RNA for generating tol-APCs in vivo and preparation method and application thereof

Abstract

The invention provides RNA (Ribonucleic Acid) for generating tol-APCs (Antigen Procalcitonin Cells) in vivo as well as a preparation method and application thereof The RNA comprises a coding region and a non-coding region, the coding region encodes an immunomodulatory molecule, and the non-coding region comprises a 5 '-cap structure, a 5'-UTR element, a 3 '-UTR element and a polynucleotide tail. The RNA is delivered to an antigen presenting cell through a delivery vector, so that the antigen presenting cell can be induced to differentiate to a tolerance phenotype so as to inhibit activation of an immune system, and autoimmune diseases are effectively treated. The sequence and functional elements of the mRNA are optimized and modified, so that the stability and translation efficiency of the RNA are further improved, and the immunogenicity is reduced.

Description

[0001] This application claims priority to the earlier Chinese application, application number 2025103416322, filed on March 21, 2025; all its contents are part of this invention. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to an RNA that generates tol-APCs in vivo, its preparation method, and its application. Background Technology

[0003] Autoimmune diseases (AIDs) are a group of diseases that seriously threaten human health, affecting approximately 8% of the global population. These diseases are caused by abnormal activation of the body's own immune system, which mistakenly attacks its own tissues and organs, affecting various tissues and organs such as the skin, joints, muscles, and blood vessels. Common examples include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). Currently, the treatment of AIDs faces many challenges. Traditional drugs for treating AIDs mainly include nonspecific immunosuppressants, anti-inflammatory drugs, targeted drugs, and biologics. Nonspecific immunosuppressants, such as glucocorticoids and cyclophosphamide, can suppress immune system activity and reduce inflammation and immune responses, but they also systemically suppress the body's immune surveillance capabilities, increasing the risk of infection, and long-term use may cause other serious side effects. Anti-inflammatory drugs, such as antirheumatic drugs (DMARDs) and nonsteroidal anti-inflammatory drugs (NSAIDs), are mainly used to reduce inflammation and pain; however, their therapeutic effects are limited and they cannot fundamentally regulate the abnormal immune system. While targeted drugs and biologics can regulate immune responses by targeting specific components of the immune system, they are not targeted at specific antigens and still carry the risk of causing systemic immunosuppression. Furthermore, some of these drugs are expensive, which limits their widespread use.

[0004] Aberrant T cell activation plays a crucial role in the pathogenesis of acute respiratory infections (AIDs) and is an important target for AID treatment. Based on this, researchers have explored various T cell-targeting therapeutic strategies. For example, CTLA4-Ig molecules, such as abatacept, can inhibit T cell activation by blocking co-stimulatory molecules on the T cell surface, showing some efficacy in the treatment of some AIDs, but individual variability and potential side effects exist. CD28 antibody therapy strategies have attracted considerable attention; although early clinical trials encountered setbacks, subsequent studies have discovered dosing regimens that preferentially activate regulatory T cells (Tregs), although their safety and efficacy still require further validation. The reverse application of tumor immune checkpoint blockade strategies, namely activating the PD1 pathway to establish immune tolerance, has shown potential in preclinical studies, but immune-mediated adverse events limit its development. In addition, some studies have attempted to train T cells to develop specific tolerance to specific antigens, but this strategy is difficult to implement for diseases whose pathogenic antigens and mechanisms are not yet fully elucidated, such as Crohn's disease and some types of rheumatoid arthritis. Tolerance antigen-presenting cells (tol-APCs) play a crucial role in maintaining immune tolerance, and tol-APC-based therapeutic strategies have brought new hope to the treatment of AIDs (anti-inflammatory diseases). Tolerance dendritic cells (tol-DCs) can maintain immune tolerance by inducing T cell dysfunction and apoptosis, and promoting Treg transformation. In a phase I clinical trial, tol-DC transfer has proven effective and safe for AIDs such as type 1 diabetes (T1D), multiple sclerosis (MS), and Crohn's disease (CD). However, traditional tol-APC transfer therapy has many limitations. Its preparation process requires isolating and expanding autologous DCs in vitro, and inducing tol-DC generation using immunosuppressants and disease-associated antigens. This process is complex, costly, and often requires customization for each patient, severely limiting the widespread application of this therapy.

[0005] In recent years, mRNA technology has made significant progress in the biomedical field, especially in vaccine development. mRNA vaccines induce a specific immune response by introducing mRNA encoding antigen proteins into the body. During the COVID-19 pandemic, mRNA COVID-19 vaccines demonstrated highly effective immune protection, and their rapid development and large-scale production capabilities made a significant contribution to the global fight against the pandemic. mRNA technology also shows broad application prospects in protein replacement therapy, gene therapy, and cell therapy. In the treatment of acute respiratory infections (AIDs), mRNA technology has also shown some potential. For example, by encoding immunomodulatory factors or autoantigen-specific mRNA vaccines, the immune system function can be modulated, alleviating AID symptoms. However, the application of mRNA technology in AID treatment still faces challenges. mRNA drugs have poor stability and are easily degraded by enzymes and the immune system; their immunogenicity also needs attention. Even if modified mRNA (such as m1ΨmRNA) can reduce immunogenicity, various factors, including the delivery vector, still need to be optimized to reduce the risk of potential immune responses.

[0006] Lipid nanoparticles (LNPs) play a crucial role in mRNA vaccine applications as primary delivery carriers. LNPs are mainly composed of ionizable lipids, cholesterol, helper lipids, and PEGylated lipids, effectively protecting mRNA and promoting its uptake by cells. However, the adjuvant properties of LNPs themselves pose some challenges. In AID therapy, the adjuvant properties of LNPs may stimulate antigen-presenting cells (APCs) and T cell activation, counteracting the intended immunosuppressive therapeutic effect and even triggering other potential risks. Although existing research has focused on optimizing the adjuvant effect of LNPs, such as adjusting their composition and size or using non-cationic thiourea lipids to replace traditional cationic lipids, further reducing the immunogenicity of LNPs and improving the safety and efficacy of mRNA delivery remains an urgent problem to be solved.

[0007] In summary, the current state of AID treatment is far from optimistic, with existing therapies exhibiting various limitations. While mRNA technology offers new insights into AID treatment, it faces numerous challenges in practical application. Developing safe, effective, and widely applicable AID treatments remains a crucial research direction in the medical field. Therefore, there is an urgent need to combine the advantages of mRNA technology, delivery vectors, and tol-APCs to develop a novel treatment strategy that overcomes the shortcomings of existing methods and provides more effective treatment options for AID patients. Summary of the Invention

[0008] This invention provides an RNA for generating tol-APCs in vivo, its preparation method, and its applications. The RNA comprises a coding region and a non-coding region. The coding region encodes an immunomodulatory molecule, and the non-coding region includes a 5'-cap structure, a 5'-UTR element, a 3'-UTR element, and a polynucleotide tail. Delivery of the RNA to antigen-presenting cells via a delivery vector can induce the differentiation of antigen-presenting cells into a tolerance phenotype, thereby inhibiting the activation of the immune system and effectively treating autoimmune diseases. This invention also optimizes and modifies the sequence and functional elements of the RNA to further improve its stability, translation efficiency, and reduce immunogenicity.

[0009] On one hand, the present invention provides an RNA comprising a coding region and a non-coding region, wherein the coding region encodes an immunomodulatory molecule capable of inducing antigen-presenting cells to differentiate into a tolerant phenotype, and the immunomodulatory molecule comprises an immunosuppressive protein, a cytokine, a transcription factor, or a functional fragment thereof.

[0010] In this invention, the RNA includes both coding and non-coding regions, and the coding region can encode immunomodulatory molecules, both of which can be used to induce antigen-presenting cells to differentiate into a tolerant phenotype.

[0011] The RNA includes, but is not limited to, one or more of messenger RNA (mRNA), self-replicating RNA (saRNA), and circular RNA (circRNA).

[0012] The RNA is preferably mRNA.

[0013] Traditional drugs for autoimmune diseases (AIDs), such as small molecule immunosuppressants, biologics (mostly protein-based drugs), and glucocorticoids, have several limitations. In terms of targeting, they lack precision, easily interfere with normal immune function, and cause side effects such as infection and liver and kidney damage. Most passively suppress excessive immunity, making it difficult to restore immune balance. Furthermore, traditional drugs have short half-lives, requiring frequent dosing and resulting in poor patient compliance. mRNA drugs, with their ability to precisely regulate pathology-related molecules to adapt to diverse needs and actively regulate immunity, are closer to a state of physiological equilibrium, showing great potential in AID treatment. Currently, mRNA drugs used to treat AIDs face several problems: poor stability, easily degraded by enzymes and the immune system; excessive immunogenicity can trigger excessive immunity, posing safety risks; and low translation efficiency affects the therapeutic effect of AIDs. The RNA provided in this invention as an AID drug solves these problems by adding functional elements to the RNA and optimizing and modifying its sequence, thereby improving its stability, translation efficiency, and reducing its immunogenicity.

[0014] The RNA is delivered to antigen-presenting cells (APCs) in vivo, where it expresses inhibitory co-stimulatory molecules and key inhibitory intracellular proteins, inducing APCs to form a population of tolerable APCs (tol-APCs). These tol-APCs can induce T cells to differentiate into regulatory T cells (Tregs), playing a role in the regulation of immune tolerance and inhibiting the activation of the immune system. The tol-APCs population exhibits ≥1.5-fold increased expression levels of inhibitory co-stimulatory molecules and key inhibitory intracellular proteins compared to native APCs; the tol-APCs population shows significantly lower expression levels of CD80, CD86, and CD40 compared to activated APCs; the tol-APCs population effectively increases the proportion of draining lymph nodes at the disease site and persists in vivo for 1-4 days; the phenotype of the tol-APCs population includes, but is not limited to, the combined effects of inhibitory co-stimulatory molecules (such as PD-L1, CTLA-4, TIM-3, etc.) and key intracellular immune tolerance proteins (such as FOXP3, STAT3, BATF, etc.).

[0015] Furthermore, the immunomodulatory molecules include immunosuppressive molecules and immunomodulatory-related proteins; the immunosuppressive molecules include one or more of human or mouse PD-L1, PD-L2, CTLA4, and IDO, and their homologs or functional fragments; the immunomodulatory-related proteins include one or more of human or mouse HDAC, DNMT3A, TGF-β, IL-27, IL-35, and IL-10, and their homologs or functional fragments.

[0016] Further, the DNA sequences of the human or mouse PD-L1, PD-L2, CTLA4, and IDO are shown in SEQ ID NO. 1-8, and the DNA sequences of the human or mouse HDAC, DNMT3A, TGF-β, IL-27, IL-35, and IL-10 are shown in SEQ ID NO. 9-20; the coding region encodes one or more immunomodulatory molecules, and when the coding region encodes multiple immunomodulatory molecules, the multiple immunomodulatory molecules are linked by adapter molecules; the coding region also encodes a signal peptide.

[0017] The RNA encodes at least one immunomodulatory molecule in its coding region.

[0018] The coding region contains an open reading frame (ORF), within which the sequence of the immunomodulatory molecule is located. This invention optimizes the codons within the ORF by replacing codons in the original coding sequence that might lead to lower translation efficiency or higher immunogenicity with codons commonly found and easily recognized in host cells. This improves RNA translation efficiency within host cells, facilitating rapid intracellular synthesis of the target protein and accelerating biological effects. Furthermore, specific codon optimization not only improves translation efficiency but also avoids the risk of activating the host immune system and reduces immunogenicity. For example, by selecting codon sequences that avoid activating specific pattern recognition receptors (such as Toll-like receptors), the optimized coding region helps reduce unwanted immune responses, which is crucial for avoiding excessive immune activation, especially in immunotherapy or vaccine applications. After codon optimization, the codon fitness index (CAI) is ≥0.8 (based on the human codon usage table), and the GC content is 55%-65%. Simultaneously, the following functional elements are introduced: silencing mutations at cryptic splicing sites (e.g., GT→GC); immunogenic motifs (e.g., UU / UA dinucleotides) with a substitution rate ≥95%.

[0019] An RNA codon fitness index (CAI) ≥ 0.8 indicates that the codons used in its open reading frame (ORF) are highly consistent with naturally occurring, highly translation-related codons in the host cell. The CAI is an indicator used to assess whether the codon usage in an RNA sequence conforms to the target cell's translation mechanism. A higher CAI value indicates a higher degree of codon optimization and higher translation efficiency in the RNA. A CAI value ≥ 0.8 indicates that the RNA has achieved high translation efficiency through codon optimization and is suitable for efficient expression in the target cell. Increasing the CAI value can make the RNA translation process in the cell more efficient, thereby increasing the expression level of the target protein. Codon optimization can reduce translation delay, increase the yield of the target protein, and shorten the therapeutic response time by replacing suboptimal codons in the original gene sequence and selecting preferred codons that are frequently used in the host cell.

[0020] A G / C ratio ≥55% for the mRNA indicates a high GC content in its base pair composition. Optimizing the G / C ratio helps improve mRNA stability and allows it to maintain a longer half-life in vivo or in vitro systems. RNAs with high GC content generally have stronger secondary structural stability, making them less susceptible to degradation in vivo. Furthermore, GC-rich regions can increase the binding affinity between RNA and translational machinery (such as ribosomes) during translation, thereby improving translation efficiency.

[0021] Optimizing GC content not only improves RNA stability and translation efficiency but also helps adjust its folding conformation, preventing premature degradation or loss of activity in vivo. For RNA therapy and vaccines, high GC content helps ensure sustained RNA expression and long-term efficacy.

[0022] The PD-L1 (programmed death ligand 1) binds to PD-1 on the surface of T cells, which can effectively inhibit the activation and proliferation of T cells and prevent them from attacking their own tissues.

[0023] The PD-L2 (programmed death ligand 2) binds to PD-1 on the surface of T cells and functions similarly to PD-L1.

[0024] The CTLA4 (cytotoxic T-lymphocyte-associated protein 4) binds to the co-stimulatory ligands CD80 and CD86 on the surface of APCs, blocking the interaction between CD80 / CD86 and CD28, and inhibiting T cell activation.

[0025] The IDO (indoleamine 2,3-dioxygenase) acts as an intracellular metabolic enzyme, catalyzing the breakdown of tryptophan into metabolites such as kynurenine: it consumes tryptophan in the local microenvironment, inhibiting T cell proliferation (T cell proliferation depends on tryptophan); the metabolites directly induce T cell apoptosis or promote the differentiation of regulatory T cells (Tregs), indirectly inhibiting the immune response.

[0026] The HDAC (histone deacetylase) is a class of epigenetic regulatory enzymes that indirectly suppress the immune response by modifying chromatin structure to inhibit gene transcription: inhibiting T cell activation, reducing the expression of pro-inflammatory cytokines such as IL-2 and IFN-γ, and reducing the proliferation and function of effector T cells; inhibiting the function of antigen-presenting cells (APCs), reducing the expression of co-stimulatory molecules such as CD80 / CD86 on the surface of dendritic cells (DCs), and weakening their antigen-presenting ability; and promoting the differentiation of immunosuppressive cells by upregulating the expression of genes such as Foxp3, thereby promoting the differentiation of regulatory T cells (Tregs).

[0027] The DNMT3A (DNA methyltransferase 3A) is an enzyme responsible for de novo DNA methylation. It suppresses the immune response by silencing immune-activating genes through epigenetic modification: silencing T cell activation genes, such as genes related to the T cell receptor (TCR) signaling pathway and cytokine (IL-2, TNF-α) genes, thereby inhibiting T cell activation and proliferation; maintaining the expression of immune tolerance-related genes, inhibiting pro-inflammatory genes through methylation, while preserving the expression of functional Treg genes (such as Foxp3), thus enhancing the immunosuppressive microenvironment.

