Use of aav-hhip in the treatment of pulmonary fibrosis

Abstract

The application provides an application of AAV-HHIP in treating pulmonary fibrosis. The application uses AAV to deliver HHIP to treat pulmonary fibrosis mice, and the results of HE staining and Masson staining show that the pulmonary fibrosis area of the mice overexpressing HHIP is less, and the lung collagen fiber is less, indicating that AAV-HHIP inhibits the progression of pulmonary fibrosis of the mice and reduces the collagen deposition in the lungs of the mice. The results of hydroxyproline detection show that the collagen of the mice injected with AAV-HHIP is reduced. The results of PDPN immunofluorescence staining show that the alveolar structure of the mice injected with AAV-HHIP is more complete than that of the control group, indicating that AAV-HHIP enhances the ability of AT2 to differentiate AT1. The results of SA-beta-gal staining for detecting the aging of the lungs of the mice show that the positive signal of cell aging in the mice injected with AAV-HHIP is reduced, that is, AAV-HHIP inhibits the cell aging of the lungs of the mice after injury. It is shown that the pulmonary fibrosis can be treated by delivering HHIP through AAV.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of AAV-HHIP in the treatment of pulmonary fibrosis. Background Technology

[0002] Pulmonary fibrosis is a chronic interstitial lung disease characterized by fibrosis and honeycomb-like areas on histopathology. Patients experience damage to the lung parenchyma and structure, reduced lung plasticity, impaired gas exchange capacity, and ultimately respiratory failure. The incidence of pulmonary fibrosis is positively correlated with age, with patients typically aged between 55 and 75 years. Survival time after diagnosis is generally only 3 to 5 years. With the increasing aging of the global population, the number of pulmonary fibrosis patients worldwide is rising annually.

[0003] Currently, the FDA-approved drugs for treating pulmonary fibrosis, pirfenidone and nintedanib, both inhibit fibroblast activation, thereby slowing the progression of fibrosis and lung function decline. However, they cannot cure or reverse pulmonary fibrosis, and they have significant side effects and tolerance issues in clinical practice. Therefore, with current treatments, pulmonary fibrosis is irreversible, and treatments can only delay disease progression. Thus, finding effective targets for treating pulmonary fibrosis is of great significance for improving the prognosis of patients with pulmonary fibrosis and improving human health. Chinese patent CN117482235A discloses the application of HHIP as a therapeutic target for pulmonary fibrosis, through studies on wild-type mice and HHIP... + / - Bleomycin was injected intratracheally into mice to induce pulmonary fibrosis. The degree of pulmonary fibrosis was detected by morphological analysis (H&E staining and Masson staining), α-SMA immunofluorescence staining, hydroxyproline analysis, and expression analysis. The results showed that after bleomycin injection to induce pulmonary fibrosis, HHIP... + / - The degree of pulmonary fibrosis in mice was significantly higher than that in wild-type mice, indicating that HHIP has a significant inhibitory function on pulmonary fibrosis and can be used as a therapeutic target for pulmonary fibrosis. However, no feasible therapeutic drugs or treatment methods have been provided. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of AAV-HHIP in the preparation of drugs for treating pulmonary fibrosis.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution: Gene delivery therapy using adeno-associated virus (AAV) vectors is becoming increasingly widespread in clinical applications, and different types of AAV vectors have been approved for clinical use. This invention describes the delivery of HHIP to mouse lung tissue via AAV, and found that overexpression of HHIP significantly reduced the degree of bleomycin-induced pulmonary fibrosis.

[0006] Specifically, this invention utilizes AAV to deliver HHIP to mice with pulmonary fibrosis for treatment. Morphological analysis was used to detect the degree of pulmonary fibrosis. HE staining and Masson staining results showed that mice overexpressing HHIP had fewer fibrotic areas and fewer collagen fibers in the lungs, indicating that AAV-HHIP inhibits the progression of pulmonary fibrosis and reduces collagen deposition in the lungs. Hydroxyproline detection was performed, showing reduced collagen in AAV-HHIP-injected mice. PDPN immunofluorescence staining showed that the alveolar structure of AAV-HHIP-injected mice was more intact than the control group, indicating that AAV-HHIP enhances the differentiation of AT2 into AT1. SA-β-gal staining was used to detect lung senescence in mice, showing reduced positive signals of cellular senescence in AAV-HHIP-injected mice, indicating that AAV-HHIP inhibits cellular senescence in the lungs of injured mice.

