CD4-targeted runx3-siRNA nanodelivery system for treating chronic inflammatory diseases

Abstract

This invention belongs to the field of nanomedicine delivery and immunotherapy technology, and provides a treatment for CD4-positive conditions including asthma, inflammatory bowel disease, and chronic sinusitis. + A CD4-targeting Runx3-siRNA nanodelivery system for T-cell-mediated chronic inflammatory diseases comprises lipid nanoparticles loaded with Runx3-siRNA and a CD4-targeting ligand coupled to the surface of the lipid nanoparticles. This system enables targeting of pathogenic CD4-mediated chronic inflammatory diseases. + T-cell-specific delivery and Runx3 gene silencing; lipid nanoparticles composed of ionic lipids, helper lipids, cholesterol, and PEG-modified lipids, with anti-CD4 antibodies anchored to the surface via maleimide-thiol coupling; this invention targets pathogenic CD4... + Specific delivery of T cells and silencing of the Runx3 gene solve the problems of side effects caused by non-specific immunosuppression and insufficient targeting of lipid nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery and immunotherapy technology, and relates to targeted nucleic acid delivery systems, specifically to a CD4-targeted Runx3-siRNA nanodelivery system for the treatment of chronic inflammatory diseases. Background Technology

[0002] The pathogenesis of chronic inflammatory diseases such as asthma, inflammatory bowel disease, and chronic sinusitis and CD4 + T cell-mediated abnormal immune responses are closely related. In asthma and chronic sinusitis, the pathogenic Th2 cell subset is overactivated, secreting large amounts of pro-inflammatory cytokines (such as IL-4, IL-5, and IL-13), leading to inflammatory damage to lung tissue and immune homeostasis imbalance. In inflammatory bowel disease, pathogenic Th1 and Th17 cells secrete large amounts of IFN-γ and IL-17A, causing intestinal mucosal damage. Current clinical treatments mainly rely on glucocorticoids, immunosuppressants, or biologics (such as anti-IgE / IL-4Ra monoclonal antibodies). However, while these methods suppress pathogenic T cells, they often affect the function of regulatory T cells (Tregs) and follicular helper T cells (Tfh), leading to adverse reactions such as immunosuppression and increased risk of infection. Therefore, there is an urgent need to develop treatments that can precisely target pathogenic CD4+. + Treatment strategies that target T cells and selectively regulate their function.

[0003] Initially, we found that CXCR6 was present in diseases such as asthma, chronic sinusitis, and inflammatory bowel disease. + PD1 + CD4 + T cells are an important group of pathogenic cells, possessing a strong capacity for cytokine secretion and a tendency to migrate to peripheral inflammatory tissues, and are closely related to the occurrence and development of the aforementioned diseases. The transcription factor Runx3 (Runt-related transcription factor 3) plays a crucial role in CXCR6. + PD1 + CD4 + T cells play a crucial role in the differentiation and functional regulation of T cells. Our study shows that Runx3-specific T cell knockout mice exhibit pathogenic CXCR6... + PD1 + CD4 + The reduction of T cells in bronchoalveolar lavage fluid and the lamina propria of the small intestine led to a reduction in lung and intestinal inflammation. Simultaneously, the humoral immune response, specifically the germinal center response, in Runx3-specific T cell knockout mice was unaffected. This evidence suggests that Runx3 is an intervention for pathogenic CD4+. + Potential targets for T-cell-mediated inflammatory responses, without affecting protective CD4+. +T cell function, such as follicular helper CD4 + T cells (Tfh).

[0004] Small interfering RNA (siRNA) technology can efficiently and specifically silence target genes, showing great promise in the field of gene therapy. However, naked siRNA is easily degraded by serum nucleases and has difficulty effectively penetrating cell membranes to enter target cells. Lipid nanoparticles (LNPs) are one of the most mature RNA delivery systems currently available. LNPs self-assemble into nanostructures using microfluidic technology, which can efficiently encapsulate siRNA and protect it from degradation. In recent years, the successful application of LNPs in mRNA vaccines (such as COVID-19 vaccines) and RNA drugs (such as Patisiran) has validated their clinical translational potential. However, traditional LNPs mainly accumulate in the liver through the hepatocyte uptake pathway mediated by apolipoprotein E (ApoE), and their targeting of immune cells (especially T cells) is insufficient, greatly limiting their application in immune-related diseases.

