WO2022148500A2 - Utilisation d'une protéine de liaison au tractus polypyrimidine dans la préparation d'un médicament pour la réparation de lésions de la moelle épinière - Google Patents

Utilisation d'une protéine de liaison au tractus polypyrimidine dans la préparation d'un médicament pour la réparation de lésions de la moelle épinière Download PDF

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WO2022148500A2
WO2022148500A2 PCT/CN2022/084398 CN2022084398W WO2022148500A2 WO 2022148500 A2 WO2022148500 A2 WO 2022148500A2 CN 2022084398 W CN2022084398 W CN 2022084398W WO 2022148500 A2 WO2022148500 A2 WO 2022148500A2
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spinal cord
ptb
binding protein
polypyrimidine
tract
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PCT/CN2022/084398
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English (en)
Chinese (zh)
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WO2022148500A3 (fr
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杨日云
潘静莹
包璟崟
夏盼慧
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南通大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • 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

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to the application of a polypyrimidine sequence-binding protein silencing agent combined with retinoic acid and purmorphamine in the preparation of a spinal cord injury repair medicine.
  • SCI Spinal cord injury
  • the regenerative capacity of the damaged neurons that have not been lost is also very limited; third, there will be a variety of inhibitory regeneration factors and inflammatory factors at the injury site, forming a chemical microenvironment that is unfavorable for axon regeneration. These unfavorable factors are coordinated with each other and ultimately lead to difficult tissue repair and functional reconstruction in SCI.
  • astrocytes When nerve tissue is injured, astrocytes will proliferate reactively and exhibit progenitor cell characteristics, indicating that they have a high degree of plasticity in cell transdifferentiation. If we manage to use reactive astrocytes that proliferate and have negative effects after SCI as target cells to directly induce reprogramming into neurons, or even motor neurons with corresponding functions in situ, we can achieve the goal of properly eliminating glia.
  • the purpose of scarring which can supplement the neurons lost due to SCI in situ, and at the same time improve the microenvironment of axon regeneration, should be a good method to solve the difficulties affecting repair and regeneration after SCI, and to replace the treatment and personality of SCI cells.
  • the idea of chemical medicine R&D has contributed to a new way of realization.
  • PTB is an RNA-binding protein and plays an important role in the induction and differentiation of neurons.
  • Current studies have found that silencing PTB can reprogram astrocytes into functional neurons and promote the corresponding disease model mice. function is restored.
  • Spinal cord injury is a common neurological trauma that is difficult to treat. After spinal cord injury, astrocytes reactively proliferate and form glial scars at the injury site, thereby inhibiting the regeneration of neurons and axons; at the same time, a large number of motor neurons are lost. , the limited regeneration ability of damaged neurons, all of which bring great difficulties to the repair of spinal cord injury.
  • the purpose of the present invention is to provide the application of a polypyrimidine sequence-binding protein silencing agent combined with retinoic acid and purmorphamine in the preparation of a spinal cord injury repair medicine.
  • the polypyrimidine sequence-binding protein silencing agent is selected from lentivirus-packaged shRNA-PTB (5'-GGGTGAAGATCCTGTTCAATA-3').
  • the present invention also provides a method for inducing the differentiation of spinal cord reactive astrocytes into motor neurons in vitro. Amines promote differentiation.
  • the present invention silences the polypyrimidine sequence binding protein (PTB) by virus in vitro, and at the same time, the small molecule retinoic acid (RA) and purmorphamine (PMA) related to the differentiation of motor neurons are added together to successfully make the spinal cord of mice reactive
  • RA small molecule retinoic acid
  • PMA purmorphamine
  • Figure 1 shows the establishment of a primary mouse spinal cord reactive astrocyte model.
  • A is the GFAP staining of primary mouse spinal cord astrocytes; the mRNA level (B) and protein level (C) of GFAP in the spinal cord astrocytes of mice treated with 10 ⁇ g/mL LPS for 24 h were significantly higher than those in the control group rise. scale bar: 200 ⁇ m.
  • Figure 2 shows the efficiency results of silencing PTB in shRNA-PTB lentivirus-infected mouse spinal cord reactive astrocytes.
  • the protein (A) and mRNA (B) levels of PTB were significantly decreased after shRNA-PTB lentivirus infection of cells for 2 days.
  • Figure 3 is a light microscope image of shRNA-PTB lentiviral reprogramming of reactive astrocytes in mouse spinal cord. scalebar: 200 ⁇ m.
  • Figure 4 is a fluorescent image of shRNA-PTB lentivirus-induced reprogrammed neurons in mouse spinal cord reactive astrocytes. scale bar: 200 ⁇ m.
  • Figure 5 is a fluorescence image of shRNA-PTB lentivirus combined with RA and PMA to reprogram reactive astrocytes into motor neurons in mouse spinal cord. scale bar: 200 ⁇ m.
  • Figure 6 shows the transdifferentiation rate of mouse spinal cord reactive astrocytes directly reprogramming motor neurons.
