Nasal spray for allergic rhinitis and its preparation method

Abstract

This invention discloses a gene-modified mesenchymal stem cell exosome, its preparation method, and its application in the treatment of allergic rhinitis. The method primarily involves constructing the IL-27 expression gene sequence using molecular biology techniques, building a mammalian cell expression vector, further transfecting mesenchymal stem cells, and obtaining the gene-modified mesenchymal stem cell exosomes. This invention demonstrates significant therapeutic effects on allergic rhinitis, possesses substantial economic and social benefits, and shows promising market prospects.

Description

[0001] This application is a divisional application of the invention patent with application number ZL202110204230.X and the invention title "Preparation and application of gene-modified mesenchymal stem cell exosomes", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biopharmaceutical technology and relates to the preparation and application of gene-modified mesenchymal stem cell exosomes, particularly to the preparation of gene-modified mesenchymal stem cell exosomes and their application in the treatment of allergic rhinitis. Background Technology

[0003] Allergic rhinitis, also known as hay fever, is a type I hypersensitivity reaction of the nasal mucosa mediated by IgE, which triggers the release of histamine, kinins, and leukotrienes. Symptoms mainly include nasal itching, congestion, sneezing, runny nose, and olfactory dysfunction. As the disease progresses, it can be accompanied by various other conditions such as memory loss and pharyngitis, severely impacting patients' work and quality of life. This disease is one of the most common chronic diseases in humans, and in recent years, due to the influence of global climate change, environmental pollution, and social and psychological stress, its incidence has been increasing year by year, making it considered a "21st-century" epidemic, affecting approximately 25% of the global population.

[0004] Currently, the treatment of allergic rhinitis mainly includes three aspects: avoiding allergens, drug therapy, and immunotherapy. Known allergens should be avoided as much as possible, but exposure to allergens such as pollen, dust mites, and certain foods is unavoidable in daily life. Immunotherapy mainly refers to allergen-specific immunotherapy, but its treatment process is cumbersome, the treatment cycle is long (often around 3 years), the effect is slow, the cost is high, most patients find it difficult to adhere to, and the safety of the treatment remains uncertain. Drug therapy remains the first-line treatment for allergic rhinitis. Clinically used drugs mainly include five categories: glucocorticoids, antihistamines, decongestants, anticholinergics, and mast cell membrane stabilizers. Treatment often focuses on glucocorticoids and antihistamines, with the other three categories of drugs used as adjuncts. Antihistamines are effective for nasal itching, sneezing, and runny nose, but not so much for nasal congestion; decongestants can effectively relieve nasal congestion, but are ineffective for other symptoms; anticholinergics are effective for relieving runny nose, but ineffective for nasal congestion and sneezing, and have side effects such as nasal dryness and bleeding; mast cell membrane stabilizers have a good effect in preventing allergic rhinitis, but do not antagonize existing allergic mediators and are not effective for acute attacks of allergic rhinitis; glucocorticoids can block the allergic inflammatory response of the nasal mucosa at multiple points, and can effectively prevent and treat the symptoms of allergic rhinitis. They are currently the most effective first-line drugs for treating allergic rhinitis, but they have a slow onset of action, taking effect after several days or weeks of use, and have the side effect of local irritation. Furthermore, the side effects and long-term efficacy of long-term local use of glucocorticoids still need to be evaluated by evidence-based medicine.

[0005] In view of the above, the present invention aims to provide a genetically modified mesenchymal stem cell exosome, a preparation method thereof, and its application to solve one or more of the above-mentioned technical problems. Summary of the Invention

[0006] In order to solve one or more technical problems in the prior art, the applicant has discovered that using IL-27 gene-modified mesenchymal stem cell exosomes has a good effect on the treatment of allergic rhinitis. By secreting and expressing IL-27 factor, the immunomodulatory and anti-allergic functions of mesenchymal stem cell exosomes are enhanced, which can effectively repair the nasal microenvironment, improve the symptoms of allergic rhinitis, and overcome the side effects and poor treatment effects of existing allergic rhinitis treatment drugs.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] According to one aspect of the present invention, a gene-modified mesenchymal stem cell exosome for allergic rhinitis is provided, characterized by being obtained through the following steps:

[0009] The nucleic acid sequence for efficient expression of IL-27 in mammalian cells was designed and obtained. The nucleic acid sequence for efficient expression of IL-27 in mammalian cells contains a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, as shown in SEQ ID NO.1.

[0010] Genetically modified mesenchymal stem cells were obtained by transferring the nucleic acid sequence of IL-27, which is highly expressed in the mammalian cells, into mesenchymal stem cells.

[0011] The genetically modified mesenchymal stem cells were cultured to obtain the genetically modified mesenchymal stem cell exosomes.

