Use of nucb1 in preventing and / or treating autoimmune diseases

Abstract

This invention provides the use of NUCB1 in the prevention and / or treatment of autoimmune diseases. Specifically, this invention provides the use of the NUCB1 gene, or its protein, or its promoter, for the preparation of compositions or formulations for the prevention and / or treatment of autoimmune diseases. The NUCB1 gene, or its protein, or its promoter of this invention can significantly prevent and / or treat autoimmune diseases, especially systemic lupus erythematosus, atopic dermatitis, rheumatoid arthritis, psoriasis, multiple sclerosis, or Crohn's disease.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to the application of NUCB1 in the prevention and / or treatment of autoimmune diseases. Background Technology

[0002] Autoimmune diseases are diseases caused by an immune response to self-antigens, leading to damage to the body's own tissues. The complex mechanisms of these diseases result in a wide variety of autoimmune diseases and a large patient population. Currently, more than 100 different autoimmune diseases have been identified, including systemic lupus erythematosus, atopic dermatitis, rheumatoid arthritis, psoriasis, multiple sclerosis, and Crohn's disease. Women have a higher incidence of these diseases compared to men.

[0003] As chronic diseases, autoimmune diseases are usually not directly fatal, but they are also difficult to cure and require costly long-term care. Diseases such as ankylosing spondylitis and systemic lupus erythematosus are often irreversible once contracted, earning them the nickname "the cancer that doesn't kill." Patients require long-term, even lifelong, medication, thus driving a consistently strong market demand for autoimmune disease drugs. Statistics show that there are nearly 500 million people worldwide suffering from various autoimmune diseases, and the global autoimmune drug market reached approximately US$132.3 billion in 2022, making it the second largest disease market after oncology and a hotbed for global drug development and blockbuster drug creation.

[0004] From a treatment perspective, strategies for treating autoimmune diseases include reducing inflammation and suppressing the immune system. While nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and antirheumatic drugs (ARTs) have good anti-inflammatory effects, they are unlikely to fundamentally stop disease progression, and relapse is common after discontinuation. Traditional non-selective immunosuppressants such as cyclosporine and methotrexate not only have slow onset of action but also carry a higher risk of cancer development. Biologic targeted therapies, as novel immunosuppressants, can selectively inhibit the immune system and are the most promising drugs for immune diseases, achieving great success. However, the medical needs remain largely unmet; most biologic targeted therapies have limited efficacy for patients with severe conditions, and long-term safety issues persist.

[0005] From a scientific perspective, our understanding of autoimmune diseases is still quite limited. Discovering new targets, exploring the mechanisms of autoimmune diseases from new perspectives, and discovering new therapeutic drugs are particularly important.

[0006] Therefore, there is an urgent need in this field to develop drugs that can effectively prevent and / or treat autoimmune diseases. Summary of the Invention

[0007] The purpose of this invention is to provide an effective medicine for the prevention and / or treatment of autoimmune diseases.

[0008] In a first aspect of the invention, there is provided the use of the NUCB1 gene, or its protein, or its promoter, for the preparation of compositions or formulations for the prevention and / or treatment of autoimmune diseases.

[0009] In another preferred embodiment, the NUCB1 gene, or its protein, or its promoter, is the sole active ingredient in a composition or formulation for the prevention and / or treatment of autoimmune diseases.

[0010] In another preferred embodiment, the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, atopic dermatitis, rheumatoid arthritis, psoriasis, multiple sclerosis, Crohn's disease, asthma, type I diabetes, autoimmune liver disease, myasthenia gravis, vitiligo, alopecia areata, pemphigus, or Sjögren's syndrome.

[0011] In another preferred embodiment, the autoimmune disease is selected from the group consisting of atopic dermatitis, psoriasis, Crohn's disease, or multiple sclerosis.

[0012] In another preferred embodiment, the promoter refers to a substance capable of increasing the activity and / or content of the NUCB1 gene or its protein in vivo or in vitro, and the substance may be a synthetic or natural compound, protein, nucleotide, etc.

[0013] In another preferred embodiment, the NUCB1 promoter includes a substance that promotes NUCB1 expression or activity.

[0014] In another preferred embodiment, the NUCB1 promoter includes a NUCB1 protein promoter and / or a NUCB1 gene promoter.

[0015] In another preferred embodiment, the promotion of NUCB1 expression or activity refers to increasing the expression or activity of the NUCB1 gene or protein by ≥20%, more preferably ≥50%, and even more preferably ≥70%.

[0016] In another preferred embodiment, the NUCB1 promoter is selected from the group consisting of small molecule compounds, NUCB1-expressing carriers, or combinations thereof.

[0017] In another preferred embodiment, the vector expressing NUCB1 includes a viral vector.

[0018] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus vectors, lentiviral vectors, or combinations thereof.

[0019] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0020] In another preferred embodiment, the NUCB1 gene, or its protein, is derived from mammals, more preferably from rodents (such as mice and rats), primates, and humans.

[0021] In another preferred embodiment, the NUCB1 protein also includes derivatives of the NUCB1 protein.

[0022] In another preferred embodiment, the NUCB1 protein derivative includes a modified NUCB1 protein, a protein molecule whose amino acid sequence is homologous to that of the natural NUCB1 protein and has the activity of the natural NUCB1 protein, a dimer or polymer of the NUCB1 protein, or a fusion protein containing the amino acid sequence of the NUCB1 protein.

[0023] In another preferred embodiment, the "protein molecule whose amino acid sequence is homologous to the natural NUCB1 protein and has the activity of the natural NUCB1 protein" means a protein molecule whose amino acid sequence has ≥85% homology to the NUCB1 protein, preferably ≥90% homology, more preferably ≥95% homology, and most preferably ≥98% homology; and has the activity of the natural NUCB1 protein.

[0024] In another preferred embodiment, the NUCB1 protein is selected from the group consisting of:

[0025] (A) A polypeptide with an amino acid sequence as shown in SEQ ID NO. 1 or 2;

[0026] (B) A NUCB1 protein derivative or its active fragment formed by substituting, deleting or adding one or more (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO.1 or 2.

[0027] (C) A NUCB1 protein derivative or its active fragment thereof with ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% homology to the amino acid sequence shown in SEQ ID NO.1 or 2.

[0028] In another preferred embodiment, the NUCB1 gene encodes the NUCB1 protein.

[0029] In another preferred embodiment, the NUCB1 gene is selected from the group consisting of:

[0030] (a) A polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1 or 2;

[0031] (b) Polynucleotides with sequences as shown in SEQ ID NO. 5 or 6;

[0032] (c) A polynucleotide whose nucleotide sequence is ≥95% homology (preferably ≥98%) to the sequence shown in SEQ ID NO. 5 or 6 and encodes the polypeptide shown in SEQ ID NO. 1 or 2;

[0033] (d) and any of the polynucleotides complementary to those described in (a)-(c).

[0034] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0035] In another preferred embodiment, the pharmaceutical composition contains (a) the NUCB1 gene, or its protein, or its promoter; and (b) a pharmaceutically acceptable carrier.

[0036] In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semi-solid.

[0037] In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, transdermal patches, capsules, oral solutions, or injections.

[0038] In another preferred embodiment, the component (a) in the pharmaceutical composition accounts for 1-99 wt% of the total weight of the pharmaceutical composition, more preferably 10-90 wt%, and more preferably 30-70 wt%.

