A mRNA lipid nanoparticle drug composition for treating birch hay fever, multiple sclerosis, and Sjögren's syndrome.

Abstract

This invention discloses an mRNA-lipid nanoparticle drug composition for treating birch hay fever, multiple sclerosis, and Sjögren's syndrome. The core component is a functionalized mRNA encoding antigenic epitopes associated with allergic or autoimmune diseases. The coding region of the mRNA contains screened and validated encoding sequences for specific antigenic epitopes related to birch hay fever, multiple sclerosis, and Sjögren's syndrome, which can be combined in tandem via flexible linkers. Simultaneously, the mRNA carries optimized 5'UTR, 3'UTR, and 5' cap structures and a polyA tail, exhibiting both high stability and efficient translational expression capabilities after codon optimization and nucleotide modification. The mRNA is delivered via lipid nanoparticles targeting hepatic sinusoidal endothelial cells, effectively inducing antigen-specific immune tolerance and significantly improving disease indicators in model animals, providing a novel targeted therapeutic approach for birch hay fever, multiple sclerosis, and Sjögren's syndrome.

Description

[0001] This application claims priority to the earlier Chinese application, application number 2025109003751, filed on July 1, 2025; all its contents are part of this invention.

[0002] 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. Technical Field

[0003] This invention belongs to the field of drug delivery, and specifically relates to an mRNA lipid nanoparticle drug composition for treating birch hay fever, multiple sclerosis, and Sjögren's syndrome. Background Technology

[0004] Autoimmune and allergic diseases pose a serious threat to human health. Current clinical treatments primarily rely on anti-inflammatory drugs, non-specific immunosuppressants, monoclonal antibodies, and conventional immunomodulatory regimens. For example, birch pollen allergy is a seasonal type I allergic disease caused by birch pollen, and most existing drugs only control symptoms and cannot cure the disease. Multiple sclerosis is a chronic autoimmune demyelinating disease of the central nervous system; it is currently impossible to completely correct immune abnormalities or eradicate the cause to achieve long-term remission, and patients with progressive disease generally respond poorly to current treatments. Sjögren's syndrome is a chronic autoimmune disease primarily affecting exocrine glands such as the lacrimal and salivary glands. Currently, clinical treatments can only control symptoms through symptomatic replacement therapy or immunomodulatory drugs, and there are no means to treat the underlying cause. While these methods can alleviate symptoms to some extent and reduce disease activity in the short term, they cannot achieve long-term control of the chronic course, let alone a cure. Patients need long-term medication and face multiple side effects such as infection and metabolic disorders. Therefore, developing novel treatment strategies that can induce antigen-specific immune tolerance and correct immune imbalances at their root has become a core issue that urgently needs to be addressed in this field. Regulatory T cells (Tregs) are key cells in maintaining immune homeostasis. Antigen-specific immune tolerance therapy, developed based on the biological characteristics of Tregs, can provide long-term remission for autoimmune diseases such as multiple sclerosis and Sjögren's syndrome, as well as allergic diseases such as birch hay fever. Biodegradable nanocarriers can encapsulate disease-related antigens and target their delivery to antigen-presenting cells (APCs), effectively initiating and maintaining immune regulatory responses, making them ideal carriers for inducing antigen-specific tolerance.

[0005] The liver possesses natural immune tolerance characteristics, preventing the body from generating abnormal immune responses to exogenous food antigens from the gastrointestinal tract and portal vein. It also exhibits immune preference in organ transplantation; liver-kidney / heart transplantation significantly reduces the incidence of rejection. This characteristic is closely related to its unique antigen presentation system. Hepatic Kupffer cells (KCs), dendritic cells (DCs), and sinusoidal endothelial cells (LSECs) together constitute a tolerance antigen presentation network. LSECs can efficiently take up nanoparticles of approximately 200 nm through clathrin-mediated endocytosis and directly regulate the immunosuppressive effects of CD4+ and CD8+ T cells by secreting TGF-β, upregulating PD-L1, and inducing antigen-specific Treg production. Therefore, LSECs are the core target cells mediating hepatic immune tolerance and treating immune-related diseases.

[0006] LSECs exhibit high expression of multiple specific receptors on their surface, providing a molecular basis for precise targeting by nanocarriers: SR-E1 / LOX-1 and SR-H1 / STABILIN-1 specifically recognize anionic ligands such as phosphatidylserine (PS) and dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS); the SR-E3 / mannose receptor (CD206) binds to glycosyl ligands such as mannose, trimannose, and L-fucose; LSECtin / CLEC4G specifically recognizes N-acetylglucosamine (GlcNAc) and mannose; and LYVE-1 is a hyaluronic acid-specific receptor. All of these receptors mediate receptor-dependent endocytosis, serving as key targets for the efficient targeted delivery of LSECs.

[0007] Lipid nanoparticles (LNPs) are important carriers for delivering antigens to induce immune tolerance. Existing technologies attempt to target LSECs by modifying LNPs with a single mannose ligand. Although this can improve uptake efficiency and Treg induction levels, single ligand targeting has defects such as insufficient specificity and easy non-specific uptake by hepatocytes and non-parenchymal cells, resulting in low antigen delivery efficiency and weak immune tolerance induction effect, which is difficult to meet the needs of clinical treatment.

[0008] In summary, there is still a lack of lipid nanoparticle delivery systems that can target LSECs with high specificity and efficiency, which prevents the precise delivery of disease-related antigens and efficient induction of antigen-specific immune tolerance, thus hindering the development of radical therapies for autoimmune and allergic diseases. Summary of the Invention

[0009] This invention discloses an mRNA-lipid nanoparticle drug composition for treating birch hay fever, multiple sclerosis, and Sjögren's syndrome. The core component is a functionalized mRNA encoding epitopes associated with autoimmune diseases. The coding region of the mRNA contains screened and validated encoding sequences for specific epitopes related to birch hay fever, multiple sclerosis, and Sjögren's syndrome, which can be combined in tandem via flexible linkers. Simultaneously, the mRNA incorporates optimized 5'UTR, 3'UTR, and 5' cap structures and a polyA tail, exhibiting both high stability and efficient translational expression after codon optimization and nucleotide modification. The mRNA is delivered via lipid nanoparticles targeting hepatic sinusoidal endothelial cells, effectively inducing antigen-specific immune tolerance and significantly improving disease indicators in model animals, providing a novel targeted therapeutic approach for birch hay fever, multiple sclerosis, and Sjögren's syndrome.

[0010] This invention provides an mRNA lipid nanoparticle pharmaceutical composition for treating birch hay fever, multiple sclerosis, and Sjögren's syndrome. The pharmaceutical composition comprises lipid nanoparticles and mRNA encapsulated within the lipid nanoparticles; the coding region of the mRNA contains a coding sequence for at least one antigenic epitope associated with allergies or autoimmune diseases, wherein the antigenic epitope is selected from at least one of the following groups: (a) Birch pollen-associated antigenic epitopes, the amino acid sequences of which are any one or more of the sequences shown in SEQ ID NO. 2 to SEQ ID NO. 7; (b) Multiple sclerosis-associated antigenic epitopes, the amino acid sequences of which are any one or more of the sequences shown in SEQ ID NO.20 to SEQ ID NO.27; (c) Sjögren's syndrome-associated epitopes, the amino acid sequences of which are any one or more of the sequences shown in SEQ ID NO.41 to SEQ ID NO.50.