[0012] According to another aspect of the present invention, a method for preparing gene-modified mesenchymal stem cell exosomes for allergic rhinitis is also provided, characterized by the following steps:

[0013] a. Design and obtain a nucleic acid sequence for efficient expression of IL-27 in mammalian cells, wherein the nucleic acid sequence for efficient expression of IL-27 in mammalian cells contains a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, as shown in SEQ ID NO.1;

[0014] b. Transform the nucleic acid sequence of the mammalian cells that efficiently express IL-27 into mesenchymal stem cells to obtain genetically modified mesenchymal stem cells;

[0015] c. Culture the genetically modified mesenchymal stem cells to obtain the genetically modified mesenchymal stem cell exosomes.

[0016] According to another aspect of the present invention, in step a, the nucleic acid sequence for the efficient expression of IL-27 in the mammalian cells is artificially synthesized, wherein the IL-27B chain and the IL-27A chain are linked by a linker composed of GGGS GGGS GGGS and the amino acid sequence shown in SEQ ID NO.2 is artificially synthesized according to the codons preferred by human cells. During the synthesis, the tPA signal peptide, the Kozak sequence and the BamHI site are introduced upstream of the gene, and the stop codon and the EcoRI site are introduced downstream of the gene.

[0017] According to another aspect of the present invention, step b specifically comprises: digesting the artificially synthesized nucleic acid sequence and the pHBAd adenovirus expression vector with BamHI and EcoRI respectively to construct the pHBAd-IL27 recombinant plasmid; transfecting the pHBAd-IL27 recombinant plasmid and the pHBAd-BHG plasmid into packaging virus 293A cells using Lipofiter transfection reagent; causing cytopathic effects and detachment from the base to obtain a mature adenovirus containing the nucleic acid sequence of the mammalian cell that efficiently expresses IL-27; and infecting the mesenchymal stem cells with the mature adenovirus to obtain genetically modified mesenchymal stem cells.

[0018] According to another aspect of the present invention, step c specifically comprises: culturing the gene-modified mesenchymal stem cells and collecting the supernatant to extract the gene-modified mesenchymal stem cell exosomes.

[0019] According to another aspect of the present invention, the application of the aforementioned genetically modified mesenchymal stem cell exosomes in the treatment of allergic rhinitis is also provided.

[0020] According to another aspect of the present invention, a method for preparing a nasal spray for allergic rhinitis is also provided, characterized by comprising the following steps:

[0021] Genetically modified mesenchymal stem cell exosomes were prepared using the aforementioned preparation method;

[0022] Take deionized water, and add glycerol, eucalyptol, EDTA-Na2, benzalkonium chloride and the genetically modified mesenchymal stem cell exosomes in sequence, stirring while adding to ensure complete dissolution. Finally, add microcrystalline cellulose-carboxymethyl cellulose sodium, stir to dissolve completely, and adjust the pH to 5.2-6.6 with HCl to obtain the nasal spray.

[0023] Each 1000ml of the nasal spray contains 5.0-15.0mg of the genetically modified mesenchymal stem cell exosomes, 3.0-7.0g of eucalyptol, 23.0-30.0g of glycerol, 12.0-18.0g of microcrystalline cellulose-sodium carboxymethyl cellulose, 0.5-1.0g of EDTA-Na2, 0.2g of benzalkonium chloride, and deionized water as the solvent.

[0024] According to another aspect of the present invention, a nasal spray for allergic rhinitis is also provided, characterized in that each 1000 ml of the nasal spray comprises:

[0025] 5.0-15.0 mg of the gene-modified mesenchymal stem cell exosomes as described in claim 1;

[0026] Eucalyptol 3.0-7.0g;

[0027] Glycerin 23.0-30.0g;

[0028] Microcrystalline cellulose - sodium carboxymethyl cellulose 12.0-18.0g;

[0029] EDTA-Na2 0.5-1.0g;

[0030] Benzalkonium chloride 0.2g;

[0031] The remainder is deionized water.

[0032] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0033] 1. This invention uses IL-27 gene-modified mesenchymal stem cell exosomes as an active ingredient for the treatment of allergic rhinitis. By secreting and expressing IL-27 factor, it enhances the immunomodulatory and anti-allergic functions of mesenchymal stem cell exosomes, effectively repairs the nasal microenvironment, improves the symptoms of allergic rhinitis, and achieves the purpose of treating allergic rhinitis. The treatment effect is significant, and the safety is good, without the side effects of dependence on hormone products.

[0034] 2. Exosomes obtained from IL-27 gene-modified mesenchymal stem cells contain active IL-27 protein along with the exosomes during their formation, giving mesenchymal stem cells stronger immunomodulatory function and thus enhancing the therapeutic effect of the secreted exosomes on allergic rhinitis.

[0035] 3. Exosomes obtained from IL-27 gene-modified mesenchymal stem cells contain active IL-27 protein along with the exosomes during their formation, thus improving the stability of the active IL-27 protein.

[0036] 4. Exosomes obtained from IL-27 gene-modified mesenchymal stem cells contain active IL-27 protein along with the exosomes during their formation. The exosomes then transmit the biological information contained therein, such as nucleic acids and proteins, thereby further improving the bioavailability of the active factor IL-27 protein. The combined effect of these factors can achieve better treatment results for allergic rhinitis.

