WO2021031025A1

WO2021031025A1 - Application of ptbp1 inhibitor in prevention and/or treatment of neurodegenerative disease

Info

Publication number: WO2021031025A1
Application number: PCT/CN2019/101201
Authority: WO
Inventors: 杨辉; 周海波
Original assignee: 中国科学院脑科学与智能技术卓越创新中心
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2021-02-25

Abstract

Provided is an application of a Ptbp1 inhibitor in prevention and/or treatment of a neurodegenerative disease. Specifically, provided is use of an inhibitor for a Ptbp1 gene or an encoded protein thereof, used for preparing a composition or preparation, the composition or preparation being used for preventing and/or treating a neurodegenerative disease. By inhibiting the expression or activity of the Ptbp1 gene or the encoded protein thereof of astrocytes in brain tissues, the astrocytes may be effectively inducted to be differentiated into excitatory neuron, thereby preventing and/or treating the neurodegenerative disease.

Description

Application of Ptbp1 inhibitor in the prevention and/or treatment of neurodegenerative diseases

Technical field

The invention relates to the field of biomedicine. More specifically, the present invention relates to the use of Ptbp1 inhibitors in the prevention and/or treatment of neurodegenerative diseases.

Background technique

Parkinson's disease (PD) is a serious neurodegenerative disease characterized by the loss of dopamine neurons in the substantia nigra of the midbrain. Previous studies have achieved the direct reprogramming of astrocytes into dopamine neurons in vitro and in animal models by simultaneously overexpressing several transcription factors. However, so far, Ptbp1 mediated neuronal reprogramming in vivo has not been reported yet.

At present, the main treatment for Parkinson's disease is drugs represented by levodopa preparations. At the same time, surgical treatment can also improve symptoms to a certain extent. It should be pointed out that all these methods can only partially alleviate the disease, but cannot achieve the effect of preventing the development of the disease.

Therefore, there is an urgent need in the art to develop targets that can effectively treat neurodegenerative diseases.

Summary of the invention

The purpose of the present invention is to provide a target that can effectively treat neurodegenerative diseases.

Another purpose of the present invention is to provide a new target Ptbp1 for the treatment of Parkinson's disease. By inhibiting the expression of Ptbp1, astrocytes in the striatum can be directly converted into dopamine neurons and the phenotype of Parkinson's disease can be restored. .

In the first aspect of the present invention, there is provided a use of a Ptbp1 gene or its encoded protein inhibitor for preparing a composition or preparation, and the composition or preparation is used to prevent and/or treat neurodegenerative diseases.

In another preferred embodiment, the composition or preparation is also used for one or more purposes selected from the following group:

(a) Inducing the transdifferentiation of astrocytes into excitatory neurons;

(b) Treatment of movement disorders in mammals.

In another preferred example, the mammal includes a mammal suffering from a neurodegenerative disease.

In another preferred embodiment, the mammal includes a human or non-human mammal.

In another preferred embodiment, the non-human mammal includes rodents (such as mice, rats, or rabbits) and primates (such as monkeys).

In another preferred embodiment, the astrocytes are derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably, the astrocytes are derived from the striatum.

In another preferred embodiment, the excitatory neurons include dopamine neurons.

In another preferred embodiment, the inhibitor is selected from the following group: antibodies, small molecule compounds, microRNA, siRNA, shRNA, gene editor, or a combination thereof.

In another preferred embodiment, the gene editor includes a DNA gene editor and an RNA gene editor.

In another preferred embodiment, the gene editor includes optional gRNA and gene editing protein.

In another preferred example, the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.

In another preferred embodiment, the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.

In another preferred embodiment, the gene editing protein is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof.

In another preferred embodiment, the source of the gene editing protein is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium ), Ruminococcus Flavefaciens, or a combination thereof.

In another preferred example, the Ptbp1 is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.

In another preferred example, the Ptbp1 gene includes wild-type Ptbp1 gene and mutant Ptbp1 gene.

In another preferred example, the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (that is, the function is the same or substantially the same as that of the wild-type encoded protein).

In another preferred example, the polypeptide encoded by the mutant Ptbp1 gene is the same or substantially the same as the polypeptide encoded by the wild Ptbp1 gene.

In another preferred example, the mutant Ptbp1 gene includes homology of ≥80% (preferably ≥90%, more preferably ≥95%, more preferably ≥98% compared with the wild Ptbp1 gene) Or 99%) polynucleotides.

In another preferred embodiment, the mutant Ptbp1 gene is included in the 5'end and/or 3'end of the wild-type Ptbp1 gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.

In another preferred example, the Ptbp1 gene includes a cDNA sequence, a genome sequence, or a combination thereof.

In another preferred embodiment, the Ptbp1 protein includes active fragments of Ptbp1 or derivatives thereof.

In another preferred example, the homology of the active fragment or its derivative with Ptbp1 is at least 90%, preferably 95%, more preferably 98%, 99%.

In another preferred example, the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% of Ptbp1 activity.

