CN110237257B - Application of Ube3a ubiquitinated PP2A activator PTPA in treatment of Angel syndrome and autism - Google Patents

Abstract

The invention relates to application of Ube3a ubiquitinated PP2A activator PTPA in treatment of Angel syndrome and autism. The invention discloses a novel pathogenesis of the Angel syndrome and/or the autism or related diseases thereof, namely, the deficiency or the over-expression of Ube3a causes the abnormity of the activity of phosphotyrosine phosphatase activating factor on protein phosphatase 2A. Therefore, these factors can be used as targets for developing drugs for alleviating or treating the angel syndrome and/or autism or related diseases thereof. These factors may also serve as markers for the diagnosis, assessment of the angel syndrome and/or autism or its related diseases.

Description

Ube3a ubiquitinates PP2A activator PTPA in the treatment of Angelman syndrome and autism application in

technical field

The invention belongs to the fields of molecular biology and pharmacy, and more specifically, the invention relates to Ube3a regulating the activity of PP2A and its application in treating Angelman syndrome and autism.

Background technique

The UBE3A gene is located in the 15q11-13 region of human chromosome 15, and its encoded product Ube3a is the first E3 ubiquitin ligase found in the HECT (Homologous with E6-assiciated protein C-Terminus) family. Because there is an E6-binding domain in its protein structure, it is also called E6AP. So far, Ube3a has two functions: one is as an E3 ligase, which mediates the ubiquitination modification of the specific substrate it recognizes, and then regulates protein degradation, transportation, localization, etc.; the other is in the nucleus, Ube3a can Binds to hormone receptors and acts as an activator to regulate gene transcription.

In neurons, Ube3a specifically expresses maternally derived genes, while paternally derived genes are not or rarely expressed due to the production of an inverted RNA. The loss of Ube3a protein ubiquitin ligase function is the most important factor in the pathogenesis of Angelman syndrome (AS). Angelman syndrome was first discovered and reported by pediatrician Harry Angelman in 1965, and is characterized by developmental delay, language impairment, intellectual disability, and movement impairment. So far, four abnormal mutations in the genome have been found: deletion of the 15q11-q13 segment of the maternal chromosome 15, diploidy of the paternal chromosome 15, and abnormalities in the imprinted regulatory region of the 15q11-q13 segment of the maternal chromosome 15 , and a mutation in the Ube3a gene located in the 15q11-q13 segment of maternal chromosome 15.

Autism spectrum disorder (ASD), also known as autism spectrum disorder, is a class of neurodevelopmental disorders with heterogeneous pathogenesis. The main features of the disorder are: social impairment, communication impairment, repetitive behaviors, narrow areas of interest, and delayed language development. Children with autism usually develop it before the age of 3, and it is four times more common in males than in females. Although the pathogenesis is not very clear, but 25% of children are accompanied by de novo gene mutation or abnormal copy number of chromosome.

Gene copy number variation is one of the important causes of autism, and 15q11-13 chromosome copy number abnormality accounts for 1%-3% of all autistic patients. Autistic phenotypes were shown in some populations with maternally derived 15q11-13 interchromosomal duplication (diploid), whereas mutations in isodicentric chromosome 15 (triple) were all autistic. This type of chromosomal abnormality is also known as 15q11-13 duplication syndrome. In this segment, UBE3A was the only gene expressing only the maternal allele. Not only that, but in the mouse model, mice with three copies of Ube3a showed typical autism features. The above studies prove that UBE3A is a susceptibility gene for autism. Therefore, too much or too little protein level of Ube3a leads to abnormal development and function of the nervous system, and the study of the pathogenic mechanism of Ube3a is of great significance for the treatment of neurodevelopmental diseases caused by Ube3a.

Although some substrates of Ube3a have been reported, none of these substrates can well explain the phenotype of neurodevelopmental diseases caused by Ube3a abnormalities, and there is no effective drug treatment to intervene in the abnormality of the gene segment where Ube3a is located. resulting disease.

Therefore, there is still a need in this field to find direct substrates of Ube3a to seek more effective interventions for the treatment of neurodevelopmental diseases caused by Ube3a.

Contents of the invention

The purpose of the present invention is to provide Ube3a regulating the activity of PP2A and its application in the treatment of Angelman syndrome and/or autism-related diseases.

In the first aspect of the present invention, the use of inhibitors of protein phosphatase 2A (PP2A) or phosphotyrosine phosphatase activator (PTPA) is provided for preparing a pharmaceutical composition for alleviating or treating Angelman syndrome.

In another preferred example, the pharmaceutical composition is also used for preventing and/or treating Angelman syndrome. In a preferred example, the inhibitor is selected from: small molecule compounds that specifically inhibit protein phosphatase 2A or phosphotyrosine phosphatase activator; specifically interfere with the activation of protein phosphatase 2A or phosphotyrosine phosphatase Molecules that interfere with gene expression of factors; gene editing reagents that specifically knock out protein phosphatase 2A or phosphotyrosine phosphatase activator; or antibodies that specifically bind to protein phosphatase 2A or phosphotyrosine phosphatase activator or ligand.

In another preferred example, the inhibitor is a small molecule compound that specifically inhibits the activity of protein phosphatase 2A, which is selected from: inhibitor LB-100.

In another preferred example, the inhibitor is a reagent that specifically up-regulates Ube3a protein (including promoting its expression or increasing its activity), which degrades phosphotyrosine phosphatase activator by up-regulating Ube3a protein, thereby inhibiting protein Phosphatase 2A.

In another preferred example, the reagent for specifically up-regulating Ube3a protein is an expression construct (expression plasmid) that overexpresses Ube3a.

In another preferred example, the inhibitor further includes Ube3a protein or a vector expressing Ube3a protein.

In another preferred example, the vector expressing Ube3a protein includes a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated viral vector, lentiviral vector, or a combination thereof.

In another preferred embodiment, the protein includes a full-length protein or a protein fragment.

In another preferred embodiment, the Ube3a protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.

In another preferred example, the Ube3a protein further includes derivatives of the Ube3a protein.

In another preferred example, the derivatives of the Ube3a protein include modified Ube3a proteins, protein molecules whose amino acid sequences are homologous to natural Ube3a proteins and have natural Ube3a protein activity, dimers or multimers of Ube3a proteins, A fusion protein containing the amino acid sequence of Ube3a protein.

In another preferred embodiment, the modified Ube3a protein is PEGylated Ube3a protein.

In another preferred example, the "protein molecule whose amino acid sequence is homologous to natural Ube3a protein and has natural Ube3a protein activity" means that its amino acid sequence has a homology of ≥85% compared with Ube3a protein, preferably ≥90% homology, more preferably ≥95% homology, most preferably ≥98% homology; and a protein molecule with natural Ube3a protein activity.

In another preference, the Ube3a protein is selected from the following group:

(A) a polypeptide having an amino acid sequence as shown in SEQ ID NO.:18;

(B) pass the amino acid sequence shown in SEQ ID NO.: 18 through one or several (usually 1-60, preferably 1-30, more preferably 1-20, optimally 1-10 A) ECM1 protein derivatives formed by substitution, deletion or addition of amino acid residues, or active fragments thereof;

(C) Compared with the amino acid sequence shown in SEQ ID NO.:18, the sequence has a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and optimally ≥99% of Ube3a protein derivatives substances, or active fragments thereof.

In another preferred example, the interfering molecule that specifically interferes with the gene expression of the phosphotyrosine phosphatase activator is shRNA.

In another preferred example, the shRNA targets: CAGTTCCAGATATGGGCAAATGGAA (SEQ ID NO: 1), CCTGGTATGCC AAACTTGATCAGGA (SEQ ID NO: 2) or TCAAGGTGTTTGATAGGTATCTT GA (SEQ ID NO: 3); preferably, the shRNA has the sense strand of SEQ ID NO:4 and the antisense strand of SEQ ID NO:5, or the sense strand of SEQ ID NO:6 and the antisense strand of SEQ ID NO:7; or the Sense strand and antisense strand of SEQ ID NO:9.

In another aspect of the present invention, the physiological function and mechanism of protein phosphatase 2A or phosphotyrosine phosphatase activating factor in cells are provided for preparing a pharmaceutical composition for preventing and/or treating autism or related diseases.

In the second aspect of the present invention, it provides the use of protein phosphatase 2A or phosphotyrosine phosphatase activator for screening drugs for alleviating or treating Angelman syndrome.

In a third aspect of the present invention, there is provided a method for screening potential substances for alleviating or treating Angelman syndrome, the method comprising: (1) treating expressed protein phosphatase 2A and/or phosphotyrosine phosphatase with a candidate substance and (2) detecting the expression or activity of protein phosphatase 2A and/or phosphotyrosine phosphatase activator in the system; wherein, if the candidate substance can reduce protein phosphatase 2A and/or The expression or activity of phosphotyrosine phosphatase activator indicates that the candidate substance is a potential substance for relieving or treating Angelman syndrome.

In a preferred example, step (1) includes: in the test group, adding the candidate substance to the system expressing protein phosphatase 2A and/or phosphotyrosine phosphatase activator; step (2) includes: detection test The expression or activity of protein phosphatase 2A and/or phosphotyrosine phosphatase activator in the system of the group, and compared with the control group, wherein the control group is the expression of protein phosphatase 2A and phosphatase 2A without adding the candidate substance and/or phosphotyrosine phosphatase activator system; if the expression or activity of protein phosphatase 2A and/or phosphotyrosine phosphatase activator in the test group is statistically lower than that in the control group, it indicates the candidate It is a potential substance for the alleviation or treatment of Angelman syndrome.

In another preferred example, the statistically lower is preferably significantly lower, such as lower than 20%, preferably lower than 50%; more preferably lower than 80%.

In another preferred example, Ube3a protein is also expressed in the system, and the method also includes: detecting the expression of Ube3a protein in the system; wherein, if the candidate substance can be inhibited by increasing the expression of Ube3a (preferably Significantly inhibit, such as inhibiting more than 20%, preferably inhibiting more than 50%; more preferably inhibiting more than 80%) protein phosphatase 2A and/or phosphotyrosine phosphatase activator, then it shows that the candidate substance is relieving or treating Potential substance for Angelman syndrome.

In another preferred embodiment, the system is selected from: cell systems (such as cells or cell cultures expressing protein phosphatase 2A, phosphotyrosine phosphatase activator and/or Ube3a), subcellular systems, solution systems , tissue system, organ system or animal system.

In another preferred example, the candidate substances include (but are not limited to): small molecular compounds designed for protein phosphatase 2A gene or protein, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands), etc. or regulatory molecules designed for protein phosphatase 2A gene or protein involved in the signaling pathway or its upstream or downstream protein (such as Ube3a, phosphotyrosine phosphatase activator).

In another preferred example, the method further includes: conducting further cell experiments and/or animal experiments on the obtained potential substances to further select and determine substances useful for alleviating or treating Angelman syndrome from candidate substances.

In the fourth aspect of the present invention, the use of a reagent specifically recognizing protein phosphatase 2A gene or protein is provided for preparing reagents or kits for diagnosing or evaluating the prognosis of Angelman syndrome.

In a preferred example, the reagent for specifically recognizing protein phosphatase 2A gene or protein is selected from: primers for specifically amplifying protein phosphatase 2A gene; probes for specifically recognizing protein phosphatase 2A gene; or specific Antibodies or ligands that specifically bind protein phosphatase 2A.

In the fifth aspect of the present invention, the use of reagents for specifically recognizing phosphotyrosine phosphatase activator genes or proteins is provided for preparing reagents or kits for diagnosing or evaluating the prognosis of Angelman syndrome.

In a preferred example, the reagent for specifically recognizing phosphotyrosine phosphatase activator gene or protein is selected from: primers for specifically amplifying phosphotyrosine phosphatase activator gene; A probe for an acid phosphatase activator gene; or an antibody or a ligand specifically binding to a phosphotyrosine phosphatase activator.

In the sixth aspect of the present invention, a use of a substance is provided for preparing a composition or preparation for preventing and/or treating autism or its related diseases, wherein the substance is selected from the group consisting of: (a) Ube3a gene or an inhibitor of its protein, (b) PP2A gene, or its protein or its promoter, (c) PTPA gene, or its protein or its promoter, or a combination thereof.

In another preferred example, the substance further includes (d) an accelerator for the assembly of PP2A holoenzyme.

In another preferred example, the PP2A protein also includes an active fragment of the PP2A protein.

In another preferred example, the autism or related diseases are selected from the group consisting of autism, 15q11-13 duplication syndrome, or a combination thereof.

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

(i) inhibit the phosphorylation level of ERK1/2;

(ii) reducing the activity of the MAPK signaling pathway;

(iii) Increase the content of PP2A holoenzyme.

In another preferred embodiment, the inhibitor of the Ube3a gene or its protein is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, or combinations thereof.

In another preferred example, the PP2A promoter or PTPA promoter refers to a substance that can increase the activity and/or content of the PP2A or PTPA gene or its protein in vivo or in vitro; the substance can be artificially synthesized or natural Compounds, proteins, nucleotides, etc.

In another preferred example, the PP2A or PTPA promoter includes substances that promote the expression of PP2A or PTPA.

In another preferred example, the PP2A promoter includes a PP2A protein promoter and/or a PP2A gene promoter.

In another preferred example, the PTPA enhancer includes a PTPA protein enhancer and/or a PTPA gene enhancer.

In another preferred example, the promotion of PP2A or PTPA expression or activity refers to increasing the expression or activity of PP2A or PTPA gene or protein by ≥10%, preferably ≥20%, more preferably ≥70%.

In another preferred embodiment, the PP2A promoter is selected from the group consisting of small molecule compounds, vectors expressing PP2A, or combinations thereof.

In another preferred embodiment, the PP2A accelerator is selected from the group consisting of FTY720, C2ceramide, or a combination thereof.

In another preferred example, the vector for expressing PP2A includes a viral vector.

In another preferred embodiment, the PTPA promoter is selected from the group consisting of small molecule compounds, vectors expressing PTPA, or combinations thereof.

In another preferred example, the vector for expressing PTPA includes a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated viral vector, lentiviral vector, or a combination thereof.

In another preferred embodiment, the protein includes a full-length protein or a protein fragment.

In another preferred embodiment, the PP2A gene or its protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.

In another preferred embodiment, the PTPA gene or its protein is derived from mammals, more preferably from rodents (such as mice, rats), primates and humans.

In another preferred example, the PP2A protein also includes derivatives of PP2A protein.

In another preferred example, the derivatives of the PP2A protein include modified PP2A proteins, protein molecules whose amino acid sequences are homologous to natural PP2A proteins and have natural PP2A protein activity, dimers or multimers of PP2A proteins, A fusion protein containing the amino acid sequence of PP2A protein.

In another preferred embodiment, the modified PP2A protein is PEGylated PP2A protein.

In another preferred example, the "protein molecule whose amino acid sequence is homologous to natural PP2A protein and has natural PP2A protein activity" means that its amino acid sequence has a homology of ≥85% compared with that of PP2A protein, preferably ≥90% homology, more preferably ≥95% homology, most preferably ≥98% homology; and a protein molecule having natural PP2A protein activity.

In another preferred example, the PP2A protein is selected from the following group:

(A) a polypeptide having an amino acid sequence as shown in SEQ ID NO.:19;

(B) pass the amino acid sequence shown in SEQ ID NO.:19 through one or more (usually 1-60, preferably 1-30, more preferably 1-20, optimally 1-10 1) PP2A protein derivatives formed by substitution, deletion or addition of amino acid residues, or active fragments thereof;

(C) Compared with the amino acid sequence shown in SEQ ID NO.:19, the sequence has a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and optimally ≥99% of PP2A protein derivatives substances, or active fragments thereof.

In another preferred example, the PP2A gene encodes PP2A protein.

