WO2023246642A1 - Composition pour le traitement d'une lésion de la moelle épinière, son procédé de préparation et son utilisation - Google Patents

Composition pour le traitement d'une lésion de la moelle épinière, son procédé de préparation et son utilisation Download PDF

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WO2023246642A1
WO2023246642A1 PCT/CN2023/100707 CN2023100707W WO2023246642A1 WO 2023246642 A1 WO2023246642 A1 WO 2023246642A1 CN 2023100707 W CN2023100707 W CN 2023100707W WO 2023246642 A1 WO2023246642 A1 WO 2023246642A1
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solution
spinal cord
injury
neural stem
thrombin
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PCT/CN2023/100707
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Chinese (zh)
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陈志国
刘素梅
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北京慧心医谷生物科技有限责任公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/225—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/383—Nerve cells, e.g. dendritic cells, Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • the present invention relates to the field of stem cells, and in particular to a composition for treating spinal cord injury and its preparation method and use.
  • SCI Spinal cord injury
  • spinal cord injuries can be divided into complete injuries and incomplete injuries.
  • animal models for studying spinal cord injury also include complete spinal cord transection injury, spinal cord hemisection injury and other types.
  • full spinal cord transection injury completely cuts off the connection between the head and tail of the corresponding spinal cord segment, which is the highest degree of injury. Treatment is the most difficult; spinal cord hemisection injury is a model in which half of the spinal cord is removed but the other half is relatively intact.
  • the degree of injury is relatively low. Due to the compensatory effect, this type of injury has a relatively small impact on the body's behavior and is relatively difficult to treat. Low.
  • biomaterial transplantation therapy emerged with the development of tissue engineering technology. Its mechanism of repairing damaged spinal cord is mainly that the material acts as a bridge to fill the cavity formed after the liquefaction and necrosis of spinal nerve cells, connecting the broken ends and helping nerve regeneration. When growing along the material to avoid disordered growth.
  • biomaterials can also be used as carriers to load substances that are beneficial to nerve growth, such as stem cells, neurotrophic factors, and drugs that stimulate regeneration.
  • Biomaterials mainly include natural tissue materials, synthetic materials, hydrogels, etc.
  • tissue materials have good tissue compatibility and their degradation products have no inflammatory reaction, but they have problems such as low mechanical strength and easy collapse after absorbing water.
  • Synthetic materials have controllable mechanical strength and degradation rate, but their degradation products produce local inflammatory reactions.
  • hydrogel is a research hotspot in the field of spinal cord injury repair. It has a three-dimensional porous network structure and swells highly after absorbing water. It can provide a microenvironment for the differentiation, regeneration and reproduction of neural stem cells, making the environment similar to cells.
  • the extramatrix allows cells to better adhere and grow; it also has good tissue compatibility and can control the degradation rate; it can be used alone or as a carrier to load cells, drugs, nerve growth factors, etc.
  • Fibrinogen is a water-soluble protein synthesized and secreted by liver cells. It can form insoluble fibrin with a network structure under the catalysis of thrombin.
  • researchers have used a combination of fibrin and thrombin as a biomaterial to treat spinal cord injury.
  • Rosenzweig et al. transplanted fibrin-thrombin-loaded human neural progenitor cells (NPCs) into the cervical spinal cord injury site of a rhesus monkey model, they matured into neurons, extended axons, and formed connections with host cells. Synapse (E.S. Rosenzweig et al., Restorative effects of human neural stem cell grafts on the primate spinal cord, Nat Med 24(4)(2018)484-490).
  • the cell line used in the method reported by Rosenzweig et al. is the 566RSC-UBQT cell line derived from the lower cervical spine and upper thoracic spinal cord of 8-week-old fetuses. Ethical restrictions, limited sources and expensive prices, and because the neural progenitor cells used are intermediate cells before complete differentiation, their differentiation potential is relatively low (the proportion of differentiated into neurons is about 50%), and the number of divisions is limited;
  • the preparation method reported by Rosenzweig et al. is to mix NPCs and fibrinogen into solution 1, and dilute thrombin into solution 2, and then draw equal volumes of solution 1 and solution 2 respectively.