[0005] Currently, for CD4 + Targeted delivery strategies for T cells mainly include antibody-conjugated nanoparticles (LCNPs) and ligand-modified carriers. Among these, anti-CD4 monoclonal antibodies are ideal targeting molecules due to their high affinity and specificity. Through maleimide-thiol conjugation, anti-CD4 antibodies can be anchored to the surface of LNPs, achieving CD4 targeting. + Specific delivery of T cells. However, existing research has focused on CAR-T cell therapy or anti-tumor immune regulation, and there are no reports of using CD4-targeted LNP delivery of Runx3-siRNA to selectively silence pathogenic T cells.

[0006] In summary, a Runx3-siRNA delivery system based on CD4-targeting LNP was developed to regulate pathogenic CD4. + The generation of T cells can provide a novel, highly effective, and low-toxicity treatment strategy for inflammatory diseases, especially asthma and enteritis. Summary of the Invention

[0007] The purpose of this invention is to provide a CD4-targeting Runx3-siRNA nanodelivery system for treating chronic inflammatory diseases such as asthma and enteritis, by targeting pathogenic CD4. + Specific delivery of T cells and silencing of the Runx3 gene to reduce inflammation.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: a CD4-targeted Runx3-siRNA nanodelivery system for treating chronic inflammatory diseases, wherein the nanodelivery system comprises lipid nanoparticles loaded with Runx3-siRNA nucleic acid drugs, and the surface of the lipid nanoparticles is coupled with CD4-targeting ligands to achieve targeted modification.

[0009] Preferably, the lipid nanoparticles are composed of ionic lipids, auxiliary lipids, cholesterol, and PEG-modified lipids, and the lipid nanoparticles are coupled to CD4 targeting ligands via covalent bonds.

[0010] Preferably, the content of the ionic lipid is 10-40 wt%, the content of the auxiliary lipid is 5-15 wt%, the content of the cholesterol is 30-40 wt%, and the content of the PEG-modified lipid is 1-5 wt%.

[0011] Preferably, the lipid nanoparticles have an average diameter of 80-120 nm, a zeta potential of -5 mV to -15 mV, and an encapsulation efficiency of not less than 85%.

[0012] Preferably, the covalent bond is a thioether bond, and the CD4 targeting ligand is an anti-CD4 monoclonal antibody or a fragment thereof.

[0013] The present invention further provides a method for constructing the above-mentioned CD4-targeted Runx3-siRNA nanodelivery system.

[0014] Preferably, the nanolipid particles are prepared by a rapid mixing method of ethanol phase and buffer phase, and the surface of the nanolipid particles is targeted by conjugation with a thiolized anti-CD4 monoclonal antibody. The specific steps are as follows:

[0015] S1: Ionic lipids, auxiliary lipids, cholesterol, and PEG-modified lipids are dissolved in ethanol to form an ethanol phase;

[0016] S2: Dissolve Runx3-siRNA in HEPES buffer at pH 7.4 to form a buffer phase;

[0017] S3: In a microfluidic device, the ethanol phase and the buffer phase are rapidly mixed at a volume ratio of 1:3 to form lipid nanoparticles loaded with Runx3-siRNA.

[0018] S4: React Traut's reagent with anti-CD4 monoclonal antibody to generate thiolized antibody;

[0019] S5: Mix the lipid nanoparticles obtained in step S3 with the thiolized antibody obtained in step S4, so that the maleimide groups on the surface of the lipid nanoparticles and the thiol groups of the antibody undergo a coupling reaction.

[0020] S6: Purify the reaction product, remove unlinked antibodies, and obtain the CD4-targeted nanodelivery system.

[0021] The present invention further provides applications of the above-described CD4-targeted Runx3-siRNA nanodelivery system.

[0022] Preferably, the CD4-targeting Runx3-siRNA nanodelivery system is used to prepare therapeutic CD4-targeting drugs. + Products for T-cell-mediated chronic inflammatory diseases, the CD4 + T-cell-mediated chronic inflammatory diseases include asthma, inflammatory bowel disease, and chronic sinusitis.

