C-terminal fragment of apolipoprotein E and its application in inhibiting γ-secretase activity

Abstract

The present invention relates to a C-terminal fragment of apolipoprotein E (ApoE) (e.g., ApoE CT) and its application in inhibiting modified ApoE2 and gamma-secretase activity. One aspect of the present invention provides an isolated ApoE construct, which comprises an ApoE polypeptide, the ApoE polypeptide comprising: (1) a fragment derived from the amino acid sequence shown in SEQ ID NO:12, the first amino acid residue at the N-terminus of the fragment being any one of amino acid residues 215-221 of SEQ ID NO:12 and the most C-terminal amino acid residue of the fragment being any one of amino acid residues 274-299 of SEQ ID NO:12; or (2) the amino acid sequence of which has at least about 70% sequence identity with the fragment of (1) and retains at least about 50% of the inhibition of gamma-secretase cleavage activity by the fragment of (1). The invention further provides coding sequences for the protein portion of said ApoE constructs, nucleic acid constructs, host cells, methods of construction, pharmaceutical compositions and uses.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims priority to Chinese Application No. CN202210113320.2, filed on January 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (217334 1PCWO.xml; size: 92,558 bytes; creation date: January 30, 2023) are incorporated herein by reference in their entirety.

[0003] Technical Field The present invention relates to a C-terminal fragment of apolipoprotein E (ApoE) and its application in inhibiting γ-secretase activity. [Background technology]

[0004] γ-secretase is an enzyme that cleaves over 100 types of type I transmembrane proteins and is involved in various biological processes. T cell and B cell differentiation abnormalities, bloody diarrhea, skin and hair defects, and Alzheimer's disease (AD) are all related to γ-secretase abnormalities. γ-secretase is a complex of four proteins: NCT, APH1, PEN2, and presenilin 1 (PS1) or presenilin 2 (PS2). The enzymatic active center of γ-secretase is PS1 or PS2. Currently, various mutations in PS1 and PS2 have been found to cause AD, and both PS1 and PS2 are pathogenic genes for AD, making γ-secretase a very important drug target, especially for the treatment of AD.

[0005] AD is the most common neurodegenerative disease in humans. AD patients mainly show symptoms such as progressive memory impairment, cognitive impairment, personality changes, and speech disorders, which further develop into loss of self-care ability and ultimately lead to death. China has the largest number of AD patients in the world. As of 2019, there are more than 10 million AD patients in China, and the number is increasing every year. The probability of AD development in people over 80 years old is more than 40%. In addition, the treatment costs of AD are very high, which places a huge burden on families and society.

[0006] In the past few decades, research on the pathogenesis of AD has made great progress, and the Aβ cascade hypothesis has been proposed. This hypothesis believes that the overproduction and abnormal accumulation of Aβ in the brain is the initial cause of a series of severe and slow diseases, which triggers a series of abnormal biological processes in the brain, resulting in synaptic degeneration and neuronal death in neurons. In the brain, amyloid precursor protein (APP) is mainly expressed by neurons, and Aβ is generated by the successive cleavage of APP by β and γ secretases, and the generated Aβ is released outside the cells, and this process is called amyloid metabolism of APP. Under normal physiological conditions, Aβ generated in the brain is mostly soluble. However, when pathology occurs, a large amount of Aβ abnormally accumulates in the brain, forming amyloid plaques that deposit around nerve cells, causing neuronal damage, further causing brain tissue failure, and ultimately causing AD. In addition, abnormal aggregation and deposition of amyloid proteins in cerebral blood vessels can cause amyloid cerebrovascular disease. APP is not only cleaved by β- and γ-secretases to generate toxic Aβ, but also by α- and γ-secretases, which are the non-amyloid metabolic pathways of APP. The cleavage site of α-secretase is located inside Aβ, so the α-cleavage product does not generate Aβ, and the cleavage fragment is called α-CTF, which is non-neurotoxic and does not cause AD. The cleavage of α- and β-enzymes compete with each other, and an increase in the enzymatic cleavage of α-secretase reduces the production of Aβ; similarly, a decrease in the enzymatic cleavage of γ-secretase reduces the production of Aβ, slows the formation of amyloid plaques, and ultimately delays and even prevents the development of AD and amyloid cerebrovascular disease. Various drugs targeting γ-secretase are being tested in clinical trials.

[0007] ApoE is a protein that is widely secreted in the human body and can be co-expressed with APP in neurons in the brain. There are three isomers of human ApoE: ApoE2, ApoE3, and ApoE4; they are very similar in structure and function and are roughly divided into three functional domains: the N-terminal domain containing the receptor binding site, the C-terminal domain containing the lipid binding site, and the central flexible hinge region connecting the two domains. ApoE2, ApoE3, and ApoE4 only differ in two amino acid residues in the N-terminal domain, and have completely identical amino acid sequences in the C-terminal domain. Among them, ApoE2 has a protective effect and can reduce the risk of developing AD; ApoE4 is the main risk factor for AD, and not only significantly increases the risk of AD, but also significantly accelerates the age of onset of AD and aggravates the condition of AD. Summary of the Invention [Problem to be solved by the invention]

[0008] So far, it remains unclear how ApoE2 and ApoE4 affect the risk of AD. [Means for solving the problem]

[0009] Contents of the invention In one aspect, the invention provides an isolated ApoE construct, comprising an ApoE polypeptide, the ApoE polypeptide comprising: (1) a fragment derived from the amino acid sequence set forth in SEQ ID NO:12, wherein the first amino acid residue at the N-terminus of the fragment (or the ApoE polypeptide) is any one of amino acid residues 215-221 of SEQ ID NO:12, and the final amino acid residue at the C-terminus of the fragment (or the ApoE polypeptide) is any one of amino acid residues 215-221 of SEQ ID NO:12. or (2) the amino acid sequence of which has at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the fragment described in (1) and retains at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5-fold, 2-fold, 5-fold, 10-fold or more) inhibition of gamma secretase cleavage activity by the fragment described in (1).

