ID NO.37 Amino acid sequence of preferred epitope of TSHR: DLLQLVLLLDLPRDLGGMG SEQ ID NO.38 Amino acid sequence of TSHR preferred epitopes: NTGLKMFPDLTKVYSTDIF SEQ ID NO.39 Amino acid sequence of TSHR preferred epitopes: DVSQTSVTALPSKGLEHLK SEQ ID NO.40 Amino acid sequence of TSHR preferred epitopes: TLKLIETHLRTIPSHAFSN SEQ ID NO.41 Amino acid sequence of TSHR preferred epitopes: YNLSKVTHIEIRNTRNLTY SEQ ID NO.42 Amino acid sequence of TSHR preferred epitopes: STQTLKLIETHLRTIPSHA SEQ ID NO.43 Amino acid sequence of TSHR preferred epitopes: LSLSFLHLTRADLSYPSHC SEQ ID NO.44 Reverse-translated cDNA sequence of TSHR preferred epitope: AACATTAGCCGCATTTATGTGAGCATTGATGTGACCCTGCAGCAGCTGGAAAGCCAT SEQ ID NO.45 Reverse-translated cDNA sequence of TSHR preferred epitope: GATCTGCTGCAGCTGGTGCTGCTGCTGGATCTGCCGCGGCATCTGGGCGGCATGGGC SEQ ID NO.46 Reverse-translated cDNA sequence of TSHR preferred epitope: AACACCGGCCTGAAAATGTTTCCGGATCTGACCAAAGTGTATAGCACCGATATTTTT SEQ ID NO.47 Reverse-translated cDNA sequence of TSHR preferred epitope: GATGTGAGCCAGACCAGCGTGACCGCGCTGCCGAGCAAAGGCCTGGAACATCTGAAA SEQ ID NO.48 Reverse-translated cDNA sequence of TSHR preferred epitope: ACCCTGAAACTGATTGAAACCCATCTGCGCACCATTCCGAGCCATGCGTTTAGCAAC SEQ ID NO.49 Reverse-translated cDNA sequence of TSHR preferred epitope: TATAACCTGAGCAAAGTGACCCATATTGAAATTCGCAACACCCGCAACCTGACCTAT SEQ ID NO.50 Reverse-translated cDNA sequence of TSHR preferred epitope: AGCACCCAGACCCTGAAACTGATTGAAACCCATCTGCGCACCATTCCGAGCCATGCG SEQ ID NO.51 Reverse-translated cDNA sequence of TSHR preferred epitope: CTGAGCCTGAGCTTTCTGCATCTGACCCGCGCGGATCTGAGCTATCCGAGCCATTGC SEQ ID NO.52 The optimized coding region of the mRNA for treating GD disease was reverse-translated into cDNA sequence. ATGGATGACCAGAGAGACCTTATCTCCAACCATGAGCAGCTGCCCATCTTAGGGAATCGGCCTCGGGAGCCAGAGCGCTGCTCTCGGGGAGCCCTGTACACAGGTGTGTCAGTGCTGGTGGCACTGCTGTTGGCCGGGCAGGCCACCACCGCCTACTTCCTTTACCAGCAGCAAGGAAGACTGGATAAACTGACCATCACTTCCCAAAACCTGCAGCTGGAGTCATTGAGGATGAAACTGGGACCCGGCCCTGGAAACATCAGCCGCATTTATGTGAGCATTGATGTGACCTTGCAGCAATTGGAAAGTCATGGACCTGGGCCTGGAGACCTCCTCCAGCTTGTACTGCTCCTGGACCTGCCTCGGGACCTGGGTGGCATGGGGGGCCCTGGCCCGGGCAACACTGGCCTGAAGATGTTTCCAGACCTGACCAAGGTCTACTCGACAGATATCTTTGGACCCGGCCCTGGTGATGTCAGCCAGACCTCTGTGACGGCGCTGCCTTCTAAGGGACTGGAGCACCTTAAGGGACCAGGCCCTGGAACCCTGAAGCTGATAGAAACACACTTGAGAACCATCCCGAGCCATGCCTTCTCCAATGGCCCTGGGCCTGGCTACAATCTGAGCAAGGTGACACACATCGAAATTAGAAACACACGTAACCTGACCTATGGTCCAGGCCCCGGCAGTACCCAGACCCTGAAGCTCATCGAGACCCACCTAAGAACAATCCCATCCCACGCAGGCCCCGGCCCCGGCCTCAGCCTGTCTTTCCTGCACCTGACACGAGCTGACCTGTCCTACCCAAGCCACTGTGGGCCGGGCCCGGGCTAA SEQ ID NO.53 Amino acid sequence of the preferred epitope of TSHR: ISRIYVSIDVTLQQL [[ID=Z]]SEQ ID NO.54 Amino acid sequence of the preferred epitope of TSHR: VSQTSVTALPSKGLE SEQ ID NO.55 Amino acid sequence of TSHR preferred epitopes: LSLSFLHLTRADLSY SEQ ID NO.56 Amino acid sequence of TSHR preferred epitopes: QLVLLLDLPRDLGGM SEQ ID NO.57 Amino acid sequence of TSHR preferred epitopes: LKLIETHLRTIPSHA SEQ ID NO.58 Amino acid sequence of TSHR preferred epitopes: TLKLIETHLRTIPSH SEQ ID NO.59 Amino acid sequence of TSHR preferred epitopes: TGLKMFPDLTKVYST SEQ ID NO.60 Amino acid sequence of TSHR preferred epitopes: NPYMTSIPVNAFQGL SEQ ID NO.61 Amino acid sequence of TSHR preferred epitopes: SLSFLHLTRADLSYP.