[0039] In another preferred embodiment, the composition further includes additional components for the prevention and / or treatment of autoimmune diseases.

[0040] In another preferred embodiment, the composition further includes the NUCB2 gene, or its protein, or a promoter thereof.

[0041] In a second aspect of the invention, a pharmaceutical composition is provided, comprising:

[0042] (a1) A first active ingredient for the prevention and / or treatment of autoimmune diseases, the first active ingredient comprising: the NUCB1 gene, or its protein, or its promoter;

[0043] (a2) A second active ingredient for the prevention and / or treatment of autoimmune diseases, the second active ingredient comprising: other medicines for the prevention and / or treatment of autoimmune diseases; and

[0044] (b) Pharmaceutically acceptable carriers.

[0045] In another preferred embodiment, the component (a1) accounts for 1-99 wt% of the total weight of the pharmaceutical composition, more preferably 10-90 wt%, and more preferably 30-70 wt%.

[0046] In another preferred embodiment, the component (a2) in the pharmaceutical composition accounts for 1-99 wt% of the total weight of the pharmaceutical composition, more preferably 10-90 wt%, and more preferably 30-70 wt%.

[0047] In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, more preferably 1:10 to 10:1.

[0048] In another preferred embodiment, the pharmaceutical composition further includes the NUCB2 gene, or its protein, or a promoter thereof.

[0049] In another preferred embodiment, the autoimmune disease is selected from the group consisting of: systemic lupus erythematosus, atopic dermatitis, rheumatoid arthritis, psoriasis, multiple sclerosis, Crohn's disease, asthma, type I diabetes, autoimmune liver disease, myasthenia gravis, vitiligo, alopecia areata, pemphigus, or Sjögren's syndrome.

[0050] In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of multiple compounds.

[0051] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament or preparation for treating or preventing autoimmune diseases.

[0052] In another preferred embodiment, the drug dosage form is an oral or non-oral dosage form.

[0053] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0054] In another preferred embodiment, the non-oral dosage form is an injection, a transdermal patch, or a syringe.

[0055] In another preferred embodiment, the total content of the active ingredients (a1) and (a2) is 1 to 99 wt% of the total weight of the composition, more preferably 5 to 90 wt%.

[0056] In a third aspect of the invention, a medicine box is provided, comprising:

[0057] (i) a first container, and the active ingredient (a1) NUCB1 gene, or its protein or its promoter, or a drug containing the active ingredient (a) located in the first container; and

[0058] (ii) A second container, and the active ingredient (a2) located in the second container, other medicines for the prevention and / or treatment of autoimmune diseases, or medicines containing the active ingredient (a2).

[0059] In another preferred embodiment, the first container and the second container may be the same or different containers.

[0060] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing the NUCB1 gene, its protein, or its promoter.

[0061] In another preferred embodiment, the drug in the second container is a single-ingredient preparation containing other drugs for the prevention and / or treatment of autoimmune diseases.

[0062] In another preferred embodiment, the dosage form of the drug is a transdermal patch, an oral dosage form, or an injectable dosage form.

[0063] In another preferred embodiment, the kit also contains instructions for administration of active ingredients (a1) and (a2) in combination to prevent and / or treat autoimmune diseases.

[0064] In another preferred embodiment, the dosage form of the preparation containing the active ingredient (a1) NUCB1 gene, or its protein or its promoter, or the preparation containing other medicaments for the prevention and / or treatment of autoimmune diseases, includes capsules, tablets, transdermal patches, suppositories, or intravenous injections.

[0065] In another preferred embodiment, in the formulation containing the active ingredient (a1) NUCB1 gene, or its protein, or its promoter, the concentration of the NUCB1 gene, or its protein, or its promoter is 0.0001-100 mg / kg body weight, more preferably 0.1-50 mg / kg body weight, and more preferably 1-20 mg / kg body weight.

[0066] In a fourth aspect of the invention, a method for screening potential therapeutic agents for autoimmune diseases is provided, comprising:

[0067] (a) In the test group, in the culture system, in the presence of the test compound, cells expressing the NUCB1 gene were cultured, and the expression level E1 of the NUCB1 gene in the culture system described in the test group was detected;

[0068] Furthermore, in a control group where the test compound was absent and all other conditions were identical, the expression level (E2) of the NUCB1 gene in the culture system of the control group was measured; and

[0069] (b) Compare E1 and E2. If E1 is significantly higher than E2, the test compound is a potential therapeutic agent for autoimmune diseases.

[0070] In another preferred embodiment, “significantly higher than” means E1 / E2 ≥ 2, more preferably ≥ 3, and even more preferably ≥ 4.

[0071] In another preferred embodiment, the cells are hepatocytes.

[0072] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0073] In another preferred embodiment, the method includes step (c): administering the potential therapeutic agent identified in step (a) to a mammal to determine its effect on an autoimmune disease in the mammal.

[0074] In another preferred embodiment, the mammal includes humans or non-human mammals.

[0075] In another preferred embodiment, the non-human mammals include rodents and primates, and more preferably, mice, rats, rabbits, and monkeys.

[0076] In a fifth aspect of the invention, a method for preventing and / or treating autoimmune diseases is provided, comprising the steps of:

[0077] The NUCB1 gene, or its protein or its promoter, the pharmaceutical composition described in the second aspect of the invention, or the cassette described in the third aspect of the invention may be administered to the recipient.

[0078] In another preferred embodiment, the application includes oral administration, injection, or topical application.

[0079] In another preferred embodiment, the object includes a human or a non-human mammal.

[0080] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0081] In another preferred embodiment, the dosage of NUCB1 or its promoter is 0.0001-100 mg / kg body weight, more preferably 1-50 mg / kg body weight, and most preferably 5-20 mg / kg body weight.

[0082] In another preferred embodiment, the NUCB1 or its promoter is applied 1-150 times per month, more preferably ≥1 time per day, and even more preferably 2 times per day.

[0083] In another preferred embodiment, the application time of NUCB1 or its promoter is 1-100 days, more preferably 5-50 days, and even more preferably 5-30 days.

[0084] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0085] Figure 1 The graph shows the correlation between NUCB1 and / or NUCB2 gene knockout mice and potential autoimmune diseases. The AC graph represents the body weight of NUCB1 and / or NUCB2 gene knockout mice (female and male) at 5 weeks of age, respectively; the DF graph represents the body weight of various tissues of NUCB1 and / or NUCB2 gene knockout female mice at 6 weeks of age, respectively.

[0086] Figure 2 The figure shows the correlation between NUCB1 and / or NUCB2 gene knockout mice and atopic dermatitis, where Figure A shows the correlation between NUCB1 and / or NUCB2 gene knockout mice and atopic dermatitis. + / + With NUCB1 - / - NUCB2 + / + With NUCB2 - / - NUCB1&2 + / + With NUCB1&2 - / - A schematic diagram of constructing atopic dermatitis (AD) in female mice; Figure B shows the specific experimental procedure for constructing the AD mouse model; Figures C and D are NUCB1. + / + With NUCB1 - / - NUCB2 + / + With NUCB2 - / - NUCB1&2 + / + With NUCB1&2 - / - Results of atopic dermatitis induced in female mice in terms of body weight, skin thickness, and severity of clinical symptoms; Figure F shows the HE staining results of mouse skin sections, and Figure G shows the spleen weight results of mice.