[0028] TGF-β (transforming growth factor-β) is a multifunctional cytokine that inhibits immune responses by directly regulating immune cell function: it inhibits T cell activation and proliferation, blocks IL-2 production and IL-2 receptor expression, and inhibits the differentiation and function of effector T cells (such as Th1, Th2, and Th17); it promotes Treg differentiation, induces Foxp3 expression, promotes the transformation of naive T cells into Tregs, and enhances immune tolerance; it inhibits APC function, inhibits dendritic cell (DC) maturation and antigen-presenting capacity, and reduces the expression of co-stimulatory molecules (CD80 / CD86) and pro-inflammatory cytokines (IL-12).

[0029] The IL-27 (interleukin-27) inhibits immune system activation in the following ways: it inhibits pro-inflammatory T cell differentiation by suppressing Th1 cell secretion of IFN-γ, blocking Th17 cell differentiation (reducing the production of IL-17 and IL-22), and limiting excessive inflammatory responses; it promotes an immunosuppressive phenotype, inducing T cells to express anti-inflammatory cytokines such as IL-10, or promoting the differentiation and function of Tregs; it inhibits APC activation, reduces the production of pro-inflammatory cytokines such as IL-12 by DCs, and weakens their ability to activate T cells.

[0030] The IL-35 (interleukin-35) inhibits immune system activation in the following ways: directly inhibiting effector T cells and inhibiting CD4+. + and CD8 + T cell proliferation reduces the secretion of pro-inflammatory cytokines such as IFN-γ and IL-2; induces immunosuppressive cells, promotes the differentiation of naive T cells into "IL-35-producing regulatory T cells" (a type of iTregs), and amplifies the immunosuppressive effect.

[0031] The IL-10 (interleukin-10) inhibits immune system activation in the following ways: it inhibits APC function, significantly reduces the antigen-presenting capacity of macrophages and DCs, and reduces the expression of co-stimulatory molecules (CD80 / CD86) and pro-inflammatory cytokines (TNF-α, IL-1, IL-12); it inhibits T cell activation, reduces the secretion of cytokines (such as IFN-γ, IL-4, IL-17) by Th1, Th2, and Th17 cells, and inhibits effector T cell function; it promotes immune tolerance, induces the differentiation and stabilization of Tregs, and enhances their immunosuppressive function.

[0032] Among the immunosuppressive molecules, PD-L1, PD-L2, and CTLA4 are inhibitory co-stimulatory molecules expressed on the surface of APCs, directly regulating T cell activity through cell surface receptor-ligand interactions. In AID treatment, these inhibitory co-stimulatory molecules can block signal transduction of abnormally activated T cells, promoting the formation of immune tolerance. The RNA molecules of this invention encode full-length inhibitory co-stimulatory molecules, or preferably fragments thereof. These fragments may contain or be composed of (functional) epitopes of the inhibitory co-stimulatory molecules. Preferably, the fragments or epitopes are expressed in host cells targeting MHC class I and MHC class II (preferably MHC class II) processing compartments and are recognized by immune cells to regulate the immune response. The selection of suitable inhibitory co-stimulatory molecules generally depends on the specific type and pathological characteristics of the AIDs. For example, in rheumatoid arthritis, PD-L1 may be a more effective inhibitory co-stimulatory molecule; while in systemic lupus erythematosus, CTLA4 may have better therapeutic effects.

[0033] The linker molecule refers to a short peptide in a fusion protein that acts as a linker between immunosuppressive molecules and immunomodulatory proteins, and is expressed in fusion with these molecules. As an indispensable component of fusion protein recombination, the linker molecule plays a crucial role in constructing stable and biologically active fusion proteins. The linker molecule is an amino acid chain that connects two fusion proteins, possessing a certain degree of flexibility to allow the proteins on either side to perform their independent functions. Protein linker molecules are generally classified into three types: flexible linker molecules, rigid linker molecules, and cleavable linker molecules. Linker molecules are generally between 10 and 15 amino acids long, and should not be too long or too short. An excessively long linker molecule sequence may reduce the yield of the fusion protein and cause immunogenicity problems; an excessively short linker molecule sequence may cause the two proteins to be too close together, affecting the folding of their higher-order structures, thus interfering with each other and leading to loss of protein function. The secondary structure in the linker molecule also restricts the stretchability of the fusion protein, thereby affecting its functional activity.

[0034] The rigid linker molecules are mostly helical in structure, rich in proline, and primarily have the structures (EAAAK)m and (XP)n. They maintain the distance between the domains of the two fusion proteins.

[0035] The cleavable linker molecule is typically composed of amino acids that can form disulfide bonds, and its sequence can be degraded by proteases. In vivo, it often separates the two linked protein components.

[0036] The flexible linker molecule is composed primarily of small, hydrophilic amino acids, with main structures of (GSSS)m and (G)n. This improves the spatial separation of the two domains, thereby ensuring the interaction of specific domains of the two fusion proteins. Preferably, the present invention selects a flexible linker molecule with the sequence GS, allowing the inhibitory co-stimulatory molecule and the terminal of the immunomodulatory protein to maintain a certain degree of freedom, preventing interference between the two proteins while significantly shortening the length of the nucleic acid sequence, avoiding redundant secondary structures, and making the nucleic acid sequence easier to express.

[0037] The signal peptide refers to the short peptide chain that guides the secretion of newly synthesized proteins into the extracellular space, and also refers to the N-terminal amino acid sequence in a newly synthesized polypeptide chain that guides the transmembrane transport of the protein. Almost all secretory proteins contain a signal sequence, which is generally 20-40 amino acids long and is eventually removed by a signal peptidase during transmembrane transport.

[0038] In this invention, signal peptides encoding RNAs of different proteins are screened, and the optimal signal peptide is matched for each protein. In fusion proteins of PD-L1 and immune-regulation-related proteins, a selectable signal peptide (such as LAMP1 or CRT) can guide the fusion protein to target lysosomes or endoplasmic reticulum, thereby optimizing protein processing and expression efficiency.

[0039] Furthermore, the non-coding region includes a 5'-cap structure, a 5'-UTR element, a 3'-UTR element, and a polynucleotide tail.

[0040] The 5'-cap structure is added early in RNA synthesis in vivo, protecting RNA from degradation by exogenous nucleases. During translation, it enhances translation efficiency, improves RNA stability, and helps RNA be recognized and bound to the translation initiation complex. The addition of the cap structure is crucial for RNA translation, especially in delivery systems, ensuring efficient intracellular translation of RNA into target proteins.

[0041] The 5'-UTR element, located before the coding region, can influence the translation initiation efficiency, stability, and ribosome binding ability of RNA. Specific 5'-UTR sequences can interact with intracellular translation-related factors, regulating the translation initiation rate and efficiency. For example, some 5'-UTR sequences contain internal ribosome entry sites (IRES), which can initiate translation independently of the 5'-cap structure, increasing the translational flexibility of artificial RNA molecules in different environments.

[0042] The 3'-UTR region, located after the coding region, has a significant impact on RNA stability, transport, and translation efficiency. Some 3'-UTR sequences contain miRNA binding sites, and through interaction with miRNAs, they can regulate RNA stability and translation levels. Furthermore, 3'-UTR sequences can also interact with intracellular transport proteins, affecting RNA localization and distribution within the cell.

[0043] The polynucleotide tail can interact with certain intracellular proteins, protecting RNA from degradation by exonucleases while promoting ribosome binding to mRNA and improving translation initiation efficiency. Appropriate polynucleotide tail lengths can be selected based on different RNA types and application scenarios to optimize the performance of artificial RNA molecules.

[0044] Further, the 5'-cap structure is one or more of m7GpppN, m7GpppNmNm, and m7,3′-O-GpppG, and the DNA sequence of the 5'-UTR element is shown in SEQ ID NO.21; the DNA sequence of the 3'-UTR element is shown in SEQ ID NO.22; the polynucleotide tail is a polyadenine tail or a polycytosine tail; the polyadenine tail contains 10-200 adenine nucleotides, and each 10 adenine nucleotides are inserted with a phosphate-thiocyanate bond; the polycytosine tail contains 10-200 cytosine nucleotides.

[0045] The 5'-cap structure of this invention is preferably m7GpppN or m7GpppNmNm. The m7GpppN cap mimics the cap structure of native RNA in cells, is recognized by intracellular translation initiation factors, thereby promoting ribosome-RNA binding and initiating the translation process. The m7GpppN cap of this invention includes anti-reverse cap analog (ARCA) modification and 2'-O-methylation modification, increasing its uncapping enzyme resistance by ≥50% (verified by in vitro uncapping experiments). The m7GpppNmNm structure further modifies the m7GpppN cap, enhancing RNA stability and translation efficiency, reducing the likelihood of RNA degradation in vivo, and increasing the expression level of artificial RNA molecules in cells.

[0046] This invention optimizes the 5'-UTR element by inserting a Kozak sequence (CCACC) into the DNA sequence of the 5'-UTR element to enhance ribosome recognition. Furthermore, the DNA sequence is optimized using mfold prediction (transcribed RNA secondary structure free energy ≤ -5 kcal / mol), increasing its affinity for the translation initiation complex and thereby improving translation efficiency by ≥1.5 times (detected by an in vitro reporter system). This specific 5'-UTR sequence design avoids the formation of unfavorable secondary structures, which helps RNA initiate the translation process more smoothly.

[0047] The present invention optimizes the 3'-UTR element by removing the DNA sequences corresponding to the miR-155 and miR-21 binding sites in the DNA sequence of the 3'-UTR element to increase the stability of the transcribed RNA (half-life extended by ≥1.5 times); removing DNA sequences that would activate the immune response to reduce the immunogenicity of the transcribed RNA; and inserting a double ARE (AU-rich element) DNA sequence to achieve expression feedback inhibition under inflammatory conditions.

[0048] The polyadenine tail of this invention is preferably a polyadenine (Poly A) tail. This tail typically consists of 50 to 200 adenosine residues, which can increase RNA stability, improve translation efficiency, and regulate RNA degradation rates. The polyadenine tail not only prevents RNA degradation but also participates in the regulation of the translation process, helping RNA to exist stably within the cell and promoting its translation into the target protein. In particular, in mRNA vaccines or therapeutic mRNAs, the length and composition of the polyadenine tail are crucial to the final efficacy.

[0049] The present invention optimizes the polyadenine tail by including 70-100 adenine nucleotides, with one phosphate thiophosphate bond inserted for every 10 adenosines (synthetic formula: A10*S1), thereby increasing the resistance to exonuclease degradation by ≥80%; the length uniformity is verified by HPLC (RSD≤5%), and the end is modified with a biotin group for purification and detection (binding efficiency≥90%).

[0050] Furthermore, the uridine modification of the RNA is one of pseudouridine, 1-methylpseudouridine, and 5-methoxyuridine; the cytidine modification of the RNA is 5-methylcytidine; and 2'-fluorination modification is introduced into the 5'-UTR and 3'-UTR elements of the mRNA.

[0051] In the RNA, chemically modified nucleotides are further introduced. These chemically modified nucleotides replace some natural nucleotides in the RNA chain to improve the stability, translation efficiency, and immune escape ability of the RNA, reduce the immune activity of exogenous RNA, enhance the expression ability of RNA, and enable it to maintain the translation process in the cell for a longer period of time, thus ensuring the continuity of the therapeutic effect.

[0052] When pseudouridine (Ψ) is used to modify RNA, its nitrogen atom replaces the hydrogen atom of ordinary uridine in the nucleotide, increasing its stability in RNA. Pseudouridine can significantly reduce RNA-induced immune responses because it reduces the recognition of RNA by host cells, making it less susceptible to recognition by pattern recognition receptors (PRRs), thereby avoiding activation of the immune system.

[0053] 1-Methylpseuuridine (m1Ψ) is a modified form of pseudouridine in which the methyl group replaces a hydrogen atom. This modification not only enhances RNA stability but also effectively reduces the immune response, as the structural change further reduces its intracellular immune recognition ability. The introduction of 1-methylpseuuridine can promote RNA translation without activating the immune response, which is particularly important for immunotherapy and vaccination.

[0054] 5-Methoxyuridine (m5U) is another chemically modified nucleotide, with a methoxy group (-OCH3) added to the carbon-5 position of uridine. This modification not only increases RNA stability but also significantly reduces its likelihood of triggering an immune response. 5-Methoxyuridine is metabolized slowly in vivo, further enhancing its intracellular persistence and contributing to improved efficacy of RNA drugs.

[0055] The modification of pyrimidineuridine in this invention is preferably 1-methylpseudouridine.

[0056] On the other hand, the present invention provides a method for preparing the RNA described above, wherein the RNA is obtained by in vitro transcription, in vivo synthesis, chemical synthesis or a combination thereof.

[0057] In some methods, the RNA is obtained through in vitro transcription, as follows:

[0058] (1) Synthesize a DNA template containing a coding region and a non-coding region; the coding region contains a DNA sequence of a signal peptide and an immunomodulatory molecule; the non-coding region contains a DNA sequence of a 5'-UTR element and a 3'-UTR element;

[0059] (2) Transcribe DNA template into RNA in vitro;

[0060] (3) After transcription is completed, the RNA is modified with a 5'-cap structure and a polynucleotide tail;

[0061] (4) Purify RNA.

[0062] In another aspect, the present invention provides an RNA drug delivery system comprising the aforementioned RNA and a delivery carrier, wherein the delivery carrier is a lipid nanoparticle, a polymer nanoparticle, a lipid-polymer hybrid nanoparticle, a liposome, an exosome, a virus-like particle, or a protein nanocage; the delivery carrier may be linked to a targeting ligand via a surface modifier; the surface modifier is selected from one or more of carbohydrates or their conjugates, antibodies or their fragments, peptides, aptamers, and small molecule ligands; the RNA drug delivery system may deliver one or more RNAs.

[0063] Taking RNA encoding non-fusion proteins as an example, each RNA can encode any one of the following immunomodulatory molecules: PD-L1, PD-L2, CTLA4, IDO, HDAC, DNMT3A, TGF-β, IL-27, IL-35, and IL-10 (nucleotide sequences shown in SEQ ID NO. 1-20), meaning one mRNA encodes one protein. The delivery vector may contain only one of these RNAs, meaning that after delivery to the target via the drug delivery system, the RNA can only encode one immunomodulatory molecule; alternatively, the delivery vector may contain multiple RNAs for co-delivery, meaning that after delivery to the target via the drug delivery system, the RNA can encode multiple different immunomodulatory molecules.

[0064] The sugars or their conjugates include, but are not limited to, one or more of mannose, galactose, fucose, glucose, glucosamine, sialic acid, sulfated sugars, polyethylene glycol-glycoconjugates, lipid-glycoconjugates and their derivatives.

[0065] Furthermore, the surface modifier is an antibody or fragment, peptide, or aptamer targeting CD11c, DEC-205, and CLEC9A.

[0066] In another aspect, the present invention provides the use of the RNA drug delivery system as described above in the preparation of formulations that enhance the formation of an immune tolerance phenotype by antigen-presenting cells.

[0067] In some methods, lipid nanoparticles (LNPs) encoding different murine or human immunomodulatory molecules (non-fusion proteins) are prepared and injected subcutaneously into mice. The levels of CD11c in APCs in the mice's lymph nodes are then measured. + and CD11b + The percentage of positive expression of corresponding immune regulatory molecules in cells was analyzed, and the results showed that mRNA encoding PD-L1 had the highest positive rate. Further detection of CD4 in lymph nodes... +The expression levels of IFN-γ, IL-17, TNF-α, and FOXP3 in T cells were measured. Results showed that tol-APCs induced by PD-L1 mRNA downregulated IFN-γ. + IL-17 + and NF-α + The proportion of T cells was the most significant, and the effect of inducing Treg cell proliferation was the best. Therefore, the preferred immunomodulatory molecule is PD-L1, an immunosuppressive molecule.

