WO2023143610A2 - 载脂蛋白e的c端片段及其在抑制γ分泌酶酶活中的应用 - Google Patents

载脂蛋白e的c端片段及其在抑制γ分泌酶酶活中的应用 Download PDF

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WO2023143610A2
WO2023143610A2 PCT/CN2023/073849 CN2023073849W WO2023143610A2 WO 2023143610 A2 WO2023143610 A2 WO 2023143610A2 CN 2023073849 W CN2023073849 W CN 2023073849W WO 2023143610 A2 WO2023143610 A2 WO 2023143610A2
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apoe
amino acid
seq
acid sequence
app
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陈椰林
侯祥龙
耿泱
张雪馨
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斯莱普泰(上海)生物医药科技有限公司
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to a C-terminal fragment of apolipoprotein E (apolipoprotein E, ApoE) and its application in inhibiting the enzyme activity of ⁇ -secretase.
  • apolipoprotein E apolipoprotein E, ApoE
  • ⁇ -secretase is a cleaving enzyme for more than 100 type I transmembrane proteins and is involved in a variety of biological processes. Abnormal differentiation of T-cells and B-cells, hemorrhagic diarrhea, skin and hair defects, and Alzheimer's disease (AD) are all associated with the abnormality of ⁇ -secretase.
  • ⁇ -secretase is a complex of four proteins: NCT, APH1, PEN2 and presenilin1 (PS1) or presenilin2 (PS2).
  • PS1 or PS2 The cleavage active center of ⁇ -secretase is PS1 or PS2.
  • AD Alzheimer's disease
  • AD patients mainly manifest symptoms such as progressive memory impairment, cognitive dysfunction, personality changes, and language barriers, and further develop into loss of self-care ability and eventually death.
  • my country is the country with the largest number of AD patients in the world. As of 2019, there are more than 10 million AD patients in my country, showing an increasing trend every year. People over the age of 80 have a 40% chance of developing AD. And the treatment cost of AD is also very high, causing great burden to the family and society.
  • APP can also be cleaved sequentially by ⁇ and ⁇ secretases, which is the non-amyloid metabolic pathway of APP.
  • ⁇ -CTF the cut fragment
  • ⁇ and ⁇ enzymes compete with each other, increasing the enzyme cleavage of ⁇ secretase, and the production of A ⁇ decreases; similarly, reducing the enzyme cleavage of ⁇ secretase will also reduce the production of A ⁇ , slow down the formation of amyloid plaques, and finally slow down even Prevention of AD and amyloid cerebrovascular disease.
  • drugs targeting ⁇ -secretase have been tested in clinical trials.
  • ApoE is a protein widely secreted in the human body and can be co-expressed with APP in neurons in the brain.
  • ApoE2, ApoE3, and ApoE4 There are three isomers of human ApoE, ApoE2, ApoE3, and ApoE4; they are highly similar in structure and function, and can be roughly divided into three functional domains, which are the N-terminal functional domain containing the receptor binding site and the lipid-binding domain. The C-terminal functional domain of the binding site, and the middle flexible hinge region connecting the functional domains at both ends.
  • ApoE2, ApoE3 and ApoE4 differ only in two amino acid residues in the N-terminal domain, and have identical amino acid sequences in the C-terminal domain.
  • ApoE2 has a protective effect and can reduce the risk of AD
  • ApoE4 is the most important risk factor for AD, which can not only significantly increase the risk of AD, but also significantly advance the age of onset of AD and aggravate AD.
  • the invention provides an isolated ApoE construct comprising an ApoE polypeptide that: (1) comprises a fragment from the amino acid sequence shown in SEQ ID NO: 12, the fragment (or the ApoE Polypeptide)
  • the first amino acid residue at the N-terminal is the amino acid residue at any one of positions 215-221 of SEQ ID NO: 12, and the last amino acid residue at the C-terminal of the fragment (or the ApoE polypeptide) is SEQ ID NO: 12
  • said ApoE polypeptide or said (1) fragment from the amino acid sequence shown in SEQ ID NO: 12 comprises (or consists of, or substantially Consisting of) any of the following sequences: (i) the amino acid sequence of SEQ ID NO: 12 amino acid residues 215-299; (ii) the amino acid sequence of SEQ ID NO: 12 amino acid residues 216-299; ( iii) SEQ ID NO: the amino acid sequence of amino acid residues 217-299 of 12; (iv) SEQ ID NO: the amino acid sequence of amino acid residues 218-299 of 12; (v) SEQ ID NO: 219- of 12 The amino acid sequence of the 299th amino acid residue; (vi) SEQ ID NO: the amino acid sequence of the 12th 220-299 amino acid residue; (vii) SEQ ID NO: the amino acid sequence of the 12th 221-299th amino acid residue; ( viii) SEQ ID NO: the amino acid sequence of 216-294 amino acid residue
  • said ApoE polypeptide or said (1) fragment from the amino acid sequence shown in SEQ ID NO: 12 comprises (or consists of, or substantially Consisting of) any amino acid sequence in SEQ ID NOs: 13, 18-34, 78 and 87.
  • the ApoE polypeptide or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in the (1) comprises (or consists of, or consists essentially of) SEQ ID NOs: 31-34, The amino acid sequence shown in any one of 78 and 87 (eg, SEQ ID NO: 33).
  • the ApoE polypeptide comprises (or consists of, or consists essentially of) the composition of SEQ ID NOs: 13, 18-34, 78 and 87 Any amino acid sequence is an amino acid sequence having at least about 70% (eg, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity.
  • the ApoE polypeptide contains a non-human primate sequence, such as a highly homologous and conserved sequence with any amino acid sequence in SEQ ID NOs: 13, 18-34, 78 and 87.
  • the ApoE polypeptide comprises a mutation (e.g., addition, deletion, lost or replaced): R226, D227, R228, L229, D230, Q235, E238, V239, R240, K242, E244, E245, Q246, A247, Q248, Q249, I250, R251, L252, Q253, A254, E255, Q275 , V280, V287, T289, S290, A291, P293, V294, P295, S296, D297, N298, H299, R251+T289, R251+T289+A291, S290+P293+V294+P295+S296+D297+N298+ H299 , R226+D227+R228+L229+D230, E238+V239+R240+K242, or E244+E245+Q246
  • the ApoE polypeptide comprises mutations at one or more amino acid positions relative to the amino acid sequence shown in SEQ ID NO: 12: R226A, D227A, R228A, L229A, D230A, Q235A, E238A, V239A, R240A , K242A, E244del, E245del, Q246del, A247del, Q248del, Q249del, I250del, R251S, L252del, Q253del, A254del, E255A, Q275A, V280A, V287A, T289A, S290A, A291T, P293A, V294A, P295A, S296A, D297A, N298A , H299A, R251A+T289A, R251S+T289A+A291T, S290A+P293A+V294A+
  • the ApoE polypeptide comprises (or consists of, or consists essentially of) SEQ ID NOs: 46-52, 57-59, 62 and 76 any amino acid sequence.
  • the ApoE construct further comprises SEQ ID NO: 12 1-167 or 1-205 or 1-205 at the N-terminal of the ApoE polypeptide.
  • the ApoE construct comprises (or consists of, or consists essentially of) the amino acid sequence shown in SEQ ID NO: 60 or 61.
  • the ApoE construct further comprises a membrane-penetrating peptide at the N-terminus or C-terminus of the ApoE polypeptide.
  • the membrane-penetrating peptide includes (or consists of, or consists essentially of) any amino acid sequence in SEQ ID NOs: 35-45, 63-65, and 84-86, for example, includes (or consists of) It consists of, or consists essentially of) the amino acid sequence shown in SEQ ID NO: 63 or 65.
  • the ApoE construct further comprises a signal peptide at the N-terminus of the ApoE polypeptide.
  • the signal peptide comprises (or consists of, or consists essentially of) any amino acid sequence selected from SEQ ID NOs: 66-68.
  • the present invention provides an isolated ApoE construct comprising element (1): a penetrating peptide, and element (2): the full-length sequence of ApoE2 (such as SEQ ID NO: 12 ) or the full-length sequence of ApoE3 (such as SEQ ID NO: 89) or at least a fragment including SEQ ID NO: 12 amino acid residues 221-274; wherein, the elements (1) and elements (2) are passed between Peptide bonds link directly, or via a linker sequence.
  • the penetrating peptide is selected from any amino acid sequence in SEQ ID NOs: 35-45, 63-65 and 84-86, such as the amino acid sequence shown in SEQ ID NO: 63 or 65.
  • the fragment of element (2) at least comprising amino acid residues at positions 221-274 of SEQ ID NO: 12 is selected from any of the fragments described in the present invention comprising positions 221-274 of SEQ ID NO: 12 Amino acid residues of the ApoE polypeptide.
  • the amino acid sequence of the fragment comprising at least amino acid residues 221-274 of SEQ ID NO: 12 described in element (2) is as shown in any one of SEQ ID NOs: 13, 18-34 and 78 .
  • the fragment comprising at least SEQ ID NO: 12 amino acid residues 221-274 described in element (2) contains the N-terminal domain of ApoE2 (SEQ ID NO: 12 amino acid residues 1-167 base) and hinge region (SEQ ID NO: 12 amino acid residues 168-205), and the C-terminus at least includes a fragment of SEQ ID NO: 12 amino acid residues 221-274.
  • the element (2) comprises any of the following sequences: SEQ ID NO: 12 1-274, 1-279, 1-280, 1-281, 1-281 282, 1-283, 1-284, 1-285, 1-286, 1-287, 1-288, 1-289, 1-290 , No. 1-291, No. 1-292, No. 1-293, No. 1-294, No. 1-295, No. 1-296, No. 1-297, No. 1-298, or Amino acid residues 1-299.
  • the present invention also provides the isolated nucleic acid and carrier (for example, cloning vector or expression vector) of the protein part of the ApoE construct described in the above separation of encoding, and the expression vector of the protein part of the ApoE construct described in the above separation A host cell, or a host cell comprising the nucleic acid or vector.
  • the vector contains a neuron-specific promoter at the 5' end of the isolated nucleic acid.
  • the neuron-specific promoter is selected from the synapsin promoter, the thy-1 promoter, and the calmodulin-dependent protein kinase II-alpha promoter.
  • the vector is a recombinant AAV or lentivirus expression vector.
  • the host cell is a neuron (eg, a human neuron, a non-human primate neuron, or a rodent neuron).
  • the ApoE construct comprises (or consists of, or consists essentially of) the full-length amino acid sequence of ApoE2 (eg, SEQ ID NO: 12) or ApoE3 (eg, SEQ ID NO: 89).
  • the present invention also provides a pharmaceutical composition, which contains (i) the isolated ApoE construct, isolated nucleic acid, vector, or host cell described in any embodiment herein, and (ii) a pharmaceutically acceptable carrier .
  • the present invention also provides an isolated ApoE construct, an isolated nucleic acid, a vector, a host cell, or a pharmaceutical composition according to any of the embodiments herein prepared for treating or preventing an individual (such as a human) with ⁇ -secretase Drug application for diseases related to enzymatic cleavage activity.
  • the present invention also provides a method for treating or preventing a disease associated with ⁇ -secretase enzyme cleavage activity in an individual (such as a human), comprising administering to the individual an effective dose of the isolated ApoE described in any embodiment herein construct, isolated nucleic acid, vector, host cell or pharmaceutical composition.
  • the medicament or method inhibits the enzymatic cleavage activity of ⁇ -secretase, thereby treating or preventing the diseases related to the enzymatic cleavage activity of ⁇ -secretase.
  • the disease is selected from the group consisting of neurodegenerative diseases, tumors or cancers, inflammatory diseases and renal diseases.
  • the disease is selected from the group consisting of Alzheimer's disease and related diseases, amyloid cerebrovascular disease, Down's syndrome, other neurodegenerative diseases associated with aging (such as frontotemporal dementia), head and neck cancers (such as head and neck squamous cell carcinoma and oral squamous cell carcinoma), breast cancer, liver cancer, pancreatic cancer (including metastatic pancreatic cancer), ovarian cancer, lung cancer (such as non-small cell lung cancer, lung adenocarcinoma and small cell lung cancer), glioma (eg, malignant glioma), fibroma, lymphoma (eg, B-cell lymphoma), osteosarcoma, gastric cancer, bladder cancer, asthma (eg, allergic asthma), pneumonia , airway inflammation, acute kidney injury, clear cell renal cell carcinoma, renal fibrosis, and obstructive nephropathy.
  • Alzheimer's disease and related diseases such as frontotemporal dementia
  • head and neck cancers such as head and neck squam
  • FIG. 1 ApoE attenuates the amyloid metabolism of APP and increases its non-amyloid metabolite ( ⁇ -CTF) through its C-terminal fragment (ApoE CT).
  • Panels A and B Overexpression of ApoE2 and ApoE3 in HEK 293T cells can significantly increase ⁇ -CTF produced by co-expressed APP via non-amyloid metabolic pathway ( ⁇ cleavage), while ApoE4 cannot.
  • Panels AG HEK 293T cells.
  • Panel H Cerebral cortex in mice ApoE CT expressed by lentivirus in primary cultured neurons of layer and hippocampus can reduce the endogenous production of A ⁇ 40 in neurons.
  • Panel I ApoE CT expressed in human neurons induced by a pluripotent stem cell line using lentivirus also reduces neuronal production of endogenous A ⁇ 40.
  • the proteins APP, ⁇ -CTF and ApoE were detected by WesternBlot, and A ⁇ was detected by ELISA.
  • FIG. 2 ApoE2 and ApoECT in culture medium do not increase non-amyloid metabolites of APP in cells.
  • Panel A ApoE2 and ApoE CT proteins contained in the culture medium were detected by western blot.
  • Panel D Immunofluorescence experiments showed that after HEK 293T cells were co-incubated with cell culture medium containing ApoE2 or ApoECT protein, a large amount of ApoE2 or ApoECT protein could not be detected in the cells, and only a small part was attached to the surface of individual cells.
  • ApoECT which was transiently expressed in HEK 293T cells using liposomes, was present in large quantities in the cells.
  • Panel E Immunofluorescence experiments showed that after co-incubation with the culture solution containing ApoE2 or ApoECT protein, the existence of a large amount of ApoE2 or ApoECT protein could not be detected in the primary cultured neurons of mice, and only a small part of ApoE was attached to individual neurons. neuron surface.
  • ApoECT expressed by lentivirus exists in large numbers in neurons. MAP2 marks neurons and DAPI marks nuclei.
  • FIG. 3 ApoECT reduces the core sequence of A ⁇ .
  • Panels A, B and C Effect of overexpression of different ApoECT mutants in HEK 293T cells on APP production of A ⁇ .
  • Panel A CT NA1/2/3/4//5: ApoE original 1, 2, 3, 4 or 5 amino acid residues were added to the N-terminus of ApoE CT.
  • CT ND3/4 Delete 3 or 4 amino acid residues at the N-terminus of ApoE CT.
  • CT CD5/10 Delete 5 or 10 amino acid residues at the C-terminus of ApoE CT.
  • ELISA measures the effect of different ApoECT mutants on A ⁇ 40.
  • Panel B CT ND1/2//5: 1, 2 or 5 amino acid residues were deleted at the N-terminus of ApoE CT.
  • ELISA measures the effect of different ApoECT mutants on A ⁇ 40.
  • Panel C CT ND3CD10/11/12/13/14/15/20/25: 10, 11, 12, 13, 14, 15 at the C-terminus while deleting 3 amino acid residues at the N-terminus of ApoE CT , 20 or 25 amino acids.
  • ELISA measures the effect of different ApoECT mutants on A ⁇ 40.
  • N 5 (panels A and B) or 7 (panel C), one sample t-test.
  • FIG. 4 ApoE CT does not change the expression of APP secretase.
  • Panel A ApoE CT does not affect the expression of APP metabolic enzyme mRNA.
  • Panel B ApoE CT does not affect the protein expression of APP metabolizing enzymes.
  • ADAM10 ⁇ -secretase of APP
  • BACE1 ⁇ -secretase of APP
  • PS1 and PS2 core active subunit proteins of ⁇ -secretase.
  • FIG. 5 ApoE CT mimics the effect of ⁇ -secretase inhibitors on APP cleavage and inhibits ⁇ -cleavage of ⁇ -CTF.
  • Panels A and B Overexpression of ⁇ -secretase active center protein PS1 can reduce ⁇ -CTF production, while expression of ApoE CT or treatment with 1 ⁇ M PF03084014 ( ⁇ -secretase inhibitor) can increase ⁇ -CTF production.
  • FIG. 6 The cleavage of APP by ApoECT requires the presence of ⁇ -secretase.
  • Panels A and D In HEK 293T cells in which the ⁇ -secretase active center proteins PS1 and PS2 were knocked out (PS1/2 dKO), ⁇ -CTF was greatly increased. At this time, the overexpressed ApoE CT did not further increase ⁇ -CTF, The statistical results are in panel D (the two columns labeled GFP1).
  • FIG. 7 ApoE2 purified in vitro directly inhibits ⁇ -cleavage of APP in a concentration-dependent manner.
  • Panel A Western blot showing isolated and purified ⁇ -CTF, ApoE2, ApoE2 NT, PS1, NCT, PEN2 and GFP.
  • Panel C Purified ApoE2 inhibits cleavage of ⁇ -CTF by ⁇ -secretase in a dose-dependent manner, reducing A ⁇ 40 production.
  • FIG. 8 ApoE CT does not inhibit ⁇ -cleavage of APLP1.
  • 1 ⁇ M PF03084014 ⁇ -secretase inhibitor
  • FIG. 9 Overexpressed ApoE CT in neurons reduces amyloid plaques in the brain of a 5xFAD mouse model.
  • Panel A Representative images of co-staining of amyloid plaques (THS markers) and ApoE CT or GFP in the subiculum region of the 5xFAD mouse model.
  • ApoE CT was expressed in neurons using the adeno-associated virus (AAV) expression system in 5xFAD mice (injection at one month old, analysis at seven month old), GFP was used as a control, and the promoter was hSyn (human synapsin).
  • AAV adeno-associated virus
  • the nucleic acid sequence encoding the polypeptide is preceded by a signal peptide encoding sequence.
  • Statistics are in panels B, C, D and E.
  • FIG. 10 ApoE2 expressed in neurons can inhibit the endogenous production of A ⁇ 40 in mouse neurons.
  • ELISA detects A ⁇ .
  • FIG. 11 ApoE2 or ApoECT with tagged proteins added at both ends still has the activity of regulating APP cleavage.
  • Panels A and B ApoE2-V5 (C-terminal tagged protein) and V5-ApoE2 (N-terminal tagged protein) containing tagged protein V5 overexpressed in HEK 293T cells can significantly increase the ⁇ -CTF produced by APP .
  • Figure 12 The mutation of ApoECT can still regulate the cleavage of APP.
  • Panels A and B ApoE CT overexpressed in HEK 293T cells, single or double site mutations in ApoE CT, CT A20, CT A40, CT A60, CT A65, CT A72, CT A80, and CT A36/74 All significantly increased ⁇ -CTF.
  • Panels C and D Overexpression of CT R10, CT LF5 and CT LM5 with multiple mutations in HEK 293T cells and ApoE CT also significantly increased ⁇ -CTF; but CT E5, CT E10, CT E20 and CT F5 lost To increase the activity of ⁇ -CTF.
  • Figure 13 Adding or deleting amino acid sequences inside ApoE2, ApoE2 can still regulate APP metabolism.
  • Panel A ApoE2-M3F co-expressed with APP in HEK 293T cells had ⁇ -CTF added.
  • Panel B ApoE2 MD10 co-expressed with APP in HEK 293T cells also increased ⁇ -CTF.
  • Figure 14 Non-human ApoE CT also regulates the cleavage of APP.
  • Panels A and B ApoE CT of non-human primate origin overexpressed in HEK 293T cells with a high sequence similarity to human ApoE CT significantly increased ⁇ -CTF, and its activity was identical to that of human ApoE CT.
  • FIG. 15 ApoE linked to a membrane-penetrating peptide still has the activity of regulating APP cleavage.
  • FIG. 16 ApoECT expressed in cells requires a signal peptide to have the activity of regulating APP cleavage.
  • FIG. 17 ApoE2 increases ⁇ -CTF and reduces A ⁇ production through its C-terminal region.
  • Panel A Representative images of co-immunofluorescence staining for ApoE and NeuN (neuronal markers) in human (top) or monkey (bottom) cortex. Arrows indicate ApoE-positive neurons.
  • Panel B Representative images of co-immunofluorescence staining for ApoE and APP in human cerebral cortex.
  • FIG. 18 ApoE CT interacts with APP and ⁇ -secretase.
  • Panel A Representative images of immunofluorescence staining for ApoE and APP in N2A cells transiently co-expressing APP-FLAG and GFP, ApoE2 or ApoE CT.
  • Panel B Representative images of immunofluorescent staining of ApoE and endogenously expressed APP in primary cultured neurons transiently expressing GFP, ApoE2-V5, or ApoE CT-V5.
  • Panel C Representative images of immunofluorescence staining of ApoE CT and endogenously expressed APP in human pluripotent stem cell-induced neurons transiently expressing ApoE CT-V5.
  • MAP2 staining Color indicates neurons, and DAPI staining indicates nuclei.
  • Panel D Western blot detection of APP-APEX2-tagged ApoE2 co-expressed in HEK 293T cells.
  • Panel E Western blot detection of APP-APEX2-tagged ApoE CT co-expressed in HEK 293T cells.
  • Panel F Western blot detection of ApoE2-APEX2-tagged APP co-expressed in HEK 293T cells.
  • Panel G Western blot detection of endogenously expressed APP in ApoE-APEX2-labeled neurons expressed in mouse neuronal cells and negative control COX2.
  • Panel H Western blot detection of ApoE2-APEX2-tagged ADAM10, BACE1, PS1 and PS2 transiently co-expressed with ADAM10, BACE1, PS1 or PS2, respectively, in HEK 293T cells.
  • Panel I Co-immunoprecipitation detection of ApoE interacting with ⁇ -CTF-FLAG transiently co-expressed with ApoE2, ApoE2 NT or ApoE CT respectively in HEK 293T cells.
  • Panel J Co-immunoprecipitation detection of ApoE interacting with ⁇ -secretase (HA-APH, V5-NCT, FLAG-PEN2) transiently co-expressed with ApoE2, ApoE2 NT or ApoE CT respectively in HEK 293T cells.
  • Panel K Co-immunoprecipitation detection of the interaction of endogenous ⁇ -secretase (NCT, PS1, PS2 and PEN2) and endogenous ApoE in human brain tissue.
  • FIG. 19 ApoE CT increased ⁇ -CTF and decreased A ⁇ by inhibiting ⁇ -cleavage of APP.
  • Panels A and B Western blot detection and statistical analysis in HEK 293T cells transiently expressing APP and GFP (lane 1); when APP and PS1 were co-expressed, 1 micromolar ⁇ -secretase inhibitor PF03084014 was treated or not (lane 2, Lane 3); co-expression of APP, PS1 and ApoE CT (lane 4); under these expression or drug treatment conditions, APP produces ⁇ -CTF.
  • N 11.
  • Panels C and D Western blot detection and statistical analysis of the effects of GFP or ApoE CT on the production of ⁇ -CTF from co-expressed APP in HEK 293T cells when treated with 1 micromolar ⁇ -secretase inhibitor PF03084014, and the control group was not treated with inhibitors.
  • N 7.
  • Panel E ELISA detection of A ⁇ 40 produced in HEK 293T cells transiently co-expressing ⁇ -CTF and GFP or ApoE CT, respectively.
  • N 4.
  • Panel F ELISA detection of A ⁇ 40 produced in HEK 293T cells transiently co-expressing ⁇ -CTF and GFP or ApoE CT, respectively.
  • Panel G and Panel H ELISA detection results of A ⁇ 40 produced by HEK 293T co-expressed by APP ⁇ AICD and GFP or ApoE CT respectively.
  • Panel G cell culture medium.
  • Panel H cell lysates.
  • Panels B, D, E, F, G and H one sample t-test.
  • Figure 20 ⁇ -secretase is essential for ApoE CT to regulate the metabolism of APP.
  • Panel A Western blot detection of protein expression of PS1 and PS2 in wild-type (WT), PS1 or PS2 single knockout (PS1 KO, PS2 KO), PS1 and PS2 double knockout (PS1/2 dKO) HEK 293T cell lines.
  • Panel B APP expression in wild-type HEK 293T cell line (lane 1); APP expression in PS1 single knockout HEK 293T cell line (lane 2); APP expression in 1 micromolar ⁇ -secretase inhibitor PF03084014 treatment in the PS1 single knockout HEK 293T cell line (lane 3); APP and PS1 were co-expressed in the PS1 single knockout HEK 293T cell line (lane 4).
  • Western blotting was used to detect the content of ⁇ -CTF in cells under different conditions.
  • Panels D, F, J, L and M one sample t-test.
  • FIG. 21 Knockdown of endogenous ApoE in mouse neurons leads to decreased content of APP metabolite CTF 14kD and increased A ⁇ .
  • Panel A Western blot detection of endogenously expressed ApoE in primary cultured mouse neurons transiently expressing shScramble (negative control), shApoE_1 (shRNA_1 of ApoE) or shApoE_2 (shRNA_2 of ApoE) using a lentiviral expression system.
  • Panel B Relative content of APP metabolite CTF 14kD in mouse neurons treated with 1 micromolar ⁇ -secretase inhibitor PF03084014.
  • Panel E Western blot detection of endogenously expressed APP metabolite CTF 14kD in primary cultured mouse neurons transiently expressing shScramble, shApoE_1 or shApoE_2 using lentiviral expression system when treated with 1 micromolar ⁇ -secretase inhibitor PF03084014 relative content.
  • Figure 22 ApoE2 directly inhibits ⁇ -cleavage of APP.
  • Panel A Western blot analysis of expression of purified ⁇ -CTF, ApoE2, ApoE2 NT, PS1, NCT, PEN2 and GFP.
  • Panel C ELISA detection and statistical analysis of A ⁇ produced by co-incubation of purified ⁇ -CTF with a series of concentration gradients of ApoE2.
  • the IC50 is approximately 1.2 ⁇ 0.48 micromolar.
  • Figure 23 Neuronally expressed ApoECT migrates to amyloid plaques and slows the formation of amyloid plaques in the brain of 5 ⁇ FAD mice.
  • Panel A Experimental schema diagram.
  • Panel B Representative images of immunofluorescent staining in mouse brains expressing AAV GFP or ApoECT in 5 ⁇ FAD mouse brain neurons for 10 weeks and amyloid plaques.
  • Panel C Statistical analysis of mean size of amyloid plaques.
  • Panel D Statistical analysis of the proportion of the total amyloid plaque area in the subiculum area.
  • Figure 24 ApoE expressed in neurons of 5 ⁇ FAD mice accumulates around amyloid plaques by CT.
  • Panel A Immunofluorescence of lentiviral ApoE2-IRES-GFP, ApoE2 NT-IRES-GFP, and ApoE CT-IRES-GFP expressed in 5 ⁇ FAD mouse cortical neurons for three months with amyloid plaques (GFP) Stain representative pictures.
  • Panel B Representative images of immunofluorescent staining of lentiviral ApoECT and human APP (hAPP) expressed in 5 ⁇ FAD mouse cortical neurons for one month.
  • Panel C Representative images of immunofluorescent staining of lentiviral ApoECT and hAPP expressed in wild-type mouse brain neurons for three months. Plaque, amyloid plaque.
  • FIG. 25 Endogenous ApoE exists in human neurons, and amyloid plaques co-localize with ApoE in the brains of AD patients.
  • Panel A Representative images of immunofluorescence co-staining for ApoE and NeuN (neuronal markers) in the cortex of non-AD patients (62 years, top) or AD patients (86 years, bottom). Arrows indicate ApoE-positive neurons.
  • Panel B Percentage of ApoE positive neurons relative to total neurons in human cerebral cortex.
  • Panel D Representative images of immunofluorescent co-staining of ApoE and GFAP (astrocyte marker) in human cerebral cortex.
  • Panel E Representative images of immunofluorescent co-staining of ApoE and amyloid plaques (6E10) in the cortex of non-AD patients (62 years, top) or AD patients (86 years, bottom).
  • FIG. 26 ApoE2 increases ⁇ -CTF and decreases A ⁇ through ApoE CT in HEK 293T cells.
  • Panel A Western blot detection of ⁇ -CTF produced by co-expressing APP and GFP or ADAM10 in HEK 293T cells, respectively.
  • Panel B Western blot detection of ⁇ -CTF produced by HEK 293T cells treated with 1 micromolar ADAM10 inhibitor GI254023X, and controls not treated with GI254023X.
  • Panels C and D Western blot detection and statistical analysis of ⁇ -CTF produced by co-expressing APP and GFP, ApoE2, ApoE3 or ApoE4 in HEK 293T cells, respectively.
  • N 12.
  • Panels N and O Western blot detection and statistical analysis of ⁇ -CTF generated by APP and GFP, ApoE2 or ApoE CT, respectively, in HEK 293T cells.
  • N 6.
  • Panels P and Q Western blot detection and statistical analysis co-expression of mouse APP (mAPP) and ⁇ -CTF generated by GFP or ApoE CT in HEK 293T cells, respectively.
  • N 6.
  • Panels D, F, H, I, J, K, M, O, Q, R, S, U, and V one sample t-test.
  • FIG. 27 ApoE2 and ApoE CT in the culture medium cannot change the metabolism of APP.
  • Panel A Western blot detection of ApoE2 and ApoECT in cell culture medium.
  • Panel D Western blot detection of ApoE2 in culture medium under different conditions. The original ApoE2-containing culture medium (lane 1), the ApoE2-containing culture medium (lane 2) after incubation with untransfected cells for 48 hours, and the ApoE2-containing culture medium separately stored in a 37°C incubator for 48 hours ( Strip 3).
  • Panel E Immunofluorescence detection of ApoE2 and ApoE CT in culture medium taken up by HEK 293T cells, and ApoE CT expressed by HEK 293T cells.
  • DAPI nuclear marker.
  • Panel F Immunofluorescence detection of ApoE2 and ApoE CT in culture medium uptake by neurons, and ApoE CT expressed by neurons. MAP2, marker of mature neurons; DAPI, nuclear marker.
  • Panel G Western blot analysis of purified ApoE2 and ApoE2 from cell lysates or cell culture medium.
  • Figure 28 AD-related mutations near the ⁇ -cleavage site of APP affect the activity of ApoECT.
  • FIG. 29 ApoE CT is still able to regulate APP metabolism when ⁇ or ⁇ secretase is inhibited.
  • Panels C and D ELISA assay and statistical analysis Western blot to examine the effect of ApoE CT on A ⁇ production from co-expressed APP and BACE1 in HEK 293T cells treated with 1 ⁇ M ADAM10 inhibitor GI254023X; Formulation handling.
  • Panel C A ⁇ 40 in cell culture medium
  • Panel D A ⁇ 40 in cell lysates.
  • N 6.
  • Panel I qRT-PCR detection and statistical analysis of the effects of expressed ApoE2 or ApoE CT on the expression of endogenous ADAM10, BACE1, PS1 and PS2 mRNA in HEK 293T cells.
  • Panel J The effects of ApoE2 or ApoE CT on the expression of endogenous ADAM10, BACE1, PS1 and PS2 proteins in HEK 293T cells were detected by Western blot.
  • Panel K Effect of ApoE CT on cell surface expression of APP co-expressed in HEK 293T cells by Western blot.
  • Cell surface proteins of HEK 293T cells expressing GFP(-) and ApoE CT(+) were labeled with sulfo-N-hydroxysuccinimide-biotin and isolated with streptavidin-coupled agarose beads.
  • Figure 30 ApoE CT cannot inhibit the ⁇ -cleavage of NOCTH and APLP1.
  • Panel A In HEK 293T cells, only N100-V5 was expressed (lane 1); co-expressed N100-V5 and ⁇ -secretase (lane 2); PF03084014, an inhibitor of ⁇ -secretase, was treated with 1 micromolar N100-V5 and ⁇ -secretase (lane 3); co-expression of N100-V5, ⁇ -secretase and ApoECT (lane 4). Cleavage of N100-V5 when expressed in the above conditions was detected by immunoblotting.
  • Panel B Western blot detection of ⁇ -CTF content when APP, ⁇ -secretase and ApoECT were co-expressed in HEK 293T cells.
  • Panel C In HEK 293T cells, only APLP-V5 was expressed (lane 1); expressed APLP-V5 was treated with 1 micromolar ⁇ -secretase inhibitor PF03084014 (lane 2); APLP1-V5 and ApoE CT were co-expressed (band 3). The metabolism of APLP1-V5 was detected by immunoblotting when the above conditions were expressed.
  • Figure 31 In vitro purified ⁇ -CTF, ApoE2, ApoE2 NT and ⁇ -secretase. Coomassie brilliant blue staining showing purified ⁇ -CTF-Myc-6 ⁇ His (panel A), ApoE2-3 ⁇ FLAG (panel B), ApoE2NT-3 ⁇ FLAG (panel C), ⁇ -secretase (panel D) (NCT -V5-6 ⁇ His; PLAG-PEN2; APH; full-length PS1, FL-PS1; N-terminal fragment of PS1, PS1 NTF; C-terminal fragment of PS1, PS1CTF) and 3 ⁇ FLAG-GFP (panel E).
  • Figure 32 The human brain information table used in this example.
  • Figure 33 Human ApoE protein structure.
  • the N-terminal domain (residues 1-167) and the C-terminal domain (residues 206-299) are connected by a flexible hinge region (residues 168-205).
  • the three isoforms are ApoE2 (Cys112; Cys158), ApoE3 (Cys112; Arg158) and ApoE4 (Arg112; Arg158).
  • Human apolipoprotein E mainly includes three subtypes, ApoE2, ApoE3 and ApoE4.
  • ApoE consists of three regions: an N-terminal domain (ApoET), a hinge region, and a conserved C-terminal domain (ApoECT).
  • the only sequence difference between the three ApoE isoforms is in their N-terminal domains ( Figure 33).
  • the amino acid sequence of exemplary ApoE2 (human source ApoE2) is as follows:
  • exemplary ApoE3 human source ApoE3
  • the invention provides an isolated ApoE construct comprising an ApoE polypeptide.
  • the ApoE polypeptide comprises a part of the sequence of the C-terminal domain of the ApoE protein (for example, amino acids 221-274 of SEQ ID NO: 12), or a modified sequence based on the sequence.
  • the present invention is based on the unexpected discovery by the inventors of said ApoE constructs (such as the ApoE polypeptides or ApoE2 of the present invention): (i) they can regulate the progenitor in neurons (either in vivo or in vitro) in a cell-autonomous manner; Cleavage of body amyloid precursor protein (amyloid precursor protein, APP); (ii) it can inhibit (for example inhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the enzymatic activity of ⁇ -secretase, such as inhibition of ⁇ -secretase-mediated APP ⁇ -cleavage, and the APP ⁇ -cleavage inhibitory effect of ⁇ -secretase inhibitors (such as PF03084014) similar; (iii) it can reduce (e.g.
  • the starch of APP like metabolism e.g., reducing (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) amyloid-beta ( A ⁇ ) (e.g., A ⁇ 40 and/or A ⁇ 42) production, or reduction (e.g., reduction by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) ⁇ -cleavage of the C-terminal fragment-beta ( ⁇ -CTF); (iv) it can be increased (for example, increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or higher) non-amyloid metabolism of APP, e.g., increased (e.g., increased by at
  • AD model Amyloid plaques in mouse brain e.g., reduced (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) ) average plaque size, total area, and/or density of amyloid plaques, reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% %, 95%, or 100%) intraneuronal A ⁇ (e.g., A ⁇ 40 and/or A ⁇ 42) production, reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) AD pathological progression in mice, increasing (e.g., increasing at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2 times, 5 times, 10 times, 20 times or higher
  • This application is the first to discover that apolipoprotein E inhibits the cleavage of ⁇ -secretase in a cell-autonomous manner through its C-terminal fragment (that is, it functions in the same cell as ⁇ -secretase), inhibits the amyloid metabolism of APP, and reduces A ⁇ Generated; and increased the non-amyloid metabolite of APP, ⁇ -CTF; slowed the size and density of amyloid plaques in AD model mouse brain.
  • the C-terminal fragment of ApoE can be used as a drug targeting ⁇ -secretase to slow down (for example slow down by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%) %, 90%, 95%, or 100% of the AD development process), treat, or prevent the occurrence and development of AD and amyloid cerebrovascular disease.
  • the C-terminal fragment of ApoE as an inhibitor of ⁇ -secretase may play a role in various biological processes.
  • the application of the C-terminal fragment of ApoE is not limited to the field of neurodegenerative diseases, and can also be used for cancer, inflammatory diseases , kidney disease and other diseases, such as any disease related to the cleavage activity of ⁇ -secretase (inhibition of the cleavage activity of ⁇ -secretase can treat or prevent the disease).
  • Conventional ⁇ -secretase inhibitors cause severe side effects in treatment, all associated with defective Notch signaling, such as immunosuppression, gastrointestinal bleeding and diarrhea, and cancerous skin lesions.
  • the ApoE construct provided by the present invention does not affect the ⁇ -cleavage of Notch, so the ApoE construct and related methods provided by the present invention may provide a novel efficient and safe treatment plan.
  • MGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQR SEQ ID NO: 78; "ApoE CT ND5CD25”
  • the shortest functional fragment of the C-terminal fragment of apolipoprotein E is amino acid sequence 220-274 of SEQ ID NO: 12 (EMGSRTRDLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQR; SEQ ID NO: 87).
  • the shortest functional fragment of the C-terminal fragment of ApoE is amino acid residues 220-279 of SEQ ID NO: 12: EMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGL (SEQ ID NO: 34).
  • treating and “treatment” mean delaying the onset, delaying or reversing the progression of the disease or condition to which the term applies or one or more symptoms of such disease or condition , to reduce its severity, or to alleviate or prevent it.
  • an amount refers to the amount and/or dosage and/or dosage regimen of one or more agents necessary to produce the desired result, e.g., an amount sufficient to reduce APP ⁇ cleavage, or to treat neurological
  • an amount sufficient to reduce the risk or delay the onset and/or reduce the eventual severity of a disease characterized by amyloid deposits in the mammalian brain e.g., a prophylactically effective amount.
  • alleviate refers to reducing or eliminating one or more symptoms of the pathology or disease, and/or reducing the rate of one or more symptoms of the pathology or disease or delaying one or more symptoms of the pathology or disease Onset or severity of symptoms and/or prevention of said pathology or disease.
  • reducing or eliminating one or more symptoms of a pathology or disease includes, but is not limited to, reducing or eliminating one or more markers (e.g., total-Tau (tTau), phosphorylated - one or more of Tau (pTau), APPneo, soluble A ⁇ 40, A ⁇ 42, pTau/A ⁇ 42 ratio and tTau/A ⁇ 42 ratio and/or CSF selected from the group consisting of A ⁇ 42/A ⁇ 40 ratio, A ⁇ 42/A ⁇ 38 ratio, sAPP ⁇ , sAPP ⁇ /sAPP ⁇ ratio, increase in levels of components of the sAPP ⁇ /A ⁇ 40 ratio, sAPP ⁇ /A ⁇ 42 ratio, etc.), and/or increase non-amyloid metabolites such as ⁇ -CTF, and/or decrease, stabilize, or reverse one or more diagnostic criteria (e.g., Clinical Dementia Rating (CDR)).
  • markers e.g., total-Tau (tTau), phosphorylated - one or more of Tau (p
  • “Individual” or “subject” includes, but is not limited to, mammals, birds, frogs, fish, fruit flies, nematodes, and the like. Mammals include, but are not limited to, primates (such as humans, or non-human primates such as monkeys), rodents (such as mice, rats, hamsters, gerbils, squirrels, guinea pigs, and rabbits), livestock (such as pigs, cattle, sheep, horses, donkeys), pets (such as cats, dogs, rabbits and hamsters), etc. In some embodiments, the individual is a human.
  • formulation or "pharmaceutical formulation” or “dosage form” or “pharmaceutical formulation” as used herein refers to a composition containing at least one therapeutic agent or drug for delivery to a subject.
  • Sequence identity between two polypeptide or nucleic acid sequences indicates the number of residues that are identical between the sequences as a percentage of the total number of residues, and the calculation of the total number of residues is determined based on the type of mutation. Mutation types include insertions (extensions) at either or both ends of the sequence, deletions (truncations) at either or both ends of the sequence, substitutions/substitutions of one or more amino acids/nucleotides, insertions within the sequence, lack of interior.
  • the mutation type is one or more of the following: one or more amino acid/nucleotide substitutions/substitutions, insertions within the sequence, and deletions within the sequence , the total number of residues is calculated as the larger of the compared molecules. If the mutation type also includes insertions (extensions) or deletions (truncations) at either or both ends of the sequence, the number of amino acids inserted or deleted at either or both ends (e.g., insertions at both ends or the number of deletions is less than 20) are not counted in the total number of residues. In calculating percent identity, the sequences being compared are aligned in such a manner as to produce the greatest match between the sequences, with gaps, if any, in the alignment being resolved by specific algorithms.
  • fragment refers to a polypeptide and is defined as any isolated portion of a given polypeptide that is unique or characteristic.
  • the term as used herein also refers to any isolated portion of a given polypeptide that retains at least a portion of the activity of the full-length polypeptide.
  • the remaining active fraction is at least 10% of the activity of the full-length polypeptide. More preferably, the remaining active portion is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity of the full-length polypeptide. More preferably, the remaining active portion is at least 95%, 96%, 97%, 98%, or 99% of the activity of the full-length polypeptide.
  • the remaining active fraction is 100% of the activity of the full-length polypeptide.
  • the term also refers to any portion of a given polypeptide that includes at least one defined sequence element of the full-length polypeptide.
  • the sequence elements span at least 4-5, more preferably at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the full-length polypeptide.
  • polypeptide refers to a chain of sequential amino acids linked by peptide bonds.
  • the term is used to refer to amino acid chains of any length, however those of ordinary skill in the art know that the term does not only refer to long chains, but can also be used to refer to the shortest chains comprising 2 amino acids linked by peptide bonds.
  • protein refers to one or more polypeptides that function as discrete units.
  • polypeptide and “protein” are used interchangeably herein if a single polypeptide is a discrete functional unit and no permanent physical association with other polypeptides is required to form that separate functional unit. If the discrete functional unit consists of more than one polypeptide physically associated with each other, the term “protein” as used herein refers to a plurality of polypeptides that are physically coupled and function together as the discrete unit.
  • Recombinantly expressed polypeptide refers to a polypeptide expressed from a host cell that has been genetically engineered to express the polypeptide.
  • the recombinantly expressed polypeptide may be the same or similar to the polypeptide normally expressed in the host cell (such as mammalian cell).
  • the recombinantly expressed polypeptide can also be foreign to the host cell, For example, heterologous to a polypeptide normally expressed by a mammalian host cell.
  • the recombinantly expressed polypeptide is a chimeric polypeptide, for example, part of the polypeptide contains the same or similar amino acid sequence as the polypeptide normally expressed in the mammalian host cell, while the other part is foreign to the host cell.
  • amino acid means the twenty common naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C ); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine ( Leu; L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F), Proline (Pro; P), Serine (Ser; S), Threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic
  • Non-critical amino acids can be substituted conservatively without affecting the normal function of the protein.
  • Conservative substitutions refer to the replacement of amino acids with chemically or functionally similar amino acids.
  • Conservative substitution tables providing similar amino acids are well known in the art. For example, in some embodiments, the groups of amino acids provided below are considered conservative substitutions for each other.
  • selected groups of amino acids considered to be mutually conservative substitutions are selected groups of amino acids considered to be mutually conservative substitutions:
  • the term "about” or “approximately” refers to a change of up to 15%, 10%, or 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% in quantity, level, value, quantity, frequency, percentage, dimension, size, volume, weight or length.
  • the term "about” or “approximately” refers to ⁇ 15%, ⁇ 10%, ⁇ 9% around a reference amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length , ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% amount, level, value, amount, frequency, percentage, scale, size, amount , weight or length range.
  • the term “substantially/essentially” means about 70%, compared to a degree, reference amount, level, value, amount, frequency, percentage, dimension, size, amount, weight or length, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater degree, amount, level, value, amount, Frequency, percentage, measure, size, volume, weight or length.
  • Consisting of means including, but limited to, anything following the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.
  • Consisting essentially of is meant to include any of the elements listed after the phrase “consisting essentially of” and is limited to activities or actions specified in the disclosure that do not interfere with or contribute to the listed elements other elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but without other Other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.
  • references to "one embodiment”, “an embodiment”, “a particular embodiment”, “a related embodiment”, “an embodiment”, “another embodiment” or “further embodiments” A combination thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention.
  • the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment.
  • the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
  • the application provides an isolated ApoE construct comprising an ApoE polypeptide that: (1) comprises a fragment from the amino acid sequence shown in SEQ ID NO: 12, the fragment (or the ApoE Polypeptide)
  • the first amino acid residue at the N-terminal is the amino acid residue at any one of positions 215-221 of SEQ ID NO: 12, and the last amino acid residue at the C-terminal of the fragment (or the ApoE polypeptide) is SEQ ID NO: 12
  • the ApoE polypeptide comprises a fragment from the amino acid sequence shown in SEQ ID NO: 12, and the first amino acid residue at the N-terminal of the ApoE polypeptide is any of positions 215-221 of SEQ ID NO: 12 An amino acid residue at a position, the last amino acid residue at the C-terminal of the ApoE polypeptide is the amino acid residue at any one of positions 274-299 (eg, 279-299) of SEQ ID NO:12.
  • the ApoE construct comprises an ApoE polypeptide
  • the ApoE polypeptide consists of a fragment from the amino acid sequence shown in SEQ ID NO: 12, and the first amino acid residue at the N-terminal of the ApoE polypeptide is SEQ ID NO: 12 No. 215-
  • the amino acid residue at any position in position 221 the last amino acid residue at the C-terminal of the ApoE polypeptide is the amino acid at any position in SEQ ID NO: 12 274-299 (eg 279-299) Residues.
  • the ApoE polypeptide has at least about 70% identity with a fragment from the amino acid sequence shown in SEQ ID NO: 12, and retains at least about 50% of the fragment's inhibition of ⁇ -secretase enzyme cleavage activity, wherein the first amino acid residue at the N-terminal of the fragment is the amino acid residue at any one of positions 215-221 of SEQ ID NO: 12, and the last amino acid residue at the C-terminal of the fragment is SEQ ID NO: 12 An amino acid residue at any one of positions 274-299 (eg, 279-299).
  • the isolated ApoE construct consists of or consists essentially of the ApoE polypeptide (eg, isolated ApoE polypeptide).
  • the present application also provides an ApoE polypeptide (eg, an isolated ApoE polypeptide).
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) consists of (1) a fragment from the amino acid sequence shown in SEQ ID NO: 12, or consists essentially of (1) a fragment from the amino acid sequence shown in SEQ ID NO: NO: Composed of a fragment of the amino acid sequence shown in 12.
  • the ApoE construct does not occur naturally.
  • the ApoE construct or the ApoE polypeptide is not ApoE2, is not ApoE3, and/or is not ApoE4 full-length protein.
  • the ApoE construct or the ApoE polypeptide is a full-length ApoE2 or ApoE3 protein (eg, a human full-length ApoE2 or ApoE3 protein), such as when used as a neural-specific expressing ApoE construct.
  • the starting point of the amino acid sequence (or the first amino acid residue at the N-terminal) of the ApoE polypeptide (such as an isolated ApoE polypeptide) or a fragment of the amino acid sequence shown in SEQ ID NO: 12 in (1) ) is any site between the 215th and 221st positions (such as the 215th, 216th, 217th, 218th, 219th, 220th or 221st) of the sequence of ApoE SEQ ID NO: 12 , such as any amino acid residue at position 215, position 216, position 219, position 220 or position 221.
  • the end point of the amino acid sequence (or the last amino acid residue at the C-terminal) of the ApoE polypeptide or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) is ApoE SEQ ID NO: 12
  • the 274th and 299th positions of the sequence for example, the 274th, 275th, 276th, 277th, 278th, 279th, 280th, 281st, 282nd, 283rd, 284th, 285th, 286th, 287th, 288th, 289th, 290th, 291st, 292nd, 293rd, 294th, 295th, 296th position, 297th, 298th or 299th
  • the amino acid residue at any position between 279-299 including the original number
  • 274th, 279th Any amino acid residue at position or position 299.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) comprises (or consists of, or consists essentially of) as follows Any sequence: No. 215-299, No. 216-299, No. 217-299, No. 218-299, No. 219-299, No. 220-299, No. 219-299, No. 220-299, No.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) comprises (or consists of, or consists essentially of) as follows Any sequence: (i) the amino acid sequence of SEQ ID NO: 12 amino acid residues 215-299; (ii) the amino acid sequence of SEQ ID NO: 12 amino acid residues 216-299; (iii) SEQ ID NO : the amino acid sequence of amino acid residues 217-299 of 12; (iv) the amino acid sequence of amino acid residues 218-299 of SEQ ID NO: 12; (v) amino acid residues of 219-299 of SEQ ID NO: 12 (vi) SEQ ID NO: the amino acid sequence of the 220-299th amino acid residue of 12; (vii) the amino acid sequence of the 221-299th amino acid residue of SEQ ID NO: 12; (viii) SEQ ID NO : the amino acid sequence of amino acid residues 216-294 of 12; (ix) the amino acid sequence of
  • the ApoE construct or ApoE polypeptide does not comprise one or more amino acid sequences in positions 211-214 of SEQ ID NO:12. In some embodiments, the ApoE construct or ApoE polypeptide does not comprise one or more (or none) of the following amino acid fragments: the 212-214, 213-214, 213-214, and 211-214, 211-215, 211-216, 211-217, 211-218, 211-219, 211-220, 211-221, 211- 222nd, 212-214th, 212-215th, 212-216th, 212-217th, 212-218th, 212-219th, 212-220th, 212-221st , No. 212-222, No. 213-214, No. 213-215, No.
  • the ApoE construct or ApoE polypeptide comprises the amino acid sequence at positions 211-214 of SEQ ID NO: 12, but the C-terminus of the amino acid sequence at positions 211-214 of SEQ ID NO: 12 is not compatible with the amino acid sequence at positions 211-214.
  • the N-terminus of the ApoE polypeptide is directly connected, for example, at least one non-ApoE-derived sequence (eg, FLAG sequence) is inserted therebetween.
  • the 3XFLAG sequence shown in SEQ ID NO: 77 is inserted between the C-terminal of the amino acid sequence at positions 211-214 of SEQ ID NO: 12 and the N-terminal of the ApoE polypeptide.
  • the ApoE construct or the ApoE polypeptide may additionally comprise one or more N-terminal and/or C-terminal fragments from the amino acid sequence shown in SEQ ID NO: 12 in (1). Amino acid residues not of ApoE origin. In some embodiments, the ApoE construct or the ApoE polypeptide may additionally comprise one or more N-terminal and/or C-terminal fragments from the amino acid sequence shown in SEQ ID NO: 12 in (1). Amino acid residue, and the amino acid residue is different from the amino acid residue at the corresponding position of SEQ ID NO: 12 when added to the N-terminal or C-terminal of the (1) fragment.
  • the first amino acid residue at the N-terminal of the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) is the 215th amino acid residue of SEQ ID NO: 12, and the ApoE construct or the The ApoE polypeptide may additionally comprise one or more amino acid residues at the N-terminus of amino acid residue 215 of SEQ ID NO: 12, but this one or more amino acid residues are the same as those of SEQ ID NO: 12 214, 213, 212 , 211 and other amino acid residues are different.
  • the ApoE construct can be additionally located at the N-terminal and/or C-terminal of the ApoE polypeptide (for example, a fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1), or an ApoE mutant). Contains one or more amino acid residues not derived from ApoE.
  • the ApoE construct may additionally comprise one or more amino acid residues at the N-terminal and/or C-terminal of the ApoE polypeptide, and the amino acid residues may be added to the N-terminal of the ApoE polypeptide. Or the C-terminus is different from the amino acid residue at the corresponding position of SEQ ID NO: 12.
  • the first amino acid residue at the N-terminal of the ApoE polypeptide is the 215th amino acid residue of SEQ ID NO: 12, and the The ApoE construct may additionally comprise one or more amino acid residues at the N-terminus of amino acid residue 215 of SEQ ID NO: 12, but this one or more amino acid residues are identical to those of SEQ ID NO: 12 214, 213, 212 , 211 and other amino acid residues are different.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) at least includes the amino acid sequence 221-274 of SEQ ID NO: 12 (SEQ ID NO: 78).
  • the ApoE polypeptide or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) may additionally include 1, 2, 3, 4, 5 at the N-terminal of the amino acid sequence shown in SEQ ID NO: 78 , or 6 amino acid residues, and/or may additionally contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 at the C-terminus , 18, 19, 20, 21, 22, 23, 24 or 25 amino acid residues.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) consists of the amino acid sequence shown in SEQ ID NO: 78, or substantially It consists of the amino acid sequence shown in SEQ ID NO:78.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) at least includes the 220-279 amino acid sequence of SEQ ID NO: 12 (SEQ ID NO: 34).
  • the ApoE polypeptide or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) may additionally include 1, 2, 3, 4, or 5 amino acid residues, and/or may additionally include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 at the C-terminus , 19 or 20 amino acid residues.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) consists of the amino acid sequence shown in SEQ ID NO: 34, or substantially It consists of the amino acid sequence shown in SEQ ID NO:34.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) at least includes the amino acid sequence 219-274 of SEQ ID NO: 12 (SEQ ID NO: 32).
  • the ApoE polypeptide or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) may additionally comprise 1, 2, 3 or 4 amino acids at the N-terminal of the amino acid sequence shown in SEQ ID NO: 32 residues, and/or may additionally contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid residues.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) consists of the amino acid sequence shown in SEQ ID NO: 32, or substantially It consists of the amino acid sequence shown in SEQ ID NO:32.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) at least includes the amino acid sequence 219-279 of SEQ ID NO: 12 (SEQ ID NO: 31).
  • the ApoE polypeptide or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) can be added at the N-terminal of the amino acid sequence shown in SEQ ID NO: 31 1, 2, 3 or 4 amino acid residues outside, and/or may additionally contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 at the C-terminus , 15, 16, 17, 18, 19 or 20 amino acid residues.
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) or the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) consists of the amino acid sequence shown in SEQ ID NO: 31, or substantially It consists of the amino acid sequence shown in SEQ ID NO:31.
  • the ApoE construct (such as an isolated ApoE construct), the ApoE polypeptide (such as an isolated ApoE polypeptide), or the amino acid sequence from SEQ ID NO: 12 in (1)
  • the fragment comprises any amino acid sequence in SEQ ID NOs: 13, 18-34, 78 and 87 (eg, SEQ ID NOs: 31-34, 78 and 87, or SEQ ID NO: 33 or 78).
  • the ApoE construct (such as an isolated ApoE construct), the ApoE polypeptide (such as an isolated ApoE polypeptide), or the amino acid sequence from SEQ ID NO: 12 in (1)
  • the fragment consists of any amino acid sequence of SEQ ID NOs: 13, 18-34, 78 and 87 (e.g., SEQ ID NOs: 31-34, 78 and 87, or SEQ ID NO: 33 or 78), or consists essentially of SEQ ID NOs: 31-34, 78 and 87, or SEQ ID NO: 33 or 78) NOs: 13, 18-34, 78 and 87 (such as SEQ ID NOs: 31-34, 78 and 87, or SEQ ID NO: 33 or 78) in any amino acid sequence composition.
  • the ApoE constructs and the ApoE polypeptides described herein also include mutants thereof (also referred to herein as "ApoE mutants").
  • the mutant compared with the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) (such as any sequence in SEQ ID NOs: 13, 18-34, 78 and 87), the mutant has one or Multiple amino acid mutations (such as additions, deletions or substitutions), including but not limited to "conservative modification to inhibit enzyme activity".
  • the mutant has at least about 70% of the fragment (such as any sequence in SEQ ID NOs: 13, 18-34, 78 and 87) from the amino acid sequence shown in SEQ ID NO: 12 in (1).
  • the mutant retains at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% %, 1.5 times, 2 times, 5 times, 10 times or more) in (1) from the fragment of the amino acid sequence shown in SEQ ID NO: 12 (such as SEQ ID NOs: 13, 18-34, 78 and 87 Any sequence) inhibits the cleavage activity of ⁇ -secretase.
  • SEQ ID NO: 12 such as SEQ ID NOs: 13, 18-34, 78 and 87 Any sequence
  • the mutant retains at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% %, 1.5 times, 2 times, 5 times, 10 times or more) ApoE2 (SEQ ID NO: 12) or ApoE3 (SEQ ID NO: 89) or the ApoE fragment shown in SEQ ID NO: 33 is digested by ⁇ -secretase Inhibition of activity.
  • ApoE2 SEQ ID NO: 12
  • ApoE3 SEQ ID NO: 89
  • Constant modification of inhibiting enzyme activity herein means not significantly affecting (for example, reducing by no more than about 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) or changing the protein construct Or the amino acid modification of the polypeptide to inhibit the cleavage of ⁇ -secretase (eg ⁇ -cleavage of APP).
  • conservative modifications that inhibit enzyme activity include amino acid substitutions, additions and/or or missing. Modifications can be introduced into protein constructs or polypeptides of the invention by standard techniques known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis.
  • the ApoE mutants compared with the fragment (eg, any one of SEQ ID NOs: 13, 18-34, 78 and 87) from the amino acid sequence shown in SEQ ID NO: 12 in (1), the ApoE mutants have one or more (e.g., within 40, within 30, within 20, within 10, within 8, within 5, within 3, 2 or 1) amino acid insertions, substitutions, and and/or deletions (including insertions and/or deletions at the N-terminus and/or C-terminus), while still retaining at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5 times, 2 times, 5 times, 10 times or more) the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) inhibits ⁇ Biological activity of secretase cleavage (eg ⁇ -cleavage of APP).
  • ⁇ Biological activity of secretase cleavage eg ⁇ -clea
  • the ApoE polypeptide (e.g., an isolated ApoE polypeptide) retains at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5 times, 2 times, 5 times, 10 times or more) ApoE2 (SEQ ID NO: 12) or ApoE3 (SEQ ID NO: 89) or ApoE shown in SEQ ID NO: 33 Inhibition of the cleavage activity of ⁇ -secretase by the fragment.
  • ApoE2 SEQ ID NO: 12
  • ApoE3 SEQ ID NO: 89
  • the ApoE polypeptide (such as an isolated ApoE polypeptide) is combined with a fragment of the amino acid sequence shown in SEQ ID NO: 12 (such as SEQ ID NOs: 13, 18-34, 78 and 87, such as SEQ ID NO: 33), has a higher (eg, at least about 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold or more) inhibition of ⁇ -secretase cleavage ( For example, the biological activity of ⁇ -digestion of APP).
  • the ApoE polypeptide comprises a mutation (eg, addition, deletion, or substitution) at one or more amino acid positions in any of the ApoE polypeptides or ApoE constructs in Example 1.
  • the invention provides any of the ApoE polypeptides or ApoE constructs (eg, isolated) of Example 1 and Example 2.
  • the ApoE polypeptide comprises mutations (such as additions, deletions or substitutions) at one or more amino acid positions relative to the amino acid sequence shown in SEQ ID NO: 12: R226, D227, R228, L229, D230 , Q235, E238, V239, R240, K242, E244, E245, Q246, A247, Q248, Q249, I250, R251, L252, Q253, A254, E255, Q275, V280, V287, T289, S290, A291, P293, V294 , P295, S296, D297, N298, H299, R251+T289, R251+T289+A291, S290+P293+V294+P295+S296+D297+N298+H299, R226+D227+R228+L229+D230, E238+ V239 +R240+K242, or E244+E
  • the ApoE polypeptide comprises mutations at one or more amino acid positions relative to the amino acid sequence shown in SEQ ID NO: 12: Q235, E255, Q275, V280, V287, P295, R251+T289, S290 +P293+V294+P295+S296+D297+N298+H299, R226+D227+R228+L229+D230, E238+V239+R240+K242, E244+E245+Q246+A247+Q248+Q249+I250+R251+L252+Q253+A254, or R251+T289+A291.
  • the mutation comprises a substitution of A, S or T at the one or more positions, for example a substitution of A.
  • said mutation comprises deletion of amino acid residues at said one or more positions.
  • the enzyme-inhibiting conservative modification is a conservative substitution.
  • Conservative substitutions refer to substitutions of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with the following side chains: basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar Side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g.
  • the ApoE polypeptide comprises mutations at one or more amino acid positions relative to the amino acid sequence shown in SEQ ID NO: 12: R226A, D227A, R228A, L229A, D230A, Q235A, E238A, V239A, R240A , K242A, E244del, E245del, Q246del, A247del, Q248del, Q249del, I250del, R251S, L252del, Q253del, A254del, E255A, Q275A, V280A, V287A, T289A, S290A, A291T, P293A, V294A, P295A, S296A, D297A, N298A , H299A, R251A+T289A, R251S+T289A+A291T, S290A+P293A+V294A+
  • the ApoE polypeptide comprises mutations at one or more amino acid positions relative to the amino acid sequence shown in SEQ ID NO: 12: Q235A, E255A, Q275A, V280A, V287A, P295A, R251A+T289A, S290A +P293A+V294A+P295A+S296A+D297A+N298A+H299A, R226A+D227A+R228A+L229A+D230A, E238A+V239A+R240A+K242A, E244del+E245del+Q246del+A247del+Q 248del+Q249del+I250del+R251del+L252del +Q253del+A254del, or R251S+T289A+A291T.
  • the ApoE polypeptide or the ApoE mutant is combined with a fragment from the amino acid sequence shown in SEQ ID NO: 12 (such as SEQ ID NOs: 13, 18-34, 78 and 87) in (1).