[0087] Figure 3 This diagram illustrates the therapeutic effect of NUCB1 protein expression via AAV vector on atopic dermatitis in mice. Figure A shows a schematic diagram of tail vein administration of AAV; Figures B-C show the detection results of NUCB1 and NUCB2 proteins in the blood and liver of mice after tail vein injection of AAV-NUCB1 and AAV-NUCB2, respectively; Figure D shows the specific procedures for constructing the AD mouse model and the timing of AAV administration; Figure E shows the mouse body weight changes; Figure F shows a photograph of mouse skin after AAV treatment; and Figure G shows the results of mouse skin thickness and clinical scores. (Figure captions refer to...) Figure 3 E.

[0088] Figure 4The following figures illustrate the therapeutic effect of purified NUCB1 protein expressed in eukaryotic cells on atopic dermatitis in mice. Figure A shows a schematic diagram of intraperitoneal injection of the purified protein drug; Figure B shows the transfection of HEK293 cells with the CDS sequences of mouse NUCB1 and NUCB2 genes constructed into the eukaryotic expression vector pCDNA3.4 to obtain a large amount of eukaryotic expressed protein; Figure C shows the Coomassie brilliant blue staining results of purified NUCB1 and NUCB2 proteins; Figure D shows the concentration changes of NUCB1 and NUCB2 proteins after intraperitoneal injection in mice; Figure E shows the specific flowchart of AD mouse model construction and protein injection time; Figure F shows the mouse body weight changes; Figure G shows photographs of mouse skin after treatment with NUCB1 and NUCB2 proteins; and Figure H shows the mouse skin thickness and clinical score results. (Figure captions refer to...) Figure 4 Figure F; I shows the HE staining results of mouse skin sections.

[0089] Figure 5 This diagram illustrates the therapeutic effects of the NUCB1 gene on psoriasis and Crohn's disease. Figure A shows the flowchart of the IMQ-induced psoriasis mouse model; Figure B shows the weight changes of mice in each group of the psoriasis model; and Figure C shows the changes in skin thickness in psoriasis mice. (Figure captions refer to...) Figure 5 Figures B and D show the HE staining results of psoriasis mouse skin; Figure E is a flowchart of the TNBS-induced Crohn's Disease mouse model; Figure F shows the survival rate results of Crohn's Disease mice.

[0090] Figure 6 This demonstrates the therapeutic effect of the NUCB1 gene on a mouse model of multiple sclerosis (EAE). Figure A shows the MOG... 35-55 A schematic diagram of EAE induction, where PTX stands for pertussis toxin; Figure B shows the clinical scores of mice after tail vein injection of AAV-NUCB1 and AAV-NUCB2; Figures CE show the running ability of mice, including running distance (C), running time (D), and exhaustion speed (E). Detailed Implementation

[0091] Through in-depth research, the inventors unexpectedly discovered for the first time that nucleoside NUCB1 can effectively treat autoimmune diseases, particularly atopic dermatitis, psoriasis, Crohn's disease, and multiple sclerosis. Specifically, experiments involving knocking out the NUCB1 gene, overexpressing NUCB1 protein, or injecting NUCB1 protein into mice demonstrated that administering NUCB1 protein to mouse models of autoimmune diseases such as atopic dermatitis, psoriasis, and Crohn's disease effectively improved the skin phenotype, reduced clinical scores, and increased survival rates in these models, while nucleoside NUCB2 showed no significant effect. Therefore, nucleoside NUCB1 and its promoters can be used to prepare a pharmaceutical formulation for treating autoimmune diseases. Based on this, the inventors completed this invention.

[0092] the term

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0094] Atopic dermatitis (AD)

[0095] Atopic dermatitis (AD), also known as atopic eczema, is a common inflammatory skin disease characterized by recurrent eczematous lesions and intense itching, accompanied by significant skin dryness. The pathogenesis of AD is complex and is believed to be closely related to genetic and environmental factors. A family history of allergic diseases, such as in parents, is a strong risk factor for this disease. Genetic factors primarily affect skin barrier function and immune balance. Patients with AD often exhibit various immunological abnormalities, with Th2 cell activation being a key characteristic. Skin barrier function may also be impaired, such as a reduction or absence of filaggrin in the epidermis. Environmental factors include climate change, lifestyle modifications, improper bathing, and exposure to infectious and allergen stimuli. Although the exact pathogenesis of AD remains unclear, current research suggests that immune abnormalities, skin microbiota dysbiosis, and skin barrier dysfunction are important components in its development.

[0096] Psoriasis

[0097] Psoriasis, commonly known as "psoriasis vulgaris", is a common disease. Its causes are complex, including infection factors, mental factors, immune factors, genetic factors, and trauma. Among them, immune factors play an important role in the occurrence and development of psoriasis. Its cause is still unclear, but the incidence is gradually increasing. It mainly presents local symptoms of the body, such as the appearance of erythema accompanied by scales on the scalp, the extensor sides of the extremities, and the trunk. This is the most common type of psoriasis vulgaris. In addition, there are relatively rare disease types, such as pustular psoriasis, which can also be accompanied by small pustules in addition to the manifestations of psoriasis vulgaris. Psoriatic arthritis is often accompanied by damage and deformity of joints, especially the small joints at the ends, in addition to psoriasis skin lesions.

[0098] Crohn’s Disease

[0099] Crohn’s disease is a special intestinal immune disease with unclear causes, which can cause corresponding manifestations such as intestinal inflammation, ulcers, and intestinal wall thickening. The cause of Crohn’s disease may be related to autoimmunity, similar to autoimmune system diseases such as rheumatoid arthritis and systemic lupus erythematosus. The body's own immune system begins to attack the intestine, causing inflammatory manifestations. If the time is longer, intestinal stenosis, fistula perforation may occur, which can perforate to surrounding organs. Intestinal cobblestone-like hyperplasia may also occur, and perianal lesions may appear, such as anal fistula and perianal abscess.

[0100] Multiple Sclerosis

[0101] Multiple sclerosis is a disease that occurs when the immune system attacks the brain and spinal cord. The most commonly affected parts of this disease are the periventricular white matter, optic nerve, spinal cord, brainstem, and cerebellum. The main clinical features are multiple scattered lesions in the central nervous system white matter and the remission and recurrence presented during the course of the disease, the spatial multiplicity of symptoms and signs, and the temporal multiplicity of the course of the disease. The cause of multiple sclerosis is still unknown, but a family history increases the risk of the disease. The symptoms of multiple sclerosis vary from person to person, depending on the location and severity of nerve fiber damage. These symptoms usually include vision problems, fatigue, difficulty walking and maintaining balance, and paralysis or weakness of the limbs.

[0102] NUCBs (Nucleobindins) protein

[0103] Nucleobindins (NUCBs) are a family of calcium-binding secretory proteins, including NUCB1 and NUCB2. The function of NUCB2 was first reported in 2006 as suppressing appetite and reducing weight, but subsequent studies showed that NUCB2 lacks appetite and weight regulation functions under physiological conditions. The biological function of NUCB1 remains unclear. Therefore, the biological functions and molecular mechanisms of the NUCB family are still poorly understood. NUCB1 and NUCB2 proteins are evolutionarily conserved and share high homology; for example, the similarity between human and rat NUCB2 proteins is 87.4%, and the similarity between rat and mouse NUCB2 proteins is 95.7%. The DNA similarity between humans and mice is as follows: mouse NUCB1 is 84% ​​similarity, and mouse NUCB2 is 83% similarity. The protein sequence similarity is as follows: mouse NUCB1 is 89% similarity, and mouse NUCB2 is 87% similarity.