In another preferred embodiment, the amino acid sequence of the Ptbp1 protein is selected from the following group:

(i) A polypeptide having the amino acid sequence shown in SEQ ID NO.:1;

(ii) The amino acid sequence shown in SEQ ID NO.: 1 is formed by the substitution, deletion or addition of one or several (e.g. 1-10) amino acid residues, which has the function of the protein and is formed by ( i) Derived polypeptide; or

(iii) The homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.:1 is ≥90% (preferably ≥95%, more preferably ≥98% or 99%), and a polypeptide having the protein function.

In another preferred example, the nucleotide sequence of the Ptbp1 gene is selected from the following group:

(a) A polynucleotide encoding the polypeptide shown in SEQ ID NO.:1;

(b) A polynucleotide whose sequence is shown in SEQ ID NO.: 2;

(c) A polynucleotide whose nucleotide sequence has a homology of ≥95% (preferably ≥98%, more preferably ≥99%) with the sequence shown in SEQ ID NO.: 2;

(d) Truncate or add 1-60 (preferably 1-30, more preferably 1-10) nuclei at the 5'end and/or 3'end of the polynucleotide shown in SEQ ID NO.: 2 Glycated polynucleotide;

(e) A polynucleotide complementary to any of the polynucleotides described in (a) to (d).

In another preferred embodiment, the ptbp1 protein is shown in SEQ ID NO.:1.

In another preferred embodiment, the nucleic acid encoding the ptbp1 protein is shown in SEQ ID NO.: 2.

In another preferred example, the region targeted by the ptbp1 gene or the inhibitor of the encoded protein (such as the gene editing protein) is the 4758-4787 and/or 5381-5410 positions of the ptbp1 gene sequence.

In another preferred embodiment, the inhibitor of the ptbp1 gene or its encoded protein inhibits the activity and/or expression of ptbp1.

In another preferred embodiment, the concentration of the inhibitor gene or its encoded protein ptbp1 (virus titer)> 1 × 10 ^13, preferably, is ^{^{1 × 10 13 -1 × 10 14}} .

In another preferred example, the inhibitory rate of the ptbp1 gene or its encoded protein inhibitor on the activity and/or expression of ptbp1 is greater than 90%, preferably, 90%-95%.

In another preferred embodiment, the inhibitor targets astrocytes in brain tissue.

In another preferred embodiment, the neurodegenerative disease includes Parkinson's disease.

The second aspect of the present invention provides a composition comprising:

(a) A gene editing protein or an expression vector thereof, the gene editing protein is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof; and

(b) gRNA or an expression vector thereof, the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.

In another preferred embodiment, the composition includes a pharmaceutical composition.

In another preferred embodiment, the composition further includes:

(c) Other drugs to prevent and/or treat neurodegenerative diseases.

In another preferred embodiment, the expression vector of the gene editing protein includes a vector targeting astrocytes of brain tissue.

In another preferred embodiment, the expression vector includes a viral vector.

In another preferred example, the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.

In another preferred embodiment, the vector is selected from the following group: lentivirus, adenovirus, adeno-associated virus (AAV), or a combination thereof, preferably, the vector is adeno-associated virus (AAV).

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the composition is a liquid preparation.

In another preferred embodiment, the dosage form of the composition is an injection dosage form.

In another preferred embodiment, other drugs for preventing and/or treating neurodegenerative diseases are selected from the following group: dopamine prodrugs, non-ergot dopamine receptor agonists, monoamine oxidase B inhibitors, or combinations thereof.

In another preferred embodiment, the composition is a cell preparation.

In another preferred example, the expression vector of the gene editing protein and the expression vector of gRNA are the same vector or different vectors.

In another preferred example, the weight ratio of the component (a) to the component (b) is 100:1 to 0.01:1, preferably, 10:1 to 0.1:1, more preferably, 2: 1-0.5:1.

In another preferred embodiment, the content of the component (a) in the composition is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

In another preferred embodiment, in the composition, the content of the component (b) is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.

In another preferred example, the content of the component (c) in the composition is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.

In another preferred example, in the composition, the component (a), component (b) and optional component (c) account for 0.01-99.99 wt% of the total weight of the composition, which is greater than Preferably 0.1-90wt%, more preferably 1-80wt%.

The third aspect of the present invention provides a medicine kit including:

(a1) The first container, and the gene editing protein or its expression vector located in the first container, or a medicine containing the gene editing protein or its expression vector, the gene editing protein is selected from the group consisting of CasRx, CRISPR/ Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof;

(b1) A second container, and gRNA or its expression vector, or a drug containing gRNA or its expression vector, in the second container, and the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.

In another preferred embodiment, the kit further includes:

(c1) The third container, and other drugs for preventing and/or treating neurodegenerative diseases, or drugs containing other drugs for preventing and/or treating neurodegenerative diseases.

In another preferred embodiment, the first container, the second container, and the third container are the same or different containers.

In another preferred embodiment, the medicine in the first container is a unilateral preparation containing gene editing protein or its expression vector.

In another preferred embodiment, the medicine in the second container is a unilateral preparation containing gRNA or its expression vector.

In another preferred embodiment, the medicine in the third container is a single preparation containing other medicines for preventing and/or treating neurodegenerative diseases.

In another preferred embodiment, the dosage form of the drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.

In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.

In another preferred embodiment, the kit also contains instructions.

