Use of dtx4 gene or protein in preparation of medicine for treating pulmonary alveolar proteinosis

Abstract

The application belongs to the technical field of biological medicine, and discloses application of DTX4 gene or a protein coded by the DTX4 gene in preparation of a medicine for treating pulmonary alveolar proteinosis. Through in-vivo and in-vitro experiment verification, DTX4 is a core factor for regulating cholesterol efflux of alveolar macrophages, and expression loss of the core factor leads to lipid metabolism disorder and surfactant deposition. In the application, a recombinant adeno-associated virus (AAV6) carrier is used to carry a CD68 promoter to drive specific expression of DTX4 in alveolar macrophages, and non-invasive delivery is realized through nose dropping or tracheal instillation. The medicine can treat autoimmune, genetic, secondary and congenital PAP. Animal experiments show that DTX4 overexpression can significantly improve lung imaging abnormalities (47.6% reduction in ground glass shadow) and reduce the cholesterol content (26.1%) in alveolar lavage fluid, and has no liver and kidney toxicity. The application provides an innovative therapy with high targeting, high safety and wide indications for PAP.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically discloses the application of the DTX4 gene or protein in the preparation of drugs for treating pulmonary alveolar proteinosis. Background Technology

[0002] Pulmonary alveolar proteinosis (PAP) is a group of rare lung diseases caused by the abnormal accumulation of surfactant in the alveoli, leading to restricted gas exchange. The core mechanism involves impaired cholesterol clearance by alveolar macrophages (AMs), resulting in disordered intracellular lipid metabolism, accumulation of large amounts of lipid droplets, and consequently, impaired clearance of surfactant. Based on etiology, PAP is classified into three types: primary, secondary, and congenital. Primary PAP accounts for over 90% of all cases and is mainly caused by GM-CSF signaling deficiency leading to alveolar macrophage dysfunction. This includes autoimmune PAP (caused by anti-GM-CSF antibodies neutralizing GM-CSF signaling) and hereditary PAP (caused by mutations in the GM-CSF receptor subunit gene). Secondary PAP arises from hematologic malignancies, inhalation of toxic particles, immunodeficiency, or infections, leading to impaired macrophage function or number. Congenital PAP is caused by mutations in the genes encoding surfactant substances or their transport proteins. It often occurs in newborns or children and progresses rapidly.

[0003] Currently, the treatment of PAP is primarily etiologically targeted. Whole lung lavage (WLL) is the first-line standard treatment for autoimmune PAP (aPAP), effectively clearing alveolar deposits and improving oxygenation. Literature shows that the overall clinical improvement rate of WLL can reach 60%–84%, with some patients achieving long-term remission after only one treatment. However, approximately 10%–30% of patients may require repeated lavage or long-term follow-up. In recent years, inhaled GM-CSF replacement therapy, as a targeted therapy, has shown good efficacy in clinical trials, with an overall response rate of approximately 40%–60% and milder side effects. It is suitable for patients with mild to moderate aPAP or those contraindicated for WLL. For hereditary and congenital PAP, WLL has limited efficacy and a low long-term remission rate, and is only used as a palliative treatment. Some patients may require hematopoietic stem cell transplantation or lung transplantation. Treatment of secondary PAP depends on the control of the primary disease. If the underlying condition is stable, some patients may experience relief of pulmonary symptoms, but the overall prognosis varies considerably. Current treatment of PAP still faces challenges such as large individual differences, high relapse rate, and poor efficacy in some patients, requiring further optimization of individualized treatment strategies and development of new targeted therapies.

[0004] Defects and shortcomings of existing technology:

[0005] Current treatment strategies for PAP mainly include the following categories, but all of them have significant limitations:

[0006] 1. Whole lung lavage (WLL) is highly invasive and cannot alter the course of the disease.

[0007] WLL is currently the standard treatment for autoimmune PAP, effectively clearing surfactant deposits in the alveoli and improving short-term oxygenation. However, this method is complex, requires general anesthesia, carries the risk of intraoperative complications, and cannot reverse or correct macrophage dysfunction, thus failing to fundamentally prevent disease progression or recurrence.

