Mucosa-based non-invasive gene therapy with recombinant vector

Abstract

The present invention provides a mucosa-based non-invasive gene therapy with a recombinant vector. The recombinant region of the recombinant vector comprises a coding sequence of at least one therapeutic gene. The recombinant vector is administered to the respiratory system, the digestive system, the genitourinary system, and the eyes via the mucosa, for preventing and / or treating a disease.

Description

Recombinant vector-based non-invasive gene therapy based on mucosa Technical Field

[0001] This invention relates to viral modification and gene therapy, and more particularly to a novel cell therapy approach. Background Technology

[0002] Current gene therapy technologies typically involve taking cells from a patient, editing the patient's own cells in vitro, and introducing foreign genes capable of treating diseases into the patient's own cells, thereby enabling the patient's own cells to have the function of treating diseases. However, this treatment method requires creating an incision in the patient to take cells and administer drugs, which often causes pain to the patient.

[0003] Therefore, there is an urgent need for a gene therapy method that can both alleviate patient suffering and save costs for the prevention and treatment of lung and other diseases. Summary of the Invention

[0004] A key advantage of this invention is that it provides a recombinant vector that enables direct drug delivery to the human body via the mucous membrane, thus avoiding the need for obtaining live cells from the human body in traditional cell therapy. This reduces the suffering caused by existing gene therapy technologies and also allows for the modification of cells to have the function of treating diseases.

[0005] Another advantage of the present invention is that it provides a recombinant vector that can be administered directly through the mucous membrane of the body. Furthermore, the recombinant region of the recombinant vector is designed to include therapeutic genes, eliminating the need for in vitro transfection steps, thereby reducing the steps of in vitro cell preparation, simplifying the process of modifying human cells in existing gene therapy, and alleviating the suffering of patients caused by cell therapy in the prior art.

[0006] In one specific embodiment, the recombinant vector includes, but is not limited to, viral vectors and non-viral vectors. The viral vectors include, at least: retrovirus vector, lentivirus vector, adenovirus vector, and adeno-associated virus vector (AAV). The non-viral vectors include, at least: liposome vector, cationic polymer vector, and cationic polymer vector.

[0007] In one specific embodiment, the mucosa includes at least the digestive tract mucosa, respiratory tract mucosa, genitourinary tract mucosa, and conjunctiva. In another specific embodiment, the digestive tract mucosa includes at least the oral mucosa, esophageal mucosa, gastric mucosa, small intestinal mucosa, and large intestinal mucosa; the respiratory tract mucosa includes at least the nasal mucosa, tracheal and bronchial mucosa, and alveolar mucosa; the genitourinary tract mucosa includes at least the urethral mucosa, bladder mucosa, vaginal mucosa, uterine mucosa, and fallopian tube mucosa; and the conjunctiva includes at least the palpebral conjunctiva, bulbar conjunctiva, and fornix conjunctiva.

[0008] In one specific embodiment, the administration method of the recombinant carrier includes, but is not limited to, topical application, spray administration, nasal drop administration, suppository administration, and irrigation administration.

[0009] Another advantage of the present invention is that it provides a recombinant vector that can be directly and non-invasively introduced into the mucosal sites of humans or animals without the need for in vitro transfection, thereby reducing the steps of in vitro cell preparation and simplifying the complexity of gene therapy.

[0010] Another advantage of this invention is that it provides a mucosal-based drug delivery method, which enables the direct delivery of adenovirus to the mucosal site of an organism, thereby achieving the purpose of non-invasive gene therapy for the prevention and treatment of diseases.

[0011] Other advantages and features of the invention will be fully apparent from the following detailed description and may be achieved by combinations of the means and apparatus specifically pointed out in the appended claims.

[0012] This invention innovatively presents the following technical effects of gene editing in cells during gene therapy:

[0013] 1. The recombinant vector disclosed in this invention improves upon the shortcomings of previous gene therapies that required taking somatic cells from patients, and can deliver the drug in a non-invasive form such as nebulization;

[0014] 2. The recombinant vector disclosed in this invention also reduces the preparation cost of gene therapy drugs. In the preparation process of the drug, there is no somatic cell preparation step, which greatly saves time and money in drug preparation.

[0015] 3. Existing cell therapy techniques involve taking the patient's own cells and performing gene editing on these cells in vitro. This gene editing method means that each batch of drug can only be administered to one patient (the patient's somatic cells become a raw material for the drug, which can only be used to treat the patient's own disease). Due to the limitations of traditional autologous cell gene editing in vitro, only a single dose of drug can be prepared each time. However, the virus prepared in this application transfers the cell transfection step to the patient's body. In other words, the recombinant vector disclosed in this application enables the mass production of gene drugs and solves the problem of gene drugs for respiratory diseases, eye diseases, reproductive system diseases, urinary system diseases, and digestive system diseases being prepared only for one person.

[0016] The further objects and advantages of the invention will become fully apparent from the following description and accompanying drawings.

[0017] These and other objects, features and advantages of the present invention will be fully realized through the following detailed description, drawings and claims. Attached Figure Description

[0018] Figure 1 is a plasmid map according to a preferred embodiment of the present invention.

[0019] Figure 2 is a verification of the effect of the Ad-IL4 / 10 recombinant vector in delivering anti-inflammatory factors IL4 and IL10 according to a preferred embodiment of the present invention.

[0020] Figure 3 is a verification of the advantages of non-invasive drug delivery and survival characteristics of recombinant vectors according to a preferred embodiment of the present invention.

