Composition for preventing or treating inflammatory bowel disease

Abstract

The present disclosure relates to a composition for preventing or treating inflammatory bowel disease, and more particularly, to a technology capable of preventing or treating inflammatory bowel disease by administering a composition comprising a vector into which one or both of the Nurr1 and Foxa2 genes are introduced.

Description

COMPOSITION FOR PREVENTING OR TREATING INFLAMMATORY BOWEL DISEASE

[0001] The present disclosure claims priority to Korean Patent Application No. 10-2024-0053327, titled "Composition for Preventing or Treating Inflammatory Bowel Disease," filed with the Korean Intellectual Property Office on April 22, 2024, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to a composition for preventing or treating inflammatory bowel disease and, more particularly, to a technology capable of preventing or treating inflammatory bowel disease by administering a composition including a vector having Nurr1 and Foxa2 genes introduced thereinto.

[0003] Inflammatory bowel disease (IBD) is an idiopathic autoimmune disease characterized by abnormal chronic inflammation in the gastrointestinal tract that repeatedly undergoes remission and relapse. The major types of IBD are ulcerative colitis and Crohn's disease, and the exact pathogenesis has not yet been clearly elucidated. IBD is known for its high recurrence rate and chronic symptoms such as diarrhea, abdominal pain, bloody stool, and weight loss. A characteristic feature of IBD is fibrosis of the intestinal mucosa due to inflammation. When intestinal fibrosis occurs, the intestines become shortened and hardened. As a result, nutrient absorption is delayed due to the altered intestinal tissue, and in severe cases, intestinal stricture may occur.

[0004] Crohn's disease is an idiopathic inflammatory disease that can affect any part of the digestive tract, from the mouth to the anus. It is accompanied by symptoms such as abdominal pain, diarrhea, and loss of appetite, which lead to weight loss, with repeated cycles of remission and exacerbation. Complications such as intestinal stricture, fistulas, and abscesses may also occur. Ulcerative colitis has no clearly identified cause. It is thought to result from a complex interaction of genetic predisposition, intestinal microbiota, and immunological abnormalities in the colonic wall. It is characterized by chronic inflammation in the mucosal and submucosal layers of the colon, along with repeated remission and flare-ups. The lesions appear continuously along the colon, and symptoms include abdominal pain, diarrhea, vomiting, and bloody stools containing pus and mucus.

[0005] For the treatment of IBD, classical therapeutic agents such as 5-aminosalicylic acid (5-ASA) that blocks the production of prostaglandins, which are inflammation-inducing substances, as well as corticosteroids and immunomodulators, have been used. In recent years, however, cytokines associated with IBD have been identified, leading to the development of biologics that selectively modulate specific molecules or pathways related to intestinal inflammation. Representative biologics include infliximab, a TNF-α inhibitor; ustekinumab, which targets IL-12 and IL-23; and vedolizumab, which inhibits integrin α4β7. More recently, darvadstrocel, an allogeneic adipose-derived stem cell therapy, has been approved for the treatment of perianal fistulas in patients with mild Crohn's disease.

[0006] Nuclear receptor related 1 protein (Nurr1), also known as NR4A2 (Nuclear Receptor Subfamily 4 Group A Member 2), is a transcription factor (TF) belonging to the nuclear receptor (NR) family. It acts as an epigenetic factor that promotes local chromatin opening, facilitating access and subsequent binding by other transcription factors (TFs).

[0007] The Nurr1 gene encodes an orphan nuclear receptor characterized as a key transcription factor involved in the initial development of midbrain dopamine (mDA) neurons, including their differentiation, maturation, and axonal pathfinding. Nurr1 continues to be expressed in mature mDA neurons, and deletion of the gene during the maturation stage leads to gradual loss of mDA neurons. In Nurr1 heterozygous mice, mDA neurons are more vulnerable to dopaminergic (DA) neurotoxins. Nurr1 levels are reduced in mDA neurons in elderly individuals and patients with Parkinson's disease, and mutations or polymorphisms that reduce Nurr1 expression are known to be associated with both familial and sporadic forms of Parkinson's disease.

[0008] Foxa transcription factors belong to a subfamily of forkhead transcription factors, which share high homology in the winged-helix DNA-binding domain. Over 170 members of the Fox family have been identified to date. Initially discovered for their DNA-binding ability in hepatic nuclear extracts of mice, they were originally referred to as hepatocyte nuclear factor 3 (HNF3). Foxa family proteins can remodel nucleosomes and function as pioneering factors to facilitate the DNA binding of other TFs. In mammals, the Foxa subfamily comprises Foxa1 (HNF-3α), Foxa2 (HNF-3β), and Foxa3 (HNF-3γ) (Li et al., 2017).

[0009] Foxa family members play important roles throughout various stages of mammalian life, from early development to organogenesis and adult metabolism and homeostasis. Foxa1 and Foxa2 cooperate to establish the competence of the anterior endoderm and are necessary for the normal development of endoderm-derived organs such as the liver, pancreas, lungs, and prostate. In particular, Foxa2 (HNF-3β) is first expressed in the ventral neural plate in the central nervous system (CNS), followed by expression in the floor plate of the neural tube and broad expression in the ventral midbrain.

[0010] Foxa2 works in concert with Nurr1 to promote the transcription of genes not only related to the dopaminergic phenotype but also specific to midbrain and substantia nigra (A9) dopaminergic neurons (Lee HS, Bae EJ, Yi SH, Shim JW, Jo AY, Kang JS, Yoon EH, Rhee YH, Park CH, Koh HC, Kim HJ, Choi HS, Han JW, Lee YS, Kim J, Li JY, Brundin P, Lee SH. Foxa2 and Nurr1 synergistically yield A9 nigral dopamine neurons exhibiting improved differentiation, function, and cell survival. Stem Cells. 2010 Mar 31;28(3):501-12). Nurr1 belongs to the family of steroid nuclear hormone receptors whose activity is primarily regulated by coregulators in the form of either corepressors (NCoR) or coactivators (NCoA) (Xu et al., 1999). This suggests that Nurr1 alone may be insufficient to achieve therapeutic effects unless a coactivator is also present. Foxa2 works in concert with Nurr1 to promote the transcription of genes not only related to the dopaminergic phenotype but also specific to midbrain and substantia nigra (A9) dopaminergic neurons. In addition, Foxa2 serves as a potent coactivator of Nurr1 for the generation of mDA neurons and midbrain development. The coexpression of Nurr1 and Foxa2 has been investigated as a mechanism to protect mDA neurons in the adult midbrain and has shown potential for the development of treatments for Alzheimer's disease (Oh SM, Chang MY, Song JJ, et al.EMBO Mol Med. 2015;7(5):510-25).

[0011] However, a composition for preventing or treating inflammatory bowel disease using Nurr1 and Foxa2 has not yet been reported.