[0010] In some embodiments of any one of the isolated ApoE constructs, the ApoE polypeptide or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above comprises (or consists of, or consists essentially of) any one of the following sequences: (i) the amino acid sequence of amino acid residues 215-299 of SEQ ID NO:12; (ii) the amino acid sequence of amino acid residues 216-299 of SEQ ID NO:12; (iii) the amino acid sequence of amino acid residues 217-299 of SEQ ID NO:12; (iv) the amino acid sequence of amino acid residues 218-299 of SEQ ID NO:12; (v) the amino acid sequence of amino acid residues 219-299 of SEQ ID NO:12; (vi) the amino acid sequence of amino acid residues 220-299 of SEQ ID NO:12; (vii) the amino acid sequence of amino acid residues 221-299 of SEQ ID NO:12; (viii) the amino acid sequence of amino acid residues 222-299 of SEQ ID NO:12; (ix) the amino acid sequence of amino acid residues at positions 216-294 of SEQ ID NO:12; (ix) the amino acid sequence of amino acid residues at positions 216-289 of SEQ ID NO:12; (x) the amino acid sequence of amino acid residues at positions 219-289 of SEQ ID NO:12; (xi) the amino acid sequence of amino acid residues at positions 219-288 of SEQ ID NO:12; (xii) the amino acid sequence of amino acid residues at positions 219-287 of SEQ ID NO:12; (xiii) the amino acid sequence of amino acid residues at positions 219-286 of SEQ ID NO:12; (xiv) the amino acid sequence of amino acid residues at positions 219-285 of SEQ ID NO:12; (xv) the amino acid sequence of amino acid residues at positions 219-284 of SEQ ID NO:12; (xvi) the amino acid sequence of amino acid residues at positions 219-279 of SEQ ID NO:12; (xvii) the amino acid sequence of amino acid residues at positions 219-279 of SEQ ID NO:12 (xviii) the amino acid sequence of amino acid residues 220-274 of SEQ ID NO:12; (xix) the amino acid sequence of amino acid residues 220-279 of SEQ ID NO:12; or (xx) the amino acid sequence of amino acid residues 221-274 of SEQ ID NO:12.

[0011] In some embodiments of any one of the isolated ApoE constructs, the ApoE polypeptide or fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above comprises (or consists of, or consists essentially of) any one of the amino acid sequences set forth in SEQ ID NO:13, 18-34, 78, and 87. In some embodiments, the ApoE polypeptide or fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above comprises (or consists of, or consists essentially of) any one of the amino acid sequences set forth in SEQ ID NO:31-34, 78, and 87 (e.g., SEQ ID NO:33).

[0012] In some embodiments of any one of the isolated ApoE constructs, the ApoE polypeptide comprises (or consists of, or consists essentially of) an amino acid sequence having at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to an amino acid sequence of any one of SEQ ID NOs: 13, 18-34, 78, and 87. In some embodiments, the ApoE polypeptide contains a highly conserved sequence homologous to a non-human primate sequence, e.g., an amino acid sequence of any one of SEQ ID NOs: 13, 18-34, 78, and 87.

[0013] In some embodiments of any one of the isolated ApoE constructs, the ApoE polypeptide comprises a mutation (e.g., an insertion, deletion, or substitution) at one or more amino acid sites relative to the amino acid sequence set forth 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, P2 93, 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+A247+Q248+Q249+I250+R251+L252+Q253+A254. Contains mutations at one or more amino acid sites relative to the amino acid sequence shown in 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+P295A+S296A+D297A+N298A+H299A, R226A+D227A+R228A+L229A+D230A, E238A+V239A+R240A+K242A, or E244del+E245del+Q246del+A247del+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del.

[0014] In some embodiments of any one of the isolated ApoE constructs, the ApoE polypeptide comprises (or consists of, or consists essentially of) the amino acid sequence of any one of SEQ ID NOs:46-52, 57-59, 62, and 76.

[0015] In some embodiments of any one of the isolated ApoE constructs, the N-terminus of the ApoE polypeptide in the ApoE construct further comprises an amino acid sequence of amino acid residues 1-167, or 1-205, or 1-214 of SEQ ID NO: 12, or a sequence having at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identity to amino acid residues 1-167, or 1-205, or 1-214 of SEQ ID NO: 12. In some embodiments, the ApoE construct comprises (or consists of, or consists essentially of) the amino acid sequence set forth in SEQ ID NO:60 or 61.

[0016] In some embodiments of any one of the isolated ApoE constructs, the N-terminus or C-terminus of the ApoE polypeptide in the ApoE construct further comprises a membrane-permeable peptide. In some embodiments, the membrane-permeable peptide comprises (or consists of, or consists essentially of) the amino acid sequence of any one of SEQ ID NOs:35-45, 63-65, and 84-86, e.g., comprises (or consists of, or consists essentially of) the amino acid sequence set forth in SEQ ID NO:63 or 65. In some embodiments, the membrane-permeable peptide (e.g., at the C-terminus) is connected via a linker sequence to the ApoE polypeptide (e.g., at the N-terminus) or to an amino acid sequence (e.g., at the N-terminus) comprising amino acid residues 1-167, 1-205, or 1-214 of SEQ ID NO:12, or to a sequence (e.g., at the N-terminus) that has at least about 70% identity to amino acid residues 1-167, 1-205, or 1-214 of SEQ ID NO:12.

[0017] In some embodiments of any one of the above isolated ApoE constructs, the N-terminus of the ApoE polypeptide in the ApoE construct further comprises a signal peptide, hi some embodiments, the signal peptide comprises (or consists of, or consists essentially of) any one of the amino acid sequences set forth in SEQ ID NOs:66-68.

[0018] In another aspect, the present invention provides an isolated ApoE construct, comprising element (1): a membrane-permeable peptide, and element (2): a full-length sequence of ApoE2 (e.g., SEQ ID NO:12) or a full-length sequence of ApoE3 (e.g., SEQ ID NO:89), or a fragment containing at least amino acid residues 221-274 of SEQ ID NO:12; provided that element (1) and element (2) are directly connected via a peptide bond or connected via a linker sequence. In some embodiments, the membrane-permeable peptide is selected from any one of the amino acid sequences of SEQ ID NO:35-45, 63-65, and 84-86, e.g., the amino acid sequence shown in SEQ ID NO:63 or 65. In some embodiments, the fragment comprising at least amino acid residues 221-274 of SEQ ID NO:12 set forth in element (2) is selected from an ApoE polypeptide comprising amino acid residues 221-274 of SEQ ID NO:12 of any one of the polypeptides described herein. In some embodiments, the amino acid sequence of the fragment comprising at least amino acid residues 221-274 of SEQ ID NO:12 set forth in element (2) is set forth in any one of SEQ ID NOs:13, 18-34, and 78. In some embodiments, the fragment comprising at least amino acid residues 221-274 of SEQ ID NO:12 set forth in element (2) includes a fragment comprising the N-terminal domain (amino acid residues 1-167 of SEQ ID NO:12), the hinge region (amino acid residues 168-205 of SEQ ID NO:12), and the C-terminus of at least amino acid residues 221-274 of SEQ ID NO:12 of ApoE2.In some embodiments, the element (2) comprises any one of the following amino acid residues: 1-274, 1-279, 1-280, 1-281, 1-282, 1-283, 1-284, 1-285, 1-286, 1-287, 1-288, 1-289, 1-290, 1-291, 1-292, 1-293, 1-294, 1-295, 1-296, 1-297, 1-298, or 1-299 of SEQ ID NO:12.

