Application of compounds in the preparation of products for enhancing metabolic activity of hematopoietic stem cells and treating metachromatic leukodystrophy

Abstract

This application discloses the use of compounds, including azoramide, in the preparation of products for enhancing the metabolic activity of hematopoietic stem cells and treating metachromatic leukodystrophy. Azoramide treatment of transfected hematopoietic stem cells significantly improved apoptosis, necrosis, and mitochondrial respiration. Therefore, azoramide is expected to be used to enhance the transfection efficiency and metabolic activity of ARSA gene-transfected hematopoietic stem cells and reduce ROS generation, thereby enhancing its effectiveness in treating MLD.

Description

Technical Field

[0001] The present application relates to the technical field of metachromatic leukodystrophy, and in particular to the use of a compound in the preparation of a product for enhancing the metabolic activity of hematopoietic stem cells and treating metachromatic leukodystrophy. Background Art

[0002] Metachromatic leukodystrophy (MLD) is a lysosomal storage disease known as a sphingolipidosis. It is caused by the accumulation of fat in the brain, spinal cord, kidneys, and spleen. MLD is inherited in an autosomal recessive manner, and most cases are caused by pathogenic mutations in the arylsulfatase A (ARSA) gene. ARSA is a lysosomal acid hydrolase that catalyzes the degradation of cerebroside 3-sulfate. To date, over 100 mutations have been identified. These mutations may reduce arylsulfatase activity, hindering its transport to lysosomes, and ultimately preventing the normal degradation of its substrate, sulfatides. Consequently, sulfatides accumulate within nervous system cells, triggering the typical symptoms and pathological changes of MLD.

[0003] Currently, treatments for MLD include hematopoietic stem cell transplantation, bone marrow transplantation, enzyme replacement therapy, and gene therapy. While gene therapy is mostly still in the animal experimental stage, a few have entered clinical trials. For example, hematopoietic stem and progenitor cell gene therapy (HSPC-GT) has shown sustained increases in ARSA activity in the peripheral blood and cerebrospinal fluid of treated children, potentially protecting cognitive function, delaying severe motor decline, and slowing brain demyelination and atrophy. Alternatively, by transfecting functional ARSA into the hematopoietic stem cells of MLD patients using a vector and then re-transplanting the ARSA-transfected hematopoietic stem cells, the patient's disease progression can be delayed, demonstrating the effectiveness of this treatment approach. However, due to limitations in vector transfection efficiency and the metabolic activity of the transfected hematopoietic stem cells, the efficacy of this approach remains to be improved. Summary of the Invention

[0004] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes the use of a compound that effectively enhances the metabolic activity of hematopoietic stem cells.

[0005] In a first aspect of the present application, there is provided use of a compound in preparing a product for enhancing the activity of hematopoietic stem cells, or use of a compound in enhancing the activity of hematopoietic stem cells, wherein the compound includes Azoramide.

[0006] Azoramide, a small molecule modulator of the unfolded protein response (UPR), has the CAS number 932986-18-0. Azoramide improves ER protein folding capacity, activates long-term ER chaperones, enhances chaperone expression, promotes ER homeostasis, protects proteins from ER stress, and enhances β-cell function and survival. Studies suggest that azoramide may require intact IRE1 and the PERK branch of the UPR response to fully enhance chaperone capacity, thereby interacting with the UPR pathway to promote resolution of ER stress and improve ER function. Currently, research on azoramide-related diseases focuses primarily on obesity. For example, it can improve glucose homeostasis, insulin sensitivity, glucose tolerance, and β-cell function in mice with genetic obesity and diet-induced obesity. However, no studies have been reported in MLD.

[0007] In some embodiments of the present application, the working concentration of Azoramide is 1 to 1000 µmol / L (µM), for example, it can be 1 µmol / L, 2 µmol / L, 3 µmol / L, 4 µmol / L, 5 µmol / L, 6 µmol / L, 7 µmol / L, 8 µmol / L, 9 µmol / L, 10 µmol / L, 20 µmol / L, 30 µmol / L, 40 µmol / L, 50 µmol / L, 60 µmol / L, 70 µmol / L, 80 µmol / L, 90 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 400 µmol / L, 500 µmol / L, 600 µmol / L, 700 µmol / L, 800 µmol / L, 900 µmol / L, or 1000 µmol / L.

[0008] The working concentration refers to the concentration actually used when mixed with hematopoietic stem cells to enhance their activity. Azoramide can be stored and transported in a higher concentration solution, which is then diluted to the appropriate working concentration upon use. It is understood that other commonly used forms, such as dry powder, can also be used for storage and transportation.

[0009] In some embodiments of the present application, the hematopoietic stem cell is a hematopoietic stem cell transfected with an exogenous nucleic acid molecule.

[0010] Among them, transfection refers to the process of artificially introducing exogenous nucleic acid molecules into eukaryotic cells.

[0011] In some embodiments of the present application, the exogenous nucleic acid molecule can be at least one of DNA and RNA. In some embodiments of the present application, the exogenous nucleic acid molecule can be single-stranded, such as ssDNA and ssRNA, or double-stranded, such as dsDNA and dsRNA. In some embodiments of the present application, the exogenous nucleic acid molecule includes plasmid DNA, siRNA, shRNA plasmid, sgRNA, antisense oligonucleotide (ASO), ssODN, nucleic acid aptamer, miRNA, miRNA mimics, mRNA, etc.

[0012] In some embodiments of the present application, transfection can be achieved by a variety of different methods, including physical, chemical, and biological methods. In some embodiments of the present application, physical methods of transfection include electroporation, microinjection, gene guns, sonoporation, etc. In some embodiments of the present application, chemical methods of transfection include calcium phosphate coprecipitation, liposomes, polymers, nanocarriers, etc. In some embodiments of the present application, biological methods of transfection include viral vectors, in which case transfection is also referred to as transduction.

[0013] In some embodiments of the present application, exogenous nucleic acid molecules are transfected via at least one of liposomes, polymers, nanoparticles, and viral vectors. In some embodiments of the present application, liposomes include cationic liposomes, ionizable lipids (LNPs), polymer-lipid hybrids, and liposome-peptide complexes. In some embodiments of the present application, polymers include linear / branched polyethyleneimine (PEI), polylysine (PLL), dendrimers (such as PAMAM), chitosan derivatives (such as trimethyl chitosan), and thermosensitive polymers. In some embodiments of the present application, nanoparticles include gold nanoparticles, magnetic nanoparticles, mesoporous silica, exosomes / extracellular vesicles, and metal-organic frameworks. In some embodiments of the present application, viral vectors include lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, herpes simplex viruses, and Newcastle disease viruses.

[0014] In some embodiments of the present application, the liposome-containing reagent includes any one of Lipofectamine 2000, Lipofectamine 3000, Lipofectamine 6000, Lipofectamine stem cell transfection reagent, etc.