[0008] 2. GM-CSF replacement therapy has limited efficacy, with varying responses and the risk of drug resistance.

[0009] Inhaled or subcutaneous GM-CSF, as a etiologically targeted therapy, has shown some efficacy in some mild to moderate cases, but its overall response rate is low, and significant individual variability and drug resistance exist. Furthermore, this treatment is only applicable to antibody-mediated aPAP and is ineffective for non-autoimmune PAP.

[0010] 3. Research on treatments targeting macrophage lipid metabolism disorders is still in its early exploratory stages.

[0011] Recent studies have revealed that the core mechanism of PAP lies not only in GM-CSF signaling impairment but also in impaired cholesterol efflux function of alveolar macrophages. This mechanism is common in autoimmune, secondary, and hereditary PAP. However, there is currently a lack of effective molecular targets or clinically available drugs that regulate macrophage cholesterol metabolism. Only drugs such as PPARγ agonists and statins are in the exploratory stage, with limited efficacy and unclear mechanisms.

[0012] In summary, current treatments are mostly supportive or non-specific, lacking targeted therapies for macrophage lipid metabolism disorders. In particular, ideal intervention targets have not yet been established at the molecular level, limiting the fundamental treatment of PAP. Summary of the Invention

[0013] To address the aforementioned problems, this invention discloses the application of the DTX4 gene or protein in the preparation of drugs for treating pulmonary alveolar proteinosis. Through in vitro and in vivo functional verification, this invention has discovered and established Deltex E3 ubiquitin ligase 4 (DTX4) as a core factor regulating cholesterol efflux from alveolar macrophages, and proposes a new strategy for developing treatments for PAP by targeting the DTX4 gene or its encoded protein.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] The use of the DTX4 gene or protein in the preparation of a medicament for treating pulmonary alveolar proteinosis, the medicament being used to correct cholesterol efflux disorder of alveolar macrophages and comprising an effective amount of the DTX4 gene or the protein encoded thereon, and a pharmaceutically acceptable carrier.

[0016] Furthermore, the application of the aforementioned DTX4 gene or protein in the preparation of a medicament for treating alveolar proteinosis, wherein the medicament is prepared by a delivery system comprising a recombinant adeno-associated virus (AAV) vector containing a CD68 promoter to drive the specific expression of the DTX4 gene in alveolar macrophages.

[0017] Furthermore, in the application of the above-mentioned DTX4 gene or protein in the preparation of a drug for treating pulmonary alveolar proteinosis, the serotype of the AAV vector is AAV6, and the nucleotide sequence of the DTX4 gene is as shown in SEQ ID NO:1, or the protein sequence encoded by it is as shown in SEQ ID NO:2.

[0018] Furthermore, the above-mentioned DTX4 gene or protein is used in the preparation of a drug for treating pulmonary alveolar proteinosis, wherein the drug is prepared as a formulation suitable for lung delivery by nasal drops or tracheal infusion.

[0019] Furthermore, the above-mentioned DTX4 gene or protein is used in the preparation of a medicament for treating pulmonary alveolar proteinosis, wherein the medicament is used to treat pulmonary alveolar proteinosis selected from the following types: autoimmune PAP, hereditary PAP, secondary PAP, or congenital PAP.

[0020] Furthermore, regarding the application of the aforementioned DTX4 gene or protein in the preparation of a medicament for treating pulmonary alveolar proteinosis, the dosage of the medicament is determined by the following parameters:

[0021] Baseline expression level of DTX4 in alveolar macrophages of patients;

[0022] The ratio of cholesterol to protein in bronchoalveolar lavage fluid;

[0023] Area of ​​ground-glass opacities on lung CT images;

[0024] GM-CSF receptor gene mutation status.

[0025] This invention discloses a pharmaceutical composition for treating pulmonary alveolar proteinosis, comprising an effective amount of the DTX4 gene or the protein encoded therein, and a pharmaceutically acceptable carrier, wherein the DTX4 gene or protein is used to correct cholesterol efflux disorder of alveolar macrophages.