[0068] In some methods, lipid nanoparticles (LNPs) co-encapsulating mRNAs encoding two immunomodulatory molecules (one of which is immobilized as the immunosuppressive molecule PD-L1) are prepared and injected subcutaneously into mice. The levels of CD4+ in the lymph nodes are then measured. + The expression levels of IFN-γ, IL-17, TNF-α, and FOXP3 on T cells were analyzed. The results showed that LNPs loaded with mouse PD-L1+IDO mRNA and human PD-L1+DNMT3A mRNA were the most effective in downregulating the proportion of pro-inflammatory T cells and enhancing Treg induction.

[0069] In another aspect, the present invention provides the use of the RNA drug delivery system as described above in the preparation of formulations for treating autoimmune diseases.

[0070] The autoimmune diseases (AIDs) mentioned include, but are not limited to, rheumatoid arthritis (RA), ulcerative colitis (UC), multiple sclerosis (MS), type 1 diabetes (T1D), and systemic lupus erythematosus (SLE). These diseases are all caused by abnormalities in the immune system, often accompanied by an overactive immune response that attacks the body's own tissues and organs. For example, rheumatoid arthritis (RA) manifests as chronic inflammation of the joints; ulcerative colitis (UC) affects the intestines, leading to diarrhea and abdominal pain; multiple sclerosis (MS) is a disease of the central nervous system; type 1 diabetes (T1D) is caused by insufficient insulin secretion; and systemic lupus erythematosus is a systemic autoimmune disease involving inflammation in multiple organs.

[0071] In some methods, lipid nanoparticles (LNPs) loaded with mRNAs encoding different immunomodulatory molecules were prepared for use in mouse models of rheumatoid arthritis (RA), ulcerative colitis (UC), type 1 diabetes (T1D), systemic lupus erythematosus (SLE), graft-versus-host disease (GvHD), and psoriasis (Ps). The treatment results showed that the lipid nanoparticles (LNPs) loaded with mRNAs encoding different immunomodulatory molecules had good therapeutic effects on all of the above-mentioned autoimmune diseases (AIDs).

[0072] Furthermore, the delivery system can be administered via subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, or nasal administration.

[0073] Furthermore, the delivery system can be prepared as an injection solution, a lyophilized powder injection, a sustained-release microsphere, or an implantable sustained-release formulation.

[0074] The aforementioned formulations also include pharmaceutically acceptable carriers and excipients, including but not limited to physiological saline, buffer solutions (such as phosphate buffer), sugars (such as mannitol and sucrose), and proteins (such as albumin). Carriers such as physiological saline, glucose solutions, or buffer solutions contribute to drug dissolution, stability, and safety. Physiological saline is compatible with the osmotic pressure of body fluids, glucose solutions provide energy, and buffer solutions maintain pH stability, ensuring effective drug delivery in the body and reducing side effects.

[0075] In another aspect, the present invention provides lipid nanoparticles targeting APCs, wherein the lipid nanoparticles include at least one ionizable lipid, at least one selected from auxiliary lipids, sterol compounds, polymer components, and functional additives; the lipid nanoparticles have a particle size of 20-500 nm and a surface potential of -10-30 mV.

[0076] In AID therapy, the adjuvant properties of lipid nanoparticles (LNPs) can stimulate antigen-presenting cells (APCs) and T cell activation, counteracting the intended immunosuppressive therapeutic effect and even triggering other potential risks. Although existing methods optimize the adjuvant effect of LNPs and reduce their immunogenicity by adjusting their composition, size, or using non-cationic thiourea lipids to replace traditional cationic lipids, the results are still unsatisfactory. This invention employs Design of Experiments (DOE) and orthogonal experiments (Taguchi method) to systematically adjust the N / P ratio and lipid molar ratio, obtaining a low-immunogenic formulation through in vivo screening. LNPs prepared with this formulation, after subcutaneous injection, significantly reduce the expression of co-stimulatory molecules (CD80, CD86, CD40) on the surface of APCs, avoiding immune activation. Furthermore, the LNPs prepared with this formulation can only be taken up by APCs, while other cells hardly take them up, exhibiting specific targeting. The surface of the LNPs can also be connected to a targeting ligand, which specifically recognizes the receptor on the surface of APCs, promotes the interaction and endocytosis between APCs and LNPs, and improves the specific targeting of LNPs.

[0077] Furthermore, the ionizable lipid is an ionizable lipid or its derivative having a pKa of 5.5–7.5, and the ionizable lipid or its derivative contains a compound with a degradable bond; the auxiliary lipid is a saturated or unsaturated phospholipid with a carbon chain length of C14–C22.

[0078] The degradable bonds include ester bonds, thioether bonds, and carbonate bonds.

[0079] Further, the ionizable lipids include at least one of SM102 and its derivatives or functional equivalents, Dlin-MC3-DMA and its derivatives or functional equivalents, ALC-0315 and its derivatives or functional equivalents, ATX-126 and its derivatives or functional equivalents, E12CA1A3 and its derivatives or functional equivalents, DOTMA, DOTAP, DOSPA, and ePC; the auxiliary lipids include at least DSPC, POPC, DLPC, DEPC, DOPC, DMPC, DPPC, DOPE, DSPE, DOPG, ESM, DPPS, and D The sterol compound includes at least one of SPA, DPPA, and DMG-PEG; the sterol compound includes at least one of cholesterol, phytosterol, ergosterol, and lanosterol and their derivatives; the polymer includes at least one of DMG-PEG2000, PEG-DMG, PEG-DSPE, DSPE-PEG5000, Ceramide(C14)-PEG2000, Ceramide(C18)-PEG2000, DMG-PEG2000, DPG-PEG2000, DSG-PEG2000, and DOPE-PEG2000.

[0080] The phytosterols include, but are not limited to, β-sitosterol and stigmasterol, and the lanosterol and its derivatives include, but are not limited to, cholesterol hemisuccinate, cholesterol-PEG and amino cholesterol.

[0081] Furthermore, the functional additives include at least one of nucleic acid drugs and hydrophobic drugs.

[0082] Furthermore, the functional additive is a nucleic acid drug that encodes an immunomodulatory molecule.

[0083] Furthermore, the nucleic acid drug includes, but is not limited to, one or more of messenger RNA (mRNA), self-replicating RNA (saRNA), and circular RNA (circRNA); the immunomodulatory molecule is selected from PD-L1, PD-L2, CTLA4, IDOHDAC, DNMT3A, TGF-β, IL-27, IL-35, and IL-10 or combinations thereof.

[0084] Further, the molar percentage of the ionizable lipid is 20%-60%, the molar percentage of the auxiliary lipid is 3%-15%, the molar percentage of the sterol compound is 20%-75%, the molar percentage of the polymer is 0%-5%, and the sum of the molar percentages of the ionizable lipid, auxiliary lipid, sterol compound, and polymer is 100%; the molar percentage of the nucleic acid drug and lipid nanoparticles is 0-2%, and the N / P ratio of the lipid component in the nucleic acid drug and lipid nanoparticles is (4-9):1.

[0085] In some methods, SM-102 was fixed as the ionizable lipid, with the remaining lipids being DSPC, DMG-PEG2000, and cholesterol. The molar ratios of these components and the molar ratio (N / P ratio) of the positively charged amino groups (N) in these components to the negatively charged phosphate groups (P) in the nucleic acid (mRNA) drug were varied to construct an A library, including 10 LNPs (A0-A9). Animal experiments were conducted to evaluate the positive rate of co-stimulatory molecule expression on the surface of antigen-presenting cells (APCs) after in vivo mRNA delivery using different LNP formulations. This positive rate served as a key indicator of LNP immunogenicity. The results showed that the A7 formulation (SM-102 molar ratio of 25%, DSPC molar ratio of 5%, DMG-PEG2000 molar ratio of 0.5%, cholesterol molar ratio of 69.5%, and N / P ratio of 4) exhibited the lowest positive rate of co-stimulatory molecules, demonstrating excellent immune tolerance. Furthermore, based on the A7 formulation, the N / P ratio of the A7 formulation was varied to construct a B library with five N / P ratio levels (4, 6, 7, 8, and 9), including B1-B4 and A7, representing five LNPs. Animal experiments were conducted to evaluate the positive rate of co-stimulatory molecule expression on the surface of antigen-presenting cells (APCs) after in vivo mRNA delivery using different LNP formulations. The results showed that the A7 formulation (N / P ratio of 4) exhibited the lowest positive rate of co-stimulatory molecules, demonstrating excellent immune tolerance. Therefore, the A7 formulation is considered the optimal formulation for preparing LNPs using SM-102 as the ionizable lipid.

[0086] In some methods, the ionizable lipid is fixed at MC3, with the remaining lipids being DSPC and DMG-PEG2000. The molar ratios of these components and the molar ratio (N / P ratio) of the positively charged amino groups (N) in these components to the negatively charged phosphate groups (P) in the nucleic acid (mRNA) drug are varied to construct a C library, including nine LNPs (C1-C9). Animal experiments were conducted to evaluate the positive rate of co-stimulatory molecule expression on the surface of antigen-presenting cells (APCs) after in vivo mRNA delivery using different LNP formulations. This positive rate serves as a key indicator of LNP immunogenicity. The results showed that the C3 formulation (MC3 molar ratio 60%, DSPC molar ratio 38%, DMG-PEG2000 molar ratio 2%, N / P ratio 6) exhibited the lowest positive rate of co-stimulatory molecules, demonstrating excellent immune tolerance. Therefore, the C3 formulation is considered the optimal formulation for preparing LNPs when ionizable lipid is MC3.

[0087] In some methods, optimal formulations A7 from libraries A and B, and optimal formulation C3 from library C, were used to prepare LNPs and encapsulate the same mRNA encoding human / mouse PD-L1 for in vitro and in vivo expression validation. Results showed that both types of LNP-encapsulated mRNAs effectively upregulated CD11c in APC cells. + Cells and CD11b + While the A7 formulation showed higher PD-L1 expression in APC cells, it was the preferred formulation for targeting APC cells.

[0088] Furthermore, the lipid nanoparticles are linked to a targeting ligand via a surface modifier; the surface modifier is selected from one or more of carbohydrates or their conjugates, antibodies or their fragments, peptides, aptamers, and small molecule ligands.

[0089] The sugar or its conjugate is selected from one or more of mannose, galactose, fucose, glucose, glucosamine, sialic acid, sulfated sugar, polyethylene glycol-sugar conjugate, lipid-sugar conjugate and its derivatives.

[0090] Furthermore, the surface modifier is any one or more of the following: an antibody targeting CD11c and its fragments, peptides, or aptamers; an antibody targeting DEC-205 and its fragments, peptides, or aptamers; and an antibody targeting CLEC9A and its fragments, peptides, or aptamers. The targeting ligand may also be chemically modified with one or more of the following: mannose, galactose, fucose, glucose, glucosamine, sialic acid, sulfated sugar, polyethylene glycol-glycoconjugates, lipid-glycoconjugates, and their derivatives.

[0091] Furthermore, the molar percentage of the surface modifier to the lipid component in the lipid nanoparticles is 0.1%-2%.

[0092] The targeted ligand can specifically bind to receptors on the surface of APCs, thereby promoting the interaction and endocytosis of target cells with LNPs. The selection of the targeted ligand is crucial to ensure the precision of drug delivery to LNPs and improve therapeutic efficacy.

[0093] CD11c is an important marker molecule on the surface of dendritic cells (DCs), widely expressed on dendritic cells, macrophages, and other APCs. Peptides or functional fragments targeting CD11c can specifically guide LNPs to bind to dendritic cells or other APCs by binding to the CD11c receptor, thereby enhancing their targeting to these cells. This allows LNPs to directly act on important cell populations in the immune system, increasing delivery efficiency.

[0094] DEC-205 (Dendritic Cell Specific ICAM-3 Grabbing Non-integrin) is a receptor highly expressed on the surface of dendritic cells, involved in dendritic cell immune surveillance and antigen presentation. Peptides or functional fragments targeting DEC-205 can specifically recognize and bind to the DEC-205 receptor, promoting efficient binding of LNPs to dendritic cells and their intracellular entry via endocytosis. The high expression of DEC-205 makes it an ideal target for dendritic cell-specific delivery systems.

[0095] CLEC9A is a dendritic cell-specific pattern recognition receptor, primarily expressed in specific subsets of dendritic cells (DCs), playing a crucial role in innate immune recognition, antigen presentation, and adaptive immune activation. Antibodies, fragments, peptides, or aptamers of CLEC9A can specifically recognize and bind to the CLEC9A receptor, promoting efficient binding of LNPs to dendritic cells and their intracellular entry via endocytosis.

[0096] The targeted ligands bind to the lipid bilayer of LNPs, enabling LNPs to more efficiently target and enter specific immune cell populations, ensuring precise delivery of mRNA drugs. LNPs targeting APCs can effectively activate the immune function of dendritic cells and macrophages, enhancing antigen presentation and immune tolerance, thereby achieving more precise and efficient immunotherapy. Modification of LNPs with anti-CD11c antibody fragments, anti-DEC-205 antibodies, or their functional fragments improves their targeting, enabling them to accurately deliver mRNA to specific APCs, activating or modulating immune responses. This targeted delivery is of great significance for immunotherapy and vaccine development, especially in the treatment of diseases requiring precise regulation of immune responses, such as cancer immunotherapy, allergic immunomodulation, or the treatment of autoimmune diseases.

[0097] In some methods, different targeting ligands were screened, and mice were injected subcutaneously with LNPs (containing mRNA expressing PD-L1) linked to different targeting ligands. The expression of surface co-stimulatory molecules and PD-L1 in CD11c+ cells in their lymph nodes was then examined. The results showed that LNPs linked to peptides with amino acid sequences as shown in SEQ ID NO.24 were most effective at targeting APCs.

[0098] On the other hand, the present invention provides a method for preparing lipid nanoparticles targeting APCs as described above, wherein the lipid nanoparticles are prepared by a process of dissolving lipid components in an organic phase and mixing them in a controlled manner with an aqueous phase containing functional additives to promote self-assembly into nanoparticles; the controlled mixing includes microfluidic mixing, ethanol dilution / solvent replacement, ultrasonic emulsification, ultra-high pressure homogenization, membrane extrusion, spray drying, electrospraying / electrospinning, or any combination of the above methods.

[0099] In another aspect, the present invention provides the use of lipid nanoparticles targeting APCs as described above in the preparation of formulations for treating autoimmune diseases.

[0100] In some methods, LNPs loaded with different immunomodulatory molecules prepared by the A7 formulation can effectively treat mouse models of rheumatoid arthritis (RA), ulcerative colitis (UC), multiple sclerosis (MS), type 1 diabetes (T1D), systemic lupus erythematosus (SLE), graft-versus-host disease (GvHD), and psoriasis (Ps).

[0101] Furthermore, the present invention also provides a reagent kit comprising the following components:

[0102] a. The delivery system and composition; the composition comprises physiological saline, glucose solution or buffer to ensure mRNA stability and delivery efficiency;

[0103] b. An injection device, which may be selected from: a pre-filled syringe or a microneedle patch; the pre-filled syringe is pre-loaded with a specific dose of drug composition to facilitate subcutaneous injection by patients or healthcare professionals; the microneedle patch is suitable for non-invasive delivery of mRNA drugs, enhancing patient compliance and reducing injection-related discomfort.

