WO2024124507A1 - Progéniteurs neuronaux striataux dans le traitement de l'encéphalopathie hypoxique-ischémique - Google Patents

Progéniteurs neuronaux striataux dans le traitement de l'encéphalopathie hypoxique-ischémique Download PDF

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WO2024124507A1
WO2024124507A1 PCT/CN2022/139429 CN2022139429W WO2024124507A1 WO 2024124507 A1 WO2024124507 A1 WO 2024124507A1 CN 2022139429 W CN2022139429 W CN 2022139429W WO 2024124507 A1 WO2024124507 A1 WO 2024124507A1
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hie
striatal
neurons
neural progenitors
cells
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PCT/CN2022/139429
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Yuejun CHEN
Man XIONG
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Center For Excellence In Brain Science And Intelligence Technology, Chinese Academy Of Sciences
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Priority to PCT/CN2023/134801 priority patent/WO2024125295A1/fr
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    • 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
    • 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
    • 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
    • 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

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  • the invention relates to the field of stem cell therapy, in particular to Striatal Neural Progenitors in treatment of hypoxic-ischemic encephalopathy.
  • mice by administrating genetically labelled human embryonic stem cell-derived striatal neural progenitors into the ipsilateral striatum of hypoxic-ischaemic encephalopathy injured mice, we found that the grafted cells gradually matured into GABA spiny projection neurons morphologically and electrophysiologically and significantly rescued the area loss of hypoxic-ischaemic encephalopathy-injured brains.
  • a composition or formulation for the preparation of a composition or formulation, said composition or formulation being used for the prevention and/or treatment of hypoxic-ischemic encephalopathy (HIE) .
  • HIE hypoxic-ischemic encephalopathy
  • composition or formulation is further used for one or more purposes selected from the group consisting of:
  • said composition is a pharmaceutical composition.
  • said pharmaceutical composition comprises a pharmaceutically acceptable carrier and (a) Striatal Neural Progenitors.
  • said component (a) comprises 60-99wt%, preferably 80-99wt%, better 90-99wt%of the total weight of said composition.
  • said composition is in liquid or semi-solid form.
  • said composition is in unit dosage form and said unit dosage form has a volume of 1-100 ml, preferably 2-100 ml, more preferably 5-100 ml.
  • said composition is an injectable formulation.
  • said composition is a liquid composition.
  • said composition has a concentration of 1.0 x 10 6 -1.0 x 10 8 cells/ml, preferably 5.0 x 10 6 -1.0 x 10 8 cells/ml, more preferably 1.0 x 10 7 -1.0 x 10 8 cells/ml of said Striatal Neural Progenitors.
  • said composition further comprises other substances for the prevention and/or treatment of hypoxic-ischemic encephalopathy (HIE) .
  • HIE hypoxic-ischemic encephalopathy
  • said carrier is selected from the group consisting of: an infusion carrier and/or an injection carrier, preferably, said carrier is one or more carriers selected from the group consisting of: saline, glucose saline, or a combination thereof.
  • said Striatal Neural Progenitors are derived from a mammal, preferably from a human, mouse, or rat.
  • said Striatal Neural Progenitors are derived from human embryonic stem cells or human induced pluripotent stem cells.
  • composition or formulation may be used alone, or in combination, in applications for the prevention and/or treatment of hypoxic-ischemic encephalopathy (HIE) .
  • HIE hypoxic-ischemic encephalopathy
  • said combined use comprises: use in combination with other substances for the prevention and/or treatment of hypoxic-ischemic encephalopathy (HIE) .
  • HIE hypoxic-ischemic encephalopathy
  • a cell reagent comprising:
  • a first pharmaceutical composition comprising (a) a first active ingredient, said first active ingredient being Striatal Neural Progenitors, and a pharmaceutically acceptable carrier.
  • a second pharmaceutical composition comprising (b) a second active ingredient, said second active ingredient being other substances for the prevention and/or treatment of hypoxic-ischemic encephalopathy (HIE) ; and a pharmaceutically acceptable carrier;
  • HIE hypoxic-ischemic encephalopathy
  • first pharmaceutical composition and said second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.
  • said cell reagent is a liquid reagent.
  • said cells in said cell reagent comprise substantially ( ⁇ 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) or entirely (a) Striatal Neural Progenitors and (b) other substances for the prevention and/or treatment of hypoxic-ischemic encephalopathy (HIE) .
  • HIE hypoxic-ischemic encephalopathy
  • said composition has a concentration of said Striatal Neural Progenitors of 1.0 x 10 6 -1.0 x 10 8 cells/ml, preferably 5.0 x 10 6 -1.0 x 10 8 cells/ml, more preferably 1.0 x 10 7 -1.0 x 10 8 cells/ml.
  • said component (i) represents 60-99 wt%, preferably 80-99 wt%, more preferably 90-99 wt%of the total weight of said cell reagent.
  • said carrier is selected from the group consisting of:an infusion carrier and/or an injection carrier, preferably, said carrier is one or more carriers selected from the group consisting of: saline, glucose saline, or a combination thereof.