The fourth aspect of the present invention provides a composition according to the second aspect of the present invention or the use of the kit according to the third aspect of the present invention to prepare a medicine for preventing and/or treating neurodegenerative diseases.

In another preferred embodiment, the composition, the concentration of the role of protein or gene expression vector editing (viral titer)> 1 × 10 ^13, ^{preferably, 1 × 10 13 -1 × 10} 14.

In another preferred embodiment, the composition or expression of the gRNA concentration of the carrier (viral titer)> 1 × 10 ^13, ^{preferably, 1 × 10 13 -1 × 10} 14.

In another preferred example, in the pharmaceutical composition, the concentration (viral titer) of the other drugs for preventing and/or treating neurodegenerative diseases is> 1×10 ¹³ , preferably, 1×10 ¹³ —1×10 ¹⁴ .

In another preferred embodiment, the composition or kit includes (a) gene editing protein or its expression vector; and (b) gRNA or its expression vector; and (c) optionally other prevention and/or treatment of nerves Drugs for degenerative diseases; and (d) pharmaceutically acceptable carriers.

In another preferred embodiment, in the composition or kit, (a) gene editing protein or its expression vector; and (b) gRNA or its expression vector; and (c) optional other prevention and/or treatment The drug for neurodegenerative diseases accounts for 0.01-99.99% by weight of the total weight of the composition or the kit, preferably 0.1-90% by weight, more preferably 1-80% by weight.

The fifth aspect of the present invention provides a method for promoting the differentiation of astrocytes into excitatory neurons, including the steps:

In the presence of the Ptbp1 gene or its encoded protein inhibitor or the composition according to the second aspect of the present invention, astrocytes are cultured to promote the differentiation of astrocytes into excitatory neurons.

In another preferred embodiment, the astrocytes include striatal astrocytes.

In another preferred embodiment, the astrocytes are astrocytes of brain tissue.

In another preferred embodiment, the astrocytes are cells in vitro.

In another preferred embodiment, the effect of the concentration of the gene or its encoded protein Ptbp1 inhibitors (viral titer)> 1 × 10 ^13, ^{preferably, 1 × 10 13 -1 × 10} 14.

In another preferred embodiment, the effect of the concentration of composition 2 (titer) 1 × 10 ^13, ^{preferably, 1 × 10 13 -1 × 10} 14 claim>.

The sixth aspect of the present invention provides a method for preventing and/or treating neurodegenerative diseases, including:

Ptbp1 gene or its encoded protein inhibitor, or the composition according to the second aspect of the present invention, or the kit according to the third aspect of the present invention is administered to a subject in need.

In another preferred embodiment, the subject includes a human or non-human mammal suffering from a neurodegenerative disease.

In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

The seventh aspect of the present invention provides a method for screening candidate compounds for the prevention and/or treatment of neurodegenerative diseases, the method comprising the steps:

(a) In the test group, add the test compound to the cell culture system, and observe the expression (E1) and/or activity (A1) of Ptbp1 in the cells of the test group; in the control group, in the same cell No test compound is added to the culture system, and the expression (E0) and/or activity (A0) of Ptbp1 in the cells of the control group is observed;

Wherein, if the expression level (E1) and/or activity (A1) of cells in the test group is significantly lower than that of the control group, it indicates that the test compound is a preventive and/or inhibitory effect on the expression and/or activity of Ptbp1 Candidate compounds for the treatment of neurodegenerative diseases.

In another preferred example, the expression level of Ptbp1 is obtained by qPCR.

In another preferred embodiment, the method further includes the steps:

(b) For the candidate compound obtained in step (a), further test its promoting effect on the differentiation of astrocytes into excitatory neurons; and/or further test whether it has a down-regulation effect on the Ptbp1 gene.

In another preferred embodiment, the method includes step (c): applying the candidate compound determined in step (a) to a mammalian model, and determining its effect on the mammal.

In another preferred embodiment, the mammal is a mammal suffering from a neurodegenerative disease.

In another preferred embodiment, the "significantly lower" means E1/E0≤1/2, preferably,≤1/3, more preferably≤1/4.

In another preferred example, the "significantly lower" means that A1/A0≤1/2, preferably,≤1/3, more preferably≤1/4.

In another preferred embodiment, the cells include astrocytes.

In another preferred embodiment, the cells include striatal astrocytes.

In another preferred embodiment, the cell is a cell cultured in vitro.

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

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

Figure 1 shows the direct conversion of astrocytes into functional neurons using CasRx-mediated Ptbp1 knockdown. among them,

(a) Schematic diagram of the process of using CasRx to mediate Ptbp1 knockdown. (b) The plasmids encoding the two gRNAs and CasRx can significantly knock down the Ptbp1 transcription level of astrocytes, and n=2 repeats. (c,d) Schematic diagram of injection process. Vector 1 (AAV-GFAP-mCherry) encodes mCherry driven by astrocyte-specific promoter GFAP, and vector 2 (AAV-CasRx-Ptbp1) carries CasRx and two gRNAs. AAV-GFAP-mCherry and AAV-CasRx-Ptbp1 targeting Ptbp1 were injected into the right striatum together. On the left side, only AAV-GFAP-mCherry was injected as a control. It takes about 5-6 weeks to complete transdifferentiation after injection. (e) Immunofluorescence of the striatum at 5 weeks after injection. NeuN is a mature neuron marker. The white arrow indicates the mCherry signal that is not co-labeled with NeuN, and the orange arrow indicates the co-labeled NeuN and mCherry signals. Scale bar: 50μm. (f) Percentage of mCherry ⁺ NeuN ⁺ ^{cells in mCherry +} cells (n=6 mice; p<0.001, t=4.72). (g) A representative of mCherry ⁺ NeuN ⁺ Tbr1 ⁺ cells. Scale bar: 20μm. (h,i) Representative of the electrical signal of an induced neuron recorded in the brain slice of the striatum (n=8 cells). All values are expressed as mean±sem. .***p<0.001, unpaired t test.