[0023] Preferably, the product comprises a formulation comprising lipid nanoparticles and pharmaceutically acceptable excipients, wherein the pharmaceutically acceptable excipients are selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and controlled-release materials.

[0024] Preferably, the formulation includes a variety of acceptable dosage forms.

[0025] Preferably, the dosage form of the preparation is any one of capsules, granules, tablets, powders, ointments, powders, pills, or liquids.

[0026] The beneficial effects of this invention are:

[0027] (1) Strong targeting: Through modification with anti-CD4 antibody, it achieves targeting of CD4. + Selective recognition and uptake by T cells;

[0028] (2) Highly efficient gene silencing: Runx3-siRNA in CD4 + Effective downregulation of Runx3 expression in T cells and inhibition of pathogenic CXCR6 + PD1 + CD4 + The production of T cells;

[0029] (3) High safety: It has no significant effect on protective T cell subsets such as Tfh, reducing immunosuppressive side effects;

[0030] (4) Broad spectrum applicability: Can be extended to various CD4-based spectrums. + Treatment of chronic inflammatory diseases centered on T cells. Attached Figure Description

[0031] Figure 1In this invention, Runx3 is used in CXCR6 + PD1 + CD4 + The role of T cells in differentiation and functional regulation (A represents CXCR6 in bronchoalveolar lavage fluid of mice in each group) + PD1 + CD4 + B shows the proportion of T cells, H&E and PAS staining and statistical graphs of mouse lung tissue in each group, TFH and GCB cell proportions in mediastinal lymph nodes in each group, body weight changes in each group, H&E staining of colon tissue in each group, colon length in each group, and CXCR6 in the lamina propria of colon tissue in each group. + PD1 + CD4 + T cell ratio, H represents the IFN-γ concentration in the lamina propria of the colon tissue of mice in each group. + CD4 + T cell ratio, I represents IL-17A concentration in the lamina propria of the colon tissue of mice in each group. + CD4 + T cell percentage.

[0032] Figure 2 This relates to the particle size and potential measurement in this invention;

[0033] Figure 3 This invention demonstrates the in vitro specific binding and inhibitory functions of LNP (A shows the in vitro binding of LNP to CD4+ T cells and CD8+ T cells, B shows the inhibition of RUNX3 on CD8+ T cells by LNP, C shows the inhibition of RUNX3 on CD4+ T cells by LNP, and D shows the inhibition of CXCR6 by LNP mediated by IL-15). + PD1 + CD4 + (Diagram showing inhibition of T cell formation.)

[0034] Figure 4 This invention relates to LNP treatment of asthmatic mice (A is an animal model diagram, B is the H&E and PAS staining and statistical graph of mouse lung tissue in each group, and C is the CXCR6 content in the bronchoalveolar lavage fluid of each group of mice). + PD1 + CD4 + The proportion of T cells, D represents the number of eosinophils and CD4+ cells in the bronchoalveolar lavage fluid of mice in each group. + T cell count, E represents the ratio of TFH and GCB cells in the mediastinal lymph nodes of mice in each group);

[0035] Figure 5This invention relates to tofacitinib treatment of asthmatic mice (A is an animal model diagram, B is H&E and PAS staining and statistical graph of mouse lung tissue in each group, C is eosinophils and CD4+ in bronchoalveolar lavage fluid of mice in each group). + (T cell count and the proportion of TFH and GCB cells in the mediastinal lymph nodes of mice in each group).

[0036] Figure 6 This invention relates to LNP treatment for DSS-induced enteritis (A is an animal model diagram, B is a diagram of weight changes in mice in each group, C is a diagram of colon length in mice in each group, D is a diagram of H&E staining of colon tissue in mice in each group, and E is a diagram of CXCR6 in the lamina propria of colon tissue in mice in each group). + PD1 + CD4 + T cell ratio, F represents the IFN-γ concentration in the lamina propria of the colon tissue of mice in each group. + CD4 + (T cell ratio)

