WO2023143425A1 - 改善认知障碍的方法 - Google Patents

改善认知障碍的方法 Download PDF

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Publication number
WO2023143425A1
WO2023143425A1 PCT/CN2023/073287 CN2023073287W WO2023143425A1 WO 2023143425 A1 WO2023143425 A1 WO 2023143425A1 CN 2023073287 W CN2023073287 W CN 2023073287W WO 2023143425 A1 WO2023143425 A1 WO 2023143425A1
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dir
amino acid
seq
acid sequence
sequence shown
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PCT/CN2023/073287
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English (en)
French (fr)
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李开诚
史海翔
攸璞
李震
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上海魁特迪生物科技有限公司
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Publication of WO2023143425A1 publication Critical patent/WO2023143425A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides

Definitions

  • This application relates to the field of biomedicine, in particular to a method for improving cognitive impairment.
  • the number of aging population in my country is increasing year by year. Due to the influence of various factors such as decreased immunity and arteriosclerosis, the elderly population is facing a variety of diseases. Dementia patients not only suffer from the pain themselves, but also put enormous pressure on their families. Therefore, it is of great significance to develop therapeutic drugs for Alzheimer's disease.
  • the present application provides a novel method for improving cognitive impairment.
  • the present application provides a regulator, which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • a regulator which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • the present application provides a regulator, which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • a regulator which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • the disease comprises a neurodegenerative disease.
  • the modulator reduces the expression level and/or biological activity of the DIR and/or functional fragments thereof in a subject.
  • said reduction comprises the difference from the original DIR and/or functional fragments thereof in said subject.
  • the expression level and/or biological activity of the DIR and/or a functional fragment thereof is reduced by at least about 10% compared to the expression level and/or biological activity.
  • the expression level comprises the expression level of the gene encoding the DIR/or its functional fragment, the transcription level of the gene encoding the DIR/or its functional fragment and/or the DIR/or its Expression levels of functional fragments.
  • said expression level is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column Chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column Chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level is measured by using a substance selected from the group consisting of primers capable of specifically amplifying genes encoding the DIR/or functional fragments thereof, and encoding the DIR/or functional fragments thereof
  • a substance selected from the group consisting of primers capable of specifically amplifying genes encoding the DIR/or functional fragments thereof, and encoding the DIR/or functional fragments thereof A nucleic acid molecule specifically binding to the gene, a nucleic acid molecule specifically binding to the DIR/or a functional fragment thereof, a small molecule specifically binding to the DIR/or a functional fragment thereof, a specific binding to the DIR/or a functional fragment thereof Specifically binding probes and polypeptides specifically binding to said DIR and/or functional fragments thereof.
  • the functional fragment of the DIR retains at least a portion of the biological activity of the DIR.
  • the biological activity includes affecting the excitatory and/or inhibitory activity of neurons.
  • the biological activity comprises the ability to reduce the frequency of excitatory postsynaptic currents (EPSCs), and/or the ability to reduce the amplitude of EPSCs.
  • ESCs excitatory postsynaptic currents
  • said reducing comprises administering said DIR and/or functional fragments thereof and/or encoding
  • the nucleic acid of the DIR and/or a functional fragment thereof reduces the frequency of an excitatory postsynaptic current (EPSC) in a subject, and/or reduces the amplitude of an EPSC in a subject.
  • ESC excitatory postsynaptic current
  • the biological activity includes affecting cognitive ability.
  • the biological activity includes participating in a signaling pathway related to A ⁇ deposition, and/or, participating in a signaling pathway related to Tau tangle generation.
  • the biological activity comprises induction of A ⁇ deposition and/or amyloid plaque formation by gelsolin.
  • the DIR and/or its functional fragments induce A ⁇ deposition and/or amyloid plaque formation by binding to gelsolin.
  • the expression level of DIR and/or its functional fragments is positively correlated with the expression level of A ⁇ .
  • said reducing comprises administering said DIR and/or functional fragments thereof and/or encoding the nucleic acid of said DIR and/or its functional fragment, Decreased the subject's cognitive abilities.
  • the DIR and/or a functional fragment thereof is of mammalian origin.
  • the DIR and/or a functional fragment thereof is derived from a primate.
  • the DIR and/or a functional fragment thereof is of human origin.
  • the DIR comprises the amino acid sequence shown in SEQ ID NO.1.
  • the functional fragment of the DIR comprises an amino acid sequence encoded by a retained intron in DDIT4L.
  • the functional fragment of the DIR comprises the amino acid sequence shown in any one of SEQ ID NO.4-5.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises amnestic MCI multicognitive domain impairment (aMCI-m).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • the neurodegenerative disease comprises Alzheimer's disease.
  • the neurodegenerative disease comprises early Alzheimer's disease, intermediate Alzheimer's disease, and/or advanced Alzheimer's disease.
  • the subject comprises a human.
  • the subject comprises a neurodegenerative disease patient and/or a cognitively impaired patient.
  • the subject includes a patient with Alzheimer's disease.
  • the subject is aged.
  • the agent is formulated for oral administration and/or injectable administration.
  • the modulators include small molecular compounds, polymers and/or biomacromolecules.
  • the modulator comprises an antibody or antigen-binding fragment thereof.
  • the modulator includes an antibody or an antigen-binding fragment thereof that specifically binds the intron retention splicing product DIR of the DNA damage-inducible transcript 4-like transcript and/or a functional fragment thereof.
  • the modulator includes an antisense oligonucleotide.
  • the modulator comprises siRNA.
  • the modulator comprises an siRNA that specifically binds to the intron retention splicing product DIR of the DNA damage-inducible transcript 4-like transcript and/or a functional fragment thereof.
  • the regulator comprises the nucleotide sequence shown in any one of SEQ ID NO.84-87.
  • the present application provides a method for preventing and/or treating cognitive impairment, which includes the following steps: retaining the intron of the DNA damage-inducible transcript 4-like transcript in a subject in need The expression level and/or biological activity of the cleavage product DIR and/or its functional fragments are reduced.
  • the present application provides a method for preventing and/or treating neurodegenerative diseases, which includes the following steps: making the intron of the DNA damage-inducible transcript 4-like transcript in a subject in need The expression level and/or biological activity of the retained cleavage product DIR and/or a functional fragment thereof is reduced.
  • said reduction comprises the expression level and/or biological activity of said DIR and/or functional fragments thereof originally in said subject, said DIR and/or functional fragments thereof The expression level and/or biological activity of is reduced by at least about 10%.
  • the expression level comprises the expression level of the gene encoding the DIR/or its functional fragment, the transcription level of the gene encoding the DIR/or its functional fragment and/or the DIR/or its Expression levels of functional fragments.
  • said expression level is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column Chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column Chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level is measured by using a substance selected from the group consisting of primers capable of specifically amplifying genes encoding the DIR/or functional fragments thereof, and encoding the DIR/or functional fragments thereof
  • a substance selected from the group consisting of primers capable of specifically amplifying genes encoding the DIR/or functional fragments thereof, and encoding the DIR/or functional fragments thereof A nucleic acid molecule specifically binding to the gene, a nucleic acid molecule specifically binding to the DIR/or a functional fragment thereof, a small molecule specifically binding to the DIR/or a functional fragment thereof, a specific binding to the DIR/or a functional fragment thereof Specifically binding probes and polypeptides specifically binding to said DIR and/or functional fragments thereof.
  • the functional fragment of the DIR retains at least a portion of the biological activity of the DIR.
  • the biological activity includes affecting the excitatory and/or inhibitory activity of neurons.
  • the biological activity comprises the ability to reduce the frequency of excitatory postsynaptic currents (EPSCs), and/or the ability to reduce the amplitude of EPSCs.
  • ESCs excitatory postsynaptic currents
  • said reducing comprises administering said DIR and/or functional fragments thereof and/or encoding
  • the nucleic acid of the DIR and/or a functional fragment thereof reduces the frequency of an excitatory postsynaptic current (EPSC) in a subject, and/or reduces the amplitude of an EPSC in a subject.
  • ESC excitatory postsynaptic current
  • the biological activity includes affecting cognitive ability.
  • the biological activity includes participating in a signaling pathway related to A ⁇ deposition, and/or, participating in a signaling pathway related to Tau tangle generation.
  • the biological activity comprises induction of A ⁇ deposition and/or amyloid plaque formation by gelsolin.
  • the DIR and/or its functional fragments induce A ⁇ deposition and/or amyloid plaque formation by binding to gelsolin.
  • the expression level of DIR and/or its functional fragments is positively correlated with the expression level of A ⁇ .
  • said reducing comprises administering said DIR and/or functional fragments thereof and/or encoding
  • the nucleic acid of the DIR and/or its functional fragment reduces the cognitive ability of the subject.
  • the DIR and/or a functional fragment thereof is of mammalian origin.
  • the DIR and/or a functional fragment thereof is derived from a primate.
  • the DIR comprises the amino acid sequence shown in SEQ ID NO.1.
  • the functional fragment of the DIR comprises an amino acid sequence encoded by a retained intron in DDIT4L.
  • the functional fragment of the DIR comprises the amino acid sequence shown in any one of SEQ ID NO.4-5.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises amnestic MCI multicognitive domain impairment (aMCI-m).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • the neurodegenerative disease comprises Alzheimer's disease.
  • the neurodegenerative disease comprises early Alzheimer's disease, intermediate Alzheimer's disease, and/or advanced Alzheimer's disease.
  • the subject includes a mammal.
  • the subject comprises a human.
  • the subject comprises a neurodegenerative disease patient and/or a cognitively impaired patient.
  • the subject includes a patient with Alzheimer's disease.
  • the subject is aged.
  • the method comprises the step of administering to a subject in need a modulating agent capable of reducing the DIR and/or a functional fragment thereof, and/or encoding the DIR and/or The expression level and/or biological activity of the nucleic acid of its functional fragment.
  • the administering comprises oral administration and/or injectable administration.
  • the modulators include small molecular compounds, polymers and/or biomacromolecules.
  • the modulator comprises an antibody or antigen-binding fragment thereof.
  • the modulator includes an antibody or an antigen-binding fragment thereof that specifically binds the intron retention splicing product DIR of the DNA damage-inducible transcript 4-like transcript and/or a functional fragment thereof.
  • the modulator includes an antisense oligonucleotide.
  • the modulator comprises siRNA.
  • the modulator comprises an siRNA that specifically binds to the intron retention splicing product DIR of the DNA damage-inducible transcript 4-like transcript and/or a functional fragment thereof.
  • the regulator comprises the nucleotide sequence shown in any one of SEQ ID NO.84-87.
  • the present application provides a method for screening drugs capable of preventing and/or treating cognitive impairment and/or treating neurodegenerative diseases, which includes the following steps: detecting the effect of candidate drugs on DIR and/or The expression level and/or biological activity of its functional fragments, wherein after the candidate drug is administered, the expression level and/or biological activity of the DIR and/or its functional fragments are reduced, then the candidate drug can Prevention and/or treatment of cognitive impairment and/or treatment of neurodegenerative diseases.
  • said reduction comprises the expression level and/or biological activity of said DIR and/or functional fragments thereof originally in said subject, said DIR and/or functional fragments thereof The expression level and/or biological activity of is reduced by at least about 10%.
  • the expression level comprises the expression level of the gene encoding the DIR/or its functional fragment, the transcription level of the gene encoding the DIR/or its functional fragment and/or the DIR/or its Expression levels of functional fragments.
  • said expression level is measured by performing an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column Chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • an assay selected from the group consisting of qPCR, qRT-PCR, hybridization analysis, Northern blot, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column Chromatography, Western blotting, immunohistochemistry, immunostaining, and mass spectrometry.
  • the expression level is measured by using a substance selected from the group consisting of primers capable of specifically amplifying genes encoding the DIR/or functional fragments thereof, and encoding the DIR/or functional fragments thereof
  • a substance selected from the group consisting of primers capable of specifically amplifying genes encoding the DIR/or functional fragments thereof, and encoding the DIR/or functional fragments thereof A nucleic acid molecule specifically binding to the gene, a nucleic acid molecule specifically binding to the DIR/or a functional fragment thereof, a small molecule specifically binding to the DIR/or a functional fragment thereof, a specific binding to the DIR/or a functional fragment thereof Specifically binding probes and polypeptides specifically binding to said DIR and/or functional fragments thereof.
  • the functional fragment of the DIR retains at least a portion of the biological activity of the DIR.
  • the biological activity includes affecting the excitatory and/or inhibitory activity of neurons.
  • the biological activity comprises the ability to reduce the frequency of excitatory postsynaptic currents (EPSCs), and/or the ability to reduce the amplitude of EPSCs.
  • ESCs excitatory postsynaptic currents
  • said reducing comprises administering said DIR and/or functional fragments thereof and/or encoding
  • the nucleic acid of the DIR and/or a functional fragment thereof reduces the frequency of an excitatory postsynaptic current (EPSC) in a subject, and/or reduces the amplitude of an EPSC in a subject.
  • ESC excitatory postsynaptic current
  • the biological activity includes affecting cognitive ability.
  • the biological activity includes participating in a signaling pathway related to A ⁇ deposition, and/or, participating in a signaling pathway related to Tau tangle generation.
  • the biological activity comprises induction of A ⁇ deposition and/or amyloid plaque formation by gelsolin.
  • the DIR and/or its functional fragments induce A ⁇ deposition and/or amyloid plaque formation by binding to gelsolin.
  • the expression level of DIR and/or its functional fragments is positively correlated with the expression level of A ⁇ .
  • said reducing comprises administering said DIR and/or functional fragments thereof and/or encoding
  • the nucleic acid of the DIR and/or its functional fragment reduces the cognitive ability of the subject.
  • the DIR and/or a functional fragment thereof is of mammalian origin.
  • the DIR and/or a functional fragment thereof is derived from a primate.
  • the DIR comprises the amino acid sequence shown in SEQ ID NO.1.
  • the functional fragment of the DIR comprises an amino acid sequence encoded by a retained intron in DDIT4L.
  • the functional fragment of the DIR comprises the amino acid sequence shown in any one of SEQ ID NO.4-5.
  • the cognitive impairment comprises cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • frontotemporal dementia frontotemporal dementia
  • vascular dementia vascular dementia
  • the inducing disease of cognitive impairment includes Alzheimer's disease, multiple infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the cognitive impairment comprises early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment.
  • the cognitive impairment comprises amnestic MCI multicognitive domain impairment (aMCI-m).
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the neurodegenerative disease includes neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function diseases, neurodegenerative diseases caused by abnormal communication between cells and/or neurodegenerative diseases caused by impaired cell motility.
  • the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) and/or Huntington's disease (HD ).
  • the neurodegenerative disease comprises Alzheimer's disease.
  • the neurodegenerative disease comprises early Alzheimer's disease, intermediate Alzheimer's disease, and/or advanced Alzheimer's disease.
  • the subject includes a mammal.
  • the subject comprises a neurodegenerative disease patient and/or a cognitively impaired patient.
  • the subject includes a patient with Alzheimer's disease.
  • the subject is aged.
  • the administering comprises oral administration and/or injectable administration.
  • the drug candidates include small molecule compounds, polymers and/or biomacromolecules.
  • the present application provides an isolated antigen-binding protein, which has the following properties: in an ELISA assay, specifically binds to human DIR and/or its functional fragments at a working concentration of about 10 ng/ml or more;
  • the antigen-binding protein comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO:74.
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 26 or 16.
  • the antigen binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:73.
  • the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:25, 52,15.
  • the antigen binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:75.
  • the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 27, 53, 67, 17.
  • the antigen binding protein comprises LCDR1, LCDR2 and LCDR3, wherein,
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:25
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:27;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:53;
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:52
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:26
  • the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:67; or
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:15
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:16
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:17.
  • the antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:70.
  • the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 20, 35, 56, 10.
  • the antigen-binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:71.
  • the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 21, 36, 42, 48, 11.
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:70.
  • the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 22, 30, 37, 43, 49, 57, 62, 12.
  • the antigen binding protein comprises HCDR1, HCDR2 and HCDR3, wherein,
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:22;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:21
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:30;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:37;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:43;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:20
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:48
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:49;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:56
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:36
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:62;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:35
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:42
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:37;
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:56
  • the HCDR2 comprises SEQ ID NO:36
  • the amino acid sequence shown and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57; or,
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:10
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11
  • the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.
  • the antigen binding protein comprises a heavy chain variable region VH comprising any one of SEQ ID NO: 13, 23, 31, 38, 44, 50, 58, 63, 65 Amino acid sequence shown.
  • the antigen binding protein comprises a light chain variable region VL, and the VL comprises any one of SEQ ID NO: 18, 28, 33, 40, 46, 54, 60, 68 amino acid sequence.
  • the antigen binding protein comprises a heavy chain variable region VH and a light chain variable region VL, wherein:
  • the VH comprises the amino acid sequence shown in SEQ ID NO:23
  • the VL comprises the amino acid sequence shown in SEQ ID NO:28;
  • VH comprises the amino acid sequence shown in SEQ ID NO:31
  • VL comprises the amino acid sequence shown in SEQ ID NO:33
  • VH comprises the amino acid sequence shown in SEQ ID NO:38
  • VL comprises the amino acid sequence shown in SEQ ID NO:40
  • the VH comprises the amino acid sequence shown in SEQ ID NO:44
  • the VL comprises the amino acid sequence shown in SEQ ID NO:46;
  • the VH comprises the amino acid sequence shown in SEQ ID NO:50
  • the VL comprises the amino acid sequence shown in SEQ ID NO:54;
  • VH comprises the amino acid sequence shown in SEQ ID NO:58
  • VL comprises the amino acid sequence shown in SEQ ID NO:60
  • the VH comprises the amino acid sequence shown in SEQ ID NO:63
  • the VL comprises the amino acid sequence shown in SEQ ID NO:60;
  • VH comprises the amino acid sequence shown in SEQ ID NO:65
  • VL comprises the amino acid sequence shown in SEQ ID NO:54;
  • VH comprises the amino acid sequence shown in SEQ ID NO:58
  • VL comprises the amino acid sequence shown in SEQ ID NO:68
  • VH comprises the amino acid sequence shown in SEQ ID NO:13
  • VL comprises the amino acid sequence shown in SEQ ID NO:18.
  • the antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region.
  • the heavy chain constant region comprises a constant region derived from a histone selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the antigen binding protein comprises a light chain constant region
  • the light chain constant region comprises a constant region derived from Ig ⁇ or a constant region derived from Ig ⁇ .
  • the light chain constant region comprises a constant region derived from human Ig ⁇ .
  • the antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, murine antibodies, and chimeric antibodies.
  • the present application provides a polypeptide comprising the isolated antigen-binding protein described herein.
  • the present application provides an immunoconjugate comprising the isolated antigen-binding protein described herein or the polypeptide described herein.
  • the present application provides an isolated nucleic acid molecule encoding the isolated antigen-binding protein described herein, or the polypeptide described herein.
  • the present application provides a vector comprising the isolated nucleic acid molecule described herein.
  • the present application provides a cell comprising the isolated antigen-binding protein described herein, the polypeptide described herein, the immunoconjugate described herein, the isolated nucleic acid molecule described herein and/or the vectors described in this application.
  • the application provides a method for preparing the isolated antigen-binding protein described in the application or the polypeptide described in the application, the method comprising making the isolated antigen-binding protein described in the application or the polypeptide described in the application.
  • the cells described in the present application are cultured under conditions in which the polypeptides described above are expressed.
  • the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein described in the present application, the polypeptide described in the present application, the immunoconjugate described in the present application, the isolated antigen-binding protein described in the present application Nucleic acid molecules, vectors described herein, cells described herein, and/or pharmaceutically acceptable adjuvants and/or excipients.
  • the application provides a use of the isolated antigen-binding protein described in the application and/or the polypeptide described in the application in the preparation of a medicament for preventing and/or treating a disease or disorder, wherein the disease or disorder Includes cognitive impairment and/or neurodegenerative disease.
  • the neurodegenerative disease includes acute neurodegenerative disease and chronic neurodegenerative disease.
  • the cognitive impairment includes early cognitive impairment (MCI), intermediate cognitive impairment and late cognitive impairment cognitive barriers.
  • FIGS 1A-1D show the identification of DIRs described in this application.
  • FIG. 2 shows a schematic diagram of the structure of the functional fragments of the DIR described in this application.
  • FIG. 3 shows the effect of functional fragments of DIR described in this application on excitatory postsynaptic currents.
  • FIG. 4 shows the biological function of the DIR described in this application.
  • FIGS 5a-5i show that DIR induces A ⁇ deposition by binding gelsolin, where:
  • Mass spectrometry identified the top 8 molecules in the ⁇ 85 kDa size interval. Gelsolin is the most abundant molecule among them.
  • Figures 6a-6d show that DIR does not directly bind to A ⁇ , but induces A ⁇ deposition.
  • FIGS 7a-7c show that DIR mediates A ⁇ plaque formation, where:
  • the A ⁇ -positive area was larger than the Thioflavin S- and DIR-positive signals.
  • Scale bar 50 ⁇ m.
  • Figure 8 shows that A ⁇ -positive and thioflavin s-negative plaques do not overlap with DIR and gelsolin in the human hippocampus.
  • FIGS 9a-9l show that plasma DIR is a potential biomarker for AD and aMCI, where:
  • Figures 10a-10b show the ROC analysis of plasma tTau. in
  • FIGS 11a-11e show the correlation analysis between plasma DIR and A ⁇ , in which:
  • Plasma DIR of normal but currently MCI patients (n 12) three years ago (2018-2021). Plasma DIR concentrations in MCI patients were higher than in blood samples collected 3 years earlier.
  • DIR-induced amyloid plaque deposition and blood secretion e. Model of DIR-induced amyloid plaque deposition and blood secretion.
  • hypoxia leads to abnormal retention of introns, which induces the translation of DIR proteins.
  • a ⁇ is produced through the proteolytic process of amyloid precursor protein (APP) and has a direct binding to gelsolin.
  • APP amyloid precursor protein
  • DIR leads to A ⁇ deposition by binding gelsolin, Amyloid plaques in the brain eventually form. DIR can also be released into the blood system.
  • FIGS 12a-12b show the correlation between plasma DIR and A ⁇ 42, and quantitatively analyze the SUV value of A ⁇ -PET. in,
  • Fig. 13 shows the detection results of the binding activity of the DIR antibody described in this application.
  • Figures 14a-14h show that DIR is an aberrant splice form of human DDIT4L in a patient in which:
  • DIR is the spliced isoform of human DDIT4L, and the intron is retained between exon 2 and exon 3.
  • Flag-DIR can be detected in cell lysate and culture medium.
  • Figures 15A-15B show the effect of reduced expression of DIR on cognitive performance.
  • FIGS 16A-16C show the effect on cognitive levels of administration of DIR antibodies described herein.
  • mice treated with DIR antibody described herein had a shorter entry latency and were less likely to stay in the target quadrant. time more long. *, p ⁇ 0.05.
  • DDIT4L generally refers to DNA damage-inducible transcript 4-like, DNA damage-inducible transcript 4-like. It may also be referred to as REDD2/RTP801L. Studies have found that DDIT4L can be associated with cardiac dysfunction. It can also be used to treat glioma.
  • accession number of human DDIT4L gene in GenBank is 115265; the accession number of human DDIT4L protein in GenBank is NP_660287.1.
  • DIR generally refers to the intron retention splicing product of DDIT4L.
  • the cleavage reaction of DDIT4L can be seen in Figure 1.
  • the amino acid sequence of the DIR can be as shown in SEQ ID NO:1.
  • expression level generally refers to the protein, RNA or mRNA level of a particular gene of interest.
  • the expression level of a particular related gene eg, human DDIT4L gene
  • the "expression” generally refers to the process of converting the information encoded by a gene into a structure that exists and operates in a cell.
  • Examples may include reverse transcription and amplification assays (e.g., PCR, ligated RT-PCR, or quantitative RT-PCT), hybridization assays, Northern blotting, dot blots, in situ hybridization, gel electrophoresis, capillary electrophoresis, Column chromatography, western blotting, immunohistochemistry, immunostaining, or mass spectrometry. Analysis can be performed directly on biological samples or on proteins/nucleic acids isolated from samples.
  • reverse transcription and amplification assays e.g., PCR, ligated RT-PCR, or quantitative RT-PCT
  • hybridization assays e.g., Northern blotting, dot blots, in situ hybridization, gel electrophoresis, capillary electrophoresis, Column chromatography, western blotting, immunohistochemistry, immunostaining, or mass spectrometry. Analysis can be performed directly on biological samples or on proteins/nucleic acids isolated from samples.
  • the term "activity" generally refers to any activity associated with a particular protein.
  • the activity may include, for example, any activity related to DIR protein.
  • the activity may include protease-associated enzymatic activity.
  • the activity may include biological activity.
  • the activity may involve binding of the protein to a receptor, for example, which binding may have measurable downstream effects.
  • the activity may include any activity that would be attributed to the protein by a person skilled in the art.
  • cognitive impairment generally refers to diseases and conditions that are believed to or do involve, or are related to, progressive loss of neuronal structure and/or function, including neuronal death.
  • the cognitive impairment can be characterized by impairment of cognition (eg, memory, attention, perception, and/or thinking). These disorders can include pathogen-induced cognitive dysfunction, such as HIV-associated cognitive dysfunction and Lyme disease-associated cognitive dysfunction. Examples of cognitive disorders may include Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), autism, early cognitive impairment traumatic brain injury (MCI), stroke, traumatic brain injury (TBI) and/or age-associated memory impairment (AAMI).
  • ALS amyotrophic lateral sclerosis
  • MCI early cognitive impairment traumatic brain injury
  • TBI traumatic brain injury
  • AAMI age-associated memory impairment
  • neurodegenerative disease generally refers to cognitive disorders such as dementia that result from the gradual loss of neuronal structure and function, including neuronal death and glial homeostasis.
  • age eg, Alzheimer's disease (AD), Parkinson's disease (PD)
  • genetic mutations that affect CNS cell function eg, Huntington's disease, early-onset AD or PD, muscular dystrophy Lateral sclerosis (ALS)
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • ALS muscular dystrophy Lateral sclerosis
  • the neurodegenerative disease may have changes and/or conditions selected from the group consisting of protein misfolding and aggregation; neuroinflammation (e.g., under signal stimuli such as toxic stimuli (such as protein aggregation), infection, traumatic injury, or autoimmunity) CNS inflammation); changes in cell signaling; acquired senescence/cell death (eg, disrupted apoptotic signaling, mitochondrial dysfunction, impaired autophagy, and activation of necrosomes by stress/inflammation); exercise Cell damage and epigenetic changes.
  • signal stimuli such as toxic stimuli (such as protein aggregation), infection, traumatic injury, or autoimmunity) CNS inflammation
  • changes in cell signaling e.g., acquired senescence/cell death (eg, disrupted apoptotic signaling, mitochondrial dysfunction, impaired autophagy, and activation of necrosomes by stress/inflammation); exercise Cell damage and epigenetic changes.
  • Alzheimer's disease generally refers to Alzheimer's disease, senile dementia, a neurodegenerative disease that develops slowly and worsens over time.
  • the most common early symptom is loss of short-term memory (difficulty remembering recent events), and as the disease progresses, at least one of the following symptoms may develop gradually: language disturbance, disorientation (for example, getting lost easily), emotional disturbance stability, loss of motivation, incapacity, and behavioral problems.
  • the true cause of Alzheimer's disease is still unknown, and its process may be related to the deposition of fibrous amyloid plaques and Tau protein in the brain. There are currently no treatments that can stop or reverse the course of the disease, and only a few methods may temporarily relieve or improve symptoms.
  • Alzheimer's disease is used interchangeably with the term “Alzheimer's disease” in this application.
  • the Alzheimer's disease may include early stage Alzheimer's disease, middle stage Alzheimer's disease and/or late stage Alzheimer's disease. For example, in the early stages of Alzheimer's disease, learning and memory impairments will become more pronounced, and in some cases, language impairments, executive impairments, cognitive impairments (agnosias), and/or skills performance impairments (apraxias) will be present. ).
  • the Alzheimer's patient in the middle stage will lose the ability to live independently and may not be able to perform most of the daily activities (in some cases, may suffer from anamia, dysphasia, and/or anosognosia) ).
  • the Alzheimer's patient in advanced stages may become dependent on a caregiver.
  • MCI middle cognitive impairment
  • the MCI may include cognitive impairment meeting criteria for dementia but exceeding normal aging.
  • MCI is diverse in clinical presentation, etiology, prognosis, and prevalence.
  • MCI can be a pathological stage of Alzheimer's disease.
  • Certain forms of cognitive impairment can be seen as early manifestations of neurodegenerative diseases that will eventually lead to dementia.
  • the MCI may comprise a subtype selected from the group consisting of: aMCI-s: amnestic MCI impaired in a single cognitive domain; aMCI-m: amnestic MCI impaired in multiple cognitive domains; naMCI-s : Impairment of a single cognitive domain in non-amnestic MCI; and naMCI-m: Impairment of multiple cognitive domains in non-amnestic MCI.
  • Cognitive impairment due to normal aging generally refers to cognitive impairment due to normal aging.
  • the positive Cognitive impairment due to aging can manifest as: memory loss, confusion about where familiar places are, taking longer than usual to complete daily tasks, or changes in mood and personality.
  • lews body dementia usually refers to Lewy Body Detmentia, dementia with Lewy bodies.
  • Dementia with Lewy bodies is characterized by abnormal accumulations of protein that form masses called Lewy bodies. Dementia with Lewy bodies causes a gradual decline in mental ability. People with dementia with Lewy bodies may experience visual hallucinations and changes in alertness and concentration. Other effects include muscle stiffness, slowness of movement, difficulty walking, and tremors.
  • Patients with Lewy bodies in the brain can also have the plaques and tangles associated with Alzheimer's disease.
  • frontotemporal dementia generally refers to Pick's disease, a progressive rare disease in which the tau protein affects only the frontal and temporal lobes of the brain.
  • People with frontotemporal dementia have difficulties with higher-level reasoning, expressive language, language perception, and memory formation.
  • the frontal and temporal lobes of the brain in people with frontotemporal dementia can shrink over time.
  • vascular dementia generally refers to problems with reasoning, judgment, and memory due to impaired blood flow to the brain.
  • the vascular dementia may include dementia due to heart disease and stroke risk factors, such as high blood pressure and high cholesterol.
  • multiple infarct generally refers to small noncortical infarcts resulting from occlusion of a single perforating branch of a large cerebral artery.
  • the multiple infarction type may be a special type of cerebral infarction, also known as ischemic stroke.
  • the multi-infarct type may manifest as hemisensory impairment, aphasia, dysarthria, slowness of movement, and clumsiness (especially difficulty in fine movements such as writing).
  • Parkinson's disease generally refers to a progressive neurodegenerative disorder.
  • the clinical features of Parkinson's disease (PD) can include motor symptoms such as tremor, bradykinesia, muscle rigidity, and postural instability, as well as neuropsychiatric and other non-motor manifestations.
  • the non-motor manifestations may include cognitive dysfunction and dementia, mood disorders (eg, depression, anxiety, apathy), and sleep disorders.
  • CJD Creutzfeldt-Jakob disease
  • CJD is a transmissible spongiform encephalopathy that occurs in humans.
  • CJD is a disease caused by prion infection.
  • CJD patients can manifest paranoid behavior, confusion, loss of appetite and weight, depression, and a few patients have visual or auditory abnormalities; in the advanced stage, it manifests as progressive neurological deterioration (such as paresthesias, language disorders, and aphasia).
  • MS multiple sclerosis
  • the term “multiple sclerosis (MS)” generally refers to a demyelinating neuropathy.
  • the insulating substance (myelin sheath) on the surface of nerve cells in the brain or spinal cord of the MS patient is damaged, and the signal transduction of the nervous system is impaired, which can lead to a series of possible symptoms, affecting the patient's activities, mind, and even spirit. state. These symptoms can include double vision, impaired vision on one side, muscle weakness, dull sensations, or trouble with coordination.
  • ALS myotrophic lateral sclerosis
  • motor neuron disease a progressive and fatal neurodegenerative disease.
  • Frontotemporal dementia can occur in a minority of ALS patients. Some ALS patients feel, The senses of sight, touch, smell, and taste deteriorate, and very rarely, people with ALS also develop dementia.
  • Huntington's disease usually refers to a genetic disorder that causes brain cell death.
  • HD patients as the disease progresses, incoordination of body movements becomes more pronounced, and abilities gradually deteriorate until movement becomes difficult and speech becomes impossible.
  • Mental capacity usually declines to dementia.
  • the term "aging stage” generally refers to the aging stage of a subject.
  • the aging stage can be above 60 years old, above 70 years old or above 75 years old; for mice, the old stage can be above 10 months old, for example, it can be above 13 months old or 18 months or older.
  • the elderly subject may have one or more symptoms of learning deficits, memory impairment, memory deficits, and/or brain dysfunction.
  • modulator generally refers to a compound that alters the expression and/or activity of a molecule.
  • a modulator can include a compound that increases or decreases the magnitude of a certain activity and/or expression of a molecule compared to the magnitude of activity and/or expression in the absence of the modulator.
  • such modulators can include inhibitors that reduce the magnitude and/or expression of one or more activities of a molecule.
  • neuron generally refers to nerve cells, which are the major functional units of the nervous system.
  • a neuron can consist of a cell body with its protruding axon and one or more dendrites. Neurons can transmit messages to other neurons or cells by releasing neurotransmitters at synapses.
  • excitatory postsynaptic potential generally refers to the flow of ions that cause an excitatory postsynaptic potential (EPSP).
  • the EPSP is a postsynaptic potential that makes it easier for a postsynaptic neuron to fire an action potential. This temporary depolarization of the postsynaptic membrane potential caused by the influx of positively charged ions into the postsynaptic cell is a consequence of the opening of ligand-gated ion channels.
  • the frequency and/or amplitude of the EPSC can be recorded with a voltage clamp.
  • cognitive ability generally refers to the mental act of acquiring knowledge and understanding through thought, experience, and feeling.
  • the cognitive concepts may not be limited to mental concepts/fields, and may include, for example, executive function, memory, perception, attention, emotion, motor control, and/or disturbance processing.
  • a ⁇ generally refers to any peptide resulting from ⁇ -secretase-mediated cleavage of the ⁇ -amyloid precursor protein (APP).
  • the A[beta] may comprise a 37, 38, 39, 40, 41, 42, and 43 amino acid peptide and extend from the [beta]-secretase cleavage site to amino acid 37, 38, 39, 40, 41, 42, or 43.
  • the A ⁇ may also be N-terminally truncated versions of the above peptides, such as the pyroglutamate forms pE3-40, pE3-42, pE3-43, pE11-42, pE11-43 and the like.