  • said Striatal Neural Progenitors are derived from a mammal, preferably from a human, mouse, or rat.
  • said Striatal Neural Progenitors are derived from human embryonic stem cells or human induced pluripotent stem cells.
  • kit comprising:
  • HIE hypoxic-ischemic encephalopathy
  • said first container and said second container may be the same and may be different.
  • hypoxic-ischemic encephalopathy HIE
  • HIE hypoxic-ischemic encephalopathy
  • said subject comprises a mammal (e.g. a human) .
  • said administered dose is 5 x 10 6 -2.5 x 10 7 Striatal Neural Progenitors/kg, preferably 1 x 10 7 -2.5 x 10 7 Striatal Neural Progenitors /kg, more preferably, 1 x 10 7 -2 x 10 7 Striatal Neural Progenitors /kg.
  • said frequency of administration is once every 7-60 days, preferably once every 7-60 days, more preferably once every 30-60 days.
  • said administration time is 5min-1h, preferably 5min-30min, more preferably 5min-10min.
  • (B) Whole-mount view of mouse brain at 7 days after hypoxic-ischaemia for 30 min, Scale bars 2 mm.
  • Figure 2 Neuronal activity of grafted neurons in the ipsilateral striatum of the HIE-injured brain.
  • B-G Typical traces of injection current- (B, E) or blue light- (C, F) induced AP and spontaneous AP (D, G) of grafted neurons 2 and 6 MPT. The numbers in the upper right corner represent the numbers of neurons showing AP among recorded cells.
  • Figure 3 Axonal outgrowth of grafted striatal neurons in the HIE-injured brain.
  • Figure 4 Synapse formation between grafts and hosts in HIE-injured brains.
  • Figure 5 Synaptic inputs from host to graft by rabies tracing in HIE-injured brain.
  • (B) Representative images show EGFP-and tdTomato-expressing neurons in the grafts. Scale bar 500 ⁇ m.
  • (C) Immunohistochemical staining show the starting cells (white arrowheads) coexpress tdTomato, EGFP, GABA and hN in the graft. Scale bar 50 ⁇ m.
  • (D) Serial sections from HIE injured brain grafted with SNPs show traced host neurons (EGFP + /tdTomato - ) distribute in extensive brain regions of the ipsilateral side 2 and 6 MPT. Scale bar 500 ⁇ m.
  • AI Agranular insular cortex
  • BA Basal amygdaloid nucleus
  • Cg Cingulate cortex
  • CM Central medial thalamic nucleus
  • DR Dorsal raphe
  • GP Globus pallidus
  • FrA Frontal association cortex
  • M1 Primary motor cortex
  • M2 Secondary motor cortex
  • MD Mediodorsal thalamic nucleus
  • MO Medial orbital cortex
  • PF Parafascicular thalamic nucleus
  • PO Posterior thalamic nucleus
  • S1 Primary somatosensory cortex
  • S2 Secondary somatosensory cortex
  • SNc Substantia nigra reticular part
  • VPL/VPM Ventral posterolateral/posteromedial thalamic nucleus.
  • Figure 6 Synaptic regulation of grafted striatal neurons in the HIE-injured brain.
  • (A) The strategy for whole-cell patch-clamp recording of synaptic regulation from the host striatum or cortex to intrastriatally grafted striatal neurons.
  • (B-C) Typical traces of sEPSCs and sIPSCs from grafted neurons at 2 and 6 MPT. The numbers in the upper right corner represent the numbers of neurons showing spikes of sIPSCs and sEPSCs among recorded cells.
  • H-K Cumulative distributions of the interevent intervals (H, J) and amplitude (I, K) of sEPSCs and sIPSCs for (B) and (C) , respectively.
  • N Typical traces of paired pulse-induced EPSCs in grafted cells responding to optogenetic activation of host cortical neurons in M1 and M2 at 6 MPT (top panel) , which are blocked by CNQX (bottom panel) .
  • FIG. 1 Grafted striatal neurons regulate the activities of host neurons from the striatum and nigra of HIE-injured brains.
  • FIG. 1 Schematic diagram showing the whole-cell patch-clamp recording of host striatal neurons.
  • B-C Typical traces of blue light-induced IPSC of striatal host cells responding to optogenetic activation of grafted cells at 2 months (B) and 6 months (C) after transplantation. The induced IPSC is blocked by PTX (bottom panel) .
  • D Schematic diagram showing the whole-cell patch-clamp recording of host cells in the SNc.
  • E-F Typical traces of blue light-induced IPSCs in nigral host cells responding to optogenetic activation of axonal terminals from grafted neurons at 2 months (E) and 6 months (F) after transplantation.
  • hypoxic-ischemic encephalopathy can be effectively treated by Striatal Neural Progenitors.
  • Striatal Neural Progenitors the inventors completed the present invention.