Figure 2 shows that CasRx-mediated Ptbp1 knockdown can reprogram astrocytes into dopamine neurons in a mouse model of 6-OHDA-induced Parkinsonism. Among them, (a) experimental program. 6-OHDA and saline were injected into bilateral MFB. About one month later, the side of the striatum where 6-OHDA was injected was injected with a mixture of AAV-CasRx-Ptbp1 and AAV-GFAP-mCherry, and immunostaining and behavioral tests were performed 3-4 weeks after the injection. (b) Confocal image of dopamine neurons. The orange arrow indicates the signal that mCherry is co-labeled with TH. Note: TH is a marker of dopamine neurons. Scale bars: 50μm (left), 10μm (right). (c) Percentage of TH ⁺ /mCherry ⁺ ^{cells in TH +} cells in the injection area (n=3 mice). All values are expressed as mean±sem.

Figure 3 shows that induction of dopamine neurons can alleviate motor dysfunction in PD mouse models. Among them, (a) the net rotation caused by apomorphine injection (number of turns/20 minutes). (b) The ratio of spontaneous contact on the same side to the total number of contacts. (c) Rotating rod test. It indicates the time (seconds) that the mouse stayed on the rotating rod before falling. Saline group, n=12 mice; 6-OHDA saline group, n=14 mice. 6-OHDA+EFS-CasRx-Ptbp1, n=12 mice. The data was collected one month after the virus injection. ANOVA.

Figure 4 shows the loss of dopamine neurons and fibers caused by unilateral injection of 6-OHDA. ^{Among them, (a) TH staining shows the reduction of TH +} neurons (red) in the substantia nigra (the same side where 6-OHDA was injected) on one side. Scale bar: 50μm. (b) Dopamine transporter (DAT) staining shows the reduction of dopamine fibers (green) on the same side as the injection of 6-OHDA. Scale bar: 1mm.

detailed description

After extensive and in-depth research, the inventors unexpectedly discovered for the first time that inhibiting the expression or activity of the ptbp1 gene or its encoded protein of astrocytes in brain tissue can effectively induce astrocytes (such as striatum Astrocytes) differentiate into excitatory neurons (such as dopamine neurons) to prevent and/or treat neurodegenerative diseases (such as Parkinson's disease). On this basis, the inventor completed the present invention.

Astrocyte

Astrocytes are the most abundant type of cells in the mammalian brain. They perform many functions, including biochemical support (such as forming a blood-brain barrier), providing nutrients for neurons, maintaining extracellular ion balance, and participating in repair and scar formation after brain and spinal cord injury. According to the content of glial filaments and the shape of cell processes, astrocytes can be divided into two types: fibrous astrocytes (fibrous astrocytes) are mostly distributed in the white matter of the brain and spinal cord, with slender protrusions and fewer branches , The cytoplasm contains a lot of glial filaments; protoplasmic astrocytes (protoplasmic astrocytes) are mostly distributed in the gray matter, with stubby cell processes and many branches.

The astrocytes that can be used in the present invention are not particularly limited, and include various astrocytes derived from the central nervous system of mammals, such as from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably , From the striatum.

Dopamine neuron

Dopaminergic neuron (dopaminergic neuron) contains and releases dopamine (dopamine, DA) as a neurotransmitter. Dopamine is a catecholamine neurotransmitter and plays an important biological role in the central nervous system. Dopaminergic neurons in the brain are mainly concentrated in the substantria nigra pars compacta (SNc) of the midbrain and the ventral cover Area (ventral tegmental area, VTA), hypothalamus and periventricular. Many experiments have confirmed that dopaminergic neurons are closely related to many diseases of the human body, the most typical being Parkinson's disease.

Neurodegenerative diseases

Neurodegenerative diseases are diseases caused by the loss of neurons in the brain and spinal cord. Neurons are the most important part of the nervous system, and their death will eventually lead to the dysfunction of the nervous system. After a patient suffers from a neurodegenerative disease, there will be mobility or cognitive impairment, and the development of the disease often leads to many complications, causing serious damage to the patient's life. Clinically, neurodegenerative diseases mainly include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis. At present, the neurodegenerative diseases can only be relieved or delayed, and cannot be completely cured. Parkinson's disease (PD) is a serious neurodegenerative disease characterized by the loss of dopamine neurons in the substantia nigra of the midbrain.

Gene editor

In the present invention, the gene editor includes a DNA gene editor and an RNA gene editor. In a preferred embodiment, the gene editor of the present invention includes a gene editing protein and optionally gRNA.