[0019] The present invention further provides isolated nucleic acids and vectors (e.g., cloning vectors or expression vectors) encoding the protein portion of any one of the isolated ApoE constructs described above, and host cells expressing or comprising the protein portion of any one of the isolated ApoE constructs described above. In some embodiments, the vector comprises a neuron-specific promoter at the 5' end of the isolated nucleic acid. In some embodiments, the neuron-specific promoter is selected from a synapsin promoter, a thy-1 promoter, and a calmodulin-dependent protein kinase II-alpha promoter. In some embodiments, the vector is a recombinant AAV or lentivirus expression vector. In some embodiments, the host cell is a neuron (e.g., a human neuron, a non-human primate neuron, or a rodent neuron). In some embodiments, the ApoE construct comprises (or consists of, or consists essentially of) the full-length amino acid sequence of ApoE2 (e.g., SEQ ID NO:12) or ApoE3 (e.g., SEQ ID NO:89).

[0020] The present invention further provides a pharmaceutical composition comprising (i) an isolated ApoE construct, isolated nucleic acid, vector, or host cell described in any one of the embodiments herein, and (ii) a pharma- ceutically acceptable carrier.

[0021] The present invention further provides the use of an isolated ApoE construct, an isolated nucleic acid, a vector, a host cell, or a pharmaceutical composition as described in any one of the embodiments herein in the preparation of a medicament for treating or preventing a disease associated with the cleavage activity of gamma-secretase in an individual (e.g., a human). The present invention further provides a method for treating or preventing a disease associated with the cleavage activity of gamma-secretase in an individual (e.g., a human), comprising administering to the individual an effective dosage of an isolated ApoE construct, an isolated nucleic acid, a vector, a host cell, or a pharmaceutical composition as described in any one of the embodiments herein. In some embodiments, the medicament or method inhibits the cleavage activity of gamma-secretase and treats or prevents a disease associated with the cleavage activity of gamma-secretase. In one or more embodiments, the disease is selected from a neurodegenerative disease, a tumor or cancer, an inflammatory disease, and a kidney disease. In one or more embodiments, the disease is selected from Alzheimer's disease and related disorders, amyloid cerebrovascular disease, Down's syndrome, other age-related neurodegenerative diseases (such as frontotemporal dementia), head and neck cancer (e.g., 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 (e.g., non-small cell lung cancer, lung adenocarcinoma and small cell lung cancer), glioma (e.g., malignant glioma), fibroma, lymphoma (e.g., B cell lymphoma), osteosarcoma, gastric cancer, bladder cancer, asthma (e.g., allergic asthma), pneumonia, airway inflammation, acute kidney injury, clear cell renal cell carcinoma, renal fibrosis, and obstructive nephropathy. [Brief description of the drawings]