In another preferred example, the PP2A gene is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.:19;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.:20;

(c) a polynucleotide whose nucleotide sequence is ≥95% (preferably ≥98%) homologous to the sequence shown in SEQ ID NO.:20 and encodes the polypeptide shown in SEQ ID NO.:19;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

In another preferred example, the PTPA protein also includes derivatives of PTPA protein.

In another preferred example, the derivatives of the PTPA protein include modified PTPA proteins, protein molecules whose amino acid sequences are homologous to natural PTPA proteins and have natural PTPA protein activity, dimers or multimers of PTPA proteins, A fusion protein containing the amino acid sequence of PTPA protein.

In another preferred embodiment, the modified PTPA protein is PEGylated PTPA protein.

In another preferred example, the "protein molecule whose amino acid sequence is homologous to natural PTPA protein and has natural PTPA protein activity" means that its amino acid sequence has a homology of ≥ 85% compared with PTPA protein, preferably ≥90% homology, more preferably ≥95% homology, most preferably ≥98% homology; and a protein molecule with natural PTPA protein activity.

In another preference, the PTPA protein is selected from the following group:

(A) a polypeptide having an amino acid sequence as shown in SEQ ID NO.:21;

(B) pass the amino acid sequence shown in SEQ ID NO.:21 through one or several (usually 1-60, preferably 1-30, more preferably 1-20, optimally 1-10 1) PTPA protein derivatives formed by substitution, deletion or addition of amino acid residues, or active fragments thereof;

(C) Compared with the amino acid sequence shown in SEQ ID NO.:21, the sequence has a homology of ≥90%, preferably ≥95%, more preferably ≥98%, and optimally ≥99% of PTPA protein derivatives substances, or active fragments thereof.

In another preferred example, the PTPA gene encodes PTPA protein.

In another preference, the PTPA gene is selected from the following group:

(a) a polynucleotide encoding a polypeptide as shown in SEQ ID NO.:21;

(b) a polynucleotide whose sequence is as shown in SEQ ID NO.:22;

(c) a polynucleotide whose nucleotide sequence is ≥95% (preferably ≥98%) homologous to the sequence shown in SEQ ID NO.:22 and encodes the polypeptide shown in SEQ ID NO.:21;

(d) A polynucleotide complementary to the polynucleotide described in any one of (a)-(c).

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

In another preferred embodiment, the pharmaceutical composition contains (i) an inhibitor of Ube3a gene or its protein; PP2A gene or its protein or its promoter; PTPA gene or its protein or its promoter; or a combination thereof and (ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semi-solid.

In another preferred example, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.

In another preferred example, in the pharmaceutical composition, the component (i) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.

In another preferred example, the composition further includes additional components for preventing and/or treating autism or its related diseases.

In another preferred example, the additional components for preventing and/or treating autism or its related diseases are selected from the group consisting of antagonist morphine drugs-Nalterxone, sodium valproate, carbamazepine, Lithium carbonate, propranolol, clonazepam, clomipramine, imipramine, serotonin reuptake inhibitors (SSRS), buspirone, chlorpromazine, sulpiride, haloperidol, risperidone (Ritalin), clozapine, olanzapine, methylphenidate (Ritalin), pemoline, or combinations thereof.

In another preferred embodiment, the serotonin reuptake inhibitor (SSRS) is selected from the group consisting of fluoxetine, sertraline, fluvoxamine (Lanshi), or a combination thereof.

In another preferred example, the composition or preparation can be used alone or in combination in the prevention and/or treatment of autism or its related diseases.

In another preferred example, the combined use includes: combined use with other drugs for preventing and/or treating autism or its related diseases.

In another preferred example, the other drugs for the prevention and/or treatment of autism or its related diseases are selected from the group consisting of: antagonistic morphine drug-Nalterxone (Nalterxone), sodium valproate, carbamazepine, lithium carbonate , propranolol, clonazepam, clomipramine, imipramine, serotonin reuptake inhibitors (SSRS), buspirone, chlorpromazine, sulpiride, haloperidol, risperidone (vitamin Siton), clozapine, olanzapine, methylphenidate (Ritalin), pemoline, or combinations thereof.

In another preferred embodiment, the serotonin reuptake inhibitor (SSRS) is selected from the group consisting of fluoxetine, sertraline, fluvoxamine (Lanshi), or a combination thereof.

In the seventh aspect of the present invention, a pharmaceutical composition is provided, comprising:

(a1) The first active ingredient for preventing and/or treating autism or its related diseases, the first active ingredient is selected from the group consisting of (i) inhibitors of Ube3a gene or its protein, (ii) PP2A gene , or a protein or a promoter thereof, (iii) the PTPA gene, or a protein or a promoter thereof, or a combination thereof; and

(b) A pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition further includes (a2) a second active ingredient for preventing and/or treating diseases related to liver fibrosis, and the second active ingredient includes: other Medications for autism or its related disorders.

In another preferred example, in the pharmaceutical composition, the component (a1) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.

In another preferred example, in the pharmaceutical composition, the component (a2) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.

In another preferred example, the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.

In another preferred example, the pharmaceutical composition further includes an additional component that inhibits the phosphorylation level of ERK1/2.

In another preferred example, the additional component that inhibits the phosphorylation level of ERK1/2 is selected from the group consisting of MK-8353, U0126, SCG772984, or a combination thereof.

In another preferred example, the pharmaceutical composition further includes an additional component that reduces the activity of the MAPK signaling pathway.

In another preferred example, the additional component that reduces the activity of the MAPK signaling pathway is selected from the group consisting of PD98059, Selumetinib, PD0325901, or a combination thereof.

In another preferred example, the other drugs for the prevention and/or treatment of autism or its related diseases are selected from the group consisting of: antagonistic morphine drug-Nalterxone (Nalterxone), sodium valproate, carbamazepine, lithium carbonate , propranolol, clonazepam, clomipramine, imipramine, serotonin reuptake inhibitors (SSRS), buspirone, chlorpromazine, sulpiride, haloperidol, risperidone (vitamin Siton), clozapine, olanzapine, methylphenidate (Ritalin), pemoline, or combinations thereof.

In another preferred embodiment, the serotonin reuptake inhibitor (SSRS) is selected from the group consisting of fluoxetine, sertraline, fluvoxamine (Lanshi), or a combination thereof.

In another preferred example, the pharmaceutical composition may be a single compound or a mixture of multiple compounds.

In another preferred example, the pharmaceutical composition is used to prepare medicines or preparations for treating or preventing autism or its related diseases.

In another preferred example, the pharmaceutical dosage form is oral administration or non-oral administration dosage form.

In another preferred example, the oral dosage form is tablet, powder, granule or capsule, or emulsion or syrup.

In another preferred example, the non-oral dosage form is injection or injection.

In another preferred example, the total content of the active ingredient (a1) and the active ingredient (a2) is 1-99wt%, more preferably 5-90wt%, of the total weight of the composition.

In the eighth aspect of the present invention, a kit is provided, comprising:

(i) the first container, and the active ingredient (a1) inhibitor of Ube3a gene or its protein in the first container; PP2A gene, or its protein or its promoter; PTPA gene, or its protein or its promoter ; or a combination thereof, or a medicament containing the active ingredient (a1).

In another preferred example, the kit also includes:

(ii) a second container, and the active ingredient (a2) located in the second container and other medicines for preventing and/or treating autism or its related diseases, or medicines containing the active ingredient (a2).

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

In another preferred example, the medicine in the first container is a single preparation containing the active ingredient (a1).

In another preferred example, the medicine in the second container is a single preparation containing the active ingredient (a2).

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

In another preferred example, the kit also contains an instruction, which records the combined administration of the active ingredient (a1) and the active ingredient (a2) so as to (i) prevent and/or treat autism or its related diseases; And/or (ii) inhibit the phosphorylation level of ERK1/2; and/or (iii) reduce the activity of MAPK signaling pathway.

In another preferred example, the dosage forms of the preparation containing the active ingredient (a1) or the preparation containing the active ingredient (a2) include capsules, tablets, suppositories, or intravenous injections, respectively.

In another preferred example, in the preparation containing the active ingredient (a1), the concentration of the inhibitor of the Ube3a gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably Preferably 0.5-2mg/kg body weight.

In another preferred example, in the preparation containing the active ingredient (a1), the concentration of the PP2A gene or its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight , more preferably 0.5-2mg/kg body weight.

In another preferred example, in the preparation containing the active ingredient (a1), the concentration of the PTPA gene or its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight , more preferably 0.5-2mg/kg body weight.

In the ninth aspect of the present invention, there is provided a method for (a) inhibiting the phosphorylation level of ERK1/2; and/or (b) reducing the activity of the MAPK signaling pathway, comprising the steps of:

In the presence of (a) inhibitor of Ube3a gene or its protein, (b) PP2A gene, or its protein or its promoter, (c) PTPA gene, or its protein or its promoter, or a combination thereof, culture Brain tissue cells, thereby (a) inhibiting the phosphorylation level of ERK1/2; and/or (b) reducing the activity of the MAPK signaling pathway.

In another preferred example, the brain tissue cells are derived from the cortex of mammals.

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

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

In another preferred example, the brain tissue cells are brain tissue cells (such as neurons) cultured in vitro.

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

In another preferred embodiment, the method is therapeutic.

In another preferred example, the concentration of the inhibitor of the Ube3a gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.

In another preferred example, the concentration of the PP2A gene, or its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.

In another preferred example, in the preparation containing the active ingredient (a1), the concentration of the PTPA gene, or its protein or its promoter is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg Body weight, more preferably 0.5-2 mg/kg body weight.

The tenth aspect of the present invention provides a method for screening potential therapeutic agents for autism or its related diseases, comprising:

(a) In the test group, in the culture system, in the presence of the test compound, cultivate the cells expressing the Ube3a gene for a period of time T1, and detect the expression level E1 of the Ube3a gene in the culture system of the test group;

And in the control group in which there is no said test compound and other conditions are the same, the expression level E2 of Ube3a gene in the culture system of the control group is detected; and

(b) Comparing E1 and E2, if E1 is significantly lower than E2, it means that the test compound is a potential therapeutic agent for autism or related diseases.

In another preferred example, the "significantly lower than" refers to E1/E2≤1/2, preferably ≤1/3, more preferably ≤1/4.

In another preferred example, the cells include brain tissue cells.

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

In another preferred example, the method includes step (c): administering the potential therapeutic agent determined in step (a) to mammals, so as to determine its effect on autism or related diseases in mammals.

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

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

The eleventh aspect of the present invention provides a method for screening potential therapeutic agents for autism or its related diseases, comprising:

(a) In the test group, in the culture system, in the presence of the test compound, cultivate cells expressing PP2A and/or PTPA genes for a period of time T1, and detect PP2A and/or PTPA in the culture system of the test group Gene expression level E1;

And in the control group in which the test compound does not exist and other conditions are the same, the expression level E2 of PP2A and/or PTPA gene in the culture system of the control group is detected; and

(b) Comparing E1 and E2, if E1 is significantly higher than E2, it means that the test compound is a potential therapeutic agent for autism or related diseases.

In another preferred example, the "significantly lower than" refers to E1/E2≥2, preferably ≥3, more preferably ≥4.

In another preferred example, the cells include brain tissue cells.

In another preferred example, the cells are brain tissue cells cultured in vitro.

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

In another preferred example, the method includes step (c): administering the potential therapeutic agent determined in step (a) to mammals, so as to determine its effect on autism or related diseases in mammals.

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

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

The twelfth aspect of the present invention provides a method for preventing and/or treating autism or its related diseases, comprising the steps of:

To a subject in need, administer (a) an inhibitor of Ube3a gene or its protein, (b) PP2A gene, or its protein or its promoter, (c) PTPA gene, or its protein or its promoter, or a combination thereof, The pharmaceutical composition described in the seventh aspect of the present invention, or the kit described in the eighth aspect of the present invention.

In another preferred example, the administration includes oral administration.

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

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

In another preferred example, the dosage of the inhibitor of Ube3a gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.

In another preferred example, the administration frequency of the inhibitor of the Ube3a gene or its protein is 15-60 times/month, preferably 20-40 days/time.

In another preferred example, the administration time of the inhibitor of Ube3a gene or its protein is 10-100 days, preferably 20-80 days, most preferably 30-50 days.

In another preferred example, the dose of the promoter of the PP2A gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.

In another preferred example, the administration frequency of the promoter of the PP2A gene or its protein is 15-60 times/month, preferably 20-40 days/time.

In another preferred example, the administration time of the promoter of the PP2A gene or its protein is 10-100 days, preferably 20-80 days, most preferably 30-50 days.

In another preferred example, the administration dose of the promoter of the PTPA gene or its protein is 0.00001-10 mg/kg body weight, preferably 0.5-5 mg/kg body weight, more preferably 0.5-2 mg/kg body weight.

In another preferred example, the administration time of the promoter of the PTPA gene or its protein is 10-100 days, preferably 20-80 days, most preferably 30-50 days.

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 embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

FIG. 1A , Western blot detection of changes in PTPA protein level in the brain of Ube3a-deficient mice.

Fig. 1B, Statistical histogram of PTPA protein level changes in the brains of WT and AS mice.

Figure 2A GST pull down and western blot to detect the interaction between UBE3A and PTPA

Figure 2B Western blot detection of ubiquitylation of PTPA by UBE3A in vitro.

Figure 3. PP2A activity detection in the brains of WT and AS mice, the dot plot of the increase of PP2A activity.

Fig. 4. Administration time diagram of PP2A inhibitor LB-100 treated by intraperitoneal injection in AS mice.

Figure 5A, PP2A inhibitor LB-100 can increase the duration of AS mice in the string suspension test.

Fig. 5B, PP2A inhibitor LB-100 can improve the motor learning ability of AS mice in the rotarod test.

Fig. 6A. Morphological images of dendritic spines in cerebellar Purkinje cells of WT, AS, WT/LB-100 and AS/LB-100 mice detected by Golgi staining.

Fig. 6B, the statistical diagram of the ratio of elongated dendritic spines of the four kinds of mice in A detected by morphological analysis.

Fig. 7A, Morphological images of dendritic spines detected in WT, AS, AS; Ptpa ^+/- and Ptpa ^+/- mouse cerebellar Purkinje cells by Golgi staining.

Fig. 7B, the statistical diagram of the ratio of three kinds of dendritic spines of the four kinds of mice in A detected by morphological analysis.

Fig. 8, Western Blot determination of the effect of three PTPA shRNAs on down-regulating the protein expression of PTPA.

Figure 9. Results of motor behavior of mice after PTPA-interfering shRNA virus injection.

FIG. 10 . The scope of infection after intracerebroventricular injection of PTPA-interfering shRNA virus.

FIG. 11A , Western Blot detection film scan of Ube3a protein level in HEK293T caused by transfer of Ube3a overexpression vector.

Figure 11B, PP2A activity detection Histogram of PP2A activity in HEK293T cells after Ube3a overexpression.

Fig. 12A, Western blot detection of the film scan and statistical bar graph of the phosphorylation level of ERK1/2 in AS mice.

FIG. 12B , Western Blot detection film scan of the phosphorylation level of ERK1/2 in HEK293T caused by transfer of Ube3a overexpression vector.

FIG. 13 . Western blot detection of the effects of PP2A inhibitors or agonists on the phosphorylation level of ERK1/2.

detailed description

After extensive and in-depth research, the inventors have revealed a new mechanism for the pathogenesis of Angelman syndrome, that is, the lack of Ube3a leads to the activation of phosphotyrosine phosphatase activator (PTPA) on protein phosphatase 2A (protein phosphatase2A, PP2A) activity. An exception occurs. Therefore, PTPA and PP2A can be used as new targets to develop drugs for alleviating or treating Angelman syndrome. PTPA and PP2A can also be used as markers for the diagnosis and prognosis of Angelman syndrome.