[0104] The kit includes detailed instructions for use, guiding patients or healthcare professionals on the correct use of the delivery system or drug composition. The instructions include the following key information: 1) Dosage range: The recommended dose is 0.1-10 μg. 1) mRNA / kg body weight, the specific dosage can be adjusted according to the individual patient's condition (such as disease severity, weight, etc.); 2) Injection frequency: It is recommended to administer subcutaneous injections 1-2 times per week. The injection frequency can be adjusted appropriately according to the clinical protocol to optimize the treatment effect and reduce adverse reactions; 3) Indications: This kit is suitable for immunomodulatory therapy of autoimmune diseases, including but not limited to: Rheumatoid arthritis (RA): By regulating abnormally activated T cells and B cells, reducing the release of inflammatory factors (such as TNF-α, IL-6), alleviating joint inflammation and delaying disease progression; Ulcerative colitis (UC): By inducing a tolerant immune response, reducing colonic mucosal inflammation, restoring intestinal immune homeostasis, and reducing the recurrence rate; Multiple sclerosis (MS): By inhibiting autoreactive T cells from attacking the central nervous system, reducing myelin demyelination damage, and slowing disease progression; Type 1 diabetes (T1D): By inducing islet antigen-specific tolerance, inhibiting the autoimmune system's attack on pancreatic β cells, delaying disease progression and preserving residual β cell function.

[0105] Given the complexity of the pathogenesis of autoimmune diseases (AIDs), a single treatment may not be sufficient to meet the treatment needs. Therefore, the artificial RNA molecules, compositions, or formulations of this invention can be used in combination with other treatment methods to enhance therapeutic effects and achieve more comprehensive immune modulation.

[0106] Furthermore, the present invention provides a combination therapy comprising the following steps:

[0107] a. Administering the delivery system and composition described above, wherein the composition includes, but is not limited to, one or more of physiological saline, buffer solutions (such as phosphate buffer), carbohydrates (such as mannitol, sucrose), and proteins (such as albumin);

[0108] b. Combined with radiotherapy, immune checkpoint inhibitors, anti-inflammatory drugs (such as TNF-α inhibitors), chemotherapy agents, or combinations thereof.

[0109] The treatment involves the combined administration of immune checkpoint inhibitors, anti-inflammatory drugs (kinase inhibitors such as JAK inhibitors or cytokine inhibitors such as IL-6 receptor antibodies or TNF-α inhibitors), chemotherapy agents, or combinations thereof, wherein the combined treatment significantly reduces the levels of inflammatory factors at the disease site, particularly IFN-γ, TNF-α, and IL-17, by at least 50%, and this reduction persists for 1 to 4 weeks after treatment. The combined therapy also increases the disease remission rate (e.g., a decrease in DAI score or a decrease in the arthritis index) by ≥30%.

[0110] The use of anti-inflammatory drugs (such as TNF-α inhibitors) can inhibit the secretion of pro-inflammatory cytokines, especially TNF-α, which is often present at high levels in autoimmune diseases and participates in the vicious cycle of inflammatory response. By inhibiting TNF-α activity, the occurrence of inflammation can be significantly reduced, alleviating symptoms such as arthritis, intestinal inflammation, and nervous system damage, thereby improving the condition. At the same time, TNF-α inhibitors can also reduce the occurrence of cytokine storms and alleviate tissue damage caused by excessive immune responses.

[0111] The treatment regimen of this invention is used in combination with traditional anti-inflammatory drugs (AIDs), such as non-specific immunosuppressants and anti-inflammatory drugs. In the initial stages of combined use, the dosage of the traditional drugs can be appropriately reduced to minimize their systemic immunosuppressive side effects. For example, in the treatment of rheumatoid arthritis, the artificial RNA preparation of this invention is used in combination with low-dose glucocorticoids. Glucocorticoids can rapidly reduce the inflammatory response, while the artificial RNA preparation fundamentally corrects immune imbalance by regulating the immune system. The combined use of both can quickly relieve symptoms and reduce the adverse reactions caused by long-term use of glucocorticoids, such as increased risk of infection and osteoporosis.

[0112] The treatment regimen of this invention, used in combination with chemotherapy agents, helps restore the balance of the immune system by reducing adverse reactions and regulating immune tolerance. Some chemotherapy agents can selectively inhibit active immune cells, reducing attacks on the body's own tissues, while some chemotherapy drugs improve the immune environment, making the patient's immune response more adaptive and stable, thereby enhancing the treatment effect.

[0113] The treatment regimen of this invention is used in combination with other biological agents, such as monoclonal antibodies. Some monoclonal antibodies can specifically block specific immune signaling pathways, while the artificial RNA molecules of this invention can modulate the overall immune environment. For example, in the treatment of systemic lupus erythematosus, monoclonal antibodies targeting specific cytokines are used in combination with the artificial RNA formulation of this invention. Monoclonal antibodies can rapidly block cytokine-mediated inflammatory signals, while the artificial RNA formulation induces tolerable antigen-presenting cells (tol-APCs) and modulates the function of T cells and B cells, thereby achieving more comprehensive immune modulation and improving treatment efficacy.

[0114] The treatment strategy of this invention combines different treatment methods to achieve synergistic effects. On the one hand, different treatment methods act on different aspects of the immune system, enabling a more comprehensive regulation of immune imbalances. On the other hand, combined use can reduce the dosage and side effects of single treatment methods, improving patient tolerance and treatment adherence. Furthermore, combined treatment can also tailor personalized treatment plans to individual patient differences, enhancing the targetedness and effectiveness of the treatment.

[0115] This combination therapy regimen further improves treatment efficacy through a multi-mechanism synergy. The use of immune checkpoint inhibitors selectively unblocks T cell suppression, restoring their normal immune response. Immune checkpoints, such as the PD-1 / PD-L1 pathway, are typically used to maintain immune tolerance and prevent overactivation of the immune system. Immune checkpoint inhibitors, by blocking these inhibitory signals, release the "brakes" on T cells, thereby enhancing the immune response of T cells against diseased cells and promoting the generation of antigen-specific tolerant T cells (Tregs). The increase in Tregs helps maintain the stability of the immune system, avoids attacking healthy tissues, and further promotes immune tolerance.

[0116] The present invention has the following beneficial effects:

[0117] 1. The RNA provided by the present invention comprises a coding region and a non-coding region. The coding region encodes an immunomodulatory molecule, and the non-coding region includes a 5'-cap structure, a 5'-UTR element, a 3'-UTR element, and a polynucleotide tail. The RNA is delivered to antigen-presenting cells via a delivery vector, which can induce antigen-presenting cells to differentiate into a tolerant phenotype to inhibit the activation of the immune system and effectively treat autoimmune diseases.

[0118] 2. The present invention further optimizes and modifies the sequence and functional elements of the RNA to further improve the stability and translation efficiency of the RNA and reduce its immunogenicity;

[0119] 3. The drug delivery system provided by this invention overcomes the problems of high cost and complex preparation of traditional tol-APCs therapy, and provides a new and broad-spectrum method for the treatment of immune diseases. Attached Figure Description

[0120] Figure 1 The results of particle size and zeta potential detection for LNPs in Library A of Example 2;

[0121] Figure 2 Example 2 is used for analyzing CD11c + Gating strategies for cell-specific activation markers;

[0122] Figure 3The targeting of LNPs to non-APC cells and APC cells by the A7 and C3 formulations of Example 2;

[0123] Figure 4 The LNPs from library A in Example 2 were used for in vivo CD11c experiments in mice. + EGFP fluorescence intensity in cells;

[0124] Figure 5 The LNPs from library A in Example 2 were used in mouse in vivo experiments to express EGFP in CD11c. + CD40 in cells + CD80 + and CD86 + Percentage of positive cells;

[0125] Figure 6 The fluorescence intensity of LNPs from library A in Example 2 was used to evaluate the fluorescence intensity of CD40, CD80, and CD86 in mouse in vivo experimental positive cells.

[0126] Figure 7 The results of multifactor analysis of the detection parameters obtained from LNPs in Library A in Example 2;

[0127] Figure 8 The results of particle size and zeta potential detection for LNPs in Library B of Example 2;

[0128] Figure 9 The B library LNPs from Example 2 were used for in vivo CD11c experiments in mice. + EGFP fluorescence intensity in cells;

[0129] Figure 10 The B library LNPs from Example 2 were used in vivo in mice to express EGFP in CD11c. + CD40 in cells + CD80 + and CD86 + Percentage of positive cells;

[0130] Figure 11 The fluorescence intensity of LNPs from Library B in Example 2 was used to evaluate the fluorescence intensity of CD40, CD80, and CD86 positive cells in mice in vivo.

[0131] Figure 12 The C library LNPs from Example 2 were used in vivo in mice to express EGFP using CD11c. + CD80 in cells + Percentage of positive cells;

[0132] Figure 13 The C library LNPs from Example 2 were used for in vivo CD11c experiments in mice. + CD40 in cells+ CD80 + and CD86 + Fluorescence intensity of CD40, CD80, and CD86 in positive cells;

[0133] Figure 14 The A7 formulation LNPs of Example 2 were used in in vitro and in vivo experiments on DC2.4 and RAW264.7 cell lines and CD11c. + and CD11b + Cellular PD-L1 expression status;

[0134] Figure 15 The C3 formulation LNPs of Example 2 were used in in vitro and in vivo experiments on DC2.4 and RAW264.7 cell lines and CD11c. + and CD11b + Cellular PD-L1 expression status;

[0135] Figure 16 The D library LNPs from Example 3 were used in vivo in mice to express EGFP using CD11c. + CD80 in cells + Percentage of positive cells;

[0136] Figure 17 The D library LNPs from Example 3 were used for in vivo CD11c experiments in mice. + CD40 in cells + CD80 + and CD86 + Fluorescence intensity of CD40, CD80, and CD86 in positive cells;

[0137] Figure 18 Example 6 illustrates the effect of LNPs loaded with different mRNAs on the downregulation of pro-inflammatory T cells after generating mouse tol-APCs in vitro.

[0138] Figure 19 Example 6 shows the effect of using LNPs loaded with different mRNAs to generate human tol-APCs in vitro and downregulating the proportion of pro-inflammatory T cells.

[0139] Figure 20 Example 6 describes the use of LNPs encoding PD-L1 mRNA in mice to investigate the CD11c content in their lymph nodes. + Cellular PD-L1 expression level;

[0140] Figure 21 Example 6 describes the use of LNPs containing mRNA encoding PD-L2 in mice to investigate the CD11c content in their lymph nodes. + Cellular PD-L2 expression level;

[0141] Figure 22 In Example 6, LNPs containing mRNA encoding IDO were used in vivo in mice to investigate the CD11c content in lymph nodes. + Cellular IDO expression level;

[0142] Figure 23 Example 6 describes the use of LNPs containing mRNA encoding HDAC in mice to investigate the CD11c content in lymph nodes. + Cellular HDAC expression level;

[0143] Figure 24 In Example 6, LNPs encoding DAMT3A mRNA were used in vivo in mice to investigate the CD11c concentration in lymph nodes. + cellular DAMT3A expression level;

[0144] Figure 25 Example 6 describes the use of LNPs (Large Nucleotides) loaded with mRNAs encoding different murine immunomodulatory proteins in mice to investigate the CD4+ levels in their lymph nodes. + Expression levels of IFN-γ, IL-17, TNF-α, and FOXP3 in T cells;

[0145] Figure 26 Example 6 describes the use of LNPs (Large Nucleotides) loaded with mRNAs encoding different human immune regulatory proteins in mice to investigate the CD4+ levels in their lymph nodes. + Expression levels of IFN-γ, IL-17, TNF-α, and FOXP3 in T cells;

[0146] Figure 27 In Example 7, LNPs encoding PD-L1 mRNA were used in vivo in mice to generate tol-APCs (tolerant CD11c) in their lymph nodes. + Cellular and tolerance CD11b + The duration of PD-L1 expression in cells;

[0147] Figure 28 Arthritis index of a mouse model of rheumatoid arthritis (RA) generated in vivo using tol-APCs in Example 8;

[0148] Figure 29 The disease activity index of a mouse model of ulcerative colitis (UC) generated in vivo using tol-APCs in Example 8;

[0149] Figure 30 The disease index of a mouse model of multiple sclerosis (MS) generated in vivo using tol-APCs in Example 8;

[0150] Figure 31The blood glucose level in a mouse model of type 1 diabetes (T1D) treated with tol-APCs generated in vivo in Example 8;

[0151] Figure 32 The disease index of a mouse model of systemic lupus erythematosus (SLE) generated in vivo using tol-APCs in Example 8;

[0152] Figure 33 The GvHD value of the tol-APCs generated in vivo to treat the graft-versus-host disease (GvHD) mouse model in Example 8 is shown.

[0153] Figure 34 The disease index of a mouse model of psoriasis (Ps) generated in vivo using tol-APCs in Example 8. Detailed Implementation

[0154] 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.

[0155] Example 1: Preparation of lipid nanoparticles targeting APCs

[0156] The raw materials for preparing lipid nanoparticles (LNPs) targeting APCs include: ionizable lipids, auxiliary lipids, polymers, sterols, functional additives, and surface modifiers. In this embodiment, SM-102 is used as an example of the ionizable lipid, DSPC as an example of the auxiliary lipid, DMG-PEG2000 as an example of the polymer, cholesterol as an example of the sterol, nucleic acid drugs as an example of the functional additive, artificial mRNA molecules as an example, wherein the artificial mRNA may encode immunosuppressive molecules and / or immunomodulatory proteins, and a peptide targeting CD11c as an example of the surface modifier.

[0157] 1. Synthetic functional additives (artificial mRNA molecules):

[0158] (1) Design and synthesize DNA templates containing coding and non-coding regions:

[0159] 1) Design and synthesis of the coding region

[0160] The coding region includes the DNA sequence of immunomodulatory molecules and the DNA sequence of signal peptides. The DNA sequence of the immunomodulatory molecules includes the DNA sequence of immunosuppressive molecules and / or immunomodulatory-related proteins. During DNA template design: codons commonly found in host cells and easily recognized are selected to replace codons in the original coding sequence that may lead to lower translation efficiency or higher immunogenicity, with a codon fitness index (CAI) ≥ 0.8 (based on the human codon usage table) and a GC content of 55%-65%, thereby improving the translation efficiency of mRNA in host cells. This facilitates rapid synthesis of the target protein within the cell, thus accelerating the occurrence of biological effects; silencing mutations at cryptic splicing sites (e.g., GT→GC); and immunogenic motifs (e.g., UU / UA dinucleotides) are used, with a substitution rate ≥ 95%.

[0161] The DNA sequences of the immunosuppressive molecules include murine or human PD-L1, PD-L2, CTLA4, and IDO, with nucleotide sequences as shown in SEQ ID NO. 1-8 (the sequences themselves contain signal peptides). The DNA sequences of the immunomodulatory proteins include human or murine HDAC, DNMT3A, TGF-β, IL-27, IL-35, and IL-10, with nucleotide sequences as shown in SEQ ID NO. 9-20 (the sequences themselves contain signal peptides). The DNA sequences of the immunomodulatory molecules can be one of SEQ ID NO. 1-20 above, or multiple sequences of SEQ ID NO. 1-20 in series. When multiple immunomodulatory molecules are tandemly linked, different DNA sequences are connected using adapter molecules, preferably flexible linkers.

[0162] 2) Design and synthesis of non-coding regions

[0163] The non-coded region includes 5'-UTR elements and 3'-UTR elements.

