WO2021228050A1 - 一种诱导胶质细胞转分化为功能性神经元的方法及其应用 - Google Patents

一种诱导胶质细胞转分化为功能性神经元的方法及其应用 Download PDF

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WO2021228050A1
WO2021228050A1 PCT/CN2021/092851 CN2021092851W WO2021228050A1 WO 2021228050 A1 WO2021228050 A1 WO 2021228050A1 CN 2021092851 W CN2021092851 W CN 2021092851W WO 2021228050 A1 WO2021228050 A1 WO 2021228050A1
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neurog2
functional
neurons
cells
mcherry
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PCT/CN2021/092851
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French (fr)
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陈如雷
刘婷
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再康医药科技(上海)有限公司
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Priority to EP21805293.4A priority Critical patent/EP4151724A1/en
Priority to US17/924,685 priority patent/US20230201304A1/en
Priority to JP2022569179A priority patent/JP2023524900A/ja
Priority to CN202180034333.4A priority patent/CN115605583A/zh
Publication of WO2021228050A1 publication Critical patent/WO2021228050A1/zh

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Definitions

  • the present invention belongs to the field of biotechnology and gene therapy. Specifically, the present invention relates to a method and application for inducing glial cells to transdifferentiate into functional neuronal cells, especially in the repair of spinal cord injury.
  • the main pathological changes caused by mammalian central nervous system damage and a variety of neurodegenerative diseases are irreversible neuronal degeneration and necrosis and the destruction of neural circuits. How to replace the dead and lost neurons in the brain or spinal cord due to injury and disease and rebuild the neural circuit is a key step in treatment. Because the central nervous system (brain and spinal cord) of adult mammals has very limited self-repair capabilities, it is difficult to make up for the loss of neuronal cells by itself.
  • the present invention provides a method for the Neurog2 functional fragment to induce the transdifferentiation of glial cells into functional neuronal cells in vitro or in vivo, and its application in the preparation of a nerve repair pharmaceutical composition.
  • Neurog2 functional fragments (i) for the preparation of a pharmaceutical composition for nerve-inducing glial cells to form functional neuronal cells; and/or (ii) for the preparation of a neurog Pharmaceutical composition for systemic diseases.
  • the glial cells are selected from human or non-human mammals.
  • the glial cells are selected from the following group: astrocytes, NG2 glial cells, oligodendrocytes, microglia or combinations thereof.
  • the glial cells are astrocytes.
  • the astrocytes include astrocytes in a normal state and in an injured state.
  • the injury state is neuronal death or apoptosis caused by mechanical trauma, stroke, neurodegenerative diseases or other neurological diseases, which results in blockage or disorder of nerve signal conduction.
  • the astrocytes are derived from spinal cord, dorsal midbrain or cerebral cortex, preferably, the astrocytes are derived from spinal cord and dorsal midbrain.
  • the functional neuron is a cell having the following characteristics: (a) having a neuron morphology, (b) having one or more neuron markers, and (c) capable of issuing actions Electric potential and form synaptic connections.
  • the functional neuron may also be a neuron cell group, specifically, the neuron cell group has one or more of the following characteristics:
  • At least 50% of neuronal cells preferably at least 60%, 70%, 80%, 90%, or 100% of neuronal cells express the mature neuron marker NeuN;
  • the functional neuron is an excitatory neuron, preferably, a VGLUT2 + excitatory neuron.
  • the functional neurons include glutamatergic neurons or a group of glutamatergic neurons.
  • the functional neuron can emit action potentials and can form synaptic connections.
  • the Neurog2 functional fragment is a functional Neurog2 protein or a nucleic acid sequence encoding a Neurog2 functional protein
  • the Neurog2 functional fragment is a protein or nucleic acid sequence that can normally perform the physiological function of the Neurogenin 2 transcription factor.
  • Neurog2 is derived from mammals, preferably from humans or non-human primate mammals.
  • GenBank number of the Neurog2 functional fragment is 11924, and the protein sequence is shown in SEQ ID NO.:1; the mRNA encoding the Neurog2 gene NCBI Reference Sequence number is NM_009718.3, and the CDS sequence As shown in SEQ ID NO.: 2.
  • GenBank number of the Neurog2 functional fragment is 63973, and the protein sequence is shown in SEQ ID NO.: 3; the mRNA encoding the Neurog2 gene NCBI Reference Sequence number is NM_024019.4, and the CDS sequence As shown in SEQ ID NO.: 4.
  • sequence of the Neurog2 functional fragment sequence and SEQ ID NO.: 1 have a sequence homology of not less than 83%; more preferably, the sequence of the Neurog2 functional fragment sequence and SEQ ID NO.
  • the sequence homology of :1 is not less than 90%; optimally, the sequence of the Neurog2 functional fragment sequence is not less than 95% of the sequence homology of SEQ ID NO.:1.
  • the functional nucleic acid sequence encoding Neurog2 has no less than 80% homology with the sequence of SEQ ID NO.: 2; more preferably, the functional nucleic acid sequence encoding Neurog2 has The sequence homology of SEQ ID NO.: 2 is not less than 90%; optimally, the sequence homology of the nucleic acid functional sequence encoding Neurog2 and the sequence of SEQ ID NO.: 2 is not less than 95%.
  • sequence of the Neurog2 functional fragment sequence and SEQ ID NO.: 3 have a sequence homology of not less than 83%; more preferably, the sequence of the Neurog2 functional fragment sequence and SEQ ID NO.
  • the sequence homology of :3 is not less than 90%; optimally, the sequence of the Neurog2 functional fragment sequence and the sequence of SEQ ID NO.: 3 are not less than 95%.
  • the functional nucleic acid sequence encoding Neurog2 has no less than 80% homology with the sequence of SEQ ID NO.: 4; more preferably, the functional nucleic acid sequence encoding Neurog2 has The sequence homology of SEQ ID NO.: 4 is not less than 90%; optimally, the sequence homology of the nucleic acid functional sequence encoding Neurog2 and the sequence of SEQ ID NO.: 4 is not less than 95%.
  • a delivery system carrying a functional fragment of Neurog2 is provided,
  • the delivery system can be applied in vitro or in vivo to induce the transdifferentiation of glial cells into functional neuronal cells; the glial cells are derived from the spinal cord, dorsal midbrain or cerebral cortex, preferably, the The glial cells are derived from the spinal cord and dorsal midbrain; the glial cells are glial cells in a normal state or in an injured state.
  • the Neurog2 functional fragment can be passively absorbed by glial cells or reach the inside of glial cells through a delivery system to take effect.
  • the delivery system carries a functional fragment of Neurog2, including but not limited to an expression vector carrying a functional fragment of Neurog2, a nanoparticle coated with a functional fragment of Neurog2, and a functional fragment of Neurog2.
  • the delivery system is an expression vector carrying a functional fragment of Neurog2.
  • the expression vector can enter glial cells and express exogenous Neurog2 protein in astrocytes.
  • the expression vector includes plasmid and virus vector.
  • the expression vector is a viral vector, including but not limited to an adenovirus vector, an adeno-associated virus vector (AAV), a retroviral expression vector or a lentiviral vector, etc., preferably an adeno-associated virus vector ( AAV).
  • AAV adeno-associated virus vector
  • the expression vector is an astrocyte-specific expression vector.
  • the expression vector carrying the functional fragment of Neurog2 also contains a glial cell-specific promoter, and the promoter includes but not limited to GFAP promoter, NG2 promoter, Aldh1L1 promoter, IBA1 promoter, CNP promoter, LCN2 promoter or promoter variants after genetic engineering.
  • the expression vector carrying the functional fragment of Neurog2 also contains one or more regulatory elements for regulating gene expression, which are used to enhance the expression level of the gene, including but not limited to the enhancer of CMV , SV40 enhancer, EN1 enhancer or genetically engineered enhancer variants, as well as SV40 polyA tailing signal, human insulin gene polyA tailing signal or WPRE (groundhog hepatitis B virus post-transcriptional regulatory element), human-derived MAR sequence or genetically engineered variants.
  • regulatory elements for regulating gene expression which are used to enhance the expression level of the gene, including but not limited to the enhancer of CMV , SV40 enhancer, EN1 enhancer or genetically engineered enhancer variants, as well as SV40 polyA tailing signal, human insulin gene polyA tailing signal or WPRE (groundhog hepatitis B virus post-transcriptional regulatory element), human-derived MAR sequence or genetically engineered variants.
  • the expression vector carrying the Neurog2 functional fragment may also contain other functional fragments, and the other functional fragments may be reporter genes or other transcription factors with reprogramming functions.
  • Functional fragments including but not limited to Ascl1, NeuroD1, etc.
  • the expression vector carrying the functional fragment of Neurog2 is a GFAP-AAV vector; the GFAP-AAV vector carries viral ITR sequence, CMV enhancer, human GFAP promoter, and Neurog2 function
  • the coding frame of the sexual fragment and the post-transcriptional regulatory element WPRE, etc.; the expression vector may also contain a reporter gene, but the reporter gene is not necessary in practical applications.
  • the GFAP-AAV expression vector is from 5'to 3
  • The'end may sequentially include the following elements: viral ITR sequence + CMV enhancer + human GFAP promoter + Neurorog2 and red fluorescent protein mCherry coding frame + post-transcriptional regulatory element WPRE + viral ITR sequence + ampicillin resistance gene promoter and The coding frame, wherein the coding frame of the red fluorescent protein mCherry and the promoter and coding frame of the ampicillin resistance gene are not necessary.
  • the expression vector carrying the functional fragment of Neurog2 is NG2-lentiviral vector; the NG2-lentiviral vector carrying the viral ITR sequence, the promoter of human NG2, the functional fragment of Neurog2
  • the coding frame and the post-transcriptional regulatory element WPRE, etc.; the expression vector can also contain a reporter gene, but the reporter gene is not necessary in practical applications.
  • the NG2-lentiviral vector can be from 5'to 3'.
  • the third aspect of the present invention provides a host cell containing exogenous Neurog2 functional fragments.
  • the chromosome of the host cell integrates a polynucleotide encoding the Neurog2 protein, or the host cell contains the expression vector described in the second aspect of the present invention.
  • the host cell is derived from glial cells, and the delivery system described in the second aspect of the present invention can be used to integrate a polynucleotide encoding the Neurog2 protein in its chromosome, or in the host cell.
  • the transfer of functional fragments of Neurog2 into cells promotes the transdifferentiation of host cells into functional neurons.
  • the host cells are derived from glial cells cultured in vitro.
  • the host cells are derived from glial cells in normal and injured states in vivo.
  • the glial cells are astrocytes.
  • the host cell is a functional neuron or a functional neuron cell group.
  • the fourth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (A) the Neurog2 functional fragment described in the first aspect of the present invention; and/or (B) the Neurog2 functional fragment described in the second aspect of the present invention
  • the fifth aspect of the present invention provides the delivery system according to the second aspect of the present invention, the host cell according to the third aspect of the present invention, and/or the use of the pharmaceutical composition according to the fourth aspect of the present invention, which can be used to prepare Drugs for nervous system damage and nerve repair.
  • the nervous system damage includes spinal cord injury, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), neuronal death or irreversible loss caused by stroke, and the like.
  • the nerve repair is achieved by the pharmaceutical composition according to the fourth aspect of the present invention to achieve the recovery of neuronal function in the injured area of the nervous system.
