Reprogramming functional fragments, combinations and uses thereof

Abstract

[Solution] We provide transcription factors and combinations of transcription factors that can synergistically promote the transdifferentiation and reprogramming of glioma cells into functional neurons or neuronoids. By promoting the expression of this combination of transcription factors in vivo or in vitro, we can effectively apply the transdifferentiation of glioma cells to repair nerve damage, or use the transdifferentiation ability of key transcription factors to suppress the progression of brain tumors derived from glioma cells. We also provide applications of the expressed transcription factors and their combinations in the preparation of therapeutic drugs for nervous system diseases.

Description

[Technical field]

[0001] The present invention belongs to the field of biotechnology and gene therapy, specifically glue A method for transdifferentiating cells derived from cells into neurons and its application to repairing nervous system damage and glue The present invention relates to a method for achieving treatment of tumors of cell origin. [Background technology]

[0002] The main pathological changes caused by damage to the mammalian central nervous system and multiple neurodegenerative diseases are irreversible neuronal degeneration and necrosis and destruction of neural circuits. How to compensate for the neurons that have died and been lost in the damaged or diseased brain or spinal cord and reconstruct the neural circuits is an important step in treatment. The self-repair ability of the adult mammalian central nervous system (brain and spinal cord) is very limited, so it is difficult to compensate for the loss of neurons on its own.

[0003] Transplantation of exogenous neurons or neurogenic cells is inefficient due to potential risks such as tumorigenicity and immunogenicity. In recent years, the emergence of cell reprogramming techniques has revolutionized regenerative medicine. In vivo reprogramming of astrocytes, which induces neuronal production through the expression of single or multiple transcription factors, is expected to be an important new strategy for neuronal replacement therapy. Summary of the Invention [Problem to be solved by the invention]

[0004] Gliomas, also known as glioblastomas, are defined in a broad sense as all tumors derived from the neuroepithelium, and in a narrow sense as any type of glueGliomas are defined as tumors arising from the cytoplasm of the brain. Gliomas are one of the most lethal malignant tumors and the most common primary tumor of the central nervous system, accounting for 30% of brain and central nervous system tumors and 80% of malignant brain tumors, posing a serious threat to human health. In the 1999 World Health Organization (WHO) classification, gliomas are classified into astrocytoma, oligodendroglioma, ependymoma, mixed glioma, choroid plexus tumor, neuroepithelial tumor of unknown origin, mixed neuronal and neuroglial tumor, pineal parenchymal tumor, embryonal tumor and neuroblastoma tumor. Gliomas grow mixed with normal nerve tissue, with unclear boundaries, tumor tissue is not easy to remove, and gliomas are prone to recurrence. At the same time, due to the presence of the blood-brain barrier, the therapeutic effect of ordinary antitumor agents is poor. At present, the treatment of glioblastoma has not yet met the clinical needs of the medical community. In recent years, several studies have found that neurogenic transcription factors or combinations of transcription factors can transform brain glioma cells into neuron-like cells in vivo or in vitro, and further restrict the proliferation ability of glioma cells. However, existing transcription factors or combinations of transcription factors have only shown poor transformation efficiency in vivo or in vitro, making practical clinical application difficult. [Means for solving the problem]

[0005] Therefore, in the body glue Searching for transcription factors or combinations of transcription factors that can induce transdifferentiation from cells to activated neurons is important for repairing the brain and spinal cord nervous system. At the same time, transcription factor reprogramming technology is glue Application of glioma cells derived from brain gliomas is also a currently urgently needed treatment option.

[0006] The present invention relates to glue The present invention provides a method for synergistically promoting the transdifferentiation and reprogramming of cells into functional neurons or neuron-like cells using a combination of transcription factors, a method for increasing the expression of the combination of transcription factors in vivo or in vitro, and the application of the combination of transcription factors in the preparation of a medicament for a neurological disease.

[0007] In a first aspect of the present invention, glueA combination of functional fragments that synergistically promotes cell transdifferentiation is provided, the functional fragments including a functional fragment that promotes expression of at least one transcription factor, the functional fragment being selected from the expression of transcription factors such as NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 and / or Otx2.

[0008] In another preferred embodiment, the "transdifferentiation" is glue This refers to the transdifferentiation or reprogramming of cells into functional neuronal cells.

[0009] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of the Ascl1 transcription factor.

[0010] In another preferred embodiment, the Ascl1 is an enhanced Ascl1 having an amino acid sequence as shown in SEQ ID NO:41.

[0011] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of NeuroD1 transcription factor.

[0012] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of Brn2 transcription factor.

[0013] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of Ngn2 transcription factor.

[0014] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of Gsx1 transcription factor.

[0015] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of the Tbr1 transcription factor.

[0016] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of Dlx2 transcription factor.

[0017] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of the Ptf1a transcription factor.

[0018] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of Pax6 transcription factor.

[0019] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of Otx2 transcription factor.

[0020] In another preferred embodiment, the combination of functional fragments comprises functional fragments that promote expression of at least two transcription factors selected from functional fragments that promote expression of transcription factors such as NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 and / or Otx2.

[0021] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of the Brn2 transcription factor and another functional fragment that promotes the expression of a transcription factor, and the other functional fragment that promotes the expression of the transcription factor is selected from a functional fragment that promotes the expression of any of NeuroD1, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, or Otx2, etc. More preferably, the other functional fragment is selected from any functional fragment that promotes the expression of the transcription factor NeuroD1, Ascl1, or Ngn2.

[0022] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of NeuroD1 transcription factor and another functional fragment that promotes the expression of a transcription factor, and the functional fragment that promotes the expression of the transcription factor is selected from any functional fragment that promotes the expression of Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, or Otx2, etc. More preferably, the other functional fragment is selected from any functional fragment that promotes the expression of the transcription factor Brn2, Ascl1, or Ngn2.

[0023] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of a Gsx1 transcription factor and another functional fragment that promotes the expression of a transcription factor, and the other functional fragment that promotes the expression of the transcription factor is selected from a functional fragment that promotes the expression of any of NeuroD1, Ascl1, Ngn2, Brn2, Tbr1, Dlx2, Ptf1a, Pax6, or Otx2, etc. More preferably, the other functional fragment that promotes the expression of a transcription factor is selected from any functional fragment that promotes the expression of a transcription factor such as Ascl1, Ngn2, or Tbr1.

[0024] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of a Tbr1 transcription factor and another functional fragment that promotes the expression of a transcription factor, and the other functional fragment that promotes the expression of the transcription factor is selected from a functional fragment that promotes the expression of any of NeuroD1, Ascl1, Ngn2, Brn2, Gsx1, Dlx2, Ptf1a, Pax6, or Otx2, etc. More preferably, the other functional fragment that promotes the expression of a transcription factor is selected from any functional fragment that promotes the expression of a transcription factor such as Ascl1, Ngn2, or Gsx1.

[0025] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of Dlx2 transcription factor and another functional fragment that promotes the expression of a transcription factor, and the other functional fragment that promotes the expression of the transcription factor is selected from a functional fragment that promotes the expression of any of NeuroD1, Ascl1, Ngn2, Brn2, Tbr1, Gsx1, Ptf1a, Pax6, or Otx2, etc. More preferably, the other functional fragment that promotes the expression of a transcription factor is selected from any functional fragment that promotes the expression of a transcription factor such as Ascl1, Ngn2, or Ptf1a.

[0026] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of a Ptf1a transcription factor and another functional fragment that promotes the expression of a transcription factor, and the other functional fragment that promotes the expression of the transcription factor is selected from any functional fragment that promotes the expression of NeuroD1, Ascl1, Ngn2, Brn2, Tbr1, Gsx1, Dlx2, Pax6, or Otx2, etc. More preferably, the other functional fragment that promotes the expression of a transcription factor is selected from any functional fragment that promotes the expression of a transcription factor, such as Ascl1, Ngn2, or Dlx2.

[0027] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of Pax6 transcription factor and another functional fragment that promotes the expression of a transcription factor, and the other functional fragment that promotes the expression of the transcription factor is selected from any functional fragment that promotes the expression of NeuroD1, Ascl1, Ngn2, Brn2, Tbr1, Gsx1, Ptf1a, Dlx2, or Otx2, etc. More preferably, the other functional fragment that promotes the expression of a transcription factor is selected from any functional fragment that promotes the expression of a transcription factor, such as Ascl1, Ngn2, or Otx2.

[0028] In another preferred embodiment, the functional fragment that promotes the expression of the transcription factor includes at least a functional fragment that promotes the expression of Otx2 transcription factor and another functional fragment that promotes the expression of a transcription factor, and the other functional fragment that promotes the expression of the transcription factor is selected from a functional fragment that promotes the expression of any of NeuroD1, Ascl1, Ngn2, Brn2, Tbr1, Gsx1, Ptf1a, Dlx2, or Pax6, etc. More preferably, the other functional fragment that promotes the expression of a transcription factor is selected from any functional fragment that promotes the expression of a transcription factor, such as Ascl1, Ngn2, or Pax6.

[0029] In another preferred embodiment, the functional fragment that promotes expression of the transcription factor includes at least a functional fragment that promotes expression of either a transcription factor Ascl1 or Ngn2, and another functional fragment that promotes expression of a transcription factor, and the other functional fragment that promotes expression of the transcription factor is selected from any functional fragment that promotes expression of a transcription factor such as NeuroD1, Brn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 or Otx2.

[0030] In another preferred embodiment, glue The above functional fragments that synergistically promote cell transdifferentiation include a functional fragment that promotes the expression of at least two transcription factors, NeuroD1 and Brn2, or a functional fragment that promotes the expression of two transcription factors, Gsx1 and Tbr1, or a functional fragment that promotes the expression of two transcription factors, Dlx2 and Ptf1a, or a functional fragment that promotes the expression of two transcription factors, Pax6 and Otx2.

[0031] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation comprises a functional fragment that promotes expression of either Ascl1 or Ngn2, glue This at least includes a combination with another functional fragment that synergistically promotes cell differentiation. glueAnother combination of the above functional fragments that synergistically promotes cell transdifferentiation is selected from a combination of functional fragments that promote the expression of both transcription factors NeuroD1 and Brn2, a combination of functional fragments that promote the expression of both transcription factors Gsx1 and Tbr1, or a combination of functional fragments that promote the expression of both transcription factors Dlx2 and Ptf1a, or a combination of functional fragments that promote the expression of both transcription factors Pax6 and Otx2.

[0032] glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes the expression of a transcription factor may be a polynucleotide encoding the transcription factor, or a small molecule drug, large molecule drug, or nucleic acid drug that promotes the expression of a functional protein, polypeptide, or transcription factor after translation of the polynucleotide, or the polynucleotide or a functional protein, polypeptide, small molecule drug, large molecule drug, or nucleic acid drug that is located upstream of the transcription factor and regulates increased expression of the transcription factor.

[0033] In another preferred embodiment, glue The functional fragment synergistically promoting cell transdifferentiation or the functional fragment promoting expression of a transcription factor is a protein of a transcription factor such as functional NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and / or Otx2, or a polynucleotide encoding a transcription factor such as NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and / or Otx2. Preferably, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes transcription factor expression is derived from a mammal, preferably from a human or non-human primate.

[0034] In a further preferred embodiment, the combination of functional fragments is (Z1) NeuroD1+Brn2, (Z2) Ascl1+Ngn2, (Z3) Ngn2+NeuroD1, (Z4) Gsxl+Tbrl, (Z5)Dlx2+Ptfla, (Z6) Pax6+Otx2, (Zn) Combination of (Z1) to (Z6) above is selected from the group consisting of:

[0035] In another preferred embodiment, said combination of functional fragments is selected from the group consisting of NeuroD1+Brn2, Gsx1+Tbrl, Dlx2+Ptfla, Pax6+Otx2, or a combination thereof.

[0036] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional NeuroD1 protein, the protein sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:2, and the polynucleotide sequence encoding the functional NeuroD1 protein is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0037] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Brn2 protein, the protein sequence of which is shown in SEQ ID NO:5 or SEQ ID NO:6, and the polynucleotide sequence encoding the functional Brn2 protein is shown in SEQ ID NO:7 or SEQ ID NO:8.

[0038] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Ascl1 protein, the protein sequence of which is shown in SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:41, and the polynucleotide sequence encoding the functional Ascl1 protein is shown in SEQ ID NO:11 or SEQ ID NO:12.

[0039] In another preferred embodiment, glueThe functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Ngn2 protein, the protein sequence of which is shown in SEQ ID NO:13 or SEQ ID NO:14, and the polynucleotide sequence encoding the functional Ngn2 protein is shown in SEQ ID NO:15 or SEQ ID NO:16.

[0040] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Gsx1 protein, the protein sequence of which is shown in SEQ ID NO: 17 or SEQ ID NO: 18, and the polynucleotide sequence encoding the Gsx1 functional protein is shown in SEQ ID NO: 19 or SEQ ID NO: 20.

[0041] In another preferred embodiment, glue The above functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Tbr1 protein, the protein sequence of which is shown in SEQ ID NO: 21 or SEQ ID NO: 22, and the polynucleotide sequence encoding the above Tbr1 functional protein is shown in SEQ ID NO: 23 or SEQ ID NO: 24.

[0042] In another preferred embodiment, glue The above functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Dlx2 protein, the protein sequence of which is shown in SEQ ID NO: 25 or SEQ ID NO: 26, and the polynucleotide sequence encoding the above Dlx2 functional protein is shown in SEQ ID NO: 27 or SEQ ID NO: 28.

[0043] In another preferred embodiment, glueThe functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Ptf1a protein, the protein sequence of which is shown in SEQ ID NO:29 or SEQ ID NO:30, and the polynucleotide sequence encoding the Ptf1a functional protein is shown in SEQ ID NO:31 or SEQ ID NO:32.

[0044] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Pax6 protein, the protein sequence of which is shown in SEQ ID NO: 33 or SEQ ID NO: 34, and the polynucleotide sequence encoding the functional Pax6 protein is shown in SEQ ID NO: 35 or SEQ ID NO: 36.

[0045] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a functional Otx2 protein, the protein sequence of which is shown in SEQ ID NO: 37 or SEQ ID NO: 38, and the polynucleotide sequence encoding the Otx2 functional protein is shown in SEQ ID NO: 39 or SEQ ID NO: 40.

[0046] In another preferred embodiment, glue The functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a modified Ascl1 functional protein, and the protein sequence is shown in SEQ ID NO:41.

[0047] In another preferred embodiment, glueWhen the functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes transcription factor expression is a functional protein, the functional protein sequence has 85% or more sequence identity to SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, and / or 41. More preferably, the functional protein sequence has 95% or more sequence identity to SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, and / or 41. Most preferably, said functional proteins have 99% or more sequence identity to SEQ ID NOs: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38 and / or 41.

[0048] In another preferred embodiment, glue When the functional fragment that synergistically promotes cell transdifferentiation or the functional fragment that promotes expression of a transcription factor is a polynucleotide encoding a functional protein, the sequence of the polynucleotide encoding the functional protein has 75% or more sequence identity to SEQ ID NO:3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, and / or 40. More preferably, the polynucleotide encoding the functional protein has 85% or more sequence identity to SEQ ID NO:3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, and / or 40. Most preferably, said polynucleotide encoding a functional protein has 95% or more sequence homology to SEQ ID NOs:3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39 and / or 40.

[0049] Preferably, the above glue The cells are astrocytes, NG2 cells, derived from human or non-human mammals. glue cyst, oligodendrocyte, small glueCells or damaged glue cell, glue These are either tumor cells derived from the cytoplasm or cells in a damaged state. glue The cells are located in the tissue or glue The surrounding area is in a state of mechanical injury, neuronal death or apoptosis due to stroke or neurodegenerative disease, and neural signaling is disrupted or disturbed. glue It is a cell. glue The tumor cells derived from the alveolar follicular tract are generally glioma tumor cells, selected from astrocytes, oligodendroglioma, ependymoma, mixed glioma, choroid plexus tumor, neuroepithelial tumor of unknown origin, neuronal and neuronal mixed glioma, pineal parenchymal tumor, embryonal tumor, neuroblastoma, from a human or non-human mammal.

[0050] Preferably, said functional neuronal or neuronal cells comprise at least one of the following characteristics:

[0051] (1) They have similar neuronal morphology. (2) Increased gene expression specific to nerve cells. The genes found to be increased include one or more of DCX, Tuj1, Map2, NeuN, and Synapsin I (antibody for synapsin 1). (3) glue Decreased cell-specific gene expression, said decreased expression genes including one or more of GFAP, S100β, Glast, Acsbg1. (4) Electrophysiological properties specific to nerve cells, i.e., the cells have a resting potential and an action potential induced by the action of excitatory or inhibitory neurotransmitters, and the resulting cell resting potential is lower than -50 mV, preferably lower than -55 mV, or lower than -60 mV, or lower than -65 mV. The excitatory neurotransmitters include, but are not limited to, glutamate or kainate, and can induce inward currents, and the inhibitory neurotransmitters include, but are not limited to, glycine or gamma aminobutyric acid (GABA), and can induce outward currents. (5) They form functional synapses that can receive excitatory or inhibitory signals and output action potentials.