[0011] The mRNA lipid nanoparticle drug composition for treating birch hay fever, multiple sclerosis, and Sjögren's syndrome described in this invention is based on the use of multivalent lipid nanoparticles targeting hepatic sinusoidal endothelial cells (LSECs) as delivery carriers to precisely encapsulate mRNA encoding disease-specific autoantigen epitopes. Through the antigen presentation pathway mediated by LSECs, antigen-specific immune tolerance is induced, thereby achieving targeted treatment for various autoimmune diseases such as birch hay fever, multiple sclerosis, and Sjögren's syndrome.

[0012] The coding region of the mRNA contains highly affinity and hydrophilic autoantigen epitopes, selected and verified by calculation, and tailored to different disease types. It incorporates a combination of core pathogenic epitopes corresponding to the specific disease: for diseases related to major birch allergens, it incorporates related epitopes such as Bet v1 protein; for diseases related to autoimmune T cell attack on myelin in the central nervous system (CNS), it incorporates epitopes such as human MBP protein, human MOG protein, and human PLP protein; and for Sjögren's syndrome, it incorporates target proteins for SS treatment such as RO60, TRIM21, lupus La, and SPTN1. All of these epitopes are immunodominant epitopes that can be efficiently presented by HLA-II molecules, effectively activating regulatory T cells (Tregs) and inhibiting abnormal activation of autoreactive T cells.

[0013] 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.

[0014] When mRNA contains multiple epitopes, the coding sequences of each epitope are tandemly linked by flexible linkers to ensure effective cleavage and presentation of the epitopes within the cell. Simultaneously, upstream fusion of targeting sequences into the coding regions guides the antigenic epitopes into the MHC-II endosomal compartment, enhancing their conversion to CD4+. + The mRNA enhances T cell presentation efficiency and improves immune tolerance induction. It undergoes codon optimization, N1-methylpseuuridine modification, 5' capping, and 3' polyA tailing, resulting in high stability and high translation efficiency, enabling continuous expression of the target antigen epitope within hepatic sinusoidal endothelial cells.

[0015] The pharmaceutical composition of the present invention, after intravenous administration, can rapidly accumulate in the liver and be taken up by LSECs, inducing Foxp3 by presenting disease-specific antigenic epitopes. + Tregs proliferate in large numbers, while simultaneously inhibiting the levels of pro-inflammatory factors such as IL-4, IFN-γ, and IL-17, suppressing the production of autoantibodies, alleviating tissue damage caused by diseases, and achieving long-term control and functional protection of allergic or autoimmune diseases.

[0016] Preferably, the linker is a glycine-serine (Gly-Ser) linker or a GGPPG linker.

[0017] Furthermore, the coding region of the mRNA contains the coding sequences of all antigenic epitopes shown in SEQ ID NO.8 to SEQ ID NO.13, and the nucleotide sequence of the coding region is shown in SEQ ID NO.16.

[0018] Furthermore, the coding region of the mRNA contains the coding sequences of all antigenic epitopes shown in SEQ ID NO.28 to SEQ ID NO.35, and the nucleotide sequence of the coding region is shown in SEQ ID NO.36.

[0019] Furthermore, the coding region of the mRNA contains the coding sequences of all antigenic epitopes shown in SEQ ID NO.51 to SEQ ID NO.60, and the nucleotide sequence of the coding region is shown in SEQ ID NO.61.

[0020] Furthermore, the mRNA also includes a 5'-cap structure, a 5'-UTR, a coding region, a 3'-UTR, and a poly A tail.

[0021] 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.

[0022] 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.

[0023] Furthermore, the sequences of the 5'-UTR and 3'-UTR are independently derived from at least one of natural and synthetic proteins.

[0024] Preferably, the natural protein includes any one of α-globulin, β-globulin, and heat shock protein HSP70.

[0025] Furthermore, the 5'-UTR contains a Kozak sequence.

[0026] 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.

[0027] The nucleotide sequence of the 5'-UTR is shown in SEQ ID NO.62, and the nucleotide sequence of the 3'-UTR is shown in SEQ ID NO.63.

[0028] Furthermore, a target sequence is inserted upstream of the coding region.

[0029] 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), whose amino acid sequence is shown in SEQ ID NO.14. 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 polypeptide epitope to enter the MHC-II from the cytoplasm for CD44 expression. + T cell presentation. The target sequence can be Ii (1-80) or transferrin receptor.

[0030] 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.

[0031] 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).

[0032] Due to the fragility of RNA as a drug, 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 loaded mRNA to bind multiple ligands to the same LNP, ensuring the specific targeting of LNPs to LSECs.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] The PS (phosphatidylserine) targets SR-E1 / LOX-1 and SR-H1 / STABILIN-1 on the surface of LSECs.

[0037] 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.

[0038] The DPPS (1,2-dipalmitoyl-sn-glycerol-3-phosphate-L-serine) targets the SR-E1 / LOX-1 receptor of LSECs.

[0039] 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.

[0040] 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.

[0041] In some embodiments, the types of target-specific auxiliary lipids were screened, and the prepared LNPs showed the best targeting effect on the liver when the auxiliary lipid was PS.

[0042] Furthermore, the ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

[0043] 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.

[0044] 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.

[0045] Preferably, when the cationic ionized lipid is SM102, the N / P ratio of SM102 is 6.

[0046] Furthermore, the lipid nanoparticles have a particle size of 80~200 nm.

[0047] The pore diameter in LSECs typically ranges from 50 to 200 nm, 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.

[0048] Furthermore, the mass ratio of mRNA to lipid nanoparticles is 1~10:20~100.

[0049] Preferably, the mass ratio of mRNA to lipid nanoparticles is 1:40.

[0050] In some methods, lipid nanoparticles are prepared using microfluidic techniques.

[0051] This invention utilizes 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 hay fever and autoimmune diseases such as multiple sclerosis and Sjögren's syndrome, but rather covers a variety of allergic and autoimmune diseases that conform to the core pathological feature 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 mRNA coding region sequence, and using the lipid nanoparticle delivery system of this invention. This invention has broad applicability and scalability.

[0052] The present invention has the following beneficial effects: 1. Precise and efficient mRNA sequence design, combining high stability and high expression activity: The mRNA of this invention is computationally screened (using tools such as IEDB and NetMHCIIpan for prediction) to obtain immunodominant antigenic epitopes that bind with high affinity to high-risk HLA types of diseases, accurately covering the core pathogenic antigens of birch pollen allergy, multiple sclerosis, and Sjögren's syndrome; multiple epitopes are tandemly linked through flexible linkers, and the antigen presentation efficiency is improved by combining them with MHC-II targeting sequences; at the same time, codon optimization, nucleotide modification, and 5' cap and 3' polyA tail optimization significantly improve the in vivo stability and translational expression efficiency of mRNA, solving the pain points of easy mRNA degradation and low expression efficiency.

[0053] 2. Precisely tailored targeted delivery system ensures efficient intracellular delivery of mRNA: This invention uses multivalent lipid nanoparticles targeting hepatic sinusoidal endothelial cells to encapsulate mRNA. Through the synergistic targeting effect of auxiliary lipids and dual ligands, it significantly enhances the specific uptake of mRNA by hepatic sinusoidal endothelial cells and reduces non-specific phagocytosis by non-target cells such as hepatocytes. This solves the problems of poor transmembrane delivery and poor targeting of mRNA, ensuring that mRNA efficiently expresses target antigen epitopes in target cells and stably exerts its therapeutic effect.