[0021] Figure 4 shows the results of verifying the anti-inflammatory and anti-fibrotic activity of Ad-IL4 / 10 according to a preferred embodiment of the present invention.

[0022] Figure 5 illustrates the preventive effect of pre-inhaled Ad-IL4 / 10 on pulmonary fibrosis according to a preferred embodiment of the present invention.

[0023] Figure 6 shows the verification results of Ad-IL4 / 10 disclosed in this application on controlling inflammation and promoting lung injury recovery according to a preferred embodiment of the present invention.

[0024] Figure 7 shows a recombinant vector according to a preferred embodiment of the present invention, verifying that the nebulized Ad-IL4 / 10 in mice can rescue LPS-induced infiltration of monocytes and neutrophils, as well as T lymphocyte depletion.

[0025] Figure 8 shows a recombinant vector according to a preferred embodiment of the present invention, which verifies that the nebulized Ad-IL4 / 10 in mice reduced LPS-induced recruitment of CD11cintCD11bhi monocytes-macrophages and restored CD11c+CD11blo alveolar macrophages. Detailed Implementation

[0026] The following description is intended to disclose the present invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.

[0027] Those skilled in the art will recognize that the various illustrative logical blocks (ILBs) and steps described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, or a combination of computer software and electronic hardware. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementations should not be considered beyond the scope of this invention.

[0028] A key advantage of this invention is that it provides a recombinant vector that enables direct drug delivery to the human body. Furthermore, the recombinant vector is designed to include a recombinant region comprising an exogenous therapeutic gene. The recombinant vector disclosed in this application allows for direct drug delivery to the mucous membranes of the body, enabling the therapeutic gene to be directly transferred into and expressed in the body's cells, thereby achieving gene therapy. This avoids the need for obtaining live cells from the human body for traditional cell therapy, reducing the pain caused by existing cell therapy technologies. Simultaneously, it also enables the modification of cells to express therapeutic genes, achieving the function of preventing and treating diseases.

[0029] In one specific embodiment, the therapeutic gene includes, but is not limited to, immune regulatory factor-related genes, growth factor-related genes, gene editing-related genes (for correcting pathogenic genes), and antimicrobial peptide genes.

[0030] Furthermore, the immunomodulatory factor-related gene can specifically be an interferon gene, such as the interferon-γ (IFN-γ) gene. Interferons have antiviral, antitumor, and immunomodulatory effects. Introducing the IFN-γ gene into the mucosa can enhance the local antiviral immune response. For some mucosal diseases caused by viral infections, such as genital herpes (caused by herpes simplex virus, mainly affecting the genital mucosa), introducing the interferon gene can activate immune cells such as natural killer cells and macrophages in the mucosal immune system, inhibiting viral replication and spread.

[0031] Furthermore, the immunomodulatory factor-related genes mentioned therein can specifically be interleukin genes, such as the interleukin-12 (IL-12) gene. IL-12 can promote the activation and proliferation of T cells and NK cells, enhancing cellular immunity. Introducing the IL-12 gene into the respiratory mucosa can improve the body's defense against respiratory viruses (such as influenza virus) and bacteria (such as Streptococcus pneumoniae), stimulate specific immune responses in mucosa-associated lymphoid tissue, and reduce pathogen colonization and infection in the respiratory mucosa.

[0032] Furthermore, the therapeutic gene can also be anti-inflammatory, such as a gene encoding an anti-inflammatory factor. The gene encoding the anti-inflammatory factor...

[0033] Furthermore, the immune-regulating factor-related genes mentioned therein can specifically be growth factor-related genes. For example, epidermal growth factor (EGF) gene: for some mucosal injury diseases, such as oral ulcers, introducing the EGF gene into the oral mucosa can promote the proliferation and migration of mucosal epithelial cells. EGF can bind to receptors on the surface of mucosal epithelial cells, activate intracellular signaling pathways, stimulate cell division and growth, and accelerate ulcer healing.

[0034] Furthermore, the immune-regulating factor-related gene can specifically be the fibroblast growth factor (FGF) gene: FGF plays an important role in angiogenesis and tissue repair. In cases of intestinal mucosal damage, such as intestinal mucosal damage caused by inflammatory bowel disease, the introduction of the FGF gene can promote the proliferation of fibroblasts in the submucosa of the intestine, helping to repair the damaged intestinal mucosal tissue structure, while also improving the blood supply to the intestinal mucosa and promoting the recovery of intestinal function.

[0035] Furthermore, the gene-editing-related gene (used to correct the pathogenic gene) can specifically be a CRISPR-Cas system-related gene: in some hereditary mucosal diseases, such as cystic fibrosis (CF), the pathogenic cause is a mutation in the cystic fibrosis transmembrane conduction regulator (CFTR) gene located in the mucosal epithelial cells. By introducing a CRISPR-Cas9 system-related gene, the mutated CFTR gene can be edited and corrected to the normal gene. This gene-editing technology can precisely locate and modify pathogenic genes, and holds promise for fundamentally treating mucosal diseases caused by gene mutations.

[0036] Furthermore, the antimicrobial peptide gene can specifically be a defensin gene: defensins are small molecule peptides with antimicrobial activity. Introducing defensin genes, such as the human β-defensin-2 (hBD-2) gene, into sites like the skin, mucous membranes, and gastrointestinal mucosa can enhance the antimicrobial capacity of the mucous membranes. hBD-2 can directly act on the bacterial cell membrane, increasing its permeability and leading to bacterial death. For mucous membrane sites prone to bacterial infection, such as the nasal mucosa (susceptible to bacteria like Staphylococcus aureus), introducing defensin genes can serve as a novel antimicrobial treatment strategy.

