Neurog2 functional fragment delivery system, host cells and pharmaceutical compositions for inducing transdifferentiation of glial cells into functional neurons

Abstract

Uses of a Neurog2 functional fragment are provided, which can induce the formation of glial cells into functional neuronal cells in vivo or in vitro, and thus not only have the function of transdifferentiation in normal tissues but also promote neural remodeling in damaged neural tissues.

Description

[Technical field]

[0001] The present invention is in the field of biotechnology and gene therapy, specifically the present invention relates to methods and uses for inducing transdifferentiation of glial cells into functional neuronal cells, especially in the repair of spinal cord injuries. [Background technology]

[0002] The main pathological changes caused by central nervous system damage and various neurodegenerative diseases in mammals are irreversible neurodegeneration and necrosis and damage to neural circuits. Methods to replace dead and lost neurons in damaged or diseased brains or spinal cords and reconstruct neural circuits are important steps in treatment. Because the central nervous system (brain and spinal cord) of adult mammals has a very limited ability to repair itself, it is difficult to compensate for the loss of neural cells on its own.

[0003] Transplantation of exogenous neurons or neurogenic cells is inefficient and has potential risks of tumorigenicity and immunogenicity. In recent years, the emergence of cell reprogramming technology has brought revolutionary changes to regenerative medicine. In vivo reprogramming of astrocytes to induce neurons by expressing single or combined multiple neurogenic transcription factors is expected to become an important new strategy for neuronal replacement therapy. Therefore, it is very important to find suitable transcription factors to induce astrocytes to transdifferentiate into active neurons in vivo for functional repair of brain and spinal cord. In particular, there is currently no effective method to restore spinal cord injury. Summary of the Invention

[0004] The present invention provides a method for inducing transdifferentiation of glial cells into functional neuronal cells in vitro or in vivo by a functional fragment of Neurog2, and its use in the preparation of a nerve repair pharmaceutical composition.

[0005] A first aspect of the present invention provides a Neurog2 functional fragment for use in (i) the preparation of a pharmaceutical composition for inducing neurally-induced glial cells to form functional neurons, and / or (ii) the preparation of a pharmaceutical composition for a nervous system disease.

[0006] In another preferred embodiment, said glial cells are selected from a human or non-human mammal.

[0007] In another preferred embodiment, said glial cells are selected from the group consisting of astrocytes, NG2 glial cells, oligodendrocytes, microglia or a combination thereof.

[0008] In another preferred embodiment, said glial cells are astrocytes.

[0009] In another preferred embodiment, said astrocytes include astrocytes in normal and injured states.

[0010] In another preferred embodiment, said injury condition is neuronal cell death or apoptosis caused by mechanical trauma, stroke, neurodegenerative diseases or other nervous system diseases, thereby blocking or disrupting neuronal signaling.

[0011] In another preferred embodiment, said astrocytes are derived from the spinal cord, the dorsal midbrain or the cerebral cortex, preferably the astrocytes are derived from the spinal cord and the dorsal midbrain.

[0012] In another preferred embodiment, the functional neuron is a cell having the following characteristics: (a) having a neuronal morphology, (b) having one or more neuronal markers, and (c) being capable of firing action potentials and forming synaptic connections.

[0013] In another preferred embodiment, said functional neurons may be a population of neurons, in particular said population of neurons exhibiting one or more properties: (a) at least 50% of said neurons, preferably at least 60%, 70%, 80%, 90%, or 100% of said neurons express the neuronal marker NeuN; (b) capable of firing action potentials and forming synaptic connections; has.

[0014] In another preferred embodiment, said functional neuron is an excitatory neuron, preferably VGLUT2 + It is an excitatory neuron.

[0015] In another preferred embodiment, the functional neuron comprises a glutamatergic neuron or a group of glutamatergic neurons.

[0016] In another preferred embodiment, the functional neurons are capable of firing action potentials and forming synaptic connections.

[0017] In another preferred embodiment, the Neurog2 functional fragment is a functional Neurog2 protein or a nucleic acid sequence encoding a Neurog2 functional protein; In another preferred embodiment, said Neurog2 functional fragment is a protein or nucleic acid sequence normally capable of performing the physiological function of said Neurogenin2 transcription factor.

[0018] In another preferred embodiment, Neurog2 is derived from a mammal, preferably a human or non-human primate mammal.

[0019] In another preferred embodiment, the GenBank number of the Neurog2 functional fragment is 11924, and said protein sequence is set forth in SEQ ID NO:1; the NCBI reference sequence of the mRNA encoding said Neurog2 gene is NM_009718.3, and the CDS sequence is set forth in SEQ ID NO:2.

[0020] In another preferred embodiment, the GenBank number of the Neurog2 functional fragment is 63973 and the protein sequence is set forth in SEQ ID NO:3; the NCBI reference sequence of the mRNA encoding the Neurog2 gene is NM_24019.4 and the CDS sequence is set forth in SEQ ID NO:4.

[0021] In another preferred embodiment, the sequence homology between the Neurog2 functional fragment and SEQ ID NO: 1 is 83% or more. More preferably, the sequence homology between the Neurog2 functional fragment sequence and SEQ ID NO: 1 is 90% or more; preferably, the sequence homology between the Neurog2 functional fragment sequence and SEQ ID NO: 1 is 95% or more.

[0022] In another preferred embodiment, the nucleotide functional sequence encoding Neurog2 has 80% or more homology to the sequence of SEQ ID NO:2, more preferably, said nucleotide functional sequence encoding Neurog2 has 90% or more homology to the sequence of SEQ ID NO:2, preferably said nucleotide functional sequence encoding Neurog2 has 95% or more homology to the sequence of SEQ ID NO:2.

[0023] In another preferred embodiment, the sequence homology between the Neurog2 functional fragment and SEQ ID NO:3 is 83% or more, more preferably the sequence homology between the Neurog2 functional fragment sequence and SEQ ID NO:3 is 90% or more, preferably the sequence homology between the Neurog2 functional fragment sequence and SEQ ID NO:3 is 95% or more.

[0024] In another preferred embodiment, the nucleotide functional sequence encoding Neurog2 has 80% or more homology to SEQ ID NO:4, more preferably, the nucleotide functional sequence encoding Neurog2 has 90% or more homology to the sequence of SEQ ID NO:4, preferably, the nucleotide functional sequence encoding Neurog2 has 95% or more homology to SEQ ID NO:4.

[0025] A second aspect of the present invention provides a delivery system containing a Neurog2 functional fragment, said delivery system being applicable to induce transdifferentiation of glial cells into functional neurons in vitro or in vivo; said glial cells originating from the spinal cord, dorsal midbrain or cerebral cortex, preferably said glial cells originating from the spinal cord and dorsal midbrain; said glial cells being in a normal state or in an injured state.

[0026] In another preferred embodiment, the functional fragment of Neurog2 can be passively taken up by glial cells or reach the interior of glial cells via a delivery system to exert its effect.

[0027] In another preferred embodiment, the delivery system contains a Neurog2 functional fragment, including, but not limited to, an expression vector containing a Neurog2 functional fragment, a nanoparticle containing a Neurog2 functional fragment, an exosome containing a Neurog2 functional fragment, a modified red blood cell or a bacterium containing an effector that targets the Neurog2 functional fragment (e.g., a glial cell-specific antibody, a polypeptide, or other targeting agent) carrying a Neurog2 functional fragment.