[0054] 3. The treatment is highly effective and can achieve long-term stable control of the disease: 4. Root cause mechanism of action and excellent therapeutic safety: This invention delivers disease-specific antigen mRNA to induce antigen-specific immune tolerance, correcting the body's immune imbalance at its root, rather than the non-specific immunosuppression of traditional therapies. This avoids the side effects of long-term use of immunosuppressants, such as systemic infection and metabolic disorders, resulting in strong therapeutic targeting and higher safety.

[0055] 5. Strong platform scalability and wide applicability: The mRNA design and delivery system of this invention has platform characteristics. It can be adapted to autoimmune diseases and allergic diseases with different pathogenic antigens by simply replacing the antigenic epitope sequence in the mRNA coding region. It covers a variety of diseases with "immune tolerance destruction" as the core pathological feature and has extremely strong clinical expansion and application value. Attached Figure Description

[0056] 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 3The 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 6 The 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 The binding of the RO60 epitope to the HLA allele in Example 8; Figure 8 This shows the binding of the TRIM21 epitope to the HLA allele in Example 8; Figure 9 This shows the binding of the lupus La epitope to the HLA allele in Example 8; Figure 10 This shows the binding of the SPTN1 epitope to the HLA allele in Example 8. Detailed Implementation

[0057] 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.

[0058] 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 loaded RNA. SM102 is a cationic ionized lipid, phosphatidylserine is an accessory lipid, and DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc are ligand-modified PEGylated lipids. The RNA serves as a drug for treating allergic or autoimmune diseases. The RNA 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 causing an allergic or autoimmune disease.

[0059] The specific preparation method is as follows: The components for preparing LNPs were prepared using a microfluidic method, with an ethanol phase to aqueous phase ratio of 1:3 (the ethanol phase contains all lipid components, and the aqueous phase contains mRNA), a total flow rate of 12 mL / min, and a molar mass ratio of SM102:PS:Chol:DSPE-PEG2K-trimannose:DSPE-PEG2K-GlcNAc of 50:10:38.5:0.75:0.75, wherein the N / P ratio of SM102 was 6, and the mass ratio of mRNA to lipid nanoparticles was 1~10:20~100 (preferably 1:40). The prepared LNPs were dialyzed using PBS.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] Furthermore, 20 μg mRNA of LNPs prepared from different cationic ionized lipids was injected into the tail vein 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.

[0065] 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.

[0066] In summary, the preferred cationic ionized lipid in LNPs is SM102, and the preferred N / P ratio of SM102 is 6.

[0067] 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).

[0068] The five LNPs were injected at a rate of 20 μg mRNA per mouse into the tail vein. Six hours later, the location of the LNPs and the expression of the 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.

[0069] The fluorescence percentages of Luc, DiR, and EGFP in various organs of the five groups of mice, and the fluorescence intensities of Luc, DiR, 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 2The 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 2 The 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 (trivalent targeting 2).

[0070] 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.

[0071] 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).

[0072] The four LNPs were injected at 5 μg / mouse into the tail vein of mice. 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.

[0073] 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.

[0074] 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.

[0075] LNPs were prepared by combining PEGylated lipids modified with different ligands, as shown in Table 3 below.

[0076] 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).

[0077] The 31 LNPs were injected at 5 μg / mouse into the tail vein of mice. 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 4 below.

[0078] 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. The combination of PEGylated lipids modified with two ligands has a better targeting effect, with DSPE-PEG2K-trimannose + DSPE-PEG2K-GlcNAc being the optimal choice. Too many ligands will reduce the targeting efficiency due to steric hindrance and receptor competition.

[0079] 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.

[0080] Example 6: Treatment of birch hay fever This embodiment verifies that the LNPs prepared in Example 1 can be used to treat hay fever caused by birch pollen.

[0081] 1. Prediction, design, and mRNA preparation of antigenic epitope peptides Bet v1 protein is a major birch allergen identified in the pollen of birch and weeping birch. Its amino acid sequence is shown in SEQ ID NO.1. It induces IgE binding in more than 95% of patients with birch pollen allergies. IgE can cause type 1 hypersensitivity reactions, which manifest as allergic rhinitis, allergic conjunctivitis, hay fever, allergic asthma, bee venom allergy, and food allergy.

[0082] MHC-II epitopes were searched using the Immune Epitope Database (IEDB) and the NetMHCIIpan 4.1 EL tool. Table 5 below lists the epitopes in the Bet v1 protein that have a high binding percentile with the aforementioned MHC-II epitopes.

[0083] Table 5. Epitopes in the Bet v1 protein that have high binding percentages to the aforementioned MHC-II epitopes. Based on HLA allele coverage, six preferred Bet v1 epitope peptides were selected from Table 5 above, as shown in Table 6 below.

[0084] Table 6. Six preferred epitope peptides of Bet v1 Table 7. Reverse translation of preferred epitope peptides Next, based on the data in Table 7 above, and taking into account hydrophilicity, hydrophobicity and water solubility, the splicing epitopes in Table 8 below were selected.

[0085] Table 8. Splicing 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. 62, and the cDNA sequence of the 3' UTR is shown in SEQ ID NO. 63.

[0086] The coding region encodes a target sequence and at least one epitope peptide or spliced ​​epitope peptide from Tables 7-8 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 endosomal 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), as shown in SEQ ID NO.14, abbreviated as Ii (1-80), and its reverse-translated cDNA sequence is shown in SEQ ID NO.15. 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 polypeptide 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.

[0087] 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. This was accomplished using the GenScript online codon optimization tool.

[0088] 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, wherein the 5' cap is CleanCap or ARCA, and the PolyA tail is 100-120 nucleotides in length.

[0089] Preferably, mRNA can be transcribed by inserting the coding region cDNA into the pTNT plasmid.

[0090] 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 is shown in SEQ ID NO. 16).

[0091] 3. LNPs are used to treat hay fever caused by birch pollen. Establishment of a mouse model of hay fever: A pollen allergy animal model was established by sensitization and immunization with 25 μg of birch pollen extract (SC). The challenge step was inhalation of 1% Bet v1 purified protein.

[0092] The monovalent, bivalent, trivalent, tetravalent, and pentavalent targeted LNPs were administered intravenously at 5 μg / mouse to hay fever model mice, respectively. The control group consisted of model mice injected with an equal amount of blank LNPs. After a period of treatment, Foxp3 levels in the lymph nodes and mucosal tissues of each group of mice were measured. + The percentage of regulatory T cells and the expression levels of inflammatory factors IL-4, IL-5 and IL-13 were detected, and the results are shown in Table 9 below.

[0093] Table 9, Foxp3 + The proportion of regulatory T cells and the expression levels of inflammatory factors IL-4, IL-5 and IL-13. LNP therapy can induce Foxp3. +The generation of regulatory T cells (Tregs) allows these Tregs to migrate to draining lymph nodes and mucosal tissues, thereby inducing tolerance responses to allergens. On the other hand, it can inhibit Th2 cells, reduce IgE production, lower the levels of IL-4, IL-5, and IL-13, and limit the degranulation of mast cells and eosinophils, thus effectively controlling allergic reactions. Table 9 shows that, compared with the control group, mice injected with targeted LNPs showed increased levels of Foxp3... + The proportions of Tregs were significantly increased, while the levels of IL-4, IL-5, and IL-13 were significantly decreased. Comparing the mice injected with targeted LNPs, the trivalent targeted LNP (same as in Example 1) Foxp3... + Tregs had the highest proportion and the lowest levels of IL-4, IL-5, and IL-13, indicating that they were the most effective in treating allergic reactions.

