WO2021031025A1 - Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie neurodégénérative - Google Patents

Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie neurodégénérative Download PDF

Info

Publication number
WO2021031025A1
WO2021031025A1 PCT/CN2019/101201 CN2019101201W WO2021031025A1 WO 2021031025 A1 WO2021031025 A1 WO 2021031025A1 CN 2019101201 W CN2019101201 W CN 2019101201W WO 2021031025 A1 WO2021031025 A1 WO 2021031025A1
Authority
WO
WIPO (PCT)
Prior art keywords
ptbp1
gene
astrocytes
another preferred
protein
Prior art date
Application number
PCT/CN2019/101201
Other languages
English (en)
Chinese (zh)
Inventor
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 (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院脑科学与智能技术卓越创新中心 filed Critical 中国科学院脑科学与智能技术卓越创新中心
Priority to PCT/CN2019/101201 priority Critical patent/WO2021031025A1/fr
Publication of WO2021031025A1 publication Critical patent/WO2021031025A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • 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.
  • Parkinson's disease 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.
  • Ptbp1 mediated neuronal reprogramming in vivo has not been reported yet.
  • the main treatment for Parkinson's disease is drugs represented by levodopa preparations.
  • 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.
  • 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.
  • Ptbp1 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. .
  • 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.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the mammal includes a mammal suffering from a neurodegenerative disease.
  • the mammal includes a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, or rabbits) and primates (such as monkeys).
  • the astrocytes are derived from the striatum, spinal cord, dorsal midbrain or cerebral cortex, preferably, the astrocytes are derived from the striatum.
  • the excitatory neurons include dopamine neurons.
  • the inhibitor is selected from the following group: antibodies, small molecule compounds, microRNA, siRNA, shRNA, gene editor, or a combination thereof.
  • the gene editor includes a DNA gene editor and an RNA gene editor.
  • the gene editor includes optional gRNA and gene editing protein.
  • the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
  • the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.
  • the gene editing protein is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof.
  • 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.
  • the Ptbp1 is derived from mammals; preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.
  • the Ptbp1 gene includes wild-type Ptbp1 gene and mutant Ptbp1 gene.
  • 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).
  • polypeptide encoded by the mutant Ptbp1 gene is the same or substantially the same as the polypeptide encoded by the wild Ptbp1 gene.
  • 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.
  • 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.
  • the Ptbp1 gene includes a cDNA sequence, a genome sequence, or a combination thereof.
  • the Ptbp1 protein includes active fragments of Ptbp1 or derivatives thereof.
  • the homology of the active fragment or its derivative with Ptbp1 is at least 90%, preferably 95%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% of Ptbp1 activity.
  • amino acid sequence of the Ptbp1 protein is selected from the following group:
  • 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
  • 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.
  • nucleotide sequence of the Ptbp1 gene is selected from the following group:
  • the ptbp1 protein is shown in SEQ ID NO.:1.
  • nucleic acid encoding the ptbp1 protein is shown in SEQ ID NO.: 2.
  • the region targeted by the ptbp1 gene or the inhibitor of the encoded protein is the 4758-4787 and/or 5381-5410 positions of the ptbp1 gene sequence.
  • the inhibitor of the ptbp1 gene or its encoded protein inhibits the activity and/or expression of ptbp1.
  • 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%.
  • the inhibitor targets astrocytes in brain tissue.
  • the neurodegenerative disease includes Parkinson's disease.
  • the second aspect of the present invention provides a composition comprising:
  • a gene editing protein or an expression vector thereof is selected from the group consisting of CasRx, CRISPR/Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or a combination thereof;
  • the gRNA is RNA that guides the gene editing protein to specifically bind to the Ptbp1 gene.
  • the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.
  • the composition includes a pharmaceutical composition.
  • composition further includes:
  • the expression vector of the gene editing protein includes a vector targeting astrocytes of brain tissue.
  • the expression vector includes a viral vector.
  • the viral vector is selected from the following group: adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpes virus, SV40, poxvirus, or a combination thereof.
  • AAV adeno-associated virus
  • adenovirus adenovirus
  • lentivirus lentivirus
  • retrovirus lentivirus
  • herpes virus SV40
  • poxvirus poxvirus
  • 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).
  • the dosage form of the composition is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the dosage form of the composition is a liquid preparation.
  • the dosage form of the composition is an injection dosage form.
  • 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.
  • the composition is a cell preparation.
  • the expression vector of the gene editing protein and the expression vector of gRNA are the same vector or different vectors.
  • 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.
  • the content of the component (a) in the composition is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.
  • the content of the component (b) is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.
  • the content of the component (c) in the composition is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.
  • 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:
  • the gRNA guide gene editing protein specifically binds to the mRNA of the Ptbp1 gene.
  • the kit further includes:
  • first container, the second container, and the third container are the same or different containers.
  • the medicine in the first container is a unilateral preparation containing gene editing protein or its expression vector.
  • the medicine in the second container is a unilateral preparation containing gRNA or its expression vector.
  • the medicine in the third container is a single preparation containing other medicines for preventing and/or treating neurodegenerative diseases.
  • the dosage form of the drug is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • 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.