  • hypoxic-ischaemic encephalopathy remains the leading cause of mortality and long-term neurological sequelae, such as mental retardation, cerebral palsy and life-long cognitive and motor disabilities in neonates.
  • Stem cell-based therapies hold great promise as potential novel treatments to restore brain function after HIE 5.
  • Transplantation of different types of multipotent stem cells such as mesenchymal stem cells and umbilical cord blood stem cells, have been shown to have preliminary positive effects in animal models. However, the therapeutic effects of these donor cells are thought to stem from the bystander effects with possible mechanisms, including neuroprotection and immunomodulation.
  • hPSC human pluripotent stem cell
  • hypoxic-ischaemic injury During neonatal hypoxic-ischaemic injury, an insufficient supply of oxygen (hypoxia) and/or poor blood flow (ischaemia) reaching a particular area of the newborn brain leads to a large number of neuronal deaths triggered by the activation of various neurotoxic molecules and death pathways.
  • Previous MRI and postmortem studies have demonstrated selective vulnerability and neuronal loss in the sensorimotor cortex, basal ganglia, thalamus and brain stem in infants with severe HIE. The lesion severity of the basal ganglia, where the striatum is the major nucleus, was found to be strongly associated with the severity of motor impairment in full-term infants with HIE.
  • striatal GABA spiny projection neurons which make up 95%of all striatal neurons and project to both the globus pallidus (GP) and the substantia nigra (SN) , but not GABA interneurons, appear to be severely affected. This evidence strongly indicates the great potential of striatal GABA spiny projection neurons as therapeutic target cells to restore the functionality of neonates with HIE.
  • hNPCs brain region-specific human neural progenitor cells
  • SNPs cortical or striatal neural progenitors
  • the high plasticity of the immature brain and massive neuronal loss accompanied by extensive astrocyte activation causing enhanced production of axonal regeneration inhibition molecules such as chondroitin sulfate proteoglycans and late onset of apoptosis entangled with microglial activation and macrophage invasion lasting for several weeks in the HIE-injured immature brain provide a more complicated microenvironment for the survival and integration of grafted human cells in the host brain in comparison with others.
  • striatum is the main input nucleus which receives excitatory afferents from cortex and forms the origin of indirect (striatum to GPe) and direct (striatum to GPi and SN) pathways, two major pathways of basal ganglia circuit involved in motor control, repairment of this circuit probably the best way to rescue motor defects in HIE patient.
  • striatum to GPe the main input nucleus which receives excitatory afferents from cortex and forms the origin of indirect (striatum to GPe) and direct (striatum to GPi and SN) pathways
  • striatum to GPe striatum to GPe
  • striatum to GPi and SN two major pathways of basal ganglia circuit involved in motor control, repairment of this circuit probably the best way to rescue motor defects in HIE patient.
  • ipsilateral striatal neuron loss accompanied by motor function impairment in HIE injured mouse.
  • the grafted neurons functionally incorporated into the host basal ganglia neural circuit and rescued the motor deficits of HIE-injured animals in the long term.
  • Our data demonstrate for the first time that the anatomy and function of damaged basal ganglia neural circuits in HIE-injured brain can be reconstructed by transplantation of striatal spiny projection neurons, which provides strong evidence of stem cell replacement therapy for the treatment of hypoxic-ischaemic immature brain injury.
  • compositions (cellular reagents)
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of Striatal Neural Progenitors; other substances for the prevention and/or treatment of hypoxic-ischemic encephalopathy, and a pharmaceutically acceptable carrier.
  • Striatal Neural Progenitors and other substances for the prevention and/or treatment of hypoxic-ischemic encephalopathy can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, such as saline, where the pH is typically about 5-8, preferably about 7-8.
  • the term "effective amount” or “effective dose” refers to an amount that is functional or active in humans and/or animals and is acceptable to humans and/or animals.
  • said effective amount is: 1.0 x 10 6 -1.0 x 10 8 cells/ml, preferably 5.0 x 10 6 -1.0 x 10 8 cells/ml, better 1.0 x 10 7 -1.0 x 10 8 cells/ml.
  • said effective amount of cells is injected in a single dose.
  • the Striatal Neural Progenitors of the present invention can be used for the preparation of a medication.
  • the Striatal Neural Progenitor of the invention may be administered to a mammal, such as a human, and may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously) , topically, and the like.
  • the Striatal Neural Progenitor can be administered alone or in combination with other pharmaceutically acceptable substances.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier) , such as sodium citrate or dicalcium phosphate, or mixed with the following components: (a) a filler or compatibilizer, for example, a starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, for example, glycerin; (d) a disintegrant such as an agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; (f) absorbing accelerators, for example, quaternary amine compounds; (g) wetting agents, such as
  • Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials. If necessary, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs.
  • the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butanediol, dimethylformamide and oils
  • compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and isosorbide dinitrate, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and isosorbide dinitrate, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.
  • compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion.
  • Suitable aqueous and nonaqueous vehicles, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms for the Striatal Neural Progenitor of the present invention for topical administration include ointments, powders, patches, propellants and inhalants.