[0028] In a preferred embodiment, the delivery system is an expression vector carrying a Neurog2 functional fragment, and the expression vector can enter glial cells and express exogenous Neurog2 protein in astrocytes.

[0029] In another preferred embodiment, the expression vectors include plasmids and viral vectors.

[0030] In another preferred embodiment, the expression vector is a viral vector, including but not limited to an adenoviral vector, an adeno-associated viral vector (AAV), a retroviral expression vector or a lentiviral vector, preferably an adeno-associated viral vector (AAV).

[0031] In another preferred embodiment, said expression vector is an astrocyte-specific expression vector.

[0032] In another preferred embodiment, the expression vector containing the Neurog2 functional fragment also contains a glial cell-specific promoter, and a promoter including, but not limited to, the GFAP promoter, the NG2 promoter, the Aldh1L1 promoter, the IBA1 promoter, the CNP promoter, the LCN2 promoter or a modified promoter variant.

[0033] In another preferred embodiment, the expression vector containing the Neurog2 functional fragment also contains one or more regulatory elements for regulating gene expression, including, but not limited to, a CMV enhancer, an SV40 enhancer, an EN1 enhancer or a genetically modified enhancer mutant, as well as an SV40 polyA tailing signal, a human insulin gene polyA tailing signal or a WPRE (woodchuck hepatitis B virus post-transcriptional regulatory element), a MAR sequence of human origin or a genetically modified mutant, to promote the expression level of the gene.

[0034] In another preferred embodiment, the expression vector containing the Neurog2 functional fragment can also contain other functional fragments, and the other functional fragments can be reporter genes or other transcription factors having functional fragments of reprogramming function, including (but not limited to) Ascl1, NeuroD1, etc.

[0035] In another preferred embodiment, the expression vector containing the Neurog2 functional fragment is a GFAP-AAV vector; the GFAP-AAV vector holds a viral ITR sequence, a CMV enhancer, a human GFAP promoter, a coding frame of the Neurog2 functional fragment and a 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, and the described GFAP-AAV expression vector may contain the following elements from the 5' to 3' end: viral ITR sequence + CMV enhancer + human GFAP promoter + coding frame of Neurog2 and red fluorescent protein mCherry + post-transcriptional regulatory element WPRE + viral ITR sequence + promoter and coding frame of ampicillin resistance gene in this order, where the coding frame of the red fluorescent protein mCherry and promoter and coding frame of ampicillin resistance gene are not essential.

[0036] In another preferred embodiment, the expression vector containing the Neurog2 functional fragment is an NG2-lentiviral vector; the NG2-lentiviral vector contains the viral ITR sequence, the human NG2 promoter, and the Neurog2 functional fragment coding frame and the post-transcriptional regulatory element WPRE, etc.; the expression vector can also carry a reporter gene, but the reporter gene is not necessary in practical applications, for example, the NG2-lentiviral vector can contain the following elements from the 5' to 3' end: viral ITR sequence + human NG2 promoter + Neurog2 and green fluorescent protein GFP coding frame + post-transcriptional regulatory element WPRE + viral ITR sequence + ampicillin resistance gene promoter and coding frame in this order, where the green fluorescent protein GFP coding frame and ampicillin resistance gene promoter and coding frame are not essential.

[0037] A third aspect of the invention provides a host cell comprising an exogenous Neurog2 functional fragment.

[0038] In another preferred embodiment, the polynucleotide encoding said Neurog2 protein is integrated into the chromosome of said host cell or said host cell contains an expression vector as described in the second aspect of the invention.

[0039] In another preferred embodiment, the host cell is derived from a glial cell and using the delivery system described in the second aspect of the invention, a polynucleotide encoding Neurog2 protein can be integrated into its chromosome, or the host cell can have a functional fragment of Neurog2 transferred into the cell, promoting transdifferentiation of the host cell into a functional neuron.

[0040] In another preferred embodiment, said host cells are derived from glial cells cultured in vitro.

[0041] In a preferred embodiment, said host cells are derived from glial cells under normal and injured conditions in vivo.

[0042] In another preferred embodiment, said glial cells are astrocytes.

[0043] In a preferred embodiment, the host cell is a functional neuron or a population of functional neuronal cells.

[0044] A fourth aspect of the present invention provides a pharmaceutical composition comprising: (A) a Neurog2 functional fragment as described in the first aspect of the invention; and / or (B) a delivery system as described in the second aspect of the invention, or (C) a host cell as described in the third aspect of the invention; and (D) a pharma- ceutical composition comprising a pharma-ceutical acceptable excipient.

[0045] A fifth aspect of the invention provides the use of a delivery system as described in the second aspect of the invention, a host cell as described in the third aspect of the invention and / or a pharmaceutical composition as described in the fourth aspect of the invention, which can be used in the preparation of a medicament for nervous system damage and neuronal repair.

[0046] In another preferred embodiment, the nervous system injury comprises neuronal cell death or irreversible loss caused by spinal cord injury, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), stroke, and the like.

[0047] In another preferred embodiment, the nerve repair is achieved by use of a pharmaceutical composition according to the fourth aspect of the invention to restore neuronal function in damaged areas of the nervous system.

[0048] A sixth aspect of the present invention provides an in vitro non-therapeutic method for transdifferentiating astrocytes into functional neuronal cells, comprising the steps of: Culturing the astrocytes in the presence of an exogenous Neurog2 functional fragment, thereby inducing the astrocytes to form functional neurons. Includes.

[0049] A seventh aspect of the present invention provides a functional neuronal cell and / or a population of neuronal cells transdifferentiated from an astrocyte, said functional neuronal cell and / or a population of neuronal cells being prepared by the method described in the sixth aspect of the present invention, and said functional neuronal cell and / or a population of neuronal cells having the following characteristics: (a) at least 50% of the neurons, preferably at least 60%, 70%, 80%, 90%, or 100% of the neurons express NeuN, a marker of mature neurons; (b) capable of firing action potentials and forming synaptic connections has one or more of the following.

[0050] An eighth aspect of the present invention provides a method for treating a nervous system disease, comprising the steps of: administering to a subject in need thereof a safe and effective amount of the pharmaceutical composition according to the fourth aspect of the present invention, thereby treating the nervous system disease. Includes.

[0051] In another preferred embodiment, the neurological disease comprises neuronal cell death or irreversible loss caused by spinal cord injury, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), stroke, and the like.

[0052] In another preferred embodiment, the subject in need thereof is a human.

[0053] In another preferred embodiment, said subject in need thereof is suffering from a nervous system disease.

[0054] A ninth aspect of the present invention provides (a) a method for screening a candidate compound for the treatment of a neurological disease, and / or (b) a method for screening a candidate compound that induces transdifferentiation of astrocytes into functional neural cells, said method comprising: (i) adding a test compound to said cell culture as a test group, and using a cell culture without the test compound as a control group; (ii) comparing the expression and / or activity E1 of the Neurog2 gene or its protein in the test group with the expression and / or activity E0 in the control group; Wherein, if E1 in the test group is significantly higher than E0, it indicates that the compound is (a) a candidate compound for the treatment of a neurological disease and / or (b) a candidate compound for inducing transdifferentiation of astrocytes into functional neuronal cells.

[0055] In another preferred embodiment, the cells are astrocytes.

[0056] In another preferred embodiment, the significantly higher means that E1 is higher than E0 and there is a statistical difference, preferably E1≧2E0.