  • Tau generally refers to the Tau protein and components of extensive Tau aggregates (eg, neurofibrillary tangles) that are involved in the stabilization of microtubules in nerve cells.
  • the Tau entanglements may include oligomeric and/or fibrous forms of Tau, which are toxic.
  • the Tau may also include all types and forms of Tau (eg, different alternatively spliced forms).
  • small molecule compound generally refers to any chemical moiety with a molecular weight below about 5000 Daltons (Da).
  • the small molecular compound may include organic or inorganic molecules that are synthesized or found in nature.
  • the small molecule compounds may include non-peptidic, non-oligomeric organic compounds.
  • the small molecule compounds may include peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, and/or nucleotide analogs.
  • polymer generally refers to a molecule composed of repeating structural units linked by covalent chemical bonds. Typically the polymers are characterized by a large number of repeat units (e.g., 10 repeat units or greater, 50 repeat units or greater, or typically 100 repeat units or greater) and a high molecular weight (greater than or equal to 50,000Da).
  • the polymers may include random copolymers, block copolymers, alternating copolymers, multi-block copolymers, graft copolymers, and/or tapered copolymers.
  • biomacromolecule generally refers to macromolecules occurring in living matter and organisms, such as nucleic acids, proteins, peptides, sugars, polysaccharides, lipids, and fats. Their respective mono/oligomers may be corresponding nucleotides, peptides, amino acids, sugars, fatty acids in their respective mono/oligomeric form.
  • the biomacromolecule may include protein, and/or nucleic acid.
  • the biomacromolecule can include an antibody.
  • isolated generally means obtained from the natural state by artificial means.
  • an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state can be called isolated. of.
  • isolated may not exclude the mixing of artificial or synthetic substances, or the presence of other impure substances that do not affect the activity of the substance.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state.
  • An "isolated antigen binding protein” of the present application may comprise a moiety that binds an antigen and, optionally, a framework or framework portion that allows the antigen binding moiety to adopt a conformation that facilitates binding of the antigen binding moiety to an antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives.
  • Such frameworks may include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, as well as fully synthetic framework regions comprising, eg, biocompatible polymers.
  • antigen-binding proteins may include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single-chain antibodies; diabodies; F(ab')2, F(ab)2, scFv, di-scFv, dAb, VHH, IgD antibody; IgE antibody; IgM antibody; IgGl antibody; IgG2 antibody; IgG3 antibody; and/or IgG4 antibody and fragments thereof.
  • CDR also referred to as “complementarity determining region” generally refers to the region in the variable domain of an antibody, the sequence of which may be highly variable and/or form a structure-defining loop.
  • an antibody may comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3).
  • HCDR1, HCDR2, HCDR3 three in the VL
  • LCDR1, LCDR2, LCDR3 three in the VL
  • LCDR1, LCDR2, LCDR3 three in the VL
  • LCDR1, LCDR2, LCDR3 three in the VL
  • LCDR1, LCDR2, LCDR3 LCDR1, LCDR2, LCDR3
  • a naturally occurring camelid antibody consisting only of heavy chains is capable of normal and stable function in the absence of light chains.
  • Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothi
  • the amino acid sequence numbering of the antigen-binding protein can be numbered according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res.27:209- 212; Ruiz et al., 2000 Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res.
  • IMGT the international ImMunoGeneTics information system@imgt.cines.fr; imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res.27:209- 212; Ruiz et al., 2000 Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res.
  • the CDRs of the antigen-binding protein can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition , USDepartment of Health and Human Services, NIH Publication No.91-3242).
  • variable domains of native heavy and light chains may each comprise four FR regions, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3 and L-FR4).
  • variable domain and “variable region” are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain.
  • the variable domains of the heavy and light chains can be referred to as “VH” and “VL,” respectively (or “VH” and “VL,” respectively). These domains generally may be the most variable part of the antibody (relative to other antibodies of the same class), and may comprise the antigen binding site.
  • variable generally means that there may be large differences in the sequence of some segments of the variable domain between antibodies.
  • the variable domains mediate what antigens can bind and determine the specificity of a particular antibody for its particular antigen. However, the variability may not be evenly distributed throughout the variable domains.
  • variable domains can be typically concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved portions of variable domains may be referred to as the framework regions (FR).
  • the variable domains of native heavy and light chains can each comprise four FR regions, most adopting a ⁇ -sheet configuration, connected by three CDRs, which form a loop connecting and, in some cases, forming a ⁇ -sheet structure. part.
  • the CDRs in each chain can be held together in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site.
  • antibody generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo.
  • the term may include, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies.
  • antibody may also include antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, VHH, dAb and those that retain antigen-binding function (e.g., specifically bind human DIR ) other antibody fragments. Typically, such fragments may include an antigen binding domain.
  • the basic 4-chain antibody unit may be a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibodies can be composed of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies can include 2-5 that can be combined with the J chain to form The basic 4-chain unit of the multivalent combination.
  • the 4-chain unit can generally be about 150,000 Daltons.
  • Each L chain can be linked to an H chain by one covalent disulfide bond, while two H chains can be linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain may also have regularly spaced intrachain disulfide bridges.
  • Each H chain may have a heavy chain variable region (VH) at the N-terminus, followed by three constant domains (CH) for the alpha and gamma chains each, and four CH structures for the mu and epsilon isotypes area.
  • Each L chain can have a light chain variable region (VL) at its N-terminus and a constant domain at its other end. VL may correspond to VH, and the light chain constant region (CL) may correspond to the first constant domain (CH1) of the heavy chain. Certain amino acid residues can be considered to form the interface between the light and heavy chain variable domains. VH and VL can pair together to form a single antigen binding site.
  • immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG such as IgGl, IgG2, IgG3, and/or IgG4, and IgM, with heavy chains designated ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, DIR).
  • the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • Fab generally refers to an antigen-binding fragment of an antibody.
  • Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab” fragment, and a residual "Fc” fragment (ie, the Fc region).
  • Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region (CH1) of the H chain (VH).
  • F(ab)2 generally refers to an antigen-binding fragment of an antibody.
  • F(ab)2 can be linked by two Fab fragments.
  • Fab' generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than the Fab fragment.
  • a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain.
  • a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.
  • F(ab')2 generally refers to antibody fragments produced by pepsin digestion of intact antibodies.
  • the F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds.
  • F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.
  • Fv fragment generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions .
  • the heavy and light chain variable regions include, for example, CDRs.
  • an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.
  • the term "scFv” generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains can be The variable regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • a scFv can have the VL and VH variable regions in any order (for example, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include VL-linker-VH or VH-Linker-VL can be included.
  • the term “dAb” generally refers to an antigen-binding fragment having a VH domain or a VL domain composition, see for example Ward et al. (Nature, 1989 Oct 12; 341(6242):544-6), see Holt et al., Trends Biotechnol., 2003, 21(11): 484-490.
  • VHH generally refers to an antibody comprising the variable antigen binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs may also be referred to as Nanobodies (Nb).
  • monoclonal antibody generally refers to a preparation of antibody molecules composed of a single molecule.
  • Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody can be directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants).
  • monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins.
  • the modifier "monoclonal” may denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from an antibody of a laboratory animal, such as a rodent ("parent antibody”), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more effective in a human individual than the parental (e.g., mouse-derived) antibody. Less likely to trigger an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen.
  • a humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” may retain antigen specificity similar to that of the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin.
  • CDR region residues in a human immunoglobulin can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region.
  • donor antibody such as mouse, rat
  • rabbit or non-human primate residue substitution in the CDR region such as mouse, rat
  • FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues.
  • humanized antibodies can contain amino acid modifications that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.
  • Fully human antibody generally refers to an antibody comprising only human immunoglobulin protein sequences.
  • Fully human antibodies may contain murine sugar chains if they are produced in mice, in mouse cells, or in hybridomas derived from mouse cells.
  • a "murine antibody”, “mouse antibody” or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.
  • Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are known in the art.
  • the term "antigen binding protein” generally refers to a protein comprising a moiety that binds an antigen, and optionally a scaffold or backbone moiety that allows the moiety that binds the antigen to adopt a conformation that facilitates binding of the antigen binding protein to the antigen.
  • antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH and/or dAb), immune Conjugates, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • An "isolated antigen binding protein" of the present application may comprise an antigen-binding moiety and, optionally, a scaffold or framework moiety that allows the antigen-binding moiety to adopt a conformation that facilitates binding of the antigen-binding moiety to antigen.
  • polypeptide molecule and “polypeptide” and “peptide” are used interchangeably and generally refer to a polymer of amino acid residues.
  • fusion protein generally refers to a polypeptide having at least two moieties covalently linked together. Each of these moieties can be a polypeptide with different properties.
  • the property may be a biological property, such as in vitro or in vivo activity.
  • the property can also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. These two moieties can be linked directly by a single peptide bond or by a peptide linker.
  • nucleic acid molecule generally refers to nucleotides of any length in isolated form, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid delivery tool into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements it carries can be expressed in the host cell.
  • a vector may contain various elements that control expression.
  • the vector may also contain an origin of replication.
  • the vector may also include components that assist its entry into the cell.
  • the term "cell” generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes the nucleic acid molecules described herein or the nucleic acid molecules described herein. the carrier described.
  • Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors described herein.
  • the term “immunoconjugate” generally refers to the conjugation of the other agent (e.g., chemotherapeutics, radioactive elements, cytostatic and cytotoxic agents) to the antibody or antigen-binding fragment thereof (e.g. , a conjugate formed by covalently linking a linker molecule), the conjugate can be specifically bound to an antigen on a target cell by the antibody or antigen-binding fragment thereof, and the other agent can be delivered to the target cell (e.g. , tumor cells).
  • the other agent e.g., chemotherapeutics, radioactive elements, cytostatic and cytotoxic agents
  • the term "pharmaceutical composition” generally refers to a composition for the prevention/treatment of a disease or condition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may also contain one or more (pharmaceutically effective) carriers and other suitable preparations. Acceptable ingredients of the compositions can be nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the term "pharmaceutically acceptable carrier” generally refers to a pharmaceutically acceptable carrier, excipient or stabilizer which is effective in the dosage and concentration employed to the cell or mammal to which it is exposed. non-toxic.
  • a physiologically acceptable carrier may include suitable substances.
  • refers to the pharmaceutically acceptable carrier (carrier) and the carrier (vector) used to insert nucleic acid in genetic engineering are usually not the same substance.
  • the term "specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an The presence of the population determines the presence of the target.
  • an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets .
  • Antibodies specifically bind epitopes on proteins that are conserved among proteins of different species.
  • specific binding can include, but does not require exclusive binding.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • the variant may be, for example, one or more substitutions, deletions or additions in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds DIR) A protein or polypeptide of amino acids.
  • the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion.
  • Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition).
  • the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration.
  • the substitutions may be conservative substitutions.
  • the variant may also be a polypeptide encompassing a functionally active fragment thereof, not limited to a polypeptide comprising a functionally active fragment of the protein produced after processing and/or modification in cells.
  • the homologue may be at least about 85% (eg, having at least about 85%) of the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds to DIR). %, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology of proteins or peptides.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • Perfectage of sequence homology can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity.
  • Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest.
  • the homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in WRPearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad.
  • antisense oligonucleotide refers to a single-stranded oligonucleotide molecule having a core complementary to a corresponding fragment of a target nucleic acid (for example, a target genomic sequence, mRNA precursor, or mRNA molecule). base sequence.
  • the antisense oligonucleotides are 12 to 30 nucleobases in length.
  • an antisense oligonucleotide is an unmodified or modified nucleic acid having a nucleotide sequence that is complementary to the sequence of a target nucleic acid (eg, DIR mRNA).
  • dsRNA refers to a complex of ribonucleic acid molecules, which has a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, known as having "Justice” and “antisense” orientations.
  • double-stranded RNA dsRNA
  • RNA interference RNA interference
  • the duplex structure may be of any length that permits specific degradation of the desired target RNA by the RISC pathway, for example may be in the range of about 19 to 36 base pairs in length, e.g., about 19-30 base pairs in length, For example, about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also included as part of this application.
  • siRNA short interfering RNA
  • siRNAs are transmitters of RNA interference (the process by which double-stranded RNA silences homologous genes).
  • siRNAs typically comprise two single-stranded RNAs of about 15-25 nucleotides in length that form a duplex that may contain single-stranded overhangs.
  • siRNAs are produced by processing of double-stranded RNA by an enzyme complex such as a polymerase, resulting in cleavage of the double-stranded RNA.
  • RNA interference (RNAi) silencing complex uses the antisense strand of siRNA to guide mRNA cleavage, thereby promoting mRNA degradation.
  • siRNAs to silence specific genes, for example, in mammalian cells, base pair regions are chosen to avoid opportunistic complementation of unrelated mRNAs.
  • the RNAi silencing complex has been identified in the art, such as, for example, by Fire et al., Nature 391:806-81 (1998) and McManus et al., Nat. Rev. Genet. 3(10):737-747 (2002) .
  • the term "antisense strand” generally refers to the strand of an siRNA that includes a region that is substantially complementary to a target sequence.
  • region of complementarity generally refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (eg, a target sequence).
  • the mismatch can be in an internal or terminal region of the molecule.
  • the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3 or 2 nucleotides of the 5' end and/or the 3' end.
  • the term "sense strand” generally refers to a strand of an siRNA that includes a region that is substantially complementary to a region that is the antisense strand as the term is defined herein.
  • the "just" strand is sometimes called the “sense” strand, the “passenger” strand or The “anti-boot” chain.
  • the antisense strand targets the desired mRNA, while the sense strand targets a different target. Therefore, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can lead to off-target effects. These off-target effects can be limited by the use of modifications on the sense strand or by the use of 5' caps.
  • the term "complementary" when used to describe a first nucleotide sequence (such as the sense strand or target mRNA) with respect to a second nucleotide sequence (such as the antisense strand) refers to a sequence that includes the first core.
  • An oligonucleotide or polynucleotide of a nucleotide sequence hybridizes (forms base-pair hydrogen bonds) to an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions and forms a duplex or double helix structural capacity.
  • Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, provided the above The need was fulfilled with regard to their ability to hybridize. "Complementary” does not necessarily have nucleobase complementarity at every nucleoside. Instead, some mismatches can be tolerated.
  • target nucleic acid or “target sequence” generally refers to the portion of an mRNA molecule formed during the transcription of a gene (DDILT4 gene) encoding the intron-retention splice product of the DNA damage-inducible transcript 4-like transcript.
  • the target portion of the sequence should be at least long enough to serve as a substrate for antisense oligonucleotide or siRNA-directed cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the DDILT4 gene.
  • the target sequence is within the protein coding region of the DIR and/or functional fragments thereof.
  • the term "comprises” generally refers to the meanings of including, encompassing, containing or encompassing. In some cases, it also means “for” and “consisting of”.
  • the term "about” generally refers to a numerical range of 20% more or less than the specified value.
  • “about X” includes a numerical range of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 2%, ⁇ 1%, ⁇ 0.5%, ⁇ 0.2%, or ⁇ 0.1% of X, where X is a value.
  • the present application provides a regulator, which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • a regulator which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • the present application provides a regulator, which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • a regulator which regulates the intron retention splicing product DIR and/or its functional fragments of DNA damage-inducible transcript 4-like transcripts, which can be used in the preparation of drugs for preventing and/or treating diseases.
  • the disease comprises a neurodegenerative disease.
  • the present application provides a method for preventing and/or treating cognitive impairment, which includes the following steps: retaining the intron of the DNA damage-inducible transcript 4-like transcript in a subject in need The expression level and/or biological activity of the cleavage product DIR and/or its functional fragments are reduced.
  • the present application provides a method for preventing and/or treating neurodegenerative diseases, which includes the following steps: making the intron of the DNA damage-inducible transcript 4-like transcript in a subject in need The expression level and/or biological activity of the retained cleavage product DIR and/or a functional fragment thereof is reduced.
  • DIR intron retention form
  • the functional fragment generally refers to a polypeptide comprising an amino acid sequence that differs from the amino acid sequence of a parent or reference polypeptide (eg DIR) by at least one amino acid residue.
  • the functional fragment may have high (eg at least 80%) homology with the parent or reference polypeptide.
  • the homology may include sequence similarity or identity.
  • said homology can be determined using standard techniques known in the art (see, for example, Smith and Waterman, Adv. Appl. Math. "Advances in Applied Mathematics"); identity shared by polynucleotide or polypeptide sequences Percentages are determined by direct comparison of sequence information between molecules by aligning the sequences and determining identity using methods known in the art.
  • NCBI National Center for Biotechnology Information
  • the expression level of DIR may include the expression level of DIR gene, the transcription level of DIR gene and/or the expression level of DIR protein.
  • the expression level may include specific genes (such as human DDIT4L gene; and/or genes encoding human DIR and/or functional fragments thereof (such as DIR-I, and/or, DIR-II) (such as human DIR gene )) Amount of polynucleotide, mRNA or amino acid product or protein.
  • the expression level can include the amount of transcribed polynucleotides, translated proteins, or post-translationally modified fragments of proteins of a particular gene.
  • QDLIR can be used interchangeably with DIR, which can be an intron retention form produced by abnormal splicing of human DDIT4L gene.
  • DIR the gene encoding the DIR may be referred to as a DIR gene.
  • the expression level of the functional fragment of DIR may include the expression level of the gene encoding the functional fragment of DIR, the transcription level of the gene encoding the functional fragment of DIR, and and/or expression levels of functional fragment proteins of DIR.
  • the expression level can include the amount of a polynucleotide, mRNA or amino acid product or protein of a particular gene (eg, a gene encoding a functional fragment of human DIR (eg, DIR-I, and/or, DIR-II)).
  • Said expression level may include fragments of transcribed polynucleotides, translated proteins, or post-translationally modified proteins of specific genes (eg, genes encoding functional fragments of human DIR (eg, DIR-I, and/or, DIR-II) amount.
  • specific genes eg, genes encoding functional fragments of human DIR (eg, DIR-I, and/or, DIR-II) amount.
  • the DIR-I may be the amino acid sequence encoded by the IR (i.e. the retained intron, its amino acid sequence (shown in SEQ ID NO.3) is an amino acid sequence consisting of the first 27 amino acids from the N-terminus.
  • the amino acid sequence of DIR-I is shown in SEQ ID NO.4.
  • the DIR-II may be an amino acid sequence of IR consisting of the last 27 amino acids from the C-terminus.
  • the amino acid sequence of DIR-II is shown in SEQ ID NO.5.
  • said reduction may comprise a reduction in the expression level of said DIR by at least about 10% compared to the original DIR expression level in said subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least About 500% or more.
  • the expression level of DIR can be measured by using a substance selected from the group consisting of primers for specifically amplifying the DIR gene, nucleic acid molecules specifically binding to the DIR gene, and nucleic acid molecules specifically binding to the DIR protein , a small molecule specifically binding to the DIR protein, a probe specifically binding to the DIR protein, and a polypeptide specifically binding to the DIR protein.
  • the expression level of the functional fragment of DIR can be measured by using a substance selected from the following group: primers for specifically amplifying the functional fragment gene of DIR , a nucleic acid molecule specifically binding to a functional fragment gene of DIR, a nucleic acid molecule specifically binding to a functional fragment of DIR (such as DIR-I, and/or, DIR-II), a functional fragment of DIR (such as DIR-I , and/or, small molecules that specifically bind to DIR-II), probes that specifically bind to functional fragments of DIR (such as DIR-I, and/or, DIR-II) and functional fragments of DIR (such as DIR -I, and/or, DIR-II) specifically bound polypeptides.
  • primers for specifically amplifying the functional fragment gene of DIR a nucleic acid molecule specifically binding to a functional fragment gene of DIR
  • a nucleic acid molecule specifically binding to a functional fragment of DIR such as DIR-I
  • the expression level of said DIR and/or its functional fragments can be measured by performing a test selected from the group consisting of reverse transcription and amplification analysis (such as PCR, ligated RT-PCR or quantitative RT-PCT), hybridization Analysis, Northern blotting, dot blot, in situ hybridization, gel electrophoresis, capillary electrophoresis, column chromatography, Western blotting, immunohistochemistry, immunostaining, or mass spectrometry.
  • the expression level of DIR described in the present application can be measured by qPCR, qRT-PCR, northern hybridization, western hybridization and/or ELISA detection.
  • the expression level of the DIR can also be measured by performing an analysis directly on a biological sample or on a protein/nucleic acid isolated from a sample.
  • the activity of DIR and/or its functional fragments may include the biological activity of DIR protein.
  • the biological activity can include affecting neuronal excitatory and/or inhibitory neuronal activity.
  • the biological activity can include inhibiting cognitive ability by inhibiting neuronal excitability and/or inhibiting neuronal activity.
  • the biological activity may include the ability to reduce the frequency of excitatory postsynaptic current (EPSC), and/or the ability to reduce the amplitude of EPSC.
  • said reducing may comprise administering said DIR and/or functional fragments thereof and/or encoding said DIR and/or functional fragments thereof compared to the original biological activity of said DIR and/or functional fragments thereof in said subject.
  • the nucleic acid of the functional fragment can reduce the frequency of an excitatory postsynaptic current (EPSC) in a subject, and/or reduce the amplitude of an EPSC in a subject.
  • the functional fragment DIR-I of the DIR can reduce the frequency of the EPSC.
  • the reduction can comprise a reduction in the frequency of EPSCs by at least about 10% following administration of the DIR-1 compared to the frequency of naive EPSCs in the subject.
  • the functional fragment DIR-II of the DIR can reduce the frequency of EPSCs.
  • the reduction can comprise a reduction of at least about 10% in magnitude of EPSCs following administration of the DIR-II compared to the magnitude of native EPSCs in the subject.
  • the biological activity may include affecting cognitive ability.
  • the biological activity may include participating in a signaling pathway related to A ⁇ deposition, and/or, participating in a signaling pathway related to Tau tangle generation.
  • the DIR and/or functional fragments thereof can inhibit cognitive performance.
  • the DIR and/or functional fragments thereof can inhibit cognitive ability by inhibiting signaling pathways associated with A ⁇ deposition, and/or, inhibiting signaling pathways associated with Tau tangle generation.
  • the reduction of the activity of the DIR and/or its functional fragments may comprise the administration of the DIR and/or its functional fragments compared to the original biological activity of the DIR and/or its functional fragments in the subject. / or its functional fragments and / or the nucleic acid encoding the DIR and / or its functional fragments reduce the cognitive ability of the subject.
  • the reduction may comprise a reduction in the biological activity of the DIR by at least about 10% compared to the biological activity of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least About 500% or more.
  • the expression level of DIR and/or its functional fragments may be positively correlated with the A ⁇ level (for example, the expression level in plasma).
  • the expression level of DIR and/or its functional fragments may be positively correlated with the expression level of A ⁇ 40.
  • the expression level of DIR and/or its functional fragments may be positively correlated with the expression level of A ⁇ 42.
  • the expression level of the DIR and/or its functional fragments can be Positively correlated with the reduction degree of the subjects' cognitive ability.
  • the expression level of the DIR and/or functional fragments thereof can increase as cognitive impairment progresses (eg, MCI and/or AD disease progression).
  • the expression level of the DIR and/or its functional fragments may be positively correlated with the A ⁇ level (for example, the A ⁇ -PET expression level in the cortex).
  • the expression level of the DIR and/or its functional fragments may be related to the formation of amyloid plaques in AD patients.
  • the expression level of the DIR and/or its functional fragments may be related to the storage of declarative memory. For example, the higher the expression level of the DIR and/or functional fragments thereof, the lower the ability to store declarative memory.
  • the expression level of the DIR and/or its functional fragments can be correlated with the storage of associative learning. For example, the higher the expression level of the DIR and/or functional fragments thereof, the lower the memory capacity of associative learning.
  • the reduction of the expression level of DIR and/or its functional fragments can improve cognitive ability.
  • the expression level of the DIR and/or its functional fragments can be reduced by gene editing.
  • the gene editing method may include knock-down (for example, the CRISPR/Cas system may be used; for example, antisense oligonucleotides may be used).
  • the reduction of the expression level may comprise a reduction of the expression level of the DIR by at least about 10% compared to the expression level of the original DIR in the subject.
  • the lower the expression level of the DIR and/or its functional fragments the higher the cognitive ability (eg, as measured by a novel object recognition behavior experiment).
  • the cognitive ability can be improved by at least about 10%.
  • the DIR and/or its functional fragments may be derived from mammals.
  • it can be derived from primates.
  • it can be derived from humans.
  • the DIR may comprise the amino acid sequence shown in SEQ ID NO.1.
  • the functional fragment of DIR may comprise the amino acid sequence encoded by the retained intron in DDIT4L.
  • the functional fragment of the DIR may comprise the amino acid sequence shown in SEQ ID NO.3.
  • the functional fragment of DIR may comprise the amino acid sequence shown in any one of SEQ ID NO.4-5.
  • the DIR and/or its functional fragments may be involved in A ⁇ deposition.
  • the DIR and/or functional fragments thereof can induce A[beta] deposition by gelsolin.
  • DIR-intron ie DIR-intron (IR), its amino acid sequence is shown in SEQ ID NO.3
  • a ⁇ may contribute to the interaction between DIR and gelsolin.
  • the DIR-intron may be involved in A ⁇ deposition.
  • the DIR and/or its functional fragments can lead to A ⁇ deposition and amyloid plaque formation by combining with gelsolin under pathological conditions.
  • the subject may include a mammal.
  • the subject can include a rodent and/or a primate, eg, the subject can include a human.
  • the subjects may include patients with cognitive impairment and/or patients with neurodegenerative diseases.
  • the neurodegenerative diseases may include acute neurodegenerative diseases and chronic neurodegenerative diseases.
  • the neurodegenerative disease may include neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function, neurodegenerative disease caused by Neurodegenerative diseases caused by abnormal communication between cells and/or caused by impaired cell motility.
  • the subjects may include patients with neurodegenerative diseases.
  • the subject can include an Alzheimer's disease patient.
  • the Alzheimer's disease patient may be in the early stage, early stage, middle stage or late stage of Alzheimer's disease.
  • the cognitive impairment may include early cognitive impairment (MCI), middle cognitive impairment and late cognitive impairment.
  • MCI early cognitive impairment
  • the cognitive impairment may include cognitive impairment due to normal aging, lewy body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lewy body dementia
  • the inducing disease of cognitive impairment may include Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the cognitive impairment may include amnestic MCI multi-cognitive domain impairment (aMCI-m).
  • the subjects may include patients with cognitive impairment.
  • the subject may suffer from early cognitive impairment (MCI) (e.g., loss of short-term memory, difficulty expressing or understanding abstract impairment and/or need for reminders for basic self-care), intermediate cognitive impairment (e.g., confusion between long-term memory and real-world situation memory, poor word expression, behavioral personality changes or emotional lability and/or need for assisted self-care) or late cognitive impairment
  • MCI early cognitive impairment
  • intermediate cognitive impairment e.g., confusion between long-term memory and real-world situation memory, poor word expression, behavioral personality changes or emotional lability and/or need for assisted self-care
  • Cognitive impairment eg, memory impairment, decline in physical activity and mental status, inability to express or communicate effectively, inability to care for oneself and/or disturbance of circadian clock.
  • the subject may suffer from a disease capable of inducing the cognitive impairment.
  • the subject may suffer from Alzheimer's disease, multiple infarcts, Parkinson's Sen's disease, AIDS and/or Creutzfeldt
  • the subject may be at an aging stage.
  • the subject has exhibited cognitive impairment caused by normal aging.
  • the subject has exhibited symptoms of early cognitive impairment (MCI).
  • MCI early cognitive impairment
  • the subject has exhibited symptoms of a neurodegenerative disease such as Alzheimer's disease.
  • the subject may suffer from Alzheimer's disease.
  • the subject can be in early Alzheimer's disease, in middle Alzheimer's disease, and/or in advanced Alzheimer's disease.
  • the present application provides a regulator that can reduce the expression level and/or biological activity of the DIR and/or its functional fragments, and/or the nucleic acid encoding the DIR and/or its functional fragments.
  • the regulator can reduce the expression level and/or biological activity of the DIR and/or its functional fragments, and/or the nucleic acid encoding the DIR and/or its functional fragments.
  • the regulator can reduce the expression level of DIR gene, the transcription level of DIR gene and/or the expression level of DIR protein.
  • the regulator can reduce, for example, the human DDIT4L gene; and/or, the gene (such as the human DIR gene) polynucleotide encoding human DIR and/or its functional fragment (such as DIR-I, and/or, DIR-II) Amount of acid, mRNA or amino acid product or protein.
  • the regulator can reduce, for example, the human DDIT4L gene; and/or, the gene (such as the human DIR gene) encoding human DIR and/or its functional fragment (such as DIR-I, and/or, DIR-II) gene transcription The amount of polynucleotides, translated proteins, or fragments of post-translationally modified proteins.
  • the gene such as the human DIR gene
  • the functional fragment such as DIR-I, and/or, DIR-II
  • the regulator can reduce the expression level of the gene encoding the functional fragment of DIR, the transcription level of the gene encoding the functional fragment of DIR and/or the expression level of the protein of the functional fragment of DIR.
  • the modulator can reduce the amount of polynucleotide, mRNA or amino acid product or protein encoding a functional fragment of human DIR (eg, DIR-I, and/or, DIR-II) gene).
  • the modulator may reduce the amount of a gene transcribed polynucleotide, translated protein, or a fragment of a post-translationally modified protein encoding a functional fragment of human DIR (eg, DIR-I, and/or, DIR-II).
  • said reduction may comprise a reduction in the expression level of said DIR by at least about 10% compared to the original DIR expression level in said subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least About 500% or more.
  • the reduction may comprise a reduction in the expression level of the functional fragment of the DIR by at least about 10% compared to the expression level of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, At least about 500% or more.
  • the regulator can reduce the biological activity of the DIR and/or its functional fragments.
  • the reduction may comprise a reduction in the biology of the DIR or a functional fragment thereof of at least about 10% compared to the biological activity of the original DIR in the subject. For example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 200%, at least About 500% or more.
  • the regulator can inhibit the signaling pathway related to A ⁇ deposition.
  • the regulator can inhibit the signaling pathway related to the generation of Tau tangles.
  • the inhibition may include expression levels and/or biological activities of molecules originally involved in signaling pathways related to A ⁇ deposition and/or signaling pathways related to Tau tangle generation in the subject. In contrast, the expression level and/or biological activity of these molecules is reduced by at least about 10% after administration of the modulator.
  • the regulator can inhibit A ⁇ deposition and/or amyloid plaque formation through gelsolin.
  • the regulator can inhibit the A ⁇ deposition and/or the formation of amyloid plaques by inhibiting the binding of the DIR/or its functional fragments to gelsolin.
  • the regulator can reduce the combination of DIR-II and gelsolin.
  • the binding level of DIR-II to gelsolin can be reduced by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, after administration of the modulating agent. at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, At least about 95%, at least about 100%, at least about 200%, at least about 500%, or more.
  • the regulator may include an inhibitor of the DIR and/or its functional fragments.
  • the regulator may include small molecular compounds, polymers and/or biomacromolecules.
  • the modulator may include an antibody or an antigen-binding fragment thereof.
  • the modulator may include an antibody or an antigen-binding fragment thereof that specifically binds to DIR and/or its functional fragment.
  • the modulator may include an antibody or an antigen-binding fragment thereof targeting DIR-II.
  • the modulator may include antisense oligonucleotides.
  • said antisense oligonucleotide comprises a substantially complementary or completely complementary to at least a portion of the mRNA encoding DIR and/or functional fragments thereof Complement the area.
  • the length of the complementary region may be less than 19 nucleotides.
  • the modulator may include dsRNA.
  • the regulator may include siRNA.
  • the regulator may include siRNA that specifically binds to the intron retention splicing product DIR of the DNA damage-inducible transcript 4-like transcript and/or its functional fragment.
  • the siRNA described herein may further comprise one or more single-stranded nucleotide overhangs, eg, 1, 2 or 3 nucleotides.
  • the overhang can be on the sense strand, the antisense strand, or any combination thereof.
  • overhang nucleotides may be present at the 5'-end, 3'-end or both ends of the antisense or sense strand of the siRNA.
  • the overhang can be caused by one strand being longer than the other, or by two strands of the same length being interleaved.
  • the overhang may form a mismatch with the target mRNA or it may be complementary to the sequence of the gene being targeted or it may be another sequence.
  • the siRNA may comprise a sense strand and an antisense strand
  • the antisense strand may comprise a complementary region substantially complementary to at least a part of the mRNA encoding DIR and/or a functional fragment thereof
  • the antisense strand may Comprising at least 15, 16, 17, 18 or 19 consecutive nucleotides selected from any one of the following sequences with a difference of no more than 3 nucleotides: SEQ ID NO: xx; the sense strand and the antisense strand can be complementary to form together Complementary regions between 17 and 21 nucleotides in length.
  • the siRNA may comprise a sense strand and an antisense strand
  • the antisense strand may comprise a complementary region substantially complementary to at least a part of the mRNA encoding DIR and/or a functional fragment thereof
  • the antisense strand may Contains any one of the following sequences with a difference of no more than 3 nucleotides: SEQ ID NO: 85, 87; the sense strand and the antisense strand can be complementary to form a complementary region between 17 and 21 nucleotides in length .
  • the siRNA may comprise a sense strand and an antisense strand
  • the antisense strand may comprise a complementary region substantially complementary to at least a part of the mRNA encoding DIR and/or a functional fragment thereof
  • the antisense The strand may comprise the nucleotide sequence shown in any one of SEQID NO.85,87; the sense strand and the antisense strand may be complementary to form a complementary region with a length between 17 and 21 nucleotides.
  • the siRNA may comprise a sense strand and an antisense strand
  • the sense strand may comprise the nucleotide sequence shown in any one of SEQ ID NO.84,86
  • the antisense strand may comprise The nucleotide sequence shown in any one of SEQ ID NO.85,87.
  • At least one (eg, 2) nucleotides may be connected to the 3' end of the sense strand of the siRNA and/or the 3' end of the antisense strand of the siRNA.
  • the linked nucleotides can be identical to the target gene.
  • the linked nucleotide may be T, for example, may be TT.
  • the sense strand and the antisense strand can be selected from any one or more combinations of the following:
  • a sense strand comprising the nucleotide sequence shown in SEQ ID NO:84, and a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO:85 The antisense strand; And, the sense strand comprising the nucleotide sequence shown in SEQ ID NO:86, and the antisense strand comprising the nucleotide sequence shown in SEQ ID NO:87;
  • each strand may be between 17 and 23 nucleotides in length.
  • no more than 3 nucleotides in each chain can be replaced by other nucleotides, while basically maintaining the ability to inhibit the expression of DIR and/or its functional fragments in transfected cells.
  • the siRNA may comprise at least one modified nucleotide.
