Application of TGM2 in preparation of medicine for treating allogenic hematopoietic stem cell transplantation complications

Abstract

The invention particularly discloses application of TGM2 in preparation of a medicine for treating allogenic hematopoietic stem cell transplantation complications, and relates to the technical field of biological medicine. According to the invention, the application of the overexpressed TGM2 extracellular vesicles in preparation of drugs for treating acute infection and graft versus host disease is provided for the first time, human HEK293T cells are transfected through a TGM2 overexpressed lentiviral vector, and the overexpressed TGM2 extracellular vesicles are obtained after screening, culture, separation and purification. Animal model experiments prove that the overexpressed TGM2 extracellular vesicles effectively relieve clinical symptoms of mice with acute graft-versus-host diseases, prolong the life cycle of the mice and have good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to the application of TGM2 in the preparation of a medicament for treating complications of allogeneic hematopoietic stem cell transplantation. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently an effective method for treating various malignant hematological diseases such as leukemia, lymphoma, and multiple myeloma. According to statistics, in 2019, nearly ten thousand cases of allogeneic hematopoietic stem cell transplantation were performed by 140 medical institutions in China, among which haploidentical hematopoietic stem cell transplantation accounted for 60%. However, infection and graft-versus-host disease (GVHD) are the main causes leading to the failure after allo-HSCT. GVHD is a serious complication after allogeneic hematopoietic stem cell transplantation, and 25%-70% of patients can induce GVHD after allogeneic hematopoietic stem cell transplantation. In the past few decades, the main clinical means for treating GVHD has been relying on inducing overall immunosuppression. However, due to the lack of precision of this method, the non-relapse mortality rate of patients secondary to infection is relatively high, and it also increases the risks of drug resistance and recurrence of blood cancer. Therefore, there is an urgent need to explore new prevention and treatment methods clinically.

[0003] In recent years, it has been found that extracellular vesicles (EVs) with a lipid bilayer structure play a significant role in intercellular signal transduction and tumor suppression. EVs are an important paracrine form outside of cell-soluble cytokines, and their main characteristics include: (1) a diameter of 30-200 nm; (2) having the cytoplasmic and cell membrane components of the parental cells; (3) containing specific proteins of their source cells. The components of EVs mainly include: proteins, nucleotides (mRNA, miRNA, mtDNA, etc.), lipids, and metabolites, etc. Proteomic analysis has found that the protein composition of EVs is relatively complex, and the protein compositions of EVs from different cell sources are also different, indicating the complexity of the functions of EVs.

[0004] EVs are gradually regarded as a superior drug delivery carrier by researchers due to their multiple unique advantages. Compared with traditional liposome-based nanomaterials, the advantages include: (1) As a natural carrier produced by endogenous cells, it has better biocompatibility, stability, and lower cytotoxicity. (2) It can not only efficiently evade macrophage phagocytosis but also freely penetrate the blood-brain barrier. (3) The surface of the lipid bilayer can be endowed with disease-targeting molecules or therapeutic reagents, and functional molecules can be loaded inside to avoid degradation. In recent years, engineered EVs can load drugs with regenerative repair and immunomodulatory functions such as nucleic acids, proteins, and nanoparticles into the interior or surface of EVs, thereby achieving tissue-specific targeting, effective tissue penetration, and precise drug delivery. With the increase in basic research on EVs and the continuous development of modern biotechnology, engineered EVs will have a broader application prospect in future regenerative medicine and tumor targeted therapy.

[0005] Human tissue transglutaminase (TGM2, Tissue Transglutaminase 2) is a multifunctional enzyme with transglutaminase crosslinking (TGase), G-protein signal transduction, and kinase activities, and is considered to play a role in various diseases. Existing studies have shown that TGM2 has anti-inflammatory, antioxidant, and tissue regeneration and repair-promoting effects, but its application in drugs for treating GVHD has not been reported. Summary of the Invention

[0006] (I) Technical problems to be solved

[0007] In view of this, one of the main purposes of the present invention is to provide the use of TGM2 protein, a nucleic acid molecule encoding the protein, or its promoter in the preparation of a drug for preventing and / or treating complications of allogeneic hematopoietic stem cell transplantation. Overexpressing TGM2 extracellular vesicles effectively alleviated the clinical symptoms of acute graft-versus-host disease mice and extended the survival cycle of the mice, showing good clinical transformation and clinical application prospects.

[0008] (II) Technical solutions

[0009] To achieve the above purpose, the present invention provides the use of TGM2 protein, a nucleic acid molecule encoding the protein, or its promoter in the preparation of a drug for preventing and / or treating complications of allogeneic hematopoietic stem cell transplantation.

[0010] In one embodiment, the promoter is selected from an overexpression vector of the TGM2 coding sequence, a precursor protein or conjugate or complex that can be converted into TGM2 in vivo.