  • the active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.
  • a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective dosage, for an individual of 60 kg body weight, the daily dose to be administered is usually from 1 to 1000 mg, preferably from 20 to 500 mg.
  • the specific dose should also consider the route of administration, the health of the individual and other factors, which are within the skill of the skilled physician.
  • said pharmaceutical composition is preferably an intravenous formulation.
  • the main advantages of the invention include:
  • hypoxic-ischemic encephalopathy can be effectively treated by Striatal Neural Progenitors.
  • hESCs line WA09 [WiCell] , passages 20-40 and gene editing cell lines from hESCs were maintained on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs) in hESCs medium consisting of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F-12) , 1 ⁇ Glutamax, 1 ⁇ Nonessential Amino Acids (NEAA) and 0.1 mM ⁇ -mercaptoethanol (Reagents information in Table S1) .
  • Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 DMEM/F-12
  • NEAA 1 ⁇ Nonessential Amino Acids
  • NEAA Nonessential Amino Acids
  • ROCK Rho-kinase
  • CM MEF-conditioned ESC culture medium
  • puromycin 0.5 mg/ml
  • G418 50-100 mg/ml
  • Individual clones were picked up after drug selection, and the integration of the transgene was identified by genomic PCR.
  • the iCre, mCherry, ChR2-EYFP or hM4Di-mCherry expression cassette was inserted into the AAVS1 locus of H9 hESCs.
  • Striatal neural progenitors were differentiated from hESCs with a modified protocol following a previous report 20 . Briefly, hESCs on MEFs (5-6 days after passaging) were digested into small clumps with dispase, and cultured with ESC medium without bFGF in T25 flask for 3 days to form embryonic bodies (EBs) . The cultured medium was changed every day. From day 4 to day 6, EBs were induced to neuroepithelium with SB431542 (2 ⁇ M) and DMH-1 (2 ⁇ M) in neural induction medium (NIM) including DMEM/F-12, 1 ⁇ N2 supplement and 1 ⁇ NEAA.
  • NIM neural induction medium
  • neural spheres were attached to 6-well plates in NIM containing 5%foetal bovine serum for 20 h, then the medium was changed with NIM every other day until day 10.
  • SAG 0.2 ⁇ M
  • SAG which is an agonist of the sonic hedgehog pathway
  • RA retinoic acid
  • mice After being returned to the dam to recover for 2 h, pups were exposed to hypoxia at 36.5 °C ⁇ 37.0 °C thermostat which perfused with humidified gas mixture (8%oxygen in nitrogen) for 30 or 60 min. Sham-control animals underwent the same procedures without occlusion of the artery.
  • Huified gas mixture 8%oxygen in nitrogen
  • mice were euthanized 7 days post-HIE surgery. Animals were perfused transcardially with 0.9%saline followed by 4%PFA for fixation. After sequential dehydration in 20%and 30%sucrose, 30- ⁇ m brain sections were obtained by a microtome and stored at -20 °C in cryoprotectant solution for further experiments.
  • brain slices (30 ⁇ m) were placed on gelatine-coated glass slides and baked for 2 h at 37 °C. Then, brain slices were dehydrated in 75%, 85%, 95%(twice) and 100% (twice) ethanol for 8 min each step, followed by immersion in 95%, 85%, 75%, 50%ethanol for 3 min each step. After a quick rinse in distilled water, the brain slices were immersed in CV staining solution for 10-15 min. Then, the stained slices were quickly rinsed in 75%, 85%, 95% (0.3%glacial acetic acid) and 100% (twice) ethanol.
  • CV Cresyl violet
  • Striatal or spinal neural progenitors were digested into very small clusters at day 32 and kept in NIM medium for another two days, and then small clusters of neural progenitors were transplanted at day 34. Briefly, animals were randomly grouped and placed into a stereotaxic apparatus 2 weeks after HIE surgery.
  • brain slices were incubated in blocking solution (10%donkey serum and 0.3%Triton X-100 in DPBS) for 1h and then incubated with primary antibodies including hNCAM, GABA, DARPP32, CTIP2, GFAP, SST, CR, Ki67 and hN (antibodies information in Table S1) at 4 °C for 1-3 nights.
  • the unbound primary antibodies were washed with DPBS.
  • fluorescent immunostaining slices were incubated with corresponding fluorophore-conjugated secondary antibodies for 1h at room temperature. Nuclei were stained with Hoechst 33342, and slices were mounted in Fluoromount-G.
  • Coronal brain slices (300 ⁇ m) were prepared from animals at 2 and 6 MPT using a vibratome (Leica VT1200S) . Slices were incubated in solution containing oxygenated (95%O2 and 5%CO2) ACSF containing (in mM) : 124 NaCl, 4.4 KCl, 2 CaCl 2 , 1 MgSO 4 , 25 NaHCO 3 , 1 NaH 2 PO 4 , and 10 glucose at room temperature for 60 min. Current and voltage signals were recorded by an Axon 700B amplifier (Axon) . Current and optogenetic stimulation-induced action potentials (APs) of grafted cells were recorded in current clamp mode.