  • NSCs neural stem cells
  • OPC oligodendrocyte precursor cells
  • transplanting only cell grafts is more suitable for hemisection injury models where the type of spinal cord injury is incomplete injury, and the injury requires a smaller hole in the spinal cord; for injury sites with larger holes, especially full transection reflecting complete injury Model, only transplanting cell grafts will cause blood flow and other fluids to wash away the grafts, resulting in greater loss of transplanted cells, which is not conducive to the fixation and orderly growth of cells.
  • the first object of the present invention is to provide a composition comprising induced neural stem cells (iNSC), fibrin, and thrombin.
  • iNSC induced neural stem cells
  • fibrin fibrin
  • thrombin thrombin
  • the induced neural stem cells are human induced neural stem cells; more preferably, the induced neural stem cells are human induced neural stem cells obtained by reprogramming human peripheral blood mononuclear cells (PBMC). neural stem cells.
  • PBMC peripheral blood mononuclear cells
  • the composition is a composition for treating spinal cord injury.
  • the composition is used to treat spinal cord injury, or to prepare medical supplies for treating spinal cord injury, or to improve the microenvironment of the spinal cord injury site.
  • the medical supplies may be medicines or medical devices.
  • the spinal cord injury described in the present invention is a complete injury and/or an incomplete injury.
  • the spinal cord injury described in the present invention is a complete injury.
  • the content of induced neural stem cells in the composition is more than 2 ⁇ 10 6 /mL, preferably 5 ⁇ 10 6 to 5 ⁇ 10 7 /mL, and most preferably about 2 ⁇ 10 7 /mL.
  • the transplanted cell concentration is too low, for example, less than 2 ⁇ 10 6 /mL, such as 1 ⁇ 10 6 /mL or 1 ⁇ 10 5 /mL, the cells rarely survive, even 5 days after injury. Cells cannot be detected before injury, so it cannot improve the microenvironment in the early stage of injury.
  • the second object of the present invention is to provide a preparation method of the composition of the present invention, which includes the following steps: (1) Dissolving fibrinogen in the first solution to form a fibrinogen solution ; (2) Dissolve thrombin in the second solution to form a thrombin solution; (3) Resuspend neural stem cells in the thrombin solution to form a neural stem cell resuspension; (4) Add the fibrinogen solution to The neural stem cell resuspension is added to form a gel composition containing neural stem cells, fibrin, and thrombin.
  • the preparation method of the composition of the present invention may also include the following steps: (1) Dissolve fibrinogen in the first solution to form a fibrinogen solution; (2) Dissolve thrombin in the second solution to form coagulation enzyme solution; (3) resuspend neural stem cells in the fibrinogen solution to form a neural stem cell resuspension; (4) add the thrombin solution to the neural stem cell resuspension to form a neural stem cell resuspension containing neural stem cells and fibers.
  • Gel-like composition of protein and thrombin wherein, the first solution is a salt solution, and the second solution is a protein solution.
  • the first solution is a solution that can dissolve fibrinogen and has good biocompatibility, such as CaCl 2 , MgCl 2 , NaCl, KCl, etc.
  • Aqueous solution preferably an aqueous solution of CaCl2 , wherein the concentration of CaCl2 is 5-40mM, preferably 10-30mM, most preferably 20mM, or preferably an aqueous solution of NaCl, wherein the concentration of NaCl is 0.9% (i.e. physiological saline) ;
  • the concentration of the fibrinogen solution is 10-200mg/mL, preferably 50-100mg/mL.
  • the second solution can be any solution that can dissolve thrombin and has good biocompatibility, such as bovine serum albumin (BSA) solution or physiological saline solution, preferably a BSA solution, wherein the concentration of BSA is 0.1-10 mg. /mL, preferably 0.5-5mg/mL, most preferably 1mg/mL; the concentration of the thrombin solution is 10-200U/mL, preferably 50-100U/mL. In the present invention, the concentration of BSA solution can also be expressed in %.
  • the preparation method of 1% BSA solution is to add 1g BSA to distilled water and dilute to 100mL. That is, the concentration of 1% BSA solution is 10mg/mL. ; For BSA solutions of other concentrations, the amount of added BSA can be adjusted proportionally or diluted with 1% solution.
  • the neural stem cells are cultured in a proliferation medium before use.
  • the proliferation medium includes, for example, DMEM/F12 (Gibco), Neurobasal A (Gibco), N2 (Gibco), B27 ( Gibco), GlutaMAX (Gibco), NEAA (Gibco), CHIR99021 (3 ⁇ M), SB431542 (2 ⁇ M), LIF (10ng/ml) culture medium.