  • Either order sequence, such as SEQ ID NO: 33 has at least about 70% sequence identity, such as at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more High (but not 100%) identity.
  • the ApoE mutant and the fragment from the amino acid sequence shown in SEQ ID NO: 12 in (1) can also inhibit the cleavage of ⁇ -secretase, or retain at least about 50% (such as at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5 times, 2 times, 5 times, 10 times or more) in (1) from the fragment of the amino acid sequence shown in SEQ ID NO: 12 to ⁇ -secretase enzyme Inhibition of cleavage activity.
  • the ApoE mutant and the fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) are also derived from humans.
  • the ApoE polypeptide comprises or is derived from a non-human sequence.
  • the sequence identity between the mutant and the fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) can be determined by methods known in the art, for example, BLASTP can be used.
  • the ApoE polypeptide comprises or is derived from a non-human sequence, and its amino acid sequence is the same as that in (1) from a fragment of the amino acid sequence shown in SEQ ID NO: 12 (such as SEQ ID NOs: 13, Any one of 18-34, 78 and 87, such as SEQ ID NO: 33) has at least about 70% sequence identity, and retains at least about 50% of the amino acid sequence shown in SEQ ID NO: 12 in (1) Inhibition of the cleavage activity of ⁇ -secretase by the fragment.
  • the ApoE polypeptide comprises or is derived from an ApoE sequence of any of the following species: mammals, birds, frogs, fish, Drosophila, nematodes and the like.
  • mammals include, but are not limited to, primates (such as humans, or non-human primates such as monkeys), rodents (such as mice, rats, hamsters, gerbils, squirrels, guinea pigs, and rabbits) , domestic animals (such as pigs, cattle, sheep, horses, donkeys), pets (such as cats, dogs, rabbits and hamsters), etc.
  • the ApoE polypeptide comprises non-naturally occurring modifications, such as further comprising amino acid modifications on the monkey ApoE sequence.
  • the ApoE polypeptide or the ApoE mutant contains a non-human primate sequence, such as a fragment from the amino acid sequence shown in SEQ ID NO: 12 (such as SEQ ID NOs: 13, 18-34, 78 and 87 in any sequence, such as SEQ ID NO: 33) highly homologous and conserved sequences.
  • the ApoE polypeptide comprises the monkey-derived ApoE C-terminal amino acid sequence shown in SEQ ID NO: 62.
  • the ApoE polypeptide consists of the amino acid sequence shown in SEQ ID NO: 62, or consists essentially of the amino acid sequence shown in SEQ ID NO: 62.
  • the ApoE polypeptide comprises one or more mutations (such as additions, deletions or substitutions) based on the amino acid sequence shown in SEQ ID NO: 33, such as mutations at any one or more of the above positions, or have at least about 70% (eg, at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) of the amino acid sequence shown in SEQ ID NO: 33 sequence identity.
  • mutations such as additions, deletions or substitutions
  • the The ApoE polypeptide comprises one or more mutations (such as additions, deletions, or substitutions) based on the amino acid sequence shown in SEQ ID NO: 33, such as mutations at any one or more of the above positions, or a mutation with the amino acid sequence shown in SEQ ID NO: 33
  • the amino acid sequences shown have at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity and retain at least About 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5 times, 2 times, 5 times, 10 times or more) the amino acid sequence shown in SEQ ID NO: 33 inhibits the cleavage activity of ⁇ -secretase.
  • the ApoE construct or the ApoE polypeptide comprises any amino acid sequence in SEQ ID NOs: 46-52, 57-59, 62 and 76. In some embodiments, the ApoE construct or the ApoE polypeptide consists of any amino acid sequence in SEQ ID NOs: 46-52, 57-59, 62 and 76, or consists essentially of SEQ ID NOs: 46-52, 57-59, 62 and 76 in any amino acid sequence composition.
  • said ApoE construct (such as an isolated ApoE construct), said ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or said (1) is derived from SEQ ID NO: 12
  • the inhibitory effect of fragments of the amino acid sequence shown for example, any one of SEQ ID NOs: 13, 18-34, 78 and 87) on ⁇ -secretase-mediated digestion (such as ⁇ -digestion of APP) includes the following One or more properties: (i) inhibit (e.g.
  • the ApoE construct (such as an isolated ApoE construct), the ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or the amino acid derived from SEQ ID NO: 12 in (1) Fragments of the amino acid sequences shown have a higher (eg, at least about 10%, 20%, 30%, 40%, 50%, 60% higher) ability to inhibit ⁇ -secretase cleavage (eg, ⁇ -cleavage of APP) , 70%, 80%, 90%, 95%, 100%, 2 times, 5 times, 10 times, 20 times or more)
  • the protein shown in SEQ ID NO: 12 or 33 or 89 is cleaved by ⁇ -secretase inhibition ability.
  • the inhibitory ability of secretase enzyme cleavage (such as the ⁇ -enzyme cleavage to APP) is the same or similar to the inhibitory ability of the protein shown in SEQ ID NO: 12 or 33 or 89 to ⁇ -secretase cleavage (for example, the difference is about 10 within %).
  • the ApoE construct, the ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or a fragment of the amino acid sequence shown in SEQ ID NO: 12 in (1) pair ⁇ - the inhibitory ability of secretase cleavage (for example to the ⁇ -enzyme cleavage of APP) is lower than the inhibitory ability of the protein shown in SEQ ID NO: 12 or 33 or 89 to ⁇ -secretase cleavage by at most about 50% (for example, lower 40% %, 30%, 20%, 10%, 5%, 1% or less).
  • said ApoE construct (such as an isolated ApoE construct), said ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or said (1) is derived from SEQ ID NO: 12
  • the fragments of the amino acid sequence shown (such as any sequence in SEQ ID NOs: 13, 18-34, 78 and 87) have the same inhibitory activity as ⁇ -secretase-mediated enzyme cleavage (such as ⁇ -enzyme cleavage to APP) and ⁇ - the secretase inhibitor (eg PF03084014) is identical or similar (eg within about 10%, 5% or 1%).
  • said ApoE construct (such as an isolated ApoE construct), said ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or said (1) is derived from SEQ ID NO: 12 Fragments of the indicated amino acid sequences have higher (eg, at least about 10%, 20%, 50%, 100%, 1.5-fold, 2-fold, 5-fold or higher) 7-secretase inhibitors (eg PF03084014).
  • said ApoE construct (such as an isolated ApoE construct), said ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or said (1) is derived from SEQ ID NO: 12 Fragments of the indicated amino acid sequences have lower inhibitory activity (eg, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) a gamma-secretase inhibitor (eg PF03084014).
  • inhibitory activity eg, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more
  • a gamma-secretase inhibitor eg PF03084014
  • the ApoE construct (such as an isolated ApoE construct), the ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or the amino acid derived from SEQ ID NO: 12 in (1) Fragments of the amino acid sequence shown (for example any sequence in SEQ ID NOs: 13, 18-34, 78 and 87) have a higher (for example higher than at least about 30%, 40%, 50%) inhibition of gamma-cleavage of APP , 60%, 70%, 80%, 90%, 95%, 100%, 2 times, 5 times, 10 times, 20 times or more) of ⁇ -digestion of non-APP proteins (such as APLP1, Notch) inhibition.
  • non-APP proteins such as APLP1, Notch
  • the ApoE construct (such as an isolated ApoE construct), the ApoE polypeptide (such as an isolated ApoE polypeptide, or an ApoE mutant), or the amino acid derived from SEQ ID NO: 12 in (1)
  • the fragments of the amino acid sequences shown only inhibit the ⁇ -cleavage of APP, but have no or almost no inhibition on the ⁇ -cleavage of non-APP proteins (such as APLP1, Notch).
  • an alpha-amylase inhibitor eg, GI254023X
  • a beta-amylase inhibitor eg, AZD3839
  • said ApoE construct such as an isolated ApoE construct
  • said ApoE polypeptide such as an isolated ApoE polypeptide, or an ApoE mutant
  • said (1) Inhibition of the activity of ⁇ -secretase cleavage such as ⁇ -cleavage of APP
  • the ApoE construct or ApoE polypeptide can still increase the level of o-CTF produced by APP and reduce the production of A ⁇ in the presence of an ⁇ -amylase inhibitor or a ⁇ -amylase inhibitor.
  • the influence on the ⁇ -secretase enzymatic cleavage (such as the ⁇ -enzymatic cleavage of APP) activity can be studied in any suitable manner in the art, such as studying the enzymatic activity of ⁇ -secretase itself, or studying the cleavage product of ⁇ -secretase ( For example, A ⁇ , such as A ⁇ 40 or A ⁇ 42) identity or quantity, or to study ⁇ -secretase substrates (such as APP, o-CTF, ⁇ -CTF, APLP1, Notch, ErbB4, E-cadherin, N-cadherin, ephrin-B2 or CD44 ) characteristics or quantity (for example, whether it is reduced by ⁇ -digestion).
  • a ⁇ such as A ⁇ 40 or A ⁇ 42
  • ⁇ -secretase substrates such as APP, o-CTF, ⁇ -CTF, APLP1, Notch, ErbB4, E-cadherin, N-
  • ⁇ -secretase activity assay kit can also be used here, such as the #MBS704513 kit from MyBioSource, or the #FP003 kit from R&D Systems. See also the study format described in Examples 1 and 2.
  • the ApoE construct is in any one of the ApoE polypeptides (e.g., SEQ ID NOs: any sequence in 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87)
  • the N-terminal also further comprises the N-terminal domain sequence or partial sequence of ApoE2 albumen (such as SEQ ID NO : 12 1-167 amino acid sequence or partial sequence; SEQ ID NO: 79), and/or hinge region sequence or partial sequence (for example SEQ ID NO: 12 168-205 amino acid sequence or partial sequence; SEQ ID NO :80), or at least about 70% (such as at least about 75%, 80%, 85%, 90%) of the N-terminal domain sequence (or partial sequence) and/or hinge region sequence (or partial sequence) of the ApoE2 protein , 95%, 96%, 97%, 98%, 99% or higher) identity (eg ApoE homologous sequences from non-human primates such as monkeys).
  • the ApoE construct is in any one of the ApoE polypeptides (eg, any sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87)
  • the N-terminal further comprises the N-terminal domain sequence or partial sequence (such as the 1-167th amino acid sequence or partial sequence; SEQ ID NO: 90) of the ApoE3 protein, and/or the hinge region sequence or partial sequence (such as the 168- 205 amino acid sequence or partial sequence; SEQ ID NO:80), or at least about 70% (for example, at least about Sequences of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher) identity (e.g.
  • the ApoE construct further comprises a part of the sequence from the ApoE C-terminal domain (SEQ ID NO: 12 206-299) at the N-terminus of the ApoE polypeptide (for example, its N-terminal partial sequence ), such as the sequence or partial sequence of amino acids 206-214 of SEQ ID NO: 12, or at least about 70% (eg, at least about 75%, 80%, 85%, 90%) of the partial sequence of the ApoE C-terminal domain %, 95%, 96%, 97%, 98%, 99% or higher) identity.
  • a linker sequence such as a protein linker sequence
  • homologous to the N-terminal domain (or partial sequence) and/or hinge region (or partial sequence) and/or C-terminal domain (or its N-terminal partial sequence) of human ApoE2 or ApoE3 protein The ApoE protein sequence is derived from any of the following species: mammals (eg, non-human primates such as monkeys, rodents, livestock, pets), birds, frogs, fish, Drosophila, nematodes, and the like.
  • the ApoE construct is in any one of the ApoE polypeptides (eg, any sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87)
  • the N-terminal further comprises the amino acid sequence of SEQ ID NO: 12 1-167 (SEQ ID NO: 79) or 1-205 (SEQ ID NO: 81) or 1-214 amino acid residues, or with An amino acid sequence having at least about 70% identity to amino acids 1-167 or 1-205 or 1-214 of SEQ ID NO:12.
  • the ApoE The construct comprises the amino acid sequence shown in SEQ ID NO:60 or 61.
  • the ApoE construct consists of, or consists essentially of, the amino acid sequence set forth in SEQ ID NO:60 or 61.
  • the ApoE construct is in any one of the ApoE polypeptides (such as any sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87)
  • the N-terminus further comprises the amino acid sequence of ApoE3 (SEQ ID NO: 89) 1-167 (SEQ ID NO: 90) or 1-205 or 1-214 amino acid residues, or with ApoE3 1- An amino acid sequence having at least about 70% identity to amino acids 167 or 1-205 or 1-214.
  • the ApoE construct comprises (or consists of, or consists essentially of) the full-length sequence of ApoE2 (e.g., human ApoE2, SEQ ID NO: 12). In some embodiments, the ApoE construct comprises (or consists of, or consists essentially of) the full-length sequence of ApoE3 (e.g., human ApoE3, SEQ ID NO: 89).
  • the ApoE construct is any sequence in any of the ApoE polypeptides (such as SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, or The N-terminal or C-terminal of ApoE2 or ApoE3 full-length sequence) further comprises a membrane-penetrating peptide.
  • the membrane-penetrating peptide comprises (or consists of, or consists essentially of) any amino acid sequence in SEQ ID NOs: 35-45, 63-65, and 84-86, such as SEQ ID NO: The amino acid sequence shown in 63 or 65.
  • the N-terminal of the penetrating peptide is further connected to the C-terminal of a signal peptide through a peptide bond or a linker sequence.
  • the penetrating peptide is located at the most N-terminal of the ApoE construct. In some embodiments, the penetrating peptide is located at the most C-terminus of the ApoE construct.
  • the present invention provides an ApoE construct (or fusion protein) comprising the following elements (1) and (2): (1) a penetrating peptide, and (2) the full length of ApoE2 A sequence (eg SEQ ID NO: 12) or a full-length sequence of ApoE3 (eg SEQ ID NO: 89) or a fragment of ApoE2 at least including amino acid residues 221-274 of SEQ ID NO: 12.
  • the element (1) and element (2) can be connected directly through a peptide bond, or through a linker sequence.
  • Membrane-penetrating peptides or cell-penetrating peptides refer to a class of short peptides that can carry macromolecular substances into cells. Membrane-penetrating peptides known in the art can be used to construct the fusion protein of the present invention. Exemplary penetrating peptides can be found in Siegmund Reissmann, Cell penetration: scope and limitations by the application of cell-penetrating peptides, J. Pept. Sci. 2014; 20: 760-784. This article incorporates its entire content into this article by reference.
  • Penetrating peptides suitable for use in the present invention include, but are not limited to, RQIKIWFQNRRMKWKK (SEQ ID NO: 35); YGRKKRRQRRR (SEQ ID NO: 36); KQAIPVAK (SEQ ID NO: 37); RRRRNRTRRNRRVR (SEQ ID NO: 38); Amino acid (that is, a fragment of 9-12 arginine residues; R 9 (SEQ ID NO: 63), R 10 (SEQ ID NO: 84), R 11 (SEQ ID NO: 85), R 12 ( SEQ ID NO: 86)); KLTRAQRRAAARKNKRNTRGC (SEQ ID NO: 39); ALWKTLLKKVLKAPKKKRKVC (SEQ ID NO: 40); RKKRRQRRR (SEQ ID NO: 41); DAATATRGRSAASRPTERPRAPARSASRPRRPVE (SEQ ID NO: 42); GWTLNSAGYLL GKINLKALAALAKKIL (SEQ ID NO: 43); AG
  • the fragment of ApoE2 described in element (2) in the fusion protein at least includes SEQ ID NO: 12 amino acid residues 221-274, can be the isolated ApoE polypeptide described in any embodiment herein , and a fragment comprising the N-terminal and hinge regions (or fragments thereof) of ApoE2 or ApoE3 and at least the 221-274th amino acid residues at the C-terminal.
  • the ApoE2 polypeptide may comprise any of the following sequences: SEQ ID NO: 12 1-274, 1-279, and 1-280, 1-281, 1 -282, 1-283, 1-284, 1-285, 1-286, 1-287, 1-288, 1-289, 1-290 bit, 1-291st, 1-292nd, 1-293rd, 1-294th, 1-295th, 1-296th, 1-297th, 1-298th or Amino acid residues 1-299.
  • the element (2) in the fusion protein is selected from any amino acid sequence in SEQ ID NOs: 12, 13, 18-34, 60, 78, 87 and 89.
  • the linker is a non-protein linker. In some embodiments, the linker is a protein linker. In some embodiments, the linker is a peptide comprising 3-25 residues, eg, a peptide comprising 3-15, 5-15, 10-20, 20-25, or 3-10 residues. Suitable examples of peptide linkers are well known in the art. Typically, linkers contain one or more tandemly repeated motifs, usually containing Gly and/or Ser.
  • the motif can be SGGS (SEQ ID NO: 69), GSSGS (SEQ ID NO: 70), GGGS (SEQ ID NO: 71), GGGGS (SEQ ID NO: 72), SSSSG (SEQ ID NO: 73 ), GSGSA (SEQ ID NO: 74) GGSGG (SEQ ID NO: 75) or GGGSGGGS (SEQ ID NO: 88).
  • the above sequences are contiguous in the linker sequence with no intervening amino acid residues between the repeats.
  • Linker sequences may consist of 1, 2, 3, 4 or 5 repeat motifs.
  • the linker sequence is a polyglycine linker sequence.
  • the number of glycines in the linker sequence is not particularly limited, usually 2-20, such as 2-15, 2-10, 2-8.
  • other known amino acid residues such as alanine, leucine, threonine, glutamic acid, phenylalanine, arginine, glutamine, etc. may be contained in the linker.
  • the linker sequence is shown in SEQ ID NO:88.
  • ApoE constructs or fusion proteins of the present invention may also include gene cloning operations, and/or in order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the detection of recombinant proteins and / or amino acid residues or sequences introduced into the fusion protein for purification, etc.
  • the ApoE construct or fusion protein of the present invention also includes signal peptide, leader peptide, terminal extension, tag protein and the like.
  • the ApoE construct further comprises a signal peptide at the N-terminus of the ApoE polypeptide, especially when it needs to be expressed in cells.
  • the C-terminus of the signal peptide is identical to any sequence in the ApoE polypeptide (such as SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, or the N-terminus of ApoE2 or ApoE3 full-length sequence), or said comprising SEQ ID NO: 12 or 89 1-167 (or its fragment) or 1-205 (or its fragment) or 1-214 (or a fragment thereof) the N-terminus of the amino acid sequence of amino acid residues, or the amino acid residues described above and SEQ ID NO: 12 or 89 1-167 (or a fragment thereof) or 1-205 (or a fragment thereof) or 1 -
  • the N-terminus of the amino acid sequence at position 214 (or a fragment thereof) having at least about 70% identity, or
  • the signal peptide is located at the most N-terminal of the ApoE construct. In some embodiments, the signal peptide is further added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 60 or 61. In some embodiments, the signal peptide is further added to the N-terminus of the full-length sequence of ApoE2 (such as SEQ ID NO: 12) or ApoE3 (such as SEQ ID NO: 89). In some embodiments, the C-terminus of the signal peptide is further linked to the N-terminus of a penetrating peptide via a peptide bond or a linker sequence (eg, a protein linker sequence). Any known signal peptide can be used here.
  • the signal peptide is homologous to the ApoE construct or the ApoE polypeptide, for example, the signal peptide from the ApoE2 (SEQ ID NO: 12) or ApoE3 (SEQ ID NO: 89) protein itself.
  • the signal peptide is heterologous to the ApoE construct or the ApoE polypeptide, eg, a signal peptide from ApoAl or ApoJ.
  • the signal peptide comprises (or consists of, or consists essentially of) any amino acid sequence selected from SEQ ID NOs: 66-68.
  • the ApoE construct comprises (or consists of, or consists essentially of) such as
  • the sequence from N-terminus to C-terminus is as follows:
  • Signal peptide-optional linker 1-penetrating peptide such as the amino acid sequence shown in SEQ ID NO: 63 or 65
  • 2-ApoE2 protein such as SEQ ID NO: 12
  • ApoE3 protein such as SEQ ID NO: 12
  • ApoE3 protein such as SEQ ID NO: 89
  • Signal peptide - optional linker - ApoE2 protein eg SEQ ID NO: 12
  • ApoE3 protein eg SEQ ID NO: 89
  • ApoE2 protein such as SEQ ID NO: 12
  • ApoE3 protein such as SEQ ID NO: 89
  • Penetrating peptide such as the amino acid sequence shown in SEQ ID NO: 63 or 65
  • optional linker - ApoE2 protein such as SEQ ID NO: 12
  • ApoE3 protein such as SEQ ID NO: 89
  • Signal peptide-optional linker 1-penetrating peptide such as the amino acid sequence shown in SEQ ID NO: 63 or 65
  • 2-any ApoE polypeptide such as SEQ ID NOs: 13, 18- Any sequence in 34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33
  • ApoE polypeptide such as SEQ ID NOs: 13, 18- Any sequence in 34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33
  • Penetrating peptide such as the amino acid sequence shown in SEQ ID NO: 63 or 65
  • linker-any ApoE polypeptide such as SEQ ID NOs: 13, 18-34, 46-52, 57-59, Any sequence in 62, 76, 78 and 87, such as SEQ ID NO: 33
  • Signal peptide-optional linker-any one of said ApoE polypeptide (such as any sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO :33);
  • ApoE polypeptides for example any sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33;
  • Signal peptide-optional linker 1-penetrating peptide such as the amino acid sequence shown in SEQ ID NO: 63 or 65
  • 2-ApoE2 or ApoE3 N-terminal domain and/or hinge region and/or The N-terminus of the C-terminal domain) sequence or fragment (such as SEQ ID NOs: 79-81 and 90 any shown aminoacid sequence or its fragment)-optional linker 3-any described ApoE polypeptide (such as SEQ ID NOs: any sequence in 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33);
  • Penetrating peptide e.g. amino acid sequence shown in SEQ ID NO: 63 or 65
  • optional linker 1 - ApoE2 or ApoE3 N-terminal domain and/or hinge region and/or (N-terminal to C-terminal domain) sequence or fragment such as SEQ ID NOs: 79-81 and 90 amino acid sequence shown in any one or its fragment
  • 2-any ApoE polypeptide such as SEQ ID NOs: 13,18-34,46 Any sequence in -52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33
  • ApoE polypeptide such as SEQ ID NOs: 13,18-34,46 Any sequence in -52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33
  • Signal peptide-optional linker 1-ApoE2 or ApoE3 N-terminal domain and/or hinge region and/or (N-terminal of C-terminal domain) sequence or fragment for example any of SEQ ID NOs: 79-81 and 90
  • the indicated amino acid sequence or its fragment Section)-optional linker 2-any one of the ApoE polypeptides for example any sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33;
  • Signal peptide-optional linker 1-penetrating peptide such as the amino acid sequence shown in SEQ ID NO: 63 or 65
  • amino acid sequence shown in SEQ ID NO: 60 or 61 amino acid sequence shown in SEQ ID NO: 60 or 61
  • Penetrating peptide such as the amino acid sequence shown in SEQ ID NO: 63 or 65
  • linker- the amino acid sequence shown in SEQ ID NO: 60 or 61
  • Signal peptide - optional linker - (amino acid sequence shown in SEQ ID NO: 60 or 61).
  • the ApoE construct further comprises a tag protein at the N-terminus and/or C-terminus of the ApoE polypeptide.
  • the tag protein is located at the most N-terminal of the ApoE construct.
  • the tag protein is located at the most C-terminus of the ApoE construct.
  • the tag protein (such as the 3XFLAG sequence shown in SEQ ID NO: 77) is located inside the ApoE construct but at the N-terminal of the ApoE polypeptide, such as with the N-terminal of the ApoE polypeptide directly connected.
  • the tag protein can be an expression purification tag (such as His-tag, FLAG-tag, GST-tag, c-Myc-tag, V5-tag, HA-tag), or a detection tag (such as GFP, RFP, V5, HA ).
  • purification tags can also be used for detection, such as immunoblotting or antibody detection.
  • the tag protein is located at the most N-terminal of the ApoE construct (e.g., ApoE2 or ApoE3, or an ApoE polypeptide comprising the sequence of SEQ ID NO: 33). In some embodiments, the tag protein is located at the most C-terminus of the ApoE construct.
  • the tag protein is the V5 tag GKPIPNPLLGLDST (SEQ ID NO: 82) or IPNPLLGLD (SEQ ID NO: 83). In some embodiments, the tag protein is a FLAG tag (e.g., SEQ ID NO: 77).
  • Amyloid-beta precursor protein APP
  • APP is a cell membrane embedded protein, mainly concentrated in neuronal synapses. It contains cleavage sites for ⁇ , ⁇ , ⁇ secretases. APP is expressed in many different species and is highly homologous.
  • the APP described herein can be from any species, such as mammals, birds, frogs, fish, fruit flies, nematodes, etc.
  • Mammals include, but are not limited to, primates (such as humans, or non-human primates such as monkeys), rodents (such as mice, rats, hamsters, gerbils, squirrels, guinea pigs and rabbits), domestic animals (such as pigs, cows, sheep, horses, donkeys), pets (such as cats, dogs, rabbits and hamsters), etc.
  • the APP is human APP.
  • Amyloid metabolism of APP can generate short amyloid beta polypeptides (A ⁇ ).
  • a ⁇ polypeptides are produced by cleavage of APP by ⁇ -secretase activity near the N-terminal position of A ⁇ and by ⁇ -secretase activity near the C-terminal position of A ⁇ .
  • APP is also cleaved by ⁇ -secretase activity, yielding a secreted, non-amyloid fragment of soluble APP ⁇ .
  • Beta-site APP-cleaving enzyme (“BACE-1”) is believed to be the major aspartyl protease responsible for the production of A ⁇ by ⁇ -secretase activity. BACE-1 inhibition can suppress A ⁇ production.
  • BACE1 is a type I transmembrane protein with a luminal active site that cleaves APP to release the extracellular domain (sAPP ⁇ ) into the extracellular space.
  • the remaining C-terminal fragment (CTF) undergoes further cleavage by ⁇ -secretase, resulting in the release of the intracellular C-terminal domain (AICD) of A ⁇ and APP.
  • Presenilin is the major enzymatic component of ⁇ -secretase, and imprecise cleavage of APP by ⁇ -secretase produces contiguous A ⁇ polypeptides at the C-terminus varying in length by several amino acids.
  • a ⁇ 40 amino acid 40
  • a ⁇ 42 42-amino acid variant
  • a ⁇ 38 38-amino acid variant
  • ADAM-10 is an alpha-secretase for APP cleavage that releases an ectodomain (sAPP ⁇ ) that exhibits neuroprotective functions
  • a ⁇ is a major component of ⁇ -amyloid fibrils and plaques, which are thought to play a role in a growing number of pathologies.
  • pathologies include, but are not limited to, Alzheimer's disease, Down's syndrome, Parkinson's disease, memory loss (including memory loss associated with Alzheimer's disease and Parkinson's disease), attention deficit Alzheimer's disease (including attention deficit disorder associated with Alzheimer's disease, Parkinson's disease and Down syndrome), dementia (including presenile dementia, senile dementia, dementia associated with Kinson's disease and Down syndrome), progressive supranuclear palsy, corticobasal degeneration, neurodegeneration, olfactory disturbances (including those associated with Alzheimer's disease, Parkinson's disease, and Down syndrome) , ⁇ -amyloid cerebrovascular disease (including cerebral amyloid cerebrovascular disease), hereditary cerebral hemorrhage, mild cognitive impairment ("MCI”), glaucoma, amyloidosis, type II diabetes, hemodialysis ( ⁇ 2 microsphere protein and its
  • the APP is wild-type APP. In some embodiments, the APP is a naturally occurring APP subtype. In some embodiments, the APP is a mutant, including but not limited to any known or unknown APP mutation.
  • APP mutations have been identified in 121 families. The most common missense mutations replace the Val717 residue with an Ile (London mutation), Phe, or Gly residue, with a Gln residue (Dutch mutation) or a Gly residue (Arctic mutation) to replace Glu 693 , or use Asn and Leu residues to replace Lys 670 and Met 671 (the double mutation is called the Swedish mutation APP SW and is numbered in the APP 770 isoform).
  • the APP is human APP comprising one or more of the following mutations: K670N, M671L, Swedish (K670N+M671L), Florida (I716V), London (V717I), APP M596V (incapable of beta cleavage), fAD mutations near ⁇ -cleavage sites (e.g. T714I, V717F, L723P), fAD mutations near ⁇ - or ⁇ -cleavage sites (e.g. KM670/671NL, E693K, E693 ⁇ ), or protective Icelandic mutations (A673T ).
  • the APP does not comprise mutations affecting ⁇ -splicing, such as mutations in the attachment of the ⁇ site.
  • the ApoE construct or ApoE polypeptide can inhibit ⁇ -cleavage of any APP (e.g., inhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%) , 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%).
  • the ApoE construct or ApoE polypeptide has an ability to inhibit ⁇ -cleavage of any of the APP mutants that is at least about 30% (eg, at least about 40%) of its ability to inhibit ⁇ -cleavage of wild-type APP.
  • the ⁇ -cleavage inhibition ability of the ApoE construct or ApoE polypeptide to APP comprising a mutation affecting ⁇ -cleavage is less than (e.g., at least about 20%, 30%, %, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100%) of its ability to inhibit wild-type APP ⁇ -cleavage.
  • the ⁇ -secretase complex consists of four separate proteins: PSEN1 (presenilin-1, PS1), nicastrin, APH-1 (anterior pharynx-defective 1), and PEN-2 (presenilin Enhancer 2, presenilin enhancer 2).
  • PSEN1 presenilin-1, PS1
  • nicastrin a protein that influences the expression of CD147
  • APH-1 anterior pharynx-defective 1
  • PEN-2 presenilin Enhancer 2, presenilin enhancer 2
  • CD147 the fifth protein of the complex, is a non-essential regulator whose deletion increases ⁇ -secretase activity.
  • the ⁇ -secretase described herein can be from any species, such as mammals, birds, frogs, fish, Drosophila, nematodes and the like.
  • the gamma secretase is from a human, or a non-human primate such as a monkey.
  • the gamma-secretase described herein is wild-type gamma-secretase, or a naturally occurring isoform.
  • the ⁇ -secretase comprises one or more mutations, such as mutations that enhance or decrease its enzymatic cleavage activity.
  • the ⁇ -secretase has one or two mutations in PS1: M146L, L286V.
  • the PS1 and/or PS2 activity of the ⁇ -secretase is reduced or absent.
  • the ApoE construct or ApoE polypeptide can inhibit the enzymatic activity of any ⁇ -secretase (eg, inhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70% %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%).
  • the inhibitory ability of the ApoE construct or ApoE polypeptide to any one of the ⁇ -secretase mutant enzyme activities is at least about 30% (for example, at least About 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5 times, 2 times, 5 times, 10 times or more).
  • the present invention also provides an isolated ApoE construct (such as any amino acid sequence of SEQ ID NOs: 12, 60, 61 and 89) encoding any of the above, a fusion protein, or an ApoE polypeptide (such as SEQ ID NOs: 13, 18 -34, 46-52, 57-59, 62, 76, 78, and 87 amino acid sequences, such as isolated nucleic acids and vectors (such as cloning vectors or expression vectors) of the protein portion of SEQ ID NO: 33), and A host cell expressing any of the above isolated ApoE constructs, or a host cell comprising the nucleic acid or vector.
  • the vector is a recombinant AAV or lentivirus expression vector.
  • the vector contains a neuron-specific promoter at the 5' end of the isolated nucleic acid.
  • the neuron-specific promoter is selected from the synapsin promoter (eg, hSYN), thy-1 promoter, and calmodulin-dependent protein kinase II-alpha promoter.
  • the host cell is a neural cell, such as a neuron (eg, a human neuron, a non-human primate neuron, or a rodent neuron). See Examples 1 and 2 for vectors, cells and methods of manufacture.
  • the present invention provides a nucleic acid or vector capable of mediating the specific expression of any one of the ApoE constructs, fusion proteins, or ApoE polypeptides in neural cells.
  • the invention provides a vector or isolated nucleic acid comprising the following 5' to 3' sequence: neuron-specific promoter (eg hSYN) - nucleotide sequence encoding signal peptide - encoding transmembrane Nucleotide sequence of peptide (eg SEQ ID NO: 63 or 65) - encoding any of said ApoE construct (eg SEQ ID NO: 12 or 89)/fusion protein/ApoE polypeptide (eg SEQ ID NO: 33) Nucleotide sequence.
  • neuron-specific promoter eg hSYN
  • nucleotide sequence encoding signal peptide - encoding transmembrane
  • Nucleotide sequence of peptide eg SEQ ID NO: 63 or 65
  • the invention provides a vector or isolated nucleic acid comprising the following 5' to 3' sequence: neuron-specific promoter (eg hSYN) - encoding a penetrating peptide (eg SEQ ID NO: 63 or 65) Nucleotide sequence - a nucleotide sequence encoding any of said ApoE construct (eg SEQ ID NO: 12 or 89)/fusion protein/ApoE polypeptide (eg SEQ ID NO: 33).
  • neuron-specific promoter eg hSYN
  • a penetrating peptide eg SEQ ID NO: 63 or 65
  • Nucleotide sequence - a nucleotide sequence encoding any of said ApoE construct (eg SEQ ID NO: 12 or 89)/fusion protein/ApoE polypeptide (eg SEQ ID NO: 33).
  • the invention provides a vector or isolated nucleic acid comprising the following 5' to 3' sequence: neuron-specific promoter (eg hSYN) - nucleotide sequence encoding signal peptide - encoding either The nucleotide sequence of the ApoE construct (eg, SEQ ID NO: 12 or 89)/fusion protein/ApoE polypeptide (eg, SEQ ID NO:33).
  • the ApoE construct comprises (or consists of, or consists essentially of) the full-length sequence of ApoE2 or ApoE3.
  • the ApoE construct comprises (or Consisting of, or consisting essentially of) the amino acid sequence of SEQ ID NOs: 60 or 61.
  • the ApoE polypeptide comprises (or consists of, or consists essentially of) any of SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87 An amino acid sequence, such as SEQ ID NO:33.
  • the present application also includes nucleic acid molecules encoding any of the above isolated ApoE constructs, fusion proteins, or ApoE polypeptides, and their complements.
  • the invention includes all forms of the nucleic acid molecules of the invention, including RNA and DNA.
  • the complementary sequence described herein refers to a complementary sequence that is substantially identical in length to the nucleic acid molecule (eg, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical in length).
  • a sequence eg, at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%. 99% or 100% complementary.
  • nucleic acid constructs comprising said nucleic acid molecule or its complement are also included within the scope of the present invention.
  • the nucleic acid construct may be an expression cassette comprising a promoter, the nucleic acid molecule and a transcription termination sequence.
  • the expression cassette may optionally contain an enhancer. Promoters, enhancers, transcription termination sequences, selectable markers and signal sequences are well known in the art, and can be appropriately selected by those skilled in the art according to the selected host cell for expressing the polypeptide. These elements of the expression cassette are operably linked to each other such that they perform their intended functions.
  • operably linking a promoter sequence to a nucleotide sequence of interest refers to linking the promoter sequence and the nucleotide sequence of interest such that the promoter sequence is capable of directing the nucleotide sequence of interest in the target cell.
  • Transcription and/or synthesis of a polypeptide encoded by a nucleotide sequence of interest can aid in the selection of host cells into which the vector has been inserted from those into which the vector has not been inserted.
  • a selectable marker can be a gene that confers antibiotic resistance.
  • the signal sequence can be chosen to allow trafficking of the expressed polypeptide to the outside of the host cell.
  • Genetic control elements can be used to regulate gene expression of polypeptides or proteins. Such genetic control elements are selected to be active in the relevant host cell. Control elements may be constitutively active or inducible under specified conditions. Inducible control elements are particularly useful when expressing a protein is toxic or otherwise adversely affects cell growth and/or viability. In such cases, regulation of polypeptide or protein expression by inducible control elements can improve cell viability, cell density and/or overall yield of expressed polypeptide or protein. A large number of control elements that can be used in the practice of the present invention are known and available in the art.
  • constitutive mammalian promoters that can be used in the present invention include, but are not limited to, hypoxanthine phosphoribosyltransferase (HPTR) promoter, adenosine deaminase promoter, pyruvate kinase promoter, beta actin promoter promoters and other constitutive promoters known to those of ordinary skill in the art.
  • HPTR hypoxanthine phosphoribosyltransferase
  • adenosine deaminase promoter adenosine deaminase promoter
  • pyruvate kinase promoter pyruvate kinase promoter
  • beta actin promoter promoters and other constitutive promoters known to those of ordinary skill in the art.
  • viral promoters capable of driving constitutive expression of coding sequences in eukaryotic cells include, for example, simian virus promoters, herpes simplex virus promoters, papillomavirus promoters, adenovirus promoters, human immunodeficiency virus (HIV ) promoter, Rous sarcoma virus promoter, cytomegalovirus (CMV) promoter, long terminus of Moloney murine leukemia virus and other retroviruses repeat sequence (LTR), herpes simplex virus thymidine kinase promoter, and other viral promoters known to those of ordinary skill in the art.
  • simian virus promoters include, for example, simian virus promoters, herpes simplex virus promoters, papillomavirus promoters, adenovirus promoters, human immunodeficiency virus (HIV ) promoter, Rous sarcoma virus promoter, cytomegalovirus (CMV
  • Inducible promoters which drive the expression of an operably linked coding sequence in the presence of an inducing agent, can also be used in accordance with the present invention.
  • the metallothionein promoter can induce transcription of downstream coding sequences in the presence of certain metal ions.
  • Other inducible promoters are recognized and/or known to those of ordinary skill in the art.
  • gene expression control elements suitable for use in the present invention are neuron-specific control elements, including promoters and enhancers.
  • neuron-specific control element refers to control elements for specifically expressing a nucleic acid molecule of interest in neurons, such as promoters and enhancers.
  • a neuron-specific promoter is a promoter used to express a transgene in a certain subtype of neurons (eg, dopaminergic neurons, excitatory neurons, lateral prefrontal cortex neurons, etc.).
  • Neuron-specific promoters and other control elements are known in the art.
  • Suitable neuron-specific promoters include, but are not limited to, the neuron-specific enolase (NSE) promoter, aromatic amino acid decarboxylase promoter, neurofilament promoter, synapsin promoter, thy-1 promoter, Serotonin receptor promoter, tyrosine hydroxylase promoter, GnRH promoter, L7 promoter, DNMT promoter, enkephalin promoter, myelin basic protein promoter, calmodulin-dependent protein kinase II - alpha promoter, and CMV enhancer/platelet-derived growth factor-beta promoter.
  • the neuron-specific promoter is selected from the synapsin promoter (eg, hSYN), thy-1 promoter, and calmodulin-dependent protein kinase II-alpha promoter.
  • gene expression sequences also include 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA box, cap sequence, CAAT sequence, etc.
  • Enhancer elements can optionally be used to enhance the expression level of a polypeptide or protein to be expressed. Examples of enhancer elements that can function in mammalian cells include the SV40 early gene enhancer (as described by Dijkema et al., EMBOJ. (1985) 4:761 ), and those from Rous sarcoma virus (RSV) (as described by Gorman et al.
  • nucleic acid constructs are vectors, including expression vectors and cloning vectors.
  • the expression vector is suitable for expressing foreign genes such as coding polypeptides of the present invention (such as ApoE2 or ApoE3 full-length sequences) in host cells or any of the ApoE constructs, ApoE polypeptides or fusion proteins described herein), including prokaryotic expression vectors and eukaryotic expression vectors.
  • Eukaryotic expression systems include yeast expression systems, mammalian cell expression systems and insect cell expression systems.
  • the cloning vector is used to amplify the target gene (such as the nucleic acid molecule encoding the polypeptide of the present invention) in the host cell.
  • Cloning vectors include plasmid vectors, phage vectors, viral vectors, and vectors that are combined with each other or with other genomic DNA. The most commonly used host cell in molecular cloning is Escherichia coli.
  • the vector is a viral vector, including but not limited to lentiviral vectors, herpes simplex viral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors. Such vectors can be prepared using standard methods in the art.
  • the vector is a lentivirus or lentiviral vector.
  • the vector is a recombinant AAV vector.
  • AAV vectors can be prepared using standard methods in the art.
  • Adeno-associated virus vectors of any serotype may be used, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16.
  • the invention provides a vector (e.g., an AAV or lentiviral vector) containing an expression cassette containing a neuron-specific promoter operably linked to a nucleic acid molecule of interest (e.g., hSyn), the nucleic acid molecule of interest is selected from: a nucleic acid molecule encoding any of the ApoE C-terminal fragments of the present invention, a nucleic acid molecule encoding ApoE2 or ApoE3 full-length protein, encoding any of the ApoE2 constructs or fusions of the present invention A nucleic acid molecule of a protein, or a nucleic acid molecule encoding any of the ApoE2 polypeptides of the present invention.
  • a vector e.g., an AAV or lentiviral vector
  • an expression cassette containing a neuron-specific promoter operably linked to a nucleic acid molecule of interest e.g., hSyn
  • the vector is a recombinant AAV vector.
  • neuron-specific promoters suitable for use herein include the synapsin promoter, the thy-1 promoter, and the calmodulin-dependent protein kinase II-alpha promoter.
  • the nucleic acid molecules of interest are encoded in any one of SEQ ID NOs: 12, 13, 18-34, 46-52, 57-59, 60-62, 76, 78, 87 and 89 amino acid sequence.
  • the nucleic acid molecule of interest also encodes an N-terminal signal peptide (such as any amino acid sequence in SEQ ID NOs: 66-68).
  • the nucleic acid molecule of interest also encodes an N-terminal and/or C-terminal penetrating peptide (such as any amino acid sequence in SEQ ID NOs: 35-45, 63-65 and 84-86). In some embodiments, the nucleic acid molecule of interest also encodes an N-terminal and/or C-terminal tag protein (eg V5, EGFP).
  • an N-terminal and/or C-terminal tag protein eg V5, EGFP.
  • the present invention also provides an expression system comprising the nucleic acid molecule, nucleic acid construct or vector disclosed herein.
  • the expression system can be, for example, a Gram-positive or Gram-negative prokaryotic host cell system, such as E. coli modified to express a polypeptide of the invention.
  • the expression system is a eukaryotic host cell system, such as a yeast, such as Pichia pastoris or Saccharomyces cerevisiae.
  • the host cell is a mammalian host cell, such as HEK293T, N2A, CHO cells, and the like.
  • the host cell is a neural Cells, including but not limited to glial cells and neurons.
  • Higher eukaryotic cells can be used for the expression of glycosylated polypeptides.
  • Suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells, and vertebrate cells.
  • nucleic acid required to express a polypeptide or protein of interest are well known in the art. See eg Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); Levinson et al.; EP 117,060; and EP 117,058, all herein cited here for reference.
  • the vector can be introduced into the host cell using any suitable method known in the art, including but not limited to DEAE-dextran-mediated delivery, calcium phosphate precipitation method, cationic lipid-mediated delivery, liposome Mediated transfection, electroporation, particle bombardment, receptor-mediated gene delivery, delivery mediated by polylysine, histones, chitosan and peptides.
  • suitable transformation methods include the calcium phosphate precipitation method of Graham and van der Erb, Virology, 52:456-457 (1978), or the lipofectamine TM of Hawley-Nelson, Focus 15:73 (1193).( Gibco BRL) method.
  • Gibco BRL Gibco BRL
  • Patent No. 4,399,216 filed August 16,1983.
  • Various techniques for transforming mammalian cells can be found in Keown et al., Methods in Enzymology (1989), Keown et al., Methods in Enzymology, 185:527-537 (1990), and Mansour et al., Nature, 336:348-352 (1988 ).
  • Non-limiting representative examples of suitable vectors for expression of polypeptides or proteins in mammalian cells include P(3)NA1; p(3), see Okayama et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneoPoly -A, see Thomas et al. (1987) Cell 51:503-512; and baculovirus vectors such as PAC373 or pAC610.
  • the polypeptide or protein of the invention is stably transfected into a host cell.
  • the present invention can be used to transiently or stably transfect host cells.
  • target protein or polypeptide there are many ways to allow the target protein or polypeptide to enter the target cells, such as using liposomes to deliver DNA (such as plasmids) or RNA encoding the target protein, or using lentivirus or adeno-associated virus encoding the target protein to infect cells.
  • liposomes to deliver DNA (such as plasmids) or RNA encoding the target protein
  • lentivirus or adeno-associated virus encoding the target protein to infect cells.
  • mammalian cell or cell type that is amenable to cell culture and expression of a polypeptide may be used in the present invention.
  • mammalian cells that can be used in the present invention include BALB/c mouse myeloma cell line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6 (CruCel, Leiden, The Netherlands )); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human fetal kidney cell line (subcloned 293 or 293 cells for growth in suspension medium, Graham et al., J.
  • the isolated host cell is cultured under conditions that permit expression of the isolated nucleic acid inserted into the vector.
  • Conditions suitable for the expression of polynucleotides may include, but are not limited to, suitable medium, suitable density of host cells in the medium, presence of necessary nutrients, presence of complementary factors, suitable temperature and humidity, and absence of microbial contaminants.
  • suitable medium suitable density of host cells in the medium
  • suitable temperature and humidity suitable temperature and humidity
  • absence of microbial contaminants One of ordinary skill in the art may appropriately modify the steps and compositions of the present invention by selecting appropriate conditions for expression purposes to optimize cell growth and/or production of any given expressed polypeptide or protein.
  • a nucleic acid sufficient for expression typically a vector containing a gene encoding a polypeptide or protein of interest and any operably linked genetic control elements
  • a nucleic acid sufficient for expression can be introduced into a host cell line by any number of well-known techniques.
  • cells are screened to determine which host cells actually acquire the vector and express the polypeptide or protein of interest.
  • Routine methods for detecting a specific polypeptide or protein of interest expressed by host cells including but not limited to immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, SDS-PAGE, Western blot, enzyme-linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) techniques, bioactivity assays and affinity chromatography.
  • ELISA enzyme-linked immunosorbent assay
  • HPLC high performance liquid chromatography
  • the one or more expressed polypeptides and/or polypeptide complexes can be collected using any suitable method.
  • One or more polypeptides and/or polypeptide complexes can be expressed inside the cell, in the periplasmic space, or secreted outside the cell into the culture medium. If the polypeptide and/or polypeptide complex is expressed intracellularly, the host cell containing the polypeptide and/or polypeptide complex can be lysed, and the polypeptide and/or can be isolated from the lysate by centrifugation or ultrafiltration to remove unwanted debris Polypeptide complex. If polypeptides and/or polypeptide complexes are secreted into the periplasmic space of E.
  • cells can be pasted in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). The material is thawed for approximately 30 minutes, and cellular debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)). If the polypeptide and/or polypeptide complex is secreted into the culture medium, cell culture supernatants can be collected and concentrated using commercially available protein concentration filters such as Amincon or Millipore Pellicon ultrafiltration devices. Protease inhibitors and/or antibiotics may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminating microorganisms.
  • agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF).
  • PMSF phenylmethylsulfonyl fluoride
  • Polypeptides or proteins can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation or differential solubility, ethanol precipitation, or any other Available protein purification techniques (see e.g. Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; Higgins, S.J. and Hames' B.D. (eds.), Protein Expression: APractical Approach, Oxford Univ Press, 1999; and Deutscher, M.P., Simon, M.1., Abelson, J.N.
  • proteins can be separated by binding them to an affinity column containing antibodies raised against the protein and immobilized on a solid support.
  • affinity column containing antibodies raised against the protein and immobilized on a solid support.
  • standard recombinant techniques can be used to attach an affinity tag such as influenza coat sequence, polyhistidine, glutathione-S-transferase to the protein to allow simple purification through a suitable affinity column.
  • Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, or aprotinin can be added at any or all stages to reduce or eliminate degradation of the polypeptide or protein during purification. Protease inhibitors are especially needed when cells must be lysed to isolate and purify expressed polypeptides or proteins. Those of ordinary skill in the art should recognize that specific purification techniques vary depending on the properties of the polypeptide or protein to be purified, the properties of the cell expressing the polypeptide or protein, and the composition of the medium for cell growth.
  • PMSF phenylmethylsulfonyl fluoride
  • leupeptin leupeptin
  • pepstatin or aprotinin
  • the present invention also provides a pharmaceutical composition, which contains (i) any of the isolated ApoE constructs described herein (such as any amino acid sequence in SEQ ID NOs: 12, 60, 61 and 89), fusion protein, Isolated ApoE polypeptide (such as any amino acid sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33), isolated nucleic acid, vector (for example, a vector for neural-specific expression of an ApoE construct (for example, full-length ApoE2 or ApoE3) or an ApoE polypeptide), or a host cell, and (ii) a pharmaceutically acceptable carrier.
  • any of the isolated ApoE constructs described herein such as any amino acid sequence in SEQ ID NOs: 12, 60, 61 and 89
  • Isolated ApoE polypeptide such as any amino acid sequence in SEQ ID NOs: 13, 18-34, 46-52, 57-59,
  • the actual dosage level of the active ingredient i.e. the ApoE construct of the present invention, ApoE polypeptide (such as ApoE C-terminal polypeptide), fusion protein or expression vector) in the pharmaceutical composition can be changed to obtain the amount of the active ingredient effective to achieve the following effects : Achieving a desired therapeutic response for a particular patient, composition and mode of administration without toxicity to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the polypeptide or expression vector used in the invention, the route of administration, time of administration, duration of treatment, other drugs used in combination with the particular composition used, Compound and/or substance, age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.
  • pharmaceutically acceptable carriers include diluents, adjuvants, excipients or vehicles, including but not limited to water, animal oil, vegetable oil, starch, glucose, lactose, sucrose, gelatin, sodium stearate, glycerol monostearate Esters, Sodium Chloride, Skimmed Milk Powder, Glycerin, Propylene, Propylene Glycol, Water, Ethanol, etc.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to the cells of the recipient to which they are exposed at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including Ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; para Alkyl hydroxybenzoates such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as
  • the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including for example any of about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0 pH range.
  • the pharmaceutical composition can also be made isotonic with blood by the addition of a suitable tonicity adjusting agent, such as glycerol.
  • compositions to be used for in vivo administration are generally formulated as sterile substantially isotonic pharmaceutical compositions and in full compliance with all Good Manufacturing Practice (GMP) regulations of the United States Food and Drug Administration. Sterility is readily achieved by filtration through sterile filtration membranes.
  • the composition is pathogen-free.
  • the pharmaceutical compositions may be in liquid solutions, eg, in physiologically compatible buffers such as Hank's solution or Ringer's solution. Additionally, the pharmaceutical composition may be in solid form and reconstituted or suspended immediately prior to use. Also included are lyophilized compositions.
  • the pharmaceutical composition may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • the polypeptide of the present invention can be prepared into a suitable dosage form according to the specific administration mode and administration route.
  • the pharmaceutical composition can be adapted for various modes of administration as described herein, including, for example, systemic or localized administration.
  • the pharmaceutical composition is formulated for intravenous administration or subcutaneous injection.
  • the pharmaceutical composition is formulated for local administration, such as local administration to a tumor site (eg, intratumoral injection), or local administration (eg, injection) to the brain (eg, cortex or hippocampus).
  • the pharmaceutical composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous, intraperitoneal or intracerebral injection.
  • injectable compositions are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizer and a local anesthetic such as lignocaine to relieve pain at the injection site.
  • the ingredients are presented separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in an airtight container such as an ampoule or sachet indicating the quantity of active agent.
  • the composition When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule containing sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • the pharmaceutical composition is suitable for administration to humans. In some embodiments, the pharmaceutical composition is suitable for administration to rodents (eg, mice, rats, rabbits) or non-human primates (eg, monkeys). In some embodiments, the pharmaceutical composition is contained in a single use vial, such as a single use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multiple-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.
  • rodents eg, mice, rats, rabbits
  • non-human primates eg, monkeys
  • the pharmaceutical composition is contained in a single use vial, such as a single use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multiple-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is cryopreserved.
  • the invention also provides unit dosage forms comprising an isolated ApoE construct, fusion protein, isolated ApoE polypeptide, isolated nucleic acid, vector, host cell, or composition thereof (such as a pharmaceutical composition) described herein.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for individuals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent or excipient. These unit dosage forms may be stored in suitable packaging in single or multiple unit doses and may be further sterilized and hermetically sealed.
  • the application also provides articles of manufacture comprising a composition described herein, such as a pharmaceutical composition, in suitable packaging.
  • Packaging suitable for the compositions described herein, such as pharmaceutical compositions are known in the art and include, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (such as sealed Mylar (Mylar) or plastic bags), etc. These articles can be further sterilized and/or sealed.
  • kits comprising an isolated ApoE construct, fusion protein, isolated ApoE polypeptide, isolated nucleic acid, vector, host cell, or pharmaceutical composition of any of the herein described.
  • the kit also includes one or more instructions for methods of using the protein, expression vector, or composition, such as the uses described herein.
  • the kits described herein may also include other materials that may be desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and other materials with respect to performing any of the methods described herein.
  • Package insert for instructions are examples of instructions.
  • the present invention also provides the isolated ApoE construct described in any embodiment (such as any amino acid sequence in SEQ ID NOs: 12, 60, 61 and 89), fusion protein, isolated ApoE polypeptide (such as SEQ ID NOs: 13 , 18-34, 46-52, 57-59, 62, 76, 78 and 87 in any amino acid sequence, such as SEQ ID NO: 33), isolated nucleic acid, vector (such as neural specific expression ApoE construct (such as Full-length ApoE2 or ApoE3) or the carrier of ApoE polypeptide), host cells or pharmaceutical compositions are prepared for the treatment or prevention of an individual (such as people) with the enzymatic cleavage activity of ⁇ -secretase (such as the ⁇ -cleavage activity to APP) ) application in medicine for related diseases.
  • isolated ApoE construct described in any embodiment such as any amino acid sequence in SEQ ID NOs: 12, 60, 61 and 89
  • fusion protein such as SEQ ID NOs: 13
  • the drug inhibits the cleavage activity of ⁇ -secretase (such as the cleavage activity of APP), so as to treat or prevent the diseases related to the cleavage activity of ⁇ -secretase.
  • the present invention provides a method for treating or preventing a disease associated with ⁇ -secretase cleavage activity (eg, APP ⁇ -cleavage activity) in an individual (eg, human), comprising administering to the individual Any isolated ApoE construct (such as any amino acid sequence in SEQ ID NOs: 12, 60, 61 and 89), fusion protein, isolated ApoE polypeptide (such as SEQ ID NOs: 13, Any amino acid sequence in 18-34, 46-52, 57-59, 62, 76, 78 and 87, such as SEQ ID NO: 33), isolated nucleic acid, vector (such as neural specific expression ApoE construct (such as whole Long ApoE2 or ApoE3) or ApoE polypeptide carrier), host
  • the method inhibits the enzymatic cleavage activity of ⁇ -secretase (eg, the cleavage activity of APP), so as to treat or prevent the diseases associated with the cleavage activity of ⁇ -secretase.
  • the disease associated with the enzymatic cleavage activity of ⁇ -secretase is selected from the group consisting of neurodegenerative diseases, tumors or cancers, inflammatory diseases and renal diseases.
  • the disease is selected from the group consisting of Alzheimer's disease and related diseases, amyloid cerebrovascular disease, Down's syndrome, other neurodegenerative diseases associated with aging (such as frontotemporal lobe dementia), head and neck cancers (such as head and neck squamous cell carcinoma and oral squamous cell carcinoma), breast cancer, liver cancer, pancreatic cancer (including metastatic pancreatic cancer), ovarian cancer, lung cancer (such as non-small cell lung cancer, lung adenocarcinoma and small cell lung cancer), glioma (eg, malignant glioma), fibroma, lymphoma (eg, B-cell lymphoma), osteosarcoma, gastric cancer, bladder cancer, asthma (eg, allergic asthma), pneumonia, airway Inflammation, acute kidney injury, clear cell renal cell carcinoma, renal fibrosis, and obstructive nephropathy.
  • the disease is AD and related diseases, such as sporadic Alzheimer's disease (
  • the medicine, treatment or prevention method provided by the present invention is applicable to any individual, including but not limited to mammals, birds, frogs, fish, fruit flies, nematodes and the like.
  • Mammals include, but are not limited to, primates (such as humans, or non-human primates such as monkeys), rodents (such as mice, rats, hamsters, gerbils, squirrels, guinea pigs, and rabbits), livestock (such as pigs, cattle, sheep, horses, donkeys), pets (such as cats, dogs, rabbits and hamsters), etc.
  • the individual is a mouse or a monkey.
  • the individual is a human.
  • suitable administration methods and administration routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, such as via injection or perfusion.
  • Parenteral administration is a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intraorbital, intracardiac, dermal Intra-, intraperitoneal, nasal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.
  • systemic administration and “systemically administered” refer to the administration of an agent or composition described herein to an individual (such as a mammal) such that the agent or composition is delivered to a site in the body via the circulatory system. point (including the target site of drug action) approach.
  • Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (eg, other than through the alimentary canal, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous).