[0037] Figure 7 This invention relates to LNP therapy for T-cell metastasis-mediated enteritis (A is an animal model diagram, B is a diagram of weight changes in mice in each group, C is a diagram of H&E staining of colon tissue in mice in each group, and D is a diagram of CXCR6 in the lamina propria of colon tissue in mice in each group). + PD1 + CD4 + T cell ratio, E represents the IFN-γ concentration in the lamina propria of the colon tissue of mice in each group. + CD4 + T cell ratio, F represents IL-17A concentration in the lamina propria of the colon tissue of mice in each group. + CD4 + T cell percentage). Detailed Implementation

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] Example 1

[0042] 1. Runx3 on CXCR6 + PD1 + CD4 + The role of T cells in the differentiation and functional regulation of T cells

[0043] (1) Method

[0044] Using T cell RUNX3 specific knockout mice (Runx3) f / f CD4-cre) and normal control mice (Runx3) f / f ), and constructed an HDM (house dust mite)-induced asthma model and a T-cell transfer-mediated mouse chronic enteritis model.

[0045] For the HDM-induced asthma model, Runx3 was used. f / f CD4-cre and Runx3 f / f Mice (6-8 weeks old) were sensitized by intranasal administration of 5 μg HDM extract (Greer Laboratories, XPB82D3A25) dissolved in 20 μL PBS for 3 consecutive days under isoflurane anesthesia (2-3%). Challenge mice were then administered intranasally under anesthesia with 50 μg HDM dissolved in 20 μL PBS on days 7-11.

[0046] For a T-cell transfer-mediated chronic enteritis model in mice: Runx3 mice aged 6-8 weeks f / f CD4-cre and Runx3 f / f Mouse spleen isolation 2×10 5 One initial CD25 - CD45RB hi CD4 + T cells were administered intravenously to Rag2 cells aged 6-8 weeks. - / - A T-cell metastatic colitis model was established in mice.

[0047] (2) Results

[0048] In an asthma model, Runx3-specific T cell knockout mice showed pathogenic CXCR6 + PD1 + CD4 + T cells decreased in bronchoalveolar lavage fluid ( Figure 1 (A), which in turn leads to a reduction in lung inflammation ( Figure 1 (B). Meanwhile, the ratio of Tfh cells and germinal center B cells in the mediastinal lymph nodes of Runx3-specific T cell knockout mice remained unchanged. Figure 1 The presence of C indicates that the humoral immune response, namely the germinal center response, is unaffected. Similarly, in a T-cell transfer-mediated mouse model of chronic enteritis, RUNX3 knockout protects mice from disease by reducing weight, alleviating colonic inflammation, and shortening of the colon. Figure 1 D- Figure 1 (middle F), and pathogenic CXCR6 in the lamina propria of the small intestine.+ PD1 + CD4 + T cells and IFN-γ + and IL-17A + The effector cell population is decreasing ( Figure 1 G- Figure 1 (I). This evidence suggests that Runx3 is an intervention for pathogenic CD4. + Potential targets for T-cell-mediated inflammatory responses, without affecting protective CD4+. + T cell function, such as follicular helper CD4 + T cells (Tfh).

[0049] 2. Preparation of CD4-targeted Runx3-siRNA nanoparticles

[0050] 2.1 Preparation method

[0051] ALC-0315:DSPC:CHOL:DSPE-PEG2000-MAL was dissolved in anhydrous ethanol at a mass ratio of 50:10:38:2 to form a lipid ethanol phase. The nanoparticle suspension was rapidly mixed in HEPES buffer (pH=7.4) containing Runx3-siRNA (1 mg / mL) at a ratio of ethanol:buffer solution = 1:3 using a microfluidic mixer (flow rate ratio 1:1). Ethanol and unencapsulated siRNA were removed by ultrafiltration to obtain Runx3-siRNA nanoparticles. Anti-CD4 monoclonal antibody (5 mg / mL) was added to Traut's reagent (1:10 molar ratio) and reacted at room temperature for 1 h to generate -SH antibody. This antibody was then reacted with DSPE-PEG2000-MAL in CD4-LNP for 4 h. Unconjugated antibody was removed by dialysis to obtain CD4-targeted Runx3-siRNA nanoparticles.