  • one or more nucleotides on the sense strand and/or the antisense strand may be modified to form modified nucleotides.
  • nucleotides of the sense strand and all nucleotides of the antisense strand may contain modifications.
  • the antisense oligonucleotides or siRNA described in this application can be introduced into brain neurons or glial cells by transfection methods well known in the art. These methods include sonication, electrical pulses, electroporation, osmotic shock, calcium phosphate precipitation and DEAE-dextran transfection, lipid-mediated delivery, passive delivery, and the like.
  • transfection includes a variety of techniques that can be used to introduce nucleic acid into mammalian cells, including electroporation, calcium phosphate precipitation, DEAE-dextran treatment, lipofection, microinjection, and/or viral infection.
  • the present application also provides the use of the regulator (such as the antisense oligonucleotide or siRNA described in the present application) in the preparation of drugs for preventing and/or treating diseases or disorders, wherein the diseases Or the condition includes cognitive impairment and/or neurodegenerative disease.
  • the regulator such as the antisense oligonucleotide or siRNA described in the present application
  • the present application also provides the regulatory agent (such as the antisense oligonucleotide or siRNA described in the present application), which is used for the prevention and/or treatment of cognitive impairment and/or neurodegenerative diseases .
  • the regulatory agent such as the antisense oligonucleotide or siRNA described in the present application
  • the present application also provides a method for preventing and/or treating cognitive impairment and/or neurodegenerative diseases, which includes the following steps: administering the regulator (such as the present invention) to a subject in need application described antisense oligonucleotide or siRNA).
  • the modulating agent may be formulated for oral administration and/or injection administration.
  • the present application provides a method for screening drugs capable of preventing and/or treating cognitive impairment and/or treating neurodegenerative diseases, which includes the following steps: detecting the effect of candidate drugs on DIR and/or The expression level and/or biological activity of its functional fragments, wherein after the candidate drug is administered, the expression level and/or biological activity of the DIR and/or its functional fragments are reduced, then the candidate drug can Prevention and/or treatment of cognitive impairment and/or treatment of neurodegenerative diseases.
  • the candidate drug may comprise the regulator described in the present application.
  • the candidate drug can include Inhibitors comprising said DIR and/or functional fragments thereof.
  • the drug candidates may include small molecular compounds, polymers and/or biomacromolecules.
  • the candidate drug may be formulated for oral administration and/or injection administration.
  • the present application also provides a system for screening drugs capable of preventing and/or treating cognitive impairment and/or treating neurodegenerative diseases, which may include a detection module.
  • the detection module can detect the impact of candidate drugs on the expression level and/or biological activity of DIR and/or its functional fragments in the subject, wherein after the candidate drug is administered, the DIR and/or its functional fragments If the expression level and/or biological activity of the drug is reduced, the candidate drug can prevent and/or treat cognitive disorders and/or treat neurodegenerative diseases.
  • the detection module may include instruments and/or reagents capable of detecting the expression level and/or biological activity of DIR and/or its functional fragments in a subject.
  • the detection module may include primers that specifically amplify the DIR gene, nucleic acid molecules that specifically bind to the DIR gene, nucleic acid molecules that specifically bind to the DIR protein, small molecules that specifically bind to the DIR protein, and DIR protein A specific binding probe and/or a polypeptide that specifically binds to the DIR protein; and/or, a primer that specifically amplifies the functional fragment gene of DIR, a nucleic acid molecule that specifically binds to the functional fragment gene of DIR, and a DIR functional fragment gene A nucleic acid molecule specifically binding to a functional fragment (such as DIR-I, and/or, DIR-II), a small molecule specifically binding to a functional fragment of DIR (such as DIR-I, and/or, DIR-II), and A probe specifically binding to a functional fragment of DIR (eg, DIR-I, and/or, DIR-II) and a polypeptide specifically binding to a functional fragment of DIR (eg,
  • the detection module may include instruments and/or reagents for specifically detecting A ⁇ deposition and/or amyloid plaque formation.
  • the detection module may include primers capable of specifically detecting a target gene (such as Tau gene) related to A ⁇ deposition and/or amyloid plaque formation, a nucleic acid molecule specifically binding to the target gene, and a target gene associated with the target gene.
  • a target gene such as Tau gene
  • a small molecule that encodes a protein that specifically binds a probe that specifically binds to the protein encoded by the target gene, and/or a polypeptide that specifically binds to the protein encoded by the target gene.
  • the detection module can output the measured value of the expression level and/or biological activity of the DIR and/or its functional fragments after administration of the candidate drug (for example, a quantitative value; another example, compared with one or more threshold values After the qualitative value).
  • the system may include a judgment module, and the judgment module may compare the expression level and/or biological activity of the DIR and/or its functional fragments outputted by the detection module after administration of the candidate drug The quantitative value of ; and the expression level and/or quantitative value of biological activity of the original DIR and/or its functional fragments in the subject. If the expression level and/or biological activity of the DIR and/or its functional fragments are reduced after administration of the candidate drug, the judgment module can output that the candidate drug can prevent and/or treat cognitive impairment and and/or judgment results for treating neurodegenerative diseases.
  • the system may include a display module, and the display module may display the judgment result of the judgment module in a quantitative and/or qualitative manner.
  • the system may include a memory; and a processor coupled to the memory, the processor configured to execute the detection module and/or the judgment based on instructions stored in the memory The step in the module of detecting the effect of the candidate drug on the expression level and/or biological activity of DIR and/or its functional fragments in the subject.
  • the present application also provides a detection kit capable of detecting the expression level and/or biological activity of DIR and/or its functional fragments.
  • the detection kit may include instructions, which describe the specific steps of how to use the detection kit to detect the expression level and/or biological activity of DIR and/or its functional fragments, and/or how to use the detection results to judge Specific steps of whether the candidate drug can prevent and/or treat cognitive impairment and/or treat subjects with neurodegenerative diseases.
  • the detection kit may also include reagents capable of detecting other targets for judging whether the candidate drug can prevent and/or treat cognitive impairment and/or treat neurodegenerative diseases.
  • the application provides an isolated antigen binding protein.
  • the isolated antigen-binding protein has the following properties: in the ELISA assay, specifically binds to human DIR and/or its functional fragments at a working concentration of about 10 ng/ml or more.
  • the isolated antigen-binding protein may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:74.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • X 1 X 2 S (SEQ ID NO.74), wherein X 1 is A or Y, and X 2 is A or Y.
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 26 or 16.
  • the isolated antigen-binding protein may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:73.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the LCDR1 may comprise LCDR1 in the light chain variable region of IR-II-1 to IR-II-10.
  • the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:25,52,15.
  • the isolated antigen-binding protein may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:75.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the LCDR3 may comprise LCDR3 in the light chain variable region of IR-II-1 to IR-II-10.
  • X 1 Q X 2 X 3 X 4 X 5 P X 6 X 7 (SEQ ID NO.75), wherein X 1 is L or Q, X 2 is S or Y, X 3 is N, S or Y, X 4 is D , E or K, X 5 is D or L, X 6 is F or R, X 7 is A or T.
  • the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:27,53,67,17.
  • the isolated antigen-binding protein may comprise LCDR3, LCDR2 and LCDR1.
  • LCDR2 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:74
  • LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:73
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:75.
  • amino acid sequence for example, an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the LCDR1-3 may comprise LCDR1-3 in the light chain variable region of IR-II-1 to IR-II-10.
  • the isolated antigen-binding protein may comprise LCDR3, LCDR2 and LCDR1.
  • the LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:25
  • the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:26
  • the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:27. amino acid sequence.
  • LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:52
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:53. amino acid sequence.
  • LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:52
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:67. amino acid sequence.
  • LCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:15
  • LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:16
  • LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:17. amino acid sequence.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the LCDR1-3 may comprise LCDR1-3 in the heavy chain variable region of IR-II-1 to IR-II-10.
  • the isolated antigen-binding protein may comprise HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:70.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the HCDR1 may comprise HCDR1 in the heavy chain variable region of IR-II-1 to IR-II-10.
  • GYTFX 1 X 2 YX 3 (SEQ ID NO.70), wherein X 1 is S or T, X 2 is E, N, R or S, and X 3 is T or W.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:20, 35, 56, and 10.
  • the isolated antigen-binding protein may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQID NO:71.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the HCDR1 may comprise HCDR2 in the heavy chain variable region of IR-II-1 to IR-II-10.
  • the HCDR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 21, 36, 42, 48, 11.
  • the isolated antigen-binding protein may comprise HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQID NO:72.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the HCDR3 may comprise HCDR3 in the heavy chain variable region of IR-II-1 to IR-II-10.
  • the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 22, 30, 37, 43, 49, 57, 62, 12.
  • the isolated antigen binding protein may comprise HCDR3, HCDR2 and HCDR1.
  • HCDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:71
  • HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO:70
  • HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO:72. amino acid sequence.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the HCDR1-3 may comprise the heavy chain variable region of IR-II-1 to IR-II-10 HCDR1-3 in.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:21
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:22. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:21
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:30. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:35
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:37. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:48
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:49. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:56
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:35
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:62. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:35
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:37. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:56
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57. amino acid sequence.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 10
  • HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 11
  • HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 12 amino acid sequence.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the HCDR1-3 may comprise HCDR1-3 in the heavy chain variable region of IR-II-1 to IR-II-10.
  • the isolated antigen binding protein may comprise HCDR3, HCDR2, HCDR1, LCDR3, LCDR2 and LCDR1.
  • the isolated antigen-binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:21
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:22
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:25
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen-binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:21
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:30
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:25
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:35
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:37
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:25
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen-binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:43
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:25
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:20
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:48
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:49
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:52
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:53.
  • the isolated antigen-binding protein HCDR1 described in the present application may comprise the amino acid sequence shown in SEQ ID NO:56
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:25
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:35
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:62
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:25
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:27.
  • the isolated antigen binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:35
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:42
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:37
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:52
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:53.
  • the isolated antigen-binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:56
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:36
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:57
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:52
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:26
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:67.
  • the isolated antigen binding protein HCDR1 described in the application may comprise the amino acid sequence shown in SEQ ID NO:10
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:11
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:12
  • the amino acid sequence of LCDR1 can include the amino acid sequence shown in SEQ ID NO:15
  • LCDR2 can include the amino acid sequence shown in SEQ ID NO:16
  • LCDR3 can include the amino acid sequence shown in SEQ ID NO:17.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDRs may be determined by the IMGT numbering scheme.
  • the HCDR1-3 may comprise HCDR1-3 in the heavy chain variable region of IR-II-1 to IR-II-10; and, the LCDR1-3 may comprise IR-II-1 to IR-II LCDR1-3 in the light chain variable region of -10.
  • the isolated antigen-binding protein may comprise H-FR1, and the C-terminus of the H-FR1 may be directly or indirectly linked to the N-terminus of the HCDR1.
  • the isolated antigen binding protein may comprise H-FR2, and the H-FR2 may be located between the HCDR1 and the HCDR2.
  • the isolated antigen binding protein may comprise H-FR3, and the H-FR3 may be located between the HCDR2 and the HCDR3.
  • the isolated antigen-binding protein may comprise H-FR4, and the N-terminus of the H-FR4 may be connected to the C-terminus of the HCDR3.
  • the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4.
  • the isolated antigen-binding protein may comprise L-FR1, and the C-terminus of the L-FR1 may be combined with the The N-terminus of LCDR1 is connected directly or indirectly.
  • the isolated antigen-binding protein may comprise L-FR1, and the C-terminus of the L-FR1 may be directly or indirectly linked to the N-terminus of the LCDR1.
  • the isolated antigen binding protein may comprise L-FR2, and the L-FR2 may be located between the LCDR1 and the LCDR2.
  • the isolated antigen binding protein may comprise L-FR3, and the L-FR3 may be located between the LCDR2 and the LCDR3.
  • the isolated antigen-binding protein may comprise L-FR4, and the N-terminus of the L-FR4 may be connected to the C-terminus of the LCDR3.
  • the isolated antigen binding protein may comprise L-FR1, L-FR2, L-FR3 and L-FR4.
  • said isolated antigen binding protein may comprise a heavy chain variable region VH, said VH may comprise any of SEQ ID NO: 13, 23, 31, 38, 44, 50, 58, 63, 65 The amino acid sequence shown in item.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the antigen binding protein of described isolation can comprise light chain variable region VL, and described VL can comprise any one in SEQ ID NO:18,28,33,40,46,54,60,68 The amino acid sequence shown.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • said isolated antigen binding protein may comprise said VH and VL.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:23
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:31
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:38
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:40.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:44
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:50
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:58
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:60.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 63
  • the VL may To comprise the amino acid sequence shown in SEQ ID NO:60.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:65
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO:58
  • the VL may comprise the amino acid sequence shown in SEQ ID NO:68.
  • the VH may comprise the amino acid sequence shown in SEQ ID NO: 13
  • the VL may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the isolated antigen binding protein may comprise at least one CDR in the VH described in the present application.
  • the isolated antigen binding protein may comprise at least one CDR in the VL described in the present application.
  • the CDR can be obtained by dividing according to any division manner.
  • the CDR may cover the CDR sequence obtained according to any CDR division method; it may also cover its variants.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3 in the VH described in the present application.
  • the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3 in the VH described in the present application.
  • the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 13, 23, 31, 38, 44, 50, 58, 63, 65.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDR may cover the CDR sequence obtained according to any CDR division method; it may also cover its variants.
  • the isolated antigen-binding protein may comprise LCDR1, LCDR2 and LCDR3 in the VL of the present application.
  • the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 18, 28, 33, 40, 46, 54, 60, 68.
  • an isolated antigen binding protein of the present application may have DIR binding ability.
  • the CDR may cover the CDR sequence obtained according to any CDR division method; it may also cover its variants.
  • the isolated antigen binding protein may comprise an antibody heavy chain constant region.
  • the antibody heavy chain constant region may be derived from a human IgG, IgA, IgD, IgE and/or IgM heavy chain constant region.
  • the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.
  • the isolated antigen binding protein can comprise an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgGl, IgG2, IgG3 and/or IgG4 heavy chain constant region.
  • the isolated antigen binding protein can comprise an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG1 heavy chain constant region.
  • the isolated antigen binding protein may comprise an antibody light chain constant region.
  • the antibody light chain constant region may comprise a constant region derived from Ig ⁇ or a constant region derived from Ig ⁇ .
  • the antibody light chain constant region may be derived from a human Ig ⁇ constant district.
  • the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may comprise a Fab, Fab', Fv fragment, F(ab') 2 , F(ab) 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibodies may comprise monoclonal antibodies. In certain embodiments, the antibodies may comprise murine antibodies and/or chimeric antibodies.
  • the isolated antigen-binding protein described in the present application may comprise heavy chain and/or light chain sequences with one or more conservative sequence modifications.
  • conservative sequence modification refers to the amino acid modification that will not significantly affect or change the binding properties of the antibody.
  • conservative modifications include amino acid substitutions, additions and deletions.
  • Modifications can be introduced into the isolated antigen binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues with similar side chains. Groups of amino acid residues having similar side chains are known in the art.
  • amino acid residues include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (for example, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains ( For example, amino acids of tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • one or more amino acid residues in the CDR regions of the isolated antigen binding proteins described herein may be replaced with other amino acid residues from the same side chain group.
  • Those skilled in the art know that some conservative sequence modifications will not abolish antigen binding, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot.Eng.10:835-41; Komissarov et al., (1997) J.Biol.Chem.272:26864-26870; Hall et al., (1992) J.Immunol.149:1605-12; Kelley and O'Connell (1993) Biochem.32:6862-35; Adib-Conquy et al., (1998) Int. Immunol.10:341-6 and Beers et al., (2000) Clin.Can.Res.6: 2835-43.
  • Antigen binding proteins described herein can be identified, screened, or characterized by various assays known in the art.
  • antigen binding proteins of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc. or the antigen-binding activity of the fusion protein.
  • ELISA enzyme-linked immunosorbent assay
  • immunoblotting e.g., Western blot
  • flow cytometry e.g., FACS
  • immunohistochemistry e.g., immunofluorescence, etc.
  • immunofluorescence e.g., fluorescence, etc.
  • the isolated antigen-binding protein can specifically bind DIR or a functionally active fragment thereof.
  • the isolated antigen binding protein can specifically bind DIR-II.
  • DIR or a functionally active fragment thereof may be full-length DIR or a functionally active fragment thereof, or Fragments that exert DIR functional activity (eg, can be DIR-II).
  • DIR or functionally active fragments thereof may be isolated DIR or functionally active fragments thereof, or a mixture of various forms of DIR or functionally active fragments thereof.
  • DIR or a functionally active fragment thereof may be human DIR or a functionally active fragment thereof.
  • the isolated antigen-binding protein can be used to determine the binding activity of the antigen-binding protein to an antigen (such as DIR-II) by Elisa method.
  • the isolated antigen binding protein may have a value of about 10 ng/ml or higher (e.g., may be at least 15 ng/ml, at least 20 ng/ml, at least 25 ng/ml, at least 30 ng/ml, at least 35 ng/ml , at least 40ng/ml, at least 45ng/ml, at least 50ng/ml, at least 55ng/ml, at least 60ng/ml, at least 65ng/ml, at least 70ng/ml, at least 75ng/ml, at least 80ng/ml, at least 85ng/ml , at least 90ng/ml, at least 95ng/ml, at least 100ng/ml, at least 500ng/ml, at least 1000ng/ml or higher) the ability to bind said
  • the present application provides a polypeptide molecule, a nucleic acid molecule, a carrier, an immunoconjugate, a cell and a pharmaceutical composition.
  • polypeptide molecules which may comprise an isolated antigen binding protein described herein.
  • the polypeptide molecule may comprise a fusion protein. In certain embodiments, the polypeptide molecule may be a fusion protein. In certain embodiments, the polypeptide molecule is a multispecific (eg, bispecific, trispecific or other multispecific) antibody.
  • the multispecific antibody may comprise: 1) an isolated antigen binding protein as described herein, and 2) one or more targeting moieties that bind other antigens, and/or bind other epitopes of the same antigen.
  • the polypeptide molecules of the present application may contain structures other than amino acids, for example, the polypeptide molecules of the present application may contain nucleic acids, polysaccharides, lipids, small molecules, and any combination of the foregoing.
  • the application provides isolated nucleic acid molecules that encode the isolated antigen binding proteins described herein.
  • it may be produced or synthesized by: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification; (ii) recombinantly produced by cloning; (iii) purified or (iv) synthetic, such as by chemical synthesis.
  • PCR polymerase chain reaction
  • the present application provides a vector, which may comprise the nucleic acid molecule described in the present application.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the vector may also include components that facilitate its entry into cells, such as viral particles, liposomes or protein coats, but not Only these substances.
  • the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application.
  • each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein.
  • each or each host cell may comprise a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like.
  • the cells can be bacterial cells (eg, E. coli), yeast cells, or other eukaryotic cells.
  • the vectors described in this application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.
  • the present application also provides an immunoconjugate, which may comprise the isolated antigen-binding protein described in the present application.
  • an isolated antigen binding protein or fragment thereof described herein can be linked to another agent, such as a chemotherapeutic agent, toxin, immunotherapeutic agent, imaging probe, spectroscopic probe, and the like.
  • the linkage can be through one or more covalent bonds, or non-covalent interactions, and can include chelation.
  • linkers which may be known in the art, can be used to form immunoconjugates.
  • the immunoconjugates can be provided in the form of fusion proteins, which can be expressed from a polynucleotide encoding the immunoconjugates.
  • the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate described in the present application, the immunoconjugate described in the present application, the A nucleic acid molecule, a vector described herein and/or a cell described herein, and optionally a pharmaceutically acceptable carrier.
  • compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutical compositions may also contain more than one active compound, generally those with complementary activities that do not adversely affect each other.
  • the type and effective amount of such drug may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with pharmaceutical administration, generally safe, nontoxic .
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the present application also provides the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the nucleic acid molecule described in the present application.
  • the present application also provides the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the nucleic acid molecule described in the present application.
  • the carrier, the cell described in this application and/or the pharmaceutical composition described in this application are used for the prevention and/or treatment of cognitive impairment and/or neurodegenerative disease.
  • the present application also provides a method for preventing and/or treating cognitive impairment and/or neurodegenerative disease, which comprises the following steps: administering the isolated antigen described in the present application to a subject in need Binding proteins, polypeptide molecules described herein, immunoconjugates described herein, nucleic acid molecules described herein, vectors described herein, cells described herein and/or pharmaceutical composition.
  • the neurodegenerative diseases may include acute neurodegenerative diseases and chronic neurodegenerative diseases.
  • the neurodegenerative disease may include neurodegenerative disease caused by neuronal death and glial cell homeostasis, neurodegenerative disease caused by aging, neurodegenerative disease caused by affected CNS cell function, neurodegenerative disease caused by Neurodegenerative diseases caused by abnormal communication between cells and/or caused by impaired cell motility.
  • the cognitive impairment may include early cognitive impairment (MCI), middle cognitive impairment and late cognitive impairment.
  • MCI early cognitive impairment
  • the cognitive impairment may include cognitive impairment due to normal aging, lews body dementia (LBD), frontotemporal dementia, and/or vascular dementia.
  • LBD lews body dementia
  • the inducing disease of cognitive impairment may include Alzheimer's disease, multi-infarct type, Parkinson's disease, AIDS and/or Creutzfeldt-Jakob disease (CJD).
  • the cognitive impairment may include amnestic MCI multi-cognitive domain impairment (aMCI-m).
  • the subjects may include patients with neurodegenerative diseases.
  • the subjects may include patients with cognitive impairment.
  • the subject may be at an aging stage.
  • the subject suffers from Alzheimer's disease.
  • Plasma A ⁇ 40, A ⁇ 42, total Tau and pTau181 were quantified using the ultrasensitive Simoa technology (Quanterix, MA, US) on the automated Simoa HD-X platform (GBIO, Hangzhou, China) according to the manufacturer's instructions.
  • Purchase multiplex Neurology 3-Plex A (Cat. No. 101995) and pTau181 V2 (Cat. No. 103714) Assay Kits from Quanterix and use accordingly.
  • plasma samples were diluted 1:4. Calibrators and quality controls were prepared in duplicate. All measurements are performed on a single-run basis. Operators were unaware of participants' disease status.
  • the plasma DIR was quantified using the enzyme-linked immunosorbent assay (ELISA) kit developed by the company.
  • ELISA enzyme-linked immunosorbent assay
  • U87 cells were lysed in pre-cooled RIPA buffer (50mM Tris, 150mM NaCl, 0.1% Triton-100, 10% glycerol, 0.5mg/mL BSA and protease inhibitors). After the lysate was centrifuged, the supernatant was mixed with the antibody overnight at 4°C, and then protein G-agarose was added to bind the antibody for 4 hours at 4°C. Then the agarose was resuspended in RIPA buffer, after washing at least 3 times, SDS buffer was added and incubated at 60°C for 20 minutes. The processed samples were used for western blot experiments.
  • RIPA buffer 50mM Tris, 150mM NaCl, 0.1% Triton-100, 10% glycerol, 0.5mg/mL BSA and protease inhibitors. After the lysate was centrifuged, the supernatant was mixed with the antibody overnight at 4°C, and then protein G-agarose was added to bind
  • gel digestion was performed using the following protocol. Treat gelatin in SDS equilibration buffer (50mM Tris-Cl (pH 8.8), 6M urea, 30% glycerol, 2% SDS and bromophenol blue) containing 1% 1,4-dithiotriitol (DTT). Glue for 15 minutes. After this step, free thiols were alkylated with SDS equilibration buffer containing 2.5% iodoacetamide for 15 minutes in the dark. It was then cut into multiple gel segments (each approximately 1.0 cm in length). Each gel slice was washed three times alternately with acetonitrile and 100 mM ammonium bicarbonate.
  • SDS equilibration buffer 50mM Tris-Cl (pH 8.8), 6M urea, 30% glycerol, 2% SDS and bromophenol blue
  • DTT 1,4-dithiotriitol
  • gel pieces were incubated in 100 mM ammonium bicarbonate for 15 min at 4 °C.
  • the gel pieces were dried by vacuum centrifugation and swelled at 4°C for 45 min in a trypsin solution containing trypsin (20 ⁇ g/ml) and ammonium bicarbonate (50 mM).
  • trypsin solution again and store at 37°C for 20h.
  • the supernatant was transferred to another vial, and the gel piece was extracted three times with 60% acetonitrile solution containing 0.1% formic acid, each time for 15 min.
  • the recovered peptide solution was dried by vacuum centrifugation, desalted and washed with a Ziptip (Millipore, Corp. Bedford, MA).
  • RP-HPLC reverse-phase high-performance liquid chromatography
  • RP-HPLC reverse-phase high-performance liquid chromatography
  • C18 chromatographic column (RP, 180 ⁇ m ⁇ 150 mm) was purchased from column Technology Inc. (Fremeont, CA).
  • the pump flow rate is 1:120 to achieve a column flow rate of 1.5 ⁇ l/min.
  • Mobile phase A was 0.1% formic acid aqueous solution
  • mobile phase B was 0.1% formic acid acetonitrile solution.
  • the tryptic peptide mixture was eluted with a 2-98% gradient of B over 180 min.
  • Mass spectra were performed on an LTQ linear ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an electrospray interface and operated in positive ion mode.
  • the capillary temperature was 170° C.
  • the spray voltage was 3.4 kV.
  • the normalized collision energy is 35%.
  • Use automatic gain control to obtain the maximum signal for each scan.
  • the mass spectrometer was set up to perform 10 MS/MS scans of the 10 most intense ions following one full MS scan. Dynamic exclusion was set to repeat count 2, repeat duration 30s, and exclusion duration 90s.
  • MS/MS spectra were obtained by searching the IPI human database using BioWorks 3.0 software (Thermo Finnigan) on an 8-node Dell PowerEdge 2650 cluster.
  • An accepted SEQUEST result has a ⁇ Cn score of at least 0.1 (regardless of charge state), a value with high confidence in a SEQUEST search.
  • Use Build Summary software to combine all output results and delete redundant data. To ensure that MS/MS spectra were of good quality, with fragment ions clearly above baseline noise, parameters reported in previous studies were referred to, and more stringent criteria for peptide identification were applied. Peptides were validated after meeting the following criteria.
  • the SEQUEST cross-correlation score must be ⁇ 1.9 for +1 tryptic peptides; ⁇ 2.2 for +2 tryptic peptides; and ⁇ 3.75 for +3 tryptic peptides.
  • the ⁇ Cn cutoff value was ⁇ 0.1 and the SP grade of the peptide was ⁇ 4.
  • AV45-PET imaging using a PET/CT system (Biograph TruePoint HD 64 PET/CT, Siemens; Erlangen, Germany). Subjects received intravenous injection of AV45 (average dose: 5.55MBq/kg [0.15mCi/kg]), and then rested quietly for 20 minutes in a darkened room. This was followed by a 10-minute PET acquisition procedure and a low-dose CT scan. After acquisition, PET images were reconstructed using a filtered back-projection algorithm with corrections for attenuation, normalization, dead time, photon attenuation, scattering, and random coincidence. The size of the reconstructed PET image matrix is 168 ⁇ 168 ⁇ 148, and the voxel size is 2.04 ⁇ 2.04 ⁇ 1.5mm 3 .
  • Regions of interest were mapped on the lateral parietal, lateral temporal, medial temporal, posterior cingulate, frontal, occipital, and precuneus cortices.
  • the standardized uptake value is the radioactivity in the region of interest (ROI), using the radioactivity of the cerebellar nodule as a reference.
  • the total SUV score was calculated as a weighted average of these ROIs.
  • the DIR antibody used in this study was produced by GL Biochem (which recognizes DIR (PPPLLCRRCHKIHLRRLLSKFSNIFSP) the last 27 AAs) and Sanyou Biopharmaceutical (Shanghai) Co., Ltd. (Sanyoubio), and the polyclonal antibodies used for immunoprecipitation were derived from rabbits. Monoclonal antibodies used for intraperitoneal injection and immunostaining were from hybridomas. The monoclonal antibody used for Elisa capture was prepared from hybridoma cells by Jill Biochemical (Shanghai) Co., Ltd., and the detection antibody was constructed by Sanyou Biopharmaceutical (Shanghai) Co., Ltd. using phage display library screening. The polyclonal antibodies used for Western blotting were from rabbits, and the polyclonal antibodies used for immunostaining were from guinea pigs.
  • Primers were designed to clone the full-length sequence of CDS. Purified product and vector using Hieff Plus One Step Cloning Kit (Yisheng Biotechnology (Shanghai) Co., Ltd.) was reorganized. DIR CDS was cloned into pCMV-flag vector, and gelsolin (NM_000177.5) was cloned into pEGFP-N3 and pcDNA3.1-myc-his plasmids. DDIT4L was constructed into the pcDNA3.1-myc-his vector. Primers are shown below.
  • HEK293T or U87MG cells were transfected with PEI40000 reagent (Yisheng Biotechnology (Shanghai) Co., Ltd.). After further culturing for 48 hours, the cells were lysed with HEPES lysis buffer (30mM HEPES, 150mM NaCl, 10mM NaF, 1% Triton X-100, 0.01% SDS), centrifuged at 12000rpm at 4°C for 10min, and the supernatant was transferred for Western blotting.
  • HEPES lysis buffer (30mM HEPES, 150mM NaCl, 10mM NaF, 1% Triton X-100, 0.01% SDS), centrifuged at 12000rpm at 4°C for 10min, and the supernatant was transferred for Western blotting.
  • HEK293T or U87MG cells were transfected with PEI40000 reagent (Yisheng Biotechnology (Shanghai) Co., Ltd.). After further culturing for 48 hours, the cells were lysed with HEPES lysis buffer (30mM HEPES, 150mM NaCl, 10mM NaF, 1% Triton X-100, 0.01% SDS), centrifuged at 12000rpm at 4°C for 10min, and the supernatant was transferred for Western blotting.
  • HEPES lysis buffer (30mM HEPES, 150mM NaCl, 10mM NaF, 1% Triton X-100, 0.01% SDS), centrifuged at 12000rpm at 4°C for 10min, and the supernatant was transferred for Western blotting.
  • DIR(31-84) and A ⁇ 42 were synthesized by Gil Biochemical (Shanghai) Co., Ltd.
  • the pcDNA3.1-gelsolin-myc-his plasmid was transfected into HEK293 cells, and the medium was collected for protein purification. Briefly, the Ni-NTA agarose resin was washed and transferred to a glass column to be used as an adsorption column.
  • the eluate was further concentrated and replaced with PBS.
  • the purified protein was diluted with PBS, and different groups were set according to solute (A ⁇ 42, A ⁇ 42/DIR, A ⁇ 42/DIR/gelsolin, etc.). A 1/10 volume solution was used as the sample solution, and the rest of the solution was rotated overnight at 4°C. Centrifuge the next day, take the supernatant for immunoprecipitation, and dissolve the precipitate with 8M urea. All components were further analyzed by immunoblotting.
  • mice were anesthetized and perfused with PBS. Brain tissue was then extracted and lysed in cold RIPA buffer. After centrifugation, the supernatant of the brain lysate was collected and stored at low temperature. Human and mouse plasma was diluted 1:10 with PBS. All samples were separated by sodium dodecyl sulfate (SDS) gel electrophoresis, then transferred to nitrocellulose membranes and blocked with 5% skim milk in TBS buffer containing Tween-20 (TBST). Membranes were incubated overnight at 4°C in primary antibody. The next day, membranes were washed three times with TBST and incubated with HRP-conjugated secondary antibodies. After further washing, imaging was performed by adding ECL buffer.
  • SDS sodium dodecyl sulfate
  • Protein bands were imaged with a GE imaging system.
  • the primary antibodies used are as follows: flag (Sigma, SAB4200071), actin (Chemicon, MAB1501), GAPDH (Proteintech, 10494), gelsolin (Invitrogen, PA518605), GFP (Roche, 11814460001).
  • mice were anesthetized and perfused with 4% paraformaldehyde (PFA). Brains were then collected and fixed in 4% PFA for 1 hour at 4°C. After washing three times in PBS, the tissue was placed in 30% sucrose/PBS at 4°C overnight and then embedded with OCT compound. Brain slices (thickness 40 ⁇ m) were obtained by cryosectioning. For immunostaining, tissue sections were blocked with PBST (0.1% Triton X-100/2.5% normal donkey serum/PBS) for 30 min at room temperature and then incubated overnight at 4°C with primary antibodies.
  • PBST 0.1% Triton X-100/2.5% normal donkey serum/PBS
  • Tissue sections were washed 3 times with PBS, incubated with Alexa fluorescence-conjugated secondary antibody (Invitrogen) for 60 min at 37°C, washed 3 times with PBS, and loaded into medium containing DAPI. Images were acquired using a Leica SP8 confocal microscope. Paraffin sections of human brain tissue were provided by the Human Brain Bank of the Chinese Academy of Medical Sciences and Peking Union Medical College, China. The distribution of DIR, gelsolin and A ⁇ was studied by immunofluorescence staining. In a single tissue section, primary antibodies raised in different species (mouse, rabbit or sheep) were used.
  • PKA Proximity Ligation Technology
  • Paraffin sections of human brain tissue were provided by the Human Brain Bank of Chinese Academy of Medical Sciences and Peking Union Medical College. Samples were blocked and then incubated in a humidity chamber at 37°C for 60 minutes. Antibodies recognizing DIR, gelsolin, and A ⁇ were then provided and samples were stained overnight at 4°C. The next day samples were incubated with positive and negative PLA probes (1:5; Sigma; DUO92001 and DUO92005) for 1 hr at 37°C, followed by additional incubation with 1X ligation buffer (1:40; Sigma; DUO92008) at 37°C 30 minutes. In addition, amplification-polymerase solution (1:80; Sigma; DUO92008) was used for 100 minutes at 37°C. Finally, samples were protected using a minimal volume of Duolink mounting medium containing DAPI dye (Sigma; DUO82040). Finally imaged with a Leica SP8 microscope.
  • the brains of adult mice were quickly removed after anesthesia, and placed in ice-cold artificial cerebrospinal fluid (ACSF) containing the following compounds (unit: mM): 117 NaCl, 3.6 KCl, 1.2 NaH 2 PO 4 2H 2 O, 2.5 CaCl 2 2H 2 O, 1.2 MgCl 2 ⁇ 6H 2 O, 25 NaHCO 3 , 11 glucose.
  • ACSF ice-cold artificial cerebrospinal fluid
  • the pH of ice-cold ACSF is 7.4.
  • TE Antigen Retrieval Solution (x): 4.84g Tris, 1.48g EDTA, 180mL distilled water, NaOH to adjust pH to 9.0, add 2mL Tween 20, dilute to 200mL, store at 4°C. Dilute to 1x when needed and store at room temperature.