  • the sixth aspect of the present invention provides a non-therapeutic in vitro method for transdifferentiating astrocytes into functional neuronal cells, including the steps:
  • astrocytes are cultured to induce astrocytes to form functional neuronal cells.
  • a functional neuron cell and/or neuron cell group transdifferentiated from astrocytes and the functional neuron cell and/or neuron cell group is derived from the present invention. It is prepared by the method described in the six directions, and the functional neuron cells and/or neuron cell populations have one or more of the following characteristics:
  • At least 50% of neuronal cells preferably at least 60%, 70%, 80%, 90%, or 100% of neuronal cells express the mature neuron marker NeuN;
  • the eighth aspect of the present invention provides a method for treating neurological diseases, including the steps:
  • a safe and effective amount of the pharmaceutical composition according to the fourth aspect of the present invention is administered to a subject in need, thereby treating neurological diseases.
  • the neurological diseases include spinal cord injury, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), neuronal death or irreversible loss caused by stroke, and the like.
  • the object in need is a human.
  • the subject in need suffers from a neurological disease.
  • the ninth aspect of the present invention provides a method of (a) screening candidate compounds for the treatment of neurological diseases; and/or (b) screening candidate compounds that induce the transdifferentiation of astrocytes into functional neuronal cells, which It is characterized by including the steps:
  • E1 in the test group is significantly higher than E0, it indicates that the tested compound is (a) a candidate compound for the treatment of neurological diseases; and/or (b) induces the transdifferentiation of astrocytes into functional neuronal cells Candidate compounds.
  • the cells are astrocytes.
  • the term significantly higher means that E1 is higher than E0 and has a statistical difference; preferably, E1 ⁇ 2E0.
  • the method further includes the steps:
  • test compound is (a) a candidate compound for the treatment of neurological diseases; and/or (b) screening for induction of astrocytes Candidate compounds for transdifferentiation of glial cells into functional neuronal cells;
  • test compound when the ratio T1 of the conversion of astrocytes to functional neurons in the test group is significantly higher than the ratio T0 of the control group, it indicates that the test compound is (a) a candidate compound for treating neurological diseases; and/or (b) Screening candidate compounds that induce the transdifferentiation of astrocytes into functional neuronal cells.
  • the neurological diseases include spinal cord injury, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), neuronal death or irreversible loss caused by stroke, and the like.
  • Figure 1 Neurog2 single factor can efficiently induce midbrain astrocytes into neurons.
  • Figures 1A and B respectively show that 3 days after the control virus AAV-mCherry (negative control) and the virus AAV-Neurog2/mCherry were injected into the dorsal midbrain of adult mice, the immunolabeling showed that neither mCherry co-localized with NeuN.
  • Figure 1C shows that 30 days after the control virus AAV-mCherry was injected into the dorsal midbrain of adult mice, mCherry still did not co-localize with NeuN.
  • Figure 1D shows that 30 days after the virus AAV-Neurog2/mCherry was injected into the dorsal midbrain of adult mice, the vast majority of mCherry co-localized with NeuN.
  • Figure 1E shows a statistical graph of the ratio of neurons in different periods. Arrows and arrows represent mCherry + NeuN + and mCherry + NeuN - cells, respectively. "***" means p ⁇ 0.001. Ruler: 50um.
  • Figure 2 The neurons induced by Neurog2 in the midbrain were confirmed to be active functional neurons through electrophysiological recordings.
  • Figure 2A shows the mCherry + electrophysiological membrane parameters recorded in slices of the midbrain 30 days after AAV-mCherry virus infection. The changes in membrane current recorded by gradient voltage stimulation in current clamp mode (above) and gradient current stimulation in voltage clamp mode Record the membrane voltage change (below).
  • Figure 2B shows the post-synaptic current signal of mCherry+ cells recorded in the midbrain section 30 days after AAV-Neurog2/mCherry virus infection. The post-synaptic current signal disappears after the blocker NBQX is added, and the post-synaptic current signal is eluted. appear again.
  • Figure 3 shows that Neurog2 midbrain reprogramming neurons are glutamatergic neurons.
  • Figures 3A and B show the results of in situ histochemical double-labeling of adult mice 30 days after AAV-Neurog2/mCherry virus infection.
  • Virus signal mCherry protein (red) and VGLUT2mRNA (A, green) and Gad1mRNA (B, green) overlapped signals are (A right) and (B right) respectively.
  • the arrow in (A) indicates mCherry + VGLUT2 + double positive cells.
  • the arrow in (B) indicates mCherry + Gad1 - cells.
  • Figure 3C shows a statistical graph of the neuron ratios of different transmitters. The nucleus is marked by DAPI. Scale: 25um.
  • Figure 4 Neurog2 single factor can efficiently induce spinal cord astrocytes into neurons.
  • Figures 4A and B respectively show that 3 days after the control virus AAV-mCherry and the virus AAV-Neurog2/mCherry were injected into the spinal cord of adult mice, the immunolabeling showed that neither mCherry co-localized with NeuN.
  • Figure 4C shows that 30 days after the control virus AAV-mCherry was injected into the spinal cord of adult mice, mCherry still did not co-localize with NeuN.
  • Figure 4D shows that 30 days after the virus AAV-Neurog2/mCherry was injected into the spinal cord of adult mice, the vast majority of mCherry co-localized with NeuN.
  • Figure 4E shows a statistical graph of the ratio of neurons in different periods. Arrows and arrows represent mCherry + NeuN + and mCherry + NeuN - cells, respectively. "***" means p ⁇ 0.001. Ruler: 50um.
  • Figure 5 Neurog2 induces neurons in the spinal cord through electrophysiological recording to confirm that they are active functional neurons, and can respond to Dorsal root ganglion (DRG) stimulation.
  • Figures 5A and B show the electrophysiological recordings of spinal cord slices derived from mCherry+ cells infected with AAV-mCherry virus (A) and AAV-Neurog2/mCherry virus (B) for 30 days, and gradients were given respectively in current clamp or voltage clamp mode Voltage or current stimulation, obtained membrane voltage (left) and cell membrane current parameters (right).
  • Figure 5C shows the electrophysiological response of neurons induced by mCherry+ on the dorsal side of the spinal cord after DRG stimulation. The green arrow represents the beginning of the stimulus.
  • Figure 6 Neurog2 single factor can efficiently induce injured spinal cord astrocytes into neurons.
  • Figures 6A and B respectively show that 3 days after the control virus AAV-mCherry and virus AAV-Neurog2/mCherry were injected to injure the spinal cord of adult mice, the immunolabeling showed that neither mCherry co-localized with NeuN.
  • Figure 6C shows that 30 days after the control virus AAV-mCherry was injected to injure the spinal cord of adult mice, mCherry still did not co-localize with NeuN.
  • Figure 6D shows that 30 days after the injection of virus AAV-Neurog2/mCherry injures the spinal cord of adult mice, most of mCherry co-localizes with NeuN.
  • Figure 6E shows a statistical graph of the ratio of neurons in different periods. Arrows and arrows represent mCherry + NeuN + and mCherry + NeuN - cells, respectively. "**" means p ⁇ 0.01. Ruler: 50
  • Figure 7 Neurog2 reprogramming neurons can promote the recovery of motor function in mice with spinal cord injury.
  • Figure 7A and Figure B respectively show the motor function of the control group (without spinal cord injury) and the injured mice injected with the virus AAV-mCherry and the virus AAV-Neurog2/mCherry 3 weeks later.
  • Figure 7A scores the motor function of the mice according to the BMS standard.
  • 7B is the time when the mouse is in an exercise state during the open field experiment. "*" means p ⁇ 0.05.
  • Figure 8 Neurog2 reprograms neurons to reduce anxiety in some mice with spinal cord injury.
  • Figures 8A and B respectively show the number of times the mice in the control group (with no spinal cord injury) and injured mice injected with the virus AAV-mCherry and the virus AAV-Neurog2/mCherry 3 weeks after crossing the central area. "*" means p ⁇ 0.05.
  • Figure 9 Neurog2 transdifferentiates astrocytes from the entorhinal cortex of AD mice into neurons in vivo.
  • Figure 9A and B respectively show that after the control virus AAV-mCherry and virus AAV-Neurog2/mCherry were injected into the entorhinal cortex of 5XFAD mice for 46 days, immune co-labeling showed that the control virus AAV-mCherry group mCherry did not co-localize with NeuN.
  • AAV-Neurog2/mCherry most mCherry co-localized with NeuN, and the two groups of animal sections were simultaneously developed by A ⁇ . Arrows represent mCherry + NeuN + cells. Ruler: 50um.
  • Figure 10 Amino acid sequence alignment of mouse and human Neurog2.
  • Neurog2 gene or its protein in glial cells can efficiently induce transdifferentiation of astrocytes into neuronal cells with normal electrophysiological functions in vivo or in vitro .
  • neurons induced by glial cells from the spinal cord can respond to the stimulation of the dorsal root ganglion (DRG) of the peripheral nervous system, showing that neurons induced by Neurog2 functional fragments can be functional Is integrated into the spinal circuit and functions. Therefore, the induction of Neurog2 functional fragments is expected to be an effective method for stimulating the production of new neuronal cells in adults, which can be widely used in the development of drugs for neurological diseases.
  • DRG dorsal root ganglion
  • the present invention mainly has the following competitive advantages:
  • Neurog2 functional fragments can not only act on glial cells in normal environments, but also act on glial cells in damaged environments, breaking through glial cells in damaged environments/ The stress state of nerve cells obtains neural connections with normal electrophysiological functions and produces nerve repair functions.
  • Neurog2 functional fragments can specifically and uniformly transdifferentiate glial cells into excitatory glutamatergic neurons or groups of glutamatergic neurons, ensuring the Manipulability of neurological disease drug development and application.
  • Neurog2 (neurogenin 2) is a bHLH transcription factor.
  • the main structural domains include DNA-binding domain, activation domain, oligomerization site, and nuclear localization signal. signal) and other functional areas.
  • bHLH basic Helix-Loop-Helix, basic helix-loop-helix
  • the bHLH domain is very conserved in mammals, including mice and humans.
  • the ID# of the Neurog2 molecule from mouse in GenBank is 11924, and its protein sequence is shown in SEQ ID NO.: 1; NCBI Reference Sequence: NM_009718.3; CDS sequence is shown in SEQ ID NO.: 2; from human
  • the ID# of the Neurog2 molecule in GenBank is 63973, and its protein sequence is shown in SEQ ID NO.: 3, in which the bHLH domain is located at positions 112 to 164; NCBI Reference Sequence: NM_024019.4; CDS sequence is shown in SEQ ID NO.: 4 shown.
  • the results of the amino acid sequence alignment of the mouse and human Neurog2 are shown in FIG. 10.
  • the amino acid/protein sequences of mouse and human Neurog2 are as follows:
  • SEQ ID No: 1 amino acid/protein sequence of mouse Neurog2
  • SEQ ID No: 3 amino acid/protein sequence of human Neurog2
  • Neurog2 functional fragments refer to Neurog2 sequence fragments that can regulate transcription and realize redifferentiation, including but not limited to: Neurog2 full-length protein, and sequences that contain the conserved Neurog2bHLH domain and have more than 83% sequence homology with wild-type Neurog2 Fragment.
  • Neurog2 functional fragments include polynucleotides derived from mammals that encode Neurogenin 2 transcription factors or fragments of expressed proteins.
  • the functional fragment of Neurog2 may also be a variant of the Neurog2 protein sequence.