[0104] The full length of the human NUCB1 gene is 1485 bp (accession number NM_006184.6), located at Gene ID: 4924.

[0105] The full length of the human NUCB2 gene is 1265 bp (accession number NM_001352661.2), located at Gene ID: 4925.

[0106] The full-length mouse NUCB1 gene is 1379 bp (accession number NM_001163662.2), located at Gene ID: 18220.

[0107] The full-length NUCB2 gene in mice is 1262 bp (accession number NM_001130479.2), located at Gene ID: 53322.

[0108] NUCB1 protein

[0109] In this invention, the terms "inventive protein," "NUCB1 protein," and "NUCB1 polypeptide" are used interchangeably and all refer to proteins or polypeptides having the NUCB1 amino acid sequence. These include NUCB1 proteins with or without a starting methionine. Furthermore, the term also includes full-length NUCB1 and fragments thereof. The NUCB1 protein referred to in this invention includes its complete amino acid sequence, its secreted protein, its mutants, and its functionally active fragments.

[0110] The proteins of this invention can be recombinant proteins, natural proteins, or synthetic proteins. The proteins of this invention can be naturally purified products, chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the proteins of this invention can be glycosylated or non-glycosylated. The proteins of this invention may or may not include an initial methionine residue.

[0111] The present invention also includes NUCB1 protein fragments and analogs having NUCB1 protein activity. As used herein, the terms “fragment” and “analyte” refer to proteins that substantially retain the same biological function or activity as the native NUCB1 protein of the present invention.

[0112] The present invention also includes polypeptides or proteins having the same or similar functions and having 50% or more (preferably 60%, 70%, 80%, more preferably 90%, more preferably 95%, most preferably 98%, such as 99%) homology to the natural NUCB1 protein of the present invention. Protein variants may be derived sequences obtained by substitution, deletion, or addition of at least one amino acid, typically 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10, of the number of amino acids, and by adding one or more (typically less than 20, preferably less than 10, more preferably less than 5) amino acids to the C-terminus and / or N-terminus. For example, in said protein, substitution with amino acids of similar or comparable properties generally does not change the function of the protein, and adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. This invention includes analogs of the natural NUCB1 protein. The difference between the natural NUCB1 protein and the natural NUCB1 protein can be a difference in amino acid sequence, a difference in modifications that do not affect the sequence, or a combination of both. These protein analogs include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to a mutagen, site-directed mutagenesis, or other known biochemical techniques. Analogs also include those having residues different from the natural L-amino acid (e.g., D-amino acids), and those having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It should be understood that the proteins of this invention are not limited to the representative proteins exemplified above.

[0113] This invention also provides a polynucleotide sequence encoding the NUCB1 protein. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes DNA, genomic DNA, or synthetically produced DNA, which can be single-stranded or double-stranded. Polynucleotides encoding mature polypeptides include: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" can include a polynucleotide encoding this polypeptide, or it can include a polynucleotide also including additional coding and / or non-coding sequences. This invention also relates to variants of the above-mentioned polynucleotides that encode fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0114] In a preferred embodiment of the present invention, the DNA sequence encoding the human NUCB1 protein encodes the NUCB1 protein shown in SEQ ID NO.1 or 2, and the polynucleotide sequence encoding the NUCB1 protein is shown in SEQ ID NO.5 or 6.

[0115] SEQ ID NO.5 is the nucleotide sequence of the mouse NUCB1 gene; SEQ ID NO.6 is the nucleotide sequence of the human NUCB1 gene.

[0116] Based on the nucleotide sequence described herein, those skilled in the art can readily prepare the coding nucleic acid of this invention using various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc., and specific methods can be found in J. Sambrook, *Molecular Cloning: A Laboratory Manual*. As one embodiment of this invention, the coding nucleic acid sequence of this invention can be constructed by segmenting the nucleotide sequence and then performing overlap extension PCR.

[0117] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%.

[0118] The proteins and polynucleotides of the present invention are preferably provided in isolated form, and more preferably, purified to homogenization.

[0119] The full-length polynucleotide sequences of this invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed nucleotide sequences, especially open reading frame sequences, according to this invention, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. Once the relevant sequences are obtained, they can be obtained in large quantities using recombination. This typically involves cloning them into a vector, transforming them into cells, and then isolating the relevant sequences from the proliferated host cells using conventional methods.

[0120] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0121] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0122] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the polynucleotides of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE (RACE-cDNA end amplification) method is preferred. Primers used for PCR can be appropriately selected based on the sequence information disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0123] NUCB1 accelerator

[0124] In this invention, the NUCB1 promoter includes substances capable of increasing the activity and / or content of the NUCB1 gene or its protein in vivo or in vitro.

[0125] NuCB1 expression can be increased by the following methods: secreting large amounts of NuCB1 protein by the tissue itself, artificially overexpressing NuCB1 protein, artificially delivering NuCB1 protein (e.g., using a viral vector, such as an adeno-associated virus vector), or NuCB1 promoters.

[0126] In this invention, the NUCB1 promoter is not particularly limited, as long as it can promote the expression of NUCB1 or enhance the activity of NUCB1 protein, it is within the scope of protection of this invention.

[0127] In a preferred embodiment, the NUCB1 promoter comprises a small molecule compound.

[0128] Pharmaceutical Compositions and Kits

[0129] This invention provides compound pharmaceutical compositions containing (a) the NUCB1 gene, or its protein, or its promoter; and (b) a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated as injections, for example, using physiological saline or an aqueous solution containing glucose and other excipients prepared by conventional methods. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under sterile conditions. The pharmaceutical compositions of this invention can also be formulated as powders for nebulized inhalation. The dosage forms of the pharmaceutical compositions of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. The dosage of the active ingredient is a therapeutically effective amount. The pharmaceutical formulations of this invention can also be formulated as sustained-release formulations. The pharmaceutical compositions of this invention are preferably injection formulations. Furthermore, the pharmaceutical compositions of this invention can be used with other therapeutic agents. And, the pharmaceutical compositions of this invention may also include additional components selected from components for the prevention and / or treatment of autoimmune diseases.

[0130] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0131] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0132] The present invention also provides a medicine box for the prevention and / or treatment of autoimmune diseases, the medicine box comprising:

[0133] (i) a first container, and the active ingredient (a) NUCB1 gene, or its protein or its promoter, or a drug containing the active ingredient (a) located in the first container; and

[0134] (ii) Instructions for use, which describe how to administer the active ingredient (a) to prevent and / or treat autoimmune diseases.

[0135] The pharmaceutical compositions and kits of the present invention are suitable for the prevention and / or treatment of autoimmune diseases.

[0136] The formulation of this invention can be taken three times a day to once every ten days, or in a sustained-release manner once every ten days. The preferred method is once daily, as this facilitates patient adherence and significantly improves patient compliance.