Gene editing protein

In the present invention, the nucleotides of the gene editing protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and the amino acid sequence can be derived from the nucleotide sequence. The source of the wild-type gene editing protein includes (but is not limited to): Ruminococcus Flavefaciens, Streptococcus pyogenes, Staphylococcus aureus, and Acidaminococcus sp. , Lachnospiraceae bacterium (Lachnospiraceae bacterium).

In a preferred embodiment of the present invention, the gene editing protein includes, but is not limited to Cas13 (such as CasRx), CRISPR/Cas9, Cpf1, SaCas9, Cas13a, Cas13b, and Cas13c.

ptbp1 protein and polynucleotide

In the present invention, the terms "protein of the present invention", "ptbp1 protein" and "ptbp1 polypeptide" are used interchangeably, and all refer to a protein or polypeptide having an amino acid sequence of ptbp1. They include ptbp1 protein with or without starting methionine. In addition, the term also includes full-length ptbp1 and fragments thereof. The ptbp1 protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.

The ptbp1 protein is a polypyrimidine domain binding protein 1, which is an RNA binding protein that regulates RNA splicing. At the same time, it also plays a very critical role in other functions of RNA.

In the present invention, the terms "ptbp1 gene" and "ptbp1 polynucleotide" are used interchangeably, and both refer to a nucleic acid sequence having a ptbp1 nucleotide sequence.

The genome of the human ptbp1 gene is 14936bp in length (NCBI GenBank accession number is 5725).

The genome of the mouse ptbp1 gene is 10004bp in length (NCBI GenBank accession number is 19205).

The similarity of human and mouse ptbp1 at the DNA level is 88%, and the similarity of protein sequence is 84%. It should be understood that when encoding the same amino acid, the substitution of nucleotides in the codon is acceptable. In addition, it should be understood that when conservative amino acid substitutions are generated by nucleotide substitutions, nucleotide changes are also acceptable.

When the amino acid fragment of ptbp1 is obtained, a nucleic acid sequence encoding it can be constructed based on it, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed according to the ptbp1 nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.

At present, the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant ptbp1 polypeptide. Generally speaking, there are the following steps:

(1) Transform or transduce suitable host cells with the polynucleotide (or variant) encoding human ptbp1 polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Separate and purify protein from culture medium or cells.

In the present invention, the ptbp1 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

The methods well known to those skilled in the art can be used to construct an expression vector containing the ptbp1 coding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.

Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture can be selected from various conventional mediums. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

Adeno-associated virus

Because Adeno-associated virus (AAV) is smaller than other viral vectors, is non-pathogenic, and can transfect dividing and undivided cells, gene therapy methods for genetic diseases based on AAV vectors have been affected. Widespread concern.

Adeno-associated virus (adeno-associated virus, AAV), also known as adeno-associated virus, belongs to the Parvoviridae dependent virus genus. It is the simplest type of single-stranded DNA-deficient virus found so far and requires a helper virus (usually Viruses) participate in replication. It encodes the cap and rep genes in the inverted repeat (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.

Recombinant adeno-associated virus vector (rAAV) is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo. Long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effects in various cells, tissues and in vivo experiments have accumulated a lot of data. In medical research, rAAV is used in the research of gene therapy for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice and other aspects.

In a preferred embodiment of the present invention, the vector is a recombinant AAV vector. AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not seem to be involved in human pathology. The AAV genome has been cloned, sequenced and characterized. AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of replication of the virus. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.

AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for in vitro and in vivo gene transfer has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos. 4,797,368, 6,596,535 and 5,139,941, and European Patent No. 0488528, they are all incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and/or cap genes are missing and replaced by the gene of interest, and these constructs are in vitro (into cultured cells) or in vivo (into the organism directly) ) The purpose of transporting the gene of interest. Replication-deficient recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (such as adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeats (ITR) Region plasmids, and plasmids carrying AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.

In some embodiments, the recombinant vector is capsidized to viral particles (e.g., including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV virus particles of AAV16). Therefore, the present disclosure includes recombinant virus particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in US Patent No. 6,596,535.

ptbp1 inhibitor and pharmaceutical composition

Using the protein of the present invention, through various conventional screening methods, substances that interact with the ptbp1 gene or protein, especially inhibitors, can be screened out.

The ptbp1 inhibitor (or antagonist) that can be used in the present invention includes any substance that can inhibit the expression and/or activity of the ptbp1 gene or its encoded protein.

For example, the inhibitor of ptbp1 includes an antibody of ptbp1, antisense RNA of ptbp1 nucleic acid, siRNA, shRNA, miRNA, gene editor, or an activity inhibitor of ptbp1. A preferred inhibitor of ptbp1 refers to a gene editor capable of inhibiting the expression of ptbp1.

Preferably, the inhibitors of ptbp1 of the present invention include inhibitors that target positions 4758-4787 and/or positions 5381-5410 of the ptbp1 gene sequence. The targets of the ptbp1 inhibitor of the present invention include astrocytes.

In a preferred embodiment, the methods and steps for inhibiting ptbp1 include using an antibody of ptbp1 to neutralize its protein, and using shRNA or siRNA or a gene editor carried by a virus (such as adeno-associated virus) to silence the ptbp1 gene.