  • the variant of the functional fragment of Neurog2 protein should have no less than 83% sequence homology with SEQ ID NO.:1; or The functional sequence of the protein encoding Neurog2 has no less than 90% homology with the sequence of SEQ ID NO.:1; or the functional sequence of the protein encoding Neurog2 has the sequence homology of SEQ ID NO.:1 Not less than 95%; or the functional sequence of the protein encoding Neurog2 has no less than 98% homology with SEQ ID NO.: 1; or the variant of the functional fragment of Neurog2 protein and SEQ ID NO.:
  • the sequence homology of 3 should not be less than 83%; or the functional sequence of the protein encoding Neurog2 should not be less than 90% with the sequence of SEQ ID NO.: 3; or the protein encoding Neurog2
  • the sequence homology between the functional sequence and SEQ ID NO.: 3 is not less than 95%; or the sequence homology between the
  • the functional fragment of Neurog2 may also be a variant of the polynucleotide sequence of Neurog2.
  • the functional sequence of the polynucleotide encoding Neurog2 has a sequence homology of not less than 80% with that of SEQ ID NO.: 2.
  • nucleic acid functional sequence encoding Neurog2 has no less than 90% homology with the sequence of SEQ ID NO.: 2; or the nucleic acid functional sequence encoding Neurog2 is similar to SEQ ID NO.: 2
  • the sequence homology is not less than 95%; or the functional sequence of the nucleic acid encoding Neurog2 is not less than 98% with the sequence of SEQ ID NO.: 2; or the polynucleic acid encoding Neurog2 is functional
  • the sequence homology with the sequence of SEQ ID NO.: 4 is not less than 80%; or the sequence homology of the nucleic acid functional sequence encoding Neurog2 and the sequence of SEQ ID NO.: 4 is not less than 90%; or The nucleic acid functional sequence encoding Neurog2 has no less than 95% homology with the sequence of SEQ ID NO.: 4; or the nucleic acid functional sequence encoding Neurog2 has sequence homology with SEQ ID NO.: 4 Not less than 98%;
  • the functional fragment of Neurog2 can also be obtained by CRISPR/dCas9 targeting DNA to activate the expression of Neurog2 gene, or by CRISPR/Cas13 targeting RNA to increase the expression of Neurog2;
  • the expression promoter of Neurog2 is not particularly limited, and can be any substance that promotes the expression and/or activity of the Neurog2 gene or its protein, such as small molecule compounds and promoting miRNA. Those skilled in the art can screen Neurog2 promoters based on existing databases. It should be understood that based on the transdifferentiation induction effect of Neurog2 on astrocytes disclosed in the present invention, those skilled in the art can reasonably foresee that any substance that promotes Neurog2 will induce transdifferentiation of astrocytes. effect.
  • Astrocytes are the most abundant type of cells in the mammalian brain. They perform many functions, including biochemical support (such as forming a blood-brain barrier), providing nutrients to neurons, maintaining extracellular ion balance, and participating in repair and scar formation after brain and spinal cord injury. According to the content of glial filaments and the shape of the cell processes, astrocytes can be divided into two types: fibrous astrocytes are mostly distributed in the white matter of the brain and spinal cord, with slender protrusions and fewer branches. , The cytoplasm contains a lot of glial filaments; protoplasmic astrocytes (protoplasmic astrocytes) are mostly distributed in the gray matter, with thick and short cell processes and many branches.
  • the astrocytes that can be used in the present invention are not particularly limited, and include various astrocytes derived from the central nervous system of mammals, such as the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably , From the dorsal midbrain or spinal cord.
  • astrocytes from various sources have higher induction transformation efficiency.
  • the specific marker of astrocytes is GFAP.
  • Astrocytes in gray matter have relatively low expression of GFAP, but express Acsbg1 or GS.
  • these astrocytes show neuronal cell-specific markers, such as NeuN.
  • the term "functional neuron” refers to neuronal cells that are transdifferentiated from astrocytes and have normal neuronal electrophysiological activities in the presence of exogenous Neurog2 genes or proteins. Generally, the functional neuron cell has the following characteristics:
  • the "functional neuron” is an excitatory neuron, especially a VGLUT2 + excitatory neuron, which is a glutamatergic neuron. Therefore, in this article, the terms “excitatory neuron”, “VGLUT2 + excitatory neuron”, and “glutamatergic neuron” are used interchangeably, and all refer to the functional neuron described in the present invention.
  • the delivery system that can be used in the present invention is not particularly limited, and may be an expression vector containing the coding sequence of Neurog2 protein that can enter astrocytes.
  • a viral vector can be any viral vector that can take advantage of the characteristics of the virus to transmit its genome and bring genetic material into other cells for infection. It can occur in whole living organisms or in cell culture. Including lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, and poxvirus vectors.
  • the delivery system can also be a new type of nanoparticle, which is used to load Neurog2 functional fragments and deliver them to target cells, such as liposomal nanoparticles, metal nanoparticles, polymer nanoparticles, etc., which can carry Neurog2 functional fragments. .
  • the delivery system can also be exosomes coated with a functional fragment of Neurog2, or modified red blood cells or bacteria coated with a functional fragment of Neurog2.
  • the delivery system can also be combined with targeted functional molecules, such as specific monoclonal antibodies and peptides targeting astrocytes, which can better improve the target of Neuorog2 functional fragments on astrocytes. Tropism, increase transdifferentiation efficiency.
  • targeted functional molecules such as specific monoclonal antibodies and peptides targeting astrocytes, which can better improve the target of Neuorog2 functional fragments on astrocytes. Tropism, increase transdifferentiation efficiency.
  • the invention also provides a method for inducing astrocytes to transdifferentiate into functional neuronal cells in vivo.
  • a delivery system containing Neurog2 can be administered (for example, injected) to a desired subject containing astrocytes, such as the dorsal midbrain, cerebral cortex, or spinal cord, which can treat undamaged and damaged nervous systems.
  • astrocytes such as the dorsal midbrain, cerebral cortex, or spinal cord
  • the tissue is injected to induce transdifferentiation of astrocytes in specific parts of the nervous system.
  • a delivery system containing Neurog2 can be administered (for example, injected) to NG2 glial cell clusters cultured in vitro to induce the differentiation of functional neurons in vitro, and then the functional neuron clusters cultured in vitro can be transplanted Transplanted into the body.
  • the present invention also provides a pharmaceutical composition which is a delivery system containing Neurog2 functional fragments or functional neuron clusters induced to be transdifferentiated by the Neurog2 functional fragments in vitro.
  • the pharmaceutical composition of the present invention includes the above-mentioned expression vector (for example, viral particles) of the present invention, or the exogenous Neurog2 protein itself, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention usually contains 10 10 -10 13 PFU/ml AAV virus particles, preferably 10 11 -10 13 PFU/ml AAV virus particles, more preferably 10 10 -10 12 PFU/ml AAV virus particles.
  • “Pharmaceutically acceptable carrier” refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents. Generally, the term refers to pharmaceutical carriers that are not essential active ingredients themselves, and do not have excessive toxicity after administration. Suitable carriers are well known to those of ordinary skill in the art.
  • the pharmaceutically acceptable carrier in the composition may contain liquid, such as water, saline, and buffer. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting agents or emulsifiers, and pH buffer substances may also be present in these carriers.
  • the vector may also contain a cell transfection reagent.
  • the pharmaceutical composition of the present invention can be obtained after mixing the expression vector and the pharmaceutically acceptable carrier.
  • composition of the present invention is not particularly limited.
  • Representative examples include (but are not limited to): intravenous injection, subcutaneous injection, brain injection, intrathecal injection, spinal cord injection, and the like.
  • the Neurog2 functional fragment of the present invention can be used to prepare drugs for inducing astrocytes to produce functional neurons, so that the newly induced neurons can be applied to various types of neurons due to decrease in the number of neurons, cell decline, apoptosis, or decline in neuron function Related diseases.
  • the diseases related to the nervous system include spinal cord injury, Alzheimer's disease (AD), Parkinson's disease (PD), neuronal death caused by stroke and the like.
  • a single transcription factor Neurog2 can transdifferentiate astrocytes in the dorsal midbrain and spinal cord of adult mice into functional neurons. These induced neurons express neuronal marker molecules, can emit action potentials, and can receive synaptic afferents from other neurons to establish synaptic connections. Therefore, this method is expected to be an effective method for stimulating the production of new neuronal cells in adults, and thus is widely used in the treatment of neurological diseases, such as neurodegenerative diseases, central nervous system traumatic diseases, and so on.
  • neurological diseases such as neurodegenerative diseases, central nervous system traumatic diseases, and so on.
  • NG2 cells For the preparation and culture of NG2 cells, the cortical tissue of the mouse 3-5 days after birth was taken out and digested with 0.25% trypsin for 15 minutes. The blown cells were cultured in DMEM/F12 solution containing 10% serum for 7-9 days.
  • Immune coloration of cultured cells refers to "Direct conversion of fibroblasts to functional neurons by defined factors" (Vierbuchen, T.et al. Nature 463, 1035-1041 (2010)). Immune coloration of tissue sections combined with in situ hybridization and immunity The double-label experiment of color development was carried out according to the published method.
  • the primary antibodies used in immunochromatography include: mouse anti-GFAP (Millipore, 1:1,000), mouse anti-NeuN (Millipore, 1:100), anti-Dsred (Clontech, 1:500), mouse anti-Dsred ( Santa Cruz,1:100), rabbit anti-Acsbg1(Abcam,1:100), rabbit anti-NG2(Millipore,1:200), rabbit anti-Iba1(Wako,1:500), mouse anti-CNPase(Abcam ,1:500),mouse anti-O4(Millipore,1:500),.
  • FITC-, Cy3- and Cy5-conjugated secondary antibodies were purchased from Jackson Immunoresearch.
  • the AAV virus was performed with reference to the mouse brain map. After the virus was injected, the back midbrain and spinal cord were collected at different time points for immunochromatography or brain slice recording. Intact spinal cord and injured spinal cord virus injection concentration, the speed is consistent with the injection volume per needle and the brain area, and the spinal cord is injected at an angle of 30°.
  • mice used for the transplantation were NOD-scid mice of seven weeks old. Accutase digests and induces the cells for 4-6 days, centrifuges to remove the supernatant so that the density of the cells after concentration is about 2 ⁇ 10 5 cells/ ⁇ l, and each mouse brain is transplanted 2 ⁇ l, which is a total of 4 ⁇ 10 5 cells. Histochemical and electrophysiological tests were performed 2-4 weeks after transplantation.
  • mice The loss of sensory afferents after thoracic spinal cord injury promotes the weakening of the inhibitory effect of the descending inhibitory system of the brainstem, and the tail is oversensitive to external stimuli.
  • the motor function of mice was scored according to the BMS standard.
  • the test method refers to Basso Mouse Scale for locomotion detection differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma, 2006.23(5): p.635-59.
  • mice in the central area of the open field are related to the degree of anxiety. Normal mice frequently shuttle through the central area of the open field (Zone Crossing), while anxious mice tend to move more around the square (Peripheral).
  • the experimental method refers to The open field as a paradigm to measure the effects of drugs on anxiety-like behavior: a review. European Journal of Pharmacology, 2003.463(1-3): p.3-33.
  • Example 1 Neurog2 transdifferentiates NG2 glial cells into functional neurons in vitro
  • NG2 cells were plated and cultured for 24 hours and then added with lentivirus. After 24 hours of infection, the medium was changed: DMEM/F12, B27, Glutamax and penicillin/streptomycin. After 6-7 infections, brain-derived neurotrophic factor (BDNF; PeproTech, 20ng/ml) was added to the culture medium every three days.