[0137] When taking this medication, in most cases the total daily dose should be lower than (or in a few cases equal to or slightly greater than) the usual daily dose of each individual drug. Of course, the effective dose of the active ingredient may vary depending on the administration method and the severity of the disease being treated.

[0138] Treatment

[0139] The present invention also provides a method for preventing and / or treating autoimmune diseases using the two active ingredients of the present invention or corresponding pharmaceuticals, comprising administering an effective amount of the active ingredient (a) NUCB1 gene, or its protein or its promoter, to a mammal, or administering a pharmaceutical composition containing said active ingredient (a).

[0140] When the active ingredient of the present invention is used for the above-described uses, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the following forms: tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspension concentrate), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or transdermal patches, or non-gastrointestinally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspension concentrate in an isotonic medium). For example, these pharmaceutical formulations may contain about 0.01-99%, more preferably about 0.1%-90% (by weight) of the active ingredient mixed with a carrier.

[0141] The active ingredient or pharmaceutical composition of the present invention can be administered via conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral, or local administration. Preferred routes of administration include oral, subcutaneous, intramuscular, or intravenous administration.

[0142] From the perspective of ease of administration, preferred pharmaceutical compositions are liquid compositions, especially injectable formulations.

[0143] Furthermore, the active ingredients or drugs of the present invention can also be used in combination with other components or drugs for the prevention and / or treatment of autoimmune diseases.

[0144] The main advantages of this invention include:

[0145] This invention is the first to discover that the NUCB1 gene, or its protein, or its promoters, can significantly prevent and / or treat autoimmune diseases, especially atopic dermatitis, psoriasis, Crohn's disease, and multiple sclerosis.

[0146] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0147] Experimental methods:

[0148] 1. Gene knockout mice

[0149] NUCB1 knockout mice, NUCB2 knockout mice, and NUCB1&2 double knockout mice were constructed by the laboratory of Professor Li Lin at the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. Standard 6-8 week old C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in an SPF-grade animal facility on a normal diet (13.5% kcal), with lights off for 12 hours (19:30-07:30) and on for 12 hours (07:30-19:30).

[0150] 2. AAV virus expressing NUCB1 and NUCB2 proteins in mice and its treatment

[0151] AAV virus includes: (AAV virus prepared by Heyuan Biotechnology (Shanghai) Co., Ltd.)

[0152] (1) AAV2 / 8-Control virus (pAAV-CAG-MCS-3×FLAG-WPRE);

[0153] (2) AAV2 / 8-NUCB1 virus (pAAV-CAG-NUCB1-3×FLAG-WPRE);

[0154] (3) AAV2 / 8-NUCB2 virus (pAAV-CAG-NUCB2-3×FLAG-WPRE). All AAV viruses were administered via tail vein injection. An appropriate dose (0.5*10E10 to 6*10E10) of AAV virus was dissolved in physiological saline, and 100 μL was precisely administered to each mouse using a mouse tail vein injection device. Specific details regarding the timing and dosage of AAV administration are provided in the examples.

[0155] 3. Preparation of eukaryotic expression proteins NUCB1 and NUCB2 and their administration

[0156] Full-length mouse NUCB1 and NUCB2 proteins, with a His tag at the C-terminus, were constructed into the pCDNA3.4 expression vector and transiently transferred into HEK293 cell expression systems (the proteins were prepared by Nanjing Detai Biotechnology Co., Ltd.). The endotoxin levels of both NUCB1 and NUCB2 proteins were less than 1 EU / mg. Specific details regarding the timing and dosage of protein administration are provided in the examples.

[0157] 4. Oxazolone-induced mouse model of atopic dermatitis

[0158] Oxazolidinone-induced atopic dermatitis in mice is a classic model. The specific method is as follows: Female C57BL / 6J mice aged 6-8 weeks were acclimatized to their environment for 4-7 days, and their backs were shaved. On day 0 (the day after shaving), 50 μL of 2% oxazolidinone (MedChemExpress, HY-126360) was applied to the back to sensitize the mice. Starting on day 7, 50 μL of 1.6% or 0.4% oxazolidinone was applied every two days (Days 7, 9, 11, 13, 15, 17, 19, 21). Skin thickness was measured using calipers, and clinical scores were given based on symptoms. Samples were collected between days 22 and 25. High concentrations of oxazolidinone were dissolved in a 4:1 (v / v) acetone / olive oil solution, while low concentrations were dissolved in 50% ethanol. Specific details of the medication administration are provided in the examples.

[0159] 5. IMQ-induced mouse model of psoriasis

[0160] Female C57BL / 6J mice aged 6-8 weeks were selected. After acclimatization, their backs were shaved 4-7 days after purchase. On day 0 (the day after hair removal), imiquimod (IMQ) ointment (Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back skin of the mice continuously for 5 days. Application was stopped on days 6 and 7. The control group received the same dose of petroleum jelly ointment. One cycle consisted of 7 days, and two cycles of treatment were performed to establish a mouse model of psoriasis. Specific details of AAV administration are provided in the examples.

[0161] 6. TNBS-induced mouse Crohn's disease model

[0162] Female C57BL / 6J mice, aged 6-8 weeks, were selected. After acclimatization for 4-7 days, their backs were shaved. On day 0 (the day after shaving), 150 μL of 1% TNBS was applied to the backs of the mice to sensitize them. On day 7, the mice were fasted for 24 hours, but allowed free access to water. On day 8, the mice were given an enema of 100 μL of 2.5% TNBS. One hour before the enema, 100 μL of mannitol was administered to promote defecation. The experiment ended on day 11. See the examples for specific details on AAV administration.

[0163] 7. MOG 35-55 Induced EAE mouse model

[0164] Female C57BL / 6J mice aged 6-8 weeks were selected and allowed to acclimatize to their environment for 4-7 days after purchase. 2 mg / ml of MOG was prepared. 35-55 The emulsion was mixed with 1 mg / ml CFA (Complete Freund's Adjuvant) and injected subcutaneously into the skin of the forelimb scapula and posterior flank, 100 μL at each site. On the same day, 500 ng of pertussis toxin (PTX) was injected via the tail vein, followed by a second injection of 500 ng of pertussis toxin via the tail vein 48 hours later. Scores were observed every 3 days, and the mice's motor ability was evaluated using a treadmill after one month. Before the formal experiment, mice underwent acclimatization training on the treadmill for 3 days, once daily, with each training session ranging from 5 m / s to 10 m / s for a total of 6 minutes.

[0165] 8. Mouse tissue sampling

[0166] Detection of AAV protein expression in mouse liver: Take an appropriate amount of mouse liver and homogenize it in strong RIPA lysis buffer. Centrifuge at 13000 rpm and collect the supernatant. Detect the expression of NUCB1 and NUCB2 by Western blot (with Flag tag).

[0167] Detection of AAV expression proteins in blood: Blood was collected from the eye margin and centrifuged at 2500 rpm to obtain serum. NuCB1 and NuCB2 proteins in the blood were detected by immunoprecipitation with mouse flag antibody and Protein A / G beads. Rabbit flag antibody was used for detection in Western blot.

[0168] Stability of fusion proteins in blood: Following intraperitoneal injection of 50 μg / animal of NUCB1 or NUCB2 protein, blood samples were collected at 30, 60, 120, and 240 minutes. His-tagged NUCB1 and NUCB2 proteins in serum were enriched using a nickel-NTA (Ni-NTA) intraperitoneal chromatography (IP) method and detected by Western blot. HE staining of skin tissue: Skin tissue was fixed and stained with HE.