Claims

1. A lipid nanoparticle characterized in that, It includes cationic ionized lipids, auxiliary lipids, ligand-modified PEGylated lipids, and cholesterol; the ligands in the ligand-modified PEGylated lipids can target receptors on the surface of hepatic sinusoidal endothelial cells, and the auxiliary lipids are selected from lipids that can target receptors on the surface of hepatic sinusoidal endothelial cells or lipids without targeting.

2. The lipid nanoparticle of claim 1, wherein, The cationic ionized lipid is SM102; the auxiliary lipid includes any one of PS, DOPS and DPPS; the ligand-modified PEGylated lipid includes at least any one of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose and DSPE-PEG2K-hyaluronic acid.

3. The lipid nanoparticle of claim 2, wherein, The auxiliary lipid is PS; the ligand-modified PEGylated lipid is any two of DSPE-PEG2K-mannose, DSPE-PEG2K-GlcNAc, DSPE-PEG2K-trimannose, DSPE-PEG2K-GalNAc-4-sulfate, DSPE-PEG2K-L-fucose, and DSPE-PEG2K-hyaluronic acid.

4. The lipid nanoparticle of claim 3, wherein, The ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

5. The lipid nanoparticle of claim 1, wherein, The molar ratio of the cationic ionized lipid, auxiliary lipid, ligand-modified PEGylated lipid, and cholesterol is 20~70: 1~15: 1~5: 25~45, and the sum of the molar ratios of each component is 100%; the N / P ratio of the cationic ionized lipid is 2~6.

6. The lipid nanoparticle of claim 1, wherein, The lipid nanoparticles have a particle size of 80-200 nm; the lipid nanoparticles encapsulate RNA drugs for treating allergic or autoimmune diseases.

7. The lipid nanoparticle of claim 6, wherein, The allergic disease is Artemisia argyi allergy, and the autoimmune disease is myasthenia gravis and toxic diffuse goiter.

8. The lipid nanoparticles as described in claim 7, characterized in that, The RNA encapsulated in the lipid nanoparticles for treating Artemisia argyi allergy has a coding region that encodes at least one of the amino acid sequences shown in SEQ ID No. 1-2; the RNA encapsulated in the lipid nanoparticles for treating myasthenia gravis has a coding region that encodes at least one of the amino acid sequences shown in SEQ ID No. 14-19 and 27-29; the RNA encapsulated in the lipid nanoparticles for treating toxic diffuse goiter has a coding region that encodes at least one of the amino acid sequences shown in SEQ ID No. 36-43.

9. Use of the lipid nanoparticles as described in claims 1 to 8 in the preparation of formulations that enhance the ability to target hepatic sinusoidal endothelial cells.

10. Use of the lipid nanoparticles as described in claims 1 to 8 in the preparation of formulations that enhance the therapeutic effect on allergic or autoimmune diseases.

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