[0007] Therefore, the present invention provides the following applications of AAV-HHIP: Application of AAV virus overexpressing HHIP (AAV-HHIP) in the preparation of drugs for treating pulmonary fibrosis.

[0008] Furthermore, the AAV virus is the AAV9 serotype virus.

[0009] Furthermore, the method for preparing the AAV virus overexpressing HHIP involves first cloning the HHIP gene into an expression vector to obtain the target plasmid, and then packaging it into an AAV virus overexpressing HHIP.

[0010] Preferably, the HHIP gene has the NCBI Gene ID: 15245.

[0011] Preferably, the expression vector is AAV-CAG-EGFP.

[0012] Preferably, the packaging involves transfecting cells with the target plasmid, AAV9, and pAdDeltaF6 at a molar ratio of 1:1:1, followed by purification to obtain AAV virus overexpressing HHIP. Specifically, the drug exerts its effects by reducing the progression of pulmonary fibrosis in the subject, reducing collagen deposition in the subject's lungs, improving the alveolar structure in the subject, enhancing the subject's ability to differentiate AT2 into AT1, and / or inhibiting the senescence of lung cells in the subject.

[0013] Preferably, the subject is selected from mammals.

[0014] More preferably, the mammal is selected from rats, cats, dogs, pigs, cattle, horses, sheep, monkeys, and humans.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the dosage form of the drug is an injection.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes AAV to deliver HHIP to mice with pulmonary fibrosis for treatment. Morphological analysis was used to assess the degree of pulmonary fibrosis. HE and Masson staining results showed that mice overexpressing HHIP had fewer fibrotic areas and less collagen fibers in their lungs, indicating that AAV-HHIP inhibits the progression of pulmonary fibrosis and reduces collagen deposition in the lungs. Hydroxyproline detection results showed reduced collagen in AAV-HHIP-injected mice. PDPN immunofluorescence staining results showed that the alveolar structure of AAV-HHIP-injected mice was more intact than that of the control group, indicating that AAV-HHIP enhances the differentiation of AT2 into AT1. SA-β-gal staining was used to assess lung senescence in mice, showing a reduction in positive cellular senescence signals in AAV-HHIP-injected mice, indicating that AAV-HHIP inhibits cellular senescence in the lungs of injured mice. This demonstrates that AAV-delivered HHIP can treat pulmonary fibrosis. Attached Figure Description

[0018] Figure 1 The experimental strategy for AAV treatment of pulmonary fibrosis in mice.

[0019] Figure 2 The results of immunofluorescence staining of GFP and SPC in AAV-injected mice.

[0020] Figure 3 The results of PDPN immunofluorescence staining in the lungs of mice in the AAV-overexpressing HHIP group and the control group 21 days after bleomycin modeling.

[0021] Figure 4 H&E staining results of lungs in mice 21 days after bleomycin modeling in the AAV-HHIP group and control group.

[0022] Figure 5Masson staining results of lungs in mice 21 days after bleomycin modeling in the AAV-HHIP group and control group.

[0023] Figure 6 The results of hydroxyproline measurement were obtained 21 days after bleomycin modeling in mice of the AAV-HHIP group and the control group.

[0024] Figure 7 The results of SA-βgal staining in the lungs of mice in the AAV-HHIP group and the control group 21 days after bleomycin modeling. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0026] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0027] Example 1 The HHIP fragment (NCBI Gene ID: 15245) was cloned into the expression vector (AAV-CAG-EGFP) to obtain the target plasmid, which was then packaged into an AAV virus overexpressing HHIP (AAV9-HHIP). The AAV9 virus packaging process specifically includes the following steps: (1) The day before transfection, 293T cells were plated at a density of approximately 1:2, allowing them to grow to 70-90% the next day. They were then plated at 900cm². 2 The culture area was calculated using a three-plasmid DNA system. The molar ratio of AAV9 (Wuhan Miaoling Biotechnology Co., Ltd., P13488), pAdDeltaF6 (Wuhan Miaoling Biotechnology Co., Ltd., pAdDeltaF6), and the target plasmid was 1:1:1. The plasmid transfection system is shown in Table 1.