[0037] In one specific embodiment, the recombinant vector includes, but is not limited to, viral vectors and non-viral vectors. The viral vectors include, at least: retrovirus vectors, lentivirus vectors, adenovirus vectors, and adeno-associated virus vectors (AAV). The non-viral vectors include, at least: liposome vectors, cationic polymer vectors, cationic polymer vectors, viroids, or artificially designed vectors with similar virus delivery functions (e.g., MEFI systems).

[0038] In one specific embodiment, the retrovirus vector is an RNA virus capable of reverse transcribing its own RNA genome into DNA and integrating it into the host cell genome. This integration characteristic allows the target gene to be stably expressed in the host cell. Based on the method disclosed in this application, the sequence of anti-inflammatory factors is integrated into the retrovirus vector to achieve the production of the anti-inflammatory factors by cells at the drug administration site, thereby achieving the purpose of treating related diseases.

[0039] Furthermore, the retrovirus vector described herein has a relatively simple genome structure and a moderate gene capacity, generally capable of inserting 7-8kb of exogenous genes. Those skilled in the art can select appropriate anti-inflammatory factors and other necessary elements as needed to promote or restrict the expression of anti-inflammatory factors for treating the disease.

[0040] In one specific embodiment, the vector constructed by the retroviral vector can introduce the gene of anti-inflammatory factor into the patient's lymphocytes, causing these cells to produce more anti-inflammatory factors, thereby alleviating the patient's symptoms and achieving better prevention or treatment effects.

[0041] In one specific embodiment, the lymphocytes may be lymphocytes from the respiratory mucosa, which is the body's first line of defense against external pathogens. A large number of lymphocytes exist in the lamina propria of the nasal cavity, trachea, and bronchi. For example, in the tracheal and bronchial mucosa, lymphocytes work in conjunction with other immune cells (such as macrophages, dendritic cells, etc.). The recombinant vector disclosed in this application can better prevent / treat respiratory diseases.

[0042] In one specific embodiment, the lymphocytes may be lymphocytes from the digestive tract mucosa. The digestive tract mucosa contains abundant lymphoid tissue, called mucosa-associated lymphoid tissue (MALT). The most typical example is gut-associated lymphoid tissue (GALT), which includes structures such as Painley's aggregated lymph nodes and solitary lymphoid follicles. These lymphoid tissues are distributed in the lamina propria and submucosa of the small and large intestine mucosa. The vector constructed using the retroviral vector can introduce anti-inflammatory factor genes into the lymphocytes of the patient's digestive system, causing these cells to produce more anti-inflammatory factors, achieving non-invasive drug delivery, thereby alleviating the patient's symptoms and achieving better preventative or therapeutic effects.

[0043] In one specific embodiment, the lymphocytes may be lymphocytes from the urogenital tract mucosa. The urogenital tract mucosa also possesses immune defense functions and contains lymphocytes. In the urethral and bladder mucosa, lymphocytes can respond to the invasion of pathogens. For example, when a bacterial infection occurs in the urinary system (such as cystitis or urethritis), the lymphocytes in the mucosa are activated, generating an immune response to clear the bacteria. The vector constructed using the retroviral vector can introduce genes for anti-inflammatory factors into the lymphocytes of the patient's urinary system, causing these cells to produce more anti-inflammatory factors, achieving non-invasive drug delivery, thereby alleviating the patient's symptoms and achieving better preventative or therapeutic effects.

[0044] The vaginal and cervical mucosa contain lymphocytes in the female reproductive tract mucosa. Under normal circumstances, these lymphocytes maintain the immune balance within the vagina, preventing the overgrowth of harmful microorganisms. Vectors constructed based on the aforementioned retroviral vectors can introduce genes for anti-inflammatory factors into the lymphocytes of the patient's reproductive system, causing these cells to produce more anti-inflammatory factors to treat and prevent gynecological inflammation.

[0045] In one specific embodiment, the lymphocytes may be lymphocytes from the conjunctiva of the eye. The conjunctiva contains immune cells such as lymphocytes, which reside in the lamina propria of the conjunctiva. The conjunctiva is directly exposed to the external environment and is susceptible to invasion by pathogens. When pathogens (such as bacteria or viruses) infect the conjunctiva, lymphocytes participate in the immune response.

[0046] In one specific embodiment, the recombinant vector is a lentivirus vector belonging to the Retroviridae family, capable of infecting both dividing and non-dividing cells. This is a significant advantage over other retroviral vectors, as many somatic cells, such as neurons and muscle cells, are in a quiescent state in vivo. Based on the core of this application, designing the sequences of the anti-inflammatory factors into the lentivirus recombinant vector can provide non-invasive treatment for diseases related to the urinary, digestive, respiratory, ocular, and reproductive systems.

[0047] In one specific embodiment, the recombinant vector is an adenovirus vector, which is a double-stranded DNA virus capable of efficiently transducing various cell types, including respiratory epithelial cells and hepatocytes. It has high infection efficiency and can express large quantities of the target gene in a short time.

[0048] For example, it is widely used in gene therapy and vaccine development. In the development of COVID-19 vaccines, adenovirus vector vaccines are an important type. The antigen gene of the novel coronavirus is introduced into human cells via an adenovirus vector, enabling the cells to express the antigen protein, thereby stimulating the body to produce an immune response. In gene therapy research for diseases such as cystic fibrosis, adenovirus vectors have also been used to introduce the normal cystic fibrosis transmembrane transport regulator (CFTR) gene into respiratory epithelial cells to improve patient symptoms.