[0015] In some embodiments of the present application, hematopoietic stem cells deliver a gene editing system containing an exogenous nucleic acid molecule via liposomes. In some embodiments of the present application, the gene editing system includes a CRISPR gene editing system, such as any one of CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13. In some embodiments of the present application, the composition of the CRISPR gene editing system includes a Cas enzyme and a guide RNA. In some embodiments of the present application, the Cas enzyme includes any one of Cas9, Cas12a, and Cas13. In some embodiments of the present application, the Cas enzyme can be a wild-type or mutant enzyme. In some embodiments of the present application, the guide RNA is sgRNA. In some embodiments of the present application, the Cas enzyme can be transfected in the form of a protein or a nucleic acid. It is understandable that when the Cas enzyme is transfected in the form of a nucleic acid, the corresponding enzyme is then generated in the cell.

[0016] In some embodiments of the present application, the gene editing method of hematopoietic stem cells through transfection of exogenous nucleic acid molecules includes at least one of knock-in, knock-out, mutation, epigenetic modification, etc.

[0017] In some embodiments of the present application, the target gene for gene editing includes ARSA.

[0018] ARSA (arylsulfatase A) is located on chromosome 22q13.33. ARSA encodes a lysosomal hydrolase of the sulfatase family. Sulfatase catalyzes the breakdown of sulfatides, maintaining myelin lipid homeostasis. Mutations in ARSA are associated with metachromatic leukodystrophy (MLD). These mutations significantly reduce or eliminate enzyme activity, preventing the breakdown of the substrate sulfatides, leading to their accumulation within lysosomes. This leads to symptoms such as progressive demyelination and glial cell damage.

[0019] In some embodiments of the present application, the exogenous nucleic acid molecule includes an sgRNA targeting ARSA. The specific sgRNA can be designed using methods well known in the art, for example, design prediction and verification can be performed using relevant website software, such as CRISPOR, GuideScan, Cas-OFFinder, CRISPRitz, etc.

[0020] In some embodiments of the present application, enhancing the activity of hematopoietic stem cells includes enhancing the activity of hematopoietic stem cells under the action of sulfatide.

[0021] In some embodiments of the present application, enhancing the activity of hematopoietic stem cells includes at least one of reducing hematopoietic stem cell apoptosis, reducing hematopoietic stem cell necrosis, and enhancing the metabolic activity of hematopoietic stem cells under the action of sulfatide.

[0022] In some embodiments of the present application, enhancing the activity of hematopoietic stem cells includes at least one of reducing hematopoietic stem cell apoptosis, reducing hematopoietic stem cell necrosis, and enhancing the metabolic activity of hematopoietic stem cells.

[0023] Among them, apoptosis refers to a programmed cell death process, which is a physiological process that occurs actively during the development of organisms, to maintain tissue homeostasis, and to eliminate damaged or potentially dangerous cells. Necrosis refers to a non-programmed, non-regulated pathological cell death, which is usually caused by severe physical and chemical stimulation or damage (such as ischemia and hypoxia, high temperature, low temperature, physical trauma, strong acid and alkali, toxins, infection, etc.), resulting in the destruction of cell membrane integrity and leakage of cell contents.

[0024] In some embodiments of the present application, enhancing the metabolic activity of hematopoietic stem cells includes at least one of enhancing mitochondrial respiration of hematopoietic stem cells, changing the energy phenotype, and reducing the generation of reactive oxygen species.

[0025] In some embodiments of the present application, changing the mitochondrial respiration of hematopoietic stem cells includes changing at least one of the basal oxygen consumption, maximum oxygen consumption, and respiratory reserve capacity of mitochondrial respiration. Wherein, the change can be an enhancement or a weakening. Therefore, in some embodiments of the present application, changing the mitochondrial respiration of hematopoietic stem cells includes enhancing or weakening the basal oxygen consumption of mitochondrial respiration, enhancing or weakening the maximum oxygen consumption of mitochondrial respiration, and enhancing or weakening the respiratory reserve capacity of mitochondrial respiration. In some embodiments of the present application, enhancing or weakening the mitochondrial respiration of hematopoietic stem cells includes enhancing or weakening the basal oxygen consumption of mitochondrial respiration, enhancing or weakening the maximum oxygen consumption of mitochondrial respiration, and enhancing or weakening the respiratory reserve capacity of mitochondrial respiration.

[0026] In some embodiments of the present application, changing the energy phenotype comprises changing the energy phenotype to any one of aerobic, high energy, resting, and glycolytic.

[0027] In some embodiments of the present application, reducing the generation of reactive oxygen species includes reducing the generation of superoxide anions.

[0028] In some embodiments of the present application, the hematopoietic stem cells are hematopoietic stem cells derived from mammals (such as monotremes, marsupials, insectivores, shrews, scaphotheres, dermoptera, chiroptera, primates, edentata, phlonotida, lagomorpha, rodents, carnivores, sirenians, hyraxes, tubulodonta, perissodactyla, artiodactyla, cetaceans, etc.), specifically including hematopoietic stem cells from at least one of rodents (such as mice, rats, hamsters, guinea pigs), lagomorphs (such as rabbits), perissodactyla (such as horses), artiodactyla (such as sheep, pigs), primates (such as monkeys, orangutans, gorillas, chimpanzees, humans), and carnivores (such as dogs). It is understandable that when the hematopoietic stem cells are non-human hematopoietic stem cells, their immunogenicity can be reduced by methods well known in the art, such as genetic engineering to knock out major xenoantigens, express human protective proteins, immune isolation (such as microencapsulation), donor / recipient pretreatment (immunosuppression and tolerance induction, such as administering immunosuppressants to the subject, etc.), and specifically any one or more of these methods can be used in combination.

[0029] In some embodiments of the present application, the hematopoietic stem cells are at least one of autologous hematopoietic stem cells, allogeneic hematopoietic stem cells, and xenogeneic hematopoietic stem cells. In some embodiments of the present application, the hematopoietic stem cells are at least one of wild-type hematopoietic stem cells and pretreated hematopoietic stem cells (e.g., genetically engineered, pretreated with small or macromolecules, or physically or chemically treated).

[0030] The second aspect of the present application provides the use of a compound in preparing a product for treating metachromatic leukodystrophy, or the use of a compound in treating metachromatic leukodystrophy, wherein the compound includes Azoramide.

[0031] In some embodiments of the present application, the working concentration of Azoramide is 1 to 1000 µmol / L (µM), for example, it can be 1 µmol / L, 2 µmol / L, 3 µmol / L, 4 µmol / L, 5 µmol / L, 6 µmol / L, 7 µmol / L, 8 µmol / L, 9 µmol / L, 10 µmol / L, 20 µmol / L, 30 µmol / L, 40 µmol / L, 50 µmol / L, 60 µmol / L, 70 µmol / L, 80 µmol / L, 90 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 400 µmol / L, 500 µmol / L, 600 µmol / L, 700 µmol / L, 800 µmol / L, 900 µmol / L, or 1000 µmol / L.