[0094] Based on the above experimental results, it is evident that trivalent targeted LNPs can significantly increase Foxp3 levels when used to treat hay fever caused by birch pollen or birch extracts. + The reduction of regulatory T cell ratio and the decrease in IL-4, IL-5, and IL-13 inflammatory factor levels can effectively alleviate hay fever or oral allergy syndrome caused by birch pollen or birch extract.

[0095] This embodiment successfully designed a multi-epitope mRNA and related products (LNPs) that can tandemly encode Bet v1 protein-related antigens, which can significantly increase Foxp3 + The reduction of regulatory T cell ratio and the decrease in IL-4, IL-5, and IL-13 inflammatory factor levels can effectively alleviate hay fever or oral allergy syndrome caused by birch pollen or birch extract.

[0096] 4. Screening of spliced ​​table positions Furthermore, trivalent targeting LNPs were prepared from the mRNAs encoding different splicing epitopes in Table 8 according to the method in Example 1.

[0097] A mouse model of hay fever was constructed using the same method as above. The mice were treated with intravenous injection of LNPs containing mRNAs with different splicing epitopes, using the same methods and detection indicators as above.

[0098] Foxp3 in lymph nodes and mucosal tissues of mice in each group + The percentage of regulatory T cells and the expression levels of inflammatory factors IL-4, IL-5 and IL-13 were detected, and the results are shown in Table 10 below.

[0099] Table 10, Foxp3 + The proportion of regulatory T cells and the expression levels of inflammatory factors IL-4, IL-5 and IL-13. Table 10 shows that mice injected with mRNA encoding LNPs spliced ​​with different epitopes all showed increased Foxp3 levels compared to control mice. + The proportion of regulatory T cells was reduced, and the levels of inflammatory factors IL-4, IL-5, and IL-13 were decreased. Among these, the LNPs group encoding splicing epitope 3 in mRNA showed the most significant effect. Therefore, the preferred mRNA coding region is the combination of epitopes encoding splicing epitope 3 in Table 8. The optimized nucleotide sequence of the mRNA coding region encoding splicing epitope 3 is shown in SEQ ID NO.16, with a GC content of 56.74%, where the sequence encoding Ii (1-80) is upstream of the sequences encoding the six epitopes.

[0100] Example 7: Treatment of Multiple Sclerosis 1. Prediction, design, and mRNA preparation of antigenic epitope peptides Multiple sclerosis (MS) is a chronic autoimmune disease in which the body's own T cells attack myelin in the central nervous system (CNS), leading to demyelination of nerve fibers and neurodegeneration.

[0101] In this embodiment, three core proteins of myelin were selected, including human MBP protein (amino acid sequence as shown in SEQ ID NO. 17), human MOG protein (amino acid sequence as shown in SEQ ID NO. 18), and human PLP protein (amino acid sequence as shown in SEQ ID NO. 19). The binding of epitope peptides in the above three core proteins to MHC-II was predicted using the NetmHciipan_el 4.1 server. Epitopes with high affinity and located in the conserved region of the antigen were selected. The results of the preferred epitopes are shown in Table 11 below.

[0102] Table 11. Preferred Epitope Screening Results for MBP, MOG, and PLP Furthermore, the reverse-translated cDNA sequences of the eight preferred epitopes in Table 11 above are shown in Table 12 below.

[0103] Table 12. Reverse-translated cDNA sequences of 8 preferred epitopes Next, based on the data in Table 11 above, and taking into account hydrophilicity, hydrophobicity and water solubility, the splicing epitopes in Table 13 below were selected.

[0104] Table 13, Segmentation Table Positions Furthermore, a complete cDNA sequence was constructed using the same method as in Example 6, and the codons of the coding region cDNA were further optimized using the same optimization method as in Example 6.

[0105] Furthermore, the optimized cDNA is transcribed into mRNA, using the same method as in Example 6.

[0106] 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 is shown in SEQ ID NO. 36).

[0107] 3. LNPs for the treatment of MS Experimental autoimmune encephalomyelitis (EAE) induced by oligodendrocyte glycoprotein (MOG) is one of the most widely used animal models. MOG is a key antigen located in the outermost layer of myelin sheath in the central nervous system, possessing strong immunogenicity and capable of inducing a specific immune attack against the myelin sheath. In establishing a mouse model of MS, mice are subcutaneously immunized with a mixture of MOG (35-55) peptide fragments and complete Freund's adjuvant (CFA), supplemented with pertussis toxin (PTX) injection to increase blood-brain barrier permeability. This model highly mimics the core pathological features of MS, including extensive inflammatory cell infiltration in the central nervous system, widespread demyelinating lesions, and the resulting progressive limb paralysis. Because the immune response in this model is mainly driven by Th1 and Th17 cells, accompanied by impaired Treg function, it has become a core system for evaluating the ability of the mRNA-LNP platform to induce immune tolerance. By targeting the liver to deliver mRNA encoding MOG epitopes, systemic antigen-specific Tregs can be effectively induced to expand, thereby inhibiting effector T cell attacks on the nervous system and restoring immune balance.

[0108] MS mouse models were treated with intravenous injections of monovalent, bivalent, trivalent, tetravalent, and pentavalent targeted LNPs, respectively, once every three weeks for a total of three times. The control group received blank LNPs. Two weeks after the last injection, peripheral blood was collected to detect autoantibody levels, and mice in each group were then sacrificed, and spleens were collected to detect Foxp3. +The results of the Treg percentage, the detection of IFN-γ, IL-17A and TNF-α levels in central nervous system tissue, and the scoring of nerve fiber damage are shown in Table 14 below.

[0109] Table 14. Peripheral blood autoantibody levels and spleen Foxp3 levels in mice of each group + The percentage of Tregs, the levels of IFN-γ, IL-17A, and TNF-α in the central nervous system, and the nerve fiber damage score As shown in Table 14, compared with the control group, the autoantibody levels in mice in the monovalent or multivalent LNP-targeting groups decreased, and Foxp3... + The proportion of Tregs increased, while the levels of IFN-γ, IL-17A, and TNF-α decreased, and the neurofibrillary damage score decreased. Among the mice, the trivalent LNP-targeted group had the lowest levels of autoantibodies, Foxp3. + The highest proportion of Tregs, the lowest levels of IFN-γ, IL-17A and TNF-α, and the lowest score of nerve fiber damage indicate that it has the strongest ability to induce antigen-specific immune tolerance, effectively controls inflammatory infiltration in the central nervous system, and significantly alleviates demyelination and axonal damage, bringing them close to normal levels.

[0110] Trivalent LNPs synergistically target multiple receptors on the surface of LSECs, significantly enhancing the specific uptake efficiency of LSECs. After efficient uptake of LNPs by LSECs, they induce Foxp3... + Tregs proliferate in large numbers and secrete anti-inflammatory factors to inhibit the activation of autoreactive T cells. On the other hand, they inhibit the secretion of pro-inflammatory factors such as IFN-γ and IL-17A by Th1 / Th17 cells, reduce the production of autoantibodies, thereby blocking demyelination and nerve fiber damage and improving the pathological process of MS.

[0111] 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.