[0057] In another preferred embodiment, the method further comprises the steps of: (iii) adding said test compound to the astrocyte culture as a test group, and using an astrocyte culture without the test compound as a control group; (iv) comparing the percentage of astrocytes transformed into functional neurons in the test groups to determine whether the test compound is (a) a candidate compound for treating a neurological disease; and / or (b) a candidate compound for screening to induce astrocytes for transdifferentiation of glial cells into functional neurons; Wherein, if the proportion T1 of astrocytes transformed into functional neurons in the test group is significantly higher than the proportion T0 in the control group, it indicates that the test compound is (a) a candidate compound for the treatment of a neurological disease; and / or (b) a candidate compound to be screened for inducing transdifferentiation of astrocytes into functional neurons.

[0058] In another preferred embodiment, the neurological disease comprises neuronal cell death or irreversible loss caused by spinal cord injury, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), stroke, and the like.

[0059] It should be understood that within the scope of the present invention, the technical features of the present invention described above and the technical features specifically described below (e.g., in the embodiments) can be combined with each other to form new or preferred technical solutions, which will not be repeated in this specification due to space limitations.

[0060] The above description is only an outline of the technical solution of the present invention, in order to make the technical means of the present invention more clearly understandable, according to the content of the above description, and in order to make the above and other objects, features and advantages of the present invention more clear and understandable, the following specific preferred embodiments are detailed as follows in conjunction with the accompanying drawings. [Brief description of the drawings]

[0061] [Figure 1] A single factor, Neurog2, can efficiently induce midbrain astrocytes into neurons. Figure 1A, B shows that 3 days after injection of the control virus AAV-mCherry (negative control) and the virus AAV-Neurog2 / mCherry into the dorsal midbrain of adult mice, immunolabeling shows that neither mCherry colocalized with NeuN. Figure 1C shows that mCherry still did not colocalize with NeuN 30 days after injection of the control virus AAV-mCherry into the dorsal midbrain of adult mice. Figure 1D shows that 30 days after injection of the virus AAV-Neurog2 / mCherry into the dorsal midbrain of adult mice, most of the mCherry colocalized with NeuN. Figure 1E shows the statistics of neuronal ratios at different times. Arrows and arrowheads represent mCherry+NeuN+ and mCherry+NeuN- cells, respectively. "***" represents p<0.001. Ruler: 50 μm. [Diagram 2] Neurog2-induced neurons in the midbrain were confirmed to be active functional neurons by electrophysiological recording. Figure 2A shows mCherry+ electrophysiological membrane parameters recorded in midbrain slices 30 days after AAV-mCherry virus infection, recorded in current clamp mode with gradient voltage stimulation (top) and recorded with gradient current stimulation in voltage clamp mode, respectively recording membrane current changes (bottom). Figure 2B shows the postsynaptic current signals of mCherry+ cells recorded 30 days after midbrain infection with AAV-Neurog2 / mCherry virus. Addition of the blocker NBQX causes the postsynaptic current signal to disappear, and the postsynaptic current signal reappears after washing. [Diagram 3] Neurog2 midbrain where neurons were reprogrammed into glutamatergic neurons. Figure 3A, B shows the results of histochemical double labeling in situ of adult mice 30 days after AAV-Neurog2 / mCherry virus infection. Overlapping signals of viral signal mCherry protein (red) and VGLUT2mRNA (A, green) and Gad1mRNA (B, green) are (right of A) and (right of B), respectively. Arrows in (A) indicate mCherry+VGLUT2+ double positive cells. Arrows in (B) indicate mCherry+Gad1- cells. Figure 3C shows statistical plots of ratios of neurons to different transmitters. Nuclei were labeled with DAPI. Ruler: 25 μm. [Figure 4] A single factor, Neurog2, can induce spinal astrocytes into neurons with high efficiency. Figure 4A,B: 3 days after injection of the control virus AAV-mCherry and the virus AAV-Neurog2 / mCherry into the spinal cord of adult mice, immunolabeling shows that neither mCherry colocalized with NeuN. Figure 4C: 30 days after injection of the control virus AAV-mCherry into the spinal cord of adult mice, mCherry still did not colocalize with NeuN. Figure 4D: 30 days after injection of the virus AAV-Neurog2 / mCherry into the spinal cord of adult mice, most of mCherry colocalized with NeuN. Figure 4E: Statistics of neuronal ratio at different times. Arrows and arrowheads represent mCherry+NeuN+ and mCherry+NeuN- cells, respectively. "***" represents p<0.001. Ruler: 50 μm. [Diagram 5]Neurog2-induced neurons in the spinal cord were confirmed to be active functional neurons by electrophysiological recordings and could respond to stimulation of the dorsal root ganglion (DRG). Figure 5A,B shows that electrophysiological recordings of spinal cord slices derived from mCherry+ cells infected with AAV-mCherry virus (A) and AAV-Neurog2 / mCherry virus (B) in current-clamp or voltage-clamp mode, respectively, for 30 days, yielded membrane voltage (left) and cell membrane current parameters (right) upon gradient voltage or current stimulation. Figure 5C shows the electrophysiological response of mCherry+-induced neurons in the dorsal spinal cord after DRG stimulation. The green arrow indicates the onset of stimulation. [Figure 6] A single factor of Neurog2 can efficiently induce astrocytes in injured spinal cord to neurons. Figure 6A,B: Three days after injection of control virus AAV-mCherry and virus AAV-Neurog2 / mCherry to injure spinal cord of adult mice, immunolabeling shows that neither mCherry still colocalized with NeuN. Figure 6C: mCherry still did not colocalize with NeuN 30 days after injection of control virus AAV-mCherry to injure spinal cord of adult mice. Figure 6D: Most of mCherry colocalized with NeuN 30 days after injection of virus AAV-Neurog2 / mCherry to injure spinal cord of adult mice. Figure 6E: Statistical graph of neuron ratio at different times. Arrow and arrowhead represent mCherry+NeuN+ and mCherry+NeuN- cells, respectively. "**" represents p<0.01. Ruler: 50 μm. [Figure 7]Neurog2-reprogrammed neurons can promote the recovery of motor function in mice with spinal cord injury. Figure 7A,B show the control group (uninjured spinal cord) and the injured motor function 3 weeks after injection of virus AAV-mCherry and virus AAV-Neurog2 / mCherry, respectively. Figure 7A scores the motor function of the mice according to the BMS standard, and Figure 7B is the time the mice were moving during the open field experiment. "*" represents p<0.05. [Figure 8] Neurog2-reprogrammed neurons reduce anxiety levels in mice with partial spinal cord injury. Figure 8A,B shows the number of mice crossing the central region in the control group (uninjured spinal cord) and mice 3 weeks after injection with virus AAV-mCherry and virus AAV-Neurog2 / mCherry, respectively. "*" indicates p<0.05. [Figure 9] Neurog2 transdifferentiates astrocytes in the entorhinal cortex of AD mice into neurons in vivo. Figure 9A,B shows that 46 days after injection of the control virus AAV-mCherry and the virus AAV-Neurog2 / mCherry to damage the entorhinal cortex of 5XFAD mice, immunolabeling showed that mCherry in the control virus AAV-mCherry group did not colocalize with NeuN, whereas in AAV-Neurog2 / mCherry, most of the mCherry colocalized with NeuN, and animal sections from the two groups were stained for Aβ simultaneously. Arrows indicate mCherry+NeuN+ cells. Ruler: 50 μm. [Figure 10] Comparison of the amino acid sequences of mouse and human Neurog2.