[0049] In one specific embodiment, the recombinant vector is an adeno-associated virus vector (AAV). AAV is a single-stranded DNA virus characterized by high safety because it is not pathogenic and typically does not elicit a significant immune response in humans. It can infect various cell types, including muscle cells and retinal cells.

[0050] Furthermore, the adeno-associated virus (AAV) vector has achieved significant results in gene therapy for ophthalmic diseases. In one specific embodiment, the adenovirus recombinant vector can be used for non-invasive treatment of hereditary retinal diseases, such as retinitis pigmentosa and Reber's congenital amaurosis. By introducing normal genes into retinal cells through the AAV vector, the patient's vision can be effectively improved. Based on the recombinant vector disclosed in this application, the therapeutic gene can be formulated into eye drops and directly applied to the affected area to achieve the purpose of gene therapy.

[0051] In one specific embodiment, the recombinant vector is a liposome vector. The liposome is a bilayer membrane vesicle structure composed of lipid components such as phospholipids. It has good biocompatibility because its composition is similar to that of the cell membrane, reducing toxicity to cells. It can encapsulate nucleic acid molecules of different sizes and introduce genes into cells through fusion with the cell membrane or endocytosis.

[0052] Furthermore, the preparation of the aforementioned lipid recombinant vector is relatively simple and easy to mass-produce. However, its transfection efficiency is relatively lower than that of viral vectors, and its stability in the in vivo environment may be poor.

[0053] In one specific embodiment, the recombinant vector is a cationic polymer vector. Furthermore, the cationic polymer can bind to negatively charged nucleic acid molecules via electrostatic interactions to form a complex, thereby protecting the nucleic acid from degradation by nucleases. It can enter cells (e.g., mucosal epithelial cells, mucosa-associated macrophages, dendritic cells, etc.) via endocytosis.

[0054] In one specific embodiment, the recombinant carrier can also be an inorganic nanoparticle vector, including various types such as gold nanoparticles, carbon nanotubes, and magnetic nanoparticles. These carriers have good stability and modifiability, and can be modified to connect target molecules, achieving targeted delivery to specific cells.

[0055] In one specific embodiment, the mucosa includes at least the digestive tract mucosa, respiratory tract mucosa, urogenital tract mucosa, and conjunctiva.

[0056] In one specific embodiment, the digestive tract mucosa includes the oral mucosa covering the inside of the oral cavity, including the lips, cheeks, tongue, palate, etc.

[0057] In one specific embodiment, the digestive tract mucosa includes esophageal mucosal epithelium which is non-keratinized stratified squamous epithelium.

[0058] In one specific embodiment, the digestive tract mucosa includes the gastric mucosa, which has a thick mucus-bicarbonate barrier on its surface, mainly composed of mucus and bicarbonate secreted by surface mucus cells. This barrier prevents gastric acid and pepsin from digesting the gastric mucosa itself. The gastric mucosal epithelium is a single layer of columnar epithelium with various cell types. For example, chief cells secrete pepsinogen; parietal cells secrete hydrochloric acid. These secretions participate in the initial digestion of proteins in food.

[0059] In one specific embodiment, the digestive tract mucosa includes the small intestinal mucosa, which has many annular folds and villi, greatly increasing the absorptive surface area of ​​the small intestine.

[0060] In one specific embodiment, the digestive tract mucosa includes the colonic mucosa, which has a relatively smooth surface and a large number of goblet cells.

[0061] In one specific embodiment, the respiratory mucosa, including the nasal mucosa, is composed of epithelium and lamina propria. The epithelium is pseudostratified ciliated columnar epithelium containing goblet cells. The mucus secreted by the goblet cells can trap foreign objects such as dust and bacteria from the air.

[0062] In one specific embodiment, the respiratory system mucosa, including the tracheal and bronchial mucosa, is also pseudostratified ciliated columnar epithelium. Its ciliary movement and mucus secretion mechanisms are similar to those of the nasal mucosa, effectively clearing foreign objects from the respiratory tract. The lamina propria contains numerous elastic fibers, allowing the trachea and bronchi to maintain appropriate elasticity during respiration. Furthermore, it contains immune cells such as lymphocytes and plasma cells, which participate in the body's immune defense; for example, when infected by pathogens, plasma cells can secrete antibodies to fight the pathogens.

[0063] In one specific embodiment, the respiratory mucosa includes alveoli, which are the primary site of gas exchange. The alveolar mucosa is composed of type I and type II alveolar cells. Type I alveolar cells are thin, facilitating gas exchange; type II alveolar cells secrete alveolar surfactant, reducing alveolar surface tension and preventing alveolar collapse. Simultaneously, the alveoli are surrounded by abundant capillaries, where oxygen and carbon dioxide are exchanged through diffusion.

[0064] In one specific embodiment, when the urethral mucosa is male, it is primarily composed of stratified squamous epithelium, connecting with structures such as the acini of the prostate gland in specific locations. Female urethral mucosa is also composed of stratified squamous epithelium. The urethral mucosa prevents the reflux of harmful substances in urine and their invasion into surrounding tissues.

[0065] In one specific embodiment, the bladder mucosa is transitional epithelium, which changes shape according to the bladder's filling state. When the bladder is empty, the mucosa has many folds; when the bladder is full, the folds disappear, and the mucosa stretches and thins.

[0066] In one specific embodiment, the reproductive system mucosa, including the female vaginal mucosa, is composed of stratified squamous epithelium, whose epithelial cell morphology and function change with factors such as the menstrual cycle and estrogen levels. The vaginal mucosa maintains a moist environment and has a certain self-cleaning function. Normal vaginal flora inhibits the growth of harmful bacteria, and the mucosa also secretes substances to maintain the vaginal pH balance.