[0169] 9. Data Statistical Analysis

[0170] Data are presented as mean ± standard error (mean ± sem). P-values ​​were obtained by t-test (studentstest), one-way ANOVA, or one-way ANOVA. Significance was expressed as *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0171] Example 1: After NUCB1 gene knockout, mice exhibited potential autoimmune diseases.

[0172] Based on the above experimental methods, it was found that mice with NUCB1 gene knockout exhibited potential autoimmune diseases. The results are as follows: Figure 1 As shown, where,

[0173] (1) Body weight of NUCB1 and / or NUCB2 gene knockout mice at 5 weeks of age:

[0174] NUCB1 gene knockout (NUCB1 - / - The body weight of both male and female mice was significantly lower than that of wild-type mice (NUCB1). + / + )weight( Figure 1 A); while NUCB2 gene knockout (NUCB2 - / - The weight of male or female mice and the corresponding wild-type mice (NUCB2) + / + There was no difference in weight. Figure 1 B); NUCB1&2 double gene knockout (NUCB1&2 - / - The body weight of both male and female mice was significantly lower than that of wild-type mice (NUCB1 & 2). + / + )weight( Figure 1 C), and the weight loss was more severe than in NUCB1 knockout mice (e.g., male mice experienced a weight loss of approximately 24% (=(1-13g / 17g)*100), see Figure 1 (A and 1C). The mouse photos vividly illustrate NUCB1 & 2. - / - The mice are relatively small in size.

[0175] (2) Tissue weights of NUCB1 and / or NUCB2 knockout female mice at 6 weeks of age:

[0176] Compared to wild-type female mice, NUCB1 knockout mice showed a significantly increased proportion of brain and spleen, while the liver, lungs, pancreas, and uterus remained unchanged. Figure 1 D); while no significant differences were found in any tissue of NUCB2 gene knockout mice ( Figure 1 E); The proportions of the brain and spleen in NUCB1&2 double gene knockout mice were significantly increased, while the liver, lungs, pancreas, and uterus remained unchanged. Figure 1 F).

[0177] This invention is the first to discover that NUCB1 gene knockout mice exhibit smaller body size and weight, but also enlarged spleens. The enlarged spleen indicates an immune response in the mice. In the human population, children with various autoimmune diseases experience growth retardation compared to their peers. For children in their growth and development stage, long-term chronic inflammation can affect their absorption and utilization of nutrients, increasing bodily waste. Pain and joint problems caused by chronic inflammation can also limit children's daily physical and social activities. For example, in systemic juvenile idiopathic arthritis (SIA), approximately 41% of affected children experience growth retardation. These phenotypes in NUCB1 gene knockout mice suggest that the NUCB1 gene may play a role in autoimmune diseases.

[0178] Example 2: Detecting the effect of NUCB1 gene knockout mice on atopic dermatitis

[0179] Atopic dermatitis (AD) is an autoimmune disease known as the "number one disease," affecting hundreds of millions of people worldwide. To investigate whether the NUCB1 gene plays a role in autoimmune diseases, the inventors first used an oxazolone atopic dermatitis mouse model.

[0180] Figure 2 Figure A shows the NUC B1. + / + With NUCB1 - / - NUCB2 + / + With NUCB2 - / - NUCB1&2 + / + With NUCB1&2 - / - A schematic diagram of constructing atopic dermatitis (AD) in female mice; the specific procedure for constructing the AD mouse model is as follows. Figure 2 As shown in B, the specific experimental procedure is the same as the experimental method described above.

[0181] The results show that, compared with NUCB1 + / + In comparison, NUCB1 - / - The AD phenotype induced in mice was more severe, with a weight loss of approximately 17% (=(1-15g / 18g)*100), increased skin thickness (e.g., an increase of approximately 26% in skin thickness at day 19 (=(1-2.8mm / 3.8mm)*100), and more severe clinical symptoms (e.g., an increase of approximately 44% in clinical score at day 17 (=(1-5 / 9)*100)). Figure 2 C); while NUCB2 + / + With NUCB2 - / - There was no difference in AD phenotype induced in mice. Figure 2 D); with NUCB1&2 + / + Compared to NUCB1 & 2 - / -The AD phenotype induced in mice was more severe, with a body weight loss of approximately 25% (=(1-15g / 20g)*100), increased skin thickness (e.g., an increase of approximately 26% in skin thickness on day 17 (=(1-2.8mm / 3.8mm)*100), and more severe clinical symptoms (e.g., an increase of approximately 44% in clinical score on day 17 (=(1-5 / 9)*100)). Figure 2 E);

[0182] HE staining results of mouse skin sections showed that, compared with NUCB1 + / + In comparison, NUCB1 - / - The skin thickness of mice increased significantly. Figure 2 F); with NUCB1 + / + In comparison, NUCB1 - / - In the mouse-induced AD model, the spleen weight of mice increased significantly by approximately 27% (=(1-0.4 / 0.55)*100), while NUCB2 + / + With NUCB2 - / - There was no difference in spleen weight between mice in the induced AD model and NUCB1&2. + / + Compared to NUCB1 & 2 - / - In a mouse-induced AD model, the spleen weight of mice increased significantly by approximately 24% (=(1-0.38 / 0.5)*100)( Figure 2 G).

[0183] Example 3: Treatment of atopic dermatitis in mice by expressing NUCB1 protein via AAV vector.

[0184] Based on the above embodiments, the inventors confirmed the important function of the NUCB1 gene in atopic dermatitis using gene knockout mice. Without the NUCB1 gene, the AD phenotype in mice was more severe. In subsequent experiments, the inventors treated or improved AD by exogenously adding NUCB1 protein to mice.

[0185] Recombinant adeno-associated virus vectors (rAAVs) are derived from non-pathogenic wild-type adeno-associated virus. Due to their good safety profile, broad host cell range, low immunogenicity, and long in vivo expression time of exogenous genes, they are considered one of the most promising gene transfer vectors and are widely used in gene therapy and vaccine research worldwide. This invention first selects the AAV vector to express the NUCB1 protein in mice to treat Alzheimer's disease (AD). Since NUCB1 and NUCB2 are secretory proteins, they are secreted into the bloodstream after tissue expression. The specific procedures for constructing the AD mouse model and the timing of AAV use are as follows: Figure 3 As described in D, please refer to the experimental method above for the specific process.

[0186] The results showed that in C57 mice with an oxazolone-induced atopic dermatitis model, after intravenous injection of AAV-NUCB1 and AAV-NUCB2 into the tail vein ( Figure 3 A), NUCB1 and NUCB2 proteins were detected in the blood. Figure 3 B); This experiment used the AAV2 / 8 subtype vector. The liver is a very important organ for AAV infection, and NUCB1 and NUCB2 proteins were also detected in the liver. Figure 3 C), oxazolone-induced AD did not affect mouse body weight, and injections of AAV-NUCB1 and AAV-NUCB2 with viral genome copy numbers of 1.5*10E10 and 6*10E10 respectively also did not affect mouse body weight. Figure 3 E); AAV treatment significantly reduced skin symptoms in mice ( Figure 3 F); AAV-NUCB1 significantly reduced skin thickness and alleviated clinical symptoms in AD mice, with lower concentrations showing better results than higher concentrations; AAV-NUCB2 also reduced skin thickness and alleviated clinical symptoms in AD mice to some extent, but its effect was far less than that of NUCB1 (F). Figure 3 G).