  • any of the isolated ApoE constructs of the present invention (such as any amino acid sequence in SEQ ID NOs: 12, 60, 61 and 89), fusion proteins, isolated ApoE polypeptides (such as SEQ ID NOs : 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87 in any amino acid sequence, such as SEQ ID NO: 33), isolated nucleic acid, vector, or pharmaceutical composition via any suitable Routes into the central nervous system, including intraventricular and intrathecal injections. Intraventricular injection can be assisted, for example, via an intracerebroventricular catheter connected to a depot, such as the Ommaya depot.
  • nucleic acid molecules encoding polypeptides of the invention can be delivered using techniques well known in the art.
  • a coding sequence for a polypeptide of the invention is delivered to a subject in need (eg, a human) in an expression vector (eg, using a viral vector, eg, AAV or lentiviral vector).
  • liposomes are used to deliver DNA or RNA molecules described herein that encode polypeptides of the invention.
  • compositions of the invention comprise liposomes together with the DNA or RNA molecules encoding the polypeptides of the invention and their expression vectors described herein.
  • the liposomes suitable for the present invention may be liposomes known in the art and suitable for human administration.
  • the ApoE construct provided by the present invention (such as full-length ApoE2 or ApoE3), fusion protein, or ApoE polypeptide is an inhibitor of ⁇ -secretase (such as specific inhibition of ⁇ -cleavage of APP), so the polypeptide of the present invention or its drug
  • the compositions are useful for the treatment or prevention of diseases benefiting from the inhibition of gamma secretase, targeting the inhibition of gamma secretase for therapeutic or prophylactic benefit (also referred to herein as gamma secretase mediated diseases or in combination with gamma secretase) Diseases associated with enzymatic cleavage activity).
  • ⁇ -secretase A variety of diseases are known to be mediated by ⁇ -secretase, including but not limited to: neurodegenerative diseases such as Alzheimer's disease and related diseases and amyloid cerebrovascular diseases (Erik D. Roberson and Lennart Mucke, 100 years and Counting: Prospects for Defeating Alzheimer's Disease, 314: 781-784 (2006); Bart De Strooper et al., Presenilins and ⁇ -Secretase: Structure, Function, and Role in Alzheimer's Disease, 2: a006304 (2012); Weiming Xia, ⁇ -Secretase and its modulators: Twenty years and beyond, Neuroscience Letters 701: 162-169 (2019)); tumors or cancers, including head and neck cancers such as head and neck squamous cell Carcinoma and oral squamous cell carcinoma (Liang Mao et al., ⁇ -Secretase inhibitor reduces immunosuppressive cells and enhance
  • the disease associated with the cleavage activity of ⁇ -secretase is a neurodegenerative disease, such as Alzheimer's disease (AD), dementia with Lewy bodies ( DLB), mild cognitive impairment (MCI), frontotemporal dementia (FTD), cerebral amyloid angiopathy (CAA), CAA-related cerebral hemorrhage, vascular cognitive impairment, Parkinson's disease (PD), multiple Tremor/ataxia syndrome associated with sclerosis (MS), traumatic brain injury (TBI), or Fragile X syndrome.
  • AD Alzheimer's disease
  • DLB dementia with Lewy bodies
  • MCI mild cognitive impairment
  • FTD frontotemporal dementia
  • CAA cerebral amyloid angiopathy
  • CAA-related cerebral hemorrhage vascular cognitive impairment
  • Parkinson's disease PD
  • MS multiple Tremor/ataxia syndrome associated with sclerosis
  • TBI traumatic brain injury
  • Fragile X syndrome Fragile X syndrome.
  • CAA is a condition characterized by the deposition of proteins,
  • the disease associated with ⁇ -secretase cleavage activity is "amyloid ⁇ -associated disease", which term refers to abnormally high levels of amyloid Any disorder characterized by the production, accumulation and/or aggregation of protein beta peptides, eg in the form of amyloid plaques, especially in the brain.
  • Amyloid- ⁇ -associated diseases include AD, Down syndrome, Fragile X syndrome, CAA, CAA-associated intracerebral hemorrhage, and sporadic inclusion body myositis.
  • AD Alzheimer's disease
  • Histopathologically AD is characterized by the accumulation of amyloid plaques containing A ⁇ peptides and NFTs made of tau protein.
  • Clinically AD is associated with progressive cognitive impairment characterized by loss of memory, function, language ability, judgment, and executive functions. AD often causes severe behavioral symptoms in its later stages.
  • FAD familial Alzheimer's disease
  • PSENI familial Alzheimer's disease
  • PSEN2 familial Alzheimer's disease
  • APP Amyloid precursor protein
  • the most common missense mutations replace the Val 717 residue with an Ile (London mutation), Phe, or Gly residue, Glu 693 with a Gln residue (Dutch mutation) or Gly residue (Arctic mutation), or Asn and Leu residues in place of Lys 670 and Met 671 (the double mutation is called the Swedish mutation and is numbered in the APP 770 isoform).
  • PSEN1 presenilin 1
  • PSEN2 presenilin 2
  • any isolated ApoE construct provided by the invention can be used to treat any disease (such as AD) related to APP ⁇ -enzyme cleavage by inhibiting the enzyme activity of ⁇ -secretase, for example, with any APP Mutational diseases (such as AD).
  • the APP is human APP comprising one or more of the following mutations: K670N, M671L, Swedish (K670N+M671L), Florida (I716V), London (V717I), APP M596V (incapable of beta cleavage), fAD mutations near ⁇ -cleavage sites (e.g. T714I, V717F, L723P), fAD mutations near ⁇ - or ⁇ -cleavage sites (e.g. KM670/671NL, E693K, E693 ⁇ ), or protective Icelandic mutations (A673T ).
  • K670N, M671L Swedish (K670N+M671L), Florida (I716V), London (V717I), APP M596V (incapable of beta cleavage)
  • fAD mutations near ⁇ -cleavage sites e.g. T714I, V717F, L723P
  • Histological analysis is usually performed postmortem. Histological analysis of A ⁇ levels can be performed using Thioflavin-S. Congo red, or anti-A ⁇ staining (e.g., 4G8, 10D5, or 6E10 antibodies) visualizes A ⁇ deposition on sectioned brain tissue (see, e.g., Holcomb et al., 1998, Nat. Med. 4:97-100; Borchelt et al., 1997, Neuron 19:939-945; Dickson et al., 1988, Am. J. Path. 132:86-101).
  • the 19 F-containing amyloidophilic Congo red-type compound FSB (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene) allows A ⁇ Plaque visualization (see, eg, Higuchi et al., 2005, Nature Neurosci. 8:527-533). Radiolabeled, putrescine-modified amyloid- ⁇ peptides label amyloid deposits in vivo in a mouse model of Alzheimer's disease (see, e.g., Wengenack et al., 2000, Nat. Biotechnol. 18:868- 872).
  • a subject suffering from Alzheimer's disease can be identified using standard diagnostic methods known in the art for Alzheimer's disease.
  • diagnosis of Alzheimer's disease is based on the patient's symptoms (eg, progressive decline in memory function, progressive abandonment and frustration of normal activities, apathy, restlessness or irritability, aggression, anxiety, sleep disturbance, annoyance, abnormal motor behavior, disinhibition, social withdrawal, decreased appetite, hallucinations, dementia), medical history, neuropsychological testing, neurological and/or physical examination.
  • Cerebrospinal fluid can also be tested for a variety of proteins associated with Alzheimer's pathology, including tau, amyloid-beta peptide, and AD7C-NTP.
  • the ApoE constructs, ApoE polypeptides, vectors, compositions, or treatment/prevention methods of the present invention can achieve one or more of the following effects: (i) in neurons (in vivo or in vitro) in a cell-autonomous manner (ii) inhibit (for example, inhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% %) Enzyme activity of ⁇ -secretase, such as inhibition of ⁇ -secretase-mediated APP ⁇ -cleavage, similar to the APP ⁇ -cleavage inhibitory effect of ⁇ -secretase inhibitors (such as PF03084014); (iii) decreased (eg Reduce at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) APP amyloid metabolism, such as reducing (such as reducing by at least about 10%
  • a ⁇ 40 and/or A ⁇ 42 generation slow down (e.g. slow down by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% %, 95%, 100%, 2-fold, 5-fold, 10-fold or more) AD pathological progression, increase (eg increase by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%) , 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or higher) individual survival and/or lifespan; (ix) neuronal local expression (e.g.
  • the ApoE constructs of the present invention can migrate from a distance to the subiculum and accumulate around amyloid plaques, thereby locally inhibiting the activity of ⁇ -secretase at these amyloidogenic hotspots.
  • Enzyme activity or (x) reduce (eg, reduce by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), eliminate, or to prevent (including slowing the onset of) one or more AD symptoms (eg, progressive decline in memory function, progressive abandonment and frustration of normal activities, apathy, restlessness or irritability, aggression, anxiety, sleep disturbance, upset, abnormal movements behavior, disinhibition, social withdrawal, decreased appetite, hallucinations, dementia).
  • AD symptoms eg, progressive decline in memory function, progressive abandonment and frustration of normal activities, apathy, restlessness or irritability, aggression, anxiety, sleep disturbance, upset, abnormal movements behavior, disinhibition, social withdrawal, decreased appetite, hallucinations, dementia.
  • the cancers associated with the enzymatic cleavage activity of ⁇ -secretase include one or more of the following: head and neck cancer (such as head and neck squamous cell carcinoma and oral squamous cell carcinoma), breast cancer, liver cancer, pancreatic cancer (including metastatic pancreatic cancer), ovarian cancer, lung cancer (such as non-small cell lung cancer, lung adenocarcinoma, and small cell lung cancer), glioma (such as malignant glioma), fibroma, lymphoma (such as B-cell lymphoma Tumor), osteosarcoma, gastric cancer, bladder cancer.
  • head and neck cancer such as head and neck squamous cell carcinoma and oral squamous cell carcinoma
  • breast cancer such as head and neck squamous cell carcinoma and oral squamous cell carcinoma
  • pancreatic cancer including metastatic pancreatic cancer
  • ovarian cancer such as non-small cell lung cancer, lung adenocarcinoma, and small cell
  • the medicaments or methods described herein for treating cancers associated with the enzymatic cleavage activity of ⁇ -secretase have one or more of the following biological activities: (1) kill cancer cells, for example at least about 30% , 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher tumor cell death rate; (2) inhibiting the proliferation of cancer cells, such as inhibiting at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) proliferation; (3) reducing tumor size, for example, by at least about 10%, 20% %, 30%, 40%, 60%, 70%, 80%, 90%, or 100% of tumor size; (4) alleviate one or more symptoms in individuals with cancer; (5) inhibit tumor metastasis (such as metastasis to lymph nodes), such as inhibiting tumor metastasis by at least about 10%, including, for example, at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100% (6) prolonging survival, e.g.
  • the disease associated with the enzymatic cleavage activity of ⁇ -secretase is an inflammatory disease, including but not limited to asthma (such as allergic asthma), pneumonia, and airway inflammation.
  • the drugs described herein can reduce (e.g., reduce by at least about any of 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) the level of inflammation-triggering cytokines Secretion and/or activity, and/or alleviating (for example alleviating at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%), eliminating or Preventing (including slowing the time of onset) one or more symptoms of a disease.
  • the disease associated with the enzymatic cleavage activity of ⁇ -secretase is renal disease, which refers to any disease, disorder or condition affecting the kidney or renal system.
  • kidney-related diseases or conditions include, but are not limited to, acute kidney disease (or failure), clear cell renal cell carcinoma, renal fibrosis, or obstructive kidney disease.
  • Conventional markers for evaluating and diagnosing renal diseases include GFR, creatinine and albumin, and any one of the markers or related methods can be used to evaluate the therapeutic effect of the present invention.
  • the medicaments or methods of treatment/prevention described herein can (i) reduce (eg, reduce by at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%), eliminate or prevent (including slow the time to onset) one or more indicators of renal disease; and/or (ii) protect or improve renal function.
  • the present invention provides a method for treating or preventing a disease mediated by ⁇ -secretase in an object (such as a human), the method comprising administering to the object a therapeutically effective amount or a preventively effective amount of any of the ApoE constructs, fusion proteins, ApoE polypeptide, nucleic acid molecule, carrier or pharmaceutical composition thereof.
  • Effective amounts can be determined by those skilled in the art based on factors such as the subject's age, sex, weight, condition, general health, and previous medical history.
  • the diseases preferably include the aforementioned degenerative diseases of the nervous system, tumors or cancers, inflammatory diseases, kidney diseases and the like.
  • Some embodiments of the present invention provide a method of inhibiting the enzyme cleavage of ⁇ -secretase, thereby inhibiting the amyloid metabolism of APP and reducing the production of A ⁇ , the method comprising producing ApoE2 ( For example, ApoE2 with a penetrating peptide at the N-terminus) or an ApoE construct (such as a C-terminal fragment of ApoE2, or full-length ApoE2 or ApoE3), a fusion protein or an ApoE polypeptide (such as SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87 amino acid sequences, such as SEQ ID NO: 33).
  • the cells are neurons.
  • the method is used to slow down (eg, slow down by at least about any of 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%), Prevent or treat the occurrence and development of AD and amyloid cerebrovascular disease.
  • Production as described herein includes expression production of the polypeptide and delivery of the polypeptide into the cell from outside the cell.
  • liposomes are used to deliver the DNA or RNA encoding the ApoE construct (such as ApoE2 and/or ApoE3 and/or its C-terminal fragment) or ApoE polypeptide into the cell, so that it can be expressed in the cell
  • the ApoE construct or ApoE polypeptide is produced by internal expression; or the ApoE construct or ApoE polypeptide is produced in the cell by infecting cells with lentivirus and adeno-associated virus; or by using cell-penetrating peptide or endocytosis An enhanced manner allows entry of the ApoE construct or ApoE polypeptide into the cell.
  • polypeptides mentioned in the ApoE constructs or fusion proteins uses and methods described in any of the embodiments herein particularly include any of SEQ ID NOs: 12, 13, 18-33 and 89 indicated polypeptide.
  • the HEK 293T cell line was cultured in DMEM medium (HyClone) containing 10% fetal bovine serum (FBS) (Life Company) and 1% penicillin/streptomycin, and cultured in a sterile environment at 37°C containing 5% CO2 . in a cell culture incubator (ThermoFisher).
  • DMEM medium HyClone
  • FBS fetal bovine serum
  • penicillin/streptomycin 1% penicillin/streptomycin
  • Primary cultured neurons were derived from the cortex or hippocampus of 16-day embryonic mice.
  • the mouse cortex or hippocampus was obtained under a stereoscope; 20U/ml papain (Worthington Company) was digested at 37°C for 10 minutes; neurons in the form of single cells were digested at 100,000 neurons per well (24-well plate) Seed the plate; culture the neurons in the neuron culture medium (Neurobasal medium; ThermoFisher Company) containing 2% B27 (ThermoFisher Company) and 1% penicillin/streptomycin; change the fresh culture medium once a week.
  • mice 0-7 months old C57 wild-type mice and AD model mice (5 ⁇ FAD mice [Tg6799], mice expressing APP and PS1 containing pathogenic mutations.
  • the expressed APP contains three mutations: K670N /M671L, I716V, V717I; and PS1 contains two mutations: M146L, L286V).
  • the mice were given sufficient food and water, and were reared at room temperature with a 12h/12h light-dark cycle, and the feeding environment reached the SPF (Specific Pathogen Free) standard.
  • SPF Specific Pathogen Free
  • Enzyme-linked immunosorbent assay kit was purchased from Invitrogen Company, human A ⁇ 40 (KHB3481), human A ⁇ 42 (KHB3441), and mouse A ⁇ 40 (KMB3481).
  • Transfection reagents were purchased from ThermoFisher's lipofectamine2000 and SignaGen's Polyjet.
  • Quantitect SYBR-Green qRT-PCR kit for qRT-PCR was purchased from Qiagen.
  • the protein expression promoter is CAG.
  • HEK 293T cell line Express the target protein in HEK 293T cell line. Cells were subcultured one day before transfection, and transfection was performed when the degree of cell adhesion reached 70%-80%.
  • the transfection reagent was lipofectamine2000 (ThermoFisher).
  • Blocking and incubating antibodies Place the transformed PVDF membrane in 5% BSA/TBST blocking solution, and block on a shaker at room temperature for 1 hour. Incubate the primary antibody at 4°C overnight on a shaker (12-16h); 1 ⁇ TBST, wash three times, wash once every 10 minutes; incubate the secondary antibody, shake at room temperature for 1-2h; 1 ⁇ TBST, wash three times, every Wash once every 10 minutes.
  • mice The brain was taken by perfusion. The mice were perfused from the heart, and the blood was first washed out with 20ml PBS, and then perfused with 20ml 4% PFA.
  • Mouse brain slices After 24 hours of dehydration, the mouse brain was cut into slices with a thickness of 40 ⁇ m, stored in frozen buffer solution or used for subsequent immunohistochemistry.
  • THS staining ddH 2 O, wash three times, 5 minutes each; 1% THS aqueous solution, soak for 10 minutes; 80% ethanol, wash twice, each 3 minutes; 95% ethanol, wash once, 3 minutes. Progressive follow-up immunohistochemistry. If THS staining is not performed, proceed directly to the following immunohistochemistry.
  • the experiment was performed according to the standard procedure in the ELISA analysis kit of Invitrogen Company.
  • HEK 293T cell line a) Expression of target protein in HEK 293T cell line. Cells were subcultured one day before transfection, and transfection was performed when the degree of cell adhesion reached 70%-80%.
  • the transfection reagent was lipofectamine2000 (ThermoFisher).
  • HEK 293T cell line a) Expression of target protein in HEK 293T cell line. Cells were subcultured one day before transfection, and transfection was performed when the degree of cell adhesion reached 70%-80%.
  • the transfection reagent was lipofectamine2000 (ThermoFisher).
  • Trizol (ThermoFisher Company) was added to each well of the 24-well plate, shaken, and kept at room temperature for 20 minutes.
  • RNA white precipitate
  • Quantitect SYBR-Green qRT-PCR kit from Qiagen was used to quantitatively analyze the relative content of mRNA. qRT-PCR primers are listed in Table 2.
  • HEK 293T cells were selected, and PS1 KO, PS2 KO and PS1/2 dKO cell lines were constructed using CRISPR/CAS9 technology according to the standard procedure published by Zhang Feng et al. in 2013 (Ran et al., 2013). Using https://zlab.bio/guide-design-resources website, sgRNAs for knocking out PS1 and PS2 specifically were designed, as shown in Table 3.
  • PS2 knockout cell lines PS2 KO cell lines.
  • the present invention constructs a PS1/PS2 dKO cell line on the basis of the PS2 KO cell line.
  • PS1 sgRNA or control empty plasmid was further expressed in PS2 KO cell line; and steps a-g were performed for DNA sequence detection and protein expression of PS1.
  • PS2 KO cell line whose DNA sequence of PS1 has been edited and no longer expresses PS1 protein is PS1 and PS2 double knockout cell line—PS1/2 KO cell line.
  • lentivirus expression plasmids lentivirus-GFP and lentivirus-ApoE CT that can be specifically expressed in neurons.
  • Adeno-associated virus AAV-CT AAV2/9-hSyn-ApoECT
  • AAV-GFP AAV2/9-hSyn-EGFP-WPRE-pA expressed in neurons were purchased from Tertu Biotechnology Company.
  • HEK 293T cell line the same expression plasmid simultaneously expressed four proteins of human ⁇ -secretase, NCT, PS1, PLAG-PEN2, APH. Cells were subcultured one day before transfection, and transfection was performed when the degree of cell adhesion reached 70%-80%.
  • the transfection reagent was lipofectamine2000 (ThermoFisher).
  • ApoE2 and ApoE2NT proteins contain fusion expressed FLAG. Cells were subcultured one day before transfection, and transfection was performed when the degree of cell adhesion reached 70%-80%.
  • the transfection reagent was lipofectamine2000 (ThermoFisher).
  • ApoE2 and ApoE2 NT were eluted with the following buffer. 25mM HEPES, pH7.4; 150mM NaCl and 500 ⁇ g/ml 3xFLAG peptide (SIGMA company).
  • E. coli was lysed with the following lysate. 30mM Tris-HCl, pH 7.5; 150mM NaCl, 1mM EDTA.
  • Ni-NTA-beads with the following 10 times Ni-NTA-bead column volume: 30mM Tris-HCl, pH 8.0; 300mM NaCl, 1mM EDTA, 6M urea, 1% TritonX-100, 1mM CaCl 2 , 1 ⁇ cocktail .
  • Ni-NTA-beads with 20 times the Ni-NTA-bead column volume as follows: 50 mM Tris-HCl, pH 8.0; 300 mM NaCl, 0.1% CHAPSO.
  • ⁇ -CTF was eluted with the following buffers: 50 mM Tris-HCl, pH 8.0; 300 mM NaCl, 0.1% CHAPSO, 300 mM imidazole.
  • the purified ⁇ -CTF was detected by immunoblotting; stored at -80°C.
  • the purification method of GFP is the same as that of 2.7.2 ApoE.
  • human APP is co-expressed with ApoE2, ApoE3 and ApoE4 respectively in HEK 293T cells, and GFP is used as a control group, and Western Blot is used to measure the production amount of ⁇ -CTF.
  • the results of western blot experiments (Fig. 1, panels A-B) showed that ApoE2 and ApoE3 co-expressed with APP could significantly increase the non-amyloid metabolite ⁇ -CTF of APP; while ApoE4 co-expressed with APP could not significantly change ⁇ -CTF (compare ⁇ -CTF of the GFP control group).
  • the present invention further explores the influence of ApoE on APP amyloid metabolic pathway.
  • a ⁇ is the product of amyloid metabolism, and the less A ⁇ is produced, the weaker the amyloid metabolism of APP is.
  • human wild-type APP and ⁇ -secretase are co-expressed with ApoE2, ApoE3 or ApoE4 in HEK 293T cells respectively, and GFP is used as a control group to detect the influence of ApoE on A ⁇ production.
  • ELISA results (Fig. 1, panel C) showed that expression of ApoE2 significantly reduced A ⁇ 40 production, but ApoE3 and ApoE4 had no such effect. Expression of ApoE2 also significantly reduced A ⁇ 40 compared to ApoE4.
  • the present invention further studies which fragments of ApoE function to weaken APP amyloid metabolism.
  • ApoE2, ApoE3 and ApoE4 differ only in two amino acid residues in the N-terminal functional domain, and have identical amino acid residue sequences in the C-terminal functional domain. Therefore, the present invention constructs the expression plasmid of the N-terminal fragment and the C-terminal fragment of human source ApoE, and the plasmid named as ApoE2NT, ApoE3NT, ApoE4NT or ApoECT protein is used for follow-up experiment respectively. When expressed in cells, the N-terminals of all constructed proteins are further linked to the signal peptide of human ApoE itself (SEQ ID NO: 66).
  • APP was co-expressed with ApoE2 NT, ApoE4 NT and ApoE CT in HEK 293T cells, and GFP was used as the control group.
  • the results of western blot experiments (Fig. 1, panels D-E) showed that only the co-expression of ApoE CT significantly increased ⁇ -CTF to more than 5 times the original level. Coexpression of either ApoE2 NT or ApoE4 NT did not alter ⁇ -CTF. Meanwhile, ELISA results showed that ApoECT significantly reduced A ⁇ 40 to ⁇ 25% of GFP control, and none of the three ApoENTs reduced A ⁇ 40 (Fig. 1, panel F); similarly, ApoECT significantly reduced A ⁇ 42 to ⁇ 25% of GFP control. 26% ( Figure 1, panel G).
  • AD is a neurological disease.
  • a ⁇ is mainly produced by cleavage of APP expressed in neurons.
  • the present invention constructs the lentivirus expressing ApoECT in neurons.
  • ELISA results (Figure 1, panel H) showed that overexpression of ApoECT in neurons could reduce endogenously produced A ⁇ 40 in primary cultured mouse hippocampal or cortical neurons to ⁇ 71% and ⁇ 73% of the control group, respectively .
  • overexpression of ApoECT in human pluripotent stem cell-induced neurons also reduced the endogenous production of A ⁇ 40 by human neurons to ⁇ 54% of that in the control group.
  • ApoE2 can increase the non-amyloid metabolite ⁇ -CTF of APP through its C-terminal fragment in a cell-autonomous manner; at the same time, it can weaken the amyloid metabolic pathway and reduce A ⁇ .
  • the ApoE CT amino acid sequence used in each experiment of the present invention is as follows:
  • HEK 293T cells expressing ApoE2 or ApoECT were cultured to obtain a culture medium, and the results showed that the culture medium contained secreted ApoE2 or ApoECT (Fig. 2, panel A).
  • HEK 293T cells expressing APP were co-incubated with this medium containing ApoE2 or ApoECT.
  • HEK 293T cells expressing APP were co-incubated with culture medium containing GFP as a negative control.
  • the results showed that ApoE2 and ApoE CT in co-incubated cultures did not significantly alter ⁇ -CTF levels compared to GFP control ( Figure 2, panels B-C). Immunofluorescence results (Fig.
  • ApoE2 and ApoE CT in the cell culture medium cannot change the production of ⁇ -CTF and A ⁇ .
  • ApoE2 and ApoE CT must be in a cell-autonomous manner, that is, must be in the same cell as APP, in order to regulate the shearing of APP, thereby achieving the effect of increasing ⁇ -CTF and reducing the production of A ⁇ .
  • there are multiple ways to allow ApoE2 and ApoE CT to enter the target cells specifically including: liposomes transfer DNA or RNA encoding such proteins.
  • the plasmid is transfected into cells to express ApoE and ApoE CT; modes such as lentivirus and adeno-associated virus infecting cells can make ApoE2 and ApoE CT expressed in cells expressing APP, and ApoE2 or ApoE CT expressed in neurons of the present invention
  • a lentivirus or adeno-associated virus expression system is adopted; cell-penetrating peptides, cell endocytosis enhancement, etc. can make ApoE2 and ApoECT proteins enter the target cells expressing APP, etc., and the present invention discusses cell-penetrating in detail later. The role of peptides.
  • the present invention adds 1, 2, 3, 4 or 5 original amino acid residues of ApoE to the N-terminus of ApoE CT to construct the expression CT NA1 and CT NA2 , CT NA3, CT NA4, and CT NA5 plasmids; delete 1, 2, 3, 4, or 5 amino acids at the N-terminal of ApoE CT to construct plasmids expressing CT ND1, CT ND2, CT ND3, CT ND4, and CT ND5:
  • the original 5 and 10 amino acids of ApoE were deleted at the C-terminus of ApoE CT, and plasmids expressing CT CD5 and CT CD10 were constructed.
  • CT NA1, CT NA2, CT NA3, CT NA4, CT NA5, CT ND1, CT ND2, CT ND3, CT ND4, CT The amino acid sequences of ND5, CT CD5 and CT CD10 are respectively shown in SEQ ID NOs: 13-24. When expressed in cells, the N-termini of all constructed proteins are further linked to the signal peptide of ApoE itself (SEQ ID NO: 66).
  • ApoEND1, ApoEND2, ApoEND3, ApoEND4 and ApoEND5 could still reduce A ⁇ 40 after deletion of 1–5 amino acid residues at the N-terminus of ApoECT. Even if 5 or 10 amino acid residues were deleted at the C-terminus of ApoE CT, CT CD5 and CT CD10 still reduced the generation of A ⁇ . These results indicated that the N-terminus of ApoECT is very important, and even a change of only 1 or 2 amino acids may affect the activity of ApoECT to inhibit A ⁇ 40 production. However, the influence of its C-terminal residues on the activity is relatively weak.
  • the present invention deletes 10, 11, 12, 13, 14, 15, 20 or 25 amino acid residues at the C-terminal of ApoECT on the basis of deleting 3 amino acid residues (CT ND3) at the N-terminal of ApoECT.
  • Amino acid residues, constructing plasmids expressing CT ND3CD10, CT ND3CD11, CT ND3CD12, CT ND3CD13, CT ND3CD14, CT ND3CD15, CT ND3CD20 and CT ND3CD25 the amino acid sequences of which are shown in SEQ ID NOs: 25-32.
  • the N-termini of all constructed proteins are further linked to the signal peptide of ApoE itself (SEQ ID NO: 66).
  • SEQ ID NO: 26 (CTND3CD11):
  • SEQ ID NO: 27 (CTND3CD12):
  • SEQ ID NO: 28 (CTND3CD13):
  • SEQ ID NO: 29 (CTND3CD14):
  • SEQ ID NO: 30 (CTND3CD15):
  • SEQ ID NO: 31 (CTND3CD20):
  • SEQ ID NO: 32 (CTND3CD25):
  • the present invention detected whether ApoE CT would change the expression level of APP key secretory enzymes.
  • the present invention uses qRT-PCR and Western Blot techniques to detect the mRNA and protein expression levels of ADAM10 (main ⁇ -secretase), BACE1 ( ⁇ -secretase), PS1 and PS2 ( ⁇ -secretase active subunits), respectively.
  • ⁇ -CTF is a substrate of ⁇ -secretase
  • a ⁇ is the cleavage product of ⁇ -secretase. Therefore, when the activity of ⁇ -secretase changes, the production of ⁇ -CTF and A ⁇ will change significantly, and ApoE CT just changes the production of ⁇ -CTF and A ⁇ . Therefore, ApoE CT may change the activity of ⁇ -secretase.
  • the present invention co-expressed APP and PS1, the active center of ⁇ -secretase, in HEK293T cells.
  • the results of western blot experiments (Fig. 5, panels AB) showed that overexpression of PS1 (which can enhance the activity of ⁇ -secretase) decreased ⁇ -CTF.
  • ApoECT expressed in cells needs ⁇ -cleavage to reduce A ⁇ , and overexpressed ApoECT mimics the effect of ⁇ -secretase inhibitors; ApoECT may increase ⁇ -CTF by inhibiting ⁇ -cleavage of APP, Reduce A ⁇ .
  • the active center of ⁇ -secretase is PS1 or PS2.
  • PS1 and PS2 PS1/2 dKO
  • ⁇ -CTF cannot be metabolized by ⁇ -secretase, so it accumulates in large quantities.
  • Western blot results (Fig. 6, panels A and D) showed that in wild-type HEK 293T cell line, expressed ApoE CT significantly increased ⁇ -CTF; in PS 1/2 dKO cells expressing GFP, compared with In the wild-type HEK 293T cell line, the content of ⁇ -CTF was significantly increased.
  • APLP1 (APP like protein 1) is a protein with a structure similar to APP, and it is also one of the substrates of ⁇ and ⁇ secretases.
  • Western blot results ( Figure 8) showed that the ⁇ -secretase inhibitor PF03084014 could inhibit the ⁇ -cleavage of APLP1 in HEK293T cells and significantly increase the ⁇ -like CTF; however, the co-expression of ApoECT did not increase the ⁇ -like CTF of APLP1 , indicating that ApoECT does not inhibit the ⁇ -cleavage of APLP1. This result indicated that ApoECT had certain substrate selectivity in inhibiting the cleavage activity of ⁇ -secretase.
  • Adeno-associated virus capable of expressing ApoECT in neurons was injected into the cortex and hippocampus of 4-week-old AD model mice (5 ⁇ FAD). After expressing ApoECT (or GFP as a negative control) in neurons for six months, its effect on amyloid plaques was examined, and ApoECT, GFP, and amyloid plaques were stained. The results showed that the area of amyloid plaques in the brains of 5 ⁇ FAD mice expressing ApoE CT (the signal peptide of human ApoE itself (SEQ ID NO: 66) connected to the N-terminus) in neurons was smaller and the fluorescence intensity was higher.
  • ApoE CT the signal peptide of human ApoE itself (SEQ ID NO: 66) connected to the N-terminus
  • ELISA results demonstrate that overexpressed ApoE2 in neurons can reduce endogenously produced A[beta]40 compared to GFP controls. Further, ApoE2 was expressed in neurons in the brain of wild-type mice (WT mouse) using the AAV expression system. ELISA results ( Figure 10, panel B) showed that ApoE2 expressed in mouse brain neurons also reduced endogenous A ⁇ 40 production compared to GFP controls. These results suggest that ApoE2, like ApoE CT, can inhibit the production of endogenous A ⁇ 40 in neurons.
  • the present invention adds tag protein V5 (SEQ ID NO: 82) respectively at the C-terminus and N-terminus of ApoE2 and ApoE CT respectively, named after ApoE2-V5 ( V5 at the C-terminus), V5-ApoE2 (V5 at the N-terminus), ApoE CT-V5 and V5-ApoE CT.
  • ApoE2-V5 V5 at the C-terminus
  • V5-ApoE2 V5 at the N-terminus
  • ApoE CT-V5 and V5-ApoE CT The N-terminals of all constructed proteins were further linked to the signal peptide (SEQ ID NO: 66) of human ApoE itself.
  • the present invention constructed and analyzed a series of mutants of ApoECT. Including single site mutations: CT A20, CT A40, CT A60, CT A65, CT A72, CT A80; double site mutations: CT A36/74; multiple site mutations: CT E5, CT E10, CT E20, CT F5 , CT R10, CT LF5, CT LM5.
  • the position of the mutation site is relative to the position of ApoECT (SEQ ID NO: 33). Its amino acid sequence is shown in SEQ ID NOs: 46-59. The N-terminals of all constructed proteins were further linked to the signal peptide (SEQ ID NO: 66) of human ApoE itself.
  • SEQ ID NO: 46 (CT A20; relative to SEQ ID NO: 33Q20A; relative to SEQ ID NO: 12Q235A):
  • SEQ ID NO: 47 (CT A40; relative to SEQ ID NO: 33E40A; relative to SEQ ID NO: 12 E255A):
  • SEQ ID NO: 48 (CT A60; relative to SEQ ID NO: 33 Q60A; relative to SEQ ID NO: 12 Q275A):
  • SEQ ID NO: 49 (CT A65; relative to SEQ ID NO: 33 V65A; relative to SEQ ID NO: 12 V280A):
  • SEQ ID NO: 50 (CT A72; relative to SEQ ID NO: 33V72A; relative to SEQ ID NO: 12 V287A):
  • SEQ ID NO: 51 (CT A80; relative to SEQ ID NO: 33 P80A; relative to SEQ ID NO: 12 P295A):
  • SEQ ID NO: 52 (CT A36/74; relative to SEQ ID NO: 33 R36A+T74A; relative to SEQ ID NO: 12 R251A+T289A):
  • SEQ ID NO: 57 (CT R10; relative to SEQ ID NO: 33S75A+P78A+V79A+P80A+S81A+D82A+N83A+H84A; relative to SEQ ID NO: 12S290A+P293A+V294A+P295A+S296A+D297A+N298A +H299A):
  • SEQ ID NO: 58 (CT LF5; relative to SEQ ID NO: 33 R11A+D12A+R13A+L14A+D15A; relative to SEQ ID NO: 12 R226A+D227A+R228A+L229A+D230A):
  • SEQ ID NO: 59 (CT LM5; relative to SEQ ID NO: 33 E23A+V24A+R25A+K 27A; relative to SEQ ID NO: 12 E238A+V239A+R240A+K242A):
  • the present invention firstly adds between the 215th and 216th amino acid residues of ApoE2 (SEQ ID NO:12)
  • the tagged protein, 3XFLAG was named ApoE2-M3F (SEQ ID NO: 60; 3XFLAG sequence in bold; underline indicates the sequence of SEQ ID NO: 33), this modification does not affect the ApoE CT sequence (SEQ ID NO: 33).
  • the present invention also constructs ApoE2 MD10 (SEQ ID NO: 61).
  • ApoE2 MD10 is obtained by deleting the 244th to 254th amino acid residues of ApoE2, and these deleted amino acids are located in the C-terminal region of ApoE2 that regulates APP metabolism, that is, in In the sequence of SEQ ID NO:33. When expressed in cells, the N-termini of all constructed proteins are further linked with the signal peptide of human ApoE itself (SEQ ID NO: 66).
  • SEQ ID NO: 61 (ApoE2 MD10) (SEQ ID NO: 12; underline indicates SEQ ID NO: 33 sequence; box indicates deleted sequence in ApoE2 MD10)
  • Non-human ApoECT can also regulate the shearing of APP
  • non-human primate-derived ApoE CT monkey CT. This sequence is highly similar to human ApoECT in amino acid sequence, with only three amino acid substitutions R36S+T74A+A76T ( Figure 14, panel C). The monkey ApoECT amino acid sequence is shown below.
  • human APP and non-human primate ApoE CT when expressed in cells, the N-terminus is further connected to the signal peptide of human ApoE itself (SEQ ID NO: 66)) co-expressed, GFP
  • human ApoE CT the signal peptide (SEQ ID NO: 66) of human ApoE itself is further connected to the N-terminus
  • Western blot results ( Figure 14, panels A and B) showed that non-human primate ApoE CT (monkey CT) and human ApoE CT also increased ⁇ -CTF. This result indicates that ApoECT of non-human primates also has the activity of regulating APP cleavage.
  • the present invention adds the membrane-penetrating peptide to ApoE.
  • the selected penetrating peptide sequence is as follows:
  • SEQ ID NO: 64 (FGF4): AAVLLPVLLAAP
  • SEQ ID NO: 65 KSVRTWNEIIPSKGCLRVGGRCHPHVNGGGRRRRRRRRR
  • the ApoE2 mutants constructed by adding R9, FGF4, and RDP to the N-terminus of human ApoE2 were named R9-ApoE2, FGF4-ApoE2, and RDP-ApoE2, respectively.
  • the signal peptide (SEQ ID NO: 66) of human source ApoE itself is further linked to the N-terminus of all protein constructs.
  • R9-ApoE2, FGF4-ApoE2, and RDP-ApoE2 were co-expressed with human APP in HEK 293T cells, and GFP was used as a negative control group.
  • the results of immunoblotting FIG.
  • the signal peptide is necessary for the activity of ApoECT to regulate APP cleavage.
  • the front part of the amino acid sequence of all the corresponding polypeptides expressed in the above experiments used to explore the function of ApoE expressed in cells contains the signal peptide of human ApoE itself (ApoE SP; SEQ ID NO: 66), and the signal peptide is located in ApoE or ApoE CT and as described above The N-terminus of each mutant of ApoE or ApoECT.
  • the present invention replaces the signal peptide of ApoE CT with the signal peptides of apolipoprotein A1 and apolipoprotein J (ApoA1 SP and ApoJ SP; SEQ ID NO: 67 and SEQ ID NO: 68). Simultaneously, the ApoECT activity not connected with the signal peptide was also analyzed.
  • SEQ ID NO: 66 (ApoE SP): MKVLWAALLVTFLAGCQA
  • SEQ ID NO: 67 (ApoAI SP): MKAAVLTLAVLFLTGSQA
  • SEQ ID NO: 68 (ApoJ SP): MMKTLLLFVGLLLTWESGQVLG
  • the ApoE CT mutant replaced with apolipoprotein A1 signal peptide was named ASP CT; the ApoE CT mutant replaced with apolipoprotein J signal peptide was named JSP CT; the ApoE CT mutant without signal peptide was named NSP CT.
  • human APP was co-expressed with each of the above-mentioned ApoECT mutants, GFP was used as a negative control group, and ApoECT was used as a positive control.
  • the present invention finds for the first time that ApoE2 can regulate the shearing of APP in a cell-autonomous manner in neurons, and this process depends on the C-terminal fragment of ApoE2.
  • the C-terminal fragment of ApoE not only increases non-amyloid metabolites of APP ⁇ -CTF; and weaken APP amyloid metabolism, reduce A ⁇ .
  • ApoE2 and its C-terminal fragment regulate APP metabolism, not through the secreted ApoE protein indirectly affecting the cleavage of APP in the cell; but through ApoE2 or its C-terminal fragment that coexists with APP in the same cell It is achieved by inhibiting the ⁇ -cleavage of APP, which is a cell-autonomous mode of action.
  • ApoE2 could directly inhibit the cleavage of ⁇ -CTF by ⁇ -secretase, while ApoEN NT lacking the C-terminus could not inhibit the cleavage of ⁇ -CTF by ⁇ -secretase.
  • the C-terminal fragment of ApoE could not inhibit the ⁇ -digestion of APLP1, indicating that the inhibitory effect of the C-terminal fragment of ApoE on ⁇ -digestion was substrate-selective.
  • the present invention also found that the C-terminal fragment of ApoE specifically expressed in neurons significantly reduces the size and density of amyloid plaques in the brains of AD model mice. ApoE specifically expressed in mouse neurons can inhibit the production of endogenous A ⁇ 40.
  • the addition of tagged proteins at the N or C terminus did not affect the activity of ApoE2 and ApoE C-terminal fragments to inhibit APP ⁇ cleavage.
  • the present invention also constructs a series of mutants of ApoE C-terminal fragments and ApoE2 internal sequences, which have the activity of inhibiting APP ⁇ digestion similar to the original ApoE CT or ApoE2.
  • the present invention also found that the addition of certain penetrating peptides to the N-terminal of ApoE will not affect its activity of inhibiting APP ⁇ enzymatic cleavage, but if the N-terminal lacks a signal peptide, it will lead to the loss of ApoE’s activity of inhibiting APP ⁇ enzymatic cleavage.
  • the discovery of the present invention is not limited to the human ApoE C-terminal fragment, and the non-human primate ApoE C-terminal fragment also has similar activity of inhibiting APP ⁇ digestion.
  • ApoE2 and its C-terminal fragment can be used as a potential drug for the prevention or treatment of AD.
  • Such potential drugs would need to act in a cell-autonomous manner in neurons.
  • Liposome delivery of DNA or RNA, lentivirus and adeno-associated virus infection of cells can make ApoE2 and/or its C-terminal fragments expressed in target cells; cell-penetrating peptides, enhanced endocytosis and other methods can also make The protein of ApoE2 and/or its C-terminal fragment exists in large quantities in the target cells; then ApoE2 and/or its C-terminal fragment inhibit the enzymatic cleavage activity of ⁇ -secretase in a cell-autonomous manner, reduce A ⁇ , and weaken the amyloid metabolism of APP .
  • AD Alzheimer's disease
  • ApoE2 and/or its C-terminal fragments inhibit the enzymatic cleavage activity of ⁇ -secretase in a cell-autonomous manner.
  • conditions such as amyloid cerebrovascular diseases are also associated with abnormalities in gamma-secretase function.
  • Down syndrome is also prone to develop into AD. Therefore, ApoE2 and/or its C-terminal fragments also have application potential in the treatment and prevention of these APP or ⁇ -secretase related diseases.
  • ⁇ -secretase activity is associated with most presenilin mutations that cause familial Alzheimer's disease (familial AD, fAD).
  • fAD familial Alzheimer's disease
  • sporadic AD sporadic AD
  • sAD Human apolipoprotein E
  • CT conserved C-terminal region
  • ApoE CT-mediated inhibitory activity is impaired to varying degrees in different ApoE subtypes, resulting in a rank order of ApoE2>ApoE3>ApoE4 potency that is inversely proportional to its associated AD risk.
  • ApoE CT expressed in neurons can migrate from other regions to amyloid plaques and slow down the formation of amyloid plaques.
  • AD Alzheimer's disease
  • a ⁇ ⁇ -amyloid
  • ⁇ -secretase Presenilin 1 (PS1) and presenilin 2 (PS2)—cause a rare form of familial AD (fAD). Based on these evidences, ⁇ -secretase is considered as a drug target in AD. Unfortunately, treatment with conventional ⁇ -secretase inhibitors causes severe side effects (Panza et al., 2019; Xia, 2019).
  • sAD has no disease-causing genes, but its risk is affected by a variety of genetic factors.
  • apolipoprotein E4 (ApoE4) is the most influential one (Corder et al., 1993; Rhinn et al., 2013).
  • the ApoE gene encodes three isoforms with only a single amino acid difference in their N-terminal region (NT): ApoE2, 3, and 4 (Kanekiyo et al., 2014).
  • ApoE4 not only increases the risk of AD, but also leads to an earlier age of onset and aggravates the severity of AD (Belloy et al., 2019; Li et al., 2020). In contrast, ApoE2 reduced AD risk.
  • ApoE In the rodent brain, under normal physiological conditions, ApoE is usually detected only in glial cells (Kanekiyo et al., 2014), whereas A ⁇ is produced by neurons. However, ApoE is also present in human neurons (Aoki et al., 2003; Han et al., 1994; Metzger et al., 1996; Strittmatter et al., 1993; Xu et al., 1999) and in injured rodent neurons (Xu et al., 2006). The metabolism of ApoE in the human brain is more similar to that of ApoE expressed in neurons than in glial cells (Brecht et al., 2004).
  • ApoE is mainly expressed by glial cells in the rodent brain, and A ⁇ is produced by the metabolism of APP expressed in neurons. Therefore, most studies have focused on how ApoE secreted by glia affects extracellular amyloid plaque formation (Huynh et al., 2017; Huynh et al., 2017; Liu et al., 2017). However, a recent study showed that ApoE can regulate A ⁇ production in a cell-autonomous manner (Wang et al., 2018). Before exploring this potential mechanism, this study first used immunofluorescence to stain human brain samples in order to verify that ApoE was indeed expressed in neurons.
  • ⁇ -CTF is an enzymatic cleavage product of the non-amyloid metabolic pathway of APP, whose production is known to depend primarily on ADAM10 ( Figure 26, panels A and B), an ⁇ -secretase (Esch et al., 1990; Haass et al. people, 1992; Mucke and Selkoe, 2012).
  • ApoE contains three regions: an N-terminal region (ApoE NT), a hinge region and a conserved C-terminal region (ApoE CT) ( Figure 33) (Chen et al., 2011). The only sequence differences between the three ApoE isoforms lie within their NTs. Experiments using HEK 293T cells with ApoE subtype truncated mutants showed that NT from ApoE2, 3 or 4 did not significantly alter the ⁇ -CTF produced by co-expressing APP ( Figure 26, panels E and F). In contrast, co-expression of the conserved ApoE CT resulted in a more than 6-fold increase in ⁇ -CTF levels ( Figure 26, panels G and H).
  • ApoE CT In addition to human APP, ApoE CT also increased ⁇ -CTF levels in HEK 293T cells transiently expressing mouse APP (mAPP) (Figure 26, panels P and Q). Experimental results in primary cultured neurons showed that endogenous A ⁇ 40 was reduced by 30% after overexpression of ApoE CT (lentiviral infection) in mouse cortical and hippocampal neurons compared to GFP controls ( Figure 17, panel K). In hiPSC-induced neurons, ApoE CT (lentiviral infection) resulted in an approximately 46% reduction in A ⁇ 40 production by human neurons (Fig. 17, panel L). Collectively, these results suggest that ApoE CT can slow endogenous A ⁇ in rodent and human neurons.
  • apolipoprotein A1 Another apolipoprotein co-expressed with APP, apolipoprotein A1 (ApoA1), failed to alter the levels of ⁇ -CTF and A ⁇ (Fig. 26, panels T, U and V), suggesting that ApoE regulates the activity of APP cleavage Not any non-specific activity of apolipoproteins.
  • ApoE2 or ApoE CT had a very similar perinuclear subcellular distribution pattern to co-expressed APP-FLAG (Fig. 18, panel A).
  • Fig. 18, panel B In cultured rat hippocampal neurons (Fig. 18, panel B) and hiPSC-derived neurons (Fig. 18, panel C), the expression of ApoE2-V5 or ApoE CT-V5 had a similar subset to the endogenously expressed APP in neurons.
  • Cellular distribution pattern (V5 is tagged protein).
  • ApoE2 and ApoE CT co-immunoprecipitated with components of ⁇ -secretase (APH, NCT and PEN2) when co-expressed in HEK 293T cells (Fig. 18, panel J; using anti-FLAG- PEN2 for immunoprecipitation).
  • endogenous ⁇ -secretases NCT, PS1, PS2 and PEN2
  • endogenous ApoE Fig. 18, panel K
  • ApoE CT reduces the gamma shearing of APP.
  • ApoE CT still increased ⁇ -CTF (Fig. 29, panels E and F).
  • ApoE CT was also able to increase ⁇ -CTF produced by APP M596V (Fig. 29, panels G and H), APP M596V , an APP mutation that cannot undergo ⁇ -cleavage (Citron et al., 1995).
  • ApoE CT also significantly decreased A ⁇ 40 ( FIG. 19 , panel E) and A ⁇ 42 ( FIG. 19 , panel F ) produced by ⁇ -CTF, suggesting that ApoE CT reduces A ⁇ production by inhibiting ⁇ -cleavage.
  • ApoE CT regulates the metabolism of APP and requires the participation of ⁇ -secretase.
  • PS1 and/or PS2 knockout (KO) HEK 293T cell lines were constructed in this study (Fig. 20, panel A). ⁇ -CTF levels were elevated in PS1 KO cells, which could be reversed by compensatory expression of PS1 (Fig. 20, panel B). PF03084014 treatment further increased ⁇ -CTF levels in PS1 KO cells ( Figure 20, panel B). Co-expressed ApoECT was still able to increase ⁇ -CTF in PS1 KO cells ( Figure 20, panels C and D) and PS2 KO cells ( Figure 20, panels E and F). Therefore, knockdown of PS1 or PS2 alone is not sufficient to completely abolish the activity of ApoECT.
  • PS1/2 double knockout cells PS1/2 double knockout cells
  • ⁇ -CTF levels were also significantly increased compared with WT cells (Fig. 20, panel G);
  • ApoE CT co-expressed in PS1/2 dKO cells Figure 20, panels G and J
  • ApoE2 Figure 20, panels K and L
  • Complementary expression of PS1 or PS2 rescued the activity of ApoE CT, which again significantly increased ⁇ -CTF (Fig. 20, panels H, I and J).
  • ApoE CT also significantly reduced A ⁇ 40 produced by ⁇ -CTF in PS1/2 dKO cells supplemented with PS2 ( FIG. 20 , panel M).
  • shRNA of mouse ApoE was constructed (shApoE_1, shApoE_2, shScramble were used as controls).
  • ShApoE_1 and shApoE_2 expressed in mouse neurons using the lentiviral system successfully reduced the protein expression of endogenous ApoE in primary cultured mouse neurons ( Figure 21, panel A).
  • shApoE_1 or shApoE_2 significantly reduced the basal levels of the CTF 14kD band in mouse neurons (Fig. 21, panels C and D), indicating that endogenous ApoE increases CTF 14kD .
  • these two shRNAs of ApoE could no longer reduce CTF by 14kD ( Figure 21, panel E), indicating that this activity of ApoE requires the presence of active gamma-secretase.
  • knockdown of ApoE also resulted in increased levels of A ⁇ produced by neurons (Fig. 21, panel F).
  • ApoE CT inhibits ⁇ -cleavage in a substrate-specific manner.
  • the present invention goes on to test whether ApoE CT also inhibits the gamma cleavage of NOTCH.
  • NOTCH is gamma secretase Substrates (Xia, 2019; Yang et al., 2019).
  • ⁇ -secretase reduced N100 (NOTCH fragment)
  • treatment with PF03084014 reversed this reduction
  • co-expressed ApoE CT failed to suppress this ⁇ -secretase-dependent reduction of N100 ( Figure 30, panel A).
  • ApoE CT still inhibited ⁇ -cleavage of APP by ⁇ -secretase ( Figure 30, panel B).
  • APP-like protein 1 is another substrate of alpha and gamma secretases (Eggert et al., 2004). Treatment with PF03084014 decreased APLP1 levels while enhancing its cleavage product ( ⁇ -like CTF); however, ⁇ -like CTF was not altered by co-expression of ApoE CT with APLP1 ( Figure 30, panel C). These results demonstrate that ApoE CT does not inhibit the ⁇ -cleavage of NOTCH and APLP1.
  • ApoE is the most important genetic factor of sAD, and the data from this experiment show for the first time that ApoE appears to reduce the risk of sAD by selectively inhibiting ⁇ -cleavage of APP, a key step in amyloidogenesis.
  • the metabolism of ApoE in the human brain is more similar to that of ApoE expressed in neurons than that of ApoE expressed in glial cells (Brecht et al., 2004). It has also been reported that human neurons carrying the ApoE4 subtype produce more A ⁇ than those with other ApoE subtypes (Wang et al., 2018), strongly supporting that endogenously expressed ApoE in human neurons can regulate Metabolism of endogenous APP. However, the role of ApoE in neurons remains largely poorly understood. The present invention finds that the protective subtype ApoE2 directly inhibits ⁇ -cleavage of APP in a cell-autonomous manner through its CT, while ApoE4 has no such activity.
  • the present invention also found that endogenous ApoE in mouse neurons significantly inhibited the ⁇ -cleavage of endogenously expressed APP; and in the human brain, there was an interaction between endogenous ApoE and endogenously expressed ⁇ -secretase .
  • AD mouse models recapitulate many of the key pathological features of AD but not the amyloid deposition of ApoE (Oakley et al., 2006).
  • the present inventors found that ApoE expressed in neurons of 5 ⁇ FAD mice migrated and accumulated around amyloid plaques, which is consistent with previous reports that ApoE was present in amyloid plaques in the brains of AD patients.
  • ⁇ -secretase is a potential drug target in AD.
  • inhibition of ⁇ -secretase activity was found to lead to severe side effects in previous drug development, which may be due to the poor selectivity of ⁇ -secretase inhibitors among different substrates (Panza et al., 2019; Roberson and Mucke, 2006; Xia, 2019).
  • non-specific inhibitors while inhibiting ⁇ -secretase to block A ⁇ production, also prevent the normal processing of ⁇ -secretase’s other substrates (Panza et al., 2019; Xia, 2019), resulting in side effects.
  • ⁇ -secretase modifiers It is a promising new strategy to selectively inhibit ⁇ -cleavage of APP (Xia, 2019).
  • the present invention finds that ApoE CT inhibits the ⁇ -cleavage of APP, but does not affect the ⁇ -cleavage of NOTCH and APLP1, indicating that the inhibitory effect of ApoE CT on ⁇ -cleavage has substrate specificity.
  • small-molecule drugs inhibit the activity of ⁇ -secretase by occupying the same position on the ⁇ -chain on PS1 that interacts with its substrates, such as APP or Notch (Yang et al., 2021).
  • ApoE CT as a structurally distinct molecule from traditional small-molecule inhibitors, may act on some allosteric sites and only affect the binding of some substrates to ⁇ -secretase, but not other substrates, thereby Achieving substrate selectivity. However, the exact mechanism remains to be resolved. Nevertheless, ApoE CT may serve as a new strategic treatment approach with potentially fewer side effects compared with conventional ⁇ -secretase inhibitors. In summary, the present invention not only provides a potential mechanistic understanding of the role of neuronal ApoE isoforms in the pathogenesis of sAD; but also identifies the ApoE CT as a potential treatment for AD.
  • the present invention also identifies ApoECT as a potent gamma-secretase inhibitor with rare substrate specificity that can migrate from distant sites to act locally in periplaque amyloidogenic hotspots.
  • mice Human APP and PS1 with five fAD mutations are expressed in neurons of 5 ⁇ FAD mice: K670N/M671L+I716V+V717I mutations in APP and M146L+L286V mutations in PS1 (Oakley et al., 2006). All mice were housed under a 12-h light-dark cycle (light from 8 am to 8 pm) with ad libitum access to food and water. All mouse procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Academy of Sciences and the National Institutes of Health.
  • HEK 293T cells were cultured in DMEM medium (Gibco) supplemented with 10% bovine serum albumin (Life Technology) and 1% penicillin/streptomycin (Life Technology).
  • DMEM medium Gibco
  • bovine serum albumin Life Technology
  • penicillin/streptomycin Life Technology
  • PS1 single knockout or PS1/2 double knockout HEK 293T cell lines the CRISPR/Cas9 genome editing method was employed (Ran et al., 2013).
  • gRNA 5'-ATTATCTAATGGACGACCCC-3' (SEQ ID NO:91) targeting PS1 exon 4 and gRNA 5'-CAAATACGGAGCGAAGCACG-3' (SEQ ID NO:92) targeting PS2 exon 4
  • PS1gRNA, PS2gRNA pSpCas9(BB)-2A-puro backbone
  • PS1gRNA, PS2gRNA pSpCas9(BB)-2A-puro backbone
  • the HEK 293T cells transfected with PS1 gRNA or PS2 gRNA were cultured in a 10 cm culture dish at a dilution ratio of 1:400 to ensure that each cell was monoclonal. After individual cells grow into cell clusters, genome-edited cell clusters are selected and verified by DNA sequencing and Western blot.
  • PS2 knockout cells were transfected with PS1 gRNA and subjected to the second round of selection.
  • Brain tissue samples were lysed with TissueLyserII (QIAGEN, 85300) in RIPA buffer (Solarbio, R0010). The lysate was boiled for 10 minutes and then centrifuged at 12,000 x g for 10 minutes. The operation steps of Western blot are the same as those of cell samples.
  • a method for detecting protein interactions in living cells by targeting APEX2 was applied as previously described (Hung et al., 2016). Approximately 30 hours after transfection and APEX2 plasmids were passed through Lipofectamine2000 (Invitrogen Technology), HEK 293T cells were incubated in 1 ml of complete medium with or without 250uM biotinphenol (BP) for 30 minutes at 37°C. Cells were treated with 1 mM H 2 O 2 for 1 min and cells were washed 3 times in quencher solution (10 mM sodium ascorbate, 5 mM Trolox and 10 mM sodium azide solution in 1 ⁇ DPBS).
  • quencher solution 10 mM sodium ascorbate, 5 mM Trolox and 10 mM sodium azide solution in 1 ⁇ DPBS.
  • HEK 293T cells were transfected and plasmids were treated with Lipofectamine2000 (Invitrogen Technology) for 48 hours, HEK 293T cells were treated in 1% CHAPSO (SIGMA), 25mM HEPES (pH7.4) (Gibco), 150mM NaCl (containing 1 ⁇ protease inhibitor cocktail) lyse, centrifuge at 14,000 ⁇ g for 20 min, and collect the supernatant.
  • SIGMA CHAPSO
  • HEPES pH7.4
  • NaCl 150mM NaCl (containing 1 ⁇ protease inhibitor cocktail) lyse
  • Neurons and HEK 293T cells cultured on coverslips in 24-well plates were washed with 1 ⁇ PBS (2.5 mM MgCl 2 and 0.5 mM CaCl 2 ) and fixed with 4% PFA+4% sucrose in 1 ⁇ PBS at room temperature for 10 minute. Next, punch the wells with 0.15% Triton-X in 1 ⁇ PBS for 15 min and block with 5% BSA in 1 ⁇ PBS for 30 min at room temperature For 10 minutes, samples were incubated overnight at 4°C with primary antibodies diluted in 5% BSA (in 1 ⁇ PBS) buffer. Samples were then washed once with 0.5 M NaCl in 1 ⁇ PBS and three times with 1 ⁇ PBS.
  • mice were anesthetized with isoflurane and perfused with 4% PFA. Dissected mouse brains were fixed overnight in 4% PFA and cryoprotected overnight in 30% sucrose. A cryostat (Leica CM3050S) was used to obtain frozen brain sections (40 ⁇ M) from fixed and cryoprotected mouse brains. Floating sections were washed 3 times with 1 ⁇ PBS and then permeabilized with 0.5% Triton-X in 1 ⁇ PBS for 30 minutes. After blocking with 5% BSA in 1 ⁇ PBS at room temperature for 1 h, the sections were incubated overnight at 4° C. in 5% BSA (in 1 ⁇ PBS) buffer containing the primary antibody.
  • 5% BSA in 1 ⁇ PBS
  • Sections were washed 3 times with 1 ⁇ PBS and incubated with secondary antibody diluted in 5% BSA (1 ⁇ PBS) buffer for 1 hour at room temperature. The samples were then washed 3 times with 1 ⁇ PBS and mounted with Prolong Gold Antifade Mountant (Life Technology).
  • mice were anesthetized with isoflurane and perfused with 4% PFA. Dissected mouse brains were fixed overnight in 4% PFA and cryoprotected overnight in 30% sucrose. A cryostat (Leica, CM3050S) was used to obtain frozen brain sections (40 ⁇ M) from fixed and cryoprotected mouse brains. Floating sections were washed 3 times with ddH2O and then incubated with 1% THS (SIGMA) in water for 5 minutes at room temperature. Next, the sections were washed twice with 80% ethanol and once with 95% ethanol for 3 minutes each. The samples were washed with ddH 2 O and 3 times with 1 ⁇ PBS before IHC.
  • SIGMA 1% THS
  • Culture media and lysates were collected 48 hours after transfection of HEK 293T cells and N2A cells, or 4.5 days after lentivirus infection of neurons.
  • a ⁇ 40 and A ⁇ 42 in the culture medium and cell lysates were detected by an A ⁇ ELISA kit as indicated in the protocol. (Invitrogen Technology; KHB3481 for human A ⁇ 40, KHB3441 for human A ⁇ 42 and KMB3481 for mouse A ⁇ 40).
  • the human ⁇ -secretase complex was purified as previously described (Lu et al., 2014), and the cDNA encoding the human ⁇ -secretase complex together with the expression vector was found in Lu et al., 2014.
  • E. coli cells were transformed with human ⁇ -CTF-Myc-6 ⁇ His plasmid, and when OD260 reached 0.6-0.8, E. coli cells were induced with 0.5 ⁇ M IPTG (Solarbio) at 37° C. for 4 hours. E. coli cells were lysed in this buffer (30 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA) and sonicated on ice.
  • the lysate was centrifuged at 25,000 ⁇ g for 10 minutes, and the lysate was dissolved in urea buffer (30 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 6M urea, 1% TritonX-100 and 1 mM CaCl 2 , 1 ⁇ Protease Inhibitor Cocktail) and rotate slowly for 3 hours.
  • urea buffer (30 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 6M urea, 1% TritonX-100 and 1 mM CaCl 2 , 1 ⁇ Protease Inhibitor Cocktail
  • urea buffer 1 (30 mM Tris-HCl, pH 8.0; 300 mM NaCl, 1 mM EDTA, 6 M urea, 1% TritonX-100, 1 mM CaCl 2 , 1 ⁇ cocktail), 20 CV of wash buffer 2 (30mM Tris-HCl, pH8.0, 300 mM NaCl, 0.1% TrionX-100) and 20CV Wash Buffer 3 (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% Sharpsol).
  • CV column volumes
  • Bound material was eluted with wash buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% CHAPSO) with an imidazole gradient of 0-300 mM. SDS-PAGE and Western blotting for visualization of purified proteins
  • mice After being deeply anesthetized with isoflurane, mice (5 weeks old) were placed in a stereotaxic apparatus (RWD Instruments). The zero point (x, y, z, 0, 0, 0) is Bregma.
  • Lentivirus lentivirus-GFP, lentivirus-ApoE and lentivirus-ApoE-IRES-GFP
  • AAV AAV2/9-hSyn-EGFP-WPRE-pA and AAV2/9-hSyn-ApoECT; purchased from Shanghai Taitu Biotechnology Co., Ltd.
  • mice were injected into the cortex (x, y, z, +/-1.6, 0, -1) or hippocampus (x, y, z, +/-1.5, -2, -1.5), each injection site Inject 1ul. The mice were then returned to their cages for rehabilitation after surgery.
  • GraphPad Prism software (v6) analyzed data using unpaired, paired, or one-sample t-tests. A ⁇ 40 and A ⁇ 42 levels were normalized to the corresponding GFP controls. ⁇ -CTF levels were normalized to FL-APP and corresponding GFP controls. All data are presented as mean ⁇ SEM. Statistical significance was defined as p ⁇ 0.05. One, two, three and four stars (*) in the graph indicate p-values ⁇ 0.05, ⁇ 0.01, ⁇ 0.001 and ⁇ 0.0001, respectively.
  • Presenilins and gamma-secretase structure, function, and role in Alzheimer Disease. Cold Spring Harbor perspectives in medicine 2, a006304.
  • APLP -1 and APLP-2 The proteolytic processing of the amyloid precursor protein gene family members APLP -1 and APLP-2 involves alpha-, beta-, gamma-, and epsilon-like cleavages: modulation of APLP-1 processing by n-glycosylation. J Biol Chem 279, 18146-18156.
  • Amyloid beta-peptide is produced by cultured cells during normal metabolism. Nature 359, 322-325.
  • Apolipoprotein E is present in hippocampal neurons without neurofibrillary tangles in Alzheimer's disease and in age-matched controls.
  • Apolipoprotein E especially apolipoprotein E4, increases the oligomerization of amyloid beta peptide.
  • APOE2 protective mechanism and therapeutic implications for Alzheimer ⁇ s disease. Molecular neurodegeneration 15, 63.
  • Neurons of the human frontal cortex display apolipoprotein E immunoreactivity: implications for Alzheimer's disease. Journal al of neuropathology and experimental neurology 55, 372-380.
  • Integrative genomics identifies APOE epsilon4 effectors in Alzheimer's disease. Nature 500, 45- 50.
  • Apolipoprotein E high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proceedings of the National Academy of Sciences of the United States of America 90, 1977-1981.
  • Presenilin 2 familial Alzheimer's disease mutations result in partial loss of function and dramatic changes in Abeta 42/40 ratios. Journal of neurochemistry 92, 294-301.
  • Presenilin- 1 knockin mice reveal loss-of-function mechanism for familial Alzheimer's disease. Neuron 85, 967-981.