  • the third object of the present invention is to provide a medical product for treating spinal cord injury, wherein the medical product is a medicine or a medical device and contains a composition according to the present invention or a medical product according to the present invention.
  • the medical device may be, for example, a biomaterial scaffold, gel, or bioink for 3D printing.
  • the fourth object of the present invention is to provide the use of the composition of the present invention in the preparation of medical supplies for treating spinal cord injury, wherein the medical supplies are medicines or medical devices and comprise the combination of the present invention or a composition obtained according to the preparation method of the present invention.
  • the medical device may be, for example, a biomaterial scaffold, gel, or bioink for 3D printing.
  • the treatment of spinal cord injury includes promoting functional recovery after spinal cord injury, reducing the injury volume caused by spinal cord injury, promoting the expression of Tuj1 and NF200 at the core site of injury, or improving the microstructure of the spinal cord injury site. environment.
  • composition of the present invention is suitable for complete and/or incomplete spinal cord injuries, and is particularly suitable for complete spinal cord injuries.
  • the composition of the present invention is suitable for total transverse injury and/or hemisection injury of the spinal cord, and is particularly suitable for total transverse injury of the spinal cord.
  • the induced neural stem cells used in the present invention can be induced neural stem cells obtained by any existing technology, and human induced neural stem cells prepared using human peripheral blood mononuclear cells as starting cells are preferred.
  • any method in the prior art that induces human induced neural stem cells from human peripheral blood mononuclear cells can be used, for example, the induction method disclosed by the inventor in Chinese Patent 201810372724.7 can be used.
  • spinal cord injury used in the present invention refers to a disease in which spinal cord damage is caused by trauma, inflammation, tumors, etc., resulting in dysfunction of movement, sensation, sphincters, autonomic nerves, etc. below the level of injury.
  • complete injury used in the present invention is also called complete spinal cord injury (complete spinal cord injury), which refers to a type of spinal cord injury in which movement, sensation, and sphincter function below the injury level are completely lost, indicating that the level of spinal cord injury has occurred. Complete transverse damage.
  • incomplete injury used in the present invention is also called incomplete spinal cord injury (incomplete spinal cord injury), which refers to a type of spinal cord injury that retains some sensory or motor functions below the injury level, indicating that the spinal cord injury level has not completely occurred. Sexual transverse damage.
  • total transection injury refers to a type of spinal cord injury in which the spinal cord is completely transected.
  • hemisection injury used in the present invention refers to the type of spinal cord injury in which the spinal cord is not completely transected, but only half-transected or partially damaged.
  • the invention provides a neural stem cell composition for treating spinal cord injury, a preparation method of the composition, and uses of the composition. Compared with existing similar technologies, the solution of the present invention has the following good technical effects:
  • the cells used in the present invention are induced neural stem cells.
  • neural stem cells Compared with neural progenitor cells or neural precursor cells used in the prior art, neural stem cells have a high differentiation potential and can differentiate into a variety of cells required for spinal cord injury repair, such as Can differentiate into nerves that are specific to the spinal cord or even specific to different spinal cord segments; can differentiate into neurons at a high rate of about 70% (the neuron differentiation rate of neural progenitor cells is only about 50%, and the recruitment of neurons is important for the treatment of spinal cord injury is the most difficult and critical), and the number of divisions of neural stem cells is much higher than that of neural progenitor cells or neural precursor cells, which is also beneficial to the treatment of spinal cord injury.
  • the neural stem cells used in the present invention are human induced neural stem cells prepared from human peripheral blood mononuclear cells. Compared with existing neural stem cells, they are easy to source and abundant, and have no tumorigenicity after testing (iNSC has been verified to be or iNSC-derived dopaminergic precursors transplanted into the brains of immunodeficient mice in vivo will not lead to tumor formation), are highly safe, are still stable after different passages, and the preparation technology is relatively simple with a short cycle and low cost. It has great advantages for in vivo transplantation and clinical application; moreover, because the patient's own peripheral blood mononuclear cells can be used to prepare induced neural stem cells, the problem of allogeneic immune rejection can be avoided.
  • the neural progenitor cells loaded with biological materials for spinal cord injury are derived from 8-week-old fetuses, which are subject to ethical restrictions, limited sources, and expensive prices.