[0026] Furthermore, in the above-mentioned pharmaceutical composition for treating pulmonary alveolar proteinosis, the sequence of the DTX4 gene is shown in SEQ ID NO:1, or the protein sequence encoded by it is shown in SEQ ID NO:2.

[0027] Furthermore, the above-mentioned pharmaceutical composition for treating pulmonary alveolar proteinosis is prepared by a delivery system comprising a recombinant adeno-associated virus (AAV) vector containing a CD68 promoter to drive the specific expression of the DTX4 gene in alveolar macrophages, and the pharmaceutical composition is prepared as a formulation suitable for pulmonary delivery by nasal or tracheal instillation.

[0028] Compared with the prior art, the present invention has the following outstanding advantages:

[0029] This invention addresses the core problems of existing treatments for pulmonary alveolar proteinosis (PAP), such as high invasiveness, insufficient targeting, and limited indications. It proposes an innovative treatment strategy based on the DTX4 gene or protein, which has the following significant advantages:

[0030] 1. Targeting the core pathological mechanism and breaking through the limitations of traditional treatments: This invention reveals for the first time that DTX4 is a key factor regulating cholesterol efflux from alveolar macrophages. Restoring its expression can directly correct lipid metabolism disorders, addressing surfactant clearance obstacles at the molecular level. Compared to the physical clearance of whole lung lavage (WLL) and signal compensation of GM-CSF replacement therapy, this invention directly intervenes in the root cause of the disease, significantly improving treatment specificity and long-term efficacy.

[0031] 2. Broad applicability, covering multiple types of PAP patients: As a downstream regulator of the GM-CSF signaling pathway, the restoration of DTX4 function is effective for autoimmune, hereditary, and secondary PAP. It is especially suitable for patients with GM-CSF receptor defects or mutations, filling the treatment gap for non-autoimmune PAP in existing therapies.

[0032] 3. Non-invasive delivery, safe and convenient: Utilizing a recombinant adeno-associated virus (AAV) vector carrying the CD68 promoter, targeted delivery to the lungs is achieved via nasal or tracheal instillation, avoiding the risks of general anesthesia and surgery. The AAV6 serotype exhibits high alveolar macrophage transfection efficiency, and experimental data show that it has no significant impact on liver and kidney function, demonstrating excellent safety.

[0033] 4. Significant clinical translational potential: In animal models, DTX4 overexpression significantly reduced ground-glass opacities in the lungs (by 47.6%), restored the cholesterol / protein ratio in bronchoalveolar lavage fluid, and pathological verification showed a 68% clearance rate of alveolar deposits. Combined with mature AAV vector technology, it has the feasibility of rapidly entering clinical trials. Attached Figure Description

[0034] Figure 1 In vitro validation of DTX4 as a core factor regulating cholesterol efflux: (A) Western blot detection of DTX4 knockdown effect, with GAPDH as an internal control; (B) Statistical graph of cholesterol efflux rate in AM cells of mice in different treatment groups; (C) Detection of central lipids in AM cells of mice in different treatment groups using BODIPY 493 / 503 staining, confocal microscopy (left) and counting of lipid droplets (right), Hoechs 33342 labeling of cell nuclei. Scale bar, 50 μm;

[0035] Figure 2 In vivo validation of DTX4 as a core factor regulating cholesterol efflux and driving PAP progression: (A) Schematic diagram of DTX4 knockdown AAV vector construction (left) and animal model (right); (B, C) Micro-CT detection of lung image changes in mice in each group at the modeling endpoint (B), segmentation of abnormal lung areas and three-dimensional reconstruction of the lungs using a 3D slicer and statistical analysis (C); (D) BALF appearance photograph of modeled mice; (E, F) BALF turbidity (E) and cholesterol content (F) statistics of modeled mice; (G) HE staining of lung tissue of modeled mice. Scale bar, 50 μm; (H, I) PAS staining of lung tissue of modeled mice (H) and statistical analysis (I), scale bar, 50 μm;

[0036] Figure 3 Map of DTX4-overexpressing AAV vectors;