[0011] In one embodiment, the nucleic acid molecule includes DNA or RNA.

[0012] In one embodiment, the nucleic acid molecule is DNA.

[0013] In one embodiment, the nucleic acid molecule is selected from:

[0014] (1) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 1; or

[0015] (2) a molecule that hybridizes with the nucleotide sequence defined in (1) under stringent conditions;

[0016] (3) a nucleic acid molecule that is homologous or has sequence identity with the nucleotide sequence shown in SEQ ID NO: 1

[0017] (for example, more than 80% homologous or having more than 80% sequence identity, such as 80%, 85%, 90%, 95%, 98%, 99%), and encodes a protein having the activity of preventing and / or treating complications of allogeneic hematopoietic stem cell transplantation and / or diseases and / or symptoms related to complications of allogeneic hematopoietic stem cell transplantation;

[0018] (4) a nucleic acid molecule obtained by substituting, deleting or adding one or several nucleotides in the nucleotide sequence of (1) or (2) or (3), and encoding a protein or polypeptide having the activity of preventing and / or treating complications of allogeneic hematopoietic stem cell transplantation and / or diseases and / or symptoms related to complications of allogeneic hematopoietic stem cell transplantation.

[0019] In one embodiment, the nucleotide sequence of the nucleic acid molecule encoding the protein is as shown in SEQ ID NO: 1.

[0020] In one embodiment, the overexpression vector includes a viral vector or a non-viral vector.

[0021] In one embodiment, the viral vector includes one or a combination of an adeno-associated virus vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a retrovirus vector, a herpesvirus vector, a poxvirus vector, and a lentivirus vector.

[0022] In one embodiment, the non-viral vector includes one or a combination of mRNA, a DNA vector, nanoparticles, cationic polymers, exosomes, extracellular vesicles, and liposomes.

[0023] In one embodiment, the overexpression vector is a viral vector.

[0024] In one embodiment, the viral vector is a lentivirus vector.

[0025] In one embodiment, the lentiviral vector elements are in the order of Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin.

[0026] It is achieved through lentiviral transfection technology. Compared with other non-transfection technologies, this method has higher efficiency in enriching proteins into EVs and better safety. In addition, this technology has low cost, high generalizability, and can quickly prepare a large number of EVs carrying different types of exogenous small molecule proteins, with extremely high clinical translational application value.

[0027] In one embodiment, the TGM2 protein, the nucleic acid molecule encoding this protein, or its promoter is delivered through extracellular vesicles.

[0028] In one embodiment, the method for preparing the extracellular vesicles is as follows:

[0029] S1: Transfect cells with lentivirus and screen to obtain a cell line stably transfected with TGM2;

[0030] S2: Amplify and culture the cell line stably transfected with TGM2, and centrifuge to obtain the culture supernatant of TGM2-overexpressing cells;

[0031] S3: Isolate and purify to obtain cell-derived extracellular vesicles overexpressing TGM2.

[0032] In one embodiment, the method for preparing the extracellular vesicles is as follows:

[0033] S1: Transfect human HEK293T cells with lentivirus overexpressing TGM2 and screen to obtain a HEK293T cell line stably transfected with TGM2;

[0034] S2: Amplify and culture the HEK293T cell line stably transfected with TGM2, and centrifuge to obtain the culture supernatant of HEK293T cells overexpressing TGM2;

[0035] S3: Isolate and purify to obtain HEK293T cell-derived extracellular vesicles overexpressing TGM2.

[0036] In one embodiment, the titer of the lentivirus overexpressing TGM2 transfected in S1 is 1×10 8 PFU / mL.

[0037] In one embodiment, differential centrifugation is used for separation in S3: that is, centrifuge at 800g for 10 min to obtain the supernatant after removing dead cells; centrifuge at 2,000g for 10 min to obtain the supernatant after removing cell debris; centrifuge at 10,000g for 30 min to obtain the supernatant after removing cell organelles; centrifuge at 100,000g for 2 h to obtain the extracellular vesicle precipitate.

[0038] In one embodiment, the allogeneic hematopoietic stem cell transplantation complications and / or diseases and / or symptoms associated with allogeneic hematopoietic stem cell transplantation complications include one or more of infection, graft-versus-host disease, hemorrhagic cystitis, hepatic veno-occlusive disease, interstitial pneumonia, and implantation failure.

[0039] In one embodiment, the allogeneic hematopoietic stem cell transplantation complication is graft-versus-host disease.

[0040] In one embodiment, the graft-versus-host disease is acute graft-versus-host disease.

[0041] In one embodiment, the graft is one or more of bone marrow cells and spleen cells.