[0112] MS model mice were constructed using the same method as above. Treatment was administered via intravenous injection of LNPs containing mRNAs with different splicing epitopes, using the same methods and detection indicators as above.

[0113] Peripheral blood autoantibody levels and spleen Foxp3 levels in mice of each group + The percentage of Tregs, the content of IFN-γ, IL-17A and TNF-α in the central nervous system, and the nerve fiber damage score were measured. The results are shown in Table 15 below.

[0114] Table 15. Peripheral blood autoantibody levels and spleen Foxp3 levels in mice of each group + The percentage of Tregs, the levels of IFN-γ, IL-17A, and TNF-α in the central nervous system, and the nerve fiber damage score Table 15 shows that mice injected with mRNA encoding LNPs spliced ​​with different epitopes were able to induce Foxp3 compared to the control group. + Tregs proliferate extensively, inhibiting the secretion of pro-inflammatory factors such as IFN-γ and IL-17A by Th1 / Th17 cells and reducing the production of autoantibodies, thereby blocking demyelination and nerve fiber damage. The LNPs group encoding splice epitope 3 showed the most significant effect. Therefore, the preferred mRNA coding region encodes the epitope combination of splice epitope 3 in Table 13. The optimized cDNA sequence of the mRNA coding region encoding splice epitope 3 is shown in SEQ ID NO.36, with a GC content of 60.37%.

[0115] Example 8: Treatment of Sjögren's Syndrome 1. Prediction, design, and mRNA preparation of antigenic epitope peptides Sjögren's syndrome (SS) is an autoimmune disease caused by the combined effects of genetic susceptibility (such as HLA alleles, including HLA-DRB1*03:01, HLA-DQA1*05:01, and HLA-A*03) and environmental triggers (such as viral infection and epigenetic modification) that break immune tolerance. Its core mechanism is the activation of specific T / B lymphocytes, which produce autoantibodies such as anti-SSA / Ro and anti-SSB / La. Immune cells infiltrate and damage exocrine glands such as salivary glands and lacrimal glands, leading to decreased glandular secretion function, accompanied by systemic inflammatory response and multi-organ involvement.

[0116] This embodiment provides four target proteins for treating SS: RO60 (amino acid sequence as shown in SEQ ID NO. 37), TRIM21 (amino acid sequence as shown in SEQ ID NO. 38), lupus La (amino acid sequence as shown in SEQ ID NO. 39), and SPTN1 (amino acid sequence as shown in SEQ ID NO. 40). The peptides (epitopes) of these four target proteins that can bind to MHC-II protein were predicted using the NetmHciipan_el 4.1 server.

[0117] The binding of epitopes to HLA alleles in RO60, TRIM21, lupus La, and SPTN1 is as follows: Figures 7-10 As shown, from Figures 7-10The preferred epitopes of four target proteins and their flanking sequences are shown in Table 16 below.

[0118] Table 16. Preferred epitopes of four target proteins and their flanking sequences The inverse cDNA sequences corresponding to the peptides with flanking sequences in Table 16 are shown in Table 17 below.

[0119] Table 17. Reverse cDNA sequences corresponding to peptides with flanking sequences Next, based on the data in Table 17 above, and taking into account hydrophilicity, hydrophobicity and water solubility, the splicing epitopes in Table 18 below were selected.

[0120] Table 18. Segmentation Table Positions Furthermore, a complete cDNA sequence was constructed using the same method as in Example 6, and the codons of the coding region cDNA were further optimized using the same optimization method as in Example 6.

[0121] Furthermore, the optimized cDNA is transcribed into mRNA, using the same method as in Example 6.

[0122] 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 is shown in SEQ ID NO. 61).

[0123] 3. LNPs treatment for SS The salivary gland (SS) mouse model is established by immunizing susceptible mice with specific exocrine gland antigens or glandular extracts, supplemented by a potent adjuvant, thereby breaking peripheral immune tolerance. A commonly used antigen is salivary gland extract (SG-extract). During modeling, the antigen is typically mixed with complete Freund's adjuvant (CFA) for multiple subcutaneous sensitizations, activating effector T cells (such as Th1 and Th17 cells) and inhibiting Treg activity, triggering chronic destructive inflammation of the lacrimal and salivary glands. This model effectively mimics the typical periglandular lymphocytic infiltration and subsequent decline in glandular secretory function seen in human SS.

[0124] SS mouse models were treated with intravenous injections of monovalent, bivalent, trivalent, tetravalent, and pentavalent targeted LNPs, respectively, once every three weeks for a total of three times. The control group received blank LNPs. Two weeks after the last injection, peripheral blood was collected to detect the levels of anti-SSA / Ro and anti-SSB / La antibodies. Mice in each group were then sacrificed, and salivary glands were collected to detect Foxp3. + The percentage of Tregs, the content of IL-17 and IFN-γ, and the detection results are shown in Table 19 below.

[0125] Table 19. Levels of anti-SSA / Ro and anti-SSB / La antibodies in mice of each group, and Foxp3 levels in the spleen. + Tregs percentage, central nervous system IFN-γ and IL-17 levels As shown in Table 19, compared with the control group, the levels of autoantibodies against SSA / Ro and SSB / La in mice targeting LNPs in monovalent or multivalent groups were decreased. + The proportion of Tregs increased, while the levels of IFN-γ and IL-17A decreased. The trivalent LNP-targeting group of mice showed the lowest levels of two autoantibodies, Foxp3. + The highest proportion of Tregs and the lowest levels of IFN-γ and IL-17A indicate that it has the strongest ability to induce antigen-specific immune tolerance, effectively controls salivary gland and systemic inflammatory infiltration, and effectively reduces autoantibody-mediated exocrine gland damage.

[0126] Trivalent LNPs synergistically target multiple receptors on the surface of LSECs, significantly enhancing the specific uptake efficiency of LSECs. After efficient uptake of LNPs by LSECs, they induce Foxp3... + Tregs proliferate in large numbers and secrete anti-inflammatory factors such as IL-10 to inhibit the activation of autoreactive T / B cells. On the other hand, they inhibit the secretion of pro-inflammatory factors such as IFN-γ and IL-17 by Th1 / Th17 cells, reduce the production of anti-SSA / Ro and anti-SSB / La antibodies, thereby blocking exocrine gland damage and systemic inflammatory response and improving the pathological process of SS.

[0127] 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.

[0128] SS model mice were constructed using the same method as above. Treatment was administered via intravenous injection of LNPs containing mRNAs with different splicing epitopes, using the same methods and detection indicators as above.

[0129] The levels of anti-SSA / Ro antibodies and anti-SSB / La antibodies in mice of each group, and the Foxp3 levels in the spleen. + The percentage of Tregs, the levels of IFN-γ and IL-17 in the central nervous system, and the detection results are shown in Table 20 below.

[0130] Table 20. Levels of anti-SSA / Ro antibodies and anti-SSB / La antibodies, and spleen Foxp3. + Tregs percentage, central nervous system IFN-γ and IL-17 levels Table 20 shows that mice injected with mRNA encoding LNPs spliced ​​with different epitopes were able to induce Foxp3 compared to the control group. + Tregs proliferate extensively, inhibiting the secretion of pro-inflammatory factors such as IFN-γ and IL-17 by Th1 / Th17 cells, reducing the production of anti-SSA / Ro and anti-SSB / La antibodies, thereby blocking exocrine gland damage and systemic inflammatory response, and improving the pathological process of SS. Among them, the LNPs group encoding splice epitope 3 in mRNA showed the most significant effect. Therefore, the mRNA coding region is preferably encoded by the epitope combination of splice epitope 3 in Table 18. The optimized cDNA sequence of the mRNA coding region encoding splice epitope 3 is shown in SEQ ID NO.61, with a GC content of 57.03%.