  • Oxon Axon 700B amplifier
  • Electrodes were filled with a solution containing (in mM) : 120 K-gluconate, 5 NaCl, 1 MgCl 2 , 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.1 Na 3 -GTP and 10 phosphocreatine disodium, adjusted to pH 7.2 with HCl.
  • APs were detected in response to depolarizing currents (0-100 pA, step 10 pA, duration 400 ms) or blue light illumination (470 nm, 2 ms, 5 Hz, duration 1000 ms 1.5 mW/mm 2 ) in current clamp mode.
  • the spontaneous excitatory postsynaptic currents (sEPSCs) , spontaneous inhibitory postsynaptic currents (sIPSCs) and optogenetic stimulation of ChR2-expressing inputs were recorded in voltage clamp mode. Electrodes were filled with a solution containing (in mM) 112 Cs-Gluconate, 5 TEA-Cl, 3.7 NaCl, 0.2 EGTA, 10 HEPES, 2 Mg ATP, 0.3 Na 3 GTP and 5 QX-314 (adjusted to pH 7.2 with CsOH) .
  • Optogenetic stimulation of Channelrhodopsin 2 (ChR2) -expressing inputs was achieved with wide-field illumination using a blue LED (470 nm, 2 ms, 1.5 mW/mm 2 ) .
  • Data were analysed offline with Clamfit and MiniAnalysis. In all cases, biocytin (0.4%) was added to the internal solution to identify the morphological properties of the recorded cells.
  • rabies-mediated tracing experiments 200 nl AAV expressing Cre-dependent TVA with a nuclear location signal (NLS-tdTomato) (AAV-DIO-TVA-2A-NLS-tdTomato, titer 1.29 ⁇ 10 12 genome copies (gc) /ml, and 200 nl AAV expressing the rabies helper (Cre-dependent rabies glycoprotein, AAV-DIO-G, titer 1.29 ⁇ 10 12 gc/ml) were coinjected into the grafted site (AP: + 0.8 mm; ML: + 1.7 mm; DV: -3.2 mm from dura) of HIE mice at 1 and 5 post transplantation (MPT) .
  • AP + 0.8 mm
  • ML + 1.7 mm
  • DV -3.2 mm from dura
  • EnvA-pseudotyped and G-deleted rabies virus-tagged EGFP (RVdG-EGFP, 400nl, titer 2 ⁇ 10 8 pfu/ml) was injected into the same site.
  • the mice were perfused and sliced coronally.
  • serial slices (30 ⁇ m, every 4th) of total brain without staining were captured directly by a 20 ⁇ objective with a fluorescence microscope (Olympus VS120) .
  • the outline of brain areas and the distribution of the traced neurons were manually labelled using Photoshop software according to the mouse brain Atlas. Quantification and comparison of the percentage of ipsilateral inputs were done by a blinded counter.
  • Some brain slices were performed immunohistochemistry staining to elucidate the cell identity.
  • a total of 400 nl AAV expressing Cre-dependent (DIO) enhanced ascorbate peroxidases 2 (APEX2) with a nuclear export signal (AAV-DIO-APEX2-NES, titer 9.86 ⁇ 10 12 g/ml) was injected into the grafted site (AP: + 0.8 mm; ML: + 1.7 mm; DV: -3.2 mm from dura) of HIE-injured mouse brains at 5 MPT.
  • AP + 0.8 mm
  • ML + 1.7 mm
  • DV -3.2 mm from dura
  • mice were prepared for immunoelectron microscopy (EM) . Briefly, animals were perfused transcardially with 0.9%saline and then fixed with 4%PFA combined with 0.8%glutaraldehyde.
  • Brain slices (100 ⁇ m) were sectioned by a vibratome, fixed in the abovementioned fixative for 4 h, and then reacted with DAB solution to reveal sites of peroxidase activity.
  • To identify the APEX2-labelled synapses ultrathin sections were cut and counterstained with lead citrate and uranyl acetate.
  • Open field test Individual mice were placed in a plastic open-field chamber (40 cm ⁇ 40 cm ⁇ 40 cm) , and the distance covered were tracked and analyzed using the Ethovision video tracking system (from Noldus Information Technology) . Activities were recorded for 10 min under normal conditions of lighting. Quantitative analysis was done on total distance.
  • SPSS version 20.0 was used for statistical analysis. The results were analysed using Student’s t test, one-way ANOVA or two-way ANOVA followed by Tukey’s multiple comparison test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001 were considered to be significant.
  • Example 1 hESC-derived SNPs mature into striatal GABA spiny projection neurons and rescue brain area loss in HIE-injured brains
  • Fig. 1A To test hESC-based cell therapy for HIE-injured immature brains, we designed an experimental procedure, as shown in Fig. 1A.