  • BDNF brain-derived neurotrophic factor
  • NG2 glial cell marker NG2 Most of the cultured mouse NG2 cells were immunopositive for the NG2 glial cell marker NG2. A small number of cells expressed oligodendrocyte marker molecules O4 and CNPase. No neuron marker molecules Tuj1 and stem cell marker molecules Sox2 and Sox2 were detected. The expression of Oct4.
  • NG2 cells Ten days after NG2 cells were transfected with hNG2-Neurog2-IRES-GFP lentivirus, most NG2 cells showed typical neuronal morphology and expressed Tuj1, the neuronal marker molecule. After 21 days of infection with lentivirus, the induced cells also express NeuN and MAP2, which are the marker molecules of mature neurons. Electrophysiological records show that induced neurons can produce action potentials, and most induced neuron cells can record spontaneous postsynaptic currents, which indicates that these neurons can form functional synapses.
  • Whether the transdifferentiated neurons induced in vitro can survive and function in vivo is the key to whether they can be used for disease treatment.
  • Neurog2 was carried out for two weeks after the neurons induced by NG2 cells were transplanted into the cerebral cortex. We found that the transplanted cells can attach to the edge of the cortex, and some cells can extend the neurites deeper into the cortex. The results of immunofluorescence co-localization experiments showed that some of the transplanted cells express the neuron marker molecule Tuj1, indicating that the induced neurons can survive and express neuron-specific marker molecules.
  • Example 2 Neurog2 transdifferentiates adult dorsal midbrain astrocytes into neurons in vivo
  • the virus AAV-mCherry or AAV-mNeurog2/mCherry was injected into the cap of one side of adult wild-type mice, and then brain tissue samples were collected at different time points.
  • Three days after virus injection whether in mice injected with the control virus AAV-mCherry ( Figure 1A) or the virus AAV-Neurog2/mCherry ( Figure 1B), immune co-labeling showed that mCherry did not co-localize with NeuN.
  • Thirty days after virus injection in mice injected with the control virus AAV-mCherry, immune co-labeling showed that mCherry still did not co-localize with NeuN.
  • mice injected with the virus AAV-Neurog2/mCherry the vast majority of mCherry co-localized with NeuN (88.2 ⁇ 6.3%). It shows that Neurog2 successfully induced astrocytes into neurons.
  • the induced neurons are functional neurons
  • the induced neurons are all excitatory neurons
  • VGLUT2/Gad1mRNA and mCherry protein were stained by using double-labeling method of in situ hybridization and immunohistochemistry.
  • Experimental results show that AAV-Neurog2/mCherry virus can reprogram midbrain astrocytes into VGLUT2 + excitatory neurons (Figure 3A, C) instead of Gad1 + inhibitory nerve cells ( Figure 3B, C). Therefore, we confirmed that Neurog2 single factor can program midbrain astrocytes into glutamatergic neurons during body weight.
  • the CDS (SEQ ID No.: 4, NCBI: NM_024019.4) derived from the human Neurog2 gene was cloned into AAV-mCherry to obtain AAV-hNeurog2/mCherry Plasmid.
  • the induced neurons are functional neurons
  • Example 4 Neurog2 transdifferentiates astrocytes from injured spinal cord of adult mice into neurons in vivo
  • SCI Spinal cord injury
  • mice T8-T10 spinal cord complete transection model was constructed (refer to McDonough A, Monterrubio A, Ariza J, et al. Calibrated Forceps Model of Spinal Cord Compression Injury. Jove-Journal of Visualized Experiments 2015.), after injury The AAV-mCherry virus and AAV-mNeurog2/mCherry (Example 2) or AAV-hNeurog2/mCherry (Example 3) were immediately injected into both sides of the injured spinal cord.
  • Neurog2 reprogrammed neurons after spinal cord injury have electrophysiological characteristics and can receive external signal input. Therefore, we used a variety of animal models to evaluate the nerve repair ability of mice with spinal cord injury after neuron induction. Using the spinal cord injury detection model, we found that Neurog2 reprogramming neurons is very helpful for the recovery of sensory and motor functions in mice with spinal cord injury.
  • mice We tested the motor function of the mice three weeks after the virus injection, and scored the motor function of the mice according to the BMS standard (Basso, D.M., et al., J Neurotrauma, 2006.).
  • the test data showed that the Ctrl group mice can basically maintain the exercise ability at 9 points, while the exercise ability of the AAV-mCherry virus group mice is 2.3 points, and the mice injected with AAV-Neurog2/mCherry rises to 4.5 points ( Figure 7A). Then the open field experiment was used to further evaluate the exercise ability of the mice.
  • mice We also used the mouse square experiment to evaluate the degree of anxiety in mice after injury, to test whether neurons reprogrammed by Neurog2 can reduce the anxiety level of some mice with spinal cord injury, and the acute/chronic pain caused by spinal cord injury, especially Chronic pain can often cause anxiety.
  • the mouse model of spinal cord injury is usually accompanied by different degrees of anxiety.
  • neurog2 reprogrammed neurons also achieved great relief of the anxiety of mice with spinal cord injury.
  • open field experiments it is generally believed that the activity of mice in the central area of the open field is related to the degree of anxiety. Normal mice will frequently shuttle through the central area of the open field, while anxious mice tend to move more around the open field.