[0187] Example 4:

[0188] This embodiment examines the therapeutic effect of purified NUCB1 protein expressed in eukaryotes on atopic dermatitis (AD) in mice. Building upon Example 3, which demonstrated the effectiveness of NUCB1 protein in treating AD via AAV vector administration, this embodiment treats AD by directly injecting the purified protein into mice. The specific experimental procedure follows the methods described above. The intraperitoneal injection of the purified protein drug is as follows: Figure 4 As shown in Figure A, the CDS sequences of the mouse NUCB1 and NUCB2 genes were constructed into the eukaryotic expression vector pCDNA3.4 and then transfected into HEK293 cells to obtain a large amount of eukaryotic expressed protein. Figure 4 B); The purified proteins were stained with Coomassie Brilliant Blue, and the results showed that NUCB1 and NUCB2 proteins had high purity. Figure 4 C).

[0189] After intraperitoneal injection of the purified NUCB1 and NUCB2 proteins into mice, the concentrations of NUCB1 and NUCB2 proteins decreased significantly over time (from 30 min to 240 min). Figure 4 D) indicates that NUCB1 and NUCB2 proteins have short half-lives in vivo.

[0190] Figure 4 E shows the specific procedure for constructing the AD mouse model and the injection time of NUCB1 and NUCB2 proteins. Due to the short half-life of the above proteins, the injection frequency of the above proteins was selected as twice a day.

[0191] The results showed that oxazolone-induced atopic dermatitis did not affect weight changes in mice. Similarly, intraperitoneal injections of 3 μg and 9 μg doses of NUCB1 or NUCB2 protein did not affect weight changes in mice with atopic dermatitis. Figure 4 F); Treatment of AD mice with intraperitoneal injection of 3 μg and 9 μg doses of NUCB1 or NUCB2 protein significantly reduced skin symptoms (F). Figure 4 G); among them, NUCB1 protein significantly reduced skin thickness and clinical symptoms in AD mice in a dose-dependent manner; NUCB2 protein also reduced skin thickness and clinical symptoms in AD mice in a dose-dependent manner, but the effect was far less than that of NUCB1 protein (G); Figure 4 H);

[0192] Similarly, HE staining of mouse skin sections showed that oxazolone-induced atopic dermatitis mice had significantly increased skin thickness. Treatment with either NUCB1 or NUCB2 protein dose-dependently reduced skin thickness in AD mice, but the reduction effect of NUCB2 protein was far less significant than that of NUCB1 protein. Figure 4 I).

[0193] In summary, the results of Examples 1-4 indicate that the NUCB1 gene plays an important role in autoimmune diseases (including at least atopic dermatitis). The absence of the NUCB1 gene resulted in more severe atopic dermatitis phenotypes in mice; and both administration methods, including AAV vector and purified protein, effectively treated mice with atopic dermatitis.

[0194] Example 5

[0195] This embodiment further explores the therapeutic role of the NUCB1 gene in more autoimmune diseases, selecting mouse models of psoriasis and Crohn's disease. The specific experimental procedure follows the methods described above. The procedure for the IMQ-induced psoriasis mouse model is as follows: Figure 5 As shown in Figure A, the procedure for the TNBS-induced Crohn's disease mouse model is as follows: Figure 5 As shown in E.

[0196] The results showed that in the IMQ-induced psoriasis mouse model, the mice's body weight gradually decreased during the first 3 days after IMQ application, but gradually returned to normal over time. Furthermore, there was no significant difference in body weight between the psoriasis mice and the control group after NUCB1 and NUCB2 protein injection. Figure 5B). Furthermore, AAV-NUCB1 at doses of 0.5*10E10 and 1*10E10 significantly reduced skin thickness, with the reduction being more significant in the second IMQ application cycle compared to the first IMQ application cycle; although AAV-NUCB2 at doses of 0.5*10E10 and 1*10E10 also significantly reduced skin thickness in psoriatic mice, the reduction was far less than that of AAV-NUCB1 (the therapeutic effect of AAV-NUCB1). Figure 5 C); Similarly, a similar effect was observed in HE staining results of psoriatic mice. Figure 5 D).

[0197] Compared with the blank control group, the survival rate of TNBS-induced Crohn's Disease model mice was reduced by 50%, while both 0.5*10E10 and 1*10E10 doses of AAV-NUCB1 significantly improved the survival rate of acute Crohn's disease mice (of which, the 0.5*10E10 dose of AAV-NUCB1 increased the survival rate of acute Crohn's disease mice to approximately 100%), while the 1*10E10 dose of AAV-NUCB2 had no significant effect on the survival rate of acute Crohn's disease mice, with a mortality rate of approximately 50%. Figure 5 F).

[0198] Example 6

[0199] This embodiment investigates the therapeutic effect of NUCB1 in multiple sclerosis (MS), an autoimmune disease. MS is a primary inflammatory demyelinating disease of the central nervous system, often resulting in impaired motor function. Experimental autoimmune encephalomyelitis (EAE) is a disease model primarily mediated by specifically sensitized CD4+ T cells and constructed through myelin protein immunization in experimental animals. It is currently recognized internationally as an ideal animal model for MS. The specific experimental procedure follows the methods described above, as illustrated in the diagram. Figure 6 As shown in Figure A.

[0200] The results showed that a dose of 0.5*10E10 of AAV-NUCB1 significantly reduced clinical scores (scores were mainly based on tail flaccidity, tail paralysis, hind limb incoordination, and the degree of hind limb paralysis), while a dose of 0.5*10E10 of AAV-NUCB2 did not improve clinical scores. Figure 6B). After 30 days, the mice's exercise capacity was evaluated using a treadmill. The results showed that a 0.5*10E10 dose of AAV-NUCB1 significantly increased exercise distance, exercise time, and speed to exhaustion (exhaustion was defined as 15 electric shocks or 5 seconds of continuous electric shocks), while a 0.5*10E10 dose of AAV-NUCB2 had no effect on exercise capacity. Figure 6 CE).

[0201] The relevant sequences involved in this invention are as follows:

[0202] SEQ ID NO.1 (Mouse NUCB1 protein sequence):

[0203] MPTSVPRGAPFLLLPPLLMLSAVLAVPVDRAAPPQEDSQATETPDTGLYYHRYLQEVINVLETDGHFREKLQAANAEDIKSGKLSQELDFVSHNVRTKLDELKRQEVSRLRMLL KAKMDAKQEPNLQVDHMNLLKQFEHLDPQNQHTFEARDLELLIQTATRDLAQYDAAHHEEFKRYEMLKEHERRRYLESLGEEQRKEAERKLQEQQRRHREHPKVNVPGSQAQLKE VWEELDGLDPNRFNPKTFFILHDINSDGVLDEQELEALFTKELEKVYDPKNEEDDMREMEEERLRMREHVMKNVDTNQDRLVTLEEFLASTQRKEFGDTGEGWKTVEMSPAYTEE ELKRFEEELAAREAELNARAQRLSQETEALGRSQDRLEAQKRELQQAVLQMEQRKQQLQEQSAPPSKPDGQLQFRADTDDAPVPAPAGDQKDVPASEKKVPEQPPELPQLDSQHL