  • SCID severe combined immunodeficiency
  • GABAergic neurons GABAergic neurons
  • CIP2 striatum GABA neuron-specific transcription factors
  • DRD1 and TAC1 characteristic genes for spiny projection neurons
  • DRD2 and PENK characteristic genes for spiny projection neurons
  • hESC-derived SNPs which show high expression of neural progenitor marker SOX2, but not any embryonic stem cells markers such as OCT4, NANOG and SSEA-4 into the ipsilateral striatum 2 weeks after HIE surgery (Supplementary Fig. 2A) .
  • Grafts were identified by immunofluorescent staining of GABA or DARPP32 with human nuclei hN or hN/CTIP2 at 2 and 6MPT, respectively (Fig. 1I, K) .
  • the grafted cells repopulated the injured striatum and significantly reduced the area loss of the total ipsilateral brain (17.61 ⁇ 2.95%) , ipsilateral striatum (31.87 ⁇ 3.90%) and ipsilateral cortex (13.94 ⁇ 1.94%) compared to the ACSF control (total ipsilateral area loss: 30.36 ⁇ 5.33%, striatum: 54.70 ⁇ 3.49%, cortex: 29.58 ⁇ 5.31%) at 6 MPT, supporting hESC-derived SNPs as a potential cell source with great therapeutic effects for HIE-injured brains.
  • Example 2 Grafted striatal neurons show functional activity in the HIE-injured striatum
  • mCherry or ChR2-EYFP was knocked into the AAVS1 locus of hESC (Fig. 2A) to specifically manipulate grafted cells after transplantation.
  • the electrophysiological properties of transplanted cells were recorded at different time points (2, 6 MPT) by whole-cell patch-clamp recordings.
  • Both current-or blue light-induced action potentials (APs) (Fig. 2B and C) and spontaneous action potentials (sAPs) (Fig. 2D) showed few spikes by 2 MPT, suggesting functional immaturity of grafted cells.
  • the grafted cells displayed sustained responses to current or blue light stimulation and intensive sAP with higher peak firing rates (Fig.
  • Example 3 Grafted striatal neurons project to their endogenous targets in HIE-injured brains
  • Targeted axonal outgrowth is essential for the reconstruction of damaged neural circuits.
  • axonal projection of the grafted SNPs derived from the hESCs-mCherry line (Fig. 3A) .
  • many human fibers stained by hNCAM or mCherry appeared in endogenous striatal targets GPe, GPi and substantial nigra, including SNc (substantia nigra compacta) and SNr (substantia nigra reticulate) , in the ipsilateral HIE-injured brain at 2 MPT (Fig. 3B-C) .
  • Example 4 Grafted striatal neurons reform synapses with host neurons in the axon targeted regions of HIE-injured brains.
  • hSYN human-specific presynaptic marker synaptophysin
  • the representative images show obvious GABA + synaptic puncta in the striatum, GP (GPe and GPi) and TH + synaptic puncta in the nigra, indicating the formation of synapses between grafted and host neurons (Fig. 4B-E) .
  • SNPs differentiated from hESC lines that genetically express Cre recombinase (iCre) in the AAVS1 site Fig. 4A
  • the grafted cells were infected with Cre-dependent adeno-associated virus (AAV) expressing a peroxidase derivative, ascorbate peroxidases (APEX2) , which label the cytoplasm of the grafted cells with high electron density while retaining a high-quality of ultrastructure 35 , thus, the synaptic vesicles of grafted neurons could be clearly detected in presynaptic terminals.
  • AAV Cre-dependent adeno-associated virus
  • APEX2 ascorbate peroxidases
  • axon terminals (at) from the grafted neuron with multiple presynaptic vesicles form typical synapses with host dendritic spines (ds) , soma and axon terminals (at) in the ipsilateral striatum and GP (GPe and GPi) of HIE-injured brain (Fig. 4F-H) .
  • axon terminals projecting from striatal grafts form synapses with host dendrite spines and axon terminals in the ipsilateral nigra of HIE-injured brains (Fig. 4I) .
  • grafted hESC-derived striatal neurons establish synaptic connections with host neurons including proximal and distal striatal targeting brain regions, suggesting reconstruction of the host basal ganglia neural circuit anatomically in HIE-injured brains.
  • Example 5 Grafted striatal neurons receive extensive synaptic inputs from different regions of the HIE-injured brain
  • grafted neurons form host-to-graft synaptic connections and their upstream origins
  • monosynaptic tracing based on “modified” EnvA-pseudotyped glycoprotein-deleted rabies virus combined with the Cre-loxP gene expression system to map direct inputs to grafted neurons (Fig. 5A) .
  • SNPs derived from the hESC-iCre line into the ipsilateral striatum of HIE mice.
  • AAV expressing the Cre-dependent TVA (the receptor of engineered surface protein EnvA which originates from the avian leukosis virus) with a nuclear location signal (NLS-tdTomato) (AAV-DIO-TVA-2A-NLS-tdTomato) and AAV expressing a Cre-dependent rabies glycoprotein (G) (AAV-DIO-G) were co-injected into the graft site to express TVA and G specifically in the transplanted cells.