[0052] In a second aspect of the present invention, glue Methods are provided for promoting cell transdifferentiation and reprogramming into functional neurons or neuron-like cells.

[0053] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0054] In another preferred embodiment, the method is an in vitro method.

[0055] In another preferred embodiment, the method is therapeutic.

[0056] In another preferred embodiment, the method further comprises: glue A functional fragment according to the first aspect of the present invention that synergistically promotes cell transdifferentiation, glue The cells are contacted with the antibody or, depending on the delivery system, introduced, thereby glue The method includes transdifferentiating and reprogramming the cells into functional neurons or neuron-like cells.

[0057] Preferably, the above glue The cells are astrocytes, NG2 cells, derived from human or non-human mammals. glue cyst, oligodendrocyte, small glue Cells or damaged glue cell, glue These are either tumor cells derived from the cytoplasm or cells in a damaged state. glue The cells are located in the tissue or glue The surrounding area is in a state of mechanical injury, neuronal death or apoptosis due to stroke or neurodegenerative disease, and neural signaling is disrupted or disturbed. glue It is a cell. glueThe tumor cells derived from the alveolar follicular tract are generally glioma cells and are selected from astrocytes, oligodendroglioma, ependymoma, mixed glioma, choroid plexus tumor, neuroepithelial tumor of unknown origin, neuronal and neuronal mixed glioma, pineal parenchymal tumor, embryonal tumor, neuroblastoma from a human or non-human mammal.

[0058] glue Any method for promoting increased expression of a transcription factor for cell transdifferentiation, comprising administering to said subject an inducing factor or a functional fragment that promotes expression of said transcription factor. glue By direct contact with or introduction into the cells, glue The present invention includes, but is not limited to, promoting an increase in the expression of any one of NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2 in the cells. glue The present invention relates to a method for promoting cells to exhibit functional neuronal or neuronal cell characteristics.

[0059] The functional fragment that promotes the expression of the inducer or transcription factor may be a polynucleotide encoding the transcription factor, or a functional protein translated from a polypeptide, or a polynucleotide, or a low molecular weight drug, or a high molecular weight drug, or a nucleic acid drug, etc. that promotes the expression of any of the transcription factors NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2. The functional fragment that promotes the expression of the inducer or transcription factor may be a discovery of a transcription factor that is upregulated upstream of any of the transcription factors NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2, a polynucleotide or a functional protein, a peptide, a low molecular weight drug, a high molecular weight drug, a nucleic acid drug, etc. glue Passive absorption through the cells, glue Passively absorbed into the cell or via a delivery system glue It is delivered inside the cells and has an effect.

[0060] The delivery system includes, but is not limited to, an expression vector of the functional fragment promoting expression of the inducer or transcription factor, a nanoparticle of the functional fragment promoting expression of the inducer or transcription factor, an exosome of the functional fragment promoting expression of the inducer or transcription factor, a cellular vector (e.g., modified red blood cells or bacteria) or a viral vector of the functional fragment promoting expression of the inducer or transcription factor, but also includes a target effector (e.g., glue The targeting agent may be a cell-specific antibody, peptide, or other targeting agent.

[0061] In another preferred example, the functional fragment promoting expression of the inducer or transcription factor is a polynucleotide encoding a transcription factor, the polynucleotide being a transcription factor functional polynucleotide selected from NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 and / or Otx2, and the polynucleotide needs to be loaded into a viral or non-viral delivery system.

[0062] In another preferred embodiment, the delivery system includes, but is not limited to, a plasmid, a virus, a cell vector, preferably a virus vector, including, but not limited to, an adenovirus vector, an adeno-associated virus vector (AAV), a retrovirus expression vector, or a lentivirus vector.

[0063] In another preferred embodiment, the expression vector for the transcription factor polynucleotide comprises: glue The present invention also carries a cell-specific 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 genetically engineered promoter variants.

[0064] In another preferred embodiment, the promoter is a GFAP promoter or a genetically engineered GFAP promoter. More preferably, the human hGFAP promoter (SEQ ID NO: 42) can be modified to a 683 bp version (SEQ ID NO: 43).

[0065] In another preferred example, the above expression vector of the transcription factor polynucleotide further comprises one or more regulatory elements for increasing the expression level of the gene, including, but not limited to, a CMV transcription factor, an SV40 transcription factor, an EN1 transcription factor, a VP16 fusion protein or a genetically engineered variant of a transcription factor, and 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 engineered variant.

[0066] In another preferred embodiment, the regulatory element for enhancing expression is the transcription activation domain of the VP16 protein from Herpes simplex virus (SEQ ID NO: 44), and the coding sequence of VP16 (SEQ ID NO: 45) is glue Expression of proteins fused to the DNA binding domain of the VP16 transcription factor is driven by a cell-specific promoter, either singly or in a daisy chain.

[0067] In another preferred embodiment, the regulatory element for enhancing expression is derived from the Simian vacuolating virus 40 (SV40) transcription factor (SEQ ID NO: 46), glue By inserting it before a cell-specific promoter, promoter activity is enhanced and the efficiency of neuronal induction is improved.

[0068] In another preferred embodiment, the expression vector of the transcription factor polynucleotide may also contain other functional fragments, which may be reporter genes or other functional fragments of transcription factors having reprogramming functions, including, but not limited to, functional fragments selected from NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2. Preferably, at least two transcription factor polynucleotide fragments may be contained in the same vector, and the two transcription factor polynucleotides may be expressed by one vector. glue Even when expressed under a cell-specific promoter, glue They may be expressed separately under a cell-specific promoter. When two or more transcription factors are in the transcript of a single promoter, the promoter is linked to the open reading frames of the multiple transcription factors by polycistronic elements, in which the transcription factors can be spaced apart using IRES or 2A polypeptide (P2A) elements, etc., to achieve the expression of multiple transcription factors (Pharmaceutics 2019, 11(11), 580; the IRES sequence used in the present invention is copied from Addgene #69550, and the P2A sequence is copied from Addgene #130692). The combinations of the two transcription factors described are selected from a combination of NeuroD1 and Brn2 transcription factors, a combination of Gsx1 and Tbr1 transcription factors, a combination of Dlx2 and Ptf1a transcription factors, a combination of Pax6 and Otx2 transcription factors, and a combination of Ascl1 and Ngn2 transcription factors, and the molar expression ratio of the two transcription factors is 4:1 to 1:4, preferably, the molar expression ratio of the two transcription factors is 2:1 to 1:2, and optimally, the optimal molar expression ratio of the two transcription factors is 1:1.

[0069] In another preferred embodiment, when at least two transcription factors are contained in the same vector and one of the transcription factors is Ascl1 or Ngn2, the molar concentration ratio of the expression levels of Ascl1 or Ngn2 is 20% or more, preferably the molar concentration ratio of the expression levels of Ascl1 or Ngn2 is 33% or more, and optimally the molar concentration ratio is 50% or more. Also includes any combination of the following transcription factors:

[0070] (1) A combination of Ascl1 and any other transcription factor, wherein the other transcription factor is selected from NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 or Otx2. (2) A combination of Ngn2 with any other transcription factor, wherein the other transcription factor is selected from NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, or Otx2; and (3) When at least three transcription factors are contained in the same vector, the combination of the transcription factors includes at least the combination of Ascl1 + NeuroD1 + Brn2, the combination of Ascl1 + Gsx1 + Tbr1, the combination of Ascl1 + Dlx2 + Ptf1a, the combination of Ascl1 + Pax6 + Otx2, the combination of Ngn2 + NeuroD1 + Brn2, the combination of Ngn2 + Gsx1 + Tbr1, the combination of Ngn2 + Dlx2 + Ptf1a, or the combination of Ngn2 + Pax6 + Otx2, among which, the molar concentration ratio of the expression amounts of the other two transcription factors other than Ascl1 or Ngn2 is 4:1 to 1:4, preferably the molar concentration ratio of the expression amounts of the two transcription factors is 2:1 to 1:2. Most preferably, the optimal molar concentration ratio of the expression amounts of the two transcription factors is 1:1.

[0071] In another preferred embodiment, one or more expression vectors containing different transcription factor polynucleotides can be used simultaneously, and the transcription factors are selected from the transcription factor functional polynucleotides of NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 and / or Otx2. Preferred vector combinations are selected from a combination of a vector containing the transcription factor NeuroD1 and a vector containing the transcription factor Brn2, a combination of a vector containing the transcription factor Gsx1 and a vector containing the transcription factor Tbr1, a combination of a vector containing the transcription factor Dlx2 and a transcription factor Ptf1a, and a combination of a vector containing the transcription factor Pax6 and a transcription factor Otx2, where the molar ratio of the expression amounts of the two transcription factors is 4:1 to 1:4, preferably, the molar ratio of the expression amounts of the two transcription factors is 2:1 to 1:2, and optimally, the optimal molar ratio of the expression amounts of the two transcription factors is 1:1.

[0072] In another preferred embodiment, one or more expression vectors containing polynucleotides of different transcription factors can be used simultaneously, including at least a combination of an expression vector containing Ascl1 or an expression vector containing Ngn2 with a vector of another transcription factor, in which the molar concentration ratio of the expression amount of Ascl1 or Ngn2 is 20% or more, preferably the molar concentration ratio of the expression amount of Ascl1 or Ngn2 is 33% or more, and optimally the molar concentration ratio of the expression amount of Ascl1 or Ngn2 is 50% or more. Also, the combination of any of the following vectors is selected.

[0073] (1) A combination of a vector containing the transcription factor Ascl1 and a vector containing another transcription factor, wherein the other transcription factor is selected from NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 or Otx2. (2) A combination of a vector containing the transcription factor Ngn2 and a vector containing another transcription factor, wherein the other transcription factor is selected from NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, or Otx2. And (3) A combination of vectors containing the transcription factor Ascl1, the transcription factor NeuroD1, and the transcription factor Brn2; a combination of vectors containing the transcription factor Ascl1, the transcription factor Gsx1, and the transcription factor Tbr1; a combination of vectors containing the transcription factor Ascl1, the transcription factor Dlx2, and the transcription factor Ptf1a; a vector containing the transcription factor Ascl1, the transcription factor Pax6, and the transcription factor Otx2; or a combination of vectors containing the transcription factor Ngn2, the transcription factor NeuroD1, and the transcription factor Brn2; a combination of the transcription factor Ngn2, the transcription factor A combination of a vector containing Gsx1 and transcription factor Tbr1, a combination of a vector containing transcription factor Ngn2, transcription factor Dlx2 and transcription factor Ptf1a, and a combination of a vector containing transcription factor Ngn2, transcription factor Pax6 and transcription factor Otx2, wherein the molar concentration ratio of the expression levels of two transcription factors other than Ascl1 or Ngn2 is 4:1 to 1:4, preferably the molar concentration ratio of the expression levels of the two transcription factors is 2:1 to 1:2, and optimally the optimal molar concentration ratio of the expression levels of the two transcription factors is 1:1.

[0074] In another preferred embodiment, the expression vector containing the functional fragment of the transcription factor polynucleotide is a lentiviral vector, which contains a viral ITR sequence, a CAG promoter, a coding frame for the functional fragment of the transcription factor polynucleotide, and a post-transcriptional regulatory element WPRE, etc., and the expression vector may also contain a reporter gene, but the reporter gene is not necessary for practical use. For example, the lentiviral vector may contain the following elements in order from the 5' to 3' end: viral ITR sequence + CAG promoter + coding frame for the transcription factor polynucleotide and green fluorescent protein GFP + post-transcriptional regulatory element WPRE + viral ITR sequence + promoter and coding frame for the ampicillin resistance gene, where the coding frame for the green fluorescent protein GFP, the promoter and coding frame for the ampicillin resistance gene are not essential. Preferably, said polynucleotide of a transcription factor is a functional polynucleotide encoding NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 and / or Otx2, in particular selected from the sequences of SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39 and / or 40.

[0075] In another preferred example, the expression vector containing the functional fragment of the transcription factor polynucleotide is a GFAP-AAV vector, which contains a viral ITR sequence, a CMV transcription promoter, a human GFAP promoter, a coding frame for the functional fragment of the transcription factor polynucleotide, and a post-transcriptional regulatory element WPRE, etc., and the expression vector may also contain a reporter gene that is not practically necessary, for example, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequence + CMV transcription promoter + human GFAP promoter + coding frame for the transcription factor polynucleotide and the red fluorescent protein mCherry + post-transcriptional regulatory element WPRE + viral ITR sequence + promoter and coding frame for the ampicillin resistance gene, where the coding frame for the red fluorescent protein mCherry and the promoter and coding frame for the ampicillin resistance gene are not essential. Preferably, said polynucleotide of a transcription factor is a functional polynucleotide encoding NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 and / or Otx2, in particular selected from the sequences of SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39 and / or 40.

[0076] In another preferred embodiment, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequence + SV40 transcription enhancer + human GFAP promoter + transcription factor polynucleotide + post-transcriptional regulator WPRE + viral ITR sequence.

[0077] In another preferred embodiment, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequences + CMV transcription enhancer + human GFAP promoter + VP16 fusion protein + transcription factor DNA binding region + post-transcriptional regulatory element WPRE + viral ITR sequences.

[0078] In another preferred embodiment, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequence + CMV transcriptional enhancer + human short GFAP promoter + transcription factor polynucleotide + post-transcriptional regulatory element WPRE + viral ITR sequence.

[0079] In another preferred embodiment, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequence + SV40 transcription enhancer + human short form GFAP promoter + VP16 fusion protein + transcription factor DNA binding region + post-transcriptional regulatory element WPRE + viral ITR sequence.

[0080] In another preferred embodiment, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequences + SV40 transcription enhancer + human short GFAP promoter + transcription factor polynucleotide + post-transcriptional control element WPRE + viral ITR sequences.

[0081] In another preferred embodiment, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequences + SV40 transcription enhancer + human GFAP promoter + VP16 fusion protein + transcription factor DNA binding region + post-transcriptional regulatory element WPRE + viral ITR sequences.

[0082] In another preferred embodiment, the GFAP-AAV expression vector may contain the following elements in order from the 5' to 3' end: viral ITR sequence + CMV transcriptional enhancer + human short GFAP promoter + VP16 fusion protein + transcription factor DNA binding region + post-transcriptional regulatory element WPRE + viral ITR sequence.

[0083] In a third aspect of the present invention, there is provided a pharmaceutical composition comprising: (A) a functional fragment that enhances the expression of a transcription factor according to the first aspect of the invention, and / or (B) Using the method according to the second aspect of the present invention glue promote the transdifferentiation of cells and their reprogramming into functional neurons or neuron-like cells; and (C) Pharmaceutically acceptable excipients.

[0084] In another preferred example, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

[0085] In another preferred embodiment, the pharmaceutical composition is an injection.

[0086] In another preferred embodiment, the pharmaceutical composition comprises: (A) a combination of functional fragments that enhance expression of a transcription factor, or a combination of functional fragments that have been treated with the combination of functional fragments; glue The cells contain functional neurons or neuron-like cells generated by transdifferentiation and reprogramming.

[0087] wherein said combination is Ascl1, or enhanced Ascl1, or Ngn2, or said combination is selected from the group consisting of: (Z1) NeuroD1+Brn2, (Z2) Ascl1+Ngn2, (Z3) Ngn2+NeuroD1, (Z4) Gsx1+Tbr1, (Z5)Dlx2+Ptf1a, (Z6) Pax6+Otx2, and (Zn) A combination of the above (Z1) to (Z6). (B) Pharmaceutically acceptable excipients.

[0088] In another preferred embodiment, the combination comprises enhanced Ascl1 and at least one selected from the group consisting of NeuroD1, Brn2, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, Otx2.

[0089] In another preferred example, the combination is Ascl1 and at least one selected from the group consisting of NeuroD1, Brn2, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2.

[0090] In another preferred embodiment, the combination is Ngn2 and at least one selected from the group consisting of NeuroD1, Brn2, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2.

[0091] In a fourth aspect of the present invention there is provided an artificially reprogrammed neuron or neuron-like cell, said neuron or neuron-like cell comprising: glue Transdifferentiation of cells results in their reprogramming into functional neurons or neuron-like cells.

[0092] In another preferred embodiment, said artificial reprogrammed neuron or neuron-like cell is prepared by the method according to the second aspect of the invention.