[0062] As a result of extensive and detailed research, the present inventors have found that regulating the expression of the Neurog2 gene or its protein in glial cells can efficiently induce and transdifferentiate astrocytes into neurons with normal electrophysiological functions in vivo or in vitro, and unexpectedly discovered that not only astrocytes under normal conditions, but also astrocytes under damaged conditions can be transdifferentiated into functional neurons that can play a role in nerve repair. Specifically, neurons derived from glial cells derived from the spinal cord can respond to stimulation of the dorsal root ganglion (DRG) of the peripheral nervous system, indicating that neurons induced by Neurog2 functional fragments can be functionally integrated into spinal cord circuits and functions. Therefore, induction of Neurog2 functional fragments is expected to be an effective method to stimulate the generation of new nerve cells in adults, which can be widely used in the development of therapeutic drugs for neurological diseases.

[0063] Compared with the prior art, the present invention mainly has the following competitive advantages: (1) Compared with other discovered transdifferentiation factors, the functional fragments of Neurog2 act not only on glial cells under normal environment but also on glial cells under damaged environment, and can overcome glial cell / glial cells under damaged environment. In the stress state of neural cells, neural connections with normal electrophysiological function can be obtained, resulting in the restoration of neural function. (2) Compared with other discovered transdifferentiation factors, functional fragments of Neurog2 can specifically and uniformly transdifferentiate glial cells into excitatory glutamatergic neurons or glutamatergic neuronal populations, ensuring the manipulation of drug development and application in neurological diseases. (3) Compared with the transplantation of neuronal clusters after in vitro neuronal induction, in vivo AAV-mediated Neurog2-induced neurons are easier to manipulate, have high neuronal induction efficiency, and the induced neurons are electrophysiologically more mature and more efficient, suitable for in vivo regenerative repair of neurological diseases. has.

[0064] Neurog2 functional fragment or its promoter Neurog2 (Neurogenin 2) is a bHLH-like transcription factor, and its main structural domains include DNA binding domain, transcriptional regulatory domain (activation domain), oligomerization site and nuclear localization signal (nuclear localization signal) and other functional regions. bHLH (basic helix-loop-helix) is the DNA binding domain of Neurog2 that specifically regulates gene expression. The bHLH domain is well conserved in mammals, including mice and humans.

[0065] The Neurog2 molecule from mouse has GenBank ID #11924 and its protein sequence is shown in SEQ ID NO:1; NCBI Reference Sequence: NM_9718.3; the CDS is shown in SEQ ID NO:2; the Neurog2 molecule from human has ID #63973 in GenBank and its protein sequence is shown in SEQ ID NO:3, where the bHLH domain is located at position 112 to 164, NCBI Reference Sequence: NM_24019.4; the CDS sequence is shown in SEQ ID NO:4. The amino acid sequence alignment results of mouse and human Neurog2 are shown in FIG.

[0066] The amino acid / protein sequences of mouse and human Neurog2 are shown below: [ka] [ka]

[0067] Neurog2 functional fragments refer to fragments of the Neurog2 sequence that have the function of regulating transcription and achieving redifferentiation, including, but not limited to, full-length Neurog2 protein and sequences that contain the conserved Neurog2 bHLH domain and have 83% or more sequence identity to wild-type Neurog2 fragments. Neurog2 functional fragments include mammalian polynucleotides encoding Neurogenin2 transcription factors or protein fragments expressed therefrom.

[0068] The functional fragment of Neurog2 can also be a variant of the Neurog2 protein sequence, specifically, the sequence homology between the variant of the Neurog2 protein functional fragment and SEQ ID NO:1 must be 83% or more; or the sequence homology between the functional sequence of the protein encoding Neurog2 and SEQ ID NO:1 must be 90% or more; or the sequence homology between the functional sequence of the protein encoding Neurog2 and SEQ ID NO:1 must be 95% or more; or the sequence homology between the functional sequence of the protein encoding Neurog2 and SEQ ID NO:1 must be 98% or more; or the sequence homology between the variant of the Neurog2 protein functional fragment and SEQ ID NO:3 must be 83% or more; or the sequence homology between the functional sequence of the protein encoding Neurog2 and SEQ ID NO:3 must be 90% or more; or the sequence homology between the functional sequence of the protein encoding Neurog2 and SEQ ID NO:3 must be 95% or more; or the sequence homology between the functional sequence of the protein encoding Neurog2 and SEQ ID NO:3 must be 98% or more; The functional fragment of Neurog2 can also be a variant of the Neurog2 polynucleotide sequence, specifically, the sequence homology between the polynucleotide functional sequence encoding Neurog2 and SEQ ID NO:2 is 80% or more; or the sequence homology between the nucleic acid functional sequence encoding Neurog2 and SEQ ID NO:2 is 90% or more; or the sequence homology between the nucleic acid functional sequence encoding Neurog2 and SEQ ID NO:2 is 95% or more; or the sequence homology between the nucleic acid functional sequence encoding Neurog2 and SEQ ID NO:2 is 98% or more; or the sequence homology between the polynucleotide functional sequence encoding Neurog2 and SEQ ID NO:4 is 80% or more; or the sequence homology between the nucleic acid functional sequence encoding Neurog2 and SEQ ID NO:4 is 90% or more; or the sequence homology between the nucleic acid functional sequence encoding Neurog2 and SEQ ID NO:4 is 95% or more; or the sequence homology between the nucleic acid functional sequence encoding Neurog2 and SEQ ID NO:4 is 98% or more; Said functional fragment of Neurog2 can also be obtained by activating the expression of Neurog2 gene by CRISPR / dCas9 targeting DNA or increasing the expression of Neurog2 by CRISPR / Cas13 targeting RNA; Said Neurog2 expression promoter is not particularly limited, and can be any substance that promotes the expression and / or activity of Neurog2 gene or its protein, such as small molecule compounds and stimulatory miRNA. Those skilled in the art can screen Neurog2 promoters according to existing databases. Based on the transdifferentiation induction effect of Neurog2 on astrocytes disclosed in the present invention, those skilled in the art should understand that any substance that promotes Neurog2 can reasonably predict that it can induce the transdifferentiation of astrocytes.

[0069] astrocytes Astrocytes are the most abundant type of cell in the mammalian brain. Astrocytes perform many functions, including biochemical support (e.g., forming the blood-brain barrier), supplying nutrients to neurons, maintaining extracellular ion homeostasis, and participating in repair and scar formation after brain and spinal cord injury. According to the content of glial fibers and the shape of cell processes, astrocytes can be divided into two types: fibrous astrocytes, which are distributed mostly in the white matter of the brain and spinal cord, have elongated processes, few branches, and many glial fibers in the cytoplasm; protoplasmic astrocytes (protoplasmic astrocytes), which are distributed mostly in the gray matter, have sparse cell processes and many branches.

[0070] Astrocytes that can be used in the present invention include, but are not limited to, various astrocytes derived from the mammalian central nervous system, such as astrocytes derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably astrocytes derived from the dorsal midbrain or spinal cord.

[0071] In the present invention, astrocytes from various sources have higher induction transformation efficiency. Typically, astrocytes are GFAP specific markers, and astrocytes in gray matter have relatively low GFAP expression, but express either Acsbg1 or GS. And when induced by the method of the present invention, these astrocytes show neuronal specific markers such as NeuN.

[0072] Functional Neurons As used herein, the term "functional neuron" refers to a neuronal cell that has normal neuronal electrophysiological activity and is transdifferentiated from an astrocyte in the presence of an exogenous Neurog2 gene or protein. Typically, the functional neuronal cell has the following properties: (a) NeuN, a marker for neuronal expression; (b) capable of firing action potentials and forming synaptic connections has.