  • Cre-dependent TVA the receptor of engineered surface protein EnvA which originates from the avian leukosis virus
  • NLS-tdTomato nuclear location signal
  • AAV-DIO-TVA-2A-NLS-tdTomato AAV-DIO-TVA-2A-NLS-tdTomato
  • AAV-DIO-G Cre-dependent rabies glycoprotein
  • EnvA-pseudotyped and G-deleted rabies virus tagged EGFP was injected into the same area 3 weeks later to infects TVA-expressing grafted neurons (tdTomato + ) .
  • the grafted cells co-express EGFP and tdTomato are the starter cells, and expression of G in these cells enable RVdG-EGFP to spread transsynaptically to their upstream presynaptic input cells which will express EGFP only 36 .
  • tdTomato was expressed only in transplants of HIE-injured brains at 2 MPT or 6 MPT, suggesting the specificity of the rabies tracing system (Fig. 5B) .
  • the starter cells were identified by coexpression of TVA-tdTomato and EnvA-GFP (tdTomato + /GFP + ) , and they were also positive for GABA and hN (Fig. 5C) .
  • the starter cells were only found in the injected site and could be easily distinguished from traced neurons in intratransplants or brain regions away from the graft (tdTomato - /EGFP + ) .
  • the host-to-graft synaptic inputs also come from GPe neurons expressing PV, midbrain SNc neurons expressing TH, thalamus including ventral posterolateral/posteromedial thalamic nucleus (VPL /VPM) , parafascicular thalamic nucleus (PF) , mediodorsal thalamic nucleus (MD) and hindbrain including dorsal raphe (DR) (Fig. 5D-E, Supplementary Fig. 4A-B) , indicating extensive synaptic inputs from these brain regions to grafted spiny projection neurons, which are strikingly similar to the circuits of their endogenous counterpart.
  • VPL /VPM ventral posterolateral/posteromedial thalamic nucleus
  • PF parafascicular thalamic nucleus
  • MD mediodorsal thalamic nucleus
  • DR dorsal raphe
  • Example 6 Grafted neurons receive synaptic regulation from the striatal and cortical neurons of the HIE-injured brain.
  • sIPSCs or sEPSCs spontaneous inhibitory or excitatory postsynaptic currents
  • Fig. 6A A total of 100% (8/8) of grafted neurons showed abundant sIPSCs or sEPSCs with high density by 6 MPT (Fig. 6C) , while only 50% (9/18) were recorded with few sIPSCs or sEPSCs by 2 MPT (Fig.
  • the paired pulse ratio which is the ratio of the amplitude of the second response to that of the first, was also tested in grafted neurons upon blue light stimulation.
  • PPR paired pulse ratio
  • 75%of the grafted cells (9/12) were recorded with PPR (Fig. 6N, upper panel) .
  • the second peak amplitude is smaller than that of the first (0.67 ⁇ 0.07) (Fig. 6N, upper panel) , suggesting established plasticity of grafted neurons in the HIE-injured brain.
  • Example 7 Intrastriatal grafted neurons activate host striatal-and long-range targeted nigra neurons in HIE-injured brains.
  • Striatal GABA neurons derived from the hESC-mCherry line were used as the control for optogenetic activation of grafted cells in the striatum of HIE-injured mice (Supplementary Fig. 6A) , which showed properties of mature neurons but no APs evoked by blue light illumination at 6 MPT (Supplementary Fig. 6B-D) .
  • HIE-injured mice For the open field test, which reveals limb movements during locomotion, the total distance covered by HIE-injured mice was significantly increased over time by intrastriatal SNPs transplantation (P ⁇ 0.001) but not by SGNPs or ACSF treatment (Fig. 8C) .
  • motor coordination and balance assessed by the rotarod test also exhibited obviously increased latency on the rotarod in HIE-injured animals grafted with SNPs but not SGNPs or ACSF (Fig. 8D) .
  • Representative images showed a large number of human cells (hN + ) stained by GABA but not DARPP32 distributed in the ipsilateral striatum of HIE-injured mice at 6 MPT (Supplementary Fig.
  • CNO treatment had no effect on ipsilateral touches in mice that received SNPs expressing mCherry (Fig. 8F) .
  • CNO treatment significantly decreased the total distance covered by mice grafted with hM4Di-SNPs compared to saline treatment or CNO withdrawal (P ⁇ 0.05) , but not in animal grafted with Cherry-SNPs (Fig. 8G) , indicating graft-dependent improvement of animal motor functions.
  • HIE-injured brains we showed hESC-derived SNPs survived and matured into striatal spiny projection neurons in HIE-injured brains. And we first proved the high feasibility and long-term therapeutic efficiency of hESC-derived striatal neural progenitors in immature HIE-injured brains, including neuronal survival and differentiation, axon projections and synapse formation, neural circuit reconstruction and functional restoration.
  • transplanted striatal progenitor cells can specifically projected to their cognate brain regions including GPe, GPi, and SN (Fig. 3) , and receive innervation from host neurons in multiple brain areas (Fig. 5) in a pattern similar to their endogenous counterparts.