[0093] In a fifth aspect of the present invention, there is provided the use of a pharmaceutical composition according to the third aspect of the present invention or an artificially reprogrammed neuron or neuron-like cell according to the fourth aspect of the present invention for the preparation of a medicament for gene therapy against a nervous system disease. Preferably, said nervous system disease is caused by nerve injury or glue It is a glioma of alveolar origin. Effect of the Invention

[0094] It should be understood that within the scope of the present invention, each of the above technical features of the present invention and each of the technical features (e.g., embodiments) specifically described below can be combined with each other to form a new or preferred technical solution, which will not be repeated here. [Brief description of the drawings]

[0095] Description of the accompanying drawings [Figure 1]Figure 1 shows that the combination of Brn2 and NeuroD1 can induce human glioma cells to become neurons. Figure 1A-C show the expression of the neuronal characteristic marker molecule Tuj1 by immunofluorescence 14 days after infection of human glioma U251 cells with the control lentivirus FUGW, the single virus FUGW-NeuroD1, and the combination of the two viruses FUGW-Brn2 and FUGW-NeuroD1, respectively. Figure 1D shows the statistical diagram showing the ratio of neurons induced by different viruses. "**" indicates P<0.01, scale is 50 μm. [Diagram 2] Figure 2 shows the molecular expression characteristics of neurons induced by the combination of Brn2 and NeuroD1. Figure 2A shows the expression of MAP2, a marker molecule for mature neurons, by induced neurons of glioma cells 21 days after lentivirus infection. Figure 2B-D shows the expression of SynapsinI, a marker molecule for mature neurons, by induced neurons. Figure 2E-H shows the expression of VGLUT1, a marker molecule for glutamatergic neurons, a neurotransmitter characteristic of induction. Scale: 20 μm. [Diagram 3] Figure 3 shows the electrophysiological properties of neurons induced by the combination of Brn2 and NeuroD1. Figure 3A shows a cell recorded with a glass electrode (with green fluorescence). Figure 3B shows the ability of the induced neuron to generate action potentials. Figure 3C shows the postsynaptic current signal detected in the induced neuron, which disappeared after the addition of the blockers CNQX and AP5. [Figure 4]Figure 4 shows that the combination of Brn2 and NeuroD1 induces neurons to exit the cell cycle. Figure 4A and 4B show that the number of BrdU-positive cells induced by the combination of Brn2 / NeuroD1 is dramatically reduced when BrdU interval doping labeling is performed at different times of virus infection. Figure 4C-F show that the number of BrdU-positive cells induced by the combination of Brn2 / NeuroD1 is significantly reduced when sequential BrdU doping labeling is performed 5 days after virus infection. The arrow in Figure 4F indicates the induced neurons that were BrdU negative. "*" represents p<0.05. "**" represents p<0.01. Scale: 50 μm. [Diagram 5] Figure 5 shows that the combination of Brn2 and NeuroD1 suppresses the proliferation of glioma cells. Figure 5A-C shows that 14 days after virus infection, the number of Ki67-positive cells was obviously decreased in the combination of Brn2 / NeuroD1 by immunocytochemical analysis for Ki67. The arrow in Figure 5B indicates that the induced neurons are Ki67-negative. Figure 5D shows the cell numbers counted at different times after virus infection. "**" indicates P<0.01, scale, 50 μm. [Figure 6] Figure 6 shows that the expression of reprogramming factors NeuroD1 and Brn2, AAV viral vectors suppress glioma growth in animals by inducing transdifferentiated neurons. Figure 6A shows the tumor volume at each time point in different treatment groups, and Figure 6B shows the real-time PCR analysis results of tumor samples from different treatment groups. "*" indicates p<0.05. [Figure 7] Figure 7 shows that the expression of reprogramming factors NeuroD1 and Brn2, and type 5 adenoviral vector suppresses glioma growth in animals by inducing transdifferentiated neurons. Figure 7A shows the tumor volume at each time point in different treatment groups, and Figure 7B shows the HE color development results of tumor samples for different treatment groups. "*" indicates p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] Specific embodiments As a result of extensive and intensive research, the present inventors have unexpectedly discovered that glue A transcription factor group or combination of transcription factors that have reprogramming function and transdifferentiate cells into neurons; and glue They have found a way to differentiate cells into neurons, which allows them to be used in vitro or in vivo. glue Based on these findings, we further investigated the application scenarios of transcription factors and their combinations. Some specific combinations of transcription factors are glue The present invention can synergistically and significantly promote the transdifferentiation of cells into neurons. When the method of the present invention is applied to nerve injury repair or glioma drug development, particularly in animal models of glioma, it is observed that reprogramming causes glioma cells to exit the cell cycle, and the animals have significantly smaller tumor size and longer survival time. Therefore, the present transcription factor group or a combination of transcription factors with conversion reprogramming function is expected to be applied to the development of nerve injury repair drugs or glioma therapeutic drugs.

[0097] Glossary The term "administering" refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including, for example, intravenous, intracerebral, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration by injection or infusion.

[0098] The term "about" may refer to a value or composition that is within an acceptable range of error for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. Typically, "about" means ±10 percent or ±20 percent. For example, about 1:1 represents (1±0.2):(1±0.2), or means (1±0.1):(1±0.1).

[0099] The term "reprogramming" as used herein generally refers to a process that modulates or alters the biological activity of a cell from one biological state to another, typically a process that alters the fate of a cell, such as differentiation (progenitor cell to terminal cell), dedifferentiation (terminal cell to pluripotent stem cell), transdifferentiation (one terminal cell to another type of terminal cell), transdifferentiation (terminal cell to progenitor cell), or transcommitment (one type of progenitor cell to another type of naturally differentiated terminal cell of a progenitor cell).

[0100] In the present invention, the term "transdifferentiation" or "reprogramming" or "transdifferentiation reprogramming" refers to the process of converting one type of terminal cell into another type of terminal cell, specifically: glue It is the process by which cells transform into functional neurons or neuronal cells.

[0101] Transcription factors The present invention provides transcription factors having reprogramming function, and these transcription factors and combinations thereof have excellent transdifferentiation ability, glue The cells can enhance the efficiency of transdifferentiation into neurons.

[0102] As used herein, the term "transcription factor of the present invention" refers to one or a group of transcription factors essential for neural cell differentiation, selected from the group consisting of NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6 and Otx2. Preferably, the transcription factor of the present invention includes at least two of the above transcription factors.

[0103] A mammalian polynucleotide encoding a NeuroD1 functional fragment, a Neurogenic differentiation 1 transcription factor or its expressed protein fragment, NeuroD1 is a bHLH (basic helix-loop-helix) transcription factor, for example, a human-derived NeuroD1 molecule is listed in GenBank under ID#4760, and its protein sequence is shown in SEQ ID NO: 1. NCBI Reference Sequence: NM_002500.5, CDS sequence is shown in SEQ ID NO: 3.

[0104] The Brn2 functional fragment is also known as POU3F2, Oct7, or N-Oct3, and is a polynucleotide derived from a mammal that encodes a Pou class 3 homeobox 2 transcription factor or an expressed protein fragment thereof, and Brn2 is a neuronal cell-specific POU-III transcription factor family. For example, the human Brn2 molecule is listed in GenBank with ID# 5454, and its protein sequence is shown in SEQ ID NO: 5. NCBI Reference Sequence: NM_005604.4, and its CDS sequence is shown in SEQ ID NO: 7.

[0105] Ascl1 functional fragment is a mammalian polynucleotide encoding an Achaete-scute homolog 1 transcription factor or its expressed protein fragment, Ascl1 is a bHLH (basic helix-loop-helix) transcription factor, for example, the human Ascl1 molecule is listed in GenBank under ID#429, and its protein sequence is shown in SEQ ID NO: 9. NCBI Reference Sequence: NM_004316.4, and its CDS sequence is shown in SEQ ID NO: 11.

[0106] The Ngn2 functional fragment is also known as Neurog2 and is a mammalian polynucleotide encoding the Neurogenin-2 transcription factor or its expressed protein fragment, Ngn2 is a bHLH (basic helix-loop-helix) transcription factor, for example, the human Ngn2 molecule is listed in GenBank under ID#63973, and its protein sequence is shown in SEQ ID NO: 13. NCBI Reference Sequence: NM_024019.4, and its CDS sequence is shown in SEQ ID NO: 15.

[0107] The Gsx1 functional fragment, also known as Gsh1, is a polynucleotide of mammalian origin encoding the GShomeobox1 transcription factor or an expressed protein fragment thereof, Gsx1 having a binding site of 5'-GC[TA][AC]ATTA[GA]-3' in the DNA sequence, for example, the Gsx1 molecule of human origin is listed in GenBank under ID#219409, its protein sequence is shown in SEQ ID NO: 17, NCBI Reference Sequence: NM_145657.3, CDS sequence is shown in SEQ ID NO: 19.

[0108] The Tbr1 functional fragment is a polynucleotide derived from a mammal that encodes T-box brain transcription factor 1 or an expressed protein fragment thereof, Tbr1 is a T-box family transcription factor, for example, the Tbr1 molecule derived from human is listed in GenBank under ID# 10716, its protein sequence is shown in SEQ ID NO: 21. NCBI Reference Sequence: NM_006593.4, its CDS sequence is shown in SEQ ID NO: 23.

[0109] The Dlx2 functional fragment is a polynucleotide derived from a mammal that encodes a distal-less homeobox2 transcription factor or an expressed protein fragment thereof, Dlx2 is a transcription factor involved in the terminal differentiation of intermediate neurons, for example, a Dlx2 molecule derived from a human is listed in GenBank under ID#1746, and its protein sequence is shown in SEQ ID NO: 25. NCBI reference sequence: NM_004405.4, and its CDS sequence is shown in SEQ ID NO: 27.

[0110] The Ptf1a functional fragment is a polynucleotide derived from a mammal encoding pancreas-related transcription factor 1a or an expressed protein fragment thereof, Ptf1a is a transcription factor involved in pancreatic development, for example, a human Ptf1a molecule is listed in GenBank under ID#256297, its protein sequence is shown in SEQ ID NO: 29. NCBI Reference Sequence: NM_178161.3, its CDS sequence is shown in SEQ ID NO: 31.

[0111] The Pax6 functional fragment is a mammalian polynucleotide encoding a paired box 6 transcription factor or its expressed protein fragment, Pax6 is an important transcription factor involved in the development of nervous tissue, for example, a human Pax6 molecule is listed in GenBank under ID#5080, its protein sequence is shown in SEQ ID NO: 33. NCBI Reference Sequence: NM_000280.5, its CDS sequence is shown in SEQ ID NO: 35.

[0112] The Otx2 functional fragment is a mammalian polynucleotide encoding an orthodenticle homeobox2 transcription factor or its expressed protein fragment, Otx2 belongs to the bicoid homology structural domain subfamily of transcription factors, for example, the human Otx2 molecule is listed in GenBank under ID#5015, its protein sequence is shown in SEQ ID NO: 37. NCBI Reference Sequence: NM_001270523.2, and its CDS sequence is shown in SEQ ID NO: 39.

[0113] The present invention is not particularly limited as long as it is a method for promoting the expression of a functional fragment of the above transcription factor, and a functional fragment that promotes the discovery of a transcription factor or an inducer. glue These methods include, but are not limited to, directly contacting or introducing the cells into the cells. glueThe present invention promotes the expression or activity increase of any one of the transcription factors NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2 in the cells, glue The functional fragment that promotes the expression of an early-phase inducer or a transcription factor is a polynucleotide that encodes a transcription factor, or a functional protein or polypeptide after the polynucleotide is translated, or a small molecule drug, a polymer drug, or a nucleic acid drug that promotes the expression of any of the transcription factors NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2, or a polynucleotide or a functional protein, polypeptide, small molecule drug, or polymer drug that increases the expression of any of the upstream transcription factors located in NeuroD1, Brn2, Ascl1, Ngn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2, glue By passive absorption by the cells or through a delivery system glue It penetrates deep into the cells and exerts its effect.

[0114] In addition, a method for promoting increased expression of the functional fragment of the above transcription factor can be obtained by activating gene expression using CRISPR / dCas9 targeting the relevant transcription factor DNA, or improving the expression of the functional protein of the transcription factor using CRISPR / Cas13 targeting the relevant transcription factor RNA.

[0115] Those skilled in the art can screen the above transcription factor promoting method using existing databases. glue Based on the transdifferentiation of cells, repair of nerve damage, and the inhibitory function against glioma cells, if the substance has a promoting effect on the above transcription factors, glue It should be understood that it can be reasonably predicted by a person skilled in the art that the present invention has functions of transdifferentiation of cells, repair of nerve damage, and suppression of glioma cells.

[0116] Preferably, the reprogramming transcription factor of the present invention is used in conjunction with a modified expression element to further enhance expression of the transcription factor of the present invention.

[0117] glue cell As used herein, "neurology" glue " or " glue The term "neurons" is used interchangeably to refer to another major class of cells in nervous tissue other than neurons, which are widespread in the central and peripheral nervous systems. In mammals, the nervous glue The ratio of cells to neurons is about 10:1. In the central nervous system, astrocytes, NG2 glue The main cells are cytoplasmic cells, oligodendrocytes, and microglia. glue It is a cell. glue Alveoli perform many physiological functions, including providing biochemical support (e.g., formation of the blood-brain barrier), supplying nutrients to neurons, and maintaining extracellular ionic homeostasis. In conditions of injury or disease, glue Although the cells are activated and can proliferate to participate in repair and scar formation after brain and spinal cord injuries, they cannot differentiate into neurons. An important feature that distinguishes them from neural stem cells is that they are self-renewing, not yet fully differentiated cells, and can give rise to neurons and various glue whereas they may differentiate into cells. glue Alveoli are terminally differentiated cells.

[0118] The present invention relates to glue The cells are astrocytes, NG2 cells, derived from human or non-human mammals. glue Cells, oligodendrocytes, microglia, or damaged cells glue cell, glue The damaged cells are either tumor cells derived from the above-mentioned glue The cells are glue A state in which neuronal signal transduction is blocked or impaired due to neuronal cell death or apoptosis caused by mechanical trauma, stroke, neurodegenerative disease, etc. in the tissue or surrounding environment of the cell. glue It is a cell. glueThe tumor cells derived from the alveolar follicular tract are generally glioma cells and are selected from astrocytes, oligodendroglioma, ependymoma, mixed glioma, choroid plexus tumor, neuroepithelial tumor of unknown origin, mixed neuronal and neuroglial tumor, pineal parenchymal tumor, germinal tumor, neuroblastoma tumor of human or non-human mammalian origin.

[0119] Glioma cells As used herein, the term "glioma" is also abbreviated as "glioma" or "glioblastoma," and broadly refers to all tumors derived from neuroepithelium, and narrowly refers to all types of glue Gliomas are one of the most lethal malignant tumors and the most common primary central nervous system tumors, accounting for 30% of brain and central nervous system tumors and 80% of malignant brain tumors, posing a serious threat to human health. According to the 1999 World Health Organization (WHO) classification, gliomas are classified as astrocytoma, oligodendroglioma, ventricular meningioma, mixed glioma, choroid plexus tumor, neuroepithelial tumor of unknown origin, neuronal and neuronal-glial tumor, pineal parenchymal tumor, embryonal tumor, and neuroblastoma.

[0120] The glioma cells that can be used in the present invention are not particularly limited, and include cells derived from various gliomas derived from the central nervous system of mammals, such as astrocytoma, oligodendroglioma, ventricular meningioma, or neuroblastoma, more preferably cells derived from astrocytoma or neuroblastoma.

[0121] In the present invention, the transcription factors and combinations of transcription factors with transdifferentiation function can induce the transformation of glioma cells into neurons / neuron-like cells, and exhibit neuronal cell-specific markers, DCX, Tuj1, Map2, NeuN, and SynapsinI, while at the same time having the function of significantly inhibiting the proliferation of glioblastoma, delaying tumor growth, and reducing malignant transformation.

[0122] Delivery System As used herein, the term "delivery system" is not particularly limited. glueThe vector may be an expression vector comprising the polynucleotide sequence encoding the transcription factor to a glioma or glioma cell. The viral vector may be any viral vector that can utilize the characteristics of the virus to deliver the genome and bring the genetic material to other cells to infect them, either in vivo or in cell culture. It includes lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, pox viral vectors, etc.

[0123] The delivery system may also be a novel nanoparticle for carrying a functional fragment of said transcription factor or a molecular entity that promotes increased expression or enhanced activity of said transcription factor for delivery to the vicinity of or to the target cell, such as bulk nanoparticles, metallic nanoparticles, polymeric nanoparticles, and the like delivery systems.

[0124] The delivery system may also be an exosome encapsulating a functional fragment of the transcription factor or a molecular entity that promotes increased expression or enhanced activity of the transcription factor, or a modified red blood cell or bacterium encapsulating a functional fragment of the transcription factor or a molecular entity that promotes increased expression or enhanced activity of the transcription factor.

[0125] Additionally, the delivery system is glue The present invention can also be combined with functional targeting molecules such as monoclonal antibodies, polypeptides, etc. specific for glioma cells or glioma cells. glue and more effectively targeting a functional fragment of said transcription factor or a molecular entity that promotes increased expression or enhanced activity of said transcription factor in tumor necrosis factor or glioma cells. glue It improves induction of cell transdifferentiation and antitumor efficacy.