[0037] Figure 4 : Targeted overexpression of DTX4 alleviates disease symptoms in PAP model mice. (A) Schematic diagram of DTX4 overexpression AAV vector construction (left) and animal model (right). (B, C) Micro-CT detection of lung image changes in each group of mice at the modeling endpoint (B), segmentation of abnormal lung areas and three-dimensional reconstruction of the lungs using a 3D slicer and statistical analysis (C), (D) BALF appearance photograph of modeled mice, (EG) BALF turbidity (E), total protein (F), and cholesterol content (G) of modeled mice, (H) HE staining of lung tissue of modeled mice. Scale bar, 50 μm. (I, J) PAS staining of lung tissue of modeled mice (I) and statistical analysis (J), scale bar, 50 μm;

[0038] Figure 5 Results of liver and kidney function tests in mice: (A) Serum ALT levels (liver function index) were measured in mice of each group 2 months after AAV injection, with an equal volume of physiological saline as a control; (B) Serum BUN levels (kidney function index) were measured in mice of each group 2 months after AAV injection, with an equal volume of physiological saline as a control. Detailed Implementation

[0039] This invention aims to provide a therapeutic strategy based on the DTX4 gene or its encoded protein to repair cholesterol metabolism disorders in alveolar macrophages and improve or reverse the pathological state of PAP. Specific objectives include:

[0040] 1. Identify and verify DTX4 as a core factor regulating cholesterol efflux;

[0041] 2. Construct a recombinant overexpression vector for DTX4 to achieve its specific expression in macrophages;

[0042] 3. Provide evidence of the application of DTX4 protein or its expression system in restoring lung function in animal models;

[0043] 4. Establish the targeted application of DTX4 in the treatment of PAP, and provide new treatment methods for clinical practice.

[0044] I. Identification and Verification of DTX4 as a Core Factor in Regulating Cholesterol Efflux

[0045] To determine the role of DTX4 in the pathogenesis of PAP, this invention employed the following experimental strategy to systematically identify and verify its function:

[0046] In vitro functional validation: Knockdown of DTX4 expression in primary mouse alveolar macrophages resulted in decreased cholesterol efflux and a significant increase in intracellular lipid droplets.

[0047] In vivo functional validation: Adenovirus AAV-CD68-shDTX4-EGFP was delivered to the lungs of mice via intratracheal injection, mediating specific knockdown of DTX4 in alveolar macrophages. PAP-like imaging and pathological changes were observed in the mice.

[0048] II. DTX4 gene and protein structure and origin

[0049] The DTX4 gene sequence (cDNA) used in this invention is based on the reference sequence (NM_172442.3) indexed in PubMed:

[0050]

[0051] The DTX4 protein sequence (amino acid sequence) used in this invention is based on the reference sequence (Q9Y2E6) included in Uniprot:

[0052] =SEQ ID No.2

[0053] III. Construction and Application of DTX4 In Vivo Expression Vectors

[0054] To achieve DTX4 expression in vivo, this invention constructs a recombinant adeno-associated virus (AAV) vector system for expressing the DTX4 gene in animal models.

[0055] 1. Carrier construction steps:

[0056] The expression vector backbone used was: pAAV-CD68-MCS-P2A-EGFP;

[0057] The mouse-derived DTX4 ORF region was cloned into the MCS region to construct pAAV-CD68-DTX4-P2A-EGFP.

[0058] DTX4 was specifically expressed in alveolar macrophages using the CD68 promoter;

[0059] Downstream EGFP is used to label infection efficiency;

[0060] AAV6 packaging in HEK293T cells using a three-plasmid system;

[0061] High-titer AAV-DTX4 virus particles were obtained by ultracentrifugation or column purification.

[0062] 2. Application method:

[0063] AAV-DTX4 was delivered to the lungs of Csf2ra- / - mice via intratracheal injection.

[0064] Achieve specific expression of the DTX4 gene in alveolar macrophages;

[0065] The study investigated its alleviating effect on the PAP phenotype.

[0066] IV. Validation of DTX4 application in PAP animal model

[0067] In the classic PAP mouse model (Csf2ra- / - mice), the following effects were observed after treatment with AAV-DTX4:

[0068] 1. Improved imaging findings:

[0069] Chest CT showed a significant reduction in ground-glass opacities in the lungs; lung transparency was significantly restored.

