Pluripotent stem cell capable of stably secreting dopamine and use thereof

Abstract

The present invention relates to the field of biomedicine, and specifically provides a nucleic acid construct, an engineered pluripotent stem cell prepared from the nucleic acid construct, and a neural stem cell, astrocyte, mesenchymal stem cell, GABAergic nerve cell and DAergic nerve cell obtained by differentiation of the engineered pluripotent stem cell. Further provided is a use of the nucleic acid construct and the various cells in treatment of diseases associated with dopaminergic neuron degeneration and / or damage.

Description

A pluripotent stem cell capable of stably secreting dopamine and its use Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to a nucleic acid construct, an engineered pluripotent stem cell prepared from the nucleic acid construct, and neural stem cells, astrocytes, mesenchymal stem cells, GABAergic neurons, and DA neurons differentiated from the engineered pluripotent stem cells. The present application also relates to the use of the nucleic acid construct and various cells in treating diseases associated with dopaminergic neuron degeneration and / or damage. Background Art

[0002] Parkinson's disease (PD) is the second most common degenerative disorder of the central nervous system worldwide. It is caused by the gradual loss of dopaminergic (DA) neurons in the midbrain. Essentially, this is due to a decrease in dopamine neurotransmitter production by DA neurons, leading to a breakdown of the nigrostriatal dopamine circuit. Dopamine supplementation is an effective strategy for treating PD. Treatment of PD has long relied on oral levodopa, which is particularly effective in the early stages of Parkinson's disease. However, long-term systemic administration and the eventual death of dopamine neurons make oral levodopa ineffective or produce significant side effects.

[0003] Dopamine (DA) is synthesized from L-tyrosine in two steps: first, tyrosine hydroxylase (TH) hydroxylates L-tyrosine to L-DOPA (L-3,4-dihydroxyphenylalanine), which is then converted to DA by aromatic L-amino acid decarboxylase (AADC). TH requires tetrahydrobiopterin (BH4) as a cofactor for its catalytic activity, which is then catalyzed by GTP-cyclohydrolase-1 (GCH1) to generate GTP. Furthermore, human embryonic stem cells (hESCs), readily available, possess the capacity for self-renewal, unlimited proliferation, and the potential to differentiate into cells of all three germ layers, allow hESCs to differentiate into various cells, tissues, and even organs throughout the human body, thus holding enormous promise for their application in tissue engineering, cell therapy, and regenerative medicine.

[0004] Studies have shown that transplanting dopamine-related neural cells of fetal origin or differentiated from pluripotent stem cells into the brain can improve patients' disease symptoms. However, there are many types of cells among the cells from these sources, and only a portion are midbrain dopaminergic neurons that can secrete dopamine neurotransmitters. Other impure cells may cause various side effects to patients. For example, serotonin neurons can cause dyskinesia, and neural stem cells can overproliferate and cause compression of brain areas. In addition, there are ethical issues with fetal-derived cells; dopamine-related neural cells differentiated from pluripotent stem cells take a long time to exert their therapeutic effects after transplantation.

[0005] Therefore, there is a need to provide a genetically engineered human embryonic stem cell line that can stably express and secrete dopamine. This could be used as a seed cell to overcome the limitations of obtaining dopamine-secreting cells after induced differentiation of fetal-derived cells and pluripotent stem cells, thus facilitating subsequent clinical research and applications.

[0006] Summary of the Invention

[0007] The inventors of the present application have, through a large number of experiments and repeated explorations, obtained nucleic acid constructs that can express and secrete dopamine neurotransmitters in large quantities. Further, the engineered human embryonic stem cell system that is stable and secretes dopamine in large quantities, as well as neural stem cells (Neural stem cell, NSC), astrocytes (astrocyte) and mesenchymal stem cells (mesenchymal stem cell, MSC) obtained by differentiation of the engineered human embryonic stem cell system, GABA energy nerve cells, DA energy nerve cells. These cells can improve the behavioral ability of mammals suffering from Parkinson's disease in a short period of time (for example, 1-3 months). Therefore, the construct of the present application and the engineered human embryonic stem cell have greater potential in preventing and / or treating Parkinson's disease.

[0008] Nucleic acid constructs

[0009] Therefore, in the first aspect, the present application provides a nucleic acid construct, which comprises, from the 5' end to the 3' end, a nucleotide sequence encoding the following elements: a first promoter, an hGCH1 gene, a first self-cleaving polypeptide, an hTH gene, a second promoter, an hAADC gene, a second self-cleaving polypeptide and a screening gene.

[0010] In certain embodiments, the first promoter and the second promoter are each independently selected from a constitutive promoter, a specific promoter, and an inducible promoter.

[0011] In certain embodiments, the first promoter and the second promoter are constitutive promoters.

[0012] In certain embodiments, the first promoter and the second promoter are each independently selected from the group consisting of: beta-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α (EF1α), phosphoglycerate kinase (PGK), ubiquitin C, simian virus 40 early promoter (sv40), or any combination thereof.

[0013] In certain embodiments, the first promoter is EF1α. In certain embodiments, the sequence of the first promoter is shown in SEQ ID NO: 1.

[0014] In certain embodiments, the second promoter is PGK. In certain embodiments, the sequence of PGK is shown in SEQ ID NO:3.

[0015] In certain embodiments, the first self-cleaving polypeptide and the second self-cleaving polypeptide are each independently selected from P2A, T2A, E2A or F2A.

[0016] In certain embodiments, the first self-cleaving polypeptide is T2A. In certain embodiments, the sequence of the first self-cleaving polypeptide is shown in SEQ ID NO:18.

[0017] In certain embodiments, the second self-cleaving polypeptide is P2A. In certain embodiments, the sequence of the first self-cleaving polypeptide is shown in SEQ ID NO:19.

[0018] In certain embodiments, the selection gene is an antibiotic resistance gene.

[0019] In certain embodiments, the selection gene is selected from the group consisting of puromycin resistance gene (PuroR), ampicillin resistance gene (ampr), tetracycline resistance gene (tetr), chloramphenicol resistance gene (Cmr), kanamycin resistance gene (kanr), or any combination thereof.

[0020] In certain embodiments, the selection gene is a puromycin or kanamycin resistance gene.

[0021] In certain embodiments, the sequence of the screening gene is shown as SEQ ID NO:7.

[0022] In certain embodiments, the nucleic acid construct comprises, in order from 5' end to 3' end: EF1α, hGCH1 gene, T2A, hTH gene, SV40 PolyA, PGK, hAADC gene, P2A and PuroR.