[0126] Guidance method The present invention also relates to in vitro and in vivo methods for the purpose of nerve repair and antitumor. glueThe present invention provides a method for inducing transdifferentiation of a cell line or a glioma cell into a neuron or a neuron-like cell. The term "inducer" refers to any molecular entity that promotes the expression or enhanced activity of a functional fragment of a transcription factor of the present invention.

[0127] In vitro, a delivery system comprising the functional fragment of the transcription factor, a molecular entity that promotes expression or activity enhancement of the functional fragment of the transcription factor, or a molecular entity that promotes expression and activity enhancement of the functional fragment of the transcription factor is administered to a target cell cultured in vitro by contact or administration (e.g., injection); glue Absorbed passively by cells or via a delivery system glue By delivering the cells to the inside of the cells, it is possible to achieve neuronal differentiation in vitro and suppress the proliferation of tumor cells. In addition, by transplanting cells that have successfully undergone transdifferentiation in vitro, it is possible to achieve nerve repair at the site of nerve damage.

[0128] In the body, a delivery system comprising the functional fragment of the transcription factor, a molecular entity that promotes expression or activity enhancement of the functional fragment of the transcription factor, or a molecular entity that promotes expression and activity enhancement of the functional fragment of the transcription factor is administered to a site of nerve damage or a site of a tumor lesion by contact or administration (e.g., injection); glue Absorbed passively by cells or via a delivery system glue It can reach the interior of the cells, achieve neuronal differentiation in vitro, and inhibit tumor cell proliferation, while direct in vivo delivery can facilitate in situ repair of neural damage and in situ inhibition of tumors.

[0129] At the same time, in combination with molecular targeting technology, the induction of neuronal transdifferentiation can be glue Specific molecular targeting of glioma cells or glioma cells can be achieved by ectopic injection with a delivery system comprising a functional fragment of the transcription factor or a molecular entity that promotes the expression and enhanced activity of a functional fragment of the transcription factor.

[0130] Pharmaceutical Compositions and Dosage Forms The present invention also provides a pharmaceutical composition, which comprises any molecular entity that promotes the expression or activity enhancement of the functional fragment of the transcription factor, or comprises a delivery system that comprises a molecular entity that promotes the expression and activity enhancement of the functional fragment of the transcription factor or the functional fragment of the transcription factor, or comprises a functional neuron population after transdifferentiation, and other pharmacologic acceptable vectors.

[0131] The pharmaceutical composition of the present invention generally contains 10 10 to 10 13 PFU of AAV virus particles, preferably 10 11 to 10 13 PFU of AAV virus particles, and more preferably 10 10 to 10 12 PFU of AAV virus particles.

[0132] The pharmaceutical composition of the present invention generally contains 10 7 to 10 10 PFU of lentiviral particles, preferably 10 7 to 10 9 PFU of lentiviral particles, and more preferably 10 8 to 10 9 PFU of lentiviral particles.

[0133] The pharmaceutical composition of the present invention generally contains 10 8 to 10 11 PFU of adenovirus particles, preferably 10 8 to 10 10 PFU of adenovirus particles, and more preferably 10 9 to 10 10 PFU of adenovirus particles.

[0134] As used herein, the term "pharmaceutically acceptable vector" refers to a vector for administration of a therapeutic agent, including various excipients and diluents. It is not an essential active ingredient itself and is not excessively toxic when administered. Suitable vectors are well known to those skilled in the art. Pharmaceutically acceptable vectors in compositions include liquids such as water, saline, and buffers. In addition, auxiliary substances such as fillers, lubricants, flow aids, wetting or emulsifying agents, pH buffering substances, etc. may be present in these vectors. The vectors may also include cell transfection reagents.

[0135] Generally, the pharmaceutical composition of the present invention is obtained by mixing the above-mentioned expression vector with a pharma- ceutically acceptable vector.

[0136] The mode of administration of the compositions described herein is not particularly limited, and representative examples include, but are not limited to, intravenous injection, subcutaneous injection, intracerebral injection, intrathecal injection, spinal injection, and the like.

[0137] Therapeutic Applications The delivery system or functional nerve group according to the present invention, comprising any molecular entity that promotes the expression or enhanced activity of a functional fragment of the transcription factor, or comprising a functional fragment of the transcription factor or a molecular entity that promotes the expression and enhanced activity of a functional fragment of a transcription factor, can be used in a medicament prepared for repairing nerve damage or inhibiting the growth and progression of glioblastoma.

[0138] Main advantages of this invention The present invention may be applicable to different scenarios by innovatively obtaining a large number of transcription factors with reprogramming functions and exploring the transdifferentiation ability of transcription factors and their combinations. For example, for repairing nerve damage, transcription factors and combinations of transcription factors with medium to high efficiency can be selectively used depending on the damage. Meanwhile, in glioblastoma, transcription factors and combinations of transcription factors with higher conversion efficiency are required to rapidly downregulate the malignancy of glioblastoma.

[0139] The present invention further improves the expression elements of transcription factors used in gene therapy, glue The combination of transcription factors used in the present invention significantly improves the efficiency of promoting the transdifferentiation of human glioma cells into neurons. In particular, the combination of transcription factors used in the present invention can induce the transdifferentiation of human glioma cells into neurons, and can cause glioma cells to exit the cell cycle and cease to proliferate. In a glioma model, the injection of an adeno-associated virus containing the combination of transcription factors can significantly reduce tumor size and extend the survival time of the animals.

[0140] The main advantages of the present invention compared to the prior art are: (1) There is no potential risk of tumorigenicity or immunogenicity; (2) glue Passively absorbed by the cells or transformed by the delivery system, and is not affected by the blood-brain barrier; (3) It has a higher conversion efficiency than existing transcription factors or combinations of transcription factors, and has the potential for clinical application.

[0141] The present invention will be further described below in relation to specific examples. It should be understood that these examples are only used to explain the present invention and do not limit the scope of the present invention. In the following embodiments, the experimental methods for which no specific conditions are shown generally follow the conditions described in Molecular Cloning: A Laboratory Manual (Sambrook et al., New York: Cold Spring Harbor Laboratory Press, 1989) or those recommended by the manufacturer. Percentages and quantity ratios are weight percentages and weight ratios unless otherwise specified.

[0142] Materials and Methods Amino acid sequence SEQ ID NO:1 (hNeuroD1 amino acid sequence) MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLETMNAEEDSLRNGGEEEDEDEDEDLEEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQ PTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD

[0143] SEQ ID NO: 2 (mNeuroD1 amino acid sequence) MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDELEAMNAEEDSLRNGGEEEEEDEDLEEEEEEEEEEDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQ PTTNLVAGCLQLNPRTFLPEQNPDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGPQSHGSIFSSGAAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD

[0144] SEQ ID NO:3 (hNeuroD1 nucleotide sequence)

[0145] SEQ ID NO:4 (mNeuroD1 nucleotide sequence)

[0146] SEQ ID NO:5 (amino acid sequence of hBrn2) MATAASNHYSLLTSSASIVHAEPPGGMQQGAGGYREAQSLVQGDYGALQSNGHPLSHAHQWITALSHGGGGGGGGGGGGGGGGGGGDGSPWSTSPLGQPDIKPSVVVQ QGGRGDELHGPGALQQQHQQQQQQQQQQQQQQQQQQQQRPPHLVHHAANHHPGPGAWRSAAAAAHLPPSMGASNGGLLYSQPSFTVNGMLGAGGQPAGLHHHGLRDAHDE PHHADHHPHPHSHPHQQPPPPPPPQGPPGHPGAHHDPHSDEDTPTSDDLEQFAKQFKQRRIKLGFTQADVGLALGTLYGNVFSQTTICRFEALQLSFKNMCKLKPLLNKWL EEADSSSGSPTSIDKIAAQGRKRKKRTSIEVSVKGALESHFLKCPKPSAQEITSLADSLQLEKEVVRVWFCNRRQKEKRMTPPGGTLPGAEDVYGGSRDTTPPHHGVQTPVQ

[0147] SEQ ID NO:6 (mBrn2 amino acid sequence) MATAASNHYSLLTSSASIVHAEPPGGMQQGAGGYREAQSLVQGDYGALQSNGHPLSHAHQWITALSHGGGGGGGGGGGGGGGGGGGDGSPWSTSPLGQPDIKPSVVVQQ GGRGDELHGPGALQQQHQQQQQQQQQQQQQQQQQQQQQRPPHLVHHAANHHPGPGAWRSAAAAAHLPPSMGASNGGLLYSQPSFTVNGMLGAGGQPAGLHHHGLRDAHD EPHHADHHPHPHSHPHQQPPPPPPPQGPPGHPGAHHDPHSDEDTPTSDDLEQFAKQFKQRRIKLGFTQADVGLALGTLYGNVFSQTTICRFEALQLSFKNMCKLKPLLNKW LEEADSSGSPTSIDKIAAQGRKRKKRTSIEVSVKGALESHFLKCPKPSAQEITSLADSLQLEKEVVRVWFCNRRQKEKRMTPPGGTLPGAEDVYGGSRDTPPHHGVQTPVQ

[0148] SEQ ID NO:7 (hBrn2 nucleotide sequence)

[0149] SEQ ID NO:8 (mBrn2 nucleotide sequence)

[0150] SEQ ID NO:9 (hAscl1 amino acid sequence) MESSAKMESGGAGQQPQPQPQQPFLPPAACFFATAAAAAAAAAAAAAQSAQQQQQQQQQQAPQLRPAADGQPSGGGHKSAPKQVKRQRSSSPELMRCKRRLNFSGFGYSLPQQQPA AVARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQAGVLSPTISPNYSNDLNSMAGSPVSSYSSDEGSYDPLSPEEQELLDFTNWF

[0151] SEQ ID NO: 10 (mAscl1 amino acid sequence) MESSGKMESGAGQQPQPPQPFLPPAACFFATAAAAAAAAAAAAQSAQQQPQAPPQQAPQLSPVADSQPSGGGHKSAAKQVKRQRSSSPELMRCKRRLNFSGFGYSLPQQQPAAV ARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQAGVLSPTISPNYSNDLNSMAGSPVSSYSSDEGSYDPLSPEEQELLDFTNWF

[0152] SEQ ID NO:11 (hAscl1 nucleotide sequence) atggaaagctctgccaagatggagagcggcggcgccggccagcagccccagccgcagccccagcagcccttcctgccgcccgcagcctgtttctttgccacggccgcagccgcggcggccgcagccgccgcagcggcagcgcagagcgcgcagcagcagcagcagcagcagcagcag cagcagcaggcgccgcagctgagaccggcggccgacggccagccctcaggggcggtcacaagtcagcgcccaagcaagtcaagcgacagcgctcgtcttcgcccgaactgatgcgctgcaaacgccggctcaacttcagcggctttggctacagcctgccgcagcagcagccggccg ccgtggcgcgccgcaacgagcgcgagcgcaaccgctcaagttggtcaacctgggctttgccacccttcgggagcacgtccccaacggcgcggccaacaagaagatgagtaaggtggagacactgcgctcggcggtcgagtacatccgcgcgctgcagcagctgctggacgagcatga cgcggtgagcgccgcccttccaggcaggcgtcctgtcgccccaccatctcccccaactactccaacgacttgaactccatggccggctcgccggtctcatcctactcgtcggacgagggctcttacgacccgctcagccccgaggacaggagcttctcgacttcaccaactggttctga

[0153] SEQ ID NO:12(mAscl1 nucleotide sequence) atggagagctctggcaagatggagagtggagccggccagcagccgcagcccccgcagcccttcctgcctcccgcagcctgcttctttgcgaccgcggcggcggcggcagcggcggcggccgcggcagctcagagcgcgcagcagcaacagccgcaggcgccgccgcagcaggcg ccgcagctgagcccggtggccgacagccagccctcagggggcggtcacaagtcagcggccaagcaggtcaagcgccagcgctcgtcctctccggaactgatgcgctgcaaacgccggctcaacttcagcggcttcggctacagcctgccacagcagcagccggccgccgtggcg cgccgcaacgagcgcgagcgcaaccgggtcaagttggtcaacctgggttttgccaccctccgggagcatgtccccaacggcgcggccaacaagaagatgagcaaggtggagacgctgcgctcggcggtcgagtacatccgcgcgctgcagcagctgctggacgagcacgacgcg gtgagcgctgcctttcaggcgggcgtcctgtcgcccaccatctcccccaactactccaacgacttgaactctatggcgggttctccggtctcgtcctactcctccgacgagggatcctacgaccctcttagcccagaggaacaagagctgctggactttaccaactggttctga

[0154] sequence number:13(hNgn2 amino acid sequence) MFVKSETLELKEEEDVLVLLGSASPALAALTPLSSSADEEEEEEEPGASGGARRQRGAEAGQGARGGVAAGAEGCRPARLLGLVHDCKRRPSRARAVSRGAKTAETVQRIKKTRRLKANNRERNRMHNLNAALDALR EVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHCGGGGGGLPGALFSEAVLLSPGGASAALSSSGDSPSPASTWSCTNSPAPSSSVSSNSTSPYSCTLSPASPAGSDMDYWQPPPPDKHRYAPHLPIARDCI

[0155] SEQ ID NO: 14 (mNgn2 amino acid sequence) MFVKSETLELKEEEEVLMLLGSASPASATLTPMSSSADEEEDEELRRPGSARGQRGAEAGQGVQGSPASGAGGCRPGRLLGLMHECKRRPSRSRAVSRGAKTAETVQRIKKTRRLKANNRERNRMHNLNAA LDALREVLPTFPEDAKLTKIETLRFAHNYIWALTETLRLADHCAGAGGLQGALFTEAVLLSPGAALGASGDSPSPPSSWSCTNSPASSSNSTSPYSCTLSPASPGSDVDYWQPPPEKHRYAPHLPLARDCI

[0156] SEQ ID NO:15 (hNgn2 nucleotide sequence) atgttcgtcaaatccgagaccttggagttgaaggaggaagaggacgtgttagtgctgctcggatcggcctccccgccttggcggccctgaccccgctgtcatccagcgccgacgaagaagaggaggaggccgggcgcgtcaggcggggcgcgtcggcagcgcggggctgaggccgggcaggggcgcgggcggcgtggct gcgggtgcggagggctgccggcccgcacggctgctgggtctggtacacgattgcaaacggcgcccttcccgggcggggccgtctcccgaggcgccaagacggccgagacggtgcagcgcatcaagaagacccgtagactgaaggccaacaaccgcgagcgaaacgcatgcacaacctcaacgcggcactggacggctcgcg aggtgctccccacgttccccgaggacgccaagctcaccaagatcgagaccctgcgcttcgcccacaactacatctgggcactcaccgagaccctgcgcctggcggatcactgcgggggcggcggcggggcctgccggggggcgctcttctccgaggcagtgttgctgagcccgggaggagccagcgccgccctgagcagcagcgg agacagcccctcgcccgcctccaccgtggagttgcaccaacagccccgcgccgtcctcctccgtgtcctccaattccacctccccctacagctgcactttatcgcccgccagcccggccgggtcagacatggactattggcagcccccacctccgcaagcaccgctatgcacctcacctccccatagccagggattgtatctag

[0157] SEQ ID NO:16(mNgn2 nucleotide sequence) atgttcgtcaaatctgagactctggagttgaaggaggaagaggaggtactgatgctgctgggctcggcttccccggcctcggcgaccctgaccccgatgtcctccagcgcggacgaggagaggacgaggagctcgcccggccgggctccgcgcgtgggcagcgtggagcggaagccgggcagggggtgcagggcagt ccggcgtcgggtgccggggggttgccggccagggcggctgctgggcctgatgcacgagtgcaagcgtcgcccgtcgcctcacgggccgtctcccgaggtgccaagacggcggagacggtgcagcgcatcaagaagacccgcaggctcaaggccaacaaccggagcgcaaccgcatgcacaacctaaacgccgcgctg gacgcgctgcgcgaggtgctgcccaccttccccgaggatgccaagctcacgaagatcgagacgctgcttcgcccacaattacatctgggcgctcaccgagactctgcgcctggcggaccactgcgccggcgccggtggcctccaggggcgctcttcacggaggcggtgctcctgagcccggagctgcgctcggc gccagcggggacagcccttctccaccttctcctggagctgcaccaacagcccggcgtcatcctccaactccacgtccccatacagctgcactttatcgcccgctagccccgggtcagacgtggactactggcagcccccacctccgggagaagcatcgttatgcgcctcacctgcccctcgccaggactgtatctag

[0158] sequence number:17(hGsx1 amino acid sequence) MPRSFLVDSLVLREAGEKKAPEGSPPPLFPYAVPPPHALHGLSPGACHARKAGLLCVCPLCVTASQLHGPGPPALPLLKASFPPFGSQYCHAPLGRQHSAVSPGVAHGPAAAAAAALYQTSYPLPDPRQF HCISVDSSSNQLPSSKRMRTAFTSTQLLELEREFASNMYLSRLRRIEIATYLNLSEKQVKIWFQNRRVKHKKEGKGSNHRGGGGGGAGGGGSAPQGCKCASLSSAKCSEDDDELPMSPSSSGKDDRDLTVTP