[0052] 2.2 Performance Testing

[0053] 2.2.1 Particle size and encapsulation efficiency determination

[0054] (1) Method

[0055] The particle size distribution and surface potential of the LNP samples were measured using a BI-200 SM dynamic light scattering system (Brookhaven Instruments) at 25°C. The scattered light was scattered at 90°... o Detection and collection were performed on an automated accelerator. A dedicated micro-detection sample cell was used when measuring surface potential to increase the accuracy of the data. For each sample group, the average of three measurements was taken.

[0056] The encapsulation ratio is calculated as follows:

[0057] Encapsulation rate (%) = Encapsulated nucleic acid amount / Total amount

[0058] (2) Results

[0059] The average particle size determined by dynamic light scattering (DLS) was 124.103.44 nm. Figure 2 The Zeta potential was 7.55 ± 1.02 mV. Figure 2 The encapsulation rate of siRNA exceeded 95%.

[0060] 2.2.2 Cellular uptake and gene silencing experiments

[0061] (1) Method

[0062] Isolated human peripheral blood CD3 + T cells, CD4-LNP / siRunx3 coupled with PE fluorescein, and 1 million CD3 cells were collected. + T cells were incubated with PE-CD4-LNP / siRunx3 for 30 minutes, followed by flow cytometry analysis of CD4. + T cells and CD8 + The binding of T cells and PE-LNP; CD4+ isolated from human peripheral blood. + T cells and CD8 + T cells, 1 million CD4 cells + T cells or CD8 + T cells were cultured with CD4-LNP / siRunx3 or CD4-LNP / siNC for 72 hours, followed by flow cytometry to detect Runx3 protein expression.

[0063] Flow cytometry assay: After LNP culture, cells were collected by centrifugation at 1500 rpm for 5 minutes, washed once with phosphate-buffered saline (PBS), and incubated with fluorescein-labeled anti-CD4, anti-CD8, anti-CXCR6, and anti-PD1 antibodies (at room temperature for 30 minutes). After washing once with PBS, the cells were then nucleated and incubated with fluorescein-labeled goat anti-rabbit secondary antibody and fluorescein-labeled goat anti-rabbit secondary antibody. After washing once with PBS, the cells were analyzed using a flow cytometer.

[0064] (2) Results

[0065] During in vitro incubation, PE-CD4-LNP / siRunx3 can effectively bind CD4. + T cells (over 82%), not CD8 + T cells (only about 7%) Figure 3 (A). Flow cytometry analysis revealed that it was associated with CD4. +Co-culture of T cells with anti-CD4-LNP / siRunx3 significantly downregulated Runx3 protein expression, while anti-CD4-LNP / siNC showed no significant difference; conversely, co-culture with CD8... + T cell co-culture, neither anti-CD4-LNP / siRunx3 nor anti-CD4-LNP / siNC could inhibit CD8. + Runx3 protein expression in T cells ( Figure 3 China B- Figure 3 (C). IL-15 can induce CXCR6 in vitro. + PD1 + CD4 + The production of T cells was significantly inhibited by the addition of anti-CD4-LNP / siRunx3, which significantly suppressed the IL-15-mediated production of this cell group. Figure 3 (D).

[0066] Application Example 1: Animal Experiment: Treatment of HDM-induced mouse asthma model

[0067] 1. Method

[0068] For HDM (house dust mite) induced asthma models ( Figure 4 Wild-type mice (6-8 weeks old, C57) were sensitized by intranasal administration of 5 μg HDM extract (Greer Laboratories, XPB82D3A25) dissolved in 20 μL PBS for 3 consecutive days under isoflurane anesthesia (2-3%). Challenge mice were then administered intranasally under anesthesia with 50 μg HDM dissolved in 20 μL PBS on days 7-11.