[0067] In one specific embodiment, the reproductive system mucosa includes the uterine mucosa (endometrium), which is divided into a functional layer and a basal layer. The functional layer undergoes cyclical changes with the menstrual cycle, thickening and shedding under the influence of estrogen and progesterone to form menstruation. The endometrium is also the site of implantation for the fertilized egg. During pregnancy, the endometrium undergoes a series of changes to provide a favorable environment for embryonic development.

[0068] In one specific embodiment, the reproductive system mucosa, including the fallopian tube mucosa, is a single layer of columnar epithelium with numerous folds that aid in the retrieval and transport of the fertilized egg. The cilia movement of the mucosal epithelial cells propels the egg towards the uterus.

[0069] In one specific embodiment, the conjunctiva is divided into the palpebral conjunctiva (covering the inner surface of the eyelids), the bulbar conjunctiva (covering the front of the eyeball), and the fornix conjunctiva (located at the transition between the palpebral and bulbar conjunctiva). The conjunctiva consists of epithelium and the lamina propria. The epithelium is stratified columnar epithelium or pseudostratified columnar epithelium, containing goblet cells that secrete mucus to keep the surface of the eyeball moist. The lamina propria contains abundant blood vessels and lymphatic vessels, as well as immune cells such as lymphocytes, which protect the surface of the eyeball and prevent the invasion of foreign bodies and pathogens. When the conjunctiva is irritated or infected, such as by bacteria or viruses, it can cause conjunctivitis, resulting in symptoms such as conjunctival congestion and increased secretions.

[0070] This invention discloses a recombinant vector comprising a recombinant region, wherein the therapeutic gene in the recombinant region is designed to express an anti-inflammatory factor. The anti-inflammatory factors in the recombinant vector are preferably IL-4 and IL-10. The recombinant vector disclosed in this application can effectively prevent and / or treat respiratory, digestive, and urinary system diseases. The adenovirus delivery method disclosed in this application achieves non-invasive gene therapy, significantly reducing production costs.

[0071] In one specific embodiment, the disease includes gastrointestinal mucosal inflammation-related lesions, such as gastric ulcers: primarily chronic ulcers formed after the gastric mucosa is digested by gastric acid and pepsin. Helicobacter pylori (Hp) infection is one of the most common causes. Hp, with its spiral structure, easily penetrates the gastric mucosal layer, triggering an inflammatory response. Inflammatory cell infiltration leads to damage to the gastric mucosal barrier, and gastric acid and pepsin further erode the submucosa and muscularis propria, forming ulcers. Patients experience periodic, rhythmic upper abdominal pain, possibly accompanied by symptoms such as acid reflux, belching, nausea, and vomiting. Optionally, those skilled in the art can select anti-inflammatory genes as needed. For example, duodenal ulcers: usually occur in the duodenal bulb. This is mainly due to excessive gastric acid secretion and Helicobacter pylori infection, which disrupts the defense mechanism of the duodenal mucosa. Based on the recombinant vector disclosed in this application, genes encoding intestinal mucosal repair are designed into the recombinant vector, enabling the body to self-repair and alleviate the pain caused by mucosal damage due to inflammatory stimulation. The pain is often characterized by pain on an empty stomach, which is relieved after eating. For example, inflammatory bowel disease (IBD): This is an idiopathic inflammatory bowel disease, mainly including ulcerative colitis (UC) and Crohn's disease (CD). For example, ulcerative colitis: The lesions primarily affect the rectal and colonic mucosa. In the early stages of inflammation, the intestinal mucosa becomes congested and edematous. As the disease progresses, ulcers form on the mucosal surface, leading to symptoms such as mucus and bloody stools, abdominal pain, and diarrhea. Persistent inflammation causes repeated damage and repair of the intestinal mucosa, resulting in polypoid hyperplasia and increasing the risk of colon cancer. For example, Crohn's disease: It can affect the entire digestive tract, but mainly the terminal ileum and colon. It is a transmural inflammation, affecting not only the mucosal layer but also the submucosa, muscularis propria, and even the serosa. The intestinal mucosa may exhibit longitudinal ulcers and cobblestone-like changes. Based on the recombinant vector disclosed in this application, therapeutic genes can be designed as anti-inflammatory factors, which can be administered to the affected area via suppositories or other forms to prevent / improve symptoms such as abdominal pain, diarrhea, and weight loss. It may also lead to intestinal obstruction due to intestinal wall fibrosis. For example, acute gastroenteritis is often caused by viral (such as rotavirus and norovirus), bacterial (such as Salmonella and Escherichia coli) or parasitic infections. These pathogens infect the gastrointestinal mucosa, causing an acute inflammatory response. Patients may experience symptoms such as nausea, vomiting, abdominal pain, and diarrhea; severe cases may lead to dehydration and electrolyte imbalance. Those skilled in the art can incorporate suitable anti-inflammatory genes as therapeutic genes into this recombinant vector as needed, and then administer the medication via suppositories or irrigation.