[0159] SEQ ID NO: 18 (mGsx1 amino acid sequence) MPRSFLVDSLVLREASDKKAPEGSPPPLFPYAVPPPHALHGLSPGACHARKAGLLCVCPLCVTASQLHGPPGPPALPLLKASFPPFGSQYCHAPLGRQHSVSPGVAHGPAAAAAAAAALYQTSYPLPDPRQ FHCISVDSSSNQLPSSKRMRTATFTSTQLLELEREFASNMYLSRLRRIEIATYLNLSEKQVKIWFQNRRVKHKKEGKGSNHRGGAGAGAGGGAPQGCCKCSSLSSAKCSEDDDELPMSPSSSGKDDRDLTVTP

[0160] SEQ ID NO:19 (hGsx1 nucleotide sequence) atgccgcctccttcctggtggactcgctagtgctgcgcgaggcgggcgagaagaaggcgcccgagggcagcccgccgccgctcttcccctacgctgtgcccccgccgcacggctccacggtctctcgcctggcgcctgccacgcgcgcaaggctggggctgctgtgcgtgtgcccgctctgcgtcaccgctcgcag ctgcatgggccccccgggccgccgccgctgccctctactcaaggcttcttcccacccttcggctcgcagtactgccacgcgcccctgggccgccagcactctgctgtgtcgccccggggtcgctcacggcccggccgccgctgctgctgccgccgcgctctaccagacctctctacccgctgcctgaccccaggcagttcc actgcatctctgtggacagcagctctaaccagctgcccagcagcaagaggatgcgcacggctttcaccagcacgcagctgctaggctggagcggagttcgcttcttaatatgtacctgtcccgcctacgtcgcatcgagatcgcgacctacctgaatctgtccgagaagcaggtgaagatctggtttcagaaccgccg agtgaagcacaagaagggagcaagggcagcaaccatcgtggcggcggcggcgggggtgccggtggtggcgggagcgcaccgcaaggctgcaagtgcgcatcgctctcctcagccaagtgctccgaggatgacgacgaattgcccatgtctccgtcctctccagggaaggacgaccgggatcttacggtcactccctag

[0161] SEQ ID NO:20(mGsx1 nucleotide sequence) atgccgcgctccttcctggtggattcccttgtgctgcgggaagccagcgacaagaaggctccggagggcagcccgcaccgctcttcccctacgcggtcccgccgccgcacgcctccacggcctctcgccgggcgcctgccacgcgcgcaaggccggcttgctgtgcgtgtgtcccctctgtgtcaccgcttcgc agctgcacgggccccccgggccgccggcactgccgctactcaaggcgtccttccctcccttcggatcgcagtactgccacgcacccctgggccgccagcactccgtgtcccctggagtcgcccacggcccggctgcggccgcagcagctgctgcactctaccagacctctactacccgctgccggatcccagacagttt cactgcatctctgtggacagcagctcgaaccagctgcccagcagcaagaggatgcggacggcgttcaccagcacacagctcctggagctggagcgagagttcgcctccaacatgtacctctcccgcctgcggcgcatcgagatcgcgacctatctgaacctgtccgagaagcaggtgaagatctggtttcagaacc gccgggtgaagcacaagaaagaaggcaaaggcagtaaccaccgcggcggagctggggcggggccggcggggcgcaccgcaaggctgcaagtgctcttcgctctcctcagccaaatgctcagaggacgacgacgaattgcccatgtctccatcttcctccgggaaggatgacagagatctcacagtcactccgtag

[0162] sequence number:21(hTbr1 amino acid sequence) MQLEHCLSPSIMLSKKFLNVSSSYPHSGGSELVLHDHPIISTTDNLERSSPLKKITRGMTNQSDTDNFPDSKDSPGDVQRSKLSPVLDGVSELRHSFDGSAADRYLLSQSSQPQSAATAPSAMFPYPGQHGPAHPAFSIGSPSRYMAHHPVITNGAYNSLLSNSSPQGYP TAGYPYPQQYGHSYQGAPFYQFSSTQPGLVPGKAQVYLCNRPLWLKFHRHQTEMIITKQGRRMFPFLSFNISGLDPTAHYNIFVDVILADPNHWRFQGGKWVPCGKADTNVQGNRVYMHPDSPNTGAHWMRQEISFGKLKLTNNKGASNNNGQMVVLQSLHKYQPRLHVVE VNEDGTEDTSQPGRVQTFFTFPETQFIAVTAYQNTDITQLKIDHNPFAKGFRDNYDTIYTGCDMDRLTPSPNDSPRSQIVPGARYAMAGSFLQDQFVSNYAKARFHPGAGAGPGPGTDRSVPHTNGLLSPQQAEDPGAPSPQRWFVTPANNRLDFAASAYDTATDFAGNAA TLLSYAAAGVKALPLQAAGCTGRPLGYYADPSGWGARSPPQYCGTKSGSVLPCWPNSAAAAARMAGANPYLGEEAEGLAAERSPLPPGAAEDAKPKDLSDSSWIETPSSIKSIDSSDSGIYEQAKRRRISPADTPVSESSSPLKSEVLAQRDCEKNCAKDISGYYGFYSHS

[0163] SEQ ID NO: 22 (mTbr1 amino acid sequence) MQLEHCLSPSIMLSKKFLNVSSSYPHSGGSELVLHDHPIISTTDNLERSSPLKKITRGMTNQSDTDNFPDSKDSPGDVQRSKLSPVLDGVSELRHSFDGSAADRYLLSQSSQPQSAATAPSAMFPYPSQHGPAHPAFSIGSPSRYMAHHPVITNGAYNSLLSNSSPQGYP TAGYPYPQQYGHSYQGAPFYQFSSTQPGLVPGKAQVYLCNRPLWLKFHRHQTEMIITKQGRRMFPFLSFNISGLDPTAHYNIFVDVILADPNHWRFQGGKWVPCGKADTNVQGNRVYMHPDSPNTGAHWMRQEISFGKLKLTNNKGASNNNGQMVVLQSLHKYQPRLHVV EVNEDGTEDTSQPGRVQTFFTFPETQFIAVTAYQNTDITQLKIDHNPFAKGFRDNYDTIYTGCDMDRLTPSPNDSPRSQIVPGARYAMAGSFLQDQFVSNYAKARFHPGAGAGPGPGTDRSVPHTNGLLSPQQAEDPGAPSPQRWFVTPANNRLDFAASAYDTATDFAGNA ATLLSYAAAGVKALPLQAAGCTGRPLGYYADPSGWGARSPPQYCGAKSGSVLPCWPNSAAAAARMAGANPYLGEEAEGLAAERSPLAPAAEDAKPKDLSDSSWIETPSSIKSIDSSDSGIYEQAKRRRISPADTPVSESSSPLKSEVLAQRDCEKNCAKDIGGYYGFYSHS

[0164] SEQ ID NO:23 (hTbr1 nucleotide sequence)

[0165] SEQ ID NO:24 (mTbr1 nucleotide sequence)

[0166] SEQ ID NO: 25 (hDlx2 amino acid sequence) MTGVFDSLVADMHSTQIAASSTYHQHQQPPSGGGAGPGGNSSSSSSLHKPQESPTLPVSTATDSSYYTNQQHPAGGGGGGGSPYAHMGSYQYQASGLNNVPYSAKSSYDLGYTAAYTSYAPYGTSSSPANNEPEKEDLEPEIRIVNGKPKKVRKPRTIYSSFQL AALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNRRSKFKKMWKSGEIPSEQHPGASASPPCASPPVSAPASWDFGVPQRMAGGGGPGSGGAGSSGSSPSSAASAFLGNYPWYHQTSGSASHLQATAPLLHPTQTPQPHHHHHHHGGGAPVSAGTIF

[0167] SEQ ID NO: 26 (mDlx2 amino acid sequence) MTGVFDSLVADMHSTQITASSTYHQHQQPPSGAGAGPGGNSNSSSSNSSLHKPQESPTLPVSTATDSSYYTNQQHPAGGGGGASPYAHMGSYQYHASGLNNVSYSAKSSYDLGYTAAYTSYAPYGTSSSPVNNEPDKEDLEPEIRIVNGKPKKVRKPRTIYSSFQ LAALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNRRSKFKKMWKSGEIPTEQHPGASASPPCASPPVSAPASWDFGAPQRMAGGGPGSGGGAGSSGSSPSSAASAFLGNYPWYHQASGSASHLQATAPLLHPSQTPQAHHHHHHHHHAGGGAPVSAGTIF

[0168] SEQ ID NO:27 (hDlx2 nucleotide sequence) atgactggagtctttgacagtctagtggctgatatgcactcgacccagatcgccgcctccagcacgtaccaccagcaccagcagcccccgagcggcggcggcgccggcccgggtggcaacagcagcagcagcagcagcctccacaagccccaggagtcgcccacccttccggtgtccaccgccaccgacagcagctactacaccaaccagcagcacccggcgggcggcggcggcggcgggggctcgccctacgcgcacatgggttcctaccagtaccaagccagcggcctcaacaacgtcccttactccgccaagagcagctatgacctgggctacaccgccgcctacacctcctacgctccctatggaaccagttcgtccccagccaacaacgagcctgagaaggaggaccttgagcctgaaattcggatagtgaacgggaagccaaagaaagtccggaaaccccgcaccatctactccagtttccagctggcggctcttcagcggcgtttccaaaagactcaatacttggccttgccggagcgagccgagctggcggcctctctgggcctcacccagactcaggtcaaaatctggttccagaaccgccggtccaagttcaagaagatgtggaaaagtggtgagatcccctcggagcagcaccctggggccagcgcttctccaccttgtgcttcgccgccagtctcagcgccggcctcctgggactttggtgtgccgcagcggatggcgggcggcggtggtccgggcagtggcggcagcggcgccggcagctcgggctccagcccgagcagcgcggcctcggcttttctgggcaactacccctggtaccaccagacctcgggatccgcctcacacctgcaggccacggcgccgctgctgcaccccactcagaccccgcagccgcatcaccaccaccaccatcacggcggcgggggcgccccggtgagcgcggggacgattttctaa

[0169] SEQ ID NO:28 (mDlx2 nucleotide sequence) atgactggagtctttgacagtctggtggctgatatgcactcgacccagatcaccgcctccagcacgtaccaccagcaccagcagcccccgagcggtgcgggcgccggccctggcggcaacagcaacagcagcagcagcaacagcagcctgcacaagccccaggagtcgccaaccctcccggtgtccacggctacggacagcagctactacaccaaccagcagcacccggcgggcggcggcggcgggggggcctcgccctacgcgcacatgggctcctaccagtaccacgccagcggcctcaacaatgtctcctactccgccaaaagcagctacgacctgggctacaccgccgcgtacacctcctacgcgccctacggcaccagttcgtctccggtcaacaacgagccggacaaggaagaccttgagcctgaaatccgaatagtgaacgggaagccaaagaaagtccggaaaccacgcaccatctactccagtttccagctggcggcccttcaacgacgcttccagaagacccagtatctggccctgccagagcgagccgagctggcggcgtccctgggcctcacccaaactcaggtcaaaatctggttccagaaccgccgatccaagttcaagaagatgtggaaaagcggcgagatacccaccgagcagcaccctggagccagcgcttctcctccttgtgcctccccgccggtctcggcgccagcatcctgggacttcggcgcgccgcagcggatggctggcggcggcccgggcagcggaggcggcggtgcgggcagctctggctccagcccgagcagcgccgcctcggcctttctgggaaactacccgtggtaccaccaggcttcgggctccgcttcacacctgcaggccacagcgccacttctgcatccttcgcagactccgcaggcgcaccatcaccaccatcaccaccaccacgcaggcgggggcgccccggtgagcgcggggacgattttctaa

[0170] SEQ ID NO: 29 (hPtf1a amino acid sequence) MDAVLLEHFPGGLDAFPSSYFDEDDFFTDQSSRDPLEDGDELLADEQAEVEFLSHQLHEYCYRDGACLLLQPAPPAAPLALAPPSSGGGLGEPDDGGGGGYCCETGAPPGGFPYSPGSPPSCLAYPCAGAAVLSPGARLRGLSGAAAAAARRRRRVRSEAELQQL RQAANVERERRMQSINDAFEGLRSHIPTLPYEKRLSKVDTLRLAIGYINFLSELVQADLPLRGGGAGGCGGPGGGGRLGGDSPGSQAQKVIICHRGTRSPSPSDPDYGLPPLAGHSLSWTDEKQLKEQNIIRTAKVWTPEDPRKLNSKSSFNNIENEPPFEFVS

[0171] SEQ ID NO: 30 (mPtf1a amino acid sequence) MDAVLLEHFPGGLDTFPSPYFDEEDFFTDQSSRDPLEDSDELLGDEQAEVEFLSHQLHEYCYRDGACLLLQPAPSAAPHALAPPPLGDPGEPEDNVSYCCDAGAPLAAFPYSPGSPPSCLAYPCAAVLSPGARLGGLNGAAAAAAARRRRRVRSEAELQQLR QAANVRERRRMQSINDAFEGLRSHIPTLPYEKRLSKVDTLRLAIGYINFLSELVQADLPLRGSGAGGCGGPGGSRHLGEDSPGNQAQKVIICHRGTRSPSPSDPDYGLPPLAGHSLSWTDEKQLKEQNIIRTAKVWTPEDPRKLNSKSFDNIENEPPFEFVS

[0172] SEQ ID NO:31 (hPtf1a nucleotide sequence) atggacgcggtgttgctggagcacttccccgggggcctagacgcctttccttcttcgtacttcgacgaggacgacttcttcaccgaccagtcttcacgggaccccctggaggacggcgatgagctgctggcggacgagcaggccgaggtggagttccttagccaccagctccacgagtactgctaccgcgacggggcgtgcctgctgctgcagcccgcgcccccggccgccccgctagcgctcgccccgccgtcctcggggggcctcggtgagccagacgacggcggcggcggcggctactgctgcgagacgggggcgcccccaggcggcttcccctactcgcccggctcgccgccctcgtgcctggcctacccgtgcgccggggcggcagtactgtctcccggggcgcggctgcgcggcctgagcggagcggcggctgcggcggcgcggcgccggcggcgggtgcgctccgaggcggagctgcagcagctgcggcaggcggccaacgtgcgcgagcggcggcgcatgcagtccatcaacgacgccttcgaggggctgcgctcgcacatccccacgctgccctacgagaagcgcctctccaaggtggacacgctgcgcctggccatcggctacatcaacttcctcagcgagctcgtgcaggccgacctgcccttgcgcggcggtggcgcgggcggctgcggggggccgggcggcggcgggcgcctgggcggggacagcccgggcagccaggcccagaaggtcatcatctgccatcggggcacccggtccccctcccccagcgaccctgattatggcctccctcccctagcaggacactctctctcatggactgatgaaaaacaactcaaggaacaaaatattatccgaacagccaaagtctggaccccagaggaccccagaaaactcaacagcaaatcttccttcaacaacatagaaaacgaaccaccatttgagtttgtgtcctga

[0173] SEQ ID NO:32 (mPtf1a nucleotide sequence) atggacgccgtactcctggagcacttccccgggggcctggacaccttcccatccccttactttgatgaggaagatttcttcaccgaccagtcctctcgggacccgctggaggacagcgacgagctgctgggggacgagcaagcagaagtagagttcctcagccaccagctacacgaatactgctaccgcgacggggcgtgcctgctgctgcaacccgcgccctcggccgccccgcacgcgctcgccccgccgcctttgggggatcctggcgagcccgaggacaacgtcagctattgctgcgatgcaggggctcctctcgctgccttcccctactcgcctggctcaccgccctcgtgcctcgcctacccgtgtgccgcggtgctgtcccccggtgcgcggctcggtggtttgaacggggctgcggcagcggcggcagcaaggcggcggcgacgcgtgcgctccgaggcggagctgcagcagctgcgacaagccgctaatgtgcgagagcggcgccgcatgcagtccatcaacgacgccttcgaggggctgcgttcgcacatccccacgctaccctacgaaaagcgcctctccaaagtagacacgctgcgcttggccataggctacattaacttcctcagcgagctggtgcaagccgacctgccgctgcgcgggagtggcgcaggtggttgcgggggcccaggtggcagccggcacctcggagaggacagtcccggtaaccaggcccagaaggttatcatctgccatcgaggcacccgttcaccctcccccagtgacccggattatggtctccctcctcttgcagggcactctctttcctggactgatgaaaaacagctcaaagaacaaaatatcatccgtacagctaaagtgtggaccccagaggaccccagaaaactcaacagtaaatctttcgacaacatagagaacgaaccaccctttgagtttgtgtcctga