[0069] For the therapeutic intervention in the HDM-induced asthma model, the following treatment was administered: Anti-CD4 / NC siRNA-LNP or anti-CD4 / Runx3 siRNA-LNP (50 μg / mouse / day) was administered intravenously 1 hour before allergen challenge on days 8-10. Figure 4 (A)

[0070] In addition, we also treated the HDM-induced asthma model with tofacitinib: tofacitinib (10 mg / kg / day, dissolved in 0.5% methylcellulose) was administered orally by gavage (200 μL / mouse) 1 hour before allergen challenge on days 8–10. Figure 5 (A)

[0071] 2. Results

[0072] 2.1 Anti-CD4 / Runx3 siRNA-LNP effectively alleviates lung inflammation

[0073] The results showed that mice treated with anti-CD4 / Runx3 siRNA-LNP exhibited significantly reduced airway inflammation and goblet cell metaplasia in their lung H&E results. Figure 4 (B). Flow cytometry results showed that after treatment with anti-CD4 / Runx3 siRNA-LNP, the proportion of pathological CD4 T cells in bronchoalveolar lavage fluid was significantly reduced. Figure 4 (C), and the total CD4 in the bronchoalveolar lavage fluid + The number of T cells and eosinophils were both significantly reduced. Figure 4 (Middle D). Conversely, in the mediastinal lymph nodes, germinal center B cells and follicular helper CD4 cells... + The proportion of T cells was not affected. Figure 4 (E).

[0074] 2.2 Comparative advantages with the positive control drug tofacitinib

[0075] Despite the positive control group of tofacitinib ( Figure 5 Tofacitinib also showed a significant inhibitory effect on pulmonary inflammation, but flow cytometry analysis revealed a key difference: while inhibiting pulmonary inflammation, tofacitinib also significantly reduced germinal center B cells and follicular helper CD4 in mediastinal lymph nodes. + The proportion of T cells ( Figure 5 (C). The anti-CD4-LNP / siRunx3 treatment group had no significant effect on the proportion of the aforementioned immune cells in the mediastinal lymph nodes. Figure 4 (E).

[0076] Application Example 2: Animal Experiment: Treatment of a Mouse Model of Enteritis

[0077] 1. Method

[0078] A mouse model of acute enteritis induced by sodium dextran sulfate (DSS) Figure 6 (A) Mice were given drinking water containing 2% (w / v) DSS for two consecutive days (referred to as Day 0 and Day 1), followed by regular drinking water on Day 3 (referred to as Day 2), constituting one cycle, for a total of three cycles. On Days 2, 4, and 6, each mouse was intravenously injected with either anti-CD4 / Runx3 siRNA-LNP or anti-CD4 / NCsiRNA-LNP (50 μg), and body weight was recorded at specified time intervals. After the model was completed, colon tissue was collected for subsequent histopathological and other analyses.

[0079] T-cell transfer-mediated mouse model of chronic enteritis ( Figure 7 (A): 2 × 10⁻⁶ spleens were isolated from wild-type mice aged 6-8 weeks. 5One initial CD25 - CD45RB hi CD4 + T cells were administered intravenously to Rag2 cells aged 6-8 weeks. - / - A T-cell metastatic colitis model was established in mice. Mice were given intravenous injections of anti-CD4 / Runx3 siRNA-LNP or anti-CD4 / NC siRNA-LNP (100 μg per mouse) once a week for three consecutive weeks.

[0080] 2. Results

[0081] In DSS-induced enteritis, anti-CD4 / Runx3 siRNA-LNP treatment protected mice from disease, manifested by weight loss, reduced colonic shortening, and remission of colonic histopathology. Figure 6 China B- Figure 6 (D). This protection is associated with CXCR6 after anti-CD4 / Runx3 siRNA-LNP treatment. + PD1 + CD4 + T cells and IFN-γ + A significant reduction in effector cell population is associated with ( Figure 6 China E- Figure 6 (Middle F).

[0082] Similarly, in T-cell metastatic colitis, anti-CD4 / Runx3 siRNA-LNP treatment reduced weight loss, alleviated colonic tissue inflammation, and decreased CXCR6 levels in the lamina propria of mice. + PD1 + CD4 + T cells and IFN-γ + and IL-17A + effector cell population ( Figure 7 China B- Figure 7 (Middle F).