[0012] Leading to the present disclosure, intensive and thorough research conducted by the present inventors resulted in the finding that when after a C57BL / 6J mouse model treated with dextran sulfate sodium (DSS), a well-established model of inflammatory bowel disease, was administered, the effects were calculated in terms of disease activity index (DAI) scores were calculated on the basis of on body weight change, stool consistency and occult blood, and rectal bleeding after administering Nurr1 and Foxa2 gene therapeutics to a C57BL / 6J mouse model treated with dextran sulfate sodium (DSS), which is a well-established model of inflammatory bowel disease, statistically significant efficacy was confirmed.

[0013] Accordingly, the present disclosure aims to provide a pharmaceutical composition for preventing or treating inflammatory bowel disease, the composition comprising a vector into which one or both Nurr1 and Foxa2 genes introduced thereinto.

[0014] To achieve the goal, the present disclosure provides a pharmaceutical composition for preventing or treating inflammatory bowel disease, the composition comprising a vector into which one or both Nurr1 and Foxa2 genes introduced thereinto.

[0015] The composition for preventing or treating inflammatory bowel disease, including a vector having Nurr1 and Foxa2 genes introduced thereinto, according to the present disclosure, can reduce diarrhea, a representative symptom of inflammatory bowel disease, improve stool consistency, and reduce rectal bleeding. In addition, administration of the composition to subjects induced with colitis resulted in the inhibition of colon length shortening. Therefore, the composition of the present disclosure can be advantageously used as an active ingredient for the prevention and treatment of inflammatory bowel disease.

[0016] FIG. 1A is a plot showing the disease activity index (DAI) of compositions (G3 and G4) containing AAV9-Nurr1 and AAV9-Foxa2 (Code name: IPS103) according to an embodiment of the present disclosure, compared with that of the negative control group (G2) (*,p<0.05; **,p<0.01; ****,p<0.0001).

[0017] FIG. 1B is a plot showing the disease activity index (DAI) of compositions (G3 and G4) containing AAV9-Nurr1 and AAV9-Foxa2 (Code name: IPS103) according to an embodiment of the present disclosure, compared with that of the untreated group (G1) (*,p<0.05; **,p<0.01; ****,p<0.0001).

[0018] FIG. 2 shows photographic images comparing the lengths of colon tissues, from the cecum to the rectum, excised from each experimental group according to an embodiment of the present disclosure.

[0019] FIG. 3 shows plots comparing the lengths of colon tissues, from the cecum to the rectum, excised according to an embodiment of the present disclosure, with those of the untreated group (G1) and the negative control group (G2) (**,p<0.01; ****,p<0.0001).

[0020] A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a vector into which one or both of Nurr1 and Foxa2 genes are introduced.

[0021] Definitions of terms used in the present disclosure are as follows.

[0022] As used herein, the term "vector" refers to a tool used for delivering genes. It can be considered analogous to a carrier for a drug. Therefore, in addition to DNA vectors composed of the same nucleic acid molecule as the transgene to be introduced, such as general plasmid vectors, the term "vector" in a broad sense also encompasses reagents used in DNA transfection, such as nanoparticles made of silica or gold; phospholipid-based structures including liposomes, extracellular vesicles, and exosomes; cationic polymers, e.g., chitosan, polyethyleneimine (PEI), and polylysine; and calcium phosphate.

[0023] As used herein, the term "subject" refers to a vertebrate animal that is the target of treatment, observation, or experimentation, preferably mammals such as cattle, pigs, horses, goats, dogs, cats, rats, mice, rabbits, guinea pigs, or humans.

[0024] As used herein, the term "prevention" refers to any action that suppresses or delays the onset of inflammatory bowel disease through the administration of the pharmaceutical composition of the present disclosure.

[0025] As used herein, the term "treatment" refers to an approach that ameliorates or alleviates the symptoms of inflammatory bowel disease, or achieves beneficial or desirable clinical outcomes through administration of the pharmaceutical composition of the present disclosure. For the purpose of the present disclosure, beneficial or desirable clinical outcomes include, without limitation: alleviation of inflammatory bowel disease symptoms; reduction in the extent of the disease; stabilization of the condition of inflammatory bowel disease (i.e., no worsening); delay or slowing of disease progression; and palliation or temporary relief and mitigation of the condition, either partially or completely, regardless of whether the disease is detectable. Additionally, "treatment" may refer to increased survival compared to the expected survival without treatment, a reduction in the extent of intestinal resection (e.g., colon or rectum), or reduced frequency of hospital visits or shorter hospitalization periods compared to the absence of treatment. The term "treatment" encompasses both therapeutic and prophylactic or preventive approaches. These treatments may address not only disorders being prevented but also those already present and requiring therapeutic intervention.

[0026] As used herein, the term "effective amount" refers to an amount sufficient to delay the onset or progression of inflammatory bowel disease, or to alleviate or halt the symptoms of the disease. The composition of the present disclosure may be administered in a pharmaceutically effective amount. It is apparent to those skilled in the art that an appropriate total daily dosage can be determined by a practitioner within the scope of proper medical judgment.

[0027] For the purposes of the present disclosure, a specific therapeutically effective amount for a given subject or patient may vary depending on various factors such as the type and extent of the desired response, whether other agents are used in combination, the specific composition, the age, weight, general health, sex, and diet of the subject (patient), the timing and route of administration, the rate of secretion of the composition, the treatment duration, and any other drugs used concurrently or in combination, along with other well-known and relevant factors in the pharmaceutical field.

[0028] Unless otherwise defined, all technical terms used in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Although preferred methods and materials are described herein, similar or equivalent methods and materials can also be used and are considered to fall within the scope of the present disclosure. All publications cited as references in the present specification are incorporated herein by reference in their entirety.

[0029] Hereinafter, the present disclosure will be described in detail.

[0030] According to an aspect thereof, the present disclosure relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a vector into which one or both of Nurr1 and Foxa2 genes introduced thereinto.

[0031] According to an aspect thereof, the present disclosure relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a vector into which Nurr1 and Foxa2 genes introduced thereinto.

[0032] In the method of the present disclosure, the phrase "introducing Nurr1 and Foxa2" refers to transfecting nucleic acids of the both genes into cells. The two genes may be introduced either separately or simultaneously. To introduce the genes encoding Nurr1 and Foxa2 into cells (or tissues), gene delivery methods known in the art may be employed, such as DNA-calcium precipitation using plasmids, liposome-mediated delivery, polyamine-based methods, electroporation, retroviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, and others.

[0033] Gene editing technology may also be used for the introduction of Nurr1 and Foxa2. Gene editing refers to the process of freely modifying the genetic information of a living organism to express a desired genetic trait. Systems that may be used for gene editing include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (CRISPR / Cas9) systems.