[0022] [Figure 1]Figure 1: ApoE attenuates the amyloid metabolism of APP and increases its nonamyloid metabolites (α-CTF) through its C-terminal fragment (ApoE CT). Panels A and B: Overexpression of ApoE2 and ApoE3 in HEK293T cells can significantly increase α-CTF produced from coexpressed APP through the nonamyloid metabolic pathway (α-cleavage), but ApoE4 cannot. The statistical results are shown in panel B, N=8, paired or one sample t-test. Panel C: Overexpression ApoE2 by transient transfection reduces total Aβ40 produced from coexpressed APP, N=8, paired or one sample t-test. Panels D and E: Immunoblot experiments show that overexpression of ApoE CT increases α-CTF produced from co-expressed APP, whereas overexpression of N-terminal fragments of ApoE2 or ApoE4 (ApoE2 NT, ApoE4 NT) does not change the production of α-CTF. The statistical results are shown in Panel E, N=8, paired or one sample t-test. Panels F and G: Overexpression of ApoE CT reduces Aβ40 and Aβ42 produced from APP, whereas overexpression of ApoE2 NT, ApoE3 NT, or ApoE4 NT does not change Aβ, N=5, one sample t-test. Panels AG: HEK 293T cells. Panel H: ApoE CT expressed using a lentivirus expression system in primary cultured neurons of mouse cerebral cortex and hippocampus can reduce the endogenous production of Aβ40 in neurons. The promoter is hSyn. N=7, one sample t-test. Panel I: ApoE CT expressed in human neurons derived from a pluripotent stem cell line using lentivirus can also reduce endogenous Aβ40 produced by neurons. The promoter is hSyn, N=5, unpaired t-test. Proteins APP, α-CTF and ApoE are detected by Western Blot and Aβ is detected by ELISA. [Diagram 2]Figure 2: ApoE2 and ApoE CT in the medium cannot increase nonamyloid metabolites of APP in cells. Panel A: ApoE2 and ApoE CT proteins in the medium are detected by immunoblotting experiments. Panels B and C: Co-incubation with cell culture medium containing ApoE2 or ApoE CT protein does not change α-CTF produced from APP in cells, and the statistical results are shown in panel C, N=9, one sample t-test. Panel D: Immunofluorescence experiments showed that after co-incubation of HEK 293T cells with cell culture medium containing ApoE2 or ApoE CT protein, no large amount of ApoE2 or ApoE CT protein was detected in the cells, and only a small portion was attached to the surface of individual cells. ApoE CT expressed by transient transfection in HEK 293T cells using liposomes is abundantly present in the cells. Panel E: Immunofluorescence experiments showed that after co-incubation with medium containing ApoE2 or ApoE CT protein, no significant amount of ApoE2 or ApoE CT protein was detected in primary cultured mouse neurons, and only a small fraction of ApoE was attached to the surface of individual neurons. Lentivirally expressed ApoE CT is abundantly present in neurons. MAP2 labels neurons and DAPI labels nuclei. [Diagram 3]Figure 3: ApoE CT reduces the Aβ core sequence. Panels A, B and C: Effect of overexpression of different ApoE CT mutants on Aβ production by APP in HEK 293T cells. Panel A: CT NA1 / 2 / 3 / 4 / / 5: Insertion of 1, 2, 3, 4 or 5 amino acid residues originally present in ApoE at the N-terminus of ApoE CT. CT ND3 / 4: Deletion of 3 or 4 amino acid residues from the N-terminus of ApoE CT. CT CD5 / 10: Deletion of 5 or 10 amino acid residues from the C-terminus of ApoE CT. The effect of different ApoE CT mutants on Aβ40 was measured by ELISA. Panel B: CT ND1 / 2 / / 5: Deletion of 1, 2 or 5 amino acid residues from the N-terminus of ApoE CT. The effect of different ApoE CT mutants on Aβ40 was measured by ELISA. Panel C: CT ND3CD10 / 11 / 12 / 13 / 14 / 15 / 20 / 25: deletion of 3 amino acid residues from the N-terminus of ApoE CT and deletion of 10, 11, 12, 13, 14, 15, 20 or 25 amino acids from the C-terminus. The effect of different ApoE CT mutants on Aβ40 was measured by ELISA. N=5 (panels A and B) or 7 (panel C), one sample t-test. [Figure 4] Figure 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 expression of APP metabolic enzyme mRNA protein. ADAM10: α-secretase of APP; BACE1: β-secretase of APP; PS1 and PS2: core active subunit proteins of γ-secretase. qRT-PCR and Western Blot were used to detect the mRNA and protein levels, respectively. Cells expressing GFP are the negative control. N=9, one sample t-test. [Diagram 5]Figure 5: ApoE CT simulates the effect of γ-secretase inhibitors on APP cleavage and inhibits γ-cleavage of β-CTF. Panels A and B: Overexpression of γ-secretase active center protein PS1 can reduce the production of α-CTF, while expression of ApoE CT or treatment with 1 μM PF03084014 (γ-secretase inhibitor) both increase the production of α-CTF. Statistical results are shown in Panel B, N=11, paired or one sample t-test. Panels C and D: Overexpression of ApoE CT can inhibit Aβ40 (N=4) and Aβ42 (N=6) produced by β-CTF, one sample t-test. Panel E: Overexpression of ApoE CT has no effect on Aβ40 produced by β-cleavage of APPΔAICD. Proteins were detected by Western Blot and Aβ was detected by ELISA. [Figure 6] Figure 6: ApoE CT affects the presence of γ-secretase required for APP cleavage. Panels A and D: In HEK 293T cells in which the γ-secretase active center proteins PS1 and PS2 were knocked out (PS1 / 2 dKO), α-CTF was increased in large amounts, and in this case, overexpression of ApoE CT did not further increase α-CTF, the statistical results are shown in panel D (two columns labeled with GFP1). Panels B, C and D: In PS1 / 2 dKO cells supplemented with expression of PS1 or PS2, overexpression of ApoE CT once again increased α-CTF, the statistical results are shown in panel D, N=8, paired t-test. Panel E: In PS1 / 2 dKO cells supplemented with expression of PS2, overexpression of ApoE CT reduced Aβ40 produced from APP, N=4, one sample t-test. Proteins are detected by Western Blot and Aβ40 is detected by ELISA. [Figure 7]Figure 7: In vitro purified ApoE2 directly inhibits γ-cleavage of APP in a concentration-dependent manner. Panel A: Immunoblots show isolated purified β-CTF, ApoE2, ApoE2 NT, PS1, NCT, PEN2 and GFP. Panel B: In the purified system, γ-secretase cleavage of β-CTF to produce Aβ40 was inhibited by 10 μM PF03084014 (γ-secretase inhibitor), N=3, paired t-tests. Panel C: Purified ApoE2 inhibits γ-secretase cleavage of β-CTF and reduces Aβ40 production in a dose-dependent manner. The half inhibitory concentration of ApoE2 is approximately 1.2 μM, N=3. Panel D: Purified ApoE2 can inhibit Aβ40 generated by γ-cleavage of β-CTF, but ApoE2 NT cannot inhibit β-CTF cleavage by γ-secretase, N=3, paired t-tests. Proteins were detected by Western Blot, and Aβ40 was detected by ELISA. [Figure 8] Figure 8: ApoE CT does not inhibit gamma cleavage of APLP1. 