[0037] 5. In every 1000ml of nasal spray, eucalyptol (3.0-7.0g) and genetically modified mesenchymal stem cell exosomes (5.0-15.0mg) work synergistically to further enhance the therapeutic effect of allergic rhinitis. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the embodiments are used only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0039] According to a preferred embodiment of the present invention, a genetically modified mesenchymal stem cell exosome is provided, characterized by being obtained through the following steps:

[0040] The nucleic acid sequence for efficient expression of IL-27 in mammalian cells was designed and obtained. The nucleic acid sequence for efficient expression of IL-27 in mammalian cells contains a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, as shown in SEQ ID NO.1.

[0041] Genetically modified mesenchymal stem cells were obtained by transferring the nucleic acid sequence of IL-27, which is highly expressed in the mammalian cells, into mesenchymal stem cells.

[0042] The genetically modified mesenchymal stem cells were cultured to obtain the genetically modified mesenchymal stem cell exosomes.

[0043] Advantageously, the present invention constructs the gene sequence for IL-27 expression using molecular biology techniques, constructs a mammalian cell expression vector, further transfects mesenchymal stem cells, and obtains gene-modified mesenchymal stem cell exosomes.

[0044] According to another preferred embodiment of the present invention, a method for preparing gene-modified mesenchymal stem cell exosomes is also provided, characterized by the following steps:

[0045] a. Design and obtain a nucleic acid sequence for efficient expression of IL-27 in mammalian cells, wherein the nucleic acid sequence for efficient expression of IL-27 in mammalian cells contains a tPA signal peptide coding sequence, an IL-27B chain coding sequence, a linker sequence and an IL-27A chain coding sequence, as shown in SEQ ID NO.1;

[0046] b. Transform the nucleic acid sequence of the mammalian cells that efficiently express IL-27 into mesenchymal stem cells to obtain genetically modified mesenchymal stem cells;

[0047] c. Culture the genetically modified mesenchymal stem cells to obtain the genetically modified mesenchymal stem cell exosomes.

[0048] According to another preferred embodiment of the present invention, in step a, the nucleic acid sequence for the efficient expression of IL-27 in the mammalian cells is artificially synthesized, wherein the IL-27B chain and the IL-27A chain are linked by a linker composed of GGGS GGGS GGGS and the amino acid sequence shown in SEQ ID NO.2 is artificially synthesized according to the codons preferred by human cells. During the synthesis, the tPA signal peptide, Kozak sequence and BamHI site are introduced upstream of the gene, and the stop codon and EcoRI site are introduced downstream of the gene.

[0049] According to another preferred embodiment of the present invention, step b specifically comprises: digesting the artificially synthesized nucleic acid sequence and the pHBAd adenovirus expression vector with BamHI and EcoRI respectively to construct the pHBAd-IL27 recombinant plasmid; transfecting the pHBAd-IL27 recombinant plasmid and the pHBAd-BHG plasmid into packaging virus 293A cells using Lipofiter transfection reagent; causing cytopathic effects and detachment from the base to obtain a mature adenovirus containing the nucleic acid sequence of mammalian cells that efficiently expresses IL-27; and infecting the mesenchymal stem cells with the mature adenovirus to obtain genetically modified mesenchymal stem cells.

[0050] According to another preferred embodiment of the present invention, step c specifically involves: culturing the gene-modified mesenchymal stem cells and collecting the supernatant to extract the gene-modified mesenchymal stem cell exosomes.

[0051] According to another preferred embodiment of the present invention, the application of the aforementioned genetically modified mesenchymal stem cell exosomes in the treatment of allergic rhinitis is also provided.

[0052] According to another preferred embodiment of the present invention, a method for preparing a nasal spray for allergic rhinitis is also provided, characterized by comprising the following steps:

[0053] Genetically modified mesenchymal stem cell exosomes were prepared using the aforementioned preparation method;

[0054] Take deionized water, and add glycerol, eucalyptol, EDTA-Na2, benzalkonium chloride and the genetically modified mesenchymal stem cell exosomes in sequence, stirring while adding to ensure complete dissolution. Finally, add microcrystalline cellulose-carboxymethyl cellulose sodium, stir to dissolve completely, and adjust the pH to 5.2-6.6 with HCl to obtain the nasal spray.

[0055] Each 1000ml of the nasal spray contains 5.0-15.0mg of the genetically modified mesenchymal stem cell exosomes, 3.0-7.0g of eucalyptol, 23.0-30.0g of glycerol, 12.0-18.0g of microcrystalline cellulose-sodium carboxymethyl cellulose, 0.5-1.0g of EDTA-Na2, 0.2g of benzalkonium chloride, and deionized water as the solvent.