[0034] As used herein, the term "RNA-guided nuclease" refers to a nuclease capable of recognizing and cleaving a specific site in the genome via guide RNA. While not limited thereto, the RNA-guided nuclease may specifically be a Cas protein derived from the microbial immune system CRISPR, and more specifically may include the Cas9 (CRISPR-associated protein 9) nuclease and its variants such as Cas9 nickase.

[0035] As used herein, the term "Cas protein" refers to a key protein component of the CRISPR / Cas system that can function as an active endonuclease. The Cas protein may form a complex with crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) to exert its activity.

[0036] The Cas9 nuclease recognizes a specific sequence in the genome of plant and animal cells, including human cells, and induces double-strand breaks (DSBs). The DSB includes both blunt ends and cohesive ends created by cutting the double-stranded DNA. Such DSBs are efficiently repaired in cells through homologous recombination (HR) or non-homologous end-joining (NHEJ), during which desired mutations can be introduced at the target site. The RNA-guided nuclease may be artificially or synthetically produced, and may be non-naturally occurring.

[0037] The Cas9 nickase includes at least one mutation in one of the catalytic domains of the Cas9 nuclease. Such mutations may be selected from the group consisting of D10A, E762A, and D986A in the RuvC domain, or from the group consisting of H840A, N854A, and N863A in the HNH domain. Unlike the Cas9 nuclease, Cas9 nickase induces single-strand breaks. Therefore, two guide RNAs are required for the Cas9 nickase to function as a pair. Each guide RNA directs sequence-specific binding of a CRISPR complex to its respective target site and induces a nick in one strand of the DNA duplex near each target site, thereby generating two nicks on different DNA strands.

[0038] Cas proteins or their genetic information may be obtained from public databases such as GenBank of the National Center for Biotechnology Information (NCBI). Specifically, the Cas protein may be a Cas9 protein. The Cas protein may be derived from various species including, but not limited to,Staphylococcus,Streptococcus,Neisseria,Pasteurella,Francisella, orCampylobacter. More specifically, it may be a Cas9 protein derived fromStaphylococcus. However, the present disclosure is not limited to the examples described above. The Cas protein used in the present disclosure may be a recombinant protein.

[0039] The nucleic acids encoding Nurr1 and Foxa2 may be any known nucleotide sequences encoding Nurr1 and Foxa2, without limitation, and may also include nucleotide sequences encoding functional equivalents of Nurr1 and Foxa2. As used herein, "functional equivalents" refer to polypeptides having at least 70%, preferably at least 80%, more preferably at least 90% sequence homology (i.e., identity) with the amino acid sequence of Nurr1 or Foxa2. For example, the functional equivalents may include polypeptides having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology. The functional equivalents may be generated by addition, substitution, or deletion of certain amino acids. Preferably, such deletions or substitutions occur in regions not directly related to the biological activity of the polypeptide of the present disclosure.

[0040] The nucleic acids encoding Nurr1 and Foxa2 may be produced by genetic engineering methods known in the art (see Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Short Protocols in Molecular Biology, John Wiley and Sons, 1992). For example, nucleic acids may be obtained via PCR amplification from genomic DNA, chemical synthesis, or cDNA production techniques.

[0041] The nucleic acids encoding Nurr1 and Foxa2 may be operably linked to expression control sequences and inserted into expression vectors. The term "operably linked" refers to a configuration in which a nucleic acid fragment is functionally connected to another such that its function or expression is affected by the other. The term "expression control sequence" refers to a DNA sequence that regulates the expression of operably linked nucleic acid sequences in a particular host cell. Such control sequences include promoters for initiating transcription, operator sequences for regulating transcription, sequences encoding appropriate mRNA ribosome binding sites, and sequences controlling transcriptional and translational termination. Collectively, these may be referred to as "DNA constructs comprising nucleic acids encoding Nurr1 and Foxa2."

[0042] As used herein, the term "expression vector" refers to a plasmid, viral vector, or other vehicle known in the art into which a nucleic acid encoding a structural gene can be inserted for expression in a host cell. Preferably, the expression vector is a viral vector. The viral vector may be, but is not limited to, an adeno-associated virus (AAV) vector, adenoviral vector, herpesvirus vector, avipoxvirus vector, or lentiviral vector. More preferably, a lentiviral vector or AAV vector is used.

[0043] The AAV vector may be produced by introducing the necessary viral components into specific cells, and the lentiviral vector may be produced through multiple steps that allow for virus production in particular cell lines. The main advantages of lentiviral and AAV vectors for gene therapy are their efficiency and stability.

[0044] The expression vector containing the nucleic acids of the present disclosure may be introduced into cells via known methods, including but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, or gene gun. For example, Nurr1 and Foxa2 may be inserted into an AAV or lentiviral vector to generate an expression vector, which may then be transfected into packaging cells. After culturing the transfected packaging cells and filtering the culture, an AAV or lentiviral solution may be obtained. Subsequently, expression of Nurr1 and Foxa2 can be confirmed using a selectable marker included in the AAV or lentiviral vector to obtain the desired vector.

[0045] According to an embodiment of the present disclosure, the vector may be a linear DNA, plasmid DNA, recombinant non-viral vector, or recombinant viral vector.

[0046] According to an embodiment of the present disclosure, the non-viral vector may include an RNA molecule, a plasmid, a liposome complex, a molecular conjugate, and / or a gene-editing protein (CRISPR, e.g., Cas9).

[0047] According to one specific embodiment of the present disclosure, the vector may be a recombinant viral vector.

[0048] According to an embodiment of the present disclosure, the recombinant viral vector may be selected from the group consisting of adeno-associated virus (AAV), retrovirus, adenovirus, herpes simplex virus, and lentivirus, and preferably may be an adeno-associated virus (AAV) vector.

[0049] The recombinant AAV vector may include any viral strain or serotype. The serotype may be selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, Rh-74, Rh-10, or AAV-2i8, with preference for the serotype AAV-9.