[0073] In the present invention, a "functional neuron" is an excitatory neuron, in particular a glutamatergic neuron, VGLUT2 + Therefore, in this specification, the terms "excitatory neuron", "VGLUT2 + "Excitatory neuron" and "glutamatergic neuron" are used interchangeably and all refer to the functional neurons of the present invention.

[0074] Delivery System The delivery system that can be used in the present invention is not particularly limited, and may be an expression vector containing the Neurog2 protein coding sequence that can enter astrocytes. For example, the viral vector can be any viral vector that can utilize the characteristics of the virus to transfer its genome and bring genetic material into other cells for infection. It occurs entirely in vivo or in cell culture. It includes lentiviral vector, adenoviral vector, adeno-associated viral vector, herpes viral vector, and pox viral vector.

[0075] The delivery system can also be a new type of nanoparticle for incorporating Neurog2 functional fragments and delivering them to target cells, such as liposomal nanoparticles, metal nanoparticles, polymeric nanoparticles, and other delivery systems capable of carrying Neurog2 functional fragments.

[0076] The delivery system can also be exosomes coated with a Neurog2 functional fragment, or modified red blood cells or bacteria coated with a Neurog2 functional fragment.

[0077] Furthermore, the delivery system can also be combined with functional targeting molecules, such as specific monoclonal antibodies targeting astrocytes, polypeptides, etc., that can better improve the targeting of Neurog2 functional fragments on astrocytes, tropism and increased transdifferentiation efficiency.

[0078] Guidance method The present invention also provides methods for inducing the transdifferentiation of astrocytes into functional neural cells in vivo. In vivo, a Neurog2-containing delivery system can be administered (e.g., injected) into a desired subject where astrocyte-containing sites, such as the dorsal midbrain, cerebral cortex, or spinal cord, can target both intact and injured nervous systems. The tissue is injected to induce astrocyte transdifferentiation in specific parts of the nervous system. In vitro, a Neurog2-containing delivery system can be administered (e.g., injected) into in vitro cultured NG2 glial cell clusters to induce in vitro differentiation of functional neurons, and the in vitro cultured functional neuronal clusters can then be transplanted into the body by transplantation.

[0079] Pharmaceutical Compositions and Administration The present invention also provides a pharmaceutical composition, which is a delivery system, containing a Neurog2 functional fragment or a functional neuronal mass after in vitro induction and transdifferentiation with a Neurog2 functional fragment.

[0080] The pharmaceutical composition of the present invention comprises the above-mentioned expression vector of the present invention (eg, viral particle) or the exogenous Neurog2 protein itself, and a pharma- ceutically acceptable carrier.

[0081] The pharmaceutical composition of the present invention is generally administered in an amount of 10 10 ~10 13 PFU / mL of AAV viral particles, preferably 10 11 ~10 13 PFU / mL of AAV viral particles, more preferably 10 10 ~10 12 Contains PFU / mL of AAV viral particles.

[0082] "Pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, and includes various excipients and diluents. In general, the term refers to a pharmaceutical carrier that is not itself an essential active ingredient and does not exhibit excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. The pharmaceutically acceptable carrier in the composition may contain liquids such as water, saline, buffer, etc. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances, etc. may also be present in these carriers. The carrier may contain a cell transfection agent.

[0083] Generally, the pharmaceutical composition of the present invention can be obtained after mixing the expression vector with a pharma- ceutically acceptable carrier.

[0084] The method of administration of the compositions of the present invention is not particularly limited, and representative examples include (but are not limited to) intravenous injection, subcutaneous injection, intracerebral injection, intrathecal injection, intraspinal injection, and the like.

[0085] Purpose The Neurog2 functional fragment of the present invention can be used to prepare a drug for inducing astrocytes to generate functional neurons, and the newly induced neurons can then be used in a variety of applications for diseases associated with decreased neuronal numbers, cell loss, apoptosis, or decreased neuronal function, including neuronal cell death caused by spinal cord injury, Alzheimer's disease (AD), Parkinson's disease (PD), and stroke.

[0086] Beneficial Effects of the Invention In the present invention, 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 characteristic molecules, can fire action potentials, and can receive synaptic afferents from other neurons to establish synaptic connections. Therefore, this method is an effective way to stimulate the generation of new neurons in adults, and is expected to be widely used in the treatment of neurological diseases such as neurodegenerative diseases, traumatic diseases of the central nervous system, etc.

[0087] Further, the present invention will be described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention, and are not used to limit the scope of the present invention. The experimental methods of unaccepted specific conditions in the following examples are according to standard conditions, such as those described in Molecular Cloning: Conditions Described in Laboratory Manual by Sambrook et al. (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions recommended by the manufacturer.

[0088] General approach NG2 cell culture For the preparation and culture of NG2 cells, cortical tissues from 3- to 5-day-old mice were removed and digested with 0.25% trypsin for 15 min. Dispersed cells were cultured for 7–9 days in DMEM / F12 solution containing 10% serum. The flasks were then shaken, the supernatant collected, and the cells were centrifuged, resuspended, and seeded on poly-D-lysine (Sigma)-coated coverslips with N2 supplement (STEMCELL™), 10 ng / mL platelet-derived growth factor (PDGF, Invitrogen), 10 ng / mL epidermal growth factor (EGF, Invitrogen), and 10 ng / mL fibroblast growth factor 2 (FGF2, Invitrogen) in serum-free DMEM medium (Gibco) at 5% CO. 2The cells were cultured in an incubator at 37°C for 3 days.

[0089] immunostaining Immunostaining of cultured cells was performed according to "Direct conversion of fibroblasts to functional neurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)). Immunostaining of tissue sections and double-labeling experiments for color development combined with in situ hybridization and immunoassay were performed according to published methods. The primary antibodies used for immunochromatography were 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), and mouse anti-O4 (Millipore, 1:500). FITC-, Cy3-, and Cy5-conjugated secondary antibodies were purchased from Jackson Immunoresearch.

[0090] Stereotaxic injection of AAV virus AAV virus was injected with reference to the mouse brain atlas. After virus injection, the dorsal midbrain and spinal cord were harvested at different time points for recording the immunostaining or brain sections. The injection concentration, speed and volume of each virus injection in intact and injured spinal cord were consistent with the brain area, and the injection in spinal cord was performed at a 30° angle.

[0091] Transplantation of induced neurons Mice used for transplantation were 7-week-old NOD-scid mice. Cells were digested with Accutase for 4–6 days, and the cell density was approximately 2 × 10 per μL after concentration. 5Remove the supernatant by centrifugation to obtain 2 x 10 cells in 2 µL of each mouse brain, for a total of 4 x 10 cells. 5 Cells were transplanted. Histochemical and electrophysiological tests were performed 2 to 4 weeks after transplantation.

[0092] Spinal Cord Injury Detection Model Loss of sensory afferents after thoracic spinal cord injury promotes the weakening of the descending inhibitory system in the brainstem, resulting in hypersensitivity of the tail to external stimuli. We used a tail flick experimental model to measure the sensory ability of mice by measuring the tail response latency of two groups of mice under thermal stimulation at 48°C and 52°C. The motor function of mice was scored according to the BMS criteria. The test method refers to the Basso Mouse Scale for locomotion, which detects differences in recovery after spinal cord injury in five common mouse strains. J Neurotrauma, 2006.23(5):p.635-59.