  • 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 .
  • 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.
  • composition or kit in another preferred embodiment, (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:
  • astrocytes are cultured to promote the differentiation of astrocytes into excitatory neurons.
  • the excitatory neurons include dopamine neurons.
  • the astrocytes include striatal astrocytes.
  • the astrocytes are astrocytes of brain tissue.
  • the astrocytes are cells in vitro.
  • the effect of the concentration of the gene or its encoded protein Ptbp1 inhibitors > 1 ⁇ 10 13, preferably, 1 ⁇ 10 13 -1 ⁇ 10 14.
  • 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.
  • the subject includes a human or non-human mammal suffering from a neurodegenerative disease.
  • 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:
  • test group 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;
  • the expression level of Ptbp1 is obtained by qPCR.
  • the method further includes the steps:
  • step (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.
  • the method includes step (c): applying the candidate compound determined in step (a) to a mammalian model, and determining its effect on the mammal.
  • the mammal is a mammal suffering from a neurodegenerative disease.
  • the "significantly lower” means E1/E0 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the "significantly lower” means that A1/A0 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the cells include astrocytes.
  • the cells include striatal astrocytes.
  • the cell is a cell cultured in vitro.
  • the method is non-diagnostic and non-therapeutic.
  • Figure 1 shows the direct conversion of astrocytes into functional neurons using CasRx-mediated Ptbp1 knockdown. among them,
  • FIG. 1 Schematic diagram of the process of using CasRx to mediate Ptbp1 knockdown.
  • FIG. 1 Schematic diagram of injection process.
  • Vector 1 AAV-GFAP-mCherry
  • 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.
  • FIG. 2 shows that CasRx-mediated Ptbp1 knockdown can reprogram astrocytes into dopamine neurons in a mouse model of 6-OHDA-induced Parkinsonism.
  • (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.
  • (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.
  • 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.
  • 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: 50 ⁇ m.
  • DAT Dopamine transporter staining shows the reduction of dopamine fibers (green) on the same side as the injection of 6-OHDA. Scale bar: 1mm.
  • astrocytes such as striatum Astrocytes
  • excitatory neurons such as dopamine neurons
  • neurodegenerative diseases such as Parkinson's disease
  • 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.
  • biochemical support such as forming a blood-brain barrier
  • 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 protru
  • 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.
  • 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 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.
  • 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.
  • the gene editor includes a DNA gene editor and an RNA gene editor.
  • the gene editor of the present invention includes a gene editing protein and optionally gRNA.
  • 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).
  • the gene editing protein includes, but is not limited to Cas13 (such as CasRx), CRISPR/Cas9, Cpf1, SaCas9, Cas13a, Cas13b, and Cas13c.
  • protein of the present invention refers 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant ptbp1 polypeptide. Generally speaking, there are the following steps:
  • the ptbp1 polynucleotide sequence can be inserted into a recombinant expression vector.
  • 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.
  • 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.
  • 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • 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.
  • 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.
  • 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.
  • 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 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 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 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.
  • 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.
  • 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.
  • ITR inverted terminal repeat
  • 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.
  • 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).
  • a human helper virus such as adenovirus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a virus such as adeno-associated virus
  • 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 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.
  • 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.
  • 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
  • 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).
  • 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.
  • a gene editor including gene editing protein and gRNA
  • the present invention found for the first time that the induction of dopamine neurons alleviated the motor dysfunction in the Parkinsonian mouse model.
  • 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.
  • 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 ⁇ g of gRNA-CasRx-GFP expressing vector was used for transient transfection. The control plasmid expresses non-targeted guidance.
  • 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 (2 ⁇ l per injection) and 1.6 ⁇ 10e13 (2-3 ⁇ l per injection). Inject AAV into the striatum (AP+0.8mm, ML ⁇ 1.6mm and DV-2.8mm).
  • 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 ⁇ m. Before immunofluorescence staining, the brain sections were washed thoroughly with 0.1M phosphate buffer (PB).
  • PB 0.1M phosphate buffer
  • Electrophysiological recordings after AAV injection electrophysiological recordings 5-6 weeks, 3 as previously described.
  • 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].
  • aCSF NMDG artificial cerebrospinal fluid
  • HEPES 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].
  • 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.
  • 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.
  • apomorphine A4393, Sigma-Aldrich
  • 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.
  • mice 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.
  • Example 1 Knockdown of Ptbp1 by CasRx can directly convert astrocytes into functional neurons
  • 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).
  • Example 2 CasRx-mediated knockdown of Ptbp1 can convert astrocytes into dopamine neurons in a 6-OHDA-induced 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.
  • 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).
  • 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.