[0164] The DNA sequence of the 5'-UTR element is shown in SEQ ID NO.21. The 5'-UTR element's DNA sequence incorporates a Kozak sequence (CCACC) that enhances ribosome recognition, and the DNA sequence is optimized using mfold prediction. The 5'-UTR element's DNA sequence precedes the signal peptide DNA sequence.

[0165] The DNA sequence of the 3'-UTR element is shown in SEQ ID NO.22. In its DNA sequence, the DNA sequences corresponding to the miR-155 and miR-21 binding sites have been removed, and a dual ARE (AU-rich element) DNA sequence has been inserted. The DNA sequence of the 3'-UTR element is located after the DNA sequence of the immunomodulatory molecule.

[0166] (2) Using RNA polymerase, the synthesized DNA template (taking one of the DNA sequences of the immunomodulatory molecule in SEQ ID NO. 1-20 as an example) is transcribed in vitro to generate an artificial mRNA molecule. During the transcription process: nucleotide modification is performed by adding modifying nucleotides (uridine is replaced with 1-methylpseudouridine (m1Ψ) and cytidine is replaced with 5-methylcytidine (m5C)); 2'-fluorination is introduced into the modification of the 5'-UTR and 3'-UTR regions (1-2 positions are modified per 10 nucleotides).

[0167] (3) After transcription, the mRNA molecule is modified with a 5'-cap structure and a PolyA tail; the 5'-cap structure is m7GpppN, which includes anti-reverse cap analog (ARCA) modification and 2'-O-methylation modification; the PolyA tail includes 70-100 adenine nucleotides, with one phosphate thiophosphate bond inserted for every 10 adenine nucleotides (synthetic formula: A10*S1), and the PolyA tail is located after the 3'-UTR element sequence. The 5'-cap structure m7GpppN can also be replaced with m7GpppNmNm and m7,3′-O-GpppG.

[0168] (4) Purify mRNA molecules, remove impurities and unreacted raw materials, improve the purity and quality of mRNA, and store at -80℃ or -20℃ for later use.

[0169] 2. Preparation of lipid nanoparticles

[0170] In this embodiment, SM102 is used as an example of ionizable lipid, DSPC is used as an example of auxiliary lipid, DMG-PEG2000 is used as an example of polymer, and cholesterol is used as an example of sterol compound.

[0171] The ionizable lipid SM102, the auxiliary lipid DSPC, cholesterol, and the polymer DMG-PEG2000 were dissolved in ethanol solution at a concentration of 10 mg / mL. SM102, DSPC, cholesterol, and DMG-PEG2000 were mixed in a molar ratio of 25:5:69.5:0.5 and anhydrous ethanol was added to control the total lipid concentration at 10 mM. The artificial mRNA molecules obtained in step 1 above (the coding region nucleotide sequences of the mRNA molecules are shown in SEQ ID NO. 1–20) were diluted in citrate buffer (pH 4.0) at a concentration of 91 μg / mL, with the N / P ratio of mRNA and lipids being 4. The prepared organic phase (lipids) and aqueous phase (mRNA) were used to prepare mRNA-loaded LNPs using a microfluidic instrument, with the flow rate of the aqueous phase at 12 mL / min and the flow rate of the organic phase at 4 mL / min. After obtaining LNPs loaded with mRNA, they were immediately diluted with sterile ultrapure water at a volume ratio of 10-fold. The diluted LNPs were then ultrafiltered using a 100 kDa ultrafiltration tube at 3000 rpm and 4°C. When the liquid was reduced to 1 mL, sterile ultrapure water was added to bring the volume to 15 mL, and centrifugation was repeated three times to remove ethanol. Besides the microfluidic method described above, lipid nanoparticles can also be prepared using ethanol dilution / solvent replacement, ultrasonic emulsification, ultra-high pressure homogenization, membrane extrusion, spray drying, electrospray / electrospinning, or any combination of these methods.

[0172] The ionizable lipid SM102 is replaced with one of the following: SM102 and its derivatives, Dlin-MC3-DMA and its derivatives, ALC-0315 and its derivatives, ATX-126 and its derivatives, E12CA1A3 and its derivatives, DOTMA, DOTAP, DOSPA, and ePC. The auxiliary lipid DSPC is replaced with one of the following: POPC, DLPC, DEPC, DOPC, DMPC, DPPC, DSPE, DOPG, ESM, DPPS, DOPE, DSPA, DPPA, and DMG-PEG. The polymer DMG-PEG200 is replaced with one of the following: The above-mentioned LNPs can be prepared by replacing 0 with one of PEG-DMG, PEG-DSPE, DSPE-PEG5000, Ceramide(C14)-PEG2000, Ceramide(C18)-PEG2000, DMG-PEG2000, DPG-PEG2000, DSG-PEG2000, and DOPE-PEG2000, and by replacing cholesterol with one of phytosterols (such as β-sitosterol and stigmasterol), ergosterol and lanosterol and their derivatives (such as cholesterol hemisuccinate, cholesterol-PEG and amino cholesterol).

[0173] The prepared lipid nanoparticles can also be linked to targeting ligands via surface modifiers. These surface modifiers are preferably antibodies or fragments, peptides, or aptamers targeting CD11c, antibodies or fragments, peptides, or aptamers targeting DEC-205, or antibodies or fragments, peptides, or aptamers targeting CLEC9A. This embodiment uses a peptide targeting CD11c as an example. A peptide fragment targeting CD11c (amino acid sequence as shown in SEQ ID NO. 24) is modified onto the surface of the lipid nanoparticles. The molar ratio of the peptide fragment targeting CD11c to the lipids in the lipid nanoparticles is 0.5%-1.5%.

[0174] Example 2: Screening of the types and proportions of ionizable lipids and the ratio of DSPC and DMG-PEG2000.

[0175] The lipid nanoparticles (LNPs) used in this embodiment are LNPs prepared according to the method in Example 1, and their surfaces are not connected to targeting ligands.

[0176] 1. The ionizable lipid is SM-102.

[0177] This experiment will modify the molar ratios of SM-102, DSPC, and DMG-PEG2000, as well as the molar ratio (N / P ratio) of positively charged amino groups (N) in the lipid component to negatively charged phosphate groups (P) in the nucleic acid (mRNA), to construct library A (as shown in Table 1 below). Simultaneously, the molar ratio of cholesterol will be varied to ensure the sum of the molar ratios of the four components is 100%. Ten different formulations of LNPs (A0-A9) from Table 1 will be prepared according to the method described in Example 1. The mRNA molecules will be mRNA molecules encoding human PD-L1 and enhanced green fluorescent protein (EGFP) (i.e., the EGFP DNA sequence will be inserted into a non-natural amino acid (such as acetylphenylalanine) site when designing and synthesizing the coding region DNA template of human PD-L1). The EGFP fluorescent protein will be used in the following experiments to track and evaluate the targeting ability of the LNPs.

[0178] Table 1. Formulas for Library A

[0179] ID SM-102mol% DSPC mol% DMG-PEG2000 mol% N / P A0 50 10 1.5 6 A1 50 20 0.5 6 A2 70 10 0.5 9 A3 50 10 4.5 4 A4 70 20 1.5 4 A5 25 20 4.5 9 A6 50 5 1.5 9 A7 25 5 0.5 4 A8 70 5 4.5 6 A9 25 10 1.5 6

[0180] Furthermore, the particle size and zeta potential of 10 different formulations of LNPs in library A were detected, and the results are as follows: Figure 1 As shown, the particle size of 10 different formulations of LNPs ranges from 120 to 200 nm, and the zeta potential ranges from 8 to 15 mV.

[0181] Furthermore, 10 LNPs formulations from library A were subcutaneously injected into 6-8 week old C57BL / 6 mice (2 μg / mouse, n=4 per group). Twenty-four hours later, inguinal lymph nodes from the injection side were collected, and single-cell suspensions were prepared. CD45, CD11c, CD40, CD80, and CD86 were labeled using flow cytometry, focusing on CD11c. + The expression levels of co-stimulatory molecules (CD40, CD80, CD86) in the APC subset (dendritic cells (DCs)) were assessed to reflect the immunogenicity of different formulations, such as... Figure 2 As shown in the figure. Empty lipid nanoparticles were used as a blank control, and correlation analysis was further used to confirm whether there were any statistically significant influencing factors.

[0182] EGFP was detected only in mouse dendritic cells (DCs), and almost undetectable in other mouse cells or tissues, indicating that LNPs prepared from library A specifically target DCs. For example, LNPs prepared using the A7 formulation target non-APC cells as follows: Figure 3 As shown, EGFP expression was not different in non-APC cells compared to the control group, but was significantly higher in APC cells (CD11c). + CD11b + The expression of LNPs in the A7 formulation was significantly different from that in the control group, indicating that the LNPs prepared by the A7 formulation did not target non-APC cells.

[0183] DCs expressing EGFP, as follows Figure 4 As shown, compared with the control group, this demonstrates that the 10 LNPs (A0-A9) successfully targeted DCs and successfully translated mRNA. In DCs expressing EGFP, CD40... + CD80 + and CD86 + Positive cells account for, for example Figure 5 As shown, the fluorescence intensity of CD40, CD80, and CD86 in these positive cells is as follows: Figure 6 As shown in the figure. The results showed that the A7 formulation exhibited the lowest positive rate and expression level of co-stimulatory molecules, demonstrating excellent immune tolerance. Therefore, the A7 formulation was identified as the optimal candidate formulation, with a molar ratio of SM-102 of 25%, DSPC of 5%, and DMG-PEG2000 of 0.5%. The detection parameters obtained from the A library were comprehensively evaluated using multivariate analysis of variance (ANOVA), principal component analysis (PCA), and correlation analysis to assess the impact of each key parameter on the performance of LNPs. The analysis results are shown in the figure. Figure 7 As shown, the results indicate that the N / P ratio has the most significant positive correlation with EGFP (i.e., targeting efficiency) (0.88), making it a core regulatory point.

[0184] Furthermore, based on formulation A7, library B was established (as shown in Table 2 below), and LNPs were prepared using the same method as library A. The N / P ratio was further optimized to improve the tolerance-inducing ability of LNPs. A central composite design (CCD) was used for systematic optimization. This design method can accurately estimate secondary effects in multi-factor experiments and establish a predictive model to optimize key parameters. Five levels of N / P ratio (low, medium, optimized, medium-high, and high) were set to comprehensively cover the possible range of influence. A regression model was established using response surface methodology (RSM) to explore the optimal N / P ratio range.

[0185] Table 2. Formulas for Library B

[0186] ID SM-102mol% DSPC mol% DMG-PEG2000 mol% N / P B1 25 5 0.5 9 B2 25 5 0.5 8 B3 25 5 0.5 7 B4 25 5 0.5 6 A7 25 5 0.5 4

[0187] Furthermore, the particle size and zeta potential of five different formulations of LNPs from library B were detected, and the results are as follows: Figure 8 As shown, the particle size of the five different formulations of LNPs ranges from 150 to 210 nm, and the zeta potential ranges from 10 to 20 mV.

[0188] Furthermore, 24 hours after subcutaneously injecting mice with the formulations of 5 LNPs (5 μg mRNA) from Library B, the expression of EGFP in DCs in mouse lymph nodes and the expression levels of co-stimulatory molecules (CD80, CD86, CD40) on the surface of DCs in APCs expressing EGFP were detected by flow cytometry, using the same detection method as for Library A.

[0189] EGFP was detected only in mouse dendritic cells (DCs), and almost undetectable in other mouse cells or tissues, indicating that LNPs prepared from library B specifically target DCs. The expression of EGFP in DCs is as follows: Figure 9 As shown, CD40 in DCs expressing EGFP + CD80 + and CD86 + Positive cells account for, for example Figure 10 The fluorescence intensity of CD40, CD80, and CD86 in these positive cells is as follows: Figure 11 As shown in the figure. The results showed that the A7 formulation in library B exhibited the lowest positive rate and expression level of co-stimulatory molecules, demonstrating excellent immune tolerance.

[0190] Therefore, based on the in vivo screening experimental data of the above-mentioned A and B libraries, the A7 formulation was determined as the optimal candidate formulation, with the molar ratio of SM-102 being 25%, the molar ratio of DSPC being 5%, the molar ratio of DMG-PEG2000 being 0.5%, and the N / P ratio being 4.

[0191] 2. The ionizable lipid is MC3.

[0192] In this experiment, the ionizable lipid SM-102 in Example 1 was replaced with MC3. Simultaneously, the molar ratios of MC3, DSPC, and DMG-PEG2000, as well as the molar ratio (N / P ratio) of positively charged amino groups (N) in the lipid components to negatively charged phosphate groups (P) in the nucleic acids (mRNA), were varied to construct a C library (as shown in Table 3 below). The molar ratio of cholesterol was also varied to ensure the sum of the molar ratios of the four components was 100%. Ten different formulations of LNPs were prepared from A0-A9 in Table 1 according to the method described in Example 1. The mRNA molecules were those encoding human PD-L1 and enhanced green fluorescent protein (EGFP) (i.e., the EGFP DNA sequence was inserted into a non-natural amino acid (such as acetylphenylalanine) site when designing and synthesizing the coding region DNA template for human PD-L1). The EGFP fluorescent protein will be used in the following experiments to track and evaluate PD-L1.

[0193] Table 3. Formulas for Library C

[0194] ID MC3 mol% DSPC mol% DMG-PEG2000 mol% N / P C1 50 48 2 8 C2 50 48 2 4 C3 60 38 2 6 C4 60 38 2 8 C5 60 38 2 4 C6 55 42 3 6 C7 70 28 2 6 C8 65 33 2 6 C9 65 33 2 8

[0195] Furthermore, the particle size and zeta potential of nine different formulations of LNPs in library C were detected. The results are shown in Table 4 below. The particle size of the nine different formulations of LNPs is between 100-200 nm, and the zeta potential is between 10-20 mV.

[0196] Table 4. Particle size and zeta potential of LNPs with 9 different formulations in Library C.

[0197]

[0198]

[0199] Furthermore, nine LNPs formulations from the C library were subcutaneously injected into 6-8 week old C57BL / 6 mice (2 μg / mouse, n=4 per group). Twenty-four hours later, inguinal lymph nodes from the injection side were collected, and single-cell suspensions were prepared. CD45, CD11c, CD40, CD80, and CD86 were labeled using flow cytometry, focusing on CD11c. + The expression levels of co-stimulatory molecules (CD40, CD80, CD86) in the APC subset (dendritic cells (DCs)) were assessed to reflect the immunogenicity of different formulations. The detection method was the same as... Figure 2 As shown, empty lipid nanoparticles were used as a blank control.

[0200] EGFP was detected only in mouse dendritic cells (DCs), and almost undetectable in other mouse cells or tissues, indicating that LNPs prepared from the C library specifically target DCs. For example, LNPs prepared using the C3 formulation target non-APC cells as follows: Figure 3 As shown, EGFP expression was not different in non-APC cells compared to the control group, but was significantly higher in APC cells (CD11c). + CD11b + The expression of LNPs in the C3 formulation was significantly different from that in the control group, indicating that the LNPs prepared by the C3 formulation did not target non-APC cells.

[0201] EGFP was successfully detected in DCs, and among the DCs expressing EGFP, the proportion of CD80-positive cells was as follows: Figure 12 As shown, compared with the control group, this demonstrates that the 10 LNPs (C1-C9) successfully targeted DCs and successfully translated mRNA. In DCs expressing EGFP, CD40... + CD80 + and CD86 + The fluorescence intensity of CD40, CD80, and CD86 in positive cells is as follows: Figure 13 As shown in the figure. The results showed that the C3 formulation exhibited the lowest positive rate and expression level of co-stimulatory molecules, demonstrating excellent immune tolerance. The C3 formulation significantly reduced the immunogenicity of LNPs by balancing the N / P ratio and the cationic lipid ratio. Therefore, the C3 formulation was identified as the optimal candidate formulation, with a molar ratio of 60% for MC3, 38% for DSPC, 2% for DMG-PEG2000, and an N / P ratio of 6.