[0131] 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.

[0132] sequence list SEQ ID NO.1 The amino acid sequence of the Bet v1 protein: MGVFNYETEATSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKYVKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQVKASKEMGETLLRAVESYLLAHSDAYN SEQ ID NO.2 Epitope amino acid sequence of Bet v1 protein: FPEGFPFKYVKDRVDEVDH SEQ ID NO.3 Epitope amino acid sequence of Bet v1 protein: NFKYNYSVIEGGPIGDTLE SEQ ID NO. 4 Epitope amino acid sequence of Bet v1 protein: EMGETLLRAVESYLLAHSD SEQ ID NO. 5 Epitope amino acid sequence of Bet v1 protein: LFKAFILDGDNLFPKVAPQ SEQ ID NO. 6 Epitope amino acid sequence of Bet v1 protein: EVDHTNFKYNYSVIEGGPI SEQ ID NO. 7 Epitope amino acid sequence of Bet v1 protein: ILKISNKYHTKGDHEVKAE SEQ ID NO. 8 cDNA sequence of epitope reverse translation of Bet v1 protein: TTTCCGGAAGGCTTTCCGTTTAAATATGTGAAAGATCGCGTGGATGAAGTGGATCAT SEQ ID NO. 9 cDNA sequence of epitope reverse translation of Bet v1 protein: AACTTTAAATATAACTATAGCGTGATTGAAGGCGGCCCGATTGGCGATACCCTGGAA SEQ ID NO. 10 cDNA sequence of epitope reverse translation of Bet v1 protein: GAAATGGGCGAAACCCTGCTGCGCGCGGTGGAAAGCTATCTGCTGGCGCATAGCGAT SEQ ID NO. 11 cDNA sequence of epitope reverse translation of Bet v1 protein: CTGTTTAAAGCGTTTATTCTGGATGGCGATAACCTGTTTCCGAAAGTGGCGCCGCAG SEQ ID NO.12 cDNA sequence of epitope reverse translation of Bet v1 protein: GAAGTGGATCATACCAACTTTAAATATAACTATAGCGTGATTGAAGGCGGCCCGATT SEQ ID NO.13 cDNA sequence of epitope reverse translation of Bet v1 protein: ATTCTGAAAATTAGCAACAAATATCATACCAAAGGCGATCATGAAGTGAAAGCGGAA SEQ ID NO. 14 The amino acid sequence of Ii (1-80): MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTAYFLYQQQGRLDK SEQ ID NO. 15 The cDNA sequence of Ii(1-80) reverse translation: ATGCATCGCCGCCGCAGCCGCAGCTGCCGCGAAGATCAGAAACCGGTGATGGATGATCAGCGCGATCTGATTAGCAACAACGAACAGCTGCCGATGCTGGGCCGCCGCCCGGGCGCGCCGGAAAGCAAATGCAGCCGCGGCGCGCTGTATACCGGCTTAGCATTCTGGTGACCCTGCTGCTGGCGGGCCAGGCGACCACCGCGTATTTTCTGTATCAGCAGCAGGGCCGCCTGGATAAA SEQ ID NO.16 Optimized nucleotide sequence of the mRNA coding region for treating hay fever caused by birch pollen: ATGGACGACCAGAGAGACCTGATCTCGAACCACGAGCAGCTGCCGATCCTGGGAAACAGACCACGGGAGCCAGAGCGCTGCTCCCGGGGGGCCCTGTACACAGGGGTATCAGTATTGGTGGCCCTGTTGCTGGCAGGCCAAGCTACCACTGCCTACTTCCTGTACCAGCAGCAAGGAAGACTGGACAAGCTCACCATCACAAGCCAGAATCTCCAGCTAGAATCTCTGAGGATGAAACTGGGCCCTGGTCCCGGCTTTCCAGAAGGCTTTCCTTTCAAGTATGTGAAAGACCGTGTGGATGAGGTGGACCATGGCCCTGGACCTGGCAACTTCAAATATAATTACAGCGTCATCGAAGGTGGCCCTATAGGAGACACTCTGGAGGGGCCTGGTCCTGGAGAGATGGGGGAGACCCTGCTTCGGGCTGTGGAGAGCTACCTGCTGGCCCACAGCGATGGCCCGGGCCCTGGCCTCTTTAAGGCCTTCATCCTGGATGGGGACAACCTCTTCCCCAAGGTGGCTCCACAGGGCCCCGGCCCAGGAGAAGTTGACCACACCAACTTTAAATACAACTACTCTGTCATTGAAGGAGGCCCCATTGGGCCCGGCCCTGGCATCCTGAAGATCTCTAATAAGTACCACACAAAAGGAGATCATGAGGTGAAGGCAGAATAA SEQ ID NO.17 Amino acid sequence of human MBP protein: MASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLSLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR SEQ ID NO.18 Amino acid sequence of human MOG protein: MASLRSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEG GFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF SEQ ID NO.19 Amino acid sequence of human PLP protein: MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERV CHCLGKWLGHPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF SEQ ID NO.20 The amino acid sequence of the preferred epitope in MBP: FLPRHRDTGILDSIGRFFG SEQ ID NO.21 The amino acid sequence of the preferred epitope in MBP: GGRASDYKSAHKGFKGVDA SEQ ID NO.22 The amino acid sequence of the preferred epitope in MBP: HKGFKGVDAQGTLSKIFKL SEQ ID NO.23 The amino acid sequence of the preferred epitope in MBP: QDENPVVHFFKNIVTPRTPPP SEQ ID NO.24 The amino acid sequence of the MOG preferred epitope: GQFRVIGPRHPIRALVGDEV SEQ ID NO.25 The amino acid sequence of the MOG preferred epitope: MEVGWYRPPFSRVVHLYRNGK SEQ ID NO.26 The amino acid sequence of the MOG preferred epitope: GGFTCFFRDHSYQEEAAME SEQ ID NO.27 The amino acid sequence of the PLP preferred epitope: AVRQIFGDYKTTICGKGLSATV SEQ ID NO.28 The reverse-translated cDNA sequence of the MBP preferred epitope: TTTCTGCCGCGCCATCGCGATACCGGCATTCTGGATAGCATTGGCCGCTTTTTTGGC SEQ ID NO.29 The reverse-translated cDNA sequence of the MBP preferred epitope: GGCGGCCGCGCGAGCGATTATAAAAGCGCGCATAAAGGCTTTAAAGGCGTGGATGCG SEQ ID NO.30 The reverse-translated cDNA sequence of the MBP preferred epitope: CATAAAGGCTTTAAAGGCGTGGATGCGCAGGGCACCCTGAGCAAAATTTTTAAACTG SEQ ID NO.31 The reverse-translated cDNA sequence of the MBP preferred epitope: CAGGATGAAAACCCGGTGGTGCATTTTTTTAAAAACATTGTGACCCCGCGCACCCCGCCGCCG SEQ ID NO.32 The reverse-translated cDNA sequence of MOG preferred epitopes: GGCCAGTTTCCGTGATTGGCCCGCGCCATCCGATTCGCGCGCTGGTGGGCGATGAAGTG SEQ ID NO.33 The reverse-translated cDNA sequence of MOG preferred epitopes: ATGGAAGTGGGCTGGTATCGCCCGCCGTTTAGCCGCGTGGTGCATCTGTATCGCAACGGCAAA SEQ ID NO.34 The reverse-translated cDNA sequence of MOG preferred epitopes: GGCGGCTTTACCTGCTTTTTTCGCGATCATAGCTATCAGGAAGAAGCGGCGATGGAA SEQ ID NO.35 Reverse translation of cDNA sequence of PLP-preferred epitopes: GCGGTGCGCCAGATTTTTGGCGATTATAAAACCACCATTTGCGGCAAAGGCCTGAGCGCGACCGTG SEQ ID NO.36 Optimized reverse-translated cDNA sequence of the coding region of mRNA for treating MS: ATGCACAGGAGAAGATCTCGCTCCTGCCGGGAAGACCAGAAGCCAGTCATGGATGATCAAAGAGACCTCATCAGCAACAATGAGCAGCTGCCCATGCTGGGAAGACGGCCGGGTGCGCCAGAGAGCAAATGCAGCAGAGGTGCCTTATACACAGGGTTCTCCATTCTGGTGACTCTGTTGCTGGCTGGGCAGGCTACCACAGCCTATTTCCTCTACCAACAGCAGGGCCGCCTGGACAAGGGCCCTGGACCTGGATTCCTGCCAAGGCATAGGGACACGGGCATTCTTGACTCCATTGGGCGCTTCTTTGGAGGACCTGGCCCAGGAGGTGGCCGCGCCTCTGACTATAAGTCTGCGCACAAGGGCTTCAAGGGTGTGGACGCAGGCCCCGGCCCAGGCCACAAAGGCTTCAAAGGAGTGGATGCCCAGGGGACTTTGTCTAAGATCTTCAAGCTGGGCCCAGGTCCTGGACAGGATGAAAACCCTGTGGTGCACTTTTTCAAGAACATCGTCACCCCTCGGACACCCCCTCCTGGCCCGGGCCCTGGGGGCCAGTTTAGAGTCATCGGTCCCAGACACCCCATCCGGGCCCTGGTGGGTGATGAGGTGGGCCCTGGGCCTGGCATGGAAGTTGGCTGGTACCGGCCACCCTTCTCACGAGTGGTCCACCTGTACAGGAATGGAAAGGGTCCTGGCCCTGGAGGAGGCTTCACCTGCTTTTTCCGGGATCATAGCTACCAGGAGGAAGCTGCCATGGAGGGACCCGGCCCTGGGGCAGTGAGACAGATCTTTGGGGACTACAAGACCACCATCTGTGGGAAGGGGCTGTCAGCCACTGTGGGCCCTGGGCCCGGTTGA SEQ ID NO.37 Amino acid sequence of RO60: MEESVNQMQPLNEKQIANSQDGYVWQVTDMNRLHRFLCFGSEGGTYYIKEQKLGLENAEALIRLIEDGRGCEVIQEIKSFSQEGRTTKQEPMLFALAICSQCSDISTKQAAFKAVSEVCRIPTHLFTFIQFKKDLKESMKCGMWGRALRKAIADWYNEKGGMALALAVTKYKQRNGWSHKDLLRLSHLKPSSEGLAIVTKYITKGWKEVHELYKEKALSVETEKLLKYLEAVEKVKRTRDELEVIHLIEEHRLVREHLLTNHLKSKEVWKALLQEMPLTALLRNLGKMTANSVLEPGNSEVSLVCEKLCNEKLLKKARIHPFHILIALETYKTGHGLRGKLKWRPDEEILKALDAAFYKTFKTVEPTGKRFLLAVDVSASMNQRVLGSILNASTVAAAMCMVVTRTEKDSYVVAFSDEMVPCPVTTDMTLQQVLMAMSQIPAGGTDCSLPMIWAQKTNTPADVFIVFTDNETFAGGVHPAIALREYRKKMDIPAKLIVCGMTSNGFTIADPDDRGMLDMCGFDTGALDVIRNFTLDMI SEQ ID NO.38 Amino acid sequence of TRIM21: MASAARLTMMWEEVTCPICLDPFVEPVSIECGHSFCQECISQVGKGGGSVCPVCRQRFLLKNLRPNRQLANMVNNLKEISQEAREGTQGERCAVHGERLHLFCEKDGKALCWVCAQSRKHRDHAMVPLEEAAQEYQEKLQVALGELRRKQELAEKLEVEIAIKRADWKKTVETQKSRIHAEFVQQKNFLVEEEQRQLQELEKDEREQLRILGEKEAKLAQQSQALQELISELDRRCHSSALELLQEVIIVLERSESWNLKDLDITSPELRSVCHVPGLKKMLRTCAVHITLDPDTANPWLILSEDRRQVRLGDTQQSIPGNEERFDSYPMVLGAQHFHSGKHYWEVDVTGKEAWDLGVCRDSVRRKGHFLLSSKSGFWTIWLWNKQKYEAGTYPQTPLHLQVPPCQVGIFLDYEAGMVSFYNITDHGSLIYSFSECAFTGPLRPFFSPGFNDGGKNTAPLTLCPLNIGSQGSTDY SEQ ID NO.39 Amino acid sequence of lupus La: MAENGDNEKMAALEAKICHQIEYYFGDFNLPRDKFLKEQIKLDEGWVPLEIMIKFNRLNRLTTDFNVIVEALSKSKAELMEISEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYIKGFPTDATLDDIKEWLEDKGQVLNIQMRRTLHKAFKGSIFVVFDSIESAKKFVETPGQKYKETDLLILFKDDYFAKKNEERKQNKVEAKLRAKQEQEAKQKLEEDAEMKSLEEKIGCLLKFSGDLDDQTCREDLHILFSNHGEIKWIDFVRGAKEGIILFKEKAKEALGKAKDANNGNLQLRNKEVTWEVLEGEVEKEALKKIIEDQQESLNKWKSKGRRFKGKGKGNKAAQPGSGKGKVQFQGKKTKFASDDEHDEHDENGATGPVKRAREETDKEEPASKQQKTENGAGDQ SEQ ID NO.40 Amino acid sequence of SPTN1: SEQ ID NO.41 Amino acid sequence of the preferred epitope of RO60: KLLKYLEAVEKVKRTRDEL SEQ ID NO.42 Amino acid sequence of the preferred epitope of RO60: RDELEVIHLIEEHRLVREH SEQ ID NO.43 Amino acid sequence of the preferred epitope of RO60: VHELYKEKALSVETEKLLK SEQ ID NO.44 The amino acid sequence of the preferred epitope of TRIM21: QRQLQELEKDEREQLRILG SEQ ID NO.45 The amino acid sequence of the preferred epitope of TRIM21: TCAVHITLDPDTANPWLIL SEQ ID NO.46 The amino acid sequence of the preferred epitope of TRIM21: KQELAEKLEVEIAIKRADW SEQ ID NO.47 The amino acid sequence of the preferred epitope of lupus La: GKKTKFASDDEHDEHDENG SEQ ID NO.48 The amino acid sequence of the preferred epitope of lupus La: ALKKIIEDQQESLNKWKSK SEQ ID NO.49 The amino acid sequence of the preferred epitope of lupus La: EVTDEYKNDVKNRSVYIKG SEQ ID NO.50 The preferred amino acid sequence of the SPTN1 epitope: YRFQFFQRDAEELEKWIQ SEQ ID NO.51 Reverse cDNA sequence of the RO60 preferred epitope: AAACTGCTGAAATATCTGGAAGCGGTGGAAAAAGTGAAACGCACCCGCGATGAACTG SEQ ID NO.52 Reverse cDNA sequence of the RO60 preferred epitope: CGCGATGAACTGGAAGTGATTCATCTGATTGAAGAACATCGCCTGGTGCGCGAACAT SEQ ID NO.53 Reverse cDNA sequence of the RO60 preferred epitope: GTGCATGAACTGTATAAAGAAAAAGCGCTGAGCGTGGAAACCGAAAAACTGCTGAAA SEQ ID NO.54 The reverse cDNA sequence of the TRIM21 preferred epitope: CAGCGCCAGCTGCAGGAACTGGAAAAAGATGAACGCGAACAGCTGCGCATTCTGGGC SEQ ID NO.55 The reverse cDNA sequence of the TRIM21 preferred epitope: ACCTGCGCGGTGCATATTACCCTGGATCCGGATACCGCGAACCCGTGGCTGATTCTG SEQ ID NO.56 The reverse cDNA sequence of the TRIM21 preferred epitope: AAACAGGAACTGGCGGAAAAACTGGAAGTGGAAATTGCGATTAAACGCGCGGATTGG SEQ ID NO.57 The reverse cDNA sequence of the lupus La preferred epitope: GGCAAAAAAACCAAATTTGCGAGCGATGATGAACATGATGAACATGATGAAAACGGC SEQ ID NO.58 The reverse cDNA sequence of the lupus La preferred epitope: GCGCTGAAAAAAATTATTGAAGATCAGCAGGAAAGCCTGAACAAATGGAAAAGCAAA SEQ ID NO.59 The reverse cDNA sequence of the lupus La preferred epitope: GAAGTGACCGATGAATATAAAAACGATGTGAAAAACCGCAGCGTGTATATTAAAGGC SEQ ID NO.60 The reverse cDNA sequence of the SPTN1 preferred epitope: TATCGCTTTCAGTTTTTTCAGCGCGATGCGGAAGAACTGGAAAAATGGATTCAG SEQ ID NO.61 The optimized reverse-translated cDNA sequence of the coding region of the mRNA for treating SS: ATGCACAGGAGACGTTCTCGGTCTTGCAGAGAAGATCAGAAGCCAGTGATGGATGACCAACGAGACCTCATCTCCAACAATGAACAGCTGCCCATGCTGGGCAGGAGACCAGGCGCCCCAGAGTCCAAATGCAGCCGGGGAGCCCTGTACACAGGATTCAGCATCCTAGTGACCCTGCTGCTGGCTGGCCAGGCCACCACGGCCTATTTCCTCTACCAGCAGCAAGGTCGGTTGGACAAGGGACCCGGTCCTGGAAAGTTACTCAAGTACCTAGAGGCTGTGGAAAAAGTGAAGAGAACAAGAGATGAACTCGGCCCAGGCCCGGGCCGAGATGAGTTGGAAGTCATCCACCTGATTGAAGAACACCGTCTGGTGAGAGAGCACGGGCCAGGCCCGGGTGTGCATGAGCTCTACAAAGAGAAGGCACTGTCTGTAGAGACCGAAAAACTGCTTAAGGGCCCCGGCCCCGGGCAGAGACAGCTCCAGGAGCTGGAGAAAGATGAGCGGGAGCAGCTGCGCATCCTTGGTGGCCCTGGTCCCGGAACCTGTGCCGTCCACATCACCCTGGACCCAGACACTGCCAACCCCTGGCTTATACTGGGTCCTGGGCCTGGGAAGCAAGAACTGGCAGAGAAGCTGGAGGTTGAGATTGCCATCAAAAGAGCTGACTGGGGCCCTGGACCTGGGGGCAAGAAGACAAAGTTTGCCAGTGACGATGAGCATGACGAGCACGACGAAAATGGAGGTCCTGGCCCCGGCGCCCTGAAGAAAATCATTGAAGACCAACAGGAGAGCCTGAACAAATGGAAGAGCAAAGGGCCTGGCCCTGGAGAAGTGACAGACGAATATAAGAATGATGTCAAGAACAGGAGCGTCTACATAAAGGGAGGACCTGGCCCAGGCTACCGTTTTCAGTTCTTCCAGAGAGATGCTGAGGAGCTGGAAAAGTGGATCCAGGGGCCTGGCCCCGGGTGA SEQ ID NO.62 cDNA sequence of 5’UTR: GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACC SEQ ID NO.63 cDNA sequence of 3’UTR: CTCTTCCTCTATGCTCTTCCTGTGCTCTTCCTCTATGCTCTTCCTCTCAAAAAAAAAAAAAAAGCATAAATAACTAAAATACCCAGTCAAGTTACTATTAGTAGATAG