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Abstract

本发明涉及载脂蛋白E（apolipoproteinE，ApoE）的C端片段（例如ApoE CT）和经修饰的ApoE2及其在抑制γ分泌酶酶活中的应用。本发明的一个方面提供分离的ApoE构建物（ApoE construct），其包含一个ApoE多肽，所述ApoE多肽：（1）包含来自SEQ ID NO：12所示氨基酸序列的一个片段，该片段N端第一个氨基酸残基为SEQ ID NO：12第215-221位中任一位置上的氨基酸残基，该片段C端最后一个氨基酸残基为SEQ ID NO：12第274-299位中任一位置上的氨基酸残基；或（2）其氨基酸序列与（1）所述片段具有至少约70％序列同一性，同时保留至少约50％（1）所述该片段对γ分泌酶酶切活性的抑制。本发明还提供所述ApoE构建物的蛋白部分的编码序列、核酸构建物、宿主细胞、构建方法、药物组合物以及应用。

Description

载脂蛋白E的C端片段及其在抑制γ分泌酶酶活中的应用
相关申请的交叉引用
本专利申请要求于2022年1月30日提交的中国申请号CN202210113320.2的优先权，其全部内容通过引用并入本文。
对电子序列表的引用
电子序列表(217334 1PCWO.xml；大小：92,558字节；创建日期：2023年1月30日)的内容通过引用整体并入本文。
技术领域
本发明涉及载脂蛋白E(apolipoprotein E，ApoE)的C端片段及其抑制γ分泌酶酶活中的应用。
背景技术
γ分泌酶是100多种I型跨膜蛋白的剪切酶，参与了多种生物学过程。T-细胞和B-细胞分化异常，出血性腹泻，皮肤和毛发缺陷，阿尔茨海默症(Alzheimer’s disease，AD)等都与γ分泌酶的异常相关。γ分泌酶是四个蛋白组成的复合物：NCT、APH1、PEN2和presenilin1(PS1)或者presenilin2(PS2)。γ分泌酶的酶切活性中心是PS1或者PS2。现在已经发现多种PS1和PS2的突变会导致AD，PS1和PS2都是AD的致病基因，因此γ分泌酶是非常重要的药物靶点，特别是针对AD的治疗。
AD是人类最常见的一种神经退行性疾病。AD患者主要表现为渐进性记忆障碍、认知功能障碍、人格改变及语言障碍等症状，进一步发展为自理能力丧失，最终死亡。我国是世界上AD患者数量最多的国家。截至2019年，我国有1000多万AD患者，呈每年递增趋势。超过80岁的人群患AD的概率超过40％。并且AD的治疗费用也非常高昂，给家庭和社会造成极大负担。
在过去的几十年里，人们对AD致病机理的研究已经取得了很大的进展，提出了Aβ级联假说。该假说认为大脑中Aβ的过度产生、异常聚集是一系列严重而缓慢病变的起始原因，引起大脑内一系列生物过程异常，导致神经元上突触退化和神经元死亡。 在大脑中，淀粉样前体蛋白(amyloid precursor protein，APP)主要由神经元表达，Aβ是由经过β和γ分泌酶依次剪切APP产生的，生成的Aβ被释放到细胞外，这一过程被称为APP的淀粉样代谢。在正常的生理状态下，大脑中生成的Aβ多是可溶性的。但是发生病变后，大脑中的Aβ大量异常聚集，形成沉积在神经元周边的淀粉样斑块，引发神经元的损伤，进一步引起脑组织的衰竭，最终导致AD。此外，淀粉样蛋白在大脑血管中异常聚集和沉淀会引发淀粉样脑血管病。APP除了被β和γ分泌酶剪切生成有毒性的Aβ外，还可以被α和γ分泌酶依次酶切，是APP的非淀粉样代谢途径。由于α分泌酶的酶切位点位于Aβ内部，所以α酶切产物不会生成Aβ，剪切片段被称为α-CTF，没有神经毒性，不会导致AD。α和β酶切相互竞争，增加α分泌酶的酶切，Aβ的生成下降；同样地，减少γ分泌酶的酶切，也会减少Aβ的生成，减缓淀粉样斑块的形成，最后减缓甚至预防AD和淀粉样脑血管病的发生。多种以γ分泌酶为靶点的药物已经做过临床试验。
ApoE是在人体中广泛分泌的蛋白，在脑中可以与APP共同表达在神经元中。人源ApoE有三种异构体ApoE2、ApoE3、ApoE4；它们在结构和功能上高度相似，大致分为三个功能域，分别是包含与受体结合位点的N端功能域，包含与脂质结合位点的C端功能域，以及连接两端功能域的中间柔性铰链区。ApoE2、ApoE3和ApoE4仅在N端功能域有两个氨基酸残基不同，在C端功能域上的氨基酸序列完全相同。其中ApoE2具有保护作用，可以降低AD的发病风险；ApoE4是AD的最主要的风险因子，不仅能够显著增加患AD的风险，还能够显著提前患AD的发病年龄，加重AD病情。
目前为止，人们依然不清楚ApoE2和ApoE4如何改变AD的患病风险。
发明内容
在一个方面，本发明提供一种分离的ApoE构建物，其包含一个ApoE多肽，所述ApoE多肽：(1)包含来自SEQ ID NO：12所示氨基酸序列的一个片段，该片段(或该ApoE多肽)N端第一个氨基酸残基为SEQ ID NO：12第215-221位中的任意一个位置上的氨基酸残基，该片段(或该ApoE多肽)C端最后一个氨基酸残基为SEQ ID NO：12第274-299位(例如第279-299位)中的任意一个位置上的氨基酸残基；或(2)其氨基酸序列与(1)所述片段具有至少约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)序列同一性，并保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)(1)所述片段对γ分泌酶酶切活性的抑制。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含(或由其组成，或基本由其组成)如下任一序列：(i)SEQ ID NO：12第215-299位氨基酸残基的氨基酸序列；(ii)SEQ ID NO：12第216-299位氨基酸残基的氨基酸序列；(iii)SEQ ID NO：12第217-299位氨基酸残基的氨基酸序列；(iv)SEQ ID NO：12第218-299位氨基酸残基的氨基酸序列；(v)SEQ ID NO：12第219-299位氨基酸残基的氨基酸序列；(vi)SEQ ID NO：12第220-299位氨基酸残基的氨基酸序列；(vii)SEQ ID NO：12第221-299位氨基酸残基的氨基酸序列；(viii)SEQ ID NO：12第216-294位氨基酸残基的氨基酸序列；(ix)SEQ ID NO：12第216-289位氨基酸残基的氨基酸序列；(x)SEQ ID NO：12第219-289位氨基酸残基的氨基酸序列；(xi)SEQ ID NO：12第219-288位氨基酸残基的氨基酸序列；(xii)SEQ ID NO：12第219-287位氨基酸残基的氨基酸序列；(xiii)SEQ ID NO：12第219-286位氨基酸残基的氨基酸序列；(xiv)SEQ ID NO：12第219-285位氨基酸残基的氨基酸序列；(xv)SEQ ID NO：12第219-284位氨基酸残基的氨基酸序列；(xvi)SEQ ID NO：12第219-279位氨基酸残基的氨基酸序列；(xvii)SEQ ID NO：12第219-274位氨基酸残基的氨基酸序列；(xviii)SEQ ID NO：12第220-274位氨基酸残基的氨基酸序列；(xix)SEQ ID NO：12第220-279位氨基酸残基的氨基酸序列；或(xx)SEQ ID NO：12第221-274位氨基酸残基的氨基酸序列。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含(或由其组成，或基本由其组成)SEQ ID NOs：13、18-34、78和87中任一氨基酸序列。在一些实施方式中，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含(或由其组成，或基本由其组成)SEQ ID NOs：31-34、78和87中任一所示的氨基酸序列(例如SEQ ID NO：33)。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE多肽包含(或由其组成，或基本由其组成)与SEQ ID NOs：13、18-34、78和87中任一氨基酸序列具有至少约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)序列同一性的氨基酸序列。在一些实施方式中，所述ApoE多肽含有非人灵长类序列，例如与SEQ ID NOs：13、18-34、78和87中任一氨基酸序列高度同源和保守的序列。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变(例如添加、缺 失或取代)：R226、D227、R228、L229、D230、Q235、E238、V239、R240、K242、E244、E245、Q246、A247、Q248、Q249、I250、R251、L252、Q253、A254、E255、Q275、V280、V287、T289、S290、A291、P293、V294、P295、S296、D297、N298、H299、R251+T289、R251+T289+A291、S290+P293+V294+P295+S296+D297+N298+H299、R226+D227+R228+L229+D230、E238+V239+R240+K242、或E244+E245+Q246+A247+Q248+Q249+I250+R251+L252+Q253+A254。在一些实施方式中，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变：R226A、D227A、R228A、L229A、D230A、Q235A、E238A、V239A、R240A、K242A、E244del、E245del、Q246del、A247del、Q248del、Q249del、I250del、R251S、L252del、Q253del、A254del、E255A、Q275A、V280A、V287A、T289A、S290A、A291T、P293A、V294A、P295A、S296A、D297A、N298A、H299A、R251A+T289A、R251S+T289A+A291T、S290A+P293A+V294A+P295A+S296A+D297A+N298A+H299A、R226A+D227A+R228A+L229A+D230A、E238A+V239A+R240A+K242A、或E244del+E245del+Q246del+A247del+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE多肽包含(或由其组成，或基本由其组成)SEQ ID NOs：46-52、57-59、62和76中任一氨基酸序列。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE构建物在ApoE多肽的N端还进一步包含SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸残基的氨基酸序列，或与SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸至少有约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)同一性的序列。在一些实施方式中，所述ApoE构建物包含(或由其组成，或基本由其组成)SEQ ID NO：60或61所示的氨基酸序列。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE构建物在所述ApoE多肽的N端或C端还进一步包含一个穿膜肽。在一些实施方式中，所述穿膜肽包括(或由其组成，或基本由其组成)SEQ ID NOs：35-45、63-65和84-86中任一氨基酸序列，例如包括(或由其组成，或基本由其组成)SEQ ID NO：63或65所示的氨基酸序列。在一些实施方式中，所述穿膜肽(例如C端)与所述ApoE多肽(例如N端)、 或所述包含SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸残基的氨基酸序列(例如N端)、或所述与SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸至少有约70％同一性的序列(例如N端)通过一个接头序列连接。
在根据以上任一所述分离的ApoE构建物的一些实施方式中，所述ApoE构建物在所述ApoE多肽的N端还进一步包含一个信号肽。在一些实施方式中，所述信号肽包含(或由其组成，或基本由其组成)选自SEQ ID NOs：66-68中任一氨基酸序列。
在另一个方面，本发明提供一种分离的ApoE构建物，该分离的ApoE构建物包括元件(1)：穿膜肽，和元件(2)：ApoE2的全长序列(例如SEQ ID NO：12)或ApoE3的全长序列(例如SEQ ID NO：89)或至少包括SEQ ID NO：12第221-274位氨基酸残基的片段；其中，所述元件(1)和元件(2)之间通过肽键直接连接，或通过接头序列连接。在一些实施方式中，所述穿膜肽选自SEQ ID NOs：35-45、63-65和84-86中任一氨基酸序列，例如SEQ ID NO：63或65所示的氨基酸序列。在一些实施方式中，元件(2)所述的至少包括SEQ ID NO：12第221-274位氨基酸残基的片段选自本发明所述的任一包括SEQ ID NO：12第221-274位氨基酸残基的ApoE多肽。在一些实施方案中，元件(2)所述的至少包括SEQ ID NO：12第221-274位氨基酸残基的片段的氨基酸序列如SEQ ID NOs：13、18-34和78中任一所示。在一些实施方案中，元件(2)所述的至少包括SEQ ID NO：12第221-274位氨基酸残基的片段含有ApoE2的N端结构域(SEQ ID NO：12第1-167位氨基酸残基)和铰链区(SEQ ID NO：12第168-205位氨基酸残基)、以及C端至少包括SEQ ID NO：12第221-274位氨基酸残基的片段。在一些实施方式中，所述元件(2)包含以下任一序列：SEQ ID NO：12第1-274位，第1-279位，第1-280位、第1-281位、第1-282位、第1-283位、第1-284位、第1-285位、第1-286位、第1-287位、第1-288位、第1-289位、第1-290位、第1-291位、第1-292位、第1-293位、第1-294位、第1-295位、第1-296位、第1-297位、第1-298位、或第1-299位氨基酸残基。
本发明还提供了编码以上任一所述分离的ApoE构建物的蛋白部分的分离的核酸和载体(例如克隆载体或表达载体)，以及表达以上任一所述分离的ApoE构建物的蛋白部分的宿主细胞，或包含该核酸或载体的宿主细胞。在一些实施方式中，所述载体在所述分离的核酸的5’端含有神经元特异性启动子。在一些实施方式中，所述神经元特异性启动子选自突触蛋白启动子、thy-1启动子和钙调素依赖型蛋白激酶II-α启动子。在一些实施方式中，所述载体为是重组AAV或lentivirus表达载体。在一些实施方式中，所述宿主细胞为神经元(例如人神经元、非人灵长类神经元、或啮齿类神经元)。在 一些实施方式中，所述ApoE构建物包含(或由其组成，或基本由其组成)ApoE2(例如SEQ ID NO：12)或ApoE3(例如SEQ ID NO：89)的全长氨基酸序列。
本发明还提供了一种药物组合物，其含有(i)本文任一实施方案所述的分离的ApoE构建物、分离的核酸、载体、或宿主细胞，和(ii)药学上可接受的载体。
本发明还提供了本文任一实施方案所述的分离的ApoE构建物、分离的核酸、载体、宿主细胞或药物组合物在制备用于治疗或预防一个个体(例如人)中与γ分泌酶的酶切活性相关的疾病的药物中的应用。本发明还提供了一种治疗或预防一个个体(例如人)中与γ分泌酶的酶切活性相关疾病的方法，包括向所述个体施用有效剂量的本文任一实施方案所述的分离的ApoE构建物、分离的核酸、载体、宿主细胞或药物组合物。在一些实施方式中，所述药物或方法抑制γ分泌酶的酶切活性，从而治疗或预防所述与γ分泌酶的酶切活性相关的疾病。在一个或多个实施方案中，所述疾病选自如下的组：神经系统退行性疾病，肿瘤或癌症，炎性疾病和肾脏疾病。在一个或多个实施方案中，所述疾病选自如下的组：阿尔茨海默病及其相关疾病、淀粉样脑血管病、唐氏综合症、与衰老相关的其他神经退行性疾病(比如额颞叶痴呆)、头颈癌(如头颈鳞细胞癌和口腔部鳞状细胞癌)、乳腺癌、肝癌、胰腺癌(包括转移性胰腺癌)、卵巢癌、肺癌(如非小细胞肺癌、肺腺癌和小细胞肺癌)、胶质瘤(如恶性神经胶质瘤)、纤维瘤、淋巴瘤(如B细胞淋巴瘤)、骨肉瘤、胃癌、膀胱癌、哮喘(如过敏性哮喘)、肺炎、气道炎症、急性肾损伤、透明细胞肾细胞癌、肾纤维化、和梗阻性肾病。
附图说明
图1：ApoE通过其C端片段(ApoE CT)减弱APP的淀粉样代谢，增加其非淀粉样代谢产物(α-CTF)。面板A和B：在HEK 293T细胞中过表达的ApoE2和ApoE3可以显著增加共表达的APP经过非淀粉样代谢通路(α剪切)产生的α-CTF，而ApoE4不可以。统计结果在面板B，N＝8，paired或者one sample t-test。面板C：通过瞬时转染过表达的ApoE2减少了共表达的APP生成的总Aβ40，N＝8，paired或者one sample t-test。面板D和E：免疫印迹实验表明过表达的ApoE CT增加了共表达的APP生成的α-CTF，而过表达ApoE2或ApoE4的N端片段(ApoE2 NT，ApoE4 NT)则不改变α-CTF的产生，统计结果在面板E，N＝8，paired或者one sample t-test。面板F和G：过表达ApoE CT降低了由APP生成的Aβ40和Aβ42，而过表达ApoE2 NT、ApoE3 NT或者ApoE4 NT均不改变Aβ，N＝5，one sample t-test。面板A-G：HEK 293T细胞。面板H：在小鼠的大脑皮 层和海马原代培养神经元中使用慢病毒表达系统(lentivirus)表达的ApoE CT可以降低神经元内源产生的Aβ40。启动子是hSyn。N＝7，one samplet-test。面板I：使用慢病毒在多能干细胞系诱导得到的人神经元中表达的ApoE CT同样可以减少神经元产生的内源性Aβ40。启动子是hSyn，N＝5，unpairedt-test。WesternBlot检测蛋白APP，α-CTF和ApoE，Aβ通过ELISA检测。
图2：培养液中的ApoE2和ApoE CT不能增加细胞中APP的非淀粉样代谢产物。面板A：免疫印迹实验检测培养液中含有的ApoE2和ApoE CT蛋白。面板B和C：与含有ApoE2或者ApoE CT蛋白的细胞培养液共孵育并不改变细胞中APP所生成的α-CTF，统计结果在面板C，N＝9，one sample t-test。面板D：免疫荧光实验显示，含有ApoE2或者ApoE CT蛋白的细胞培养液共孵育HEK 293T细胞之后在细胞内无法检测到ApoE2或者ApoE CT蛋白的大量存在，只有很少一部分附着于个别细胞表面。而利用脂质体在HEK 293T细胞中瞬转表达的ApoE CT则大量存在于细胞内。面板E：免疫荧光实验显示，与含有ApoE2或者ApoE CT蛋白的培养液共孵育后，小鼠原代培养神经元内无法检测到ApoE2或者ApoE CT蛋白的大量存在，ApoE只有很少一部分附着于个别神经元表面。而利用慢病毒表达的ApoE CT则大量存在于神经元内。MAP2标记神经元，DAPI标记细胞核。
图3：ApoE CT减少Aβ的核心序列。面板A、B和C：在HEK 293T细胞中过表达不同的ApoE CT突变体对APP生成Aβ的影响。面板A：CT NA1/2/3/4//5：在ApoE CT的N端添加ApoE原有的1、2、3、4或5个氨基酸残基。CT ND3/4：在ApoE CT的N端删除3或4个氨基酸残基。CT CD5/10：在ApoE CT的C端删除5或10个氨基酸残基。ELISA测量不同ApoE CT突变体对Aβ40的影响。面板B：CT ND1/2//5：在ApoE CT的N端删除1、2或5个氨基酸残基。ELISA测量不同ApoE CT突变体对Aβ40的影响。面板C：CT ND3CD10/11/12/13/14/15/20/25：在ApoE CT的N端删除3个氨基酸残基的同时，在C端删除10、11、12、13、14、15、20或25个氨基酸。ELISA测量不同ApoE CT突变体对Aβ40的影响。N＝5(面板A和B)或7(面板C)，one sample t-test。
图4：ApoE CT并不改变APP分泌酶的表达量。面板A：ApoE CT不影响APP代谢酶mRNA的表达。面板B：ApoE CT不影响APP代谢酶的蛋白表达量。ADAM10：APP的α分泌酶；BACE1：APP的β分泌酶；PS1和PS2：γ分泌酶的核心活性亚基蛋白。使用qRT-PCR和Western Blot分别检测mRNA和蛋白水平。表达GFP的细胞为负对照。N＝9，one sample t-test。
图5：ApoE CT模拟了γ分泌酶抑制剂对APP剪切的影响，抑制了β-CTF的γ剪切。面板A和B：过表达的γ分泌酶活性中心蛋白PS1可以降低α-CTF的生成，而表达的ApoE CT或者使用1μM PF03084014(γ分泌酶抑制剂)处理均会增加α-CTF的生成。统计结果在面板B，N＝11，paired或者one sample t-test。面板C和D：过表达的ApoE CT可以抑制由β-CTF生成的Aβ40(N＝4)和Aβ42(N＝6)，one sample t-test。面板E：过表达的ApoE CT不影响APPΔAICD经过β酶切而生成的Aβ40，N＝4，one sample t-test。Western Blot检测蛋白，ELISA检测Aβ。
图6：ApoE CT影响APP的剪切需要γ分泌酶的存在。面板A和D：γ分泌酶活性中心蛋白PS1和PS2被敲除的HEK 293T细胞中(PS1/2 dKO)，α-CTF大量增加，此时过表达的ApoE CT不再进一步增加α-CTF，统计结果在面板D(标注GFP1的两个柱子)。面板B、C和D：在补充表达PS1或者PS2的PS1/2 dKO细胞中，过表达的ApoE CT再次增加了α-CTF，统计结果在面板D，N＝8，pairedt-test。面板E：在补充表达PS2的PS1/2 dKO细胞中，过表达ApoE CT可以减少APP生成的Aβ40，N＝4，one sample t-test。Western Blot检测蛋白，ELISA检测Aβ40。
图7：体外纯化的ApoE2以浓度依赖性的方式直接抑制APP的γ酶切。面板A：免疫印迹显示分离纯化的β-CTF，ApoE2，ApoE2 NT，PS1，NCT，PEN2和GFP。面板B：在纯化的系统中，γ分泌酶剪切β-CTF生成Aβ40的反应可以被10μM PF03084014(γ分泌酶抑制剂)抑制，N＝3，pairedt-tests。面板C：纯化的ApoE2以剂量依赖性的方式抑制γ分泌酶对β-CTF剪切，减少Aβ40的生成。ApoE2的半抑制浓度约为1.2μM，N＝3。面板D：纯化的ApoE2可以抑制β-CTF经过γ酶切而生成的Aβ40，但是ApoE2 NT不能抑制γ分泌酶对β-CTF剪切，N＝3，paired t-tests。Western Blot检测蛋白，ELISA检测Aβ40。
图8：ApoE CT不抑制APLP1的γ酶切。1μM PF03084014(γ分泌酶抑制剂)抑制了HEK 293T细胞中外源表达的APLP1的γ剪切，增多了APLP1经过α剪切生成的α-like CTF；但是ApoE CT并不会增多APLP1产生的α-like CTF。
图9：神经元中过表达的ApoE CT减少了5xFAD小鼠模型脑中的淀粉样斑块。面板A：5xFAD小鼠模型subiculum区域淀粉样斑块(THS标记)和ApoE CT或者GFP的共染色代表性图片。在5xFAD小鼠中使用腺相关病毒(AAV)表达系统在神经元中表达ApoE CT(一月龄注射，七月龄分析)，GFP作为对照，启动子是hSyn(human synapsin)。编码多肽的核酸序列前面加有信号肽编码序列。统计结果在面板B、C、D和E。面板B：表达ApoE CT的小鼠subiculum区域每个淀粉样斑块 (plaque)的平均面积更小(面板B)，平均荧光强度更弱(面板C)，总面积中所占比例更小(面板D)，斑块的密度也更低(面板E)。THS用来标记淀粉样斑块。N＝33，unpaired t-test。
图10：神经元中表达的ApoE2可以抑制小鼠神经元内源产生的Aβ40。面板A：在小鼠的原代培养神经元中使用慢病毒表达系统(lenti-virus)表达的ApoE2可以降低小鼠神经元(mouse neurons)产生的内源性Aβ40，GFP作为对照。N＝7，pairedt-test。面板B：在野生型小鼠(WT mouse)大脑中利用AAV表达的ApoE2降低了小鼠脑中的内源Aβ40，GFP作为对照。N＝5，unpaired t-test。使用hsyn启动子保证神经元特异性表达。ELISA检测Aβ。
图11：在两端添加标签蛋白的ApoE2或ApoE CT依然具有调控APP剪切的活性。面板A和B：在HEK 293T细胞中过表达的含有标签蛋白V5的ApoE2-V5(C端添加标签蛋白)和V5-ApoE2(N端添加标签蛋白)都可以显著增加APP所产生的α-CTF。统计结果在面板B。N＝6，one sample t-test。面板C和D：在HEK 293T细胞中过表达的在ApoE CT的C端或者N端添加标签蛋白V5的ApoE CT-V5和V5-ApoE CT都可以显著增加APP所产生的α-CTF，统计结果在面板D。N＝6，one sample t-test。Western Blot检测APP及其剪切片段。
图12：ApoE CT的突变依然可以调控APP的剪切。面板A和B：在HEK 293T细胞中过表达的ApoE CT，ApoE CT的单个位点或者双位点突变，CT A20、CT A40、CT A60、CT A65、CT A72、CT A80和CT A36/74都显著地增加了α-CTF。统计结果在面板B。N＝6，one sample t-test。面板C和D：在HEK 293T细胞中过表达多位点突变的CT R10、CT LF5和CT LM5和ApoE CT同样能够显著增加α-CTF；但是CT E5、CT E10、CT E20和CT F5则失去了增加α-CTF的活性。统计结果在面板D。N＝4，one sample t-test。Western Blot检测蛋白。
图13：在ApoE2的内部添加或者删除氨基酸序列，ApoE2依然可以调控APP代谢。面板A：在HEK 293T细胞中与APP共表达的ApoE2-M3F曾加了α-CTF。面板B：在HEK 293T细胞中与APP共表达的ApoE2 MD10也增加了α-CTF。
图14：非人源的ApoE CT同样调控APP的剪切。面板A和B：在HEK 293T细胞中过表达的与人源ApoE CT序列高度相似的非人灵长类来源的ApoE CT显著增加了α-CTF，并且其活性与人源ApoE CT的活性相同。统计结果在面板B。N＝6，one sample t-test。Western Blot检测蛋白。人源与猴源ApoE CT序列比对在面板C。
图15：连有穿膜肽的ApoE依然具有调控APP剪切的活性。面板A和B：在HEK 293T细胞中将APP与R9-ApoE2、FGF4-ApoE2或者RDP-ApoE2共表达，GFP作为阴性对照组。共表达的R9-ApoE2和RDP-ApoE2显著增加了α-CTF。统计结果在面板B。N＝5，one sample t-test。Western Blot检测蛋白。
图16：在细胞中表达的ApoE CT需要连有信号肽才能具有调控APP剪切的活性。面板A和B：在HEK 293T细胞中过表达的在N端连有载脂蛋白A1信号肽的ApoE CT突变体(ASP CT)和含有载脂蛋白J信号肽的ApoE CT突变体(JSP CT)依然可以显著增加α-CTF。在细胞中表达没有连接信号肽的NSP CT则不能调控APP剪切而增加α-CTF。统计结果在面板B。N＝6，one sample t-test。Western Blot检测蛋白。
图17：ApoE2通过其C末端区域增加α-CTF并减少Aβ的产生。面板A：人类(顶部)或猴子(底部)皮质中ApoE和NeuN(神经元标志物)的共免疫荧光染色的代表图像。箭头表示ApoE阳性神经元。面板B：人脑皮层中ApoE和APP的共免疫荧光染色的代表性图像。面板C和D：瞬时共表达APP和GFP、ApoE2、ApoE3或ApoE4的N2A细胞中的全长APP(FL-APP)、α-CTF和ApoE信号的Westernblot和相应的统计分析。N＝8，one sample t-test。面板E：瞬时共表达APP、BACE1和GFP、ApoE2、ApoE3或ApoE4的HEK 293T细胞中的Aβ40的归一化统计分析。N＝8，one sample t-test。面板F和G：瞬时共表达APP和GFP、ApoE CT、ApoE2 NT或ApoE4 NT的N2A细胞中的全长APP(FL-APP)、α-CTF和ApoE信号的Western blot和相应的统计分析。N＝8，one sample t-test。面板H：瞬时共表达APP、BACE1和GFP、ApoE CT的N2A细胞中的Aβ40的归一化统计分析。N＝4，one sample t-test。面板I和J：瞬时共表达APP和GFP、ApoE2或ApoE CT的N2A细胞中的全长APP(FL-APP)、α-CTF和ApoE信号的Western blot和相应的统计分析。N＝6，one sample t-test。面板K：利用慢病毒系统瞬时表达GFP或者ApoE CT的小鼠皮层(cortical neurons)或者(hippocampal neurons)神经元中的Aβ40归一化统计分析。启动子是hSyn。N＝7，one sample t-test。面板L：利用慢病毒系统瞬时表达GFP或者ApoE CT的人源多能干细胞诱导的神经元(human neurons)中的Aβ40归一化统计分析。启动子是hSyn。N＝5，unpaired t-test。
图18：ApoE CT与APP和γ分泌酶相互作用。面板A：瞬时共表达APP-FLAG和GFP、ApoE2或者ApoE CT的N2A细胞中ApoE和APP的免疫荧光染色的代表性图像。面板B：瞬时表达GFP、ApoE2-V5或者ApoE CT-V5的原代培养神经元中ApoE和内源表达的APP的免疫荧光染色的代表性图像。面板C：瞬时表达ApoE CT-V5的人源多能干细胞诱导的神经元中ApoE CT和内源表达的APP的免疫荧光染色的代表性图像。MAP2染 色表示神经元，DAPI染色表示细胞核。面板D：免疫印迹检测在HEK 293T细胞中共表达的APP-APEX2标记的ApoE2。面板E.免疫印迹检测在HEK 293T细胞中共表达的APP-APEX2标记的ApoE CT。面板F.免疫印迹检测在HEK 293T细胞中共表达的ApoE2-APEX2标记的APP。面板G：免疫印迹检测在小鼠神经元细胞中表达的ApoE-APEX2标记的神经元中内源表达的APP和阴性对照COX2。面板H：免疫印迹检测在HEK 293T细胞中与ADAM10、BACE1、PS1或者PS2分别瞬时共表达的ApoE2-APEX2标记的ADAM10、BACE1、PS1和PS2。面板I：免疫共沉淀检测在HEK 293T细胞中与ApoE2、ApoE2 NT或者ApoE CT分别瞬时共表达的β-CTF-FLAG相互作用的ApoE。面板J：免疫共沉淀检测在HEK 293T细胞中与ApoE2、ApoE2 NT或者ApoE CT分别瞬时共表达的γ分泌酶(HA-APH，V5-NCT，FLAG-PEN2)相互作用的ApoE。面板K：免疫共沉淀检测人脑组织中内源性γ分泌酶(NCT、PS1、PS2和PEN2)和内源性ApoE的相互作用。
图19：ApoE CT通过抑制APP的γ酶切增加α-CTF和减少Aβ。面板A和B：免疫印迹检测和统计分析在HEK 293T细胞瞬时表达APP和GFP(条带1)；共表达APP和PS1时，1微摩尔γ分泌酶抑制剂PF03084014处理与否(条带2，条带3)；共表达APP、PS1和ApoE CT(条带4)；这几种表达或者药物处理条件下，APP产生α-CTF的情况。N＝11。面板C和D，免疫印迹检测和统计分析1微摩尔γ分泌酶抑制剂PF03084014处理时，在HEK 293T细胞中GFP或者ApoE CT对共表达APP生成α-CTF的影响，对照组不用抑制剂处理。N＝7。面板E：ELISA检测分别瞬时共表达β-CTF和GFP或ApoE CT的HEK 293T细胞中生成的Aβ40。N＝4。面板F：ELISA检测分别瞬时共表达β-CTF和GFP或ApoE CT的HEK 293T细胞中生成的Aβ40。N＝6。面板G和面板H：ELISA检测APPΔAICD和GFP或者ApoE CT分别共表达的HEK 293T生成Aβ40的结果。面板G，细胞培养基。面板H，细胞裂解液。面板B、D、E、F、G和H，one sample t-test。
图20：γ分泌酶对于ApoE CT调节APP的代谢是必需的。面板A：免疫印迹检测野生型(WT)、PS1或者PS2单敲除(PS1 KO，PS2 KO)、PS1和PS2双敲除(PS1/2 dKO)HEK 293T细胞系中PS1和PS2的蛋白表达。面板B：APP表达在野生型HEK 293T细胞系中(条带1)；APP表达在PS1单敲除HEK 293T细胞系中(条带2)；APP表达在1微摩尔γ分泌酶抑制剂PF03084014处理的PS1单敲除HEK 293T细胞系中(条带3)；APP和PS1共表达在PS1单敲除HEK 293T细胞系中(条带4)。免疫印迹检测不同条件下，细胞中α-CTF的含量。面板C和D：分别在野生型(WT)和PS1单敲除(PS1 KO)HEK 293T细胞系中免疫印迹检测和统计分析ApoE CT对共表达的APP经酶切产生的α-CTF 的影响，GFP作为对照。N＝9。面板E和F：分别在野生型和PS2单敲除(PS2 KO)HEK 293T细胞系中免疫印迹检测和统计分析ApoE CT对共表达的APP经酶切产生的α-CTF的影响，GFP作为对照。N＝9。面板G和J：分别在野生型(WT)和PS1和PS2双敲除(PS1/2 dKO)HEK 293T细胞系中免疫印迹检测和统计分析ApoE CT对共表达的APP产生α-CTF的影响，GFP作为对照。N＝8。面板H和J：免疫印迹检测和统计分析在PS1和PS2双敲除(PS1/2 dKO)HEK 293T细胞中分别共表达APP、PS1和GFP或者ApoE CT生成的α-CTF。N＝8。面板I和J：免疫印迹检测和统计分析在PS1和PS2双敲除(PS1/2 dKO)HEK 293T细胞中分别共表达APP、PS2和GFP或者ApoE CT生成的α-CTF。N＝8。面板K和L：免疫印迹检测和统计分析在PS1和PS2双敲除(PS1/2 dKO)HEK 293T细胞中分别共表达APP和GFP或者ApoE2生成的α-CTF。N＝9。面板M：ELISA检测在PS1和PS2双敲除(PS1/2 dKO)HEK 293T细胞中分别共表达β-CTF、PS2和GFP或者ApoE CT生成的Aβ40。N＝4。面板D、F、J、L和M，one sample t-test。
图21：小鼠神经元中敲低内源性ApoE导致APP的代谢产物CTF14kD含量降低和Aβ升高。面板A：免疫印迹检测利用慢病毒表达系统瞬时表达shScramble(阴性对照)、shApoE_1(ApoE的shRNA_1)或shApoE_2(ApoE的shRNA_2)的原代培养小鼠神经元中内源表达的ApoE。面板B：1微摩尔γ分泌酶的抑制剂PF03084014处理时，小鼠神经元中APP的代谢物CTF14kD的相对含量。面板C和D：免疫印迹检测和统计分析利用慢病毒表达系统瞬时表达shScramble、shApoE_1或shApoE_2的原代培养小鼠神经元中内源表达的APP的代谢物CTF14kD的相对含量。N＝5，one sample t-test。面板E：1微摩尔γ分泌酶的抑制剂PF03084014处理时，免疫印迹检测利用慢病毒表达系统瞬时表达shScramble、shApoE_1或shApoE_2的原代培养小鼠神经元中内源表达的APP的代谢物CTF14kD的相对含量。面板F：ELISA分析利用慢病毒表达系统瞬时表达shScramble、shApoE_1或shApoE_2的原代培养小鼠神经元中内源表达的APP产生的Aβ40。N＝5，one sample t-test。
图22：ApoE2直接抑制APP的γ酶切。面板A：免疫印迹分析表达纯化的β-CTF、ApoE2、ApoE2 NT、PS1、NCT、PEN2和GFP。面板B：ELISA检测和统计分析纯化的β-CTF、纯化的β-CTF和γ分泌酶共孵育、以及1微摩尔γ分泌酶的抑制剂PF03084014处理时纯化的β-CTF和γ分泌酶共孵育，三种不同条件下生成的Aβ水平。N＝3，student′s ttest。面板C：ELISA检测和统计分析纯化的β-CTF与一系列浓度梯度的ApoE2共孵育生成的Aβ。IC50大约是1.2±0.48微摩尔。面板D：ELISA检测和统计分析纯化的β-CTF 分别与2.5微摩尔纯化的GFP、ApoE2和ApoE2 NT共孵育生成的Aβ。N＝3，student′s t test。
图23：神经元表达的ApoE CT迁移至淀粉样斑块并减缓5×FAD小鼠脑中淀粉样斑块的形成。面板A：实验模式图。面板B：在5×FAD小鼠脑神经元中表达10周的腺相关病毒GFP或者ApoE CT和淀粉样斑块(plaque)在小鼠脑中的免疫荧光染色代表性图片。面板C：统计分析淀粉样斑块的平均大小。面板D：统计分析淀粉样斑块总面积在subiculum区域所占的比例。面板E：统计分析淀粉样斑块的密度。N＝21。
图24：在5×FAD小鼠神经元中表达的ApoE通过CT在淀粉样斑块周边聚集。面板A：在5×FAD小鼠皮层神经元中表达三个月的慢病毒ApoE2-IRES-GFP、ApoE2 NT-IRES-GFP和ApoE CT-IRES-GFP与淀粉样斑块(GFP)的免疫荧光染色代表性图片。面板B：在5×FAD小鼠皮层神经元中表达一个月的慢病毒ApoE CT与人源APP(hAPP)的免疫荧光染色代表性图片。面板C：在野生型小鼠脑神经元中表达三个月的慢病毒ApoE CT与hAPP的免疫荧光染色代表性图片。Plaque，淀粉样斑块。
图25：人神经元中存在内源性ApoE，AD患者脑中淀粉样斑块与ApoE共定位。面板A：非AD患者(62岁，顶部)或者AD患者(86岁，底部)皮质中ApoE和NeuN(神经元标志)的免疫荧光共染色的代表图像。箭头表示ApoE阳性神经元。面板B：ApoE阳性神经元相对于人脑皮层总神经元的百分比。面板C：ApoE阳性神经元相对于所有ApoE阳性细胞的百分比。年轻(young)：16-23岁，n＝3；老年(old)：＞60岁，n＝3；AD：＞60岁，n＝6。面板D：人脑皮层中ApoE和GFAP(星形胶质细胞标志)的免疫荧光共染色的代表图像。面板E：非AD患者(62岁，顶部)或者AD患者(86岁，底部)皮质中ApoE和淀粉样斑块(6E10)的免疫荧光共染色的代表性图像。
图26：在HEK 293T细胞中，ApoE2通过ApoE CT增加α-CTF和减少Aβ。面板A：免疫印迹检测在HEK 293T细胞中分别共表达APP和GFP或者ADAM10生成的α-CTF。面板B：免疫印迹检测1微摩尔ADAM10的抑制剂GI254023X处理的HEK 293T细胞生成的α-CTF，对照没有用GI254023X处理。面板C和D：免疫印迹检测和统计分析在HEK 293T细胞中分别共表达APP和GFP、ApoE2、ApoE3或ApoE4生成的α-CTF。N＝12。面板E和F：免疫印迹检测和统计分析在HEK 293T细胞中分别共表达APP和GFP、ApoE2 NT、ApoE3 NT或ApoE4 NT生成的α-CTF。N＝12。面板G和H.免疫印迹检测和统计分析在HEK 293T细胞中分别共表达APP和GFP或ApoE CT生成的α-CTF。N＝18。面板I：ELISA检测和统计分析在HEK 293T细胞中分别共表达APP、BACE1和GFP、ApoE CT、ApoE2 NT、ApoE3 NT或ApoE4 NT产生的分泌到培养基中的Aβ40。N＝4。面板J：ELISA 检测在HEK 293T细胞中分别共表达APP、BACE1和GFP或ApoE CT产生的细胞裂解液中的Aβ40。N＝5。面板K：ELISA检测在HEK 293T细胞中分别共表达APP、BACE1和GFP或ApoE CT产生的Aβ42和Aβ42/Aβ40。N＝6。面板L和M：免疫印迹检测和统计分析在HEK 293T细胞中分别将APP、BACE1分别与GFP或ApoE CT共表达生成的β-CTF。N＝13。面板N和O：免疫印迹检测和统计分析在HEK 293T细胞中分别共表达APP和GFP、ApoE2或ApoE CT生成的α-CTF。N＝6。面板P和Q：免疫印迹检测和统计分析在HEK 293T细胞中分别共表达mouse APP(mAPP)和GFP或者ApoE CT生成的α-CTF。N＝6。面板R和S：ELISA检测和统计在HEK 293T细胞中分别共表达APPSW和GFP或者ApoE CT生成的Aβ40；面板R，培养基中的Aβ40；面板S，细胞裂解液中的Aβ40。N＝6。面板T和U：免疫印迹检测和统计分析在HEK 293T细胞中分别共表达APP和GFP或ApoA1生成的α-CTF。N＝6。面板V：ELISA检测和统计分析在HEK 293T细胞中分别共表达APP、BACE1和GFP或ApoA1生成的Aβ40。N＝3。面板D、F、H、I、J、K、M、O、Q、R、S、U和V，one sample t-test。
图27：培养液中的ApoE2和ApoE CT不能改变APP的代谢。面板A：免疫印迹检测细胞培养液中的ApoE2和ApoE CT。面板B和C：免疫印迹检测和统计分析培养液中GFP、ApoE2或者ApoE CT对共表达在HEK 293T细胞中APP生成α-CTF的影响。N＝9。面板D：免疫印迹检测不同条件下培养液中的ApoE2。原始含有ApoE2的培养液(条带1)，与未转染细胞孵育48小时后的含有ApoE2的培养液(条带2)，在37℃培养箱中单独保存48小时的含有ApoE2的培养液(条带3)。面板E：免疫荧光检测HEK 293T细胞摄取的培养液中的ApoE2和ApoE CT，以及HEK 293T细胞表达的ApoE CT。DAPI，细胞核标志物。面板F：免疫荧光检测神经元摄取的培养基中的ApoE2和ApoE CT，以及神经元表达的ApoE CT。MAP2，成熟神经元的标志物；DAPI，细胞核标志物。面板G：免疫印迹分析纯化的ApoE2和来自细胞裂解物或细胞培养基的ApoE2。
图28：在APP的γ酶切位点附近的AD相关突变影响ApoE CT的活性。面板A和B：免疫印迹检测统计分析在HEK 293T细胞中分别共表达APP不同的AD突变和GFP或者ApoE CT生成的α-CTF。N＝7，one sample and student’s t-test。
图29：当α或β分泌酶被抑制时，ApoE CT仍然能够调节APP的代谢。面板A：免疫印迹检测和分析1微摩尔ADAM10抑制剂GI254023X处理时，在HEK 293T细胞中ApoE CT对共表达的APP生成α-CTF的影响；对照组不用抑制剂处理。N＝9。面板C和D：ELISA检测和统计分析免疫印迹检测1微摩尔ADAM10抑制剂GI254023X处理时，在HEK 293T细胞中ApoE CT对共表达的APP和BACE1生成Aβ的影响；对照组不用抑 制剂处理。面板C，细胞培养基中的Aβ40；面板D，细胞裂解液中的Aβ40。N＝6。面板E和F：免疫印迹检测和统计分析5微摩尔BACE1抑制剂AZD3839处理时，在HEK 293T细胞中ApoE CT对共表达的APP生成α-CTF的影响；对照组不用抑制剂处理。N＝10。面板G和H：免疫印迹检测和统计分析在HEK 293T细胞中分别共表达APPM596V和GFP或者ApoE CT生成的α-CTF。N＝7。面板I：qRT-PCR检测和统计分析表达的ApoE2或者ApoE CT对HEK 293T细胞内源性ADAM10、BACE1、PS1和PS2 mRNA表达量的影响。面板J：免疫印迹检测ApoE2或者ApoE CT对HEK 293T细胞内源性ADAM10、BACE1、PS1和PS2蛋白表达量的影响。面板K：免疫印迹检测ApoE CT对共表达在HEK 293T细胞中的APP在细胞表面表达的影响。表达GFP(-)和ApoE CT(+)的HEK 293T细胞的细胞表面蛋白用磺基-N-羟基琥珀酰亚胺-生物素标记，并用链霉亲和素偶联的琼脂糖珠分离。
图30：ApoE CT无法抑制NOCTH和APLP1的γ酶切。面板A：在HEK 293T细胞中，只表达N100-V5(条带1)；共表达的N100-V5和γ分泌酶(条带2)；1微摩尔γ分泌酶的抑制剂PF03084014处理共表达的N100-V5和γ分泌酶(条带3)；共表达N100-V5、γ分泌酶和ApoE CT(条带4)。免疫印迹检测以上条件表达时N100-V5的剪切。面板B：免疫印迹检测在HEK 293T细胞中共表达APP、γ分泌酶和ApoE CT时α-CTF的含量。面板C：在HEK 293T细胞中，只表达APLP-V5(条带1)；1微摩尔γ分泌酶的抑制剂PF03084014处理表达的APLP-V5(条带2)；共表达APLP1-V5和ApoE CT(条带3)。免疫印迹检测以上条件表达时APLP1-V5的代谢。
图31：体外纯化的β-CTF、ApoE2、ApoE2 NT和γ分泌酶。考马斯亮蓝染色显示纯化的β-CTF-Myc-6×His(面板A)、ApoE2-3×FLAG(面板B)、ApoE2NT-3×FLAG(面板C)、γ分泌酶(面板D)(NCT-V5-6×His；PLAG-PEN2；APH；全长PS1，FL-PS1；PS1的N端片段，PS1 NTF；PS1的C端片段，PS1CTF)和3×FLAG-GFP(面板E)。
图32：本实例所用人脑信息表。
图33：人ApoE蛋白结构。N端结构域(残基1-167)和C端结构域(残基206-299)由柔性铰链区(残基168-205)连接。三种亚型为ApoE2(Cys112；Cys158)，ApoE3(Cys112；Arg158)和ApoE4(Arg112；Arg158)。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
人源载脂蛋白E(ApoE)主要包括ApoE2、ApoE3和ApoE4三种亚型。ApoE包含三个区域：一个N端结构域(ApoE NT)、一个铰链区域和一个保守的C端结构域(ApoE CT)。三种ApoE亚型之间的唯一序列差异在于它们的N端结构域(图33)。示例性的ApoE2(人源ApoE2)的氨基酸序列如下所示：
(SEQ ID NO：12，全长299氨基酸残基；下划线序列为216-299位点ApoE CT SEQ ID NO：33；方框为1-167位点N端结构域；斜体为168-205位点铰链区；残基206-299为C端结构域)。
(SEQ ID NO：79；ApoE2 N端结构域)
(SEQ ID NO：80；ApoE2或ApoE3铰链区)
(SEQ ID NO：81；ApoE2 N端结构域+铰链区)
示例性的ApoE3(人源ApoE3)的氨基酸序列如下所示：
(SEQ ID NO：89，全长299氨基酸残基；下划线序列为216-299位点ApoE CT SEQ ID NO：33；方框为1-167位点N端结构域；斜体为168-205位点铰链区；残基206-299为C端结构域)。
(SEQ ID NO：90；ApoE3 N端结构域)
本发明提供了一种分离的ApoE构建物，其包含一个ApoE多肽。该ApoE多肽包含ApoE蛋白C端结构域的一部分序列(例如SEQ ID NO：12第221-274位氨基酸)、或基于该序列的改造序列。本发明是基于发明人对所述ApoE构建物(例如本发明所述的ApoE多肽或ApoE2)的意料发现：(i)其可以在神经元中(体内或体外)以细胞自主性的方式调控前体淀粉蛋白蛋白质(amyloid precursor protein，APP)的剪切；(ii)其可以抑制(例如抑制至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)γ-分泌酶的酶活，例如抑制γ-分泌酶介导的APPγ-剪切，与γ-分泌酶抑制剂(例如PF03084014)的APPγ-剪切抑制效果相似；(iii)其可以降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)APP的淀粉样代谢，例如减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)淀粉样蛋白-β(Aβ)(例如Aβ40和/或Aβ42)的生成，或降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)对C末端片段-β(β-CTF)的γ-剪切；(iv)其可以提高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)APP的非淀粉样代谢，例如增加(例如增加至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)C末端片段-α(α-CTF)的生成；(v)其不影响(或影响不超过约30％、20％、10％、5％或更少)α-分泌酶、β-分泌酶、和/或γ-分泌酶(或其亚基)(例如ADAM10、BACE1、PS1和/或PS2)的mRNA和/或蛋白质表达；(vi)其不影响(或影响不超过约30％、20％、10％、5％或更少)β-分泌酶介导的APPβ-剪切，和/或α-分泌酶介导的APPα-剪切；(vii)其不抑制(或抑制不超过约30％、20％、10％、 5％或更少)对非APP蛋白(例如APLP1(APP like protein 1)或Notch)或其片段的γ-剪切，不增加(或增加不超过约30％、20％、10％、5％或更少)非APP蛋白γ-剪切产生的α-CTF，说明所述ApoE构建物以底物特异性抑制γ-剪切，而γ-分泌酶抑制剂则没有此底物特异性；(viii)所述ApoE构建物可以减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)AD模型小鼠脑中的淀粉样斑块，例如减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)淀粉样斑块的平均斑块大小、总面积、和/或密度，减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)神经元内Aβ(例如Aβ40和/或Aβ42)生成，降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)小鼠的AD病理进程，提高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)AD小鼠存活率和/或寿命；(ix)在所述ApoE构建物的N端和/或C端添加标签蛋白(例如纯化标签或检测标签)，和在某些所述ApoE构建物(例如ApoE2或ApoE3)的内部添加或删除氨基酸残基(例如ApoE2-M3F或ApoE2 MD10)，不会影响(或影响不超过约30％、20％、10％、5％或更少)该ApoE构建物抑制γ-分泌酶酶活，例如抑制γ-分泌酶介导的APPγ-剪切；(x)在所述ApoE构建物的N端或C端添加穿膜肽(cell-penetrating peptide，CPP)可以提高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)所述ApoE构建物的抑制γ-分泌酶酶活的效果(如以上所述一个或多个效果)；(xi)在细胞内，所述ApoE构建物的N端需要有信号肽才能调控γ分泌酶对APP的剪切，但对信号肽的来源没有严格要求；(xii)本发明所述的某些ApoE构建物(例如ApoE CT(SEQ ID NO：33))甚至比ApoE2(例如SEQ ID NO：12)和/或ApoE3(例如SEQ ID NO：89)全长蛋白有更高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)的抑制γ-分泌酶介导的APPγ-剪切的活性；(xiii)所述ApoE构建物对多种表达APP变体的细胞，例如致病性瑞典APP突变体(APPsw)、保护性冰岛APP突变(A673T)、APPM596V、或APP在β-或α-切割位点附近的突变(KM670/671NL、E693K、E693Δ)，也有抑制APPγ-剪切的效果，例如减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)Aβ的生成；(xiv)所述ApoE构建物可以与细胞内源APP呈现重叠的核周亚细胞分布模式，与细胞内源的APP、α-分泌酶和γ-分泌酶或其组分相互作用；(xv)在AD模 型小鼠中，神经元局部表达(例如在皮质、齿状回(DG)和海马体CA3表达)的所述ApoE构建物可以从远处迁移到下托并积聚在淀粉样斑块周围，从而在这些淀粉样蛋白生成热点局部抑制γ-分泌酶的酶活；和/或(xvi)与本发明所述ApoE构建物具有高度同源性(例如至少约90％、95％、96％、97％、98％、99％或更高)的非人源ApoE构建物(例如猴ApoE C端序列)也具有类似的以上所述一个或多个特性。
本申请首次发现载脂蛋白E通过它的C端片段以细胞自主性的方式抑制γ分泌酶的酶切(即与γ分泌酶在同一细胞内发挥作用)，抑制APP的淀粉样代谢，减少Aβ生成；并且增加了APP的非淀粉样代谢产物，α-CTF；在AD模型小鼠脑中减缓淀粉样斑块的大小和密度。因此ApoE的C端片段可作为一个以γ分泌酶为靶点的药物，用于减缓(例如减慢至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％的AD发展进程)、治疗、或者预防AD和淀粉样脑血管病的发生和发展。此外，ApoE的C端片段作为一种γ分泌酶的抑制剂可能在多种生物过程发挥着作用，ApoE的C端片段的应用不限于神经退行性疾病领域，还可以用于癌症、炎性疾病、肾脏疾病等多种疾病，例如任何与γ分泌酶的酶切活性相关的疾病(抑制γ分泌酶的酶切活性可以治疗或预防所述疾病)。常规γ分泌酶抑制剂在治疗中会引起严重的副作用，均与Notch信号缺陷相关，例如免疫抑制、胃肠道出血和腹泻以及癌性皮肤损伤。本发明提供的ApoE构建物不影响Notch的γ-剪切，因此本发明提供的ApoE构建物和相关方法可能可以提供一种新型的高效且安全的治疗方案。
本申请进一步发现，载脂蛋白E的C端片段中SEQ ID NO：12第221-274位氨基酸序列
(MGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQR；SEQ ID NO：78；“ApoE CT ND5CD25”)是该C端片段发挥生物功能(例如抑制γ-分泌酶介导的APPγ-剪切)所必须含有的氨基酸序列。在一些实施方式中，载脂蛋白E的C端片段中起作用的最短片段是SEQ ID NO：12第220-274位氨基酸序列(EMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQR；SEQ ID NO：87)。在一些实施方式中，ApoE的C端片段中起作用的最短片段是SEQ ID NO：12第220-279位氨基酸残基：EMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGL(SEQ ID NO：34)。
1.定义
除非另有特别指出，否则本发明的实施将采用在本领域技术内的化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学、细胞生物学、干细胞方案、细胞培养和转基因生物学的常规方法，其中许多在下文描述用于举例说明的目的。这样的技术已经在文献中充分说明。本文引用的所有公开文件、专利和专利申请通过引入全文并入本文。
除非另有说明，本文使用的所有技术和科学术语具有本发明所属的技术领域的普通技术人员通常理解的含义。为了本发明的目的，定义以下术语，以同本技术领域通常理解的含义保持一致。
如本文所用，术语“治疗(treating)”和“治疗(treatment)”是指延迟术语适用的疾病或病患或此类疾病或病患的一种或多种症状的发作、延迟或逆转其进展、降低其严重度或对其的缓解或预防。
术语“有效量”或“药学有效量”是指必需产生所需结果的一种或多种试剂的量和/或剂量和/或剂量方案，如，足以降低APPγ剪切的量，或治疗神经退行性疾病(例如AD)相关的一种或多种症状的量，或足以减轻特征为哺乳动物脑中的淀粉样沉积物的疾病的严重度或延迟所述疾病进展的量(如，治疗有效量)，足以降低特征为哺乳动物脑中的淀粉样沉积物的疾病的风险或延迟所述疾病的发作和/或降低最终的严重度的量(如，预防有效量)。
术语“减轻”是指减少或消除该病理或疾病的一种或多种症状，和/或减少该病理或疾病的一种或多种症状的速率或延迟该病理或疾病的一种或多种症状的发作或严重度和/或预防所述病理或疾病。在某些实施方案中，减少或消除病理或疾病的一种或多种症状包括但不限于减少或消除为病理或疾病特征的一个或多个标记(如，总-Tau(tTau)、磷酸化-Tau(pTau)、APPneo、可溶性Aβ40、Aβ42、pTau/Aβ42比率和tTau/Aβ42比率和/或CSF中一个或多个选自Aβ42/Aβ40比率、Aβ42/Aβ38比率、sAPPα、sAPPα/sAPPβ比率、sAPPα/Aβ40比率、sAPPα/Aβ42比率等的组分的水平的增加)，和/或增加非淀粉样代谢产物如α-CTF，和/或减少、稳定或逆转一个或多个诊断标准(如，临床痴呆评定(CDR))。
“个体”或“受试者”包括但不限于哺乳动物、鸟类、蛙类、鱼类、果蝇、线虫等。哺乳动物包括但不限于灵长类(例如人类，或非人类灵长类例如猴子)，啮齿类(例如小鼠、大鼠、仓鼠、沙鼠、松鼠、豚鼠和兔子)，家畜(例如猪、牛、羊、马、驴)，宠物(例如猫、狗、兔和仓鼠)等。在一些实施方式中，所述个体是人类。
如本文所用的术语“制剂”或“药物制剂”或“剂型”或“药物制剂”是指含有至少一种用于递送至受试者的治疗剂或药物的组合物。
两条多肽或核酸序列之间的“序列同一性”表示所述序列之间相同的残基的数目占残基总数的百分比，且基于突变类型确定残基总数的计算。突变类型包括在序列任一端或两端的插入(延伸)、在序列任一端或两端的缺失(截短)、一个或多个氨基酸/核苷酸的置换/替代、在序列内部的插入、在序列内部的缺失。举多肽为例(核苷酸同理)，如果突变类型为以下中的一种或多种：一个或多个氨基酸/核苷酸的置换/替代、在序列内部的插入和在序列内部的缺失，则残基总数以比较的分子中较大者来计算。如果突变类型还包括在序列任一端或两端的插入(延伸)或在序列任一端或两端的缺失(截短)，则在任一端或两端插入或缺失的氨基酸的数量(例如，在两端插入或缺失的数量小于20个)并不计入残基总数中。在计算同一性百分数时，将正在比较的序列以产生序列之间最大匹配的方式比对，通过特定算法解决比对中的空位(如果存在的话)。
此处所用的术语“片段”指多肽并且被定义为给定多肽中特有的或特征性的任意分离部分。此处所用的该术语也指给定多肽中保留至少部分全长多肽活性的任意分离部分。优选保留的活性部分至少为全长多肽活性的10％。更优选保留的活性部分至少为全长多肽活性的20％、30％、40％、50％、60％、70％、80％或90％。更优选保留的活性部分至少为全长多肽活性的95％、96％、97％、98％或99％。最优选保留的活性部分为全长多肽活性的100％。此处所用的该术语也指包括全长多肽中至少一个确定序列元件的指定多肽的任意部分。优选该序列元件跨越全长多肽的至少4-5个，更优选至少约10、15、20、25、30、35、40、45、50或更多氨基酸。
此处所用的术语“多肽”是指通过肽键相连的顺序氨基酸链。该术语用以指任意长度的氨基酸链，然而本领域普通技术人员知道该术语不仅指代长链，也可用于指含有通过肽键相连的2个氨基酸的最短链。
此处所用的术语“蛋白质”是指作用如离散单位的一个或多个多肽。如果单个多肽即为离散的功能单位，且不需要与其他多肽的持久物理缔合以形成该独立的功能单位，则此处术语“多肽”和“蛋白质”可互换使用。如果该离散功能单位由多于一个彼此物理缔合的多肽组成，则此处所用的术语“蛋白质”指物理偶联的并且共同作为离散单位起作用的多个多肽。
“重组表达多肽”、“重组多肽”和“经修饰的多肽”(或蛋白)是指从经遗传工程修饰以表达该多肽的宿主细胞中表达的多肽。重组表达多肽可以与宿主细胞(如哺乳动物细胞)内正常表达的多肽相同或相似。重组表达多肽也可为宿主细胞的外源物， 例如异源于哺乳动物宿主细胞正常表达的多肽。或者重组表达多肽为嵌合多肽，例如部分多肽含有与哺乳动物宿主细胞内正常表达的多肽相同或相似的氨基酸序列，而其他部分为宿主细胞外源物。
术语“氨基酸”意指二十种常见的天然存在的氨基酸。天然存在的氨基酸包括丙氨酸(Ala；A)、精氨酸(Arg；R)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、半胱氨酸(Cys；C)；谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、蛋氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
可以保守性置换非关键的氨基酸而不影响蛋白质的正常功能。保守性置换意指用化学或功能相似的氨基酸置换氨基酸。提供相似氨基酸的保守性置换表是本领域熟知的。举例来说，在一些实施方式中，以下提供的氨基酸组被认为是相互的保守性置换。
在某些实施方式中，被认为是相互保守性置换的氨基酸的所选组：
在某些实施方式中，被认为是相互的保守性置换的氨基酸的其他所选组：
在某些实施方式中，被认为是相互的保守性置换的氨基酸的其他所选组：