In addition, the present inventors have unexpectedly found that autism-related diseases can be prevented and/or treated by down-regulating the expression or activity of Ube3a, and/or up-regulating the expression or activity of PP2A, and/or up-regulating the expression or activity of PTPA, and , can also (i) inhibit the phosphorylation level of ERK1/2; and/or (ii) reduce the activity of MAPK signaling pathway. On this basis, the present inventors have completed the present invention.

As used herein, the structural formula of the term "FTY720" is

As used herein, the structural formula of the term "C2ceramide" is

As used herein, the structural formula of the term "MK-8353" is

如本文所用，术语“SCG772984”的结构式为

As used herein, the structural formula of the term "U0126" is

As used herein, the structural formula of the term "SCG772984" is

As used herein, the structural formula of the term "PD98059" is

As used herein, the structural formula of the term "Selumetinib" is

As used herein, the structural formula of the term "PD0325901" is

Ube3a protein and its encoding nucleic acid

The UBE3A gene is located in the 15q11-13 region of human chromosome 15, and its encoded product UBE3A is the first E3 ubiquitin ligase found in the HECT (Homologous with E6-assiciated protein C-Terminus) family. Because there is an E6-binding domain in its protein structure, it is also called E6AP. In neurons, UBE3A is an imprinted gene whose main expression is derived from the allele on the maternal chromosome; while the allele on the paternal chromosome is not expressed due to the influence of reverse transcribed non-coding RNA. The 11-13 region of the long arm of human chromosome 15 is homologous to the 7C segment sequence on mouse chromosome 7.

In mice, specific deletion of Ube3a can well mimic the human Angelman syndrome phenotype. In this AS model mouse, studies have found changes in the phosphorylation of some proteins, such as Ca/calmodulin-dependent protein kinase II (CaMKII) and ERK1/2.

The present invention relates to a Ube3a protein and its variants. In a preferred example of the present invention, the amino acid sequence of the Ube3a protein is shown in SEQ ID NO.:18. The Ube3a protein of the present invention or its promoter can be used as an inhibitor of PP2A and/or PTPA for the prevention, alleviation and/or treatment of Angelman syndrome; in addition, the Ube3a gene of the present invention or its protein inhibitor can also be used for ( a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; (c) reducing the activity of MAPK signaling pathway.

The present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably More than 98%, such as 99%) homologous polypeptides or proteins with the same or similar functions.

Among them, SEQ ID NO.: 18 is the mouse Ube3a protein (95% homology with the human Ube3a protein); SEQ ID NO.: 23 is the human Ube3a protein.

The "same or similar function" mainly refers to: preventing, alleviating and/or treating Angelman syndrome.

The protein of the present invention can be recombinant protein, natural protein, synthetic protein. The proteins of the present invention may be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the proteins of the invention may be glycosylated, or may be non-glycosylated. The proteins of the invention may or may not include an initial methionine residue.

The present invention also includes Ube3a protein fragments and analogs having Ube3a protein activity. As used herein, the terms "fragment" and "analogue" refer to proteins that substantially maintain the same biological function or activity of the native Ube3a protein of the present invention.

The mutein fragments, derivatives or analogs of the present invention may be (i) muteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a mutein with a substitution group in one or more amino acid residues, or (iii) a mature mutein combined with another compound (such as an elongated mutein A mutein formed by fusion of a compound with a half-life, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the mutein sequence (such as a leader sequence or secretory sequence or used to purify the mutein The sequence or protein sequence, or the fusion protein formed with the antigen IgG fragment). Such fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein. In the present invention, the conservatively substituted amino acid is preferably produced by performing amino acid substitution according to Table I.

Table I

initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The present invention also includes 50% or more (preferably more than 60%, more than 70%, more than 80%, more preferably more than 90%, more preferably more than 95%, most preferably more than 98%, such as 99% of the natural Ube3a protein of the present invention. %) homologous polypeptides or proteins with the same or similar functions. The protein variant can be obtained through several (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) substitutions, deletions or additions of at least one amino acid Derivative sequence, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the protein, when amino acids with similar or similar properties are used for substitution, the function of the protein will not generally be changed, and the addition of one or several amino acids at the C-terminal and/or \terminal will generally not change the function of the protein. The difference between the natural Ube3a protein analog and the natural Ube3a protein included in the present invention may be a difference in amino acid sequence, or a difference in a modified form that does not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis, or other techniques known to be divided into biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins listed above.

Modified (usually without altering primary structure) forms include: in vivo or in vitro chemical derivatization of proteins such as acetylation or carboxylation. Modifications also include glycosylation, such as those carried out in protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). In addition, the muteins of the present invention can also be modified. Modified (usually without altering primary structure) forms include: chemically derivatized forms of muteins such as acetylation or carboxylation in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications during the synthesis and processing of the mutein or during further processing steps. This modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

The present invention also provides a polynucleotide sequence encoding Ube3a protein. A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include: DNA, genomic DNA, or synthetic DNA, and DNA can be single-stranded or double-stranded. A polynucleotide encoding a mature polypeptide includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optionally additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences. The present invention also relates to variants of the above polynucleotides, which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides without substantially altering the function of the polypeptide it encodes .

Based on the nucleotide sequences described herein, those skilled in the art can conveniently use various known methods to prepare the encoding nucleic acid of the present invention. These methods are, for example but not limited to: PCR, artificial DNA synthesis, etc. For specific methods, please refer to J. Sambrook, "Molecular Cloning Experiment Guide". As an embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by synthesizing nucleotide sequences in segments and then performing overlap extension PCR.

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) to the polynucleotides of the present invention. In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.

The proteins and polynucleotides of the invention are preferably provided in isolated form, more preferably, purified to homogeneity.

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

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The method of amplifying DNA/RNA using PCR technique is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used, and the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, And can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

PP2A protein or its coding gene

PP2A is a highly conserved serine/threonine phosphatase that accounts for nearly 1% of the total protein content of cells. PP2A is a heterotrimer. Among them, the 35kD catalytic subunit C (PP2Ac) and the 65kD structural subunit A (PR65) constitute the core dimer of PP2A, and the core dimer combines a regulatory subunit B through the A subunit to form the PP2A holoenzyme Complex.

The present invention relates to a PP2A catalytic subunit PP2Ac protein and its variants. In a preferred example of the present invention, the amino acid sequence of the PP2A protein is shown in SEQ ID NO.:19. The PP2A protein of the present invention or its promoter can be used for (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; (c) reducing the activity of MAPK signaling pathway .

The present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably More than 98%, such as 99%) homologous polypeptides or proteins with the same or similar functions.

Among them, SEQ ID NO.:19 is the murine PP2A protein (99% homology with the human PP2A protein); SEQ ID NO.:24 is the human PP2A protein.

The "same or similar function" mainly refers to: (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; (c) reducing the activity of MAPK signaling pathway active.

PTPA protein or its coding gene

PTPA, encoded by the PPP2R4 gene, is an ATP/Mg ²⁺ -dependent peptide proamide trans/cis isomerase commonly found in mammalian tissues and cells. PTPA enhances the activity of PP2A phosphatase by changing the conformation of proline 190 of PP2Ac subunit and increasing the methylation level of Leu309.

The present invention relates to a PTPA protein and its variants. In a preferred example of the present invention, the amino acid sequence of the PTPA protein is shown in SEQ ID NO.:21. The PTPA protein of the present invention or its promoter can be used for (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; (c) reducing the activity of MAPK signaling pathway .

The present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably More than 98%, such as 99%) homologous polypeptides or proteins with the same or similar functions.

Among them, SEQ ID NO.: 21 is a murine PTPA protein (87% homology with human PTPA protein); SEQ ID NO.: 25 is a human PTPA protein.

The "same or similar function" mainly refers to: (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; (c) reducing the activity of MAPK signaling pathway active.

Accelerator

In the present invention, the accelerator includes substances capable of increasing the activity and/or content of PP2A or PTPA gene or its protein in vivo or in vitro.

Among them, the expression of PP2A or PTPA can be increased by the following methods: the tissue itself secretes a large amount of PP2A or PTPA protein or artificially overexpresses PP2A or PTPA protein, or artificially transports PP2A or PTPA protein (for example, using viral vectors, such as adeno-associated virus carrier) or PP2A or PTPA promoters.

In the present invention, the PP2A or PTPA promoter is not particularly limited, as long as it can promote the expression of PP2A or PTPA or enhance the activity of PP2A or PTPA protein, it is within the protection scope of the present invention.

In a preferred embodiment, the PP2A or PTPA enhancer comprises a small molecule compound.

angel syndrome

In 1965, a British pediatrician, Harry Angelman, first reported three children with Angelman syndrome, whom he dubbed "the puppet children." The incidence of Angelman syndrome (AS) is 1:12,000. The main clinical manifestations are developmental delay, mental retardation, aphasia, poor motor coordination, frequent laughter, and seizures. After more than 30 years of research, it was found that UBE3A in the segment of maternal chromosome 15q11-13 is the causative gene of Angelman syndrome. The four forms of genetic mutations leading to Angelman syndrome are: deletion of maternally derived chromosome 15q11-13 (75%); diploidy of paternally derived chromosome 15 (2%); deletion of imprinted center of chromosome 15 ( imprinting defect, ID) (2%); mutation of UBE3A gene (20%). These chromosomal abnormalities lead to a decrease in the level or activity of the UBE3A protein, which in turn leads to the occurrence of Angelman syndrome.

Autism or its related disorders

In the present invention, autism or its related diseases include but not limited to: autism, 15q11-13 duplication syndrome.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders with heterogeneous pathogenesis. The main features of the disorder are: social impairment, communication impairment, repetitive behaviors, narrow areas of interest, and delayed language development. Children with autism usually develop it before the age of 3, and it is four times more common in males than in females. Although the pathogenesis is not very clear, but 25% of children are accompanied by de novo gene mutation or abnormal copy number of chromosome.

Gene copy number variation is one of the important causes of autism, and 15q11-13 chromosome copy number abnormality accounts for 1%-3% of all autistic patients. Autistic phenotypes were shown in some populations with maternally derived 15q11-13 interchromosomal duplication (diploid), whereas mutations in isodicentric chromosome 15 (triple) were all autistic. This type of chromosomal abnormality is also known as 15q11-13 duplication syndrome. In this segment, UBE3A was the only gene expressing only the maternal allele. Not only that, but in the mouse model, mice with three copies of Ube3a showed typical autism features. In a genome-wide study, the researchers found an autistic phenotype in a patient with a UBE3A point mutation that resulted in increased UBE3A activity. The above studies prove that UBE3A is a susceptibility gene for autism.

Loss of UBE3A leads to Angelman Syndrome, while abnormal excess of UBE3A leads to autism. Thus, more or less strains of UBE3A contribute to the development of neurodevelopmental disorders.

Regulator

Based on the above new discovery of the present inventors, the present invention provides the use of an inhibitor of PP2A or PTPA for the preparation of a pharmaceutical composition for preventing, alleviating and/or treating Angelman syndrome.

As used herein, the PP2A or PTPA inhibitors include down-regulators, antagonists, blockers, blockers, degradation agents and the like.

The inhibitor of PP2A or PTPA gene or protein refers to any activity that can reduce PP2A or PTPA protein, reduce the stability of PP2A or PTPA gene or protein, down-regulate the expression of PP2A or PTPA protein, reduce the effective effect of PP2A or PTPA protein Time, or substances that inhibit the transcription and translation of PP2A or PTPA genes, these substances can be used in the present invention, as useful substances for down-regulating PP2A or PTPA, so as to alleviate or treat Angelman syndrome. For example, the inhibitors are: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of PP2A or PTPA genes; or antibodies or ligands that specifically bind to proteins encoded by PP2A or PTPA genes, and so on.

In a preferred embodiment, the inhibitor of the present invention can also simultaneously inhibit the expression or activity of PP2A and PTPA genes or their proteins.

As a preferred mode of the present invention, the inhibitor is a small molecule compound against PP2A or PTPA. Those skilled in the art can use conventional screening methods in this field to screen such small molecule compounds. For example, the small molecular compound is the PP2A inhibitor LB-100.

As an option, the inhibitor can be a PP2A or PTPA-specific interfering RNA molecule (shRNA). The use of interfering RNA molecules can significantly down-regulate PP2A or PTPA, and play a role in alleviating or treating diseases. The present invention has no particular limitation on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis method, in vitro transcription method and the like. The interfering RNA can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells by various techniques known in the art.

Alternatively, the CRISPR/Cas9 system can be used for targeted gene editing to specifically knock out the PP2A or PTPA genes. A common method for knocking out the PP2A or PTPA gene includes: co-transferring sgRNA or the nucleic acid capable of forming the sgRNA, Cas9 mRNA or the nucleic acid capable of forming the Cas9 mRNA into the targeted region or targeted cells. After the target site is determined, known methods can be used to introduce sgRNA and Cas9 into the cells. The nucleic acid that can form the sgRNA is a nucleic acid construct or an expression vector, or the nucleic acid that can form the Cas9mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into the cell, so that in the cell Form active sgRNA and Cas9 mRNA.

Based on the inventor's new discovery, the inhibitor is a reagent that specifically up-regulates Ube3a protein (including promoting its expression or increasing its activity), and it degrades PTPA by up-regulating Ube3a protein, thereby inhibiting the activity of PP2A.

The up-regulator of Ube3a refers to any substance that can increase the activity of Ube3a, improve the stability of Ube3a, up-regulate the expression of Ube3a, and increase the effective action time of Ube3a. It is used to inhibit (including degrade) PTPA, thereby inhibiting PP2A. They can be chemical compounds, small chemical molecules, biological molecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.

As a preferred option of the present invention, the Ube3a up-regulator is an expression system or an expression construct expressing Ube3a. The expression system or expression construct is not particularly limited, and any expression system capable of expressing activities similar to Ube3a or its analogs and mimics can be applied in the present invention. For example, the expression system or expression construct includes, but is not limited to: expression vector, host cell or virus containing the expression vector; the virus includes adenovirus, adeno-associated virus, lentivirus, etc. Those skilled in the art know how to express glycerol kinase or its analogs and mimetics, and there are already some commercialized expression systems, which can all be applied in the present invention.

drug screening

After knowing that increasing the expression of Ube3a or inhibiting the expression of PP2A or PTPA can alleviate or treat Angelman syndrome, substances that increase the expression of Ube3a or inhibit the expression or activity of PP2A or PTPA can be screened based on this feature. Really useful medicines for alleviating or treating Angelman syndrome can be found from said substances.

Therefore, the present invention provides a method for screening potential substances for alleviating or treating Angelman syndrome, said method comprising: treating a system expressing PP2A with a candidate substance; and detecting the expression or activity of PP2A in said system; if said The candidate substance can inhibit the expression or activity of PP2A, which indicates that the candidate substance is a potential substance for alleviating or treating Angelman syndrome. The system for expressing PP2A is preferably a cell (or cell culture) system, and the cells may be cells expressing PP2A endogenously; or cells expressing PP2A recombinantly.

In a preferred mode of the present invention, the Ube3a protein is also expressed in the system, and the method also includes: detecting the expression of the Ube3a protein in the system; wherein, if the candidate substance can inhibit the protein by increasing the expression of Ube3a Phosphatase 2A indicates that the candidate substance is a potential substance for alleviating or treating Angelman syndrome.

In a preferred mode of the present invention, PTPA is also expressed in the system, and the method also includes: detecting the expression of PTPA in the system; wherein, if the candidate substance can inhibit PP2A by inhibiting the expression of PTPA, then It is indicated that the candidate substance is a potential substance for alleviating or treating Angelman syndrome.