[0050] According to an embodiment of the present disclosure, the concentration of the vector may be in the range of 5.0 x 106vg / mL to 5.0 x 1016vg / mL, 5.0 x 106vg / mL to 5.0 x 1015vg / mL, 5.0 x 1014vg / mL to 5.0 x 1013vg / mL, 5.0 x 1014vg / mL to 5.0 x 1012vg / mL, 5.0 x 107vg / mL to 5.0 x 1016vg / mL, 5.0 x 107vg / mL to 5.0 x 1015vg / mL, 5.0 x 107vg / mL to 5.0 x 1014vg / mL, 5.0 x 107vg / mL to 5.0 x 1013vg / mL, 5.0 x 107vg / mL to 5.0 x 1012vg / mL, 5.0 x 108vg / mL to 5.0 x 1016vg / mL, 5.0 x 108vg / mL to 5.0 x 1015vg / mL, 5.0 x 108vg / mL to 5.0 x 1014vg / mL, 5.0 x 108vg / mL to 5.0 x 1013vg / mL, 5.0 x 108vg / mL to 5.0 x 1012vg / mL, 5.0 x 109vg / mL to 5.0 x 1016vg / mL, 5.0 x 109vg / mL to 5.0 x 1015vg / mL, 5.0 x 109vg / mL to 5.0 x 1014vg / mL, 5.0 x 109vg / mL to 5.0 x 1013vg / mL, 5.0 x 109vg / mL to 5.0 x 1012vg / mL, 5.0 x 1010vg / mL to 5.0 x 1016vg / mL, 5.0 x 1010vg / mL to 5.0 x 1015vg / mL, 5.0 x 1010vg / mL to 5.0 x 1014vg / mL, 5.0 x 1010vg / mL to 5.0 x 1013vg / mL, 5.0 x 1010vg / mL to 5.0 x 1012vg / mL, 5.0 x 1011vg / mL to 5.0 x 1016vg / mL, 5.0 x 1011vg / mL to 5.0 x 1015vg / mL, 5.0 x 1011vg / mL to 5.0 x 1014vg / mL, 5.0 x 1011vg / mL to 5.0 x 1013vg / mL, for example, 5.0 Х 10¹¹ vg / mL to 5.0 Х 10¹² vg / mL.

[0051] According to an embodiment of the present disclosure, the concentration of the vector may be 5.0 x 106vg / mL or 5.0 x 1016vg / mL, and preferably 5.0 x 1011vg / mL or 5.0 x 1012vg / mL.

[0052] According to an embodiment of the present disclosure, the inflammatory bowel disease may be at least one selected from the group consisting of Crohn's disease, ulcerative colitis, Behcet's syndrome, ulcerative proctitis, lymphocytic colitis, ischemic colitis, diversion colitis, intestinal tuberculosis, and irritable bowel syndrome.

[0053] According to an embodiment of the present disclosure, the method for preventing or treating inflammatory bowel disease using the composition may comprise administering the composition to a subject or patient via a general route suitable for introducing the desired substance.

[0054] According to an embodiment of the present disclosure, the composition may be administered via a non-oral route selected from intraperitoneal, intradermal, subcutaneous, intramuscular, rectal, or intravenous administration, or via an oral route selected from enteric-coated capsules, enteric-coated tablets, or direct administration to the gastrointestinal tract.

[0055] According to an embodiment of the present disclosure, the composition may be administered via the intraperitoneal route.

[0056] According to an embodiment of the present disclosure, the composition may reduce diarrhea and improve stool consistency in the subject.

[0057] According to an embodiment of the present disclosure, the composition may reduce rectal bleeding in the subject.

[0058] According to an embodiment of the present disclosure, the composition may suppress shortening of colon length in the subject.

[0059] As used herein, the term "stool consistency" refers to the consistency of stool and indicates symptoms of diarrhea in the subject. The term "rectal bleeding" refers to bleeding from the rectum or anus of the subject, which may appear as hematochezia, melena, or occult bleeding. The term "colon length" refers to the length from the appendix and colonic junction to the upper rectum of the subject. In inflammatory bowel disease models, the colon tends to shorten and inflammatory lesions are observed; therefore, suppression of colon shortening can serve as an indicator of symptom improvement.

[0060] According to an embodiment of the present disclosure, the composition may reduce the disease activity index (DAI) of inflammatory bowel disease.

[0061] As used herein, the term "disease activity index" (DAI) refers to an index for evaluating disease severity in inflammatory bowel disease, based specifically on stool consistency, rectal bleeding, and colon length shortening. A higher DAI indicates more severe disease, and a lower DAI indicates milder symptoms. Specifically, a DAI score of 0 to 1 indicates normal, 2 to 4 indicates mild, 5 to 7 indicates severe, and 8 to 10 indicates very severe inflammatory bowel disease. Preferably, the pharmaceutical composition may reduce the DAI by 2 to 3 points or more, thereby alleviating severe or very severe inflammatory bowel disease to a mild form.

[0062] The pharmaceutical composition of the present disclosure may be administered using any method or formulation that allows the active substance to reach the target cells. Preferred administration methods and formulations include intraperitoneal, intravenous, subcutaneous, intradermal, intramuscular, or infusion injections. The injections may be prepared using aqueous solvents such as saline or Ringer's solution, or non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate), or alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin). The formulation may further include stabilizers for preventing degeneration (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, BHA, tocopherol, EDTA), emulsifiers, buffers for pH control, and preservatives for inhibiting microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol). Preferably, the method for treating inflammatory bowel disease using the therapeutic composition of the present disclosure includes administering the composition in a pharmaceutically effective amount. The pharmaceutically effective amount may be easily determined by those skilled in the art based on factors well known in the medical field, such as the type of disease, the age, weight, health, sex, and drug sensitivity of the subject (patient), administration route and method, frequency, treatment duration, and drugs used in combination or concurrently.

[0063] The composition of the present disclosure may be formulated into appropriate preparations using pharmaceutically acceptable carriers depending on the administration method. Suitable formulations for each route of administration are well known and typically include carriers that facilitate transport or absorption through membranes. For example, pharmaceutically acceptable carriers may be sterile and biocompatible and include saline, sterile water, Ringer's solution, buffered saline, albumin for injection, dextrose solution, maltodextrin solution, glycerol, ethanol, or combinations thereof. Optionally, additional ingredients such as antioxidants, buffers, and bacteriostatic agents may be added. The composition may also be formulated into injections such as aqueous solutions, suspensions, emulsions, and so on, pills, capsules, granules, or tablets by adding diluents, dispersing agents, surfactants, binders, and lubricants. To ensure specificity for the target organ, the carrier may be combined with a target-specific antibody or ligand.

[0064] Also, the composition of the present disclosure may be used in general pharmaceutical dosage forms. For non-oral administration, the composition may be prepared in the form of sterile solutions, non-aqueous solvents, suspensions, emulsions, or lyophilized preparations. For oral administration, it may be formulated as tablets, lozenges, capsules, elixirs, suspensions, syrups, or wafers. In the case of injections, the composition may be provided in unit-dose ampoules or multi-dose formats. The therapeutic composition of the present disclosure may be administered with a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, or flavoring agents may be used. For injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers may be added. For topical administration, bases, excipients, lubricants, and preservatives may be included.

[0065] The pharmaceutical composition of the present disclosure may be administered in combination with other therapeutic agents. In such cases, the pharmaceutical composition of the present disclosure and the other therapeutic agents may be administered simultaneously, sequentially, or separately. The other therapeutic agents may include, but are not limited to, compounds or proteins having preventive, therapeutic, or ameliorative effects on inflammatory bowel disease.