[0032] In a third aspect of the present application, a composition is provided, comprising hematopoietic stem cells and Azoramide.

[0033] In some embodiments of the present application, the working concentration of Azoramide is 1 to 1000 µmol / L (µM), for example, it can be 1 µmol / L, 2 µmol / L, 3 µmol / L, 4 µmol / L, 5 µmol / L, 6 µmol / L, 7 µmol / L, 8 µmol / L, 9 µmol / L, 10 µmol / L, 20 µmol / L, 30 µmol / L, 40 µmol / L, 50 µmol / L, 60 µmol / L, 70 µmol / L, 80 µmol / L, 90 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 400 µmol / L, 500 µmol / L, 600 µmol / L, 700 µmol / L, 800 µmol / L, 900 µmol / L, or 1000 µmol / L.

[0034] In some embodiments of the present application, hematopoietic stem cells and Azoramide are premixed or separately provided.

[0035] In some embodiments of the present application, the hematopoietic stem cells are hematopoietic stem cells derived from mammals (such as monotremes, marsupials, insectivores, shrews, scaphotheres, dermoptera, chiroptera, primates, edentata, phlonotida, lagomorpha, rodents, carnivores, sirenians, hyraxes, tubulodonta, perissodactyla, artiodactyla, cetaceans, etc.), specifically including hematopoietic stem cells from at least one of rodents (such as mice, rats, hamsters, guinea pigs), lagomorphs (such as rabbits), perissodactyla (such as horses), artiodactyla (such as sheep, pigs), primates (such as monkeys, orangutans, gorillas, chimpanzees, humans), and carnivores (such as dogs). It is understandable that when the hematopoietic stem cells are non-human hematopoietic stem cells, their immunogenicity can be reduced by methods well known in the art, such as genetic engineering to knock out major xenoantigens, express human protective proteins, immune isolation (such as microencapsulation), donor / recipient pretreatment (immunosuppression and tolerance induction, such as administering immunosuppressants to the subject, etc.), and specifically any one or more of these methods can be used in combination.

[0036] In some embodiments of the present application, the hematopoietic stem cells are at least one of autologous hematopoietic stem cells, allogeneic hematopoietic stem cells, and xenogeneic hematopoietic stem cells. In some embodiments of the present application, the hematopoietic stem cells are at least one of wild-type hematopoietic stem cells and pretreated hematopoietic stem cells (e.g., genetically engineered, pretreated with small or macromolecules, or physically or chemically treated).

[0037] In some embodiments of the present application, the hematopoietic stem cell is a hematopoietic stem cell transfected with an exogenous nucleic acid molecule.

[0038] In some embodiments of the present application, the exogenous nucleic acid molecule can be at least one of DNA and RNA. In some embodiments of the present application, the exogenous nucleic acid molecule can be single-stranded, such as ssDNA and ssRNA, or double-stranded, such as dsDNA and dsRNA. In some embodiments of the present application, the exogenous nucleic acid molecule includes plasmid DNA, siRNA, shRNA plasmid, sgRNA, antisense oligonucleotide (ASO), ssODN, nucleic acid aptamer, miRNA, miRNA mimics, mRNA, etc.

[0039] In some embodiments of the present application, transfection can be achieved by a variety of different methods, including physical, chemical, and biological methods. In some embodiments of the present application, physical methods of transfection include electroporation, microinjection, gene guns, sonoporation, etc. In some embodiments of the present application, chemical methods of transfection include calcium phosphate coprecipitation, liposomes, polymers, nanocarriers, etc. In some embodiments of the present application, biological methods of transfection include viral vectors, in which case transfection is also referred to as transduction.

[0040] In some embodiments of the present application, exogenous nucleic acid molecules are transfected via at least one of liposomes, polymers, nanoparticles, and viral vectors. In some embodiments of the present application, liposomes include cationic liposomes, ionizable lipids (LNPs), polymer-lipid hybrids, and liposome-peptide complexes. In some embodiments of the present application, polymers include linear / branched polyethyleneimine (PEI), polylysine (PLL), dendrimers (such as PAMAM), chitosan derivatives (such as trimethyl chitosan), and thermosensitive polymers. In some embodiments of the present application, nanoparticles include gold nanoparticles, magnetic nanoparticles, mesoporous silica, exosomes / extracellular vesicles, and metal-organic frameworks. In some embodiments of the present application, viral vectors include lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, herpes simplex viruses, and Newcastle disease viruses.

[0041] In some embodiments of the present application, the liposome-containing reagent includes any one of Lipofectamine 2000, Lipofectamine 3000, Lipofectamine 6000, Lipofectamine stem cell transfection reagent, etc.

[0042] In some embodiments of the present application, hematopoietic stem cells deliver a gene editing system containing an exogenous nucleic acid molecule via liposomes. In some embodiments of the present application, the gene editing system includes a CRISPR gene editing system, such as any one of CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13. In some embodiments of the present application, the composition of the CRISPR gene editing system includes a Cas enzyme and a guide RNA. In some embodiments of the present application, the Cas enzyme includes any one of Cas9, Cas12a, and Cas13. In some embodiments of the present application, the Cas enzyme can be a wild-type or mutant enzyme. In some embodiments of the present application, the guide RNA is sgRNA. In some embodiments of the present application, the Cas enzyme can be transfected in the form of a protein or a nucleic acid. It is understandable that when the Cas enzyme is transfected in the form of a nucleic acid, the corresponding enzyme is then generated in the cell.

[0043] In some embodiments of the present application, the gene editing method of hematopoietic stem cells through transfection of exogenous nucleic acid molecules includes at least one of knock-in, knock-out, mutation, epigenetic modification, etc.

[0044] In some embodiments of the present application, the target gene for gene editing includes ARSA.

[0045] In some embodiments of the present application, the exogenous nucleic acid molecule includes an sgRNA targeting ARSA. The specific sgRNA can be designed using methods well known in the art, for example, design prediction and verification can be performed using relevant website software, such as CRISPOR, GuideScan, Cas-OFFinder, CRISPRitz, etc.

[0046] In some embodiments of the present application, hematopoietic stem cells are seeded in a hematopoietic stem cell culture medium. In some embodiments of the present application, the hematopoietic stem cell culture medium is a serum-containing culture medium or a serum-free culture medium. Because serum-containing culture medium has certain immunological issues, in some embodiments of the present application, the hematopoietic stem cell culture medium is a serum-free culture medium.

[0047] In some embodiments of the present application, the seeding amount of hematopoietic stem cells in the hematopoietic stem cell culture medium is 1×10 3 ~1×10 6 / mL, for example, it can be 1×10 3 / mL, 2×10 3 / mL, 3×10 3 / mL, 4×10 3 / mL, 5×10 3 / mL, 6×10 3 / mL, 7×10 3 / mL, 8×10 3 / mL, 9×10 3 / mL, 1×10 4 / mL, 2×10 4 / mL, 3×10 4 / mL, 4×10 4 / mL, 5×10 4 / mL, 6×10 4 / mL, 7×10 4 / mL, 8×10 4 / mL, 9×10 4 / mL, 1×10 5 / mL, 2×10 5 / mL, 3×10 5 / mL, 4×10 5 / mL, 5×10 5 / mL, 6×10 5 / mL, 7×10 5 / mL, 8×10 5 / mL, 9×10 5 / mL, 1×10 6 pieces / mL.