  • the solution of the present invention is applicable to more comprehensive types of spinal cord injuries, and is particularly suitable for severe spinal cord injuries, such as complete injuries or total transection injuries. Of course, it is also applicable to incomplete injuries or half-cut injuries with lighter degrees of injury.
  • the present invention prepares the composition in advance as a soft gel-like solid, which is easy to clamp during transplantation, and the cells are evenly distributed and will not When blood flow disperses cells, the transplanted cells will suffer less loss and will not grow disorderly, which is more conducive to spinal cord regeneration at the injured site.
  • the “SCI control group” refers to the SCI rats administered a placebo (vehicle); the “biomaterial alone group” refers to the SCI rats administered only biological materials (i.e., fibrin and thrombin); and the “iNSCs+biomaterial group” refers to SCI rats administered iNSCs and biomaterials (i.e., fibrin and thrombin); the “normal group” refers to rats that did not undergo SCI.
  • “dpi” refers to days post injury (days post injury), that is, the number of days after spinal cord injury modeling. Since the graft is transplanted immediately after spinal cord injury, dpi also refers to days after transplantation.
  • Figure 1 shows iNSC survival 5 days after transplantation.
  • A Schematic diagram of the experimental process
  • B Operational process diagram of establishing a complete spinal cord transection injury model
  • C Schematic diagram of iNSC migrating to the outside of the spinal cord injury site 5 days after transplantation.
  • iNSCs are represented by dots, and biomaterials are represented by grid lines.
  • D Human nuclear antigen (Hu) expressed by transplanted iNSC in and around the injury site 5 days after transplantation, DAPI (blue) represents the nucleus of the host and graft, and Hu (green) represents Nuclei of transplanted human iNSCs; E: Transplanted iNSCs co-express Hu and Sox2 (a marker of NSCs) in the lesion center at 5 days post-transplantation; asterisks indicate biomaterial; blue is DAPI, green is Hu, red is GFAP, white is Sox2; F: staining diagram of human fibrinogen/fibrin on the whole spinal cord section of SCI animals sacrificed on the 5th day after transplantation (5dpi), blue is DAPI, green is fibrinogen, and red for GFAP.
  • the scale bars of panels D, E, and F are all 100 ⁇ m.
  • Figure 2 shows that transplantation of iNSCs and biomaterials can promote electrophysiological recovery and motor improvement in SCI rats 7 months after transplantation.
  • A BBB scores of each group starting from SCI (day 0) until 7 months after SCI, determined by double-blind independent observers (compared with the biomaterial transplantation alone group, ⁇ means p ⁇ 0.05, ⁇ ⁇ means p ⁇ 0.01, two-way analysis of variance);
  • B The latency and amplitude of MEP (motor evoked potential) (compared with the SCI control group, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, one-way analysis of variance);
  • C In MEPs were recorded in the left and right legs of different treatment groups and control groups.
  • Figure 3 shows that transplantation of iNSCs and biomaterials resulted in reduced lesion volume 7 months post-transplantation compared to SCI controls or biomaterials alone.
  • A Representative images of the spinal cord in each group, the injury site is indicated in the box; B: Injury volume analysis; C: H&E (hematoxylin-eosin) staining of the spinal cord tissue in each group, the left picture shows the entire spinal cord section Low-magnification image of the horizontal section. The right image represents the high-magnification image within the black box in the left image.
  • D LFB (luxol fast blue) staining of spinal cord tissue in each group. The left image shows the low-magnification image of the horizontal section of the entire spinal cord section. Magnified image, the right image represents the high-magnification image within the black box in the left image.
  • Figure 4 shows fibrin degradation in the injury center and Tuj1 and NF200 staining in whole spinal cord sections at different time points.
  • A Fibrinogen staining at different time points after SCI. A small amount of fibrin was detected from the 30th day to the 7th month after SCI. Blue is DAPI, green is human fibrinogen (human fibrinogen), and red is GFAP; B: Tuj1 (neuronal class III ⁇ -tubulin) and GFAP staining 7 months after SCI, a higher degree of Tuj1 expression was detected at the injury site in the iNSC + biomaterial transplantation group, blue is DAPI, green is Tuj1, Red is GFAP; C.