[0174] SEQ ID NO: 33 (hPax6 amino acid sequence) MQNSHSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGARPCDISRILQVSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPEVVSKIAQYKRECPSIFAWEIRDR LLSEGVCTNDNIPSVSSINRVLRNLASEKQQMGADGMYDKLRMLNGQTGSWGTRPGWYPGTSVPGQPTQDGCQQQEGGGENTNSISSNGEDSDEAQMRLQLKRKLQ RNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQVWFSNRRAKWRREEKLRNQRRQASNTPSHIPISSSFSTSVYQPIPQPTTPVSSFTSGSMLG RTDTALTNTYSALPPMPSFTMANNLPMQPPVPSQTSSYSCMLPTSPSVNGRSYDTYTPPHMQTHMNSQPMGTSGTTSTGLISPGVSVPVQVPGSEPDMSQYWPRLQ

[0175] SEQ ID NO: 34 (mPax6 amino acid sequence) MQNSHSGVNQLGGVFVNGRPLPDSTRQKIVELAHSGARPCDISRILQTHADAKVQVLDNENVSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPEVVSKIAQYKRECP SIFAWEIRDRLLSEGVCTNDNIPSVSSINRVLRNLASEKQQMGADGMYDKLRMLNGQTGSWGTRPGWYPGTSVPGQPTQDGCQQQEGGGENTNSISSNGEDSDEAQMRL QLKRKLQRNRTSFTQEQIEALEKEFERTHYPDVFARERLAAKIDLPEARIQVWFSNRRAKWRREEKLRNQRRQASNTPSHIPISSSFSTSVYQPIPQPTTPVSSFTSGS MLGRTDTALTNTYSALPPMPSFTMANNLPMQPPVPSQTSSYSCMLPTSPSVNGRSYDTYTPPHMQTHMNSQPMGTSGTTSTGLISPGVSVPVQVPGSEPDMSQYWPRLQ

[0176] SEQ ID NO:35 (hPax6 nucleotide sequence)

[0177] SEQ ID NO:36 (mPax6 nucleotide sequence)

[0178] SEQ ID NO: 37 (hOtx2 amino acid sequence) MMSYLKQPPYAVNGLSLTTSGMDLLHPSVGYPATPRKQRRERTTFTRAQLDVLEALFAKTRYPDIFMREEVALKINLPESRVQVWFKNRRAKCRQQQQQQQNGGQNKVRPAKKKTSPAREVSSESGTSGQFTPPSSTSVPTIAS SSAPVSIWSPASISPLSDPLSTSSSCMQRSYPMTYTQASGYSQGYAGSTSYFGGMDCGSYLTPMHHQLPGPGATLSPMGTNAVTSHLNQSPASLSTQGYGASSLGFNSTTDCLDYKDQTASWKLNFNADCLDYKDQTSSWKFQVL

[0179] SEQ ID NO: 38 (mOtx2 amino acid sequence) MMSYLKQPPYAVNGLSLTTSGMDLLHPSVGYPGPWASCPAATPRKQRRERTTFTRAQLDVLEALFAKTRYPDIFMREEVALKINLPESRVQVWFKNRRAKCRQQQQQQQNGGQNKVRPAKKKSSPAREVSSESGTSGQFSPPSSTSVP TIASSSAPVSIWSPASISPLSDPLSTSSCMQRSYPMTYTQASGYSQGYAGSTSYFGGMDCGSYLTPMHHQLPGPGATLSPMGTNAVTSHLNQSPASLSTQGYGASSLGFNSTTDCLDYKDQTASWKLNFNADCLDYKDQTSSWKFQVL

[0180] SEQ ID NO:39 (hOtx2 nucleotide sequence) atgatgtcttatcttaagcaaccgccttacgcagtcaatgggctgagtctgaccacttcgggtatggacttgctgcacccctccgtgggctacccggccaccccccggaaacagcgccgggagaggacgacgttcactcgggcgcagctagatgtgctggaagcactgtttgccaagacccggtaccagacatcttcatgcgagagggtggcac tgaaaatcaacttgcccgagtcgagggtgcaggtatggtttaagaatcgaagagctaagtgccgccaacaacagcaacaacagcagaatggaggtcaaaacaaagtgagacctgccaaaaaagaagacatctccagctcgggaagttcagagagtggaacaagtggccaattcactccccctctagcacctcagtcccgaccattgccagcagc agtgctcctgtgtctatctggagcccagcttccatctccccactgtcagatcccttgtccacctcctcttcctgcatgcagaggtcctatcccatgacctatactcaggcttcaggttatagtcaaggatatgctggctcaacttcctactttggggggcatggactgtggatcatatttgaccctatgcatcaccagcttcccggaccaggggcca cactcagtcccatgggtaccaatgcagtcaccagccatctcaatcagtccccagcttctctttccacccagggatatggagcttcaagcttgggttttaactcaaccactgattgcttggattataaggaccaaactgcctcctggaagcttaacttcaatgctgactgcttggattataaaagatcagacatcctcgtggaaattccaggtttgtga

[0181] SEQ ID NO:40(mOtx2 nucleotide sequence) atgatgtcttatctaaagcaaccgccttacgcagtcaatgggctgagtctgaccacttcgggtatggacttgctgcatccctccgtgggctaccccgggccctgggcttcttgtcctgcagccaccccccggaaacagcgaagggagaggacgacatttactagggcacagctcgacgttctggaagctctgtttgccaagacccggtacccagacatcttcatgagggaagaggtggcactgaaaatcaacttgccagaatccagggtgcaggtatggtttaagaatcgaagagctaagtgccgccaacagcagcagcagcagcagaatggaggtcagaacaaagtgaggcctgccaagaagaagagctctccagctcgggaagtgagttcagagagtggaacaagtggccagttcagtcccccctctagtacctcagtcccaaccattgccagcagcagtgctccagtgtctatctggagcccagcgtccatctccccactgtctgaccccttgtccacttcctcctcctgcatgcagaggtcctatcccatgacctatactcaggcttcaggttatagtcaaggctatgctggctcaacttcctactttgggggcatggactgtggatcttatttgacccctatgcatcaccagcttcctggaccaggggccacactcagtcccatgggtaccaatgctgttaccagccatctcaatcagtccccagcttctctttccacccagggatatggagcttcaagcttgggttttaactcaaccactgattgcttggattataaggaccaaactgcctcttggaagcttaacttcaatgctgactgcttggattataaagatcagacgtcctcatggaaattccaggttttgtga

[0182] Accession number: 41 (SA-hAscl1 amino acid sequence) MESSAKMESGGAGQQPQPQPQQPFLPPAACFFATAAAAAAAAAAAAAQSAQQQQQQQQQQAPQLRPAADGQPSGGGHKSAPKQVKRQRSSAPELMRCKRRLNFSGFGYSLPQQQPA AVARRNERERNRVKLVNLGFATLREHVPNGAANKKMSKVETLRSAVEYIRALQQLLDEHDAVSAAFQAGVLAPTIAPNYSNDLNSMAGAPVSSYSSDEGSYDPLAPEEQELLDFTNWF

[0183] SEQ ID NO:42 (hGFP nucleotide sequence)

[0184] Accession number: 43 (shorthGFP nucleotide sequence) aacatatcctggtgtggagtaggggacgctgctctgacagaggctcgggggcctgagctggctctgtgagctggggaggaggcagacagccaggccttgtctgcaagcagacctggcagcattgggctggccgccccccagggcctcctcttcatgcccagtgaatgactcaccttggcacagacacaatgttcggggtgggcacagtgcctgcttcccgccgcaccccagcccccctcaaatgccttccgagaagcccattgagcagggggcttgcattgcaccccagcctgacagcctggcatcttgggataaaagcagcacagccccctaggggctgcccttgctgtgtggcgccaccggcggtggagaacaaggctctattcagcctgtgcccaggaaaggggatcaggggatgcccaggcatggacagtgggtggcagggggggagaggagggctgtctgcttcccagaagtccaaggacacaaatgggtgaggggagagctctccccatagctgggctgcggcccaaccccaccccctcaggctatgccagggggtgttgccaggggcacccgggcatcgccagtctagcccactccttcataaagccctcgcatcccaggagcgagcagagccagagcaggttggagaggagacgcatcacctccgctgctcgcgg

[0185] Accession number: 44 (VP16 amino acid sequence) MLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG

[0186] Accession number: 45 (VP16 nucleotide sequence) atgttggggattgagcagatgtttaccgatgcccttggaattgacgagtacggtggg

[0187] SEQ ID NO: 46 (SV40 nucleotide sequence) cgatggagcggagaatgggcggaactgggcggagttaggggcgggatgggcggagttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttctgcctgctg gggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacaccctaactgacacacattccacagc

[0188] Common methods Human glioma cell culture Human glioma cell lines U251 and U87 cells (purchased from the Shanghai Institute of Biological Sciences Cell Bank, Chinese Academy of Sciences) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin in an incubator at 37°C and 5% CO2. After infection with lentivirus, the culture medium was changed to induction medium (DMEM, 2% B-27, 1% PS) 12 hours later, and then changed again to neural culture medium DMEM / F-12, 2% B-27, 1% PS, 20ng / ml BDNF, and 20ng / ml GDNF 48 hours later. Half of the culture medium was then replaced every 3 days.

[0189] immunostaining For immunostaining of cultured cells, see "Direct conversion of fibroblasts to functional neurons by defined factors" (Vierbuchen, T. et al. Nature 463, 1035-1041 (2010)). For immunostaining of tissue sections, follow published methods. Primary antibodies used for immunostaining included: mouse anti-NeuN (Millipore, 1:100), rabbit anti-Dsred (Clontech, 1:500), mouse anti-Tuj1 (Covance, 1:500), mouse anti-Map2 (Sigma, 1:500), rabbit anti-GFP (Invitrogen, 1:1,000), chicken anti-GFP (Invitrogen, 1:1,000), rabbit anti-Synapsin I (Millipore, 1:1,000), rabbit anti-VGLUT1 (Synaptic Systems, 1:500), rabbit anti-Ki67 (1:200, RM-9106, Thermo Fisher Scientific), mouse anti-BrdU (1:200, B2531, Sigma). FITC-, Cy3-, and Cy5-conjugated secondary antibodies were purchased from Jackson Immunoresearch.

[0190] BrdU (5-bromodeoxyuridine) labeling and cell proliferation experiments Cultured human glioma cells were incubated with 10 mM BrdU (Sigma) for 2 h or continuously as required by the experiment, and BrdU color development was immunodetected with anti-BrdU antibody. Proliferating cells were detected with Ki67 antibody. Furthermore, cell numbers were counted and statistically analyzed in 24-well plates (5 × 104 cells / well) at different time points (days 0, 3, 7, 14, and 21) for evaluation of glioma cell proliferation assay.

[0191] glue In vitro cell differentiation model (1) Plasmid construction and viral infection In the FUGW-IRES-EGFP vector template (vector information see, "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)), the human NG2 promoter was cloned in place of the CAG promoter to generate the hNG2-transcription factor-IRES-EGFP lentiviral plasmid, and the polynucleotide fragment of the transcription factor was constructed in the lentiviral vector. For lentiviral packaging, see "Production and purification of lentiviral vectors" (Tiscornia, G., Singer, O. & Verma, IMNat. Protoc. 1, 241-245 (2006)). NG2 cells are plated and cultured for 24 h before addition of lentivirus, and 24 h post-infection, the medium is changed: DMEM / F12, B27, Glutamax, and penicillin / streptomycin. 6-7 days after infection, brain-derived neurotrophic factor (BDNF; PeproTech, 20 ng / ml) is added to the medium every 3 days.

[0192] (2) Transdifferentiation of NG2 cells into neurons The majority of cultured mouse NG2 cells are NG2 glueThe cells were immunopositive for the cytoplasmic marker NG2, and a small number of cells expressed the oligodendrocyte marker molecules O4 and CNPase, while the neuronal marker Tuj1 and the stem cell marker molecules Sox2 and Oct4 were undetectable. NG2 cells were transfected with hNG2 transcription factor-IRES-GFP lentivirus 10 days after transfection, and the neuronal morphology of NG2 cells and the neuronal marker molecule Tuj1 were examined. 21 days after lentivirus infection of NG2 cells, both mature neuronal markers NeuN and MAP2 were examined simultaneously, and the neuronal morphology and positive marker cells were recorded to be able to generate action potentials by electrophysiology. Recording spontaneous postsynaptic currents indicates that the neurons can form functional synapses.

[0193] (3) NG2 cell-derived neurons can survive transplantation in vivo Whether the transdifferentiated neurons obtained by in vitro induction survive and function in vivo is crucial for their potential use in disease treatment. Two weeks after transplanting NG2 cell-induced neurons into the cerebral cortex, we performed immunohistochemistry experiments for transcription factors to detect whether the transplanted cells attached to the edge of the cerebral cortex and formed neurites extending deep into the cerebral cortex. At the same time, we performed immunofluorescence collocalization assays to confirm whether the transplanted cells expressed the neuronal markers Tuj1, NeuN, and MAP2.

[0194] In addition, other than the NG2 promoter glue Although there are some differences in the transformation efficiency, cell-specific promoters can exert similar transdifferentiation functions. For uniform screening, glue The in vitro cell differentiation transformation model is carried out using one type of vector and one type of promoter.

[0195] glue In vivo model of cell differentiation transformation (1) Construction of adeno-associated virus plasmid and virus infection The GFAP promoter was cloned into the vector template of AAV-FLEX-Arch-GFP (Addgene, #22222) to replace it with the CAG promoter, and the CMV transcription factor was retained. After replacing the GFP with the mCherry coding frame, the AAV-mCherry plasmid (control) was obtained. The AAV-mNeurog2 / mCherry plasmid was obtained by cloning the above transcription factor into the AAV-mCherry plasmid, and the target gene can be specifically introduced into astrocytes by the action of the GFAP promoter.

[0196] (2) Transdifferentiation of astrocytes into neurons We injected the virus AAV-mCherry or AAV-transcription factor / mCherry unilaterally into the parietal region of adult wild-type mice, after which brain tissue samples were harvested at different time points. On the 3rd and 30th days after virus injection, we observed whether mCherry colocalized with NeuN in mice administered with viral AAV-transcription factor / mCherry, but not with the control viral AAV-mCherry. To demonstrate that the induced neurons were functional active neurons, we simultaneously performed electrophysiological recordings in the midbrain infected with AAV virus. We recorded inward Na+ and outward K+ currents in voltage clamp mode, counted the ratio of recorded action potentials to postsynaptic currents, and determined whether the induced neurons had integrated into neural circuits and established synaptic connections by whether the postsynaptic current signal disappeared with the blocker NBQX and whether the postsynaptic current signal appeared after washing out, to determine whether the induced neurons were functional neurons.

[0197] The described AAV virus was referenced to the mouse brain atlas. After virus injection, the midbrain and spinal cord were collected at different time points for immunostaining or brain slice recording. The virus injection concentration, injection rate, and injection volume per injection in the intact and injured spinal cord were consistent with the brain region, and the spinal cord was injected at a 30° angle.

[0198] Nerve injury repair model (1) Spinal cord injury and viral transfection We established a mouse T8-T10 spinal cord complete transection model (see McDonough A, Monterrubio A, Ariza J, et al. Calibrated Forceps Model of Spinal Cord Compression Injury. Jove-Journal of. Visualized Experiments 2015.) and injected AAV-mCherry virus and AAV-transcription factor / mCherry into both sides of the spinal cord immediately after injury. Whether mCherry colocalized with NeuN was observed 3 days after virus injection and 30 days after virus injection.

[0199] (2) Spinal Cord Injury Repair Assay It is said that the loss of sensory afferents after thoracic spinal cord injury weakens the inhibitory effect of the descending inhibitory system in the brainstem, resulting in hypersensitivity of the tail to external stimuli. Therefore, using a tail flick experimental model, the tail reaction latency of two groups of mice was measured under thermal stimulation at 48°C and 52°C to measure the sensory ability of the mice. The motor function of the mice was scored according to the BMS criteria. The test method was based on "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.

[0200] Glioma Models The mice used for glioma model transplantation were 7-week-old NOD-scid mice. Human glioma cells induced for 3 days or uninduced were digested with 0.25% trypsin and concentrated to a density of approximately 2.5×105 cells / μl by centrifugation to remove the supernatant. 2 μl, i.e., 5×105 cells, were transplanted into each mouse brain striatum. Histochemistry was performed 3 weeks after transplantation or 1 week after virus injection, followed by immunohistochemistry.

[0201] Example 1 Functional fragments of a single transcription factor glue First, in vitro glue Preliminary screening was performed using a cell differentiation induction model. glue We obtained transcription factors that can induce differentiation of cells into neural cells. The coding sequences of the transcription factors used and their conversion efficiencies are shown in Table 1.

[0202] In vitro differentiation efficiency % = (number of virus-infected fluorescent expression positive cells that are positive for the neuronal marker Tuj1 and capable of electrophysiologically detecting spontaneous postsynaptic currents / total number of virus-infected fluorescent expression positive cells) x 100%, and an average of more than 100 Tuj1-positive transdifferentiated cells that were capable of detecting spontaneous postsynaptic currents were detected per transcription factor.