In a preferred mode of the present invention, when screening, in order to more easily observe changes in the expression or activity of Ube3a, PP2A or PTPA, a control group can also be set, and the control group can be without adding the candidate substance Systems expressing Ube3a, PP2A or PTPA.

As a preferred mode of the present invention, the method further includes: conducting further cell experiments and/or animal experiments on the obtained potential substances, so as to further select and determine substances that are really useful for alleviating or treating Angelman syndrome.

On the other hand, the present invention also provides potential substances obtained by the screening method. These initially screened substances can constitute a screening library, so that people can finally screen out substances that can inhibit the expression and activity of PP2A or PTPA, and then alleviate or treat Angelman syndrome.

pharmaceutical composition

The present invention also provides a pharmaceutical composition for alleviating or treating Angelman syndrome, which contains an effective amount (such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably, 0.0001-10wt%) of all The above-mentioned PP2A or PTPA gene or protein inhibitor, and a pharmaceutically acceptable carrier. Any of the aforementioned inhibitors of PP2A or PTPA genes or proteins may be used in the preparation of the composition.

As a preferred mode of the present invention, a composition for alleviating or treating Angelman syndrome is provided, the composition contains an effective amount of the inhibitor LB-100 of the present invention, and a pharmaceutically acceptable Carrier.

As used herein, the "effective amount" refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for the administration of therapeutic agents, including various excipients and diluents. The term refers to pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in compositions may contain liquids, such as water, saline, buffers. In addition, there may also be auxiliary substances in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and the like. The carrier may also contain cell transfection reagents.

After knowing the use of the inhibitor of the PP2A or PTPA gene or protein, various methods well known in the art can be used to administer the inhibitor or its coding gene or its pharmaceutical composition to mammals . Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, local administration, implantation, sustained-release administration, etc.; preferably, the administration method is parenteral administration.

Administration may be by means of gene therapy. For example, the inhibitor of PP2A or PTPA can be directly administered to the subject through methods such as injection; or, the expression unit carrying the inhibitor of PP2A or PTPA (such as an expression vector or virus, etc., or siRNA) to the target, and make it express an active PP2A or PTPA inhibitor, the specific situation depends on the type of the inhibitor, which are well known to those skilled in the art.

The effective amount of the PP2A or PTPA gene or protein inhibitor of the present invention can vary with the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the inhibitor of the PP2A or PTPA gene or protein such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, The immune status of the patient, the route of administration, etc.

diagnostic use

Based on the above new findings of the present inventors, PP2A or PTPA can be used as markers for the diagnosis or prognosis of Angelman syndrome: (i) carry out typing and/or differential diagnosis of Angelman syndrome; disease risk, drug efficacy, prognosis, and selection of appropriate treatment. For example, people with abnormal expression of PP2A or PTPA genes can be isolated, so that more targeted treatment can be carried out.

According to the new findings of the present inventors, the prognosis of Angelman syndrome of the subject providing the sample to be evaluated can be predicted by judging the expression or activity of PP2A or PTPA in the sample to be evaluated, and an appropriate drug can be selected for treatment. Usually, a threshold value of PP2A or PTPA can be specified, and when the expression of PP2A or PTPA is higher than the specified threshold value, the regimen of inhibiting PP2A or PTPA should be considered for treatment. The threshold is easy to determine for those skilled in the art, for example, it can be obtained by comparing the expression of PP2A or PTPA in the microenvironment of normal human tissues with the expression of PP2A or PTPA in the microenvironment of patients. Threshold for abnormal expression of PP2A or PTPA.

Various techniques known in the art can be used to detect the presence or absence and expression of the PP2A or PTPA gene, and these techniques are included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination.

The present invention also provides reagents for detecting the presence or absence and expression of PP2A or PTPA gene in an analyte. Preferably, when performing gene level detection, primers for specific amplification of PP2A or PTPA can be used; or probes for specific recognition of PP2A or PTPA are used to determine the presence or absence of PP2A or PTPA genes; In this case, the expression of PP2A or PTPA protein can be determined by using an antibody or ligand that specifically binds to the protein encoded by PP2A or PTPA.

The design of specific probes for PP2A or PTPA genes is a technique well known to those skilled in the art, for example, preparing a probe that can specifically bind to a specific site on the PP2A or PTPA genes, but not to PP2A or PTPA Other genes than genes specifically bind, and the probes carry a detectable signal.

The method of detecting the expression of PP2A or PTPA protein in an analyte by using an antibody that specifically binds to PP2A or PTPA protein is also well known to those skilled in the art.

The present invention also provides a kit for detecting the presence or absence and expression of PP2A or PTPA gene in an analyte, the kit comprising: primers for specific amplification of PP2A or PTPA gene; specific recognition of PP2A or PTPA gene Probes; or antibodies or ligands that specifically bind to proteins encoded by PP2A or PTPA genes.

In addition, the kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution , Chromogenic solution, lotion, etc.

In addition, the kit may also include instructions for use and/or nucleic acid sequence analysis software and the like.

Compound pharmaceutical composition and kit

The present invention provides an inhibitor containing active ingredient (a) Ube3a gene or its protein; PP2A gene, or its protein or its promoter; PTPA gene, or its protein or its promoter; or its combination; and (b) pharmaceutical Compound pharmaceutical composition on an acceptable carrier. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or 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 are preferably manufactured under sterile conditions. The pharmaceutical combination of the present invention can also be made into powder for nebulized inhalation. The dosage forms of the pharmaceutical composition of the present invention are injections, oral preparations (tablets, capsules, oral liquids), transdermal agents, and sustained release agents. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparations of the present invention can also be made into sustained-release preparations. The pharmaceutical composition of the present invention is preferably an injection preparation. In addition, the pharmaceutical compositions of the present invention can also be used together with other therapeutic agents. And, the pharmaceutical composition of the present invention can also include additional components, and the additional components are selected from the group: other (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting The phosphorylation level of ERK1/2; and/or (c) substances that reduce the activity of MAPK signaling pathway, such as MK-8353, U0126 and SCG772984, PD98059, Selumetinib and PD0325901, etc.

The effective amount of the active ingredient in the present invention may vary with the mode of administration, the severity of the disease to be treated, and the like. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, drug administration way etc. Usually, satisfactory effects can be obtained when the active ingredient of the present invention is administered at a daily dose of about 0.00001 mg-10 mg/kg animal body weight (preferably, 0.5 mg-5 mg/kg animal body weight). For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.

The pharmaceutically acceptable carrier of the present invention includes (but not limited to): water, saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or its combination. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

The present invention also provides a drug that can be used for (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; and/or (c) reducing the MAPK signaling pathway Active kit, the kit contains:

(i) the first container, and the active ingredient (a1) inhibitor of Ube3a gene or its protein in the first container; PP2A gene, or its protein or its promoter; PTPA gene, or its protein or its promoter ; or a combination thereof, or a medicament containing active ingredient (a1); and

(ii) instructions for administering the active ingredient (a1) to (i) prevent and/or treat liver fibrosis-related diseases; and/or (ii) maintain liver homeostasis.

The pharmaceutical composition and kit of the present invention are suitable for (a) preventing and/or treating autism or its related diseases; and/or (b) inhibiting the phosphorylation level of ERK1/2; and/or (c) reducing MAPK signal pathway activity.

The formulation of the present invention can be taken three times a day to once every ten days, or once every ten days in a sustained release form. The preferred way is to take it once a day, because it is easy for patients to persist in this way, thereby significantly improving the compliance of patients taking medicine.

When taking it, the total daily dose in most cases should be lower than (or in a small number of cases, equal to or slightly greater than) the usual daily dose of each single drug. Of course, the effective dose of the active ingredient used can vary with the mode of administration and the dose to be treated. Depending on the severity of the disease being treated, etc.

treatment method

The present invention also provides the use of the active ingredients of the present invention or corresponding drugs to (a) prevent and/or treat autism or its related diseases; and/or (b) inhibit the phosphorylation level of ERK1/2; and/or ( c) A method for reducing the activity of the MAPK signaling pathway, which includes administering to mammals an effective amount of active ingredient (a1) an inhibitor of Ube3a gene or its protein; PP2A gene, or its protein or its promoter; PTPA gene, or its promotor; A protein or an accelerator thereof; or a combination thereof, or administering a pharmaceutical composition containing the active ingredient (a1).

When the active ingredient of the present invention is used for the above purposes, it can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be orally administered in the following forms: tablet , pills, capsules, dispersible powders, granules or suspensions (containing e.g. about 0.05-5% suspending agent), syrups (containing e.g. about 10-50% sugar), and elixirs (containing e.g. about 20-50% ethanol), or Parenteral administration is in the form of sterile injectable solutions or suspensions containing about 0.05-5% suspending agent in an isotonic medium. For example, these pharmaceutical formulations may contain about 0.01-99%, more preferably about 0.1%-90% by weight of the active ingredient in admixture with a carrier.

The two active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration . Preferred routes of administration include oral, intramuscular or intravenous administration.

From the standpoint of ease of administration, preferred pharmaceutical compositions are liquid compositions, especially injections.

In addition, the active ingredient or drug of the present invention can also be combined with other (a) prevention and/or treatment of autism or its related diseases; and/or (b) inhibition of the phosphorylation level of ERK1/2; and/or (c) reduction of Active components or drugs of the MAPK signaling pathway (such as MK-8353, U0126, SCG772984, PD98059, Selumetinib, and PD0325901, etc.) are used in combination.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions such as edited by J. Sam Brook, Molecular Cloning Experiment Guide, Science Press, 2002 (New York: Cold Spring Harbor Laboratory Press, 2002) described in conditions, or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The materials and reagents used in the examples are all commercially available products unless otherwise specified.

Example 1, Detection of PTPA protein level in Ube3a deficient mice

First, 30-day-old (P30) mice were taken out: wild-type (WT) and littermate AS mice with maternal deletion of Ube3a (obtained from Duke University, a termination was introduced under exon 2 of Ube3a codon, resulting in the non-expression of Ube3a), anesthetized, and injected 0.7% sodium pentobarbital intraperitoneally according to the standard of 70mg/kg; Put it into pre-cooled 0.9% normal saline, wash away the blood with normal saline, put it on sterile filter paper, use tweezers and hippocampal spoon to dissect the brain tissue, put half of the cerebellum tissue into a 2ml Axygen centrifuge tube, and then Put 400ul of RIPA lysate with 1mM PMSF into it, put a 3mm diameter magnetic bead into a tissue homogenizer, break it at 60Hz for 30s, take it out, and put it on ice for 30min. Afterwards, in a refrigerated centrifuge at 4°C, centrifuge at 14000 g for 20 min, then take the supernatant into a new 1.5 ml centrifuge tube, and place it on ice.

The protein concentration in tissues was determined using Tiangen BCA protein quantification kit. First take out 5ul of the protein lysate and dilute it 10 times. According to the method in the instruction manual, mix the A and B solutions in the kit at 1:50, prepare 50ul of BSA protein standard, and separate the sample and the standard 25 ul and 200 ul of the AB mixture were mixed and put into a 96-well plate (produced by Corning, USA). After reacting at 37° for 30 min, it was taken out and put into a microplate reader to measure the absorbance at 562 nm. After calculating the standard curve, bring the obtained absorbance value of the sample into the formula to calculate the corresponding concentration, and then enlarge it by 10 times to obtain the concentration of the measured sample. After the determination, adjust the sample uniformly to a concentration of 2ug/ul, mix the protein sample with an equal volume of 2×protein loading buffer (dithiothreitol (DTT): 0.1572g bromophenol blue; 0.01g Tris-HCl ( 1M pH 6.8): 0.5ml 10% SDS: 2ml glycerin: 1ml H ₂ O: dilute to 10ml) and mix, boil at 80°C for 7min.

Preparation method of Tris (1M pH 6.8) buffer solution: Weigh 24.228g of Tris Base and dissolve in about 160ml of ultrapure water, stir well to dissolve, adjust the pH to 6.8 with hydrochloric acid, and dilute to 200ml with ultrapure water.

Detection of protein expression by Western Blot method:

Prepare the reagents needed to prepare the gel as follows: 30% acrylamide mix was purchased from Generay;

Tris (1.5M pH 8.8) preparation method: Weigh 36.342g of Tris Base and dissolve in about 160ml of ultrapure water, stir well to dissolve, adjust the pH to 8.8 with hydrochloric acid, and dilute to 200ml with ultrapure water at the same time;

Preparation method of Tris (0.5M pH 6.8) buffer solution: Weigh 12.114g of Tris Base and dissolve in about 160ml of ultrapure water, stir well to dissolve, adjust the pH to 6.8 with hydrochloric acid, and at the same time, dilute to 200ml with ultrapure water;

The preparation method of 10% SDS: Weigh 5g sodium dodecyl sulfate (sodium dodecyl sulfate, SDS), dissolve in ultrapure water to 50ml;

The preparation method of 10% APS: weigh 1g ammonium persulfate (Ammonium persulfate, APS), dissolve in ultrapure water to 10ml;

TEMED refers to N,N,N',N'-Tetramethylethylenediamine, the Chinese name is N,N,N',N'-Tetramethyldiethylamine.

Prepare the electrophoresis device and the glass plate for preparing 1.5mm thick gel. Wash the glass plate, rinse it again with deionized water, and dry it in an electric blast drying oven. After the glass plate is completely dry, assemble the gel mixing device and prepare 10% separating gel (ddH ₂ O: 4.0ml 30% acrylamide mix: 3.3ml Tris (PH 8.8 1.5M): 2.5ml 10% SDS: 100μl 10% APS: 100μl TEMED: 4 μl), inject it into a glass plate, and seal the upper surface with 500 μl of absolute ethanol after adding.

Place at room temperature for more than 30 minutes, and when the two-phase interface between ethanol and solidified separating gel becomes clear, prepare 5% stacking gel (ddH ₂ O: 2.7ml 30% acrylamide mix: 0.67ml Tris (PH 6.8 0.5M): 0.5ml 10% SDS: 40 μl 10% APS: 40 μl TEMED: 4 μl), pour off the alcohol in the glass plate, inject the stacking gel, insert a comb, leave it at room temperature for 20 minutes and wait for it to solidify before use.

Electrophoresis: prepare 10× electrophoresis solution (Tris: 30.3g, glycine: 144.0g, SDS: 10.0g deionized water, set to 1000ml), mix 100ml 10× electrophoresis solution with 900ml H ₂ O ₂ to get 1000ml 1× Electrophoretic fluid.

Install the electrophoresis device and rinse it with clean water, and pull out the comb. Pour 1000ml of electrophoresis solution to keep the electrophoresis tank full of electrophoresis solution. The sample is collected by centrifugation at 6000 rpm for 1 second and then added to the swimming lane. Add a pre-stained marker (manufactured by Fermentas, Canada, Cat. No. SM0671) to one of the blank wells adjacent to the sample. ). Restricted conditions for power supply during electrophoresis: 80V for 40min, then 110V for 90min. Stop electrophoresis when the indicator shows that the sample is close to the lower boundary of the gel.

Membrane transfer: Prepare 10× transfer solution (Tris: 30.3g glycine: 144.0g H ₂ O: dilute to 1000ml). Next, prepare 1×transfer solution (10×transfer solution: 80ml methanol: 160ml H ₂ O: dilute to 800ml). Prepare the membrane transfer device, two sets of 8.5×8cm filter paper, two sheets for each set. Soak the PVDF membrane in methanol for about 1 min until it is soaked without white spots, then put it in the transfer solution for equilibrium, and then transfer it to a small box containing the transfer solution for later use. Then use a plastic sheet to gently cut off the concentrated gel, and then cut the two ends of the separating gel and the surface in contact with the glass. Use a plastic sheet dipped in a little transfer solution, stuff it under the separation gel to make it loose, then turn it upside down into the plastic basin containing the transfer solution and shake gently, the separation gel will be separated from the glass into the transfer solution. Open the plastic plate for membrane transfer, immerse it in the membrane transfer solution, and assemble it in the following order in the membrane transfer solution: Negative plate (black) put sponge → a set of filter paper (three sheets) → separation gel → PVDF membrane → a set Filter paper (three sheets) → sponge → positive plate. Then clamp the device, pour the transfer solution, ensure that the sandwich-like structure of the transfer film is completely submerged in the transfer solution, cover the cover with the power cord, and place the entire electrophoresis device in a plastic basin. The ice-water mixture is ready for transfer. Restricted conditions of power supply during film transfer: I=350mA; t=120min.