[0066] Additionally, the pharmaceutical composition of the present disclosure may be formulated for administration simultaneously, sequentially, or separately with other therapeutic agents. For example, the vector into which the Nurr1 and Foxa2 genes are introduced and the other therapeutic agents may be administered in a single formulation, or they may be administered as separate formulations simultaneously, sequentially, or separately. To achieve simultaneous, sequential, or separate administration, the vector into which the Nurr1 and Foxa2 genes are introduced and the other therapeutic agents included in the pharmaceutical composition of the present disclosure may be formulated in separate containers or in the same container. Moreover, the vector into which the Nurr1 and Foxa2 genes are introduced and the other therapeutic agents may be administered in the same or different therapeutically effective amounts, at the same or different times, intervals, routes of administration, or treatment durations.

[0067] The term "therapeutically effective amount" as used herein refers to an amount sufficient to inhibit or alleviate a physiological effect caused by inflammatory bowel disease in a subject or patient. The therapeutic effective amount used may depend on the needs of the subject (patient), the age, physiological condition, and health of the subject (patient), the desired therapeutic effect, the size and area of the target tissue, the severity of the lesion, and the selected route of delivery.

[0068] According to one aspect of the present disclosure, the present disclosure relates to a method for treating inflammatory bowel disease, comprising the step of: administering to a subject a therapeutically effective amount of a composition comprising a vector into which one or both of Nurr1 and Foxa2 genes introduced thereto.

[0069] According to one aspect of the present disclosure, the present disclosure relates to a method for treating inflammatory bowel disease, comprising the step of: administering to a subject a therapeutically effective amount of a composition comprising a vector having Nurr1 and Foxa2 genes introduced thereto.

[0070] The term "introduction of the Nurr1 and Foxa2 genes" is as described above.

[0071] The term "therapeutically effective amount" is also as described above.

[0072] According to an embodiment of the present disclosure, the vector may be a linear DNA, plasmid DNA, recombinant non-viral vector, or recombinant viral vector.

[0073] According to an embodiment of the present disclosure, the recombinant viral vector may be selected from the group consisting of adeno-associated virus, retrovirus, adenovirus, herpes simplex virus, and lentivirus, with preference for an adeno-associated virus (AAV) vector.

[0074] In the method, the recombinant AAV vector may include any viral strain or serotype. The serotype may be selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, Rh-74, Rh-10, or AAV-2i8, and preferably may be AAV-9.

[0075] According to an embodiment of the present disclosure, the concentration of the vector may range from 5.0 x 106vg / mL to 5.0 x 1016vg / mL, and preferably may be 5.0 x 1011vg / mL or 5.0 x 1012vg / mL.

[0076] According to an embodiment of the present disclosure, the inflammatory bowel disease may be at least one selected from the group consisting of Crohn's disease, ulcerative colitis, Behcet's syndrome, ulcerative proctitis, lymphocytic colitis, ischemic colitis, diversion colitis, intestinal tuberculosis, and irritable bowel syndrome.

[0077] According to an embodiment of the present disclosure, the method may include administration via a non-oral route selected from intraperitoneal, intradermal, subcutaneous, intramuscular, rectal, and intravenous routes, or via an oral route selected from enteric-coated capsules, enteric-coated tablets, or direct administration to the gastrointestinal tract.

[0078] According to one specific embodiment of the present disclosure, the method may include administration via the intraperitoneal route.

[0079] According to an embodiment of the present disclosure, the method may reduce diarrhea, improve stool consistency, reduce rectal bleeding, and suppress the shortening of colon length in the subject.

[0080] The terms "stool consistency," "rectal bleeding," and "colon length" are as described above.

[0081] According to an embodiment of the present disclosure, the method may reduce the disease activity index (DAI) in the subject.

[0082] The term "disease activity index (DAI)" is as described above.

[0083] To avoid undue complexity in the specification, overlapping content between the method for treating inflammatory bowel disease and the above-described composition is omitted.

[0084] Unless otherwise defined, all technical terms used in the present disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Although preferred methods and samples are described in this specification, similar or equivalent alternatives are also within the scope of the present disclosure. All publications cited as references in this specification are incorporated herein by reference.

[0085] Hereinafter, the present disclosure will be described in further detail with reference to the following examples. These examples are provided solely for illustrative purposes and are not intended to limit the scope of the present disclosure, as it will be apparent to those of ordinary skill in the art.

[0086] EXAMPLE 1. Production of Adeno-Associated Virus (AAV)

[0087] 1.1.1 Culture of HEK293 Cells

[0088] HEK293 cells, which are host cells used for packaging and producing AAV, were seeded into a cell culture vessel (CellFactory or CellSTACK) at a density of 5,000 to 80,000 cells / cm2in DMEM medium supplemented with 10% fetal bovine serum (FBS), and cultured at 37°C in a 5% CO2incubator. The HEK293 cells were cultured for 1 to 10 days to reach 30% to 90% confluency at the bottom surface of the culture vessel.

[0089] 1.1.2 Preparation of Plasmids for Transfection

[0090] Plasmids for introducing AAV production-related genes into the HEK293 cells cultured in Example 1.1.2 were prepared.

[0091] Specifically,pNurr1, which is a plasmid carrying an expression cassette for expression of the therapeutic gene Nurr1 flanked by AAV inverted terminal repeats (ITRs),pFoxa2, which is a plasmid containing an expression cassette for expression of the therapeutic gene Foxa2 flanked by ITRs,pSero9, which is a plasmid for introducing capsid and replication proteins for serotype 9 AAV (AAV9), andpHelper, which is a plasmid for introducing adenoviral genes E2A and E4, which are required for large-scale production of progeny viruses derived from the initially introduced AAV production genes, were constructed.

[0092] To construct each plasmid, kanamycin antibiotic was added to sterilized LB medium, and bacteria containing the respective plasmids were inoculated and cultured with shaking at 200 to 300 rpm for 15 to 18 hours at 37°C. Thereafter, the bacteria were harvested by centrifugation. The bacterial cell structure was lysed and denatured by treatment with alkaline and acidic solutions. Plasmid purification and isolation were then performed from the lysate using anion exchange chromatography, hydrophobic interaction chromatography, ultrafiltration, and diafiltration techniques.

[0093] Subsequently, each plasmid was added to a bacterial (e.g., E.coli DH5α) culture medium (e.g., LB medium) containing a selection marker (e.g., ampicillin or kanamycin) and incubated for 15 to 20 hours. After culture, the medium was centrifuged at 2,000 x g to 5,000 x g for 10 to 30 minutes to harvest the bacteria. The bacteria were lysed using an alkaline solution (e.g., NaOH), and the lysate thus obtained was neutralized using an acidic solution (e.g., acetic acid). The neutralized lysate was subjected to gel filtration, ion exchange chromatography, and ultrafiltration to obtain plasmid solutions containing the desired plasmids.