[0048] In some embodiments of the present application, the culture time of hematopoietic stem cells in the hematopoietic stem cell culture medium is 1 hour to 30 days, for example, it can be 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 15 days, 16 days, 18 days, 20 days, 25 days, or 30 days.

[0049] In some embodiments of the present application, the culture temperature of hematopoietic stem cells in the hematopoietic stem cell culture medium is 30-40°C, for example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0050] In some embodiments of the present application, hematopoietic stem cells are cultured in a cell culture vessel.

[0051] In some embodiments of the present application, the cell culture container includes a cell culture plate (such as a 4-well, 6-well, 8-well, 12-well, 24-well, 48-well, 96-well, 384-well, or 1536-well culture plate), a cell culture dish (such as a 35 mm, 60 mm, 100 mm, or 150 mm culture dish), a cell culture flask (such as a T25, T75, T175, or T225 culture flask), a cell factory (such as a 1-layer, 2-layer, 3-layer, 4-layer, 5-layer, 10-layer, or 40-layer cell factory), and the like.

[0052] In some embodiments of the present application, the raw material for preparing the cell culture container includes a polymer, such as polystyrene.

[0053] In some embodiments of the present application, during the culture process of hematopoietic stem cells in the cell culture container, the culture medium is replaced and the cell culture container is subcultured every 1 to 5 days, for example, every 1, 2, 3, 4, or 5 days.

[0054] The fourth aspect of the present application provides use of a composition in preparing a product for treating metachromatic leukodystrophy, or use of a composition in treating metachromatic leukodystrophy, wherein the composition comprises hematopoietic stem cells and Azoramide.

[0055] In some embodiments of the present application, the working concentration of Azoramide is 1 to 1000 µmol / L (µM), for example, it can be 1 µmol / L, 2 µmol / L, 3 µmol / L, 4 µmol / L, 5 µmol / L, 6 µmol / L, 7 µmol / L, 8 µmol / L, 9 µmol / L, 10 µmol / L, 20 µmol / L, 30 µmol / L, 40 µmol / L, 50 µmol / L, 60 µmol / L, 70 µmol / L, 80 µmol / L, 90 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 400 µmol / L, 500 µmol / L, 600 µmol / L, 700 µmol / L, 800 µmol / L, 900 µmol / L, or 1000 µmol / L.

[0056] In some embodiments of the present application, hematopoietic stem cells and Azoramide are premixed or separately provided.

[0057] In some embodiments of the present application, the hematopoietic stem cells are hematopoietic stem cells derived from mammals (such as monotremes, marsupials, insectivores, shrews, scaphotheres, dermoptera, chiroptera, primates, edentata, phlonotida, lagomorpha, rodents, carnivores, sirenians, hyraxes, tubulodonta, perissodactyla, artiodactyla, cetaceans, etc.), specifically including hematopoietic stem cells from at least one of rodents (such as mice, rats, hamsters, guinea pigs), lagomorphs (such as rabbits), perissodactyla (such as horses), artiodactyla (such as sheep, pigs), primates (such as monkeys, orangutans, gorillas, chimpanzees, humans), and carnivores (such as dogs). It is understandable that when the hematopoietic stem cells are non-human hematopoietic stem cells, their immunogenicity can be reduced by methods well known in the art, such as genetic engineering to knock out major xenoantigens, express human protective proteins, immune isolation (such as microencapsulation), donor / recipient pretreatment (immunosuppression and tolerance induction, such as administering immunosuppressants to the subject, etc.), and specifically any one or more of these methods can be used in combination.

[0058] In some embodiments of the present application, the hematopoietic stem cells are at least one of autologous hematopoietic stem cells, allogeneic hematopoietic stem cells, and xenogeneic hematopoietic stem cells. In some embodiments of the present application, the hematopoietic stem cells are at least one of wild-type hematopoietic stem cells and pretreated hematopoietic stem cells (e.g., genetically engineered, pretreated with small or macromolecules, or physically or chemically treated).

[0059] In some embodiments of the present application, the hematopoietic stem cell is a hematopoietic stem cell transfected with an exogenous nucleic acid molecule.

[0060] In some embodiments of the present application, the exogenous nucleic acid molecule can be at least one of DNA and RNA. In some embodiments of the present application, the exogenous nucleic acid molecule can be single-stranded, such as ssDNA and ssRNA, or double-stranded, such as dsDNA and dsRNA. In some embodiments of the present application, the exogenous nucleic acid molecule includes plasmid DNA, siRNA, shRNA plasmid, sgRNA, antisense oligonucleotide (ASO), ssODN, nucleic acid aptamer, miRNA, miRNA mimics, mRNA, etc.

[0061] In some embodiments of the present application, transfection can be achieved by a variety of different methods, including physical, chemical, and biological methods. In some embodiments of the present application, physical methods of transfection include electroporation, microinjection, gene guns, sonoporation, etc. In some embodiments of the present application, chemical methods of transfection include calcium phosphate coprecipitation, liposomes, polymers, nanocarriers, etc. In some embodiments of the present application, biological methods of transfection include viral vectors, in which case transfection is also referred to as transduction.

[0062] In some embodiments of the present application, exogenous nucleic acid molecules are transfected via at least one of liposomes, polymers, nanoparticles, and viral vectors. In some embodiments of the present application, liposomes include cationic liposomes, ionizable lipids (LNPs), polymer-lipid hybrids, and liposome-peptide complexes. In some embodiments of the present application, polymers include linear / branched polyethyleneimine (PEI), polylysine (PLL), dendrimers (such as PAMAM), chitosan derivatives (such as trimethyl chitosan), and thermosensitive polymers. In some embodiments of the present application, nanoparticles include gold nanoparticles, magnetic nanoparticles, mesoporous silica, exosomes / extracellular vesicles, and metal-organic frameworks. In some embodiments of the present application, viral vectors include lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, herpes simplex viruses, and Newcastle disease viruses.

[0063] In some embodiments of the present application, the liposome-containing reagent includes any one of Lipofectamine 2000, Lipofectamine 3000, Lipofectamine 6000, Lipofectamine stem cell transfection reagent, etc.