Claims

1. A mRNA lipid nanoparticle drug composition for treating birch hay fever, multiple sclerosis, and Sjögren's syndrome, characterized in that, The pharmaceutical composition comprises lipid nanoparticles and mRNA encapsulated within the lipid nanoparticles; the coding region of the mRNA contains a coding sequence for at least one epitope associated with allergy or autoimmune disease, wherein the epitope is selected from at least one of the following: (a) Birch pollen-associated antigenic epitopes, the amino acid sequences of which are any one or more of the sequences shown in SEQ ID NO.2 to SEQ ID NO.7; (b) Multiple sclerosis-associated antigenic epitopes, the amino acid sequences of which are any one or more of the sequences shown in SEQ ID NO.20 to SEQ ID NO.27; (c) Sjögren's syndrome-associated antigenic epitopes, the amino acid sequences of which are any one or more of the sequences shown in SEQ ID NO.41 to SEQ ID NO.

50.

2. The pharmaceutical composition according to claim 1, characterized in that, The coding region of the mRNA contains the coding sequences of all antigenic epitopes shown in SEQ ID NO. 8 to SEQ ID NO. 13, and the nucleotide sequence of the coding region is shown in SEQ ID NO.

16.

3. The pharmaceutical composition according to claim 1, characterized in that, The coding region of the mRNA contains the coding sequences of all antigenic epitopes shown in SEQ ID NO. 28 to SEQ ID NO. 35, and the nucleotide sequence of the coding region is shown in SEQ ID NO.

36.

4. The pharmaceutical composition according to claim 1, characterized in that, The coding region of the mRNA contains the coding sequences of all antigenic epitopes shown in SEQ ID NO. 51 to SEQ ID NO. 60, and the nucleotide sequence of the coding region is shown in SEQ ID NO.

61.

5. The pharmaceutical composition according to claim 1, characterized in that, 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.

6. The pharmaceutical composition according to claim 5, characterized in that, 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.

7. The pharmaceutical composition according to claim 6, characterized in that, 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.

8. The pharmaceutical composition according to claim 7, characterized in that, The ligand-modified PEGylated lipids are DSPE-PEG2K-trimannose and DSPE-PEG2K-GlcNAc.

9. The pharmaceutical composition according to claim 5, characterized in that, 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.

10. The pharmaceutical composition according to claim 5, characterized in that, The lipid nanoparticles have a particle size of 80~200nm.

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