  • the sections were added to PBS containing 3% goat serum and blocked for 1 hour.
  • Configure the True Black working solution (380ul 70% EtOH, 20ul preheated 20x True Black), drop it on the tissue, incubate at room temperature for 1 minute, suck off the True Black, wash with PBS 3 times. Place the slices in a wet box, cover the surface of the specimen with the diluted antibody, and put the wet box in a 4°C refrigerator for overnight Incubate overnight.
  • the slices were soaked in PBS and washed three times, and the diluted fluorescent secondary antibody was covered on the surface of the sample, and incubated at room temperature for 1 hour. Then wash the sections with PBS three times, add 50% ethanol solution containing 0.05% Thioflavin S, incubate in the dark for 8 minutes, and wash the sections twice with 80% ethanol for 10 seconds each time. Finally, the slices were washed three times with PBS, sealed with a drop of fluorescent mounting medium, and observed and recorded under a microscope.
  • Cas9mRNA and sgRNA (SEQ ID NO: 88) for knocking in were obtained; then, a homologous recombination vector as a donor was constructed, which contained a 5' homology arm of about 3 kb (SEQ ID NO: 89), the nucleic acid sequence (SEQ ID NO:90) of 64 amino acids behind the encoding human QDLIR, and the 3' homology arm (SEQ ID NO:91) of about 3kb.
  • the mRNA, sgRNA and supplying The F0 generation mice were obtained by microinjecting the body carrier into the fertilized eggs of C57BL/6J mice.
  • the F0 generation mice identified as positive by PCR were mated with the C57BL/6J mice to obtain the positive F1 generation mice. Mice among mice can obtain F2 generation wild-type, heterozygous and homozygous mice.
  • mice were acclimated to the experimental room 30 minutes earlier on the day of the experiment.
  • the experiment is divided into adaptation period, training period and experiment period.
  • Adaptation period Take the mice out of the breeding cage, place them in the center of the plastic box, and freely explore the experimental site for 10 minutes.
  • the training period is carried out after 12 to 24 hours of the adaptation period; training period: place 2 identical batteries at a distance of 5 cm from the wall. Place the mice in the center of the field and explore freely for 10 minutes.
  • the experimental period was carried out within 3 to 4 hours after the end of the training period; during the experimental period: a battery was randomly replaced by a toy, and the placement was the same as the original object.
  • the mice were placed in the center of the arena to explore freely for 10 minutes.
  • mice were acclimated to the experimental room 30 minutes earlier on the day of the experiment.
  • the experiment was divided into training day and experiment day.
  • the training day is 5-6 days, and the experiment day is 1 day.
  • fill the water tank with water at a depth of 30 cm, maintain the water temperature at 19-22°C, and add talcum powder to the water to make it turbid.
  • the water tank was divided into four directions: N, S, E and W, the platform was placed in the NE quadrant, and the daily water entry point of the mice was selected from a non-repeated combination of S, W, NW and SE.
  • the room setting remained unchanged during the experiment, and specific patterns were posted around the water maze as distal cues. Room lighting is indirect lighting.
  • the whole experiment was recorded by video camera.
  • the experiment will be carried out 24 hours after the end of the training day. Remove platform. Mice entered the water from the SW quadrant, facing the tank wall. After 1 minute, the mouse was taken out, dried, and returned to the cage. After the experiment, the EthoVision XT 14 software was used to analyze the mouse behavior videos. The collected data included: the escape latency of entering the target quadrant, the number of times crossing the platform position, and the residence time in the target and non-target quadrants.
  • mice were allowed to acclimatize to the feeding environment for one week.
  • the mice were acclimatized to the experimental room 30 minutes earlier on the day of the experiment.
  • the mice were placed in the starting arm A of the Y-shaped maze facing the wall, and freely explored the maze for 10 minutes.
  • mice can choose any direction to enter.
  • Cognitively normal mice tended to enter the unexplored arm during the experiment. The whole process is recorded by video camera. After the experiment, the mice were returned to their cages. 75% alcohol was used to clean the experimental apparatus between experiments.
  • AD Alzheimer's disease
  • the normal splice body of the human DDIT4L gene will partially excise the intron (Intron) and connect two adjacent exons (Exon) during the formation of mRNA.
  • Intron intron
  • Exon two adjacent exons
  • IR intron retention
  • DIR DIR receptor RI 1
  • SEQ ID NO.1 DIR protein
  • the open reading frame (SEQ ID NO.8) of DIR was constructed into the pCMV-flag vector, and the Flag-DIR plasmid and the control flag plasmid were respectively transfected into HEK293 cells, and the cell culture medium was taken after 48 hours
  • the results are shown in Figure 1C.
  • the plasmid capable of expressing DIR was transfected into U87 cells, and the cells were lysed after 48 hours, and the cell lysate was equally divided into two parts, and equal amounts of IgG and DIR antibodies were added for co-immunoprecipitation experiments, and the experimental products were separated by SDS-PAGE , and soak the separated PAGE gel in Coomassie Brilliant Blue staining solution for 1 hour, and then wash off the non-specifically attached dye solution on the surface of the PAGE gel with decolorization solution until the bands can be clearly displayed in the PAGE gel. Then, the specific band was cut and mass spectrometry was performed to obtain the candidate molecule gelsolin.
  • the brain tissue of adult C57BL/6 mice was taken to make brain slices, and the brain slices of the hippocampus were taken for electrophysiological recording.
  • the changes of post-synaptic excitatory currents in brain slices were recorded before and after IR peptide administration.
  • DIR (i.e. QDLIR, its amino acid sequence is shown in SEQ ID NO.1) comprises the amino acid sequence encoded by the first exon (its amino acid sequence is shown in SEQ ID NO.2) and the retained intron encoded Amino acid sequence (its amino acid sequence is shown in SEQ ID NO.3).
  • the amino acid sequence (IR) encoded by the retained intron can be divided into two parts, which are respectively IR-I composed of the first 27 amino acids from the N-terminus (ie, DIR-I, whose amino acid sequence Shown in SEQ ID NO.4) and the DIR-II (being DIR-II, its aminoacid sequence shown in SEQ ID NO.5) that last 27 amino acids form.
  • IR-I composed of the first 27 amino acids from the N-terminus
  • DIR-III being DIR-II, its aminoacid sequence shown in SEQ ID NO.5
  • IR-I and DIR-II fragments were synthesized separately to screen functional regions.
  • the results showed that IR-I could reduce the frequency of EPSC but not the amplitude of EPSC; although DIR-II had no significant effect on the amplitude and frequency of EPSC.
  • DIR-II has a tendency to reduce the EPSC amplitude, and has a tendency to increase the EPSC frequency (see Figure 3).
  • IR-I can reduce the frequency of EPSC
  • DIR-II can reduce the amplitude of EPSC.
  • Example 4 DIR participates in A ⁇ deposition
  • DIR mainly co-localized with A ⁇ in the hippocampus (see Figure 5a). Interaction between DIR and A ⁇ was observed in transfected HEK293T cells, but DDIT4L (including DIR-exon) could not interact with A ⁇ (see Fig. 5b, Fig. 6a). These results indicated that the DIR-intron (the amino acid sequence encoded by the retained intron, whose amino acid sequence is shown in SEQ ID NO.3) may be the main region where DIR interacts with A ⁇ .
  • PKA Proximity ligation analysis
  • DIR was transfected into a human brain cell line, and cell lysates were immunoprecipitated with a rabbit-derived DIR antibody (purchased from Jill Biochemical (Shanghai) Co., Ltd.). The results showed that an immunoreactive band with a molecular weight of about 85 kDa was co-immunoprecipitated (see Figure 5d). Bands were extracted and further analyzed by mass spectrometry. This analysis identified 12-15 peptides that matched human gelsolin (85 kDa) and covered 35% of the gelsolin sequence (see Figure 5e).
  • Gelsolin has been shown to interact with A ⁇ 17 to form a complex that is delivered to the circulation. Studies have shown that DIR interacts with gelsolin in DIR knock-in mice, HEK293T cells, and human tissues (see Figure 5f,g, Figure 6c).
  • the DIR-intron is involved in the deposition of A ⁇ .
  • Thioflavine S (thioflavine S) is a specific marker of A ⁇ plaques in the brains of AD patients.
  • Thioflavin S was unable to label amyloid plaques in the hippocampus of DIR knock-in mice (see Fig. 7a), as the mouse A ⁇ sequence could not be detected with Thioflavin.
  • amyloid plaques in hippocampal slices of DIR knock-in mice incubated with human A ⁇ 40 peptide for 6 h could be stained by Thioflavin S (see Figure 7a), which was consistent with the results of Thioflavin S staining of human A ⁇ plaques.
  • DIR may lead to the deposition of A ⁇ , and under pathological conditions, it can lead to the subsequent formation of amyloid plaques by combining with gelsolin.
  • DIR may be the initiator of A[beta] deposition and amyloid plaque formation (see Figure 11e).
  • Plasma DIR level as a diagnostic indicator
  • naMCI non-amnestic MCI
  • aMCI amnestic MCI
  • DIR concentrations in plasma were progressively elevated in naMCI, aMCI and AD patients compared to cognitively normal controls (see Figure 9a). Furthermore, DIR concentrations in plasma were found to be positively correlated with those of pTau181 but negatively correlated with the A ⁇ 42/40 ratio and with the severity of cognitive decline across the clinical range from normal cognition to naMCI, aMCI, and AD negative correlation.
  • An integrated model combining DIR, pTau, A ⁇ 42/40 demonstrated the potential to distinguish AD patients from cognitively normal controls with an AUC of 96%, which was higher than that obtained using pTau and A ⁇ 42 (88%)/40 (see Figure 9h) .
  • DIR concentrations were examined in 12 MCI patients three years ago when they were cognitively normal (as a baseline sample) and in plasma after the disease. DIR plasma concentrations were found to be higher in samples from these MCI patients than in their baseline samples (Fig. 11a).
  • a ⁇ -PET levels were significantly correlated with A ⁇ -PET levels in the whole cortex, and plasma DIR levels were strongly correlated with A ⁇ -PET levels in the temporal lobe, suggesting that A ⁇ Accumulation (see Figure 11c, Figure 12b).
  • the spleens of the mice immunized with the antigen were taken and digested into single cells. Then the B cells were isolated and cultured in an in vitro system, and the B cells were fused with myeloma cells using chemical inducers. The fused cells were cultured in 96-well plates by flow sorting technology. Finally, the culture medium was collected and incubated into a well plate coated with antigen to detect the specificity of the DIR antibody.
  • the binding activity of the above-mentioned IR-II-1 ⁇ IR-II-10 antibodies to the DIR-II fragment was detected.
  • the specific method is:
  • Embodiment 7 utilizes DIR antibody to detect DIR
  • Antibodies targeting 30 amino acids of the translated sequence of exon 2 of DIR were prepared and tested in patient tissues. Immunoblot of GBM tissue showed two bands ( ⁇ 12kDa and ⁇ 22kDa) (see Figure 14c). Since the molecular weight of DDIT4L is about 22kDa, it is speculated that the 12kDa band may be DIR.
  • an antibody (a polyclonal antibody derived from guinea pig, purchased from Jill Biochemical (Shanghai) Co., Ltd.) that only recognizes 54 amino acids of the DIR-specific intron translation sequence was obtained and tested in patient tissues. A protein of about 12 kDa was only detected in GBM tissue (see Figure 14d).
  • a heterologous expression plasmid containing the DIR open reading frame (its nucleotide sequence is shown in SEQ ID NO.1) was constructed, and the plasmid was transfected into HEK293T cells. DIR could be detected in cell lysates and conditioned media (see Figure 14e), suggesting that DIR can be translated and secreted in vivo.
  • Example 8 Reduced DIR levels can improve cognition
  • A2D10 0.5 mg/day
  • IgG obtained in Example 6 were injected intraperitoneally into homozygous DIR-KI mice for 5 days.
  • mice administered A2D10 had a shorter latency to enter the target quadrant and stayed longer.

Abstract

本申请涉及一种改善认知障碍的方法。所述方法包括施用调控剂，该调控剂调控DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段，从而可以预防和/或治疗疾病，其中所述疾病包括认知障碍和/或神经退行性疾病。

Description

改善认知障碍的方法 技术领域
本申请涉及生物医药领域，具体的涉及一种改善认知障碍的方法。
背景技术
我国老龄化人口数量逐年增加，老年人群由于免疫力下降、血管硬化等多种因素的影响，面临着多种疾病的困扰，其中老年痴呆症的发病率在当前老年人群中的比例已经不容忽视。痴呆症患者不仅仅自身受到病痛的折磨，也会让各自的家庭负担巨大的压力。因此，针对老年痴呆症开发治疗性药物具有极其重要的意义。
在多年的研究过程中，越来越多老年痴呆症相关的基因被报导，例如APP、PSEN1等等。这些基因突变会加速神经元损伤，从而造成认知障碍。基于这些靶点的药物，尤其是针对Aβ的单克隆抗体药物已经有多条研发生产管线，并且今年也有被FDA批准的药物上市。然而，这类单抗药物尽管可以有效清除人脑中Aβ的累积，但是对于患者认知能力的改善并不理想。
可见亟待寻求改善认知障碍的新方法。
发明内容
本申请提供了一种新型的改善认知障碍的方法。
一方面，本申请提供一种调控剂，其调控DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段，在制备用于预防和/或治疗疾病的试剂中的用途，其中所述疾病包括认知障碍。
一方面，本申请提供一种调控剂，其调控DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段，在制备用于预防和/或治疗疾病的试剂中的用途，其中所述疾病包括神经退行性疾病。
在某些实施方式中，所述调控剂使受试者中，所述DIR和/或其功能片段的表达水平和/或生物学活性降低。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的 表达水平和/或生物学活性相比，所述DIR和/或其功能片段的表达水平和/或生物学活性降低至少约10％。
在某些实施方式中，所述表达水平包括编码所述DIR/或其功能片段的基因的表达水平、编码所述DIR/或其功能片段的基因的转录水平和/或所述DIR/或其功能片段的表达水平。
在某些实施方式中，所述表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述表达水平通过利用选自下组的物质衡量：能够特异性扩增编码所述DIR/或其功能片段的基因的引物、与编码所述DIR/或其功能片段的基因特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的小分子、与所述DIR/或其功能片段特异性结合的探针和与所述DIR/或其功能片段特异性结合的多肽。
在某些实施方式中，所述DIR的功能片段保留所述DIR的至少部分的生物学活性。
在某些实施方式中，所述生物学活性包括影响神经元的兴奋性和/或抑制神经元的活性。
在某些实施方式中，所述生物学活性包括能够降低兴奋性突触后电流(EPSC)的频率，和/或能够降低EPSC的幅度。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者中的兴奋性突触后电流(EPSC)的频率降低，和/或使受试者中的EPSC的幅度降低。
在某些实施方式中，所述生物学活性包括影响认知能力。
在某些实施方式中，所述生物学活性包括参与与Aβ沉积相关的信号通路，和/或，参与与Tau缠结产生相关的信号通路。
在某些实施方式中，所述生物学活性包括通过gelsolin诱导Aβ沉积和/或淀粉样斑块形成。
在某些实施方式中，所述DIR/或其功能片段通过与gelsolin结合诱导Aβ沉积和/或淀粉样斑块形成。
在某些实施方式中，所述DIR和/或其功能片段的表达水平与Aβ的表达水平正相关。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸， 使受试者的认知能力降低。
在某些实施方式中，所述DIR/或其功能片段来源于哺乳动物。
在某些实施方式中，所述DIR/或其功能片段来源于灵长类动物。
在某些实施方式中，述DIR/或其功能片段来源于人。
在某些实施方式中，所述DIR包含SEQ ID NO.1所示的氨基酸序列。
在某些实施方式中，所述DIR的功能片段包含DDIT4L中滞留的内含子所编码的氨基酸序列。
在某些实施方式中，所述DIR的功能片段包含SEQ ID NO.4-5中任一项所示的氨基酸序列。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括遗忘型MCI多认知域受损(aMCI-m)。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
在某些实施方式中，所述神经退行性疾病包括早老性痴呆。
在某些实施方式中，所述神经退行性疾病包括早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。
在某些实施方式中，所述受试者包括哺乳动物。
在某些实施方式中，所述受试者包括人。
在某些实施方式中，所述受试者包括神经退行性疾病患者和/或认知障碍患者。
在某些实施方式中，所述受试者包括阿尔兹海默症患者。
在某些实施方式中，所述受试者处于老龄阶段。
在某些实施方式中，所述试剂被配制为适于口服施用和/或注射施用。
在某些实施方式中，所述调控剂包括小分子化合物、聚合物和/或生物大分子。
在某些实施方式中，所述调控剂包括抗体或其抗原结合片段。
在某些实施方式中，所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的抗体或其抗原结合片段。
在某些实施方式中，所述调控剂包括反义寡核苷酸。
在某些实施方式中，所述调控剂包括siRNA。
在某些实施方式中，所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的siRNA。
在某些实施方式中，所述调控剂包含SEQ ID NO.84-87中任一项所示的核苷酸序列。
另一方面，本申请提供一种预防和/或治疗认知障碍的方法，其包括以下的步骤：使有需要的受试者中，DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的表达水平和/或生物学活性降低。
另一方面，本申请提供一种预防和/或治疗神经退行性疾病的方法，其包括以下的步骤：使有需要的受试者中，DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的表达水平和/或生物学活性降低。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的表达水平和/或生物学活性相比，所述DIR和/或其功能片段的表达水平和/或生物学活性降低至少约10％。
在某些实施方式中，所述表达水平包括编码所述DIR/或其功能片段的基因的表达水平、编码所述DIR/或其功能片段的基因的转录水平和/或所述DIR/或其功能片段的表达水平。
在某些实施方式中，所述表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述表达水平通过利用选自下组的物质衡量：能够特异性扩增编码所述DIR/或其功能片段的基因的引物、与编码所述DIR/或其功能片段的基因特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的小分子、与所述DIR/或其功能片段特异性结合的探针和与所述DIR/或其功能片段特异性结合的多肽。
在某些实施方式中，所述DIR的功能片段保留所述DIR的至少部分的生物学活性。
在某些实施方式中，所述生物学活性包括影响神经元的兴奋性和/或抑制神经元的活性。
在某些实施方式中，所述生物学活性包括能够降低兴奋性突触后电流(EPSC)的频率，和/或能够降低EPSC的幅度。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者中的兴奋性突触后电流(EPSC)的频率降低，和/或使受试者中的EPSC的幅度降低。
在某些实施方式中，所述生物学活性包括影响认知能力。
在某些实施方式中，所述生物学活性包括参与与Aβ沉积相关的信号通路，和/或，参与与Tau缠结产生相关的信号通路。
在某些实施方式中，所述生物学活性包括通过gelsolin诱导Aβ沉积和/或淀粉样斑块形成。
在某些实施方式中，所述DIR/或其功能片段通过与gelsolin结合诱导Aβ沉积和/或淀粉样斑块形成。
在某些实施方式中，所述DIR和/或其功能片段的表达水平与Aβ的表达水平正相关。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者的认知能力降低。
在某些实施方式中，所述DIR/或其功能片段来源于哺乳动物。
在某些实施方式中，所述DIR/或其功能片段来源于灵长类动物。
在某些实施方式中，所述DIR包含SEQ ID NO.1所示的氨基酸序列。
在某些实施方式中，所述DIR的功能片段包含DDIT4L中滞留的内含子所编码的氨基酸序列。
在某些实施方式中，所述DIR的功能片段包含SEQ ID NO.4-5中任一项所示的氨基酸序列。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括遗忘型MCI多认知域受损(aMCI-m)。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
在某些实施方式中，所述神经退行性疾病包括早老性痴呆。
在某些实施方式中，所述神经退行性疾病包括早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。
在某些实施方式中，所述受试者包括哺乳动物。
在某些实施方式中，所述受试者包括人。
在某些实施方式中，所述受试者包括神经退行性疾病患者和/或认知障碍患者。
在某些实施方式中，所述受试者包括阿尔兹海默症患者。
在某些实施方式中，所述受试者处于老龄阶段。
在某些实施方式中，所述方法包括以下步骤：向有需要的受试者施用调控剂，所述调控剂能够降低所述DIR和/或其功能片段，和/或编码所述DIR和/或其功能片段的核酸的表达水平和/或生物学活性。
在某些实施方式中，所述施用包括口服施用和/或注射施用。
在某些实施方式中，所述调控剂包括小分子化合物、聚合物和/或生物大分子。
在某些实施方式中，所述调控剂包括抗体或其抗原结合片段。
在某些实施方式中，所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的抗体或其抗原结合片段。
在某些实施方式中，所述调控剂包括反义寡核苷酸。
在某些实施方式中，所述调控剂包括siRNA。
在某些实施方式中，所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的siRNA。
在某些实施方式中，所述调控剂包含SEQ ID NO.84-87中任一项所示的核苷酸序列。
另一方面，本申请提供一种筛选能够预防和/或治疗认知障碍和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中DIR和/或其功能片段的表达水平和/或生物学活性的影响，其中在施用所述候选药物后，所述DIR和/或其功能片段的表达水平和/或生物学活性降低，则所述候选药物能够预防和/或治疗认知障碍和/或治疗神经退行性疾病。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的表达水平和/或生物学活性相比，所述DIR和/或其功能片段的表达水平和/或生物学活性降低至少约10％。
在某些实施方式中，所述表达水平包括编码所述DIR/或其功能片段的基因的表达水平、编码所述DIR/或其功能片段的基因的转录水平和/或所述DIR/或其功能片段的表达水平。
在某些实施方式中，所述表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
在某些实施方式中，所述表达水平通过利用选自下组的物质衡量：能够特异性扩增编码所述DIR/或其功能片段的基因的引物、与编码所述DIR/或其功能片段的基因特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的小分子、与所述DIR/或其功能片段特异性结合的探针和与所述DIR/或其功能片段特异性结合的多肽。
在某些实施方式中，所述DIR的功能片段保留所述DIR的至少部分的生物学活性。
在某些实施方式中，所述生物学活性包括影响神经元的兴奋性和/或抑制神经元的活性。
在某些实施方式中，所述生物学活性包括能够降低兴奋性突触后电流(EPSC)的频率，和/或能够降低EPSC的幅度。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者中的兴奋性突触后电流(EPSC)的频率降低，和/或使受试者中的EPSC的幅度降低。
在某些实施方式中，所述生物学活性包括影响认知能力。
在某些实施方式中，所述生物学活性包括参与与Aβ沉积相关的信号通路，和/或，参与与Tau缠结产生相关的信号通路。
在某些实施方式中，所述生物学活性包括通过gelsolin诱导Aβ沉积和/或淀粉样斑块形成。
在某些实施方式中，所述DIR/或其功能片段通过与gelsolin结合诱导Aβ沉积和/或淀粉样斑块形成。
在某些实施方式中，所述DIR和/或其功能片段的表达水平与Aβ的表达水平正相关。
在某些实施方式中，所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者的认知能力降低。
在某些实施方式中，所述DIR/或其功能片段来源于哺乳动物。
在某些实施方式中，所述DIR/或其功能片段来源于灵长类动物。
在某些实施方式中，所述DIR包含SEQ ID NO.1所示的氨基酸序列。
在某些实施方式中，所述DIR的功能片段包含DDIT4L中滞留的内含子所编码的氨基酸序列。
在某些实施方式中，所述DIR的功能片段包含SEQ ID NO.4-5中任一项所示的氨基酸序列。
在某些实施方式中，所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