[0056] According to another preferred embodiment of the present invention, a nasal spray for allergic rhinitis is also provided, characterized in that each 1000 ml of the nasal spray comprises:

[0057] 5.0-15.0 mg of the gene-modified mesenchymal stem cell exosomes as described in claim 1;

[0058] Eucalyptol 3.0-7.0g;

[0059] Glycerin 23.0-30.0g;

[0060] Microcrystalline cellulose - sodium carboxymethyl cellulose 12.0-18.0g;

[0061] EDTA-Na2 0.5-1.0g;

[0062] Benzalkonium chloride 0.2g;

[0063] The remainder is deionized water.

[0064] Advantageously, eucalyptol, a colorless to pale yellow oily liquid, is the main component of eucalyptus oil. It has camphor and a cool herbal odor and has the effects of relieving wind and heat, removing dampness and detoxifying, and antibacterial and antiviral properties. Its synergistic effect with gene-modified mesenchymal stem cell exosomes further enhances the therapeutic effect of allergic rhinitis.

[0065] According to another preferred embodiment of the present invention, a spray for treating allergic rhinitis containing mesenchymal stem cell exosomes is also provided, having a pH value of 5.2-6.6, and containing 5.0-15.0 mg of mesenchymal stem cell exosomes, 3.0-7.0 g of eucalyptol, 23.0-30.0 g of glycerol, 12.0-18.0 g of microcrystalline cellulose-sodium carboxymethyl cellulose, 0.5-1.0 g of EDTA-Na2, 0.2 g of benzalkonium chloride, and deionized water as the solvent.

[0066] In a preferred embodiment of the present invention, each 1000ml contains 10.0mg of mesenchymal stem cell exosomes, 5.0g of eucalyptol, 26.0g of glycerol, 15.0g of microcrystalline cellulose-sodium carboxymethyl cellulose, 0.5g of EDTA-Na2, 0.2g of benzalkonium chloride, and deionized water as the solvent.

[0067] Preferably, the gene-modified mesenchymal stem cells are mesenchymal stem cells transfected with the IL-27 gene and capable of secreting and expressing functional IL-27.

[0068] Preferably, the vector transfecting the IL-27 gene is a viral vector such as adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, or a non-viral vector such as pcDNA3.1.

[0069] More preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and placental mesenchymal stem cells.

[0070] According to another preferred embodiment of the present invention, a method for preparing a mesenchymal stem cell exosome nasal spray is also provided, comprising the following steps:

[0071] (1) Using mammalian cell expression vectors, an IL-27 secretory expression vector was designed and constructed;

[0072] (2) Isolation, culture, purification and identification of mesenchymal stem cells;

[0073] (3) Transform the IL-27 secretion expression vector into mesenchymal stem cells, continue culturing for at least 3 days, collect the culture supernatant, and extract the stem cell exosome precipitate.

[0074] Preferably, the preparation method further includes step (4): according to the formula, eucalyptol, glycerol, microcrystalline cellulose-carboxymethyl cellulose sodium, EDTA-Na2, benzalkonium chloride and mesenchymal stem cell exosomes are added to deionized water, the pH value is adjusted, and then the solution is filtered through a 0.45μm sterile filter membrane to remove bacteria, thereby obtaining the allergic rhinitis treatment spray containing mesenchymal stem cell exosomes.

[0075] Preferably, the construction of the IL-27 secretory expression vector involves subcloning the expression cassette of the IL-27 protein into a mammalian cell expression vector using molecular biology and genetic engineering techniques.

[0076] In a preferred embodiment of the present invention, the expression vector is an adenovirus vector.

[0077] Further preferably, step (1) comprises: artificially synthesizing a nucleic acid sequence (as shown in SEQ ID NO.1) suitable for efficient expression of IL-27 in mammalian cells, comprising a Kozak sequence, a tPA signal peptide sequence, an IL-27B chain (EBI3) sequence, a linker sequence, and an IL-27A chain (p28) sequence. The nucleic acid sequence suitable for efficient expression of IL-27 in mammalian cells is ligated into a pHBAd adenovirus expression vector using BamHI and EcoRI restriction enzymes. The nucleic acid sequence suitable for efficient expression of IL-27 in mammalian cells may encode an amino acid sequence as shown in SEQ ID NO.2.

[0078] In a preferred embodiment of the present invention, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0079] More preferably, step (2) is as follows: take a fresh umbilical cord, wash it with sterile saline to remove blood, then remove the veins, capsule and artery, cut Wharton's jelly into small pieces and inoculate it into a culture dish, add mesenchymal stem cell culture medium, and culture it in a 37°C, 5% CO2 cell culture incubator for 6-8 days. A large number of spindle cells can be seen crawling out of the tissue block, which are the mesenchymal stem cells.

[0080] The various parts of the present invention will now be described in further detail with reference to embodiments. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0081] Example 1: Artificial synthesis of DNA sequence encoding IL-27 (as shown in SEQ ID NO.1)

[0082] The mature B-chain protein sequence of IL-27 contains 209 amino acid residues, and the mature A-chain protein sequence contains 215 amino acid residues. The B and A chains are linked by a linker consisting of GGGS GGGS GGGS. The above amino acid sequences are artificially synthesized based on codons preferred by human cells. During synthesis, the tPA signal peptide (MDAMKRGLCCVLLLCGAVFVSP), the Kozak sequence, and the BamHI site are introduced upstream of the gene, and a stop codon and the EcoRI site are introduced downstream of the gene.