[0083] In summary, the CD4-targeting Runx3-siRNA nanodelivery system provided by this invention can effectively target and silence CD4 in an HDM-induced asthma model. +This invention targets the Runx3 gene in T cells, significantly reducing airway inflammation with efficacy comparable to the clinical drug tofacitinib. Importantly, unlike tofacitinib's broad suppression of the immune system, the delivery system of this invention exhibits higher targeting specificity. While effectively treating local lung pathological reactions, it does not affect key immune cells (germinal center B cells and follicular helper T cells) responsible for normal humoral immune responses in the mediastinal lymph nodes. This suggests a potentially superior safety profile, avoiding systemic immunosuppression-related side effects (such as potential impact on vaccine efficacy) that may be associated with traditional immunosuppressants. This provides a strong candidate strategy for developing new therapies for autoimmune diseases and allergic inflammation in the clinical setting.

[0084] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A CD4-targeting Runx3-siRNA nanodelivery system for the treatment of chronic inflammatory diseases, characterized in that: The nanodelivery system includes lipid nanoparticles loaded with Runx3-siRNA nucleic acid drugs, and the surface of the lipid nanoparticles is coupled with CD4 targeting ligands to achieve targeted modification.

2. The CD4-targeted Runx3-siRNA nanodelivery system according to claim 1, characterized in that: The lipid nanoparticles are composed of ionic lipids, auxiliary lipids, cholesterol and PEG-modified lipids, and the lipid nanoparticles are coupled to CD4 targeting ligands through covalent bonds.

3. The CD4-targeted Runx3-siRNA nanodelivery system according to claim 2, characterized in that: The content of the ionic lipid is 10-40 wt%, the content of the auxiliary lipid is 5-15 wt%, the content of cholesterol is 30-40 wt%, and the content of the PEG-modified lipid is 1-5 wt%.

4. The CD4-targeted Runx3-siRNA nanodelivery system according to claim 2, characterized in that: The lipid nanoparticles have an average diameter of 80-120 nm, a zeta potential of -5 mV to -15 mV, and an encapsulation efficiency of not less than 85%.

5. The CD4-targeted Runx3-siRNA nanodelivery system according to claim 2, characterized in that: The covalent bond is a thioether bond, and the CD4 targeting ligand is an anti-CD4 monoclonal antibody or a fragment thereof.

6. The method for constructing the CD4-targeted Runx3-siRNA nanodelivery system according to claim 1, characterized in that: The lipid nanoparticles were prepared by a rapid mixing method of ethanol and buffer phases. The surface of the lipid nanoparticles was targeted by conjugation with a thiolized anti-CD4 monoclonal antibody. The specific steps are as follows: S1: Ionic lipids, auxiliary lipids, cholesterol, and PEG-modified lipids are dissolved in ethanol to form an ethanol phase; S2: Dissolve Runx3-siRNA in a solution with a pH value of [missing value]. A buffer phase is formed in HEPES buffer at 7.4°C; S3: In a microfluidic device, the ethanol phase and the buffer phase are rapidly mixed at a volume ratio of 1:3 to form lipid nanoparticles loaded with Runx3-siRNA. S4: React Traut's reagent with anti-CD4 monoclonal antibody to generate thiolized antibody; S5: Mix the lipid nanoparticles obtained in step S3 with the thiolized antibody obtained in step S4, so that the maleimide groups on the surface of the lipid nanoparticles and the thiol groups of the antibody undergo a coupling reaction. S6: Purify the reaction product, remove unlinked antibodies, and obtain the CD4-targeted nanodelivery system.

7. The application of the CD4-targeted Runx3-siRNA nanodelivery system according to any one of claims 1-6, characterized in that: The CD4-targeting Runx3-siRNA nanodelivery system is used to prepare therapeutic CD4 + Products for T-cell-mediated chronic inflammatory diseases, the CD4 + T-cell-mediated chronic inflammatory diseases include asthma, inflammatory bowel disease, and chronic sinusitis.

8. The application according to claim 7, characterized in that: The product includes a formulation comprising lipid nanoparticles and pharmaceutically acceptable excipients selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, osmotic pressure regulators, stabilizers, suspending agents, coating materials, anti-adhesives, binding agents, penetration enhancers, pH adjusters, buffers, surfactants, absorbents, diluents, filter aids, and controlled-release materials.

9. The application according to claim 8, characterized in that: The formulation includes a variety of acceptable dosage forms.

10. The application according to claim 9, characterized in that: The dosage form of the preparation is any one of capsules, granules, tablets, powders, ointments, powders, pills, or liquids.

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