在某些实施方式中，所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
在某些实施方式中，所述认知障碍包括遗忘型MCI多认知域受损(aMCI-m)。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
在某些实施方式中，所述神经退行性疾病包括早老性痴呆。
在某些实施方式中，所述神经退行性疾病包括早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。
在某些实施方式中，所述受试者包括哺乳动物。
在某些实施方式中，所述受试者包括神经退行性疾病患者和/或认知障碍患者。
在某些实施方式中，所述受试者包括阿尔兹海默症患者。
在某些实施方式中，所述受试者处于老龄阶段。
在某些实施方式中，所述施用包括口服施用和/或注射施用。
在某些实施方式中，所述候选药物包括小分子化合物、聚合物和/或生物大分子。
另一方面，本申请提供一种分离的抗原结合蛋白，其具有以下性质：在ELISA测定中，以约10ng/ml以上的工作浓度与人DIR和/或其功能片段特异性结合；
在某些实施方式中，所述的抗原结合蛋白包含LCDR2，所述LCDR2包含SEQ ID NO:74所示的氨基酸序列。
在某些实施方式中，所述LCDR2包含SEQ ID NO:26或16所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含LCDR1，所述LCDR1包含SEQ ID NO:73所示的氨基酸序列。
在某些实施方式中，所述LCDR1包含SEQ ID NO:25、52、15中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含LCDR3，所述LCDR3包含SEQ ID NO:75所示的氨基酸序列。
在某些实施方式中，所述LCDR3包含SEQ ID NO:27、53、67、17中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白，其包含LCDR1、LCDR2和LCDR3，其中，
a)所述LCDR1包含SEQ ID NO:25所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:27所示的氨基酸序列；
b)所述LCDR1包含SEQ ID NO:52所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:53所示的氨基酸序列；
c)所述LCDR1包含SEQ ID NO:52所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:67所示的氨基酸序列；或者，
d)所述LCDR1包含SEQ ID NO:15所示的氨基酸序列，所述LCDR2包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含HCDR1，所述HCDR1包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述HCDR1包含SEQ ID NO:20、35、56、10中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含HCDR2，所述HCDR2包含SEQ ID NO:71所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO:21、36、42、48、11中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含HCDR3，所述HCDR3包含SEQ ID NO:70所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO:22、30、37、43、49、57、62、12中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，其中，
a)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序列；
b)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:30所示的氨基酸序列；
c)所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:37所示的氨基酸序列；
d)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:42所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:43所示的氨基酸序列；
e)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:48所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:49所示的氨基酸序列；
f)所述HCDR1包含SEQ ID NO:56所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:57所示的氨基酸序列；
g)所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:62所示的氨基酸序列；
h)所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:42所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:37所示的氨基酸序列；
i)所述HCDR1包含SEQ ID NO:56所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36 所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:57所示的氨基酸序列；或者，
j)所述HCDR1包含SEQ ID NO:10所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含重链可变区VH，所述VH包含SEQ ID NO:13、23、31、38、44、50、58、63、65中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含轻链可变区VL，所述VL包含SEQ ID NO:18、28、33、40、46、54、60、68中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含重链可变区VH和轻链可变区VL，其中：
a)所述VH包含SEQ ID NO:23所示的氨基酸序列，且所述VL包含SEQ ID NO:28所示的氨基酸序列；
b)所述VH包含SEQ ID NO:31所示的氨基酸序列，且所述VL包含SEQ ID NO:33所示的氨基酸序列；
c)所述VH包含SEQ ID NO:38所示的氨基酸序列，且所述VL包含SEQ ID NO:40所示的氨基酸序列；
d)所述VH包含SEQ ID NO:44所示的氨基酸序列，且所述VL包含SEQ ID NO:46所示的氨基酸序列；
e)所述VH包含SEQ ID NO:50所示的氨基酸序列，且所述VL包含SEQ ID NO:54所示的氨基酸序列；
f)所述VH包含SEQ ID NO:58所示的氨基酸序列，且所述VL包含SEQ ID NO:60所示的氨基酸序列；
g)所述VH包含SEQ ID NO:63所示的氨基酸序列，且所述VL包含SEQ ID NO:60所示的氨基酸序列；
h)所述VH包含SEQ ID NO:65所示的氨基酸序列，且所述VL包含SEQ ID NO:54所示的氨基酸序列；
i)所述VH包含SEQ ID NO:58所示的氨基酸序列，且所述VL包含SEQ ID NO:68所示的氨基酸序列；或者，
j)所述VH包含SEQ ID NO:13所示的氨基酸序列，且所述VL包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含重链恒定区，且所述重链恒定区包括源自IgG的恒定区。
在某些实施方式中，所述重链恒定区包含源自选自下组蛋白的恒定区：IgG1、IgG2、IgG3和IgG4。
在某些实施方式中，所述的抗原结合蛋白包含轻链恒定区，且所述轻链恒定区包括源自Igκ的恒定区或源自Igλ的恒定区。
在某些实施方式中，所述轻链恒定区包括源自人Igκ的恒定区。
在某些实施方式中，所述的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
在某些实施方式中，所述抗体选自下组：单克隆抗体、鼠源抗体和嵌合抗体。
另一方面，本申请提供一种多肽，其包含本申请所述的分离的抗原结合蛋白。
另一方面，本申请提供一种免疫缀合物，其包含本申请所述的分离的抗原结合蛋白或本申请所述的多肽。
另一方面，本申请提供一种分离的核酸分子，其编码本申请所述的分离的抗原结合蛋白，或者本申请所述的多肽。
另一方面，本申请提供一种载体，其包含本申请所述的分离的核酸分子。
另一方面，本申请提供一种细胞，其包含本申请所述的分离的抗原结合蛋白，本申请所述的多肽，本申请所述的免疫缀合物，本申请所述的分离的核酸分子和/或本申请所述的载体。
另一方面，本申请提供一种制备本申请所述的分离的抗原结合蛋白或本申请所述的多肽的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白或本申请所述的多肽表达的条件下，培养本申请所述的细胞。
另一方面，本申请提供一种药物组合物，其包含本申请所述的分离的抗原结合蛋白，本申请所述的多肽，本申请所述的免疫缀合物，本申请所述的分离的核酸分子，本申请所述的载体，本申请所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。
另一方面，本申请提供一种本申请所述的分离的抗原结合蛋白和/或本申请所述的多肽在制备预防和/或治疗疾病或病症的药物中的用途，其中所述疾病或病症包括认知障碍和/或神经退行性疾病。
在某些实施方式中，所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
在某些实施方式中，所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认 知障碍。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1A-图1D显示的是本申请所述DIR的鉴定。
图2显示的是本申请所述DIR的功能片段的结构示意图。
图3显示的是本申请所述DIR的功能片段对兴奋性突触后电流的影响。
图4显示的是本申请所述DIR的生物学功能。
图5a-5i显示的是DIR通过结合gelsolin诱导Aβ沉积，其中：
a.DIR存在于AD患者海马体中硫磺素s阳性斑块(箭头)中(n＝3)。标尺＝50μm。
b.转染表达Flag-DIR质粒并添加人工合成的Aβ42的HEK293T细胞裂解液中，在用Flag抗体沉淀的蛋白中发现Aβ(n＝3)。
c.PLA实验显示，在AD患者的海马体中DIR不能与Aβ结合。比例尺＝50μm。
d.考马斯蓝染色显示，在转染Flag-DIR质粒的U87MG细胞裂解液中，DIR抗体可以沉淀出分子量约为85kDa的蛋白(n＝3)。
e.质谱鉴定了～85kDa大小区间的前8个分子。gelsolin是其中含量最多的分子。
f.在DIR-knock-in小鼠的海马体裂解液中，用DIR和Aβ抗体沉淀出的蛋白中发现了gelsolin(n＝3)。
g.PLA实验显示，gelsolin在AD患者海马体中与Aβ(箭头)结合。比例尺＝50μm。
h.在表达Flag-DIR的HEK293T细胞裂解液中，添加不同剂量的Aβ42，在Flag抗体沉淀出的蛋白中发现gelsolin，并且高剂量Aβ42组中，两者的结合水平增加(10μg,n＝3)。
i.在gelsolin-his与合成Aβ42或DIR(31-84)的混合物中，DIR(31-84)通过gelsolin引起Aβ42的不溶性(n＝3)。
图6a-6d显示的是DIR不直接与Aβ结合，而是诱导Aβ沉积。其中，
a.在转染表达myc-DIR质粒或DDIT4L-myc的HEK293T细胞裂解液中，添加人工合成的Aβ42，然后分别用myc抗体做免疫沉淀实验。其中只有表达myc-DIR-的裂解液能够沉淀出Aβ(n＝3)。
b.用合成的DIR(31-84)和Aβ42做免疫共沉淀实验，结果显示两者之间没有直接相互作用(n＝3)。
c.在共转染表达Flag-DIR和gelsolin-GFP质粒的HEK293T细胞裂解液中，发现用Flag抗体沉淀的蛋白中存在gelsolin-GFP(n＝3)。
d.在HEK293T细胞裂解液中加入合成的Aβ42或DIR(31-84)，DIR(31-84)通过gelsolin诱导Aβ42不溶(n＝3)。
图7a-7c显示的是DIR介导Aβ斑块形成，其中：
a.在DIR-KI小鼠(n＝3)海马体切片中添加合成的人Aβ40(100μM)，孵育6h后出现硫磺素s阳性斑块(箭头)，并与DIR和gelsolin共标。比例尺＝100μm。
b.在AD患者(n＝3)，而非对照组(n＝3)中，硫磺素s阳性致密核斑块与DIR和gelsolin(箭头)共同定位于海马体DG区。比例尺＝50μm。定量分析显示，在大脑皮层中86.3％的致密核斑块是DIR、gelsolin和硫磺素s阳性，在海马体中这一比例可以达到92.8％。
c.DIR存在于AD患者海马体中的Aβ-和硫磺素S阳性斑块(箭头)中(n＝3)。Aβ阳性区域大于硫磺素S-和DIR-阳性信号。比例尺＝50μm。
图8显示的是在人海马体中，Aβ阳性且硫磺素s-阴性斑块未与DIR和gelsolin重叠。其中，
a.在AD患者中(n＝3)，而在对照组中(n＝3)，Aβ阳性且硫磺素s-阴性弥漫性斑块(箭头)未与DIR和gelsolin共同定位于海马DG区。比例尺＝50μm。
图9a-9l显示的是血浆DIR是AD和aMCI的潜在生物标志物，其中：
a.与认知正常对照组(NC,n＝33)相比，naMCI(n＝44)、aMCI(n＝42)或AD(n＝31)患者血浆DIR逐渐升高。**，p<0.01，***，p<0.001。
b.与NC(n＝33)相比，naMCI(n＝44)、aMCI(n＝42)或AD(n＝31)患者血浆Aβ42/Aβ40逐渐降低。*，p<0.05，**，p<0.01。
c.与NC(n＝33)相比，naMCI(n＝44)、aMCI(n＝42)或AD(n＝31)患者血浆pTau181逐渐升高。*，p<0.05，***，p<0.001。
d.与对照组(n＝33)相比，naMCI(n＝44)、aMCI(n＝42)、AD(n＝31)患者血浆 tTau无明显变化。
e.NC(n＝33)和AD(n＝31)患者血浆DIR的受试者工作特征(ROC)分析。
f.对NC(n＝33)和AD(n＝31)的血浆pta181 ROC分析。
g.对NC(n＝33)和AD(n＝31)血浆Aβ42/Aβ40 ROC分析。
h.结合血浆DIR、pTau181和Aβ42/Aβ40对NC(n＝33)和AD(n＝31)进行ROC分析(红色)。结合血浆pTau181和Aβ42/Aβ40对NC(n＝33)和AD(n＝31)进行ROC分析(蓝色)。
i.NC(n＝33)和aMCI(n＝42)血浆DIR的ROC分析。
j.NC(n＝33)和aMCI(n＝42)血浆pTau的ROC分析。
k.对NC(n＝33)和aMCI(n＝42)血浆Aβ42/Aβ40的ROC分析。
l.结合血浆DIR、pta181和Aβ42/Aβ40对NC(n＝33)和aMCI(n＝42)进行ROC分析(红色)。结合血浆pta181和Aβ42/Aβ40对NC(n＝33)和aMCI(n＝42)进行ROC分析(蓝色)。
图10a-10b显示的是血浆tTau的ROC分析。其中，
a.NC(n＝33)和AD(n＝31)血浆tTau的ROC分析。
b.NC(n＝33)和aMCI(n＝42)血浆tTau的ROC分析。
图11a-11e显示的是血浆DIR与Aβ的相关性分析，其中：
a.三年前(2018-2021年)正常但目前是MCI患者(n＝12)的血浆DIR。MCI患者血浆DIR浓度高于3年前采集的血液样本。
b.MCI和AD患者(n＝117)和认知正常对照组(n＝33)血浆DIR与血浆Aβ40呈正相关。
c.AD/MC患者I或NC中淀粉样蛋白18F-AV-45 SUV的三维可视化图像。颜色条表示从淀粉样蛋白18F-AV-45图像中得到的SUV指数。AD/MCI患者的血浆DIR值与皮层阳性信号相关，尤其是颞叶阳性信号。
d.与淀粉样PET阴性个体(n＝26)相比，淀粉样PET阳性个体(n＝11)的血浆DIR明显升高。淀粉样PET阳性个体(n＝11)和淀粉样PET阴性个体(n＝26)血浆DIR的ROC分析。**，p<0.01。
e.DIR诱导的淀粉样斑块沉积和血液分泌的模型。在几种局部微脑血管循环的病理情况下，缺氧导致内含子异常滞留，从而引起DIR蛋白的翻译。Aβ是通过淀粉样前体蛋白(APP)的蛋白水解过程产生的，与gelsolin有着直接的结合。DIR通过结合gelsolin，导致Aβ沉积， 最终形成脑内的淀粉样斑块。DIR也可以释放进入血液系统。
图12a-12b显示的是血浆DIR与Aβ42的相关性，定量分析Aβ-PET的SUV值。其中，
a.MCI和AD患者(n＝117)和认知正常对照组(n＝33)血浆DIR与血浆Aβ42呈正相关。
b.AD/MCI患者或NC SUV值的定量分析。*，p<0.05。
图13显示的是本申请所述DIR抗体的结合活性检测结果。
图14a-14h显示的是DIR是患者中人DDIT4L的异常剪接体，其中：
a.利用针对DDIT4L mRNA引物从患者1(G2913)的GBM mRNA中发现了两个产物(～200bp和～2000bp)，但在LGG患者1(L1002)的低级别胶质瘤(LGG)组织中没有。
b.DIR是人DDIT4L的剪接亚型，内含子保留在外显子2和外显子3之间。
c.GBM裂解液的免疫印迹显示，靶向DDIT4L N端的抗体检测到两条免疫反应条带(～22kDa和～12kDa，箭头)，但未被预吸收(pre-absorbed)的抗体检测到。
d.在两名患者(G2913,G1359)的GBM裂解液中，使用靶向内含子翻译区的DIR抗体检测～12kDa的条带。
e.转染表达Flag-DIR质粒的HEK293T细胞，在细胞裂解液和培养基中均可检测到Flag-DIR。
f.患者大脑磁共振成像显示，胶质母细胞瘤侵袭海马体区(海马体区，G8098 in g)或未发生胶质母细胞瘤的海马体区(G8409 in g)。
g.仅在侵袭海马体的GBM患者(G8098和G7200)血浆中检测到DIR。
h.AD患者血浆中存在DIR，命名为A7098等。
图15A-15B显示了DIR表达水平降低后对认知水平的影响。
图16A-16C显示了施用本申请所述的DIR抗体对认知水平的影响。
a.Y迷宫测试表明，与用IgG处理的小鼠(n＝13)相比，纯合DIR-KI小鼠(n＝17)在本申请所述的DIR抗体理后工作记忆发生逆转。*，p<0.05。
b.与用IgG处理的小鼠(n＝11)相比，在同源DIR-KI小鼠(n＝14)中，本申请所述的DIR抗体处理逆转了新物体识别的能力。**，p<0.01。
c.Morris水迷宫测试表明，与同源DIR-KI小鼠(n＝9)中的IgG治疗相比，施用本申请所述的DIR抗体(n＝11)治疗减少了5天训练期后的逃避潜伏期。在评估空间记忆的探测测试中，用IgG处理的小鼠没有表现出对目标象限的偏好，而用本申请所述的DIR抗体处理的小鼠进入的潜伏期更短，并且在目标象限中停留的时间更 长。*，p<0.05。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
术语“DDIT4L”通常是指DNA damage-inducible transcript 4-like，DNA损伤诱导转录本4样。其也可以称为REDD2/RTP801L。研究发现，DDIT4L可以与心脏功能障碍相关。也可以用于治疗胶质瘤。人DDIT4L基因在GenBank的登录号为115265；人DDIT4L蛋白在GenBank的登录号为NP_660287.1。
术语“DIR”通常是指DDIT4L的内含子滞留剪切产物。DDIT4L的剪切反应可以参见图1。在本申请中，所述DIR的氨基酸序列可以如SEQ ID NO:1所示。
术语“表达水平”通常是指特定相关基因的蛋白质、RNA或mRNA水平。可以采用本领域中已知的任何方法来测定特定相关基因(例如人DDIT4L基因)的表达水平。在本申请中，所述“表达”通常是指将基因编码的信息转变成存在于细胞中并在细胞中操作的结构的过程。例如可以包括逆转录和扩增分析(例如PCR、连接RT-PCR或定量RT-PCT)、杂交分析、RNA印迹法(Northern blotting)、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色或质谱。可以直接对生物样品或对从样品中分离出来的蛋白质/核酸执行分析。
术语“活性”通常是指与特定蛋白质相关的任何活性。在本申请中，所述活性可以包括例如DIR蛋白相关的任何活性。所述活性可以包括蛋白酶相关的酶促活性。在某些情况下，所述活性可以包括生物活性。在某些情况下，所述活性可以包括蛋白质与受体的结合，例如，该结合可能产生可测量的下游效应。在本申请中，所述活性可以包括将被本领域技术人员归因于该蛋白的任何活性。
术语“认知障碍”通常是指被认为或确实牵涉于神经元结构和/或功能的进行性损失(包括神经元死亡)或与上述有关的疾病和病症。例如，所述认知障碍的特征可以包括认知(例如，记忆、注意力、感知和/或思维)的损伤。这些障碍可以包括病原体-诱导的认知功能障碍，例如HIV相关的认知功能障碍和莱姆病相关的认知功能障碍。认知障碍的实例可以包括阿尔茨海默病、亨廷顿病、帕金森病、肌萎缩侧索硬化(ALS)、孤独症、早期认知功能损 害(MCI)、中风、创伤性脑损伤(TBI)和/或与年龄相关的记忆损害(AAMI)。
术语“神经退行性疾病”通常是指由神经元结构和功能逐渐丧失，包括神经元死亡和胶质细胞平衡，所导致的痴呆等认知障碍。在某些情况下，年龄(例如阿尔茨海默病(AD)、帕金森病(PD))或影响CNS细胞功能的基因突变(例如亨廷顿氏舞蹈病、早发性AD或PD、肌萎缩性脊髓侧索硬化症(ALS))可以引起所述神经退行性疾病。所述神经退行性疾病可以具有选自以下的变化和/或病症：蛋白错误折叠和聚集；神经炎症(例如在毒刺激(如蛋白聚集)、感染、创伤性损伤或自身免疫等信号刺激下出现的CNS炎症)；细胞信号转导的改变；获得性衰老/细胞死亡(例如被中断的凋亡信号转导、线粒体功能障碍、自噬受损以及坏死小体被应激/炎症激活)；运动细胞受损以及表观遗传学变化。
术语“阿尔兹海默症”通常是指早老性痴呆、老年痴呆，是一种发病进程缓慢、随着时间不断恶化的神经退化性疾病。最常见的早期症状为丧失短期记忆(难以记住最近发生的事)，当疾病逐渐进展，可能逐渐出现下述症状中的至少一种：语言障碍、定向障碍(例如，容易迷路)、情绪不稳、丧失动机、无法自理和行为问题。阿尔茨海默病的真正成因至今仍然不明，其进程可能与大脑中的纤维状类淀粉蛋白质斑块沉积和Tau蛋白有关。目前并没有可以阻止或逆转病程的治疗，只有少数方法或许可以暂时缓解或改善症状。
术语“早老性痴呆”在本申请中可以与术语“阿尔兹海默症”互换使用。所述早老性痴呆可以包括早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。例如，所述早老性痴呆早期患者的学习与记忆障碍会愈见明显，在某些情况下，会具有语言障碍、执行障碍、认识障碍(失认症)和/或技能执行障碍(失用症)。例如，所述早老性痴呆中期患者将失去独立生活的能力，并且可能无法进行大部分的日常活动(在某些情况下，可以患有命名不能症、言语错乱症、和/或病觉缺失症)。例如，所述早老性痴呆晚期患者可能会晚期依赖照护者。例如，可能完全失去了语言能力。例如，可能无法自行进食。
术语“早期认知障碍(MCI)”通常是指一种介于正常认知与认知障碍的中间临床状态。在某些情况下，所述MCI可以包括满足痴呆标准但程度超过正常衰老的认知损害。MCI在临床表现、病因、预后和患病率方面存在多样化。在某些情况下，MCI可以为阿尔茨海默症的一个病理阶段。某些形式的认知损害可视为神经变性疾病的早期表现，最终将导致痴呆。在某些情况下，所述MCI可以包括选自下组的亚型：aMCI-s：遗忘型MCI单认知域受损；aMCI-m：遗忘型MCI多认知域受损；naMCI-s：非遗忘型MCI单认知域受损；以及naMCI-m：非遗忘型MCI多认知域受损。
术语“正常衰老引起的认知障碍”通常是指由于正常衰老引起的认知损害。例如，所述正 常衰老引起的认知障碍可以表现为：记忆丧失、对熟悉地方的位置感到困惑、完成日常工作需要比平时更长时间、或者情绪和性格的变化。
术语“lews身体痴呆(LBD)”通常是指Lewy Body Detmentia，路易体痴呆。路易体痴呆的特征是蛋白质异常积聚成为称为路易体的肿块。路易体痴呆症导致心智能力逐渐下降。路易体痴呆症患者可能会出现幻视以及警觉性和注意力的变化。其他影响包括肌肉僵硬、运动缓慢、行走困难和颤抖。大脑中有路易体的患者也可以有与阿尔茨海默病相关的斑块和缠结。
术语“额颞叶痴呆”通常是指皮克病，其是一种tau蛋白只影响大脑的额叶和颞叶的渐进性的罕见病。额颞叶痴呆患者在更高层次的推理、表达语言、语言感知和记忆形成方面都有困难。额颞叶痴呆患者的大脑的额叶和颞叶可以随着时间推移而萎缩。
术语“血管性痴呆”通常是指由于大脑血液流动受损而导致的推理、判断和记忆方面的问题。例如，所述血管性痴呆可以包括由于心脏病和中风风险的因素，如高血压和高胆固醇而导致的痴呆。
术语“多发性梗死型”通常是指由大型脑动脉的单个穿通支闭塞引起的非皮质性小梗死。所述多发性梗死型可以是脑梗塞的一种特殊类型，又称缺血性中风。所述多发性梗死型可以表现为偏身感觉障碍、失语、构音不全、动作缓慢、笨拙(尤以精细动作如书写更为困难)。
术语“帕金森氏症”通常是指一种进行性神经变性疾病。所述帕金森氏症(PD)的临床特征可以包括运动症状(例如震颤、运动徐缓、肌强直和姿势不稳)，以及神经精神性及其他非运动表现。例如，所述非运动表现可以包括认知功能障碍和痴呆、心境障碍(例如抑郁、焦虑、情感淡漠)和睡眠障碍。
术语“克雅氏病(CJD)”通常是指一种发生在人类身上的传染性海绵状脑病。CJD是由朊病毒感染引起的疾病。CJD患者可以表现为偏执行为，意识模糊、食欲及体重下降、抑郁，少数患者有视觉或听觉异常；在进展期，表现为进行性的神经系统病情恶化(例如感觉异常、语言障碍和失语)。
术语“多发性硬化(MS)”通常是指一种脱髓鞘性神经病变。所述MS患者脑或脊髓中的神经细胞表面的绝缘物质(即髓鞘)受到破坏，神经系统的信号转导受损，可以导致一系列可能发生的症状，影响患者的活动、心智、甚至精神状态。这些症状可以包括复视、单侧视力受损、肌肉无力、感觉迟钝，或协调障碍。
术语“肌萎缩性脊髓侧索硬化症(ALS)”通常是指渐冻人症、运动神经元病，是一种渐进且致命的神经退行性疾病。其中少数ALS患者可以出现额颞叶痴呆。部分ALS患者的觉、 视觉、触觉、嗅觉和味觉会出现退化，极少数肌萎缩侧索硬化症患者会同时出现痴呆症。
术语“亨廷顿病(HD)”，即亨廷顿舞蹈症，通常是指一种会导致脑细胞死亡的遗传性疾病。HD患者随着疾病的进展，身体运动的不协调变得更加明显，能力逐渐恶化直到运动变得困难，无法说话。心智能力则通常会衰退为痴呆症。
术语“老龄阶段”通常是指受试者的老龄化阶段。例如，对人而言，所述老龄阶段可以为60岁以上，70岁以上或75岁以上；对小鼠而言，所述老龄阶段可以为10月龄以上，例如可以为13月龄以上或者18月龄以上。在某些情况下，所述老龄阶段的受试者可以具有学习缺陷、记忆力障碍、记忆力缺陷和/或脑功能障碍中的一种或多种症状。
术语“调控剂”通常是指改变分子表达量和/或活性的化合物。例如，调控剂可以包括这样的化合物，其使得分子的某种活性强度和/或表达量与没有该调控剂时的活性强度和/或表达量相比增大或下降。例如，所述调控剂可以包括抑制剂，其降低分子的一种或多种活性的强度和/或表达量。
术语“神经元”通常是指神经细胞，其是神经系统主要的功能单元。神经元可以由细胞体及其突起轴突以及一个或多个树突组成。神经元可以通过在突触处释放神经递质向其他神经元或细胞传递信息。
术语“兴奋性突触后电流(EPSC)”通常是指引起兴奋性突触后电位(excitatory postsynaptic potential，EPSP)的离子流。所述EPSP是突触后电位，使突触后神经元更容易触发一个动作电位。这种由带正电荷的离子流入突触后细胞引起的突触后膜电位的暂时去极化是打开配体门控离子通道的结果。所述EPSC的频率和/或振幅可以用电压夹具记录。
术语“认知能力”通常是指通过思想、经验和感觉获得知识和理解的心理行为的能力。所述认知的概念可以不限于心理概念/领域，例如可以包括执行功能、记忆、感知、注意力、情绪、运动控制和/或干扰处理。
术语“Aβ”通常是指β分泌酶介导β淀粉样前体蛋白(APP)裂解所得到的任何的肽。例如，所述Aβ可以包括37、38、39、40、41、42以及43个氨基酸的肽，并且从β分泌酶裂解位点延长至氨基酸37、38、39、40、41、42或43。所述Aβ还可以上述肽的N-末端截短类型，诸如焦谷氨酸形式pE3-40、pE3-42、pE3-43、pE11-42、pE11-43及类似形式。
术语“Tau”通常是指与神经细胞中微管的稳定化有关的Tau蛋白及广泛的Tau聚集体(例如，神经原纤维缠结)的组份。所述Tau缠结可以包括Tau的寡聚体形式和/或纤维形式，其具有毒性。所述Tau还可以包括所有类型和形式(例如不同的选择性剪接形式)的Tau。
术语“小分子化合物”通常是指分子量低于约5000道尔顿(Da)的任何化学部分。在本 申请中，所述小分子化合物可以包括合成的或在自然界发现的有机或无机分子。所述小分子化合物可以包括非肽、非低聚有机化合物。所述小分子化合物可以包括，肽，肽类似物(peptidomimetic)，氨基酸，氨基酸类似物，多核苷酸，多核苷酸类似物，适体，核苷酸，和/或核苷酸类似物。
术语“聚合物”通常是指由通过共价化学键连接的重复结构单元组成的分子。通常所述聚合物的特征在于重复单元的大的数量(例如，等于或大于10个重复单元，等于或大于50个重复单元，或者通常等于或大于100个重复单元)以及高的分子重量(大于或者等于50,000Da)。所述聚合物可以包括无规共聚物、嵌段共聚物、交替共聚物、多嵌段共聚物、接枝共聚物、和/或递变(tapered)共聚物。
术语“生物大分子”通常是指发生在生命物质和有机体中的大分子，例如核酸类、蛋白质类、肽类、糖类、多糖类、脂质类，和脂肪类。它们各自的单/低聚物可以是相应的以其各自的单/低聚物形式的核苷酸类、肽类、氨基酸类、糖类、脂肪酸类。在本申请中，所述生物大分子可以包括蛋白质，和/或核酸。例如，所述生物大分子可以包括抗体。
在本申请中，术语“分离的”通常是指从天然状态下经人工手段获得的。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽可以称之为分离的。术语“分离的”可以不排除混有人工或合成的物质，也可以不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“分离的抗原结合蛋白”通常是指脱离了其天然存在状态的具有抗原结合能力的蛋白。本申请“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的CDR或CDR衍生物的备选蛋白框架区或人工框架区。此类框架可以包括，但不限于包含被引入例如以稳定抗原结合蛋白的三维结构的突变的抗体来源的框架区以及包含例如生物相容性聚合物的完全合成的框架区。抗原结合蛋白的实例可以包括但不限于：人抗体、人源化抗体；嵌合抗体；重组抗体；单链抗体；双功能抗体；三功能抗体；四功能抗体；Fab，Fab’，Fv片段，F(ab’)2，F(ab)2，scFv，di-scFv，dAb，VHH，IgD抗体；IgE抗体；IgM抗体；IgG1抗体；IgG2抗体；IgG3抗体；和/或IgG4抗体以及其片段。
在本申请中，术语“CDR”也称“互补决定区”，通常是指抗体可变结构域中的区域，其序列可以是高度可变的和/或形成结构定义环。例如，抗体可以包括六个CDR；在VH中三个(HCDR1、HCDR2、HCDR3)，和在VL中三个(LCDR1、LCDR2、LCDR3)。在某些实 施方案中，仅由重链组成的天然存在的骆驼抗体在缺乏轻链的情况下，其功能也能够正常且稳定。抗体CDR可以通过多种编码系统来确定，如CCG、Kabat、Chothia、IMGT、综合考虑Kabat/Chothia等。这些编码系统为本领域内已知，具体可参见，例如，www.bioinf.org.uk/abs/index.html#kabatnum。例如，所述抗原结合蛋白的氨基酸序列编号可以按照IMGT编号方案(IMGT,the international ImMunoGeneTics information system@imgt.cines.fr；imgt.cines.fr；Lefranc等,1999,Nucleic Acids Res.27:209-212；Ruiz等,2000 Nucleic Acids Res.28:219-221；Lefranc等,2001,Nucleic Acids Res.29:207-209；Lefranc等,2003,Nucleic Acids Res.31:307-310；Lefranc等,2005,DevComp Immunol 29:185-203)。例如，所述抗原结合蛋白的CDR可以根据Kabat编号系统确定(参见例如Kabat EA&Wu TT(1971)Ann NY AcadSci 190:382-391和Kabat EAet al.,(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)。
在本申请中，术语“FR”通常是指抗体可变结构域的更高度保守的部分，其被称为框架区。例如，天然重链和轻链的可变结构域各自可以包含四个FR区，即在VH中四个(H-FR1，H-FR2，H-FR3和H-FR4)，和在VL中四个(L-FR1，L-FR2，L-FR3和L-FR4)。
在本申请中，术语“可变结构域”与“可变区”可以互换使用，通常是指抗体重链和/或轻链的一部分。重链和轻链的可变结构域可以分别称为“VH”和“VL”(或者分别称为“VH”和“VL”)。这些结构域通常可以是抗体的变化最大的部分(相对于相同类型的其它抗体)，且可以包含抗原结合位点。在本申请中，术语“可变”通常是指在抗体之间可变结构域的某些区段在序列上可能存在较大差异。可变结构域介导抗原可以结合并决定特定抗体对其特定抗原的特异性。然而，可变性可以并非在整个可变结构域范围内均匀分布。它可以通常集中在轻链和重链可变结构域中称为高变区(CDR或HVR)的三个区段中。可变结构域的更高度保守的部分可以称为框架区(FR)。天然重链和轻链的可变结构域各自可以包含四个FR区，大多数采用β-折叠构型，通过三个CDR连接，其形成环形连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDR可以通过FR区紧密靠近地保持在一起，并且来自另一条链的CDR一同促进抗体的抗原结合位点的形成。
在本申请中，术语“抗体”通常是指免疫球蛋白或其片段或其衍生物，涵盖包括抗原结合位点的任何多肽，无论其是在体外还是体内产生的。该术语可以包括但不限于多克隆的、单克隆的、单特异性的、多特异性的、非特异性的、人源化的、单链的、嵌合的、合成的、重组的、杂化的、突变的和移植的抗体。除非另外被术语“完整的”修饰，如在“完整的抗体” 中，为了本申请的目的，术语“抗体”也可以包括抗体片段，比如Fab、F(ab')2、Fv、scFv、Fd、VHH、dAb和保持抗原结合功能(例如，特异性结合人DIR)的其它抗体片段。通常，这样的片段可以包括抗原结合结构域。基本的4链抗体单元可以是由两个相同的轻(L)链和两个相同的重(H)链组成的异四聚体糖蛋白。IgM抗体可以由5个基本的异四聚体单元与另外一个称为J链的多肽组成，且含有10个抗原结合位点，而IgA抗体可以包括2-5个可以与J链相结合聚合形成多价组合的基本4链单元。就IgG而言，4链单元一般可以为约150,000道尔顿。每个L链可以通过一个共价二硫键与H链连接，而两个H链可以通过一个或多个取决于H链同种型的二硫键相互连接。每个H和L链还可以具有规则间隔的链内二硫化桥键。每个H链在N末端可以具有重链可变区(VH)，对于α和γ链各自继之以三个恒定结构域(CH)、对于μ和ε同种型继之以四个CH结构域。每个L链在N末端可以具有轻链可变区(VL)，在其另一端可以具有恒定结构域。VL与VH可以对应，且轻链恒定区(CL)与重链的第一恒定结构域(CH1)可以相对应。特定的氨基酸残基可以被认为在轻链和重链可变结构域之间形成界面。VH和VL可以配对一起形成单个抗原结合位点。来自任何脊椎动物物种的L链可以基于其恒定结构域的氨基酸序列被分为两种明显不同的类型中的一种，称为κ和λ。根据重链恒定区(CH)恒定结构域的氨基酸序列，可以将免疫球蛋白分为不同的类别或同种型。目前存在五类免疫球蛋白：IgA、IgD、IgE、IgG例如IgG1、IgG2、IgG3和/或IgG4，和IgM，具有分别被命名为α、δ、ε、γ和μ的重链。
在本申请中，术语“抗原结合片段”通常是指具有特异结合抗原(例如，DIR)能力的一个或多个片段。在本申请中，所述抗原结合片段可以包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2，scFv，di-scFv，VHH和/或dAb。
在本申请中，术语“Fab”通常是指抗体的抗原结合片段。如上所述，可以使用木瓜蛋白酶消化完整的抗体。抗体经木瓜蛋白酶消化后产生两个相同的抗原结合片段，即“Fab”片段，和残余的“Fc”片段(即Fc区)。Fab片段可以由一条完整的L链与一条重链的可变区和该H链(VH)的第一恒定区(CH1)组成。
在本申请中，术语“F(ab)2”通常是指抗体的抗原结合片段。例如，F(ab)2可以是由两个Fab片段连接的。
在本申请中，术语“Fab′”通常是指人单克隆抗体的单价抗原结合片段，该片段比Fab片段稍大。例如，Fab′片段可以包括所有轻链，所有重链可变区以及重链的所有或部分第一和第二恒定区。例如，Fab′片段还可包括重链的部分或所有的220-330个氨基酸残基。
在本申请中，术语“F(ab')2”通常是指通过胃蛋白酶消化完整抗体所产生的抗体片段。F(ab')2片段含有由二硫键维持在一起的两个Fab片段和部分铰链区。F(ab')2片段具有二价抗原结合活性并且能够交联抗原。
在本申请中，术语“Fv片段”通常是指人单克隆抗体的单价抗原结合片段，包括所有或部分重链可变区和轻链可变区，并且缺乏重链恒定区和轻链恒定区。重链可变区和轻链可变区包括例如CDR。例如，Fv片段包括重链和轻链的约110个氨基酸的所有或部分氨基端可变区。
在本申请中，术语“scFv”通常是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非特别说明，否则如本申请中使用的那样，scFv可以以任何顺序(例如相对于多肽的N末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
在本申请中，术语“dAb”通常是指具有VH域或VL域组成的抗原结合片段，参考例如Ward等人(Nature,1989Oct 12；341(6242)：544-6)，参考Holt等人,Trends Biotechnol.,2003,21(11)：484-490。
在本申请中，术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体(参见Vanlandschoot P.等人，2011，Antiviral Research 92，389-407)。VHH也可称为纳米抗体(Nanobody)(Nb)。
在本申请中，术语“单克隆抗体”通常是指单分子组成的抗体分子制备物。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体可以是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”可以表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，本申请使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的小的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”可以保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可以包含在受体抗体中或在供体抗体中没有的氨基酸修饰。进行这些修饰可以是为了进一步改进抗体的性能，诸如结合亲和力。
在本申请中，术语“全人源抗体”通常是指仅包含人类免疫球蛋白蛋白质序列的抗体。如果其是在小鼠中、在小鼠细胞中或在衍生自小鼠细胞的杂交瘤中生产，那么全人源抗体可能含有鼠糖链。类似地，“鼠源抗体”、“小鼠抗体”或“大鼠抗体”分别指仅包含小鼠或大鼠免疫球蛋白序列的抗体。可通过噬菌体展示或其它分子生物学方法，在人体内、在具有人类免疫球蛋白种系序列的转基因动物体内生成全人源抗体。可用于制造抗体的示例性技术是本领域已知的。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。本申请的“分离的抗原结合蛋白”可以包含结合抗原的部分和任选地，允许抗原结合部分采用促进所述抗原结合部分结合抗原的构象的支架或构架部分。
在本申请中，术语“多肽分子”和“多肽”、“肽”可以互换使用，通常是指氨基酸残基的聚合物。术语“融合蛋白”通常是指具有至少两个共价连接在一起的部分的多肽。其中每个部分可以是具有不同特性的多肽。该特性可以是生物学性质，例如体外或体内活性。该性质也可以是简单的化学或物理性质，例如与靶分子的结合，反应的催化等。这两个部分可以通过单个肽键或通过肽接头直接连接。
在本申请中，术语“核酸分子”通常是指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常是指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可以通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。一种载体可能含有多种控制表达的元件。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分。
在本申请中，术语“细胞”通常是指可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物，其包括本申请所述的核酸分子或本申请所述的载体。细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。细胞可包括用本申请所述的载体在体外转染的细胞。
在本申请中，术语“免疫缀合物”通常是指所述其他试剂(例如，化疗剂、放射性元素、细胞生长抑制剂和细胞毒性剂)与所述抗体或其抗原结合片段缀合(例如，通过连接分子共价相连)而形成的缀合物，该缀合物可以通过所述抗体或其抗原结合片段与靶细胞上的抗原特异性结合，将所述其他试剂递送至靶细胞(例如，肿瘤细胞)。