[0083] Example 2: Construction of expression vector

[0084] The synthesized gene fragment and pHBAd adenovirus expression vector were digested with BamHI and EcoRI, respectively, and incubated overnight at 37°C. The digestion products were excised using agarose gel electrophoresis to remove the target fragment, which was then recovered using an agarose gel DNA recovery kit. The recovered digested target gene and vector were mixed at a 3:1 molar ratio and ligated overnight using a T4 DNA Ligase ligase PCR instrument at 16°C to construct the pHBAd-IL27 recombinant plasmid. The ligation product was transformed into competent E. coli DH5α cells. The transformed competent cells were evenly spread on LB agar plates containing ampicillin (100 μg / mL) and incubated upside down at 37°C for 12-16 h after complete absorption. Using a sterile pipette tip, 4-8 healthy single colonies were picked from the transformation plate and inoculated into 5 mL of LB agar containing ampicillin (100 μg / mL), and incubated overnight at 37°C with vigorous shaking (225 rpm). Plasmid DNA was extracted using a rapid plasmid miniprep kit. The recombinant plasmid pHBAd-IL27 was identified by double digestion with BamHI and EcoRI. After digestion at 37°C for 3 hours, the correct clones were identified based on the restriction enzyme profile. Clones with correct restriction patterns were selected and sent to a sequencing company for sequencing verification. Using a large-scale plasmid miniprep kit, a large quantity of the recombinant plasmid pHBAd-IL27 was extracted from the correctly sequenced clones. Then, the pHBAd-IL27 and pHBAd-BHG plasmids were transfected into packaging virus 293A cells using Lipofiter transfection reagent. Virus was harvested when most cells showed pathotropic effects and detached from the base. After three freeze-thaw cycles, the cells were centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected to obtain a large quantity of mature adenovirus containing the aforementioned IL-27 sequence. The viral titer was calculated, and the virus was stored at -80°C for subsequent experiments.

[0085] Example 3: Obtaining Umbilical Cord Mesenchymal Stem Cells

[0086] Fresh umbilical cord is taken, washed with sterile saline to remove blood, and cut into 4cm segments. Then, the veins, capsule, and two arteries are removed. Wharton's jelly is cut into small pieces and seeded into 10cm culture dishes. Mesenchymal stem cell culture medium is added, and the dishes are cultured at 37°C in a 5% CO2 cell culture incubator for 6-8 days. A large number of spindle-shaped cells can be seen crawling out of the tissue block, which are the mesenchymal stem cells. The obtained umbilical cord mesenchymal stem cells are then passaged, expanded, purified, and identified.

[0087] Example 4: Transfection of umbilical cord mesenchymal stem cells

[0088] Umbilical cord mesenchymal stem cells were seeded in T175 culture flasks. When the cell density reached approximately 80%, the umbilical cord mesenchymal stem cells were infected with an adenovirus that secretes and expresses IL-27 with a multiplicity of infection (MOI) of 200. The medium was changed after 6 hours, and the cells were cultured for another 72 hours. The supernatant was collected to extract exosomes, and 2 ml of the supernatant was used to detect the expression of IL-27 by ELISA.

[0089] Example 5: Preparation of exosomes from umbilical cord mesenchymal stem cells

[0090] Exosomes were extracted using differential centrifugation, with the following steps: Centrifuge at 2000×g for 10 minutes at 4℃, and collect the supernatant. Centrifuge at 10000×g for 30 minutes at 4℃, and collect the supernatant. Centrifuge at 100000×g for 120 minutes at 4℃, discard the supernatant, resuspend the remaining precipitate in PBS, and centrifuge again at 100000×g for 120 minutes to obtain the exosome precipitate. After resuspending in physiological saline, quantification was performed using the BCA method.

[0091] Example 6: Preparation of an allergic rhinitis spray

[0092] The above-mentioned spray for treating allergic rhinitis contains an aqueous solution of mesenchymal stem cell exosomes, eucalyptol, glycerin, microcrystalline cellulose-carboxymethyl cellulose sodium, EDTA-Na2, and benzalkonium chloride.

[0093] In nasal sprays, sodium carboxymethyl cellulose mainly functions as a suspending agent and thickener, which helps the active ingredients to be distributed and remain in the nasal cavity for longer periods, thereby improving drug utilization. Its concentration is 0.91-2.1%, and the preferred concentration used in this formulation is 1.5%.