[0202] LNPs (each encapsulating 0.5 μg of mRNA encoding human or murine PD-L1) prepared using the optimal formulation A7 from libraries A and B, and the optimal formulation C3 from library C, were further co-cultured with DC2.4 and RAW264.7 cell lines for 24 h. PD-L1 expression in DC2.4 and RAW264.7 cells was then detected by flow cytometry. In vivo experiments were also conducted. Twenty-four h after subcutaneous injection of different LNPs (2 μg / mouse) into C57 mice, lymph nodes were isolated, and APC cells (CD11c cells) were detected by flow cytometry. + CD11b + PD-L1 expression. Flow cytometry results for in vitro and in vivo cell experiments using formulations A7 and C3 are as follows: Figure 14-15 As shown in the figure. The results showed that LNPs prepared by the two formulations, which encapsulate PD-L1 mRNA, can be used in in vitro cell lines (DC2.4, RAW264.7) and in vivo APC cells (CD11c). + CD11b+ All formulations effectively upregulated PD-L1 expression, but compared to APC cells, the A7 formulation resulted in a higher overall PD-L1 expression level. Therefore, the preferred formulation is A7.

[0203] Example 3: Polymer Screening

[0204] This embodiment is based on the components and proportions of the optimal formulation A7 selected in Example 2, except for the type of polymer DMG-PEG2000 in the A7 formulation, to construct library D. Different types of polymers are shown in Table 5 below. The preparation method of LNPs and the mRNA they encapsulate are the same as in Example 2.

[0205] Table 5. D Library Formula

[0206] ID polymer D1 DSPE-PEG2000 D2 DSPE-PEG5000 D3 Ceramide(C14)-PEG2000 D4 Ceramide(C18)-PEG2000 D5 DMG-PEG2000 D6 DPG-PEG2000 D7 DSG-PEG2000 D8 DOPE-PEG2000

[0207] First, the particle size and zeta potential of eight different formulations of LNPs from library D were measured. The particle size was between 120-150 nm, and the zeta potential was between 8-15 mV. The encapsulation efficiency of mRNA was above 80%.

[0208] Furthermore, eight LNPs formulations from library D were subcutaneously injected into 6-8 week old C57BL / 6 mice (2 μg / mouse, n=4 per group). Twenty-four hours later, inguinal lymph nodes from the injection side were collected, and single-cell suspensions were prepared. CD45, CD11c, CD40, CD80, and CD86 were labeled using flow cytometry, focusing on CD11c. + The expression levels of co-stimulatory molecules (CD40, CD80, CD86) in the APC subset (dendritic cells (DCs)) were assessed to reflect the immunogenicity and delivery efficiency of different formulations.

[0209] In the D library, EGFP was detected only in mouse DCs, and almost undetectable in other mouse cells or tissues, indicating that LNPs prepared from the D library specifically target DCs. CD80 in DCs expressing EGFP... + Positive cells account for, for example Figure 16 As shown, CD40 in DCs expressing EGFP + CD80 + and CD86 + The fluorescence intensity of CD40, CD80, and CD86 in these positive cells is as follows: Figure 17 As shown in the figure. The results showed that the D5 formulation exhibited the lowest positive rate and expression level of co-stimulatory molecules, demonstrating excellent immune tolerance. Therefore, the preferred polymer is DMG-PEG2000.

[0210] Example 4: Screening of surface modifiers and optimization of their molar ratio with lipids

[0211] The lipid nanoparticles (LNPs) used in this embodiment are LNPs prepared according to the method in Example 1, and their surfaces are connected to the targeting ligands through surface modifiers.

[0212] 1. Screening of target ligand types

[0213] This experiment will change the targeting ligands modified on the surface of lipid nanoparticles in Example 1. The different targeting ligands are shown in Table 6 below.

[0214] Table 6. Different Targeting Ligands

[0215] ID Targeted ligands amino acid sequence Ligand 1 Targeting CD11c peptide fragment 1 SEQ ID NO.23 Ligand 2 Targeting CD11c peptide fragment 2 SEQ ID NO.24 Ligand 3 Targeting DEC-205 peptide fragment 1 SEQ ID NO.25 Ligand 4 Targeting DEC-205 peptide fragment 2 SEQ ID NO.26 Ligand 5 Targeting DEC-205 peptide fragment 3 SEQ ID NO.27 Comparison ligand-free ——

[0216] The targeting ligands in Table 6 above were prepared into different LNPs according to the method in Example 1, wherein the mRNA molecules were the same as those in Example 2, which were mRNA molecules that could encode human PD-L1 and enhanced green fluorescent protein (EGFP).

[0217] Furthermore, following the method of subcutaneous injection into mice in Example 2, the positive rate of co-stimulatory molecules (CD40, CD80, and CD86) after LNPs targeted APC cells was detected to assess immunogenicity, and the PD-L1 expression level of APC cells was detected to assess the targeting effect. The detection results are shown in Tables 7-8 below.

[0218] Table 7. Positive rate of cells expressing co-stimulatory molecules after targeting APC cells with different modified LNPs.

[0219] LNPs ligands <![CDATA[CD40 + in EGFP + CD11c + (%)]]> <![CDATA[CD80 + in EGFP + CD11c + (%)]]> <![CDATA[CD86 + in EGFP + CD11c + (%)]]> Ligand 1 44.3 56.3 49.6 Ligand 2 42.9 55.4 53.3 Ligand 3 33.8 49.3 51.9 Ligand 4 51.2 53.9 60.2 Ligand 5 49.3 60.2 55.4 Comparison 42.3 46.0 51.3

[0220] Table 8. PD-L1 expression levels after targeting APC cells with different modified LNPs

[0221] LNPs ligands <![CDATA[PD-L1 + in CD11c + (%)]]> Ligand 1 82.5 Ligand 2 91.2 Ligand 3 79.3 Ligand 4 83.2 Ligand 5 76.3 Comparison 67.8

[0222] According to the experimental results in Table 7, LNPs modified with different ligands were used in mouse in vivo experiments. The number of cells positive for surface costimulatory molecules CD40, CD80 and CD86 in EGFPCD11c positive cells in mouse lymph nodes was not significantly different from that in the control group, indicating that ligand-modified LNPs also have low immunogenicity. However, when the target ligands were ligands 1 to 3, the level of surface costimulatory molecules expressed by APC cells was relatively lower.

[0223] Based on the experimental results in Table 8, the levels of PD-L1 expression in APC cells were compared after LNPs modified with different ligands were targeted. Compared with the control, the LNPs modified with ligands resulted in higher levels of PD-L1 expression in APC cells. When the targeting ligand was ligand 2, the level of PD-L1 expression in APC cells was the highest, and the targeting effect was the best.

[0224] Based on the above analysis of immunogenicity and targeting effect, the preferred targeting ligand is CD11c polypeptide fragment 2, whose amino acid sequence is shown in SEQ ID NO.24.

[0225] 2. Optimization of the molar ratio of targeting ligand to lipids

[0226] In this experiment, the molar ratio of the targeting ligand to lipid in the lipid nanoparticles in Example 1 will be varied, with the molar ratios being 0.1%, 0.5%, 1%, 1.5%, and 2%. Different LNPs will be prepared using the preferred molar ratio of the targeting ligand 2 to lipid in Table 6 above, following the method described in Example 1. The mRNA molecules will be the same as in Example 2, encoding human PD-L1 and enhanced green fluorescent protein (EGFP).

[0227] Furthermore, the particle size, zeta potential, encapsulation efficiency, positive rate of target cell co-stimulatory molecules (CD40, CD80, and CD86), and PD-L1 expression level of LNPs were detected according to the method in Example 2. The results of particle size, zeta potential, and encapsulation efficiency are shown in Table 9 below, and the results of the positive rate and expression level of target cell co-stimulatory molecules are shown in Tables 10-11 below.

[0228] Table 9. Particle size and zeta potential of LNPs prepared with different molar ratios of targeting ligand to lipid.

[0229] Molar ratio of ligand to lipid Particle size (nm) Zeta potential (mV) Encapsulation efficiency (%) 0.1％ 122 13.4 82 0.5％ 135 15.6 87 1％ 140 17.2 81 1.5％ 142 16.3 76 2％ 162 16.9 72

[0230] Table 10. Positive rate of cells expressing co-stimulatory molecules after LNPs targeted APC cells at different molar ratios of targeting ligands and lipids.

[0231]

[0232] Table 11. Levels of PD-L1 expression in APC cells after LNPs targeting different molar ratios of targeting ligands and lipids.

[0233] Molar ratio of ligand to lipid <![CDATA[PD-L1 + in EGFP + CD11c + (%)]]> 0.1％ 78.2 0.5％ 82.3 1％ 83.6 1.5％ 86.8 2％ 87.2

[0234] According to the results in Table 9, the particle size of LNPs increases with the increase of the molar ratio of ligand to lipid; its zeta potential first increases with the increase of the molar ratio of ligand to lipid, and decreases but remains high after the molar ratio reaches 1.5%; its encapsulation efficiency remains above 80% when the molar ratio of ligand to lipid is in the range of 0.1% to 1%, and decreases after the molar ratio exceeds 1%.

[0235] According to the results in Table 10, when the molar ratio of ligand to lipid is in the range of 0.1%-1.5%, EGFP expressing the co-stimulatory molecule... +CD11c + The proportion of positive cells was low, indicating low immunogenicity.

[0236] According to the results in Table 11, within the molar ratio of ligand to lipid of 0.1%-2%, the expression level of PD-L1 gradually increased with increasing molar ratio, indicating that the targeting effect of LNPs gradually improved. However, considering the data on particle size and potential, a higher molar ratio may lead to the rupture or instability of the lipid bilayer molecules, thereby reducing the encapsulation efficiency of mRNA and ultimately affecting the targeting effect. Therefore, the preferred molar ratio of surface modifier to lipid is 0.5%-1.5%.

[0237] Example 5: Optimization of DNA Template

[0238] The lipid nanoparticles (LNPs) used in this embodiment are LNPs prepared according to the method in Example 1, and their surfaces are not connected to targeting ligands.

[0239] 1. Optimization of coding region DNA template sequence

[0240] 1) Base optimization

[0241] The DNA sequences of the 10 immunomodulatory molecules provided in Example 1 have been optimized, with a codon fitness index (CAI) ≥ 0.8 (based on the human codon usage table) and a GC content of 55%-65%. Simultaneously, the following functional elements were introduced: silencing mutations at cryptic splicing sites (e.g., GT→GC); and immunogenic motif substitution rates (e.g., UU / UA dinucleotides) ≥ 95%.

[0242] In this experiment, two types of mRNA were prepared according to the method in Example 1. The DNA template of one LNP mRNA was optimized according to the method described above. The optimized DNA template was taken as the human PD-L1 DNA template, and its sequence is shown in SEQ ID NO.1. The DNA template of the other mRNA was the unoptimized human PD-L1 DNA template.

[0243] This experiment will use HEK-Blue TM The reporting system detected the TLR7 / 8 activation levels and in vitro protein expression levels of mRNA transcribed from optimized and unoptimized DNA templates. The TLR7 / 8 activation level detection results showed that the TLR7 / 8 activation level of mRNA transcribed from optimized DNA templates was significantly lower than that of mRNA transcribed from unoptimized DNA templates, indicating that optimizing the DNA template reduces immunogenicity and improves safety. The in vitro protein expression level detection results are shown in Table 12 below.

[0244] Table 12. In vitro protein expression levels of mRNA transcribed from different DNA templates

[0245] DNA template In vitro protein expression levels optimization 1.9 Unoptimized 1

[0246] According to the results in Table 12, the in vitro protein expression levels of mRNA transcribed from optimized DNA templates were significantly higher than those of mRNA transcribed from unoptimized DNA templates, indicating that optimizing the DNA template improves translation efficiency.

[0247] Furthermore, the optimized and unoptimized DNA templates were prepared into LNPs according to the method of Example 1, wherein the mRNA molecule was further inserted with an enhanced green fluorescent protein (EGFP) sequence according to the method of Example 2. The LNPs formulation was subcutaneously injected into 6-8 week old C57BL / 6 mice (2 μg / mouse, n=4 per group). Twenty-four hours later, inguinal lymph nodes from the injection side were collected, and single-cell suspensions were prepared. After labeling the target cells and target proteins with the corresponding antibodies, CD11c was detected by flow cytometry. + The percentage of PD-L1-positive cells on the cell surface was measured, and CD4+ levels in lymph nodes were also detected. + The percentage of T cells expressing the three inflammatory factors IFN-γ, IL-17, and TNF-α, and CD4+ + The percentage of T cells expressing the transcription factor FOXP3 was positive. The results are shown in Table 13-14 below.

[0248] Table 13. CD11c of mRNA transcribed from different DNA templates targeting APCs + PD-L1 in cells + percentage of positive cells

[0249] DNA template <![CDATA[PD-L1 + in CD11c + cells(%)]]> optimization 79.2 Unoptimized 63.8

[0250] Table 14. CD4 in lymph nodes after mRNA transcribed from different DNA templates targets APCs. + IFN-γ in T cells + IL-17 + TNF-α + and FOXP3 + percentage of positive cells

[0251]

[0252] Based on the results in Table 13-14, comparing the experimental results before and after DNA template optimization, it is shown that the mRNA prepared from the optimized DNA template, after delivery via LNPs, exhibits lower CD11c levels. + PD-L1 in cells + A higher percentage of positive cells indicates that the mRNA derived from the optimized DNA template has higher translation efficiency; CD4 in lymph nodes + FOXP3 in T cells +The higher percentage of positive cells indicates that the mRNA derived from the optimized DNA template induced tol-APCs to promote Treg amplification more effectively.

[0253] 2. Optimization of non-coding region DNA template sequence

[0254] (1) Optimization of 5'-UTR components

[0255] The DNA template provided in Example 1 contains an optimized 5'-UTR element in its non-coding region. The DNA template containing the 5'-UTR element incorporates a Kozak sequence (CCACC) that enhances ribosome recognition, and the DNA template sequence is optimized using mfold prediction.

[0256] Three types of PD-L1 mRNA were prepared according to the method in Example 1. The first mRNA contained an optimized 5'-UTR element (nucleotide sequence as shown in SEQ ID NO. 21), the second mRNA contained an unoptimized 5'-UTR element (nucleotide sequence as shown in SEQ ID NO. 28), and the third mRNA did not contain a 5'-UTR element. The in vitro translation efficiency of the three PD-L1 mRNAs was tested, with the in vitro translation efficiency of the mRNA containing the unoptimized 5'-UTR element being defined as "1". The test results are shown in Table 15 below.

[0257] Table 15. In vitro translation efficiency of three PD-L1 mRNAs

[0258] PD-L1 mRNA In vitro translation efficiency Contains optimized 5'-UTR ≥1.5 Contains unoptimized 5'-UTR 1 Without 5'-UTR 0.75

[0259] According to the results in Table 15, the translation efficiency of mRNA containing the optimized 5'-UTR was increased by ≥1.5 times, indicating that adding the 5'-UTR element to mRNA and optimizing the 5'-UTR element can significantly improve the translation efficiency.

[0260] Further analysis of the secondary structure free energy of the three mRNAs after transcription revealed that the secondary structure free energy of the mRNA containing the optimized 5'-UTR was ≤-5 kcal / mol, indicating that the optimized mRNA has a more stable secondary structure and stronger resistance to degradation.