[0023] In certain embodiments, the various elements of the nucleic acid construct are optionally connected by a linker (eg, a flexible peptide linker) or not.

[0024] In certain embodiments, the nucleic acid construct further comprises additional regulatory elements.

[0025] In certain embodiments, the additional regulatory element is selected from a promoter, a self-cleaving polypeptide, an internal ribosome entry site (IRES), a signal peptide, or any combination thereof.

[0026] In certain embodiments, the nucleic acid construct further comprises a nucleotide sequence encoding a detectable label. In certain embodiments, the detectable label is a fluorescent protein.

[0027] In certain embodiments, the detectably labeled fluorescent protein is red fluorescent protein (RFP), green fluorescent protein (GFP), blue fluorescent protein (BFP), or any combination thereof.

[0028] In certain embodiments, the sequence of the hGCH1 gene is shown in SEQ ID NO:16.

[0029] In certain embodiments, the sequence of the hTH gene is shown in SEQ ID NO:14.

[0030] In certain embodiments, the sequence of the hAADC gene is shown in SEQ ID NO:15.

[0031] In certain embodiments, the sequence of the SV40 PolyA is shown in SEQ ID NO:17.

[0032] In certain embodiments, the length of the nucleic acid construct is 100-10000 bp; for example, 100-10000 bp, 500-9000 bp, 1000-8000 bp, 2000-7000 bp, 3000-6000 bp, 4000-5000 bp.

[0033] In certain embodiments, the sequence of the nucleic acid construct is shown in SEQ ID NO:12.

[0034] carrier

[0035] In a second aspect, the present application provides a vector comprising the nucleic acid construct as described in the first aspect.

[0036] In certain embodiments, the vector is a cloning vector or an expression vector. In certain embodiments, the vector is a SB transposon vector, or a vector comprising a nucleotide sequence encoding a Cas protease (eg, Cas9) and a guide RNA sequence.

[0037] host cells

[0038] In a third aspect, the present application provides a host cell comprising the nucleic acid construct as described in the first aspect or the vector as described in the second aspect.

[0039] In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are pluripotent stem cells. In certain embodiments, the pluripotent stem cells are embryonic stem cells. In certain embodiments, the cells are human embryonic stem cells.

[0040] Engineered pluripotent stem cells and preparation methods thereof

[0041] In a fourth aspect, the present application provides a method for preparing engineered pluripotent stem cells, the method comprising:

[0042] (1) Providing embryonic stem cells;

[0043] (2) Providing the nucleic acid construct described in the first aspect or the vector described in the second aspect;

[0044] (3) contacting the nucleic acid construct or vector with embryonic stem cells under conditions enabling transformation;

[0045] Wherein, the steps (1) and (2) are performed in any order (for example, performed sequentially in any order, and / or performed simultaneously).

[0046] In certain embodiments, after step (2), the vector is amplified.

[0047] In certain embodiments, the vector is obtained by constructing the nucleic acid construct of the first aspect into a donor vector.

[0048] In certain embodiments, in step (3), the nucleic acid construct or vector is transformed into embryonic stem cells by a gene editing system. In certain embodiments, the method further provides a Cas protease (e.g., Cas9) and a guide RNA, and inserts the nucleic acid construct into embryonic stem cells by a homologous recombination method.

[0049] In certain embodiments, in step (3), the nucleic acid construct or vector is transformed into embryonic stem cells using a transposon method. In certain embodiments, the method further provides an SB-transposase vector, and the SB-transposase vector is contacted with the embryonic stem cells together with the vector described in the second aspect.

[0050] In a fifth aspect, the present application provides an engineered pluripotent stem cell, which is prepared by the method described in the fourth aspect.

[0051] In certain embodiments, the engineered pluripotent stem cells express the hGCH1 gene, the hTH gene, and the hAADC gene.

[0052] In certain embodiments, the genome of the engineered pluripotent stem cell comprises a nucleotide sequence encoding the hGCH1 gene, the hTH gene, and the hAADC gene.

[0053] In certain embodiments, the engineered pluripotent stem cells possess one or more characteristics selected from the group consisting of:

[0054] (1) Possess the ability to differentiate into cells of the three germ layers;

[0055] (2) The differentiated cells can stably express dopamine synthetase TH, AADC, and GCH;

[0056] (3) The engineered pluripotent stem cells or cells differentiated therefrom are capable of stably secreting dopamine neurotransmitter.

[0057] In a sixth aspect, the present application provides a differentiated cell obtained by differentiation of the engineered pluripotent stem cell according to the fifth aspect.

[0058] In certain embodiments, the differentiated cells are selected from neural stem cells, astrocytes, mesenchymal stem cells, GABAergic neural cells, DAergic neural cells, or any combination thereof.

[0059] In certain embodiments, the differentiated cells express the hGCH1 gene, the hTH gene, and the hAADC gene.

[0060] In certain embodiments, the genome of the differentiated cell comprises nucleotide sequences encoding the hGCH1 gene, the hTH gene, and the hAADC gene.

[0061] Pharmaceutical composition

[0062] In a seventh aspect, the present application provides a pharmaceutical composition comprising at least one of the following (1) to (5) (e.g., 1, 2, 3, 4, 5):

[0063] (1) The nucleic acid construct described in the first aspect;

[0064] (2) the vector described in the second aspect;

[0065] (3) the host cell described in the third aspect;

[0066] (4) The engineered pluripotent stem cells described in the fifth aspect;

[0067] (5) The differentiated cells described in the sixth aspect;

[0068] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0069] Methods and uses

[0070] In the eighth aspect, the present application provides the use of the nucleic acid construct described in the first aspect, or the vector described in the second aspect, or the host cell described in the third aspect, or the engineered pluripotent stem cell described in the fifth aspect, or the differentiated cell described in the sixth aspect for preparing a drug or reagent for preventing and / or treating a disease associated with dopaminergic neuron degeneration and / or damage in a subject.

[0071] On the other hand, the present application provides a method for preventing and / or treating a disease associated with dopaminergic neuron degeneration and / or damage in a subject, the method comprising: administering the nucleic acid construct described in the first aspect, or the vector described in the second aspect, or the host cell described in the third aspect, or the engineered pluripotent stem cell described in the fifth aspect, or the differentiated cell described in the sixth aspect to the subject by transplantation (e.g., intracerebral striatal transplantation) or injection (e.g., intravenous injection).

[0072] In certain embodiments, the disease associated with dopaminergic neuron degeneration and / or damage is a neurodegenerative disease.