如本文使用的，术语”约”或”约“是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度相比较，改变多达15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。在一个实施方式中，术语“约”或“大约”是指围绕参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度±15％、±10％、±9％、±8％、±7％、±6％、±5％、±4％、±3％、±2％或±1％的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。
如本文使用的，术语”基本上(substantially/essentially)“是指与程度、参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度相比较，是约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高的程度、数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。
数值范围，包含该范围的端点值，以及范围的每个具体数值，例如“16至100个氨基酸之间”包括16和100，以及16至100之间的每一个具体数值。
在本说明书全文，除非上下文另有要求，否则术语″包含″，“包括”、“含有”和“具有”应理解为暗示包括所述步骤或要素或者步骤或要素组，但不排除任何其他步骤或要素或者步骤或要素组。在特定实施方式中，术语″术语″包含″，“包括”、“含有”和“具有”同义使用。
“由……组成”意指包括但限于在短语”由……组成”后的任何。因此，短语”由……组成“是指示所列出的要素是需要的或强制性的，并且没有其他要素是可以存在的。
″基本上由……组成″意指包括在短语″基本上由……组成″后列出的任何要素，并且限于不干扰或贡献于所列出的要素的公开内容中指定的活动或动作的其他要素。因此，短语″基本上由……组成”是指示所列出的要素是需要的或强制性的，但没有其 他要素是任选的，并且取决于它们是否影响所列出的要素的活动或动作而可以存在或不存在。
冠词″一个/一种(a/an)″和″该/所述(the)″在本文中用于指一个/一种或超过一个/一种(即至少一个/一种)所述冠词的语法对象。例如，″要素″意指一个/一种要素或超过一个/一种要素。
替代(例如″或″)的使用应理解为意指替代方案中任一、两者或其任何组合。
术语″和/或″应理解为意指替代方案中任一或两者。
在本说明书全文，提到″一个实施方式″、″实施方式″、″特定实施方式″、″相关实施方式″、″某个实施方式″、″另外的实施方式″或″进一步的实施方式″或其组合意指与所述实施方式结合描述的特定特征、结构或特性被包括在本发明的至少一个实施方式中。因此，前述短语在本说明书全文的各个地方的出现不一定全部指相同实施方式。此外，特定特征、结构或特性可以以任何合适方式在一个或多个实施方式中组合。
2.ApoE构建物和ApoE多肽
在一个方面，本申请提供一种分离的ApoE构建物，其包含一个ApoE多肽，所述ApoE多肽：(1)包含来自SEQ ID NO：12所示氨基酸序列的一个片段，该片段(或该ApoE多肽)N端第一个氨基酸残基为SEQ ID NO：12第215-221位中的任意一个位置上的氨基酸残基，该片段(或该ApoE多肽)C端最后一个氨基酸残基为SEQ ID NO：12第274-299位(例如第279-299位)中的任意一个位置上的氨基酸残基；或(2)其氨基酸序列与(1)所述片段具有至少约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)序列同一性，并保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)(1)所述片段对γ分泌酶酶切活性的抑制。应理解，本文所述的“片段”是指来自相应蛋白的连续的一段氨基酸序列。
在一些实施方式中，所述ApoE多肽包含来自SEQ ID NO：12所示氨基酸序列的一个片段，该ApoE多肽N端第一个氨基酸残基为SEQ ID NO：12第215-221位中的任意一个位置上的氨基酸残基，该ApoE多肽C端最后一个氨基酸残基为SEQ ID NO：12第274-299位(例如第279-299位)中的任意一个位置上的氨基酸残基。在一些实施方式中，所述ApoE构建物包含一个ApoE多肽，所述ApoE多肽由一个来自SEQ ID NO：12所示氨基酸序列的片段组成，该ApoE多肽N端第一个氨基酸残基为SEQ ID NO：12第215- 221位中的任意一个位置上的氨基酸残基，该ApoE多肽C端最后一个氨基酸残基为SEQ ID NO：12第274-299位(例如第279-299位)中的任意一个位置上的氨基酸残基。
在一些实施方式中，所述ApoE多肽与一个来自SEQ ID NO：12所示氨基酸序列的片段具有至少约70％同一性，并保留至少约50％该片段对γ分泌酶酶切活性的抑制，其中该片段的N端第一个氨基酸残基为SEQ ID NO：12第215-221位中的任意一个位置上的氨基酸残基，并且该片段C端最后一个氨基酸残基为SEQ ID NO：12第274-299位(例如第279-299位)中的任意一个位置上的氨基酸残基。
在一些实施方式中，所述分离的ApoE构建物由所述ApoE多肽(例如分离的ApoE多肽)组成或基本上由所述ApoE多肽组成。因此在一些实施方式中，本申请还提供一种ApoE多肽(例如分离的ApoE多肽)。
在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)由(1)所述来自SEQ ID NO：12所示氨基酸序列的一个片段组成，或基本上由所述(1)来自SEQ ID NO：12所示氨基酸序列的一个片段组成。
在一些实施方式中，所述ApoE构建物不是自然存在的。比如，在一些实施方式中，所述ApoE构建物或所述ApoE多肽不是ApoE2，不是ApoE3，和/或不是ApoE4全长蛋白。
在一些实施方式中，所述ApoE构建物或所述ApoE多肽是全长ApoE2或ApoE3蛋白(例如人全长ApoE2或ApoE3蛋白)，比如在用作神经特性表达的ApoE构建物的时候。
在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段的氨基酸序列起点(或N端第一个氨基酸残基)为ApoE SEQ ID NO：12序列的第215-221位间(例如第215位、第216位、第217位、第218位、第219位、第220位或第221位)任意一个位点的氨基酸残基，例如第215位、第216位、第219位、第220位或第221位上任意一个氨基酸残基。在一些实施方式中，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段的氨基酸序列终点(或C端最后一个氨基酸残基)为ApoE SEQ ID NO：12序列第274至第299位间(例如第274位、第275位、第276位、第277位、第278位、第279位、第280位、第281位、第282位、第283位、第284位、第285位、第286位、第287位、第288位、第289位、第290位、第291位、第292位、第293位、第294位、第295位、第296位、第297位、第298位或第299位)任意一个位点的氨基酸残基，例如第279-299位间(包括本数)的任一位置的氨基酸残基，例如第274位、第279位或第299位的任一氨基酸残基。
在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含(或由其组成，或基本由其组成)如下任一序列：SEQ ID NO：12所示序列的第215-299位、第216-299位、第217-299位、第218-299位、第219-299位、第220-299位、第221-299位、第215-274位、第216-274位、第217-274位、第218-274位、第219-274位、第220-274位、第221-274位、第215-279位、第216-279位、第217-279位、第218-279位、第219-279位、第220-279位、第221-279位、第215-294位、第216-294位、第217-294位、第218-294位、第219-294位、第220-294位、第221-294位、第215-289位、第216-289位、第217-289位、第218-289位、第219-289位、第220-289位、第221-289位、第215-288位、第216-288位、第217-288位、第218-288位、第219-288位、第220-288位、第221-288位、第215-287位、第216-287位、第217-287位、第218-287位、第219-287位、第220-287位、第221-287位、第215-286位、第216-286位、第217-286位、第218-286位、第219-286位、第220-286位、第221-286位、第215-285位、第216-285位、第217-285位、第218-285位、第219-285位、第220-285位、第221-285位、第215-284位、第216-284位、第217-284位、第218-284位、第219-284位、第220-284位、或第221-284位的氨基酸残基。
在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含(或由其组成，或基本由其组成)如下任一序列：(i)SEQ ID NO：12第215-299位氨基酸残基的氨基酸序列；(ii)SEQ ID NO：12第216-299位氨基酸残基的氨基酸序列；(iii)SEQ ID NO：12第217-299位氨基酸残基的氨基酸序列；(iv)SEQ ID NO：12第218-299位氨基酸残基的氨基酸序列；(v)SEQ ID NO：12第219-299位氨基酸残基的氨基酸序列；(vi)SEQ ID NO：12第220-299位氨基酸残基的氨基酸序列；(vii)SEQ ID NO：12第221-299位氨基酸残基的氨基酸序列；(viii)SEQ ID NO：12第216-294位氨基酸残基的氨基酸序列；(ix)SEQ ID NO：12第216-289位氨基酸残基的氨基酸序列；(x)SEQ ID NO：12第219-289位氨基酸残基的氨基酸序列；(xi)SEQ ID NO：12第219-288位氨基酸残基的氨基酸序列；(xii)SEQ ID NO：12第219-287位氨基酸残基的氨基酸序列；(xiii)SEQ ID NO：12第219-286位氨基酸残基的氨基酸序列；(xiv)SEQ ID NO：12第219-285位氨基酸残基的氨基酸序列；(xv)SEQ ID NO：12第219-284位氨基酸残基的氨基酸序列；(xvi)SEQ ID NO：12第219-279位氨基酸残基的氨基酸序列；(xvii)SEQ ID NO：12第219-274位氨基酸残基的氨基酸序列；(xviii)SEQ ID NO：12第220-274位氨基酸残基的氨基酸序列；(xix)SEQ ID NO：12第220-279位氨基酸残基的氨基酸序列；或(xx)SEQ ID NO：12第221-274位氨基酸残基的氨基酸序列。
在一些实施方式中，所述ApoE构建物或ApoE多肽不包含SEQ ID NO：12第211-214位中一个或多个氨基酸序列。在一些实施方式中，所述ApoE构建物或ApoE多肽不包含以下一个或多个(或不包含任何一个)氨基酸片段：SEQ ID NO：12的第212-214位、第213-214位、第211-214位、第211-215位、第211-216位、第211-217位、第211-218位、第211-219位、第211-220位、第211-221位、第211-222位、第212-214位、第212-215位、第212-216位、第212-217位、第212-218位、第212-219位、第212-220位、第212-221位、第212-222位、第213-214位、第213-215位、第213-216位、第213-217位、第213-218位、第213-219位、第213-220位、第213-221位、第213-222位、第214-215位、第214-216位、第214-217位、第214-218位、第214-219位、第214-220位、第214-221位、或第214-222位。
在一些实施方式中，所述ApoE构建物或ApoE多肽包含SEQ ID NO：12第211-214位的氨基酸序列，但是该SEQ ID NO：12第211-214位的氨基酸序列的C端不与所述ApoE多肽的N端直接连接，比如之间至少插入一个非ApoE来源的序列(例如FLAG序列)。在一些实施方式中，所述SEQ ID NO：12第211-214位的氨基酸序列的C端与所述ApoE多肽的N端之间插入SEQ ID NO：77所示的3XFLAG序列。
在一些实施方式中，所述ApoE构建物或所述ApoE多肽可以在所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段的N端和/或C端额外包含一个或多个非ApoE来源的氨基酸残基。在一些实施方式中，所述ApoE构建物或所述ApoE多肽可以在所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段的N端和/或C端额外包含一个或多个氨基酸残基，并且该氨基酸残基在添加到所述(1)片段N端或C端时与SEQ ID NO：12对应位点的氨基酸残基不一样。比如，所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段的N端第一个氨基酸残基为SEQ ID NO：12的第215位氨基酸残基，所述ApoE构建物或所述ApoE多肽可以在SEQ ID NO：12第215位氨基酸残基的N端额外包含一个或多个氨基酸残基，但这一个或多个氨基酸残基与SEQ ID NO：12第214、213、212、211等位点的氨基酸残基不同。
在一些实施方式中，所述ApoE构建物可以在所述ApoE多肽(例如(1)中来自SEQ ID NO：12所示氨基酸序列的片段，或ApoE突变体)的N端和/或C端额外包含一个或多个非ApoE来源的氨基酸残基。在一些实施方式中，所述ApoE构建物可以在所述ApoE多肽的N端和/或C端额外包含一个或多个氨基酸残基，并且该氨基酸残基在添加到所述ApoE多肽的N端或C端时与SEQ ID NO：12对应位点的氨基酸残基不一样。比如，所述ApoE多肽的N端第一个氨基酸残基为SEQ ID NO：12的第215位氨基酸残基，所述 ApoE构建物可以在SEQ ID NO：12第215位氨基酸残基的N端额外包含一个或多个氨基酸残基，但这一个或多个氨基酸残基与SEQ ID NO：12第214、213、212、211等位点的氨基酸残基不同。
在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段至少包括SEQ ID NO：12第221-274位氨基酸序列(SEQ ID NO：78)。例如，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段在SEQ ID NO：78所示氨基酸序列的N端可以额外包含1、2、3、4、5、或6个氨基酸残基，和/或在C端可以额外包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25个氨基酸残基。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段由SEQ ID NO：78所示氨基酸序列组成，或基本由SEQ ID NO：78所示氨基酸序列组成。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段至少包括SEQ ID NO：12第220-279位氨基酸序列(SEQ ID NO：34)。例如，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段在SEQ ID NO：34所示氨基酸序列的N端可以额外包含1、2、3、4、或5个氨基酸残基，和/或在C端可以额外包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个氨基酸残基。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段由SEQ ID NO：34所示氨基酸序列组成，或基本由SEQ ID NO：34所示氨基酸序列组成。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段至少包括SEQ ID NO：12第219-274位氨基酸序列(SEQ ID NO：32)。例如，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段在SEQ ID NO：32所示氨基酸序列的N端可以额外包含1、2、3或4个氨基酸残基，和/或在C端可以额外包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25个氨基酸残基。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段由SEQ ID NO：32所示氨基酸序列组成，或基本由SEQ ID NO：32所示氨基酸序列组成。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段至少包括SEQ ID NO：12第219-279位氨基酸序列(SEQ ID NO：31)。例如，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段在SEQ ID NO：31所示氨基酸序列的N端可以额 外包含1、2、3或4个氨基酸残基，和/或在C端可以额外包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个氨基酸残基。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段由SEQ ID NO：31所示氨基酸序列组成，或基本由SEQ ID NO：31所示氨基酸序列组成。
在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含SEQ ID NOs：13、18-34、78和87(例如SEQ ID NOs：31-34、78和87，或SEQ ID NO：33或78)中任一氨基酸序列。在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段由SEQ ID NOs：13、18-34、78和87(例如SEQ ID NOs：31-34、78和87，或SEQ ID NO：33或78)中任一氨基酸序列组成，或基本由SEQ ID NOs：13、18-34、78和87(例如SEQ ID NOs：31-34、78和87，或SEQ ID NO：33或78)中任一氨基酸序列组成。
本申请所述ApoE构建物和所述ApoE多肽也包括其突变体(在本文中也称“ApoE突变体”)。例如，与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列)相比，该突变体具有一个或多个氨基酸突变(例如添加、缺失或取代)，包括但不限于“抑制酶活保守修饰”。再例如，该突变体与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列)具有至少约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)序列同一性。在一些实施方式中，所述突变体保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列)对γ分泌酶酶切活性的抑制。在一些实施方式中，所述突变体保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)ApoE2(SEQ ID NO：12)或ApoE3(SEQ ID NO：89)或SEQ ID NO：33所示ApoE片段对γ分泌酶酶切活性的抑制。
本文中“抑制酶活保守修饰”指不显著影响(例如降低不多于约50％、40％、30％、20％、10％、5％、1％或更少)或改变该蛋白构建物或多肽抑制γ分泌酶的酶切(例如对APP的γ-酶切)的氨基酸修饰。此类抑制酶活保守修饰包括氨基酸替换、添加和/ 或缺失。可通过本领域已知的标准技术，如定点诱变或PCR介导的诱变来向本发明的蛋白构建物或多肽中引入修饰。在一些实施方案中，与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列)相比，所述ApoE突变体具有1个或多个(如40个以内、30个以内、20个以内、10个以内、8个以内、5个以内、3个以内、2个或1个)氨基酸插入、取代和/或缺失(包括在N端和/或C端进行插入和/或删除)，同时仍保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段抑制γ分泌酶的酶切(例如对APP的γ-酶切)的生物学活性。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)ApoE2(SEQ ID NO：12)或ApoE3(SEQ ID NO：89)或SEQ ID NO：33所示ApoE片段对γ分泌酶酶切活性的抑制。在一些实施方式中，所述ApoE多肽(例如分离的ApoE多肽)与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列，比如SEQ ID NO：33)相比，具有更高的(例如至少约1.2倍、1.5倍、2倍、5倍、10倍或更多)抑制γ分泌酶的酶切(例如对APP的γ-酶切)的生物学活性。
在一些实施方式中，所述ApoE多肽包含实施例1中任何ApoE多肽或ApoE构建物中的一个或多个氨基酸位点的突变(例如添加、缺失或取代)。本发明提供实施例1中和实施例2中任何ApoE多肽或ApoE构建物(例如分离的)。
在一些实施方式中，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变(例如添加、缺失或取代)：R226、D227、R228、L229、D230、Q235、E238、V239、R240、K242、E244、E245、Q246、A247、Q248、Q249、I250、R251、L252、Q253、A254、E255、Q275、V280、V287、T289、S290、A291、P293、V294、P295、S296、D297、N298、H299、R251+T289、R251+T289+A291、S290+P293+V294+P295+S296+D297+N298+H299、R226+D227+R228+L229+D230、E238+V239+R240+K242、或E244+E245+Q246+A247+Q248+Q249+I250+R251+L252+Q253+A254。在一些实施方式中，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变：Q235、E255、Q275、V280、V287、P295、R251+T289、S290+P293+V294+P295+S296+D297+N298+H299、R226+D227+R228+L229+D230、 E238+V239+R240+K242、E244+E245+Q246+A247+Q248+Q249+I250+R251+L252+Q253+A254、或R251+T289+A291。在一些实施方式中，所述突变包括在所述一个或多个位点取代为A、S或T，例如取代为A。在一些实施方式中，所述突变包括删除所述一个或多个位点的氨基酸残基。
在一些实施方式中，所述抑制酶活保守修饰是保守取代。保守取代指氨基酸残基被具有相似侧链的氨基酸残基替换的替换。已经在本领域中定义了具有相似侧链的氨基酸残基家族。这些家族包括带有以下侧链的氨基酸：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)、不带电的极性侧链(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-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
在一些实施方式中，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变：R226A、D227A、R228A、L229A、D230A、Q235A、E238A、V239A、R240A、K242A、E244del、E245del、Q246del、A247del、Q248del、Q249del、I250del、R251S、L252del、Q253del、A254del、E255A、Q275A、V280A、V287A、T289A、S290A、A291T、P293A、V294A、P295A、S296A、D297A、N298A、H299A、R251A+T289A、R251S+T289A+A291T、S290A+P293A+V294A+P295A+S296A+D297A+N298A+H299A、R226A+D227A+R228A+L229A+D230A、E238A+V239A+R240A+K242A、或E244del+E245del+Q246del+A247del+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del。在一些实施方式中，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变：Q235A、E255A、Q275A、V280A、V287A、P295A、R251A+T289A、S290A+P293A+V294A+P295A+S296A+D297A+N298A+H299A、R226A+D227A+R228A+L229A+D230A、E238A+V239A+R240A+K242A、E244del+E245del+Q246del+A247del+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del、或R251S+T289A+A291T。
在一些实施方案中，所述ApoE多肽或所述ApoE突变体与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序 列，比如SEQ ID NO：33)具有至少约70％的序列同一性，例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高(但不是100％)的同一性。在一些实施方式中，所述ApoE突变体与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列，比如SEQ ID NO：33)相比同样能抑制γ分泌酶的酶切，或保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对γ分泌酶酶切活性的抑制。在一些实施方式中，所述ApoE突变体与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段同样来源于人。在一些实施方式中，所述ApoE多肽包含或源自非人源序列。可采用本领域周知的方法测定突变体与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段之间的序列同一性，例如，可采用BLASTP测定。
在一些实施方式中，所述ApoE多肽包含或源自非人源序列，并且其氨基酸序列与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列，比如SEQ ID NO：33)具有至少约70％序列同一性，并保留至少约50％所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对γ分泌酶酶切活性的抑制。在一些实施方式中，所述ApoE多肽包含或源自以下任一物种的ApoE序列：哺乳动物、鸟类、蛙类、鱼类、果蝇、线虫等。在一些实施方式中，哺乳动物包括但不限于灵长类(例如人类，或非人类灵长类例如猴子)，啮齿类(例如小鼠、大鼠、仓鼠、沙鼠、松鼠、豚鼠和兔子)，家畜(例如猪、牛、羊、马、驴)，宠物(例如猫、狗、兔和仓鼠)等。在一些实施方式中，所述ApoE多肽包含非天然存在的修饰，例如在猴ApoE序列上进一步包括氨基酸修饰。
在一些实施方式中，所述ApoE多肽或所述ApoE突变体含有非人灵长类序列，例如与所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列，比如SEQ ID NO：33)高度同源和保守的序列。在一些实施方式中，所述ApoE多肽包含SEQ ID NO：62所示猴源ApoE C端氨基酸序列。在一些实施方式中，所述ApoE多肽由SEQ ID NO：62所示氨基酸序列组成，或基本由SEQ ID NO：62所示氨基酸序列组成。
在一些实施方式中，所述ApoE多肽包含基于SEQ ID NO：33所示氨基酸序列的一个或多个突变(例如添加、缺失或取代)，例如在上述任何一个或多个位点的突变，或与SEQ ID NO：33所示氨基酸序列具有至少约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)的序列同一性。在一些实施方式中，所 述ApoE多肽包含基于SEQ ID NO：33所示氨基酸序列的一个或多个突变(例如添加、缺失或取代)，例如在上述任何一个或多个位点的突变，或与SEQ ID NO：33所示氨基酸序列具有至少约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)的序列同一性，且保留至少约50％(例如至少约60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)SEQ ID NO：33所示氨基酸序列对γ分泌酶酶切活性的抑制。
在一些实施方式中，所述ApoE构建物或所述ApoE多肽包含SEQ ID NOs：46-52、57-59、62和76中任一氨基酸序列。在一些实施方式中，所述ApoE构建物或所述ApoE多肽由SEQ ID NOs：46-52、57-59、62和76中任一氨基酸序列组成，或基本由SEQ ID NOs：46-52、57-59、62和76中任一氨基酸序列组成。
在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列)对于γ分泌酶介导的酶切(例如对APP的γ-酶切)的抑制作用包括下面一个或多个特性：(i)抑制(例如抑制至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)γ-分泌酶介导的APPγ-剪切；(ii)降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)APP的淀粉样代谢；(iii)减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)Aβ(例如Aβ40和/或Aβ42)的生成；(iv)降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)对β-CTF的γ-剪切；(v)提高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)APP的非淀粉样代谢；(vi)增加(例如增加至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)α-CTF的生成；(vii)不抑制(或抑制不超过约30％、20％、10％、5％或更少)对非APP蛋白(例如APLP1或Notch)或其片段的γ-剪切；(viii)不增加(或增加不超过约30％、20％、10％、5％或更少)非APP蛋白(例如APLP 1、Notch、ErbB4、E-cadherin、N-cadherin、ephrin-B2或CD44)γ-剪切产生的α-CTF；(ix)减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)患AD个体脑中的淀粉样斑块，例如减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)淀粉样斑块的平均斑块大小、总面积、和/或密度；(x)减少(例如减少至少约10％、20％、 30％、40％、50％、60％、70％、80％、90％、95％、或100％)患AD个体神经元内Aβ(例如Aβ40和/或Aβ42)生成；(xi)降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)患AD个体病理进程；(xii)提高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)患AD个体存活率和/或寿命；和/或(xiii)不影响(或影响不超过约30％、20％、10％、5％或更少)α-分泌酶、β-分泌酶、和/或γ-分泌酶(或其亚基)(例如ADAM10、BACE1、PS1和/或PS2)的mRNA和/或蛋白质表达。
在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对γ-分泌酶酶切(例如对APP的γ-酶切)的抑制能力高于(例如高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更多)SEQ ID NO：12或33或89所示蛋白对γ-分泌酶酶切的抑制能力。在一些实施方式中，所述ApoE构建物、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对γ-分泌酶酶切(例如对APP的γ-酶切)的抑制能力与SEQ ID NO：12或33或89所示蛋白对γ-分泌酶酶切的抑制能力相同或相似(例如差别在约10％以内)。在一些实施方式中，所述ApoE构建物、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对γ-分泌酶酶切(例如对APP的γ-酶切)的抑制能力比SEQ ID NO：12或33或89所示蛋白对γ-分泌酶酶切的抑制能力低最多约50％(例如低40％、30％、20％、10％、5％、1％或更少)。
在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列)对于γ分泌酶介导的酶切(例如对APP的γ-酶切)的抑制活性与γ-分泌酶抑制剂(例如PF03084014)相同或相似(例如差别在约10％、5％或1％之内)。在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对于γ分泌酶介导的酶切(例如对APP的γ-酶切)的抑制活性高于(例如高至少约10％、20％、50％、100％、1.5倍、2倍、5倍或更高)7-分泌酶抑制剂(例如PF03084014)。 在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对于γ分泌酶介导的酶切(例如对APP的γ-酶切)的抑制活性低于(例如低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％或更多)γ-分泌酶抑制剂(例如PF03084014)。
在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段(例如SEQ ID NOs：13、18-34、78和87中任一序列)对APP的γ-酶切的抑制高于(例如高至少约30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更多)对非APP蛋白(例如APLP1、Notch)的γ-酶切的抑制。在一些实施方式中，所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段只对APP的γ-酶切抑制，而对非APP蛋白(例如APLP1、Notch)的γ-酶切没有或近乎没有抑制。
在一些实施方式中，α-淀粉酶抑制剂(例如GI254023X)和/或β-淀粉酶抑制剂(例如AZD3839)不会影响，或影响不超过约50％(例如不超过约40％、30％、20％、10％、5％、1％或更少)所述ApoE构建物(例如分离的ApoE构建物)、所述ApoE多肽(例如分离的ApoE多肽，或ApoE突变体)、或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段对γ分泌酶酶切(例如对APP的γ-酶切)活性的抑制。比如，在α-淀粉酶抑制剂或β-淀粉酶抑制剂存在下，所述ApoE构建物或ApoE多肽依然可以提高由APP产生的o-CTF水平并减少Aβ的产生。
对γ分泌酶酶切(例如对APP的γ-酶切)活性的影响可以用任何本领域内适合的方式进行研究，比如研究γ分泌酶的酶活本身，或研究γ分泌酶酶切产物(例如Aβ，如Aβ40或Aβ42)特性或数量，或研究γ分泌酶底物(例如APP、o-CTF、β-CTF、APLP1、Notch、ErbB4、E-cadherin、N-cadherin、ephrin-B2或者CD44)特性或数量(例如是否因被γ酶切而减少)。这些研究方法包括但不限于qRT-PCR、BCA、流式细胞分选、蛋白质组学、代谢组学、ELISA、Western blot、免疫共沉淀、蛋白荧光标记、免疫染色等，可以是体内方法，也可以是体外方法(细胞或非细胞实验)。任何γ分泌酶活性检测试剂盒也可在此使用，例如MyBioSource的#MBS704513试剂盒、或R&D Systems的#FP003试剂盒。另参见实施例1和2中所述研究方式。
在一些实施方式中，所述ApoE构建物在任一所述ApoE多肽(例如SEQ ID  NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列)的N端还进一步包含ApoE2蛋白的N端结构域序列或部分序列(例如SEQ ID NO：12第1-167位氨基酸序列或部分序列；SEQ ID NO：79)，和/或铰链区序列或部分序列(例如SEQ ID NO：12第168-205位氨基酸序列或部分序列；SEQ ID NO：80)，或与ApoE2蛋白的N端结构域序列(或部分序列)和/或铰链区序列(或部分序列)至少有约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)同一性的序列(例如来自非人灵长类(比如猴)的ApoE同源序列)。在一些实施方式中，所述ApoE构建物在任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列)的N端还进一步包含ApoE3蛋白的N端结构域序列或部分序列(例如第1-167位氨基酸序列或部分序列；SEQ ID NO：90)，和/或铰链区序列或部分序列(例如第168-205位氨基酸序列或部分序列；SEQ ID NO：80)，或与ApoE3蛋白的N端结构域序列(或部分序列)和/或铰链区序列(或部分序列)至少有约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)同一性的序列(例如来自非人灵长类(比如猴)的ApoE同源序列)。在一些实施方式中，所述ApoE构建物在所述ApoE多肽的N端还进一步包含来自ApoE C端结构域(SEQ ID NO：12第206-299位)的一部分序列(例如其N端部分序列)，例如SEQ ID NO：12第206-214位氨基酸的序列或部分序列，或与ApoE C端结构域的该一部分序列至少有约70％(例如至少约75％、80％、85％、90％、95％、96％、97％、98％、99％或更高)同一性的序列。在一些实施方式中，所述ApoE2或ApoE3蛋白(或非人同源蛋白)N端结构域序列或部分序列的C端，或(N端结构域+铰链区)序列或部分序列的C端，或(N端结构域+铰链区+C端结构域的部分N端序列)序列的C端，与所述ApoE多肽的N端通过肽键或接头序列(例如蛋白接头序列)连接。在一些实施方式中，与人ApoE2或ApoE3蛋白的N端结构域(或部分序列)和/或铰链区(或部分序列)和/或C端结构域(或其N端部分序列)同源的ApoE蛋白序列源自以下任一物种的序列：哺乳动物(例如非人类灵长类比如猴子、啮齿类、家畜、宠物)、鸟类、蛙类、鱼类、果蝇、线虫等。
在一些实施方式中，所述ApoE构建物在任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列)的N端还进一步包含SEQ ID NO：12第1-167位(SEQ ID NO：79)或第1-205位(SEQ ID NO：81)或第1-214位氨基酸残基的氨基酸序列，或与SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸至少有约70％同一性的氨基酸序列。在一些实施方式中，所述ApoE 构建物包含SEQ ID NO：60或61所示的氨基酸序列。在一些实施方式中，所述ApoE构建物由SEQ ID NO：60或61所示的氨基酸序列组成，或基本由SEQ ID NO：60或61所示的氨基酸序列组成。
在一些实施方式中，所述ApoE构建物在任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列)的N端还进一步包含ApoE3(SEQ ID NO：89)第1-167位(SEQ ID NO：90)或第1-205位或第1-214位氨基酸残基的氨基酸序列，或与ApoE3第1-167位或第1-205位或第1-214位氨基酸至少有约70％同一性的氨基酸序列。
在一些实施方式中，所述ApoE构建物包含(或由其组成、或基本由其组成)ApoE2(例如人ApoE2，SEQ ID NO：12)的全长序列。在一些实施方式中，所述ApoE构建物包含(或由其组成、或基本由其组成)ApoE3(例如人ApoE3，SEQ ID NO：89)的全长序列。
在一些实施方式中，所述ApoE构建物在任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，或ApoE2或ApoE3全长序列)的N端或C端还进一步包含一个穿膜肽。在一些实施方式中，所述穿膜肽包含(或由其组成、或基本由其组成)SEQ ID NOs：35-45、63-65和84-86中任一氨基酸序列，例如SEQ ID NO：63或65所示的氨基酸序列。在一些实施方式中，所述穿膜肽(例如C端)与所述ApoE多肽(例如N端)、或所述包含SEQ ID NO：12或89第1-167位(或其片段)或第1-205位(或其片段)或第1-214位(或其片段)氨基酸残基的氨基酸序列(例如N端)、或所述与SEQ ID NO：12或89第1-167位(或其片段)或第1-205位(或其片段)或第1-214位(或其片段)氨基酸至少有约70％同一性的序列(例如N端)通过肽键或接头序列(例如蛋白接头序列)连接。在一些实施方式中，所述穿膜肽的N端进一步和一个信号肽的C端通过肽键或接头序列连接。在一些实施方式中，所述穿膜肽位于所述ApoE构建物的最N端。在一些实施方式中，所述穿膜肽位于所述ApoE构建物的最C端。
在一些实施方案中，本发明提供一种ApoE构建物(或融合蛋白)，该融合蛋白包括以下元件(1)和元件(2)：(1)穿膜肽，和(2)ApoE2的全长序列(例如SEQ ID NO：12)或ApoE3的全长序列(例如SEQ ID NO：89)或ApoE2的至少包括SEQ ID NO：12第221-274位氨基酸残基的片段。所述元件(1)和元件(2)之间可通过肽键直接连接，也可通过接头序列连接。
穿膜肽或细胞穿膜肽指一类能携带大分子物质进入细胞的短肽。可采用本领域周知的穿膜肽来构建本发明的融合蛋白。示例性的穿膜肽可参见Siegmund Reissmann，Cell penetration：scope and limitations by the application of cell-penetrating peptides，J.Pept.Sci.2014；20：760-784。本文将其全部内容以引用的方式纳入本文。适用于本发明的穿膜肽包括但不限于RQIKIWFQNRRMKWKK(SEQ ID NO：35)；YGRKKRRQRRR(SEQ ID NO：36)；KQAIPVAK(SEQ ID NO：37)；RRRRNRTRRNRRRVR(SEQ ID NO：38)；寡精氨酸(即长9-12个精氨酸残基的片段；R9(SEQ ID NO：63)，R10(SEQ ID NO：84)，R11(SEQ ID NO：85)，R12(SEQ ID NO：86))；KLTRAQRRAAARKNKRNTRGC(SEQ ID NO：39)；ALWKTLLKKVLKAPKKKRKVC(SEQ ID NO：40)；RKKRRQRRR(SEQ ID NO：41)；DAATATRGRSAASRPTERPRAPARSASRPRRPVE(SEQ ID NO：42)；GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO：43)；AGYLLGKINLKALAALAKKIL(SEQ ID NO：44)；YTAIAWVKAFIRKLRK(SEQ ID NO：45)；和KSVRTWNEIIPSKGCLRVGGRCHPHVNGGGRRRRRRRRR(SEQ ID NO：65)。
在一些实施方案中，融合蛋白中元件(2)所述的ApoE2的至少包括SEQ ID NO：12第221-274位氨基酸残基的片段，可以是本文任一实施方案所述的分离的ApoE多肽，以及含有ApoE2或ApoE3的N端和铰链区(或其片段)以及C端至少第221-274位氨基酸残基的片段。在一些实施方式中，所述的ApoE2多肽可以包含如下任一序列：SEQ ID NO：12第1-274位，第1-279位，以及第1-280位、第1-281位、第1-282位、第1-283位、第1-284位、第1-285位、第1-286位、第1-287位、第1-288位、第1-289位、第1-290位、第1-291位、第1-292位、第1-293位、第1-294位、第1-295位、第1-296位、第1-297位、第1-298位或第1-299位氨基酸残基。
在一些实施方案中，所述融合蛋白中的元件(2)选自：SEQ ID NOs：12、13、18-34、60、78、87和89中任一氨基酸序列。
在一些实施方式中，所述接头是非蛋白接头。在一些实施方式中，所述接头是蛋白接头。在一些实施方式中，所述接头是包含3～25个残基的肽，例如包含3～15、5～15、10～20、20-25、或3-10个残基的肽。肽接头的适合的实例是本领域中公知的。通常，接头含有一个或多个前后重复的基序，该基序通常含有Gly和/或Ser。例如，该基序可以是SGGS(SEQ ID NO：69)、GSSGS(SEQ ID NO：70)、GGGS(SEQ ID NO：71)、GGGGS(SEQ ID NO：72)、SSSSG(SEQ ID NO：73)、GSGSA(SEQ  ID NO：74)GGSGG(SEQ ID NO：75)或GGGSGGGS(SEQ ID NO：88)。在一些实施方式中，以上序在接头序列中是相邻的，在重复之间没有插入氨基酸残基。接头序列可以包含1、2、3、4或5个重复基序组成。在某些实施方案中，接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制，通常为2～20个，例如2～15、2～10、2～8个。除甘氨酸和丝氨酸外，接头中还可含有其它已知的氨基酸残基，例如丙氨酸、亮氨酸、苏氨酸、谷氨酸、苯丙氨酸、精氨酸、谷氨酰胺等。在一些实施方式中，所述接头序列如SEQ ID NO：88所示。
本发明的ApoE构建物或融合蛋白中还可能包括因基因克隆操作、和/或为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的检测和/或纯化等而在融合蛋白中引入的氨基酸残基或序列。在一些实施方式中，本发明的ApoE构建物或融合蛋白中还包括信号肽、前导肽、末端延伸、标签蛋白等。
在一些实施方式中，所述ApoE构建物在所述ApoE多肽的N端还进一步包含一个信号肽，特别是需要在细胞中表达时。在一些实施方式中，所述信号肽的C端与所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，或ApoE2或ApoE3全长序列)的N端、或所述包含SEQ ID NO：12或89第1-167位(或其片段)或第1-205位(或其片段)或第1-214位(或其片段)氨基酸残基的氨基酸序列的N端、或所述与SEQ ID NO：12或89第1-167位(或其片段)或第1-205位(或其片段)或第1-214位(或其片段)氨基酸至少有约70％同一性的序列的N端、或穿膜肽的N端通过肽键或接头序列(例如蛋白接头序列)连接。在一些实施方式中，所述信号肽位于所述ApoE构建物的最N端。在一些实施方式中，所述信号肽进一步添加于SEQ ID NO：60或61所示氨基酸序列的N端。在一些实施方式中，所述信号肽进一步添加于ApoE2(例如SEQ ID NO：12)或ApoE3(例如SEQ ID NO：89)全长序列的N端。在一些实施方式中，所述信号肽的C端进一步与一个穿膜肽的N端通过肽键或接头序列(例如蛋白接头序列)连接。任何周知的信号肽均可在此使用。在一些实施方式中，所述信号肽与所述ApoE构建物或所述ApoE多肽同源，例如是来自ApoE2(SEQ ID NO：12)或ApoE3(SEQ ID NO：89)蛋白本身的信号肽。在一些实施方式中，所述信号肽与所述ApoE构建物或所述ApoE多肽异源，例如是来自ApoA1或ApoJ的信号肽。在一些实施方式中，所述信号肽包含(或由其组成，或基本由其组成)选自SEQ ID NOs：66-68中任一氨基酸序列。
在一些实施方式中，所述ApoE构建物包含(或由其组成、或基本由其组成)如 下从N端到C端的序列：
信号肽-可选的接头1-穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头2-ApoE2蛋白(例如SEQ ID NO：12)或ApoE3蛋白(例如SEQ ID NO：89)；
信号肽-可选的接头-ApoE2蛋白(例如SEQ ID NO：12)或ApoE3蛋白(例如SEQ ID NO：89)；
ApoE2蛋白(例如SEQ ID NO：12)或ApoE3蛋白(例如SEQ ID NO：89)；
穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头-ApoE2蛋白(例如SEQ ID NO：12)或ApoE3蛋白(例如SEQ ID NO：89)；
信号肽-可选的接头1-穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头2-任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头-任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
信号肽-可选的接头-任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
信号肽-可选的接头1-穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头2-ApoE2或ApoE3 N端结构域和/或铰链区和/或(C端结构域的N端)序列或片段(例如SEQ ID NOs：79-81和90中任一所示氨基酸序列或其片段)-可选的接头3-任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头1-ApoE2或ApoE3 N端结构域和/或铰链区和/或(C端结构域的N端)序列或片段(例如SEQ ID NOs：79-81和90中任一所示氨基酸序列或其片段)-可选的接头2-任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
信号肽-可选的接头1-ApoE2或ApoE3 N端结构域和/或铰链区和/或(C端结构域的N端)序列或片段(例如SEQ ID NOs：79-81和90中任一所示氨基酸序列或其片 段)-可选的接头2-任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
ApoE2或ApoE3 N端结构域和/或铰链区和/或(C端结构域的N端)序列或片段(例如SEQ ID NOs：79-81和90中任一所示氨基酸序列或其片段)-可选的接头-任一所述ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一序列，比如SEQ ID NO：33)；
信号肽-可选的接头1-穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头2-(SEQ ID NO：60或61所示的氨基酸序列)；
穿膜肽(例如SEQ ID NO：63或65所示的氨基酸序列)-可选的接头-(SEQ ID NO：60或61所示的氨基酸序列)；
SEQ ID NO：60或61所示的氨基酸序列；或
信号肽-可选的接头-(SEQ ID NO：60或61所示的氨基酸序列)。
在一些实施方式中，所述ApoE构建物在所述ApoE多肽的N端和/或C端还进一步包含一个标签蛋白。在一些实施方式中，所述标签蛋白位于所述ApoE构建物的最N端。在一些实施方式中，所述标签蛋白位于所述ApoE构建物的最C端。在一些实施方式中，所述标签蛋白(例如SEQ ID NO：77所示的3XFLAG序列)位于所述ApoE构建物的内部但是位于所述ApoE多肽的N端，例如与所述ApoE多肽的N端直接相连。所述标签蛋白可以是表达纯化标签(例如His-tag，FLAG-tag、GST-tag、c-Myc-tag、V5-tag、HA-tag)，或检测标签(例如GFP、RFP、V5、HA)。在一些实施方式中，纯化标签也可用于检测，例如免疫印迹或抗体检测。在一些实施方式中，所述标签蛋白位于所述ApoE构建物(例如ApoE2或ApoE3，或包含SEQ ID NO：33序列的ApoE多肽)的最N端。在一些实施方式中，所述标签蛋白位于所述ApoE构建物的最C端。在一些实施方式中，所述标签蛋白是V5标签GKPIPNPLLGLDST(SEQ ID NO：82)或IPNPLLGLD(SEQ ID NO：83)。在一些实施方式中，所述标签蛋白是FLAG标签(例如SEQ ID NO：77)。
淀粉样蛋白前体蛋白(Amyloid-beta precursor protein，APP)和水解
APP是细胞膜内嵌蛋白，主要集中于神经元突触。其含有α、β、γ分泌酶的酶切位点。APP表达于很多不同物种且高度同源。本文所述APP可以来自任何物种，例如哺乳动物、鸟类、蛙类、鱼类、果蝇、线虫等。哺乳动物包括但不限于灵长类(例如人类，或非人类灵长类例如猴子)，啮齿类(例如小鼠、大鼠、仓鼠、沙鼠、松鼠、 豚鼠和兔子)，家畜(例如猪、牛、羊、马、驴)，宠物(例如猫、狗、兔和仓鼠)等。在一些实施方式中，所述APP是人APP。
APP的淀粉样代谢可以产生短的β淀粉样多肽(Aβ)。Aβ多肽由在接近Aβ的N-末端位置处通过β-分泌酶活性和在接近Aβ的C-末端位置处通过γ分泌酶活性的APP切割制备。APP也由α-分泌酶活性切割，产生可溶性APPα的分泌的、非淀粉样片段。β位点APP切割酶(″BACE-1″)被认为是负责通过β-分泌酶活性产生Aβ的主要的天冬氨酰蛋白酶。BACE-1抑制可以抑制Aβ的产生。BACE1为具有切割APP以将胞外域(sAPPβ)释放至细胞外间隙的腔活性位点的I型跨膜蛋白。剩余的C-末端片段(CTF)通过γ-分泌酶经历进一步切割，导致Aβ和APP细胞内C-末端结构域(AICD)的释放。早老蛋白为γ-分泌酶的主要酶组分，γ-分泌酶的APP的不精确切割在C-末端处产生长度变化若干氨基酸的连续的Aβ多肽。大部分Aβ通常在氨基酸40(Aβ40)处结束，但是已示出42-氨基酸变体(Aβ42)更易受聚合的影响，并且已假设成核老年斑形成。γ-分泌酶的调节也可导致38-氨基酸变体(Aβ38)的增加。竞争α-分泌酶途径为α-分泌酶和γ-分泌酶相继切割的结果。ADAM-10为用于APP切割的α-分泌酶，该切割释放胞外域(sAPPα)，所述胞外域显示神经保护功能(Lammich等，(1999)Proc.Natl.Acad.Sci.USA，96：3922-3927)。83-氨基酸CTF(C83)的随后切割释放p3(其为非淀粉样的)和AICD(Furukawa等，(1996)J.Neurochem.，67：1882-1896)。
Aβ为β淀粉样纤维和斑块的主要组分，所述β淀粉样纤维和斑块被认为在越来越多的病理学中发挥作用。此类病理学的实例包括但不限于阿尔茨海默氏病、唐氏综合征、帕金森病、记忆丧失(包括与阿尔茨海默氏病和帕金森病相关的记忆丧失)、注意力缺陷症(包括与阿尔茨海默氏病、帕金森病和唐氏综合征相关的注意力缺陷症)、痴呆(包括老年前期痴呆，老年性痴呆，与阿尔茨海默氏病(AD)、帕金森病和唐氏综合征相关的痴呆)、进行性核上麻痹、皮质基底变性、神经变性、嗅觉障碍(包括与阿尔茨海默氏病、帕金森病和唐氏综合征相关的嗅觉障碍)、β-淀粉样脑血管病(包括大脑淀粉样脑血管病)、遗传性脑出血、轻度认知障碍(″MCI″)、青光眼、淀粉样变性、II型糖尿病、血液透析(β2微球蛋白和由其产生的并发症)、神经变性疾病诸如痒病、牛海绵状脑炎、克雅氏病(Creutzfeld Jakobdisease)、外伤性脑损伤等。
在一些实施方式中，所述APP是野生型APP。在一些实施方式中，所述APP是自然存在的APP亚型。在一些实施方式中，所述APP是突变体，包括但不限于任何已知或未知APP突变。已在121个家族中识别出APP突变。最常见的错义突变利用Ile(伦敦突变)、Phe或Gly残基替换Val717残基，利用Gln残基(荷兰突变)或Gly残基 (北极突变)替换Glu693，或利用Asn和Leu残基替换Lys670和Met671(双重突变称为瑞典突变APPSW，在APP770异构体中编号)。在一些实施方式中，所述APP是人APP，其包含下述一个或多个突变：K670N、M671L、瑞典(K670N+M671L)、佛罗里达(I716V)、伦敦(V717I)、APPM596V(不能进行β切割)、γ切割位点附近的fAD突变(例如T714I、V717F、L723P)、β-或α-切割位点附近的fAD突变(例如KM670/671NL、E693K、E693Δ)、或保护性冰岛突变(A673T)。在一些实施方式中，所述APP不包含影响γ-剪切的突变，例如γ位点附件的突变。
在一些实施方式中，所述ApoE构建物或ApoE多肽可以对任一APP的γ-切割进行抑制(例如抑制至少约10％、20％、30％、40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％、99％或100％)。在一些实施方式中，所述ApoE构建物或ApoE多肽对任一所述APP突变体的γ-切割抑制能力为其对野生型APPγ-切割的抑制能力的至少约30％(例如至少约40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)。在一些实施方式中，所述ApoE构建物或ApoE多肽对包含影响γ-剪切的突变的APP(例如T714I、V717F、L723P)的γ-切割抑制能力低于(例如低至少约20％、30％、40％、50％、60％、70％、80％、85％、90％、95％、99％或100％)其对野生型APPγ-切割的抑制能力。
γ分泌酶
γ分泌酶复合物由四种单独的蛋白质组成：PSEN1(早老素1，presenilin-1，PS1)、nicastrin、APH-1(前咽缺陷1，anterior pharynx-defective 1)和PEN-2(早老素增强子2，presenilin enhancer 2)。最近的证据表明，CD147是复合物的第五种蛋白，是非必需调节剂，其缺失会增加γ分泌酶活性。
本文所述γ分泌酶可以来自任何物种，例如哺乳动物、鸟类、蛙类、鱼类、果蝇、线虫等。在一些实施方式中，所述γ分泌酶来自人类，或非人类灵长类例如猴子。
在一些实施方式中，本文所述γ分泌酶是野生型γ分泌酶，或自然存在的亚型。在一些实施方式中，所述γ分泌酶包含一个或多个突变，例如增强或降低其酶切活性的突变。在一些实施方式中，所述γ分泌酶在PS1上有一个或两个突变：M146L，L286V。在一些实施方式中，所述γ分泌酶的PS1和/或PS2活性降低或缺失。
在一些实施方式中，所述ApoE构建物或ApoE多肽可以对任一γ分泌酶的酶活进行抑制(例如抑制至少约10％、20％、30％、40％、50％、60％、70％、80％、85％、 90％、95％、96％、97％、98％、99％或100％)。在一些实施方式中，所述ApoE构建物或ApoE多肽对任一所述γ分泌酶突变体酶活的抑制能力为其对野生型γ分泌酶酶活的抑制能力的至少约30％(例如至少约40％、50％、60％、70％、80％、85％、90％、95％、96％、97％、98％、99％、100％、1.5倍、2倍、5倍、10倍或更多)。
3.ApoE构建物的制造方法
本发明还提供了编码以上任一所述分离的ApoE构建物(例如SEQ ID NOs：12、60、61和89任一氨基酸序列)、融合蛋白、或ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33)的蛋白部分的分离的核酸和载体(例如克隆载体或表达载体)，以及表达以上任一所述分离的ApoE构建物的宿主细胞，或包含该核酸或载体的宿主细胞。在一些实施方式中，所述载体为是重组AAV或lentivirus表达载体。在一些实施方式中，所述载体在所述分离的核酸的5’端含有神经元特异性启动子。在一些实施方式中，所述神经元特异性启动子选自突触蛋白启动子(例如hSYN)、thy-1启动子和钙调素依赖型蛋白激酶II-α启动子。在一些实施方式中，所述宿主细胞为神经细胞，例如神经元(比如人神经元、非人灵长类神经元、或啮齿类神经元)。可参见实施例1和2的载体、细胞和制造方法。
在一些实施方式中，本发明提供一种可以介导神经细胞特异性表达任一所述ApoE构建物、融合蛋白、或ApoE多肽的核酸或载体。在一些实施方式中，本发明提供一种载体或分离的核酸，其包含如下5’至3’序列：神经元特异性启动子(例如hSYN)-编码信号肽的核苷酸序列-编码穿膜肽(例如SEQ ID NO：63或65)的核苷酸序列-编码任一所述ApoE构建物(例如SEQ ID NO：12或89)/融合蛋白/ApoE多肽(例如SEQ ID NO：33)的核苷酸序列。在一些实施方式中，本发明提供一种载体或分离的核酸，其包含如下5’至3’序列：神经元特异性启动子(例如hSYN)-编码穿膜肽(例如SEQ ID NO：63或65)的核苷酸序列-编码任一所述ApoE构建物(例如SEQ ID NO：12或89)/融合蛋白/ApoE多肽(例如SEQ ID NO：33)的核苷酸序列。在一些实施方式中，本发明提供一种载体或分离的核酸，其包含如下5’至3’序列：神经元特异性启动子(例如hSYN)-编码信号肽的核苷酸序列-编码任一所述ApoE构建物(例如SEQ ID NO：12或89)/融合蛋白/ApoE多肽(例如SEQ ID NO：33)的核苷酸序列。在一些实施方式中，所述ApoE构建物包含(或由其组成，或基本由其组成)ApoE2或ApoE3全长序列。在一些实施方式中，所述ApoE构建物包含(或 由其组成，或基本由其组成)SEQ ID NOs：60或61的氨基酸序列。在一些实施方式中，所述ApoE多肽包含(或由其组成，或基本由其组成)SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33。
本申请也包括编码以上任一所述分离的ApoE构建物、融合蛋白、或ApoE多肽的核酸分子及其互补序列。本发明包括本发明核酸分子的所有形式，包括RNA和DNA。应理解，本文所述的互补序列指长度与该核酸分子基本一致(例如长度至少为约85％、90％、95％、96％、97％、98％。99％或100％相同)的互补序列，例如至少约70％、75％、80％、85％、90％、95％、96％、97％、98％。99％或100％互补。
含有所述核酸分子或其互补序列的核酸构建物也包括在本发明的范围内。核酸构建物可以是一表达盒，其含有启动子、所述核酸分子以及转录终止序列。根据表达所需，表达盒中还可任选地含有增强子。启动子、增强子、转录终止序列、可选择标记和信号序列为本领域所周知，并可由本领域技术人员根据所选择的用于表达所述多肽的宿主细胞而做出适当的选择。表达盒中的这些元件相互之间可操作连接，以使它们发挥它们预期的功能。例如，可操作连接启动子序列至感兴趣的核苷酸序列是指连接启动子序列和感兴趣的核苷酸序列，以使启动子序列能够指导感兴趣的核苷酸序列在目标细胞中进行转录和/或感兴趣的核苷酸序列编码的多肽的合成。可选择标记可以帮助从未插入载体的那些宿主细胞中选择出插入有载体的宿主细胞，举例来说，可选择标记可为赋予抗生素抗性的基因。可选择信号序列以允许所表达多肽运输至宿主细胞的外部。
遗传控制元件可用于调控多肽或蛋白质的基因表达。选择此类遗传控制元件使其在相关宿主细胞中具有活性。控制元件可具有组成型活性或为指定条件下可诱导。可诱导型控制元件在表达蛋白质具毒性或对细胞生长和/或存活力具其他不良影响时特别有用。在这种情况下，通过可诱导型控制元件调控多肽或蛋白质的表达，可以改善细胞存活力、细胞密度和/或表达多肽或蛋白质的总产率。可用于实施本发明的大量控制元件为本领域公知且可获取。
可用于本发明的代表性组成型哺乳动物启动子包括但不仅限于，次黄嘌呤磷酸核糖转移酶(HPTR)启动子、腺苷脱氨酶启动子、丙酮酸激酶启动子、β肌动蛋白启动子以及其他本领域普通技术人员已知的组成型启动子。此外，能驱动真核细胞中编码序列的组成型表达的病毒启动子包括，如猿猴病毒启动子、单纯疱疫病毒启动子、乳头瘤病毒启动子、腺病毒启动子、人免疫缺陷病毒(HIV)启动子、劳斯肉瘤病毒启动子、巨细胞病毒(CMV)启动子、莫洛尼鼠白血病病毒及其他逆转录病毒的长末端 重复序列(LTR)、单纯疱疹病毒胸苷激酶启动子，以及其他本领域普通技术人员已知的病毒启动子。
诱导型启动子可在存在诱导剂时驱动有效连接的编码序列的表达，也可根据本发明使用。例如在哺乳动物细胞内，金属硫蛋白启动子在某些金属离子存在时能诱导下游编码序列的转录。其他诱导型启动子为本领域普通技术人员所识别和/或公知。
在一些实施方式中，，适用于本发明的基因表达控制元件为神经元特异性控制元件，包括启动子和增强子。本文中，术语“神经元特异性”的控制元件指用于在神经元中特异性表达感兴趣的核酸分子的控制元件，如启动子和增强子。在一些实施方案中，神经元特异性启动子是用于在某个亚型的神经元(例如多巴胺能神经元、兴奋性神经元、侧前额叶皮质神经元等)中表达转基因的启动子。
本领域已知神经元特异性启动子和其它控制元件(如增强子)。适合的神经元特异性启动子包括但不限于神经元特异性烯醇酶(NSE)启动子、芳香族氨基酸脱羧酶启动子、神经丝启动子、突触蛋白启动子、thy-1启动子、血清素受体启动子、酪氨酸羟化酶启动子、GnRH启动子、L7启动子、DNMT启动子、脑啡肽启动子、髓鞘碱性蛋白启动子、钙调素依赖型蛋白激酶II-α启动子、和CMV增强子/血小板源生长因子-β启动子。在一些实施方式中，所述神经元特异性启动子选自突触蛋白启动子(例如hSYN)、thy-1启动子和钙调素依赖型蛋白激酶II-α启动子。
一般而言，基因表达序列也包括分别涉及转录和翻译起始的5’非转录和5’非翻译序列，如TATA盒、帽子序列、CAAT序列等。增强子元件可任选用于增强欲表达多肽或蛋白质的表达水平。可在哺乳动物细胞中作用的增强子元件的实例包括SV40早期基因增强子(如Dijkema等人，EMB0J.(1985)4：761所述)、以及来自劳斯肉瘤病毒(RSV)(如Gorman等人，Proc.Natl.Acad.Sc1.USA(1982b)79：6777所述)和人巨细胞病毒(如Boshart等人，Cell(1985)41：521所述)长末端重复序列的增强子/启动子。
将控制元件连接到编码序列的系统为本领域公知(普通分子生物学和重组DNA技术，如Sambrook，Fritsch和Maniatis，Molecular Cloning：ALaboratory Manual，第二版，Cold Spring Harbor Laboratory Press，ColdSpring Harbor，NY，1989，此处引用作为参考)。适用于插入优选编码序列以在多种生长和诱导条件下的多种哺乳动物细胞中表达的商业载体也为本领域所公知。
在一些实施方案中，核酸构建物为载体，包括表达载体和克隆载体。表达载体是适合在宿主细胞中表达外源基因如编码本发明所述多肽(如ApoE2或ApoE3全长序 列或任一本文所述的ApoE构建物、ApoE多肽或融合蛋白)的核酸分子的载体，包括原核表达载体和真核表达载体。真核表达系统有酵母表达系统，哺乳细胞表达系统和昆虫细胞表达系统等。克隆载体用于在宿主细胞中进行目的基因(如编码本发明所述多肽的核酸分子)的扩增。克隆载体包括质粒载体、噬菌体载体、病毒载体、以及由它们互相组合或者与其他基因组DNA组合而成的载体。分子克隆中最常用的宿主细胞是大肠杆菌。
在一些实施方案中，载体为病毒载体，包括但不限于慢病毒载体、单纯疱疹病毒载体、腺病毒载体和腺相关病毒(AAV)载体。可使用本领域的标准方法制备此类载体。在一些实施方式中，所述载体为lentivirus或lentiviral载体。
在一些实施方案中，所述载体为重组AAV载体。可使用本领域的标准方法制备AAV载体。可使用任何血清型的腺相关病毒载体，包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15和AAV16。
在一些实施方案中，本发明提供一种载体(例如AAV或慢病毒载体)，其含有一表达盒，该表达盒含有与感兴趣的核酸分子可操作性连接的神经元特异性启动子(例如hSyn)，该感兴趣的核酸分子选自：编码本发明任一所述ApoE C端片段的核酸分子、编码ApoE2或ApoE3全长蛋白的核酸分子、编码本发明任一所述ApoE2构建物或融合蛋白的核酸分子、或编码本发明任一所述ApoE2多肽的核酸分子。优选地，该载体是重组AAV载体。优选地，适用于本文的神经元特异性启动子包括突触蛋白启动子、thy-1启动子和钙调素依赖型蛋白激酶II-α启动子。在一些实施方案中，所述感兴趣的核酸分子编码SEQ ID NOs：12、13、18-34、46-52、57-59、60-62、76、78、87和89中任一所示的氨基酸序列。在一些实施方案中，所述感兴趣的核酸分子还编码一个N端的信号肽(例如SEQ ID NOs：66-68中任一氨基酸序列)。在一些实施方案中，所述感兴趣的核酸分子还编码一个N端和/或C端穿膜肽(例如SEQ ID NOs：35-45、63-65和84-86中任一氨基酸序列)。在一些实施方案中，所述感兴趣的核酸分子还编码一个N端和/或C端的标签蛋白(例如V5、EGFP)。
本发明还提供一种表达系统，其包含本文公开的核酸分子、核酸构建物或载体。表达系统可为例如革兰氏阳性或革兰氏阴性原核宿主细胞系统，例如经修饰以表达本发明多肽的大肠杆菌。在一些实施方案中，表达系统是真核宿主细胞系统，例如酵母，例如巴斯德毕赤酵母或酿酒酵母。在一些实施方案中，所述宿主细胞为哺乳动物宿主细胞，例如HEK293T、N2A、CHO细胞等。在一些实施方案中，所述宿主细胞为神 经细胞，包括但不限于胶质细胞和神经元。高等真核细胞，特别是源于多细胞生物体的那些，可用于糖基化多肽的表达。适合高等真核细胞包括但不限于无脊椎动物细胞和昆虫细胞、以及脊椎动物细胞。
适于将表达目的多肽或蛋白质所需的足量核酸引入宿主细胞(例如哺乳动物宿主细胞)内的方法为本领域所公知。见如Gething等人，Nature，293：620-625(1981)；Mantei等人，Nature，281：40-46(1979)；Levinson等人；EP 117，060；以及EP 117，058，均在此处引用作为参考。可使用本领域中已知的任何适合方法将载体引入至宿主细胞中，所述方法包括但不限于DEAE-右旋糖酐介导的递送、磷酸钙沉淀方法、阳离子脂质介导的递送、脂质体介导的转染、电穿孔、微粒轰击、受体介导的基因递送、由聚赖氨酸、组蛋白、壳聚糖和肽介导的递送。对于哺乳动物细胞而言，优选转化方法包括，Graham和van der Erb，Virology，52：456-457(1978)的磷酸钙沉淀法，或Hawley-Nelson，Focusl5：73(1193)的lipofectamineTM.(Gibco BRL)法。哺乳动物细胞宿主系统转化的一般方面已由Axel述于1983年8月16日递交的美国专利号4，399，216。转化哺乳动物细胞的多种技术可见Keown等人，Methods inEnzymology(1989)、Keown等人，Methods in Enzymology，185：527-537(1990)，以及Mansour等人，Nature，336：348-352(1988)。在哺乳动物细胞中表达多肽或蛋白质的合适载体的非限制性代表实例包括P(3)NAl；p(3)，见Okayama等人.(1985)Mol.Cell Biol.5：1136-1142；pMClneoPoly-A，见Thomas等人·(1987)Cell 51：503-512；以及杆状病毒载体如PAC373或pAC610。
在一些实施方式中，本发明所述多肽或蛋白质稳定转染入宿主细胞中。然而，本领域普通技术人员应当认识到，本发明可用于瞬时或稳定转染宿主细胞。
有多种方式可以实现让目标蛋白或多肽进入目标细胞，例如使用脂质体传递编码目标蛋白的DNA(例如质粒)或者RNA，或使用编码目标蛋白的慢病毒或和腺相关病毒侵染细胞。
本发明中可应用任何易于细胞培养及表达多肽的哺乳动物细胞或细胞类型。可用于本发明的哺乳动物细胞的非限制性实例，包括BALB/c小鼠骨髓瘤细胞系(NSO/1，ECACC No：85110503)；人成视网膜细胞(PER.C6(CruCelI，Leiden，The Netherlands))；SV40转化的猴肾CVl系(COS-7，ATCC CRL 1651)；人胎肾细胞系(亚克隆的293或293细胞用于在悬浮培养基中生长，Graham等人，J.Gen Virol.，36：59(1977))；幼仓鼠肾细胞(BHK，ATCC CCL 10)；中国仓鼠卵巢细胞+/-DHFR(CH0，Urlaub和Chasin，Proc.Natl.Acad.Sc1.USA，77：4216(1980))；小鼠支持 细胞(TM4，Mather，Biol.R印rod.，23：243-251(1980))；猴肾细胞(CVIATCC CCL70)；非洲绿猴肾细胞(VER0-76，ATCC CRL-1587)；人宫颈癌细胞(HeLa，ATCC CCL 2)；狗肾细胞(MDCK，ATCC CCL 34)；布法罗大鼠肝细胞(BRL3A，ATCC CRL 1442)；人肺细胞(Wl38，ATCC CCL 75)；人肝细胞(Hep G2，HB 8065)；小鼠乳腺肿瘤(MMT 060562，ATCC CCL51)；TRI细胞(Mather等人，Annals N.Y.Acad.Sc1.，383：44-68(1982))；MRC 5细胞；FS4细胞和人肝癌细胞系(Hep G2)；小鼠神经元细胞N2A。
在允许插入载体中的经分离核酸的表达的条件下培养经分离宿主细胞。适于多核苷酸的表达的条件可包括但不限于适合培养基、宿主细胞在培养基中的适合密度、存在必要营养物、存在补充性因子、适合温度和湿度、以及不存在微生物污染物。本领域普通技术人员可出于表达的目的酌情选择适合条件，适当修饰本发明的步骤和组合物，从而优化细胞生长和/或任意给定表达的多肽或蛋白质的产生。
制备宿主细胞(例如哺乳动物细胞)的多种方法为本领域公知。例如，可通过任意数目的公知技术将足以进行表达的核酸(一般为含有编码目的多肽或蛋白质基因及任何有效连接的遗传控制元件的载体)引入宿主细胞系内。一般而言，筛查细胞以确定哪些宿主细胞实际获得了载体并表达目的多肽或蛋白质。检测宿主细胞表达的某一特定目的多肽或蛋白质的常规方法，包括但不仅限于免疫组化、免疫沉淀、流式细胞术、免疫荧光显微术、SDS-PAGE，Western印迹、酶联免疫吸附测定(ELISA)、高效液相色谱(HPLC)技术、生物活性测定和亲和层析。本领域普通技术人员将会知道检测表达多肽或蛋白质的其他合适技术。如果多个宿主细胞表达目的多肽或蛋白质，可以使用某些或所有上述技术确定哪些细胞表达多肽或蛋白质的水平最高。
一种或多种所表达多肽和/或多肽复合物可使用任何适合方法加以收集。一种或多种多肽和/或多肽复合物可在细胞内，在周质间隙中表达，或被分泌至细胞的外部而进入培养基中。如果多肽和/或多肽复合物在细胞内表达，那么可使含有多肽和/或多肽复合物的宿主细胞溶解，并且可通过离心或超滤移除非所要碎片来从溶解产物分离多肽和/或多肽复合物。如果多肽和/或多肽复合物被分泌至大肠杆菌的周质间隙中，那么可在诸如乙酸钠(pH 3.5)、EDTA和苯基甲基磺酰氟(PMSF)的剂存在下使细胞糊状物解冻约30分钟，并且可通过离心移除细胞碎片(Carter等人，BioTechnology 10：163-167(1992))。如果多肽和/或多肽复合物被分泌至培养基中，那么可收集细胞培养物的上清液，并且使用可商购获得的蛋白质浓缩过滤器例如Amincon或Millipore Pellicon超滤装置加以浓缩。可在收集和浓缩步骤中包括蛋白酶抑制剂和/或抗生素以抑制蛋白质降解和/或污染微生物的生长。
可以通过标准方法分离和纯化多肽或蛋白质，包括但不限于色谱法(例如离子交换、亲和力、尺寸排阻和羟基磷灰石层析)、凝胶过滤、离心或差异溶解度、乙醇沉淀或任何其他可用的纯化蛋白质技术(见如Scopes，Protein Purification Principles and Practice第二版，Springer-Verlag，NewYork，1987；Higgins，S.J.和Hames’B.D.(编辑)，Protein Expression：APractical Approach，Oxford Univ Press，1999；以及Deutscher，M.P.，Simon，M.1.，Abelson，J.N.(编辑)，Guide to Protein Purification：Methods inEnzymology(Methods in Enzymology Series，Vol 182)，Academic Press，1997，均在此处引用作为参考)。具体对于免疫亲和层析而言，通过将蛋白质结合于含有抗体的亲和柱可以分离蛋白质，所述抗体为针对该蛋白质所产生并固定于固态支持物上。或者可利用标准重组技术将亲和标记如流感外壳序列、多聚组氨酸、谷胱甘肽-S-转移酶连接于蛋白质，以使得可通过合适的亲和柱进行简单纯化。可在任意或所有阶段加入蛋白酶抑制剂如苯甲基磺酰氟(PMSF)、亮抑蛋白酶肽、胃蛋白酶抑制剂或抑酶肽，以减少或消除纯化过程中多肽或蛋白质的降解。当必须裂解细胞以分离和纯化表达多肽或蛋白质时，尤其需要蛋白酶抑制剂。本领域普通技术人员应当认识到根据待纯化多肽或蛋白质的特性、表达多肽或蛋白质的细胞特性，以及细胞生长的培养基组成，具体纯化技术有所不同。
4.药物组合物和试剂盒
本发明还提供了一种药物组合物，其含有(i)本文任一所述的分离的ApoE构建物(例如SEQ ID NOs：12、60、61和89中任一氨基酸序列)、融合蛋白、分离的ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33)、分离的核酸、载体(例如神经特异性表达ApoE构建物(例如全长ApoE2或ApoE3)或ApoE多肽的载体)、或宿主细胞，和(ii)药学上可接受的载体。
药物组合物中活性成分(即本发明的ApoE构建物、ApoE多肽(如ApoE C端多肽)、融合蛋白或表达载体)的实际剂量水平可以改变，以获得有效达到下述效果的活性成分的量：达到对具体患者、组合物和施用模式的所需治疗反应，且对患者无毒性。所选择的剂量水平将取决于多种药代动力学因素，包括本发明所用的多肽或者表达载体的活性、施用途径、施用时间、治疗持续时间、用于与所用特定组合物组合的其它药物、化合物和/或物质、所治疗患者的年龄、性别、体重、病症、一般健康以及先前医疗史，以及医学领域内熟知的类似因素。
本文中，可药用载体包括稀释剂、佐剂、赋形剂或溶媒，包括但不限于水、动物油、植物油、淀粉、葡萄糖、乳糖、蔗糖、明胶、硬脂酸钠、单硬脂酸甘油酯、氯化钠、脱脂奶粉、甘油、丙烯、丙二醇、水、乙醇等。如果需要，该组合物还可以包含少量的湿润剂或乳化剂，或pH缓冲剂。通常，生理上可接受的载体是pH缓冲水溶液。可接受的载体、赋形剂或稳定剂在采用的剂量和浓度下对接受者暴露于其的细胞无毒，并且包括缓冲剂，诸如磷酸盐、柠檬酸盐和其他有机酸；抗氧化剂，包括抗坏血酸和甲硫氨酸；防腐剂(诸如十八烷基二甲基苯甲基氯化铵；氯化六甲双铵；苯扎氯铵、苄索氯铵；苯酚、丁醇或苯甲醇；对羟基苯甲酸烷基酯诸如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；以及间甲酚)；低分子量(小于约10个残基)多肽；蛋白质，诸如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯烷酮；氨基酸，诸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸；单糖、二糖以及其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合剂，诸如EDTA；糖，诸如蔗糖、甘露糖醇、海藻糖或山梨糖醇；成盐反离子，诸如钠；金属络合物(例如Zn-蛋白质络合物)；和/或非离子表面活性剂，诸如TWEENTM、PLURONICSTM或聚乙二醇(PEG)。
在一些实施方案中，药物组合物被配制以具有在约4.5至约9.0的范围内的pH，包括例如约5.0至约8.0、约6.5至约7.5、或约6.5至约7.0中的任一者的pH范围。在一些实施方案中，还可通过添加适合张力调节剂诸如甘油来使药物组合物与血液等张。
待用于体内施用的药物组合物通常被配制成无菌的大致上等张药物组合物，并且完全符合美国食品与药物管理局的所有良好制造规范(GMP)规定。通过经无菌过滤膜过滤，易于实现无菌性。在一些实施方案中，组合物不含病原体。对于注射，药物组合物可呈液体溶液的形式，例如处于生理上可相容的缓冲液诸如汉克斯溶液(Hank′s solution)或林格氏溶液(Ringer′s solution)中。此外，药物组合物可呈固体形式，并且在使用之前即刻加以再溶解或混悬。还包括冻干组合物。
本文中，药物组合物可以为溶液、混悬液、乳剂、片剂、丸剂、胶囊、粉末、缓释制剂等形式。可根据具体的给药方式和给药途径将本发明的多肽制备成合适的剂型。
药物组合物可适于本文所述的多种施用模式，包括例如全身性或局部化施用。在一些实施方案中，药物组合物被配制用于静脉内施用或皮下注射。在一些实施方案中，药物组合物被配制用于局部施用，例如局部施用至肿瘤部位(例如肿瘤内注射)、或局部施用(如注射)到大脑(例如皮层或海马体)。
在一些实施方案中，药物组合物根据常规程序被配制成适合于静脉内、腹膜内或脑内注射的药物组合物。通常，注射用组合物是处于无菌等张水性缓冲液中的溶液。必要时，组合物还可包括增溶剂和局部麻醉剂(诸如利多卡因(lignocaine))以减轻注射部位处的疼痛。通常，成分以分开方式或以一起混合成单位剂型的方式加以供给，例如作为干燥冻干粉末或无水浓缩物处于指示活性剂的数量的气密容器诸如安瓿或药囊中。当组合物待通过输注施用时，它可用含有无菌药品级水或盐水的输注瓶加以分配。当组合物通过注射施用时，可提供含无菌注射用水或盐水的安瓿以使成分可在施用之前加以混合。
在一些实施方案中，所述药物组合物适于施用至人。在一些实施方案中，所述药物组合物适于施用至啮齿动物(例如小鼠、大鼠、兔)或非人灵长类动物(例如猴)。在一些实施方案中，所述药物组合物含于单次使用小瓶诸如单次使用密封小瓶中。在一些实施方案中，所述药物组合物含于多次使用小瓶中。在一些实施方案中，所述药物组合物散装含于容器中。在一些实施方案中，所述药物组合物是经低温保存的。
本发明还提供了包含本文任一所述的分离的ApoE构建物、融合蛋白、分离的ApoE多肽、分离的核酸、载体、宿主细胞、或其组合物(诸如药物组合物)的单位剂型。术语“单位剂型”是指适合作为单位剂量用于个体的物理离散单元，每个单元含有被计算以产生所需治疗作用的预定量的活性物质，以及适合药物载体、稀释剂或赋形剂。这些单位剂型可以单一或多个单位剂量储存在适合包装中，并且还可加以进一步灭菌和密封。
本申请还提供了在适合包装中包含本文所述的组合物(诸如药物组合物)的制品。适于本文所述的组合物(诸如药物组合物)的包装在本领域中是已知的，并且包括例如小瓶(诸如密封小瓶)、容器、安瓿、瓶、罐、柔性包装(例如密封迈拉(Mylar)或塑料袋)等。这些制品可进一步加以灭菌和/或密封。
本发明还提供了包含本文任一所述的分离的ApoE构建物、融合蛋白、分离的ApoE多肽、分离的核酸、载体、宿主细胞、或药物组合物的试剂盒。在一些实施方式中，所述试剂盒还包括关于使用所述蛋白、表达载体、或组合物的方法诸如本文所述的用途的一个或多个说明。本文所述的药盒可还包括从商业和使用者观点来看可合乎需要的其他材料，包括其他缓冲剂、稀释剂、过滤器、针、注射器、以及具有关于进行本文所述的任何方法的说明的包装插页。
5.疾病治疗方法
本发明还提供了任一实施方案所述的分离的ApoE构建物(例如SEQ IDNOs：12、60、61和89中任一氨基酸序列)、融合蛋白、分离的ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33)、分离的核酸、载体(例如神经特异性表达ApoE构建物(例如全长ApoE2或ApoE3)或ApoE多肽的载体)、宿主细胞或药物组合物在制备用于治疗或预防一个个体(例如人)中与γ分泌酶的酶切活性(例如对APP的γ酶切活性)相关的疾病的药物中的应用。在一些实施方式中，所述药物抑制γ分泌酶的酶切活性(例如对APP的γ酶切活性)，从而治疗或预防所述与γ分泌酶的酶切活性相关的疾病。在一些实施方式中，本发明提供了一种治疗或预防一个个体(例如人)中与γ分泌酶的酶切活性(例如对APP的γ酶切活性)相关疾病的方法，包括向所述个体施用有效剂量的本发明所述任一分离的ApoE构建物(例如SEQ ID NOs：12、60、61和89中任一氨基酸序列)、融合蛋白、分离的ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33)、分离的核酸、载体(例如神经特异性表达ApoE构建物(例如全长ApoE2或ApoE3)或ApoE多肽的载体)、宿主细胞或药物组合物。在一些实施方式中，所述方法抑制γ分泌酶的酶切活性(例如对APP的γ酶切活性)，从而治疗或预防所述与γ分泌酶的酶切活性相关的疾病。在一些实施方式中，所述与γ分泌酶的酶切活性相关的疾病选自如下的组：神经系统退行性疾病，肿瘤或癌症，炎性疾病和肾脏疾病。在一些实施方式中，所述疾病选自如下的组：阿尔茨海默病及其相关疾病、淀粉样脑血管病、唐氏综合症、与衰老相关的其他神经退行性疾病(比如额颞叶痴呆)、头颈癌(如头颈鳞细胞癌和口腔部鳞状细胞癌)、乳腺癌、肝癌、胰腺癌(包括转移性胰腺癌)、卵巢癌、肺癌(如非小细胞肺癌、肺腺癌和小细胞肺癌)、胶质瘤(如恶性神经胶质瘤)、纤维瘤、淋巴瘤(如B细胞淋巴瘤)、骨肉瘤、胃癌、膀胱癌、哮喘(如过敏性哮喘)、肺炎、气道炎症、急性肾损伤、透明细胞肾细胞癌、肾纤维化、和梗阻性肾病。在一些实施方式中，所述疾病为AD及其相关疾病，例如散发性阿尔茨海默病(sporadic Alzheimer′s disease，sAD)。
本发明提供的所述药物、治疗或预防方法适用于任何个体，包括但不限于哺乳动物、鸟类、蛙类、鱼类、果蝇、线虫等。哺乳动物包括但不限于灵长类(例如人类，或非人类灵长类例如猴子)、啮齿类(例如小鼠、大鼠、仓鼠、沙鼠、松鼠、豚鼠和兔子)、家畜(例如猪、牛、羊、马、驴)、宠物(例如猫、狗、兔和仓鼠)等。在一些实施方式中，所述个体是小鼠或猴。在一些实施方式中，所述个体是人类。
本文中，合适的给药方式和给药途径为本领域所周知，合适的施用途径包括静脉内、肌内、真皮内、腹膜内、皮下、脊柱或其它胃肠外施用途径，例如经注射或灌注。“胃肠外给药”是除了肠内和局部施用以外的施用模式，通常经注射，并包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、真皮内、腹膜内、鼻腔、经气管、皮下、表皮下、关节内、囊下、蛛网膜下腔、脊柱内、硬膜外和胸骨内注射和灌注。术语“全身施用(systemic administration)”和“全身施用(systemicallyadministered)”是指将本文描述的试剂或组合物施用于个体(如哺乳动物)使得所述试剂或组合物经由循环系统被递送至体内位点(包括药物作用的靶向位点)的方法。全身施用包括但不限于口服、鼻内、直肠和胃肠外(如，除了通过消化道之外，诸如肌内、静脉内、动脉内、透皮和皮下)施用。
在一些实施方案中，将本发明任一所述分离的ApoE构建物(例如SEQ ID NOs：12、60、61和89中任一氨基酸序列)、融合蛋白、分离的ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33)、分离的核酸、载体、或药物组合物经任何适合的途径引入中枢神经系统，包括脑室内和鞘内注射。脑室内注射可经例如与贮库(例如Ommaya贮库)连接的脑内室导管辅助。
在一些实施方案中，可采用本领域周知的技术递送编码本发明多肽的核酸分子，如本发明多肽的编码序列，包括RNA和DNA序列。在一些实施方案中，以表达载体(如利用病毒载体，例如AAV或慢病毒载体)的方式向需要的对象(例如人)递送本发明多肽的编码序列。在一些实施方案中，采用脂质体递送本文所述的可以编码本发明多肽的DNA或者RNA分子。因此，在这些实施方案中，本发明的药物组合物含有脂质体以及本文所述的编码本发明多肽的DNA或者RNA分子及其表达载体。适用于本发明的脂质体可以是本领域周知的适合于人体给药的脂质体。
本发明提供的ApoE构建物(例如全长ApoE2或ApoE3)、融合蛋白、或ApoE多肽是γ分泌酶的抑制剂(例如对APP的γ酶切特异性抑制)，因此本发明的多肽或其药物组合物可用于治疗或预防受益于γ分泌酶被抑制的疾病，以抑制γ分泌酶为靶标而获得治疗或预防益处的疾病(本文也称为γ分泌酶介导的疾病或与γ分泌酶的酶切活性相关的疾病)。已知有多种疾病由γ分泌酶介导，包括但不限于：神经系统退行性疾病，如阿尔茨海默病及相关疾病和淀粉样脑血管病(Erik D.Roberson和Lennart Mucke，100 years and Counting：Prospects for Defeating Alzheimer’s Disease，314：781-784(2006)；Bart De Strooper等，Presenilins and γ-Secretase：Structure，Function，and  Role in Alzheimer’s Disease，2：a006304(2012)；Weiming Xia，γ-Secretase and its modulators：Twenty years and beyond，Neuroscience Letters 701：162-169(2019))；肿瘤或癌症，包括头颈癌如头颈鳞细胞癌和口腔部鳞状细胞癌(Liang Mao等，γ-Secretase inhibitor reduces immunosuppressive cells and enhances tumour immunity in head and neck squamous cell carcinoma，Int.J.Cancer：142，999-1009(2018))，乳腺癌和肝细胞癌(Hui Jia等，γ-Secretase inhibitors for breast cancer and hepatocellular carcinoma：From mechanism to treatment，Life Sciences 268(2021)119007)，胰腺癌(包括转移性胰腺癌)(Ana De Jesus-Acosta等，A Phase II Study of the Gamma Secretase Inhibitor RO4929097 in Patients with Previously Treated Metastatic Pancreatic Adenocarcinoma，Invest New Drugs.2014August；32(4)：739-745)，卵巢癌(Zhaoyi Feng，Inhibition of gamma-secretase in Notch1 signaling pathway as a novel treatment for ovarian cancer，Oncotarget，2017，Vol.8，(No.5)，pp：8215-8225)，肺癌(如非小细胞肺癌或肺腺癌和小细胞肺癌)(Antonio Maraver，Therapeutic effect of γ-secretase inhibition in KrasG12V-driven non-small cell lung carcinoma through derepression of DUSP1 phosphatase and inhibition of ERK，Cancer Cell.2012 August 14；22(2)；Katherine M.Morgan等，Gamma secretase inhibition by BMS-906024 enhances efficacy of paclitaxel in lung adenocarcinoma，Mol Cancer Ther.2017 December；16(12)：2759-2769)，胶质瘤如恶性神经胶质瘤(Edward Pan等，Phase I Study of RO4929097 with Bevacizumab in Patients with Recurrent Malignant Glioma，Neurooncol.2016 December；130(3)：571-579)，纤维瘤(Shivaani Kummar等，Clinical Activity of the g-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors(Aggressive Fibromatosis)，JOURNAL OF CLINICAL ONCOLOGY，VOLUME 35，NUMBER 14，MAY 10，2017)，淋巴瘤如B细胞淋巴瘤(Shuji Tohda，Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors，Leukemia Research 30(2006)1385-1390)，胃癌(Hyun-Woo Lee等，Targeting Notch signaling by c-secretase inhibitor I enhances the cytotoxic effect of 5-FU in gastric cancer，Clin Exp Metastasis(2015)32：593-603)，膀胱癌(YIBING WANG等，γ-secretase inhibitor inhibits bladder cancer cell drug resistance and invasion by reducing epithelial-mesenchymal transition，MOLECULAR MEDICINE REPORTS 12：2821-2827，2015)，骨肉瘤(M Tanaka等，Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation，British Journal of Cancer(2009)100，1957-1965)；炎性疾病，包括哮喘，如过敏性哮喘(Weixi Zhang等，γ-Secretase Inhibitor  Alleviates Acute Airway Inflammation of Allergic Asthma in Mice by Downregulating Th17 Cell Differentiation，Mediators of Inflammation，Volume 2015，Article ID 258168，7 pages)，气道炎症如过敏性气道炎症(Weixi Zhang等，γ-Secretase Inhibitor Alleviates Acute Airway Inflammation of Allergic Asthma in Mice by Downregulating Th17 Cell Differentiation，Mediators of Inflammation，Volume 2015，Article ID 258168)，肺炎(Jin Hyun Kang等，g-Secretase Inhibitor Reduces Allergic Pulmonary Inflammation by Modulating Th1 and Th2 Responses，AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 179，2009)等；肾脏疾病，如急性肾损伤(Jean-Christophe Wyss等，Targetedγ-secretase inhibition of Notch signaling activation in acute renal injury，Am J Physiol Renal Physiol 314：F736-F746，2018)。
在一些实施方式中，所述与γ分泌酶的酶切活性(例如对APP的γ酶切活性)相关的疾病是神经退行性疾病，例如阿尔茨海默氏病(AD)、路易体痴呆(DLB)、轻度认知障碍(MCI)、额颞叶痴呆(FTD)、脑淀粉样血管病(CAA)、CAA-相关脑出血、血管性认知障碍、帕金森病(PD)、多发性硬化症(MS)、创伤性脑损伤(TBI)或脆性X(Fragile X syndrome)相关性震颤/共济失调综合征。CAA是一种以蛋白质(主要是淀粉样蛋白β)沉积在脑血管壁为特征的病症。CAA在AD患者中很常见，但在没有AD证据的情况下也会发生。
在一些实施方案中，所述与γ分泌酶的酶切活性(例如对APP的γ酶切活性)相关的疾病是“淀粉样蛋白β相关疾病”，该术语是指以异常高水平的淀粉样蛋白β肽的产生、积累和/或聚集为特征的任何病症，例如，在淀粉样斑块的形式，特别是在大脑中。淀粉样蛋白β相关疾病包括AD、唐氏综合征、脆性X综合征(Fragile X syndrome)、CAA、CAA相关脑出血和散发性包涵体肌炎。
AD是在发达国家中最常见的神经退化性疾病。在组织病理学上，AD的特征在于包含Aβ肽的淀粉样蛋白斑的积聚和由tau蛋白制成的NFT。在临床上，AD与特征在于记忆、功能、语言能力、判断、以及执行功能的损失的进行性认知障碍相关联。AD经常在其后期阶段导致严重的行为症状。
大致一半的早发型阿尔茨海默氏病例(即，在65岁之前诊断)为家族性阿尔茨海默氏病(FAD)，并且以常染色体显性方式遗传。FAD由至少3个基因中的一者的突变造成：APP突变、PSENI突变和PSEN2突变。已在121个家族中识别出淀粉样蛋白前体蛋白质(APP)突变。最常见的错义突变利用Ile(伦敦突变)、Phe或Gly残基替换Val717残基，利用Gln残基(荷兰突变)或Gly残基(北极突变)替换Glu693，或利用Asn 和Leu残基替换Lys670和Met671(双重突变称为瑞典突变，在APP770异构体中编号)。已在480个家族中识别出219个早老素1(PSEN1)，其中γ分泌酶裂解减弱、一些突变的氧化应力增加且对DNA损伤诱发的死亡的神经元敏感性增加。已在34个家族中识别出13个早老素2(PSEN2)，其中γ分泌酶裂解减弱且一些突变的氧化应力增加。
在一些实施方式中，本发明提供的任一分离的ApoE构建物(例如SEQ ID NOs：12、60、61和89任一氨基酸序列)、融合蛋白、分离的ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33)、分离的核酸、载体(例如神经特异性表达ApoE构建物(例如全长ApoE2或ApoE3)或ApoE多肽的载体)、宿主细胞或药物组合物可以通过抑制γ分泌酶酶活而用于治疗任何与APPγ-酶切相关的疾病(如AD)，例如带有任何APP突变的疾病(如AD)。在一些实施方式中，所述APP是人APP，其包含下述一个或多个突变：K670N、M671L、瑞典(K670N+M671L)、佛罗里达(I716V)、伦敦(V717I)、APPM596V(不能进行β切割)、γ切割位点附近的fAD突变(例如T714I、V717F、L723P)、β-或α-切割位点附近的fAD突变(例如KM670/671NL、E693K、E693Δ)、或保护性冰岛突变(A673T)。
用于评估阿尔茨海默病表型、用于表征治疗剂和评价治疗的一些行为和组织病理学测试是本领域已知的。组织学分析通常在死后进行。可使用硫磺素-S进行Aβ水平的组织学分析。刚果红，或抗-Aβ染色(例如，4G8、10D5或6E10抗体)将切片的脑组织上的Aβ沉积可视化(参见，例如，Holcomb等人，1998，Nat.Med.4：97-100；Borchelt等人，1997，Neuron19：939-945；Dickson等人，1988，Am.J.Path.132：86-101)。将转基因小鼠中的Aβ沉积物可视化的体内方法也已得到描述。BSB((反式，反式)-1-溴-2，5-双-(3-羟基羰基-4-羟基)苯乙烯基苯)和PET示踪物11C-标记的Pittsburgh化合物-B(PIB)与Aβ斑块结合(参见，例如，Skovronsky等人，2000，Proc.Natl.Acad.Sci.USA 97：7609-7614；Klunk等人，2004，Ann.Neurol.55：306-319)。含19F的亲淀粉样蛋白刚果红型化合物FSB((E，E)-1-氟-2，5-双-(3-羟基羰基-4-羟基)苯乙烯基苯)允许通过MRI将Aβ斑块可视化(参见，例如，Higuchi等人，2005，Nature Neurosci.8：527-533)。放射性标记的、腐胺修饰的淀粉样蛋白-β肽体内标记阿尔茨海默病的小鼠模型中的淀粉样沉积物(参见，例如，Wengenack等人，2000，Nat.Biotechnol.18：868-872)。
可使用针对阿尔茨海默病的本领域已知的标准诊断方法来鉴别罹患阿尔茨海默病的受试者。通常，阿尔茨海默病的诊断是基于患者的症状(例如，记忆功能进行性下降、正常活动的逐步放弃和受挫、冷漠、躁动或易怒、攻击性、焦虑、睡眠障碍、心 烦、异常运动行为、去抑制、社交退缩、食欲下降、幻觉、痴呆)、病史、神经心理学测试、神经和/或身体检查。还可以测试脑脊髓液中与阿耳茨海默病理学有关的多种蛋白质，包括tau、淀粉样β肽和AD7C-NTP。基因测试也可用于早发性家族性阿尔茨海默病(eFAD)、常染色体显性遗传性疾病。临床基因测试可用于具有AD症状的个体或具有早发型疾病患者的风险家族成员。在美国，可根据临床实验室改进修正案，在临床或联邦政府批准的临床实验室中测试PS2的突变和APP。PS1突变的商业测试也是可用的(Elan Pharmaceuticals)。
可以通过上述任何所述方法或指标来评估本发明的治疗效果。在一些实施方式中，本发明的ApoE构建物、ApoE多肽、载体、组合物、或治疗/预防方法可以达到以下一个或多个效果：(i)在神经元中(体内或体外)以细胞自主性的方式调控APP的剪切；(ii)抑制(例如抑制至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)γ-分泌酶的酶活，例如抑制γ-分泌酶介导的APPγ-剪切，与γ-分泌酶抑制剂(例如PF03084014)的APPγ-剪切抑制效果相似；(iii)降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)APP的淀粉样代谢，例如减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)淀粉样蛋白-β(Aβ)(例如Aβ40和/或Aβ42)的生成，或降低(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)对C末端片段-β(β-CTF)的γ-剪切；(iv)提高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)APP的非淀粉样代谢，例如增加(例如增加至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)C末端片段-α(α-CTF)的生成；(v)不影响(或影响不超过约30％、20％、10％、5％或更少)α-分泌酶、β-分泌酶、和/或γ-分泌酶(或其亚基)(例如ADAM10、BACE1、PS1和/或PS2)的mRNA和/或蛋白质表达；(vi)不影响(或影响不超过约30％、20％、10％、5％或更少)β-分泌酶介导的APPβ-剪切，和/或α-分泌酶介导的APPα-剪切；(vii)不抑制(或抑制不超过约30％、20％、10％、5％或更少)对非APP蛋白(例如APLP1(APP like protein 1)或Notch)或其片段的γ-剪切，不增加(或增加不超过约30％、20％、10％、5％或更少)非APP蛋白γ-剪切产生的α-CTF；(viii)减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)或预防脑中淀粉样斑块，例如减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)淀粉样斑块的平 均斑块大小、总面积、和/或密度，减少(例如减少至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)或预防神经元内Aβ(例如Aβ40和/或Aβ42)生成，减缓(例如减缓至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍或更多)AD病理进程，提高(例如提高至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、100％、2倍、5倍、10倍、20倍或更高)个体存活率和/或寿命；(ix)神经元局部表达(例如在皮质、齿状回(DG)和海马体CA3表达)的本发明所述ApoE构建物可以从远处迁移到下托并积聚在淀粉样斑块周围，从而在这些淀粉样蛋白生成热点局部抑制γ-分泌酶的酶活；或(x)减轻(例如降低至少约10％、20％、30％、40％、50％、60％、70％、80％、90％、95％、或100％)、消除、或预防(包括减缓发生时间)一个或多个AD症状(例如，记忆功能进行性下降、正常活动的逐步放弃和受挫、冷漠、躁动或易怒、攻击性、焦虑、睡眠障碍、心烦、异常运动行为、去抑制、社交退缩、食欲下降、幻觉、痴呆)。
在一些实施方案中，所述与γ分泌酶的酶切活性相关的癌症包括如下一个或多个：头颈癌(如头颈鳞细胞癌和口腔部鳞状细胞癌)、乳腺癌、肝癌、胰腺癌(包括转移性胰腺癌)、卵巢癌、肺癌(如非小细胞肺癌、肺腺癌和小细胞肺癌)、胶质瘤(如恶性神经胶质瘤)、纤维瘤、淋巴瘤(如B细胞淋巴瘤)、骨肉瘤、胃癌、膀胱癌。
在一些实施方案中，本文所述的治疗与γ分泌酶的酶切活性相关癌症的药物或方法具有以下生物活性中的一者或多者：(1)杀灭癌细胞，例如至少约30％、40％、50％、60％、70％、80％、90％、95％或更高中的任一者的肿瘤细胞死亡率；(2)抑制癌细胞的增殖，例如抑制至少约10％(包括例如至少约20％、30％、40％、60％、70％、80％、90％或100％中的任一者)增殖；(3)降低肿瘤尺寸，例如降低至少约10％、20％、30％、40％、60％、70％、80％、90％或100％肿瘤尺寸；(4)缓和患有癌症的个体中的一种或多种症状；(5)抑制肿瘤转移(例如向淋巴结的转移)，例如抑制至少约10％(包括例如至少约20％、30％、40％、60％、70％、80％、90％或100％中的任一者)的肿瘤转移；(6)延长存活期，例如延长至少1、2、3、4、5、6、7、8、9、10、11、12、18或24个月或更长时间中的任一者；(7)预防癌症或延长达到癌症进展的时间，例如使达到癌症进展的时间延长至少1、2、3、4、5、6、7、8、9、10、11或12个月或更长时间中的任一者；以及(8)预防、抑制或降低癌症复发的可能性。
在一些实施方案中，所述与γ分泌酶的酶切活性相关的疾病是炎性疾病，包括但不限于哮喘(如过敏性哮喘)、肺炎、气道炎症。在一些实施方案中，本文所述的药 物或治疗/预防方法可以降低(例如降低至少约10％、20％、30％、40％、60％、70％、80％、90％或100％中的任一者)炎症引发细胞因子的分泌和/或活性，和/或减轻(例如减轻至少约10％、20％、30％、40％、60％、70％、80％、90％或100％中的任一者)、消除或预防(包括减缓发生时间)一种或多种疾病症状。
在一些实施方案中，所述与γ分泌酶的酶切活性相关的疾病是肾脏疾病，指影响肾脏或肾脏系统的任意疾病、病症或病况。肾脏相关疾病或病况的实例包括，但不限于，急性肾脏疾病(或衰竭)、透明细胞肾细胞癌、肾纤维化、或梗阻性肾病。评估和诊断肾脏疾病的常规标记物包括GFR、肌酸酐和白蛋白，可以使用任一种标记物或相关方法评估本发明治疗效果。在一些实施方案中，本文所述的药物或治疗/预防方法可以(i)降低(例如降低至少约10％、20％、30％、40％、60％、70％、80％、90％或100％中的任一者)、消除或预防(包括减缓发生时间)一个或多个肾脏疾病指标；和/或(ii)保护或改善肾功能。
本发明提供一种治疗或预防对象(例如人)γ分泌酶介导的疾病的方法，该方法包括给予该对象治疗有效量或预防有效量的本发明任一所述ApoE构建物、融合蛋白、ApoE多肽、核酸分子、载体或其药物组合物。有效量可根据对象年龄、性别、体重、病症、一般健康以及先前医疗史等因素由本领域技术人员确定。所述疾病优选包括前文所述的神经系统退行性疾病、肿瘤或癌症、炎性疾病以及肾脏疾病等。
本发明的一些实施方案提供一种抑制γ分泌酶的酶切，从而抑制APP的淀粉样代谢，减少Aβ生成的方法，该方法包括在表达γ分泌酶的细胞内(例如神经元)产生ApoE2(例如N端带有穿膜肽的ApoE2)或本文任一实施方案所述的ApoE构建物(例如ApoE2的C端片段，或全长ApoE2或ApoE3)、融合蛋白或ApoE多肽(例如SEQ ID NOs：13、18-34、46-52、57-59、62、76、78和87中任一氨基酸序列，比如SEQ ID NO：33)。在一些实施方案中，所述细胞为神经元。在一些实施方案中，所述方法用于减缓(例如减缓至少约10％、20％、30％、40％、60％、70％、80％、90％或100％中的任一者)、预防或治疗AD和淀粉样脑血管病的发生和发展。本文所述的产生包括表达产生多肽和从细胞外部将多肽递送进入细胞内。在一些实施方案中，采用脂质体将编码所述ApoE构建物(例如ApoE2和/或ApoE3和/或其C端片段)或ApoE多肽的DNA或者RNA递送到所述细胞内，使其在细胞内表达产生所述ApoE构建物或ApoE多肽；或者采用慢病毒和腺相关病毒侵染细胞的方式在所述细胞内产生所述ApoE构建物或ApoE多肽；或采用细胞穿膜肽或细胞胞吞增强的方式使所述ApoE构建物或ApoE多肽进入所述细胞内。
在一些实施方案中，本文任一所述实施方案所述的ApoE构建物或融合蛋白、用途和方法中提到的多肽特别包括SEQ ID NOs：12、13、18-33和89中任一所示的多肽。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，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。
实施例中，数据使用GraphPad Prism软件分析，所有的数据都展示为mean±SEM。下述数据分析使用了unpaired，paired或者one sample t-test。显著性差异由星号(*)展示，一个星号(*)代表P值小于0.05；两个星号(**)代表P值小于0.01；三个星号(***)代表P值小于0.001；四个星号(****)代表P值小于0.0001。
实施例1
1.主要试剂与材料
HEK 293T细胞系培养在含有10％胎牛血清(FBS)(Life公司)和1％青霉素/链霉素的DMEM培养液(HyClone公司)中，并培养在含有5％CO2的37℃无菌细胞培养箱(ThermoFisher公司)中。
原代培养的神经元来自于16天胚胎小鼠的皮层或者海马。小鼠皮层或海马在体视镜下取得；20U/ml木瓜蛋白酶(Worthington公司)在37℃消化10分钟；消化后以单细胞形式存在的神经元以每孔(24孔板)100000个神经元种板；在含有2％B27(ThermoFisher公司)和1％青霉素/链霉素的神经元培养液中(Neurobasal medium；ThermoFisher公司)培养该神经元；每周换一次新鲜培养液。
0-7月龄C57野生型小鼠和AD模型小鼠(5×FAD小鼠[Tg6799]，表达含有致病性突变的APP和PS1的小鼠。所表达的APP上包含有三个突变：K670N/M671L、I716V、V717I；而PS1则包含有两个突变：M146L，L286V)。给予小鼠充足的食物和水，并在室温下以12h/12h的明暗周期饲养，饲养环境达到SPF(Specific Pathogen Free)级标准。
酶联免疫吸附剂测定盒(ELISA)购自Invitrogen公司，人源Aβ40(KHB3481)，人源Aβ42(KHB3441)，小鼠源Aβ40(KMB3481)。
转染试剂是购自ThermoFisher公司的lipofectamine2000和SignaGen公司的Polyjet。
用于qRT-PCR的Quantitect SYBR-Green qRT-PCR试剂盒购自Qiagen公司。
表1：抗体信息
2.实验方法
2.1蛋白样品制备及免疫印迹(Western Blot)
a)构建在哺乳动物细胞中表达目的蛋白的质粒，蛋白表达启动子是CAG。
b)在HEK 293T细胞系中表达目的蛋白。在转染前一天进行细胞传代，当细胞的贴壁程度达到70％-80％时进行转染。转染试剂是lipofectamine2000(ThermoFisher公司)。
c)转染4-6小时后，更换为新鲜的细胞培养液。
d)转染36-48小时后，裂解细胞(10％1M tris-HCl(pH＝6.8)，4％SDS，20％甘油，0.01％溴酚蓝)，收集蛋白样品，之后进入免疫印迹实验或者冻存在-80℃冰箱中。
e)聚丙烯酰胺凝胶电泳。蛋白上样之前，98℃煮沸10min，然后12000rpm离心10min。电泳：上层浓缩胶80V；下层分离胶120V。
f)转膜。滤纸使用前在电转液中浸湿，PVDF膜放在甲醇中10s激活，从电泳槽中取出电泳结束的胶，按滤纸-PVD膜-胶-滤纸从下到上的顺序放好，放入转膜仪，恒流转膜，0.1A，70min.
g)封闭和孵育抗体：将转好的PVDF膜放在5％BSA/TBST封闭液中，于摇床上室温封闭1h。孵育一抗，4℃，摇床过夜(12-16h)；1×TBST，洗三遍，每10分钟清洗一次；孵育二抗，室温摇床1-2h；1×TBST，洗三遍，每10分钟清洗一次。
h)显影成像：ECL化学发光超敏显色试剂显影。
2.2脑片制备和免疫组化
a)灌流取脑。从心脏处对小鼠灌流，先用20mlPBS冲洗掉血液，之后用20ml 4％PFA进行灌流。
b)固定和脱水。先用4％PFA在4℃固定24小时，再用30％蔗糖在4℃脱水。
c)鼠脑切片。脱水24小时后，将小鼠脑子切成厚度为40μm的脑片，放在冰冻缓存液中储存或进入后续免疫组化。
d)THS染色。ddH2O，洗三遍，每次5分钟；1％的THS水溶液，浸泡10分钟；80％乙醇，洗两遍，每次3分钟；95％乙醇，洗一遍，3分钟。进性后续免疫组化。如果不进行THS染色，则直接进行下面的免疫组化。
e)免疫组化。1×PBS，洗三遍，每次10分钟；使用0.1％的Triton-X100进行细胞破膜；使用5％的驴或者山羊血清进行封闭；之后孵育一抗，4℃，摇床过夜(12-16h)；1×PBS，洗三遍，每次10分钟；孵育二抗，室温摇床1-2h；1×PBS，洗三遍，每次10分钟。
f)贴片及封片。
g)呈像，在封片24小时后。
h)使用Image J处理图像及分析信号密度。
2.3酶联免疫吸附剂测定(ELISA)
按照Invitrogen公司的ELISA分析试剂盒中的标准流程进行实验。
a)在HEK 293T细胞系中表达目的蛋白。在转染前一天进行细胞传代，当细胞的贴壁程度达到70％-80％时进行转染。转染试剂是lipofectamine2000(ThermoFisher公司)。
b)转染4-6小时后，更换新鲜的培养液；
c)转染36-48小时后，收集细胞培养液，之后进入ELISA流程或者冻存在-80℃冰箱中。
d)50μl标准样品和实验样品分别添加到96孔板每孔中。
e)50μl人源Aβ40或Aβ42或小鼠源Aβ40检测抗体添加到96孔板每孔中。
f)轻轻摇晃，室温放置3小时。
g)去除孔中液体，1×wash缓冲液清洗4次。
h)添加100μl的含有1×anti-Rabbit IgG HRP的缓冲液。
i)轻轻摇晃，室温放置30分钟。
j)去除孔中液体，1×wash缓冲液清洗4次。
k)每孔中添加100μl的Stabilized Chromogen，避光室温放置30分钟。
l)每孔中添加100μl的Stop Solution。
m)酶标仪测量450nm吸光度。
2.4 qRT-PCR
按照TRIzolTM Reagent(ThermoFisher公司)的操作手册提取处理的细胞的RNA。
a)在HEK 293T细胞系中表达目的蛋白。在转染前一天进行细胞传代，当细胞的贴壁程度达到70％-80％时进行转染。转染试剂是lipofectamine2000(ThermoFisher公司)。
b)转染6小时后，更换新鲜的培养液。
c)转染48小时后，提取RNA。
d)24孔板的每孔加入500μl Trizol(ThermoFisher公司)，摇晃，室温20分钟。
e)收集细胞悬液至离心管中。
f)加入250μl氯仿，彻底颠倒混匀，室温静置3分钟。
g)12000g，4℃离心15分钟。离心后，溶液出现明显分层，底部为氯仿含苯酚(粉色)，中间为DNA和蛋白质，最上层为含有RNA的水相(透明澄清)，转移上清至新的离心管。
h)加入等体积(上清)的异丙醇，颠倒混匀，室温放置20分钟。
i)12000g，4℃离心15分钟。除去上清，底部可见少量白色沉淀(RNA)。
j)加入400μl 75％的乙醇。12000g，4℃离心5分钟。
k)尽量除去乙醇，通风厨内彻底干燥，但避免过干燥。加入适量DEPC水。
l)去除DNA后，-80℃保存用于后续实验。
使用Qiagen公司的Quantitect SYBR-Green qRT-PCR试剂盒定量分析mRNA的相对含量。qRT-PCR引物如表2。
表2：用于qRT-PCR的引物