[0028] Table 1 Plasmid Transfection System

[0029] Tube 1: Divide into half of the Opti-MEM and add plasmid; Tube 2: Divide into half of the Opti-MEM and add PEI; mix well and let stand for 5 min; mix the two tubes by inverting, let stand for 15 min, and then drop into a culture dish; 6 h after transfection, remove the culture medium and replace with fresh, preheated culture medium; collect cells and supernatant approximately 72 h after medium replacement. Centrifuge at 1000g for 5 min, combine the supernatants, add PEG, shake well, and let stand overnight; the next day, centrifuge at 4000g for 30 min at 4℃, discard the supernatant, and resuspend the precipitate in lysis buffer (PBS + 0.001% PF-68). The cell pellet was resuspended in lysis buffer, frozen at -80°C for at least 30 minutes, thawed in a 37°C water bath for 6-10 minutes, and subjected to three freeze-thaw cycles to completely lyse the cells. After the last cycle, the pellet was stored at -80°C and thawed at 37°C the next day. The pellet was then centrifuged at 3000g at 4°C for 5 minutes, and the supernatant was collected. The two fractions were combined; 5 mM MgCl2 and Benzonase were added and mixed thoroughly, then incubated at 37°C for 30 minutes. Then, 12.5% ​​w / v sodium deoxycholate was added, mixed thoroughly, and incubated for another 30 minutes. The pellet was then centrifuged at 2500g at 4°C for 10 minutes, and the supernatant was collected.

[0030] (2) Virus purification: Prepare the iodixanol density gradient according to Table 2 and perform ultracentrifugation.

[0031] Table 2 Iodixanol Preparation System

[0032] PBS MK: 2.8mM MgCl2, 2mM KCl in PBS. Simply weigh 13.2mg MgCl2 and 7.45mg KCl and dissolve them in 50mL PBS. 1M NaCl in PBS MK: 1M NaCl, 2.8mM MgCl2, 2mM KCl in PBS. Simply weigh 1.46g of NaCl and dissolve it in 25mL of PBS MK. Using a pipette, slowly add 60% iodixanol, 40% iodixanol, 25% iodixanol, 15% iodixanol, and virus solution to the bottom of an ultracentrifuge tube. Centrifuge at 39,000 rpm for 3 hours at 4°C. After centrifugation, the AAV virus will be located in the 40% layer. Slowly aspirate the fraction using a syringe. Transfer the virus solution from the syringe to a 15 mL centrifuge tube, add lysis buffer to dilute the iodixanol, and mix by inverting. Add an appropriate amount of lysis buffer to the column and centrifuge at 3000g for 5 minutes at 4°C to equilibrate the column. Pour off the filtrate and add the diluted virus solution. Centrifuge at 3000g for 5 minutes at 4°C. Repeat the column loading process until all the virus solution has been loaded. After centrifugation, observe the liquid volume and determine the final volume based on the packaging quantity and experimental results. This yields adeno-associated virus (AAV-GFP) and adeno-associated virus (AAV-HHIP), respectively.

[0033] Wild-type mice were intratracheally injected with AAV9-HHIP at a dose of 1*10^12 vg (Vector Genomes) and AAV9-GFP as a control group. Seven days after injection, bleomycin was administered intratracheally at a dose of 2 mg / kg to induce pulmonary fibrosis in the mice. The degree of pulmonary fibrosis was detected by morphological analysis after 21 days. Figure 1 The detailed experimental method is as follows: 1. Endotracheal injection: (1) Use isoflurane to anesthetize mice with gas. After anesthetizing the mice, hook the mice’s incisors with a thin rope and hang the mice up. Use tweezers to pull the mice’s tongue out so that the mice cannot swallow.

[0034] (2) Each mouse was given bleomycin at a dose of 2 mg / kg, in a volume of 50 μL to 100 μL. If AAV was injected, the AAV was diluted with sterile PBS to a 75 μl solution with a viral load of 1*10^12 vg (Vector Genomes). The drug was injected into the mouse's mouth, and the position was maintained for 30 seconds after injection. The mouse was observed to choke on the drug at least 15 times.

[0035] 2. Preparation of paraffin sections of mouse tissue: (1) After the lung tissue was removed from the mouse, it was placed in a vial containing 4% PFA as soon as possible and incubated at room temperature on a shaker for 1 hour.

[0036] (2) Discard the PFA, add an appropriate amount of 1×PBS solution, shake at room temperature for 15 min, and repeat twice.

[0037] (3) Discard the 1×PBS solution, add an appropriate amount of 70% ethanol, and place it in a refrigerator at 4°C overnight.

[0038] (3) Discard 70% ethanol, add 95% ethanol, shake at room temperature for 30 minutes, and repeat twice.

[0039] (4) Discard 95% ethanol, add 100% ethanol, shake at room temperature for 1 hour, and repeat twice.

[0040] (5) Discard 100% ethanol, add an appropriate amount of xylene to clear the tissue, and shake at room temperature for 1 hour.