[0070] 2. Pathological and cytological changes:

[0071] The clarity of bronchoalveolar lavage fluid (BALF) was improved, and the cholesterol-to-protein ratio was restored; foamy lipid droplets in macrophages were reduced; and the deposition of surfactant was significantly reduced.

[0072] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0073] Example 1

[0074] Identification and Validation of DTX4 as a Core Factor in Regulating Cholesterol Efflux

[0075] 1.1 In vitro functional validation: DTX4 knockdown affects the cholesterol efflux capacity of macrophages

[0076] Eight-week-old male C57BL / 6 mice (SPF grade) were used for bronchoalveolar lavage via repeated intratracheal infusion of sterile PBS (0.5 mL each time, for a total of 5 times). The lavage fluid was collected and centrifuged at 250 × g for 10 minutes to obtain alveolar macrophages. Cells were seeded in 6-well plates and cultured in RPMI-1640 medium (Gibco) with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. After cell adhesion, 50 nM siRNA targeting mouse DTX4 (purchased from GenePharma) was transfected into the cells using Lipofectamine RNAiMAX transfection reagent (Invitrogen) for 6 hours, followed by 18 hours of culture with fresh medium. To assess cholesterol efflux capacity, transfected macrophages were treated with BODIPY-labeled cholesterol and incubated for 16 hours. Then, 10 μg / mL ApoA-I (Sigma) was added as a cholesterol receptor, and incubation continued for another 4 hours to induce cholesterol efflux. After the experiment, intracellular neutral lipids were stained using BODIPY staining, and lipid droplet numbers were quantitatively analyzed using microscopy and ImageJ software. The results showed that, compared with the control group, the cholesterol efflux capacity of macrophages in the DTX4 knockdown group was decreased. BODIPY staining showed an increase of approximately 45% in the number of intracellular lipid droplets and a significantly increased degree of foaming, indicating that DTX4 plays a key regulatory role in maintaining cholesterol metabolic homeostasis in alveolar macrophages. Figure 1 ).

[0077] siRNA4 sequence:

[0078] Sense strand:5'-CCAAC AUGU AAGAC CAUUU dTdT-3'=SEQ IDNo.3;

[0079] siRNA5 sequence:

[0080] Sense strand:5'-CCACA UCUA CCACA UCUAC dTdT-3'=SEQ ID No.4;

[0081] The above siRNA4 and siRNA5 sequences are both siRNA sequences that target the mouse DTX4 gene.

[0082] 1.2 In vivo functional validation: DTX4 knockdown induces PAP-like phenotype

[0083] This invention constructs an adeno-associated virus vector targeting mouse DTX4.

[0084] AAV6-CD68-shDTX4-EGFP was used to drive the specific expression of shRNA in macrophages via the mouse CD68 promoter, and infection efficiency was assessed by EGFP labeling. The control virus was AAV6-CD68-shScramble-EGFP containing shRNA without the target sequence. Both viruses were packaged in HEK293T cells using a three-plasmid system and purified by Iodixanol density gradient ultracentrifugation to a final concentration of 1×10⁻⁶. 12 vg / mL. Eight-week-old male C57BL / 6 mice (SPF grade) were selected, with eight mice in each group. They were anesthetized with 1.5% isoflurane inhalation before the experiment. At week 0 and week 8, 50 μL of virus solution (containing 1×10⁻⁶ g / mL) was injected intratracheally under aseptic conditions. 11 VG virus particles). Continue feeding the animals until week 16 after virus injection, during which time their health status should be monitored regularly. Figure 2 A). At the end of the experiment, lung structural changes were assessed using a small animal microCT imaging system. The results showed that the shDTX4 group of mice had obvious ground-glass opacities in their lungs, and the area of ​​high-density material deposition in the alveoli increased by approximately 11%. Figure 2 B, C). Subsequently, bronchoalveolar lavage fluid (BALF) was collected. Visually, the fluid was significantly turbid, and its cholesterol concentration increased from 10.4 ng / μL to 72.1 ng / μL compared to the control group. Figure 2 DF). Histological analysis showed that HE staining revealed a large amount of pink protein-like material deposited in the alveolar spaces of mice in the shDTX4 group, and PAS staining was strongly positive, indicating abnormal accumulation of surfactant in the alveoli, presenting typical PAP-like lesion characteristics. Figure 2 The above results indicate that the absence of DTX4 in alveolar macrophages can induce a PAP-like phenotype, further validating its crucial role in maintaining pulmonary cholesterol homeostasis and surfactant clearance.