[0094] In this embodiment, the preparation is carried out according to the following prescription:

[0095] Table 1. Composition of Mesenchymal Stem Cell Exosome Allergic Rhinitis Spray Formulation

[0096]

[0097] Take the prescribed amount of deionized water, and add glycerin, eucalyptol, EDTA-Na2, benzalkonium chloride, and mesenchymal stem cell exosomes in sequence while stirring to ensure complete dissolution. Finally, add microcrystalline cellulose-sodium carboxymethyl cellulose and stir to ensure complete dissolution. Adjust the pH to approximately 6.0 using HCl, then filter through a 0.45μm sterile filter membrane for sterilization. Pour the solution into a sterile nasal spray bottle to obtain the allergic rhinitis treatment spray containing mesenchymal stem cell exosomes.

[0098] Example 7: Study on the therapeutic effect of an allergic rhinitis spray on allergic rhinitis in rats.

[0099] 1) Establishment of a rat model of allergic rhinitis

[0100] Sixty male Wistar rats, weighing 180-200g, were randomly selected as a control group (n=10). The remaining rats were challenged by instilling 5μl of a 10% toluene diisocyanate (TDI) solution in olive oil into each nostril using a micropipette, once daily for 7 consecutive days. The control group received the same amount of olive oil in the same manner. Thirty minutes after the last challenge, the success of the model was determined by observing the rats' nose scratching, sneezing, and runny nose. The scoring criteria were as follows: 1-2 nose scratches, 1-3 sneezes, and runny nose extending into the nostrils each scored 1 point; multiple nose scratches, 4-10 sneezes, and runny nose extending beyond the anterior nostrils each scored 2 points; and persistent scratching of the nose and face, rubbing, 11 or more sneezes, and runny nose covering the face each scored 3 points. The scores for each symptom were added together, and a total score exceeding 5 points indicated successful model establishment.

[0101] 2) Experimental study on the treatment of a rat model of allergic rhinitis

[0102] 2.1 Grouping and administration of rats

[0103] After successful modeling, rats were divided into a blank control group, a positive control group, and low-, medium-, and high-dose exosome spray groups, with 10 rats in each group. The drug administration experiment was then conducted. The blank control group received 15 μl of physiological saline per nostril using a micropipette, the positive control group received 15 μl of mometasone furoate nasal spray per nostril using a micropipette, and the low-, medium-, and high-dose exosome spray groups received 5 μl, 10 μl, and 15 μl of exosome spray per nostril, respectively, using a micropipette. Simultaneously, the model group rats were maintained sensitized by receiving 5 μl of 10% toluene diisocyanate (TDI) in olive oil solution per nostril every other day. The normal rat blank control group received olive oil in the same manner and at the same dose until the end of the experiment.

[0104] 2.2 Observation Indicators and Results

[0105] 2.2.1 Rhinitis behavioral indicators and scores:

[0106] At the time of successful model establishment and grouping, on day 7 and day 14 of drug administration, behavioral indicators of rhinitis symptoms in rats, such as nose scratching, sneezing, and rhinorrhea, were evaluated. The results were statistically analyzed and calculated according to the above scoring criteria, and are shown in the table below:

[0107] Table 2. Behavioral scores of allergic rhinitis symptoms in rats

[0108]

[0109] * indicates a significant difference compared to the model group (p < 0.01).

[0110] 2.2.2 Pathological results of HE staining of nasal mucosa tissue

[0111] Nasal mucosa respiratory areas of rats in each group were collected, fixed in tissue fixative, sectioned into paraffin blocks, stained with hematoxylin and eosin (HE), and the inflammation of the nasal mucosa was observed under a light microscope. Pathological results were graded according to the following criteria: "-" No inflammatory reaction or exudate in the nasal mucosa epithelium and submucosal tissue; "+" Submucosal congestion with a small amount of inflammatory cell infiltration; "++" Abundant inflammatory cell infiltration in the submucosa, with inflammatory exudate in the nasal cavity; "+++" Abundant inflammatory cell infiltration in the submucosa, submucosal tissue hyperplasia, mucosal shedding, inflammatory exudate and hemorrhage in the nasal cavity.

[0112] Table 3. Pathological results of HE staining of rat mucosal tissue

[0113]

[0114] These results demonstrate that the nasal spray prepared from a genetically modified mesenchymal stem cell exosome according to the present invention has a significant therapeutic effect on allergic rhinitis.

[0115] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0116] 1. This invention uses IL-27 gene-modified mesenchymal stem cell exosomes as an active ingredient for the treatment of allergic rhinitis. By secreting and expressing IL-27 factor, it enhances the immunomodulatory and anti-allergic functions of mesenchymal stem cell exosomes, effectively repairs the nasal microenvironment, improves the symptoms of allergic rhinitis, and achieves the purpose of treating allergic rhinitis. The treatment effect is significant, and the safety is good, without the side effects of dependence on hormone products.

[0117] 2. Exosomes obtained from IL-27 gene-modified mesenchymal stem cells contain active IL-27 protein along with the exosomes during their formation, giving mesenchymal stem cells stronger immunomodulatory function and thus enhancing the therapeutic effect of the secreted exosomes on allergic rhinitis.

[0118] 3. Exosomes obtained from IL-27 gene-modified mesenchymal stem cells contain active IL-27 protein along with the exosomes during their formation, thus improving the stability of the active IL-27 protein.