[0073] In certain embodiments, the neurodegenerative disease is selected from Parkinson's disease, depression, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, or any combination thereof.

[0074] In certain embodiments, the subject is a mammal, such as a human.

[0075] In certain embodiments, the drug or agent is administered to the subject by transplantation (eg, intracerebral striatal transplantation) or injection (eg, intravenous injection).

[0076] In another aspect, the present application provides the use of the nucleic acid construct described in the first aspect, or the vector described in the second aspect, or the host cell described in the third aspect, or the engineered pluripotent stem cell described in the fifth aspect for preparing differentiated cells.

[0077] In certain embodiments, the differentiated cells are selected from neural stem cells, astrocytes, mesenchymal stem cells, GABAergic neural cells, DAergic neural cells, or any combination thereof.

[0078] In certain embodiments, the differentiated cells express the hGCH1 gene, the hTH gene, and the hAADC gene.

[0079] Definition of terms

[0080] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0081] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0082] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0083] As used herein, the term "prevention" refers to the method implemented in order to prevent or delay the occurrence of disease or illness or symptom in a subject. As used herein, the term "treatment" refers to the method implemented in order to obtain beneficial or required clinical results. For the purposes of the present invention, beneficial or required clinical results include but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (that is, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease and alleviating symptoms (no matter in part or in whole), no matter it is detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival (if not receiving treatment), extending the survival.

[0084] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) has a tumor, or is at risk of having a tumor.

[0085] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0086] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, and papillomaviruses. A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site.

[0087] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0088] As used herein, the terms "element" or "regulatory element" have the same meaning and are intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA (1990). In some cases, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in the desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). In some cases, regulatory elements can also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; the CMV enhancer; the R-U 5' segment in the LTR of HTLV-I ((Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0089] As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that can initiate expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0090] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0091] Advantageous Effects of the Invention

[0092] The present application has obtained a nucleic acid construct capable of high expression and secretion of the dopamine neurotransmitter. Furthermore, an engineered human embryonic stem cell line capable of stable and high-volume dopamine secretion has been obtained, as well as neural stem cells, astrocytes, and mesenchymal stem cells, GABAergic neurons, and DAergic neurons derived from the engineered human embryonic stem cell line.

[0093] These cells can significantly improve the behavioral abilities of mammals suffering from Parkinson's disease within a short period of time (e.g., 1-3 months). Therefore, the constructs, engineered human embryonic stem cells, and differentiated cells of the present application have great potential for preventing and / or treating diseases associated with dopaminergic neuron degeneration and / or damage. For example, the cells have the potential to be used as seed cells to develop cell-based drugs for the treatment of Parkinson's disease.

[0094] Below will be described in detail by example embodiments of the present invention, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, rather than to limit the scope of the invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 shows the original plasmid vector map used in this application and the constructed pSB-TH-AADC-GCH1-Puro-Transposon transposon system plasmid map. Figure 1a shows the SB transposon vector map, Figure 1b shows the SB transposase vector map, Figure 1c shows the pLV3-EF1a-MCS-PGK-EGFP-Puro vector, and Figure 1d shows the constructed SB transposon system plasmid map containing the three key genes hTH, hAADC, and hGCH1.

[0096] Figure 2 shows the results of immunofluorescence staining (Oct4, Nanog, TH).

[0097] FIG3 shows the results of dopamine neurotransmitter secretion in six cell types obtained after transfection of six different nucleic acid constructs comprising hTH, hAADC and hGCH1 genes constructed by the present invention.

[0098] Figure 4 shows the results of in vitro differentiation of DA-GTA-ESCs into engineered neural stem cells (DA-GTA-NSCs). Figure 4a shows NBs formed from DA-ESCs in suspension culture using neural induction medium. NBs can efficiently differentiate into DA-NSCs after attachment in neural stem cell culture medium. Figure 4b shows immunofluorescence staining of DA-NSCs (TH, NESTIN, SOX1, SOX2, and PAX6).

[0099] Figure 5 shows the results of in vitro differentiation of DA-GTA-ESCs into engineered astrocytes (DA-GTA-astrocytes). Figure 5a shows NBs formed by DA-ESCs in suspension culture using neural induction medium. NBs can efficiently differentiate into DA-NPCs after attachment in neural progenitor cell culture medium. DA-NPCs can then differentiate into DA-astrocytes in astrocyte induction culture. Figure 5b shows immunofluorescence staining of DA-NPCs (TH, NESTIN, SOX2, PAX6) following induced differentiation. Figure 5c shows immunofluorescence staining of DA-astrocytes (TH, GFAP, S100β, EAAT1) following induced differentiation.

[0100] Figure 6 shows the results of in vitro differentiation of DA-GTA-ESCs into engineered mesenchymal stem cells (DA-MSCs). Figure 6a shows EBs formed by DA-ESCs in suspension culture using embryoid body medium. EBs rapidly differentiated into DA-MSCs exhibiting fibroblast morphology and swirling growth when cultured in adherent MSC induction medium. Figure 6b shows immunofluorescence staining (TH) of the differentiated DA-MSCs. Figure 6c shows the tri-lineage differentiation (osteoblastic, adipogenic, and chondrogenic) of the differentiated DA-MSCs, along with flow cytometry results.

[0101] Figure 7 shows the results of dopamine transmitter secretion during in vitro differentiation of DA-ESCs into engineered cells, passage expansion (culture). Figure 7a shows the results of dopamine transmitter secretion during in vitro differentiation of DA-ESCs into neural stem cells (DA-NSCs) and engineered mesenchymal stem cells (DA-MSCs). Figure 7b shows the results of dopamine transmitter secretion during in vitro differentiation of DA-ESCs into engineered astrocytes (DA-astrocytes).

[0102] Figure 8a shows the behavioral improvement results of rats with Parkinson's disease after transplantation of the differentiated cells (DA-NSC, DA-astrocyte or DA-MSC) of the present application and control cells; Figure 8b shows the behavioral improvement results of rats with Parkinson's disease before and after transplantation of the differentiated cells (DA-NSC, DA-astrocyte or DA-MSC) of the present application.

[0103] Sequence information

[0104] Information on the partial sequences involved in the present invention is provided in Table 1 below.

[0105] Table 1: Description of sequences DETAILED DESCRIPTION

[0106] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0107] Unless otherwise specified, the experiments and methods described in the embodiments are carried out substantially according to conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (editor F M Ausubel et al., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL METHOD. APPROACH) (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

[0108] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0109] Example 1. Preparation of cells

[0110] Construction method of SB vector plasmid

[0111] 1 Experimental Materials

[0112] 1.1 Competent cells: trans5α (Beijing Quanshijin Biotechnology Co., Ltd.)