Blocking: prepare 10×TBS buffer solution (1L containing 24.23g Tris, 80.06g NaCl, adjust pH to 7.4 with HCl), then dilute 10 times with deionized water to obtain 1×TBS, then add 0.5% (v/v) Tween-20 formulated as TBST. Prepare 5% skimmed milk, and dissolve 5g skimmed milk (China Inner Mongolia Yili Industrial Group Co., Ltd.) in 100ml TBST solution, which is the blocking solution. After the film transfer is completed, take out the PVDF film and put it into a plastic box, pour 30ml of 5% skimmed milk, and shake it on a decolorizing shaker for one hour.

Primary antibody incubation: After removing the PVDF membrane from the shaker, take PTPA antibody (Cell signaling company, 3330) at a dilution ratio of 1:2000, take Ube3a antibody (Sigma company, E8655) and antibody HRP conjugate at a dilution ratio of 1:5000 Combined Actin (Santa Cruz Company, sc-1615HRP) was added to 3ml blocking solution, and the antibody working solution was obtained by shaking well. Put the membrane into a ziplock bag, pour in the prepared antibody working solution, and incubate overnight at 4°C on a medium-speed decolorization shaker. After the primary antibody incubation, if the primary antibody has been labeled with horseradish peroxidase (such as the antibody of the internal reference protein GAPDH), skip directly to the development step, if not, you need to incubate with the secondary antibody.

Secondary antibody incubation: Wash 3 times with 1×TBST, 10 minutes each time. Draw horseradish peroxidase-labeled rabbit immunoglobulin antibody (manufactured by Santa Cruz, with a dilution rate of 1:3000) or mouse immunoglobulin antibody (manufactured by Santa Cruz, with a dilution rate of 1:3000), and add to 3ml Shake well in blocking solution. Put the washed membrane into a ziplock bag, pour in the prepared secondary antibody working solution, seal it, and shake it on a shaker for 120 minutes.

Imaging: After incubation with the secondary antibody, wash 3 times with 1×TBST, 10 minutes each time. The following operations were completed under the assistance of red light in the developing darkroom. Pipette 800 μl of developing substrates A and B (produced by China Tiangen Biochemical Technology Co., Ltd.) respectively, mix well, and equilibrate to room temperature. Put the PVDF membrane on the inner surface of the clean cut ziplock bag. Quickly add the developing substrate and incubate for 1 min. If obvious bands are visible to the naked eye, the incubation is terminated. Discard the developing substrate liquid, transfer the film to a ziplock bag cut on three sides, close the ziplock bag, carefully absorb the excess liquid with toilet paper, put it into the developing cassette, and fix it with insulating tape. Put the film on, after the film is exposed for a certain period of time (5-30 seconds), put in the developing solution and the fixer solution for 20 seconds each, and then put the film in clear water. Finally, wash the film under tap water, dry it, scan it to the computer, and get the test result.

The results of Western Blot are shown in Figure 1 (A, B), indicating that Ube3a regulates the protein expression of PTPA and presents a negative correlation.

Example 2, UBE3A ubiquitination of PTPA

1) GST pull-down

Purification of GST-tagged protein: The constructed prokaryotic expression plasmid (PGEX-4T-1 or Pgex-4T-1-UBE3A) was transformed into a competent Rosetta expression strain, and spread on an ampicillin-resistant plate. After the colonies grow out, pick the colonies and place them in 5 mL of LB bacterial culture medium containing antibiotics, culture them overnight at 37 degrees Celsius and 250 rpm. The overnight cultured bacteria were inserted into the LB bacterial medium to which antibiotics had been added at a ratio of 1:100, and cultured at 37°C until OD600=0.6-0.8 (about 2.5 hours). Then add IPTG (0.168mM) and induce for 5 hours at 27°C. The induced bacterial liquid was centrifuged at 12000rpm for 2 minutes and collected into a 15mL centrifuge tube. Add 5 mL of GST lysis buffer (PBS+1% TritonX-100, 1 mM PMSF) to resuspend the cells. Ultrasonic at 80-100W for 5 seconds, with an interval of 15 seconds until the bacterial liquid becomes clear. After the sonication, centrifuge at 12000g, 4°C for 20 minutes, and transfer the supernatant to a new centrifuge tube. Add the supernatant to a chromatographic column equipped with an appropriate amount of glutathione-bound beads (GE Healthcare), wash five times with a certain volume of GST lysis buffer, and finally elute the protein with an appropriate amount of elution buffer Down, stored at -80 degrees Celsius. The buffers used above need to add proteasome inhibitors in advance.

Preparation of IPTG (isopropylthio-β-D-galactoside): 2g of IPTG was dissolved in 10mL of water, sterilized by filtration, subpackaged, and stored at -20°C.

Preparation of PBS: KH2PO4 0.24g, Na2HPO4 1.44g, NaCl 8.0g, KCl 0.2g, add water to 1000mL.

Preparation of elution buffer: 0.615g glutathione, 10mL Tris-HCl (1M, pH 8.0), 90mL double distilled water.

The purification steps of the His-tagged protein are similar to the purification steps of the GST protein, Ni-NTA beads (Qiagen) are used in this purification, and the purification steps are operated according to the instructions.

Put the purified GST fusion protein and His fusion protein in pull-down buffer (50mM Tris-Cl, (pH 8.0), 200mM NaCl, 1mM EDTA, 1%NP-40, 1mM DTT, 10mM MgCl2), 4 Incubate for 2 hours at °C. Then, after washing the beads four times with pull-down buffer, 2X protein loading buffer was added for western blotting.

2) In vitro ubiquitination reaction

The in vitro ubiquitination reaction was performed according to the instructions provided in the E6AP (UBE3A) Ubiquitin Ligation Kit (Boston Biochem, K-240). The specific reaction was as follows: Mix 3 μL 10X E1, 3 μL 10X E2, 3 μL 10X His6-UBE3A, 3 μL 10X Mg2+-ATP, 3 μL 10X His6-PTPA enzyme (2 μg), 3 μL 10X reaction buffer and 6 μL water. In the negative control group, water was substituted for Mg2+-ATP. The reaction started when 3 μL of 10X ubiquitin solution was added. The reaction was carried out at 37°C. After 1.5 hours, the reaction was stopped by adding loading buffer, and the ubiquitination level of PTPA was detected by western blotting.

The results of UBE3A ubiquitination of PTPA are shown in Figure 2 (A, B), indicating that UBE3A directly interacts with PTPA and makes PTPA ubiquitination.

Example 3, Detection of PP2A activity in Ube3a deficient mice

The whole brain of the mouse was taken out according to the method in Example 1, it was put into a pre-cooled glass homogenizer, and 1ml of imidazole lysate (20mM imidazole hydrochloride (PH7.0), 2mM EDTA, 2mM EGTA, 1mM PMSF , 5ul Protease Inhibitor Cocktail (P8430)), homogenate about 20 times, transfer the lysate to a 1.5ml centrifuge tube with a pipette, and insert it into an ice box. Put the centrifuge tube into a refrigerated centrifuge, centrifuge at 3000g for 5min at 4°C. Take out the supernatant into a new centrifuge tube, and measure the protein concentration according to the method in Example 1.

Take 500ug of protein into a new 1.5ml centrifuge tube, make up the volume to 500ul with imidazole lysate, and complete the detection of PP2A activity according to the steps provided by the PP2A activity detection kit (Moker Millipore, #17-313). Specifically, 4ug of Anti i-PP2A, C (#05-421) antibody and 40ul of Protein A agarose beads provided in the kit were added to 500ug of protein lysate, and placed in a mixer in a 4°C freezer ( H30 mixer) and rotate for 2 hours. Remove the centrifuge tube, centrifuge at 3000g at 4°C for 3min, remove the supernatant, and leave the agarose beads. Add 700ul of 1×TBS, mix well, centrifuge again at 3000g, 4°C for 3min. repeat three times. Finally, wash once again with 500ul Ser/Thr reaction buffer provided by the kit, and finally leave the agarose beads. Add 60ul of phosphorylated polypeptide (750uM) and 20ul of Ser/Thr reaction buffer, and mix well. Incubate in a water bath at 30°C for 10 minutes, take it out and mix well before putting it back in. After the reaction, centrifuge at 10000g for 30s, take 25ul into the 96-well plate provided by the kit, then add 100ul of malachite green phosphoric acid detection solution, react at room temperature for 15min, measure the fluorescence at 650nm in a microplate reader value. Relative tissue phosphatase activity was derived.

The results of phosphatase activity are shown in FIG. 3 , indicating that in the Ube3a-deleted AS mouse model, PP2A activity was significantly increased.

Example 4, Effect of PP2A Inhibitors on Dyskinesia in Angelman Syndrome Mice

LB-100 is a water-soluble PP2A inhibitor, the solubility in water is 53mg/mL, its molecular formula is C ₁₃ H ₂₀ N ₂ O ₄ , and its molecular structure is as follows:

In this example, the LB-100 used was purchased from Selleck Company with a purity of 95%. The drug was dissolved in physiological saline, and the concentration of the mother solution was prepared to be 10mg/ml. After 20ul aliquots, it was stored in a -20°C refrigerator.

AS model mice are from Duke University, and a stop codon was introduced under exon 2 of Ube3a, resulting in non-expression of Ube3a. The mice that do not express Ube3a are model mice for Angelman syndrome.

Take 8 wild-type (WT) mice and 8 AS mice each, and divide them into 4 groups: WT is given normal saline group (WT), AS is given normal saline group (AS), WT is given LB-100 drug group (WT/LB- 100), AS to the LB-100 drug group (AS/LB-100). On the 14th day after birth, the drug injection was started, and the concentration of LB-100 injected per kilogram of body weight was administered according to the weight of the mouse. Now take the prepared LB-100 mother solution out of the refrigerator, and after thawing, dilute it to a working concentration of 50ug/ml, weigh the mice, and inject them intraperitoneally. Injections were given every two days until 30 days after birth, when behavioral testing began.

The dosing steps are shown in Figure 4, starting from the 14th day after birth (P14), and giving the medicine every two days until the 30th day after birth (P30), the behavioral detection was carried out.

Thin rope suspension experiment: This experiment is mainly to detect the muscle strength and movement coordination of mice. Take a thin rope with a diameter of 0.2 cm and a length of 54.4 cm, fix it on the brackets at both ends, adjust its height to 50 cm from the ground, and put a foam cushion under it to prevent the mice from falling and falling . The mice were placed in the room to adapt to the environment for 30 minutes before the experiment began. During the experiment, after the mice were lifted, the forelimbs were hung on the strings and then the hands were released, and the time the mice did not fall on the wires was recorded, and the maximum time was recorded to 120s. Each mouse repeated three times, each time interval of 15min.

Rotarod test: This test is mainly to test the motor coordination and learning ability of mice. The experiment needs to be carried out for six days: on the first day, the mouse is placed on the rotarod instrument at 4 revolutions per minute, and after 2 minutes of exercise, it is taken out and put into the cage; the formal experiment starts on the second day: the setting of the rotarod instrument is adjusted to a gradual acceleration program , from 5 rpm to 30 rpm, the acceleration time is 5min. This experiment records the time it moves on the rotarod instrument. If the mouse falls or holds the column and rotates twice, it is recorded as a failure, and the time recorded at this time is the time the mouse stopped on the rotarod instrument. Continue this step for five days. The five-day performance of each mouse was plotted into a curve to compare the exercise capacity of the mice.

The motor behavior results of the four groups of mice after administration are shown in Figure 5, A-B, indicating that the PP2A inhibitor of the present invention can improve the muscle strength and motor coordination of Angelman syndrome mice.

Example 5. Effects of PP2A inhibitors on abnormal dendritic spines in mice with Angelman syndrome

After the mice in Example 4 completed the behavioral experiment, Golgi silver staining was performed to observe the dendritic spines of Purkinje cells in the mouse cerebellum. The staining kit used in this experiment is FD Golgi stain kit (Consulting & Services Company, PK401). First, mix A solution and B solution in the kit 1:1 one day in advance, and store at room temperature in the dark. On the second day, the mice were anesthetized, and the method was as in Example 1. The brain tissue of the mice was put into ddH ₂ O, rinsed briefly, and the blood and sundries on the surface were removed. Put the mouse brain tissue into a 15 ml centrifuge tube (manufactured by Corning) containing 5 ml of the AB mixture, and soak the brain tissue. After 24 hours of storage in the dark, replace with 5ml of the AB mixture, and continue to store in the dark at room temperature for 14 days. After 14 days, replace the AB mixture with solution C in the kit, and store at 4°C in the dark. After 24 hours, replace solution C, and continue to store at 4°C for 3 days. After 3 days, the cerebellum in the brain tissue was excised, placed sagittally into a 24-well plate, poured into pre-dissolved 3% agar, and placed in the refrigerator until it solidified.

Preparation of 3% agar solution: Weigh 3g of agar powder and put it into a glass conical flask, add 110ml of water, heat it in a microwave oven, after it boils once, take it out and shake it a few times, put it in the microwave oven again, Let it boil again. Finally, after the solution was in a uniform state, quickly pour it into a 50ml centrifuge tube (purchased by Cornin), and put it into a water bath at 60°C to keep it in a dissolved state.

Embedded brain tissue was removed and placed on a vibratome (Leica) for sectioning. Specifically, after the brain tissue was taken out and trimmed, it was fixed on the base of the microtome with superglue, liquid C was quickly poured in, the slice thickness of the microtome was adjusted to 150um, and the excised brain slices were directly Mount on gelatin-coated slides, one slide per cerebellum. When finished, put it in a dark box and let it dry. After about 2 days, Golgi staining was performed. Wash the brain slices with ultrapure water twice, 4 minutes each time, then place the brain slices in the mixture of D+E+water (50ml:50ml:100ml) for 10 minutes for staining, and then wash the brain slices with ultrapure water 4 times, each time 4min. Then use 50%, 75%, 95% dehydration step by step, each brain slice is placed for 4min. Finally, dehydration was carried out four times with absolute ethanol, 4 min each time. Finally, degrease with xylene 3 times, 4 min each time. Finally, after adding 200ul of resin dropwise to each slide, cover with a cover slip and mount the slide. After sealing, the slides were stored at room temperature and protected from light until they were photographed.

The film to be shot was shot under the bright field of a Nikon A1 inverted confocal microscope with a 60x objective lens. After adjusting the light source to the optimal level, the dendrites of Purkinje cells away from the cell body were selected for shooting. Finally, the dendritic spines in the obtained pictures are divided into three types according to their shape: mushroom, stubby, and thin, and the three types of dendritic spines in each dendritic trunk are calculated. The number of , and finally calculate the proportion of each category.

After administration, the category analysis of dendritic spines of Purkinje cells in the cerebellum of the four groups of mice is shown in Figure 6, A-B, indicating that PP2A inhibitors can significantly improve the Purkinje cell tree in Angelman syndrome mice Abnormal maturity of spinous processes.