  • NF200 neuroofilament-200
  • GFAP GFAP staining 7 months after SCI
  • blue is DAPI
  • green is NF200
  • Red is GFAP
  • D Recognize fibrin by anti-fibrinogen antibodies, convert fibrinogen into fibrin by mixing with thrombin at different times in vitro, and stain the mixture with anti-fibrinogen antibodies, the results are displayed at 5 minutes , the staining signal at 30 minutes and 1 hour is relatively stable, indicating that the anti-fibrinogen antibody can recognize fibrinogen and fibrin.
  • Green indicates human fibrinogen.
  • the upper picture is an immunofluorescence image
  • the lower picture is a white light image.
  • the scale bar of panel A is 100 ⁇ m
  • the scale bar of panels B and C is 2000 ⁇ m
  • the scale bar of panel D is 250 ⁇ m.
  • Figure 5 shows the expression of Tuj1 and NF200 around the injury core and injury boundary in the SCI control group, biomaterial transplantation alone group, and iNSC + biomaterial transplantation group 7 months after SCI.
  • A The expression of Tuj1 in the damage core and around the damage boundary in each group, blue is DAPI, green is Tuj1, and red is GFAP;
  • B The proportion of Tuj1 + cells in the damage core and around the damage boundary in each group.
  • C Expression of NF200 in the damage core and around the damage boundary in each group, blue is DAPI, green is Tuj1, red is GFAP, white is NF200;
  • D Ratio of Tuj1 + /NF200 + cells around the damage core and damage boundary in each group .
  • E Synaptic staining of the injury site. Neurons with synaptic connections were detected at the injury core of the iNSC+biomaterial transplantation group. Blue is DAPI, green is Synapsin, and red is MAP2, indicating mature neurons. Left picture It is a low-magnification image, and the image on the right is an enlarged image within the frame in the left image.
  • the scale bar of images A, C, and E (left) is 100 ⁇ m, and the scale bar of image E (right) is 25 ⁇ m. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01 (one-way analysis of variance).
  • Figure 6 shows the expression of BDNF, TGF ⁇ and TNF ⁇ in the SCI control group, biomaterial transplantation alone group, and iNSC + biomaterial transplantation group.
  • A BDNF (brain-derived neurotrophic factor), TGF ⁇ (transforming growth factor- ⁇ ) and TNF ⁇ (tumor necrosis factor- ⁇ ) staining in each group at 15 dpi.
  • the iNSC+ biomaterial transplantation group showed higher levels of BDNF at the injury site.
  • TGF ⁇ and lower level TNF ⁇ expression blue is DAPI
  • green is BDNF
  • red GFAP
  • B The proportion of BDNF, TGF ⁇ , and TNF ⁇ positive cells in each group at 15dpi.
  • the scale bar of panel A is 100 ⁇ m. * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01 (one-way analysis of variance).
  • Figure 7 shows that the iNSC+biomaterial transplantation group showed reduced inflammatory response after SCI.
  • the scale bars of panels A and B are both 100 ⁇ m.
  • Figure 8 shows that the iNSC+biomaterial transplantation group does not change the type of scar tissue 7 months after SCI.
  • the scale bar of panels A and B is 2000 ⁇ m.
  • Example 1 Culture and preparation of human induced neural stem cells
  • the human induced neural stem cells used in the present invention can be human induced neural stem cells obtained according to any known method (for example, the induction method disclosed by the inventor in Chinese Patent 201810372724.7).
  • the method reported by Y. Yuan et al. was used to prepare human iNSCs (Y. Yuan et al., Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model, Theranostics 8(17)(2018)4679-4694).
  • PBMCs peripheral blood mononuclear cells
  • iNSCs induced neural stem cells
  • NSC medium made of DMEM/ F12 :Neurobasal (1:1), 1 ⁇ N2, 1 ⁇ B27, 2mM GlutaMAX, 1% NEAA (Life Technologies) at a density of 2 ⁇ 10 5 /well ), 10 ng/mL recombinant human leukemia inhibitory factor (rhLIF, Millpore, Billerica, USA), 3 ⁇ M CHIR99021 and 2 ⁇ M SB431542 (both from Gene Operation, Michigan, USA). The medium was changed every other day. After ten days, epithelial cells Like clones appeared in the culture.