[0042] In another aspect, the present invention also provides a pharmaceutical composition, which comprises:

[0043] (1) A therapeutically effective amount of TGM2 protein, a nucleic acid molecule encoding the protein, and / or its promoter; wherein, the promoter is selected from an overexpression vector of the TGM2 coding sequence, a precursor protein or conjugate or complex that can be converted into TGM2 in vivo;

[0044] (2) Pharmaceutically acceptable excipients.

[0045] In one embodiment, the TGM2 protein, the nucleic acid molecule encoding the protein, and / or its promoter in the pharmaceutical composition account for 0.001-99.9 wt% of the total weight of the pharmaceutical composition.

[0046] In another aspect, the present invention also provides a pharmaceutical preparation, which contains the above-mentioned pharmaceutical composition.

[0047] In another aspect, the present invention also provides the use of the above-mentioned pharmaceutical composition and / or pharmaceutical preparation in the preparation of a drug for preventing and / or treating allogeneic hematopoietic stem cell transplantation complications.

[0048] Beneficial effects

[0049] Compared with the prior art, the beneficial effects of the present invention are:

[0050] 1. The overexpressed TGM2 extracellular vesicles provided by the present invention significantly prolong the survival of mice with acute graft-versus-host disease and effectively relieve the clinical symptoms of the mice.

[0051] 2. Preparation of overexpressed TGM2 extracellular vesicles is achieved through lentiviral transfection technology. Compared with other non-transfection technologies, this method has higher efficiency in enriching proteins into EVs and better safety. It can be stored at -80°C and can be used immediately after thawing, with easy control of the usage time. In addition, this technology has low cost, high popularization potential, and can quickly prepare a large number of EVs carrying different types of exogenous small molecule proteins, with extremely high clinical translational application value.

[0052] 3. Derived from human HEK293T cells, it has the advantage of lower in vivo transplantation immunogenicity. Brief Description of the Drawings

[0053] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0054] Figure 1 It is the map of the lentiviral vector for overexpression of TGM2;

[0055] Figure 2 It is the preparation flow chart of overexpressed engineered extracellular vesicles of TGM2 (TGM2-OE EVs);

[0056] Figure 3 It is the fluorescence expression efficiency of TGM2 protein detected by inverted fluorescence microscope after lentiviral transfection of HEK293T cells with overexpression of TGM2;

[0057] Figure 4 It is the fluorescence expression efficiency of TGM2 protein detected by flow cytometry after lentiviral transfection of HEK293T cells with overexpression of TGM2 and screening with puromycin;

[0058] Figure 5 It is the overexpression efficiency of TGM2 protein detected by Western blot after lentiviral transfection of HEK293T cells with overexpression of TGM2;

[0059] Figure 6 It is the particle size and concentration of EVs detected by NTA;

[0060] Figure 7 It is the morphology and size of EVs detected by TEM;

[0061] Figure 8 It is the surface characteristic markers of EVs detected by Western blot;

[0062] Figure 9Schematic diagram of the intervention of TGM2-OE-EVs in a mouse model of acute graft-versus-host disease;

[0063] Figure 10 Survival curve of mice after intervention with TGM2-OE-EVs in acute graft-versus-host disease;

[0064] Figure 11 Clinical symptom scores of mice after intervention with TGM2-OE-EVs in acute graft-versus-host disease;

[0065] Figure 12 Pathological changes in small intestine tissues of mice after intervention with TGM2-OE-EVs in acute graft-versus-host disease detected by HE staining. Detailed implementation mode

[0066] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0067] As used herein, "comprising", "having", or "including" includes "containing", "consisting essentially of...", "consisting substantially of...", and "consisting of"; "consisting essentially of...", "consisting substantially of...", and "consisting of" are subordinate concepts of "comprising", "having", or "including".

[0068] The term TGM2 protein (polypeptide) of the present invention refers to human tissue transglutaminase.