Example 6. Knocking down the expression of PTPA gene can improve the abnormal morphology of neurons in Angelman syndrome mice

According to the aforementioned research results, the inventors combined female paternal Ube3a-deleted heterozygous mice (Ube3a ^m+/p- ) (obtained from Duke University) with male Ptpa-deleted heterozygous mice (Ptpa ^+/- ) (Institute of Neuroscience, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences, obtained by modifying Ptpa with CRISPR/Cas9 technology) mating, which produced four offspring: wild type (WT), AS mice with maternal Ube3a deletion, and heterozygous mice with Ptpa deletion (Ptpa ^+/- ), and Ube3a and Ptpa double knockout mice (AS; Ptpa ^+/- ). Thirty days after birth, according to the method in Example 5, the mice with four genes were stained with Golgi respectively, and the dendritic spines of Purkinje cells in the mouse cerebellum were observed. The dendritic spines are divided into three types according to their morphology: mushroom, stubby and thin. Count the proportions of the three kinds of dendritic spines in different mice.

The category analysis of the dendritic spines of the Purkinje cells in the cerebellum of the four groups of mice is shown in Figure 7, indicating that reducing the protein level of PTPA in the present invention can significantly improve the dendritic spines of the Purkinje cells in Angelman syndrome mice. Very mature.

Example 7, shRNA targeting PTPA gene and specifically inhibiting PTPA gene expression can alleviate the abnormal movement of Angelman syndrome model mice

1. Construction and sequence screening of shRNA vectors that specifically inhibit PTPA gene expression

Download the complementary DNA (Complementary DNA, cDNA) sequence information of the mRNA (GenebankAccession: NM_138748.5) of mouse PTPA from NCBI website (http://www.ncbi.nlm.nih.gov/), in Invitrogen website (https: //rnaidesigner.thermofisher.com/rnaiexpress/) designed shRNA targeting PTPA, and selected 3 target sequences in the early stage. The comparison analysis of the target sequence requires that the target sequence has no high homology with other human mRNA genes, and can be used as an interference target that specifically interferes with PTPA. Then artificially design shRNA according to these sequences in the order of sense-loop-antisense, sense refers to the sense strand of the target sequence, antisense refers to the antisense strand of the target sequence, loop refers to the sequence forming a loop, and TTCAAGAGA is used here. Add the sequences recognized by the restriction endonucleases BglII and HindIII to both ends to obtain two shRNA sequences targeting one target as follows: sense strand: 5'-GATCCCC-sense-TTCAAGAGA-antisense-TTTTTA-3' ; antisense strand: 5'-AGCTTAAAAA-sense-TCTCTTGAA-antisense-GGG-3'.

The sequences of the three shRNAs are as follows:

Sense strand of sh1# (sh1#-F): 5'-GATCCCCCAGTTCCAGATATGGGCAAATGGAATTCAAGAGAattccatttgcccatatctggaactgTTTTTA-3' (SEQ ID NO: 4);

Antisense strand of sh1# (sh1#-R): 5'-AGCTTAAAAACAGTTCCAGATATGGGCAAATGGAATCTCTTGAAttccatttgcccatatctggaactgGGG-3' (SEQ ID NO: 5);

Sense strand of sh3# (sh1#-F): 5'-GATCCCCCCTGGTATGCCAAACTTGATCAGGATTCAAGAGAtcctgatcaagtttggcataccaggTTTTTA-3' (SEQ ID NO: 6);

Antisense strand of sh3# (sh1#-R): 5'-AGCTTAAAAACCTGGTATGCCAAACTTGATCAGGATCTCTTGAAtcctgatcaagtttggcataccaggGGG-3' (SEQ ID NO: 7);

Sense strand of sh4# (sh1#-F): 5'-GATCCCCTCAAGGTGTTTGATAGGTATCTTGATTCAAGAGAtcaagatacctatcaaacaccttgaTTTTTA-3' (SEQ ID NO: 8);

Antisense strand of sh4# (sh1#-R): 5'-AGCTTAAAAATCAAGGTGTTTGATAGGTATCTTGATCTCTTGAAtcaagatacctatcaaacaccttgaGGG-3' (SEQ ID NO: 9);

The primer information was submitted to Invitrogen Company for synthesis. DNA Oligo was dissolved by adding ddH ₂ O at a concentration of 3 mg/ml. Take shRNA sense strand and antisense strand 2ul of DNA oligo and 5ul of 10× annealing buffer (composition: 100mM Tris-HCl (pH 7.5), 10mM EDTA, 1mM NaCl), mix them in water bath at 95°C for 12min, and take them out. Cool to room temperature at room temperature. At 25°C, use T4 ligase (produced by NEB, USA) to ligate the complementary double strands to the pSuper basic vector digested with BglII and HindIII at 16°C overnight. Enzyme 2 μl, ddH ₂ O 10 μl).

Take the ligation product and add it to a 1.5ml centrifuge tube filled with Escherichia coli DH5α competent cells.

It was mixed, placed on ice for 30 minutes, then placed in a 42 °C water bath for 90 seconds, and then placed back on ice for 2 minutes

Add 800 μl of antibiotic-free culture medium, and shake in a 37°C bacterial incubator for 1 hour. 4500 rpm/

Separation and collection of bacteria were applied to a solid LB medium plate and cultivated overnight in a bacterial incubator at 37°C

until colonies grow. Pick a single clone and place it in a 1.5ml centrifuge tube for culture. Use PCR to identify whether the transformation is successful. The primers used for identification are:

T7primer: 5'-AATACGACTCACTATAG-3' (SEQ ID NO: 16);

T3primer: 5'-TTAACCCTCACTAAAGG-3' (SEQ ID NO: 17).

The successfully transformed samples were sent for sequencing (the sequencing service was provided by Huada Genomics Co., Ltd.). The primer used for the sequencing was: T7primer: 5'-AATACGACTCACTATAG-3' (SEQ ID NO.: 16). Take the correctly sequenced bacteria and 5ml of antibiotic-added medium and add them to a 50ml centrifuge tube for 8 hours, then pour them together with 400ml of antibiotic-added medium into a 1000ml culture bottle to expand the culture, and use the QIAGEN Plasmid Maxi Kit to pump out reagents Kit (produced by Kaijie Biotechnology Co., Ltd., Germany) to extract the plasmid, and the plasmid extraction was performed according to the instructions of the Plasmid Maxi Kit.

The brain tissue of E18 or POSD rats was taken out according to the animal operating procedures, transferred to a 10cm petri dish filled with dissection fluid (Hank'sbuffer; NaHCO ₃ 0.35g/L; HEPES 10mM, pH 7.3; Glucose 33.3mM), and the Meninges, isolated from the cerebral cortex. The cortex was transferred to a 35 mm Petri dish filled with dissection solution, cut into pieces with scissors, and then digested with Trypsin (0.14%) at 37°C for 15 minutes. The digestion of Trypsin was terminated by adding plating medium (95% MEM, Invitrogen, USA; 5% FBS (v/v), Invitrogen, USA; 0.4% Glucose (w/v), Sigma, USA). Carefully dissipate the digested tissue into a single-cell suspension with a Pasteur pipette or 1ml pipette tip. Centrifuge at a speed of 500g for 5 minutes in a centrifuge, the cell debris is suspended in the supernatant, and the cells sink to the bottom. Discard the supernatant, wash the pelleted cells once with plating medium, and centrifuge at 500g for 5 minutes to pellet the cells. Resuspend the cells in plating medium again. The cell suspension was diluted 10 times and counted with a hemocytometer. All petri dishes and slides were coated with polylysine (PDL) (0.1 mg/ml for slides, 0.01 mg/ml for petri dishes) before use. The cells were cultured in a 5% CO ₂ , 37°C cell culture incubator. After cell attachment (about 1-2 hours), use Neurobasal cell culture medium (97% Neurobasal medium (v/v), Invitrogen, the U.S.; 2% B-27supplement (v/v), Invitrogen, the U.S.; 1% GlutaMAX (v/v), Invitrogen, USA) for medium replacement.

Use lonza's nucleofection system to electroporate neurons before plating: mix 200 μl electrotransfer solution, 15 μg constructed PTPA shRNA plasmids and control plasmids with 1×10 ⁷ cells respectively, add to the electroporation cup, and use The electroporator (Lonza) selects the O-03 program for electric shock, then quickly adds 1 mL of medium to resuspend the cells, and spreads them on the culture plate at an appropriate density. After 2 hours, the serum-free N27 medium (500ml Neural basal+10ml B27+500μl Glutamax+10μl gentamicin) was replaced. The cells were cultured in a 5% CO ₂ incubator at 37°C.

Three days later, add RIPA lysate (purchased by Biyuntian Company) and lyse on ice for 30 minutes, then in a refrigerated centrifuge (supplied by Eppendorf), centrifuge at 14,000g for 20 minutes at 4°C and transfer the supernatant to a new 1.5ml centrifuge tube , and then carry out protein concentration determination and Western blot detection PTPA protein level according to the method in embodiment 1.

The electrotransfer fluid is composed of liquid A and liquid B, which should be mixed at a ratio of 1:50 before use.

Electroporation solution A: 2g ATP-disodium salt, sigma A2383; 1.2g MgCl ₂ ·6H ₂ O, sigma M2393; dilute to 10ml with double-distilled water, filter and aliquot through a 0.22 micron filter, and store at -20°C.

Electroporation solution B: 6g KH ₂ PO ₄ , sigma P5655; 0.6g NaHCO ₃ , sigma S5761; 0.2g Glucose, sigma G6152, adjust the pH to 7.4 after dissolving, dilute to 500ml, filter through a 0.22 micron filter, Store at -20°C.

The results of Western Blot are shown in Figure 8, indicating that the above three PTPA shRNAs can down-regulate the protein expression level of PTPA.

2. PTPA interference shRNA alleviates the motor phenotype of Angelman syndrome

The sh4# sequence of PTPA was sent to Shanghai Taiertu Biotechnology Co., Ltd. to construct an AAV virus, and the sequence was inserted into the p-CMV-bGlobin-Egfp-H1 (Shanghai Taiertu Biotechnology Co., Ltd.) vector to obtain the serotype AAV9-type empty vector control virus and virus particles that interfere with PTPA shRNA. 1ul (1×10 ¹² VG/ml) of the two constructed viruses were injected into the lateral ventricles of WT and AS mice on the first day of birth using a 10ul micro-injector (Shanghai Gaoge) respectively. Mice were examined for rotarod behavior after four weeks. The operating steps of this behavior are shown in Example 5.

After virus injection, the motor behavior results of the mice are shown in Figure 9, indicating that the PTPA-interfering shRNA virus of the present invention can improve the motor coordination and learning ability of Angelman syndrome mice.

After the behavioral experiment was over, 0.7% pentobarbital sodium was injected intraperitoneally according to the standard of 70 mg/kg; After cutting the left and right costal arches and raising them, the heart can be seen. Cut the right atrial appendage with scissors, and you can see the blood flowing out. Quickly inject 20ml of normal saline through the left ventricle and use the body fluid circulation to flush out the blood. At this time, you can see that the liver turns white. Then use 20ml of 4% paraformaldehyde (PFA) (4g paraformaldehyde, 8g NaCl, 0.2g KCl, 3.58g Na ₂ HPO ₄ ·12H ₂ O, 0.24g KH ₂ PO ₄ , distilled water to make up to 1000ml ) immobilized mice. After fixation, the brain tissue of the mouse was taken out, placed in 5 ml of 4% PFA solution, and placed at 4° C. overnight for post-fixation. On the second day, change to 15% sucrose solution (15g sucrose prepared in 1×PBS solution) and put it back into 4°C for dehydration), and on the third day, change to 30% sucrose solution (30g sucrose prepared in 1×PBS solution, 4 dehydrate overnight.

Take out the completely dehydrated mouse brain, blot the peripheral liquid on the filter paper, cut the mouse brain along the middle seam, use the cut surface as the bottom surface, place it on the base of the microtome, and embed it with OCT. Freeze in a cryostat for about 30 minutes. Place the frozen brain together with the base on a cryostat (CM1950, Leica), adjust the slice thickness button, and cut out a 30-micron sagittal brain slice. Brain slices were collected in 12-well dishes filled with PBS.

After all the brain slices were cut, 4 brain slices were picked out, and the inventors placed the selected brain slices in PBS containing the nuclear antibody Hoechst (Biyuntian) for 15 minutes for staining. Finally, after rinsing twice with PBS, mount the slide, and mount the slide with 90% glycerol (90ml glycerol, 10ml PBS). Then, proceed to observe the extent of viral infection. Because the virus vector used has the GFP gene. Therefore, the present inventors used the location information of GFP to represent the infection range of the virus. The same use of OlympusVS120 high-throughput fluorescence imaging microscope, with 20 × objective lens for shooting.

After the virus was injected into the lateral ventricle, the scope of infection is shown in Figure 10, indicating that the virus infection in the present invention is very extensive.

Embodiment 8, screening method

1. PP2A is the screening target

set up:

Test group: HEK 293T cells (where PP2A is expressed), and the candidate substances are administered;

Control group: HEK 293T cells (in which PP2A is expressed), no candidate substance was administered.

The expression of PP2A in the test group and the control group were respectively detected and compared. If the expression of PP2A in the test group is statistically lower (eg, 30% lower or lower) than the control group, it indicates that the candidate is a potential substance for alleviating or treating Angelman syndrome.

2. PTPA is the screening target

set up:

Test group: HEK 293T cells (where PTPA is expressed), and the candidate substances are administered;

Control group: HEK 293T cells (in which PTPA is expressed), to which no candidate substance was administered.

The expression of PTPA in the test group and the control group were respectively detected and compared. If the expression of PTPA in the test group is statistically lower (eg, 30% lower or lower) than the control group, it indicates that the candidate is a potential substance for alleviating or treating Angelman syndrome.

3. PP2A is the screening target, PTPA and Ube3a are involved

set up:

Test group: HEK 293T cells (where Ube3a, PTPA, PP2A are simultaneously expressed), and given candidate substances;

Control group: HEK 293T cells (in which Ube3a, PTPA, PP2A are simultaneously expressed), no candidate substance was administered.

The expression of Ube3a and PP2A in the test group and the control group were respectively detected and compared. A candidate is indicated if the expression of Ube3a in the test group is statistically higher (eg, 30% higher or higher) than that in the control group, accompanied by degradation of PTPA and a significant decrease (eg, 30% or lower) in PP2A It is a potential substance for the alleviation or treatment of Angelman syndrome.

Example 9, Detection of PP2A activity after Ube3a overexpression

1. Construction of Ube3a overexpression vector

Amplification of Ube3a fragment: Ube3a sequence of mouse (NM_011668), obtain its coding sequence from NCBI, design PCR primers, and amplify it, the primer sequence is as follows:

Ube3a-F: 5'-atgaagcgagcagctgcaaagc-3' (SEQ ID NO: 10);

Ube3a-R: 5'-ttacagcatgccaaatcctttggc-3' (SEQ ID NO: 11);

Submit primer information to Invitrogen Company for synthesis. The obtained primers were dissolved in ddH ₂ O at a concentration of 10 uM. Using the PCR method, using the mouse cDNA library as a template, specifically amplify the CDS of Ube3a, the specific reaction system is: (1ulUbe3a-F, 1ul Ube3a-R, 10ul dNTP, 1ul KOD, 25ul reaction buffer, 2ul cDNA library , and finally fill up to 50ul with ddH ₂ O), and amplify a fragment with a length of 2613bp according to the reaction specificity and instructions of KOD enzyme (KFX-101). The obtained fragment is amplified again with new primers, and its two ends are added to the sequences at both ends of the connected vector for the following recombination connection. The primer sequences are as follows:

Ube3a-F-infusion:

5'-GAGCCCGGGCGGATCCatggccacagcttgtaaaagatc-3' (SEQ ID NO: 12);

Ube3a-R-infusion:

5'-GGTATCGATAAGCTTttacagcatgccaaatcctttggc-3' (SEQ ID NO: 13);

The PCR fragment obtained for the first time was used as a template, and PCR was performed again, and then the vector pCAG-tag2B empty vector (purchased from Stratagene Company) after digesting with BamHI and HindIII was digested with recombinase (ClonExpress II One Step Cloning Kit, vazyme) at 37 After recombination at ℃ for 30 minutes, add the ligation product into a 1.5ml centrifuge tube containing Escherichia coli DH5α competent cells for mixing, put it on ice for 30 minutes, then put it in a water bath at 42℃ for 90 seconds, and then put it back on ice, - After 2 minutes, 800 μl of antibiotic-free medium was added, and placed in a bacterial incubator at 37° C. for shaking for 1 hour. The obtained bacteria were collected by centrifugation at 4500 rpm and then coated with a solid LB medium plate, and cultivated overnight in a 37°C bacterial incubator until colonies grew.