[0094] Finally, endotoxin levels in the obtained plasmid solutions were measured using a colorimetric method among optical techniques. Bacterial-derived DNA or protein contents were analyzed using commercially available ELISA kits. The supercoiled DNA content was analyzed by agarose gel electrophoresis. Plasmid solutions with low levels of impurities and 70% to 95% supercoiled DNA were used in subsequent transfection steps.

[0095] 1.1.3 Introduction of Nurr1 and Foxa2 Genes through Plasmid-Based Transfection

[0096] The HEK293 host cells cultured in Example 1.1.1 were transfected with the plasmids prepared in Example 1.1.2 To produce the recombinant AAV9-Nurr1 vector, the three plasmids pNurr1, pSero9, and pHelper were used. To produce the recombinant AAV9-Foxa2 vector, the three plasmids pFoxa2, pSero9, and pHelper were used.

[0097] The transfection solution containing plasmids for transfection was prepared. In this regard, 1 to 10 mg of polyethyleneimine (PEI) was diluted in 30 to 300 mL of serum-free medium (e.g., Opti-MEM). Separately, 1 to 10 mg of each plasmid set (pNurr1 or pFoxa2, pSero9, and pHelper) to be used for production of each solution was also diluted in 30 to 300 mL of serum-free medium. After allowing both solutions to stand for several minutes, they were combined, gently inverted and swirled several times to mix, and then allowed to stand again at room temperature for 15 to 30 minutes to complete the transfection solution.

[0098] For delivery of the gene for recombinant AAV production, on the day of transfection, the medium of the HEK293 cells cultured as in Example 1.1.1 was replaced with fresh medium at 0.1 to 0.3 mL / cm2. After 3 to 5 hours, the same volume of additional fresh medium was added to the HEK293culture and mixed well.

[0099] Finally, the transfection solution was added to the HEK293 culture medium to deliver the plasmid sets into the cells. The transfected HEK293 host cells were cultured for 2 to 8 days to induce production of recombinant AAV.

[0100] Afterward, the cultures for AAV were all collected from the cell culture vessels. The collected cultures were filtered through a 0.2 μm pore-size filter. To isolate and purify the AAV, ultrafiltration, diafiltration / dialysis, ion exchange chromatography, ultracentrifugation, and affinity chromatography were sequentially conducted.

[0101] 1.2 Isolation and Purification of AAV

[0102] 1.2.1 Cell Lysis and Filtration

[0103] After the transfected HEK293 host cells were cultured, the culture was completely collected from the cell culture vessel to recover AAV present both inside and outside the cells. The collected medium was filtered through a 0.2 μm pore size filter.

[0104] The cultured cells were washed twice with D-PBS, and then lysed either by treating with a cell lysis buffer containing a surfactant (e.g., Triton X-100) or by trypsinization followed by repeated freeze-thaw cycles of the harvested cells. The resulting lysates were centrifuged at 3,000 to 5,000 Х g for 10 minutes or filtered through filters with pore sizes of 0.8 μm, 0.45 μm, or 0.22 μm to remove cell debris.

[0105] The filtered cell lysates and culture supernatants containing recombinant AAV were then sequentially subjected to the following purification steps described in Examples 1.2.2 to 1.2.6: ultrafiltration, diafiltration / dialysis, ion exchange chromatography, ultracentrifugation, and affinity chromatography.

[0106] 1.2.2 Ultrafiltration

[0107] After the cell lysates and culture supernatants containing AAV were collected, tangential flow filtration (TFF) or direct flow filtration (DFF) was performed to concentrate the solution or exchange the buffer. Membranes with molecular weight cut-off (MWCO) corresponding to 200 kDa, 100 kDa, 50 kDa, or 30 kDa were used for filtration.

[0108] 1.2.3 Diafiltration or Dialysis

[0109] To exchange the buffer or enhance the stability of the AAV-containing solution, TFF or dialysis was employed. Similar to ultrafiltration, membranes with MWCOs of 200 kDa, 100 kDa, 50 kDa, or 30 kDa were used. The process involved continuous addition of a diafiltration buffer at the same flow rate as the filtrate to maintain a constant volume of the AAV solution.

[0110] 1.2.4 Ion Exchange Chromatography

[0111] For separation and purification of the produced AAV, ion exchange chromatography was performed based on the principle that AAV capsids possess unique dissociation constants (Ka) depending on the polar amino acids on their surfaces. By adjusting the pH of the buffer, the net surface of the AAV capsid was controlled to be positively or negatively charged, thus allowing binding and elution from the ion exchange resin.

[0112] Anion exchange resins (e.g., resins with ammonium / amine, diethylaminoethyl [DEAE], or diethylaminopropyl functional groups) were equilibrated with a buffer containing Tris (10 to 50 mM, pH 7.0 to 8.0), NaCl (100 to 500 mM), and MgCl2(1 to 100 mM). The recombinant AAV-containing solution was loaded and bound to the resin which was then washed with 10 volumes of the same buffer. The recombinant AAV bound to the resin was then eluted by lowering the pH (e.g., Tris buffer at pH .0 to 4.0).

[0113] 1.2.5 Ultracentrifugation

[0114] To further separate and purify recombinant AAV from the AAV-containing sample prepared, density gradient ultracentrifugation was performed using cesium chloride or iodixanol. For example, a cesium chloride density gradient was prepared in an ultracentrifuge tube (approximately 40 mL) by layering 10 mL of light cesium chloride solution (3.88 M CsCl) over 10 mL of heavy CsCl solution (5.93 M CsCl). The recombinant AAV-containing sample was added to the top, and the tube was sealed and centrifuged at 40,000 to 60,000 rpm for 16 to 24 hours.

[0115] After completion of the centrifugation, a hole was made at the bottom of the centrifugal tube to collect fractions in order to isolate the layer AAV-containing layer, or a needle was inserted at the appropriate layer containing AAV to separate and purify AAV.

[0116] 1.2.6 Affinity Chromatography

[0117] For separation and purification of the produced AAV, affinity chromatography was performed based on the fact that specific binding between the AAV capsid and the ligand bound to the resin can be made at nanomolar-range dissociation constant on the basis of high specificity therebetween.

[0118] Commercially available resins such as AVIPureTM(Repligen) and POROSTMCaptureSelectTM(Thermo Fisher Scientific) were used. The AAV-containing solution was injected into the chromatography column to allow binding of the AAV to the resin's ligand, washed with buffer, and then eluted to recover AAV.

[0119] 1.3 Quantification of Produced AAV

[0120] Each purified AAV was quantitatively analyzed. The quantification was performed using quantitative PCR (qPCR) targeting the AAV genome DNA (gDNA) with specific primers. The number of AAV vectors was expressed as vector genome (vg) copies.