Abstract

L'invention concerne une application d'un inhibiteur de Ptbp1 dans la prévention et/ou le traitement d'une maladie neurodégénérative. Plus particulièrement, l'invention concerne l'utilisation d'un inhibiteur pour un gène Ptbp1 ou une protéine codée correspondante, utilisé pour préparer une composition ou une préparation, la composition ou la préparation étant utilisée pour prévenir et/ou traiter une maladie neurodégénérative. En inhibant l'expression ou l'activité du gène Ptbp1 ou de la protéine codée correspondantes dans des astrocytes du tissu cérébral, les astrocytes peuvent être efficacement induits pour être différenciés en neurone excitateur, ce qui permet de prévenir et/ou de traiter la maladie neurodégénérative.
PCT/CN2019/101201 2019-08-16 2019-08-16 Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie neurodégénérative WO2021031025A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/101201 WO2021031025A1 (fr) 2019-08-16 2019-08-16 Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie neurodégénérative

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/101201 WO2021031025A1 (fr) 2019-08-16 2019-08-16 Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie neurodégénérative

Publications (1)

Publication Number Publication Date
WO2021031025A1 true WO2021031025A1 (fr) 2021-02-25

Family

ID=74660441

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/101201 WO2021031025A1 (fr) 2019-08-16 2019-08-16 Application d'un inhibiteur de ptbp1 dans la prévention et/ou le traitement d'une maladie neurodégénérative

Country Status (1)

Country Link
WO (1) WO2021031025A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023018856A1 (fr) * 2021-08-11 2023-02-16 Arbor Biotechnologies, Inc. Systèmes d'édition génétique comprenant un arn guide ciblant la protéine de liaison du tractus polypyrimidine 1 (ptbp1) et leurs utilisations

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950557A (zh) * 2016-04-22 2016-09-21 中国科学院生物物理研究所 一种控制人神经细胞重编程的信号通路及其应用
WO2018208998A1 (fr) * 2017-05-10 2018-11-15 The Regents Of The University Of California Édition dirigée d'arn cellulaire par administration nucléaire de crispr/cas9

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950557A (zh) * 2016-04-22 2016-09-21 中国科学院生物物理研究所 一种控制人神经细胞重编程的信号通路及其应用
WO2018208998A1 (fr) * 2017-05-10 2018-11-15 The Regents Of The University Of California Édition dirigée d'arn cellulaire par administration nucléaire de crispr/cas9