[0064] In some embodiments of the present application, hematopoietic stem cells deliver a gene editing system containing an exogenous nucleic acid molecule via liposomes. In some embodiments of the present application, the gene editing system includes a CRISPR gene editing system, such as any one of CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13. In some embodiments of the present application, the composition of the CRISPR gene editing system includes a Cas enzyme and a guide RNA. In some embodiments of the present application, the Cas enzyme includes any one of Cas9, Cas12a, and Cas13. In some embodiments of the present application, the Cas enzyme can be a wild-type or mutant enzyme. In some embodiments of the present application, the guide RNA is sgRNA. In some embodiments of the present application, the Cas enzyme can be transfected in the form of a protein or a nucleic acid. It is understandable that when the Cas enzyme is transfected in the form of a nucleic acid, the corresponding enzyme is then generated in the cell.

[0065] In some embodiments of the present application, the gene editing method of hematopoietic stem cells through transfection of exogenous nucleic acid molecules includes at least one of knock-in, knock-out, mutation, epigenetic modification, etc.

[0066] In some embodiments of the present application, the target gene for gene editing includes ARSA.

[0067] In some embodiments of the present application, the exogenous nucleic acid molecule includes an sgRNA targeting ARSA. The specific sgRNA can be designed using methods well known in the art, for example, design prediction and verification can be performed using relevant website software, such as CRISPOR, GuideScan, Cas-OFFinder, CRISPRitz, etc.

[0068] In some embodiments of the present application, hematopoietic stem cells are seeded in a hematopoietic stem cell culture medium. In some embodiments of the present application, the hematopoietic stem cell culture medium is a serum-containing culture medium or a serum-free culture medium. Because serum-containing culture medium has certain immunological issues, in some embodiments of the present application, the hematopoietic stem cell culture medium is a serum-free culture medium.

[0069] In some embodiments of the present application, the seeding amount of hematopoietic stem cells in the hematopoietic stem cell culture medium is 1×10 3 ~1×10 6 / mL, for example, it can be 1×10 3 / mL, 2×10 3 / mL, 3×10 3 / mL, 4×10 3 / mL, 5×10 3 / mL, 6×10 3 / mL, 7×10 3 / mL, 8×10 3 / mL, 9×10 3 / mL, 1×10 4 / mL, 2×10 4 / mL, 3×10 4 / mL, 4×10 4 / mL, 5×10 4 / mL, 6×10 4 / mL, 7×10 4 / mL, 8×10 4 / mL, 9×10 4 / mL, 1×10 5 / mL, 2×10 5 / mL, 3×10 5 / mL, 4×10 5 / mL, 5×10 5 / mL, 6×10 5 / mL, 7×10 5 / mL, 8×10 5 / mL, 9×10 5 / mL, 1×10 6 pieces / mL.

[0070] In some embodiments of the present application, the culture time of hematopoietic stem cells in the hematopoietic stem cell culture medium is 1 hour to 30 days, for example, it can be 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 15 days, 16 days, 18 days, 20 days, 25 days, or 30 days.

[0071] In some embodiments of the present application, the culture temperature of hematopoietic stem cells in the hematopoietic stem cell culture medium is 30-40°C, for example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0072] In some embodiments of the present application, hematopoietic stem cells are cultured in a cell culture vessel.

[0073] In some embodiments of the present application, the cell culture container includes a cell culture plate (such as a 4-well, 6-well, 8-well, 12-well, 24-well, 48-well, 96-well, 384-well, or 1536-well culture plate), a cell culture dish (such as a 35 mm, 60 mm, 100 mm, or 150 mm culture dish), a cell culture flask (such as a T25, T75, T175, or T225 culture flask), a cell factory (such as a 1-layer, 2-layer, 3-layer, 4-layer, 5-layer, 10-layer, or 40-layer cell factory), and the like.

[0074] In some embodiments of the present application, the raw material for preparing the cell culture container includes a polymer, such as polystyrene.

[0075] In some embodiments of the present application, during the culture process of hematopoietic stem cells in the cell culture container, the culture medium is replaced and the cell culture container is subcultured every 1 to 5 days, for example, every 1, 2, 3, 4, or 5 days.

[0076] In a fifth aspect of the present application, a method for enhancing the activity of hematopoietic stem cells is provided, the method comprising the following steps: contacting the hematopoietic stem cells with Azoramide.

[0077] In some embodiments of the present application, the working concentration of Azoramide is 1 to 1000 µmol / L (µM), for example, it can be 1 µmol / L, 2 µmol / L, 3 µmol / L, 4 µmol / L, 5 µmol / L, 6 µmol / L, 7 µmol / L, 8 µmol / L, 9 µmol / L, 10 µmol / L, 20 µmol / L, 30 µmol / L, 40 µmol / L, 50 µmol / L, 60 µmol / L, 70 µmol / L, 80 µmol / L, 90 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 400 µmol / L, 500 µmol / L, 600 µmol / L, 700 µmol / L, 800 µmol / L, 900 µmol / L, or 1000 µmol / L.

[0078] In some embodiments of the present application, the hematopoietic stem cells are hematopoietic stem cells under sulfatide stress conditions, for example, they can be hematopoietic stem cells under sulfatide stress conditions of 1 to 1000 µmol / L, for example, they can be 1 µmol / L, 2 µmol / L, 3 µmol / L, 4 µmol / L, 5 µmol / L, 6 µmol / L, 7 µmol / L, 8 µmol / L, 9 µmol / L, 10 µmol / L, 20 µmol / L, 30 µmol / L, 40 µmol / L, 50 µmol / L, 60 µmol / L, 70 µmol / L, 80 µmol / L, 90 µmol / L, 100 µmol / L, 200 µmol / L, 300 µmol / L, 400 µmol / L, 500 µmol / L, 600 µmol / L, 700 µmol / L, 800 µmol / L, 900 µmol / L, 1000 Hematopoietic stem cells under µmol / L sulfatide stress conditions.

[0079] In some embodiments of the present application, the hematopoietic stem cells are hematopoietic stem cells derived from mammals (such as monotremes, marsupials, insectivores, shrews, scaphotheres, dermoptera, chiroptera, primates, edentata, phlonotida, lagomorpha, rodents, carnivores, sirenians, hyraxes, tubulodonta, perissodactyla, artiodactyla, cetaceans, etc.), specifically including hematopoietic stem cells from at least one of rodents (such as mice, rats, hamsters, guinea pigs), lagomorphs (such as rabbits), perissodactyla (such as horses), artiodactyla (such as sheep, pigs), primates (such as monkeys, orangutans, gorillas, chimpanzees, humans), and carnivores (such as dogs).

[0080] In some embodiments of the present application, the hematopoietic stem cells are at least one of autologous hematopoietic stem cells, allogeneic hematopoietic stem cells, and xenogeneic hematopoietic stem cells. In some embodiments of the present application, the hematopoietic stem cells are at least one of wild-type hematopoietic stem cells and pretreated hematopoietic stem cells (e.g., genetically engineered, pretreated with small or macromolecules, or physically or chemically treated).

[0081] In some embodiments of the present application, the hematopoietic stem cell is a hematopoietic stem cell transfected with an exogenous nucleic acid molecule.