Pick a single clone and place it in a 1.5ml centrifuge tube for culture. Use PCR to identify whether the transformation is successful. The primers used for identification are:

pCAG-tag2B-F: 5'-ggcaaagaagctagcg-3' (SEQ ID NO: 14);

pCAG-tag2B-R: 5'-gggcgatcgagtgaattgtaatac-3' (SEQ ID NO: 15);

The successfully transformed samples were sent for sequencing (the sequencing service is provided by Huada Genomics Co., Ltd.). The primers used for the sequencing are: pCAG-tag2B-F: ggcaaagaagctagcg. Take the correctly sequenced bacteria and 5ml of antibiotic-added medium and add them to a 50ml centrifuge tube for 8 hours, then pour them together with 400ml of antibiotic-added medium into a 1000ml culture bottle to expand the culture, and use the QIAGEN Plasmid Maxi Kit to pump out reagents Kit (produced by Kaijie Biotechnology Co., Ltd., Germany) to extract the plasmid, and the plasmid extraction was performed according to the instructions of the Plasmid Maxi Kit.

Culture HEK 293T cells (hereinafter referred to as 293T, purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) in a cell culture incubator at 37 °C with 5% _CO2 , and the medium used was DMEM supplemented with 10% fetal bovine serum (Gibco produced by the company) culture medium. Subculture 293T cells in a petri dish with a diameter of 6 cm. When the cells grow to a confluence of about 70%, operate according to the instructions of Lipofectamine 2000 (produced by Invitrogen, USA), and prepare 6 ug of the above-obtained pCAG-tag2B-Ube3a plasmid (or Empty vector plasmid without Ube3a) transfection mix. The mixture was added to the cells for transfection, and the cells were collected after 24 hours of culture, and the PP2A activity was measured according to the method in Example 3.

The results of phosphatase activity are shown in Figure 11, A-B, indicating that in HEK293T cells overexpressing Ube3a, PP2A activity was significantly reduced.

Example 10, UBE3A regulates the activity of MAPK signaling pathway

After taking out the mice 30 days after birth, the wild-type (WT) and littermate AS mice were anesthetized, and then the brain tissue was taken for western blot detection. The specific method was the same as in Example 1.

While in UBE3A overexpressed HEK293T, the phosphorylation level of ERK1/2 was upregulated.

The results of Western blot are shown in Figure 12 (A, B), indicating that in UBE3A-deficient AS mice, the phosphorylation level of ERK1/2 decreased; after UBE3A was overexpressed, the phosphorylation level of ERK1/2 increased. The two are positively correlated.

Example 11, PP2A is used as a screening target, and ERK1/2 is used as an index for screening

Using the immunoblotting method (described in detail above), add PP2A inhibitor Okadaic acid (OA, Cell signaling, #5934) (10nM, 2 hours) or CalyculinA (CYA) to neurons cultured for 10 days in vitro (Sigma, C5552) (10nM, 2 hours), the phosphorylation of ERK1/2 increased, and when the PP2A agonist FTY720 (Sigma, SML0700) (10uM, 8 hours) was added, the phosphorylation of ERK1/2 decreased. It shows that autism caused by overexpression of UBE3A can be improved by agonists of PP2A. At the same time, FTY720 may also become a potential drug for the treatment of autism.

The specific experimental methods and steps are as follows:

Primary cultured rat cortical and hippocampal neurons were obtained from Sprague-Dawley (SD) rats within 24 hours of birth. The specific steps are roughly as follows: soak the dissecting instruments in 75% alcohol in advance, and irradiate them with ultraviolet rays for 30 minutes in a sterile operating table. The rats were sprayed with 75% disinfectant alcohol for disinfection, the scalp and skull were carefully torn apart with scissors and tweezers, the brain was peeled off and placed in dissecting fluid (Hank's buffer; NaHCO ₃ 0.35g/L; HEPES 10mM, pH 7.3; Glucose33.3mM )middle. Use scissors and tweezers in the dissection solution to cut off the connection between the cortex and other structures such as the brainstem and thalamus, and carefully peel off the meninges of the cerebral hemispheres under a stereomicroscope. Hippocampal tissue was harvested with ophthalmic scissors. The cortical tissue and hippocampal tissue from which the meninges were removed were cut into small pieces, and digested with freshly prepared papain in a 37°C incubator for 7 minutes, gently pipetting once in the middle, and digested again for 7 minutes. After the digestion, an equal volume of plating medium (450 mL MEM, 50 mL FBS, 0.4% Glucose) was added to stop the digestion, and the tissue pieces were blown gently with a pipette. The entire solution was filtered through a 70 μm pore size filter to remove undigested tissue pieces. The filtered product was centrifuged at 500 g for 5 minutes to obtain the cells, and the supernatant was removed. The cells were resuspended in culture medium, centrifuged, and the supernatant removed. The resulting cells were resuspended in culture medium, counted with a hemocytometer and plated in cell wells at the desired density. All Petri dishes and slides were coated with polylysine (PDL) (0.1 mg/mL for slides, 0.01 mg/mL for Petri dishes) before use. The cells were cultured in a 5% CO2, 37°C cell incubator. After 2-4 hours, the culture medium was replaced with Nuerobasal medium.

Dissecting fluid: Hank's buffer; NaHCO ₃ 0.35g/L; HEPES 10mM, pH 7.3; Glucose33.3mM.

Plating medium: 450mL MEM, 50mL HBS, 2g Glucose.

Nuerobasal medium culture: 500mL Neurobasal A medium, 5mL B-27supplement, 5mL GlutaMAX.

Okadaic acid and Calyculin A were dissolved in DMSO, and FTY720 was dissolved in water.

The results of Western Blot are shown in Figure 13, indicating that PP2A inhibitors can increase the phosphorylation level of ERK1/2 and activate the MAPK pathway; PP2A agonist FTY720 can inhibit the phosphorylation level of ERK1/2 and inhibit the MAPK pathway.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by 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.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> Ube3a ubiquitinates PP2A activator PTPA in the treatment of Angelman syndrome and autism

<130> P2018-1542

<150> CN 201810195972.9

<151> 2018-03-09

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Ala Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp Asn Arg Ile Arg Met

485 490 495

Tyr Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser Leu Val Gln Gly Gln

500 505 510

Gln Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg Arg Asp His Ile Ile

515 520 525

Asp Asp Ala Leu Val Arg Leu Glu Met Ile Ala Met Glu Asn Pro Ala

530 535 540

Asp Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu Gly Glu Gln Gly Val

545 550 555 560

Asp Glu Gly Gly Val Ser Lys Glu Phe Phe Gln Leu Val Val Glu Glu

565 570 575

Ile Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr Asp Glu Ala Thr Lys

580 585 590

Leu Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr Glu Gly Gln Phe Thr

595 600 605

Leu Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr Asn Asn Cys Ile Leu

610 615 620

Asp Val His Phe Pro Met Val Val Tyr Arg Lys Leu Met Gly Lys Lys

625 630 635 640

Gly Thr Phe Arg Asp Leu Gly Asp Ser His Pro Val Leu Tyr Gln Ser

645 650 655

Leu Lys Asp Leu Leu Glu Tyr Glu Gly Ser Val Glu Asp Asp Met Met

660 665 670

Ile Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe Gly Asn Pro Met Met

675 680 685

Tyr Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro Ile Thr Asn Glu Asn

690 695 700

Arg Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr Ile Leu Asn Lys Ser

705 710 715 720

Val Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly Phe His Met Val Thr

725 730 735

Asn Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro Glu Glu Ile Glu Leu

740 745 750

Leu Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln Ala Leu Glu Glu Thr

755 760 765

Thr Glu Tyr Asp Gly Gly Tyr Thr Arg Glu Ser Val Val Ile Arg Glu

770 775 780

Phe Trp Glu Ile Val His Ser Phe Thr Asp Glu Gln Lys Arg Leu Phe

785 790 795 800

Leu Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro Val Gly Gly Leu Gly

805 810 815

Lys Leu Lys Met Ile Ile Ala Lys Asn Gly Pro Asp Thr Glu Arg Leu

820 825 830

Pro Thr Ser His Thr Cys Phe Asn Val Leu Leu Leu Pro Glu Tyr Ser

835 840 845

Ser Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys Ala Ile Thr Tyr Ala

850 855 860

Lys Gly Phe Gly Met Leu

865 870

<210> 19

<211> 309

<212> PRT

<213> Mouse (Mus musculus)

<400> 19

Met Asp Glu Lys Leu Phe Thr Lys Glu Leu Asp Gln Trp Ile Glu Gln

1 5 10 15

Leu Asn Glu Cys Lys Gln Leu Ser Glu Ser Gln Val Lys Ser Leu Cys

20 25 30

Glu Lys Ala Lys Glu Ile Leu Thr Lys Glu Ser Asn Val Gln Glu Val

35 40 45

Arg Cys Pro Val Thr Val Cys Gly Asp Val His Gly Gln Phe His Asp

50 55 60

Leu Met Glu Leu Phe Arg Ile Gly Gly Lys Ser Pro Asp Thr Asn Tyr

65 70 75 80

Leu Phe Met Gly Asp Tyr Val Asp Arg Gly Tyr Tyr Ser Val Glu Thr

85 90 95

Val Thr Leu Leu Val Ala Leu Lys Val Arg Tyr Arg Glu Arg Ile Thr

100 105 110

Ile Leu Arg Gly Asn His Glu Ser Arg Gln Ile Thr Gln Val Tyr Gly

115 120 125

Phe Tyr Asp Glu Cys Leu Arg Lys Tyr Gly Asn Ala Asn Val Trp Lys

130 135 140

Tyr Phe Thr Asp Leu Phe Asp Tyr Leu Pro Leu Thr Ala Leu Val Asp

145 150 155 160

Gly Gln Ile Phe Cys Leu His Gly Gly Leu Ser Pro Ser Ile Asp Thr

165 170 175

Leu Asp His Ile Arg Ala Leu Asp Arg Leu Gln Glu Val Pro His Glu

180 185 190

Gly Pro Met Cys Asp Leu Leu Trp Ser Asp Pro Asp Asp Arg Gly Gly

195 200 205

Trp Gly Ile Ser Pro Arg Gly Ala Gly Tyr Thr Phe Gly Gln Asp Ile

210 215 220

Ser Glu Thr Phe Asn His Ala Asn Gly Leu Thr Leu Val Ser Arg Ala

225 230 235 240

His Gln Leu Val Met Glu Gly Tyr Asn Trp Cys His Asp Arg Asn Val

245 250 255

Val Thr Ile Phe Ser Ala Pro Asn Tyr Cys Tyr Arg Cys Gly Asn Gln

260 265 270

Ala Ala Ile Met Glu Leu Asp Asp Thr Leu Lys Tyr Ser Phe Leu Gln

275 280 285

Phe Asp Pro Ala Pro Arg Arg Gly Glu Pro His Val Thr Arg Arg Thr

290 295 300

Pro Asp Tyr Phe Leu

305

<210> 20

<211> 930

<212>DNA

<213> Mouse (Mus musculus)

<400> 20

atggacgaga agttgttcac caaggagctg gaccagtgga tcgagcagct gaacgagtgc 60

aagcagctct ccgagtccca ggtcaagagc ctctgcgaga aggctaaaga aatcctgaca 120

aaagaatcca acgttcaaga ggttcgatgt ccagtcactg tgtgtggaga tgtacatggg 180

caatttcatg atctcatgga actctttaga attggtggta aatcaccaga tacaaattac 240

ctgtttatgg gagactatgt ggacagagga tattactctg ttgaaacagt tacactgctt 300

gtagctctta aggttcgtta ccgagagcgc atcaccatac tccgagggaa tcacgagagc 360

agacagatca cacaggttta tgggttctac gacgagtgtt taaggaaata cggaaatgca 420

aatgtttgga aatacttcac agaccttttt gactatcttc ctctcactgc cttggtggat 480

gggcagatct tctgtctaca cggtggtctg tcaccatcca tagacacact ggatcacatc 540

cgagcactcg atcgcctaca ggaagttcct catgagggtc caatgtgtga cttgctgtgg 600

tcagatccag atgaccgtgg tggctggggg atatctcctc ggggagctgg ttataccttt 660

ggccaagata tttctgagac atttaatcat gccaatggcc tcacgttggt gtccagagct 720

caccagctgg tgatggaggg atataactgg tgccatgacc ggaacgtagt aacaattttc 780

agtgctccaa actattgcta tcgttgtggt aaccaagctg caatcatgga acttgacgac 840

actcttaagt attctttctt gcagtttgac ccagcacctc gtagaggcga gccacatgtc 900

actcgtcgta ccccagacta cttcctgtaa 930

<210> 21

<211> 323

<212> PRT

<213> Mouse (Mus musculus)

<400> 21

Met Ala Glu Gly Glu Arg Gln Pro Pro Pro Asp Ser Ser Glu Glu Thr

1 5 10 15

Pro Pro Thr Thr Gln Asn Phe Ile Ile Pro Lys Lys Glu Ile His Thr

20 25 30

Val Pro Asp Met Gly Lys Trp Lys Arg Ser Gln Ala Tyr Ala Asp Tyr

35 40 45

Ile Gly Phe Ile Leu Thr Leu Asn Glu Gly Val Lys Gly Lys Lys Leu

50 55 60

Thr Phe Asp Tyr Lys Val Ser Glu Ala Ile Glu Lys Leu Val Ala Leu

65 70 75 80

Leu Asp Thr Leu Asp Arg Trp Ile Asp Glu Thr Pro Pro Val Asp Gln

85 90 95

Pro Ser Arg Phe Gly Asn Lys Ala Tyr Arg Thr Trp Tyr Ala Lys Leu

100 105 110

Asp Gln Glu Ala Glu Asn Leu Val Ala Thr Val Val Pro Thr His Leu

115 120 125

Ala Ala Ala Val Pro Glu Val Ala Val Tyr Leu Lys Glu Ala Val Gly

130 135 140

Asn Ser Thr Arg Ile Asp Tyr Gly Thr Gly His Glu Ala Ala Phe Ala

145 150 155 160

Ala Phe Leu Cys Cys Leu Cys Lys Ile Gly Val Leu Arg Val Asp Asp

165 170 175

Gln Val Ala Ile Val Phe Lys Val Phe Asp Arg Tyr Leu Glu Val Met

180 185 190

Arg Lys Leu Gln Lys Thr Tyr Arg Met Glu Pro Ala Gly Ser Gln Gly

195 200 205

Val Trp Gly Leu Asp Asp Phe Gln Phe Leu Pro Phe Ile Trp Gly Ser

210 215 220

Ser Gln Leu Ile Asp His Pro His Leu Glu Pro Arg His Phe Val Asp

225 230 235 240

Glu Lys Ala Val Ser Glu Asn His Lys Asp Tyr Met Phe Leu Gln Cys

245 250 255

Ile Leu Phe Ile Thr Glu Met Lys Thr Gly Pro Phe Ala Glu His Ser

260 265 270

Asn Gln Leu Trp Asn Ile Ser Ala Val Pro Ser Trp Ser Lys Val Asn

275 280 285

Gln Gly Leu Ile Arg Met Tyr Lys Ala Glu Cys Leu Glu Lys Phe Pro

290 295 300

Val Ile Gln His Phe Lys Phe Gly Ser Leu Leu Pro Ile His Pro Val

305 310 315 320

Thr Ser Gly

<210> 22

<211> 978

<212>DNA

<213> Mouse (Mus musculus)