[0261] (2) Optimization of 3'-UTR components

[0262] In the DNA template of the 3'-UTR element, the DNA sequences corresponding to the miR-155 and miR-21 binding sites were removed; and the DNA sequence of the double ARE (AU-rich element) was inserted.

[0263] Three types of PD-L1 mRNA were prepared according to the method in Example 1. The first mRNA contained an optimized 3'-UTR element (nucleotide sequence as shown in SEQ ID NO. 22), the second mRNA contained an unoptimized 3'-UTR element (nucleotide sequence as shown in SEQ ID NO. 29), and the third mRNA did not contain a 3'-UTR element. The half-life of the three PD-L1 mRNAs was detected, with the half-life of the mRNA containing the unoptimized 3'-UTR element being "1". The detection results are shown in Table 16 below.

[0264] Table 16. Half-life of three PD-L1 mRNAs

[0265] PD-L1 mRNA half life Contains optimized 3'-UTR ≥1.5 Contains unoptimized 3'-UTR 1 Without 3'-UTR 0.68

[0266] According to the results in Table 16, the half-life of mRNA containing optimized 3'-UTR can be extended by ≥1.5 times compared with unoptimized 3'-UTR and mRNA without 3'-UTR. This indicates that the stability of mRNA containing optimized 3'-UTR is significantly improved, which can increase its lifespan in cells and ensure that it maintains a sufficient concentration for a long time.

[0267] Further through HEK-Blue TM The reporting system detected the TLR7 / 8 activation levels of three mRNAs. The results showed that the mRNAs containing optimized 3'-UTRs had lower TLR7 / 8 activation levels compared to those without optimized 3'-UTRs or without 3'-UTRs. This indicates that the immunogenicity of mRNAs containing optimized 3'-UTRs is significantly reduced, thus avoiding potential immune side effects.

[0268] Example 6: Effects of LNPs delivering one or more mRNAs on the in vitro and in vivo generation of tol-APCs.

[0269] 1. The effect of LNPs delivering mRNA encoding an immunomodulatory molecule on the induction of tol-APCs in vivo and in vitro.

[0270] In this embodiment, LNPs were prepared according to the method provided in Example 1. Each LNP encapsulates an mRNA (the mRNA encodes a human or mouse immunomodulatory molecule (inhibitory co-stimulatory molecule or immunomodulatory related protein), and the nucleotide sequence of the mRNA is shown in SEQ ID NO. 1-20). The mRNAs encapsulating different proteins in the LNPs are shown in Table 17 below.

[0271] Table 17. LNPs encapsulate mRNAs encoding different non-fusion proteins

[0272] LNPs number mRNA encodes a human or mouse non-fusion protein. LNPs-1 PD-L1 LNPs-2 PD-L2 LNPs-3 CTLA-4 LNPs-4 IDO LNPs-5 HDAC LNPs-6 DNMT3A LNPs-7 TGF-β LNPs-8 IL-27 LNPs-9 IL-35 LNPs-10 IL-10

[0273] (1) LNPs encapsulate mRNA encoding a murine immunomodulatory molecule for in vitro induction of tol-APCs.

[0274] 1) T cell sorting and activation: Single-cell suspensions were prepared from the spleens of C57BL / 6 mice, and CD3 cells were obtained by sorting using a negative selection magnetic bead kit. + T cells; the sorted T cells were resuspended in complete RPMI-1640 medium containing anti-mouse CD3 (1 μg / mL) and CD28 (1 μg / mL) antibodies and incubated at 37°C and 5% CO2 for 12 hours to induce initial activation.

[0275] 2) Isolation and induction of bone marrow-derived dendritic cells (BMDCs): Bone marrow cells were extracted from the femur and tibia of C57BL / 6 mice. After erythrocyte lysis, the cells were resuspended in complete RPMI-1640 medium containing GM-CSF (20 ng / mL) and IL-4 (10 ng / mL) and cultured in 6-well plates to induce differentiation into BMDCs. Fresh cytokines were replaced every two days during the culture process. Suspension cells were collected on day 6 and used as mature BMDCs for subsequent transfection.

[0276] 3) mRNA transfection of BMDCs and co-culture with T cells: BMDCs induced on day 6 were transfected with LNPs (LNPs in Table 17) carrying mouse immunomodulatory molecule mRNA; after transfection, they were incubated for another 6 hours to ensure sufficient expression of the target protein; then, the activated T cells and the transfected tolerable BMDCs were co-cultured at a ratio of 1:10 and incubated at 37°C and 5% CO2 for 24 hours.

[0277] 4) T cell function analysis: After co-culture, T cells were collected and analyzed using multiparameter flow cytometry as follows: Treg cell detection: CD4 and CD25 were stained on the surface, and FOXP3 was stained intracellularly to analyze CD4. + FOXP3 + The proportion of regulatory T cells was assessed to evaluate the ability of transfected BMDCs to induce / expand Tregs. Intracellular cytokine detection: Monensin, a protein transport inhibitor, was added to the end of the co-culture medium and incubated for 4-6 hours to promote cytokine accumulation. Intracellular staining was then performed to detect the expression levels of inflammatory factors such as IFN-γ, IL-17, and TNF-α, and to assess the activation status of effector T cells and their regulation by BMDCs.

[0278] The expression levels of inflammatory factors and the activation status of effector T cells, such as Figure 18As shown, the results indicate that the designed LNPs / mRNA delivery systems exhibit good immunomodulatory effects in in vitro experiments. Compared with the control group, the proportion of pro-inflammatory T cells in disease-related draining lymph nodes was significantly reduced after co-culturing LNPs loaded with different mRNAs and T cells, among which IFN-γ... + TNF-α + and IL-17 + T cells were significantly reduced. Meanwhile, CD4+ cells were significantly reduced in the transfection group. + FOXP3 + The proportion of regulatory T cells (Tregs) was significantly increased. Among the immunosuppressive factors tested, mRNAs encoding PD-L1, PD-L2, IDO, HDAC, and DNMT3A showed the most significant effects, outperforming other constructs in multiple indicators, including downregulation of pro-inflammatory cytokine expression and induction of Treg amplification, demonstrating stronger immunomodulatory potential and therapeutic value. Specifically, LNPs encapsulating PD-L1 mRNA exhibited the strongest effects in inhibiting inflammatory cytokine secretion and enhancing Treg induction.

[0279] (2) LNPs encapsulate mRNA encoding a human immune regulatory molecule for in vitro induction of tol-APCs.

[0280] 1) Isolation and APC induction of peripheral blood mononuclear cells (PBMCs): PBMCs were isolated from healthy donor peripheral blood by Ficoll density gradient centrifugation. Subsequently, PBMCs were seeded in 6-well plates and cultured for 4-6 hours to promote mononuclear cell adhesion. After removing suspension cells, RPMI-1640 complete medium containing rhGM-CSF (50 ng / mL) and rhIL-4 (20 ng / mL) was added to the adherent cells to induce their differentiation into immature dendritic cells (moDCs). APCs obtained by induction were collected on days 5-6 for mRNA transfection.

[0281] 2) Transfection of immunosuppressive factor mRNA with LNPs: The mRNA of the human immunosuppressive factor to be tested (such as PD-L1, IL-10, IDO1, etc.) is encapsulated in lipid nanoparticles (LNPs) and transfected into the above-induced moDCs; the transfection dose and reaction time are optimized according to the preliminary experiment (e.g. 0.5-1 μg mRNA, incubation for 6 hours); after transfection, continue to culture for 6-12 hours to ensure protein expression.

[0282] 3) Sorting and activation of peripheral blood-derived T cells: CD3+ cells were obtained from donor PBMCs using magnetic beads. + T cells were pre-activated for 12 hours with anti-human CD3 / CD28 antibody (1 μg / mL) to simulate the state of T cells after antigen stimulation.

[0283] 4) Co-culture and functional analysis: Activated human T cells and transfected moDCs were co-cultured at a DC:T ratio of 1:5 at 37°C and 5% CO2 for 24 hours. After co-culture, multi-parameter flow cytometry was used for the following analysis: Treg ratio detection: CD4 was assessed by surface staining with CD4 and CD25, combined with intracellular staining with FOXP3. + CD25 + FOXP3 + Induction effect of regulatory T cells; detection of inflammatory factor expression: Protein transport inhibitors (such as Brefeldin A) were added to the end of the co-culture and incubated for 6 hours. Intracellular staining was then performed to detect the expression levels of cytokines such as IFN-γ, IL-2, and TNF-α in T cells, so as to evaluate the regulatory ability of APC cells transfected with different immunomodulatory factors on T cell function.

[0284] The expression levels of inflammatory factors and the activation status of effector T cells, such as Figure 19 As shown, the co-culture experiment results indicated that DCs treated with LNPs delivering human immunomodulatory molecule mRNA significantly regulated T cell functional status. Specifically, this manifested as: IFN-γ + IL-17 + TNF-α + The significantly decreased expression levels of pro-inflammatory cytokines indicate that the activity of inflammatory T cells is suppressed. Meanwhile, CD4+ expression levels are also significantly reduced. + CD25 + FOXP3 + The significantly increased proportion of Treg cells suggests that regulatory T cells were effectively induced or expanded, thus promoting immune tolerance. Among the human immunomodulatory molecules tested, gene constructs of PD-L1, PD-L2, IDO1, HDAC1, and DNMT3A showed outstanding performance in multiple indicators, effectively inhibiting the secretion of inflammatory factors and enhancing Treg induction, demonstrating strong potential for human immunomodulation. Among these, LNPs carrying PD-L1 mRNA exhibited the strongest effects in inhibiting inflammatory factor secretion and enhancing Treg induction.

[0285] (3) LNPs encapsulate mRNA encoding a human / mouse immunomodulatory molecule for use in experiments to induce the generation of tol-APCs in mice.

[0286] To evaluate whether the LNPs / mRNA system encapsulating a human / mouse mRNA could effectively transfect APCs in vivo and convert them into tolerant APCs, this example involved subcutaneous injection of the LNPs / mRNAs listed in Table 19 into mice. 24 hours later, mouse lymph nodes were collected, and the target cells and proteins were labeled with the corresponding antibodies. CD11c was then detected by flow cytometry. + and CD11b + The expression levels of corresponding immunomodulatory molecules in these two cell groups were measured to verify the delivery efficiency of the system. Simultaneously, CD4+ expression in lymph nodes was detected. + Expression levels of IFN-γ, IL-17, TNF-α, and FOXP3 in T cells.

[0287] Table 19 shows the expression results of immunomodulatory molecules detected by targeting LNPs-1~2 and LNPs-4~6 mRNA to APCs. Figure 20-24 As shown, subcutaneously injected LNPs / mRNA can effectively express APCs in lymph nodes, significantly increasing the expression levels of murine and human PD-L1, PD-L2, IDO on the surface of APCs and intracellular HDAC and DNMT3A, leading to the generation of tol-APCs in vivo. CD4+ in lymph nodes... + The expression levels of IFN-γ, IL-17, TNF-α, and FOXP3 in T cells were as follows: Figure 25-26 As shown in the figure. The results indicate that regardless of whether LNPs encode human or mouse mRNA, they can induce the generation of tol-APCs and IFN-γ. + IL-17 + and NF-α + The proportion of T cells was significantly downregulated, indicating that it effectively downregulated the proportion of pro-inflammatory T cells; FOXP3 + The significantly increased proportion of T cells indicates the induction of Treg cell expansion and maintenance of immune tolerance. Among them, LNPs carrying human or murine PD-L1 mRNA showed the strongest effects in inhibiting the secretion of inflammatory factors and enhancing Treg induction.

[0288] 2. The effect of LNPs delivering mRNAs encoding multiple immunomodulatory molecules on the induction of tol-APCs in vivo and in vitro.

[0289] Experiment 1 above demonstrated that LNPs of human or murine PD-L1 mRNA had the strongest effect in inhibiting the secretion of inflammatory factors and enhancing the induction of Tregs. In this experiment, LNPs were encapsulated with mRNAs encoding two different human or murine immunomodulatory molecules (one encoding PD-L1 and the other an immunomodulatory molecule), as shown in Table 18 below.

[0290] Table 18. LNPs encapsulate mRNAs encoding two different immune regulatory molecules.

[0291]

[0292]

[0293] (1) LNPs encapsulate mRNAs encoding two murine immunomodulatory molecules for in vitro induction of tol-APCs.

[0294] The experimental procedures and detection methods were the same as in Experiment 1 above. The results of using LNPs loaded with two different mRNAs to generate murine tol-APCs in vitro, which downregulated the proportion of pro-inflammatory T cells and induced the generation of Treg cells, are shown in Table 19 below, and are compared with LNPs loaded with mRNA that only encodes murine PD-L1.

[0295] Table 19. The proportion of pro-inflammatory T cells downregulated after LNPs carrying two different mRNAs were used to generate murine tol-APCs in vitro.

[0296]

[0297] Table 19 shows that LNPs encapsulating two murine immunomodulatory molecules, one encoding murine PD-L1 and the other encoding only murine PD-L1, were more effective at downregulating the proportion of pro-inflammatory T cells and enhancing Treg induction. Specifically, downregulation of pro-inflammatory IFN-γ was observed. + The optimal proportion of T cells was observed in LNPs carrying murine PD-L1+IDO, PD-L1+DNMT3A, and PD-L1+IL-27 mRNA; pro-inflammatory IL-17 was downregulated. + The optimal proportion of T cells was found in LNPs carrying murine PD-L1+PD-L2, PD-L1+IDO, PD-L1+HDAC, and PD-L1+DNMT3A mRNA; downregulation of pro-inflammatory TNF-α was observed. + The LNPs with the best T cell proportions were those loaded with murine PD-L1+PD-L2, PD-L1+IDO, PD-L1+HDAC, and PD-L1+DNMT3AmRNA; and the LNPs with the best Treg induction ability were loaded with murine PD-L1+PD-L2, PD-L1+IDO, PD-L1+HDAC, and PD-L1+TGF-β mRNA. However, comprehensive analysis showed that the LNPs loaded with murine PD-L1+IDO mRNA were the most effective in downregulating pro-inflammatory T cells and enhancing Treg induction.

[0298] (2) LNPs carrying mRNAs encoding two human immune regulatory molecules were used in an in vitro experiment to induce the generation of tol-APCs.

[0299] The experimental procedures and detection methods were the same as in Experiment 1 above. The results of the detection of the proportion of pro-inflammatory T cells downregulated and the proportion of Treg cells induced after the use of LNPs loaded with two different mRNAs to generate human tol-APCs in vitro are shown in Table 20 below, and are compared with LNPs loaded with mRNA that only encodes human PD-L1.

[0300] Table 20. The proportion of pro-inflammatory T cells downregulated after LNPs carrying two different mRNAs were used to generate human tol-APCs in vitro.

[0301]

[0302] Table 20 shows that LNPs encapsulating two human immunomodulatory molecules, one encoding human PD-L1 and the other encoding only human PD-L1, were more effective at downregulating the proportion of pro-inflammatory T cells and enhancing Treg induction. Specifically, downregulation of pro-inflammatory IFN-γ was observed. + The LNPs with the best T cell ratio were those carrying human PD-L1+HDAC, PD-L1+DNMT3A, and PD-L1+TGF-β mRNA; pro-inflammatory IL-17 was downregulated. + The optimal proportion of T cells was found in LNPs containing human PD-L1+PD-L2, PD-L1+HDAC, PD-L1+DNMT3A, and PD-L1+TGF-β; pro-inflammatory TNF-α was downregulated. + The LNPs with the best T cell ratio were those carrying human PD-L1+PD-L2, PD-L1+IDO, and PD-L1+DNMT3Am RNA; and the LNPs with the best Treg induction ability were those carrying human PD-L1+PD-L2, PD-L1+IDO, PD-L1+DNMT3Am RNA, and PD-L1+IL-10 mRNA. However, comprehensive analysis showed that the LNPs carrying human PD-L1+DNMT3A mRNA were the most effective in downregulating pro-inflammatory T cells and enhancing Treg induction.