在本申请中，术语“药物组合物”通常是指用于预防/治疗疾病或病症的组合物。所述药物组合物可以包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。此外，所述药物组合物还可以包含一种或多种(药学上有效的)载剂等合适的制剂。组合物的可接受成分在所用剂量和浓度下可以对接受者无毒。本申请的药物组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“药学上可接受的载剂”通常是指药剂学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒的。生理学可接受的载体可包括合适的物质。指药剂学可接受的载体(carrier)与基因工程中用于插入核酸的载体(vector)通常并不是同一物质。
在本申请中，术语“特异性结合”或“特异性的”通常是指可测量的和可再现的相互作用，例如靶标和抗体之间的结合，可在分子(包括生物分子)的异质群体存在的情况决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体可以是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体。在某些实施方案中， 抗体特异性结合蛋白质上的表位，所述表位在不同种属的蛋白质中是保守的。在某些实施方案中，特异性结合可以包括但不要求排他性地结合。
在本申请中，术语“受试者”通常是指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，所述变体可以为，例如在所述蛋白质和/或所述多肽(例如，特异性结合DIR的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述功能性变体可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的蛋白质或多肽。所述功能性变体可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述功能性变体可保持改变之前的所述蛋白质或所述多肽的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。例如，所述变体也可以为涵盖其功能活性片段的多肽，不限于在细胞中发生的加工和/或修饰后产生的包含所述蛋白功能活性片段的多肽。
在本申请中，所述同源物可以为与所述蛋白质和/或所述多肽(例如，特异性结合DIR的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435- 1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，术语“反义寡核苷酸”是指单链寡核苷酸分子，其具有与靶核酸(例如，目标基因组序列，mRNA前体，或mRNA分子)的相应片段互补的核碱基序列。在某些实施方案中，反义寡核苷酸的长度为12至30个核碱基。在某些实施方案中，反义寡核苷酸是具有与靶核酸(如DIR mRNA)序列互补的核苷酸序列的未经修饰或经修饰的核酸。
在本申请中，术语“dsRNA”指核糖核酸分子的复合物，其具有包含两个反向平行且基本上互补的核酸链的双链体结构，称为具有相对于靶RNA(例如DIR基因)的“正义”和“反义”取向。在本申请的一些实施方式中，双链RNA(dsRNA)通过转录后基因沉默机制(本申请中称为RNA干扰或RNAi)触发靶RNA(例如，mRNA)的降解。双链体结构可为容许所需的靶RNA通过RISC途径的特异性降解的任何长度，例如可在约19至36碱基对的长度范围内，例如，约19-30碱基对的长度，例如，约19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或36碱基对的长度。上述范围与长度中间的范围与长度也包括为本申请的部分。
在本申请中，术语“短干扰RNA(siRNA)”是指干扰基因表达的小的双链RNAs。siRNAs是RNA干扰(双链RNA沉默同源基因的过程)的传递者。siRNAs通常包含两条长为约15-25个核苷酸的单链RNA，其形成双链体，其可以包含单链突出。通过酶复合体例如，聚合酶对双链RNA的加工，产生双链RNA的切割，从而产生siRNAs。RNA干扰(RNAi)沉默复合体使用siRNA的反义链引导mRNA切割，从而促进mRNA降解。为了使用siRNAs例如，在哺乳动物细胞中沉默特定基因，选择碱基对区域以避免对不相关mRNA的机会性互补。在本领域中已经比如，例如，由Fire等人，Nature391：806-81(1998)和McManus等人，Nat.Rev.Genet.3(10)：737-747(2002)鉴定了RNAi沉默复合体。
在本申请中，术语“反义链”通常是指siRNA的包括与靶序列实质上互补的区域的链。在本申请中使用时，术语“互补性区域”通常指反义链上与本申请定义的序列(例如靶序列)实质上互补的区域。当互补性区域与靶序列不完全互补时，错配可以在分子的内部或末端区域。通常，最被容许的错配在末端区域，例如，在5’末端和/或3’末端的5、4、3或2个核苷酸内。
在本申请中，术语“正义链”通常是指siRNA的这样一条链，所述链包括与作为在此定义的术语反义链的区域基本互补的区域。“正义”链有时被称为“有义”链，“过客”链或 “反引导”链。借助它们的序列，反义链靶向所希望的mRNA，同时正义链靶向不同靶标。因此，如果反义链被掺入RISC中，则正确的靶标被靶向。正义链的掺入可以导致脱靶效应。这些脱靶效应可以通过在正义链上使用修饰或使用5’端帽加以限制。
在本申请中，术语“互补”当用于描述就第二核苷酸序列(如反义链)而言的第一核苷酸序列(如正义链或靶mRNA)时是指包含第一核苷酸序列的寡核苷酸或多核苷酸在一定条件下与包含第二核苷酸序列的寡核苷酸或多核苷酸杂交(形成碱基对氢键)并形成双链体或双螺旋结构的能力。互补序列包括沃森-克里克碱基对(Watson-Crick base pairs)或非沃森-克里克碱基对，并且包括天然或经修饰的核苷酸或核苷酸模拟物，只要以上关于它们的杂交能力而言的需求得以实现。“互补”不必需在每个核苷上均具有核碱基互补性。相反，可以容忍一些错配。
在本申请中，术语“靶核酸”或“靶序列”通常指在编码DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物的基因(DDILT4基因)转录期间所形成mRNA分子的核苷酸序列的连续部分，包括作为主要转录产物的RNA加工产物的mRNA。序列的靶部分应至少足够长以作为在DDILT4基因转录期间形成的mRNA分子的核苷酸序列的该部分位置处或附近反义寡核苷酸或siRNA指导的切割的底物。在一个实施方式中，该靶序列在DIR和/或其功能片段的蛋白质编码区内。
在本申请中，术语“包含”通常是指包括、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
术语“约”通常是指在比特定值多或少20％的数值范围。例如，“约X”包括为X的±20％、±10％、±5％、±2％、±1％、±0.5％、±0.2％或±0.1％的数值范围，其中X为数值。
发明详述
一方面，本申请提供一种调控剂，其调控DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段，在制备用于预防和/或治疗疾病的试剂中的用途，其中所述疾病包括认知障碍。
一方面，本申请提供一种调控剂，其调控DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段，在制备用于预防和/或治疗疾病的试剂中的用途，其中所述疾病包括神经退行性疾病。
另一方面，本申请提供一种预防和/或治疗认知障碍的方法，其包括以下的步骤：使有需要的受试者中，DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的表达水平和/或生物学活性降低。
另一方面，本申请提供一种预防和/或治疗神经退行性疾病的方法，其包括以下的步骤：使有需要的受试者中，DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的表达水平和/或生物学活性降低。
DIR与其功能片段
人DDIT4L异常剪切产生的一种内含子滞留形式(DIR)。通过序列比对，发现该核酸序列在灵长类中具有保守性，其中与人类亲缘关系较近的黑猩猩具有高度保守性，而在猕猴中的保守性则相对较差。但是在大鼠、小鼠、狗、猪等常用的实验动物中无保守性。
在本申请中，所述功能片段通常是指包含与亲本或参考多肽(例如DIR)的氨基酸序列相差至少一个氨基酸残基的氨基酸序列的多肽。在本申请中，所述功能片段可以与所述亲本或参考多肽具有较高的(例如至少80％)的同源性。所述同源性可以包括序列的相似性或同一性。在本申请中，所述同源性可以用本领域已知的标准技术确定(参见例如Smith和Waterman，Adv.Appl.Math.《应用数学进展》)；多核苷酸或多肽序列共有的同一性百分数通过分子之间序列信息的直接比较测定，所述比较通过序列比对并使用本领域已知的方法确定同一性来进行。适用于确定序列相似性的算法的例子是BLAST算法(参见Altschul等人，J.Mol.Biol.《分子生物学杂志》，215:403-410[1990])。用于进行BLAST分析的软件可通过国家生物技术信息中心(NCBI)公开获得。
在本申请中，所述DIR的表达水平可以包括DIR基因的表达水平、DIR基因的转录水平和/或DIR蛋白的表达水平。例如，所述表达水平可以包括特定基因(例如人DDIT4L基因；和/或，编码人DIR和/或其功能片段(例如DIR-I，和/或，DIR-II)的基因(例如人DIR基因))多核苷酸、mRNA或氨基酸产物或蛋白质的量。所述表达水平可以包括特定基因转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的量。在本申请中，QDLIR可以与DIR替换使用，其可以为人DDIT4L基因表达时异常剪切产生的一种内含子滞留形式。在本申请中，编码所述DIR的基因可以称为DIR基因。
在本申请中，所述DIR的功能片段(例如DIR-I，和/或，DIR-II)的表达水平可以包括编码DIR的功能片段基因的表达水平、编码DIR的功能片段基因的转录水平和/或DIR的功能片段蛋白的表达水平。例如，所述表达水平可以包括特定基因(例如编码人DIR的功能片段(例如DIR-I，和/或，DIR-II)的基因)的多核苷酸、mRNA或氨基酸产物或蛋白质的量。所述表达水平可以包括特定基因(例如编码人DIR的功能片段(例如DIR-I，和/或，DIR-II)的基因)转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的量。
在本申请中，所述DIR-I可以为IR(即滞留的内含子所编码的氨基酸序列，其氨基酸序 列如SEQ ID NO.3所示)由自N端起前27个氨基酸组成的氨基酸序列。DIR-I的氨基酸序列如SEQ ID NO.4所示。所述DIR-II可以为IR由自C端起后27个氨基酸组成的氨基酸序列。DIR-II的氨基酸序列如SEQ ID NO.5所示。
在本申请中，所述降低可以包括与所述受试者中原始DIR的表达水平相比，所述DIR的表达水平降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述DIR的表达水平可以通过利用选自下组的物质衡量：特异性扩增DIR基因的引物、与DIR基因特异性结合的核酸分子、与DIR蛋白特异性结合的核酸分子、与DIR蛋白特异性结合的小分子、与DIR蛋白特异性结合的探针和与DIR蛋白特异性结合的多肽。
在本申请中，所述DIR的功能片段(例如DIR-I，和/或，DIR-II)的表达水平可以通过利用选自下组的物质衡量：特异性扩增DIR的功能片段基因的引物、与DIR的功能片段基因特异性结合的核酸分子、与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的核酸分子、与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的小分子、与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的探针和与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的多肽。
在本申请中，所述DIR和/或其功能片段的表达水平可以通过实施选自下组的试验衡量：逆转录和扩增分析(例如PCR、连接RT-PCR或定量RT-PCT)、杂交分析、RNA印迹法(Northern blotting)、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色或质谱。例如，可以通过qPCR、qRT-PCR、northern杂交、western杂交和/或ELISA检测对本申请所述的DIR的表达水平进行测量。所述DIR的表达水平也可以通过直接对生物样品或对从样品中分离出来的蛋白质/核酸执行分析来进行测量。
在本申请中，所述DIR和/或其功能片段的活性可以包括DIR蛋白的生物活性。例如，所述生物学活性可以包括影响神经元的兴奋性和/或抑制神经元的活性。例如，所述生物学活性可以包括通过抑制神经元的兴奋性和/或抑制神经元的活性而抑制认知能力。
在本申请中，所述生物学活性可以包括能够降低兴奋性突触后电流(EPSC)的频率，和/或能够降低EPSC的幅度。例如，所述降低可以包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其 功能片段的核酸，可以使受试者中的兴奋性突触后电流(EPSC)的频率降低，和/或使受试者中的EPSC的幅度降低。
例如，所述DIR的功能片段DIR-I可以降低EPSC的频率。例如，所述降低可以包括与所述受试者中原始EPSC的频率相比，施用所述DIR-I后，EPSC的频率降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。例如，所述DIR的功能片段DIR-II可以降低EPSC的频率。例如，所述降低可以包括与所述受试者中原始EPSC的幅度相比，施用所述DIR-II后，EPSC的幅度降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述生物学活性可以包括影响认知能力。在本申请中，所述生物学活性可以包括参与与Aβ沉积相关的信号通路，和/或，参与与Tau缠结产生相关的信号通路。例如，所述DIR和/或其功能片段可以抑制认知能力。例如，所述DIR和/或其功能片段可以通过抑制与Aβ沉积相关的信号通路，和/或，抑制与Tau缠结产生相关的信号通路而抑制认知能力。
在本申请中，所述DIR和/或其功能片段的活性的降低可以包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者的认知能力降低。
在本申请中，所述降低可以包括与所述受试者中原始DIR的生物学活性相比，所述DIR的生物学活性降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述DIR和/或其功能片段的表达水平(例如在血浆中的表达水平)可以与Aβ水平(例如在血浆中的表达水平)正相关。例如，所述DIR和/或其功能片段的表达水平可以与Aβ40的表达水平正相关。例如，所述DIR和/或其功能片段的表达水平可以与Aβ42的表达水平正相关。
在本申请中，所述DIR和/或其功能片段的表达水平(例如在血浆中的表达水平)可以 与受试者的认知能力的降低程度正相关。例如，所述DIR和/或其功能片段的表达水平能够随着认知障碍的进展(例如MCI和/或AD的疾病进展)而提高。
在本申请中，所述DIR和/或其功能片段的表达水平(例如在血浆中的表达水平)可以与Aβ水平(例如在皮质的Aβ-PET表达水平)正相关。在本申请中，所述DIR和/或其功能片段的表达水平可以与AD患者的淀粉样斑块形成相关。
在本申请中，所述DIR和/或其功能片段的表达水平可以与陈述性记忆的存储相关。例如，所述DIR和/或其功能片段的表达水平越高，陈述性记忆的存储的能力越低。在本申请中，所述DIR和/或其功能片段的表达水平可以与联想学习的存储相关。例如，所述DIR和/或其功能片段的表达水平越高，联想学习的存储的能力越低。
在本申请中，所述DIR和/或其功能片段的表达水平的降低可以提高认知能力。在本申请中，可以通过基因编辑的方法降低所述DIR和/或其功能片段的表达水平。例如，所述基因编辑的方法可以包括knock-down(例如，可以借助CRISPR/Cas系统；例如，可以借助反义寡核苷酸)。在本申请中，所述表达水平的降低可以包括与所述受试者中原始DIR的表达水平相比，所述DIR的表达水平降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述DIR和/或其功能片段的表达水平越低，认知能力(例如通过新物体识别行为实验衡量的认知能力)越高。例如，当所述表达水平的降低为与所述受试者中原始DIR的表达水平相比，所述DIR的表达水平降低了至少约10％时，所述认知能力可以提高至少约10％。例如，可以提高至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述DIR/或其功能片段可以来源于哺乳动物。例如，可以来源于灵长类动物。例如，可以来源于人。
在本申请中，所述DIR可以包含SEQ ID NO.1所示的氨基酸序列。
在本申请中，所述DIR的功能片段可以包含DDIT4L中滞留的内含子所编码的氨基酸序列。在本申请中，所述DIR的功能片段可以包含SEQ ID NO.3所示的氨基酸序列。
在本申请中，所述DIR的功能片段可以包含SEQ ID NO.4-5中任一项所示的氨基酸序列。
在本申请中，所述DIR/或其功能片段可以参与Aβ沉积。例如，所述DIR/或其功能片段可以通过gelsolin诱导Aβ沉积。
其中，DDIT4L中滞留的内含子(即DIR-内含子(IR)，其氨基酸序列如SEQ ID NO.3所示)可以为DIR与Aβ相互作用的主要区域。Aβ可以有助于DIR与gelsolin之间的相互作用。在本申请中，所述DIR-内含子可以参与Aβ沉积。
在本申请中，所述DIR/或其功能片段能够在病理条件下通过与gelsolin结合，而导致Aβ沉积和淀粉样斑块形成。
受试者和适应症
在本申请中，所述受试者可以包括哺乳动物。例如，所述受试者可以包括啮齿类动物和/或灵长类动物，例如所述受试者可以包括人。
在本申请中，所述受试者可以包括认知障碍患者和/或神经退行性疾病患者。
在本申请中，所述神经退行性疾病可以包括急性神经退行性疾病和慢性神经退行性疾病。例如，所述神经退行性疾病可以包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在本申请中，所述受试者可以包括神经退行性疾病患者。例如，所述受试者可以包括阿尔兹海默症患者。例如所述阿尔兹海默症患者可以处于阿尔兹海默症的前期、早期、中期或晚期。
在本申请中，所述认知障碍可以包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。例如，所述认知障碍可以包括正常衰老引起的认知障碍、lewy身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。例如，所述认知障碍的诱导疾病可以包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。在本申请中，所述认知障碍可以包括遗忘型MCI多认知域受损(aMCI-m)。
在本申请中，所述受试者可以包括认知障碍患者。例如，所述受试者可以患有早期认知障碍(MCI)(例如，失去短期记忆、表达或理解抽象事情时感困难、情绪或行为变幻无常、学习新事物及跟随复杂指令感到困难、判断力减退和/或基本自理需要旁人提醒)、中期认知障碍(例如，混淆远期记忆和现实情况记忆、词不达意、行为性格转变或情绪不稳和/或需要旁人协助自理)或晚期认知障碍(例如，记忆缺损、身体活动及精神状况出现衰退、不能有效表达或沟通、不能自理和/或生物钟混乱)。在本申请中，所述受试者可以患有能够引起诱导所述认知障碍的疾病。例如，所述受试者可以患有阿尔兹海默症、多发性梗死型、帕金 森氏症、艾滋病和/或克雅氏病(CJD)。
在本申请中，所述受试者可以处于老龄阶段。例如，所述受试者已表现出了正常衰老引起的认知障碍。例如，所述受试者已表现出了早期认知障碍(MCI)的症状。例如，所述受试者已表现出了神经退行性疾病(例如阿尔兹海默症)的症状。
在本申请中，所述受试者可以患有早老性痴呆。例如，所述受试者可以处于早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。
调控剂
本申请提供了一种可以降低所述DIR和/或其功能片段，和/或编码所述DIR和/或其功能片段的核酸的表达水平和/或生物学活性的调控剂。
在本申请中，所述调控剂可以降低所述DIR和/或其功能片段，和/或编码所述DIR和/或其功能片段的核酸的表达水平和/或生物学活性。
在本申请中，所述调控剂可以降低DIR基因的表达水平、DIR基因的转录水平和/或DIR蛋白的表达水平。例如，所述调控剂可以降低例如人DDIT4L基因；和/或，编码人DIR和/或其功能片段(例如DIR-I，和/或，DIR-II)的基因(例如人DIR基因)多核苷酸、mRNA或氨基酸产物或蛋白质的量。例如，所述调控剂可以降低例如人DDIT4L基因；和/或，编码人DIR和/或其功能片段(例如DIR-I，和/或，DIR-II)的基因(例如人DIR基因)基因转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的量。
在本申请中，所述调控剂可以降低编码DIR的功能片段基因的表达水平、编码DIR的功能片段基因的转录水平和/或DIR的功能片段蛋白的表达水平。例如，所述调控剂可以降低编码人DIR的功能片段(例如DIR-I，和/或，DIR-II)的基因)的多核苷酸、mRNA或氨基酸产物或蛋白质的量。例如，所述调控剂可以降低编码人DIR的功能片段(例如DIR-I，和/或，DIR-II)的基因转录的多核苷酸、翻译的蛋白质或翻译后修饰的蛋白质的片段的量。
在本申请中，所述降低可以包括与所述受试者中原始DIR的表达水平相比，所述DIR的表达水平降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述降低可以包括与所述受试者中原始DIR的表达水平相比，所述DIR的功能片段的表达水平降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至 少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述调控剂可以降低所述DIR和/或其功能片段的生物学活性。在本申请中，所述降低可以包括与所述受试者中原始DIR的生物学活性相比，所述DIR或其功能片段的生物学降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述调控剂可以抑制与Aβ沉积相关的信号通路。在本申请中，所述调控剂可以抑制与Tau缠结产生相关的信号通路。在本申请中，所述抑制可以包括与所述受试者中原始参与与Aβ沉积相关的信号通路和/或与Tau缠结产生相关的信号通路的分子的表达水平和/或生物学活性相比，经施用所述调控剂后，这些分子的表达水平和/或生物学活性降低了至少约10％。例如，可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。在本申请中，所述调控剂可以通过gelsolin抑制Aβ沉积和/或淀粉样斑块形成。例如，所述调控剂可以通过抑制所述DIR/或其功能片段与gelsolin的结合，从而抑制Aβ沉积和/或淀粉样斑块形成。
在本申请中，所述调控剂可以降低DIR-II与gelsolin的结合。例如，经施用所述调控剂后，DIR-II与gelsolin的结合水平可以降低至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约100％、至少约200％、至少约500％或更多。
在本申请中，所述调控剂可以包括所述DIR和/或其功能片段的抑制剂。
在本申请中，所述调控剂可以包括小分子化合物、聚合物和/或生物大分子。在本申请中，所述调控剂可以包括抗体或其抗原结合片段。在本申请中，所述调控剂可以包括特异性结合DIR和/或其功能片段的抗体或其抗原结合片段。在本申请中，所述调控剂可以包括靶向靶向DIR-II的抗体或其抗原结合片段。
在本申请中，所述调控剂可以包括反义寡核苷酸。在某些实施方式中，其中所述反义寡核苷酸包含与编码DIR和/或其功能片段的mRNA的至少一部分基本上互补或完全互补的互 补区域。
在本申请中，所述互补区域的长度可以小于19个核苷酸。
在本申请中，所述调控剂可以包括dsRNA。在本申请中，所述调控剂可以包括siRNA。
在本申请中，所述调控剂可以包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的siRNA。
本申请所述siRNA可进一步包括一个或多个单链核苷酸突出端，例如，1、2或3个核苷酸。该突出端可在正义链、反义链或其任何组合上。此外，突出端的核苷酸可存在于siRNA的反义或正义链的5’-端、3’-端或两端。该突出端可由一条链长于另一条链所造成，或由相同长度的两条链交错造成。该突出端可与靶mRNA形成错配或其可与所靶向的基因序列互补或可为另一个序列。
在本申请中，所述siRNA可以包含正义链和反义链，所述反义链可以包含与编码DIR和/或其功能片段的mRNA的至少一部分基本上互补的互补区域，且反义链可以包含选自以下序列中任意一个相差不超过3个核苷酸的至少15、16、17、18或19个连续核苷酸：SEQ IDNO:xx；所述正义链和反义链可以互补共同形成长度介于17到21个核苷酸的互补区域。
在本申请中，所述siRNA可以包含正义链和反义链，所述反义链可以包含与编码DIR和/或其功能片段的mRNA的至少一部分基本上互补的互补区域，且反义链可以包含选自以下序列中任意一个相差不超过3个核苷酸：SEQ ID NO:85、87；所述正义链和反义链可以互补共同形成长度介于17到21个核苷酸的互补区域。
在本申请中，所述siRNA可以包含正义链和反义链，所述反义链可以包含与编码DIR和/或其功能片段的mRNA的至少一部分基本上互补的互补区域，且所述反义链可以包含SEQID NO.85、87中任一项所示的核苷酸序列；所述正义链和反义链可以互补共同形成长度介于17到21个核苷酸的互补区域。
在本申请中，所述siRNA可以包含正义链和反义链，所述正义链可以包含SEQ ID NO.84、86中任一项所示的核苷酸序列，且所述反义链可以包含SEQ ID NO.85、87中任一项所示的核苷酸序列。
在本申请中，所述siRNA的正义链的3'端和/或所述siRNA的反义链的3'端可以连接至少一个(例如2个)核苷酸。例如，所连接的核苷酸可以与靶基因一致。又例如，所连接的核苷酸可以为T，例如，可以为TT。例如，所述正义链和反义链可以选自下述任意一种或多种组合：
包含SEQ ID NO:84所示核苷酸序列的正义链，和包含SEQ ID NO:85所示核苷酸序列 的反义链；以及，包含SEQ ID NO:86所示核苷酸序列的正义链，和包含SEQ ID NO:87所示核苷酸序列的反义链；
在本申请中，每个链的长度可以介于17和23个核苷酸之间。
在本申请中，每条链中不超过3个核苷酸可以分别被其它核苷酸所替代，同时基本保持了抑制被转染的细胞中DIR和/或其功能片段的表达能力。
在本申请中，所述siRNA可以包含至少一个修饰的核苷酸。
在本申请中，所述正义链和/或反义链上的一个或多个核苷酸可以被修饰以形成修饰的核苷酸。
在本申请中，所述正义链的所有核苷酸和所述反义链的所有核苷酸可以包含修饰。
可通过本领域熟知的转染方法将本申请所述的反义寡核苷酸或siRNA引入脑神经细胞或者胶质细胞。这些方法包括超声处理、电脉冲、电穿孔、渗透压冲击、磷酸钙沉淀和DEAE葡聚糖转染、脂质介导的递送、被动递送等。术语“转染”包括可用于将核酸引入哺乳动物细胞的多种技术，包括电穿孔、磷酸钙沉淀、DEAE-葡聚糖处理、脂质转染、显微注射和/或病毒感染。
另一方面，本申请还提供了所述的调控剂(例如本申请所述的反义寡核苷酸或siRNA)在制备预防和/或治疗疾病或病症的药物中的用途，其中所述疾病或病症包括认知障碍和/或神经退行性疾病。
另一方面，本申请还提供了所述的调控剂(例如本申请所述的反义寡核苷酸或siRNA)，其用于备预防和/或治疗认知障碍和/或神经退行性疾病。
另一方面，本申请还提供了一种预防和/或治疗认知障碍和/或神经退行性疾病的方法，其包括以下步骤：向有需要的受试者施用所述的调控剂(例如本申请所述的反义寡核苷酸或siRNA)。
在本申请中，所述调控剂可以被配制为适于口服施用和/或注射施用。
药物筛选方法/系统
另一方面，本申请提供一种筛选能够预防和/或治疗认知障碍和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中DIR和/或其功能片段的表达水平和/或生物学活性的影响，其中在施用所述候选药物后，所述DIR和/或其功能片段的表达水平和/或生物学活性降低，则所述候选药物能够预防和/或治疗认知障碍和/或治疗神经退行性疾病。
在本申请中，所述候选药物可以包含本申请所述的调控剂。例如，所述候选药物可以包 括所述DIR和/或其功能片段的抑制剂。所述候选药物可以包括小分子化合物、聚合物和/或生物大分子。
在本申请中，所述候选药物可以被配制为适于口服施用和/或注射施用。
本申请还提供了一种用于筛选能够预防和/或治疗认知障碍和/或治疗神经退行性疾病的药物的系统，其可以包括检测模块。所述检测模块可以检测候选药物对受试者中DIR和/或其功能片段的表达水平和/或生物学活性的影响，其中在施用所述候选药物后，所述DIR和/或其功能片段的表达水平和/或生物学活性降低，则所述候选药物能够预防和/或治疗认知障碍和/或治疗神经退行性疾病。
所述检测模块可以包括能够检测受试者中DIR和/或其功能片段的表达水平和/或生物学活性的仪器和/或试剂。
例如，所述检测模块可以包括特异性扩增DIR基因的引物、与DIR基因特异性结合的核酸分子、与DIR蛋白特异性结合的核酸分子、与DIR蛋白特异性结合的小分子、与DIR蛋白特异性结合的探针和/或与DIR蛋白特异性结合的多肽；和/或，特异性扩增DIR的功能片段基因的引物、与DIR的功能片段基因特异性结合的核酸分子、与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的核酸分子、与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的小分子、与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的探针和与DIR的功能片段(例如DIR-I，和/或，DIR-II)特异性结合的多肽。
例如，所述检测模块可以包括特异性检测Aβ沉积和/或淀粉样斑块形成的仪器和/或试剂。例如，所述检测模块可以包括能够特异性检测Aβ沉积和/或淀粉样斑块形成相关的目标基因(例如Tau基因)的引物、与该目标基因特异性结合的核酸分子、与该目标基因所编码蛋白特异性结合的小分子、与该目标基因所编码蛋白特异性结合的探针和/或与该目标基因所编码蛋白特异性结合的多肽。
所述检测模块可以输出施用所述候选药物后，所述DIR和/或其功能片段的表达水平和/或生物学活性的测定值(例如，定量值；又例如，与一个或多个阈值比较后的定性值)。
在本申请中，所述系统可以包括判断模块，所述判断模块可以比较所述检测模块输出的施用所述候选药物后，所述DIR和/或其功能片段的表达水平和/或生物学活性的定量值；与受试者中原始DIR和/或其功能片段的表达水平和/或生物学活性的定量值的大小。如果在施用所述候选药物后，所述DIR和/或其功能片段的表达水平和/或生物学活性降低，则所述判断模块能够输出所述候选药物能够预防和/或治疗认知障碍和/或治疗神经退行性疾病的判断结果。
在本申请中，所述系统可以包括显示模块，所述显示模块可以以定量和/或定性的方式显示所述判断模块的判断结果。
在本申请中，所述系统可以包含存储器；和耦接至所述存储器的处理器，所述处理器被配置为基于存储在所述存储器中的指令执行所述检测模块和/或所述判断模块中检测候选药物对受试者中DIR和/或其功能片段的表达水平和/或生物学活性的影响的步骤。
本申请还提供了一种可以检测DIR和/或其功能片段表达水平和/或生物学活性的检测试剂盒。所述检测试剂盒可以包含说明书，其中记载了如何采用所述检测试剂盒对DIR和/或其功能片段的表达水平和/或生物学活性进行检测的具体步骤，和/或如何利用检测结果判断所述候选药物能否预防和/或治疗认知障碍和/或治疗神经退行性疾病受试者的具体步骤。
在本申请中，所述检测试剂盒还可以包括能够检测其他判断所述候选药物能否预防和/或治疗认知障碍和/或治疗神经退行性疾病的靶点的试剂。
抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白。
在本申请中，所述分离的抗原结合蛋白具有以下性质：在ELISA测定中，以约10ng/ml以上的工作浓度与人DIR和/或其功能片段特异性结合。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR2，所述LCDR2可以包含SEQ ID NO:74所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。
X1X2S(SEQ ID NO.74)，其中X1为A或Y，X2为A或Y。
在本申请中，所述LCDR2可以包含SEQ ID NO:26或16所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR1，所述LCDR1可以包含SEQ ID NO:73所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述LCDR1可以包含IR-II-1～IR-II-10的轻链可变区中的LCDR1。
Q X1X2D X3X4X5X6X7Y(SEQ ID NO.73)，其中X1为缺失或S，X2为缺失或V，X3为缺失或Y，X4为缺失或D，X5为G或I，X6为D、E或S，X7为N或S。
在本申请中，所述LCDR1可以包含SEQ ID NO:25、52、15中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3，所述LCDR3可以包含SEQ ID NO:75所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例 如，所述CDR可以由IMGT编号方案来确定。例如，所述LCDR3可以包含IR-II-1～IR-II-10的轻链可变区中的LCDR3。
X1Q X2 X3 X4 X5P X6 X7(SEQ ID NO.75)，其中X1为L或Q，X2为S或Y，X3为N、S或Y，X4为D、E或K，X5为D或L，X6为F或R，X7为A或T。
在本申请中，所述LCDR3可以包含SEQ ID NO:27、53、67、17中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3，LCDR2和LCDR1。例如，所述分离的抗原结合蛋白的LCDR2可以包含SEQ ID NO:74所示的氨基酸序列，LCDR1可以包含SEQ ID NO:73所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:75所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述LCDR1-3可以包含IR-II-1～IR-II-10的轻链可变区中的LCDR1-3。
在本申请中，所述分离的抗原结合蛋白可以包含LCDR3，LCDR2和LCDR1。例如，所述分离的抗原结合蛋白的LCDR1可以包含SEQ ID NO:25所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:27所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的LCDR1可以包含SEQ ID NO:52所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:53所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的LCDR1可以包含SEQ ID NO:52所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:67所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的LCDR1可以包含SEQ ID NO:15所示的氨基酸序列，LCDR2可以包含SEQ ID NO:16所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:17所示的氨基酸序列。
例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述LCDR1-3可以包含IR-II-1～IR-II-10的重链可变区中的LCDR1-3。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR1，所述HCDR1可以包含SEQ ID NO:70所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。 例如，所述CDR可以由IMGT编号方案来确定。例如，所述HCDR1可以包含IR-II-1～IR-II-10的重链可变区中的HCDR1。
GYTFX1X2YX3(SEQ ID NO.70)，其中X1为S或T，X2为E、N、R或S，X3为T或W。
在本申请中，所述HCDR1可以包含SEQ ID NO:20、35、56、10中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR2，所述HCDR2可以包含SEQID NO:71所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述HCDR1可以包含IR-II-1～IR-II-10的重链可变区中的HCDR2。
I X1P X2 X3 X4 X5 X6T(SEQ ID NO.71)，其中X1为L或Y，X2为G或H，X3为G、N或S，X4为缺失或Y、X5为G或Y、X6为G、N、S或T。
在本申请中，所述HCDR2可以包含SEQ ID NO:21、36、42、48、11中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，所述HCDR3可以包含SEQID NO:72所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述HCDR3可以包含IR-II-1～IR-II-10的重链可变区中的HCDR3。
X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15DY(SEQ ID NO.72)，其中X1为缺失或A，X2为缺失或R，X3为缺失、S或T，X4为缺失、D、G或Y，X5为缺失、D、I、M或V，X6为缺失、G或I，X7为缺失、T或Y，X8为缺失、A、L或T，X9为缺失、R、S或T，X10为缺失、G或T，X11为缺失、D或E，X12为D或Y，X13为F、L或Y，X14为A、T或V，X15为F或M。
在本申请中，所述HCDR3可以包含SEQ ID NO:22、30、37、43、49、57、62、12中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，HCDR2和HCDR1。例如，所述分离的抗原结合蛋白的HCDR2可以包含SEQ ID NO:71所示的氨基酸序列，HCDR1可以包含SEQ ID NO:70所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:72所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述HCDR1-3可以包含IR-II-1～IR-II-10的重链可变区 中的HCDR1-3。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:21所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:22所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:21所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:30所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:35所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:37所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:42所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:43所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:48所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:49所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:56所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:57所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:35所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:62所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:35所示的氨基酸序列，HCDR2可以包含SEQ ID NO:42所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:37所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:56所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:57所示的氨基酸序列。
例如，所述分离的抗原结合蛋白的HCDR1可以包含SEQ ID NO:10所示的氨基酸序列，HCDR2可以包含SEQ ID NO:11所示的氨基酸序列，且HCDR3可以包含SEQ ID NO:12所示 的氨基酸序列。
例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述HCDR1-3可以包含IR-II-1～IR-II-10的重链可变区中的HCDR1-3。