[0072] In one specific embodiment, the diseases mentioned include respiratory mucosal inflammation-related lesions, such as the common cold: primarily caused by viral infections such as rhinovirus and coronavirus, resulting in upper respiratory tract mucosal inflammation. After viral infection, the nasal mucosa is initially stimulated, becoming congested and edematous, producing a large amount of secretions. Patients experience symptoms such as nasal congestion, runny nose, sneezing, and sore throat. For example, influenza: an acute respiratory infectious disease caused by the influenza virus. The influenza virus attacks the respiratory mucosa, triggering a strong inflammatory response. In addition to common cold symptoms, it may also be accompanied by systemic symptoms such as high fever, headache, body aches, and fatigue. Because the influenza virus is highly pathogenic, it may lead to more serious complications, such as pneumonia. For example, acute bronchitis: often caused by viral or bacterial infections, but may also be caused by physical or chemical irritants. Under inflammatory stimulation, the bronchial mucosa becomes congested and edematous, and ciliary movement is weakened. Patients experience symptoms such as cough and sputum production, initially a dry cough, which progresses to mucoid or purulent sputum. For example, pneumonia: Although pneumonia is primarily an inflammation of the lung parenchyma, the inflammation often begins in the respiratory mucosa. For instance, bacterial pneumonia occurs when bacteria colonize the respiratory mucosa, breach the mucosal barrier, and enter the alveoli, causing inflammation. Patients may experience symptoms such as fever, cough, sputum production, chest pain, and difficulty breathing. Those skilled in the art can incorporate suitable anti-inflammatory genes or interferons as therapeutic genes into this recombinant vector as needed, and then administer the medication via methods such as nebulized inhalation. Furthermore, the respiratory diseases mentioned also include diseases such as pulmonary fibrosis. The therapeutic genes in the recombinant region of the recombinant vector disclosed in this application are designed as anti-inflammatory factors such as IL-4 and IL-10, thereby achieving the purpose of disease prevention / treatment.

[0073] In one specific embodiment, the disease includes inflammatory lesions related to the mucosa of the genitourinary tract. For example, urethritis: primarily an inflammation of the urethral mucosa, which can be divided into gonococcal urethritis and non-gonococcal urethritis. Gonococcal urethritis is caused by infection with Neisseria gonorrhoeae, while non-gonococcal urethritis is mainly caused by infections such as Chlamydia trachomatis and Ureaplasma urealyticum. In an inflammatory state, the urethral mucosa is congested and edematous, and patients will experience urinary tract irritation symptoms such as urinary frequency, urgency, and dysuria; purulent discharge may also be present at the urethral opening. For example, cystitis: usually caused by bacterial infection of the bladder mucosa (such as Escherichia coli). Under inflammatory stimulation, the bladder mucosa becomes more sensitive, resulting in bladder irritation symptoms such as urinary frequency, urgency, and dysuria, and may also be accompanied by hematuria. Severe cystitis may lead to ulceration and bleeding of the bladder mucosa. For example, vaginitis: there are various types, such as bacterial vaginosis, fungal vaginitis, and trichomonal vaginitis. For example, bacterial vaginosis is an inflammation caused by an imbalance of the normal vaginal flora and the overgrowth of anaerobic bacteria such as Gardnerella vaginalis. The vaginal mucosa will become congested and edematous, with increased discharge and a fishy odor. For example, yeast vaginitis is mainly caused by Candida albicans infection. The vaginal mucosa is red and swollen, with white, cottage cheese-like discharge, and the patient will experience vulvar itching and burning. For example, trichomonal vaginitis is caused by Trichomonas vaginalis infection. The vaginal mucosa shows scattered bleeding points, and the discharge is thin, purulent, and frothy, accompanied by vulvar itching. For example, cervicitis is mostly caused by pathogens infecting the cervical mucosa, such as Neisseria gonorrhoeae and Chlamydia trachomatis. In an inflammatory state, the cervical mucosa will become red, swollen, and prone to bleeding; the patient may experience increased vaginal discharge and postcoital bleeding. Those skilled in the art can, as needed, incorporate suitable anti-inflammatory genes or interferons as therapeutic genes into this recombinant vector, and then administer the medication via suppositories or irrigation methods.

[0074] In a preferred embodiment, the recombinant vector has a recombinant region encoding at least one anti-inflammatory factor; the recombinant vector is administered to the respiratory system via nebulization through the mouth or nose for the prevention and / or treatment of respiratory diseases. The anti-inflammatory factors of the recombinant vector are preferably IL-4 and IL-10. The recombinant vector disclosed in this application can effectively prevent respiratory diseases, and the adenovirus delivery method disclosed in this application achieves non-invasive gene therapy, significantly reducing production costs. This invention discloses a recombinant vector comprising a recombinant region designed to express a therapeutic gene.

[0075] In addition to the therapeutic gene, the recombinant region further includes a first expression control element, a target protein, and a tag. Furthermore, the recombinant region may also include a second expression control element and the structure of a fluorescent protein, more specifically, the fluorescent protein being used to track the recombinant adenovirus disclosed in this application.

[0076] Furthermore, the first expression control element is used to control the expression of the therapeutic gene, wherein the second expression control element is used to control the expression of the fluorescent protein.

[0077] More specifically, both the first expression control element and the second expression control element may include: a promoter and a gene that enhances expression.

[0078] More specifically, the therapeutic gene includes at least one coding sequence for expressing an anti-inflammatory factor. When the therapeutic gene includes coding sequences for two or more anti-inflammatory factors, the coding sequences of the plurality of anti-inflammatory factors are tandemly linked by a linker structure. More specifically, the coding sequences of the plurality of anti-inflammatory factors may also include expression-enhancing elements for enhancing the expression of the anti-inflammatory factors.

[0079] Furthermore, the anti-inflammatory factors include, but are not limited to, IL-4, IL-6, IL-10, IL-11, and IL-13. More specifically, the anti-inflammatory factor is preferably IL-4 or IL-10.