[0119] 4. Exosomes obtained from IL-27 gene-modified mesenchymal stem cells contain active IL-27 protein along with the exosomes during their formation. The exosomes then transmit the biological information contained therein, such as nucleic acids and proteins, thereby further improving the bioavailability of the active factor IL-27 protein. The combined effect of these factors can achieve better treatment results for allergic rhinitis.

[0120] 5. In every 1000ml of nasal spray, eucalyptol (3.0-7.0g) and genetically modified mesenchymal stem cell exosomes (5.0-15.0mg) work synergistically to further enhance the therapeutic effect of allergic rhinitis.

[0121] The above descriptions are specific and detailed embodiments of the present invention, but they should not be construed as limiting the scope of the invention. For example, the gene-modified mesenchymal stem cell exosomes of the present invention can also be used to treat asthma, pulmonary fibrosis, or rheumatoid arthritis. 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 modifications and improvements all fall within the scope of protection of the present invention.

[0122]

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[0124] sequence list <110> Cyp Biotechnology (Changchun) Co., Ltd. <120> Preparation and application of gene-modified mesenchymal stem cell exosomes <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1401 <212> DNA <213> Artificial Sequence <400> 1 ggatccgccg ccaccatgga cgccatgaag agaggcctgt gctgcgtgct gctgctgtgc 60 ggcgctgtgt ttgtgagccc tagaaagggg cctcctgccg ctctgaccct gcctagagtg 120 cagtgcagag ccagcaggta tcccatcgcc gtcgattgca gctggaccct gcccccttgct 180 cccaacagca catctcccgt gagcttcatc gccacctaca gactgggaat ggctcccaga 240 ggccacagct ggccctgcct gcagcagaca cccacttcta caagctgcac catcaccgac 300 gtgcagctgt tcagcatggc tccctacgtg ctgaacgtga ccgccgtgca cccctggggc 360 tctagctcta gcttcgtgcc cttcatcacc gagcacatca tcaagcccga ccctcccgag 420 ggcgtgaggc tgtctcctct ggccgagaga cagcttcaag tacagtggga acctcccggc 480 agctggccct tccccgagat cttcagcctg aagtactgga tcagatacaa gagacaaggc 540 gctgccaggt tccacagagt gggccctatc gaggccacaa gcttcatcct gagagccgtc 600 agacctagag ctatagacta cgtgcaagtg gctgcccaag acctcaccga ctacggcgag 660 ttaagcgatt ggtctctgcc tgccaccgca accatgtcat taggcaaagg aggcgggagc 720 ggcggtgggt ctggcggcgg aagctttcct agacctcccg gcagacctca gctgagcctg 780 caagagctga ggagagagtt caccgtgtct ctgcacctgg ctagaaagct gctgagcgag 840 gtgagaggcc aagcccacag gttcgccgag agccacctgc ctggcgtgaa cctgtacctg 900 ctgcctctgg gcgagcagct gcccgacgtg agcctgacct tccaagcctg gaggagactg 960 agcgaccccg agagactgtg cttcatctct accactctgc agcccttcca cgccctgctg 1020 ggcggcttag gtacccaagg cagatggacc aacatggaga gaatgcagct gtgggccatg 1080 aggctggacc tgagagacct gcagagacac ctgagattcc aagtgctggc tgctggcttc 1140 aacctgcccg aggaggagga ggaggaggag gaggaggaag aggaggaaag aaagggactg 1200 ctccctggtg ccttaggctc tgcactacaa ggccctgccc aagtgagctg gcctcagctg 1260 ctgtctacct acaggttgct gcacagcctg gagctggtgc tgagcagagc agtgagggaa 1320 ctgctcctcc tgagcaaggc tgggcacagc gtgtggcctc tgggcttccc cactctgagc 1380 cctcagccct gataagaatt c 1401 <210> 2 <211> 458 <212> PRT <213> Artificial Sequence <400> 2 Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu 20 25 30 Pro Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys 35 40 45 Ser Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe 50 55 60 Ile Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro 65 70 75 80 Cys Leu Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val 85 90 95 Gln Leu Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His 100 105 110 Pro Trp Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile 115 120 125 Ile Lys Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu 130 135 140 Arg Gln Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro 145 150 155 160 Glu Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala 165 170 175 Ala Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu 180 185 190 Arg Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln 195 200 205 Asp Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr 210 215 220 Ala Thr Met Ser Leu Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly 225 230 235 240 Gly Gly Ser Phe Pro Arg Pro Pro Gly Arg Pro Gln Leu Ser Leu Gln 245 250 255 Glu Leu Arg Arg Glu Phe Thr Val Ser Leu His Leu Ala Arg Lys Leu 260 265 270 Leu Ser Glu Val Arg Gly Gln Ala His Arg Phe Ala Glu Ser His Leu 275 280 285 Pro Gly Val Asn Leu Tyr Leu Leu Pro Leu Gly Glu Gln Leu Pro Asp 290 295 300 Val Ser Leu Thr Phe Gln Ala Trp Arg Arg Leu Ser Asp Pro Glu Arg 305 310 315 320 Leu Cys Phe Ile Ser Thr Thr Leu Gln Pro Phe His Ala Leu Leu Gly 325 330 335 Gly Leu Gly Thr Gln Gly Arg Trp Thr Asn Met Glu Arg Met Gln Leu 340 345 350 Trp Ala Met Arg Leu Asp Leu Arg Asp Leu Gln Arg His Leu Arg Phe 355 360 365 Gln Val Leu Ala Ala Gly Phe Asn Leu Pro Glu Glu Glu Glu Glu Glu 370 375 380 Glu Glu Glu Glu Glu Glu Glu Arg Lys Gly Leu Leu Pro Gly Ala Leu 385 390 395 400 Gly Ser Ala Leu Gln Gly Pro Ala Gln Val Ser Trp Pro Gln Leu Leu 405 410 415 Ser Thr Tyr Arg Leu Leu His Ser Leu Glu Leu Val Leu Ser Arg Ala 420 425 430 Val Arg Glu Leu Leu Leu Leu Ser Lys Ala Gly His Ser Val Trp Pro 435 440 445 Leu Gly Phe Pro Thr Leu Ser Pro Gln Pro 450 455