  • iNSC proliferation media examples include DMEM/F12 (Gibco), Neurobasal A (Gibco), N2 (Gibco), B27 (Gibco), glutaMAX (Gibco), NEAA (Gibco), CHIR99021 (3 ⁇ M), SB431542 (2 ⁇ M) , LIF (10ng/ml) culture medium. Medium was replaced half way every 2 days and passaged every 4-6 days.
  • Preparation of iNSC + biomaterial grafts Prepare iNSC grafts (containing 4 ⁇ 10 6 cells) wrapped by fibrin-thrombin. The specific method is: add Accutase TM to the iNSCs in Example 1, and incubate at 37°C.
  • the inventor also tried to use other concentrations of human fibrinogen solution, such as 200 mg/mL human fibrinogen solution and 200 U/mL thrombin as raw materials to mix with iNSC grafts, and found that the resulting gel-like soft mixture was as good as the one used in this example.
  • human fibrinogen solution such as 200 mg/mL human fibrinogen solution and 200 U/mL thrombin
  • the products obtained by using 100 mg/mL human fibrinogen solution and 100 U/mL thrombin as raw materials are very similar, and there is no difference in the material cross-linking time, softness and flexibility after cross-linking. From the perspective of cost reduction, the product with the above concentration in this example (that is, the product obtained using 100 mg/mL human fibrinogen solution and 100 U/mL thrombin as raw materials) was selected for testing below.
  • the experimental animals used in this invention are female SD rats (210-230 grams), purchased from Vital River (China), and raised in a temperature- and humidity-controlled animal area with a 12-hour light/dark cycle. All animal experiments were performed in accordance with the guidelines of the Ministry of Public Health of China and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All research procedures involving animals were approved by the Ethics Committee of Xuanwu Hospital, Capital Medical University.
  • Spinal cord injury modeling and transplantation method uses a complete spinal cord injury model.
  • the modeling method is: intraperitoneally injecting ketamine (25mg/mL), xylazine (1.3g/mL) and acepromazine (0.25mg/mL). mL) to deeply anesthetize the animals at a dose of 2 mL/kg.
  • ketamine 25mg/mL
  • xylazine 1.3g/mL
  • acepromazine 0.25mg/mL
  • mL acepromazine
  • the tissues were grouped as mentioned above and subjected to different treatments (such as transplantation of iNSC + biomaterials or biomaterials or no transplantation, use of hemostatic sponges at the transection site to control bleeding), suturing of muscle tissue, suturing of skin and application of medical iodophor, and finally all rats were placed Return to his home cage for recovery.
  • Transplantation method Before transplantation, prepare compositions according to the method of Example 2 - namely, a composition containing iNSC and biological material group (for iNSC transplantation group), and a composition containing only biological material (material alone group) , and mix each gently but thoroughly to cross-link each to produce a soft clot. Take 200 ⁇ L of each of the two biomaterials (with or without iNSC cells), which is enough to fill the injury gap, that is, the hole formed by the total spinal cord injury model.
  • a graft containing a composition containing iNSCs and biomaterials or a composition containing only biomaterials is placed at the injury site of SCI.
  • all rats were treated with the antibiotic ampicillin for one week.
  • daily injections of cyclosporine D (10 mg/kg) were started one week before transplantation until the animals were sacrificed.
  • the rats' bladders were manually emptied twice a day until sacrifice (see Figure 1A and Figure 1B).
  • Rats were anesthetized with a combination of ketamine (25 mg/mL), xylazine (1.3 g/mL), and acepromazine (0.25 mg/mL) at a dose of 2 mL/kg. Rats were perfused transcardially with cold 0.9% saline. Spinal cords from T6-T10 were dissected and fixed in 4% paraformaldehyde (PFA) for 48 h at 4°C, then transferred to 20% sucrose for 24 h (4°C), followed by transfer to 30% sucrose for 24 h (4 °C). These fragments were then embedded in OCT embedding medium (optimal cutting temperature compound), cut into 20 ⁇ m thick sections (Leica Microsystems), and stored at -80°C.
  • PFA paraformaldehyde
  • Functional recovery was assessed weekly throughout the study. Functional analysis was performed by double-blind observers in an open-field experiment using the Basso, Beattie, and Bresnahan (BBB) motor rating scale to evaluate the recovery of hindlimb motor function in rats after SCI.