2.5 PS1 KO、PS2 KO和PS1/2 dKO细胞系的构建
本发明选用HEK 293T细胞，按照2013年张锋等发表的标准程序(Ran等人.，2013)使用CRISPR/CAS9技术构建PS1 KO、PS2 KO和PS1/2 dKO细胞系。使用https：//zlab.bio/guide-design-resources网站设计了特异性敲除PS1和PS2的sgRNA，如表3。
a)在HEK 293T细胞中表达PS2 sgRNA和对照空质粒。在转染前一天进行细胞传代，当细胞的贴壁程度达到70％-80％时进行转染。转染试剂是lipofectamine2000(ThermoFisher公司)。
b)转染6小时后，更换新鲜的培养液。
c)转染18小时后，更换为含有3μg/ml嘌呤霉素(Sellect公司)的新鲜培养液。
d)当转染空质粒的HEK 293T细胞系，在3μg/ml嘌呤霉素的作用下全部死亡后，收集转染了PS2 sgRNA的HEK 293T细胞。
e)以1∶400的比例将转染了PS2 sgRNA的HEK 293T细胞传代到10cm细胞培养皿中，保证细胞以单克隆的形式繁殖。
f)当单克隆繁殖的细胞生长到细胞团后，收集单个细胞团，并将单个细胞团传代到12孔板中。
g)等细胞团生长铺满整个12孔板，收集该细胞。一半细胞用于提取DNA和蛋白质，检测PS2的DNA序列和蛋白质表达量。另一部分细胞用于传代。
h)PS2的DNA序列已经被编辑和不再表达PS2蛋白质的细胞即是PS2敲除细胞系-PS2 KO细胞系。
i)本发明在PS2 KO细胞系基础上构建了PS1/PS2 dKO细胞系。在PS2 KO细胞系中进一步表达PS1 sgRNA或对照空质粒；并进行a-g步骤，进行PS1的DNA序列检测和蛋白质表达。
j)PS1的DNA序列已经被编辑和不再表达PS1蛋白质的PS2 KO细胞系即是PS1和PS2双敲除细胞系-PS1/2 KO细胞系。
表3：sgRNA
2.6病毒制备及注射。
2.6.1慢病毒制备
a)构建可以特异性在神经元中表达的慢病毒表达质粒，lentivirus-GFP和lentivirus-ApoE CT。
b)将慢病毒表达载体与外壳蛋白载体(PSPAX2和PMD2G)共转染在HEK 293T细胞系中。在转染前一天进行细胞传代，当细胞的贴壁程度达到70％-80％时进行转染。转染试剂是Polyjet(SignaGen公司)。
c)转染36-48小时后，收集细胞培养液，0.22μm的滤膜过滤。
d)过滤后的细胞培养液超速离心。20000rpm，4℃离心2小时5分钟。
e)离心后，去除上清，每3个10厘米细胞培养液的沉淀用50μl的PBS重悬。
f)重悬的病毒分装，保存到-80℃冰箱中用于后续实验。
2.6.2腺相关病毒制备
表达在神经元中的腺相关病毒AAV-CT(AAV2/9-hSyn-ApoE CT)和AAV-GFP(AAV2/9-hSyn-EGFP-WPRE-pA)，购自泰尔图生物科技公司。
2.6.3病毒注射
利用立体定位注射仪将上述1ul制备的腺相关病毒注射到1月龄的小鼠的大脑皮层(x，y，z，+/-1.6，0，-1)和海马体(x，y，z，+/-1.5，-2，-1.5)中。
2.7蛋白纯化
2.7.1γ分泌酶的纯化
人源γ分泌酶的表达载体参见Lu等人，2014。
a)在HEK 293T细胞系中表达人源γ分泌酶(同一个表达质粒同时表达人源γ分泌酶的四个蛋白，NCT，PS1，PLAG-PEN2，APH)。在转染前一天进行细胞传代，当细胞的贴壁程度达到70％-80％时进行转染。转染试剂是lipofectamine2000(ThermoFisher公司)。
b)转染4-6小时后，更换为新鲜的细胞培养液。
c)转染48小时后，冰1×PBS清洗一次，用细胞刮刀将细胞刮下来，离心收集细胞，800g，5min。
d)用裂解液[25mM HEPES，pH 7.4；150mM NaCl，5mM MgCl2，1mM二硫苏糖醇，0.05％(v/v)Tween-20，1×cocktail]裂解重悬细胞。
e)超声处理裂解的细胞，20个循环(3秒开，3秒关)，45％power output。
f)3000g，4℃离心10分钟，收集上清至新的离心管。
g)15000g，4℃离心1小时，弃上清。
h)用1ml裂解液重悬沉淀，重悬后向其中加入1％CHAPSO，在四度孵育2小时。15000g，离心30分钟，收集上清至新的离心管。
i)上清中加入100μl FLAG珠子(SIGMA公司)，在4℃孵育2小时。
j)用下述缓冲液清洗三次。25mM HEPES，pH7.4；150mM NaCl，0.1％digitonin。
k)用下述缓冲液洗脱下人源γ分泌酶。25mM HEPES，pH 7.4；150mM NaCl，0.1％digitonin和500μg/ml 3xFLAG peptide(SIGMA公司)。
l)免疫印迹检测纯化得到的人源γ分泌酶；-80℃保存。
2.7.2 ApoE的纯化
a)在HEK 293T细胞系中表达ApoE2和ApoE2 NT。ApoE2和ApoE2NT蛋白上含有融合表达的FLAG。在转染前一天进行细胞传代，当细胞的贴壁程度达到70％-80％时进行转染。转染试剂是lipofectamine2000(ThermoFisher公司)。
b)转染4-6小时后，更换为新鲜的细胞培养液。
c)转染48小时后，冰1×PBS清洗一次，用细胞刮刀将细胞刮下来，离心收集细胞，800g，5min。
d)用裂解液[25mM HEPES，pH 7.4；150mM NaCl，1×cocktail]裂解重悬细胞。
e)超声处理裂解的细胞，20个循环(3秒开，3秒关)，45％power output。
f)离心得到含有蛋白的上清，12000g，15分钟，4℃，收上清至新的离心管。
g)含有蛋白的上清孵育anti-FLAGM2亲和树脂(SIGMA公司)90分钟，4℃。孵育后ApoE2和ApoE2NT转移到anti-FLAG M2亲和树脂上。
h)含有ApoE2和ApoE2 NT的anti-FLAG M2亲和树脂用下述缓冲液清洗三次。25mM HEPES，pH 7.4；150mM NaCl，5mM MgCl2，1mM DTT，0.05％(v/v)Tween-20。
i)ApoE2和ApoE2 NT用下述缓冲液洗脱下来。25mM HEPES，pH7.4；150mM NaCl和500μg/ml 3xFLAG peptide(SIGMA公司)。
j)免疫印迹检测纯化得到的ApoE2和ApoE2NT；-80℃保存。
2.7.3β-CTF的纯化
a)在E.coli大肠杆菌中表达β-CTF，表达质粒为pET22β-CTF-Myc-6xHis
b)0.5mM IPTG诱导，37℃表达4小时后，E.coli大肠杆菌用下述裂解液裂解。30mM Tris-HCl，pH 7.5；150mM NaCl，1mM EDTA。
c)25000g，室温离心10分钟。去除上清。
d)用下述缓冲液重悬沉淀，在室温缓慢混匀。30mM Tris-HCl，pH 8.0；300mM NaCl，1mM EDTA，6M urea，1％TritonX-100，1mM CaCl2，1×cocktail。
e)40000g，离心30分钟，弃掉沉淀，0.45mM滤膜过滤上清。
f)过滤后的上清，与Ni-NTA-珠室温孵育2h，而后将孵育好的Ni-NTA-珠与上清液同时装载空的层析柱，使其在重力的作用下流出。收集流穿液体再使其与珠结合一次。
g)以下述10倍Ni-NTA-珠柱体积清洗Ni-NTA-珠：30mM Tris-HCl，pH 8.0；300mM NaCl，1mM EDTA，6M urea，1％TritonX-100，1mM CaCl2，1×cocktail。
h)以下述20倍Ni-NTA-珠柱体积清洗Ni-NTA-珠：30mM Tris-HCl，pH 8.0；300mM NaCl，0.1％Trion X-100。
i)以下述20倍Ni-NTA-珠柱体积清洗Ni-NTA-珠：50mM Tris-HCl，pH 8.0；300mM NaCl，0.1％CHAPSO。
j)以下述缓冲液洗脱β-CTF：50mM Tris-HCl，pH 8.0；300mM NaCl，0.1％CHAPSO，300mM咪唑。
k)免疫印迹检测纯化得到的β-CTF；-80℃保存。
2.7.4 GFP的纯化
GFP的纯化方式同2.7.2 ApoE的纯化方式。
3.实验结果
3.1三种ApoE异构体和ApoE不同蛋白片段对APP代谢的影响
本发明在HEK 293T细胞中将人源的APP分别与ApoE2、ApoE3和ApoE4共表达，GFP作为对照组，使用WesternBlot测定α-CTF的生成量。免疫印迹实验结果(图1，面板A-B)显示与APP共表达的ApoE2和ApoE3可以显著增加APP的非淀粉样代谢产物α-CTF；而与APP共表达的ApoE4则不能显著改变α-CTF(对比GFP对照组的α-CTF)。本发明进一步探究了ApoE对APP淀粉样代谢通路的影响。Aβ是淀粉样代谢的产物，Aβ的生成量越少，APP的淀粉样代谢越弱。本发明将人源的野生型APP和β分泌酶分别与ApoE2、ApoE3或者ApoE4在HEK 293T细胞中共表达，GFP作为对照组，检测ApoE对Aβ生成的影响。ELISA结果(图1，面板C)显示ApoE2的表达显著减少了Aβ40的生成，但是ApoE3和ApoE4则没有此效果。相比于ApoE4，ApoE2的表达也显著减少了Aβ40。这些结果表明与APP在同一种细胞中表达的ApoE2可以减弱APP的淀粉样代谢，减少Aβ40；增加APP的非淀粉样代谢产物α-CTF。而ApoE4并不具备这一能力。
本发明进一步研究ApoE的哪些片段发挥了减弱APP淀粉样代谢的功能。ApoE2、ApoE3和ApoE4仅在N端功能域有两个氨基酸残基不同，在C端功能域的氨基酸残基序列完全相同。因此，本发明构建了人源ApoE的N端片段和C端片段的表达质粒，并分别命名为ApoE2NT、ApoE3NT、ApoE4NT或者ApoE CT蛋白的质粒用于后续实验。在细胞中表达时，所有构建蛋白的N端都进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66)。在HEK 293T细胞中将APP分别与ApoE2 NT、ApoE4 NT和ApoE CT共表达，GFP作为对照组。免疫印迹实验结果(图1，面板D-E)显示只有共表达ApoE CT显著地增加了α-CTF到原有水平的5倍以上。共表达ApoE2 NT或ApoE4 NT都没有改变α-CTF。同时，ELISA实验结果显示ApoE CT显著地降低Aβ40到GFP对照的～25％，三种ApoE NT都没有减少Aβ40(图1，面板F)；同样地，ApoE CT显著地降低Aβ42到GFP对照的～26％(图1，面板G)。
AD是一种神经系统疾病，在脑中Aβ主要由神经元中表达的APP剪切产生。本发明构建了在神经元中表达ApoE CT的慢病毒。ELISA结果(图1，面板H)表明在神经元中过表达ApoE CT可以使原代培养的小鼠海马或者皮层神经元中內源生成的Aβ40分别降低到对照组的～71％和～73％。同样(图1，面板I)，在人源多能干细胞诱导的神经元中过表达的ApoE CT也使人神经元內源生成的Aβ40降低到对照组的～54％。
这些结果说明，在多种细胞中，ApoE2均能以细胞自主式的方式通过它的C端片段增加APP的非淀粉样代谢产物α-CTF；同时减弱淀粉样代谢通路，减少Aβ。
本发明各实验所使用的ApoE CT氨基酸序列如下：
3.2分泌在培养液中的ApoE2和ApoE CT不影响APP的剪切
培养表达ApoE2或者ApoE CT的HEK 293T细胞，获得培养液，结果显示培养液中含有分泌出来的ApoE2或者ApoE CT(图2，面板A)。将表达APP的HEK 293T细胞与这种含有ApoE2或ApoE CT的培养液共孵育。表达APP的HEK 293T细胞与含有GFP的培养液共孵育作为阴性对照。结果显示，与GFP对照相比，共孵育的培养液中的ApoE2和ApoE CT并未显著改变α-CTF水平(图2，面板B-C)。免疫荧光结果(图2，面板D-E)显示，即使经过长时间的共孵育，培养液中的ApoE2和ApoE CT也主要粘附在细胞表面，几乎不进入细胞内。而在HEK 293T细胞(图2，面板D)或小鼠原代培养神经元(图2，面板E)中表达的ApoE2和ApoE CT则可以在细胞内大量存在。
这一结果说明，细胞培养液中的ApoE2和ApoE CT并不能改变α-CTF和Aβ的生成。ApoE2和ApoE CT必须要以细胞自主性的方式，即必须与APP处于同一细胞中，才能调控APP的剪切，从而达到增加α-CTF并且降低Aβ的生成的效果。其中，有多种方式可以实现让ApoE2和ApoE CT进入目标细胞，具体包括：脂质体传递编码此类蛋白的DNA或者RNA，如果不加特殊说明，本发明采用脂质体将编码相应蛋白的质粒转染到细胞中表达ApoE和ApoE CT；慢病毒和腺相关病毒侵染细胞等方式都可以使ApoE2和ApoE CT在表达APP的细胞中表达，本发明在神经元中表达的ApoE2或者ApoE CT采用了慢病毒或者腺相关病毒表达系统；细胞穿膜肽，细胞胞吞增强等方式都可以使ApoE2和ApoE CT蛋白进入表达APP的目标细胞内等，本发明在后文详细探讨了细胞穿膜肽的作用。
3.3 ApoE CT减弱APP淀粉样代谢的核心序列
为探究ApoE CT上的哪些氨基酸是改变APP代谢的核心序列，本发明在ApoE CT的N端添加ApoE原有的1、2、3、4或5个氨基酸残基，构建表达CT NA1、CT NA2、CT NA3、CT NA4、CT NA5的质粒；在ApoE CT的N端删除1、2、3、4或5个氨基酸，构建表达CT ND1、CT ND2、CT ND3、CT ND4、CT ND5的质粒：在ApoE CT的C端删除ApoE原有的5、10个氨基酸，构建表达CT CD5和CT CD10的质粒。上述CT NA1、CT NA2、CT NA3、CT NA4、CT NA5、CT ND1、CT ND2、CT ND3、CT ND4、CT  ND5、CT CD5和CT CD10的氨基酸序列分别如SEQ ID NOs：13-24所示。在细胞中表达时，所有构建蛋白的N端都进一步连接了ApoE本身的信号肽(SEQ ID NO：66)。
SEQ ID NO：13(CT NA1)：
SEQ ID NO：14(CT NA2)：
SEQ ID NO：15(CT NA3)：
SEQ ID NO：16(CT NA4)：
SEQ ID NO：17(CT NA5)：
SEQ ID NO：18(CT ND1)：
SEQ ID NO：19(CT ND2)：
SEQ ID NO：20(CT ND3)：
SEQ ID NO：21(CT ND4)：
SEQ ID NO：22(CT ND5)：
SEQ ID NO：23(CT CD5)：
SEQ ID NO：24(CT CD10)：
在HEK 293T细胞中将人源的APP和β分泌酶与上述每个质粒分别共表达，GFP作为对照组，使用ELISA测定上述蛋白对Aβ40生成的影响。ELISA结果(图3，面板A-B)表明在ApoE CT的N端添加1个其本身原有的氨基酸残基后，CT NA1依然减少Aβ40生成；但是添加两个或者以上的氨基酸残基，CT NA2、CT NA3、CT NA4和CT NA5就完全丧失了减少Aβ的能力。在ApoE CT的N端删除1-5个氨基酸残基后，ApoEND1，ApoEND2，ApoEND3，ApoEND4和ApoEND5依然可以减少Aβ40。在ApoE CT的C端即使删除5或10个氨基酸残基，CT CD5、CT CD10依然减少Aβ的生成。这些结果说明，ApoE CT的N端非常重要，即使只有1或者2个氨基酸的改变都有可能影响ApoE CT抑制Aβ40生成的活性。但是其C端残基对活性的影响则相对较弱。
根据以上结果，本发明在ApoE CT的N端删除3个氨基酸残基(CT ND3)的基础上，在ApoE CT的C端分别删除10、11、12、13、14、15、20或25个氨基酸残基，构建了表达CT ND3CD10、CT ND3CD11、CT ND3CD12、CT ND3CD13、CT ND3CD14、CT ND3CD15、CT ND3CD20和CT ND3CD25的质粒，其氨基酸序列如SEQ ID NOs：25-32所示。在细胞中表达时，所有构建蛋白的N端都进一步连接了ApoE本身的信号肽(SEQ ID NO：66)。
SEQ ID NO：25(CT ND3CD10)：
SEQ ID NO：26(CT ND3CD11)：
SEQ ID NO：27(CT ND3CD12)：
SEQ ID NO：28(CT ND3CD13)：
SEQ ID NO：29(CT ND3CD14)：
SEQ ID NO：30(CT ND3CD15)：
SEQ ID NO：31(CT ND3CD20)：
SEQ ID NO：32(CT ND3CD25)：
在HEK 293T细胞中将人源的APP、β分泌酶与上述每个质粒分别共表达，GFP作为对照组，使用ELISA测定上述蛋白对细胞中Aβ生成的影响。ELISA结果(图3，面板C)表明ApoE CT的N端删除3个氨基酸残基并且C端删除10、11、12、13、14、15、20或25个氨基酸后，CT ND3CD10、CT ND3CD11、CT ND3CD12、CT ND3CD13、CT ND3CD14、CT ND3CD15、CT ND3CD20或者CT ND3CD25依然可以显著减少Aβ40。这些结果表明ApoE CT抑制Aβ生成的核心序列结束位置在ApoE蛋白第274位残基到最末尾氨基酸残基。
3.4 ApoE CT通过γ剪切影响APP的剪切
为了研究ApoE CT影响APP剪切的机制，本发明检测了ApoE CT是否会改变APP关键分泌酶的表达水平。本发明利用qRT-PCR和Western Blot技术分别检测了ADAM10(主要的α分泌酶)、BACE1(β分泌酶)、PS1和PS2(γ分泌酶活性亚基)的mRNA和蛋白表达量。qRT-PCR(图4，面板A)和Western Blot(图4，面板B)结果显示，与GFP对照相比，在细胞中过表达ApoE CT并不会改变ADAM10、BACE1、 PS1和PS2的mRNA和蛋白表达量，说明ApoE CT不是通过改变APP代谢酶的表达量来影响APP剪切。
α-CTF是γ分泌酶的一个底物，而Aβ则是γ分泌酶的剪切产物。所以γ分泌酶活性发生变化时，α-CTF和Aβ的生成量会显著变化，而ApoE CT正是改变了α-CTF和Aβ的生成量。因此ApoE CT有可能改变了γ分泌酶的活性。为验证这一猜想，本发明在HEK293T细胞中共表达APP和γ分泌酶的活性中心PS1。免疫印迹实验结果(图5，面板A-B)显示，过表达PS1(可以增强γ分泌酶活性)降低了α-CTF。而表达ApoE CT或者用1μMγ分泌酶抑制剂PF03084014处理都使α-CTF显著增多(图5，面板A-B)。这一结果提示，在细胞中表达的ApoE CT对APP剪切的影响与抑制γ分泌酶的效果相似。β-CTF是APP经过β分泌酶切的产物，也是γ分泌酶的底物，只需经过一步γ酶切就可以生成Aβ。ELISA结果(图5，面板C-D)显示表达ApoE CT不仅使β-CTF生成的Aβ40下降到原有水平的～80％，也使Aβ42下降到原有水平的～8％；说明ApoE CT降低Aβ的能力可能是通过调节γ酶切来实现的。本发明又进一步将去掉APP胞内域(APP intracellular C-terminal domain)的APP(APPΔAICD)与BACE1(β分泌酶)、ApoE CT在HEK 293T细胞中共表达。APPΔAICD不需要经过γ酶切，只要经过一步β酶切就可以产生Aβ。ELISA结果(图5，面板E)显示，表达ApoE CT不再降低由APPΔAICD所生成的Aβ，说明ApoE CT并不影响β酶切。
这些结果说明在细胞中表达的ApoE CT降低Aβ需要γ酶切，过表达的ApoE CT模拟了γ分泌酶抑制剂的效果；ApoE CT可能是通过抑制APP的γ酶切，从而增加α-CTF，减少Aβ。
3.5ApoE CT调控APP的剪切需要γ分泌酶参与
γ分泌酶的活性中心是PS1或者PS2，在敲除PS1和PS2后(PS1/2 dKO)，α-CTF无法通过γ分泌酶酶切进行代谢，因此大量聚集。免疫印迹结果(图6，面板A和D)显示，在野生型HEK 293T细胞系中，表达的ApoE CT显著地增加了α-CTF；在表达GFP的PS 1/2 dKO细胞中，相比于野生型HEK 293T细胞系，α-CTF含量显著增加。在PS1/2 dKO细胞中表达ApoE CT并不进一步增加α-CTF，说明在没有γ分泌酶时，ApoE CT不再能够增加α-CTF。在补充表达PS1或者PS2的PS1/2 dKO细胞中表达的ApoE CT则可以显著增加了α-CTF(图6，面板B-D)，说明ApoE CT增加α-CTF依赖于PS1或者PS2。同样地，在补充表达PS2的PS1/2 dKO细胞系中，过表达ApoE CT可以减少Aβ40(图6，面板E)。这些结果说明ApoE CT只有在γ分泌酶存在的情况下才能调控APP的代谢。
3.6 ApoE在纯化体系中直接抑制γ分泌酶对β-CTF的剪切
为了进一步验证ApoE是否直接抑制γ分泌酶，本发明纯化了β-CTF，ApoE2，ApoE2NT和γ分泌酶，免疫印迹验证了这些纯化的蛋白(图7，面板A)。其中PS1、NCT和PEN2是γ分泌酶的组成蛋白。ELISA结果(图7，面板B)显示，γ分泌酶与β-CTF共孵育可产生Aβ，此时加入的10μMγ分泌酶抑制剂(PF03084014)可以抑制Aβ的生成。在此反应体系中加入纯化的ApoE2以浓度依赖性的方式抑制了Aβ40的产生，其半抑制浓度(IC50)为～1.2μM(图7，面板C)。另外，在同等浓度条件下(2.5μM)，ApoE2可以显著抑制Aβ产生，但是ApoE2NT无法抑制Aβ40的产生(图7，面板D)。这些结果表明，ApoE2可以直接抑制γ分泌酶对β-CTF剪切，而ApoE CT对ApoE的该活性是必须的，提示ApoE CT是ApoE2抑制γ分泌酶剪切APP的关键片段。
3.7 ApoE CT不抑制APLP1的γ酶切
APLP1(APP like protein 1)是一种和APP结构相似的蛋白，同样也是α和γ分泌酶的底物之一。免疫印迹结果(图8)显示γ分泌酶的抑制剂PF03084014可以抑制HEK293T细胞中APLP1的γ剪切，使α-like CTF显著增多；但是共表达的ApoE CT并不会增加APLP1的α-like CTF，说明ApoE CT并不抑制APLP1的γ酶切。这一结果表明ApoE CT抑制γ分泌酶的酶切活性具有一定的底物选择性。
3.8在神经元中表达的ApoE CT减少了AD模型小鼠脑内的淀粉样斑块
在4周龄的AD模型小鼠(5×FAD)皮层和海马体中注射能够在神经元中表达ApoE CT的腺相关病毒。在神经元中表达ApoE CT(或GFP作为阴性对照)六个月后，检测其对淀粉样斑块的影响，对ApoE CT、GFP和淀粉样斑块进行染色。结果显示，在神经元中表达ApoE CT(N端连接了人源ApoE本身的信号肽(SEQ ID NO：66))的5×FAD小鼠脑中淀粉样斑块的面积更小、荧光强度更弱、淀粉样斑块的总面积在大脑中所占比例更小、淀粉样斑块的密度也减小(图9，面板A-E)。这些结果表明表达在神经元中的ApoE CT抑制了5×FAD小鼠脑中淀粉样斑块的沉积，减弱了小鼠的AD病理进程。
3.9在神经元中表达的ApoE2可以抑制小鼠内源Aβ40的产生
上述实验已经表述了ApoE CT是ApoE2抑制γ分泌酶活性的关键功能域，并且ApoE CT可以抑制小鼠神经元产生Aβ40。接下来探究了ApoE2是否同样抑制神经元中 内源Aβ40的产生(图10)。在小鼠的原代培养神经元中分别使用慢病毒表达系统(lentivirus)和神经元特异性启动子表达GFP或者ApoE2。ApoE2的N端进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66)。ELISA结果(图10，面板A)展示，与GFP对照相比，在神经元中过表达的ApoE2可以降低內源生成的Aβ40。进一步，利用AAV表达系统将ApoE2表达在野生型小鼠(WT mouse)大脑中的神经元中。ELISA结果(图10，面板B)显示，与GFP对照相比，在小鼠大脑神经元内表达的ApoE2同样降低了内源Aβ40的生成。这些结果说明ApoE2与ApoE CT一样可以抑制神经元中内源Aβ40的产生。
3.10在两端添加标签蛋白的ApoE依然拥有调控APP剪切的活性
为探究在ApoE上添加的标签蛋白是否会破坏ApoE的活性，本发明分别在ApoE2和ApoE CT的C端和N端分别添加标签蛋白V5(SEQ ID NO：82)，分别命名为ApoE2-V5(V5在C端)、V5-ApoE2(V5在N端)、ApoE CT-V5和V5-ApoE CT。所有构建蛋白的N端都进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66)。并将这些表达质粒与APP在HEK 293T细胞中共表达。免疫印迹结果显示(图11)，不论是ApoE2-V5和V5-ApoE2(图11，面板A-B)，还是ApoE CT-V5和V5-ApoE CT(图11，面板C-D)都显著地增加α-CTF，且活性与ApoE2或ApoE CT相近。这说明在两端添加标签蛋白的ApoE2和ApoE CT依然具有调控APP剪切的活性。
3.11 ApoE CT的突变体依然可以调控APP的剪切
为探究ApoE CT的突变是否会改变ApoE CT调控APP剪切的活性，本发明构建并分析了一系列的ApoE CT的突变体。包括单个位点突变：CT A20，CT A40，CT A60，CT A65，CT A72，CT A80；双位点突变：CT A36/74；多位点突变：CT E5，CT E10，CT E20，CT F5，CT R10，CT LF5，CT LM5。所述突变位点位置是相对于ApoE CT(SEQ ID NO：33)的位置。其氨基酸序列如SEQ ID NOs：46-59所示。所有构建蛋白的N端都进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66)。
SEQ ID NO：46(CT A20；相对于SEQ ID NO：33Q20A；相对于SEQ ID NO：12Q235A)：
SEQ ID NO：47(CT A40；相对于SEQ ID NO：33E40A；相对于SEQ ID NO：12 E255A)：
SEQ ID NO：48(CT A60；相对于SEQ ID NO：33 Q60A；相对于SEQ ID NO：12 Q275A)：
SEQ ID NO：49(CT A65；相对于SEQ ID NO：33 V65A；相对于SEQ ID NO：12 V280A)：
SEQ ID NO：50(CT A72；相对于SEQ ID NO：33V72A；相对于SEQ ID NO：12 V287A)：
SEQ ID NO：51(CT A80；相对于SEQ ID NO：33 P80A；相对于SEQ ID NO：12 P295A)：
SEQ ID NO：52(CT A36/74；相对于SEQ ID NO：33 R36A+T74A；相对于SEQ ID NO：12 R251A+T289A)：
SEQ ID NO：53(CT E5)：
SEQ ID NO：54(CT E10)：
SEQ ID NO：55(CT E20)：