[0069] As used herein, "TGM2" and "TGM2 protein (polypeptide)" are used interchangeably and refer to human tissue transglutaminase. The human tissue transglutaminase disclosed in the present invention can be SEQ ID No. 2 or its homologous sequence, variant or modified form. For example, the TGM2 can be selected from the amino acid sequence shown in SEQ ID No. 2; or a protein or polypeptide having the same function after substitution, deletion or addition of one or several amino acids in the amino acid sequence shown in SEQ ID No. 2. SEQ ID No. 2: MAEELVLERCDLELET NGRDHHTADLCREKLVVRRGQPFWLTLHFEGRNYEASVDSLTFSVVTGPAPSQEAGTKARFPLRDAVEEGDWTATVVDQQDCTLSLQLTTPANAPIGLYRLSLEASTGYQGSSFVLGHFILLFNAWCPADAVYLDSEEERQEYVLTQQGFIYQGSAKFIKNIPWNFGQFEDGILDICLILLDVNPKFLKNAGRDCSRRSSPVYVGRVVSGMVNCNDDQGVLLGRWDNNYGDGVSPMSWIGSVDILRRWKNHGCQRVKYGQCWVFAAVACTVLRCLGIPTRVVTNYNSAHDQNSNLLIEYFRNEFGEIQGDKSEMIWNFHCWVESWMTRPDLQPGYEGWQALDPTPQEKSEGTYCCGPVPVRAIKEGDLSTKYDAPFVFAEVNADVVDWIQQDDGSVHKSINRSLIVGLKISTKSVGRDEREDITHTYKYPEGSSEEREAFTRANHLNKLAEKEETGMAMRIRVGQSMNMGSDFDVFAHITNNTAEEYVCRLLLCARTVSYNGILGPECGTKYLLNLNLEPFSEKSVPLCILYEKYRDCLTESNLIKVRALLVEPVINSYLLAERDLYLENPEIKIRILGEPKQKRKLVAEVSLQNPLPVALEGCTFTVEGAGLTEEQKTVEIPDPVEAGEEVKVRMDLLPLHMGLHKLVVNFESDKLKAVKGFRNVIIGPA. The protein or polypeptide disclosed in the present invention can be a natural purified product, a chemically synthesized product, or a product produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher animals, insects, and mammalian cells). The TGM2 protein or polypeptide in the present disclosure is preferably encoded by the human TGM2 gene or its homologous gene or family gene.

[0070] Nucleic acid molecule encoding TGM2

[0071] As used herein, the terms "TGM2 gene", "TGM2-encoding gene", "TGM2 protein-encoding gene", or "nucleic acid molecule encoding TGM2" are used interchangeably, and all refer to a nucleotide sequence encoding the TGM2 protein or polypeptide disclosed in the present invention. Such a nucleotide sequence can be, for example, the nucleotide sequence shown in SEQ ID NO:1, a molecule that hybridizes with these sequences under stringent conditions, or a family gene molecule highly homologous to the above-mentioned molecules.

[0072] Stringent conditions

[0073] As used herein, the term "stringent conditions" refers to: (1) hybridization and washing at a lower ionic strength and a higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90%, and more preferably more than 95%.

[0074] It should be understood that the TGM2 gene disclosed in the present invention is preferably obtained from humans, and other genes highly homologous to the human TGM2 gene obtained from other animals (such as having a sequence identity of more than 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, and more preferably more than 85%, such as 85%, 90%, 95%, 98%, or even 99% or more) are also within the equivalent scope preferably considered in the present disclosure. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

[0075] Genes are also within the equivalent scope preferably considered in the present disclosure. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

[0076] Promoter of TGM2 or TGM2-encoding sequence

[0077] The present disclosure also relates to the "promoter" of TGM2 or the TGM2-encoding sequence. The terms "promoter" or "promoter of TGM2 or its encoding sequence" are used interchangeably, and refer to a substance that can increase the level or activity of TGM2 or its encoding nucleic acid molecule. Promoters that can be used in the present disclosure include, but are not limited to: overexpression vectors of the TGM2-encoding sequence, precursor proteins or conjugates or complexes that can be converted into TGM2 in vivo.

[0078] Vector, host

[0079] The present disclosure also relates to vectors comprising the TGM2 gene, and host cells genetically engineered with such vectors.

[0080] Using conventional recombinant DNA techniques (Science, 1984; 224: 1431), the coding sequences disclosed herein can be used to express or produce recombinant TGM2 protein. Generally, the following steps are involved:

[0081] (1) Transforming or transducing a suitable host cell with the polynucleotide (or variant) encoding the TGM2 protein disclosed herein, or with a recombinant expression vector containing such polynucleotide;

[0082] (2) Culturing the host cell in a suitable medium;

[0083] (3) Isolating and purifying the protein or polypeptide from the medium or the cells.

[0084] As used herein, in the present disclosure, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, phages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is usually that it contains an origin of replication, a promoter, a marker gene and translation control elements.

[0085] Methods well known to those skilled in the art can be used to construct expression vectors containing the TGM2 coding sequence and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0086] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0087] Vectors containing the appropriate DNA sequences described above and appropriate promoters or control sequences can be used to transform appropriate host cells to enable them to express proteins or polypeptides. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell. Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells, etc. In the present disclosure, human HEK293T cells are preferably used as host cells.

[0088] The recombinant polypeptides in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatments, treatment with protein precipitants (salting-out methods), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography, and various other liquid chromatography techniques, as well as combinations of these methods.

[0089] Therapeutically effective amount

[0090] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose levels include factors such as the type and severity of the subject, age, gender, drug activity, sensitivity to the drug, administration time, administration route, and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

[0091] Pharmaceutically acceptable

[0092] As used herein, the term "pharmaceutically acceptable" means physiologically acceptable and, when administered to humans, generally does not cause gastrointestinal disorders, allergic reactions (such as dizziness), or similar reactions.