[0041] (6) Discard the xylene, add the pre-melted paraffin, place it in the tissue embedding machine for paraffin infiltration, and change the paraffin every 1 hour for a total of 4 times.

[0042] (7) Take out the tissue that has been soaked in wax, put it into the embedding mold, add new paraffin wax for embedding, and after solidification, it can be sectioned with paraffin wax.

[0043] (8) Paraffin sections were prepared with a thickness of 7 μm. The prepared paraffin sections were placed on a 37°C baking machine for 2 hours or left at room temperature overnight before the experiment was conducted.

[0044] 3. Preparation of frozen sections of mouse tissue: (1) After the lung tissue was removed from the mouse, it was placed in a vial containing 4% PFA as soon as possible and incubated at room temperature on a shaker for 1 hour.

[0045] (2) Discard the PFA, add an appropriate amount of 1×PBS solution, shake at room temperature for 15 min, and repeat twice.

[0046] (3) Discard the 1×PBS solution, add an appropriate amount of 30% sucrose, and place it in a refrigerator at 4°C overnight.

[0047] (4) Wipe the surface moisture of the mouse lung tissue with a paper towel, put the tissue into a mold, drip ice embedding agent into the mold until the tissue is completely submerged, and slowly sink the mold into liquid nitrogen to solidify.

[0048] (5) Frozen sections were prepared with a thickness of 10 μm, and the frozen sections were brought back to room temperature for at least 30 min.

[0049] 4. Dewaxing and rehydration of paraffin sections: (1) Immerse the slices in xylene for 5 minutes to dewax, and repeat twice.

[0050] (2) Transfer the slice from xylene into 100% ethanol for 5 min.

[0051] (3) Transfer the slices to the second container of 100% ethanol for 1 min, 95% ethanol for 1 min, and 80% ethanol for 1 min.

[0052] (4) Transfer the slice into ddH2O for 2 min, and repeat twice.

[0053] 5. Immunofluorescence (1) After dewaxing and rehydration of paraffin sections, antigen retrieval was performed: sodium citrate microwave retrieval, boiling for 75 seconds, and then placed at room temperature for 1 hour to return to room temperature.

[0054] (2) Washing: Wash with ddH2O for 2 minutes, repeat twice.

[0055] (3) Removal of endogenous horseradish peroxidase (this step is to be performed when the triple antibody SA-hrp needs to be incubated later): 0.3% H2O2in methanol, room temperature for 15 min.

[0056] (4) Washing: Wash twice with dd water for 2 min, then wash twice with PBS for 5 min.

[0057] (5) Permeabilization: 0.5% PBST (0.5% Triton-X100 in PBS) at room temperature for 10 min.

[0058] (6) Blocking non-specific antigens: Antigen blocking solution (5% donkey serum + 1% BSA + 0.1% Triton X-100 in PBS) was used to block for 1 hour at room temperature.

[0059] (7) Incubate with primary antibody at 4°C overnight.

[0060] (8) Washing: Wash 3 times with PBS for 5 min each time.

[0061] (9) Incubate the secondary antibody at room temperature for 1 hour.

[0062] (10) Washing: Wash 3 times with PBS for 5 min each time.

[0063] (11) (If signal amplification is required) Triple resistance: 30 min at room temperature (12) Washing: Wash 3 times with PBS for 5 min.

[0064] (13) (If SA-hrp is selected for triple antibody amplification) TSA working solution 15s.

[0065] (14) Washing: Wash 3 times with PBS for 5 min each time.

[0066] (15) Stain cell nuclei with DAPI at room temperature for 5 min.

[0067] (16) Seal the slide with anti-fluorescence quenching agent and take a picture.

[0068] (17) Immunofluorescence assay was used to detect AAV infection in the lungs. The results showed that AAV could infect most cells in the lungs, including AT2 ( Figure 2 ).

[0069] Immunofluorescence was used to detect PDPN expression in mice to reflect alveolar integrity. The results showed that the alveolar structure of mice overexpressing HHIP was more intact than that of the control group. Figure 3 ).

[0070] 6. HE staining (1) After dewaxing and rehydrating the paraffin sections, soak them in hematoxylin for 5 minutes.

[0071] (2) Rinse with tap water 3 times, then rinse with running water for 5 minutes.

[0072] (3) 80% ethanol for 1 min.

[0073] (4) ddH2O 1min.

[0074] (5) Eosin in 45s.

[0075] (6) Rinse 5 times with 95% ethanol.