[0085] shRNA sequence: 5'-CCAACATGTAAGACCATTT TTCAAGAGA AAATGGTCTTACATGTTGG TTTTT-3'=SEQ ID No. 5.

[0086] Example 2

[0087] 2.1 DTX4 gene sequence origin and synthesis

[0088] The mouse DTX4 gene (Deltex E3 ubiquitin ligase 4) open reading frame (ORF) sequence used in this invention is based on a publicly available sequence in the NCBI database (Gene ID: 207521), with a full length of 1848 bp. The DTX4 gene was fully synthesized by a commercial company (Sangon) based on the reference sequence and verified to be mutation-free by sequencing. Restriction enzyme sites (EcoRI and XbaI) were designed at both ends of the synthesized fragment for subsequent cloning operations. The synthesized gene was provided in lyophilized form, dissolved in RNase-free water at a concentration of 100 ng / μL, and stored at -20°C.

[0089] Example 3

[0090] DTX4 overexpression preparation of recombinant AAV

[0091] 3.1 Materials and Reagents

[0092] Backbone vector: pAAV-CD68-MCS-P2A-EGFP (Cresbio);

[0093] Restriction endonucleases: EcoRI, XbaI (NEB);

[0094] Ligase: T4 DNA ligase (Takara);

[0095] Competent cells: DH5α;

[0096] Plasmid extraction kit: Qiagen;

[0097] Sequencing services: Sangon;

[0098] Transfected cells: HEK293T cells (ATCC CRL-3216);

[0099] Transfection reagent: PEI (Polysciences, #23966-1);

[0100] Triple plasmid system: pAAV-CD68-DTX4-P2A-EGFP, pHelper (Kress Biotech), pAAV-RC6 (Kress Biotech);

[0101] Culture medium: High-glucose DMEM (Gibco) + 10% FBS;

[0102] Virus purification: Iodixanol density gradient centrifugation (Optiprep);

[0103] Titer determination: qPCR method.

[0104] 3.2 AAV vector cloning steps

[0105] The pAAV-CD68-MCS-P2A-EGFP backbone vector was double-digested with restriction endonucleases EcoRI and XbaI, and the artificially synthesized mouse DTX4 open reading frame (ORF) gene fragment was also digested with the same enzymes to obtain a matching insert. The double-digested linearized vector and DTX4 insert fragment were then recovered and ligated at a molar ratio of 3:1 (insert fragment:vector). T4 DNA ligase was added, and the reaction was carried out at 16°C for 16 hours. The ligation product was used to transform chemically competent DH5α *E. coli* strains. After transformation, the samples were plated on LB agar plates containing 100 μg / mL ampicillin and incubated at 37°C for 12–16 hours. Single colonies were picked and inoculated into liquid LB medium, and after 12 hours of shaking culture at 37°C, plasmid DNA was extracted. Sanger sequencing was used to verify the sequence of the cloned fragment, confirming that the DTX4 sequence was correct, the orientation was correct, and there were no mutations. The constructed recombinant expression plasmid was named...

[0106] pAAV-CD68-DTX4-P2A-EGFP( Figure 3 (This is used for subsequent AAV virus packaging and in vivo delivery experiments.)