[0093] Excipient

[0094] As used herein, the term "excipient" includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-caking agents, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be further included.

[0095] Formulation

[0096] As used herein, the term "formulation" includes powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injectable solution, and sterile powder forms.

[0097] The composition can be administered by various routes and can be administered by general systemic administration or local administration (e.g., by subcutaneous injection, intra-synovial injection, intraperitoneal injection, intramuscular injection, or intravenous injection). However, it is not limited thereto.

[0098] Reagent description

[0099] High-glucose DMEM, fetal bovine serum (product of Gibco), trypsin (Sigma), antibiotics (product of Sigma), lentiviral vector for overexpression of TGM2, lentiviral vector of green fluorescent protein (Shanghai GeneChem Co., Ltd.), BCA protein quantification kit, goat anti-rabbit IgG secondary antibody labeled with horseradish peroxidase (HRP) (Beijing ComWin Biotech Co., Ltd.), premixed HRP chemiluminescent substrate, 0.22 μm sterile filter membrane (product of Millipore, USA).

[0100] Example 1

[0101] Preparation of lentivirus for overexpression of TGM2:

[0102] Entrust GeneChem Co., Ltd. to construct and prepare the lentiviral vector for overexpression of TGM2 and the control lentiviral vector of green fluorescent protein; as Figure 1

[0103] The present invention has no special limitation on the source of the TGM2 gene, and the well-known sources of the TGM2 gene in the art can be used. The present invention has no special limitation on the source of the lentivirus, and it can be obtained by purchasing through the well-known commercial channels of lentivirus in the art. The construction of the TGM2 overexpression lentivirus is consistent with the steps of constructing a conventional lentiviral vector, and there are no special requirements.

[0104] Example 2

[0105] Preparation of extracellular vesicles (the preparation flow chart is as Figure 2 shown):

[0106] S1: Culture HEK293T cells in a 10% high-glucose medium and maintain them in an incubator at 37°C and 5% CO2 with saturated humidity;

[0107] S2: Transfect human HEK293T cells with the TGM2 overexpression lentiviral vector and the control green fluorescent protein lentiviral vector described in Example 1 at a titer of 10 8 PFU / mL respectively, culture them in an incubator at 37°C and 5% CO2 with saturated humidity, and screen through puromycin for 1 week to obtain the TGM2 stable transfection HEK293T cell line and the green fluorescent protein stable transfection HEK293T cell line;

[0108] S3: Inoculate the TGM2 stable transfection HEK293T cell line and the green fluorescent protein stable transfection HEK293T cell line into high-glucose DMEM medium containing 10% fetal bovine serum respectively, and perform the first-round amplification culture in an incubator at 37°C and 5% CO2 with saturated humidity. When the cells are fused to 70-80%, passage them to a 10 cm 2 dish containing high-glucose DMEM with 10% fetal bovine serum. When the cells are fused to 50%-60%, add high-glucose medium containing 10% fetal bovine serum without serum extracellular vesicles for amplification culture. After 48 hours, collect the cell culture supernatant and centrifuge to remove floating live cells to obtain the HEK293T cell supernatant;

[0109] S4: Centrifuge the supernatant of the HEK293T cells at 4°C and 800×g for 10 min to obtain a supernatant free of dead cells; centrifuge the supernatant at 4°C and 2,000×g for 10 min to obtain a supernatant free of cell debris; centrifuge the supernatant at 4°C and 10,000×g for 30 min to obtain a supernatant free of organelles; centrifuge the supernatant at 100,000×g for 2 h and then discard the supernatant to obtain a precipitate of extracellular vesicles; resuspend the precipitate of extracellular vesicles with PBS and centrifuge at 100,000×g for 2 h to obtain a precipitate of extracellular vesicles derived from HEK293T cells overexpressing TGM2 (TGM2-OE-EVs) and a precipitate of extracellular vesicles derived from HEK293T cells overexpressing GFP (GFP-EVs). Finally, resuspend with PBS, filter through a 0.22-μm sterile filter membrane to sterilize, aliquot, store at -80°C, and perform protein quantification detection using the BCA protein quantification kit method.

[0110] Detection and characterization:

[0111] (1) Detect the fluorescence expression efficiency of TGM2 protein after transfection of HEK293T cells with lentivirus overexpressing TGM2 by inverted fluorescence microscopy (as Figure 3 shown).

[0112] (2) Detect the fluorescence expression efficiency of TGM2 protein after transfection of HEK293T cells with lentivirus overexpressing TGM2 and screening with puromycin by flow cytometry (as Figure 4 shown): The transfection efficiency is above 99%.