[0113] 1.2 Plasmid: pLV[Exp]-EGFP:T2A:Puro-EF1A>TH[NM_000360.3], pLV[Exp]-EGFP:T2A:Neo-EF1A>ADDC(NM_001082971.1):T2A:GCHⅠ(NM_001024024.1) (for details, see Li J, et al. Genetically engineered mesenchymal stem cells with dopamine synthesis for Parkinson's disease in animal models.NPJ Parkinson's Dis.2022;8(1):175.doi:10.1038 / s41531-022-00440-6), SB transposon vector (pSB-Transposon, addgene, pSBtet-GP, #60495), SB transposase vector (pSB-Transposase, addgene, pCMV(CAT)T7-SB100, #34879), pLV3-EF1a-MCS-PGK-EGFP-Puro vector (Miaoling, P50556). The original plasmid vector map used in this application and the constructed pSB-TH-AADC-GCH1-Puro-Transposon transposon system plasmid map are shown in Figure 1. Among them, a in Figure 1 is the SB transposon vector map, b in Figure 1 is the SB transposase vector map, c in Figure 1 is the pLV3-EF1a-MCS-PGK-EGFP-Puro vector, and d in Figure 1 is the constructed SB transposon system plasmid map containing the three key genes hTH, hAADC, and hGCH1.

[0114] Vector construction

[0115] Step 1: Construct vector 1: Use homologous recombination to replace the promoter tight TRE promoter in the SB transposon vector with EF-1α promoter (SEQ ID NO: 1) to obtain vector 1.

[0116] Step 2: Construction of Vector 2: PCR amplify hTH (SEQ ID NO: 2) from the pLV[Exp]-EGFP:T2A:Puro-EF1A>TH[NM_000360.3] vector, and replace the luciferase in vector 1 with hTH by homologous recombination to obtain vector 2.

[0117] Step 3: Construction of Vector 3: PCR amplify the PGK promoter from the pLV3-EF1a-MCS-PGK-EGFP-Puro vector. Homologous recombination replaced feature 21 in Vector 1 with the PGK promoter (SEQ ID NO: 3). Overlapping PCR replaced the RPBSA-EGFP-rTetR in Vector 2 with mPGK-hDDC-T2A- hGCH (PCR template from pLV[Exp]-EGFP:T2A:Neo-EF1A>ADDC[NM_001082971.1]:T2A:GCHⅠ[NM_001024024.1]). This resulted in the transposon SB-TH-AADC-GCH1-Puro-Transposon containing the three key genes hTH, hAADC, and hGCH1, i.e., Vector 3.

[0118] Refer to Molecular Cloning (4th edition) to transform competent cells, pick single clones, amplify and extract ultrapure plasmids.

[0119] Transfection method

[0120] Nucleofection reagent: recommended kit p3 primary cell 4D-nucleofector X Kit (Lonza, V4XP-3032).

[0121] Nuclear transfection instrument: LONZA Nucleofector 4D.

[0122] Digest adherent hES cells (ES cell line (h1) established in our laboratory, for detailed methods, see: Ai Z, et al. Modulation of Wnt and Activin / Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells. Biomaterials. 2020 Aug; 249: 120015. doi: 10.1016 / j.biomaterials.2020.120015), centrifuge and remove the supernatant. 4D- Basic Protocol for Human Stem Cells: Add the plasmids pSB-TH-AADC-GCH1-Puro-Transposon and pSB-Transposase into the Resuspend the digested hESC cell pellet in Solution with Supplement (Lonza, Catalog #: V4XP-3032) and transfer to a 16-well plate. Place the strips (Lonza, Catalog #: V4XP-3032) in an electroporator and select the program to begin electroporation. After completion of the electroporation, resuspend the cells in pre-warmed 37°C culture medium and inoculate them into a VTN-coated culture dish. Incubate in a 37°C, saturated humidity, 5% CO2 incubator.

[0123] The six SB vector plasmids and cells constructed

[0124] Referring to the above method, three key genes, hTH (nucleotide sequence shown in SEQ ID NO: 2, amino acid sequence shown in SEQ ID NO: 14), hAADC (nucleotide sequence shown in SEQ ID NO: 5, amino acid sequence shown in SEQ ID NO: 15), and hGCH1 (nucleotide sequence shown in SEQ ID NO: 6, amino acid sequence shown in SEQ ID NO: 16), were ligated into the SB transposon system vector plasmid by genetic engineering means to construct a triple gene SB transposon (SB-TH-AADC-GCH1-Puro-Transposon) containing the Puro drug screening gene and a double plasmid transduction system plasmid vector of the SB-Transposase (SB-Transposase).

[0125] Furthermore, the connection order of the three key genes hTH, hAADC, and hGCH1 on the SB transposon system vector plasmid includes the following six combinations:

[0126] ① hTH-hAADC-hGCH1, the sequence of all elements from the 5' end to the 3' end is EF1α-hTH-SV40 PolyA-PGK-hAADC-T2A-hGCH1-P2A-PuroR; the nucleotide sequence of this fragment is shown in SEQ ID NO: 8;

[0127] ② hTH-hGCH1-hAADC, the sequence of all elements from the 5' end to the 3' end is EF1α-hTH-T2A-hGCH1-SV40 PolyA-PGK-hAADC-P2A-PuroR; the nucleotide sequence of this fragment is shown in SEQ ID NO: 9;

[0128] ③ hAADC-hTH-hGCH1, the sequence of all elements from the 5' end to the 3' end is EF1α-hAADC-SV40 PolyA-PGK-hTH-T2A-hGCH1-P2A-PuroR; the nucleotide sequence of this fragment is shown in SEQ ID NO: 10;

[0129] ④ hAADC-hGCH1-hTH, the sequence of all elements from the 5' end to the 3' end is EF1α-hAADC-SV40 PolyA-PGK-hGCH1-T2A-hTH-P2A-PuroR; the nucleotide sequence of this fragment is shown in SEQ ID NO: 11;

[0130] ⑤ hGCH1-hTH-hAADC, the sequence of all elements from the 5' end to the 3' end is EF1α-hGCH1-T2A-hTH-SV40 PolyA-PGK-hAADC-P2A-PuroR; the nucleotide sequence of this fragment is shown in SEQ ID NO: 12;

[0131] ⑥ hGCH1-hAADC-hTH, the sequence of all elements from the 5' end to the 3' end is EF1α-hGCH1-T2A-hAADC-SV40 PolyA-PGK-hTH-P2A-PuroR; the nucleotide sequence of this fragment is shown in SEQ ID NO: 13;

[0132] Among them, EF1α and PGK in the above construct are promoters; SV40 PolyA (Simian Vacuolar Virus PolyA) functions to terminate transcription and add a PolyA tail to the transcribed mRNA; P2A and T2A are self-cleaving polypeptides; PuroR is a puromycin resistance gene.