1 μM PF03084014 (gamma secretase inhibitor) inhibits gamma cleavage of exogenously expressed APLP1 in HEK 293T cells and increases α-like CTFs produced by α-cleavage of APLP1; however, ApoE CT does not increase α-like CTFs produced by APLP1. [Figure 9]Figure 9: Overexpression of ApoE CT in neurons reduces amyloid plaques in the brain of 5xFAD mouse model. Panel A: Representative images of amyloid plaques (marked with THS) in the subiculum of 5xFAD mouse model and co-staining with ApoE CT or GFP. In 5xFAD mice, ApoE CT was expressed in neurons using an adeno-associated virus (AAV) expression system (injected at 1 month of age and analyzed at 7 months of age), GFP was used as a control, and the promoter was hSyn (human synapsin). The nucleic acid sequence encoding the polypeptide is preceded by a sequence encoding a signal peptide. The statistical results are shown in panels B, C, D, and E. Panel B: In the subiculum of mice expressing ApoE CT, the average area per amyloid plaque was smaller (panel B), the average fluorescence intensity was weaker (panel C), the percentage of the total area was smaller (panel D), and the density of plaques was lower (panel E). Amyloid plaques are marked by THS. N=33, unpaired t-test. [Figure 10] Figure 10: Neuronally expressed ApoE2 inhibits endogenously produced Aβ40 in mouse neurons. Panel A: ApoE2 expressed by lenti-virus in primary cultured mouse neurons reduces endogenous Aβ40 produced in mouse neurons, GFP as control. N=7, paired t-test. Panel B: ApoE2 expressed by AAV in WT mouse brain reduces endogenous Aβ40 in mouse brain, GFP as control. N=5, unpaired t-test. The hsyn promoter is used to ensure neuron-specific expression. Aβ is detected by ELISA. [Figure 11]Figure 11: ApoE2 or ApoE CT with tag proteins inserted at both ends still have activity in regulating APP cleavage. Panels A and B: Overexpression of ApoE2-V5 (tagged protein inserted at the C-terminus) and V5-ApoE2 (tagged protein inserted at the N-terminus) containing the tag protein V5 in HEK 293T cells both significantly increased α-CTF produced from APP. The statistical results are shown in Panel B. N=6, one sample t-test. Panels C and D: Overexpression of ApoE CT-V5 and V5-ApoE CT with the tag protein V5 inserted at the C-terminus or N-terminus of ApoE CT in HEK 293T cells both significantly increased α-CTF produced from APP. The statistical results are shown in Panel D. N=6, one sample t-test. APP and its cleavage fragments were detected by Western Blot. [Figure 12] Figure 12: ApoE CT mutations can still control APP cleavage. Panels A and B: Overexpression of ApoE CT, CT A20, CT A40, CT A60, CT A65, CT A72, CT A80 and CT A36 / 74 as single or double site mutations of ApoE CT in HEK 293T cells both significantly increase α-CTF. Statistical results are shown in Panel B. N=6, one sample t-test. Panels C and D: Overexpression of CT R10, CT LF5 and CT LM5 as multi-site mutations in HEK 293T cells significantly increase α-CTF as well as ApoE CT; however, CT E5, CT E10, CT E20 and CT F5 lose the function of increasing the activity of α-CTF. Statistical results are shown in Panel D. N=4, one sample t-test. Proteins detected by Western Blot. [Figure 13]Figure 13: When amino acid sequences are inserted or deleted within ApoE2, ApoE2 can still regulate APP metabolism. Panel A: ApoE2-M3F co-expressed with APP in HEK 293T cells increases α-CTF. Panel B: ApoE2 MD10 co-expressed with APP in HEK 293T cells increases α-CTF. [Figure 14] Figure 14: Non-human ApoE CT also regulates APP cleavage. Panels A and B: Overexpression of non-human primate ApoE CT, which is highly similar to the human ApoE CT sequence, in HEK 293T cells significantly increases α-CTF and its activity is the same as that of human ApoE CT. Statistical results are shown in Panel B. N=6, one sample t-test. Proteins are detected by Western Blot. Alignment of human and monkey ApoE CT sequences is shown in Panel C. [Figure 15] Figure 15: ApoE linked to a membrane-permeable peptide still has activity in regulating APP cleavage. Panels A and B: APP was co-expressed with R9-ApoE2, FGF4-ApoE2 or RDP-ApoE2 in HEK 293T cells, and GFP was used as the negative control group. Co-expression of R9-ApoE2 and RDP-ApoE2 significantly increased α-CTF. Statistical results are shown in Panel B. N=5, one sample t-test. Proteins were detected by Western Blot. [Figure 16]Figure 16: ApoE CT expressed in cells needs to be connected to a signal peptide to have the activity of regulating APP cleavage. Panels A and B: Overexpression of ApoE CT mutants connected to apolipoprotein A1 signal peptide at the N-terminus (ASP CT) and ApoE CT mutants containing apolipoprotein J signal peptide (JSP CT) in HEK 293T cells still significantly increases α-CTF. Expression of NSP CT not connected to a signal peptide in cells cannot increase α-CTF by regulating APP cleavage. Statistical results are shown in Panel B. N=6, one sample t-test. Proteins detected by Western Blot. [Figure 17]Figure 17: ApoE2 increases α-CTF and reduces Aβ production through its C-terminal region. Panel A: Representative images of co-immunofluorescence staining of ApoE and NeuN (neuronal marker) in human (top) or monkey (bottom) cortex. Arrows indicate ApoE-positive neurons. Panel B: Representative images of co-immunofluorescence staining of ApoE and APP in human brain cortex. Panels C and D: Western blots of full-length APP (FL-APP), α-CTF and ApoE signals in N2A cells transiently co-expressing APP with GFP, ApoE2, ApoE3 or ApoE4, and corresponding statistical analysis. N=8, one sample t-test. Panel E: Normalized statistical analysis of Aβ40 in HEK 293T cells transiently co-expressing APP, BACE1 with GFP, ApoE2, ApoE3 or ApoE4. N=8, one sample t-test. Panels F and G: Western blots of full-length APP (FL-APP), α-CTF and ApoE signals in N2A cells where APP was transiently co-expressed with GFP, ApoE CT, ApoE2 NT or ApoE4 NT, and corresponding statistical analysis. N=8, one sample t-test. Panel H: Normalized statistical analysis of Aβ40 in N2A cells where APP, BACE1 were transiently co-expressed with GFP and ApoE CT. N=4, one sample t-test. Panels I and J: Western blots of full-length APP (FL-APP), α-CTF and ApoE signals in N2A cells where APP was transiently co-expressed with GFP, ApoE2 or ApoE CT, and corresponding statistical analysis. N=6, one sample t-test. Panel K: Aβ40 normalization statistical analysis in mouse cortical or hippocampal neurons transiently expressing GFP or ApoE CT using a lentivirus system. The promoter is hSyn. N=7, one sample t-test. Panel L: Aβ40 normalization statistical analysis in human neurons induced by human pluripotent stem cells transiently expressing GFP or ApoE CT using a lentivirus system. The promoter is hSyn. N=5, unpaired t-test. [Figure 18] Figure 18: ApoE CT interacts with APP and gamma-secretase. Panel A: Representative images of immunofluorescence staining of ApoE and APP in N2A cells transiently co-expressing APP-FLAG with GFP, ApoE2, or ApoE CT. Panel B: Representative images of immunofluorescence 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 neurons induced by human pluripotent stem cell-derived cells transiently expressing ApoE CT-V5. MAP2 staining represents neurons, and DAPI staining represents cell nuclei. Panel D: Immunoblot detecting APP-APEX2-labeled ApoE2 co-expressed in HEK 293T cells. Panel E: Immunoblot detecting APP-APEX2 labeled ApoE CT coexpressed in HEK 293T cells. Panel F: Immunoblot detecting ApoE2-APEX2 labeled APP coexpressed in HEK 293T cells. Panel G: Immunoblot detecting ApoE-APEX2 labeled endogenously expressed APP in neurons and negative control COX2 expressed in mouse neuronal cells. Panel H: Immunoblot detecting ApoE2-APEX2 labeled ADAM10, BACE1, PS1 and PS2 transiently coexpressed with ADAM10, BACE1, PS1 or PS2, respectively, in HEK 293T cells. Panel I: Co-immunoprecipitation detecting β-CTF-FLAG interacting ApoE transiently coexpressed with ApoE2, ApoE2 NT or ApoE CT, respectively, in HEK 293T cells. Panel J: Co-immunoprecipitation detects ApoE interacting with γ-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 detects endogenous