SEQ ID NO：56(CT F5)：
SEQ ID NO：57(CT R10；相对于SEQ ID NO：33S75A+P78A+V79A+P80A+S81A+D82A+N83A+H84A；相对于SEQ ID NO：12S290A+P293A+V294A+P295A+S296A+D297A+N298A+H299A)：
SEQ ID NO：58(CT LF5；相对于SEQ ID NO：33 R11A+D12A+R13A+L14A+D15A；相对于SEQ ID NO：12 R226A+D227A+R228A+L229A+D230A)：
SEQ ID NO：59(CT LM5；相对于SEQ ID NO：33 E23A+V24A+R25A+K 27A；相对于SEQ ID NO：12 E238A+V239A+R240A+K242A)：
在HEK 293T细胞中将人源的APP与上述每个质粒分别共表达，GFP作为阴性对照组，ApoE CT作为阳性对照。免疫印迹结果显示，构建的ApoE CT的单位点突变和双位点突变(图12，面板A-B)，CT A20、CT A40、CT A60、CT A65、CT A72、CT A80和CT A36/74都显著地增加了α-CTF；并且相比于ApoE CT，这些突变增加α-CTF的程度没有降低(CT A72、CT A80和CT A36/74增加α-CTF的程度甚至更高)。多位点突变CT E5、CT E10、CT E20和CT F5则不再能增加α-CTF；但是CT R10、CT LF5和CT LM5依然显著地增加了α-CTF，并且这些突变和ApoE CT增加了相似水平的α-CTF(图12，面板C-D)。这些数据表明ApoE CT的突变体(如CT A20、CT A40、CT A60、CT A65、CT A72、CT A80、CT A36/74、CT R10、CT LF5和CT LM5)同样具备与原始ApoE CT类似的、调节APP剪切的活性。
3.12在ApoE2内部添加或删除氨基酸残基依然能够调控APP代谢
本实验探究在ApoE2的内部添加或者删除氨基酸残基是否会影响ApoE的活性。本发明首先在ApoE2(SEQ ID NO：12)第215位和第216位氨基酸残基之间添加了 标签蛋白，3XFLAG，并命名为ApoE2-M3F(SEQ ID NO：60；3XFLAG序列加粗；下划线指示SEQ ID NO：33序列)，该修饰不影响ApoE CT序列(SEQ ID NO：33)。本发明还构建了ApoE2 MD10(SEQ ID NO：61)，ApoE2 MD10是删除ApoE2的第244至254位氨基酸残基得到的，并且删除的这些氨基酸位于ApoE2发挥调控APP代谢的C端区域，即位于SEQ ID NO：33序列中。在细胞中表达时，所有构建蛋白的N端都进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66)。
SEQ ID NO：60(ApoE2-M3F)
(SEQ ID NO：12；下划线指示SEQ ID NO：33序列；加粗指示3XFLAG序列，SEQ ID NO：77)
SEQ ID NO：61(ApoE2 MD10)

(SEQ ID NO：12；下划线指示SEQ ID NO：33序列；方框指示ApoE2 MD10中删除的序列)
ARMEEMGSRTRDRLDEVKEQVAEVRAKLEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH(SEQ ID NO：76；删除ApoE2的第244至254位氨基酸残基的ApoE CT)
GSGGSDYKDHDGDYKDHDIDYKDDDDKSGSGS(SEQ ID NO：77：3XFLAG)
在HEK 293T细胞中将人源的APP分别与ApoE2-M3F、ApoE2 MD10共表达，GFP作为阴性对照组。免疫印迹结果显示(图13)，不论是在ApoE2中间位置添加氨基酸残基的ApoE2-M3F(不影响ApoE CT序列)，还是删除C端功能区域中部氨基酸残基的ApoE2 MD10都依然显著地增加了α-CTF。这一结果表明在ApoE2内部添加或者删除某些氨基酸序列，ApoE2依然可以调控APP代谢。
3.13非人源的ApoE CT也可以调控APP的剪切
为进一步探究非人源的ApoE是否也具有同样的活性，本实验分析了非人灵长类来源的ApoE CT(monkey CT)。该序列与人源的ApoE CT在氨基酸序列上高度相似，仅有三个氨基酸取代R36S+T74A+A76T(图14，面板C)。猴ApoE CT氨基酸序列如下所示。
SEQ ID NO：62(monkey CT)：
在HEK 293T细胞中将人源的APP与非人灵长类ApoE CT(在细胞中表达时，N端都进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66))共表达，GFP作为阴性对照组，人源ApoE CT(N端都进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66))作为阳性对照。免疫印迹结果(图14，面板A和B)显示非人灵长类ApoE CT(monkey CT)和人源的ApoE CT同样增多了α-CTF。这一结果表明非人灵长类的ApoE CT同样具有调控APP剪切的活性。
3.14添加穿膜肽的ApoE具有调控APP剪切的活性
为探究穿膜肽是否改变ApoE的活性，本发明在ApoE上添加穿膜肽。所选用的穿膜肽序列如下：
SEQ ID NO：63(R9)：RRRRRRRRR
SEQ ID NO：64(FGF4)：AAVLLPVLLAAP
SEQ ID NO：65(RDP)：KSVRTWNEIIPSKGCLRVGGRCHPHVNGGGRRRRRRRRR
在人源ApoE2的N端添加R9、FGF4、RDP构建的ApoE2的突变体分别被命名为R9-ApoE2、FGF4-ApoE2、RDP-ApoE2。所有蛋白构建体N端都进一步连接了人源ApoE本身的信号肽(SEQ ID NO：66)。将R9-ApoE2、FGF4-ApoE2、RDP-ApoE2分别与人源的APP在HEK 293T细胞中共表达，GFP作为阴性对照组。免疫印迹结果(图15)显示虽然FGF4-ApoE2没有增加α-CTF，但是R9-ApoE2和RDP-ApoE2都显著地增加了α-CTF。这一结果说明连有穿膜肽的ApoE2依然可以调控APP的剪切。
3.15信号肽对于ApoE CT调控APP剪切的活性是必须的。
上述实验用来探究细胞中表达的ApoE功能的所有表达相应多肽的氨基酸序列前段包含有人源ApoE本身的信号肽(ApoE SP；SEQ ID NO：66)，信号肽位于ApoE或ApoE CT和前文所述ApoE或ApoE CT各突变体的N端。为探究信号肽是否会影响ApoE CT的活性，本发明更换了ApoE CT的信号肽，分别更换为载脂蛋白A1和载脂蛋白J的信号肽(ApoA1 SP和ApoJ SP；SEQ ID NO：67和SEQ ID NO：68)。同时还分析了不连接信号肽的ApoE CT活性。
SEQ ID NO：66(ApoE SP)：MKVLWAALLVTFLAGCQA
SEQ ID NO：67(ApoAI SP)：MKAAVLTLAVLFLTGSQA
SEQ ID NO：68(ApoJ SP)：MMKTLLLFVGLLLTWESGQVLG
更换为载脂蛋白A1信号肽的ApoE CT突变体，命名为ASP CT；更换为载脂蛋白J信号肽的ApoE CT突变体，命名为JSP CT；不含有信号肽的ApoE CT突变体，命名为NSP CT。在HEK 293T细胞中将人源的APP与上述每个ApoE CT的突变体分别共表达，GFP作为阴性对照组，ApoE CT作为阳性对照。免疫印迹结果(图16)显示ASPCT和JSP CT依然可以增加α-CTF，但增加α-CTF的程度不如带有ApoE本身信号肽的ApoE CT；删除信号肽后，NSP CT不再增加α-CTF。这一结果表明在细胞中表达的ApoE CT需要连有信号肽才能调控γ分泌酶对APP的剪切，但是这个信号肽可以是不同的信号肽，并不局限于ApoE原有的信号肽。
4.实验结论
本发明首次发现ApoE2可以在神经元中以细胞自主性的方式调控APP的剪切，这一过程依赖于ApoE2的C端片段。ApoE的C端片段不仅增加APP的非淀粉样代谢产物 α-CTF；而且减弱APP的淀粉样代谢，减少Aβ。还发现ApoE2及其C端片段调控APP代谢，并不是通过分泌出来的ApoE蛋白在细胞外间接影响细胞中APP的剪切；而是通过与APP共存于同一细胞中的ApoE2或其C端片段直接抑制APP的γ酶切来实现的，这是一种细胞自主性作用方式。在使用纯化蛋白完成的实验中也发现ApoE2可以直接抑制γ分泌酶对β-CTF的酶切，而缺失C端的ApoE NT则不能抑制γ分泌酶对β-CTF的酶切。此外，ApoE的C端片段不可以抑制APLP1的γ酶切，说明ApoE的C端片段对γ酶切的抑制作用是有一定的底物选择性的。本发明还发现，神经元中特异性表达的ApoE的C端片段显著降低AD模型小鼠脑中淀粉样斑块的大小和密度。小鼠神经元细胞中特异性表达的ApoE可以抑制内源Aβ40的产生。此外，在N或C端添加标签蛋白不会影响ApoE2和ApoE C端片段抑制APPγ酶切的活性。本发明还构建了一系列ApoE C端片段和ApoE2内部序列的突变体，具备与原始ApoE CT或ApoE2相似的抑制APPγ酶切的活性。本发明还发现ApoE的N端添加某些穿膜肽不会影响其抑制APPγ酶切的活性，但是如果N端缺乏信号肽会导致ApoE抑制APPγ酶切活性的缺失。本发明的发现不局限于人源ApoE C端片段，非人灵长类的ApoE C端片段也有相似的抑制APPγ酶切的活性。
这些结果说明ApoE2及其C端片段(或基于ApoE2或C端片段的突变体)可以作为一种潜在药物用于AD的预防或者治疗。而此类潜在药物需要在神经元中以细胞自主性的方式起作用。脂质体传递DNA或者RNA，慢病毒和腺相关病毒侵染细胞等方式都可以使ApoE2和/或其C端片段在目标细胞中表达；细胞穿膜肽，细胞胞吞增强等方式也可以使ApoE2和/或其C端片段的蛋白在目标细胞内大量存在；进而ApoE2和/或其C端片段以细胞自主性的方式抑制γ分泌酶的酶切活性，减少Aβ，减弱APP的淀粉样代谢。
上述实验仅是以AD疾病为例说明ApoE2和/或其C端片段以细胞自主性的方式抑制γ分泌酶的酶切活性。此外，诸如淀粉样脑血管病也与γ分泌酶功能的异常相关。而唐氏综合症也容易发展成AD。因此，ApoE2和/或其C端片段在这些APP或γ分泌酶相关疾病的治疗和预防中也有应用潜力。
实施例2.ApoE对γ分泌酶的精确抑制是其预防阿尔茨海默病的基础
1.概要
异常低的γ分泌酶活性与大多数早老素突变有关，而这些突变会导致家族性阿尔茨海默病(familial AD，fAD)。然而，γ分泌酶在更普遍的散发性AD(sporadic AD， sAD)中的作用仍未得到解决。人类载脂蛋白E(ApoE)是sAD最重要的遗传风险因素，本发明报告了ApoE与γ分泌酶相互作用，并通过其保守的C末端区域(CT)以细胞自主方式以底物特异性抑制γ分泌酶的活性。这种ApoE CT介导的抑制活性在不同的ApoE亚型中受到不同程度的损害，导致ApoE2＞ApoE3＞ApoE4效力等级顺序与其相关的AD风险成反比。有趣的是，在AD小鼠模型中，神经元中表达的ApoE CT可以从其他区域迁移至淀粉样版块，并减缓淀粉样斑块的形成。总之，本实验的数据揭示了ApoE具有底物特异性的γ分泌酶抑制剂的活性，并强烈表明ApoE的这种对γ分泌酶的精确抑制可以防止sAD的风险。
2.介绍
阿尔茨海默病(AD)是最常见的神经退行性疾病，影响全球近5000万人(Prince，2015年)。β淀粉样蛋白(Aβ)假说将AD归因于Aβ的过度聚集，Aβ是一种由β分泌酶和γ分泌酶连续切割淀粉样前体蛋白(APP)产生的多肽(De Strooper和Karran，2016年；De Strooper等人，1998年；Haass和Selkoe，1993年；Hardy和Selkoe，2002年；Wolfe等人，1999年)。γ分泌酶的酶活催化中心——早老素1(presenilin 1，PS1)和早老素2(PS2)——的突变会导致罕见的家族性AD(fAD)。基于这些证据，γ分泌酶被认为是AD的药物靶标。不幸的是，使用常规γ分泌酶抑制剂治疗会引起严重的副作用(Panza等人，2019年；Xia，2019年)。最近的研究发现，与fAD相关的早老素突变通常与γ分泌酶的整体活性降低有关(Shen和Kelleher，2007年；Sun等人，2017年；Walker等人，2005年；Xia等人，2015年)，这与基于Aβ假设的γ分泌酶的预测作用相悖。鉴于基于Aβ的药物研发工作的结果不尽如人意(Karran和De Strooper，2016年)，迫切需要揭示γ分泌酶在更为普遍的散发性AD(sAD)中发挥的作用。
sAD没有致病基因，但其患病风险受多种遗传因素影响。其中，载脂蛋白E4(ApoE4)是最具影响力的一种(Corder等人，1993；Rhinn等人，2013)。ApoE基因编码三种亚型，仅在其N末端区域(NT)具有单个氨基酸差异：ApoE2、3和4(Kanekiyo等人，2014年)。ApoE4不仅会增加AD的风险，还会导致发病年龄提前，加重AD的严重程度(Belloy等人，2019；Li等人，2020)。相反，ApoE2降低了AD风险。在啮齿动物的大脑中，在正常生理条件下，ApoE通常仅在胶质细胞中检测到(Kanekiyo等人，2014)，而Aβ则由神经元产生。然而，ApoE也在人类神经元(Aoki等人，2003年；Han等人，1994年；Metzger等人，1996年；Strittmatter等人，1993年； Xu等人，1999年)和受伤的啮齿动物神经元中表达(Xu等人，2006年)。人脑中ApoE的代谢更类似于神经元中表达的ApoE而不是胶质细胞中表达的ApoE的代谢(Brecht etal.，2004)。最近一项使用携带不同ApoE亚型的人诱导多能干细胞(hiPSC)产生的神经元的研究表明，ApoE4阳性神经元中Aβ的产生增加，表明神经元中的ApoE会以细胞自主性的方式调节APP的代谢(Wang等人，2018年)。这些证据表明神经元中的ApoE有着非常关键的生理作用，但是这一点从未被充分研究过。
本实验报告了ApoE直接与γ分泌酶相互作用，并通过其C末端区域特异性抑制APP的γ酶切。值得兴奋的是，这种抑制活性在不同的ApoE亚型中受到不同的损害，与ApoE亚型所对应的AD风险呈现负相关性。此外，神经元中表达的ApoE CT在淀粉样斑块周围积聚，具有在该淀粉样蛋白生成热点区域内实现局部γ抑制的潜在可能性(Jordà-Siquier等人，2022年)。总之，本实验的数据表明ApoE对γ分泌酶的精确抑制可防止sAD的风险，并将ApoE CT鉴定为具有前所未有特异性的新型γ分泌酶抑制剂。
3.实验结果
3.1.内源性ApoE和APP在人类皮层神经元中共表达，具有重叠的亚细胞分布模式
ApoE在啮齿动物大脑中主要由胶质细胞表达，Aβ则由神经元中表达的APP代谢产生。因此，大多数研究都集中在胶质细胞分泌的ApoE如何影响胞外的淀粉样斑块形成(Huang等人，2017年；Huynh等人，2017年；Liu等人，2017年)。然而，最近的一项研究表明ApoE能够以细胞自主方式调节Aβ的产生(Wang等人，2018年)。本研究在探寻此潜在机制之前，首先使用免疫荧光对人脑样本进行染色，以期验证ApoE确实在神经元中表达。实验结果与之前报道的人脑数据一致(Aoki et al.，2003；Belonwu et al.，2022；Han et al.，1994；Zalocusky et al.，2021)，在人脑神经元和胶质细胞中均检测到ApoE。具体而言，年轻对照组(年轻，16-23岁；图17，面板A；图25，面板B；以及图32人脑供体的信息)、非AD患者的年龄匹配对照组(老年，＞60岁)和AD患者(AD，＞60岁；图25，面板A和B)的大脑样本中30-50％的皮层神经元呈ApoE阳性；同时，40-62％的ApoE阳性细胞是人类皮层样本中的神经元(图17，面板A；图25，面板A和C)。此外，人脑皮层星形胶质细胞也表达ApoE(图25，面板D)。在猴脑皮层中，大多数可检测到的ApoE信号均位于神经元中(图17，面板A)。在人类AD患者大脑的淀粉样蛋白斑块中可检测到很强的ApoE染色信号，但在没有罹患AD的对照大脑样本的相同区域则无法检测到ApoE信号 (图25，面板E)。重要的是，免疫染色显示内源性ApoE和APP在人类皮层神经元中共存，并且具有重叠的亚细胞信号模式(图17，面板B)。
3.2.ApoE2以细胞自主方式增加α-CTF和减少Aβ
为了研究ApoE是否能以细胞自主方式来调控APP的代谢，本实验将APP与ApoE在小鼠神经元细胞系(N2A；图17，面板C)和HEK 293T细胞(图26，面板C)中共表达。α-CTF是APP的非淀粉样代谢途径的酶切产物，已知其产生主要取决于ADAM10(图26，面板A和B)，一种α-分泌酶(Esch等人，1990年；Haass等人，1992年；Mucke和Selkoe，2012年)。在N2A和HEK293T细胞中，ApoE2或ApoE3显著增加了共表达APP产生的α-CTF水平(图17，面板C和D，N2A细胞；图26，面板C和D，HEK 293T细胞)。此外，ApoE2也显著降低了共表达APP产生的Aβ40水平(图17，面板E)。有趣的是，ApoE4和APP的共表达未能改变α-CTF(图17，面板C和D)和Aβ40水平(图17，面板E)。这些结果表明ApoE2以细胞自主方式增加α-CTF。
3.3.ApoE2的C端区域比全长ApoE2在提高α-CTF水平方面活性更强
ApoE包含三个区域：一个N末端区域(ApoE NT)、一个铰链区域和一个保守的C末端区域(ApoE CT)(图33)(Chen et al.，2011)。三种ApoE亚型之间的唯一序列差异位于它们的NT之内。使用具有ApoE亚型截短突变体的HEK 293T细胞进行的实验表明，来自ApoE2、3或4的NT并未显著改变共表达APP所产生的α-CTF(图26，面板E和F)。相反，共表达保守的ApoE CT却导致α-CTF水平升高了6倍以上(图26，面板G和H)。
引人注目的是，实验显示与APP一起共表达ApoE CT使得分泌到细胞培养基中的Aβ40减少了约75％(图26，面板I)，细胞裂解物中的Aβ40减少了约42％(图26，面板J)，并且还显著减少了约74％的Aβ42(图26，面板K)，但是Aβ42/Aβ40的比率保持不变(图26，面板K)。但与APP和BACE1共表达的ApoE CT并未改变β-CTF水平(图26，面板L和M)。所有ApoE NT均未能显著降低Aβ40(图26，面板I)。类似地，使用N2A细胞进行的实验显示，只有与APP共表达ApoE CT才能显著增加α-CTF，而共表达ApoE NT则不可以(图17，面板F和G)；此外，ApoE CT也显著减少了总Aβ40(图17，面板H)。
实验发现与ApoE2做对比，ApoE CT同样显著增加了α-CTF水平(图17，面板 I和J，N2A细胞；图26，面板N和O，HEK 293T细胞)，这表明ApoE CT调节APP代谢的活性显然比全长ApoE2更高。这些结果表明ApoE CT这一ApoE的保守区域可以提高α-CTF水平，并表明ApoE的NT可能以某种方式阻碍ApoE CT的这一活性。
为了研究分泌的ApoE是否也会影响APP的酶切，本研究用含有分泌的ApoE或ApoE CT的培养液培养仅表达APP的HEK 293T细胞(图27)。实验表明培养液中游离的ApoE2或ApoE CT不会改变细胞中APP生成的α-CTF(图27，面板A、β和C)。值得注意的是，ApoE2在37℃的条件培养基中能够稳定存在48小时(图27，面板D)，这可排除由ApoE降解带来的可能干扰因素。对于用条件培养基处理的HEK 293T细胞和原代培养的大鼠神经元，ApoE2或ApoE CT主要在细胞膜外检测到(图27，面板E和F)。相比之下，细胞或神经元表达的ApoE CT要分布在细胞或神经元内(图27，面板E和F)。比较细胞裂解液中的ApoE2与分泌在培养基中ApoE2的相对量，发现两者含有相近的ApoE2(图27，面板G)。此外，来自条件培养基的少量ApoE在共孵育后可被细胞或神经元内吞(图27，面板E和F)。因此，ApoE调节APP代谢更可能是由细胞内ApoE以细胞自主的方式所介导的，而不是由分泌的ApoE介导的。
3.4.神经元中表达的ApoE CT减少了内源性Aβ。
除了人类APP，ApoE CT还提高了瞬时表达小鼠APP(mAPP)的HEK 293T细胞中的α-CTF水平(图26，面板P和Q)。原代培养神经元的实验结果表明，与GFP对照相比，在小鼠皮质和海马神经元中过表达ApoE CT(慢病毒感染)后，内源性Aβ40减少了30％(图17，面板K)。在hiPSC诱导的神经元中，ApoE CT(慢病毒感染)导致人神经元产生的Aβ40约减少了46％(图17，面板L)。总的来说，这些结果表明ApoE CT可以减缓啮齿动物和人类神经元中的内源性Aβ。此外，ApoE CT的表达还减缓了表达致病性瑞典APP突变体(APPSW)(Citron等人，1992)的HEK 293T细胞中Aβ40的产生：培养基中的Aβ40约减少了76％(图26，面板R)，在细胞裂解物中的Aβ40约减少了43％(图26，面板S)，表明ApoE CT可减少APPSW产生的Aβ。
与APP共表达的另一种载脂蛋白，载脂蛋白A1(ApoA1)，未能改变α-CTF和Aβ的水平(图26，面板T、U和V)，表明ApoE调节APP剪切的活性不是任意载脂蛋白的非特异性活性。
3.5.ApoE2通过CT与APP和γ分泌酶相互作用。
在N2A细胞中，ApoE2或者ApoE CT与共表达的APP-FLAG有着非常相似的核周亚细胞分布模式(图18，面板A)。在培养的大鼠海马神经元(图18，面板B)和hiPSC衍生神经元(图18，面板C)中，表达的ApoE2-V5或者ApoE CT-V5与神经元内源表达的APP有类似亚细胞分布模式(V5为标签蛋白)。这些结果表明ApoE和ApoE CT与APP具有相似的亚细胞分布模式。
为了检测ApoE和APP或APP分泌酶之间的潜在相互作用，本实验使用了蛋白质临近标记技术(Hung等人，2016年)。当在HEK 293T细胞中共表达时，ApoE2和ApoE CT被APP-APEX2(抗坏血酸过氧化物酶2，连接到APP的C末端；图18，面板D和E)生物素化。同时，FL-APP(全长APP)和α-CTF也可以在同一系统中被ApoE2-APEX2生物素化(图18，面板F)。在原代培养的大鼠神经元中，ApoE2-APEX2生物素化内源表达的APP(图18，面板G)。此外，ApoE2-APEX2生物素化共表达的ADAM10(α-分泌酶)、PS1和PS2(γ分泌酶的酶切催化活性)，但不生物素化β-分泌酶BACE1(图18，面板H)。此外，当在HEK293T细胞中共表达时，ApoE2和ApoE CT，而不是ApoE2 NT，能够与β-CTF-FLAG免疫共沉淀(图18，面板I)。类似地，当在HEK 293T细胞中共表达时，ApoE2和ApoE CT(但不是ApoE2 NT)与γ分泌酶的成分(APH、NCT和PEN2)发生免疫共沉淀(图18，面板J；使用抗FLAG-PEN2进行免疫沉淀)。有趣的是，在人脑样本中，内源性γ分泌酶(NCT、PS1、PS2和PEN2)与内源性ApoE发生免疫共沉淀(图18，面板K)。上述结果表明只有ApoE2和ApoE CT(不是ApoE NT)能够与APP、γ分泌酶和α-分泌酶相互作用，但它们不与BACE1相互作用，并且人脑中存在ApoE与γ分泌酶的内源性相互作用。
3.6.ApoE CT减少了APP的γ剪切。
为了进一步研究潜在机制，本研究进一步探索了与AD相关的APP突变体与ApoE CT的互作情况。ApoE CT增加了所有测试的APP突变体产生的α-CTF(图28)。有趣的是，与野生型APP(WT APP)相比，在表达γ切割位点附近发生fAD突变的APP变体的细胞中(T714I、V717F和L723P)(Kumar-Singh等人，2000年；Kwok等人，2000年；Murrell等人，1991年)，ApoE CT提高α-CTF水平的能力显著降低(图28)。在检查WTAPP和在β-或α-切割位点附近产生fAD突变的APP亚 型(KM670/671NL、E693K、E693A)(Bugiani等人，2010年；Citron等人，1992年；Tomiyama等人，2008年；Xia等人，2021年)时，则没有检测到ApoE CT对α-CTF水平产生具有统计学差异的影响(图28)；共表达具有减少AD发病几率、保护性的冰岛突变(A673T)(Xia等人，2021年)的APP变体，也没有检测到ApoE CT对α-CTF水平的差异性影响(图28)。综合来看，APP的γ位点附近发生突变时，ApoE CT活性遭到破坏，这一现象提示γ剪切与ApoE CT调节APP代谢的活性有关。由于α-CTF被γ剪切而降解(De Strooper等人，2012年；De Strooper等人，1998年；Haass等人，1992年)，这些结果提示ApoE CT可能会影响α-CTF降解。
接下来，本研究根据γ分泌酶在APP代谢过程中的作用进行深入探究。与之前α-CTF通过γ酶切的报道一致(De Strooper等人，2012年；De Strooper等人，1998年；Haass等人，1992年)，过表达的PS1显著将α-CTF的水平降低至50％(图19，面板A和B)；使用γ分泌酶抑制剂PF03084014(1μM)处理时，过表达的PS1不再降低α-CTF，PF03084014也增加了α-CTF；γ分泌酶抑制剂处理时，α-CTF的水平与共表达ApoE CT的HEK 293T细胞中的α-CTF水平相似(图19，面板A、B、C和D)。有趣的是，在PF03084014存在的情况下，ApoE CT未能进一步增加α-CTF(图19，面板C和D)，表明通过抑制γ分泌酶可以阻断ApoE CT活性。此外，使用ADAM10抑制剂GI254023X(1μM)可以将α-CTF减少至约45％(图29，面板A和B)。在GI254023X存在的情况下，ApoE CT仍然可以增加α-CTF(约4倍；图29，面板A和B)和减少Aβ40(图29，面板C和D)。此外，当β-分泌酶被其抑制剂AZD3839(5μM)抑制时，ApoE CT仍会增加α-CTF(图29，面板E和F)。ApoE CT还能够提高APPM596V所产生的α-CTF(图29，面板G和H)，APPM596V是一个不能进行β酶切的APP突变(Citron等人，1995年)。此外，ApoE CT也显著降低了β-CTF产生的Aβ40(图19，面板E)和Aβ42(图19，面板F)，表明ApoE CT通过抑制γ酶切减少了Aβ的产生。APP的γ剪切产物APPΔAICD只需要经过β酶切就可以生成Aβ40。有趣的是，ApoE CT未能减APPΔAICD产生的Aβ40(图19，面板G和H)。这些结果共同支持了ApoE CT抑制了APP的γ酶切，但不直接调节α或β酶切的假设。
此外，ApoE2和ApoE CT均未改变ADAM10、BACE1、PS1或PS2的mRNA或蛋白质表达水平(图29，面板I和J)，表明APP分泌酶的表达水平没有改变。此外，ApoE CT也没有改变APP在细胞表面的表达(图29，面板K)。
3.7.ApoE CT调节APP的代谢需要γ分泌酶参与。
为了确定ApoE CT调节APP的代谢是否需要γ分泌酶，本研究构建了PS1和/或PS2敲除(KO)HEK 293T细胞系(Lu等人，2014年)(图20，面板A)。PS1 KO细胞中的α-CTF水平升高，这可以通过PS1的代偿性表达来逆转(图20，面板B)。PF03084014处理进一步增加了PS1 KO细胞中的α-CTF水平(图20，面板B)。共表达的ApoE CT仍然能够增加PS1 KO细胞(图20，面板C和D)和PS2 KO细胞(图20，面板E和F)中的α-CTF。因此，单独敲除PS1或PS2不足以完全消除ApoECT的活性。
在PS1/2双敲除细胞(PS1/2 dKO)中，与WT细胞相比，α-CTF水平也显著升高(图20，面板G)；在PS1/2 dKO细胞中共表达的ApoE CT(图20，面板G和J)或ApoE2(图20，面板K和L)没有进一步提高α-CTF水平。PS1或PS2的补充表达挽救了ApoE CT的活性，ApoE CT再次显著增加了α-CTF(图20，面板H、I和J)。同时，在补充表达PS2的PS1/2 dKO细胞中，ApoE CT也显著减少了β-CTF产生的Aβ40(图20，面板M)。这些结果共同表明，ApoE CT或ApoE2需要γ分泌酶来增加α-CTF。
3.8.小鼠神经元内源表达的ApoE调节内源APP的γ酶切。
为了探索神经元中内源性ApoE的生理活性，构建了小鼠ApoE的shRNA(shApoE_1、shApoE_2、shScramble作为对照)。在小鼠神经元中利用慢病毒系统表达的shApoE_1和shApoE_2成功降低了原代培养的小鼠神经元中内源性ApoE的蛋白表达量(图21，面板A)。用γ分泌酶抑制剂PF03084014处理后，神经元中APP代谢产生的～14kD CTF(C端片段，CTF14kD)水平显著增加，表明该CTF14kD也可以被γ分泌酶剪切(图21，面板B)。shApoE_1或shApoE_2显著降低了小鼠神经元中CTF14kD条带的基础水平(图21，面板C和D)，表明内源性ApoE增加了CTF14kD。当存在γ分泌酶抑制剂PF03084014时，ApoE的这两个shRNA不能再降低CTF14kD(图21，面板E)，表明ApoE的这种活性需要有活性的γ分泌酶的存在。此外，ApoE的敲低还导致神经元产生的Aβ水平上升(图21，面板F)。这些结果共同表明，神经元中内源性的ApoE调控了内源APP的γ酶切。
3.9.ApoE CT以底物特异性的方式抑制γ酶切。
本发明继续检测ApoE CT是否也抑制NOTCH的γ酶切。NOTCH是γ分泌酶的 底物(Xia，2019年；Yang等人，2019年)。在HEK 293T细胞中，共表达的γ分泌酶减少N100(NOTCH片段)，并且PF03084014的处理逆转了这种降低(图30，面板A)。有趣的是，共表达的ApoE CT未能抑制N100的这种γ分泌酶依赖性减少(图30，面板A)。然而，当与γ分泌酶共表达时，ApoE CT仍然抑制γ分泌酶对APP的γ剪切(图30，面板B)。APP样蛋白1(APP-like protein 1，APLP1)是α和γ分泌酶的另一个底物(Eggert等人，2004年)。用PF03084014处理降低了APLP1的水平，同时增强了它的切割产物(α-like CTF)；但是α-like CTF不会因ApoE CT与APLP1的共表达而改变(图30，面板C)。这些结果证ApoE CT不抑制NOTCH和APLP1的γ酶切。
3.10.ApoE2直接抑制APP的γ酶切。
为了确定ApoE是否直接抑制β-CTF的γ剪切，本发明纯化了β-CTF、ApoE2、ApoE2 NT和γ分泌酶(Lu et al.，2014)(图22，面板A；图31)以供进一步测试。纯化的γ分泌酶与β-CTF一起孵育导致Aβ40的产生；在没有γ分泌酶存在的情况下，β-CTF不产生Aβ40；并且γ分泌酶与β-CTF一起孵育生成的Aβ40，可以被PF03084014完全阻断(图22，面板B)。纯化的ApoE2以剂量依赖性方式抑制Aβ40的产生，IC50为1.2μM(图22，面板C)。相反，ApoE2 NT未能抑制Aβ40的产生(图22，面板D)。这些结果表明ApoE2通过其CT直接抑制β-CTF的γ酶切。
3.11.神经元表达的ApoE CT迁移至淀粉样斑块并减轻5×FAD小鼠的斑块负担。
为了研究ApoE CT是否可以延迟AD小鼠模型中的淀粉样斑块的生成，本实验将ApoE CT通过AAV载体在4周龄5×FAD小鼠的皮质和海马中的神经元中表达(图23，面板A)。10周后，与GFP对照相比，表达ApoE CT的5×FAD小鼠下托(subiculum)脑区内的平均斑块大小、总淀粉样斑块面积百分比和斑块密度均显著降低(图23，面板B、C、D和E)。
有趣的是，在这些5×FAD小鼠中，虽然ApoE CT表达局限于皮质、海马体的齿状回(DG)和CA3区域的神经元中，但它迁移了到subiculum并积聚在淀粉样斑块周围(图23，面板B)；而神经元表达的GFP未能做到这一点。正如本发明(图25，面板E)和其他人报道的数据所示(Metzger等人，1996年)，神经元中表达的ApoECT可以迁移到淀粉样蛋白斑块以实现在这些淀粉样蛋白生成热点(Jordà-Siquier等人，2022年)中对γ分泌酶的局部调节。在用慢病毒载体(lentiviral vectors)在5×FAD 小鼠中表达ApoE2和ApoE CT时，淀粉样斑块周围也检测到类似的分布模式(图24，面板A，箭头，淀粉样斑块中的ApoE；无柄箭头，神经元中的ApoE)；而GFP和ApoE2 NT未能在斑块周围积聚(图24，面板A)。在未产生淀粉样斑块的1个月大5×FAD小鼠(图24，面板B)和WT小鼠(图24，面板C)中，ApoE CT仅存在于神经元胞体中，并与表达的人源APP(hAPP)亚细胞分布部分共定位。这些结果表明ApoE积累在淀粉样斑块周围需要CT。
4.讨论
以Aβ为靶点治疗AD的努力持续失败，使得Aβ在AD中的作用受到质疑(Liu等人，2019年；Panza等人，2019年；Roberson和Mucke，2006年)。不可避免地，这种失败也挑战了对AD本身的许多基本理解。例如，尽管γ分泌酶的功能障碍导致fAD仍然是毫无疑问的，这将Aβ失调与fAD联系起来，并具有强大的人类遗传证据(Selkoe，2001)。然而，支持Aβ在sAD中作用的证据要弱得多。不能完全排除sAD是一种恰好与fAD具有相同病理特征的不同疾病的可能性(Armstrong，2013；De Strooper和Karran，2016年)。事实上，fAD和sAD除了潜在的遗传因素外，在其他方面也存在差异，包括发病年龄、详细的病理异常和症状等(Dorszewska等人，2016年；Roher等人，2016年)。
ApoE是sAD最重要的遗传因子，本实验的数据首次显示，ApoE似乎通过选择性抑制APP的γ剪切(淀粉样蛋白生成的关键步骤)来降低sAD的风险。本发明的这些结果解释了为什么与ApoE2携带者相比，ApoE4携带者会产生更高的淀粉样蛋白负荷并且具有更少的来自非淀粉样蛋白生成途径的代谢产物(Corder等人，1993年；Hashimoto等人，2012年；Olsson等人，2003年；Schmechel等人，1993年)。总之，本实验的数据支持精确抑制γ分泌酶可以预防sAD的风险，因此通过抑制γ分泌酶来治疗sAD是合理的。值得注意的是，最近的研究发现，大多数fAD的早老素突变会导致γ分泌酶活性降低(Sun等人，2017年；Xia等人，2015年)。这表明尽管γ分泌酶的功能障碍在sAD和fAD的发病机制中起着核心作用，但在这两种不同形式的AD中，γ分泌酶活性的变化却是相反的。从这个角度来看，γ分泌酶抑制可能不适用于具有早老素功能丧失突变的fAD。它提出了有趣的问题，即为什么较高和较低的γ分泌酶活性都会导致AD，以及是否应根据早老素突变如何影响γ分泌酶活性将fAD分为不同的组。
同时，考虑到尚未测试的广谱γ底物(Güner和Lichtenthaler，2020年)，APP可能不是唯一一种其切割被ApoE抑制的底物，因此ApoE对APP的γ酶切的影响可能不是导致sAD风险的唯一原因，但仍然是合理的。因此，对γ分泌酶的抑制可能不会导致与Aβ 清除相同的效果。这是一个特别值得关注的问题，因为它可能有助于解释为什么单独使用Aβ清除策略无法得到令人满意的临床效果。通过分析哪些γ分泌酶的底物受ApoE影响，本发明的研究将提供一个难得的机会来探讨除Aβ外其他γ分泌酶的底物在AD病因学中的作用。
在小鼠模型中，ApoE主要在神经胶质细胞中表达(Xu et al.，2006)。由于Aβ是在神经元中产生的，因此大多数研究都集中在神经胶质细胞分泌的ApoE如何影响胞外淀粉样进程(Holtzman等人，1999年；Huang等人，2017年；Huynh等人，2017年；Liu等人，2017年；Yu等人，2014年)。实际上，来自不同实验室的许多论文独立地表明，ApoE的mRNA和蛋白质都在人类神经元中检测到(Aoki等人，2003年；Han等人，1994年；Metzger等人，1996年；Strittmatter等人，1993；Xu等人，1999)。最近的单核RNA-seq数据也表明，可以在神经元中检测到ApoE，高达28％的AD患者神经元高水平表达ApoE(Belonwu等人，2022年；Zalocusky等人，2021年)，这与我们观察到的一致，即大约30-40％的神经元在人类皮层中表达ApoE。此外，ApoE在人脑中的代谢更类似于神经元中表达的ApoE，而不是胶质细胞中表达的ApoE的代谢(Brecht etal.，2004)。还有报道称，携带ApoE4亚型的人类神经元产生的Aβ多于拥有其他ApoE亚型的人类神经元(Wang等人，2018年)，这强烈支持人类神经元中内源表达的ApoE可以调节内源APP的代谢。然而，ApoE在神经元中的作用在很大程度上仍未得到充分理解。本发明发现保护性亚型ApoE2通过其CT以细胞自主方式直接抑制APP的γ酶切，而ApoE4没有此活性。重要的是，本发明还发现小鼠神经元中内源性的ApoE显著抑制内源表达的APP的γ酶切；并且人大脑中，内源的ApoE和内源表达的γ分泌酶存在相互作用。此外，AD小鼠模型概括了AD的许多关键病理特征，但没有概括ApoE的淀粉样蛋白沉积(Oakley等人，2006年)。令人惊讶的是，本发明发现在5×FAD小鼠的神经元中表达的ApoE迁移并聚集在淀粉样蛋白斑块周围，这与先前报道的ApoE存在于AD患者大脑的淀粉样蛋白斑块中一致(Han et al.，1994；Strittmatter et al，1993)，表明斑块中的ApoE具有神经元起源。这些发现强调了神经元中的ApoE在AD中发挥着非常关键的作用，并为ApoE2和ApoE4对AD发病机制的不同贡献提供了新的机制解释。
γ分泌酶是AD的潜在药物靶点。然而，在过往的药物研发中发现抑制γ分泌酶的活性会导致严重的副作用，这可能是由于γ分泌酶抑制剂在不同底物中的选择性较差的缘故(Panza等人，2019年；Roberson和Mucke，2006年；Xia，2019年)。广泛的、非特异的抑制剂在抑制γ分泌酶阻断Aβ生成的同时，也会阻止γ分泌酶其他底物的正常加工(Panza等人，2019年；Xia，2019年)，从而导致副作用。开发γ分泌酶修饰剂 来选择性抑制APP的γ剪切是一种很有前途的新策略(Xia，2019)。本发明发现ApoE CT抑制APP的γ酶切，但不影响NOTCH和APLP1的γ酶切，表明ApoE CT对γ剪切的抑制作用具有底物特异性。正如之前报道的那样，小分子药物通过占据PS1上与其底物(例如APP或Notch(Yang等人，2021))相互作用的β链的相同位置来抑制γ分泌酶的活性。ApoE CT作为一种结构上与传统小分子抑制剂截然不同的分子，可能作用于一些变构位点，仅影响某些底物与γ分泌酶的结合，而不影响其他底物的结合，从而实现底物选择性。然而，确切的机制仍有待解决。尽管如此，与传统的γ分泌酶抑制剂相比，ApoE CT可能作为一种新的战略性治疗方法，副作用可能更小。综上所述，本发明不仅提供了潜在的机制理解神经元ApoE亚型在sAD发病机制中的作用；而且还通过选择性抑制AD相关的γ分泌酶底物的γ剪切，确定了ApoE CT作为AD的潜在治疗方法。
这些结果支持γ分泌酶的精确抑制可防止sAD的风险，这与通常与γ分泌酶活性降低相关的fAD相反。本发明还将ApoE CT鉴定为一种有效的γ分泌酶抑制剂，ApoE CT具有罕见的底物特异性，可以从远处迁移到斑块周围的淀粉样蛋白生成热点中局部起作用。
5.实验材料和方法
动物
5×FAD小鼠的神经元中表达有五个fAD突变的人源APP和PS1：APP中的K670N/M671L+I716V+V717I突变和PS1中的M146L+L286V突变(Oakley等人，2006)。所有小鼠都被安置在12小时的光暗循环(从早上8点到晚上8点光照)下，随意获取食物和水。所有小鼠程序均按照中国科学院和美国国立卫生研究院的实验动物护理和使用指南进行。
人和猴脑组织
所有人类脑组织均来自中国湖北中国脑库(CBBC)和浙江大学医学院以及中国浙江国家健康与疾病人类脑库。图32总结了捐赠者的信息。人脑组织实验前-80℃保存，4％PFA固定过夜，切片前30％蔗糖冷冻过夜。以下步骤与在小鼠脑切片上执行的IHC相同。
成年恒河猴固定脑片来自中国科学院神经科学研究所，中国上海(Wu等人，2021)。
原代神经元的培养
如前人所述制备原代分离的神经元培养物(Chen等人，2014年)。从第16天野生型胚胎中取出整个海马体或皮质小鼠(C57BL/6)或大鼠(SD)，并在37℃下用木瓜蛋白酶(Worthington)处理10分钟，并解离。将分离的细胞以150,000个细胞/孔(24孔板)接种在B27神经元生长培养基。神经元在37℃和5％CO2培养箱中培养，并且每周添加一次新鲜培养基。
PS1KO、PS2KO和PS1/2dKO细胞系的构建
HEK 293T细胞在补充有10％牛血清白蛋白(Life Technology)和1％青霉素/链霉素(Life Technology)的DMEM培养基(Gibco)中培养。为了制备PS1、PS2单敲除或PS1/2双敲除HEK 293T细胞系，采用了CRISPR/Cas9基因组编辑方法(Ran等人，2013)。靶向PS1外显子4的向导RNA(gRNA)5′-ATTATCTAATGGACGACCCC-3′(SEQ ID NO：91)和靶向PS2外显子4的gRNA5′-CAAATACGGAGCGAAGCACG-3′(SEQ ID NO：92)分别亚克隆到pSpCas9(BB)-2A-puro骨架(PS1gRNA、PS2gRNA)中。使用Lipofectamine2000(Invitrogen Technology)将含有gRNA的质粒转染到HEK 293T细胞中。转染PS1 gRNA或PS2 gRNA 18小时后，HEK 293T细胞用3μg/ml嘌呤霉素(Selleck)处理。当转染空白质粒的HEK 293T细胞完全死亡后，将转染PS1 gRNA或PS2 gRNA的HEK 293T细胞按1∶400稀释度在10cm培养皿中培养，保证每个细胞均为单克隆。在单个细胞长成细胞团后，选择基因组编辑的细胞团并通过DNA测序和Western blot验证。用于构建PS1/2dKO细胞系用PS1 gRNA转染PS2已经敲除细胞并进行第二轮选择。
蛋白质印迹(WB)
在HEK 293T或N2A细胞中转染质粒36-48小时，然后在细胞裂解缓冲液[10％1Mtris-HCl(pH＝6.8)，4％SDS，20％甘油，0.01％溴酚蓝]中裂解。裂解物在室温下摇动10分钟，然后煮沸10分钟，并以12,000×g离心10分钟。蛋白质在8％或12％Tris-甘氨酸SDS-PAGE上运行并转移到PVDF膜(Life Technologies)。将样品在5％BSA缓冲液(在1×TBST中)稀释的一抗在4℃孵育过夜。用1×TBST洗涤3次后，将它们与稀释在5％BSA缓冲液(在1×TBST中)的二抗在室温下孵育2小时。最后用1×TBST洗涤3次后，通过ECL蛋白质印迹底物(Solarbio，PE0010)检测信号。
脑组织样本用TissueLyserII(QIAGEN，85300)并在RIPA缓冲液(Solarbio，R0010)中裂解。裂解物煮沸10分钟，然后以12,000×g离心10分钟。Western blot的操作步骤同细胞样本。
用于蛋白质印迹的APEX标记蛋白的制备
如前人所述，应用了一种通过APEX2靶向检测活细胞中蛋白质相互作用的方法(Hung等人，2016年)。转染后约30小时和APEX2质粒通过Lipofectamine2000(Invitrogen Technology)，将HEK 293T细胞在含有或不含250uM生物素苯酚(BP)的1ml完全培养基中于37℃孵育30分钟。细胞用1mM H2O2处理1分钟，细胞在猝灭剂溶液(10mM抗坏血酸钠、5mM Trolox和10mM叠氮化钠溶液的1×DPBS溶液)中洗涤3次。细胞在RIPA裂解缓冲液(1×蛋白抑制剂混合物、1mM PMSF和10mM抗坏血酸钠、5mM Trolox和10mM叠氮化钠)中裂解。裂解物在冰上超声并离心(12,000×g，4℃，10分钟)，上清液与链霉亲和素磁珠在4℃孵育过夜。使用磁性架收集珠子并用一系列缓冲液洗涤(两次使用RIPA缓冲液，一次使用1MKCl，一次使用0.1MNa2CO3，两次使用2M尿素的10mM Tris-HCl，pH8.0，并使用RIPA裂解缓冲液两次)。通过在含有2mM生物素和20mM DTT的加载缓冲液中煮沸每个样品10分钟，从珠子上洗脱生物素化的蛋白质，收集洗脱液。样本继续进行蛋白质印迹。
免疫共沉淀(co-IP)
HEK 293T细胞转染的和质粒通过Lipofectamine2000(Invitrogen Technology)处理48小时，HEK 293T细胞在1％CHAPSO(SIGMA)、25mM HEPES(pH7.4)(Gibco)、150mMNaCl(含1×蛋白酶抑制剂混合物)中裂解，并以14,000×g离心20min，收集上清液。将上清液与抗FLAG(SIGMA)或抗IgG(Santacruz)抗体在4℃下孵育3小时，并使用ProteinG Sepharose beads(Gen Script)pulldown，然后在0.1％洋地黄皂苷-HEPES缓冲液中洗涤3次，每次15分钟，并收集在细胞裂解缓冲液[10％1Mtris-HCl(pH＝6.8)，4％SDS，20％甘油，0.01％溴酚蓝]。样本继续进行蛋白质印迹。
免疫荧光染色和免疫组织化学(IHC)
在24孔板的盖玻片上培养的神经元和HEK 293T细胞用1×PBS(2.5mMMgCl2和0.5mMCaCl2)洗涤，并在室温下用4％PFA+4％蔗糖在1×PBS中固定10分钟。接下来，用0.15％Triton-X在1×PBS中打孔15分钟，并在室温下用5％BSA在1×PBS中封闭30分 钟，将样品与稀释在5％BSA(在1×PBS中)缓冲液中的一抗一起孵育4℃过夜。然后用1×PBS中的0.5M NaCl洗涤样品一次，并用1×PBS洗涤三次。然后将样品与稀释在5％BSA(在1×PBS中)缓冲液中的二抗一起孵育室温静置1h。在用1×PBS进行三次最终洗涤步骤后，用Prolong Gold Antifade Mountant(Life Technology)封固样品。所有图像均以盲法拍摄和分析。
小鼠用异氟醚麻醉并灌注4％PFA。解剖的小鼠大脑在4％PFA中固定过夜，并在30％蔗糖中冷冻保护过夜。低温切片机(LeicaCM3050S)用于从固定和冷冻保护的小鼠大脑中获取冷冻脑切片(40μM)。漂浮切片用1×PBS洗涤3次，然后用0.5％Triton-X的1×PBS透化30分钟。1×PBS中的5％BSA室温封闭1h后，切片在含有一抗的5％BSA(1×PBS中)缓冲液中4℃孵育过夜。切片用1×PBS洗涤3次，并与稀释在5％BSA(1×PBS)缓冲液中的二抗在室温下孵育1小时。然后用1×PBS洗涤样品3次并用Prolong Gold Antifade Mountant(LifeTechnology)封片。
THS染色
小鼠用异氟醚麻醉并灌注4％PFA。解剖的小鼠大脑在4％PFA中固定过夜，并在30％蔗糖中冷冻保护过夜。低温切片机(Leica，CM3050S)用于从固定和冷冻保护的小鼠大脑中获取冷冻脑切片(40μM)。漂浮切片用ddH2O洗涤3次，然后在室温下用1％THS(SIGMA)的水溶液孵育5分钟。接下来，切片用80％乙醇洗涤两次，一次用95％乙醇，每次3分钟。样品经ddH2O洗涤，1×PBS洗涤3次后，即可进行IHC。
定量实时PCR(qRT-PCR)
使用TRIzol试剂(Invitrogen)从HEK 293T细胞中提取总RNA。使用QuantiTect SYBR-Green RT-PCR(逆转录-PCR)试剂盒(Qiagen)进行逆转录。GAPDH用作内部对照。ADAM10 mRNA的正向引物5′-AGAGCAACATCTGGGGACAAA-3′(SEQ ID NO：93)和反向引物5′-TCCACAAATAGGTTGGCCAGAT-3′(SEQ ID NO：94)，BACE1 mRNA的正向引物5′-AACGAATTGGCTTTGCTGTC(SEQ ID NO：95)和反向引物5′-AGGCTATGGTCATGAGGGTTG-3′(SEQ ID NO：96)，正向引物5′-TGACTCTCTGCATGGTGGTG-3′(SEQ ID NO：97)和用于PS1 mRNA的反向引物5′-TCAGAATTGAGTGCAGGGCT-3′(SEQ ID NO：98)，用于PS2mRNA的正向引物5′-TTTGAGCCTCCCTTGACTGG-3′(SEQ ID NO：99)和反向引物5′- GGTATTCCAGTCCCCGCTG-3′(SEQ ID NO：100)用于qRT-PCR。使用SYBR绿色荧光信号(Thermo Fisher Scientific的应用生物系统)的比较阈值循环测量来测试量化。
酶联免疫吸附试验(ELISA)
HEK 293T细胞和N2A细胞转染48小时，或慢病毒感染神经元4.5天后，收集培养基和裂解物。按照方案的指示，通过Aβ酶联免疫吸附测定试剂盒检测培养基和细胞裂解物中的Aβ40和Aβ42。(Invitrogen Technology；KHB3481用于人Aβ40，KHB3441用于人Aβ42和KMB3481用于小鼠Aβ40)。
蛋白纯化
人的γ-分泌酶复合物如前所述纯化(Lu等人，2014)，编码人γ-分泌酶复合物的cDNA连同表达载体参见Lu等人，2014。
人ApoE蛋白的纯化。用人ApoE质粒(ApoE2-3×FLAG或ApoE2NT-3×FLAG)转染细胞48小时后，将转染的细胞在冷的1×PBS中洗涤一次，收获并以800g离心。然后将细胞沉淀重悬于含有25mM HEPES、pH7.4、150mMNaCl、5mM MgCl2、1mM二硫苏糖醇(DTT)、0.05％(v/v)Tween-20和1×蛋白酶抑制剂混合物的5ml裂解缓冲液中。细胞裂解通过超声处理实现，细胞碎片通过离心分离(12,000×g，4℃，15分钟)。将上清液与抗FLAGM2亲和凝胶(SIGMA)在4℃下孵育90分钟。然后用含有25mM HEPES、pH7.4、150mM NaCl、5mM MgCl2、1mM DTT和0.05％(v/v)Tween-20的缓冲液洗涤树脂3次。ApoE蛋白用含有25mM HEPES、pH7.4、150mM NaCl和500μg/ml3×FLAG肽(SIGMA)的缓冲液洗脱。SDS-PAGE和蛋白质印迹用于显示纯化的蛋白质。
β-CTF的纯化。用人β-CTF-Myc-6×His质粒转化大肠杆菌细胞，当OD260达到0.6-0.8时，用0.5μM IPTG(Solarbio)在37℃下诱导大肠杆菌细胞4小时。大肠杆菌细胞在该缓冲液(30mM Tris-HCl，pH7.5，150mM NaCl，1mM EDTA)中裂解，并在冰上超声处理。将裂解物以25,000×g离心10分钟，将裂解物溶解在尿素缓冲液(30mM Tris-HCl，pH8.0，300mM NaCl，1mM EDTA，6M尿素，1％TritonX-100和1mM CaCl2，1×蛋白酶抑制剂混合物)并缓慢旋转3小时。将重悬的裂解物以40,000×g、30分钟离心两次，并将上清液与Ni-NTA珠(GEhealthcare)在室温下孵育2小时。用10柱体积(CV)的尿素缓冲液1(30mM Tris-HCl，pH8.0；300mM NaCl，1mM EDTA，6M尿素，1％TritonX-100，1mM CaCl2，1×cocktail)、20CV洗涤缓冲液2(30mM Tris-HCl、pH8.0、 300mM NaCl、0.1％TrionX-100)和20CV洗涤缓冲液3(50 mMTris-HCl、pH8.0、300mM NaCl、0.1％夏普索)。结合的材料用洗涤缓冲液(50mM Tris-HCl，pH8.0，300mM NaCl，0.1％CHAPSO)洗脱，咪唑梯度为0-300mM。SDS-PAGE和蛋白质印迹用于可视化纯化的蛋白质
病毒立体定位注射
在用异氟醚深度麻醉后，将小鼠(5周大)置于立体定向装置(RWD Instruments)中。零点(x，y，z，0，0，0)是Bregma。慢病毒(lentivirus-GFP、lentivirus-ApoE和lentivirus-ApoE-IRES-GFP)和AAV(AAV2/9-hSyn-EGFP-WPRE-pA和AAV2/9-hSyn-ApoECT；购自上海泰图生物科技有限公司)被注射到皮层(x，y，z，+/-1.6，0，-1)或海马体(x，y，z，+/-1.5，-2，-1.5)，每个注射位点注射1ul。然后在手术后将小鼠放回笼子中进行康复。
量化和统计分析
GraphPadPrism软件(v6)使用未配对、配对或一个样本t检验分析数据。Aβ40和Aβ42水平针对相应的GFP对照进行标准化。将α-CTF水平标准化为FL-APP和相应的GFP对照。所有数据均以平均值±SEM表示。统计显着性定义为p＜0.05。图表中的一颗、二颗、三颗和四颗星(*)分别表示p值＜0.05、＜0.01、＜0.001和＜0.0001。
6.参考文献
Aoki，K.，Uchihara，T.，Nakamura，A.，Komori，T.，Arai，N.，and Mizutani，T.(2003).Expression of apolipoprotein E in ballooned neurons-comparative immunohistochemical study on neurodegenerative disorders and infarction.Acta Neuropathol 106，436-440.
Armstrong，R.A.(2013).What causes alzheimer′s disease？Folia Neuropathol 51，169-188.
Belloy，M.E.，Napolioni，V.，and Greicius，M.D.(2019).A Quarter Century of APOE and Alzheimer′s Disease：Progress to Date and the Path Forward.Neuron 101，820-838.
Belonwu，S.A.，Li，Y.，Bunis，D.，Rao，A.A.，Solsberg，C.W.，Tang，A.，Fragiadakis，G.K.，Dubal，D.B.，Oskotsky，T.，and Sirota，M.(2022).Sex-Stratified Single-Cell RNA-SeqAnalysis Identifies Sex-Specific and Cell Type-Specific Transcriptional Responses in Alzheimer′s Disease Across Two Brain Regions.Molecular neurobiology 59，276-293T.
Brecht，W.J.，Harris，F.M.，Chang，S.，Tesseur，I.，Yu，G.Q.，Xu，Q.，Dee Fish，J.，Wyss- Coray，T.，Buttini，M.，Mucke，L.，et al.(2004).Neuron-specific apolipoprotein e4proteolysis is associated with increased tau phosphorylation in brains of transgenic mice.The Journal of neuroscience：the official journal of the Society for Neuroscience 24，2527-2534.
Bugiani，O.，Giaccone，G.，Rossi，G.，Mangieri，M.，Capobianco，R.，Morbin，M.，Mazzoleni，G.，Cupidi，C.，Marcon，G.，Giovagnoli，A.，et al.(2010).Hereditary cerebral hemorrhage with amyloidosis associated with the E693K mutation of APP.Archives of neurology 67，987-995.
Chen，J.，Li，Q.，and Wang，J.(2011).Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions.Proceedings of the National Academy of Sciences of the United States of America 108，14813-14818.
Chen，Y.，Wang，Y.，Modrusan，Z.，Sheng，M.，and Kaminker，J.S.(2014).Regulation of neuronal gene expression and survival by basal NMDA receptor activity：a role for histone deacetylase 4.The Journal of neuroscience：the official journal of the Society for Neuroscience 34，15327-15339.
Citron，M.，Oltersdorf，T.，Haass，C.，McConlogue，L.，Hung，A.Y.，Seubert，P.，Vigo-Pelfrey，C.，Lieberburg，I.，and Selkoe，D.J.(1992).Mutation of the beta-amyloid precursor protein in familial Alzheimer′s disease increases beta-protein production.Nature 360，672-674.
Citron，M.，Teplow，D.B.，and Selkoe，D.J.(1995).Generation of amyloid beta protein from its precursor is sequence specific.Neuron 14，661-670.
Corder，E.H.，Saunders，A.M.，Strittmatter，W.J.，Schmechel，D.E.，Gaskell，P.C.，Small，G.W.，Roses，A.D.，Haines，J.L.，and Pericak-Vance，M.A.(1993).Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer′s disease in late onset families.Science 261，921-923.
De Strooper，B.，Iwatsubo，T.，and Wolfe，M.S.(2012).Presenilins and gamma-secretase：structure，function，and role in Alzheimer Disease.Cold Spring Harbor perspectives in medicine 2，a006304.
De Strooper，B.，and Karran，E.(2016).The Cellular Phase of Alzheimer′s Disease.Cell164，603-615.
De Strooper，B.，Saftig，P.，Craessaerts，K.，Vanderstichele，H.，Guhde，G.，Annaert，W.， Von Figura，K.，and Van Leuven，F.(1998).Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.Nature 391，387-390.
Dorszewska，J.，Prendecki，M.，Oczkowska，A.，Dezor，M.，and Kozubski，W.(2016).Molecular Basis of Familial and Sporadic Alzheimer′s Disease.Current Alzheimer research13，952-963.
Eggert，S.，Paliga，K.，Soba，P.，Evin，G.，Masters，C.L.，Weidemann，A.，and Beyreuther，K.(2004).The proteolytic processing of the amyloid precursor protein gene family members APLP-1 and APLP-2 involves alpha-，beta-，gamma-，and epsilon-like cleavages：modulation of APLP-1 processing by n-glycosylation.J Biol Chem 279，18146-18156.
Esch，F.S.，Keim，P.S.，Beattie，E.C.，Blacher，R.W.，Culwell，A.R.，Oltersdorf，T.，McClure，D.，and Ward，P.J.(1990).Cleavage of amyloid beta peptide during constitutive processing of its precursor.Science 248，1122-1124.
Güner，G.，and Lichtenthaler，S.F.(2020).The substrate repertoire ofγ-secretase/presenilin.Seminars in cell&developmental biology 105，27-42.
Haass，C.，Schlossmacher，M.G.，Hung，A.Y.，Vigo-Pelfrey，C.，Mellon，A.，Ostaszewski，B.L.，Lieberburg，I.，Koo，E.H.，Schenk，D.，Teplow，D.B.，et al.(1992).Amyloid beta-peptide is produced by cultured cells during normal metabolism.Nature 359，322-325.
Haass，C.，and Selkoe，D.J.(1993).Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide.Cell 75，1039-1042.
Han，S.H.，Hulette，C.，Saunders，A.M.，Einstein，G.，Pericak-Vance，M.，Strittmatter，W.J.，Roses，A.D.，and Schmechel，D.E.(1994).Apolipoprotein E is present in hippocampal neurons without neurofibrillary tangles in Alzheimer′s disease and in age-matched controls.Experimental neurology 128，13-26.
Hardy，J.，and Selkoe，D.J.(2002).The amyloid hypothesis of Alzheimer′s disease：progress and problems on the road to therapeutics.Science 297，353-356.
Hashimoto，T.，Serrano-Pozo，A.，Hori，Y.，Adams，K.W.，Takeda，S.，Banerji，A.O.，Mitani，A.，Joyner，D.，Thyssen，D.H.，Bacskai，B.J.，et al.(2012).Apolipoprotein E，especially apolipoprotein E4，increases the oligomerization of amyloid beta peptide.The Journal of neuroscience：the official journal of the Society for Neuroscience 32，15181-15192.
Holtzman，D.M.，Bales，K.R.，Wu，S.，Bhat，P.，Parsadanian，M.，Fagan，A.M.，Chang， L.K.，Sun，Y.，and Paul，S.M.(1999).Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer′s disease.The Journal of clinical investigation 103，R15-r21.
Huang，Y.A.，Zhou，B.，Wernig，M.，and Sudhof，T.C.(2017).ApoE2，ApoE3，and ApoE4 Differentially Stimulate APP Transcription and Abeta Secretion.Cell 168，427-441e421.
Hung，V.，Udeshi，N.D.，Lam，S.S.，Loh，K.H.，Cox，K.J.，Pedram，K.，Carr，S.A.，and Ting，A.Y.(2016).Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2.Nature protocols 11，456-475.
Huynh，T.V.，Liao，F.，Francis，C.M.，Robinson，G.O.，Serrano，J.R.，Jiang，H.，Roh，J.，Finn，M.B.，Sullivan，P.M.，Esparza，T.J.，et al.(2017).Age-Dependent Effects of apoE Reduction Using Antisense Oligonucleotides in a Model of beta-amyloidosis.Neuron 96，1013-1023 el014.
Jordà-Siquier，T.，Petrel，M.，Kouskoff，V.，Smailovic，U.，Cordelières，F.，Frykman，S.，Müller，U.，Mulle，C.，and Barthet，G.(2022).APP accumulates with presynaptic proteins around amyloid plaques：A role for presynaptic mechanisms in Alzheimer′s disease？Alzheimer′s&dementia：the journal of the Alzheimer′s Association.
Kanekiyo，T.，Xu，H.，and Bu，G.(2014).ApoE and Abeta in Alzheimer′s disease：accidental encounters or partners？Neuron 81，740-754.
Karran，E.，and De Strooper，B.(2016).The amyloid cascade hypothesis：are we poised for success or failure？Journal of neurochemistry 139 Suppl 2，237-252.
Kumar-Singh，S.，De Jonghe，C.，Cruts，M.，Kleinert，R.，Wang，R.，Mercken，M.，De Strooper，B.，Vanderstichele，H.，A.，Vanderhoeven，I.，et al.(2000).Nonfibrillar diffuse amyloid deposition due to a gamma(42)-secretase site mutation points to an essential role for N-truncated A beta(42)in Alzheimer′s disease.Human molecular genetics 9，2589-2598.
Kwok，J.B.，Li，Q.X.，Hallupp，M.，Whyte，S.，Ames，D.，Beyreuther，K.，Masters，C.L.，and Schofield，P.R.(2000).Novel Leu723Pro amyloid precursor protein mutation increases amyloid beta42(43)peptide levels and induces apoptosis.Ann Neurol 47，249-253.
Li，Z.，Shue，F.，Zhao，N.，Shinohara，M.，and Bu，G.(2020).APOE2：protective mechanism and therapeutic implications for Alzheimer′s disease.Molecular  neurodegeneration 15，63.
Liu，C.C.，Zhao，N.，Fu，Y.，Wang，N.，Linares，C.，Tsai，C.W.，and Bu，G.(2017).ApoE4Accelerates Early Seeding of Amyloid Pathology.Neuron 96，1024-1032 e1023.
Liu，P.P.，Xie，Y.，Meng，X.Y.，and Kang，J.S.(2019).History and progress of hypotheses and clinical trials for Alzheimer′s disease.Signal transduction and targeted therapy 4，29.
Lu，P.，Bai，X.C.，Ma，D.，Xie，T.，Yan，C.，Sun，L.，Yang，G.，Zhao，Y.，Zhou，R.，Scheres，S.H.W.，et al.(2014).Three-dimensional structure of humanγ-secretase.Nature 512，166-170.
Metzger，R.E.，LaDu，M.J.，Pan，J.B.，Getz，G.S.，Frail，D.E.，and Falduto，M.T.(1996).Neurons of the human frontal cortex display apolipoprotein E immunoreactivity：implications for Alzheimer′s disease.Journal ofneuropathology and experimental neurology55，372-380.
Mucke，L.，and Selkoe，D.J.(2012).Neurotoxicity of amyloid beta-protein：synaptic and network dysfunction.Cold Spring Harb Perspect Med 2，a006338.
Murrell，J.，Farlow，M.，Ghetti，B.，and Benson，M.D.(1991).A mutation in the amyloid precursor protein associated with hereditary Alzheimer′s disease.Science 254，97-99.
Oakley，H.，Cole，S.L.，Logan，S.，Maus，E.，Shao，P.，Craft，J.，Guillozet-Bongaarts，A.，Ohno，M.，Disterhoft，J.，Van Eldik，L.，et al.(2006).Intraneuronal beta-amyloid aggregates，neurodegeneration，and neuron loss in transgenic mice with five familial Alzheimer′s disease mutations：potential factors in amyloid plaque formation.The Journal of neuroscience：the official journal of the Society for Neuroscience 26，10129-10140.
Olsson，A.，K.，M.，Andreasen，N.，Minthon，L.，Lannfelt，L.，Buerger，K.，H.-J.，Hampel，H.，Davidsson，P.，et al.(2003).Measurement of t-andβ-secretase cleaved amyloid precursor protein in cerebrospinal fluid from Alzheimer patients.Experimental Neurology 183，74-80.
Panza，F.，Lozupone，M.，Logroscino，G.，and Imbimbo，B.P.(2019).A critical appraisal of amyloid-beta-targeting therapies for Alzheimer disease.Nature reviews Neurology 15，73-88.
Prince，M.(2015).World Alzheimer report 2015：the global impact of dementia.
Ran，F.A.，Hsu，P.D.，Wright，J.，Agarwala，V.，Scott，D.A.，and Zhang，F.(2013). Genome engineering using the CRISPR-Cas9 system.Nature protocols 8，2281-2308.
Rhinn，H.，Fujita，R.，Qiang，L.，Cheng，R.，Lee，J.H.，and Abeliovich，A.(2013).Integrative genomics identifies APOE epsilon4 effectors in Alzheimer′s disease.Nature 500，45-50.
Roberson，E.D.，and Mucke，L.(2006).100 years and counting：prospects for defeating Alzheimer′s disease.Science 314，781-784.
Roher，A.E.，Maarouf，C.L.，and Kokjohn，T.A.(2016).Familial Presenilin Mutations and Sporadic Alzheimer′s Disease Pathology：Is the Assumption of Biochemical Equivalence Justified？J Alzheimers Dis 50，645-658.
Schmechel，D.E.，Saunders，A.M.，Strittmatter，W.J.，Crain，B.J.，Hulette，C.M.，Joo，S.H.，Pericak-Vance，M.A.，Goldgaber，D.，and Roses，A.D.(1993).Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease.Proc Natl Acad Sci U S A 90，9649-9653.
Selkoe，D.J.(2001).Alzheimer′s disease：genes，proteins，and therapy.Physiological reviews 81，741-766.
Shen，J.，and Kelleher，R.J.，3rd(2007).The presenilin hypothesis of Alzheimer′s disease：evidence for a loss-of-function pathogenic mechanism.Proceedings of the National Academy of Sciences of the United States of America 104，403-409.
Strittmatter，W.J.，Saunders，A.M.，Schmechel，D.，Pericak-Vance，M.，Enghild，J.，Salvesen，G.S.，and Roses，A.D.(1993).Apolipoprotein E：high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease.Proceedings of the National Academy of Sciences of the United States of America 90，1977-1981.
Sun，L.，Zhou，R.，Yang，G.，and Shi，Y.(2017).Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production ofAβ42 and Aβ40 peptides byγ-secretase.Proc Natl Acad Sci U S A 114，E476-e485.
Tomiyama，T.，Nagata，T.，Shimada，H.，Teraoka，R.，Fukushima，A.，Kanemitsu，H.，Takuma，H.，Kuwano，R.，Imagawa，M.，Ataka，S.，et al.(2008).A new amyloid beta variant favoring oligomerization in Alzheimer′s-type dementia.Ann Neurol 63，377-387.
Walker，E.S.，Martinez，M.，Brunkan，A.L.，and Goate，A.(2005).Presenilin 2 familial Alzheimer′s disease mutations result in partial loss of function and dramatic changes in  Abeta 42/40 ratios.Journal of neurochemistry 92，294-301.
Wang，C.，Najm，R.，Xu，Q.，Jeong，D.E.，Walker，D.，Balestra，M.E.，Yoon，S.Y.，Yuan，H.，Li，G.，Miller，Z.A.，et al.(2018).Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector.Nat Med 24，647-657.
Wolfe，M.S.，Xia，W.，Ostaszewski，B.L.，Diehl，T.S.，Kimberly，W.T.，and Selkoe，D.J.(1999).Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity.Nature 398，513-517.
Wu，S.-H.，Li，X.，Qin，D.-D.，Zhang，L.-H.，Cheng，T.-L.，Chen，Z.-F.，Nie，B.-B.，Ren，X.-F.，Wu，J.，Wang，W.-C.，et al.(2021).Induction of core symptoms of autism spectrum disorder by in vivo CRISPR/Cas9-based gene editing in the brain of adolescent rhesus monkeys.Science Bulletin 66，937-946.
Xia，D.，Watanabe，H.，Wu，B.，Lee，S.H.，Li，Y.，Tsvetkov，E.，Bolshakov，V.Y.，Shen，J.，and Kelleher，R.J.，3rd(2015).Presenilin-1 knockin mice reveal loss-of-function mechanism for familial Alzheimer′s disease.Neuron 85，967-981.
Xia，Q.，Yang，X.，Shi，J.，Liu，Z.，Peng，Y.，Wang，W.，Li，B.，Zhao，Y.，Xiao，J.，Huang，L.，et al.(2021).The Protective A673T Mutation of Amyloid Precursor Protein(APP)in Alzheimer′s Disease.58，4038-4050.
Xia，W.(2019).gamma-Secretase and its modulators：Twenty years and beyond.Neuroscience letters 701，162-169.
Xu，P.-T.，Gilbert，J.R.，Qiu，H.-L.，Ervin，J.，Rothrock-Christian，T.R.，Hulette，C.，and Schmechel，D.E.(1999).Specific Regional Transcription of Apolipoprotein E in Human Brain Neurons.The American Journal of Pathology 154，601-611.
Xu，Q.，Bernardo，A.，Walker，D.，Kanegawa，T.，Mahley，R.W.，and Huang，Y.(2006).Profile and regulation of apolipoprotein E(ApoE)expression in the CNS in mice with targeting of green fluorescent protein gene to the ApoE locus.The Journal of neuroscience：the official journal of the Society for Neuroscience 26，4985-4994.
Yang，G.，Zhou，R.，Guo，X.，Yan，C.，Lei，J.，and Shi，Y.(2021).Structural basis ofγ-secretase inhibition and modulation by small molecule drugs.Cell 184，521-533.e514.
Yang，G.，Zhou，R.，Zhou，Q.，Guo，X.，Yan，C.，Ke，M.，Lei，J.，and Shi，Y.(2019).Structural basis of Notch recognition by humanγ-secretase.Nature 565，192-197.
Yu，J.T.，Tan，L.，and Hardy，J.(2014).Apolipoprotein E in Alzheimer′s disease：an update.Annu Rev Neurosci 37，79-100.
Zalocusky，K.A.，Najm，R.，Taubes，A.L.，Hao，Y.，Yoon，S.Y.，Koutsodendris，N.，Nelson，M.R.，Rao，A.，Bennett，D.A.，Bant，J.，et al.(2021).Neuronal ApoE upregulates MHC-I expression to drive selective neurodegeneration in Alzheimer′s disease.Nature neuroscience 24，786-798.