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Dopaminergic neurons conjured from astrocytes restore motion", 5 July 2019 (2019-07-05), pages 1 - 4, XP055782134, Retrieved from the Internet <URL:https://www.alzforum.org/news/conference-coverage/dopaminergic-neurons-conjured-astrocytes-restore-motion> *
ANONYMOUS: "Getting rid of alpha-synuclein-active and passive immunotherapy, or maybe targeting LRRK2", 8 October 2018 (2018-10-08), pages 1 - 4, XP055782956, Retrieved from the Internet <URL:https://progress.im/en/content/getting-rid-alpha-synuclein-%E2%80%93-active-and-passive-immunotherapy-or-maybe-targeting-lrrk2> *
GASCON SERGIO; MASSERDOTTI GIACOMO; RUSSO GIANLUCA LUIGI; GOTZ MAGDALENA: "Direct neuronal reprogramming:achievements, hurdles, and new roads to success.", CELL STEM CELL, vol. 21, no. 1, 6 July 2017 (2017-07-06), pages 18 - 34, XP085122158, ISSN: 1934-5909, DOI: 10.1016/j.stem.2017.06.011 *
HU JING , QIAN HAO , XUE YUANCHAO , FU XIANG-DONG: "PTB/nPTB:master regulators of neuronal fate in mammals.", BIOPHYSICS REPORTS, vol. 4, no. 4, 1 August 2018 (2018-08-01), pages 204 - 214, XP055782111, ISSN: 2364-3439, DOI: 10.1007/s41048-018-0066-y *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023018856A1 (fr) * 2021-08-11 2023-02-16 Arbor Biotechnologies, Inc. Systèmes d'édition génétique comprenant un arn guide ciblant la protéine de liaison du tractus polypyrimidine 1 (ptbp1) et leurs utilisations

Similar Documents

Publication Publication Date Title
WO2021031810A1 (fr) Application d&#39;un inhibiteur de ptbp1 dans la prévention et/ou le traitement d&#39;une maladie du système nerveux liée à la mort neuronale fonctionnelle
KR20200039617A (ko) 아데노-연관 바이러스 캡시드 변이체 및 이의 사용 방법
KR20160010617A (ko) 캡시드-변형된 raav3 벡터 조성물, 및 인간 간암의 유전자 요법에서의 용도
KR20120006073A (ko) 신경변성 질환에 대한 유전자 요법
CN117321213A (zh) 具有优选表达水平的腺相关病毒组合物
WO2021032068A1 (fr) Application d&#39;un inhibiteur de ptbp1 dans la prévention et/ou le traitement d&#39;une maladie du système nerveux liée à la mort neuronale fonctionnelle
CN113383010A (zh) 具有经工程化改造的启动子的共济蛋白表达构建体及其使用方法
CN109843913B (zh) 神经肽表达载体以及用于治疗癫痫的方法
US20210301305A1 (en) Engineered untranslated regions (utr) for aav production
WO2020211843A1 (fr) Nouveau type de composition enzymatique
CN111601620A (zh) 用于21-羟化酶缺乏症的腺相关病毒基因疗法
CN112386699A (zh) Ptbp1抑制剂在预防和/或治疗功能性神经元死亡相关的神经***疾病中的应用
WO2021031025A1 (fr) Application d&#39;un inhibiteur de ptbp1 dans la prévention et/ou le traitement d&#39;une maladie neurodégénérative
WO2023104028A1 (fr) Traitement de maladie neurologique médiée par un arn non codant
WO2022171167A1 (fr) Utilisation d&#39;une transdifférenciation de cellules gliales en neurones pour prévenir ou traiter des maladies associées à une perte de la fonction neuronale ou à la mort de neurones
JP2022512621A (ja) Ａａｖ産生タンパク質をコードする操作された核酸コンストラクト
WO2021081826A1 (fr) Applications d&#39;inhibiteur de ptbp1 dans la prévention et/ou le traitement de maladies rétiniennes.
JP2017123840A (ja) グルコーストランスポーター１発現用アデノ随伴ウイルスベクター
WO2024046393A1 (fr) Procédé de trans-différenciation de cellules non neuronales en neurones et son utilisation
WO2023184688A1 (fr) VARIANT FONCTIONNEL DE β-GALACTOSIDASE, VECTEUR D&#39;EXPRESSION DE β-GALACTOSIDASE HUMAINE MÉDIÉ PAR VAA ET UTILISATION ASSOCIÉE
KR20230123925A (ko) Neurod1 및 dlx2 벡터
JP2023543360A (ja) Ｄｌｘ２ベクター
CN116761812A (zh) Neurod1和dlx2载体
JP2023544264A (ja) 核酸構築物、ウイルスベクター及びウイルス粒子
KR20240028976A (ko) 뇌에서 높은 발현 수준을 갖는 aav 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19942544

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19942544

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19942544

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 02.09.2022)

122 Ep: pct application non-entry in european phase

Ref document number: 19942544

Country of ref document: EP

Kind code of ref document: A1