[0082] In some embodiments of the present application, the exogenous nucleic acid molecule can be at least one of DNA and RNA. In some embodiments of the present application, the exogenous nucleic acid molecule can be single-stranded, such as ssDNA and ssRNA, or double-stranded, such as dsDNA and dsRNA. In some embodiments of the present application, the exogenous nucleic acid molecule includes plasmid DNA, siRNA, shRNA plasmid, sgRNA, antisense oligonucleotide (ASO), ssODN, nucleic acid aptamer, miRNA, miRNA mimics, mRNA, etc.

[0083] In some embodiments of the present application, transfection can be achieved by a variety of different methods, including physical, chemical, and biological methods. In some embodiments of the present application, physical methods of transfection include electroporation, microinjection, gene guns, sonoporation, etc. In some embodiments of the present application, chemical methods of transfection include calcium phosphate coprecipitation, liposomes, polymers, nanocarriers, etc. In some embodiments of the present application, biological methods of transfection include viral vectors, in which case transfection is also referred to as transduction.

[0084] In some embodiments of the present application, exogenous nucleic acid molecules are transfected via at least one of liposomes, polymers, nanoparticles, and viral vectors. In some embodiments of the present application, liposomes include cationic liposomes, ionizable lipids (LNPs), polymer-lipid hybrids, and liposome-peptide complexes. In some embodiments of the present application, polymers include linear / branched polyethyleneimine (PEI), polylysine (PLL), dendrimers (such as PAMAM), chitosan derivatives (such as trimethyl chitosan), and thermosensitive polymers. In some embodiments of the present application, nanoparticles include gold nanoparticles, magnetic nanoparticles, mesoporous silica, exosomes / extracellular vesicles, and metal-organic frameworks. In some embodiments of the present application, viral vectors include lentiviruses, adenoviruses, adeno-associated viruses, retroviruses, herpes simplex viruses, and Newcastle disease viruses.

[0085] In some embodiments of the present application, the liposome-containing reagent includes any one of Lipofectamine 2000, Lipofectamine 3000, Lipofectamine 6000, Lipofectamine stem cell transfection reagent, etc.

[0086] In some embodiments of the present application, hematopoietic stem cells deliver a gene editing system containing an exogenous nucleic acid molecule via liposomes. In some embodiments of the present application, the gene editing system includes a CRISPR gene editing system, such as any one of CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas13. In some embodiments of the present application, the composition of the CRISPR gene editing system includes a Cas enzyme and a guide RNA. In some embodiments of the present application, the Cas enzyme includes any one of Cas9, Cas12a, and Cas13. In some embodiments of the present application, the Cas enzyme can be a wild-type or mutant enzyme. In some embodiments of the present application, the guide RNA is sgRNA. In some embodiments of the present application, the Cas enzyme can be transfected in the form of a protein or a nucleic acid. It is understandable that when the Cas enzyme is transfected in the form of a nucleic acid, the corresponding enzyme is then generated in the cell.

[0087] In some embodiments of the present application, the gene editing method of hematopoietic stem cells through transfection of exogenous nucleic acid molecules includes at least one of knock-in, knock-out, mutation, epigenetic modification, etc.

[0088] In some embodiments of the present application, the target gene for gene editing includes ARSA.

[0089] In some embodiments of the present application, the exogenous nucleic acid molecule includes an sgRNA targeting ARSA. The specific sgRNA can be designed using methods well known in the art, for example, design prediction and verification can be performed using relevant website software, such as CRISPOR, GuideScan, Cas-OFFinder, CRISPRitz, etc.

[0090] In some embodiments of the present application, hematopoietic stem cells are seeded in a hematopoietic stem cell culture medium. In some embodiments of the present application, the hematopoietic stem cell culture medium is a serum-containing culture medium or a serum-free culture medium. Because serum-containing culture medium has certain immunological issues, in some embodiments of the present application, the hematopoietic stem cell culture medium is a serum-free culture medium.

[0091] In some embodiments of the present application, the seeding amount of hematopoietic stem cells in the hematopoietic stem cell culture medium is 1×10 3 ~1×10 6 / mL, for example, it can be 1×10 3 / mL, 2×10 3 / mL, 3×10 3 / mL, 4×10 3 / mL, 5×10 3 / mL, 6×10 3 / mL, 7×10 3 / mL, 8×10 3 / mL, 9×10 3 / mL, 1×10 4 / mL, 2×10 4 / mL, 3×10 4 / mL, 4×10 4 / mL, 5×10 4 / mL, 6×10 4 / mL, 7×10 4 / mL, 8×10 4 / mL, 9×10 4 / mL, 1×10 5 / mL, 2×10 5 / mL, 3×10 5 / mL, 4×10 5 / mL, 5×10 5 / mL, 6×10 5 / mL, 7×10 5 / mL, 8×10 5 / mL, 9×10 5 / mL, 1×10 6 pieces / mL.

[0092] In some embodiments of the present application, the culture time of hematopoietic stem cells in the hematopoietic stem cell culture medium is 1 hour to 30 days, for example, it can be 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 15 days, 16 days, 18 days, 20 days, 25 days, or 30 days.

[0093] In some embodiments of the present application, the culture temperature of hematopoietic stem cells in the hematopoietic stem cell culture medium is 30-40°C, for example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0094] In some embodiments of the present application, hematopoietic stem cells are cultured in a cell culture vessel.

[0095] In some embodiments of the present application, the cell culture container includes a cell culture plate (such as a 4-well, 6-well, 8-well, 12-well, 24-well, 48-well, 96-well, 384-well, or 1536-well culture plate), a cell culture dish (such as a 35 mm, 60 mm, 100 mm, or 150 mm culture dish), a cell culture flask (such as a T25, T75, T175, or T225 culture flask), a cell factory (such as a 1-layer, 2-layer, 3-layer, 4-layer, 5-layer, 10-layer, or 40-layer cell factory), and the like.

[0096] In some embodiments of the present application, the raw material for preparing the cell culture container includes a polymer, such as polystyrene.

[0097] In some embodiments of the present application, during the culture process of hematopoietic stem cells in the cell culture container, the culture medium is replaced and the cell culture container is subcultured every 1 to 5 days, for example, every 1, 2, 3, 4, or 5 days.

[0098] In some embodiments of the present application, the method for enhancing the activity of hematopoietic stem cells is a non-disease diagnosis or treatment method.

[0099] The present application also provides a method for treating metachromatic leukodystrophy, comprising hematopoietic stem cell therapy, wherein the hematopoietic stem cells are the hematopoietic stem cells contacted with Azoramide as described above.

[0100] According to the embodiments of the present application, at least the following beneficial effects are achieved:

[0101] During the experiment, the applicant found that when Azoramide was used to treat the transfected hematopoietic stem cells, their apoptosis, cell necrosis and mitochondrial respiration were significantly improved. Therefore, it is expected that Azoramide can be used to enhance the transfection efficiency and metabolic activity of ARSA gene-transfected hematopoietic stem cells and reduce ROS generation, thereby enhancing its effectiveness in treating MLD.