  • PTB Polypyrimidine Binding Protein
  • RA small molecule retinoic acid
  • PMA Purmorphamine
  • the invention silences PTB through virus in vitro, and at the same time, the small molecule retinoic acid and purmorphamine related to the differentiation of motor neurons are added together, and the reactive astrocytes of the mouse spinal cord are successfully reprogrammed into motor neurons.
  • the in vivo study of the reprogramming strategy of PTB combined with small molecules in the repair of spinal cord injury provides help, and then achieves better repair and functional reconstruction of spinal cord injury.
  • Spinal cord tissue was obtained from neonatal mice.
  • the spinal cord was placed in a petri dish filled with ice D-Hanks solution and rinsed twice. The meninges and blood vessels on the surface were carefully stripped off with microsurgical forceps. D-Hanks solution was rinsed 2-3 times and transferred to another.
  • a petri dish chop the spinal cord to a chyle shape, transfer the chopped spinal cord block in the petri dish into a 15 mL centrifuge tube, then add an equal amount of 0.25% trypsin, digest it in a 37 °C water bath for 15 min, and mix by pipetting every 5 min.
  • the medium was changed in full after every 2 days to remove the dead cell debris that did not adhere to the wall, so that the glial cells could fully grow. Observe under the microscope during each medium change. After about 1 week, the cells covered the bottom of the bottle, and then further purified and cultured . When the glial cells are cultured for 7 to 9 days, after the cells are covered with the bottom of the culture flask, place them on a constant temperature shaking culture bed at 37°C, with a rotation speed of 280 rmp/min, for 16 to 18 hours (the constant temperature culture bed is UV sterilized in advance).
  • LPS lipopolysaccharide
  • shRNA-PTB silences the PTB of reactive astrocytes in the spinal cord of mice
  • the lentivirus-packaged shRNA-PTB (sequence 5'-GGGTGAAGATCCTGTTCAATA-3'SEQ ID NO.1) produced by Shanghai Heyuan Biotechnology Co., Ltd. was infected with reactive astrocytes 2d, and real-time RT-PCR was used.
  • the mRNA and protein expression changes of PTB were detected by techniques such as Western blot to determine the silencing efficiency of shRNA-PTB.
  • the protein (A) and mRNA (B) levels of PTB were significantly decreased after shRNA-PTB lentivirus infected cells for 2 days, indicating that shRNA-PTB lentivirus could reduce the reactive astrocytes in mouse spinal cord in vitro PTB expression, and the silencing efficiency is about 50%
  • the packaged shRNA-PTB lentivirus was used to infect reactive astrocytes, and after 2 days, the infection medium was replaced with induction medium (N3/basal medium: Insulin, sodium selenite, Retinoic acid, putrescine, ChIR99021, SB431542, Db-cAMP, FGF-basic, GDNF), half-change medium for induction medium every 2 d, induction culture for 7 d, 14 d, 16 d, 21 d, 28 d; immunocytochemical technique was used to detect MAP2 pan-neuronal markers and ChAT movement Neuronal marker expression.
  • induction medium N3/basal medium: Insulin, sodium selenite, Retinoic acid, putrescine, ChIR99021, SB431542, Db-cAMP, FGF-basic, GDNF
  • the cell bodies of the shCtrl group were flat, and the shape was the same as that of astrocytes; while the cell bodies of the shPTB group gradually took on a spherical or pyramidal shape, with different numbers and lengths protruding from the cell bodies. protrusions, showing a neuron-like morphology.
  • shRNA-PTB lentivirus can gradually change the morphology of reactive astrocytes in mouse spinal cord to neuron-like, and directly reprogram them into mature neurons of MAP2+ and motor neurons of ChAT+.
  • shRNA-PTB lentivirus combined with RA and PMA to reprogram mouse spinal cord reactive astrocytes
  • the packaged shRNA-PTB lentivirus was used to infect reactive astrocytes, and after 2 days, the infection medium was replaced with induction medium (N3/basal medium + 1 ⁇ M RA + 0.5 ⁇ M PMA, that is, N3/basal per 1000 mL).
  • induction medium N3/basal medium + 1 ⁇ M RA + 0.5 ⁇ M PMA, that is, N3/basal per 1000 mL.
  • 1 micromolar RA and 0.5 micromolar PMA were added to the medium, and the medium was half-changed every 2 d, and the culture medium was induced for 7d, 14d, 16d, 21d, and 28d; immunocytochemical techniques were used to detect MAP2 pan-neural
  • the expression of meta-markers and ChAT motor neuron markers, and the transdifferentiation rate of motor neurons was calculated.
  • the invention Based on the current situation of spinal cord injury repair and the progress of inducing in situ reprogramming, the invention achieves silencing of PTB in vitro, and simultaneously adds small molecules RA and PMA, and finally successfully reprograms the reactive astrocytes of the spinal cord into motor neurons; It provides a theoretical basis for the in vivo application of the reprogramming strategy of PTB combined with small molecules, and strives to achieve a better effect of spinal cord injury repair and functional reconstruction.
  • the present invention combines the two issues of suppressing hyperproliferative reactive astrocytes and replenishing lost motor neurons, and designs a method that can not only replenish the number of motor neurons lost due to spinal cord injury in situ, but also Simultaneously reducing the number of reactive astrocytes activated and proliferating due to spinal cord injury to alleviate the rapid proliferation of glial scars and improve the chemical microenvironment for axon regeneration, ultimately promoting functional recovery after spinal cord injury. .