  • mice We also analyzed the performance of the three groups of experimental mice. The results showed that the Ctrl group mice frequently shuttled through the central area of the open field at 3 weeks, while the AAV-mCherry group mice obviously tended to exercise more around the open field, AAV-Neurog2/ The mice in the mCherry group shuttled more frequently than the mice in the AAV-mCherry group ( Figure 8). These results indicate that Neurog2 reprogramming neurons can reduce the anxiety level of some mice with spinal cord injury.
  • Example 5 Neurog2 transdifferentiates the astrocytes of the entorhinal cortex of AD mice into neurons in vivo
  • Neurog2 successfully transdifferentiated the astrocytes in the entorhinal cortex of AD mice into neurons in vivo and reduced the expression of A ⁇ . Furthermore, these results indicate that Neurog2, a transcription factor with transdifferentiation, provides new technologies and drugs for the treatment of Alzheimer's disease.

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Abstract

提供了一种Neurog2功能性片段的用途，该功能性片段可在体内或体外诱导胶质细胞形成功能性神经元细胞，不仅可以在正常组织起到转分化的作用，还可以促进受损神经组织的神经重建。

Description

一种诱导胶质细胞转分化为功能性神经元的方法及其应用 技术领域
本发明属于生物技术和基因治疗领域，具体地，本发明涉及一种诱导胶质细胞转分化为功能性神经元细胞的方法及应用，特别是在脊髓损伤修复中的应用。
背景技术
哺乳动物的中枢神经系统损伤和多种神经退行性疾病引起的主要病理变化是不可逆的神经元变性坏死和神经环路破坏。如何补充替代损伤和疾病的大脑或脊髓中死亡丢失的神经元并重建神经环路是治疗的关键步骤。由于成年哺乳动物的中枢神经系统(大脑和脊髓)自我修复的能力非常有限，很难靠自身来弥补神经元细胞的丢失。
由于外源性神经元或神经源性细胞的移植的效率较低，而且有致瘤性和免疫原性的潜在风险。近年来，细胞重编程技术的出现给再生医学带来了革命性的变化。通过表达单个或组合的多个神经原性转录因子实现体内重编程星形胶质细胞诱导产生神经元有望成为神经元替代疗法的重要新策略。因此，寻找合适的转录因子，在体内诱导星形胶质细胞转分化为有活性的神经元，对于大脑和脊髓功能修复非常地重要。特别是目前，尚无有效逆转脊髓损伤的神经元修复方法。
发明内容
本发明提供了Neurog2功能性片段在诱导体外或体内胶质细胞转分化为功能性神经元细胞的方法，以及其在制备神经修复药物组合物方面的应用。
本发明第一方面，提供了一种Neurog2功能性片段的用途，(i)用于制备神经诱导胶质细胞形成功能性神经元细胞的药物组合物；和/或(ii)用于制备针对神经系统疾病的药物组合物。
在另一优选例中，所述的胶质细胞选自来源于人或非人哺乳动物。
在另一优选例中，所述的胶质细胞选自下组：星形胶质细胞、NG2胶质细胞、少突胶质细胞、小胶质细胞或其组合。
在另一优选例中，所述的胶质细胞为星形胶质细胞。
在另一优选例中，所述的星形胶质细胞包括正常状态和损伤状态下的星形胶质细胞。
在另一优选例中，所述的损伤状态为机械性的创伤、中风、神经退行性疾病或其他神经系统疾病引起的神经元死亡、凋亡从而致使神经信号传导受阻或紊乱。
在另一优选例中，所述的星形胶质细胞来源于脊髓、背侧中脑或大脑皮层，较佳地，所述的星形胶质细胞来源于脊髓和背侧中脑。
在另一优选例中，所述的功能性神经元为具有以下特征的细胞：(a)具有神经 元形态、(b)具有一种或多种神经元标记物、和(c)能够发放动作电位并形成突触联系。
在另一优选例中，所述的功能性神经元还可以是神经元细胞群，具体地，所述神经元细胞群具有以下一种或多种特征：
(a)至少50％的神经元细胞，优选至少60％、70％、80％、90％、或100％的神经元细胞表达成熟神经元的标志物NeuN；
(b)能够发放动作电位并能够形成突触联系。
在另一优选例中，所述的功能性神经元为兴奋性神经元，较佳地，为VGLUT2 +兴奋性神经元。
在另一优选例中，所述的功能性神经元包括谷氨酸能神经元或谷氨酸能神经元群。
在另一优选例中，所述的功能性神经元能够发放动作电位并能够形成突触联系。
在另一优选例中，所述的Neurog2功能性片段为功能性的Neurog2蛋白或编码Neurog2功能性蛋白的核酸序列；
在另一优选例中，所述的Neurog2功能性片段为可正常执行Neurogenin 2转录因子生理功能的蛋白或核酸序列。
在另一优选例中，Neurog2来源于哺乳动物，较佳地，来源于人或非人灵长类哺乳动物。
在另一优选例中，所述Neurog2功能性片段的GenBank号为11924，蛋白序列如SEQ ID NO.:1所示；编码所述的Neurog2基因的mRNA NCBI Reference Sequence号为NM_009718.3，CDS序列如SEQ ID NO.:2所示。
在另一优选例中，所述Neurog2功能性片段的GenBank号为63973，蛋白序列如SEQ ID NO.:3所示；编码所述的Neurog2基因的mRNA NCBI Reference Sequence号为NM_024019.4，CDS序列如SEQ ID NO.:4所示。
在另一优选例中，所述的Neurog2功能性片段序列与SEQ ID NO.:1的序列同源性不低于83％；更优地，所述的Neurog2功能性片段序列与SEQ ID NO.:1的序列同源性不低于90％；最优地，所述的Neurog2功能性片段序列与SEQ ID NO.:1的序列同源性不低于95％。
在另一优选例中，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于80％；更优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于90％；最优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于95％。
在另一优选例中，所述的Neurog2功能性片段序列与SEQ ID NO.:3的序列同源性不低于83％；更优地，所述的Neurog2功能性片段序列与SEQ ID NO.:3的序列同源性不低于90％；最优地，所述的Neurog2功能性片段序列与SEQ ID NO.: 3的序列同源性不低于95％。
在另一优选例中，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于80％；更优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于90％；最优地，所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于95％。
本发明第二方面，提供了载有Neurog2功能性片段的递送系统，
所述的递送系统可以应用于体外或应用于体内诱导胶质细胞转分化为功能性神经元细胞；所述的胶质细胞来源于脊髓、背侧中脑或大脑皮层，较佳地，所述的胶质细胞来源于脊髓和背侧中脑；所述的胶质细胞为正常状态或损伤状态下的胶质细胞。
在另一优选例中，所述的Neurog2功能性片段可以依靠胶质细胞被动吸收或通过递送系统到达胶质细胞内部起效。
在另一优选例中，所述的递送系统载有Neurog2功能性片段，包括但不限于载有Neurog2功能性片段的表达载体、包裹有Neurog2功能性片段的纳米颗粒、包裹有Neurog2功能性片段的外泌体、包裹有Neurog2功能性片段的改造红细胞或细菌、挂载有Neurog2功能性片段的靶向效应物(如胶质细胞特异性抗体、多肽或其他靶向物质等)。
在一优选例中，所述的递送系统为载有Neurog2功能性片段的表达载体，所述的表达载体可进入胶质细胞，并在星形胶质细胞中表达外源的Neurog2蛋白。
在另一优选例中，所述的表达载体包括质粒、病毒载体。
在另一优选例中，所述的表达载体为病毒载体，包括但不限于腺病毒载体、腺相关病毒载体(AAV)、逆转录病毒表达载体或慢病毒载体等，优选为腺相关病毒载体(AAV)。
在另一优选例中，所述的表达载体是星形胶质细胞特异性表达载体。
在另一优选例中，所述的载有Neurog2功能性片段的表达载体中还载有胶质细胞特异性的启动子，所述的启动子，包括但不限于GFAP启动子、NG2启动子、Aldh1L1启动子、IBA1启动子、CNP启动子、LCN2启动子或经过基因工程改造后的启动子变体。
在另一优选例中，所述的载有Neurog2功能性片段的表达载体中还载有一个或多个调控基因表达的调控元件，用于增强基因的表达水平，包括但不限于CMV的增强子、SV40增强子、EN1增强子或经过基因工程改造后的增强子变体，以及SV40polyA加尾信号、人胰岛素基因polyA加尾信号或者WPRE(土拨鼠乙肝病毒转录后调控元件)、人源性MAR序列或经过基因工程改造后的变体。
在另一优选例中，所述的载有Neurog2功能性片段的表达载体中还可以载有其他的功能性片段，所述的其他功能性片段可以是报告基因或其他具有重编程功 能的转录因子功能性片段，包括但不限于Ascl1、NeuroD1等。
在另一优选例中，所述的载有Neurog2功能性片段的表达载体为GFAP-AAV载体；所述GFAP-AAV载体载有病毒ITR序列、CMV的增强子、人GFAP的启动子、Neurog2功能性片段的编码框以及转录后调控元件WPRE等；所述的表达载体还可以载有报告基因，但报告基因在实际应用中不是必需的，如所述的GFAP-AAV表达载体自5'到3'端可依次包括以下元件：病毒ITR序列+CMV的增强子+人GFAP的启动子+Neurog2和红色荧光蛋白mCherry的编码框+转录后调控元件WPRE+病毒ITR序列+氨苄抗性基因的启动子和编码框，其中红色荧光蛋白mCherry的编码框与氨苄抗性基因的启动子和编码框不是必需的。
在另一优选例中，所述的载有Neurog2功能性片段的表达载体为NG2-慢病毒载体；所述NG2-慢病毒载体载有病毒ITR序列、人NG2的启动子、Neurog2功能性片段的编码框以及转录后调控元件WPRE等；所述的表达载体还可以载有报告基因，但报告基因在实际应用中不是必需的，如所述的NG2-慢病毒载体自5'到3'端可依次包括以下元件：病毒ITR序列+人NG2的启动子+Neurog2和绿色荧光蛋白GFP的编码框+转录后调控元件WPRE+病毒ITR序列+氨苄抗性基因的启动子和编码框，其中绿色荧光蛋白GFP的编码框与氨苄抗性基因的启动子和编码框不是必需的。
本发明的第三方面，提供了一种包含有外源的Neurog2功能性片段的宿主细胞。
在另一优选例中，所述的宿主细胞的染色体整合有编码Neurog2蛋白的多核苷酸，或所述的宿主细胞含有本发明第二方面中所述的表达载体。
在另一优选例中，所述的宿主细胞来源于胶质细胞，可以利用本发明第二方面所述的递送系统，在其染色体整合有编码Neurog2蛋白的多核苷酸，或在所述的宿主细胞中转入Neurog2的功能性片段，促使宿主细胞转分化为有功能的神经元。
在另一优选例中，所述的宿主细胞源自体外培养的胶质细胞。
在一优选例中，所述的宿主细胞源自体内正常状态和损伤状态下的胶质细胞。
在另一优选例中，所述的胶质细胞为星形胶质细胞。
在一优选例中，所述的宿主细胞为有功能的神经元或有功能的神经元细胞群。
本发明的第四方面，提供了一种药物组合物，所述的药物组合物包括(A)本发明第一方面所述的Neurog2功能性片段；和/或(B)本发明第二方面所述的递送系统，或(C)本发明第三方面所述的宿主细胞；和(D)药学上可接受的辅料。
本发明第五方面，提供了本发明第二方面所述的递送系统、本发明第三方面 所述的宿主细胞、和/或本发明第四方面所述的药物组合物的用途，可用于制备针对神经系统损伤与神经修复的药物。
在另一优选例中，所述的神经系统损伤包括脊髓损伤、癫痫、阿尔兹海默症(AD)、帕金森病(PD)、中风引起的神经元死亡或不可逆的丧失等。
在另一优选例中，所述的神经修复则是通过本发明第四方面所述的药物组合物，实现对神经系统损伤区域的神经元功能的恢复。
本发明第六方面，提供了一种体外非治疗性的将星形胶质细胞转分化为功能性神经元细胞的方法，包括步骤：
在外源性Neurog2功能性片段存在下，培养星形胶质细胞，从而诱导星形胶质细胞形成功能性神经元细胞。
本发明第七方面，提供了一种由星形胶质细胞转分化的功能性神经元细胞和/或神经元细胞群，所述的功能性神经元细胞和/或神经元细胞群由本发明第六方所述的方法制备获得，且所述的功能性神经元细胞和/或神经元细胞群具有以下一种或多种特征：