[0093] An open field experiment was used to evaluate the anxiety level of animals. In general, mouse activity in the central area of ​​the open field is thought to be related to the degree of anxiety. Normal mice frequently move back and forth to the central area of ​​the open field (Zone Crossing), while anxious mice tend to move around the periphery of the field (Peripheral). The experimental method refers to the open field as a paradigm for measuring the effects of drugs on anxiety-like behavior: a review. European Journal of Pharmacology, 2003. 463(1-3): p.3-33. Embodiment 1: Neurog2 transdifferentiates NG2 glial cells into functional neurons in vitro. (1) Plasmid construction and viral infection On the vector template of FUGW-IRES-EGFP (Vector information reference. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 93:11382-11388), human NG2 promoter (SEQ ID NO: 5) was cloned to replace the CAG promoter, and CDS (SEQ ID NO: 4) fragment from human Neurog2 gene was constructed into this lentiviral vector to generate hNG2-hNeurog2-IRES-EGFP lentiviral plasmid. For packaging of lentiviral vectors, see "Production and purification of lentiviral vectors" (Tiscornia, G., Singer, O. & Verma, IM Nat. Protoc. 1, 241-245 (2006)). NG2 cells were cultured for 24 h before adding lentivirus, and 24 h after infection, the medium was changed to DMEM / F12, B27, Glutamax, and penicillin / streptomycin. Six to seven days after infection, brain-derived neurotrophic factor (BDNF; Pepro Tech, 20 ng / mL) was added to the medium every 3 days. (2) Neurog transdifferentiates NG2 cells into neurons. Most of the cultured mouse NG2 cells were immunopositive for the NG2 glial cell marker NG2, a minority of cells expressed the oligodendrocyte marker molecules O4 and CNPase, and expression of the neuronal marker molecule Tuj1 and the stem cell marker molecules Sox2 and Oct4 was undetectable. Ten days after NG2 cells were transfected with hNG2-Neurog2-IRES-GFP lentivirus, most NG2 cells exhibited typical neuronal morphology and expressed the neuronal marker molecule Tuj1. Twenty-one days after lentiviral infection, the induced cells also expressed the mature neuronal marker molecules NeuN and MAP2. Electrophysiological recordings showed that the induced neurons were able to generate action potentials, and spontaneous postsynaptic currents could be recorded from the majority of the induced neurons, suggesting that these neurons could form functional synapses. (3) Neurons induced by NG2 cells can survive transplantation in vivo. Whether in vitro induced transdifferentiated neurons can survive and function in vivo is the key to whether they can be used to treat diseases. Two weeks after transplanting NG2 cell-induced neurons into the cerebral cortex, we performed Neurog2 immunohistochemistry experiments. We found that the transplanted cells could attach to the edge of the cortex, and some cells could extend neurites deep into the cortex. The results of the immunofluorescence colocalization experiment showed that some of the transplanted cells expressed the neuronal marker molecule Tuj1, indicating that the induced neurons could survive and express neuron-specific marker molecules.

[0094] Embodiment 2: Neurog2 transdifferentiates adult dorsal midbrain astrocytes into neurons in vivo. (1) Adeno-associated virus plasmid construction and virus infection The GFAP promoter (SEQ ID NO:6) was cloned onto the vector template of AAV-FLEX-Arch-GFP (Addgene, #22222) to replace the CAG promoter and retain the CMV enhancer, and then GFP was replaced into the coding frame of mCherry. AAV-mCherry plasmid (control). The CDS from mouse Neurog2 gene (SEQ ID NO:2, NCBI:NM_009718.3) was cloned into the AAV-mCherry plasmid to obtain the AAV-mNeurog2 / mCherry plasmid. The target gene can be specifically targeted under the action of the GFAP promoter astrocytes. (2) Neurog2 transdifferentiates astrocytes into neurons. The virus AAV-mCherry or AAV-mNeurog2 / mCherry was injected into the lateral tectum of adult wild-type mice, and then brain tissue samples were taken at different time points. Co-immunolabeling showed that mCherry colocalized with NeuN 3 days after injection in either mice injected with the control virus AAV-mCherry (Figure 1A) or mice injected with the virus AAV-Neurog2 / mCherry (Figure 1B). In mice injected with the control virus AAV-mCherry 30 days after virus injection, co-immunolabeling showed that mCherry still did not colocalize with NeuN. However, in mice injected with the virus AAV-Neurog2 / mCherry, the majority of mCherry colocalized with NeuN (88.2 ± 6.3%). This indicates that Neurog2 successfully guides astrocytes into neurons. (3) The induced neurons are functional neurons. To demonstrate that the induced neurons were functionally active neurons, we performed electrophysiological recordings. We recorded mCherry neurons in slices 30 days after infection of the midbrain with the AAV-mCherry virus. +We found that cells did not fire action potentials (Figure 2A), suggesting that targeting astrocytes in vivo with the AAV-mCherry virus does not alter their electrophysiological properties. When recording from brain slices infected with the Neurog2 / mCherry virus, 19 of 20 recorded cells induced inward Na in the voltage clamp mode. + Current and outward K + We found that Neurog2-induced neurons exhibited signal transduction in the neural circuits that were in agreement with the neurog2-induced state (19 / 20). Moreover, 19 recorded cells were able to fire multiple action potentials (19 / 20). Importantly, 17 of the recorded cells were able to generate excitatory postsynaptic currents (EPSCs) (Figure 2B) (17 / 20), which disappeared after addition of the blocker NBQX, and after washing, postsynaptic current signaling reappeared. This indicates that Neurog2-induced neurons are functional neurons, having integrated into neural circuits and established synaptic connections. (4) All induced neurons are excitatory neurons. We characterized the neurotransmitters induced by Neurog2. VGLUT2 / Gad1 mRNA and mCherry protein were stained by in situ hybridization and immunohistochemistry. The experimental results showed that 30 days after infection, AAV-Neurog2 / mCherry virus induced VGLUT2 in midbrain astrocytes. + Although Gad1 can be reprogrammed into excitatory neurons (Fig. 3A, C), + We showed that Neurog2 was unable to reprogram midbrain astrocytes into inhibitory neurons (Figure 3B,C). Thus, we demonstrated that a single factor, Neurog2, can reprogram midbrain astrocytes into glutamatergic neurons.