[0080] Furthermore, the fluorescent protein used to track the recombinant vector can be at least one of EGFP and Nluc.

[0081] Furthermore, the adenovirus disclosed in this application is preferably at least one of ad5, ad35, and ad50.

[0082] The technical solution described in this invention also provides a non-invasive gene therapy. The gene therapy involves applying the recombinant vector disclosed in this application to the affected area.

[0083] In one specific embodiment, the diseases are colds, influenza, pneumonia, tuberculosis, chronic obstructive pulmonary disease, pulmonary fibrosis, bronchial asthma, pharyngitis, rhinitis, tonsillitis, gastritis, gastric ulcer, gastric cancer, esophagitis, enteritis, irritable bowel syndrome, colorectal cancer, hepatitis, cirrhosis, liver cancer, prostatitis, benign prostatic hyperplasia, epididymitis, vaginitis, cervicitis, uterine fibroids, urethritis, cystitis, kidney stones, conjunctivitis, and dry eye.

[0084] In one specific embodiment, the recombinant vector is used in the prevention and / or treatment of respiratory diseases, wherein the recombinant region of the recombinant vector is the sequence of SEQ ID No. 1.

[0085] To verify the effectiveness of the technical solution disclosed in this application, the inventors selected the recombinant vector disclosed in this application for adenovirus atomization to administer the drug to the respiratory system.

[0086] The inventors constructed the plasmid shown in Figure 1. Furthermore, the recombination region of the recombinant vector is part of Figure 2A.

[0087] We use AdMax TM A recombinant adenovirus plasmid, Ad-IL4 / 10, was successfully constructed. This plasmid stably expresses Nluc and EGFP for easy tracking, as shown in Figure 1A. To verify that IL4 and IL10 can be expressed in cells transfected with the Ad-IL4 / 10 recombinant adenovirus plasmid, we used Western blotting or ELISA to detect the supernatant and lysis buffer of the transfected 293FT cells. The results are shown in Figures 2B to 3C. Furthermore, RAW264.7 cells were infected with the Ad-IL4 / 10 recombinant adenovirus plasmid at different MOIs. Nluc and EGFP were detected by flow cytometry and fluorescence. The expression of Nluc and EGFP in the infected cells was higher than that in the control, as shown in Figures 2D to F. Further, C57BL / 6 mice were inhaled with the Ad-IL4 / 10 adenovirus vector (1x10⁹ IFU / mouse), and the fluorescent expression of EGFP and Nluc in heart, liver, and lung tissues was detected after inhalation at different times (detected at 2 / 5 / 7 / 12 days). Compared with control mice, mice inhaling Ad-IL4 / 10 exhibited stronger bilateral lung lobes bioluminescent signals in in vivo imaging, which persisted until day 12 post-inhalation (Figures 2G to 2I). Finally, lung tissue was collected at four time points, and ELISA analysis revealed significantly increased expression levels of IL-4 and IL-10 in lung tissue after Ad-IL4 / 10 inhalation (Figures 1J and 1K). These data validate the effectiveness of the Ad-IL4 / 10 recombinant vector disclosed in this study in delivering the anti-inflammatory factors IL-4 and IL-10.

[0088] Furthermore, the Ad-IL4 / 10 recombinant carrier inhalant disclosed in this application exhibits good delivery and survival characteristics in the lungs. To verify its biological effects, the researchers designed a series of experiments (see Figure 3A for a schematic diagram of the experimental design). ELISA results showed that the expression levels of IL-4 and IL-10 in mouse lung homogenate were significantly increased (Figures 3B and 3C). In addition, bioluminescence imaging of mouse lung tissue (Figure 3D) showed the luminescence intensity (in [p / sec / cm]). 2 The enhancement of / sr] was confirmed by quantitative analysis (Figures 3E and 3F).

[0089] To further evaluate the role of this recombinant vector in preventing lung injury, additional experiments were conducted. Results showed that prophylactic inhalation of Ad-IL4 / 10 significantly reduced LPS-induced weight loss in mice (Figure 4A) and decreased the wet / dry ratio of lung tissue in an acute lung injury (ALI) model (Figure 4B). Furthermore, compared to mice pretreated with Ad-Con, Ad-IL4 / 10 pretreatment suppressed the increase in total protein levels in BALF, although this difference was not statistically significant (Figure 4C). HE-stained lung tissue section analysis revealed significant inflammatory infiltration and alveolar structural damage in the lung tissue of mice in the PBS+LPS group, while Ad-IL4 / 10 pretreatment significantly alleviated the inflammatory response and reduced alveolar structural damage (Figures 4D, 4E). Masson staining results showed that pre-inhalation of Ad-IL4 / 10 reduced the degree of LPS-induced pulmonary fibrosis and accelerated tissue repair (Figures 4F, 4G). These results indicate that Ad-IL4 / 10 possesses significant anti-inflammatory and anti-fibrotic activities. Further analysis of the ELISA results (Figures 5A-F) showed that prophylactic inhalation of Ad-IL4 / 10 reduced the levels of airway inflammatory markers (TNF-α, IL-6, and IL-1β) and fibrosis-related factors (TGF-β1, PDGF, and BMP7) in LPS-induced lung homogenate and BALF (Figures 5G and 5H). This result validates the effectiveness of the recombinant vector Ad-IL4 / 10 disclosed in this application in preventing LPS-induced inflammation and fibrosis, further supporting its application in the prevention and treatment of pulmonary fibrosis. Furthermore, since LPS-induced ALI is closely related to inflammatory cell infiltration, we further analyzed the effects of Ad-IL4 / 10 inhalation on LPS-induced inflammatory cells in lung tissue and BALF. Flow cytometry analysis (Figure 6) showed that during LPS-induced ALI, CD11b+Ly6C+ monocytes, CD11b+Ly6G+ neutrophils, and CD3+ T cells were significantly increased, while Ad-IL4 / 10 inhalation effectively inhibited the infiltration of these inflammatory cells, thereby controlling inflammation and promoting lung injury repair. Furthermore, Figure 7 further confirmed that Ad-IL4 / 10 inhalation reduced LPS-induced monocyte and neutrophil infiltration while restoring T lymphocyte levels. Finally, Figure 8 showed that prophylactic inhalation of Ad-IL4 / 10 reduced LPS-induced CD11cintCD11bhi monocyte-macrophage recruitment and restored normal levels of CD11c+CD11blo alveolar macrophages. This finding further supports the reparative capacity of Ad-IL4 / 10 in regulating lung inflammation and repairing lung injury. In summary, based on the recombinant adenovirus vector disclosed in this application, by designing appropriate anti-inflammatory factors into the therapeutic gene, the prevention of pulmonary fibrosis has been achieved, effective control has been verified, and lung damage has been restored.