在本申请中，所述分离的抗原结合蛋白可以包含HCDR3，HCDR2，HCDR1，LCDR3，LCDR2和LCDR1。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:21所示的氨基酸序列，HCDR3可以包含SEQ ID NO:22所示的氨基酸序列，LCDR1可以包含SEQ ID NO:25所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:27所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:21所示的氨基酸序列，HCDR3可以包含SEQ ID NO:30所示的氨基酸序列，LCDR1可以包含SEQ ID NO:25所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:27所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:35所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，HCDR3可以包含SEQ ID NO:37所示的氨基酸序列，LCDR1可以包含SEQ ID NO:25所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:27所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:42所示的氨基酸序列，HCDR3可以包含SEQ ID NO:43所示的氨基酸序列，LCDR1可以包含SEQ ID NO:25所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:27所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:20所示的氨基酸序列，HCDR2可以包含SEQ ID NO:48所示的氨基酸序列，HCDR3可以包含SEQ ID NO:49所示的氨基酸序列，LCDR1可以包含SEQ ID NO:52所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:53所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:56所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，HCDR3可以包含SEQ ID NO:57所示的氨基酸序列，LCDR1可以包含SEQ ID NO:25所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:27所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:35所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，HCDR3可以包含SEQ ID NO:62所示的氨基酸序列，LCDR1可以包含SEQ ID NO:25所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:27所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:35所示的氨基酸序列，HCDR2可以包含SEQ ID NO:42所示的氨基酸序列，HCDR3可以包含SEQ ID NO:37所示的氨基酸序列，LCDR1可以包含SEQ ID NO:52所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:53所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:56所示的氨基酸序列，HCDR2可以包含SEQ ID NO:36所示的氨基酸序列，HCDR3可以包含SEQ ID NO:57所示的氨基酸序列，LCDR1可以包含SEQ ID NO:52所示的氨基酸序列，LCDR2可以包含SEQ ID NO:26所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:67所示的氨基酸序列。
例如，本申请所述的分离的抗原结合蛋白HCDR1可以包含SEQ ID NO:10所示的氨基酸序列，HCDR2可以包含SEQ ID NO:11所示的氨基酸序列，HCDR3可以包含SEQ ID NO:12所示的氨基酸序列，LCDR1可以包含SEQ ID NO:15所示的氨基酸序列，LCDR2可以包含SEQ ID NO:16所示的氨基酸序列，且LCDR3可以包含SEQ ID NO:17所示的氨基酸序列。
例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。例如，所述CDR可以由IMGT编号方案来确定。例如，所述HCDR1-3可以包含IR-II-1～IR-II-10的重链可变区中的HCDR1-3；且，所述LCDR1-3可以包含IR-II-1～IR-II-10的轻链可变区中的LCDR1-3。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR1，所述H-FR1的C末端可以与所述HCDR1的N末端直接或间接相连。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR2，所述H-FR2可以位于所述HCDR1与所述HCDR2之间。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR3，所述H-FR3可以位于所述HCDR2与所述HCDR3之间。
在本申请中，所述分离的抗原结合蛋白可以包含H-FR4，所述H-FR4的N末端可以与所述HCDR3的C末端相连。
在本申请中，所述抗原结合蛋白可以包含H-FR1，H-FR2，H-FR3和H-FR4。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，所述L-FR1的C末端可以与所 述LCDR1的N末端直接或间接相连。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，所述L-FR1的C末端可以与所述LCDR1的N末端直接或间接相连。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR2，所述L-FR2可以位于所述LCDR1与所述LCDR2之间。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR3，所述L-FR3可以位于所述LCDR2与所述LCDR3之间。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR4，所述L-FR4的N末端可以与所述LCDR3的C末端相连。
在本申请中，所述分离的抗原结合蛋白可以包含L-FR1，L-FR2，L-FR3和L-FR4。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH，所述VH可以包含SEQ ID NO:13、23、31、38、44、50、58、63、65中任一项所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。
在本申请中，所述分离的抗原结合蛋白可包含轻链可变区VL，所述VL可以包含SEQ ID NO:18、28、33、40、46、54、60、68中任一项所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。
在本申请中，所述分离的抗原结合蛋白可包含所述VH和VL。在某些实施方式中，所述VH可以包含SEQ ID NO:23所示的氨基酸序列，且所述VL可以包含SEQ ID NO:28所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:31所示的氨基酸序列，且所述VL可以包含SEQ ID NO:33所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:38所示的氨基酸序列，且所述VL可以包含SEQ ID NO:40所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:44所示的氨基酸序列，且所述VL可以包含SEQ ID NO:46所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:50所示的氨基酸序列，且所述VL可以包含SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:58所示的氨基酸序列，且所述VL可以包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:63所示的氨基酸序列，且所述VL可 以包含SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:65所示的氨基酸序列，且所述VL可以包含SEQ ID NO:54所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:58所示的氨基酸序列，且所述VL可以包含SEQ ID NO:68所示的氨基酸序列。
在某些实施方式中，所述VH可以包含SEQ ID NO:13所示的氨基酸序列，且所述VL可以包含SEQ ID NO:18所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。
在本申请中，所述分离的抗原结合蛋白可以包含本申请所述的VH中的至少一个CDR。在本申请中，所述分离的抗原结合蛋白可以包含本申请所述的VL中的至少一个CDR。所述CDR可以根据任何划分方式划分得到。在本申请中，所述CDR可以涵盖根据任何CDR划分方式划分得到的CDR序列；也可以涵盖其变体。
在本申请中，所述分离的抗原结合蛋白可以包含本申请所述VH中的HCDR1，HCDR2和HCDR3。
在本申请中，所述分离的抗原结合蛋白可以包含本申请所述VH中的HCDR1，HCDR2和HCDR3。所述VH可以包含SEQ ID NO:13、23、31、38、44、50、58、63、65中任一项所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。在本申请中，所述CDR可以涵盖根据任何CDR划分方式划分得到的CDR序列；也可以涵盖其变体。
在本申请中，所述分离的抗原结合蛋白可以包含本申请所述VL中的LCDR1，LCDR2和LCDR3。所述VL可以包含SEQ ID NO:18、28、33、40、46、54、60、68中任一项所示的氨基酸序列。例如，本申请的分离的抗原结合蛋白可以具有DIR结合能力。在本申请中，所述CDR可以涵盖根据任何CDR划分方式划分得到的CDR序列；也可以涵盖其变体。
在本申请中，所述分离的抗原结合蛋白可以包括抗体重链恒定区。所述抗体重链恒定区可以源自人IgG、IgA、IgD、IgE和/或IgM重链恒定区。所述抗体重链恒定区可以源自人IgG重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG1、IgG2、IgG3和/或IgG4重链恒定区。在某些实施方式中，所述分离的抗原结合蛋白可以包括抗体重链恒定区，且所述抗体重链恒定区可以源自人IgG1重链恒定区。
在本申请中，所述分离的抗原结合蛋白可以包括抗体轻链恒定区。所述抗体轻链恒定区可以包括源自Igκ的恒定区或源自Igλ的恒定区。所述抗体轻链恒定区可以源自人Igκ恒定 区。
在本申请中，所述分离的抗原结合蛋白可以包含抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段可以包括Fab，Fab’，Fv片段，F(ab’)2，F(ab)2，scFv，di-scFv，VHH和/或dAb。
在某些实施方式中，所述抗体可以包括单克隆抗体。在某些实施方式中，所述抗体可以包括鼠源抗体和/或嵌合抗体。
此外，需要说明的是，本申请所述分离的抗原结合蛋白可以包含与其存在一个或多个保守序列修饰的重链和/或轻链序列。所谓“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请所述分离的抗原结合蛋白中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。在某些实施方式中，本申请所述分离的抗原结合蛋白的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换。本领域内的技术人员知道，一些保守序列修改不会使抗原结合性消失，具体可以参见，例如，Brummell et al.,(1993)Biochem 32:1180-8；de Wildt et al.,(1997)Prot.Eng.10:835-41；Komissarov et al.,(1997)J.Biol.Chem.272:26864-26870；Hall et al.,(1992)J.Immunol.149:1605-12；Kelley and O'Connell(1993)Biochem.32:6862-35；Adib-Conquy et al.,(1998)Int.Immunol.10:341-6 and Beers et al.,(2000)Clin.Can.Res.6:2835-43。
可以通过本领域已知的各种测定鉴别、筛选或表征本申请所述的抗原结合蛋白。
例如，可以通过已知方法诸如酶联免疫吸附测定(ELISA)、免疫印迹(例如，蛋白质印迹)、流式细胞术(例如，FACS)、免疫组织化学、免疫荧光等来测试本申请抗原结合蛋白或融合蛋白的抗原结合活性。
在本申请中，所述分离的抗原结合蛋白能够特异性结合DIR或其功能活性片段。在本申请中，所述分离的抗原结合蛋白能够特异性结合DIR-II。
在某些实施方式中，DIR或其功能活性片段可以是全长的DIR或其功能活性片段，或是 发挥DIR功能活性的片段(例如，可以为DIR-II)。在某些实施方式中，DIR或其功能活性片段可以是分离的DIR或其功能活性片段，或是各种形式DIR或其功能活性片段的混合物。例如，DIR或其功能活性片段可以是人DIR或其功能活性片段。
在某些实施方式中，所述分离的抗原结合蛋白可以通过Elisa法测定抗原结合蛋白与抗原(例如DIR-II)的结合活性。例如，所述分离的抗原结合蛋白可以具有以约10ng/ml或更高的值(例如，可以为至少15ng/ml、至少20ng/ml、至少25ng/ml、至少30ng/ml、至少35ng/ml、至少40ng/ml、至少45ng/ml、至少50ng/ml、至少55ng/ml、至少60ng/ml、至少65ng/ml、至少70ng/ml、至少75ng/ml、至少80ng/ml、至少85ng/ml、至少90ng/ml、至少95ng/ml、至少100ng/ml、至少500ng/ml、至少1000ng/ml或更高)结合所述或其功能片段的能力。例如，
一方面，本申请提供一种多肽分子、核酸分子、载体、免疫缀合物、细胞和药物组合物。
另一方面，本申请提供了多肽分子，其可以包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，所述多肽分子可以包含融合蛋白。在某些实施方式中，所述多肽分子可以为融合蛋白。在某些实施方式中，所述多肽分子为多特异性(例如，双特异性，三特异性或其他多特异性)抗体。所述多特异性抗体可以包含：1)本申请所述的分离的抗原结合蛋白，以及2)一个或多个结合其他抗原、和/或结合相同抗原的其他表位的靶向部分。在某些实施方式中，本申请的多肽分子可以包含氨基酸以外的结构，例如，本申请的多肽分子可以包含核酸、多糖、脂质、小分子，以及前述的任意组合。
另一方面，本申请提供了分离的核酸分子，其可以编码本申请所述的分离的抗原结合蛋白。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的；(ii)通过克隆重组产生的；(iii)纯化的，例如通过酶切和凝胶电泳分级分离；或者(iv)合成的，例如通过化学合成。
另一方面，本申请提供了一种载体，其可以包含本申请所述的核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不 仅仅只有这些物质。
另一方面，本申请提供了一种细胞，其可以包含本申请所述的核酸分子或本申请所述的载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如真核细胞，如来自植物的细胞、真菌或酵母细胞等。在某些实施方式中，所述细胞可以是细菌细胞(例如，大肠杆菌)、酵母细胞或其它真核细胞。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。
另一方面，本申请还提供了免疫缀合物，其可以包含本申请所述的分离的抗原结合蛋白。
在某些实施方式中，可以将本申请所述的分离的抗原结合蛋白或其片段与另一试剂，如化学治疗剂、毒素、免疫治疗剂、成像探针、分光镜探针等的连接。该连接可以通过一个或多个共价键，或非共价相互作用，并且可以包括螯合作用。可以使用多种接头(所述接头可以为本领域所知)以形成免疫缀合物。此外，可以以融合蛋白质的形式提供免疫缀合物，其可以由编码免疫缀合物的多核苷酸表达。
另一方面，本申请还提供了药物组合物，其可以包含本申请所述的分离的抗原结合蛋白、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载剂。
本申请的药物组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方案中，所述药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量可以取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。
在某些实施方案中，所述药学上可接受的载剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。
另一方面，本申请还提供了本申请所述的分离的抗原结合蛋白、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备预防和/或治疗疾病或病症的药物中的用途，其中所述 疾病或病症包括认知障碍和/或神经退行性疾病。
另一方面，本申请还提供了本申请所述的分离的抗原结合蛋白、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物，其用于备预防和/或治疗认知障碍和/或神经退行性疾病。
另一方面，本申请还提供了一种预防和/或治疗认知障碍和/或神经退行性疾病的方法，其包括以下步骤：向有需要的受试者施用本申请所述的分离的抗原结合蛋白、本申请所述的多肽分子、本申请所述的免疫缀合物、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物。
在本申请中，所述神经退行性疾病可以包括急性神经退行性疾病和慢性神经退行性疾病。例如，所述神经退行性疾病可以包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
在本申请中，所述认知障碍可以包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。例如，所述认知障碍可以包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。例如，所述认知障碍的诱导疾病可以包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。在本申请中，所述认知障碍可以包括遗忘型MCI多认知域受损(aMCI-m)。
在本申请中，所述受试者可以包括神经退行性疾病患者。在本申请中，所述受试者可以包括认知障碍患者。在本申请中，所述受试者可以处于老龄阶段。在本申请中，所述受试者患有早老性痴呆。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的蛋白片段、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
临床研究的参与者
本研究的参与者来自于上海老龄化研究(SAS)项目。SAS的详细研究设计和招募程序已在其他地方发表(参见Ding,D.et al.The Shanghai Aging Study:study design,baseline characteristics,and prevalence of dementia.Neuroepidemiology 43,114-122(2014))。在这项研究中，根据2018年至2021年间的随访诊断，选择的参与者是：(1)年龄≥60岁，(2)能够配合体检和神经心理学测试，以及(3)同意抽血。排除标准是(1)显著不稳定的、会使参与研究变得困难的全身性疾病，如晚期癌症、器官衰竭或严重的慢性全身性疾病并发症； (2)当前严重的酒精或物质滥用；(3)重大精神疾病。
本研究获得复旦大学附属华山医院伦理委员会批准。从每个参与者那里获得了书面知情同意书。
定量检测Aβ40、Aβ42、Tau和pTau181
根据制造商的说明，在自动化Simoa HD-X平台(GBIO，中国杭州)上使用超灵敏的Simoa技术(Quanterix，MA，US)对血浆Aβ40、Aβ42、总Tau和pTau181进行定量。从Quanterix购买multiplex Neurology 3-Plex A(货号101995)和pTau181 V2(货号103714)检测试剂盒并相应地使用。在所有测定中，血浆样品均以1：4的比例稀释。校准品和质控品一式两份。所有测量均在单次运行的基础上进行。操作人员不知道参与者的疾病状况。
定量检测DIR
根据说明书，使用公司自研的酶联免疫吸附测定(ELISA)试剂盒对血浆DIR进行定量。在所有测定中，血浆样品均以1：4的比例稀释。校准品和质控品一式两份。所有数据均在ELISA试剂盒的推荐范围内。
免疫沉淀
将U87细胞在预冷的RIPA缓冲液(50mM Tris，150mM NaCl，0.1％Triton-100，10％甘油，0.5mg/mL BSA和蛋白酶抑制剂)中裂解。裂解液离心后取上清与抗体在4℃混合过夜，然后加入蛋白G-琼脂糖在4℃下结合抗体4h。接着将琼脂糖重悬于RIPA缓冲液中，洗涤至少3次后，添加SDS缓冲液，并在60℃下孵育20分钟。处理后的样品做免疫印迹实验。
质谱(MS)
对于质谱分析，凝胶消化使用以下操作步骤进行。用含有1％1,4-二硫代三糖醇(DTT)的SDS平衡缓冲液(50mM Tris-Cl(pH 8.8)，6M尿素，30％甘油，2％SDS和溴酚蓝)中处理凝胶15分钟。这一步之后，用含有2.5％碘乙酰胺的SDS平衡缓冲液将游离巯基烷基化，此步骤需避光处理15分钟。然后将其切成多个凝胶段(每段长度约1.0cm)。每个凝胶切片用乙腈和100mm碳酸氢铵交替洗涤三次。在最后一次清洗中，凝胶片在100mM碳酸氢铵中孵育15分钟，温度保持在4℃。凝胶片通过真空离心干燥并在含有胰蛋白酶(20μg/ml)和碳酸氢铵(50mM)的胰蛋白酶溶液中膨胀45min，温度保持在4℃。再次加入胰蛋白酶溶液，37℃保存20h。离心后将上清液转移到另一个小瓶中，用含有0.1％甲酸的60％乙腈溶液中提取凝胶片3次，每次15min。回收的肽溶液通过真空离心干燥，用Ziptip(Millipore,Corp.Bedford,MA)脱盐和清洗。
通过反相高效液相色谱(RP-HPLC)分离肽混合物中的各组分，然后进行串联质谱分析。 RP-HPLC在测量器LC系统(Thermo Finnigan,San Jose,CA)上进行。C18色谱柱(RP,180μm×150mm)购自column Technology Inc.(Fremeont,CA)。泵流量按1:120，达到1.5μl/min的柱流量。流动相A为0.1％甲酸水溶液，流动相B为0.1％甲酸乙腈溶液。用2-98％梯度的B在180分钟内洗脱胰蛋白酶肽混合物。
质谱在LTQ线性离子阱质谱仪(Thermo Finnigan,San Jose,CA)上进行，配有电喷雾接口，并在正离子模式下操作。毛细管温度为170℃，喷雾电压为3.4kV。归一化碰撞能量为35％。采用自动增益控制，获得每次扫描的最大信号。质谱仪的设置是，在一次完整的MS扫描之后，对10个最强烈的离子进行10次MS/MS扫描。动态排除设置为重复计数2，重复持续时间30s，排除持续时间90s。
在8节点Dell PowerEdge 2650集群上，使用BioWorks 3.0软件(Thermo Finnigan)在IPI人类数据库中搜索获得的MS/MS谱。一个被接受的SEQUEST结果的ΔCn分数至少为0.1(不管电荷状态)，这个值在SEQUEST搜索中具有较高的置信度。使用Build Summary软件将所有输出结果组合在一起，删除冗余数据。为了确保MS/MS谱具有良好的质量，片段离子明显高于基线噪声，参考了以前研究中报道的参数，并应用了更严格的肽识别标准。肽符合以下标准后进行验证。对于+1胰蛋白酶肽，SEQUEST交叉相关评分必须≥1.9；对于+2胰蛋白酶肽，评分≥2.2；对于+3胰蛋白酶肽，评分≥3.75。此外，ΔCn截断值≥0.1，肽的SP等级≤4。
PET
所有受试者使用PET/CT系统进行AV45-PET成像(Biograph TruePoint HD 64 PET/CT，西门子；德国Erlangen)。受试者静脉注射AV45(平均剂量:5.55MBq/kg[0.15mCi/kg])，然后在昏暗的房间安静休息20分钟。随后进行10分钟PET采集程序，并进行低剂量CT扫描。采集后，使用滤波后的反投影算法对PET图像进行重构，并对衰减、归一化、死区时间(dead time)、光子衰减、散射和随机巧合进行校正。重建的PET图像矩阵大小为168×168×148，体素大小为2.04×2.04×1.5mm3。
图像分析
感兴趣区域(ROI)在外侧顶叶、外侧颞叶、内侧颞叶、后扣带(posterior cingulate)、额叶、枕叶和楔前叶皮层上绘制。标准摄取值(SUV)是感兴趣区域(ROI)的放射性，以小脑结节的放射性作为参考。总SUV得分是通过这些ROI的加权平均计算出来的。
抗体
本研究使用的DIR抗体由吉尔生化(上海)有限公司(GL Biochem)(其识别了DIR (PPLPLCRRCHKIHLRRLLSKFSNIFSP)的最后27个AAs)和三优生物医药(上海)有限公司(Sanyoubio)生产，用于免疫沉淀的多克隆抗体来源于家兔。用于腹腔注射和免疫染色的单克隆抗体来自杂交瘤。用于Elisa捕获的单克隆抗体由吉尔生化(上海)有限公司从杂交瘤细胞中制备，检测抗体由三优生物医药(上海)有限公司利用噬菌体展示库筛选构建。用于Western blotting的多克隆抗体来源于家兔，用于免疫染色的多克隆抗体来源于豚鼠。
为检测抗体的特异性，将抗体与抗原(10-5或10-6M)于4℃下孵育12h做吸收实验，然后将吸收的和未吸收的抗体溶液孵育-含有相同裂解物样品的硝酸纤维素膜。并用hrp连接的第二抗体孵育以及成像。此外，还对同种DIR-KI小鼠脑切片进行免疫染色，检测抗体的特异性。
质粒的构建与表达
引物设计用于克隆CDS全长序列。纯化产物与载体使用Hieff Plus One Step Cloning Kit(翌圣生物科技(上海)有限公司)进行重组。DIR CDS克隆到pCMV-flag载体上，gelsolin(NM_000177.5)克隆到pEGFP-N3和pcDNA3.1-myc-his质粒上。DDIT4L构建至pcDNA3.1-myc-his载体。引物如下所示。
用PEI40000试剂(翌圣生物科技(上海)有限公司)转染HEK293T或U87MG细胞。进一步培养48小时，用HEPES裂解缓冲液(30mM HEPES,150mM NaCl,10mM NaF,1％Triton X-100,0.01％SDS)裂解细胞，4℃以12000rpm转速离心10min，转移上清进行Western blotting。
针对pCMV-Flag-DIR:
Forward:5’-CCATGGAGGCCCGAATTCGGATGGTTGCAACTGGC-3’(SEQ ID NO.76),
Reverse:5’-ACTCATCAATGTATCTTATCTTATGGAGAGAAGATGTTAGAAAA-3’(SEQ ID NO.77)；
针对pcDNA3.1-DDIT4L-myc-his:
Forward:5’-GCCACCATGGTTGCAACTGGCAGTTTGAGC-3’(SEQ ID NO.78),
Reverse:5’-GAATTCCACCACACTGGACTAGTGGATCCG-3’(SEQ ID NO.79)；
针对gelsolin-pEGFP:
Forward:5’-TCAAGCTTCGAATTCATGGCTCCGCACCGCCC-3’(SEQ ID NO.80),
Reverse:5’-CGGGCCCGCGGTACCGGCAGCCAGCTCAGCCATGG-3’(SEQ ID NO.81)；
针对pcDNA3.1-gelsolin-myc-his:
Forward:5’-GTGGAATTCGCCACCATGGCTCCGCACCGCCC-3’(SEQ ID NO.82),
Reverse:5’-CCCTCTAGACTCGAGGGCAGCCAGCTCAGCCATGG-3’(SEQ ID NO.83)；
用PEI40000试剂(翌圣生物科技(上海)有限公司)转染HEK293T或U87MG细胞。进一步培养48小时，用HEPES裂解缓冲液(30mM HEPES,150mM NaCl,10mM NaF,1％Triton X-100,0.01％SDS)裂解细胞，4℃以12000rpm转速离心10min，转移上清进行Western blotting。
体外聚合实验
DIR(31-84)和Aβ42由吉尔生化(上海)有限公司合成。将pcDNA3.1-gelsolin-myc-his质粒转染HEK293细胞，收集培养基进行蛋白纯化。简单地说，将Ni-NTA琼脂糖树脂洗净转移到玻璃柱中用作吸附柱。然后将培养基与等体积的裂解缓冲液(50mM NaH2PO4,300mM NaCl,10mM咪唑，PH＝8.0)混合，混合液通过吸附柱，洗涤(50mM NaH2PO4,300mM NaCl,20mM咪唑，PH＝8.0)。然后用洗脱缓冲液(50mM NaH2PO4,300mM NaCl,250mM咪唑，PH＝8.0)从树脂中释放目的蛋白。洗脱液进一步浓缩，并用PBS置换。将纯化后的蛋白用PBS稀释，按溶质(Aβ42、Aβ42/DIR、Aβ42/DIR/gelsolin等)设置不同的组。以1/10体积溶液作为上样液，其余溶液在4℃下旋转过夜。次日离心，取上清做免疫沉淀，用8M尿素溶解沉淀。用免疫印迹法进一步分析所有成分。
免疫印迹
成年小鼠麻醉后灌注PBS。然后提取脑组织，并在预冷的RIPA缓冲液中裂解。离心后，收集脑裂解液上清液低温保存。人和小鼠的血浆用PBS按1:10的比例稀释。所有样品经十二烷基硫酸钠(SDS)凝胶电泳分离，接着转移到硝酸纤维素膜上，并在含有Tween-20(TBST)的TBS缓冲液中用5％脱脂牛奶封闭。膜孵育在第一抗体中4℃过夜。第二天，膜用TBST冲洗三次，并与HRP连接的第二抗体孵育。在进一步洗涤后，通过添加ECL缓冲液进行成像。蛋白条带用GE成像系统成像。所使用的第一抗体在如下:flag(Sigma,SAB4200071)，actin(Chemicon,MAB1501)，GAPDH(Proteintech,10494)，gelsolin(Invitrogen,PA518605)，GFP(Roche,11814460001)。
免疫组织化学
小鼠被麻醉并灌注4％多聚甲醛(PFA)。然后收集脑组织，在4％的PFA中4℃固定1小时。在PBS中洗涤三次后，将组织置于30％蔗糖/PBS中4℃过夜，然后用OCT化合物包埋。通过冷冻切片得到脑切片(厚度40μm)。为了进行免疫染色，组织切片用PBST(0.1％Triton X-100/2.5％正常驴血清/PBS)在室温下封闭30分钟，然后在4℃下用一抗孵育过夜。 组织切片用PBS洗涤3次，Alexa荧光偶联二抗(Invitrogen)37℃孵育60min,PBS洗涤3次，并装入含DAPI的培养基。图像采集使用徕卡SP8共聚焦显微镜。人脑组织石蜡切片由中国医学科学院人类脑库、中国北京协和医学院提供。采用免疫荧光染色研究DIR、gelsolin和Aβ的分布。在单个组织切片中，使用不同物种(小鼠、兔子或绵羊)中产生的一抗。二抗(抗小鼠，抗兔或抗羊)偶联到不同的荧光色素(Alexa Fluor 488,Cy3或Cy5)。染色后，用封闭液覆盖荧光标记的部分(Vector Labs)。
邻位连接技术(PLA)
人脑组织石蜡切片由中国医学科学院、北京协和医学院人类脑库提供。样品经过封闭，然后在37℃的湿度室中孵育60分钟。接着提供识别DIR、gelsolin和Aβ的抗体，在4℃下对样品进行过夜染色。第二天用正负PLA探针孵育样品(1:5；Sigma；DUO92001和DUO92005)，37℃1小时，然后用1X连接缓冲液(1:40；Sigma；DUO92008)，在37℃下额外孵育30分钟。此外，用扩增-聚合酶溶液(1:80；Sigma；DUO92008)在37℃下放置100分钟。最后使用最小体积的Duolink封片剂保护样品，封片剂中含有DAPI染料(Sigma；DUO82040)。最后用徕卡SP8显微镜成像。
脑组织切片制备
将成年小鼠麻醉后迅速取出大脑，置于含有以下化合物(单位:mM)的冰冷人工脑脊液(ACSF)中:117 NaCl,3.6 KCl,1.2 NaH2PO4·2H2O,2.5 CaCl2·2H2O,1.2 MgCl2·6H2O,25NaHCO3,11葡萄糖。当含95％O2和5％CO2时，冰冷的ACSF的pH值为7.4。将大脑粘在徕卡VT1200S振动仪的台上，切下350μm厚的海马体横切片。切片在含氧(95％O2和5％CO2)的ACSF中室温孵育至少1小时。
石蜡切片免疫荧光染色
将人脑组织石蜡切片浸泡在二甲苯中10分钟进行脱蜡，共两次；再将切片浸泡在纯乙醇中10分钟，共两次；并依次浸泡在95％、90％、80％和70％的乙醇中各5分钟进行复水，然后使用PBS清洗三次。最后将切片放置在1x TE抗原修复缓冲液中，100℃修复20分钟，接着让切片自然冷却至室温，使用PBS清洗3次。TE抗原修复液(20x)：4.84g Tris、1.48g EDTA、180mL蒸馏水、NaOH调pH至9.0，加入2mL Tween 20，定容至200mL，4℃保存。使用时按需稀释成1x，常温保存。
将切片加入含有3％羊血清的PBS，封闭1小时。配置True Black工作液(380ul 70％EtOH，20ul预热的20x True Black)，滴加在组织上，室温孵育1分钟后吸去True Black，PBS清洗3次。切片放在湿盒中，将稀释好的抗体覆盖在标本表面，将湿盒放入4℃冰箱过 夜孵育。
第二天，将切片浸泡在PBS中洗涤三次，并将稀释好的荧光二抗覆盖在样品表面，室温孵育1小时。接着将PBS清洗切片三次，加入含0.05％硫磺素S的50％乙醇溶液，避光孵育8分钟，并使用80％的乙醇清洗切片两次，每次10秒。最后用PBS洗涤切片三次，滴上荧光封片剂封片，使用显微镜观察记录。
DIR-KI小鼠的制备
通过体外转录的方式，获得Cas9mRNA和用于敲入的sgRNA(SEQ ID NO:88；然后，构建作为供体的同源重组载体，该载体包含约3kb的5’同源臂(SEQ ID NO:89)、编码人QDLIR后64个氨基酸的核酸序列(SEQ ID NO:90)，以及约3kb的3’同源臂(SEQ ID NO:91)。紧接着，将编码Cas9的mRNA、sgRNA和供体载体显微注射到C57BL/6J小鼠的受精卵中，获得F0代小鼠。最后将通过PCR鉴定为阳性的F0代小鼠与C57BL/6J小鼠交配获得阳性F1代小鼠。F1代小鼠间进行交配可获得F2代野生型、杂合子以及纯合子小鼠。
新物体识别实验
实验当天提前30分钟使小鼠适应实验房间。实验分为适应期、训练期和实验期。适应期：将小鼠从饲养笼取出，置于塑料箱中央，自由探索实验场地10分钟。在适应期的12～24小时后进行训练期；训练期：将2个相同的电池置于距离墙壁5厘米处。将小鼠置于场地中央，自由探索10分钟。在训练期结束的3～4小时之内进行实验期；实验期：将玩具随机替代1个电池，摆放位置与原物体相同。将小鼠置于场地中央自由探索10分钟。实验结束后，小鼠归笼。整个过程采用摄像头录像记录。实验间隔中采用75％的酒精清理实验装置。实验结束后，由人工对录像中小鼠探索新旧物体的时间进行计数并计算新物体的时间识别指数。当小鼠的鼻尖距离物体2厘米范围内，且小鼠嗅或触碰物体视为探索物体的行为，啃咬或爬上物体不视作探索行为。小鼠对新物体的识别指数＝(探索新物体的时间-探索旧物体的时间)/(探索新物体的时间+探索旧物体的时间)。小鼠探索总时间小于15秒的数据将被排除。实验操作者和数据分析者为双盲。
Morris水迷宫实验
实验当天提前30分钟使小鼠适应实验房间。实验分为训练日和实验日。训练日为5～6天，实验日为1天。实验前在水箱中装入30厘米深度的水，水温维持在19～22℃，水中加入滑石粉使之浑浊。水箱分为四个方向：N、S、E和W，平台放置在NE象限，小鼠每日的入水点选择S、W、NW和SE的不重复组合。房间设置在实验期间保持不变，在水迷宫四周贴有特定图案作为远端线索。房间灯光为非直射灯光。实验全程采用摄像头录像记录，实验日 的录像使用Etho Vision XT 14软件分析结果。训练日：将平台高度置于水下0.5厘米处。将小鼠从指定入水点入水，面朝水箱壁，同时开始计时1分钟。当小鼠到达平台时停止计时，若在1分钟内小鼠没有到达平台，则将小鼠置于平台上或引导上平台。待小鼠在平台停留30秒后，取出小鼠，擦干，归笼。在下一次入水前需间隔30分钟。将小鼠置于新的入水点，重复上述实验3次。实验人员记录每次实验中小鼠上平台的用时。第二天，重复以上实验。每天训练4次，共训练5～6天。实验日：训练日结束的24小时之后进行实验。移除平台。小鼠从SW象限入水，面朝水箱壁。1分钟后取出小鼠，擦干，归笼。实验结束后采用EthoVision XT 14软件分析小鼠行为录像，采集数据包括：进入目标象限的逃避潜伏期、穿越平台位置的次数、在目标和非目标象限的停留时间。
Y迷宫实验
开展行为学实验前，使小鼠适应饲养环境一周。实验当天提前30分钟让小鼠适应实验房间。实验开始后，将小鼠面朝墙壁置于Y形迷宫起始臂A，自由探索迷宫10分钟。当位于迷宫中心时，小鼠可选择任意方向进入。认知正常的小鼠在实验过程中会倾向进入未被探索过的臂。整个过程采用摄像头录像记录。实验结束后，小鼠归笼。实验间隔中采用75％的酒精清理实验装置。实验结束后，人工记录小鼠探索迷宫臂的顺序，用于计算总进臂次数和交替比例(交替比例＝实际交替入臂次数/(总进臂次数-2))。实验操作者和数据分析者为双盲。
统计分析
数值数据(如生物标记物浓度)的组间差异用Welch's t检验或单向方差分析进行分析。采用线性回归模型进行相关分析。分析受试者工作特征(ROC)曲线，以曲线下面积(AUC)作为生物标志物的预测价值。
使用PRISM(GraphPad Software)进行统计分析。使用R(4.0.2版)中的包ggplot2生成盒图和roc。p<0.05时认为差异显著(*p<0.05，**p<0.01，***p<0.001)。
实施例1 DIR的鉴定
通过对老年痴呆患者(AD)的转录本进行分析，发现了DDIT4L基因的一个可变剪切形式在患者中高表达，这种剪切导致DDIT4L第2个内含子发生了滞留，从而产生了一种新的转录本DIR。
具体而言，人源DDIT4L基因的正常剪切体在形成mRNA过程中会将内含子(Intron)部分切除，并连接相邻的两个外显子(Exon)。而在异常剪切过程中，造成了内含子滞留 (IR)，形成一种全新的DIR的mRNA形式(参见图1A，其中NS表示正常剪切体，IR表示异常剪切形成的DIR)。
鉴定正常人(NC)与老年痴呆症患者(AD)DDIT4L和DIR mRNA：取正常人和老年痴呆症患者捐献的部分脑组织，抽提RNA并反转成cDNA后，通过PCR的方法鉴定DDIT4L和DIR mRNA。引物设计(正向引物：5’-tgctggactgtggctatcac3’(SEQ ID NO.6)；反向引物5’-acaaggacctttgagcaacca-3’(SEQ ID NO.7))在两个外显子中，由于异常剪切导致内含子滞留，因此DIR的PCR产物(约2000bp)要大于正常剪切的DDIT4L(约200bp)。DDIT4L和DIR的mRNA鉴定结果参见图1B。
克隆出DIR的开放阅读框并做外源表达，发现DIR可以在体外正常翻译出对应的DIR蛋白(其氨基酸序列如SEQ ID NO.1所示)，并且该蛋白能够被分泌出细胞外。
具体而言，将DIR的开放阅读框(SEQ ID NO.8)构建到pCMV-flag载体中，并将Flag-DIR质粒和对照flag质粒分别转染到HEK293细胞中，48小时后取细胞培养基和细胞裂解液做Western Blot，用DIR特异性抗体检测其表达，actin则用作为对照。结果如图1C所示。
对人血液中的DIR进行检测。取2例正常人和7例不同年龄的AD患者(年龄分别为82、55、65、53、78、68和58岁)血液做Western Blot，用DIR特异性抗体检测其表达，丽春红染色条带用做上样对照。结果如图1D所示。可见DIR可以特异性存在于AD患者中。
实施例2 DIR的生物学功能
2.1 DIR与gelsolin有相互作用
将能够表达DIR质粒转染至U87细胞中，48小时后裂解细胞，并将细胞裂解液平均分成两份，分别加入等量的IgG和DIR抗体做免疫共沉淀实验，实验产物通过SDS-PAGE分离，并将分离好的PAGE胶浸泡考马斯亮蓝染液1小时，然后用脱色液洗去PAGE胶表面非特异性附着的染液，直至PAGE胶中能够清晰显示条带为止。接着切割下特异条带后做质谱分析，得到候选分子gelsolin。
然后将gelsolin的开放阅读框(SEQ ID NO.9)克隆到pEGFP-N3载体得到Gelsolin-GFP质粒，并将gelsolin-GFP与Flag或者Flag-DIR质粒共同转染至HEK293细胞中，48小时后裂解细胞，取得裂解液用Flag抗体做免疫共沉淀实验，最后通过GFP的抗体检测到gelsolin与DIR的结合。
2.2 DIR抑制兴奋性突触后电流幅度
取成年C57BL/6小鼠的脑组织制成脑片，取海马体部分的脑片做电生理记录。记录在给予IR肽段前后，脑片的突触后兴奋性电流变化。
结果如图4所示。结果显示施用所述DIR后，电流幅度发生了降低，而电流发放的频率并没有发生变化。
实施例3 DIR的功能片段及其生物学功能
DIR(即QDLIR，其氨基酸序列如SEQ ID NO.1所示)包含第一外显子所编码的氨基酸序列(其氨基酸序列如SEQ ID NO.2所示)和滞留的内含子所编码的氨基酸序列(其氨基酸序列如SEQ ID NO.3所示)。
其中，所述滞留的内含子所编码的氨基酸序列(IR)可以为分为2个部分，分别是由自N端起前27个氨基酸组成的IR-I(即DIR-I，其氨基酸序列如SEQ ID NO.4所示)和后27个氨基酸组成的DIR-II(即DIR-II，其氨基酸序列如SEQ ID NO.5所示)。上述结构的具体参见图2。
通过电生理记录的方法来验证上述IR片段的功能。
首先，合成所述IR片段，孵育野生型小鼠的脑片，记录兴奋性突触后电流(EPSC)(具体方法参见Pavel等人，Curr Biol.《当代生物学》，26:2194-2201[2016])。结果显示IR可以降低EPSC的幅度，尽管没有观察到EPSC频率的显著性下降，但是可以看出有下降的趋势。
分别合成所述IR-I和DIR-II片段来筛选功能区域。结果显示，IR-I可以降低EPSC的频率但是不影响EPSC的幅度；尽管DIR-II对于EPSC的幅度和频率都没有显著影响。但是从统计结果可以看出，DIR-II有降低EPSC幅度的趋势，并且有升高EPSC频率的趋势(参见图3)。
由上述的结果可知，IR有两个功能区域。其中IR-I可以降低EPSC的频率，而DIR-II可以降低EPSC幅度。