[0095] Embodiment 3: Neurog2 transdifferentiates normal spinal cord astrocytes into neurons in adult mice in vivo. (1) Plasmid construction and viral infection Using the adeno-associated virus vector AAV-mCherry plasmid described in embodiment 2, the CDS from the human Neurog2 gene (SEQ ID NO: 4, NCBI: NM_24019.4) was cloned into AAV-mCherry to obtain the AAV-hNeurog2 / mCherry plasmid. (2) Neurog2 transdifferentiates astrocytes into neurons. We injected AAV-mCherry and AAV-hNeurog2 / mCherry viruses into the dorsal spinal cord of adult mice pectoral muscle (thorax, T8-T10) and found that 3 days later, both viruses could specifically infect astrocytes. Co-immunolabeling showed that mCherry did not co-localize with NeuN 3 days after injection in either mice injected with the control virus AAV-mCherry (Figure 4A) or mice injected with the virus AAV-Neurog2 / mCherry (Figure 4B). In mice injected with the control virus AAV-mCherry, 30 days after virus injection, co-immunolabeling showed that mCherry still did not co-localize with NeuN (Figure 4C). However, in mice injected with the virus AAV-Neurog2 / mCherry, most of the mCherry co-localized with NeuN (Figure 4D). This indicated that Neurog2 successfully guided spinal astrocytes into neurons. (3) Induced neurons are functional neurons We analyzed the electrophysiological properties of neurons in the spinal cord reprogrammed with Neurog2. Through whole-cell recordings in brain slices, 30 days after AAV-mCherry viral infection, mCherry + We found that the cells were not only unable to generate action potentials when stimulated with intracellular gradient currents, but also unable to generate inward currents even when stimulated in voltage-clamp mode (Figure 5A). These characteristics are consistent with those reported for astrocytes. Meanwhile, 30 days after AAV-Neurog2 / mCherry virus infection, most mCherry+ cells were able to fire multiple action potentials under gradient current stimulation and generated inward Na currents under voltage-clamp mode stimulation.+ Current and outward K + In addition, we tested whether the Neurog2-reprogrammed neurons could integrate into existing neural circuits. The spinal cord was removed together with the DRG section for electrophysiological testing. When the DRG was stimulated, excitatory postsynaptic current (EPSC) and excitatory postsynaptic potential (EPSP) signals from the induced neurons in the dorsal spinal cord could be detected (FIG. 5C), indicating that Neurog2 not only reprograms neurons, but also reprogrammed neurons in the intact spinal cord can integrate into neural circuits and exert potential functions.

[0096] Embodiment 4: Neurog2 transdifferentiates adult mouse spinal cord astrocytes into neurons in vivo. Spinal cord injury (SCI) is a type of central nervous system injury disease that is accompanied by the death of spinal cord neurons and the formation of glial scars. In vivo neural cell reprogramming to convert astrocytes into neurons may alleviate the damage caused by SCI and is expected to become a new treatment method. (1) Neurog2 transdifferentiates astrocytes into neurons. We attempted to use Neurog2 to reprogram glial cells into neurons under injury conditions. First, we established a mouse T8-T10 spinal cord transection model after immediate injection of AAV-mCherry virus and AAV-mNeurog2 / mCherry (Example 2) or AAV-hNeurog2 / mCherry (Example 3) into both sides of the injured spinal cord (see Methods in McDonough A, Monterrubio A, Ariza J, et al.Calibrated Forceps Model of Spinal Cord Compression Injury.Jove-Journal of Visualized Experiments 2015). Co-immunolabeling showed that mCherry did not co-localize with NeuN 3 days after injection in either mice injected with the control virus AAV-mCherry (Figure 6A) or the virus AAV-mNeurog2 / mCherry (Figure 6B). In mice injected with the control virus AAV-mCherry, co-immunolabeling showed that mCherry still did not co-localize with NeuN 30 days after virus injection (Figure 6C). However, in mice injected with the virus AAV-mNeurog2 / mCherry, some mCherry was found to be able to co-localize with NeuN (Figure 6D). This indicated that Neurog2 could successfully guide injured spinal cord astrocytes into neurons. Similarly, the adeno-associated virus vector plasmid AAV-hNeurog2 / mCherry loaded with human-derived Neurog2 could also successfully guide injured spinal cord astrocytes into neurons. (2) Repair of spinal cord injury model Neurog2 reprogrammed neurons after spinal cord injury have electrophysiological properties and can receive external signal input. Therefore, we used various animal models to evaluate the neural repair ability of mice with spinal cord injury after neural induction. Using a spinal cord injury detection model, we found that Neurog2 reprogrammed neurons greatly contributed to the recovery of sensory and motor functions in mice with spinal cord injury.

[0097] We also used a spinal cord T8-T10 transection model and injected AAV-mCherry and AAV-Neurog2 / mCherry viruses into both sides of the injured spinal cord to serve as experimental and control groups, and six age-matched untreated male mice were selected as the reference group (Ctrl).

[0098] Three weeks after virus injection, the motor function of the mice was tested and scored according to the BMS criteria (Basso, DM, et al., J Neurotrauma, 2006.). The results showed that the mice in the Ctrl group could basically maintain a motor ability of 9 points, while the motor ability of the mice in the AAV-mCherry virus group was 2.3 points, and the mice injected with AAV-Neurog2 / mCherry increased to 4.5 points (Figure 7A). Then, the motor ability of the mice was further evaluated by the open field test. Through the analysis, it was found that the two groups of mice injected with the virus had a shorter motor time within 15 minutes than the Ctrl group, but the motor time between the two groups of mice injected with the virus was statistically significantly different (Figure 7B). This indicates that neurons reprogrammed with Neurog2 can promote the recovery of motor function in mice with spinal cord injury.

[0099] We also used the mouse square assay to assess the level of anxiety in mice after injury to test whether Neurog2-reprogrammed neurons could reduce the level of anxiety in mice with partial spinal cord injury, acute / chronic pain due to spinal cord injury, especially since chronic pain often leads to anxiety. In a mouse model of spinal cord injury that usually involves various degrees of anxiety, we found that neurog2-reprogrammed neurons also achieved a greater reduction in anxiety in spinal cord-injured mice. In open field experiments, mouse activity in the central area of ​​the open field is generally thought to be related to the degree of anxiety. Normal mice frequently shuttle between the central area of ​​the open field, while anxious mice tend to move more around the periphery of the open field.

[0100] We also analyzed the performance of the three groups of experimental mice. The results showed that after 3 weeks, the mice in the Ctrl group frequently shuttled around the central area of ​​the open field, while the mice in the AAV-mCherry group tended to move around the periphery of the open field more. The mice in the AAV-Neurog2 / mCherry group shuttled around more frequently than the mice in the AAV-mCherry group (Figure 8). These results suggest that Neurog2-reprogrammed neurons can reduce anxiety levels in some mice with spinal cord injury.

[0101] Embodiment 5: Neurog2 transdifferentiates astrocytes in the AD mouse entorhinal cortex into neurons in vivo. We injected AAV-mCherry virus as well as AAV-hNeurog2 / mCherry virus into the entorhinal cortex of 5xFAD, a 6-month-old AD model mouse. In mice with mCherry, immunolabeling showed that mCherry did not colocalize with NeuN. 46 days after virus injection, in mice injected with the control virus AAV-mCherry, coimmunolabeling showed that mCherry still did not colocalize with NeuN (Figure 9A). However, in mice injected with the virus AAV-Neurog2 / mCherry, most of the mCherry colocalized with NeuN (Figure 9B), but the extent of Aβ expression was reduced.

[0102] These results demonstrate that Neurog2 successfully transdifferentiates astrocytes into neurons in the entorhinal cortex of AD mice in vivo and reduces the expression of Aβ. Furthermore, these results suggest that the transdifferentiation transcription factor Neurog2 provides a new technique and drug for the treatment of Alzheimer's disease.