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La divulgation concerne une utilisation d'un silenceur de protéine de liaison au tractus polypyrimidine combiné à de l'acide rétinoïque et à de la purmorphamine dans la préparation d'un médicament pour la réparation de lésions de la moelle épinière, se rapportant au domaine technique de la biomédecine. Dans la présente invention, un virus est utilisé in vitro pour inactiver la protéine de liaison au tractus polypyrimidine (PTB), et de l'acide rétinoïque (RA) à petites molécules et de la purmorphamine (PMA) associés à la différenciation des neurones moteurs sont ajoutés en combinaison, ce qui permet de reprogrammer avec succès les astrocytes réactifs de la moelle épinière d'une souris en neurones moteurs et de faciliter la recherche sur l'effet de stratégies de reprogrammation de PTB à petites molécules combinées dans la réparation après lésions de la moelle épinière, permettant ainsi une meilleure réparation de lésions de la moelle épinière et une meilleure reconstruction fonctionnelle.
PCT/CN2022/084398 2021-04-16 2022-03-31 Utilisation d'une protéine de liaison au tractus polypyrimidine dans la préparation d'un médicament pour la réparation de lésions de la moelle épinière WO2022148500A2 (fr)

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CN202110413331.8 2021-04-16
CN202110413331.8A CN113171369A (zh) 2021-04-16 2021-04-16 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用

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CN113171369A (zh) * 2021-04-16 2021-07-27 南通大学 多聚嘧啶序列结合蛋白在制备脊髓损伤的修复药物中的应用

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