ApoE interacting with endogenous γ-secretase (NCT, PS1, PS2 and PEN2) in human brain tissue. [Figure 19]Figure 19: ApoE CT increases α-CTF and reduces Aβ by inhibiting γ-cleavage of APP. Panels A and B: Immunoblots were used to detect and statistically analyze the production of α-CTF from APP in HEK 293T cells under the following conditions: transient expression of APP and GFP (band 1); co-expression of APP and PS1 with or without treatment with 1 micromolar γ-secretase inhibitor PF03084014 (bands 2, 3); co-expression of APP, PS1 and ApoE CT (band 4). N=11. Panels C and D: Immunoblots were used to detect and statistically analyze the effect of GFP or ApoE CT on α-CTF production by APP co-expression in HEK293T cells treated with 1 micromolar γ-secretase inhibitor PF03084014 and control group without inhibitor treatment. N=7. Panel E: ELISA was performed to detect Aβ40 produced in HEK 293T cells transiently co-expressing β-CTF with GFP or ApoE CT, respectively. N=4. Panel F: ELISA was performed to detect Aβ40 produced in HEK 293T cells transiently co-expressing β-CTF with GFP or ApoE CT, respectively. N=6. Panels G and H: ELISA was performed to detect Aβ40 produced in HEK 293T cells co-expressing APPΔAICD with GFP or ApoE CT, respectively. Panel G, cell culture medium. Panel H, cell lysate. Panels B, D, E, F, G, and H, one sample t-test. [Figure 20]Figure 20: γ-secretase is required for ApoE CT to regulate APP metabolism. Panel A: Immunoblots detected protein expression of PS1 and PS2 in wild-type (WT), PS1 or PS2 single knockout (PS1 KO, PS2 KO), and PS1 and PS2 double knockout (PS1 / 2 dKO) HEK 293T cell lines. Panel B: Expression of APP in wild-type HEK 293T cell line (band 1); Expression of APP in PS1 single knockout HEK 293T cell line (band 2); Expression of APP in PS1 single knockout HEK 293T cell line treated with 1 micromolar γ-secretase inhibitor PF03084014 (band 3); Co-expression of APP and PS1 in PS1 single knockout HEK 293T cell line (band 4). Immunoblots detected cellular α-CTF content under different conditions. Panels C and D: Immunoblot analysis of the effect of ApoE CT on α-CTF produced by cleavage of co-expressed APP in wild-type (WT) and PS1 single knockout (PS1 KO) HEK 293T cell lines, respectively; GFP was used as a control. N=9. Panels E and F: Immunoblot analysis of the effect of ApoE CT on α-CTF produced by cleavage of co-expressed APP in wild-type and PS2 single knockout (PS2 KO) HEK 293T cell lines, respectively; GFP was used as a control. N=9. Panels G and J: Immunoblot analysis of the effect of ApoE CT on α-CTF produced by APP co-expression in wild-type (WT) and PS1 and PS2 double knockout (PS1 / 2 dKO) HEK 293T cell lines, respectively; GFP was used as a control. N=8. Panels H and J: Immunoblot analysis of α-CTF produced by co-expression of APP, PS1 and GFP or ApoE CT in PS1 and PS2 double knockout (PS1 / 2 dKO) HEK 293T cells. N=8. Panels I and J: Immunoblot analysis of α-CTF produced by co-expression of APP, PS2 and GFP or ApoE CT in PS1 and PS2 double knockout (PS1 / 2 dKO) HEK 293T cells. N=8.N=8. Panels K and L: Immunoblotting to detect α-CTF produced by co-expression of APP and GFP or ApoE2, respectively, in PS1 and PS2 double knockout (PS1 / 2 dKO) HEK 293T cells and statistical analysis. N=9. Panel M: ELISA to detect Aβ40 produced by co-expression of β-CTF, PS2 and GFP or ApoE CT, respectively, in PS1 and PS2 double knockout (PS1 / 2 dKO) HEK 293T cells. N=4. Panels D, F, J, L, and M, one sample t-test. [Figure 21]Figure 21: Knockdown of endogenous ApoE in mouse neurons led to decreased levels of the APP metabolite CTF14kD and increased Aβ. Panel A: Immunoblot detection of endogenously expressed ApoE in primary mouse neurons transiently expressing shScramble (negative control), shApoE_1 (shRNA_1 for ApoE) or shApoE_2 (shRNA_2 for ApoE) by lentiviral expression system. Panel B: Relative content of the APP metabolite CTF14kD in mouse neurons treated with 1 micromolar γ-secretase inhibitor PF03084014. Panels C and D: Immunoblot detection of the relative content of the APP metabolite CTF14kD in primary mouse neurons transiently expressing shScramble, shApoE_1 or shApoE_2 by lentiviral expression system. Statistical analysis. N=5, one sample t-test. Panel E: Immunoblot analysis of the relative content of CTF14kD, a metabolite of endogenously expressed APP, in primary cultured mouse neurons transiently expressing shScramble, shApoE_1, or shApoE_2 by lentiviral expression system after treatment with 1 micromolar γ-secretase inhibitor PF03084014. Panel F: ELISA analysis of Aβ40 produced by endogenously expressed APP in primary cultured mouse neurons transiently expressing shScramble, shApoE_1, or shApoE_2 by lentiviral expression system. N=5, one sample t-test. [Figure 22]Figure 22: ApoE2 directly inhibits gamma cleavage of APP. Panel A: Immunoblots analyzed expressed and purified β-CTF, ApoE2, ApoE2 NT, PS1, NCT, PEN2, and GFP. Panel B: ELISA was used to detect and statistically analyze Aβ levels produced in three different conditions: purified β-CTF, co-incubation of purified β-CTF with γ-secretase, and co-incubation of purified β-CTF with γ-secretase when treated with 1 micromolar of the γ-secretase inhibitor PF03084014. N=3, student's t test. Panel C: ELISA was used to detect and statistically analyze Aβ produced in co-incubation of purified β-CTF with a series of gradient concentrations of ApoE2. The IC50 is approximately 1.2±0.48 micromolar. Panel D: ELISA showed that purified β-CTF detected Aβ produced by co-incubation with 2.5 μmol of purified GFP, ApoE2, and ApoE2 NT, respectively. N=3, student's t test. [Figure 23] Figure 23: ApoE CT expressed in neurons translocated to amyloid plaques and delayed amyloid plaque formation in the brains of 5×FAD mice. Panel A: Schematic of the experimental model. Panel B: Representative images of immunofluorescence staining of adeno-associated virus GFP or ApoE CT in mouse brain neurons and amyloid plaques expressed in 5×FAD mice for 10 weeks. Panel C: Statistical analysis of the average size of amyloid plaques. Panel D: Statistical analysis of the percentage of the total area of ​​amyloid plaques in the subiculum area. Panel E: Statistical analysis of the density of amyloid plaques. N=21. [Figure 24]Figure 24: ApoE expressed in neurons of 5×FAD mice aggregated around amyloid plaques via CT. Panel A: Representative images of immunofluorescence staining of lentiviral ApoE2-IRES-GFP, ApoE2 NT-IRES-GFP and ApoE CT-IRES-GFP expressed in cortical neurons of 5×FAD mice for three months and amyloid plaques (GFP). Panel B: Representative images of immunofluorescence staining of lentiviral ApoE CT and human APP (hAPP) expressed in 5×FAD mouse cortical neurons for one month. Panel C: Representative images of immunofluorescence staining of lentiviral ApoE CT and hAPP expressed in wild-type mouse cortical neurons for three months. Plaque, amyloid plaques. [Diagram 25] Figure 25: Endogenous ApoE is present in human neurons; amyloid plaques in the brains of AD patients colocalize with ApoE. Panel A: Representative images of co-immunofluorescent staining of ApoE and NeuN (neuronal marker) in the cortex of a non-AD patient (62 years old, top) or an AD patient (86 years old, bottom). Arrows indicate ApoE-positive neurons. Panel B: Percentage of ApoE-positive neurons to total neurons in the human cerebral cortex. Panel C: Percentage of ApoE-positive neurons to