[0133] The constructed SB-TH-AADC-GCH1-Puro-Transposon (including the six transposons with different linkage sequences) and SB-Transposase plasmids were co-transfected into ESCs. Puro (Meilun Biotech, MB2005) was then added to the cell culture medium for several days to obtain ESC lines stably transfected with the TH, AADC, and GCH1 genes. These lines were named DA-TAG-ESC, DA-TGA-ESC, DA-ATG-ESC, DA-AGT-ESC, DA-GTA-ESC, and DA-GAT-ESC, respectively. Single clones were selected as first-generation cells and cultured and subcultured to obtain second, third, fourth, fifth, and even further generations of cells.

[0134] In addition to the above-mentioned cell preparation methods, this example also transfected the six constructs described above into ESC cells using the Crispr-Cas9 system (the nucleotide sequence of Cas9 is shown in SEQ ID NO: 20). By homologous recombination, ESC cell lines stably transfected with the TH, AADC, and GCH1 genes were obtained. The transfection method is as follows:

[0135] Nuclear transfection reagent: Highgene (abclonol, RM09014).

[0136] hES cells were cultured on VTNs in a 37°C, saturated humidity, 5% CO2 incubator. When the density of hES cells adherent to the six-well plate reached 70%, 1.5 μg of spCas9 KI Cas9, 3 μg of spCas9-DONAR-TH[NM_000360.3]-GCH1[NM_001024024.1]-ADC[NM_001082971.1], and 8 μl of HighGene reagent were added to the cell culture medium. After 12 hours, the medium was replaced and the cells continued to be cultured on VTNs in a 37°C, saturated humidity, 5% CO2 incubator.

[0137] Example 2. Detection of pluripotent stem cells

[0138] After several days of cell culture, a culture medium containing a certain concentration of PuroP was added to the culture dish and cultured continuously for several days to screen out cells that were stably resistant to PuroP. The cells were then passaged and expanded, and the supernatant of the culture medium during the stable passage and expansion process was collected to measure the dopamine concentration in the supernatant.

[0139] First, the screened and expanded cells were preliminarily identified using immunofluorescence staining (refer to Li J, et al. Genetically engineered mesenchymal stem cells with dopamine synthesis for Parkinson's disease in animal models. NPJ Parkinson's Dis. 2022; 8(1):175. doi:10.1038 / s41531-022-00440-6). Then, the dopamine secretion levels of the cells of each passage obtained in Example 1 were measured using a dopamine ELISA kit (Abnova, Catalog Number KA1887) to screen for cell lines that can stably secrete dopamine.

[0140] The above experiments revealed that the three key genes, TH, AADC, and GCH1, were consistently and stably expressed at high levels in the screened DA-TAG-ESC, DA-TGA-ESC, DA-ATG-ESC, DA-AGT-ESC, DA-GTA-ESC, and DA-GAT-ESC cell lines. Furthermore, as shown in Figures 2 and 3, the six cell lines can stably synthesize and secrete dopamine neurotransmitters. Among them, the synthesis and secretion of dopamine neurotransmitters by DA-TAG-ESC, DA-TGA-ESC, DA-ATG-ESC, and DA-GTA-ESC were significantly higher than those of the other two cell lines. Among these four cell lines, DA-GTA-ESC demonstrated the most outstanding effect.

[0141] Example 3. Differentiation and detection of NSCs

[0142] The engineered DA-GTA-ESC cells identified above were differentiated. The neural stem cell (NSC) differentiation method was performed according to the method in Zhao, S. et al. Generation of cortical neurons through large-scale expanding neuroepithelial stem cells from human pluripotent stem cells. Stem Cell Res Ther, 11, 431, (2020).

[0143] The specific steps are as follows:

[0144] Step 1. Tryple the adherent DA-GTA-ESCs into single cells. Transfer the single cells into a 15 ml centrifuge tube, centrifuge at 150 g for 3 min, and discard the supernatant.

[0145] Step 2. Resuspend the cell pellet in neural induction medium and seed it into an ultra-low attachment culture dish. After 6 days of continuous suspension culture, neuron bodies (NBs) will form, as shown in Figure 4(a).

[0146] The neural induction medium formula is as follows: Advance DMEM / F12 (Gibco, 10565-018): Neurobasal media (Gibco, 21103-049) (1:1) supplemented with 1% N2 (Gibco, 17502-048), 2% B27 (Gibco, 17504-044), 1% Glutmax (Gibco, 35050-061), 10 ng / mL OsrbFGF (Oryza sativa recombinant human basic fibroblast growth factor, Wuhan Healthgen, China, HYC005M01), 3 μM CHIR99021 (Selleck, S2924), 5 μM SB431542 (Cellagen technology, C7243), 0.2 μM Compound E (Calbiochem, 565790), 0.1 μM LDN193189 (Selleck, S2618), and 0.1mM β-mercaptoethanol (Sigma, M3148).

[0147] Step 3. Collect the NB-containing culture medium from Step 2 into a centrifuge tube. Let it sit for 3 minutes until the NBs settle to the bottom, then discard the supernatant. Resuspend the NBs in neural stem cell culture medium and plate them onto a VTN-coated culture plate. They will rapidly adhere and proliferate, as shown in Figure 4, a.

[0148] Step 4. When the neural stem cells from Step 3 adhere to the culture medium and reach 80% confluence, designate them as DA-GTA-NSC-P0. Tryply digest the DA-GTA-NSC-P0 cells into single cells, transfer them to a 15 ml centrifuge tube, centrifuge at 150 g for 3 minutes, and discard the supernatant. Resuspend the cells in fresh neural stem cell culture medium and subculture to obtain DA-GTA-NSC-P1, DA-GTA-NSC-P2, DA-GTA-NSC-P3, DA-GTA-NSC-P4, DA-GTA-NSC-P5, and so on.