[0203] [Table 1]

[0204] Both human and mouse transcription factors were expressed in vitro. glue It has the ability to transdifferentiate cells into neural cells.

[0205] In further studies, human Ascl1 protein was targeted and five conserved serine-proline (SP) phosphorylation sites in its protein sequence (positions 93, 190, 194, 207, and 223 in the protein sequence) were mutated to alanine-proline (AP) (enhanced Ascl1 (SA-hAscl1), protein sequence SEQ ID NO: 41), further increasing the conversion efficiency to a level of 85.5%.

[0206] Example 2 Functional fragmentation of a single transcription factor in the dorsal midbrain glue Promotes cell transdifferentiation glue Based on an in vivo model of cellular transdifferentiation, the transcription factors screened in Example 1 were used to glue When we attempted to induce cells, we found that the transformation efficiency significantly differed depending on the transcription factor, as shown in the table below (Table 2).

[0207] In vivo transdifferentiation efficiency was characterized by the percentage of neuronal colocalization, defined as % in vivo transdifferentiation efficiency = (number of virally-infected fluorescent-positive cells positive for the neuronal marker NeuN and capable of electrophysiologically detecting spontaneous postsynaptic currents / total number of virally-infected fluorescent-positive cells) × 100%, and an average of 100 or more transdifferentiated cells positive for NeuN and capable of detecting spontaneous postsynaptic currents were tested per transcription factor.

[0208] [Table 2]

[0209] Based on this in vivo model, we further investigated the expression elements of the AAV expression vector in detail. glue We have made at least three technical improvements that can significantly improve cell transformation efficiency.

[0210] (1) Insertion of VP16 fusion protein VP16 is the activation domain (SEQ ID NO: 45) of the VP16 protein derived from Herpes simplex virus, and the gene sequence was cloned into the AAV-transcription factor / mCherry plasmid to obtain the AAV-VP16-transcription factor / mCherry plasmid. VP16 can be alone or in a chain. This plasmid translates the fusion protein VP16-transcription factor, enhancing the activation gene expression function of the transcription factor. AAV-VP16-transcription factor / mCherry induces nerve cells remarkably efficiently and quickly (see Table 3).

[0211] (2) Shortening the promoter When the human-derived hGFAP promoter 2.2 kb (SEQ ID NO: 42) was changed to 683 bp (SEQ ID NO: 43), the changed promoter did not affect the target astrocyte specificity, while improving the AAV packaging efficiency, reducing the empty shell rate of the virus packaging, and obtaining a higher purity AAV titer. During induction in vivo, Short-hGFAP-AAV-transcription factor / mCherry increased the virus transdifferentiation efficiency, reduced the number of cell deaths, and made the induction process safer (see Table 3).

[0212] (3) Insertion of a transcription factor Simian vacuolating virus 40 transcription factor (SEQ ID NO: 46) was inserted into the hGFAP-AAV-transcription factor / mCherry plasmid to obtain SV40-hGFAP-AAV-transcription factor / mCherry. The SV40 transcription factor can greatly enhance the activity of the hGFAP promoter, allowing the target gene to be expressed efficiently in vivo, improving the induction efficiency of neural cells (see Table 3).

[0213] The above three expression enhancement methods can be used alone, in combination, or all three can be used simultaneously. The transcription factors described in this example can all improve induction efficiency. Take human Ascl1 as an example, see Table 3.

[0214] [Table 3]

[0215] Similarly, for other transcription factors, any of the three technical solutions above can significantly improve the transduction efficiency or AAV titer, and Table 4 shows the average transduction efficiency of other transcription factors after using the above modification strategies.

[0216] [Table 4]

[0217] Example 3 Combination of functional fragments of transcription factors glue Further increasing the efficiency of cell transdifferentiation glue Based on the in vitro and in vivo transdifferentiation model of cells, and in conjunction with the vector modification strategy described in Example 2, we first randomly combined selected transcription factors into two-part combinations. Here, different transcription factors can be expressed simultaneously in the same vector or in different expression vectors, and the expression ratios listed in the table below are the molar concentration ratios of the functional proteins expressed in the actual study. Among the transcription factors NeuroD1, Brn2, Gsx1, Tbr1, Dlx2, Ptf1a, Pax6, and Otx2, we unexpectedly obtained several combinations of transcription factors that were not highly efficient alone, but could be synergistically and significantly more efficient when combined, and the closer the molar concentration ratio of the expressed functional proteins, the higher the conversion efficiency obtained (see Table 5).

[0218] [Table 5]

[0219] For example, NeuroD1 and Brn2 showed 42.30% and 8.70% transdifferentiation efficiency, respectively, when used alone (Table 1), and showed a synergistic transdifferentiation efficiency of 76.2% when used in combination (1:1). Similarly, Gsx1+Tbr1, Dlx2+Ptf1a, and Pax6+Otx2 also significantly improved the transdifferentiation efficiency in a synergistic manner.

[0220] In addition, Ascl1 and Ngn2 were found to be important transcription factors that significantly improve transcription efficiency and exert cumulative enhancement and synergistic functions when combined with the above-mentioned transcription factors or combinations of transcription factors (see Tables 6 and 7).

[0221] [Table 6]

[0222] [Table 7]

[0223] In the above test, both human and mouse transcription factors and their combinations glue It was confirmed that this improves the efficiency of differentiation of cells into neural cells.

[0224] Example 4 Application of transcription factors and their combinations in spinal cord injury repair Spinal cord injury (SCI) is a central nervous system disorder accompanied by the death of spinal cord neurons and the formation of glial scars. In vivo neuronal reprogramming, which converts astrocytes into neurons, has the potential to alleviate the damage caused by SCI and is therefore a promising new therapeutic approach.

[0225] According to the nerve injury repair model, the transcription factors or combinations of transcription factors described in Examples 1 to 3 with a conversion efficiency of 50% or more were found to be effective in repairing damaged areas. glueIt was found that these transcription factors reprogrammed neural cells to acquire electrophysiological properties and have the ability to receive external signal input. Based on a spinal cord injury assay model, it was found that these transcription factor reprogrammed neural cells contribute greatly to the recovery of sensory and motor functions of spinal cord injured mice, and it was found that the transcription factors or combinations thereof with a conversion efficiency of 75% or more described in Examples 1 to 3 are particularly preferable. Examples of preferable transcription factors and combinations thereof are shown in Table 8.

[0226] [Table 8]

[0227] Example 5 In vitro transdifferentiation of glioma cells into neurons by combination of functional fragments of transcription factors In addition, by obtaining the transcription factors and their combinations as in Examples 1 to 3, and considering the application of transcription factors or combinations of transcription factors with high conversion efficiency that can promote the conversion of glioma cells to nerve cells, a method for promoting the conversion of glioma cells to nerve cells and an in vivo or in vitro method for the same can be obtained. glue This was found to be similar to the cell differentiation transdifferentiation model, and was carried out as follows, taking the combination of NeuroD1 and Brn2 factors as an example.

[0228] (1) Plasmid construction and virus infection In the vector template of FUGW-IRES-EGFP (for vector information see "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), a polynucleotide functional fragment is constructed to generate a lentiviral plasmid carrying the polynucleotide functional fragment. In one embodiment, a fragment from human NeuroD1 transcription factor (SEQ ID NO:3) is constructed on a lentiviral vector to generate hNeuroD1-IRES-EGFP lentiviral plasmid. Lentiviral packaging is described in "Production and purification of lentiviral vectors" (Tiscornia, G., Singer, O. & Verma, IM Nat. Protoc. 1, 241 -245 (2006)). -245 (2006)).

[0229] Human glioma cells were plated and cultured for 24 hours before addition of lentivirus, and 24 hours after infection, the medium was changed to DMEM / F12, B27, Glutamax, and penicillin / streptomycin. 6-7 days after infection, brain-derived neurotrophic factor (BDNF; PeproTech, 20 ng / ml) was added to the medium every 3 days.

[0230] (2) NeuroD1-mediated transdifferentiation of glioma cells into neurons After infecting cultured human glioma U251 cells with hNeuroD1-IRES-EGFP lentivirus for 14 days, we confirmed by cell immunofluorescence that some Tuj1-positive cells appeared and exhibited neuronal morphology (Figure 1B to D). This indicates that NeuroD1 alone can convert glioma cells into neurons, with a conversion efficiency of 5.1%.

[0231] (3) Co-expression of NeuroD1 and Brn2 improved the transdifferentiation efficiency of glioma cells into neurons. NeuroD1 alone was able to differentiate glioma cells into neurons, but the induction efficiency was not very high. In order to improve the induction efficiency, other transcription factors were tested, and it was found that the combination of NeuroD1 and Brn2 (SEQ ID NO: 7) was able to differentiate glioma cells U251 into neurons very efficiently, and the cells showed the morphology of mature neurons (Figure 1C, D), with a conversion efficiency of 58.3%. In addition, when another human glioma cell, U87, was also tested, NeuroD1 and Brn2 were also able to differentiate glioma cells U87 into neurons very efficiently, with a conversion efficiency of 61.5%.

[0232] (4) Molecular expression characteristics of neurons transdifferentiated from glioma cells Twenty-one days after infecting glioma cells U251 with the lentiviruses hNeuroD1-IRES-EGFP and hBrn2-IRES-EGFP, the induced neurons expressed the mature neuronal marker molecules MAP2 (Fig. 2A) and synapsin I (Fig. 2B-D), and the glutamatergic neuronal marker molecule VGLUT1 (Fig. 2E-H), indicating that the induced neurons were predominantly excitatory neurons.

[0233] (5) Electrophysiological properties of glioma cell-transdifferentiated neurons Twenty-eight days after infecting glioma cells U251 with lentiviruses hNeuroD1-IRES-EGFP and hBrn2-IRES-EGFP, electrophysiological recordings showed that the induced neurons were able to emit multiple action potentials (Figure 3A-B) and postsynaptic current signals were detected in the induced neurons (Figure 3C). However, after addition of blockers CNQX and AP5, the postsynaptic current signals disappeared, indicating that the induced neurons were able to receive synaptic signals and were functional neurons.

[0234] (6) Transdifferentiation by induction of NeuroD1 and Brn2 induces cell cycle exit in glioma cells Neurons are cells that have exited the cell cycle and stopped dividing, and if NeuroD1 and Brn2 can induce glioma cells to become neurons, it will lead to glioma cells exiting the cell cycle. To further support this, we performed BrdU labeling for 2 hours at different times (days 1, 3, and 5) after lentivirus infection, followed by immunocytochemical analysis (Figure 4A). The ratio of BrdU positive counts in glioma cells expressing NeuroD1 and Brn2 lentiviruses was dramatically reduced compared to the control group (Figure 4B), suggesting that reprogramming of neurons by NeuroD1 and Brn2 leads to the exit of the cell cycle in glioma cells. Furthermore, 5 days after lentivirus infection, BrdU labeling paper was administered continuously for 14 days, and immunocytochemical analysis was performed, and a significant decrease in the number of BrdU positive glioma cells was observed due to the expression of NeuroD1 and Brn2 lentiviruses (Figure 4C-E).

[0235] (7) Transdifferentiation by NeuroD1 and Brn2 suppresses proliferation of glioma cells Furthermore, immunofluorescence staining of Ki67, an endogenous molecular marker for proliferating cells, revealed that the number of Ki67-positive glioma cells expressing NeuroD1 and Brn2 lentiviruses was significantly reduced (Figure 5A and B). Quantitative statistics of the number of cells at different virus infection times showed that the proliferation of glioma cells infected with NeuroD1 and Brn2 lentiviruses reached a plateau on day 7 and no longer proliferated significantly (Figure 5C).

[0236] These results demonstrated that NeuroD1 and Brn2 could induce malignant, proliferating glioma cells into terminally differentiated neurons, enabling glioma cells to exit the cell cycle and stop proliferation / growth.

[0237] (8) Glioma cells expressing NeuroD1 and Brn2 exhibit significantly reduced tumorigenicity in vivo. NeuroD1 and Brn2 can induce in vitro cultured glioma cells to become neurons and can terminate the cell cycle of glioma cells, which may reduce the tumorigenicity of these induced glioma cells in vivo. Therefore, we performed an orthotopic tumor cell transplantation experiment to evaluate the size of tumor burden after 21 days by injecting human glioma U251 cells (5 × 105) infected with NeuroD1 and Brn2 lentiviruses for 3 days into the striatum of NOD-scid mice. As a result, glioma cells infected with NeuroD1 and Brn2 lentiviruses formed significantly smaller tumor tissues than controls, indicating that the tumorigenicity of these glioma cells was significantly reduced.

[0238] Next, we selected transcription factors or combinations of transcription factors with a transdifferentiation efficiency of 50% or more, and tested the transdifferentiation ability and the proliferation inhibition ability of these transcription factors in glioma cells. As a result, we found that the transcription factors or combinations of transcription factors with a transdifferentiation efficiency of 75% or more were glue It was found that the transcription factors are most effective in suppressing cell-derived tumors. Examples of preferred transcription factors and combinations thereof are shown in Table 9.

[0239] [Table 9]

[0240] Example 6: Inhibitory effect of in vivo reprogramming using a combination of functional fragments of transcription factors on the proliferation of brain glioma cells Based on Example 5, and taking the combination of NeuroD1 and Brn2 as an example, we attempted to verify the effects of transcription factors in a mouse transplanted tumor model as follows.

[0241] (1) Construction of AAV plasmids Using the template of the AAV-FLEX-Arch-GFP (Addgene, #22222) vector, a fragment derived from human NeuroD1 (SEQ ID NO: 3) was constructed to obtain AAV-hNeuroD1-P2A-GFP. P2A is a self-cleaving peptide that can efficiently co-express hNeuroD1 and GFP. By constructing a CDS (SEQ ID NO: 7) fragment derived from the human Brn2 gene into this vector, AAV-hBrn2-P2A-GFP was obtained.

[0242] (2) Inhibitory effect of AAV viral vectors on the proliferation of brain glioma cells using NeuroD1 and Brn2 To confirm whether reprogramming by induction of NeuroD1 and Brn2 has the ability to treat glioma cells, we first performed intracerebral transplantation of glioma cells (5 × 105 cells). Seven days after transplantation, AAV viral vectors of NeuroD1 and Brn2 were injected orthotopically. Thirty days after viral injection, immunohistochemical analysis showed that the virus-infected cells expressed the neural marker molecule Tuj1 and had neuronal morphology. This was accompanied by a significant reduction in tumor volume. More importantly, the survival time of mice injected with AAV viruses of NeuroD1 and Brn2 was significantly extended. In addition, other transcription factors or combinations of transcription factors with transdifferentiation efficiency of 75% or more have also been reported. glueIt was observed that the compound showed a significant tumor-suppressing effect in a tumor suppressor model.

[0243] (3) AAV viral vectors expressing NeuroD1 and Brn2 suppress tumor cell proliferation in an ectopic inoculation model of human glioma U87 BALB / CA-nu mice. Human U87 glioma cells were cultured and passaged twice, and then inoculated into the armpits of nude mice in the logarithmic growth phase. Tumor masses were removed from tumor-bearing mice under aseptic conditions, cut into uniformly sized rice grain-like pieces, and subcutaneously inoculated into the armpits of nude mice using a block inoculation needle. When the tumors reached approximately 100 mm3, the nude mice were randomly divided into groups, and administration began after grouping. All samples were dissolved in PBS, and the intratumoral injection volume was 50 μl / tumor. The length and width of the tumor mass were measured every 3 days, and the tumor volume was calculated using the following formula: Tumor volume calculation formula Volume = (length x width2) / 2 The tumor inhibition rate was calculated using the following formula. Tumor inhibition rate% = (V model group - V administration group) / V model group × 100% The animals were then sacrificed, and the tumor mass was measured and biochemical and molecular tests were performed. The results showed that the expression of AAV-mediated reprogramming factors significantly reduced the tumor volume (Figure 6A). Real-time PCR analysis of tumor samples revealed that the cells in the experimental group expressed the early neural marker molecule DCX (Figure 6B), indicating that glioma cells in the animals were induced to become neural cells, resulting in tumor growth inhibition.

[0244] Example 7 Application of other delivery systems in the delivery of functional fragments of transcription factors In addition to the lentivirus vectors and adeno-associated virus vectors described in the above examples, other types of delivery systems can achieve similar functions. In this example, we found that the type 5 adenovirus vector expressing NeuroD1 and Brn2 also suppressed tumor cell growth in an ectopic inoculation model of human glioma U87 BALB / CA-nu mice.

[0245] Because adeno-associated virus does not replicate autonomously in vivo, we designed a type 5 adenovirus vector that efficiently and rapidly expresses reprogramming factors, and by utilizing the specificity of type 5 adenovirus to proliferate in tumor cells, we realized in vivo transdifferentiation therapy to suppress glioma recurrence.