total ApoE-positive cells. YOUNG: 16-23 years old, n=3; OLD: 60 years old or older, n=3; AD: 60 years old or older, n=6. Panel D: Representative images of co-immunofluorescent staining of ApoE and GFAP (astrocyte marker) in the human cerebral cortex. Panel E: Representative images of co-immunofluorescent staining of ApoE and amyloid plaques (6E10) in the cortex of a non-AD patient (age 62, top) or an AD patient (age 86, bottom). [Figure 26]Figure 26: ApoE2 increases α-CTF and reduces Aβ in HEK 293T cells via ApoE CT. Panel A: Immunoblots showing α-CTF produced in HEK 293T cells co-expressing APP with GFP or ADAM10, respectively. Panel B: Immunoblots showing α-CTF produced in HEK 293T cells treated with 1 micromolar ADAM10 inhibitor GI254023X or control without GI254023X treatment. Panels C and D: Immunoblots showing α-CTF produced in HEK 293T cells co-expressing APP with GFP, ApoE2, ApoE3 or ApoE4, respectively, and statistical analysis. N=12. Panels E and F: Immunoblot analysis of α-CTF produced by co-expression of APP with GFP, ApoE2 NT, ApoE3 NT, or ApoE4 NT in HEK 293T cells; N=12. Panels G and H: Immunoblot analysis of α-CTF produced by co-expression of APP with GFP or ApoE CT in HEK 293T cells; N=18. Panel I: ELISA analysis of Aβ40 produced and secreted into the medium in HEK 293T cells co-expressed with APP, BACE1, GFP, ApoE CT, ApoE2 NT, ApoE3 NT, or ApoE4 NT; N=4. Panel J: ELISA analysis of Aβ40 produced in cell lysates in HEK 293T cells co-expressed with APP, BACE1, GFP, or ApoE CT; N=5. Panel K: ELISA to detect Aβ42 and Aβ42 / Aβ40 produced by co-expression of APP, BACE1, and GFP or ApoE CT in HEK 293T cells, respectively. N=6. Panels L and M: Immunoblot to detect β-CTF produced by co-expression of APP, BACE1, and GFP or ApoE CT in HEK 293T cells, respectively. N=13. Panels N and O: Immunoblot to detect α-CTF produced by co-expression of APP, GFP, ApoE 2 or ApoE CT in HEK 293T cells, respectively. N=6.Panels P and Q: Immunoblot analysis of α-CTF produced by co-expression of mouse APP (mAPP) with GFP or ApoE CT in HEK 293T cells, respectively. N=6. Panels R and S: ELISA analysis of Aβ40 produced by co-expression of APPSW with GFP or ApoE CT in HEK 293T cells, respectively; Panel R, Aβ40 in medium; Panel S, Aβ40 in cell lysate, respectively. N=6. Panels T and U: Immunoblot analysis of α-CTF produced by co-expression of APP with GFP or ApoEA1 in HEK 293T cells, respectively. N=6. Panel I: ELISA analysis of Aβ40 produced by co-expression of APP, BACE1 with GFP or ApoA1 in HEK 293T cells, respectively. N=3. Face plate D, F, H, I, J, K, M, O, Q, R, S, U and V,one sample t-test. [Figure 27] Figure 27: ApoE2 and ApoE CT in the medium do not change the metabolism of APP. Panel A: Immunoblotting to detect ApoE2 and ApoE CT in the cell medium. Panels B and C: Immunoblotting to detect and statistically analyze the effect of GFP, ApoE2, or ApoE CT in the medium on the production of α-CTF from APP coexpressed in HEK 293T cells. N=9. Panel D: Immunoblotting to detect ApoE2 in the medium under different conditions: original ApoE2-containing medium (band 1), ApoE2-containing medium after 48 hours of incubation with non-transfected cells (band 2), and ApoE2-containing medium stored alone in a 37°C incubator for 48 hours (band 3). Panel E: Immunofluorescence to detect ApoE2 and ApoE CT in the medium introduced into HEK 293T cells, and ApoE CT expressed in HEK 293T cells. DAPI, nuclear marker. Panel F: Immunofluorescence detected ApoE2 and ApoE CT in the medium delivered to neurons and ApoE CT expressed in neurons. MAP2, a marker for mature neurons; DAPI, nuclear marker. Panel G: Immunoblotting analyzed purified ApoE2 and ApoE2 derived from cell lysates or cell medium. [Figure 28] Figure 28: AD-associated mutations near the γ-cleavage site of APP affect the activity of ApoE CT. Panels A and B: Immunoblot analysis of α-CTF produced by co-expressing different AD mutations of APP with GFP or ApoE CT in HEK 293T cells. N=7, one sample and student's t-test. [Figure 29]Figure 29: ApoE CT can still regulate APP metabolism when α- or β-secretase is inhibited. Panel A: Immunoblot detection and statistical analysis of the effect of ApoE CT on α-CTF generation by APP co-expression in HEK293T cells when treated with 1 micromolar ADAM10 inhibitor GI254023X, and control group was not treated with inhibitors. N=9. Panels C and D: ELISA and immunoblot detection and statistical analysis of the effect of ApoE CT on Aβ generation by APP and BACE1 co-expression in HEK293T cells when treated with 1 micromolar ADAM10 inhibitor GI254023X, and control group was not treated with inhibitors. Panel C, Aβ40 in cell medium; Panel D, Aβ40 in cell lysate. N=6. Panels E and F: Immunoblot analysis of the effect of ApoE CT on α-CTF production by APP co-expression in HEK293T cells treated with 5 micromolar BACE1 inhibitor AZD3839 and control group without inhibitor treatment. N=10. Panels G and H: Immunoblot analysis of α-CTF production by APPM596V co-expressed with GFP or ApoE CT, respectively, in HEK 293T cells. N=7. Panel I: qRT-PCR analysis of the effect of ApoE2 or ApoE CT expression on endogenous ADAM10, BACE1, PS1, and PS2 mRNA expression levels in HEK 293T cells. Panel J: Immunoblot analysis of the effect of ApoE2 or ApoE CT on endogenous ADAM10, BACE1, PS1, and PS2 protein expression levels in HEK 293T cells. Panel K: Immunoblot detection of the effect of ApoE CT on cell surface expression of APP coexpressed in HEK 293T cells. Cell surface proteins of HEK 293T cells expressing GFP(-) and ApoE CT(+) were labeled with sulfo-N-hydroxysuccinimide-biotin and separated with streptavidin-conjugated agarose beads. [Diagram 30]Figure 30: ApoE CT cannot inhibit gamma cleavage of NOCTH and APLP1. Panel A: HEK 293T cells expressing only N100-V5 (band 1); co-expressed N100-V5 and gamma secretase (band 2); co-expressed N100-V5 and gamma secretase treated with 1 micromolar gamma secretase inhibitor PF03084014 (band 3); co-expressed N100-V5, gamma secretase and ApoE CT (band 4). Immunoblots detected the cleavage of N100-V5 when expressed under the above conditions. Panel B: Immunoblots detected the content of α-CTF when co-expressing APP, gamma secretase and ApoE CT in HEK 293T cells. Panel C: HEK 293T cells expressing only APLP-V5 (band 1); expressing APLP-V5 upon treatment with 1 micromolar γ-secretase inhibitor PF03084014 (band 2); co-expressing APLP1-V5 and ApoE CT (band 3). Immunoblots were used to detect the metabolism of APLP1-V5 when expressed under these conditions. [Diagram 31] Figure 31: In vitro purified β-CTF, ApoE2, ApoE2 NT and γ-secretase. Caucasus blue staining shows purified β-CTF-Myc-6×His (panel A), ApoE2-3×FLAG (panel B), ApoE2NT-3×FLAG (panel C), γ-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, PS1 CTF) and 3×FLAG-GFP (panel E). [Diagram 32] Figure 32: Information table about the human brain used in this example. [Diagram 33] Figure 33: Structure of human ApoE protein. The N-terminal structural domain (residues 1-167) and the C-terminal structural domain (residues 206-299) are connected by a flexible hinge region (residues 168-205). The three subtypes are ApoE2 (Cys112; Cys158), ApoE3 (Cys112; Arg158), and ApoE4 (Arg112; Arg158). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Specific embodiments It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as examples) can be combined with each other to form preferred technologies.