[0107] 3.3 AAV Virus Packaging and Purification

[0108] HEK293T cells were seeded in 15cm culture dishes and cultured at 37℃ and 5% CO2 until 50% confluence was achieved before transfection. A three-plasmid system was used for co-transfection: pAAV-CD68-DTX4-P2A-EGFP expression plasmid, pAAV-RC6 packaging plasmid, and pHelper helper plasmid, with a total plasmid amount of 30μg per dish. Polyethyleneimine (PEI) was used as the transfection reagent, with a DNA to PEI mass ratio of 1:4. 48–72 hours after transfection, cells and culture supernatant were collected. Cells were lysed using three freeze-thaw cycles or sonication to obtain crude virus extract. Viral particles were separated by ultracentrifugation at 175,000×g for 2 hours in a Beckman SW41 Ti rotor using an Iodixanol density gradient (15%, 25%, 40%, 60%), and the viral layer at the 40%–60% interface was recovered. The viral solution was dialyzed through an Amicon ultrafiltration tube (100 kDa) to remove Iodixanol, and the medium was replaced with PBS. Finally, the viral titer was quantitatively determined using qPCR, and the results showed that the prepared AAV viral titer was approximately 1 × 10⁻⁶. 12 The concentration of vg / mL can meet the needs of in vivo delivery in subsequent animal experiments.

[0109] Example 4

[0110] Application Validation of DTX4 in PAP Model

[0111] 4.1 Animal models and virus delivery

[0112] Choose 8-week-old Csf2ra - / - Mice (sex or non-sex, SPF grade, purchased from a reputable laboratory animal center) were used as a classic PAP animal model. Mice were randomly divided into two groups: the treatment group was inoculated with AAV6-CD68-DTX4-P2A-EGFP virus, and the control group was inoculated with the pAAV-CD68-MCS-P2A-EGFP empty vector. The virus solution was diluted with PBS to 50 μL per mouse, with a viral load of approximately 1 × 10⁻⁶. 11 After anesthetizing mice with 1.5% isoflurane by inhalation, the virus solution was slowly injected intratracheally under aseptic conditions to ensure uniform distribution in the lungs. Mice were fed for 8 weeks post-injection, during which their general condition was observed daily, and changes in weight, respiratory rate, and any adverse reactions were recorded as preliminary indicators of virus safety. Figure 4 A).

[0113] 4.2 Detection Indicators

[0114] In terms of imaging, lung scans were performed using a small animal micro-CT imaging system (Bruker SkyScan) after intravenous injection of contrast agent to analyze ground-glass opacities and changes in lung density. Following pulmonary function assessment, bronchoalveolar lavage (BAL) was performed. Each mouse was injected with and recovered 1 mL of sterile PBS, and the supernatant was collected after centrifugation for biochemical analysis. Cholesterol content was determined using a cholesterol assay kit (Applygen), and total protein content was quantified using the BCA method. Lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. HE staining was performed to observe alveolar structural integrity, and PAS staining was used to assess the degree of surfactant deposition. All tissue images were semi-quantitatively analyzed using ImageJ software.

[0115] Experimental results showed that in mice treated with DTX4, the percentage of ground-glass opacities in the lungs decreased from 39.1% to 20.5% on micro-CT imaging, and lung transparency improved. Figure 4 B, C); The cholesterol / protein ratio in BALF recovered, and the total cholesterol level decreased from 124 to 91.6 ng / μL (B, C); Figure 4 DG); the area of ​​PAS staining positive area decreased significantly, the score dropped from 280.6 to 191.9, and the deposition of surfactant in the alveolar space was significantly reduced (DG); Figure 4 HJ). In contrast, the control group mice did not show significant improvement in any of the above indicators, further validating the significant efficacy of DTX4 in alleviating the pathological changes of PAP. Figure 4Furthermore, compared with the saline control group, there were no statistically significant differences in serum ALT and BUN levels between the DTX4-overexpressing AAV treatment group and the empty AAV control group. Figure 5 The results indicate that the above treatment had no significant effect on the liver and kidney function of mice and was biosafe.

[0116] Summary of Examples: The above examples verified the function of DTX4 in regulating cholesterol efflux (45% increase in lipid droplets) through in vitro knockdown. In vivo experiments showed that DTX4 deficiency induced a PAP-like phenotype (11% increase in alveolar deposits). The AAV6-CD68-DTX4 vector was successfully constructed and delivered intratracheally to Csf2ra- / - mice, significantly improving lung imaging (47.6% reduction in ground-glass opacities) and pathological indicators (26.1% reduction in cholesterol), without abnormal liver and kidney function. The data confirm that DTX4-targeted therapy can effectively reverse the pathological progression of PAP and has clinical translational potential.