[0204] SEQ ID NO.2 (human NUCB1 protein sequence):

[0205] MPPSGPRGTLLLLPLLLLLLLRAVLAVPLERGAPNKEETPATESPDTGLYYHRYLQEVIDVLETDGHFREKLQAANAEDIKSGKLSRELDFVSHHVRTKLDELKRQEVSRLRMLLKAKMDAEQDPNVQVDHLNLLKQFEHLDPQNQHTFEARDLELLIQTATRDLAQYDAAHHEEFKRYEMLKEHERRRYLESLGEEQRKEAERKLEEQQRRHREHPKVNVPGSQAQLKEVWEELDGLDPNRFNPKTFFILHDINSDGVLDEQELEALFTKELEKVYDPKNEEDDMREMEEERLRMREHVMKNVDTNQDRLVTLEEFLASTQRKEFGDTGEGWETVEMHPAYTEEELRRFEEELAAREAELNAKAQRLSQETEALGRSQGRLEAQKRELQQAVLHMEQRKQQQQQQQGHKAPAAHPEGQLKFHPDTDDVPVPAPAGDQKEVDTSEKKLLERLPEVEVPQHL

[0206] SEQ ID NO.3 (Mouse NUCB2 protein sequence):

[0207] MRWRIIQVQYCFLLVPCMLTALEAVPIDVDKTKVHNTEPVENARIEPPDTGLYYDEYLKQVIEVLETDPHFREKLQKADIEEIRSGRLSQELDLVSHKVRTRLDELKRQEVGRLRMLIKAKLDALQDTGMNHHLLLKQFEHLNHQNPNTFESRDLDMLIKAATADLEQYDRTRHEEFKKYEMMKEHERREYLKTLSEEKRKEEESKFEEMKRKHEDHPKVNHPGSKDQLKEVWEETDGLDPNDFDPKTFFKLHDVNNDGFLDEQELEALFTRELEKVYNPQNAEDDMIEMEEERLRMREHVMSEIDNNKDRLVTLEEFLRATEKKEFLEPDSWETLDQQQLFTEDELKEYESIIAIQENELKKRAEELQKQKEDLQRQHDHLEAQKQEYHQAVQHLEQKKLQQGIAPSGPAGELKFEPHT

[0208] SEQ ID NO.4 (Human NUCB2 Protein Sequence):

[0209] MRWRTILLQYCFLLITCLLTALEAVPIDIDKTKVQNIHPVESAKIEPPDTGLYYDEYLKQVIDVLETDKHFREKLQKADIEEIKSGRLSKELDLVSHHVRTKLDELKRQEVGRLRMLIKAKLDSLQDIGMDHQALLKQFDHLNHLNPDKFESTDLDMLIKAATSDLEHYDKTRHEEFKKYEMMKEHERREYLKTLNEEKRKEEESKFEEMKKKHENHPKVNHPGSKDQLKEVWEETDGLDPNDFDPKTFFKLHDVNSDGFLDEQELEALFTKELEKVYDPKNEEDDMVEMEEERLRMREHVMNEVDTNKDRLVTLEEFLKATEKKEFLEPDSWETLDQQQFFTEEELKEYENIIALQENELKKKADELQKQKEELQRQHDQLEAQKLEYHQVIQQMEQKKLQQGIPPSGPAGELKFEPHI

[0210] SEQ ID NO.5 Mouse NUCB1 Protein-Coding Nucleotide Sequence (>NM_001163662.2:78-1457):

[0211]

[0212] SEQ ID NO.6 Human NUCB1 protein encoding nucleotide sequence (>NM_006184.6:73-1458):

[0213]

[0214] SEQ ID NO.7 Mouse NUCB2 protein encoding nucleotide sequence (>NM_001130479.2:288-1550):

[0215]

[0216] SEQ ID NO.8 Human NUCB2 protein encoding nucleotide sequence (>NM_001352661.2:453-1718):

[0217]

[0218] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a NUCB1 gene, or its protein, or its promoter, for the preparation of a composition or formulation for the prevention and / or treatment of autoimmune diseases.

2. Use according to claim 1, characterized in that, The autoimmune diseases mentioned are selected from the following group: systemic lupus erythematosus, atopic dermatitis, rheumatoid arthritis, psoriasis, multiple sclerosis, Crohn's disease, asthma, type I diabetes, autoimmune liver disease, myasthenia gravis, vitiligo, alopecia areata, pemphigus, or Sjögren's syndrome.

3. Use according to claim 1, characterized in that, The NUCB1 protein is selected from the following group: (A) A polypeptide with an amino acid sequence as shown in SEQ ID NO. 1 or 2; (B) A NUCB1 protein derivative or its active fragment formed by substituting, deleting or adding one or more (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO.1 or 2. (C) A NUCB1 protein derivative or its active fragment thereof with ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% homology to the amino acid sequence shown in SEQ ID NO.1 or 2.

4. The use as described in claim 1, characterized in that, The NUCB1 gene mentioned above is selected from the following group: (a) A polynucleotide encoding a polypeptide as shown in SEQ ID NO. 1 or 2; (b) Polynucleotides with sequences as shown in SEQ ID NO. 5 or 6; (c) A polynucleotide whose nucleotide sequence is ≥95% homology (preferably ≥98%) to the sequence shown in SEQ ID NO. 5 or 6 and encodes the polypeptide shown in SEQ ID NO. 1 or 2; (d) and any of the polynucleotides complementary to those described in (a)-(c).

5. A pharmaceutical composition, characterized in that, include: (a1) A first active ingredient for the prevention and / or treatment of autoimmune diseases, the first active ingredient comprising: the NUCB1 gene, or its protein, or its promoter; (a2) A second active ingredient for the prevention and / or treatment of autoimmune diseases, the second active ingredient comprising: other medicines for the prevention and / or treatment of autoimmune diseases; and (b) Pharmaceutically acceptable carriers.

6. A medicine box, comprising: (i) a first container, and the active ingredient (a1) NUCB1 gene, or its protein or its promoter, or a drug containing the active ingredient (a) located in the first container; and (ii) A second container, and the active ingredient (a2) located in the second container, other medicines for the prevention and / or treatment of autoimmune diseases, or medicines containing the active ingredient (a2).

7. The medicine box as described in claim 6, characterized in that, The drug is available in the form of a transdermal patch, oral medication, or injection.

8. The medicine box as described in claim 6, characterized in that, The medicine box also contains instructions for use, which describe how to administer active ingredients (a1) and (a2) in combination to prevent and / or treat autoimmune diseases.

9. A method for screening potential therapeutic agents for autoimmune diseases, comprising: (a) In the test group, in the culture system, in the presence of the test compound, cells expressing the NUCB1 gene were cultured, and the expression level E1 of the NUCB1 gene in the culture system described in the test group was detected; Furthermore, in the control group where the test compound was absent and all other conditions were the same, the expression level E2 of the NUCB1 gene in the culture system of the control group was detected; and (b) Compare E1 and E2. If E1 is significantly higher than E2, the test compound is a potential therapeutic agent for autoimmune diseases.

10. The method as described in claim 9, characterized in that, The term "significantly higher than" means E1 / E2 ≥ 2, preferably ≥ 3, even better, ≥ 4.

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