<400> 22

atggccgagg gcgagcggca gccgccgcca gattcttcag aagagacccc tccaactact 60

cagaacttta tcattccaaa aaaggagatc cacacagttc cagatatggg caaatggaag 120

cgctctcagg catatgctga ctacattggc ttcatcctta ccctcaatga aggtgtgaag 180

gggaagaagc tgaccttcga ctacaaagtc tctgaggcca tcgagaagct ggtggcactt 240

cttgatacgc tggataggtg gattgatgaa accccgccag tggaccagcc ttcccggttt 300

gggaacaaag cctacagaac ctggtatgcc aaacttgatc aggaagcaga aaacttggtg 360

gccacagtgg tccccaccca cctggctgct gctgtgcctg aagtggcagt ttacctgaag 420

gaggctgtgg ggaactccac acgaattgac tatggcacag ggcatgaggc tgcctttgct 480

gctttcctct gttgtctctg caagattggt gtactccggg tggacgacca ggtggctatt 540

gtcttcaagg tgtttgatag gtatcttgag gttatgcgga agttgcagaa gacatacagg 600

atggagcctg caggcagcca gggcgtatgg ggtctggatg acttccagtt cctgcccttc 660

atctggggca gctcacagct cataggtact aaccacccccc acctggagcc cagacatttc 720

gtggatgaga aggcggtgag cgagaaccac aaggactaca tgtttctcca gtgcatcctg 780

ttcatcactg agatgaagac tggccccttt gcggaacact ccaaccagct gtggaacatc 840

agtgctgtcc cctcctggtc taaagtgaac cagggcctca ttcgaatgta taaggcagag 900

tgcctggaga agttccctgt gatccagcac ttcaagttcg ggagcctgct gcccatccat 960

cccgtcacat caggctag 978

<210> 23

<211> 875

<212> PRT

<213> Homo sapiens

<400> 23

Met Glu Lys Leu His Gln Cys Tyr Trp Lys Ser Gly Glu Pro Gln Ser

1 5 10 15

Asp Asp Ile Glu Ala Ser Arg Met Lys Arg Ala Ala Ala Lys His Leu

20 25 30

Ile Glu Arg Tyr Tyr His Gln Leu Thr Glu Gly Cys Gly Asn Glu Ala

35 40 45

Cys Thr Asn Glu Phe Cys Ala Ser Cys Pro Thr Phe Leu Arg Met Asp

50 55 60

Asn Asn Ala Ala Ala Ile Lys Ala Leu Glu Leu Tyr Lys Ile Asn Ala

65 70 75 80

Lys Leu Cys Asp Pro His Pro Ser Lys Lys Gly Ala Ser Ser Ala Tyr

85 90 95

Leu Glu Asn Ser Lys Gly Ala Pro Asn Asn Ser Cys Ser Glu Ile Lys

100 105 110

Met Asn Lys Lys Gly Ala Arg Ile Asp Phe Lys Asp Val Thr Tyr Leu

115 120 125

Thr Glu Glu Lys Val Tyr Glu Ile Leu Glu Leu Cys Arg Glu Arg Glu

130 135 140

Asp Tyr Ser Pro Leu Ile Arg Val Ile Gly Arg Val Phe Ser Ser Ala

145 150 155 160

Glu Ala Leu Val Gln Ser Phe Arg Lys Val Lys Gln His Thr Lys Glu

165 170 175

Glu Leu Lys Ser Leu Gln Ala Lys Asp Glu Asp Lys Asp Glu Asp Glu

180 185 190

Lys Glu Lys Ala Ala Cys Ser Ala Ala Ala Met Glu Glu Asp Ser Glu

195 200 205

Ala Ser Ser Ser Arg Ile Gly Asp Ser Ser Gln Gly Asp Asn Asn Leu

210 215 220

Gln Lys Leu Gly Pro Asp Asp Val Ser Val Asp Ile Asp Ala Ile Arg

225 230 235 240

Arg Val Tyr Thr Arg Leu Leu Ser Asn Glu Lys Ile Glu Thr Ala Phe

245 250 255

Leu Asn Ala Leu Val Tyr Leu Ser Pro Asn Val Glu Cys Asp Leu Thr

260 265 270

Tyr His Asn Val Tyr Ser Arg Asp Pro Asn Tyr Leu Asn Leu Phe Ile

275 280 285

Ile Val Met Glu Asn Arg Asn Leu His Ser Pro Glu Tyr Leu Glu Met

290 295 300

Ala Leu Pro Leu Phe Cys Lys Ala Met Ser Lys Leu Pro Leu Ala Ala

305 310 315 320

Gln Gly Lys Leu Ile Arg Leu Trp Ser Lys Tyr Asn Ala Asp Gln Ile

325 330 335

Arg Arg Met Met Glu Thr Phe Gln Gln Leu Ile Thr Tyr Lys Val Ile

340 345 350

Ser Asn Glu Phe Asn Ser Arg Asn Leu Val Asn Asp Asp Asp Ala Ile

355 360 365

Val Ala Ala Ser Lys Cys Leu Lys Met Val Tyr Tyr Ala Asn Val Val

370 375 380

Gly Gly Glu Val Asp Thr Asn His Asn Glu Glu Asp Asp Glu Glu Pro

385 390 395 400

Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu

405 410 415

Arg Arg Asn Lys Lys Gly Pro Arg Val Asp Pro Leu Glu Thr Glu Leu

420 425 430

Gly Val Lys Thr Leu Asp Cys Arg Lys Pro Leu Ile Pro Phe Glu Glu

435 440 445

Phe Ile Asn Glu Pro Leu Asn Glu Val Leu Glu Met Asp Lys Asp Tyr

450 455 460

Thr Phe Phe Lys Val Glu Thr Glu Asn Lys Phe Ser Phe Met Thr Cys

465 470 475 480

Pro Phe Ile Leu Asn Ala Val Thr Lys Asn Leu Gly Leu Tyr Tyr Asp

485 490 495

Asn Arg Ile Arg Met Tyr Ser Glu Arg Arg Ile Thr Val Leu Tyr Ser

500 505 510

Leu Val Gln Gly Gln Gln Leu Asn Pro Tyr Leu Arg Leu Lys Val Arg

515 520 525

Arg Asp His Ile Ile Asp Asp Ala Leu Val Arg Leu Glu Met Ile Ala

530 535 540

Met Glu Asn Pro Ala Asp Leu Lys Lys Gln Leu Tyr Val Glu Phe Glu

545 550 555 560

Gly Glu Gln Gly Val Asp Glu Gly Gly Val Ser Lys Glu Phe Phe Gln

565 570 575

Leu Val Val Glu Glu Ile Phe Asn Pro Asp Ile Gly Met Phe Thr Tyr

580 585 590

Asp Glu Ser Thr Lys Leu Phe Trp Phe Asn Pro Ser Ser Phe Glu Thr

595 600 605

Glu Gly Gln Phe Thr Leu Ile Gly Ile Val Leu Gly Leu Ala Ile Tyr

610 615 620

Asn Asn Cys Ile Leu Asp Val His Phe Pro Met Val Val Tyr Arg Lys

625 630 635 640

Leu Met Gly Lys Lys Gly Thr Phe Arg Asp Leu Gly Asp Ser His Pro

645 650 655

Val Leu Tyr Gln Ser Leu Lys Asp Leu Leu Glu Tyr Glu Gly Asn Val

660 665 670

Glu Asp Asp Met Met Ile Thr Phe Gln Ile Ser Gln Thr Asp Leu Phe

675 680 685

Gly Asn Pro Met Met Tyr Asp Leu Lys Glu Asn Gly Asp Lys Ile Pro

690 695 700

Ile Thr Asn Glu Asn Arg Lys Glu Phe Val Asn Leu Tyr Ser Asp Tyr

705 710 715 720

Ile Leu Asn Lys Ser Val Glu Lys Gln Phe Lys Ala Phe Arg Arg Gly

725 730 735

Phe His Met Val Thr Asn Glu Ser Pro Leu Lys Tyr Leu Phe Arg Pro

740 745 750

Glu Glu Ile Glu Leu Leu Ile Cys Gly Ser Arg Asn Leu Asp Phe Gln

755 760 765

Ala Leu Glu Glu Thr Thr Glu Tyr Asp Gly Gly Tyr Thr Arg Asp Ser

770 775 780

Val Leu Ile Arg Glu Phe Trp Glu Ile Val His Ser Phe Thr Asp Glu

785 790 795 800

Gln Lys Arg Leu Phe Leu Gln Phe Thr Thr Gly Thr Asp Arg Ala Pro

805 810 815

Val Gly Gly Leu Gly Lys Leu Lys Met Ile Ile Ala Lys Asn Gly Pro

820 825 830

Asp Thr Glu Arg Leu Pro Thr Ser His Thr Cys Phe Asn Val Leu Leu

835 840 845

Leu Pro Glu Tyr Ser Ser Lys Glu Lys Leu Lys Glu Arg Leu Leu Lys

850 855 860

Ala Ile Thr Tyr Ala Lys Gly Phe Gly Met Leu

865 870 875

<210> 24

<211> 309

<212> PRT

<213> Homo sapiens

<400> 24

Met Asp Glu Lys Val Phe Thr Lys Glu Leu Asp Gln Trp Ile Glu Gln

1 5 10 15

Leu Asn Glu Cys Lys Gln Leu Ser Glu Ser Gln Val Lys Ser Leu Cys

20 25 30

Glu Lys Ala Lys Glu Ile Leu Thr Lys Glu Ser Asn Val Gln Glu Val

35 40 45

Arg Cys Pro Val Thr Val Cys Gly Asp Val His Gly Gln Phe His Asp

50 55 60

Leu Met Glu Leu Phe Arg Ile Gly Gly Lys Ser Pro Asp Thr Asn Tyr

65 70 75 80

Leu Phe Met Gly Asp Tyr Val Asp Arg Gly Tyr Tyr Ser Val Glu Thr

85 90 95

Val Thr Leu Leu Val Ala Leu Lys Val Arg Tyr Arg Glu Arg Ile Thr

100 105 110

Ile Leu Arg Gly Asn His Glu Ser Arg Gln Ile Thr Gln Val Tyr Gly

115 120 125

Phe Tyr Asp Glu Cys Leu Arg Lys Tyr Gly Asn Ala Asn Val Trp Lys

130 135 140

Tyr Phe Thr Asp Leu Phe Asp Tyr Leu Pro Leu Thr Ala Leu Val Asp

145 150 155 160

Gly Gln Ile Phe Cys Leu His Gly Gly Leu Ser Pro Ser Ile Asp Thr

165 170 175

Leu Asp His Ile Arg Ala Leu Asp Arg Leu Gln Glu Val Pro His Glu

180 185 190

Gly Pro Met Cys Asp Leu Leu Trp Ser Asp Pro Asp Asp Arg Gly Gly

195 200 205

Trp Gly Ile Ser Pro Arg Gly Ala Gly Tyr Thr Phe Gly Gln Asp Ile

210 215 220

Ser Glu Thr Phe Asn His Ala Asn Gly Leu Thr Leu Val Ser Arg Ala

225 230 235 240

His Gln Leu Val Met Glu Gly Tyr Asn Trp Cys His Asp Arg Asn Val

245 250 255

Val Thr Ile Phe Ser Ala Pro Asn Tyr Cys Tyr Arg Cys Gly Asn Gln

260 265 270

Ala Ala Ile Met Glu Leu Asp Asp Thr Leu Lys Tyr Ser Phe Leu Gln

275 280 285

Phe Asp Pro Ala Pro Arg Arg Gly Glu Pro His Val Thr Arg Arg Thr

290 295 300

Pro Asp Tyr Phe Leu

305

<210> 25

<211> 358

<212> PRT

<213> Homo sapiens

<400> 25

Met Ala Glu Gly Glu Arg Gln Pro Pro Pro Asp Ser Ser Glu Glu Ala

1 5 10 15

Pro Pro Ala Thr Gln Asn Phe Ile Ile Pro Lys Lys Glu Ile His Thr

20 25 30

Val Pro Asp Met Gly Lys Trp Lys Arg Ser Gln Ala Tyr Ala Asp Tyr

35 40 45

Ile Gly Phe Ile Leu Thr Leu Asn Glu Gly Val Lys Gly Lys Lys Leu

50 55 60

Thr Phe Glu Tyr Arg Val Ser Glu Met Trp Asn Glu Val His Glu Glu

65 70 75 80

Lys Glu Gln Ala Ala Lys Gln Ser Val Ser Cys Asp Glu Cys Ile Pro

85 90 95

Leu Pro Arg Ala Gly His Cys Ala Pro Ser Glu Ala Ile Glu Lys Leu

100 105 110

Val Ala Leu Leu Asn Thr Leu Asp Arg Trp Ile Asp Glu Thr Pro Pro

115 120 125

Val Asp Gln Pro Ser Arg Phe Gly Asn Lys Ala Tyr Arg Thr Trp Tyr

130 135 140

Ala Lys Leu Asp Glu Glu Ala Glu Asn Leu Val Ala Thr Val Val Pro

145 150 155 160

Thr His Leu Ala Ala Ala Val Pro Glu Val Ala Val Tyr Leu Lys Glu

165 170 175

Ser Val Gly Asn Ser Thr Arg Ile Asp Tyr Gly Thr Gly His Glu Ala

180 185 190

Ala Phe Ala Ala Phe Leu Cys Cys Leu Cys Lys Ile Gly Val Leu Arg

195 200 205

Val Asp Asp Gln Ile Ala Ile Val Phe Lys Val Phe Asn Arg Tyr Leu

210 215 220

Glu Val Met Arg Lys Leu Gln Lys Thr Tyr Arg Met Glu Pro Ala Gly

225 230 235 240

Ser Gln Gly Val Trp Gly Leu Asp Asp Phe Gln Phe Leu Pro Phe Ile

245 250 255

Trp Gly Ser Ser Gln Leu Ile Asp His Pro Tyr Leu Glu Pro Arg His

260 265 270

Phe Val Asp Glu Lys Ala Val Asn Glu Asn His Lys Asp Tyr Met Phe

275 280 285

Leu Glu Cys Ile Leu Phe Ile Thr Glu Met Lys Thr Gly Pro Phe Ala

290 295 300

Glu His Ser Asn Gln Leu Trp Asn Ile Ser Ala Val Pro Ser Trp Ser

305 310 315 320

Lys Val Asn Gln Gly Leu Ile Arg Met Tyr Lys Ala Glu Cys Leu Glu

325 330 335

Lys Phe Pro Val Ile Gln His Phe Lys Phe Gly Ser Leu Leu Pro Ile

340 345 350

His Pro Val Thr Ser Gly

355

Claims (1)

1. The use of an inhibitor of protein phosphatase 2A or an inhibitor of phosphotyrosine phosphatase activator, for preparing a pharmaceutical composition for alleviating or treating Angelman syndrome, The inhibitor of protein phosphatase 2A is a small molecular compound that specifically inhibits the activity of protein phosphatase 2A, which is selected from: inhibitor LB-100; The inhibitor of the phosphotyrosine phosphatase activator is an interfering molecule that specifically interferes with the gene expression of the phosphotyrosine phosphatase activator, and the interference molecule that specifically interferes with the gene expression of the phosphotyrosine phosphatase activator Molecules are shRNAs.

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