  • transplanted striatal neurons can form functional synaptic connection with host neurons both pre-and post-synaptically (Fig. 6 and 7) .
  • chemogenetic tools we demonstrated that the functional recovery of HIE model mice depends on the graft activity.
  • striatal grafted SNPs send axons as early as 1 month to endogenous target areas after cell transplantation in HIE-injured brains, similar like the SNPs grafted in adult HD mouse brains, indicating cell intrinsic properties probably determine the targeted projection of grafted human neurons.
  • grafted neuron should form synaptic connections with host neurons.
  • the grafted SNPs receive synaptic inputs from extensive brain regions, similar to their endogenous counterparts, by 2 MPT.
  • hESCs may confront potential ethical issues in some countries, and personalized iPSC-derived neural progenitors have been proved effective in the treatment of some neurological diseases without detectable immune rejection in clinical studies of human participants 53, 54 . Further investigation about therapeutic effects of striatal neural progenitors derived from iPSCs in the treatment of HIE need to be done in the future.

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Abstract

L'invention concerne des progéniteurs neuronaux striataux dans le traitement de l'encéphalopathie hypoxique-ischémique, et en particulier une utilisation de progéniteurs neuronaux striataux pour la préparation d'une composition ou d'une formulation, ladite composition ou formulation étant utilisée pour la prévention et/ou le traitement de l'encéphalopathie hypoxique-ischémique (HIE). L'encéphalopathie hypoxique-ischémique peut être efficacement traitée par des progéniteurs neuronaux striataux.
PCT/CN2022/139429 2022-12-15 2022-12-15 Progéniteurs neuronaux striataux dans le traitement de l'encéphalopathie hypoxique-ischémique WO2024124507A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1273534A (zh) * 1997-08-04 2000-11-15 加利福尼亚大学董事会 治疗神经缺损的方法
US20030060398A1 (en) * 1997-09-19 2003-03-27 Gluckman Peter David Neuronal rescue agent
US20090196858A1 (en) * 2006-05-19 2009-08-06 Aichi Prefecture Ameliorating agent for brain damage
US20100158880A1 (en) * 2008-12-19 2010-06-24 Ethicon, Incorporated Regeneration and repair of neural tissue following injury
CN107864626A (zh) * 2015-05-28 2018-03-30 生物组织再生和修复公司 用于治疗脑损伤的组合物

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1273534A (zh) * 1997-08-04 2000-11-15 加利福尼亚大学董事会 治疗神经缺损的方法
US20030060398A1 (en) * 1997-09-19 2003-03-27 Gluckman Peter David Neuronal rescue agent
US20090196858A1 (en) * 2006-05-19 2009-08-06 Aichi Prefecture Ameliorating agent for brain damage
US20100158880A1 (en) * 2008-12-19 2010-06-24 Ethicon, Incorporated Regeneration and repair of neural tissue following injury
CN107864626A (zh) * 2015-05-28 2018-03-30 生物组织再生和修复公司 用于治疗脑损伤的组合物

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JI XIAOLI, ZHOU YINGYING, GAO QINQIN, HE HUI, WU ZIYAN, FENG BAN, MEI YUTING, CHENG YAN, ZHOU WENHAO, CHEN YUEJUN, XIONG MAN: "Functional reconstruction of the basal ganglia neural circuit by human striatal neurons in hypoxic–ischaemic injured brain", BRAIN, OXFORD UNIVERSITY PRESS, GB, vol. 146, no. 2, 13 February 2023 (2023-02-13), GB , pages 612 - 628, XP093180826, ISSN: 0006-8950, DOI: 10.1093/brain/awac358 *
L. AUBRY, BUGI A., LEFORT N., ROUSSEAU F., PESCHANSKI M., PERRIER A. L.: "Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 105, no. 43, 28 October 2008 (2008-10-28), pages 16707 - 16712, XP055225840, ISSN: 0027-8424, DOI: 10.1073/pnas.0808488105 *
MIN YANG, JIANG FENG, YANG JIAN-HUA,, WANG YU, MA JIE: "Differentiation of Neural Progenitor Cells Originated from Embryonic Stem Cells in Hippocampus of Hypoxic-Ischemic Brain Damage in Mice", JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY(MEDICAL SCIENCE), vol. 26, no. 7, 25 July 2006 (2006-07-25), pages 733 - 737, XP093180822 *
PEDRO M. PIMENTEL-COELHO: "Human Cord Blood Transplantation in a Neonatal Rat Model of Hypoxic–Ischemic Brain Damage: Functional Outcome Related to Neuroprotection in the Striatum", STEM CELLS AND DEVELOPMENT, MARY ANN LIEBERT, INC. PUBLISHERS, US, vol. 19, no. 3, 1 March 2010 (2010-03-01), US , pages 351 - 358, XP093154478, ISSN: 1547-3287, DOI: 10.1089/scd.2009.0049 *

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