(a)至少50％的神经元细胞，优选至少60％、70％、80％、90％、或100％的神经元细胞表达成熟神经元的标志物NeuN；
(b)能够发放动作电位并能够形成突触联系。
本发明第八方面，提供了一种治疗神经系统疾病的方法，包括步骤：
向需要的对象施用安全有效量的本发明第四方面所述的药物组合物，从而治疗神经系统疾病。
在另一优选例中，所述的神经系统疾病包括脊髓损伤、癫痫、阿尔兹海默症(AD)、帕金森病(PD)、中风引起的神经元死亡或不可逆的丧失等。
在另一优选例中，所述需要的对象为人类。
在另一优选例中，所述需要的对象患有神经系统疾病。
本发明第九方面，提供了一种(a)筛选治疗神经系统疾病的候选化合物；和/或(b)筛选诱导星形胶质细胞转分化为功能性神经元细胞的候选化合物的方法，其特征在于，包括步骤：
(i)将测试化合物加入细胞培养体系作为测试组，并将未加入测试化合物的细胞培养体系作为对照组；
(ii)比较测试组中Neurog2基因或其蛋白的表达量和/或活性E1与对照组中的表达量和/或活性E0；
其中，当测试组中E1显著高于E0，则表明所试化合物为(a)治疗神经系统疾 病的候选化合物；和/或(b)诱导星形胶质细胞转分化为功能性神经元细胞的候选化合物。
在另一优选例中，所述的细胞为星形胶质细胞。
在另一优选例中，所述的显著高于指的是E1高于E0，且具有统计学差异；优选地，为E1≥2E0。
在另一优选例中，所述的方法还包括步骤：
(iii)将测试化合物加入星形胶质细胞培养体系作为测试组，并将未加入测试化合物的星形胶质细胞培养体系作为对照组；
(iv)比较测试组中星形胶质细胞向功能性神经元转化的比例，从而确定所述测试化合物是否为(a)治疗神经系统疾病的候选化合物；和/或(b)筛选诱导星形胶质细胞转分化为功能性神经元细胞的候选化合物；
其中，当测试组中星形胶质细胞向功能性神经元转化的比例T1显著高于对照组的比例T0，则表明所述测试化合物为(a)治疗神经系统疾病的候选化合物；和/或(b)筛选诱导星形胶质细胞转分化为功能性神经元细胞的候选化合物。
在另一优选例中，所述的神经系统疾病包括脊髓损伤、癫痫、阿尔兹海默症(AD)、帕金森病(PD)、中风引起的神经元死亡或不可逆的丧失等。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1Neurog2单因子能将中脑星形胶质细胞高效率诱导成神经元。图1A，B分别显示在对照病毒AAV-mCherry(阴性对照)和病毒AAV-Neurog2/mCherry注射成年小鼠背侧中脑3天后，免疫共标显示mCherry均不与NeuN共定位。图1C显示在对照病毒AAV-mCherry注射成年小鼠背侧中脑30天后，mCherry仍不与NeuN共定位。图1D显示在病毒AAV-Neurog2/mCherry注射成年小鼠背侧中脑30天后，绝大多数mCherry与NeuN共定位。图1E显示的是是不同时期神经元比率的统计图。箭与箭头分别代表mCherry +NeuN +以及mCherry +NeuN -细胞。"***"代表p<0.001。标尺:50um。
图2Neurog2在中脑诱导的神经元通过电生理记录证实是有活性的功能性神经元。图2A显示AAV-mCherry病毒感染中脑30天后切片记录的mCherry +电生理膜参数，分别在在电流钳模式下给予梯度电压刺激记录的膜电流变化(上面)和电压钳模式下给予梯度电流刺激记录膜电压变化(下面)。图2B显示的是AAV-Neurog2/mCherry病毒感染中脑30天后切片记录的mCherry +细胞的突触后 电流信号，加入阻断剂NBQX后突触后电流信号消失，洗脱后突触后电流信号重新出现。
图3显示Neurog2中脑重编程神经元为谷氨酸能神经元。图3A，B显示的是AAV-Neurog2/mCherry病毒感染成年小鼠30天后，原位组化双标显色结果。病毒信号mCherry蛋白(红色)与VGLUT2mRNA(A,绿色)以及Gad1mRNA(B,绿色)重叠后的信号分别为(A右边)与(B右边)。(A)中的箭指示的是mCherry +VGLUT2 +双阳性的细胞。(B)中的箭头指示的是mCherry +Gad1 -细胞。图3C显示的是不同递质神经元比率的统计图。细胞核被DAPI所标记。标尺：25um。
图4Neurog2单因子能将脊髓星形胶质细胞高效率诱导成神经元。图4A，B分别显示在对照病毒AAV-mCherry和病毒AAV-Neurog2/mCherry注射成年小鼠脊髓3天后，免疫共标显示mCherry均不与NeuN共定位。图4C显示在对照病毒AAV-mCherry注射成年小鼠脊髓30天后，mCherry仍不与NeuN共定位。图4D显示在病毒AAV-Neurog2/mCherry注射成年小鼠脊髓30天后，绝大多数mCherry与NeuN共定位。图4E显示的是是不同时期神经元比率的统计图。箭与箭头分别代表mCherry +NeuN +以及mCherry +NeuN -细胞。"***"代表p<0.001。标尺:50um。
图5Neurog2在脊髓诱导神经元通过电生理记录证实是有活性的功能性神经元，并能够响应背根神经节(Dorsal root ganglion,DRG)的刺激。图5A，B显示的是脊髓切片电生理记录来源于AAV-mCherry病毒(A)和AAV-Neurog2/mCherry病毒(B)感染30天的mCherry+细胞，在电流钳或者电压钳模式下，分别给予梯度电压或者电流刺激，得到的膜电压(左)以及细胞膜电流参数(右)。图5C显示的是在DRG刺激后，脊髓背侧mCherry+诱导神经元的电生理反应。绿色箭头代表刺激的开始。
图6Neurog2单因子能将损伤脊髓星形胶质细胞高效率诱导成神经元。图6A，B分别显示在对照病毒AAV-mCherry和病毒AAV-Neurog2/mCherry注射损伤成年小鼠脊髓3天后，免疫共标显示mCherry均不与NeuN共定位。图6C显示在对照病毒AAV-mCherry注射损伤成年小鼠脊髓30天后，mCherry仍不与NeuN共定位。图6D显示在病毒AAV-Neurog2/mCherry注射损伤成年小鼠脊髓30天后，绝大多数mCherry与NeuN共定位。图6E显示的是是不同时期神经元比率的统计图。箭与箭头分别代表mCherry +NeuN +以及mCherry +NeuN -细胞。"**"代表p<0.01。标尺:50um。
图7Neurog2重编程神经元对脊髓损伤小鼠运动功能的恢复有促进作用。图7A，B分别显示对照组(未损伤脊髓)以及损伤小鼠注射病毒AAV-mCherry和病毒AAV-Neurog2/mCherry 3周后的运动功能，图7A根据BMS标准对小鼠运动功能进行评分，图7B是小鼠在旷场实验时处于运动状态的时间。"*"代表p<0.05。
图8Neurog2重编程神经元减缓部分脊髓损伤小鼠焦虑水平。图8A，B分别显示对照组(未损伤脊髓)以及损伤小鼠注射病毒AAV-mCherry和病毒AAV-Neurog2/mCherry 3周后的小鼠穿越中心区域的次数。"*"代表p<0.05。
图9Neurog2在体内将AD小鼠内嗅皮层的星型胶质细胞在体转分化为神经元。图9A，B分别显示在对照病毒AAV-mCherry和病毒AAV-Neurog2/mCherry注射损伤5XFAD小鼠内嗅皮层46天后，免疫共标显示对照病毒AAV-mCherry组mCherry均不与NeuN共定位，组病毒AAV-Neurog2/mCherry绝大多数mCherry与NeuN共定位，两组动物切片同时进行Aβ显色。箭头代表mCherry +NeuN +细胞。标尺:50um。
图10小鼠与人Neurog2的氨基酸序列比对。
具体实施方式
本发明人经过广泛而深入的研究，发现调控胶质细胞中的Neurog2基因或其蛋白表达，能够在体内或体外高效地将星形胶质细胞诱导转分化为具有正常电生理功能的神经元细胞，而且意外发现，不仅是正常状态下的星形胶质细胞，即使是处于损伤环境下的星形胶质细胞，也可以被转分化为功能性的神经元，起到神经修复的功能。特别地，来源于脊髓的胶质细胞诱导产生的神经元，可以响应外周神经系统的背根神经节(Dorsal root ganglion,DRG)的刺激，显示出经过Neurog2功能性片段诱导的神经元能够功能性的整合到脊髓环路中并发挥功能。因此，Neurog2功能性片段的诱导作用，有望成为在成人体内刺激产生新神经元细胞的有效方法，从而广泛可应用于神经系统疾病的药物开发中。
与现有技术相比，本发明主要存在以下竞争性优势：
(1)相对于其他已发现的转分化因子，Neurog2功能性片段不仅可以作用在正常环境下的胶质细胞中，还可以作用在损伤环境下的胶质细胞，突破损伤环境下胶质细胞/神经细胞的应激状态，获得具有正常电生理功能的神经连接，产生神经修复的功能。
(2)相对于其他已发现的转分化因子，Neurog2功能性片段可以特异性地将胶质细胞均一地转分化为兴奋性谷氨酸能神经元或谷氨酸能神经元群，保证了在 神经系统疾病药物开发和应用的可操控性。
(3)相对于体外诱导神经元后的神经元团的移植，体内AAV介导的Neurog2诱导神经元的操作更简便，神经元诱导效率更高，诱导的神经元电生理上更为成熟，更适合用于神经系统疾病的体内再生修复。
Neurog2功能性片段或其促进剂
Neurog2(neurogenin 2)为bHLH类转录因子，主要的结构域包括DNA结合区(DNA-binding domain)、转录调控区(activation domain)、寡聚化位点(oligomerization site)以及核定位信号(nuclear localization signal)等功能区域。bHLH(basic Helix-Loop-Helix,碱性螺旋-环-螺旋)是Neurog2与DNA结合特异性调控基因表达的结构域。哺乳动物包括小鼠和人中bHLH结构域非常保守。
来自小鼠的Neurog2分子在GenBank的ID#为11924，其蛋白序列如SEQ ID NO.:1所示；NCBI Reference Sequence:NM_009718.3；CDS序列如SEQ ID NO.:2所示；来自人的Neurog2分子在GenBank的ID#为63973，其蛋白序列如SEQ ID NO.:3所示，其中bHLH结构域位于第112至164位；NCBI Reference Sequence:NM_024019.4；CDS序列如SEQ ID NO.:4所示。所述小鼠与人Neurog2的氨基酸序列比对结果如图10所示。小鼠与人Neurog2的氨基酸/蛋白序列如下所示：
SEQ ID No:1(小鼠Neurog2的氨基酸/蛋白序列)
Figure PCTCN2021092851-appb-000001
SEQ ID No:3(人Neurog2的氨基酸/蛋白序列)
Figure PCTCN2021092851-appb-000002
Neurog2功能性片段是指具有调控转录，实现再分化功能的Neurog2序列片段，包括但不限于：Neurog2全长蛋白，以及含有保守Neurog2bHLH结构域且与野生型Neurog2具有83％以上序列同源性的序列片段。Neurog2功能性片段，包括来源于哺乳动物、编码Neurogenin 2转录因子的多核苷酸或其表达蛋白片段。
所述的Neurog2的功能性片段还可以是Neurog2蛋白序列的变体，特别地，Neurog2蛋白功能性片段的变体与SEQ ID NO.:1的序列同源性不应低于83％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于90％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于 95％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:1的序列同源性不低于98％；或Neurog2蛋白功能性片段的变体与SEQ ID NO.:3的序列同源性不应低于83％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于90％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于95％；或所述的编码Neurog2的蛋白功能性序列与SEQ ID NO.:3的序列同源性不低于98％
所述的Neurog2的功能性片段还可以是Neurog2多核苷酸序列的变体，特别地，所述的编码Neurog2的多核酸功能性序列与SEQ ID NO.:2的序列同源性不低于80％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于90％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于95％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:2的序列同源性不低于98％；或所述的编码Neurog2的多核酸功能性序列与SEQ ID NO.:4的序列同源性不低于80％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于90％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于95％；或所述的编码Neurog2的核酸功能性序列与SEQ ID NO.:4的序列同源性不低于98％；