[0303] Based on the above analysis, the LNPs loaded with murine PD-L1+IDO mRNA and human PD-L1+DNMT3A mRNA were the most effective in downregulating the proportion of pro-inflammatory T cells and enhancing Treg induction.

[0304] Example 7: Duration of in vivo generation of tol-APCs

[0305] In this embodiment, LNPs (unmodified target ligands) encoding PD-L1 mRNA were prepared according to the method in Example 1. Mice were subcutaneously injected with LNPs / mPD-L1 on days 1, 3, 5, and 6 as a paradigm to characterize the maintenance time of tol-APCs in vivo. On day 7, the expression level of PD-L1 on APCs was detected by separating inguinal lymph nodes.

[0306] Test results as follows Figure 27 As shown, after drug treatment, the expression of PD-L1 on the surface of APCs reached its peak on day 1 and then gradually decreased, with the longest duration lasting until day 4, indicating that the duration of tol-APCs generated in vivo is controllable.

[0307] Example 8: In vivo generation of tol-APCs to treat mouse models of immune diseases

[0308] LNPs were prepared according to the method in Example 1, and mRNAs encoding PD-L1, PD-L2, DNMT3A, HDAC and IDO were encapsulated in them, respectively. The therapeutic effects of LNPs / mRNA on autoimmune diseases were then evaluated.

[0309] 1. Treatment of RA mouse model

[0310] Rheumatoid Arthritis (RA) Modeling in Mice: Eight-week-old DBA / 1 mice were inoculated with type II collagen on days 0 and 21. On day 28 post-modeling, mice were randomly divided into eight groups and injected subcutaneously with 2 μg of LNPs / mRNA solution on the posterior back at days 28, 32, 36, and 40. Control mice were injected with the same volume of PBS or empty LNPs at the same time points. Arthritis indices were assessed periodically after treatment initiation.

[0311] Test results as follows Figure 28 As shown, the treatment group significantly reduced the arthritis index and alleviated disease progression in the RA mouse model, showing a significant difference compared with the PBS group and the LNPs group.

[0312] 2. Treatment of UC mouse model

[0313] Ulcerative colitis (UC) modeling in mice: C57BL / 6 mice were given free access to drinking water containing 3% DSS for 7 consecutive days to induce UC pathogenesis. DSS-induced mice exhibited typical UC symptoms, including weight loss, bloody stools, diarrhea, and colonic tissue damage. UC-modeled mice were randomly divided into eight groups: on days 1, 3, and 5 after modeling, mice were subcutaneously injected with 2 μg of LNPs / mRNA solution in the posterior dorsal region. Control group mice were injected with the same volume of PBS or empty LNPs at the same time points. After treatment began, the disease activity index of the mice was assessed regularly, particularly by observing changes in body weight, the degree of intestinal bleeding, the frequency of diarrhea, and the severity of clinical symptoms.

[0314] Experimental results are as follows Figure 29 As shown, in a mouse model of ulcerative colitis (UC), the treatment group using tol-APCs (tolerance antigen-presenting cells) significantly reduced the disease activity index and effectively alleviated disease progression. Mice in the LNPs / mRNA treatment group showed significantly reduced disease activity index and significantly improved clinical symptoms on days 7, 14, and 21 post-treatment, particularly in weight recovery, reduced diarrhea, and decreased intestinal bleeding. Compared to mice in the PBS and LNPs groups, the LNPs / mRNA group showed a significantly lower disease activity index and a longer duration of symptom relief. Mice in the PBS and LNPs groups continued to exhibit severe diarrhea, weight loss, and intestinal bleeding, indicating that mRNA-induced tol-APCs have a significant therapeutic effect in the UC mouse model.

[0315] 3. Treatment of MS mouse models

[0316] Multiple sclerosis (MS) modeling in mice: C57BL / 6 mice can be used to establish an experimental autoimmune encephalomyelitis (EAE) model by subcutaneous injection of the autoimmune myelin antigen MOG35-55 (emulsified with complete Freund's adjuvant) to simulate MS-related immune-mediated neurological damage. Each mouse is typically injected subcutaneously with 100 μg MOG35-55 into the scapular region, and simultaneously injected intraperitoneally with diphtheria-tetanus-pertussis toxin (PTX) to enhance immune activation and blood-brain barrier permeability. Approximately 7–10 days post-immunization, mice exhibit typical neurological damage symptoms, such as tail relaxation or paralysis, hind limb motor dysfunction, and weight loss. The degree of neurological damage can be quantitatively assessed using a clinical score (0–5 points). MS modeling mice are randomly divided into four groups. On days 2, 5, 8, and 11 post-modeling, mice are subcutaneously injected with 2 μg of LNPs / mRNA solution in the posterior back. Control group mice are injected with the same amount of PBS or empty LNPs at the same time points. After treatment began, the disease progression in mice was assessed regularly, particularly by evaluating the symptoms and extent of neurological damage in MS using a clinical scoring system and histological analysis.

[0317] Experimental results are as follows Figure 30 As shown, in a mouse model of multiple sclerosis (MS), treatment with mRNA drugs that generate tolerable antigen-presenting cells (tol-APCs) significantly reduced the disease index and effectively alleviated disease progression. Compared with the PBS and LNPs groups, mice in the LNPs / mRNA group showed a significantly lower disease index, especially on days 14 and 28 after treatment, with significant improvement in clinical scores and better recovery of motor function. Mice in the PBS and LNPs groups still showed higher disease indices and continued deterioration of motor function, indicating that the therapeutic effect of mRNA-induced tol-APCs in the MS mouse model was significantly better than that in the control group.

[0318] 4. Treatment of T1D mouse model

[0319] Modeling Type 1 Diabetes (T1D) in Mice: C57BL / 6 mice were injected with a small dose of streptozotocin (STZ) to destroy pancreatic β cells, leading to elevated blood glucose levels. This method induced the characteristic persistently elevated blood glucose levels in T1D mice, mimicking the pathology of clinical type 1 diabetes. T1D modeling mice were randomly divided into eight groups. On days 2, 4, and 6 after modeling, 2 μg of LNPs / mRNA solution was subcutaneously injected into the posterior back. Control group mice were injected with the same amount of PBS or empty LNPs at the same time points. After treatment began, blood glucose levels were monitored regularly, and clinical symptoms and pancreatic function were assessed.

[0320] Experimental results are as follows Figure 31 As shown, in a mouse model of type 1 diabetes (T1D), treatment with mRNA drugs that generate tolerogenic antigen-presenting cells (tol-APCs) significantly reduced blood glucose levels and effectively mitigated disease progression. Mice in the LNPs / mRNA group showed a significant improvement in blood glucose levels compared to the PBS and LNPs groups. Particularly on days 14 and 28 post-treatment, the blood glucose levels in the LNPs / mRNA group were significantly lower than those in the PBS and LNPs groups. This indicates that tol-APCs induced by mRNA drugs play a crucial role in effectively regulating immune tolerance, inhibiting the immune system's attack on pancreatic β-cells, thereby improving pancreatic islet function and reducing blood glucose levels.

[0321] 5. Treatment of SLE mouse models

[0322] Establishing a systemic lupus erythematosus (SLE) mouse model: C57BL / 6 mice (6-8 weeks old) were selected for the experiment; nuclear antigen (such as U1-snRNP or dsDNA) was dissolved in PBS at a concentration of 1-2 mg / mL, and emulsified thoroughly with an equal volume of complete Freund's adjuvant (CFA, containing inactivated Mycobacterium tuberculosis) to form a stable emulsion. 100 μL was injected subcutaneously into each mouse (50 μL injected into each side under the left and right scapulae) as the primary immunization; 2-3 weeks after immunization, the same nuclear antigen and incomplete Freund's adjuvant (IFA) could be used for emulsification. Boosting immunizations were administered at the same dose each time, and this could be repeated 2-3 times to continuously stimulate the immune system and induce the production of autoantibodies. In some experiments, 200 ng / mouse of diphtheria-tetanus-pertussis toxin (PTX) could be injected as an adjunct to enhance immune activation and blood-brain barrier permeability. After repeated immune stimulation, the level of autoantibodies in the mouse serum significantly increased (such as anti-dsDNA and antinuclear antibodies), and proteinuria, glomerular immune complex deposition, and nephritis appeared simultaneously, accompanied by clinical symptoms such as weight loss and decreased activity, thus mimicking the immune abnormalities and multi-organ damage characteristics of SLE patients. SLE model mice were randomly divided into eight groups. On days 2, 4, and 6 after successful modeling, 2 μg of LNPs / mRNA solution was subcutaneously injected into the posterior back of the mice. Control group mice were injected with the same amount of PBS or empty LNPs at the same time points. Before the experiment, the mice underwent preliminary tests, including weight, skin lesions, urinary protein, and antinuclear antibody (ANA) levels, to assess the clinical characteristics and pathological development of SLE. After treatment began, the clinical symptoms of the mice were recorded regularly, and the SLE disease index was calculated, which was scored based on indicators such as weight loss, skin lesions, renal dysfunction, and antibody levels.

[0323] Treatment results as follows Figure 32 As shown, mice in the LNPs / mRNA group exhibited a significant reduction in SLE disease indices compared to the PBS and LNPs groups. In the LNPs / mRNA group, skin lesions were significantly reduced, urinary protein levels were significantly decreased, and renal function was effectively protected. Furthermore, the level of antinuclear antibodies (ANA) in the blood was significantly lower in the LNPs / mRNA group than in the control group, indicating that inducing the production of tol-APCs can effectively alleviate the SLE-specific autoimmune response.

[0324] 6. Treatment of GvHD mouse model

[0325] Establishment of a graft-versus-host disease (GvHD) mouse model: Recipient BALB / c mice were irradiated whole-body (7.5 Gy, disrupting the host immune system). GvHD model mice were randomly divided into eight groups. On day one, donor cells (5 × 10⁻⁶) were injected via tail vein: bone marrow cells. 6 ) and spleen cells (5×106 Starting on day 10, mice were subcutaneously injected with 2 μg of LNPs / mRNA (once every 2 days, for a total of 3 times). Control group mice were injected with the same amount of PBS or empty LNPs at the same time points. After treatment began, the clinical symptoms of the mice were observed and recorded regularly, including changes in weight, activity level, and skin lesions, and the GvHD index was calculated. The GvHD index is a standardized indicator for assessing the clinical severity of graft-versus-host disease, and its score is based on the degree of damage to the skin, liver, and intestines.

[0326] Test results as follows Figure 33 As shown, mice in the LNPs / mRNA group exhibited a significant decreasing trend in the GvHD index. Compared with the PBS and LNPs groups, the LNPs / mRNA group had a significantly lower GvHD index, indicating that inducing the generation of tol-APCs effectively alleviates the clinical manifestations of GvHD and reduces the attack of the immune system on host tissues. The difference between the LNPs / mRNA group and the PBS and LNPs groups was statistically significant (P<0.05), further demonstrating that this strategy is superior to lipid nanoparticle delivery alone and the PBS control group. Therefore, treatment with mRNA drugs that generate in vivo tolerable antigen-presenting cells (tol-APCs) significantly improved the clinical manifestations of the disease, markedly reduced the GvHD index, and alleviated disease progression.

[0327] 7. Treatment of Ps mouse model

[0328] Establishing a psoriasis (Ps) mouse model: C57BL / 6 mice were shaved on their backs and treated with imiquimod cream (62.5 mg, 5% concentration) daily from day 0 to day 6. Ps-model mice were randomly divided into eight groups, and every other day, mice were subcutaneously injected with 2 μg of LNPs / mRNA. Control group mice were injected with the same amount of PBS or empty LNPs at the same time points. After treatment began, the disease index of the mice was assessed periodically.

[0329] Test results as follows Figure 34 As shown, the treatment group significantly reduced the disease index and alleviated psoriasis progression in the Ps mouse model, showing a significant difference compared with the PBS group and the LNPs group.

[0330] 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.

[0331]

[0332]

[0333]

[0334]

[0335]

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[0337]

Claims

1. An RNA, characterized in that, The RNA includes coding and non-coding regions, the coding regions encoding immunomodulatory molecules that can induce antigen-presenting cells to differentiate into a tolerant phenotype, the immunomodulatory molecules including immunosuppressive proteins, cytokines, transcription factors, or functional fragments thereof.

2. The RNA as described in claim 1, characterized in that, The immunomodulatory molecules include immunosuppressive molecules and immunomodulatory-related proteins; the immunosuppressive molecules include one or more of human or mouse PD-L1, PD-L2, CTLA4, and IDO, and their homologs or functional fragments; the immunomodulatory-related proteins include one or more of human or mouse HDAC, DNMT3A, TGF-β, IL-27, IL-35, and IL-10, and their homologs or functional fragments.

3. The RNA as described in claim 2, characterized in that, The DNA sequences of the human or mouse PD-L1, PD-L2, CTLA4, and IDO are shown in SEQ ID NO. 1-8, and the DNA sequences of the human or mouse HDAC, DNMT3A, TGF-β, IL-27, IL-35, and IL-10 are shown in SEQ ID NO. 9-20. The coding region encodes one or more immunomodulatory molecules, and when the coding region encodes multiple immunomodulatory molecules, the multiple immunomodulatory molecules are linked by adapter molecules. The coding region also encodes a signal peptide.

4. The RNA as described in claim 3, characterized in that, The non-coding region includes a 5'-cap structure, a 5'-UTR element, a 3'-UTR element, and a polynucleotide tail.

5. The RNA as described in claim 4, characterized in that, The 5'-cap structure is one or more of m7GpppN, m7GpppNmNm, and m7,3′-O-GpppG, and the DNA sequence of the 5'-UTR element is shown in SEQ ID NO.21; the DNA sequence of the 3'-UTR element is shown in SEQ ID NO.22; the polynucleotide tail is a polyadenine tail or a polycytosine tail; the polyadenine tail contains 10-200 adenine nucleotides, and each 10 adenine nucleotides are inserted with a phosphate-thiocyanate bond; the polycytosine tail contains 10-200 cytosine nucleotides.

6. The RNA as described in claim 5, characterized in that, The uridine modification of the RNA is one of pseudouridine, 1-methylpseudouridine, and 5-methoxyuridine; the cytidine modification of the RNA is 5-methylcytidine; and 2'-fluorination is introduced into the 5'-UTR and 3'-UTR elements of the RNA.

7. The method for preparing RNA according to any one of claims 1-6, characterized in that, The RNA is obtained by in vitro transcription, in vivo synthesis, chemical synthesis, or a combination thereof.

8. An RNA drug delivery system, characterized in that, The invention includes the RNA and delivery vector as described in any one of claims 1-6, wherein the delivery vector is a lipid nanoparticle, a polymer nanoparticle, a lipid-polymer hybrid nanoparticle, a liposome, an exosome, a virus-like particle, or a protein nanocage; the delivery vector may be linked to a targeting ligand via a surface modifier; the surface modifier is selected from one or more of carbohydrates or their conjugates, antibodies or their fragments, peptides, aptamers, and small molecule ligands; and the RNA drug delivery system may deliver one or more RNAs.

9. Use of the RNA drug delivery system of claim 8 in the preparation of formulations that enhance the formation of an immune tolerance phenotype by antigen-presenting cells.

10. Use of the RNA drug delivery system of claim 8 in the preparation of formulations for treating autoimmune diseases.

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