[0102] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 The following table shows the cytotoxicity of small molecule compounds at different concentrations on hematopoietic stem cells (HSCs) in an example of this application. (a) Sulfatides, 10 µM to 100 µM; (b) Azoramide, 10 µM to 1000 µM.

[0104] Figure 2This is the cell death rate after untransfected and transfected hematopoietic stem cells are treated with different concentrations of sulfatide in an embodiment of the present application.

[0105] Figure 3 This is an example of the present application showing changes in the cell death rates of untransfected and transfected hematopoietic stem cells treated with S (Sulfatides) alone, A (Azoramide) alone, or A+Z combined treatment.

[0106] Figure 4 In one embodiment of the present application, flow cytometry was used to detect changes in cell apoptosis and necrosis under different treatment conditions.

[0107] Figure 5 This is an example of a mitochondrial stress test in one embodiment of the present application to examine the OCR of transfected and untransfected hematopoietic stem cells. (a) is the OCR of untransfected HSCs, (b) is the OCR of transfected HSCs, (c) is the OCR of basal oxygen consumption, (d) is the OCR of maximal oxygen consumption, (e) is the respiratory potential (respiratory reserve capacity), and (f) is the percentage of respiratory potential.

[0108] Figure 6 In one embodiment of the present application, the ECAR rates of transfected and untransfected hematopoietic stem cells were tested using a Mito stress assay, wherein (a) is the ECAR rate of untransfected HSCs, and (b) is the ECAR rate of transfected HSCs.

[0109] Figure 7 The energy phenotypes of transfected and untransfected hematopoietic stem cells after sulfatide treatment in one embodiment of the present application are shown in Figure 1. (a) shows the energy phenotype of untransfected HSCs, and (b) shows the energy phenotype of transfected HSCs.

[0110] Figure 8 This is an example of a mitochondrial stress test in an embodiment of the present application, examining the OCR of transfected and untransfected hematopoietic stem cells under azoramide treatment. (a) is the OCR of untransfected HSCs, (b) is the OCR of transfected HSCs, (c) is the OCR of basal oxygen consumption, (d) is the OCR of maximal oxygen consumption, (e) is the respiratory potential (respiratory reserve capacity), and (f) is the percentage of respiratory potential.

[0111] Figure 9 The energy phenotypes of transfected and untransfected hematopoietic stem cells after azoramide treatment in one embodiment of the present application are shown in Figure 1. (a) shows the energy phenotype of untransfected HSCs, and (b) shows the energy phenotype of transfected HSCs.

[0112] Figure 10This is the quantitative analysis result of mitochondrial superoxide anions in hematopoietic stem cells in one embodiment of the present application.

[0113] Figure 11 This is the quantitative analysis result of mitochondrial superoxide anion in hematopoietic stem cells after treatment with Azoramide in one embodiment of the present application.

[0114] Figure 12 The following are the microscopic examination results of untransfected and transfected cells in an embodiment of the present application, wherein (a) is an untransfected hematopoietic stem cell, and (b) is a transfected hematopoietic stem cell.

[0115] All data are presented as mean ± standard deviation. Statistical differences between the two groups were analyzed using an unpaired t-test. A one-way analysis of variance with the Bonferroni test was used to evaluate the differences between the two groups. In the figures, *: p < 0.05, **: p < 0.01, ***: p < 0.001. DETAILED DESCRIPTION

[0116] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0117] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0118] In the description of this application, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0119] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0120] Throughout the description of this application, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0121] Example 1: Cell viability and cytotoxicity assays of hematopoietic stem cells (HSCs)

[0122] (1) Thawing and culture

[0123] After thawing, human spinal cord hematopoietic stem cells (Lonza) were assessed for viability using 0.4% trypan blue staining in a Neubauer counting chamber. Following the manufacturer's instructions, they were cultured in 24-well plates using the accompanying serum-free hematopoietic stem cell culture medium (Lonza) in a cell culture incubator at 37°C and 5% CO2. Twenty-four hours after seeding, the cultured HSCs reached a density of approximately 2 × 10 4 cells / well.

[0124] (2) Cell transfection

[0125] Hematopoietic stem cells cultured in 24-well plates were trypsinized and then transfected with the CRISPR / Cas9 complex using Lipofectamine Stem Cell Transfection Reagent according to the manufacturer's instructions. The sgRNA sequence, GACCGUGGCCGAAGU, is homologous to exon 2 of the ARSA gene and directs Cas9 nuclease activity. Ribonucleoprotein complexes were assembled at a 1.3:1 ratio using a custom single guide RNA containing the targeting sequence (sgRNA). GeneArt Platinum Cas9 nuclease was used as the nuclease.

[0126] Transfected HSCs were incubated in a cell culture incubator at 37°C and 5% CO2 for 2 days. Clonal expansion was performed by limiting dilution and continued until 80% confluence was achieved. The cells were then transferred to 60 mm culture dishes for subsequent assays. Both untransfected and transfected HSCs were assayed in triplicate.

[0127] Untransfected HSCs were seeded in 96-well plates (2 × 10 4Cells were plated in a 100 μL volume per well (100 cells / well) and incubated in a cell culture incubator at 37°C with 5% CO2 for 24 hours. Group 1, in which the culture medium was supplemented with sulfatides (Sigma-Aldrich) at final concentrations of 10 μM, 25 μM, 50 μM, and 100 μM, and Group 2, in which the culture medium was supplemented with azoramide (Selleck) at final concentrations of 10 μM, 25 μM, 50 μM, 100 μM, 300 μM, and 1000 μM. Both groups 1 and 2 were supplemented with DMSO and water as controls.

[0128] After 24 hours of incubation, the cells were replaced with serum-free hematopoietic stem cell culture medium containing 1 mg / mL MTT. The cells were incubated in a cell culture incubator for another 1 hour. The medium was then removed and replaced with 100 μL of dimethyl sulfoxide. The absorbance was measured at 570 nm using a microplate reader (Thermo Fisher Scientific).

[0129] The cell viability of untransfected hematopoietic stem cells was taken as 100%, and the survival rates of cells under different treatment conditions in Group 1 and Group 2 were calculated. Figure 1 As shown in the figure, it can be seen that within the above concentration range, the addition of different concentrations of sulfatide or azoramide will not cause the death of untransfected hematopoietic stem cells. In addition, the cell microscopy results before and after transfection are as follows Figure 12 shown.

[0130] Example 2: Cell Death Assessment

[0131] (1) Individual evaluation

[0132] Untransfected and transfected HSCs were seeded in 96-well plates (2 × 10 4 Cells were pre-incubated for 2 hours in a 37°C, 5% CO2 incubator with 100 μL of sulfatide per well. Sulfatide was then added to each well at varying concentrations (10 μM, 25 μM, 50 μM, and 100 μM) and incubated for an additional 24 hours. 100 μL of serum-free hematopoietic stem cell culture medium (containing 0.05 μM SYTOX Green nucleic acid dye) was added to each well and incubated for 15 minutes. Fluorescence intensity was measured at 520 nm using a microplate reader. Wells containing lysed cells in the presence of 0.1% Triton X-100 served as positive controls, while wells containing culture medium alone served as negative controls. Increased fluorescence intensity correlated with increased cell death.