[0113] (3) Detect the overexpression efficiency of TGM2 protein after transfection of HEK293T cells with lentivirus overexpressing TGM2 by Western blot: Prepare a 15% SDS-PAGE electrophoresis gel, fully lyse the HEK293T cells described in S3 of Example 2, add 1 / 4 volume of 5×SDS loading buffer, boil for 5 min, load the sample according to a total protein amount of 200 μg, electrotransfer (350 mA, 120 min) to transfer the protein to a PVDF membrane, block at room temperature with TBS / T containing 50 g / L skim milk for 1 h, react with rabbit anti-human CD9 antibody and rabbit anti-human CD81 antibody (1:500) at 4°C overnight, wash the membrane 3 times with TBS / 0.5% Tween20 the next day, incubate with HRP-labeled goat anti-rabbit IgG secondary antibody at 37°C for 1 h. After washing the membrane 3 times with TBS / 0.5% Tween20, add a premixed HRP chemiluminescent substrate, and detect by a chemiluminescent gel imaging system. The results are as Figure 5As shown, both TGM2-modified HEK293T and non-TGM2-modified HEK293T express TGM2. At the same time, it can be found that the protein expression level in TGM2-modified HEK293T is significantly increased, proving that the establishment of the TGM2-overexpressing HEK293T stable cell line is successful.

[0114] (4) NTA detection of the particle size and concentration of EVs: Dilute 1 μL of TGM2-OE-EVs and GFP-EVs with pure drinking water from C'estbon, and adjust the concentration of TGM2-OE-EVs and GFP-EVs to 1.0×108 - 2.5×109 particles / mL according to the detection concentration range of the instrument. Use a 5 mL disposable sterile syringe to aspirate the diluted TGM2-OE-EVs and GFP-EVs and inject them into the sample detection chamber. The instrument will perform video capture, particle statistics, and potential analysis at 11 sites on the TGM2-OE-EVs and GFP-EVs in stable Brownian motion according to the internal processing software. The separation and identification results of TGM2-OE-EVs and GFP-EVs are as Figure 6 shown, and the peak concentrations of TGM2-OE-EVs and GFP-EVs are concentrated at approximately 120 nm.

[0115] (5) TEM detection of the morphology and size of EVs: Take 20 μL each of TGM2-OE-EVs and GFP-EVs, mix them well, and then drop them onto a copper specimen grid with a diameter of 2 mm. After standing at room temperature for 5 min, gently suck off the residual liquid at the edge of the copper grid with filter paper. Then, invert the copper grid onto a droplet of 30 g / L phosphotungstic acid (pH 6.8) and perform negative staining at room temperature for 5 min. Finally, dry the copper grid under an incandescent lamp and observe and take pictures under a transmission electron microscope. The separation and identification results of TG M2-OE-EVs and GFP-EVs are as Figure 7 shown, and the diameters of TGM2-OE-EVs and GFP-EVs are about 150 nm vesicular structures.

[0116] (6) Detection of surface characteristic markers of EVs by Western blot: Prepare a 15% SDS-PAGE electrophoresis gel. After fully lysing TGM2-OE-EVs and GFP-EVs, add 1 / 4 volume of 5× SDS loading buffer, boil for 5 min, load the sample according to a total protein amount of 200 μg, and electrotransfer (350 mA, 120 min) the protein onto a PVDF membrane. Block the membrane at room temperature for 1 h with TBS / T containing 50 g / L skim milk, and react with rabbit anti-human TGM 2 antibody, rabbit anti-human HSP70 antibody, rabbit anti-human GAPDH antibody, and rabbit anti-human Calnexin antibody (1:500) overnight at 4°C. The next day, wash the membrane 3 times with TBS / 0.5% Tween 20, then incubate with HRP-labeled goat anti-rabbit IgG secondary antibody at 37°C for 1 h. After washing the membrane 3 times with TBS / 0.5% Tween 20, add a premixed HRP chemiluminescent substrate, and detect it using a chemiluminescent gel imaging system. The results are as Figure 8 shown. TGM2-OE-EVs and GFP-EVs were positive for the markers HSP70 and GAPDH. At the same time, it was found that the expression level of TGM2 protein in TGM2-OE-EVs was significantly increased, demonstrating that the overexpressed TGM2 lentiviral vector was effective in modifying extracellular vesicles of HEK293T cells.

[0117] Example 3

[0118] Animal model experiments of extracellular vesicle therapy for infection and graft-versus-host disease

[0119] 8-week-old Balb / c mice (wild type, male, H-2Kd / H-2Dd) and 6-8-week-old C57BL / 6J (wild type, male, H-2Kb / H-2Db) were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. The mice were housed in a specific pathogen-free (SPF) grade platform, maintained at a constant temperature of 23 ± 2°C, humidity of 50 - 60%, and a 12-h day-night rhythm environment, and were able to move, eat, and drink freely. The 3R principle of laboratory animals was strictly followed throughout the experiment. All animal experiments were approved by the Ethics Committee of the University of Science and Technology of China (USTCACUC24120122042) and were carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health of the United States.