[0121] For a standard material for standard curve preparation in qPCR, plasmid solutions containing the target gene used in transfection were quantified using an ultraviolet spectrophotometer, and the molar amount was calculated based on the plasmid's molecular weight. Using Avogadro's number based on the molar amount of the plasmid, the number of plasmid copies per unit volume was determined to prepare a stock solution. This stock was serially diluted 10-fold to generate standard solutions with various concentrations (e.g., plasmid copies per mL), which were used to generate a standard curve from Ct values obtained in qPCR.

[0122] The qPCR was performed using the TaqMan probe method in a total reaction volume of 20 μL. The reaction mixture included 2 μL of 10- to 200-fold diluted AAV sample, 1 μL each of forward and reverse primers, PCR buffer (1x concentration), and distilled water. The thermal cycling conditions of PCR included 2 minutes at 50°C and 10 minutes at 95°C, followed by 30 to 40 cycles of 15 seconds at 95°C and 1 minute at 60°C.

[0123] For efficacy testing against inflammatory bowel disease, the recombinant vectors AAV9-Nurr1 and AAV9-Foxa2 to be introduced into animals were purified and quantified from cell lysates and culture supernatants obtained from transfected host cells, and then used for administration to animals.

[0124]

[0125] EXAMPLE 2. Experimental Materials and Methods

[0126] In this example, an inflammatory bowel disease (IBD) model was induced by administration of dextran sodium sulfate (DSS). The DSS-induced inflammatory bowel disease model is a well-established and widely used model that reliably reproduces lesion phenotypes representative of IBD. In this experiment, C57BL / 6J mice were used, a strain known to be sensitive to DSS-induced inflammatory bowel disease.

[0127] 1. Prepared Compositions

[0128] The AAV9-Nurr1 and AAV9-Foxa2 gene therapeutics prepared in Example 1 were formulated into two dosage groups (Low-dose group: 5.0 Х 1011vg / mL each vector, High-dose group: 5.0 Х 1012vg / mL each vector). The formulations were stored in an ultra-low temperature freezer at -70°C and thawed immediately prior to use. A volume of 0.1 mL per subject was administered.

[0129] 2. Experimental Design

[0130] 2.1 Animal Acquisition and Acclimation

[0131] A total of 44 male C57BL / 6J mice (6 weeks old, weighing 17-19 g) were obtained from Orient Bio (322 Galmachi-ro, Seongnam-si, Gyeonggi-do, Korea) on November 9, 2023. During a 5-day acclimation period, clinical observations were performed once daily.

[0132] 2.2 Experimental Groups

[0133] Following acclimation, mice were grouped such that body weights were balanced across groups prior to treatment. Each group consisted of 11 animals (G1, G2, G3, G4). The administration route was intraperitoneal (IP). The group composition is shown in Table 1 below.

[0134] No.GroupRouteSubstanceDoseNo. of Animals (subject no.)G1Untreated---11 (1101-1111)G2Negative controlIPPBS10 mL / kg11 (1201-1211)G3Low doseIPAAV9-Nurr1 5.0x1011vg / mL,AAV9-Foxa2 5.0x1011vg / mL0.1 mL11 (1301-1311)G4High doseIPAAV9-Nurr1 5.0x1012vg / mL,AAV9-Foxa2 5.0x1012vg / mL0.1 mL11 (1401-1411)

[0135] 2.3 Animal Husbandry

[0136] According to separation of groups and individuals, each animal was assigned a unique identification number, which was marked on the ear. Cages were labeled with experiment number, animal number, and group.

[0137] Mice were housed in individually ventilated cage systems (IVCS; 391W Х 199D Х 160H mm) with 3-4 mice per cage. The animal room was maintained at 22 ± 1°C, 50 ± 10% relative humidity, with 10-15 air changes per hour, and a 12-hour light / dark cycle (07:00-19:00). Illumination was maintained at 150-300 lux. Cages and feed were changed weekly. Cages were washed using an automated washer and sterilized using an autoclave. Feed from Woojung Bio was provided ad libitum in the form of solid pellets. Drinking water, sourced from the Daegu municipal water supply, was filtered using a reverse osmosis (RO) system and provided ad libitum. Water quality was tested for all parameters under the "Standards and Testing Methods for Drinking Water Quality" (Ministry of Environment Ordinance No. 621, Nov. 23, 2015), and the analysis was performed twice per year to ensure compliance with established limits.

[0138] 2.4 Experimental Schedule

[0139] The experiment was conducted according to the schedule shown in Table 2. On the day after group separation, G2 (PBS), G3 (low-dose), and G4 (high-dose) groups received a single intraperitoneal injection of the respective test substance. Seven days later, 3% DSS was administered in drinking water to G2, G3, and G4 for five consecutive days. The G1 group received regular drinking water throughout the study period.

[0140] Day-7-6-5-4-3-2-10123456789G1UntreatedNormal waterNecropsyG2PBS3% DSS waterG3Low dose3% DSS waterG4High dose3% DSS waterMeasure-mentDAIDAIDAIDAIDAIDAIDAIDAIDAIDAI

[0141] 2.5 Animal Observation

[0142] 2.5.1 Clinical Signs

[0143] All animals were monitored daily for fur condition, food and water intake, activity, mortality, and injury. If abnormal symptoms were observed, veterinary recommendations were followed. To evaluate the disease activity index (DAI), stool consistency and the presence of rectal bleeding or occult blood were examined daily after DSS administration.

[0144] 2.5.2 Body Weight

[0145] Body weight was measured once before DSS treatment and then recorded daily from the start of DSS treatment until necropsy.

[0146] 2.6 Statistical Analysis

[0147] Statistical significance of the experimental results was analyzed using Prism 10 (GraphPad). All data were presented as mean ± standard deviation (SD). After testing for normal distribution, statistical analysis was performed using two-way ANOVA or one-way ANOVA with Dunnett's multiple comparisons test. Statistical significance between groups was evaluated atp< 0.05.

[0148] 2.7 Euthanasia

[0149] At the end of the experiment, all animals were euthanized using CO2gas.

[0150]

[0151] EXAMPLE 3. Results

[0152] 3.1 Evaluation of Disease Activity Index (DAI)

[0153] The disease activity index (DAI) was assessed daily from Day 0 (the start of DSS administration) through Day 9 (prior to necropsy). DAI evaluation was conducted in accordance with the reference (CR Mantyh et al.,Gut, 2003; 52:713-710), and the scores were calculated based on stool consistency, occult or gross rectal bleeding, and weight loss, as summarized in Table 3.

[0154] ScoreStool consistencyOccult / gross rectal bleedingWeight loss0NormalNormalNone1loose stoolsHemoccult1 - 5 %25 - 10 %310 -20 %4DiarrheaGross bleeding> 20 %

[0155] A comparative analysis of stool consistency among DAI evaluation items is shown in Table 4. Compared to the negative control group (G2), the low-dose group (G3) exhibited significantly improved stool consistency on Days 1-5 and Day 8. The high-dose group (G4) showed statistically significant improvements on Days 3-5 and Days 8-9.