[0076] (7) Rinse 5 times with 100% ethanol (8) Soak in 100% ethanol for 2 minutes.

[0077] (9) Xylene for 5 min, repeat twice.

[0078] (10) Take a drop of neutral resin and gently drop it onto the sample surface, then slowly place the coverslip down to seal the sample.

[0079] HE staining was used to detect pulmonary fibrosis in mice. The results showed that the pulmonary fibrosis area in mice overexpressing HHIP was reduced compared with the control group. Figure 4 ).

[0080] 7. Masson staining (1) After dewaxing and rehydration of paraffin sections, stain with Ponceau Rhizoma and Magenta for 3-5 seconds.

[0081] (2) Wash with 0.2% acetic acid for 1 min.

[0082] (3) Wash with phosphomolybdic acid solution for 1 min. Wash with 0.2% acetic acid for 30 s.

[0083] (4) Stain with aniline blue solution for 90 seconds, then wash with 0.2% acetic acid for 30 seconds.

[0084] (5) ddH2O 1min.

[0085] (6) Rinse 5 times with 95% ethanol.

[0086] (7) Rinse 5 times with 100% ethanol.

[0087] (8) Soak in 100% ethanol for 2 minutes.

[0088] (9) Xylene for 5 min, repeat twice.

[0089] (10) Covering the film.

[0090] Masson staining was used to detect collagen deposition in the lungs of mice. The results showed that mice overexpressing HHIP had fewer collagen fibers than the control group. Figure 5 ).

[0091] 8. Hydroxyproline content detection First, the entire left lung lobe of AAV-mCherry mice and AAV-HHIP mice injected with the drug was harvested and weighed. 10 μl of 2.5 mol / L NaOH solution was added to each 1 mg of tissue to prepare a 10% tissue homogenate, which was then homogenized at 4°C. The absorbance values ​​were then measured at wavelengths of 560 and 630 nm. A standard curve was plotted based on the measurement results, and the hydroxyproline content of the sample was calculated, expressed in μg / mg.

[0092] Test results as follows Figure 6 As shown in the results, the hydroxyproline content in mice injected with AAV-HHIP was significantly lower than that in mice injected with AAV-mCherry, further demonstrating that AAV-HHIP inhibits pulmonary fibrosis.

[0093] 9. SA-beta gal staining (1) Wash frozen sections twice with sufficient PBS for about 30 seconds.

[0094] (2) Add sufficient fixative and incubate at room temperature for 5 minutes. (Strict time required; prolonged incubation will destroy SAbeta-gal activity).

[0095] (3) Remove the fixative and wash twice with PBS for 30 seconds.

[0096] (4) Add staining solution and incubate overnight at 37°C (12-16h). Do not incubate in a CO2 incubator (otherwise it will lead to overstaining). Blue may be detectable within 2h, but staining will reach its maximum after 12-16h.

[0097] (5) After incubation, wash twice with PBS for 30 seconds each time, rinse once with methanol, and allow the petri dish to air dry. (If protected from light, it can be stored at room temperature for several months) (6) Observe with a bright field microscope.

[0098] (7) SA-β-gal staining was used to detect lung aging in mice. The results showed that the positive signal of cellular senescence was reduced in mice overexpressing HHIP compared with the control group. Figure 7 This indicates that HHIP can be treated by delivering it via AAV.

Claims

1. Application of AAV virus overexpressing HHIP in the preparation of drugs for treating pulmonary fibrosis.

2. The application according to claim 1, characterized in that, The AAV virus in question is the AAV9 serotype virus.

3. The application according to claim 1, characterized in that, The method for preparing the AAV virus overexpressing HHIP involves first cloning the HHIP gene into an expression vector to obtain the target plasmid, and then packaging it into an AAV virus overexpressing HHIP.

4. The application according to claim 3, characterized in that, The HHIP gene has the NCBI GeneID number 15245.

5. The application according to claim 3, characterized in that, The expression vector is AAV-CAG-EGFP.

6. The application according to claim 3, characterized in that, The packaging involves transfecting cells with the target plasmid, AAV9, and pAdDeltaF6 at a molar ratio of 1:1:1, followed by purification to obtain AAV virus overexpressing HHIP.

7. The application according to claim 1, characterized in that, The drug works by reducing the progression of pulmonary fibrosis in subjects, reducing collagen deposition in subjects' lungs, improving alveolar structure in subjects, enhancing subjects' ability to differentiate AT2 into AT1, and / or inhibiting lung cell senescence in subjects.

8. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The application according to claim 1, characterized in that, The drug is in the form of an injection.

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