[0149] The differentiated DA-GTA-NSCs were identified by immunofluorescence staining. The specific identification method is as follows: 1. Fixation and permeabilization: Cells cultured adherently in well plates were fixed with 4% PFA (Sigma, Paraformaldehyde, STBJ9683) at room temperature for 10 minutes, then washed with PBS and discarded, and permeabilized for 30 minutes with PBS containing 10% donkey serum (Solarbio, SL050) and 0.4% Triton X-100 (Sigma, SLBW6818). 2. Wash with PBS and discarded, then incubate with primary antibody diluted in PBS containing 2.5% donkey serum and 0.4% Triton X-100 at 4°C overnight. 3. Wash with PBS and discarded, then incubate with secondary antibody diluted in 5% donkey serum in PBS and in the dark for 2 hours at room temperature. DAPI (Thermo Scientific, 62248) was added. 4. Observation and photography were performed. After washing with PBS and discarding, 30 μl of PBS was added, and the cells were observed and photographed under a confocal microscope. TH (aves, TYH1727980), Nestin (millpore, MAB5326), Sox1 (R&D, AF3369), Sox2 (millpore, AB5603), PAX6 (biolegend Covance, 901301) were used.

[0150] The experimental results, shown in Figure 4, show that the differentiated cells highly expressed classic neural stem cell (NSC) markers, including Nestin, Sox1, Sox2, and PAX6, as well as TH. This result demonstrates the successful differentiation of DA-GTA-NSC into neural stem cells (NSCs), and the resulting cells were named DA-GTA-NSC.

[0151] Example 4. Astrocyte differentiation and detection

[0152] The engineered cells DA-GTA-ESC identified above were differentiated. The astrocyte differentiation method was based on the method described in Perriot S, Differentiation of functional astrocytes from human-induced pluripotent stem cells in chemically defined media. STAR Protoc. 2(4): 100902, (2021).

[0153] The specific steps are as follows:

[0154] Step 1-Step 2 are the same as in Example 2.

[0155] Step 3. The culture medium used was neural progenitor cell culture medium with the following formula: Advance DMEM / F12 (Gibco, 10565-018), 1% N2 supplement, 2% B27 supplement, 10 ng / mL FGF-2, and 10 ng / mL EGF. Other steps were the same as those in step 3 of Example 2.

[0156] Step 4. Change the neural progenitor cell culture medium every other day. When the cells adhere to the wall and grow to a confluence of 80%-90%, they are recorded as DA-GTA-NPC-P0. Then, subculture and expand the cells to obtain DA-GTA-NPC-P1, DA-GTA-NPC-P2, DA-GTA-NPC-P3, DA-GTA-NPC-P4, DA-GTA-NPC-P5, etc.

[0157] Step 5. Differentiate DA-GTA-NPCs into astrocytes (see Figure 5, a). Transfer the single cells to a 15 ml centrifuge tube, centrifuge at 150 g for 3 minutes, discard the supernatant, and resuspend in astrocyte induction medium. The astrocyte induction medium recipe is as follows: Advance DMEM / F12, 1% N2 supplement, 2% B27 supplement, 10 ng / mL LIF, and 10 ng / mL EGF.

[0158] The culture was continued for 14 days, with the medium changed every other day. When the cells adhered to the wall and grew to a confluence of 80%-90%, the cells were digested with Tryple and passaged. On D14, the medium was changed to astrocyte medium + CNTF (20 ng / mL).

[0159] Step 6. From D14 to D19, change the culture medium every other day. When the cells adhere to the culture medium and reach 80%-90% confluence, use Tryptophan to digest and passage the cells. The differentiated astrocytes are designated as DA-GTA-astrocytes.

[0160] DA-GTA-NPCs described in step 4 were identified by immunofluorescence staining. The specific identification method is as follows: 1. Fixation and permeabilization: Cells cultured in adherent well plates were fixed with 4% PFA (Sigma, Paraformaldehyde, STBJ9683) at room temperature for 10 minutes, then washed with PBS and discarded, and permeabilized with PBS containing 10% donkey serum (Solarbio, SL050) and 0.4% Triton X-100 (Sigma, SLBW6818) for 30 minutes. 2. Wash with PBS and discarded, then incubate with primary antibody diluted in PBS containing 2.5% donkey serum and 0.4% Triton X-100 at 4°C overnight. 3. Wash with PBS and discarded, then incubate with secondary antibody diluted in 5% donkey serum in PBS and in the dark for 2 hours at room temperature. 4. Observe and photograph. After washing with PBS and discarding, 30 μl of PBS was added, and the cells were observed and photographed under a confocal microscope. Antibody information is as follows: TH (aves, TYH1727980), Nestin (millpore, MAB5326), GFAP (millpore, MAB360), Sox2 (millpore, AB5603), PAX6 (biolegend Covance, 901301), S100β (abcam, ab52642), EAAT1 (abcam, ab181036).

[0161] The experimental results, shown in Figure 5, show that the differentiated cells highly expressed classic astrocyte markers, including GFAP, S100β, and EAAT1, as well as TH. This result confirms the successful differentiation of DA-GTA-ESCs into astrocytes, and the resulting cells were named DA-GTA-astrocytes. Figure 7(b) shows that during the differentiation process from DA-GTA-ESCs to DA-GTA-NSCs, dopamine neurotransmitter secretion was consistently and stably expressed.

[0162] Example 5. Differentiation and detection of MSCs

[0163] The engineered cells DA-GTA-ESC identified above were differentiated. The MSC differentiation method was carried out according to the method in Hwang, NS et al. In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells. Proc Natl Acad Sci USA, 105, 20641-20646, (2008).

[0164] The specific steps are as follows:

[0165] Step 1. Tryple the adherent DA-GTA-ESCs into single cells. Transfer the single cells into a 15 ml centrifuge tube, centrifuge at 150 g for 3 min, and discard the supernatant.

[0166] Step 2. Resuspend the cell pellet after centrifugation in embryoid body culture medium and seed it into an ultra-low attachment culture dish. EBs will form after 7 days of continuous suspension culture, as shown in Figure 6(a). The embryoid body medium formula is as follows: Advance DMEM / F12 (Gibco, 10565-018), 1% N2 supplement, 2% B27 supplement, 0.5% GlutaMAX (Thermo Fisher Scientific, 35,050-061), 1% nonessential amino acids (Thermo Fisher Scientific, 11,140-050), 0.1mM β-mercaptoethanol (Sigma, M7522), 50μg / ml L-ascorbic acid 2-phosphate (Sigma, A8960), 0.5ml Chemically Defined Lipid Concentrate (Thermo Fisher Scientific, 11,905-031), 12.5μg / ml Insulin (Roche, 11,376,497,001), 0.25mM Sodium Pyruvate (Thermo Fisher Scientific, 11,360-070), 0.2μg / ml Progesterone (Sigma, P8783), 7.5% HAS (Recombinant Human Serum Albumin, Wuhan Healthgen, China, HYC002M01).