Claims

1. A method for preparing a nasal spray for allergic rhinitis, characterized in that... Includes the following steps: Genetically modified mesenchymal stem cell exosomes; Take deionized water, and add glycerol, eucalyptol, EDTA-Na2, benzalkonium chloride and the genetically modified mesenchymal stem cell exosomes in sequence, stirring while adding to ensure complete dissolution. Finally, add microcrystalline cellulose-carboxymethyl cellulose sodium, stir to dissolve completely, and adjust the pH to 5.2-6.6 with HCl to obtain the nasal spray. Each 1000ml of the nasal spray contains 10mg of the genetically modified mesenchymal stem cell exosomes, 5g of eucalyptol, 26g of glycerol, 15g of microcrystalline cellulose-carboxymethyl cellulose sodium, 0.5g of EDTA-Na2, 0.2g of benzalkonium chloride, and deionized water as the solvent. The genetically modified mesenchymal stem cells are mesenchymal stem cells modified with the nucleic acid sequence shown in SEQ ID NO.1; Among them, the secretion and expression of IL-27 factor enhances the immunomodulatory and anti-allergic functions of the gene-modified mesenchymal stem cell exosomes, which can be used to repair the nasal microenvironment; while forming exosomes, active IL-27 protein is also loaded into the exosomes, giving mesenchymal stem cells stronger immunomodulatory functions, improving the stability of active IL-27 protein and the bioavailability of active factor IL-27 protein. The preparation of gene-modified mesenchymal stem cell exosomes specifically includes the following steps: a. Design and obtain a nucleic acid sequence for efficient expression of IL-27 in mammalian cells. The nucleic acid sequence for efficient expression of IL-27 in mammalian cells includes a tPA signal peptide coding sequence, an IL-27 B chain coding sequence, a linker sequence, and an IL-27 A chain coding sequence. The gene-modified mesenchymal stem cells are shown in SEQ ID NO.

1. b. Transform the nucleic acid sequence of the mammalian cells that efficiently express IL-27 into mesenchymal stem cells to obtain genetically modified mesenchymal stem cells; c. Culture the gene-modified mesenchymal stem cells to obtain the gene-modified mesenchymal stem cell exosomes; In step a, the nucleic acid sequence for efficiently expressing IL-27 in mammalian cells is artificially synthesized. The IL-27 B chain and IL-27 A chain are linked by a linker composed of GGGS GGGS GGGS, and the amino acid sequence shown in SEQ ID NO.2 of the gene-modified mesenchymal stem cells is artificially synthesized according to the codons preferred by human cells. During synthesis, the tPA signal peptide, Kozak sequence and BamHI site are introduced upstream of the gene, and the stop codon and EcoRI site are introduced downstream of the gene.

2. A nasal spray for allergic rhinitis, characterized in that... Each 1000ml of the nasal spray contains: 10 mg of gene-modified mesenchymal stem cell exosomes prepared according to the method of claim 1; 5g of eucalyptol; 26g of glycerin; 15g of microcrystalline cellulose-sodium carboxymethyl cellulose; EDTA-Na2 0.5-1.0g; Benzalkonium chloride 0.2g; The remainder is deionized water; in, The genetically modified mesenchymal stem cells are mesenchymal stem cells modified with the nucleic acid sequence shown in SEQ ID NO.1; Among them, the secretion and expression of IL-27 factor enhances the immunomodulatory and anti-allergic functions of the gene-modified mesenchymal stem cell exosomes, which can be used to repair the nasal microenvironment; while forming exosomes, active IL-27 protein is also loaded into the exosomes, giving mesenchymal stem cells stronger immunomodulatory functions, improving the stability of active IL-27 protein and the bioavailability of active factor IL-27 protein.

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