  • BBB Bresnahan
  • Electrophysiological studies were performed on each group (n 6 rats/group). Before examination, animals were anesthetized with a combination of ketamine (25 mg/mL), xylazine (1.3 g/mL), and acepromazine (0.25 mg/mL) at 2 mL/kg. Then Keypoint-II dual-channel evoked potential/electromyography was used to measure the motor evoked potential (MEP).
  • the stimulating electrode needle electrode
  • the recording electrode is inserted into the gastrocnemius muscle of the contralateral hind limb.
  • lesion volume was calculated from GFAP-stained sections, which provides a more accurate means of determining lesions. Specifically, the areas of three manually traced sections per section were averaged, the areas of 20 to 25 consecutive sections collected at defined intervals per spinal cord were summed, and the known distance between each section was used to The lesion volume (expressed in cubic millimeters) was calculated for each spinal cord.
  • PBMCs peripheral blood mononuclear cells
  • iNSCs were expanded in proliferation medium for several generations, dissociated into single cells, and then mixed with biomaterials (fibrinogen and thrombin) to form soft clots, which were then transplanted into the fully transverse spinal cord injury site ( Figure 1A and Figure 1B). Once mixed, the biomaterial and cells solidify rapidly at room temperature (typically within 3 seconds). For each rat, 4 ⁇ 10 6 cells-loaded biomaterials were transplanted to the injury site of the spinal cord (T8-T9). SCI rats were euthanized at different time points after injury, and the spinal cords were sectioned and stained for analysis.
  • biomaterials fibrinogen and thrombin
  • Hu + human nuclei, a marker of human cells
  • Some transplanted iNSC cells were also observed outside the injury site, mainly located in the caudal region of the injury boundary, covering a distance of up to 2 mm ( Figures 1C and 1D).
  • Hu + cells co-labeled with the NSC marker Sox2 but were negative for GFAP (glial fibrillary acidic protein, a marker for astrocytes that typically stains at injury sites and can be used to define injury boundaries) negative (Figure 1E).
  • GFAP glial fibrillary acidic protein, a marker for astrocytes that typically stains at injury sites and can be used to define injury boundaries
  • Hu + cells were no longer detectable on day 15 post-injury (data not shown).
  • electrophysiological analyzes were performed by placing a stimulating electrode in the brain's motor cortex and a recording electrode in the gastrocnemius muscle of the hindlimb.
  • the electrophysiological signals of the left and right hindlimbs were recorded separately.
  • electrical stimulation of the motor cortex induced a signal wave with a latency of approximately 4.69 ms and an amplitude of 4.13 mv in the hind limbs ( Figures 2B and 2C).
  • Fig. 3C tissue morphology
  • fibrin-thrombin biomaterials biomaterial transplantation alone group
  • iNSC gel grafts wrapped with fibrin-thrombin transplantation of iNSC gel grafts wrapped with fibrin-thrombin.
  • iNSC + biomaterial transplantation group could reverse this phenomenon, but the ability of transplanting iNSC gel grafts wrapped with fibrin-thrombin to reverse this phenomenon was significantly higher ( Figure 3C).
  • LFB Longer Fast Blue staining used to examine the level of myelination showed that compared with the SCI control group, by transplanting fibrin-thrombin biomaterial (biomaterial transplantation alone group), or transplanting wrapped in fibrin - Thrombin iNSC gel graft (iNSC+biomaterial transplantation group) both made the myelination area at the injury site larger, but the myelination area in the iNSC+biomaterial transplantation group was significantly larger than the biomaterial transplantation group alone (Figure 3D ).
  • the results showed that transplantation of iNSC gel grafts encapsulated in fibrin-thrombin reduced lesion volume and improved axonal myelination, which may be responsible for functional recovery.
  • the iNSC+biomaterial transplantation group showed good cell survival rate on day 5, but gradually decreased over time. From day 15 onwards, no transplanted iNSCs were detected in the spinal cord. In order to study the reason why the iNSC + biomaterial transplantation group can still maintain long-term functional benefits 7 months after transplantation, the inventors studied the kinetics of the biomaterials used.
  • Thrombin and fibrinogen were used because of their good biocompatibility and the use of fibrinogen-specific antibodies to indicate fibrin (KGSharp et al., Salmon fibrin treatment of spinal cord injury promotes functional recovery and density of serotonergic innervation , Exp Neurol 235(1)(2012)345-56), thereby examining the dynamics of biomaterials ( Figure 4A and Figure 4D). Specifically, significant amounts of fibrin remained at the implantation site 5 days after injury/transplantation (Fig. 4A). The material then gradually degrades, with the quantity significantly reduced at 15dpi. Starting from 30 dpi, only a small amount of material can be detected ( Figure 4A). The presence of neuronal axons at the site of injury was also studied.