[0090] The corresponding structures, actions, behaviors, and equivalent forms of all device elements or step elements in this disclosure are intended to include any structure, action, or behavior for performing functions in conjunction with other claimed components, such as those specifically claimed. Various preferred embodiments of this disclosure have been described for disclosure purposes, intended to cover various variations and equivalent arrangements, and not as an exhaustive or limiting approach. The terminology used herein has been chosen to best explain the principles of the embodiments, the practical application of technology superior to that found in the market, or technical improvements, and the objectives of the invention have been fully and effectively achieved. The functional and structural principles of the invention have been shown and illustrated in the embodiments, and any variations or modifications may be made to the implementation of the invention without departing from these principles.

Claims

1. A recombinant vector based mucosal non-invasive gene therapy, characterized in that, The recombinant vector includes a recombinant region, wherein the recombinant region is designed to include a therapeutic gene; The recombinant vector is administered via a mucosal membrane; The recombinant vector is used to transfect mucosal cells of the organism, causing the mucosal cells to express the therapeutic gene, thereby preventing / treating the disease.

2. The recombinant vector based mucosal non-invasive gene therapy of claim 1, wherein, The therapeutic gene is at least one or any combination of immune regulatory factor-related genes, growth factor-related genes, gene editing-related genes (used to correct pathogenic genes), antimicrobial peptide genes, and anti-inflammatory factor genes.

3. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 2, characterized in that, The immunomodulatory factor-related genes include interferon, growth factor, and interleukin.

4. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 2, characterized in that, The anti-inflammatory factors include at least one of IL-10 and IL-4.

5. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 1, characterized in that, The recombinant vector is selected from retrovirus vector, lentivirus vector, adenovirus vector, and adeno-associated virus vector (AAV), wherein the non-viral vector includes at least one of liposome vector, cationic polymer vector, and cationic polymer vector.

6. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 1, characterized in that, When the recombinant vector is an adenovirus, the adenovirus may be selected from at least one of ad5, ad35, and ad50.

7. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 2, characterized in that, The anti-inflammatory factors also include IL-6, IL-11, and IL-13.

8. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 1, characterized in that, The mucosa includes at least the digestive tract mucosa, respiratory tract mucosa, urogenital tract mucosa, and conjunctiva of the eye; The digestive tract mucosa mentioned therein includes at least the oral mucosa, esophageal mucosa, gastric mucosa, small intestinal mucosa, and large intestinal mucosa; The respiratory mucosa mentioned above includes at least the nasal mucosa, tracheal and bronchial mucosa, and alveolar mucosa; The urogenital tract mucosa includes at least the urethral mucosa, bladder mucosa, vaginal mucosa, uterine mucosa, and fallopian tube mucosa; The conjunctiva mentioned therein includes at least the palpebral conjunctiva, bulbar conjunctiva, and fornix conjunctiva.

9. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 1, characterized in that, The administration methods of the recombinant vector include, but are not limited to, topical application, spray administration, nasal drop administration, suppository administration, and irrigation administration.

10. The recombinant vector-based non-invasive gene therapy based on mucosa according to claim 1, characterized in that, The recombination region of the recombinant vector also includes the coding sequence of a fluorescent protein; wherein the fluorescent protein of the recombinant vector is EGFP and Nluc.

11. The application of recombinant vectors in the prevention and / or treatment of diseases, characterized in that, The recombinant vector is the recombinant vector of claims 1-10, and the diseases are colds, influenza, pneumonia, tuberculosis, chronic obstructive pulmonary disease, pulmonary fibrosis, bronchial asthma, pharyngitis, rhinitis, tonsillitis, gastritis, gastric ulcer, gastric cancer, esophagitis, enteritis, irritable bowel syndrome, colorectal cancer, hepatitis, cirrhosis, liver cancer, prostatitis, benign prostatic hyperplasia, epididymitis, vaginitis, cervicitis, uterine fibroids, urethritis, cystitis, kidney stones, conjunctivitis, and dry eye.

12. The use of a recombinant vector in the prevention and / or treatment of respiratory diseases, wherein the recombinant vector is the recombinant vector of claim 11.

13. The use of the recombinant vector according to claim 12 in the prevention and / or treatment of respiratory diseases, wherein the recombinant region of the recombinant vector is the sequence of SEQ ID No. 1.

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