The inhibition rate of ptbp1 is generally at least 50% or more inhibition, preferably 60%, 70%, 80%, 90%, 95% inhibition, which can be based on conventional techniques, such as flow cytometry, fluorescent quantitative PCR or Western Methods such as blot control and detect the inhibition rate of ptbp1.

The inhibitor of the ptbp1 protein of the present invention (including antibodies, antisense nucleic acids, gene editors and other inhibitors), when administered (administered) therapeutically, can inhibit the expression and/or activity of the ptbp1 protein, thereby inducing stars The glial cells differentiate into dopamine neurons, thereby preventing and/or treating neurodegenerative diseases. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): local, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical administration, autologous cell extraction and culture and reinfusion Wait.

The present invention also provides a pharmaceutical composition, which contains a safe and effective amount of the inhibitor of the present invention (such as antibody, gene editor, antisense sequence (such as siRNA), or inhibitor) and a pharmaceutically acceptable carrier or excipient Shape agent. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg-10 mg/kg body weight per day.

The main advantages of the present invention include:

(1) The present invention found for the first time that reducing the expression or activity of the Ptbp1 gene or its encoded protein in astrocytes can induce the differentiation of astrocytes into dopamine neurons, thereby preventing and/or treating neurodegeneration Diseases (such as Parkinson's disease).

(2) The present invention finds for the first time that using a gene editor (including gene editing protein and gRNA) to inhibit the expression of ptbp1 in astrocytes can make astrocytes transdifferentiate into dopamine neurons, which in turn is Parkinson’s Treatment provides a potential way.

(3) The present invention found for the first time that the induction of dopamine neurons alleviated the motor dysfunction in the Parkinsonian mouse model.

(4) The present invention finds for the first time that the RNA-targeted CRISPR system CasRx can avoid the risk of permanent DNA changes caused by traditional CRISPR-Cas9 editing. Therefore, CasRx-mediated RNA editing provides an effective means for the treatment of various diseases.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

General method

Animal ethics: The use and feeding of animals conform to the guidelines of the Biomedical Research Ethics Committee of the Institute of Neuroscience, Chinese Academy of Sciences.

GRNA sequence: The gRNA sequence targeting Ptbp1 is: gRNA1: 5'-tgtagatgggctgtccacgaagcactggcg-3'; gRNA2: 5'-gcttggagaagtcgatgcgcagcgtgcagc-3'.

Transient transfection of astrocytes and qPCR: isolated and cultured as previously described ¹ astrocytes. In short, astrocytes were seeded in 6-well plates. Using Lipofectamine 3000 (Thermo Fisher Scientific) according to standard procedures, 3 μg of gRNA-CasRx-GFP expressing vector was used for transient transfection. The control plasmid expresses non-targeted guidance. 1-2 days after transient transfection, GFP-positive cells were collected by flow fluorescence cell sorting (FACS) and lysed for qPCR analysis: first use Trizol (Ambion) to extract RNA, and then use reverse transcription kit (HiScript for qPCR) Q RT SuperMix, Vazyme, Biotech) reverse transcription of RNA into cDNA. The amplification was followed by AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech). Ptbp1 qPCR primers are: forward, 5'-AGAGGAGGCTGCCAACACTA-3'; reverse, 5'-GTCCAGGGTCACTGGGTAGA-3'.

Stereotactic injection: AAV8 (Figure 1) and AAV-PhP.eb (Figures 2 and 3) were used in this study. Stereotactic injection (C57BL / 6,1-3 months) ² method as described above. The titers of AAV-CasRx-Ptbp1 in Figure 1 and Figures 2, 3 are about 5×10e12 (2μl per injection) and 1.6×10e13 (2-3μl per injection). Inject AAV into the striatum (AP+0.8mm, ML±1.6mm and DV-2.8mm).

Immunofluorescence staining: Immunofluorescence staining was performed 5-6 weeks (Figure 1) or 3-4 weeks (Figures 2 and 3) after injection. After the mice were perfused, their brains were taken and fixed with 4% paraformaldehyde (PFA) overnight, and kept in 30% sucrose for at least 12 hours. The sections were frozen after embedding, and the section thickness was 35 μm. Before immunofluorescence staining, the brain sections were washed thoroughly with 0.1M phosphate buffer (PB). Primary antibodies: rabbit polyclonal NeuN antibody (Brain, 1:500, #ABN78, Millipore), rabbit polyclonal Tbr1 antibody (#NG1854874, Millipore), mouse TH antibody (1:300, MAB318, Millipore) and rabbit DAT antibody (1:100, MAB369, Millipore). Secondary antibodies: Donkey anti-mouse (#715-545-150, Jackson ImmunoResearch) and donkey anti-rabbit (#711-545-152, Jackson ImmunoResearch) were used in this study. After the antibody incubation, the sections were washed and covered with mounting media (Life Technology).