  • Tuj1 a marker of immature neurons in the early stages of neuronal differentiation
  • NF200 a marker of mature neurons
  • Transplanted iNSCs showed good survival at 5 dpi but gradually decreased over time. From day 15 onwards, no transplanted iNSCs were detected. However, long-lasting effects of transplanted iNSCs and biomaterials on motor function and pathology were observed.
  • an adverse microenvironment may result in critical steps involved in endogenous axonal regeneration and/or neurogenesis not occurring. Modulation of the microenvironment at early stages may have long-term effects on pathology and function. To test this possibility, we examined the microenvironmental components on spinal cord tissue sections at 15 dpi.
  • Microglia are resident immune cells in the central nervous system (CNS) and can be broadly divided into M1 and M2 phenotypes.
  • the M2 phenotype is generally considered to be beneficial to CNS repair.
  • chronic activation of M1 microglia is part of the inflammatory response after SCI and can trigger further loss of neural tissue.
  • the results showed that compared with the SCI control group and the biomaterial transplantation group alone, the number of CD206 + /Iba1 + cells (M2 microglia) was greater in the iNSC + biomaterial transplantation group at 15 dpi ( Figures 7A and 7C).
  • the inventors also studied infiltrating immune cells by staining for CD45 and CD68, and the results showed that the number of CD45 + and CD68 + cells in the iNSC transplantation group was significantly reduced relative to the SCI control group and the biomaterial transplantation group alone ( Figures 7B and 7D ).
  • Previous studies reported that the extracellular molecule laminin stimulates neurite outgrowth of dorsal root ganglion (DRG) neurons.
  • Laminin and GFAP staining (Fig. 8) showed that the graft did not alter the subtype of activated astrocytes at the injury site.
  • inflammation begins in the acute phase, with the activation of glial cells and immune cells following blood vessel rupture and swelling and compression of the cord tissue. of infiltration.
  • the dead cells and released substances further enhance the inflammatory response and invasion of immune cells, causing further damage to the spinal cord.
  • This reinforcing feed-forward loop reinforces itself and peaks during the subacute phase and gradually weakens during the intermediate/chronic phase.
  • the magnitude and extent of these early inflammatory cascades may determine the level of subsequent long-term functional impairment. In turn, intervention at these early stages may dampen the amplified cascade and have some lasting effects.
  • iNSCs can produce neurotrophic factors and other soluble cytokines/chemokines, some of which help reshape the microenvironment at the site of injury into one that is conducive to the regenerative process.
  • engraftment of iNSCs and biomaterials was associated with a microglia-biased M2 phenotype, a decrease in the pro-inflammatory cytokine TNF ⁇ and an increase in the anti-inflammatory cytokine TGF ⁇ , and a decrease in the number of immune cells infiltrating into the site of injury.
  • the inventor transplanted a composition composed of induced neural stem cells obtained by reprogramming human PBMC, thrombin, and fibrin into the injured spinal cord. Compared with no treatment or transplantation of only thrombin and fibrin, for the group of protein compositions, it promotes the recovery of motor and electrophysiological functions after spinal cord injury to a greater extent, and improves the microenvironment of the spinal cord injury site, which plays a key role in functional recovery after spinal cord injury.

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Abstract

L'invention concerne une composition pour traiter une lésion de la moelle épinière, son procédé de préparation et son utilisation. La composition comprend des cellules souches neurales induites, de la fibrine et de la thrombine, et est particulièrement applicable pour des lésions sévères de la moelle épinière complètement blessées et complètement transversales. La composition facilite l'opération de transplantation, et la perte de cellules transplantées est plus faible ; les cellules souches neurales induites ont un potentiel de différenciation élevé et sont exemptes de tumorigénicité, ayant un rapport de différenciation élevé, une source large et une excellente sécurité.
PCT/CN2023/100707 2022-06-24 2023-06-16 Composition pour le traitement d'une lésion de la moelle épinière, son procédé de préparation et son utilisation WO2023246642A1 (fr)

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