Claims (29)

  1. 一种分离的ApoE构建物，其包含一个ApoE多肽，所述ApoE多肽：
    (1)包含来自SEQ ID NO：12所示氨基酸序列的一个片段，该片段N端第一个氨基酸残基为SEQ ID NO：12第215-221位中的任意一个位置上的氨基酸残基，该片段C端最后一个氨基酸残基为SEQ ID NO：12第274-299位(优选第279-299位)中的任意一个位置上的氨基酸残基；或
    (2)其氨基酸序列与(1)所述片段具有至少约70％序列同一性，并保留至少约50％(1)所述片段对γ分泌酶酶切活性的抑制。
  2. 如权利要求1所述的分离的ApoE构建物，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含如下任一序列：
    (i)SEQ ID NO：12第215-299位氨基酸残基的氨基酸序列；
    (ii)SEQ ID NO：12第216-299位氨基酸残基的氨基酸序列；
    (iii)SEQ ID NO：12第217-299位氨基酸残基的氨基酸序列；
    (iv)SEQ ID NO：12第218-299位氨基酸残基的氨基酸序列；
    (v)SEQ ID NO：12第219-299位氨基酸残基的氨基酸序列；
    (vi)SEQ ID NO：12第220-299位氨基酸残基的氨基酸序列；
    (vii)SEQ ID NO：12第221-299位氨基酸残基的氨基酸序列；
    (viii)SEQ ID NO：12第216-294位氨基酸残基的氨基酸序列；
    (ix)SEQ ID NO：12第216-289位氨基酸残基的氨基酸序列；
    (x)SEQ ID NO：12第219-289位氨基酸残基的氨基酸序列；
    (xi)SEQ ID NO：12第219-288位氨基酸残基的氨基酸序列；
    (xii)SEQ ID NO：12第219-287位氨基酸残基的氨基酸序列；
    (xiii)SEQ ID NO：12第219-286位氨基酸残基的氨基酸序列；
    (xiv)SEQ ID NO：12第219-285位氨基酸残基的氨基酸序列；
    (xv)SEQ ID NO：12第219-284位氨基酸残基的氨基酸序列；
    (xvi)SEQ ID NO：12第219-279位氨基酸残基的氨基酸序列；
    (xvii)SEQ ID NO：12第219-274位氨基酸残基的氨基酸序列；
    (xviii)SEQ ID NO：12第220-274位氨基酸残基的氨基酸序列；
    (xix)SEQ ID NO：12第220-279位氨基酸残基的氨基酸序列；或
    (xx)SEQ ID NO：12第221-274位氨基酸残基的氨基酸序列。
  3. 如权利要求1或2所述的分离的ApoE构建物，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含SEQ ID NOs：13、18-34、78和87中任一氨基酸序列。
  4. 如权利要求1-3中任一项所述的分离的ApoE构建物，所述ApoE多肽或所述(1)中来自SEQ ID NO：12所示氨基酸序列的片段包含SEQ ID NOs：31-34、78和87中任一所示的氨基酸序列。
  5. 如权利要求1-4中任一项所述的分离的ApoE构建物，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变：R226、D227、R228、L229、D230、Q235、E238、V239、R240、K242、E244、E245、Q246、A247、Q248、Q249、I250、R251、L252、Q253、A254、E255、Q275、V280、V287、T289、S290、A291、P293、V294、P295、S296、D297、N298、H299、R251+T289、R251+T289+A291、S290+P293+V294+P295+S296+D297+N298+H299、R226+D227+R228+L229+D230、E238+V239+R240+K242、或E244+E245+Q246+A247+Q248+Q249+I250+R251+L252+Q253+A254。
  6. 如权利要求1-5中任一项所述的分离的ApoE构建物，所述ApoE多肽包含相对于SEQ ID NO：12所示氨基酸序列的一个或多个氨基酸位点的突变：R226A、D227A、R228A、L229A、D230A、Q235A、E238A、V239A、R240A、K242A、E244del、E245del、Q246del、A247del、Q248del、Q249del、I250del、R251S、L252del、Q253del、A254del、E255A、Q275A、V280A、V287A、T289A、S290A、A291T、P293A、V294A、P295A、S296A、D297A、N298A、H299A、R251A+T289A、R251 S+T289A+A291T、S290A+P293A+V294A+P295A+S296A+D297A+N298A+H299A、R226A+D227A+R228A+L229A+D230A、E238A+V239A+R240A+K242A、或E244del+E245del+Q246del+A247del+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del。
  7. 如权利要求1-6中任一项所述的分离的ApoE构建物，所述ApoE多肽包含SEQ ID NOs：46-52、57-59、62和76中任一氨基酸序列。
  8. 如权利要求1-7中任一项所述的分离的ApoE构建物，所述ApoE构建物在ApoE多肽的N端还进一步包含SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸残基的氨基酸序列，或与SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸至少有约70％同一性的序列。
  9. 如权利要求8所述的分离的ApoE构建物，所述ApoE构建物包含SEQ ID NO：60或61所示的氨基酸序列。
  10. 如权利要求1-9中任一项所述的分离的ApoE构建物，所述ApoE构建物在所述ApoE多肽的N端或C端还进一步包含一个穿膜肽。
  11. 如权利要求10所述的分离的ApoE构建物，所述穿膜肽包括SEQ ID NOs：35-45、63-65和84-86中任一氨基酸序列。
  12. 如权利要求10或11所述的分离的ApoE构建物，所述穿膜肽包括SEQ ID NO：63或65所示的氨基酸序列。
  13. 如权利要求10-12中任一项所述的分离的ApoE构建物，所述穿膜肽与所述ApoE多肽、或所述包含SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸残基的氨基酸序列、或所述与SEQ ID NO：12第1-167位或第1-205位或第1-214位氨基酸至少有约70％同一性的序列通过一个接头序列连接。
  14. 如权利要求1-13中任一项所述的分离的ApoE构建物，所述ApoE构建物在所述ApoE多肽的N端还进一步包含一个信号肽。
  15. 如权利要求14所述的分离的ApoE构建物，所述信号肽包含选自SEQ ID NOs：66-68中任一氨基酸序列。
  16. 一种分离的核酸，其编码如权利要求1-15中任一项所述的分离的ApoE构建物的蛋白部分。
  17. 一种载体，其包含权利要求16所述的分离的核酸。
  18. 如权利要求17所述的载体，所述载体在所述分离的核酸的5’端含有神经元特异性启动子。
  19. 如权利要求18所述的载体，所述神经元特异性启动子选自突触蛋白启动子、thy-1启动子和钙调素依赖型蛋白激酶II-α启动子。
  20. 如权利要求17-19中任一项所述的载体，所述载体为是重组AAV或lentivirus表达载体。
  21. 一种宿主细胞，其表达权利要求1-15中任一项所述的分离的ApoE构建物、含有权利要求16所述的分离的核酸、或含有权利要求17-20中任一项所述的载体。
  22. 如权利要求21所述的宿主细胞，所述宿主细胞为神经元。
  23. 一种药物组合物，其含有(i)权利要求1-15中任一项所述的分离的ApoE构建物、含有权利要求16所述的分离的核酸、含有权利要求17-20中任一项所述的载体、或含有权利要求21或22所述的宿主细胞，和(ii)药学上可接受的载体。
  24. 权利要求1-15中任一项所述的分离的ApoE构建物、权利要求16所述的分离的核酸、权利要求17-20中任一项所述的载体、权利要求21或22所述的宿主细胞、或权利要求23所述的药物组合物在制备用于治疗或预防一个个体中与γ分泌酶的酶切活性相关的疾病的药物中的应用。
  25. 一种治疗或预防一个个体中与γ分泌酶的酶切活性相关疾病的方法，包括向所述个体施用有效剂量的权利要求1-15中任一项所述的分离的ApoE构建物、权利要求16所述的分离的核酸、权利要求17-20中任一项所述的载体、权利要求21或22所述的宿主细胞、或权利要求23所述的药物组合物。
  26. 如权利要求24所述的应用或25所述的方法，所述药物或方法抑制γ分泌酶的酶切活性，从而治疗或预防所述与γ分泌酶的酶切活性相关的疾病。
  27. 如权利要求24-26中任一项所述的应用或方法，所述疾病选自如下的组：神经系统退行性疾病，癌症，炎性疾病和肾脏疾病。
  28. 如权利要求24-27中任一项所述的应用或方法，
    (i)所述神经系统退行性疾病选自如下的组：阿尔茨海默病及其相关疾病、淀粉样脑血管病、唐氏综合症、和与衰老相关的其他神经退行性疾病(如额颞叶痴呆)；
    (ii)所述癌症选自如下的组：头颈癌、乳腺癌、肝癌、胰腺癌、卵巢癌、肺癌、胶质瘤、纤维瘤、淋巴瘤、骨肉瘤、胃癌、和膀胱癌；
    (iii)所述炎性疾病选自如下的组：哮喘、肺炎、和气道炎症；和/或
    (iv)所述肾脏疾病选自如下的组：急性肾损伤、透明细胞肾细胞癌、肾纤维化、和梗阻性肾病。
  29. 如权利要求24-28中任一项所述的应用或方法，所述个体是人。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
EP0117058A2 (en) 1983-01-19 1984-08-29 Genentech, Inc. Methods for producing mature protein in vertebrate host cells
EP0117060A2 (en) 1983-01-19 1984-08-29 Genentech, Inc. Methods of screening and amplification in eukaryotic host cells, and nucleotide sequences and expression vectors for use therein

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6787519B2 (en) * 2000-11-03 2004-09-07 The Regents Of The University Of California Methods of treating disorders related to apoE
US20100204103A1 (en) * 2002-05-08 2010-08-12 The Regents Of The University Of California Helical synthetic peptides that stimulate cellular cholesterol efflux
WO2005058938A2 (en) * 2003-12-15 2005-06-30 The Regents Of The University Of California Helical synthetic peptides that stimulate cellular cholesterol efflux

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4399216A (en) 1980-02-25 1983-08-16 The Trustees Of Columbia University Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials
EP0117058A2 (en) 1983-01-19 1984-08-29 Genentech, Inc. Methods for producing mature protein in vertebrate host cells
EP0117060A2 (en) 1983-01-19 1984-08-29 Genentech, Inc. Methods of screening and amplification in eukaryotic host cells, and nucleotide sequences and expression vectors for use therein

Non-Patent Citations (109)

* Cited by examiner, † Cited by third party
Title
ANA DE JESUS-ACOSTA ET AL.: "A Phase II Study of the Gamma Secretase Inhibitor RO4929097 in Patients with Previously Treated Metastatic Pancreatic Adenocarcinoma", INVEST NEW DRUGS, vol. 32, no. 4, August 2014 (2014-08-01), pages 739 - 745, XP035906255, DOI: 10.1007/s10637-014-0083-8
ANTONIO MARAVER: "Therapeutic effect of γ-secretase inhibition in KrasG12V-driven non-small cell lung carcinoma through derepression of DUSP1 phosphatase and inhibition of ERK", CANCER CELL, vol. 22, no. 2, 14 August 2012 (2012-08-14)
AOKI, K.UCHIHARA, T.NAKAMURA, A.KOMORI, T.ARAI, N.MIZUTANI, T.: "Expression of apolipoprotein E in ballooned neurons-comparative immunohistochemical study on neurodegenerative disorders and infarction", ACTA NEUROPATHOL, vol. 106, 2003, pages 436 - 440
ARMSTRONG, R.A.: "What causes alzheimer's disease?", FOLIA NEUROPATHOL, vol. 51, 2013, pages 169 - 188
BART DE STROOPER ET AL., PRESENILINS AND Γ-SECRETASE: STRUCTURE, FUNCTION, AND ROLE IN ALZHEIMER'S DISEASE, vol. 2, 2012, pages a006304
BELLOY, M.E.NAPOLIONI, V.GREICIUS, M.D.: "A Quarter Century of APOE and Alzheimer's Disease: Progress to Date and the Path Forward", NEURON, vol. 101, 2019, pages 820 - 838, XP085620114, DOI: 10.1016/j.neuron.2019.01.056
BELONWU, S.A.LI, Y.BUNIS, D.RAO, A.A.SOLSBERG, C.W.TANG, A.FRAGIADAKIS, G.K.DUBAL, D.B.OSKOTSKY, T.SIROTA, M.: "Sex-Stratified Single-Cell RNA-Seq Analysis Identifies Sex-Specific and Cell Type-Specific Transcriptional Responses in Alzheimer's Disease Across Two Brain Regions", MOLECULAR NEUROBIOLOGY, vol. 59, 2022, pages 276 - 293T
BORCHELT ET AL., NEURON, vol. 182, 1997, pages 939 - 945
BOSHART ET AL., CELL, vol. 41, 1985, pages 521
BRECHT, W.J.HARRIS, F.M.CHANG, S.TESSEUR, I.YU, G.Q.XU, Q.DEE FISH, J.WYSS-CORAY, T.BUTTINI, M.MUCKE, L. ET AL.: "Neuron-specific apolipoprotein e4 proteolysis is associated with increased tau phosphorylation in brains of transgenic mice", THE JOURNAL OF NEUROSCIENCE: THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 24, 2004, pages 2527 - 2534
BUGIANI, O.GIACCONE, G.ROSSI, G.MANGIERI, M.CAPOBIANCO, R.MORBIN, M.MAZZOLENI, G.CUPIDI, C.MARCON, G.GIOVAGNOLI, A. ET AL.: "Hereditary cerebral hemorrhage with amyloidosis associated with the E693K mutation of APP", ARCHIVES OF NEUROLOGY, vol. 67, 2010, pages 987 - 995
CARTER ET AL., BIOTECHNOLOGY, vol. 10, 1992, pages 163 - 167
CHEN, J.LI, Q.WANG, J.: "Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 108, 2011, pages 14813 - 14818
CHEN, Y.WANG, Y.MODRUSAN, Z.SHENG, M.KAMINKER, J.S.: "Regulation of neuronal gene expression and survival by basal NMDA receptor activity: a role for histone deacetylase 4", THE JOURNAL OF NEUROSCIENCE: THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 34, 2014, pages 15327 - 15339
CITRON, M.OLTERSDORF, T.HAASS, C.MCCONLOGUE, L.HUNG, A.Y.SEUBERT, P.VIGO-PELFREY, C.LIEBERBURG, I.SELKOE, D.J.: "Mutation of the beta-amyloid precursor protein in familial Alzheimer's disease increases beta-protein production", NATURE, vol. 360, 1992, pages 672 - 674, XP002013249, DOI: 10.1038/360672a0
CITRON, M.TEPLOW, D.B.SELKOE, D.J.: "Generation of amyloid beta protein from its precursor is sequence specific", NEURON, vol. 14, 1995, pages 661 - 670, XP000577165, DOI: 10.1016/0896-6273(95)90323-2
CORDER, E.H.SAUNDERS, A.M.STRITTMATTER, W.J.SCHMECHEL, D.E.GASKELL, P.C.SMALL, G.W.ROSES, A.D.HAINES, J.L.PERICAK-VANCE, M.A.: "Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families", SCIENCE, vol. 261, 1993, pages 921 - 923, XP000619874, DOI: 10.1126/science.8346443
DE STROOPER, B.IWATSUBO, T.WOLFE, M.S.: "Presenilins and gamma-secretase: structure, function, and role in Alzheimer Disease", SPRING HARBOR PERSPECTIVES IN MEDICINE, vol. 2, 2012, pages a006304
DE STROOPER, B.KARRAN, E.: "The Cellular Phase of Alzheimer's Disease", CELL, vol. 164, 2016, pages 603 - 615
DE STROOPER, B.SAFTIG, P.CRAESSAERTS, K.VANDERSTICHELE, H.GUHDE, G.ANNAERT, W.VON FIGURA, K.VAN LEUVEN, F.: "Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein", NATURE, vol. 391, 1998, pages 387 - 390, XP001010557, DOI: 10.1038/34910
DICKSON ET AL., AM. J. PATH., vol. 132, 1988, pages 86 - 101
DIJKEMA ET AL., EMBOJ, vol. 4, 1985, pages 761
DORSZEWSKA, J.PRENDECKI, M.OCZKOWSKA, A.DEZOR, M.KOZUBSKI, W.: "Molecular Basis of Familial and Sporadic Alzheimer's Disease", CURRENT ALZHEIMER RESEARCH, vol. 13, 2016, pages 952 - 963
EDWARD PAN ET AL.: "Phase I Study of R04929097 with Bevacizumab in Patients with Recurrent Malignant Glioma", NEUROONCOL, vol. 130, no. 3, December 2016 (2016-12-01), pages 571 - 579, XP036103835, DOI: 10.1007/s11060-016-2263-1
EGGERT, S.PALIGA, K.SOBA, P.EVIN, G.MASTERS, C.L.WEIDEMANN, A.BEYREUTHER, K.: "The proteolytic processing of the amyloid precursor protein gene family members APLP-1 and APLP-2 involves alpha-, beta-, gamma-, and epsilon-like cleavages: modulation of APLP-1 processing by n-glycosylation", J BIOL CHEM, vol. 279, 2004, pages 18146 - 18156
ESCH, F.S.KEIM, P.S.BEATTIE, E.C.BLACHER, R.W.CULWELL, A.R.OLTERSDORF, T.MCCLURE, D.WARD, P.J.: "Cleavage of amyloid beta peptide during constitutive processing of its precursor", SCIENCE, vol. 248, 1990, pages 1122 - 1124, XP000573985, DOI: 10.1126/science.2111583
FURUKAWA ET AL., J. NEUROCHEM., vol. 67, 1996, pages 1882 - 1896
GETHING ET AL., NATURE, vol. 293, 1981, pages 620 - 625
GIINER, G.LICHTENTHALER, S.F.: "The substrate repertoire of γ-secretase/presenilin", SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY, vol. 105, 2020, pages 27 - 42
GORMAN ET AL., HUMAN, PROC.NATL.ACAD.SCL.USA, vol. 79, 1982, pages 6777
GRAHAM ET AL., J. GEN VIROL ., vol. 36, 1977, pages 59
GRAHAMVAN DER ERB, VIROLOGY, vol. 52, 1978, pages 456 - 457
HAASS, C.SCHLOSSMACHER, M.G.HUNG, A.YVIGO-PELFREY, C.MELLON, A.OSTASZEWSKI, B.L.LIEBERBURG, I.KOO, E.H.SCHENK, D.TEPLOW, D.B. ET A: "Amyloid beta-peptide is produced by cultured cells during normal metabolism", NATURE, vol. 359, 1992, pages 322 - 325
HAASS, C.SELKOE, D.J.: "Cellular processing of beta-amyloid precursor protein and the genesis of amyloid beta-peptide", CELL, vol. 75, 1993, pages 1039 - 1042
HAN, S.H.HULETTE, C.SAUNDERS, A.M.EINSTEIN, G.PERICAK-VANCE, M.STRITTMATTER, W.J.ROSES, A.D.SCHMECHEL, D.E.: "Apolipoprotein E is present in hippocampal neurons without neurofibrillary tangles in Alzheimer's disease and in age-matched controls", EXPERIMENTAL NEUROLOGY, vol. 128, 1994, pages 13 - 26
HARDY, J.SELKOE, D.J.: "The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics", SCIENCE, vol. 297, 2002, pages 353 - 356
HASHIMOTO, T.SERRANO-POZO, A.HORI, Y.ADAMS, K.W.TAKEDA, S.BANERJI, A.O.MITANI, A.JOYNER, D.THYSSEN, D.H.BACSKAI, B.J. ET AL.: "Apolipoprotein E, especially apolipoprotein E4, increases the oligomerization of amyloid beta peptide", THE JOURNAL OF NEUROSCIENCE: THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 32, 2012, pages 15181 - 15192, XP055198388, DOI: 10.1523/JNEUROSCI.1542-12.2012
HIGUCHI ET AL., NATURE NEUROSCI., vol. 8, 2005, pages 527 - 533
HOLCOMB ET AL., NAT. MED., vol. 4, 1998, pages 97 - 100
HOLTZMAN, D.M.BALES, K.R.WU, S.BHAT, P.PARSADANIAN, M.FAGAN, A.M.CHANG, L.K.SUN, Y.PAUL, S.M.: "Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer's disease", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 103, 1999, pages R15 - r21
HUANG, Y.A.ZHOU, B.WERNIG, M.SUDHOF, T.C.: "ApoE2, ApoE3, and ApoE4 Differentially Stimulate APP Transcription and Abeta Secretion", CELL, vol. 168, 2017, pages 427 - 441
HUI JIA ET AL.: "γ-Secretase inhibitors for breast cancer and hepatocellular carcinoma: From mechanism to treatment", LIFE SCIENCES, vol. 268, 2021, pages 119007, XP086492948, DOI: 10.1016/j.lfs.2020.119007
HUNG, V.UDESHI, N.D.LAM, S.S.LOH, KH.COX, K.J.PEDRAM, K.CARR, S.A.TING, A.Y.: "Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2", NATURE PROTOCOLS, vol. 11, 2016, pages 456 - 475, XP093135014, DOI: 10.1038/nprot.2016.018
HUYNH, T.V.LIAO, F.FRANCIS, C.M.ROBINSON, G.O.SERRANO, J.R.JIANG, H.ROH, J.FINN, M.B.SULLIVAN, P.M.ESPARZA, T.J. ET AL.: "Age-Dependent Effects of apoE Reduction Using Antisense Oligonucleotides in a Model of beta-amyloidosis", NEURON, vol. 96, 2017, pages 1013 - 1023
HYUN-WOO LEE ET AL.: "Targeting Notch signaling by c-secretase inhibitor I enhances the cytotoxic effect of 5-FU in gastric cancer", CLIN EXP METASTASIS, vol. 32, 2015, pages 593 - 603, XP035513988, DOI: 10.1007/s10585-015-9730-5
JEAN-CHRISTOPHE WYSS ET AL.: "Targeted γ-secretase inhibition of Notch signaling activation in acute renal injury", AM J PHYSIOL RENAL PHYSIOL, vol. 314, 2018, pages F736 - F746
JIN HYUN KANG ET AL.: "g-Secretase Inhibitor Reduces Allergic Pulmonary Inflammation by Modulating Th1 and Th2 Responses", AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, vol. 179, 2009, XP055120487, DOI: 10.1164/rccm.200806-893OC
JORDÀ-SIQUIER, T., PETREL, M., KOUSKOFF, V., SMAILOVIC, U., CORDELIÈRES, F., FRYKMAN, S.,MULLER, U., MULLE, C., AND BARTHET, G.: "APP accumulates with presynaptic proteins around amyloid plaques: A role for presynaptic mechanisms in Alzheimer's disease?", ALZHEIMER'S & DEMENTIA: THE JOURNAL OF THE ALZHEIMER'S ASSOCIATION, 2022
KANEKIYO, T.XU, H.BU, G.: "ApoE and Abeta in Alzheimer's disease: accidental encounters or partners?", NEURON, vol. 81, 2014, pages 740 - 754
KARRAN, E.DE STROOPER, B.: "The amyloid cascade hypothesis: are we poised for success or failure?", JOURNAL OF NEUROCHEMISTRY, vol. 139, 2016, pages 237 - 252
KATHERINE M. MORGAN ET AL.: "Gamma secretase inhibition by BMS-906024 enhances efficacy of paclitaxel in lung adenocarcinoma", MOL CANCER THE R, vol. 16, no. 12, December 2017 (2017-12-01), pages 2759 - 2769, XP055651326, DOI: 10.1158/1535-7163.MCT-17-0439
KEOWN ET AL., METHODS IN ENZYMOLOGY, 1989
KEOWN ET AL., METHODS IN ENZYMOLOGY, vol. 185, 1990, pages 527 - 537
KLUNK ET AL., ANN. NEUROL., vol. 55, 2004, pages 306 - 319
KUMAR-SINGH, S.DE JONGHE, C.CRUTS, M.KLEINERT, R.WANG, R.MERCKEN, M.DE STROOPER, B.VANDERSTICHELE, H.LÖFGREN, A.VANDERHOEVEN, I. E: "Nonfibrillar diffuse amyloid deposition due to a gamma (42)-secretase site mutation points to an essential role for N-truncated A beta(42) in Alzheimer's disease", HUMAN MOLECULAR GENETICS, vol. 9, 2000, pages 2589 - 2598
KWOK, J.B.LI, Q.X.HALLUPP, M.WHYTE, S.AMES, D.BEYREUTHER, K.MASTERS, C.L.SCHOFIELD, P.R.: "Novel Leu723Pro amyloid precursor protein mutation increases amyloid beta42(43) peptide levels and induces apoptosis", ANN NEUROL, vol. 47, 2000, pages 249 - 253, XP008061862, DOI: 10.1002/1531-8249(200002)47:2<249::AID-ANA18>3.0.CO;2-8
LAMMICH ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 3922 - 3927
LI, Z.SHUE, F.ZHAO, N.SHINOHARA, M.BU, G.: "APOE2: protective mechanism and therapeutic implications for Alzheimer's disease", MOLECULAR NEURODEGENERATION, vol. 15, 2020, pages 63, XP093043522, DOI: 10.1186/s13024-020-00413-4
LIANG MAO ET AL.: "γ-Secretase inhibitor reduces immunosuppressive cells and enhances tumor immunity in head and neck squamous cell carcinoma", INT.J.CANCER, vol. 142, 2018, pages 999 - 1009
LIU, C.C.ZHAO, N.FU, Y.WANG, N.LINARES, C.TSAI, C.W.BU, G.: "ApoE4 Accelerates Early Seeding of Amyloid Pathology", NEURON, vol. 96, 2017, pages 1024 - 1032
LIU, P.P.XIE, Y.MENG, X.Y.KANG, J.S.: "History and progress of hypotheses and clinical trials for Alzheimer's disease", SIGNAL TRANSDUCTION AND TARGETED THERAPY, vol. 4, 2019, pages 29
LU, P.BAI, X.C.MA, D.XIE, T.YAN, C.SUN, L.YANG, G.ZHAO, Y.ZHOU, R.SCHERES, S.H.W. ET AL.: "Three-dimensional structure of human y-secretase", NATURE, vol. 512, 2014, pages 166 - 170
M TANAKA ET AL.: "Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation", BRITISH JOURNAL OF CANCER, vol. 100, 2009, pages 1957 - 1965
MANSOUR ET AL., NATURE, vol. 336, 1988, pages 348 - 352
MANTEI ET AL., NATURE, vol. 281, 1979, pages 40 - 46
MATHER ET AL., ANNALS N.Y. ACAD. SCI., vol. 383, 1982, pages 44 - 68
MATHER, BIOL. REPROD., vol. 23, 1980, pages 243 - 251
METZGER, R.E.LADU, M.J.PAN, J.B.GETZ, G.S.FRAIL, D.E.FALDUTO, M.T.: "Neurons of the human frontal cortex display apolipoprotein E immunoreactivity: implications for Alzheimer's disease", JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY, vol. 55, 1996, pages 372 - 380
MUCKE, L.SELKOE, D.J.: "Neurotoxicity of amyloid beta-protein: synaptic and network dysfunction", COLD SPRING HARB PERSPECT MED, vol. 2, 2012, pages a006338
MURRELL, J.FARLOW, M.GHETTI, B.BENSON, M.D.: "A mutation in the amyloid precursor protein associated with hereditary Alzheimer's disease", SCIENCE, vol. 254, 1991, pages 97 - 99, XP001249309, DOI: 10.1126/science.1925564
OAKLEY, H.COLE, S.L.LOGAN, S.MAUS, E.SHAO, P.CRAFT, J.GUILLOZET-BONGAARTS, A.OHNO, M.DISTERHOFT, J.VAN ELDIK, L. ET AL.: "Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation", THE JOURNAL OF NEUROSCIENCE: THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 26, 2006, pages 10129 - 10140, XP002688009, DOI: 10.1523/​JNEUROSCI.1202-06.2006
OKAYAMA ET AL., MOL. CELL BIOL., vol. 5, 1985, pages 1136 - 1142
OLSSON, A.HOGLUND, K.SJOGREN, M.ANDREASEN, N.MINTHON, L.LANNFELT, L.BUERGER, K.MOLLER, H.-J.HAMPEL, H.DAVIDSSON, P. ET AL.: "Measurement of α- and β-secretase cleaved amyloid precursor protein in cerebrospinal fluid from Alzheimer patients", EXPERIMENTAL NEUROLOGY, vol. 183, 2003, pages 74 - 80
PANZA, F.LOZUPONE, M.LOGROSCINO, G.IMBIMBO, B.P.: "A critical appraisal of amyloid-beta-targeting therapies for Alzheimer disease", NATURE REVIEWS NEUROLOGY, vol. 15, 2019, pages 73 - 88
PRINCE, M., WORLD ALZHEIMER REPORT 2015: THE GLOBAL IMPACT OF DEMENTIA, 2015
RAN, F.A.HSU, P.D.WRIGHT, J.AGARWALA, V.SCOTT, D.A.ZHANG, F.: "Genome engineering using the CRISPR-Cas9 system", NATURE PROTOCOLS, vol. 8, 2013, pages 2281 - 2308, XP009174668, DOI: 10.1038/nprot.2013.143
RHINN, H.FUJITA, R.QIANG, L.CHENG, R.LEE, J.H.ABELIOVICH, A.: "Integrative genomics identifies APOE epsilon4 effectors in Alzheimer's disease", NATURE, vol. 500, 2013, pages 45 - 50
ROBERSON, E.D.MUCKE, L.: "100 years and counting: prospects for defeating Alzheimer's disease", SCIENCE, vol. 314, 2006, pages 781 - 784
ROHER, A.E.MAAROUF, C.L.KOKJOHN, T.A.: "Familial Presenilin Mutations and Sporadic Alzheimer's Disease Pathology: Is the Assumption of Biochemical Equivalence Justified?", J ALZHEIMERS DIS, vol. 50, 2016, pages 645 - 658
SCHMECHEL, D.E.SAUNDERS, A.M.STRITTMATTER, W.J.CRAIN, B.J.HULETTE, C.M.JOO, S.H.PERICAK-VANCE, M.A.GOLDGABER, D.ROSES, A.D.: "Increased amyloid beta-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease", PROC NATL ACAD SCI U S A, vol. 90, 1993, pages 9649 - 9653, XP000198199, DOI: 10.1073/pnas.90.20.9649
SCOPES: "Protein Purification Principles and Practice", 1987, SPRINGER-VERLAG
SELKOE, D.J.: "Alzheimer's disease: genes, proteins, and therapy", PHYSIOLOGICAL REVIEWS, vol. 81, 2001, pages 741 - 766
SHEN, J.KELLEHER, R.J., 3RD: "The presenilin hypothesis of Alzheimer's disease: evidence for a loss-of-function pathogenic mechanism", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 104, 2007, pages 403 - 409
SHIVAANI KUMMAR ET AL.: "Clinical Activity of the g-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors (Aggressive Fibromatosis", JOURNAL OF CLINICAL ONCOLOGY, vol. 35, no. 14, 10 May 2017 (2017-05-10), XP055655190, DOI: 10.1200/JCO.2016.71.1994
SHUJI TOHDA: "Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors", LEUKEMIA RESEARCH, vol. 314, 2006, pages 1385 - 1390, XP055559000, DOI: 10.1016/j.leukres.2006.05.003
SIEGMUND REISSMANN: "Cell penetration: scope and limitations by the application of cell-penetrating peptides", J. PEPT. SCI., vol. 20, 2014, pages 760 - 784, XP055407090, DOI: 10.1002/psc.2672
SKOVRONSKY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 97, 2000, pages 7609 - 7614
STRITTMATTER, W.J.SAUNDERS, A.M.SCHMECHEL, D.PERICAK-VANCE, M.ENGHILD, J.SALVESEN, G.S.ROSES, A.D.: "Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 90, 1993, pages 1977 - 1981, XP000619618, DOI: 10.1073/pnas.90.5.1977
SUN, L.ZHOU, R.YANG, G.SHI, Y.: "Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of A042 and Aβ40 peptides by y-secretase", PROC NATL ACAD SCI U S A, vol. 114, 2017, pages E476 - e485
THOMAS ET AL., CELL, vol. 51, 1987, pages 503 - 512
TOMIYAMA, T.NAGATA, T.SHIMADA, H.TERAOKA, R.FUKUSHIMA, A.KANEMITSU, H.TAKUMA, H.KUWANO, R.IMAGAWA, M.ATAKA, S. ET AL.: "A new amyloid beta variant favoring oligomerization in Alzheimer's-type dementia", ANN NEUROL, vol. 63, 2008, pages 377 - 387, XP055239846, DOI: 10.1002/ana.21321
WALKER, E.S.MARTINEZ, M.BRUNKAN, A.L.GOATE, A.: "Presenilin 2 familial Alzheimer's disease mutations result in partial loss of function and dramatic changes in Abeta 42/40 ratios", JOURNAL OF NEUROCHEMISTRY, vol. 92, 2005, pages 294 - 301
WANG, C.NAJM, R.XU, Q.JEONG, D.E.WALKER, D.BALESTRA, M.E.YOON, S.Y.YUAN, H.LI, G.MILLER, Z.A. ET AL.: "Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector", NAT MED, vol. 24, 2018, pages 647 - 657, XP036901060, DOI: 10.1038/s41591-018-0004-z
WEIMING XIA: "Secretase and its modulators: Twenty years and beyond", NEUROSCIENCE LETTERS, vol. 701, 2019, pages 162 - 169, XP085666612, DOI: 10.1016/j.neulet.2019.02.011
WEIXI ZHANG ET AL.: "γ-Secretase Inhibitor Alleviates Acute Airway Inflammation of Allergic Asthma in Mice by Downregulating Th17 Cell Differentiation", MEDIATORS OF INFLAMMATION, vol. 2015, pages 7
WENGENACK ET AL., NAT. BIOTECHNOL., vol. 18, 2000, pages 868 - 872
WOLFE, M.S., XIA, W., OSTASZEWSKI, B.L., DIEHL, T.S., KIMBERLY, W.T., AND SELKOE, D.J.: "Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity", NATURE, vol. 398, 1999, pages 513 - 517, XP002186848, DOI: 10.1038/19077
WU, S.-H.LI, X.QIN, D.-D.ZHANG, L.-H.CHENG, T.-L.CHEN, Z.-F.NIE, B.-B.REN, X.-F.WU, J.WANG, W.-C. ET AL.: "Induction of core symptoms of autism spectrum disorder by in vivo CRISPR/Cas9-based gene editing in the brain of adolescent rhesus monkeys", SCIENCE BULLETIN, vol. 66, 2021, pages 937 - 946, XP093095716, DOI: 10.1016/j.scib.2020.12.017
XIA, D.WATANABE, H.WU, B.LEE, S.H.LI, Y.TSVETKOV, E.BOLSHAKOV, V.Y.SHEN, J.KELLEHER, R.J., 3RD: "Presenilin-1 knockin mice reveal loss-of-function mechanism for familial Alzheimer's disease", NEURON, vol. 85, 2015, pages 967 - 981
XIA, Q., YANG, X., SHI, J., LIU, Z., PENG, Y., WANG, W., LI, B., ZHAO, Y., XIAO, J., HUANG, L., THE PROTECTIVE A673T MUTATION OF AMYLOID PRECURSOR PROTEIN (APP) IN ALZHEIMER'S DISEASE, vol. 58, 2021, pages 4038 - 4050
XIA, W.: "gamma-Secretase and its modulators: Twenty years and beyond", NEUROSCIENCE LETTERS, vol. 701, 2019, pages 162 - 169, XP085666612, DOI: 10.1016/j.neulet.2019.02.011
XU, P.-T.GILBERT, J.R.QIU, H.-L.ERVIN, J.ROTHROCK-CHRISTIAN, T.R.HULETTE, C.SCHMECHEL, D.E.: "Specific Regional Transcription of Apolipoprotein E in Human Brain Neurons", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 154, 1999, pages 601 - 611
XU, Q.BERNARDO, A.WALKER, D.KANEGAWA, T.MAHLEY, R.W.HUANG, Y.: "Profile and regulation of apolipoprotein E (ApoE) expression in the CNS in mice with targeting of green fluorescent protein gene to the ApoE locus", THE JOURNAL OF NEUROSCIENCE: THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 26, 2006, pages 4985 - 4994
YANG, G.ZHOU, R.GUO, X.YAN, C.LEI, J.SHI, Y.: "Structural basis of γ-secretase inhibition and modulation by small molecule drugs", CELL, vol. 184, 2021, pages 521 - 533
YANG, G.ZHOU, R.ZHOU, Q.GUO, X.YAN, C.KE, M.LEI, J.SHI, Y.: "Structural basis of Notch recognition by human γ-secretase", NATURE, vol. 565, 2019, pages 192 - 197, XP036668893, DOI: 10.1038/s41586-018-0813-8
YIBING WANG ET AL.: "γ-secretase inhibitor inhibits bladder cancer cell drug resistance and invasion by reducing epithelial-mesenchymal transition", MOLECULAR MEDICINE REPORTS, vol. 12, 2015, pages 2821 - 2827
YU, J.T.TAN, L.HARDY, J.: "Apolipoprotein E in Alzheimer's disease: an update", ANNU REV NEUROSCI, vol. 37, 2014, pages 79 - 100
ZALOCUSKY, K.A.NAJM, R.TAUBES, A.L.HAO, Y.YOON, S.Y.KOUTSODENDRIS, N.NELSON, M.R.RAO, A.BENNETT, D.A.BANT, J. ET AL.: "Neuronal ApoE upregulates MHC-I expression to drive selective neurodegeneration in Alzheimer's disease", NATURE NEUROSCIENCE, vol. 24, 2021, pages 786 - 798, XP037471301, DOI: 10.1038/s41593-021-00851-3
ZHAOYI FENG: "Inhibition of gamma-secretase in Notch1 signaling pathway as a novel treatment for ovarian cancer", ONCOTARGET, vol. 8, no. 5, 2017, pages 8215 - 8225

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