[0120] (1) BALB / c recipient mice started drinking sterile acidified water containing 250 mg / L erythromycin (powder dissolved in absolute ethanol) and 320 mg / L gentamicin (powder dissolved in sterile double-distilled water) 5 days before transplantation (changed every three days until the mice died or the experimental period ended). On the day of transplantation, the mice received whole-body irradiation with 8.0 Gy of 137Cs source γ-rays at a radiation rate of 1.47 Gy / min;

[0121] (2) Sacrifice C57BL / 6 donor mice. After soaking them in 75% medical alcohol for 5 min, transfer the bone marrow cells and spleen cells aseptically isolated in a biosafety cabinet. For the isolation of bone marrow cells: Remove the muscles outside the bones, cut both ends of the bones, aspirate 1×D-Hank's buffer with a 1 mL syringe to blow out the bone marrow, transfer it to a 70 μm filter mesh for grinding, rinse the filter mesh with 1×D-Hank's buffer, centrifuge at 4°C, 500×g for 5 min, resuspend with red blood cell lysate to remove red blood cells, keep at room temperature for 3 - 4 min, wait until the color of the cell suspension turns light red, then add RPMI1640 basal medium restored to room temperature to terminate the reaction, centrifuge at 4°C, 500×g for 5 min, resuspend with 1×D-Hank's buffer, filter through a 70 μm filter mesh, centrifuge at 4°C, 500×g for 5 min, resuspend with 1×D-Hank's buffer, and count; For the isolation of spleen cells: Carefully remove the attached fat on the spleen, transfer it to a 70 μm filter mesh for grinding, follow the same treatment as bone marrow cells, after treatment with red blood cell lysate and filtration, resuspend with 1×D-Hank's buffer and count;

[0122] (3) Randomly divide the irradiated BALB / c recipient mice into 3 groups. TBI group: Only irradiated without cell re-infusion; BM group: Only re-infuse 5×10 6 bone marrow cells after irradiation; aGVHD group: Give 5×10 6 bone marrow cells plus 2×10 6 spleen cells simultaneously after irradiation. The cells are transplanted into BALB / c recipient mice through tail vein injection within 4 - 6 h after irradiation.

[0123] (4) For the prevention and treatment of the aGVHD model: In the EVs treatment group, starting from the day before transplantation, inject 200 μL of EVs (TGM2-OE-EVs or GFP-EVs) with a particle number of 2×10 9 / mouse through the tail vein every other day until the 5th day; In the PBS group, starting from the day before transplantation, inject 200 μL of 1×DPBS through the tail vein every other day as a negative control group; In the DEX group (dexamethasone, a first-line therapeutic drug for aGVHD, starting from the first day after re-infusion, inject a dose of 5 mg / kg DEX through the tail vein every other day until the 5th day as a positive control group (the experimental process is as Figure 9 shown).

[0124] Detection and Results

[0125] (1) Survival curve of mice with acute graft-versus-host disease intervened by TGM2-OE-EVs: To evaluate whether TGM2-OE-EVs have a therapeutic effect on aGVHD mice, the survival rates of mice in each group after treatment were observed. The results are as Figure 10 shown. Mice in the BM graft group survived for a long time; while all mice in the aGVHD group died from the 7th day to the 43rd day after transplantation. Before death, symptoms such as weight loss, poor hair luster, diarrhea, eye diseases, bloody stools, hair loss, arched back, and decreased activity were successively observed in the mice; Mice in the Dex treatment group did not show symptoms for a period of time after transplantation, but gradually showed symptoms similar to those in the PBS group after the 15th day, and the progression was rapid. Deaths occurred successively from the 17th day to the 39th day, and the remaining 2 mice survived for a long time; Mice in the GFP-EVs treatment group died successively and all died from the 9th day to the 41st day; Mice in the TGM2-OE-EVs treatment group began to die on the 17th day after transplantation, and the remaining 4 mice survived for a long time. This indicates that TGM2-OE-EVs have a significant therapeutic effect and can effectively prolong the survival of aGVHD mice.