[0167] Step 3. Collect the culture medium containing EBs from Step 2 into a centrifuge tube and allow the EBs to settle to the bottom for 3 minutes, then discard the supernatant. Resuspend the EBs in MSC induction medium and plate them onto a VTN-coated culture plate for adherent culture. After 5-7 days of induction, cells emerging from the EBs will have reached 80%-90% confluence (see Figure 6, a). This is designated as DA-GTA-MSC-P0.

[0168] The MSC induction medium formula is as follows: IMDM (Basal Media, L610KJ), 1% N2 supplement, 2% B27 supplement, 0.5% GlutaMAX (Thermo Fisher Scientific, 35,050-061), 1% nonessential amino acids (Thermo Fisher Scientific, 11,140-050), 5 ng / mL bFGF and 5 ng / mL TGF-β (Peprotech, 96-100-21-10).

[0169] Step 4. Subculture and expand MSC-P0 to obtain DA-GTA-MSC-P1, DA-GTA-MSC-P2, DA-GTA-MSC-P3, DA-GTA-MSC-P4, DA-GTA-MSC-P5...

[0170] Step 5. Identify the expression of the target gene in DA-MSCs by immunofluorescence staining (see Figure 6(b)). The specific identification method is as follows: 1. Fixation and permeabilization: Cells cultured in adherent microplates were fixed with 4% PFA (Sigma, Paraformaldehyde, STBJ9683) for 10 minutes at room temperature, then washed with PBS and discarded, and permeabilized for 30 minutes with PBS containing 10% donkey serum (Solarbio, SL050) and 0.4% Triton X-100 (Sigma, SLBW6818). 2. Wash with PBS and discarded, then incubate with primary antibody diluted in PBS containing 2.5% donkey serum and 0.4% Triton X-100 overnight at 4°C. 3. Wash with PBS and discarded, then incubate with secondary antibody diluted in PBS containing 5% donkey serum and DAPI (Thermo Scientific, 62248) at room temperature in the dark for 2 hours. 4. Observe and photograph. After washing with PBS and discarding, 30 μl of PBS was added, and the cells were observed and photographed under a confocal microscope. Antibody information is as follows: TH (aves, TYH1727980). The results showed that the differentiated MSCs still stably and highly expressed TH.

[0171] Step 6. Use the human MSC Analysis Kit (BD biosciences, 562245) to identify the hallmark markers of the differentiated DA-GTA-MSCs by flow cytometry, as shown in Figure 6 (c).

[0172] Step7.Use human mesenchymal stem cell functional identification kit( HUXXC-90021, HUXXC-90031, HUXXC-90041) were differentiated into adipogenic, osteogenic, and chondrogenic cells according to the instructions ( Figure 6 c ).

[0173] For more specific dopa concentration determination methods, immunofluorescence staining methods, and flow cytometry identification methods, please refer to the literature Li, J. et al. Genetically engineered mesenchymal stem cells with dopamine synthesis for Parkinson's disease in animal models. NPJ Parkinson's Dis, 8, 175, (2022).

[0174] The experimental results (Figure 6) showed that the differentiated cells adhered to the wall and exhibited the typical swirl-shaped morphology of mesenchymal stem cells (MSC) (Figure 6a), possessed the ability to differentiate into osteoblasts, adipocytes, and chondrocytes (Figure 6c), and expressed classic MSC markers, including CD44, CD73, and CD105. This confirmed the successful differentiation of DA-GTA-ESCs into mesenchymal stem cells (MSCs), and the resulting cells were named DA-GTA-MSCs. Figure 7a shows that during the differentiation process from DA-GTA-ESCs to DA-GTA-MSCs, the secretion of dopamine neurotransmitter was continuously and stably expressed.

[0175] Example 6. Therapeutic effect on Parkinson's disease

[0176] The differentiated cells obtained in Examples 3-5 were tested in this example. In order to verify the role of differentiated cells in the treatment of Parkinson's disease, we prepared a rat Parkinson's disease model by 6-OHDA (6-hydroxydopamine) injection and evaluated the transplanted cells. Apomorphine-induced rotation test was performed for 3 consecutive months after cell transplantation to detect behavioral ability. Among them, the modeling method, cell transplantation and behavioral detection methods refer to the method described in the literature Li, J. et al. Genetically engineered mesenchymal stem cells with dopamine synthesis for Parkinson's disease in animal models. NPJ Parkinson's Dis, 8, 175, (2022).

[0177] Cell transplantation procedure: The differentiated cells obtained in Examples 3-5 were collected by tryple digestion and centrifuged at 300×g for 4 minutes. The cell pellets were resuspended in transplantation medium (Advance DMEM / F12; Gibco, 10565-018) and the viable cell concentration was adjusted to 100,000 ± 10,000 cells / μl. With bregma as reference, 4 μl of single cell suspension was placed at approximately 4×10 5 The control rats were transplanted with NSCs, astrocytes, or MSCs differentiated from ES (h1) cells. Cyclosporine 15 mg / kg was injected intraperitoneally daily starting 24 hours before cell transplantation and continued until death.

[0178] The results in Figure 8a show that compared to control cells (NSC, astrocyte, or MSC differentiated from h1 cells), transplantation of DA-GTA-NSC, DA-GTA-astrocyte, or DA-GTA-MSC cells prepared in the present application can significantly reduce the number of rotations in rats within a short period of time (e.g., 1-3 months), with the number of rotations per minute in rats all reduced by at least half! The results in Figure 8b show that compared to before transplantation, transplantation of DA-GTA-NSC, DA-GTA-astrocyte, or DA-GTA-MSC cells prepared in the present application can significantly reduce the number of rotations in rats within a short period of time (e.g., 1-3 months), with the number of rotations per minute in rats all reduced by at least half!

[0179] The above results show that transplanting NSC, astrocyte or MSC cells derived from in vitro differentiation of DA-GTA-ESC cells can effectively improve the behavioral ability of Parkinson's disease animal models, which indicates that these cells have preventive and therapeutic prospects for Parkinson's disease.

[0180] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A nucleic acid construct, which comprises, from the 5' end to the 3' end, a nucleotide sequence encoding the following elements: a first promoter, a hGCH1 gene, a first self-cleaving polypeptide, a hTH gene, a second promoter, a hAADC gene, a second self-cleaving polypeptide and a screening gene.