Electrophysiological recordings: after AAV injection electrophysiological recordings 5-6 weeks, ³ as previously described. In short, the mice were anesthetized and perfused into the heart, and their brains were put into carbon dioxide-filled NMDG artificial cerebrospinal fluid (aCSF) [NMDG aCSF(mM): NMDG 92, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, bicarbonate Sodium 30, HEPES 20, glucose 25, thiourea 2, sodium ascorbate 5) at room temperature, sodium pyruvate 3, calcium chloride 0.5, magnesium sulfate 10]. After perfusion, the brain was extracted and placed in an ice-cold NMDG aCSF solution for 30 seconds. Trim the brain and slice at a thickness of 250-350 μm at a speed of 0.04-0.05 mm/s. Move the brain slices into a dish filled with carbon dioxide NMDG aCSF and keep it at 32-34°C for ≤12 minutes. Transfer the sections to a new dish of HEPES aCSF filled with carbon dioxide at room temperature [HEPES, which contains aCSF (mM): sodium chloride 92, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, sodium bicarbonate 30, HEPES 20, Glucose 25, Thiourea 2, Sodium Ascorbate 5, Sodium Pyruvate 3, Calcium Chloride 2, Magnesium Sulfate 2]. After 1 hour, the slices were transferred to a recording dish containing a recording buffer [record aCSF (mM): sodium chloride 119, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, sodium bicarbonate 24, glucose 12.5, chloride Calcium 2, magnesium sulfate 2]. The neuron-like mCherry positive cells were recorded under a microscope (Olympus BX51WI), and Clampex 10 was used to obtain the data.

6-OHDA PD mouse model

The study was based on previous studies ^4. Adult C57BL/6 mice were injected intraperitoneally (7-10 weeks). Inject 25mg/kg desipramine hydrochloride (D3900, Sigma-Aldrich) half an hour before anesthesia. After anesthesia, 3μg 6-OHDA (H116, Sigma-Aldrich) or normal saline was injected into the right medial forebrain tract of the mouse according to the following coordinates: anteroposterior (A/P) = -1.2mm, medial and lateral (M/L) = -1.1mm, dorsoventricular (D/V) = -5mm. One hour after the operation, all mice were injected subcutaneously with 1 ml of 4% glucose-saline solution. The mice consumed the soaked food pellets for the next 3 weeks.

Apomorphine-induced rotation test

Mice were intraperitoneally injected with 0.5 mg/kg apomorphine (A4393, Sigma-Aldrich) 10 minutes before the test. After that, each of them was placed in an opaque cylinder (30 cm in diameter) and recorded on it by a camera for 20 minutes. Rotation is defined as a whole body turning with one hind paw as the center and no head orientation is switched. Calculate the number of rotations on the injection side and the contralateral side. The data was quantified as the number of contralateral reversals within 20 minutes.

Cylinder test

Each mouse was gently put into a glass beaker (1000 ml), and the camera was recorded for 10 minutes in front of it. Calculate the number of wall touches on the injection side and the contralateral paw respectively, and quantify the data as the ratio of the number of wall touches on the same side to the total number of wall touches.

Rotating rod test

All mice were trained for 2 days and tested on the third day. On the first day, the mice were trained 4 times on a rotating rod at a fixed speed of 4 laps/min, each for 300 seconds. On the 2nd and 3rd days, the mice were trained or tested 4 times at an acceleration of 4 to 40 laps/min. The time the mouse stayed on the rod before falling off was recorded as the stay period, and the average of the 3 longest stay periods was used for analysis.

Statistical analysis: set error bars by sem, and calculate statistical significance by unpaired two-tailed t test or one-way analysis of variance (p<0.05). All experiments were randomized to develop, not to use statistical methods to determine the pre-sample size, but our sample size reference ⁵ reported previously in the literature. It is assumed that the data distribution is normal but not formally tested. Data collection and analysis were not performed under blind experimental conditions.

Example 1: Knockdown of Ptbp1 by CasRx can directly convert astrocytes into functional neurons

In order to achieve effective knockdown of Ptbp1, two gRNAs targeting exons 4 and 7 of Ptbp1 were designed respectively, and these two gRNAs were driven by a U6 promoter. Next, the two gRNAs and CasRx genes were loaded onto a separate vector, and it was found that the vector can effectively down-regulate Ptbp1mRNA in cultured astrocytes 2 days after transfection (Figure 1a, b). In order to confirm whether neurotransdifferentiation can be achieved in vivo, AAV-CasRx-Ptbp1 and AAV-GFAP-mCherry (used to label astrocytes) were mixed and injected into the striatum (Figure 1c, d). 5-6 weeks after injection, NeuN+/mCherry+ cells (16.2±3%, SEM) were found in the striatum of AAV-CasRx-Ptbp1 injection, but no AAV-CasRx-Ptbp1 in the striatum when the contralateral side was not injected NeuN+/mCherry+ cells (0%, SEM), which shows that CasRx successfully mediates the transformation of astrocytes into neurons (Figure 1e, f). In addition, most of the transformed neurons (NeuN+/mCherry+) were co-stained with the excitatory neuron marker Tbr1 (75% in avarege, n=2 mice) (Figure 1g), indicating that most of the transformed neurons are excited Sex neuron. In order to check whether the induced neurons are functional, a whole-cell electrophysiological record was made on the successfully induced mCherry+ neuron-like cells. It was found that all recorded cells (n=8 cells) could generate action potentials in response to the depolarizing current injection in the current clamp mode (Figure 1h), and showed spontaneous action in the voltage clamp mode (Figure 1i). Postsynaptic current, which indicates that the transformed neurons can receive functional synaptic signal input.