[0156] DayG1G2G3G4P valueG2 vs G3P valueG2 vs G400±00±00±00±0--10±00.45±0.520.09±0.300.27±0.410.02963*0.1873120.18±0.400.73±0.470.36±0.390.55±0.690.03109*0.2382830.18±0.250.73±0.470.50±0.810.64±0.920.00015***0.00115**40±02.91±0.831.27±0.791.82±0.750.00006***0.00210**50±03.09±0.941.55±0.692.00±1.000.00014***0.00800**60±02.73±0.912.27±0.902.27±1.190.126210.1627270±03.18±0.872.45±1.212.27±1.010.061210.1360580±03.55±0.822.64±1.122.55±0.930.02103*0.00739**90±03.00±1.052.45±1.132.18±0.870.133940.03345*

[0157] *,p<0.05; **,p<0.01; ***,p<0.0001: Statistically significant improvement

[0158]

[0159] Among DAI evaluation, the rectal bleeding scores were compared and analyzed according to group and the results are shown in Table 5. Compared to G2 (negative control), G3 (low-dose group) showed significant alleviation in rectal bleeding on Days 3-6, and G4 (high-dose group) showed alleviation on Day 4.

[0160] DayG1G2G3G4P valueG2 vs G3P valueG2 vs G400±00±00±00±0--10±00±00±00±0--20±00.50±0.220.55±0.270.59±0.300.335780.2156530±01.23±0.790.50±0.631.00±0.710.01399*0.2420940±02.55±0.690.91±0.701.27±1.010.00001***0.00125**50±02.55±0.691.73±0.792.09±0.830.00860**0.0887860±01.55±1.041.00±0.001.09±0.300.04802*0.0887870±01.36±0.921.18±0.401.09±0.300.278400.1816680±00.45±1.210.27±0.471.09±0.300.323920.1731690±00.10±0.320.09±0.300±00.473470.15309

[0161] *,p<0.05; **,p<0.01; ***,p<0.0001: Statistically significant improvement

[0162]

[0163] The overall DAI scores showed that both G3 [low-dose group of AAV9-Nurr1 and AAV9-Foxa2 (IPS103)] and G4 [high-dose group of IPS103] had significantly lower DAI values compared to G2 (negative control group, PBS-treated), with statistically significant differences (see FIGS. 1a and 1b; *,p<0.05; **,p<0.01; ***,p<0.0001).

[0164] 3.2 Evaluation of Colon Tissue (Colon Length Reduction)

[0165] Colon shortening and morphological changes are known to correlate with inflammation, and colon length is widely used as a morphological parameter indicative of inflammation severity.

[0166] To assess the effects of the compositions on colon length in mice administered G3 (low-dose group) and G4 (high-dose group), the entire colon including the cecum and rectum was harvested at necropsy (Day 9), and photographs were taken. Colon length was measured for each subject, and the results are summarized in Table 6.

[0167] Group (unit)G1(cm)G2(cm)G3(cm)G4(cm)pvalueG2 vs G3pvalueG2 vs G4018.85.35.55.50.00529**0.00616**027.56.05.86.5037.64.87.26.0047.54.97.05.4058.06.06.17.9068.55.76.26.7078.05.76.75.6088.35.06.16.7098.55.35.95.0108.35.46.06.3118.5-6.26.8Ave. Length8.15.46.26.2Std. Dev.0.450.430.520.83

[0168] **:p<0.01Statistically significant improvement.

[0169] As a result, both the G3 (low dose) and G4 (high dose) groups showed statistically significant suppression of colon shortening compared to the G2 (negative control) group.

[0170] Photographs of individual colon tissues from each experimental group are presented in FIG. 2. Compared to G2 (negative control), the G3 (low dose) and G4 (high dose) groups showed markedly less colon shortening, despite some individual variation (FIG. 2).

[0171] These results suggest that treatment with the gene therapy comprising AAV-Nurr1 and AAV-Foxa2 may contribute to recovery of colon length damaged by inflammatory bowel disease. Accordingly, the composition for preventing or treating inflammatory bowel disease according to an embodiment of the present disclosure exhibits an effect of restoring the length of the damaged colon tissue in affected subjects.

[0172]

[0173] EXAMPLE 4. Conclusion

[0174] The above experiment was conducted using a DSS-treated C57BL / 6J mouse model, which is a well-established model of inflammatory bowel disease (IBD), to evaluate the therapeutic efficacy of a single intraperitoneal (IP) administration of the gene therapies AAV9-Nurr1 and AAV9-Foxa2. After gene therapy administration, 3% DSS was provided in drinking water for 5 days to induce colitis. The results demonstrated that, compared to the negative control group (G2, PBS-treated), both the low-dose group (G3) and high-dose group (G4) exhibited lower disease activity index (DAI) scores and preservation of colon length, with statistically significant improvements. These findings confirm that the gene therapy of the present disclosure may be effectively utilized as a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease.

[0175] Although the present disclosure has been described in detail with reference to specific examples, it will be understood by those skilled in the art that various modifications and changes may be made without departing from the scope of the invention as defined in the appended claims. Therefore, the scope of the present disclosure should not be limited to the described examples but rather construed to include all modifications and equivalents falling within the scope of the appended claims.

[0176] The present disclosure is directed to providing a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease, the composition comprising a vector into which one or both of the Nurr1 and Foxa2 genes are introduced.

Claims

1.A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising a vector into which one or both of the Nurr1 and Foxa2 genes are introduced.2.The composition of claim 1, wherein the vector is selected from the group consisting of linear DNA, plasmid DNA, a recombinant non-viral vector, and a recombinant viral vector.3.The composition of claim 2, wherein the recombinant viral vector is selected from the group consisting of adeno-associated virus, retrovirus, adenovirus, herpes simplex virus, and lentivirus.4.The composition of claim 1, wherein the concentration of the vector is in the range of 5.0 Х 106vg / mL to 5.0 Х 1016vg / mL.5.The composition of claim 1, wherein the inflammatory bowel disease is one or more selected from the group consisting of Crohn's disease, ulcerative colitis, Behcet's syndrome, ulcerative proctitis, lymphocytic colitis, ischemic colitis, diversion colitis, intestinal tuberculosis infection, and irritable bowel syndrome.6.The composition of claim 1, wherein the composition is administered via a non-oral route selected from the group consisting of intraperitoneal, intradermal, subcutaneous, intramuscular, rectal, and intravenous administration.7.The composition of claim 1, wherein the composition reduces diarrhea and improves stool consistency in a subject.8.The composition of claim 1, wherein the composition reduces rectal bleeding in a subject.9.The composition of claim 1, wherein the composition suppresses colon length reduction in a subject.10.The composition of claim 1, wherein the composition reduces the disease activity index (DAI) of inflammatory bowel disease.

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