[0246] A fragment derived from human NeuroD1 (SEQ ID NO: 3) and a fragment of the CDS of human Brn2 gene (SEQ ID NO: 7) were constructed in an Adeno-Cas9 (Addgene, #64072) vector template via AgeI / SpeI double cleavage sites to obtain Ad5-hNeuroD1-P2A-hBrn2 (Ad5-AN). P2A is a self-shearing polypeptide, which achieved efficient co-expression of hNeuroD1 and hBrn2.

[0247] Using an ectopic inoculation model of human glioma U87 BALB / CA-nu mice, cultured human glioma U87 cells were inoculated into the armpits of nude mice in the logarithmic growth phase, and when the tumor size grew to approximately 100 mm3, nude mice with appropriate tumor volume were selected and randomly grouped, and treatment was started after grouping. The control group was the PBS group, the Ad5-AN-low group was administered 3×108 PFU, and the Ad5-vector-high group was administered 1×109 PFU once every two days for five consecutive doses. Tumor volumes were measured and calculated every three days, and at the later stage, animals were sacrificed and tumor volumes were weighed for biochemical and molecular biological detection.

[0248] As a result, the tumor volume was reduced by 32.25% in the Ad5-AN-low group and 67.49% in the Ad5-AN-high group compared to the control PBS group (Figure 7A), confirming that tumor cell proliferation was significantly suppressed by glioma reprogramming. HE staining also confirmed the suppression of glioma proliferation (Figure 7B). These results suggest that in vivo glioma transdifferentiation mediated by reprogramming factors leads to the suppression of tumor cell proliferation.

[0249] Exosomes derived from mesenchymal microcells or glioblastoma cells (GBM-Exo) were also used. The exosomes were extracted from cell culture supernatants by density gradient centrifugation and molecular exclusion, and the expression of the exosome marker protein CD63 was confirmed by Western blot. The shape characteristics and particle size of exosomes were examined by transmission electron microscopy and dynamic light scattering, and the exosome concentration was measured by BCA protein quantification.

[0250] For glioblastoma, Ascl1-mRNA (NCBI Reference Sequence: NM_004316.4) or other combinations of transcription factors described herein were introduced into exosomes by endogenous expression or exogenous introduction. Glioblastoma-derived exosome drugs specifically infect human glioblastoma cell lines U251 and U87, and it can be observed that the induction efficiency of human glioblastoma cell lines U251 and U87 into neural cells varies with the concentration gradient of the exosome drug during the logarithmic growth phase of the cells, and at the same time, the proliferation rate of the associated tumor cells is proportional to the efficiency of neural cell induction.

[0251] All documents mentioned in this application are incorporated by reference in the same manner as if each document was individually incorporated. Furthermore, it will be understood that after reading the above teachings of the present invention, various changes or modifications may be made to the present invention by those skilled in the art, which are equivalent and also fall within the scope defined by the claims appended hereto.

Claims

1. A set of functional fragments that synergistically promote transdifferentiation of glial cells, The functional fragment comprises at least two functional fragments that promote expression of a transcription factor; The functional fragment is (1) A combination of a functional fragment that promotes the expression of Ascl1 and a functional fragment that promotes the expression of a transcription factor selected from NeuroD1, Brn2, Ngn2, Gsx1, Tbr1, Dlx2, or Ptf1a; (2) a combination of a functional fragment that promotes the expression of Ngn2 and a functional fragment that promotes the expression of a transcription factor selected from NeuroD1, Brn2, or Ascl1; (3) A combination of functional fragments that promotes the expression of Ascl1, NeuroD1, and Brn2 transcription factors; (4) A combination of functional fragments that promotes the expression of Ascl1, Gsx1, and Tbr1 transcription factors; (5) A combination of functional fragments that promotes the expression of Ascl1, Dlx2, and Ptf1a transcription factors; (6) A combination of functional fragments that promotes the expression of Ascl1, Pax6, and Otx2 transcription factors; (7) A combination of functional fragments that promotes expression of Ngn2, NeuroD1, and Brn2 transcription factors; (8) A combination of functional fragments that promotes the expression of Ngn2, Gsx1, and Tbr1 transcription factors; (9) A combination of functional fragments that promotes the expression of Ngn2, Dlx2, and Ptf1a transcription factors; (10) A combination of functional fragments that promotes the expression of Ngn2, Pax6, and Otx2 transcription factors; is selected from the functional fragment is selected from a functional protein of a transcription factor having 99% or more sequence identity to SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, and / or 41, or selected from a polynucleotide encoding a transcription factor having 95% or more sequence identity to SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, and / or 40; The functional fragment that promotes expression of the transcription factor is contacted directly with glial cells or introduced via a delivery system, increases the promotion of expression of the transcription factor associated with glial cells and exhibits characteristics of functional neuronal or neuroid cells; The expression systems of the functional fragments that promote the expression of the transcription factor may be constructed under the same expression vector, or may be expressed separately using different expression vectors; (1) When any two functional fragments that promote the expression of a transcription factor are present, the molar concentration ratio of the expression levels of the two transcription factors is 4:1 to 1:4; or (2) when there are two or more functional fragments that promote the expression of a transcription factor, and one of the functional fragments that promote the expression of a transcription factor is a functional fragment that promotes the expression of a transcription factor Ascl1 or Ngn2, the molar concentration ratio of the expression amount of Ascl1 or Ngn2 is 20% or more; A set of functional fragments that synergistically promote the transdifferentiation of glial cells, characterized in that "transdifferentiation" refers to the transdifferentiation or reprogramming of glial cells into functional neuronal cells.

2. A set of functional fragments that synergistically promote transdifferentiation of glial cells, comprising: The functional fragment comprises at least two functional fragments that promote expression of a transcription factor; The functional fragment is (1) A combination of a functional fragment that promotes the expression of Ascl1 and a functional fragment that promotes the expression of a transcription factor selected from NeuroD1, Brn2, Ngn2, Gsx1, Tbr1, Dlx2, or Ptf1a; (2) a combination of a functional fragment that promotes the expression of Ngn2 and a functional fragment that promotes the expression of a transcription factor selected from NeuroD1, Brn2, or Ascl1; (3) A combination of functional fragments that promotes the expression of Ascl1, NeuroD1, and Brn2 transcription factors; (4) A combination of functional fragments that promotes the expression of Ascl1, Gsx1, and Tbr1 transcription factors; (5) A combination of functional fragments that promotes the expression of Ascl1, Dlx2, and Ptf1a transcription factors; (6) A combination of functional fragments that promotes the expression of Ascl1, Pax6, and Otx2 transcription factors; (7) A combination of functional fragments that promotes expression of Ngn2, NeuroD1, and Brn2 transcription factors; (8) A combination of functional fragments that promotes the expression of Ngn2, Gsx1, and Tbr1 transcription factors; (9) A combination of functional fragments that promotes the expression of Ngn2, Dlx2, and Ptf1a transcription factors; (10) A combination of functional fragments that promotes the expression of Ngn2, Pax6, and Otx2 transcription factors; is selected from the functional fragment is selected from a functional protein of a transcription factor having 99% or more sequence identity to SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, and / or 41, or selected from a polynucleotide encoding a transcription factor having 95% or more sequence identity to SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, and / or 40; The Ascl1 is an enhanced Ascl1 having the amino acid sequence set forth in SEQ ID NO: 41; A set of functional fragments that synergistically promote the transdifferentiation of glial cells, characterized in that "transdifferentiation" refers to the transdifferentiation or reprogramming of glial cells into functional neuronal cells.

3. A set of functional fragments that synergistically promote transdifferentiation of glial cells as described in claim 1, characterized in that the functional fragment is a polynucleotide encoding the transcription factor, or a small molecule drug, large molecule drug, or nucleic acid drug that promotes expression of a functional protein, polypeptide, or transcription factor after translation of the polynucleotide, or a polynucleotide or functional protein, polypeptide, small molecule drug, or large molecule drug located upstream of the transcription factor that regulates increased expression of the transcription factor.

4. A set of functional fragments that synergistically promote transdifferentiation of glial cells, comprising: The functional fragment comprises at least two functional fragments that promote expression of a transcription factor; The functional fragment is (1) A combination of a functional fragment that promotes the expression of Ascl1 and a functional fragment that promotes the expression of a transcription factor selected from NeuroD1, Brn2, Ngn2, Gsx1, Tbr1, Dlx2, or Ptf1a; (2) a combination of a functional fragment that promotes the expression of Ngn2 and a functional fragment that promotes the expression of a transcription factor selected from NeuroD1, Brn2, or Ascl1; (3) A combination of functional fragments that promotes the expression of Ascl1, NeuroD1, and Brn2 transcription factors; (4) A combination of functional fragments that promotes the expression of Ascl1, Gsx1, and Tbr1 transcription factors; (5) A combination of functional fragments that promotes the expression of Ascl1, Dlx2, and Ptf1a transcription factors; (6) A combination of functional fragments that promotes the expression of Ascl1, Pax6, and Otx2 transcription factors; (7) A combination of functional fragments that promotes expression of Ngn2, NeuroD1, and Brn2 transcription factors; (8) A combination of functional fragments that promotes the expression of Ngn2, Gsx1, and Tbr1 transcription factors; (9) A combination of functional fragments that promotes the expression of Ngn2, Dlx2, and Ptf1a transcription factors; (10) A combination of functional fragments that promotes the expression of Ngn2, Pax6, and Otx2 transcription factors; is selected from The functional fragment is selected from a functional protein of a transcription factor having 99% or more sequence identity to SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, and / or 41, or selected from a polynucleotide encoding a transcription factor having 95% or more sequence identity to SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, and / or 40; (1) When any two functional fragments that promote the expression of a transcription factor are present, the molar concentration ratio of the expression levels of the two transcription factors is 4:1 to 1:4; or (2) when there are two or more functional fragments that promote the expression of a transcription factor, and one of the functional fragments that promote the expression of a transcription factor is a functional fragment that promotes the expression of a transcription factor Ascl1 or Ngn2, the molar concentration ratio of the expression amount of Ascl1 or Ngn2 is 20% or more; A set of functional fragments that synergistically promote the transdifferentiation of glial cells, characterized in that "transdifferentiation" refers to the transdifferentiation or reprogramming of glial cells into functional neuronal cells.

5. A set of functional fragments that synergistically promote transdifferentiation of glial cells, comprising: The functional fragment comprises at least two functional fragments that promote expression of a transcription factor; The functional fragment is (1) A combination of functional fragments that promotes the expression of Ascl1, NeuroD1, and Brn2 transcription factors; (2) combinations of functional fragments that promote expression of Ascl1, Gsx1, and Tbr1 transcription factors; (3) A combination of functional fragments that promotes the expression of Ascl1, Dlx2, and Ptf1a transcription factors; (4) A combination of functional fragments that promotes the expression of Ascl1, Pax6, and Otx2 transcription factors; (5) A combination of functional fragments that promotes expression of the transcription factors Ngn2, NeuroD1, and Brn2; (6) A combination of functional fragments that promotes the expression of Ngn2, Gsx1, and Tbr1 transcription factors; (7) A combination of functional fragments that promotes the expression of Ngn2, Dlx2, and Ptf1a transcription factors; (8) Combination of functional fragments that promote expression of Ngn2, Pax6, and Otx2 transcription factors is selected from the molar concentration ratio of the expression levels of two transcription factors other than Ascl1 or Ngn2 is 4:1 to 1:4; The functional fragment is selected from a functional protein of a transcription factor having 99% or more sequence identity to SEQ ID NO: 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29, 30, 33, 34, 37, 38, and / or 41, or selected from a polynucleotide encoding a transcription factor having 95% or more sequence identity to SEQ ID NO: 3, 4, 7, 8, 11, 12, 15, 16, 19, 20, 23, 24, 27, 28, 31, 32, 35, 36, 39, and / or 40; A set of functional fragments that synergistically promote the transdifferentiation of glial cells, characterized in that "transdifferentiation" refers to the transdifferentiation or reprogramming of glial cells into functional neuronal cells.

6. The glial cells are selected from any of the following: astrocytes, NG2 glial cells, oligodendrocytes, microglia, or damaged glial cells, tumor cells of glial origin, etc.; or The delivery system is selected from an expression vector of a functional fragment that enhances expression of a transcription factor, a nanoparticle of a functional fragment that enhances expression of a transcription factor, an exosome of a functional fragment that enhances expression of a transcription factor, a viral or cellular vector of a functional fragment that enhances expression of a transcription factor, a targeted effector carrying a functional fragment of a transcription factor enhancing expression, or The set of functional fragments that synergistically promote transdifferentiation of glial cells described in claim 1, characterized in that the delivery system is equipped with a glial cell-specific promoter or expression regulatory element.

7. The expression systems of the functional fragments that promote the expression of the transcription factor may be constructed under the same expression vector, or may be expressed separately using different expression vectors; (1) When any two functional fragments that promote the expression of a transcription factor are present, the molar concentration ratio of the expression levels of the two transcription factors is 2:1 to 1:2; or (2) A set of functional fragments that synergistically promote transdifferentiation of glial cells as described in claim 1, characterized in that when there are two or more functional fragments that promote the expression of a transcription factor, and one of the functional fragments that promote the expression of a transcription factor is a functional fragment that promotes the expression of a transcription factor Ascl1 or Ngn2, the molar concentration ratio of the expression levels of Ascl1 or Ngn2 is 33% or more.

8. The expression systems of the functional fragments that promote the expression of the transcription factor may be constructed under the same expression vector, or may be expressed separately using different expression vectors; (1) When any two functional fragments that promote the expression of a transcription factor are present, the optimal molar ratio of the expression levels of the two transcription factors is 1:1; or (2) A set of functional fragments that synergistically promote transdifferentiation of glial cells as described in claim 1, characterized in that when there are two or more functional fragments that promote the expression of a transcription factor, and one of the functional fragments that promote the expression of a transcription factor is a functional fragment that promotes the expression of a transcription factor Ascl1 or Ngn2, the molar concentration ratio of the expression levels of Ascl1 or Ngn2 is 50% or more.

9. The functional fragment that promotes expression of the transcription factor is (1) A combination of functional fragments that promotes the expression of Ascl1, NeuroD1, and Brn2 transcription factors; (2) combinations of functional fragments that promote expression of Ascl1, Gsx1, and Tbr1 transcription factors; (3) A combination of functional fragments that promotes the expression of Ascl1, Dlx2, and Ptf1a transcription factors; (4) A combination of functional fragments that promotes the expression of Ascl1, Pax6, and Otx2 transcription factors; (5) A combination of functional fragments that promotes expression of the transcription factors Ngn2, NeuroD1, and Brn2; (6) A combination of functional fragments that promotes the expression of Ngn2, Gsx1, and Tbr1 transcription factors; (7) A combination of functional fragments that promotes the expression of Ngn2, Dlx2, and Ptf1a transcription factors; (8) Combination of functional fragments that promote expression of Ngn2, Pax6, and Otx2 transcription factors The present invention is characterized in that the user is allowed to select from the following: A set of functional fragments that synergistically promotes transdifferentiation of glial cells as described in claim 1, characterized in that the molar concentration ratio of the expression levels of two other transcription factors other than Ascl1 or Ngn2 is 4:1 to 1:

4.

10. A set of functional fragments that synergistically promote transdifferentiation of glial cells as described in claim 9, characterized in that the molar concentration ratio of the expression levels of two other transcription factors other than Ascl1 or Ngn2 is 2:1 to 1:

2.

11. A set of functional fragments that synergistically promote transdifferentiation of glial cells as described in claim 9, characterized in that the molar concentration ratio of expression levels of two other transcription factors other than Ascl1 or Ngn2 is approximately 1:

1.

12. A set of functional fragments that synergistically promote transdifferentiation of glial cells, comprising: The functional fragment comprises at least two functional fragments that promote expression of a transcription factor; The set of functional fragments comprises: (Z1) Ascl1+Ngn2, (Z2) Ngn2+NeuroD1, is selected from the group consisting of the functional fragment is selected from a functional protein of a transcription factor having 99% or more sequence identity to SEQ ID NO: 1, 2, 9, 10, 13, and / or 14, or selected from a polynucleotide encoding a transcription factor having 95% or more sequence identity to SEQ ID NO: 3, 4, 11, 12, 15, and / or 16; A set of functional fragments that synergistically promote the transdifferentiation of glial cells, characterized in that "transdifferentiation" refers to the transdifferentiation or reprogramming of glial cells into functional neuronal cells.

13. A use of a functional fragment that synergistically promotes transdifferentiation of glial cells according to any one of claims 1 to 12 in the preparation of a therapeutic drug for a nervous system disease, characterized in that the nervous system disease is a nervous system disease, injury, or glioma derived from glial cells.

14. (A) a set of functional fragments that synergistically promote transdifferentiation of glial cells according to any one of claims 1 to 12; (B) pharma- ceutically acceptable excipients; and A pharmaceutical composition comprising:

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