[0103] Although the preferred embodiments of the present invention have been specifically described above, the present invention is not limited to the described embodiments, and those skilled in the art may make various equivalent modifications or substitutions within the scope defined by the claims of this application. (Additional Note) (Appendix 1) below: (i) preparing a pharmaceutical composition for neuronal induction of glial cells to form functional neurons; and / or (ii) Preparation of a pharmaceutical composition for treating nervous system disorders Neurog functional fragments for use in (Appendix 2) A Neurog2 functional fragment for use according to claim 1, wherein said glial cell is selected from a human or non-human mammalian astrocyte, an NG2 glial cell, an oligodendrocyte or a microglial cell; preferably, said glial cell is an astrocyte. (Appendix 3) A Neurog2 functional fragment for use according to appendix 2, wherein the glial cells are derived from the spinal cord or the dorsal midbrain or the cerebral cortex; preferably, the glial cells are derived from the spinal cord and the dorsal midbrain; preferably, the glial cells are derived from the spinal cord and the dorsal midbrain; wherein the glial cells are glial cells in a normal or injured state. (Appendix 4) A Neurog2 functional fragment for use according to appendix 1, wherein said Neurog2 functional fragment is a functional Neurog2 protein or a nucleotide sequence encoding Neurog2; wherein the sequence homology between the Neurog2 protein sequence and SEQ ID NO:1 or SEQ ID NO:3 is 83% or more; more preferably, the sequence homology between the Neurog2 protein sequence and SEQ ID NO:1 is 90% or more; preferably, the sequence homology between the Neurog2 protein and SEQ ID NO:1 is 95% or more; wherein said nucleic acid sequence encoding Neurog2 has 80% or more homology to SEQ ID NO:2 or SEQ ID NO:4. (Appendix 5) A Neurog2 functional fragment for use according to claim 1, wherein said functional neuron comprises a glutamate neuron or a group of glutamate neurons. (Appendix 6) A Neurog2 functional fragment delivery system, wherein the Neurog2 functional fragment delivery system can be used in vivo or in vitro to induce glial cells to differentiate into functional neurons; the glial cells are in a normal state or in an injured state. (Appendix 7) A Neurog2 functional fragment delivery system as described in Appendix 6, wherein the Neurog2 functional fragment delivery system is an expression vector comprising a Neurog2 functional fragment, wherein the expression vector can enter glial cells and express exogenous Neurog2 protein in astrocytes. (Appendix 8) A Neurog2 functional fragment delivery system according to appendix 6 or 7, wherein the Neurog2 functional fragment delivery system is a viral vector, and the viral vector is selected from the group consisting of an adeno-associated virus vector, an adenovirus vector, a retrovirus vector and a lentivirus vector. (Appendix 9) A Neurog2 functional fragment delivery system according to claim 8, wherein the viral vector comprises a glial cell-specific promoter and the Neurog2 functional fragment. (Appendix 10) A Neurog2 functional fragment delivery system as described in Appendix 9, wherein the promoter of the viral vector is selected from the group consisting of GFAP promoter, NG2 promoter, Aldh1L1 promoter, IBA1 promoter, CNP promoter, LCN2 promoter, and genetically engineered promoter mutants. (Appendix 11) A Neurog2 functional fragment delivery system according to any one of claims 8 to 10, wherein the viral vector is a GFAP-AAV vector or an NG2-lentiviral vector. (Appendix 12) A host cell containing an exogenous Neurog2 functional fragment, wherein the host cell is derived from an in vitro cultured glial cell or a glial cell in a normal or injured state, and the host cell undergoes transdifferentiation to form a functional neuron or a population of functional neuronal cells. (Appendix 13) 13. The host cell of claim 12, wherein the functional neuron has the following characteristics: (a) expresses the neuronal marker NeuN; (b) is capable of firing action potentials and forming synaptic connections. (Appendix 14) 13. The host cell of claim 12, wherein the population of functional neuronal cells has the following characteristics: (a) at least 50% of the neurons, preferably at least 60%, 70%, 80%, 90%, or 100% of the neurons, express the neuronal marker NeuN; (b) are capable of firing action potentials and forming synaptic connections. (Appendix 15) Use of a Neurog2 functional fragment delivery system according to any one of appendices 6 to 11 or a host cell according to any one of appendices 12 to 14, wherein the Neurog2 functional fragment delivery system or the host cell can be used to prepare a pharmaceutical composition for nervous system damage or nerve repair, the pharmaceutical composition further comprising a pharma- ceutical acceptable carrier. (Appendix 16) The use of claim 15, wherein the nervous system injury comprises neuronal cell death caused by spinal cord injury, epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), stroke or irreversible loss. (Appendix 17) A pharmaceutical composition comprising: (A) a Neurog2 functional fragment; and / or (B) a delivery system as claimed in the appendix; or (C) a host cell as claimed in the appendix; and (D) a pharma- ceutical composition comprising a pharma-ceutical acceptable excipient.

Claims

1. A Neurog2 functional fragment delivery system, wherein said Neurog2 functional fragment delivery system can be used in vivo or in vitro to induce glial cells to differentiate into functional neurons; said glial cells are in a normal state or in an injured state; The Neurog2 functional fragment delivery system is an expression vector comprising a single transcription factor Neurog2 functional fragment, wherein the expression vector can enter glial cells and express exogenous Neurog2 protein in astrocytes; The Neurog2 functional fragment is a nucleic acid encoding a Neurog2 protein, and the sequence identity of the nucleic acid encoding the Neurog2 protein with the sequence of SEQ ID NO: 2 or SEQ ID NO: 4 is 90% or more; the expression vector is a viral vector, the viral vector being selected from an adeno-associated viral vector or a lentiviral vector; the viral vector comprises a glial cell-specific promoter and the Neurog2 functional fragment; The promoter of the viral vector is selected from the group consisting of GFAP promoter, NG2 promoter, Aldh1L1 promoter, IBA1 promoter, CNP promoter, LCN2 promoter, and genetically engineered promoter mutants; The expression vector comprising the Neurog2 functional fragment also contains one or more regulatory elements for enhancing gene expression levels and regulating gene expression, including, but not limited to, CMV enhancer, SV40 enhancer, EN1 enhancer or a genetically modified enhancer mutant, SV40 polyA tailing signal, human insulin gene polyA tailing signal or WPRE (woodchuck hepatitis B virus post-transcriptional regulatory element), a human-derived MAR sequence or a genetically modified mutant, a Neurog2 functional fragment delivery system.

2. 2. The Neurog2 functional fragment delivery system of claim 1, wherein the glial cells are selected from human or non-human mammalian astrocytes, NG2 glial cells, oligodendrocytes or microglial cells.

3. The Neurog2 functional fragment delivery system of claim 2, wherein the glial cells are derived from the spinal cord, the dorsal midbrain or the cerebral cortex; and the glial cells are glial cells in a normal state or in an injured state.

4. The Neurog2 functional fragment delivery system according to claim 1, wherein the functional neuron comprises a glutamate neuron or a group of glutamate neurons.

5. The Neurog2 functional fragment delivery system according to any one of claims 1 to 4, wherein the viral vector is a GFAP-AAV vector or an NG2-lentiviral vector.

6. A host cell containing an exogenous Neurog2 functional fragment, wherein the host cell comprises a Neurog2 functional fragment delivery system according to claims 1 to 5; The host cells are derived from glial cells cultured in vitro or from glial cells under normal or injured conditions, and the host cells undergo transdifferentiation to form a functional neuron or a population of functional neuronal cells.

7. 7. The host cell of claim 6, wherein the functional neuron has the following characteristics: (a) expresses the neuronal marker NeuN; and (b) is capable of firing action potentials and forming synaptic connections.

8. 7. The host cell of claim 6, wherein the population of functional neuronal cells has the following characteristics: (a) at least 50% of the neurons express the neuronal marker NeuN; and (b) are capable of firing action potentials and forming synaptic connections.

9. A pharmaceutical composition comprising the Neurog2 functional fragment delivery system according to claims 1 to 5 or the host cell according to claims 6 to 8 and a pharma- ceutically acceptable excipient.

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