所述的Neurog2的功能性片段还可以是通过CRISPR/dCas9靶向DNA激活Neurog2基因的表达获得，或者通过CRISPR/Cas13靶向RNA提高Neurog2的表达获得；
Neurog2的表达促进剂没有特殊限制，可以为任何促进Neurog2基因或其蛋白表达和/或活性的物质，例如小分子化合物、促进性的miRNA。本领域技术人员可以根据现有的数据库对Neurog2促进剂进行筛选。应理解，基于本发明公开的Neurog2对星形胶质细胞的转分化诱导作用，本领域技术人员能够合理地预见任何对Neurog2具有促进作用的物质均会对星形胶质细胞具有转分化的诱导作用。
星形胶质细胞
星形胶质细胞，是哺乳动物脑内数量最多的一类细胞。它们执行许多功能，包括生化支撑(例如形成血-脑屏障)，为神经元提供营养，维持细胞外离子平衡，并参与脑和脊髓损伤后的修复和瘢痕形成。根据胶质丝的含量以及胞突的形状可将星形胶质细胞分为两种：纤维性星形胶质细胞(fibrous astrocyte)多分布在脑和脊髓的白质，突起细长，分支较少，胞质中含大量胶质丝；原浆性星形胶质细胞(protoplasmic astrocyte)，多分布在灰质，细胞突起粗短，分支多。
可用于本发明的星形胶质细胞没有特别限制，包括哺乳动物中枢神经系统来源的各种星形胶质细胞，例如来源于纹状体、脊髓、背侧中脑或大脑皮层，较佳地，来源于背侧中脑或脊髓。
在本发明中，各个来源的星形胶质细胞所具有的诱导转化效率均较高。通常，星形胶质细胞的特异性标志物为GFAP，灰质中的星形胶质细胞GFAP表达相对 较低，但表达Acsbg1或GS。而当通过本发明方法的诱导后，这些星形胶质细胞表现出神经元细胞特有的标志物，例如NeuN。
功能性神经元
如本文所用，术语“功能性神经元”指的是在外源性Neurog2基因或蛋白存在下，由星形胶质细胞转分化而成的、具有正常神经元电生理活动的神经元细胞。通常，所述的功能性神经元细胞具有如下特性：
(a)表达神经元的标志物NeuN；
(b)能够发放动作电位并能够形成突触联系。
在本发明中，所述“功能性神经元”是兴奋性神经元，特别是VGLUT2 +兴奋性神经元，它是一种谷氨酸能神经元。因此，在本文中，术语“兴奋性神经元”、“VGLUT2 +兴奋性神经元”、“谷氨酸能神经元”可互换使用，均指本发明所述的功能性神经元。
递送系统
可用于本发明的递送系统没有特殊限制，可以是含有Neurog2蛋白编码序列的能够进入星形胶质细胞的表达载体。例如病毒载体，其可以是任何能够利用病毒具有传送其基因组的特点，将遗传物质带入其他细胞，进行感染的病毒载体。可发生于完整活体或是细胞培养中。包括慢病毒载体、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体。
递送系统还可以是新型纳米颗粒，用于装载Neurog2功能性片段，并递送到目标细胞中，如脂质体纳米颗粒、金属纳米颗粒、高分子纳米颗粒等可以携带有Neurog2功能性片段的递送系统。
递送系统还可以是包裹有Neurog2功能性片段的外泌体，或者是包裹有Neurog2功能性片段的改造红细胞或细菌。
此外，递送系统还可以结合上靶向功能的分子，如靶向星形胶质细胞的特异性单克隆抗体、多肽等，可以更好地提高Neuorog2功能性片段在星形胶质细胞上的靶向性，增加转分化效率。
诱导方法
本发明还提供了在体内将星形胶质细胞诱导转分化为功能性神经元细胞的方法。
在体内，可将含有Neurog2的递送系统施用(例如注射)到所需对象含有星形胶质细胞的部位，例如背侧中脑、大脑皮层或者脊髓，可以对未受损和受损的神经系统组织进行注射，从而诱导该神经系统特定部位中的星形胶质细胞进行转分化。
在体外，可将含有Neurog2的递送系统施用(例如注射)到体外培养的NG2胶质细胞团中，诱导有功能的神经元的体外分化，再通过移植的方式将体外培养的功能性神经元团移植到体内。
药物组合物以及给药方式
本发明还提供的一种药物组合物为含有Neurog2功能性片段的递送系统或在体外经Neurog2功能性片段诱导转分化后的功能性神经元团。
本发明药物组合物包括本发明上述的表达载体(例如病毒颗粒)、或外源性Neurog2蛋白本身，和药学上可接受的载体。
在本发明的药物组合物，通常含有10 10-10 13PFU/ml的AAV病毒颗粒，较佳地10 11-10 13PFU/ml的AAV病毒颗粒，更佳地10 10-10 12PFU/ml的AAV病毒颗粒。
“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。通常，该术语指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。所述的载体中还可以含有细胞转染试剂。
通常，将所述表达载体和药学上可接受的载体混合后，即可获得的本发明的药物组合物。
本发明所述的组合物的给药方式没有特别限制，代表性的例子包括(但并不限于)：静脉注射、皮下注射、脑部注射、鞘内注射和脊髓注射等。
应用
本发明Neurog2功能性片段可用于制备诱导星形胶质细胞产生功能性神经元的药物，从而将新诱导的神经元应用于各种由于神经元数量减少、细胞衰退、凋亡或神经元功能下降相关的疾病。其中，所述的神经系统相关疾病包括脊髓损伤、阿尔兹海默症(AD)、帕金森病(PD)、中风引起的神经元死亡等。
本发明有益效果
本发明将单个转录因子Neurog2能够将成年小鼠背侧中脑以及脊髓的星形胶质细胞转分化为功能性的神经元。这些诱导的神经元表达神经元的标志分子，能够发放动作电位，并能够接受其他神经元的突触传入建立突触联系。因此，该方法有望成为在成人体内刺激产生新神经元细胞的有效方法，从而广泛应用于神经系统疾病的治疗，例如神经退行性病变、中枢神经创伤性疾病等等。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
通用方法
NG2细胞培养
对于NG2细胞的制备培养，取出出生后3-5天小鼠的皮质组织，并用0.25％的胰酶消化15分钟。吹散的细胞置于含10％血清的DMEM/F12配液中培养7-9天。然后振荡烧瓶并收集上清，离心重悬细胞并接种在多聚-D-赖氨酸(Sigma)包被的盖玻片上，在37℃下在含5％CO2的培养箱中含有N2补充剂(STEMCELLTM)、10ng/ml血小板衍生生长因子(PDGF,Invitrogen)、10ng/mL表皮生长因子(EGF,Invitrogen)和10ng/mL成纤维细胞生长因子2(FGF2,Invitrogen)的无血清DMEM培养基(Gibco)中培养细胞3天。
免疫显色
培养细胞的免疫显色参照“Direct conversion of fibroblasts to functional neurons by defined factors”(Vierbuchen,T.et al.Nature 463,1035-1041(2010)).组织切片的免疫显色结合原位杂交及免疫显色的双标实验参照已发表的方法进行。免疫显色用到的一抗包括：mouse anti-GFAP(Millipore,1:1,000),mouse anti-NeuN(Millipore,1:100),anti-Dsred(Clontech,1:500),mouse anti-Dsred(Santa Cruz,1:100)，rabbit anti-Acsbg1(Abcam,1:100),rabbit anti-NG2(Millipore,1:200),rabbit anti-Iba1(Wako,1:500),mouse anti-CNPase(Abcam,1:500),mouse anti-O4(Millipore,1:500),。FITC-,Cy3-以及Cy5-偶联的二抗购自Jackson Immunoresearch。
AAV病毒的立体定位注射
AAV病毒参照小鼠脑图谱进行。注射病毒后，在不同时间点收集背部中脑、脊髓做免疫显色或脑片记录。完整脊髓以及损伤脊髓病毒注射浓度，速度与每针注射量与脑区一致，在脊髓是以30°夹角的方位注射的。
诱导神经元的移植
移植所使用的小鼠为七周的NOD-scid小鼠。Accutase消化诱导4-6天的细胞，离心去除上清使得细胞浓缩后密度大约为2×10 5cells/μl，每只小鼠大脑移植2μl即总共4×10 5细胞。移植后2-4周进行组化以及电生理检测。
脊髓损伤检测模型
脊髓胸段损伤后导致感觉传入的缺失促使脑干的下行抑制系统的抑制作用减弱导致尾巴对外界刺激过度敏感。我们利用甩尾实验模型，通过测量两组小鼠尾巴在48℃以及52℃的热刺激下反应延迟时间来检测小鼠的感觉能力。根据BMS标准对小鼠运动功能进行评分。试验方法参照Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.J Neurotrauma,2006.23(5):p.635-59。
Open field旷场实验
被用于动物焦虑水平的评估。通常认为，小鼠在旷场中心区域的活动与焦虑程度相关，正常小鼠会频繁穿梭旷场中心区域(Zone Crossing)，而焦虑小鼠倾 向于更多在广场四周运动(Peripheral)。实验方法参照The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors:a review.European Journal of Pharmacology,2003.463(1-3):p.3-33。
实施例1 Neurog2在体外将NG2胶质细胞转分化为功能性神经元
(1)质粒构建与病毒感染
在FUGW–IRES–EGFP的载体模板(载体信息参考文献Efficient transfer,integration,and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.Proc Natl Acad Sci U S A 93:11382–11388)上克隆人NG2启动子(SEQ ID No.:5)替换CAG启动子，再将源自人Neurog2基因的CDS(SEQ ID No.:4)片段构建到该慢病毒载体上，以生成hNG2-hNeurog2-IRES-EGFP慢病毒质粒。慢病毒的包装参照文献“Production and purification of lentiviral vectors”(Tiscornia,G.,Singer,O.&Verma,I.M.Nat.Protoc.1,241-245(2006))。
NG2细胞铺板培养24小时后加入慢病毒，感染24小时后更换培养基：DMEM/F12，B27，Glutamax和青霉素/链霉素。感染6-7后，每三天在培养基中加入脑源性神经营养因子(BDNF；PeproTech公司，20ng/ml)。(2)Neurog将NG2细胞转分化为神经元
培养的小鼠NG2细胞大多数对NG2胶质细胞标记物NG2为免疫阳性，少量的细胞表达少突胶质细胞的标志分子O4和CNPase，没有检测到神经元标志分子Tuj1以及干细胞标志分子Sox2和Oct4的表达。
NG2细胞转染hNG2-Neurog2-IRES-GFP慢病毒10天后，大部分NG2细胞呈现典型的神经元形态，表达神经元的标志分子Tuj1。感染慢病毒21天后，诱导细胞细胞还表达成熟神经元的标志分子NeuN和MAP2。电生理记录表明诱导的神经元能产生动作电位，而且绝大多数的诱导神经元细胞能记录到自发的突触后电流，这说明这些神经元能形成功能性突触。
(3)NG2细胞诱导的神经元移植到体内可以存活
体外诱导得到的转分化神经元能否在体内存活并发挥功能是其是否可用于疾病治疗的关键。Neurog2在NG2细胞诱导的神经元移植到大脑皮层后，两周进行免疫组化实验，我们发现移植的细胞能够依附在皮层边缘，且部分细胞能够将神经突起伸向皮层更深处。免疫荧光共定位实验结果显示移植的细胞有一部分表达神经元标志分子Tuj1，说明诱导的神经元能够存活并表达神经元特异的标志分子。
实施例2 Neurog2在体内将成年背侧中脑星形胶质细胞转分化为神经元
(1)腺相关病毒质粒构建与病毒感染
在AAV–FLEX–Arch–GFP(Addgene，#22222)的载体模板上克隆GFAP启动子(SEQ ID No.:6)替换CAG启动子并保留CMV增强子，再用mCherry的编码框取代GFP后得到AAV-mCherry质粒(对照组)。将源自小鼠Neurog2基因的CDS(SEQ ID No.:2,NCBI:NM_009718.3)克隆进AAV-mCherry质粒后得到AAV-mNeurog2/mCherry质粒，目的基因在GFAP启动子作用下可以特异靶向星形胶质细胞。
(2)Neurog2将星形胶质细胞转分化为神经元
将病毒AAV-mCherry或AAV-mNeurog2/mCherry注射到成年野生型小鼠的一侧顶盖，然后在不同的时间点收集脑组织样品。在病毒注射3天后，无论是在注射对照病毒AAV-mCherry(图1A)，还是病毒AAV-Neurog2/mCherry(图1B)的小鼠中，免疫共标显示mCherry均不与NeuN共定位。在注射病毒后30天，注射对照病毒AAV-mCherry的小鼠中，免疫共标显示mCherry仍不与NeuN共定位。然而，在注射病毒AAV-Neurog2/mCherry的小鼠中，绝大多数mCherry与NeuN共定位(88.2±6.3％)。说明Neurog2成功将星形胶质细胞诱导为神经元。
(3)经诱导的神经元为有功能的神经元