Example 2 CasRx-mediated knockdown of Ptbp1 can convert astrocytes into dopamine neurons in a 6-OHDA-induced Parkinson's mouse model

In addition, in order to explore the therapeutic potential of CasRx-mediated neuronal reprogramming, the possibility of the transformation of astrocytes into dopamine neurons was further studied in the Parkinson's mouse model. The Parkinson's model used in the present invention is to inject 6-OHDA unilaterally into the right medial fascicle (MFB) to promote the loss of dopamine neurons in the ventral midbrain on the ipsilateral side and dedopaminergic neuronization of the striatum (Figure 4) Figure 4a shows that TH staining proves that dopamine neurons are killed in the substantia nigra on the same side of OHDA injection; Figure 4b shows that DAT staining proves that dopamine neurons are denervated in the striatum. Three weeks after 6-OHDA injection, AAV-CasRx-Ptbp1 and AAV-GFAP-mCherry were mixed and injected into the striatum on the same side (same side as 6-OHDA injection) (Figure 2a). 3-4 weeks after AAV injection, the striatum Th+ cells began to appear in the body (Figure 2b). More than 80% (80±5%, S.E.M.) of Th+ cells were positive for mCherry (Figure 2c), indicating that knocking out Ptbp1 with CasRx can induce astrocytes to transform into dopamine neurons.

Example 3 The induced dopamine neurons can alleviate movement disorders in a mouse model of Parkinson's

The present invention studies whether the induced dopamine neurons can alleviate the motor symptoms of Parkinson in the 6-OHDA-induced mouse Parkinson's model. The motor function of mice was evaluated by drug induction and spontaneous exercise: We first tested apomorphine-induced rotation behavior, which is a behavioral paradigm widely used to test symptoms after unilateral dopamine neuron loss. After three weeks, it was found that compared with untreated mice after 6-OHDA injection, the apomorphine-induced rotation of the treated mice was significantly reduced (Figure 3a); then the cylindrical test and the rotating rod test were used to test separately Asymmetry and coordination of the forelimb movement, it was found that the treated mice showed a lower percentage of contact on the same side of the cylinder (Figure 3b) and a longer duration of rotation (Figure 3c).

These results indicate that the induction of dopamine neurons in the striatum indeed alleviates the motor dysfunction in the Parkinson's mouse model.

to sum up

The present invention found that only by knocking down the mRNA of PTBP1, striatum astrocytes can be effectively transformed into dopamine neurons, thereby reducing Parkinson-like symptoms. The RNA-targeted CRISPR system CasRx can avoid the risk of permanent DNA changes caused by traditional CRISPR-Cas9 editing. Therefore, CasRx-mediated RNA editing provides an effective means for the treatment of various diseases.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A use of Ptbp1 gene or its encoded protein inhibitor is characterized in that it is used to prepare a composition or preparation, and the composition or preparation is used to prevent and/or treat neurodegenerative diseases.
The use according to claim 1, wherein the composition or preparation is further used for one or more uses selected from the following group:

(a) Inducing the transdifferentiation of astrocytes into excitatory neurons;

(b) Treatment of movement disorders in mammals.
The use according to claim 2, wherein the astrocytes are derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably, the astrocytes are derived from Striatum.
The use according to claim 2, wherein the excitatory neurons comprise dopamine neurons.
The use according to claim 1, wherein the inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, gene editors, or combinations thereof.
A composition characterized by comprising:

(a) A gene editing protein or an expression vector thereof, the gene editing protein is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof; and

(b) gRNA or its expression vector, the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
A medicine box is characterized in that it comprises:

(a1) The first container, and the gene editing protein or its expression vector located in the first container, or a medicine containing the gene editing protein or its expression vector, the gene editing protein is selected from the group consisting of CasRx, CRISPR/ Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof;

(b1) A second container, and gRNA or its expression vector, or a drug containing gRNA or its expression vector, in the second container, and the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
A use of the composition according to claim 6 or the kit according to claim 7, characterized in that it is used to prepare a medicine for preventing and/or treating neurodegenerative diseases.
A method for promoting the differentiation of astrocytes into excitatory neurons is characterized in that it comprises the steps:

In the presence of the Ptbp1 gene or its encoded protein inhibitor or the composition of claim 6, astrocytes are cultured to promote the differentiation of astrocytes into excitatory neurons.
A method for screening candidate compounds for the prevention and/or treatment of neurodegenerative diseases, characterized in that the method comprises the steps:

(a) In the test group, add the test compound to the cell culture system, and observe the expression (E1) and/or activity (A1) of Ptbp1 in the cells of the test group; in the control group, in the same cell No test compound is added to the culture system, and the expression (E0) and/or activity (A0) of Ptbp1 in the cells of the control group is observed;

Wherein, if the expression level (E1) and/or activity (A1) of cells in the test group is significantly lower than that of the control group, it indicates that the test compound is a preventive and/or inhibitory effect on the expression and/or activity of Ptbp1 Candidate compounds for the treatment of neurodegenerative diseases.