[0126] (2) Clinical symptom score: The aGVHD score was determined by analyzing the weight, body posture, activity, hair, skin, diarrhea, and eye diseases of recipient mice, and the evaluation indicators are as follows. Each item with good condition was recorded as 0 point, poor as 1 point, and very poor as 2 points. According to the evaluation indicators, each mouse was scored separately and cumulatively, and the total score was the sum of 7 scores, ranging from 0 to 14 points. The evaluation indicators are shown in the following table:

[0127] Type / Rating 0 1 2 Weight loss <10％ 10-25％ >25％ Posture Normal Moderate kyphosis Severe kyphosis Mobility Normal Mild to moderate reduction Activity after stimulation, with limited mobility Hair texture Smooth and shiny Mild to moderate curl, poor gloss Severe curl, no gloss Hair integrity Normal Mild hair loss (limbs, anus) Severe hair loss (whole body) Digestive tract Normal Loose stools Blood in stools Eyelid adhesion None Unilateral Bilateral

[0128] As Figure 11 shown, BM graft mice had transient clinical symptoms in the early stage of transplantation and recovered quickly in the later stage; while aGVHD mice successively showed continuously aggravated clinical symptoms including weight loss, arched back, decreased activity, curly hair, diarrhea with bloody stools, eyelid adhesion, etc.; Mice in the Dex treatment group did not show symptoms for a period of time after transplantation, but began to show clinical symptoms two weeks after transplantation; Mice in the GFP-EVs treatment group also began to show clinical symptoms similar to those of aGVHD mice on the 5th day after transplantation, but the situation was slightly better than that of aGVHD mice; Mice in the TGM2-OE-EVs treatment group began to show symptoms such as diarrhea and eye diseases on the 5th day after transplantation. Long-term scoring showed that TGM2-OE-EVs significantly improved the quality of life of aGVHD mice. This indicates that TGM2-OE-EVs can effectively relieve the clinical symptoms of aGVHD mice and improve the quality of life of transplanted mice.

[0129] (3) HE staining of small intestine tissue: As Figure 12As shown, the HE staining results of the small intestine showed that on the 7th day after transplantation, compared with the mice in the BM group, the small intestinal villi of the mice in the PBS group became shorter and shed, abscesses appeared in the crypts, the number was significantly reduced, and lymphocyte infiltration occurred in the lamina propria of the small intestine. On the 7th day after transplantation, the GFP-EVs intervention group was slightly better than the PBS group, with extensive necrosis and shedding of small intestinal epithelial cells, and serious damage or even disappearance of the small intestinal villi and crypt structures. The TGM2-OE-EVs group showed shorter villus length and a small amount of lymphocyte infiltration in the lamina propria on the 7th day after transplantation, and the damage to the small intestinal tissue was significantly improved. This indicates that TGM2-OE-EVs prolong the survival of aGVHD mice and improve the clinical symptoms of mice partly by improving the damage and inflammatory infiltration of the small intestinal tissue and promoting the regeneration of the damaged small intestine.

[0130] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the existence of additional identical elements in the process, method, article or device comprising the element.

[0131] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of TGM2 protein, nucleic acid molecule encoding the protein or its promoter in the preparation of a medicament for preventing and / or treating complications of allogeneic hematopoietic stem cell transplantation; wherein, The promoter is selected from an overexpression vector of the TGM2 coding sequence, a precursor protein or conjugate or complex capable of being converted into TGM2 in vivo.

2. The application according to claim 1, characterized in that, The nucleic acid molecule includes DNA or RNA.

3. The application according to claim 2, wherein The nucleic acid molecule is DNA; the nucleic acid molecule is the nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:

1.

4. The application according to claim 1, wherein The overexpression vector includes a viral vector or a non-viral vector.

5. The application according to claim 4, wherein The viral vector includes one or a combination of an adeno-associated virus vector, an adenovirus vector, an alphavirus vector, a herpes simplex virus vector, a vaccinia virus vector, a Sendai virus vector, a flavivirus vector, a rhabdovirus vector, a retrovirus vector, a herpesvirus vector, a poxvirus vector or a lentivirus vector; the non-viral vector includes one or a combination of mRNA, a DNA vector, nanoparticles, cationic polymers, exosomes, extracellular vesicles or liposomes.

6. The application according to claim 5, wherein The overexpression vector is a viral vector; the viral vector is a lentivirus vector.

7. The application according to any one of claims 1-6, characterized in that The TGM2 protein, the nucleic acid molecule encoding the protein or its promoter is delivered by extracellular vesicles.

8. The application according to claim 1, wherein The allogeneic hematopoietic stem cell transplantation complications include one or more of infection, graft-versus-host disease, hemorrhagic cystitis, hepatic veno-occlusive disease, interstitial pneumonia, and implantation failure.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes: (1) A therapeutically effective amount of the TGM2 protein, the nucleic acid molecule encoding the protein, and / or its promoter; wherein, the promoter is selected from an overexpression vector of the TGM2 coding sequence, a precursor protein or conjugate or complex capable of being converted into TGM2 in vivo; (2) A pharmaceutically acceptable excipient.

10. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation contains the pharmaceutical composition described in claim 9.

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