为了证明诱导神经元是有功能的活性神经元，我们进行了电生理记录。我们发现AAV-mCherry病毒感染中脑30天后切片记录的mCherry +细胞并不能产生动作电位(图2A)，这说明AAV-mCHerry病毒体内靶向了星形胶质细胞没有改变它们的电生理特性。而在记录Neurog2/mCherry病毒感染的脑切片时，我们发现20个被记录细胞中，19个细胞在电压钳模式下表现出了内向Na +电流以及外向K +电流(19/20)。而且19个被记录的细胞能够发放多个动作电位(19/20)。重要的是，17个被记录的细胞能产生兴奋性突触后电流(Excitatory postsynaptic currents,EPSCs)(17/20)(图2B)，加入阻断剂NBQX后突触后电流信号消失，洗脱后突触后电流信号重新出现。这说明Neurog2诱导的神经元整合到神经环路中建立了突触联系，是功能性的神经元。
(4)经诱导的神经元均为兴奋性神经元
我们对Neurog2诱导的神经元递质属性进行了鉴定。通过使用原位杂交与免疫组化双标的方法对VGLUT2/Gad1mRNA与mCherry蛋白进行染色。实验结果显示，AAV-Neurog2/mCherry病毒感染30天后能将中脑星形胶质细胞重编程为VGLUT2 +兴奋性神经元(图3A，C)，而非Gad1 +抑制性神经细胞(图3B，C)。因此，我们证实Neurog2单因子可以将中脑星形胶质细胞在体重编程为谷氨酸能神经元。
实施例3 Neurog2在体内将成年小鼠正常脊髓星形胶质细胞转分化成神经元(1)质粒构建与病毒感染
利用实施例2所述的腺相关病毒载体AAV-mCherry质粒，将源自人Neurog2基因的CDS(SEQ ID No.:4,NCBI:NM_024019.4)克隆进AAV-mCherry后得到 AAV-hNeurog2/mCherry质粒。
(2)Neurog2将星形胶质细胞转分化为神经元
我们将AAV-mCherry病毒以及AAV-hNeurog2/mCherry病毒注射到成年小鼠胸段(Thoracic，T8-T10)脊髓背侧，3天后发现两个病毒都能特异性感染星形胶质细胞。在病毒注射3天后，无论是在注射对照病毒AAV-mCherry(图4A)，还是病毒AAV-Neurog2/mCherry(图4B)的小鼠中，免疫共标显示mCherry均不与NeuN共定位。在注射病毒后30天，注射对照病毒AAV-mCherry的小鼠中，免疫共标显示mCherry仍不与NeuN共定位(图4C)。然而，在注射病毒AAV-Neurog2/mCherry的小鼠中，绝大多数mCherry与NeuN共定位(图4D)。说明Neurog2成功将脊髓星形胶质细胞诱导为神经元。
(3)经诱导的神经元为有功能的神经元
我们对Neurog2在脊髓重编程神经元进行电生理特性分析。通过脑片全细胞记录的方法发现，AAV-mCherry病毒感染30天后，mCherry +细胞在给予胞内梯度电流刺激下不能产生动作电位，而且在电压钳模式下刺激也不能产生内向电流(图5A)。这些特征与报道的星形胶质细胞一致。而AAV-Neurog2/mCherry病毒感染30天后，大部分mCherry +细胞在梯度电流刺激下能够发放多个动作电位，而且在电压钳模式刺激下能够产生内向Na +电流以及外向K +电流(图5B)。我们进一步检测Neurog2重编程神经元是否整合到现有的神经环路。我们将脊髓连同DRG部分一起取出进行电生理检测。当我们刺激背根神经节DRG时，能够检测到脊髓背侧诱导的神经元产生的兴奋性突触后电流(Excitatory postsynaptic currents,EPSC)和兴奋性突触后电位(Excitatory postsynaptic potential,EPSP)信号(图5C)，这说明Neurog2不仅可重编程神经元，而且在完整脊髓重编程的神经元能够整合到神经环路并发挥潜在的功能。
实施例4 Neurog2在体内将成年小鼠损伤脊髓星形胶质细胞转分化成神经元
脊髓损伤(Spinal cord injury，SCI)是一类中枢神经受损疾病，伴随着脊髓神经元的死亡以及胶质疤痕的形成。在体神经元重编程将星形胶质细胞转化为神经元，有可能可以缓解SCI造成的伤害，有望成为新的治疗手段。
(1)Neurog2将星形胶质细胞转分化为神经元
我们尝试了使用Neurog2将损伤环境下的胶质细胞重编程为神经元。首先，构造了小鼠T8-T10脊髓全断模型(参照McDonough A,Monterrubio A,Ariza J,et al.Calibrated Forceps Model of Spinal Cord Compression Injury.Jove-Journal of Visualized Experiments 2015.的方法),损伤后立即将AAV-mCherry病毒以及AAV-mNeurog2/mCherry(实施例2)或AAV-hNeurog2/mCherry(实施例3)分别注射到损伤脊髓两侧。在病毒注射3天后，无论是在注射对照病毒AAV-mCherry(图6A)，还是病毒AAV-mNeurog2/mCherry(图6B)的小鼠中，免疫共标显示mCherry均不与NeuN共定位。在注射病毒后30天，注射对照病毒AAV- mCherry的小鼠中，免疫共标显示mCherry仍不与NeuN共定位(图6C)。然而，在注射病毒AAV-mNeurog2/mCherry的小鼠中，能发现部分mCherry与NeuN共定位(图6D)。说明Neurog2成功将损伤脊髓星形胶质细胞诱导为神经元。同样地，由于载有人源的Neurog2的腺相关病毒载体质粒AAV-hNeurog2/mCherry也能成功地将损伤的脊髓星形胶质细胞诱导为神经元。
(2)脊髓损伤模型的修复
由于脊髓损伤后的Neurog2重编程神经元具备电生理特征，而且能够接受外来信号输入。因此，我们使用了多种动物模型来评测脊髓损伤小鼠经过神经元诱导后的神经修复能力。应用脊髓损伤检测模型，我们发现Neurog2重编程神经元对脊髓损伤小鼠感觉功能以及运动功能的恢复有很大帮助。
我们同样采用了脊髓T8-T10横断模型，将AAV-mCherry病毒以及AAV-Neurog2/mCherry病毒注射到损伤脊髓两侧，分别作为实验组以及对照组，另外取同龄段6只雄性未处理小鼠作为参考组(Ctrl)。
我们在病毒注射后三周对小鼠运动功能进行测试，根据BMS标准对小鼠运动功能进行评分(Basso,D.M.,et al.,J Neurotrauma,2006.)。检测数据显示，Ctrl组小鼠基本上能够维持在9分运动能力，而AAV-mCherry病毒组小鼠运动能力2.3分，注射AAV-Neurog2/mCherry小鼠上升到4.5分(图7A)。然后利用旷场实验对小鼠运动能力进一步评估，通过分析发现注射病毒的两组小鼠在15min内运动的时间都比Ctrl组要少，但这两组病毒注射小鼠之间运动时间方面具有统计学差异(图7B)。说明Neurog2重编程神经元对脊髓损伤小鼠运动功能的恢复有促进作用。
我们还使用小鼠广场实验来评估小鼠损伤后的焦虑程度，来检测经Neurog2重编程的神经元能否能减缓部分脊髓损伤小鼠焦虑水平，由于脊髓损伤造成的急/慢性疼痛，特别是慢性疼痛通常能导致焦虑。脊髓损伤的小鼠模型，通常伴随着不同程度的焦虑，我们发现，在经Neurog2重编程神经元对脊髓损伤小鼠的焦虑的也实现了极大的缓解。在旷场实验中通常认为，小鼠在旷场中心区域的活动与焦虑程度相关，正常小鼠会频繁穿梭旷场中心区域，而焦虑小鼠倾向于更多在旷场四周运动。
我们同样分析了三组实验小鼠的表现，结果显示3周时Ctrl组小鼠频繁穿梭旷场中心区域，而AAV-mCherry组小鼠明显倾向于更多在旷场四周运动，AAV-Neurog2/mCherry组小鼠比AAV-mCherry组小鼠穿梭次数更多(图8)。这些结果说明Neurog2重编程神经元能减缓部分脊髓损伤小鼠焦虑水平。
实施例5 Neurog2在体内将AD小鼠内嗅皮层的星型胶质细胞在体转分化为神经元
我们将AAV-mCherry病毒以及AAV-hNeurog2/mCherry病毒注射到6个月 AD模型小鼠5xFAD的内嗅皮层，在病毒注射3天后，无论是在注射对照病毒AAV-mCherry，还是病毒AAV-Neurog2/mCherry的小鼠中，免疫共标显示mCherry均不与NeuN共定位。在注射病毒后46天，注射对照病毒AAV-mCherry的小鼠中，免疫共标显示mCherry仍不与NeuN共定位(图9A)。然而，在注射病毒AAV-Neurog2/mCherry的小鼠中，绝大多数mCherry与NeuN共定位(图9B)，与此同时Aβ的表达范围降低。
这些结果说明Neurog2成功将AD小鼠内嗅皮层的星型胶质细胞在体转分化为神经元，并降低了Aβ表达。进一步，这些结果说明具有转分化的转录因子Neurog2为阿尔茨海默病的治疗提供新的技术和药物。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述的实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (17)

  1. 一种Neurog2功能性片段的用途，(i)用于制备神经诱导胶质细胞形成功能性神经元细胞的药物组合物；和/或(ii)用于制备针对神经系统疾病的药物组合物。
  2. 如权利要求1所述的用途，其特征在于，所述的胶质细胞选自人或非人哺乳动物的星形胶质细胞、NG2胶质细胞、少突胶质细胞、小胶质细胞等；优选地，所述的胶质细胞为星形胶质细胞。
  3. 如权利要求2所述的用途，其特征在于，所述的胶质细胞来源于脊髓、背侧中脑或大脑皮层；较佳地，所述的胶质细胞来源于脊髓和背侧中脑；其中，所述的胶质细胞为正常状态或损伤状态下的胶质细胞。
  4. 如权利要求1所述的用途，其特征在于，所述的Neurog2功能性片段为具有功能性的Neurog2蛋白或编码Neurog2的核酸序列：
    其中，所述的Neurog2蛋白序列与SEQ ID NO.:1或SEQ ID NO.:3的序列同源性不低于83％；更优地，所述的Neurog2蛋白序列与SEQ ID NO.:1的序列同源性不低于90％；最优地，所述的Neurog2蛋白序列与SEQ ID NO.:1的序列同源性不低于95％；
    所述的编码Neurog2的核酸序列与SEQ ID NO.:2或SEQ ID NO.:4的序列同源性不低于80％。
  5. 如权利要求1所述的用途，其特征在于，所述的功能性神经元包括谷氨酸能神经元或谷氨酸能神经元群。
  6. 一种载有Neurog2功能性片段的递送系统，其特征在于，所述的递送系统可应用于体内或应用于体外，诱导胶质细胞转分化为功能性神经元；所述的胶质细胞为正常状态或损伤状态下的胶质细胞。
  7. 如权利要求6所述的递送系统，其特征在于，所述的递送系统为载有Neurog2功能性片段的表达载体，所述的表达载体可进入胶质细胞，并在星形胶质细胞中表达外源的Neurog2蛋白。
  8. 如权利要求6或7所述的递送系统，其特征在于，所述的递送系统为病毒载体，所述病毒载体选自下组：腺相关病毒载体、腺病毒载体、逆转录病毒载体或慢病毒载体。
  9. 如权利要求8所述的递送系统，其特征在于，所述病毒载体至少应包含胶质细胞特异性的启动子和Neurog2功能性片段。
  10. 如权利要求9所述的递送系统，其特征在于，所述的病毒载体的启动子选自GFAP启动子、NG2启动子、Aldh1L1启动子、IBA1启动子、CNP启动子、LCN2启动子或经过基因工程改造后的启动子变体。
  11. 如权利要求8-10任一所述的递送系统，其特征在于，所述的病毒载体为GFAP-AAV载体或NG2-慢病毒载体。
  12. 一种载有外源的Neurog2功能性片段的宿主细胞，其特征在于，所述的宿主细胞来源于体外培养的胶质细胞或体内正常状态下或损伤状态下的胶质细胞，所述宿主细胞转分化，形成了有功能的神经元或有功能的神经元细胞群。
  13. 如权利要求12所述的宿主细胞，其特征在于，所述有功能的神经元具有以下特征：
    (a)表达神经元的标志物NeuN；
    (b)能够发放动作电位并能够形成突触联系。
  14. 如权利要求12所述的宿主细胞，其特征在于，所述有功能的神经元细胞群具有以下特征：
    (a)至少50％的神经元细胞，优选至少60％、70％、80％、90％、或100％的神经元细胞表达成熟神经元的标志物NeuN；
    (b)能够发放动作电位并能够形成突触联系。
  15. 如权利要求6-11所述的任一递送系统或如权利要求12-14任一所述的宿主细胞的应用，其特征在于，所述的递送系统或所述的宿主细胞可用于制备针对神经系统损伤与神经修复的药物组合物，所述的药物组合物还包含有药学上可接受的载体。
  16. 如权利要求15所述的应用，其特征在于，所述的神经系统损伤包括脊髓损伤、癫痫、阿尔兹海默症(AD)、帕金森病(PD)、中风引起的神经元死亡或不可逆的丧失。
  17. 一种药物组合物，所述的药物组合物包括(A)Neurog2功能性片段；和/或(B)如权利要求所述的递送系统，或(C)如权利要求所述的宿主细胞；和(D)药学上可接受的辅料。
PCT/CN2021/092851 2020-05-12 2021-05-10 一种诱导胶质细胞转分化为功能性神经元的方法及其应用 WO2021228050A1 (zh)

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