实施例4 DIR参与Aβ沉积
4.1 DIR通过gelsolin诱导Aβ沉积
在AD患者样本中，DIR主要与海马体中的Aβ共定位(参见图5a)。在转染的HEK293T细胞中观察到DIR和Aβ之间的相互作用，但DDIT4L(包括DIR-外显子)不能与Aβ相互作用(参见图5b，图6a)。这些结果表明DIR-内含子(即滞留的内含子所编码的氨基酸序列，其氨基酸序列如SEQ ID NO.3所示)可能是DIR与Aβ相互作用的主要区域。
邻近连接分析(PLA)显示DIR不直接与Aβ相互作用(参见图5c)。使用合成的DIR-内含子和Aβ蛋白质，结果发现DIR-内含子不能直接与Aβ结合(参见图6b)，表明DIR有 另一个结合靶点。
将DIR转染到人脑细胞系中，并用兔源DIR抗体(购自吉尔生化(上海)有限公司)对细胞裂解物进行免疫沉淀。结果显示共免疫沉淀了一个分子量约为85kDa的免疫反应条带(参见图5d)。提取条带并通过质谱法进一步分析。该分析确定了12-15个与人gelsolin(85kDa)匹配的肽，并且覆盖了gelsolin序列的35％(参见图5e)。
gelsolin已被证明与Aβ17相互作用，形成一种复合物，被输送到循环系统。研究表明，DIR与DIR敲入小鼠、HEK293T细胞和人体组织中的gelsolin相互作用(参见图5f、g、图6c)。
在用DIR和内源表达的gelsolin转染的HEK293T细胞裂解物中，Aβ42的添加以剂量依赖性方式增强了DIR与gelsolin的相互作用，表明Aβ有助于DIR和gelsolin之间的相互作用(参见图5h).此外，将合成的DIR-内含子和Aβ42蛋白添加到纯化的gelsolin或HEK293T细胞裂解物中，DIR-内含子明显增强了不溶性的Aβ42的形成，而不溶性的Aβ42是淀粉样斑块的主要成分(参见图5i，图6d)。
由此可见，DIR-内含子参与Aβ的沉积。
4.2 DIR介导Aβ淀粉样斑块形成
硫磺素S(thioflavine S)是AD患者大脑中Aβ斑块的特异性标志物。然而，硫磺素S无法标记DIR敲入小鼠海马体中的淀粉样斑块(参见图7a)，因为无法用硫磺素检测到小鼠Aβ序列。
然而，与人Aβ40肽孵育6小时的DIR敲入小鼠海马体切片中的淀粉样斑块可以被硫磺素S染色(参见图7a)，并且与硫磺素S染色人Aβ斑块的结果一致。
并且，三重免疫染色显示，在AD患者的海马体和皮质区域，DIR与gelsolin和Aβ在致密核心斑块中的共定位程度高于弥漫性斑块，而在不患有AD的对照对象中没有明显的DIR或淀粉样蛋白斑块染色(参见图7b，图8a)。
在AD患者海马体和皮质区的致密核心斑块中，Aβ信号强于硫磺素S和DIR，这表明致密核心斑块是不可溶的；此外，可溶性Aβ肽存在于周边区域(参见图7c)。
由上结果可知，DIR表达的增加可能导致Aβ沉积，并在病理条件下通过与gelsolin结合而导致随后的淀粉样斑块形成。DIR可能是Aβ沉积和淀粉样斑块形成的引发剂(参见图11e)。
实施例5 DIR与疾病相关
5.1血浆DIR水平作为诊断指标
检测33名认知正常人(作为对照组)、44名非遗忘性MCI(non-amnestic MCI，naMCI)患者、42名遗忘性MCI(aMCI)患者和31名临床诊断为AD的患者血浆中的DIR水平。
与认知正常的对照组相比，naMCI、aMCI和AD患者的血浆中的DIR浓度逐渐升高(参见图9a)。此外，发现血浆中的DIR浓度与pTau181的浓度呈正相关，但是与Aβ42/40比率呈负相关，并且与从正常认知到naMCI、aMCI和AD的整个临床范围内认知能力下降的严重程度呈负相关。
然而，血浆中的总Tau(tTau)的浓度没有观察到这种趋势(参见图9b-d)。
血浆中的DIR和pTau181水平显示出区分AD患者和认知正常个体的潜力，其AUC分别为AUCDIR＝0.86和AUCpTau181＝0.86(参见图9e和f)；
血浆中AUC Aβ42/40比率仅达到71％，用于区分AD患者和正常对照(参见图9g)。而血浆tTau水平不能有效区分AD患者和对照组(AUC＝0.55)(参见图10a)。结合DIR、pTau、Aβ42/40的综合模型展示了区分AD患者和认知正常对照组的潜力，AUC为96％，高于使用pTau和Aβ42获得的AUC(88％)/40(参见图9h)。
与pTau181(AUC＝0.63)、Aβ42/40(AUC＝0.59)和tTau(AUC＝0.56)相比，血浆中的DIR水平在区分aMCI患者和认知正常患者(AUC＝0.73)方面也表现出更好的性能(图9i-k，图10b)。结合DIR、pTau、Aβ42/40的综合模型在区分aMCI个体和认知正常对照组方面表现出更好的效果，AUC为81％，高于pTau和Aβ42获得的AUC(75％)/40(图9l)。
5.2血浆DIR水平与疾病进程和血浆Aβ水平相关
分别检查12个MCI患者在三年前认知正常时的血浆(作为基线样本)和患病后的血浆中的DIR浓度。发现这些MCI患者样本中的DIR血浆浓度高于其基线样本(图11a)。
并且，无论是MCI患者还是认知正常人(作为对照组)的血浆中DIR水平，均与Aβ40和Aβ42的水平正相关(参见图11b，图12a)。
由此可见，血浆中的DIR浓度的升高随着认知障碍的进展而产生，并且与Aβ的水平相关。
5.3血浆DIR水平与Aβ-PET水平相关
检测血浆DIR和Aβ-PET水平之间的关联。在AD/MCI患者中，血浆DIR水平与整个皮质的Aβ-PET水平显著相关，血浆DIR水平与颞叶中的Aβ-PET水平密切相关，这表明Aβ 积聚(参见图11c，图12b)。Aβ阳性个体(n＝11)血浆中DIR浓度高于Aβ阴性个体(n＝26)(参见图11d)。血浆中的DIR水平对Aβ-PET阳性具有良好的预测价值(AUC＝0.90，参见图11d)。
这些结果显示，血浆中DIR的存在强烈反映了AD患者的淀粉样斑块形成。因此，DIR是AD的潜在生物标志物。
实施例6 DIR抗体及其生物学功能
6.1 DIR抗体的制备
1)杂交瘤的制备和DIR抗体的获得
经过抗原免疫后的小鼠，取其脾脏消化成单个细胞。然后分离其中的B细胞于体外系统中培养，并用化学诱导剂将B细胞与骨髓瘤细胞融合。融合后的细胞通过流式分选技术于96孔板中培养。最后将培养基收集，孵育到包被有抗原的孔板中，检测DIR抗体的特异性。
2)抗体测序
按照PrimeScriptTM第一链cDNA合成试剂盒(Takara，目录号6110A)的技术手册，使用反义引物将总RNA逆转录成cDNA。然后按照Biointron Biologic Inc.的SOP扩增VH和VL的抗体片段，并将PCR片段克隆到TA/Blunt-Zero Cloning载体中。进行菌落PCR以筛选克隆并且不少于5个阳性克隆被测序。
分别测得了IR-II-1～IR-II-10(IR-II-10即A2D10)的测序结果，结果如下所示：
表1

6.2 DIR抗体的结合活性检测
检测上述IR-II-1～IR-II-10抗体与DIR-II片段的结合活性。具体方法为：
将目的片段DIR-II利用包被缓冲液包被在96孔板上，并用封闭缓冲液封闭。再将单克隆抗体(1mg/mL)分别等比例稀释后(1:2k，1:6k，1:18k，1:54k，1:162k，1:486k，1:1458k，1:4374k)，移液器加至单个孔中，37℃均匀晃动2小时，并用清洗缓冲液洗三次。然后加入带有辣根过氧化物酶的二抗(1:5k)，37℃孵育。待清洗三次后，加入适当体积的TMB底物溶液，37℃孵育一定时间后置于酶标仪下记录450nm波长下样品的吸光度数值。
结果如表2和图13所示，由此结果可知上述IR-II-1～IR-II-10抗体与DIR-II片段以约10ng/ml以上的工作浓度特异性结合。
表2
实施例7利用DIR抗体检测DIR
7.1
制备靶向DIR外显子2翻译序列的30个氨基酸的抗体，并在患者组织中进行了测试。GBM组织的免疫印迹显示两条带(～12kDa和～22kDa)(参见图14c)。由于DDIT4L的分子量约为22kDa，因此推测12kDa条带可能是DIR。
进一步地，获得一种仅识别DIR特有的内含子翻译序列的54个氨基酸的抗体(豚鼠来源的多克隆抗体，购自吉尔生化(上海)有限公司)，并在患者组织中进行了测试。仅在GBM组织中检测到约12kDa的蛋白质(参见图14d)。
此外，构建了含有DIR开放阅读框(其核苷酸序列如SEQ ID NO.1所示)的异源表达质粒，并将该质粒转染到HEK293T细胞中。可以在细胞裂解物和条件培养基中检测到DIR(参见图14e)，表明DIR可以在体内翻译和分泌。
7.2
制备靶向DIR C端27个氨基酸(即DIR-II)的抗体，以检测GBM患者血液中是否存在DIR。免疫印迹显示，肿瘤侵袭海马体的患者血液中存在高水平的DIR(参见图14f，g)。具体而言，DIR存在于71.4％侵袭海马体的GBM患者(n＝7)的血液样本中，但只有15.4％未侵袭海马体的GBM患者(n＝13)的血液样本中存在DIR。
这表明DIR的存在和产生与海马体肿瘤侵袭之间的相关性，而这对于陈述性记忆和联想学习的存储非常重要。
实施例8 DIR水平降低可以提高认知水平
8.1利用siRNA
(1)在HEK293细胞中转染Flag-DIR质粒，6小时后，分别转染50nM siRNA(其中，DIR siRNA1的正义链的核苷酸序列如SEQ ID NO.84所示，siRNA1的反义链的核苷酸序列如SEQ ID NO.85所示；DIR siRNA2的正义链的核苷酸序列如SEQ ID NO.86所示，siRNA2的反义链的核苷酸序列如SEQ ID NO.87所示)。在质粒转染48小时后，裂解细胞离心收集上清做免疫印迹实验检测DIR的表达。
结果如图15A所示，结果发现给予siRNA后，检测到DIR的表达水平显著下降。
(2)利用脑立体定位仪对3月龄DIR-KI小鼠海马区进行注射给药，分别给与siRNA2(1.25mM)和对照药物PBS。分三次给药，每次给予4μl，间隔三天。给药完成后让小鼠恢复三天后做新物体识别行为实验。在图15B中，O、N分别为旧物体和新物体。
结果如图15B所示，结果发现向DIR-KI小鼠海马注射siRNA后，其新物体识别的行为学显示认知功能显著提高(n≥9)。
8.2利用IR-II抗体
为了检查针对IR-II抗体是否可以改善AD等疾病的病情，将实施例6获得的A2D10(0.5mg/天)或IgG腹膜注射到纯合的DIR-KI小鼠中5天。
对注射后的小鼠进行行为学试验检测，结果如图16所示。其中O、N分别为旧物体和新物体。
图16A-16B的结果显示，施用A2D10改善了工作记忆和新物体识别能力。图16C的结果显示，施用A2D10减少了Morris水迷宫测试训练阶段的逃避潜伏期。在用于评估空间记忆的探测测试(probe test)中，施用A2D10的小鼠进入目标象限的潜伏期更短，停留时间更长。
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作 为参考那样。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (159)

  1. 调控剂，其调控DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段，在制备用于预防和/或治疗疾病的试剂中的用途，其中所述疾病包括认知障碍。
  2. 调控剂，其调控DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段，在制备用于预防和/或治疗疾病的试剂中的用途，其中所述疾病包括神经退行性疾病。
  3. 根据权利要求1-2中任一项所述的用途，其中所述调控剂使受试者中，所述DIR和/或其功能片段的表达水平和/或生物学活性降低。
  4. 根据权利要求3所述的用途，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的表达水平和/或生物学活性相比，所述DIR和/或其功能片段的表达水平和/或生物学活性降低至少约10％。
  5. 根据权利要求3-4中任一项所述的用途，其中所述表达水平包括编码所述DIR/或其功能片段的基因的表达水平、编码所述DIR/或其功能片段的基因的转录水平和/或所述DIR/或其功能片段的表达水平。
  6. 根据权利要求3-5中任一项所述的用途，其中所述表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  7. 根据权利要求3-6中任一项所述的用途，其中所述表达水平通过利用选自下组的物质衡量：能够特异性扩增编码所述DIR/或其功能片段的基因的引物、与编码所述DIR/或其功能片段的基因特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的小分子、与所述DIR/或其功能片段特异性结合的探针和与所述DIR/或其功能片段特异性结合的多肽。
  8. 根据权利要求1-7中任一项所述的用途，其中所述DIR的功能片段保留所述DIR的至少部分的生物学活性。
  9. 根据权利要求8所述的用途，其中所述生物学活性包括影响神经元的兴奋性和/或抑制神经元的活性。
  10. 根据权利要求8-9中任一项所述的用途，其中所述生物学活性包括能够降低兴奋性突触后电流(EPSC)的频率，和/或能够降低EPSC的幅度。
  11. 根据权利要求10所述的用途，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者中的兴奋性突触后电流(EPSC)的频率降低，和/或使受试者中的EPSC的幅度降低。
  12. 根据权利要求8-11中任一项所述的用途，其中所述生物学活性包括影响认知能力。
  13. 根据权利要求8-12中任一项所述的用途，其中所述生物学活性包括参与与Aβ沉积相关的信号通路，和/或，参与与Tau缠结产生相关的信号通路。
  14. 根据权利要求8-13中任一项所述的用途，其中所述生物学活性包括通过gelsolin诱导Aβ沉积和/或淀粉样斑块形成。
  15. 根据权利要求14所述的用途，其中所述DIR/或其功能片段通过与gelsolin结合诱导Aβ沉积和/或淀粉样斑块形成。
  16. 根据权利要求8-15中任一项所述的用途，其中所述DIR和/或其功能片段的表达水平与Aβ的表达水平正相关。
  17. 根据权利要求8-16中任一项所述的用途，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者的认知能力降低。
  18. 根据权利要求1-17中任一项所述的用途，其中所述DIR/或其功能片段来源于哺乳动物。
  19. 根据权利要求1-18中任一项所述的用途，其中所述DIR/或其功能片段来源于灵长类动物。
  20. 根据权利要求1-19中任一项所述的用途，其中所述DIR/或其功能片段来源于人。
  21. 根据权利要求1-20中任一项所述的用途，其中所述DIR包含SEQ ID NO.1所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的用途，其中所述DIR的功能片段包含DDIT4L中滞留的内含子所编码的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的用途，其中所述DIR的功能片段包含SEQ ID NO.4-5中任一项所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的用途，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  25. 根据权利要求1-24中任一项所述的用途，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  26. 根据权利要求1-25中任一项所述的用途，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  27. 根据权利要求1-26中任一项所述的用途，其中所述认知障碍包括遗忘型MCI多认知域受损(aMCI-m)。
  28. 根据权利要求2-27中任一项所述的用途，其中所述神经退行性疾病包括急性神经退行性 疾病和慢性神经退行性疾病。
  29. 根据权利要求2-28中任一项所述的用途，其中所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  30. 根据权利要求2-29中任一项所述的用途，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
  31. 根据权利要求2-30中任一项所述的用途，其中所述神经退行性疾病包括早老性痴呆。
  32. 根据权利要求2-31中任一项所述的用途，其中所述神经退行性疾病包括早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。
  33. 根据权利要求3-32中任一项所述的用途，其中所述受试者包括哺乳动物。
  34. 根据权利要求3-33中任一项所述的用途，其中所述受试者包括人。
  35. 根据权利要求3-34中任一项所述的用途，其中所述受试者包括神经退行性疾病患者和/或认知障碍患者。
  36. 根据权利要求3-35中任一项所述的用途，其中所述受试者包括阿尔兹海默症患者。
  37. 根据权利要求3-36中任一项所述的用途，其中所述受试者处于老龄阶段。
  38. 根据权利要求1-37中任一项所述的用途，其中所述试剂被配制为适于口服施用和/或注射施用。
  39. 根据权利要求1-38中任一项所述的用途，其中所述调控剂包括小分子化合物、聚合物和/或生物大分子。
  40. 根据权利要求1-39中任一项所述的用途，其中所述调控剂包括抗体或其抗原结合片段。
  41. 根据权利要求1-40中任一项所述的用途，其中所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的抗体或其抗原结合片段。
  42. 根据权利要求1-41中任一项所述的用途，其中所述调控剂包括反义寡核苷酸。
  43. 根据权利要求1-42中任一项所述的用途，其中所述调控剂包括siRNA。
  44. 根据权利要求1-43中任一项所述的用途，其中所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的siRNA。
  45. 根据权利要求1-44中任一项所述的用途，其中所述调控剂包含SEQ ID NO.84-87中任一项所示的核苷酸序列。
  46. 预防和/或治疗认知障碍的方法，其包括以下的步骤：使有需要的受试者中，DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的表达水平和/或生物学活性降低。
  47. 预防和/或治疗神经退行性疾病的方法，其包括以下的步骤：使有需要的受试者中，DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的表达水平和/或生物学活性降低。
  48. 根据权利要求46-47中任一项所述的方法，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的表达水平和/或生物学活性相比，所述DIR和/或其功能片段的表达水平和/或生物学活性降低至少约10％。
  49. 根据权利要求46-48中任一项所述的方法，其中所述表达水平包括编码所述DIR/或其功能片段的基因的表达水平、编码所述DIR/或其功能片段的基因的转录水平和/或所述DIR/或其功能片段的表达水平。
  50. 根据权利要求46-49中任一项所述的方法，其中所述表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  51. 根据权利要求46-50中任一项所述的方法，其中所述表达水平通过利用选自下组的物质衡量：能够特异性扩增编码所述DIR/或其功能片段的基因的引物、与编码所述DIR/或其功能片段的基因特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的小分子、与所述DIR/或其功能片段特异性结合的探针和与所述DIR/或其功能片段特异性结合的多肽。
  52. 根据权利要求46-51中任一项所述的方法，其中所述DIR的功能片段保留所述DIR的至少部分的生物学活性。
  53. 根据权利要求46-52中任一项所述的方法，其中所述生物学活性包括影响神经元的兴奋性和/或抑制神经元的活性。
  54. 根据权利要求46-53中任一项所述的方法，其中所述生物学活性包括能够降低兴奋性突触后电流(EPSC)的频率，和/或能够降低EPSC的幅度。
  55. 根据权利要求54所述的方法，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者中的兴奋性突触后电流(EPSC)的频率降低，和/或使受试者中的EPSC的幅度降低。
  56. 根据权利要求46-55中任一项所述的方法，其中所述生物学活性包括影响认知能力。
  57. 根据权利要求46-56中任一项所述的方法，其中所述生物学活性包括参与与Aβ沉积相关的信号通路，和/或，参与与Tau缠结产生相关的信号通路。
  58. 根据权利要求46-57中任一项所述的方法，其中所述生物学活性包括通过gelsolin诱导Aβ沉积和/或淀粉样斑块形成。
  59. 根据权利要求58所述的方法，其中所述DIR/或其功能片段通过与gelsolin结合诱导Aβ沉积和/或淀粉样斑块形成。
  60. 根据权利要求46-59中任一项所述的方法，其中所述DIR和/或其功能片段的表达水平与Aβ的表达水平正相关。
  61. 根据权利要求46-60中任一项所述的方法，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者的认知能力降低。
  62. 根据权利要求46-61中任一项所述的方法，其中所述DIR/或其功能片段来源于哺乳动物。
  63. 根据权利要求46-62中任一项所述的方法，其中所述DIR/或其功能片段来源于灵长类动物。
  64. 根据权利要求46-63中任一项所述的方法，其中所述DIR包含SEQ ID NO.1所示的氨基酸序列。
  65. 根据权利要求46-64中任一项所述的方法，其中所述DIR的功能片段包含DDIT4L中滞留的内含子所编码的氨基酸序列。
  66. 根据权利要求46-65中任一项所述的方法，其中所述DIR的功能片段包含SEQ ID NO.4-5中任一项所示的氨基酸序列。
  67. 根据权利要求46-66中任一项所述的方法，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  68. 根据权利要求46-67中任一项所述的方法，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  69. 根据权利要求46-68中任一项所述的方法，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  70. 根据权利要求46-69中任一项所述的方法，其中所述认知障碍包括遗忘型MCI多认知域受损(aMCI-m)。
  71. 根据权利要求47-70中任一项所述的方法，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  72. 根据权利要求47-71中任一项所述的方法，其中所述神经退行性疾病包括由神经元死亡和 胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  73. 根据权利要求47-72中任一项所述的方法，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
  74. 根据权利要求47-73中任一项所述的方法，其中所述神经退行性疾病包括早老性痴呆。
  75. 根据权利要求47-74中任一项所述的方法，其中所述神经退行性疾病包括早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。
  76. 根据权利要求46-75中任一项所述的方法，其中所述受试者包括哺乳动物。
  77. 根据权利要求46-76中任一项所述的方法，其中所述受试者包括人。
  78. 根据权利要求46-77中任一项所述的方法，其中所述受试者包括神经退行性疾病患者和/或认知障碍患者。
  79. 根据权利要求46-78中任一项所述的方法，其中所述受试者包括阿尔兹海默症患者。
  80. 根据权利要求46-79中任一项所述的方法，其中所述受试者处于老龄阶段。
  81. 根据权利要求46-80中任一项所述的方法，其中所述方法包括以下步骤：向有需要的受试者施用调控剂，所述调控剂能够降低所述DIR和/或其功能片段，和/或编码所述DIR和/或其功能片段的核酸的表达水平和/或生物学活性。
  82. 根据权利要求81所述的方法，其中所述施用包括口服施用和/或注射施用。
  83. 根据权利要求81-82中任一项所述的方法，其中所述调控剂包括小分子化合物、聚合物和/或生物大分子。
  84. 根据权利要求46-83中任一项所述的方法，其中所述调控剂包括抗体或其抗原结合片段。
  85. 根据权利要求46-84中任一项所述的方法，其中所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的抗体或其抗原结合片段。
  86. 根据权利要求46-85中任一项所述的方法，其中所述调控剂包括反义寡核苷酸。
  87. 根据权利要求46-86中任一项所述的方法，其中所述调控剂包括siRNA。
  88. 根据权利要求46-87中任一项所述的方法，其中所述调控剂包括特异性结合DNA损伤诱导性转录物4样转录物的内含子滞留剪切产物DIR和/或其功能片段的siRNA。
  89. 根据权利要求46-88中任一项所述的方法，其中所述调控剂包含SEQ ID NO.84-87中任一项所示的核苷酸序列。
  90. 筛选能够预防和/或治疗认知障碍和/或治疗神经退行性疾病的药物的方法，其包括以下的步骤：检测候选药物对受试者中DIR和/或其功能片段的表达水平和/或生物学活性的影响，其中在施用所述候选药物后，所述DIR和/或其功能片段的表达水平和/或生物学活性降低，则所述候选药物能够预防和/或治疗认知障碍和/或治疗神经退行性疾病。
  91. 根据权利要求90所述的方法，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的表达水平和/或生物学活性相比，所述DIR和/或其功能片段的表达水平和/或生物学活性降低至少约10％。
  92. 根据权利要求90-91中任一项所述的方法，其中所述表达水平包括编码所述DIR/或其功能片段的基因的表达水平、编码所述DIR/或其功能片段的基因的转录水平和/或所述DIR/或其功能片段的表达水平。
  93. 根据权利要求90-92中任一项所述的方法，其中所述表达水平通过实施选自下组的试验衡量：qPCR、qRT-PCR、杂交分析、RNA印迹法、斑点印迹法、原位杂交、胶凝电泳、毛细管电泳、柱色谱、蛋白质印迹法、免疫组织化学、免疫染色和质谱。
  94. 根据权利要求90-93中任一项所述的方法，其中所述表达水平通过利用选自下组的物质衡量：能够特异性扩增编码所述DIR/或其功能片段的基因的引物、与编码所述DIR/或其功能片段的基因特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的核酸分子、与所述DIR/或其功能片段特异性结合的小分子、与所述DIR/或其功能片段特异性结合的探针和与所述DIR/或其功能片段特异性结合的多肽。
  95. 根据权利要求90-94中任一项所述的方法，其中所述DIR的功能片段保留所述DIR的至少部分的生物学活性。
  96. 根据权利要求95所述的方法，其中所述生物学活性包括影响神经元的兴奋性和/或抑制神经元的活性。
  97. 根据权利要求90-96中任一项所述的方法，其中所述生物学活性包括能够降低兴奋性突触后电流(EPSC)的频率，和/或能够降低EPSC的幅度。
  98. 根据权利要求90-97中任一项所述的方法，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者中的兴奋性突触后电流(EPSC)的频率降低，和/或使受试者中的EPSC的幅度降低。
  99. 根据权利要求90-98中任一项所述的方法，其中所述生物学活性包括影响认知能力。
  100. 根据权利要求90-99中任一项所述的方法，其中所述生物学活性包括参与与Aβ沉积相关的信号通路，和/或，参与与Tau缠结产生相关的信号通路。
  101. 根据权利要求90-100中任一项所述的方法，其中所述生物学活性包括通过gelsolin诱导Aβ沉积和/或淀粉样斑块形成。
  102. 根据权利要求101所述的方法，其中所述DIR/或其功能片段通过与gelsolin结合诱导Aβ沉积和/或淀粉样斑块形成。
  103. 根据权利要求90-102中任一项所述的方法，其中所述DIR和/或其功能片段的表达水平与Aβ的表达水平正相关。
  104. 根据权利要求90-103中任一项所述的方法，其中所述降低包括与所述受试者中原始的所述DIR和/或其功能片段的生物学活性相比，施用所述DIR和/或其功能片段和/或编码所述DIR和/或其功能片段的核酸，使受试者的认知能力降低。
  105. 根据权利要求90-104中任一项所述的方法，其中所述DIR/或其功能片段来源于哺乳动物。
  106. 根据权利要求90-105中任一项所述的方法，其中所述DIR/或其功能片段来源于灵长类动物。
  107. 根据权利要求90-106中任一项所述的方法，其中所述DIR包含SEQ ID NO.1所示的氨基酸序列。
  108. 根据权利要求90-107中任一项所述的方法，其中所述DIR的功能片段包含DDIT4L中滞留的内含子所编码的氨基酸序列。
  109. 根据权利要求90-108中任一项所述的方法，其中所述DIR的功能片段包含SEQ ID NO.4-5中任一项所示的氨基酸序列。
  110. 根据权利要求90-109中任一项所述的方法，其中所述认知障碍包括正常衰老引起的认知障碍、lews身体痴呆(LBD)、额颞叶痴呆和/或血管性痴呆。
  111. 根据权利要求90-110中任一项所述的方法，其中所述认知障碍的诱导疾病包括阿尔兹海默症、多发性梗死型、帕金森氏症、艾滋病和/或克雅氏病(CJD)。
  112. 根据权利要求90-111中任一项所述的方法，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
  113. 根据权利要求90-112中任一项所述的方法，其中所述认知障碍包括遗忘型MCI多认知域受损(aMCI-m)。
  114. 根据权利要求90-113中任一项所述的方法，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  115. 根据权利要求90-114中任一项所述的方法，其中所述神经退行性疾病包括由神经元死亡和胶质细胞稳态引起的神经退行性疾病、由衰老引起的神经退行性疾病、由CNS细 胞功能被影响引起的神经退行性疾病、由细胞间异常通信引起的神经退行性疾病和/或由细胞运动受损引起的神经退行性疾病。
  116. 根据权利要求90-115中任一项所述的方法，其中所述神经退行性疾病包括阿尔兹海默症、帕金森氏症、多发性硬化(MS)、肌萎缩性脊髓侧索硬化症(ALS)和/或亨廷顿病(HD)。
  117. 根据权利要求90-116中任一项所述的方法，其中所述神经退行性疾病包括早老性痴呆。
  118. 根据权利要求90-117中任一项所述的方法，其中所述神经退行性疾病包括早老性痴呆早期、早老性痴呆中期和/或早老性痴呆晚期。
  119. 根据权利要求90-118中任一项所述的方法，其中所述受试者包括哺乳动物。
  120. 根据权利要求90-119中任一项所述的方法，其中所述受试者包括神经退行性疾病患者和/或认知障碍患者。
  121. 根据权利要求90-120中任一项所述的方法，其中所述受试者包括阿尔兹海默症患者。
  122. 根据权利要求90-121中任一项所述的方法，其中所述受试者处于老龄阶段。
  123. 根据权利要求90-122中任一项所述的方法，其中所述施用包括口服施用和/或注射施用。
  124. 根据权利要求90-123中任一项所述的方法，其中所述候选药物包括小分子化合物、聚合物和/或生物大分子。
  125. 分离的抗原结合蛋白，其具有以下性质：在ELISA测定中，以约10ng/ml以上的工作浓度与人DIR和/或其功能片段特异性结合。
  126. 根据权利要求125所述的抗原结合蛋白，其包含LCDR2，所述LCDR2包含SEQ ID NO:74所示的氨基酸序列。
  127. 根据权利要求126所述的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:26或16所示的氨基酸序列。
  128. 根据权利要求125-127中任一项所述的抗原结合蛋白，其包含LCDR1，所述LCDR1包含SEQ ID NO:73所示的氨基酸序列。
  129. 根据权利要求128所述的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:25、52、15中任一项所示的氨基酸序列。
  130. 根据权利要求125-129中任一项所述的抗原结合蛋白，其包含LCDR3，所述LCDR3包含SEQ ID NO:75所示的氨基酸序列。
  131. 根据权利要求130所述的抗原结合蛋白，所述LCDR3包含SEQ ID NO:27、53、67、 17中任一项所示的氨基酸序列。
  132. 根据权利要求125-131中任一项所述的抗原结合蛋白，其包含LCDR1、LCDR2和LCDR3，其中，
    a)所述LCDR1包含SEQ ID NO:25所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:27所示的氨基酸序列；
    b)所述LCDR1包含SEQ ID NO:52所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:53所示的氨基酸序列；
    c)所述LCDR1包含SEQ ID NO:52所示的氨基酸序列，所述LCDR2包含SEQ ID NO:26所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:67所示的氨基酸序列；或者，
    d)所述LCDR1包含SEQ ID NO:15所示的氨基酸序列，所述LCDR2包含SEQ ID NO:16所示的氨基酸序列，且所述LCDR3包含SEQ ID NO:17所示的氨基酸序列。
  133. 根据权利要求125-132中任一项所述的抗原结合蛋白，其包含HCDR1，所述HCDR1包含SEQ ID NO:70所示的氨基酸序列。
  134. 根据权利要求133所述的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:20、35、56、10中任一项所示的氨基酸序列。
  135. 根据权利要求125-134中任一项所述的抗原结合蛋白，其包含HCDR2，所述HCDR2包含SEQ ID NO:71所示的氨基酸序列。
  136. 根据权利要求135所述的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:21、36、42、48、11中任一项所示的氨基酸序列。
  137. 根据权利要求125-136中任一项所述的抗原结合蛋白，其包含HCDR3，所述HCDR3包含SEQ ID NO:70所示的氨基酸序列。
  138. 根据权利要求137所述的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:22、30、37、43、49、57、62、12中任一项所示的氨基酸序列。
  139. 根据权利要求125-138中任一项所述的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，其中，
    a)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:22所示的氨基酸序 列；
    b)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:21所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:30所示的氨基酸序列；
    c)所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:37所示的氨基酸序列；
    d)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:42所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:43所示的氨基酸序列；
    e)所述HCDR1包含SEQ ID NO:20所示的氨基酸序列，所述HCDR2包含SEQ ID NO:48所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:49所示的氨基酸序列；
    f)所述HCDR1包含SEQ ID NO:56所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:57所示的氨基酸序列；
    g)所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:62所示的氨基酸序列；
    h)所述HCDR1包含SEQ ID NO:35所示的氨基酸序列，所述HCDR2包含SEQ ID NO:42所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:37所示的氨基酸序列；
    i)所述HCDR1包含SEQ ID NO:56所示的氨基酸序列，所述HCDR2包含SEQ ID NO:36所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:57所示的氨基酸序列；或者，
    j)所述HCDR1包含SEQ ID NO:10所示的氨基酸序列，所述HCDR2包含SEQ ID NO:11所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:12所示的氨基酸序列。
  140. 根据权利要求125-139中任一项所述的抗原结合蛋白，其包含重链可变区VH，所述VH包含SEQ ID NO:13、23、31、38、44、50、58、63、65中任一项所示的氨基酸序列。
  141. 根据权利要求125-140中任一项所述的抗原结合蛋白，其包含轻链可变区VL，所述 VL包含SEQ ID NO:18、28、33、40、46、54、60、68中任一项所示的氨基酸序列。
  142. 根据权利要求125-141中任一项所述的抗原结合蛋白，其包含重链可变区VH和轻链可变区VL，其中：
    a)所述VH包含SEQ ID NO:23所示的氨基酸序列，且所述VL包含SEQ ID NO:28所示的氨基酸序列；
    b)所述VH包含SEQ ID NO:31所示的氨基酸序列，且所述VL包含SEQ ID NO:33所示的氨基酸序列；
    c)所述VH包含SEQ ID NO:38所示的氨基酸序列，且所述VL包含SEQ ID NO:40所示的氨基酸序列；
    d)所述VH包含SEQ ID NO:44所示的氨基酸序列，且所述VL包含SEQ ID NO:46所示的氨基酸序列；
    e)所述VH包含SEQ ID NO:50所示的氨基酸序列，且所述VL包含SEQ ID NO:54所示的氨基酸序列；
    f)所述VH包含SEQ ID NO:58所示的氨基酸序列，且所述VL包含SEQ ID NO:60所示的氨基酸序列；
    g)所述VH包含SEQ ID NO:63所示的氨基酸序列，且所述VL包含SEQ ID NO:60所示的氨基酸序列；
    h)所述VH包含SEQ ID NO:65所示的氨基酸序列，且所述VL包含SEQ ID NO:54所示的氨基酸序列；
    i)所述VH包含SEQ ID NO:58所示的氨基酸序列，且所述VL包含SEQ ID NO:68所示的氨基酸序列；或者，
    j)所述VH包含SEQ ID NO:13所示的氨基酸序列，且所述VL包含SEQ ID NO:18所示的氨基酸序列。
  143. 根据权利要求125-142中任一项所述的抗原结合蛋白，其包含重链恒定区，且所述重链恒定区包括源自IgG的恒定区。
  144. 根据权利要求143所述的抗原结合蛋白，其中所述重链恒定区包含源自选自下组蛋白的恒定区：IgG1、IgG2、IgG3和IgG4。
  145. 根据权利要求125-144中任一项所述的抗原结合蛋白，其包含轻链恒定区，且所述轻链恒定区包括源自Igκ的恒定区或源自Igλ的恒定区。
  146. 根据权利要求145所述的抗原结合蛋白，其中所述轻链恒定区包括源自人Igκ的恒定区。
  147. 根据权利要求125-146中任一项所述的抗原结合蛋白，其包括抗体或其抗原结合片段。
  148. 根据权利要求147所述的抗原结合蛋白，其中所述抗原结合片段选自下组：Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv，VHH和/或dAb。
  149. 根据权利要求147-148中任一项所述的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、鼠源抗体和嵌合抗体。
  150. 多肽，其包含权利要求125-149中任一项所述的分离的抗原结合蛋白。
  151. 免疫缀合物，其包含权利要求125-149中任一项所述的分离的抗原结合蛋白或权利要求150所述的多肽。
  152. 分离的核酸分子，其编码权利要求125-149中任一项所述的分离的抗原结合蛋白，或者权利要求150所述的多肽。
  153. 载体，其包含权利要求152所述的分离的核酸分子。
  154. 细胞，其包含权利要求125-149中任一项所述的分离的抗原结合蛋白，权利要求150所述的多肽，权利要求151所述的免疫缀合物，权利要求152所述的分离的核酸分子和/或权利要求153所述的载体。
  155. 制备权利要求125-149中任一项所述的分离的抗原结合蛋白或权利要求150所述的多肽的方法，所述方法包括在使得权利要求125-149中任一项所述的分离的抗原结合蛋白或权利要求150所述的多肽表达的条件下，培养权利要求154所述的细胞。
  156. 药物组合物，其包含权利要求125-149中任一项所述的分离的抗原结合蛋白，权利要求150所述的多肽，权利要求151所述的免疫缀合物，权利要求152所述的分离的核酸分子，权利要求153所述的载体，权利要求154所述的细胞，和/或药学上可接受的佐剂和/或赋形剂。
  157. 权利要求125-149中任一项所述的分离的抗原结合蛋白和/或权利要求150所述的多肽在制备预防和/或治疗疾病或病症的药物中的用途，其中所述疾病或病症包括认知障碍和/或神经退行性疾病。
  158. 根据权利要求157所述的用途，其中所述神经退行性疾病包括急性神经退行性疾病和慢性神经退行性疾病。
  159. 根据权利要求157-158中任一项所述的用途，其中所述认知障碍包括早期认知障碍(MCI)、中期认知障碍和晚期认知障碍。
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