[0117] In summary, the present invention has the following specific advantages and practical applications:

[0118] 1. A non-invasive, highly targeted gene therapy or protein replacement strategy has been developed.

[0119] This invention restores the cholesterol efflux function of macrophages by reconstructing DTX4 expression, thereby improving the clearance of surfactant substances from the source. It has a clear mechanistic basis and feasible intervention approach, and is expected to replace or combine with WLL, reduce treatment risks and improve long-term efficacy.

[0120] 2. Expand the indications for PAP treatment and overcome the limitations of GM-CSF dependence.

[0121] Unlike existing GM-CSF replacement therapies, DTX4 acts downstream of the PPARγ pathway, and its function is independent of its E3 ubiquitin ligase activity, suggesting that it may play a role in different types of PAP (including GM-CSF receptor mutants, secondary, and even some congenital types), thus achieving coverage of a wider patient population.

[0122] 3. To provide a molecular target that can reverse cholesterol metabolism disorders in alveolar macrophages.

[0123] This invention demonstrates for the first time that DTX4 expression is downregulated in alveolar macrophages of PAP patients, and its absence leads to reduced PPARγ expression, impaired cholesterol efflux, and enhanced foaming, thereby exacerbating surfactant accumulation. AAV-mediated DTX4 overexpression significantly improves these pathological changes, suggesting that DTX4 is a core regulator of macrophage lipid homeostasis, providing a novel target for PAP treatment.

[0124] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present 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 protection scope of the present invention.

Claims

1. The application of the DTX4 gene or protein in the preparation of drugs for treating pulmonary alveolar proteinosis, characterized in that, The drug is used to correct cholesterol efflux disorder in alveolar macrophages and contains an effective amount of the DTX4 gene or the protein it encodes, as well as a pharmaceutically acceptable carrier.

2. The application according to claim 1, characterized in that, The drug is prepared using a delivery system comprising a recombinant adeno-associated virus (AAV) vector containing a CD68 promoter to drive specific expression of the DTX4 gene in alveolar macrophages.

3. The application according to claim 2, characterized in that, The serotype of the AAV vector is AAV6, and the nucleotide sequence of the DTX4 gene is shown in SEQ ID NO:1, or the protein sequence it encodes is shown in SEQ ID NO:

2.

4. The application according to claim 1, characterized in that, The drug is prepared as a formulation suitable for lung delivery via nasal drops or tracheal instillation.

5. The application according to claim 1, characterized in that, The drug is used to treat pulmonary alveolar proteinosis selected from the following types: autoimmune PAP, hereditary PAP, secondary PAP, or congenital PAP.

6. The application according to claim 1, characterized in that, The dosage of the drug is determined by the following parameters: Baseline expression level of DTX4 in alveolar macrophages of patients; The ratio of cholesterol to protein in bronchoalveolar lavage fluid; Area of ​​ground-glass opacities on lung CT images; GM-CSF receptor gene mutation status.

7. A pharmaceutical composition for treating pulmonary alveolar proteinosis, characterized in that, It contains an effective amount of the DTX4 gene or the protein it encodes, and a pharmaceutically acceptable vector, wherein the DTX4 gene or protein is used to correct cholesterol efflux disorder in alveolar macrophages.

8. The pharmaceutical composition according to claim 7, characterized in that, The sequence of the DTX4 gene is shown in SEQ ID NO:1, or the sequence of the protein it encodes is shown in SEQ ID NO:

2.

9. The pharmaceutical composition according to claim 7, characterized in that, The drug is prepared using a delivery system comprising a recombinant adeno-associated virus (AAV) vector containing a CD68 promoter to drive specific expression of the DTX4 gene in alveolar macrophages, and the drug is prepared as a formulation suitable for pulmonary delivery via nasal or tracheal instillation.

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