2. The nucleic acid construct according to claim 1, wherein The first promoter and the second promoter are each independently selected from a constitutive promoter, a specific promoter and an inducible promoter; Preferably, the first promoter and the second promoter are constitutive promoters; Preferably, the first promoter and the second promoter are each independently selected from: β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α (EF1α), phosphoglycerate kinase (PGK), ubiquitin C, simian virus 40 early promoter (sv40), or any combination thereof; Preferably, the first promoter is EF1α; preferably, the sequence of the first promoter is as shown in SEQ ID NO: 1; The second promoter is PGK; preferably, the sequence of the PGK is shown in SEQ ID NO:

3.

3. The nucleic acid construct according to claim 1 or 2, wherein The first self-cleaving polypeptide and the second self-cleaving polypeptide are each independently selected from P2A, T2A, E2A or F2A; Preferably, the first self-cleaving polypeptide is T2A; preferably, the sequence of the first self-cleaving polypeptide is as shown in SEQ ID NO: 18; Preferably, the second self-cleaving polypeptide is P2A; preferably, the sequence of the first self-cleaving polypeptide is as shown in SEQ ID NO:

19.

4. The nucleic acid construct according to any one of claims 1 to 3, wherein The screening gene is an antibiotic resistance gene; Preferably, the screening gene is selected from the group consisting of puromycin resistance gene (PuroR), ampicillin resistance gene (ampr), tetracycline resistance gene (tetr), chloramphenicol resistance gene (Cmr), kanamycin resistance gene (kanr), or any combination thereof; Preferably, the screening gene is a puromycin or kanamycin resistance gene; preferably, the sequence of the screening gene is shown in SEQ ID NO:

7.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein The nucleic acid construct comprises, from the 5' end to the 3' end, EF1α, hGCH1 gene, T2A, hTH gene, SV40 PolyA, PGK, hAADC gene, P2A and PuroR; Preferably, the nucleic acid construct has one or more characteristics selected from the following: (1) The various elements of the nucleic acid construct are optionally connected by a linker (e.g., a flexible peptide linker) or not; (2) the nucleic acid construct further comprises an additional regulatory element; Preferably, the additional regulatory element is selected from a promoter, a self-cleaving polypeptide, an internal ribosome entry site (IRES), a signal peptide, or any combination thereof; (3) The nucleic acid construct further comprises a nucleotide sequence encoding a detectable marker; preferably, the detectable marker is a fluorescent protein; Preferably, the sequence of the hGCH1 gene is as shown in SEQ ID NO: 16; Preferably, the sequence of the hTH gene is as shown in SEQ ID NO: 14; Preferably, the sequence of the hAADC gene is as shown in SEQ ID NO: 15; Preferably, the sequence of the nucleic acid construct is as shown in SEQ ID NO: 12; Preferably, the sequence of the SV40 PolyA is as shown in SEQ ID NO:

17.

6. A vector comprising the nucleic acid construct according to any one of claims 1 to 5; preferably, the vector is a cloning vector or an expression vector; Preferably, the vector is an SB transposon vector, or a vector comprising a nucleotide sequence encoding a Cas protease (eg, Cas9) and a guide RNA sequence.

7. A host cell comprising the nucleic acid construct according to any one of claims 1 to 5 or the vector according to claim 6; Preferably, the cell is a mammalian cell; Preferably, the cells are pluripotent stem cells; Preferably, the pluripotent stem cells are embryonic stem cells; Preferably, the cells are human embryonic stem cells.

8. A method for preparing engineered pluripotent stem cells, the method comprising: (1) Providing embryonic stem cells; (2) providing the nucleic acid construct according to any one of claims 1 to 5 or the vector according to claim 6; (3) contacting the nucleic acid construct or vector with embryonic stem cells under conditions that allow transformation; Wherein, steps (1) and (2) are performed in any order (for example, performed sequentially in any order, and / or, performed simultaneously); Preferably, in step (3), the nucleic acid construct or vector is transformed into embryonic stem cells by a gene editing system; preferably, the method further provides a Cas protease (e.g., Cas9) and a guide RNA; preferably, the nucleic acid construct is inserted into embryonic stem cells by a homologous recombination method; Preferably, in step (3), the nucleic acid construct or vector is transformed into embryonic stem cells by a transposon method; preferably, the method further provides an SB transposase vector, and the SB transposase vector is contacted with the embryonic stem cells together with the vector according to claim 6.

9. An engineered pluripotent stem cell prepared by the method of claim 8; Preferably, the engineered pluripotent stem cells express the hGCH1 gene, the hTH gene and the hAADC gene; Preferably, the genome of the engineered pluripotent stem cell comprises a nucleotide sequence encoding the hGCH1 gene, the hTH gene and the hAADC gene.

10. A differentiated cell obtained by differentiation of the engineered pluripotent stem cell according to claim 9; Preferably, the differentiated cells are selected from neural stem cells, astrocytes, mesenchymal stem cells, GABAergic neural cells, DAergic neural cells, or any combination thereof; Preferably, the differentiated cells express hGCH1 gene, hTH gene and hAADC gene.

11. A pharmaceutical composition comprising at least one of the following (1) to (5) (e.g., 1, 2, 3, 4, 5): (1) The nucleic acid construct according to any one of claims 1 to 5; (2) The vector according to claim 6; (3) The host cell according to claim 7; (4) The engineered pluripotent stem cell of claim 9; (5) The differentiated cell according to claim 10; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

12. Use of the nucleic acid construct according to any one of claims 1 to 5, or the vector according to claim 6, or the host cell according to claim 7, or the engineered pluripotent stem cell according to claim 9, or the differentiated cell according to claim 10 for preparing a drug or agent for preventing and / or treating a disease associated with dopaminergic neuron degeneration and / or damage in a subject; Preferably, the disease associated with dopaminergic neuron degeneration and / or damage is a neurodegenerative disease; Preferably, the neurodegenerative disease is selected from Parkinson's disease, depression, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, or any combination thereof; Preferably, the subject is a mammal, such as a human; Preferably, the drug or agent is administered to the subject by transplantation (eg, intracerebral striatal transplantation) or injection (eg, intravenous injection).

13. Use of the nucleic acid construct according to any one of claims 1 to 5, or the vector according to claim 6, or the host cell according to claim 7, or the kit according to claim 8, or the engineered pluripotent stem cell according to claim 10 for preparing differentiated cells; Preferably, the differentiated cells are selected from neural stem cells, astrocytes, mesenchymal stem cells, GABAergic neural cells, DAergic neural cells, or any combination thereof; Preferably, the differentiated cells express hGCH1 gene, hTH gene and hAADC gene.

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