[0133] The results are as follows Figure 2As shown in the figure, when the concentration of sulfatide reaches 100 μM, the difference in cell death between transfected and untransfected hematopoietic stem cells is significant (p=0.0124) ( Figure 2 Therefore, sulfatide concentrations of 100 μM can lead to cell death in transfected hematopoietic stem cells induced by sulfatide accumulation.

[0134] (2) Joint comparison

[0135] Referring to the experimental method in (1), 100 μM S, 100 μM S+500 μM A combined treatment, and 500 μM A treatment were added after pre-incubation, and the cells were incubated for 24 hours before the fluorescence intensity was detected.

[0136] The results are as follows Figure 3 As shown in the figure, it can be seen that the difference in cell death rate after three treatments in transfected hematopoietic stem cells is statistically significant, with a p value of 0.0124. The p values ​​of 100 μM S and 500 μM A are 0.0241 and 0.0042, respectively. This shows that 500 μM Azoramide can reduce cell death caused by sulfatide accumulation in transfected hematopoietic stem cells and enhance the transfection efficiency of hematopoietic stem cells.

[0137] Example 3: Apoptosis

[0138] The detection of Caspase-3 / 7 and annexin V is used to distinguish between apoptosis and necrosis, thereby evaluating cell apoptosis. The specific process is as follows:

[0139] Referring to Example 2, untransfected and transfected hematopoietic stem cells were seeded into hematopoietic stem cell serum-free medium supplemented with 100 µM sulfatide, 500 µM azoramide, or 100 µM sulfatide + 500 µM azoramide, and incubated in a cell culture incubator at 37 ° C., 5% CO 2 for 24 hours. 4 µM doxorubicin was used as a positive control, and no inducer was added as a negative control.

[0140] The activity of caspase-3 / 7 was then determined using the CellEvent Caspase-3 / 7 Green flow cytometry assay kit (Life Technologies) according to the instructions. The number of live, apoptotic, and necrotic cells was determined and standardized.

[0141] The results are as follows Figure 4As shown, treatment with 100 µM sulfatide did not activate Caspase-3 / 7 in untransfected hematopoietic stem cells, but induced an increase in necrotic cell death in transfected hematopoietic stem cells. The results showed that treatment with 100 µM S, 100 µM S + 500 µM A, and 500 µM A all resulted in both apoptosis and necrosis in transfected hematopoietic stem cells, with 100 µM S + 500 µM A showing a statistically significant difference between apoptotic and viable cells.

[0142] Example 4: Mitochondrial Bioenergetics

[0143] Untransfected and transfected HSCs were seeded into 24-well plates (Agilent) at 5 × 10 cells per well. 4 cells and added 100 μL of hematopoietic stem cell serum-free medium. Different groups were set up at the same time, and sulfatide was added at final concentrations of 10 μM, 25 μM, 50 μM, and 100 μM, respectively. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The experiment was then performed using the XF Cell Mitochondrial Stress Test Kit (Agilent) according to its workflow.

[0144] OCR results are as follows Figure 5 As shown in the figure, untransfected hematopoietic stem cells exhibited a metabolic response, manifested by an increase in maximal respiration rate, when treated with 10 µM, 25 µM, and 50 µM sulfatide. However, this response was absent when untransfected hematopoietic stem cells were treated with 100 µM sulfatide, as their basal oxygen consumption, maximal respiration rate, and reserve capacity decreased. Comparing the results with untransfected and transfected hematopoietic stem cells, transfected hematopoietic stem cells showed an increase in maximal respiration rate even without sulfatide treatment. However, as sulfatide concentrations increased, both respiration and basal respiration were affected.

[0145] ECAR results are as follows Figure 6 As shown in the figure, under the treatment conditions of 10 μM, 25 μM, 50 μM and 100 μM sulfatide, the ECAR levels of untransfected hematopoietic stem cells were 2.10, 1.95, 2.01 and 1.81 mpH minμg, respectively. -1 , while those of transfected hematopoietic stem cells were 4.34, 3.47, 3.47 and 2.41 mPH min µg -1 The above results showed that the ECAR rate of transfected hematopoietic stem cells was increased compared with that of untransfected hematopoietic stem cells.

[0146] Energy phenotype diagram Figure 7As shown in the figure, it can be seen that the energy phenotypes of untransfected hematopoietic stem cells and transfected hematopoietic stem cells are significantly different, and the activity of the glycolysis pathway is increased, which is a response to mitochondrial stressors.

[0147] Example 5: Azoramide treatment improves mitochondrial energy biology of transfected hematopoietic stem cells

[0148] The experiment was conducted with reference to Example 4, and different groups were set up, including a group to which sulfatide was added alone at a final concentration of 100 μM, a group to which sulfatide was added together with azoramide at a final concentration of 100 μM and a group to which 500 μM azoramide was added alone.

[0149] OCR results are as follows Figure 8 As shown by Figure 8 As can be seen in (a), for untransfected hematopoietic stem cells in 100µM Sulfatides, the addition of Azoramide does not affect mitochondrial respiration. Figure 8 As can be seen in (b), for transfected hematopoietic stem cells, the additional addition of Azoramide to 100µM Sulfatides can improve their mitochondrial energy production.

[0150] Energy phenotype results such as Figure 9 As shown, untransfected hematopoietic stem cells exhibited an energy phenotype. However, after treatment with 100µM sulfatide, the metabolic changes in transfected hematopoietic stem cells became more pronounced, tending towards a quiescent state. Simultaneous treatment with 100µM sulfatide and 500µM azoramide restored the transfected hematopoietic stem cells' initial energy phenotype. Furthermore, treatment with only 500µM azoramide significantly enhanced glycolysis in the transfected hematopoietic stem cells.

[0151] Example 6: Detection of Reactive Oxygen Species

[0152] Referring to Examples 4 and 5, untransfected and transfected hematopoietic stem cells were treated with different concentrations of sulfatide and azoramide, respectively. Mitochondrial reactive oxygen species (ROS) levels were measured using the MitoSOX mitochondrial superoxide indicator (Invitrogen) according to the manufacturer's instructions. Fluorescence intensity was measured at 510 / 580 nm using an inverted microscope (Nikon).

[0153] The results are as follows Figure 10 and Figure 11As shown in the figure, the difference in reactive oxygen species levels between untransfected and transfected hematopoietic stem cells is statistically significant, with the latter having much higher levels than the former. However, treatment with azoramide, and the combination of azoramide and azoramide, prevented the formation of superoxide anions in transfected cells.

[0154] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.

Claims

1. Use of the compound Azoramide in the preparation of a product for treating metachromatic leukodystrophy.

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