[0024] Human-derived apolipoprotein E (ApoE) mainly includes three subtypes, ApoE2, ApoE3 and ApoE4. ApoE contains three regions: an N-terminal domain (ApoE NT), a hinge region and a conserved C-terminal domain (ApoE CT). The only sequence difference between the three ApoE subtypes is in their N-terminal domains (Figure 33). The amino acid sequence of an exemplary ApoE2 (human-derived ApoE2) is shown below:

[0025] [Table 1]

[0026] The amino acid sequence of an exemplary ApoE3 (human-derived ApoE3) is shown below:

[0027] [Table 2]

[0028] The present invention provides an isolated ApoE construct, which comprises an ApoE polypeptide comprising a partial sequence of the C-terminal domain of the ApoE protein (e.g., amino acids 221-274 of SEQ ID NO:12), or a sequence modified based on said sequence. The present invention is based on the inventors' unexpected discovery that the ApoE constructs (e.g., the ApoE polypeptides or ApoE2 of the present invention) described above: (i) are capable of cell-autonomously controlling the cleavage of amyloid precursor protein (APP) in neurons (in vivo or in vitro); (ii) are capable of inhibiting (e.g., inhibiting at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the enzymatic activity of γ-secretase, e.g., γ-secretase-mediated APP γ-cleavage, and (iii) are capable of inhibiting (e.g., inhibiting at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the cleavage of APP by γ-secretase inhibitors (e.g., PF03084014). (iii) it can reduce amyloid metabolism of APP (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), e.g., reduce production of amyloid protein β (Aβ) (e.g., Aβ40 and / or Aβ42) (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), or C-terminal fragments. (iv) it can increase the non-amyloid metabolism of APP (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more), for example by increasing the production of the C-terminal fragment α (α-CTF) (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more);(v) it does not affect (or affects only about 30%, 20%, 10%, 5% or less) the mRNA and / or protein expression of α-secretase, β-secretase, and / or γ-secretase (or subunits thereof) (e.g., ADAM10, BACE1, PS1 and / or PS2); (vi) it does not affect (or affects only about 30%, 20%, 10%, 5% or less) the β-secretase-mediated APP β-cleavage and / or α-secretase-mediated APP α-cleavage; (vii) it inhibits (or inhibits only about 30%, 20%, 10%, 5% or less) the mRNA and / or protein expression of non-APP proteins (e.g., APP like protein 1 (APLP1) 1) does not inhibit (or inhibits by only about 30%, 20%, 10%, 5% or less) γ-cleavage of ApoE or Notch or fragments thereof, and does not increase (or increases by only about 30%, 20%, 10%, 5% or less) α-CTFs produced by γ-cleavage of non-APP proteins, indicating that the ApoE construct inhibits γ-cleavage with substrate specificity, whereas γ-secretase inhibitors do not have this substrate specificity; (viii) the ApoE construct can reduce (e.g., reduce by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) amyloid plaques in the AD model mouse brain, e.g., reduce the average plaque size, total area, and / or density of amyloid plaques. (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), reduce production of Aβ (e.g., Aβ40 and / or Aβ42) in neurons (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), and reduce AD ​​pathological processes in mice. 5% or more);(ix) A tag protein (e.g., a purification tag or detection tag) inserted at the N-terminus and / or C-terminus of the ApoE construct, and insertion or deletion of amino acid residues (e.g., ApoE2-M3F or ApoE2 MD10) within some of the ApoE constructs (e.g., ApoE2 or ApoE3) do not affect (or only affect by about 30%, 20%, 10%, 5% or less) the inhibition of γ-secretase enzymatic activity by the ApoE construct, e.g., inhibition of γ-secretase-mediated APP γ-cleavage; (x) A cell-penetrating peptide (CPE) inserted at the N-terminus or C-terminus of the ApoE construct. (xi) in order to control cleavage of APP by γ-secretase in a cell, the ApoE construct must have a signal peptide at its N-terminus, but there is no strict requirement as to the origin of the signal peptide; (xii) some ApoE constructs described in the present invention (e.g., ApoE CT (SEQ ID NO:33)) can enhance the effect of the ApoE construct in inhibiting γ-secretase enzymatic activity (e.g., one or more of the effects) (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more); (xiii) in inhibiting γ-secretase-mediated APP γ-cleavage, some ApoE constructs described in the present invention (e.g., ApoE CT (SEQ ID NO:33)) can enhance the effect of the ApoE construct in inhibiting γ-secretase enzymatic activity (e.g., one or more of the effects) (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more); (xiii) the ApoE construct has a higher activity (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more) than the full-length protein of ApoE (NO:89); (xiv) the ApoE construct has a higher activity (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more) than the full-length protein of ApoE (NO:89); SW(xiv) the ApoE constructs exhibit a perinuclear subcellular distribution pattern that overlaps with endogenous APP in cells and can interact with endogenous APP, α-secretase and γ-secretase or components thereof in cells; (xv) the ApoE constructs are expressed locally in neurons in AD model mice; (xvi) the ApoE constructs expressed (e.g., expressed in the cortex, dentate gyrus (DG), hippocampal CA3) migrate from distant sites to the subiculum and accumulate around amyloid plaques, thereby locally inhibiting γ-secretase enzymatic activity in these amyloidogenic hotspots; (xvi) non-human derived ApoE constructs (e.g., monkey ApoE C-terminal sequences) highly homologous (e.g., at least about 90%, 95%, 96%, 97%, 98%, 99% or more) to the ApoE constructs described in the present invention have properties similar to one or more of the above properties.

[0029] This application is the first to discover that ApoE inhibits γ-secretase cleavage cell-autonomously (i.e., functions in the same cells as γ-secretase) via its C-terminal fragment, inhibits amyloid metabolism of APP, reduces Aβ production; increases α-CTF, a non-amyloid metabolite of APP; and alleviates the increase in size and density of amyloid plaques in the brain of AD model mice. Therefore, the C-terminal fragment of ApoE can be used as a drug targeting γ-secretase to alleviate (e.g., alleviate at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the AD progression process), treat, or prevent the development and progression of AD and amyloid cerebrovascular disease. It should be noted that the C-terminal fragment of ApoE may play a role in multiple biological processes as an inhibitor of gamma-secretase, and the application of the C-terminal fragment of ApoE is not limited to the field of neurodegenerative diseases, but may be used in multiple diseases such as cancer, inflammatory diseases, and kidney diseases, for example, any disease associated with the cleavage activity of gamma-secretase (disease that can be treated or prevented by inhibiting the cleavage activity of gamma-secretase). General gamma-secretase inhibitors cause severe side effects during treatment, which are all related to defects in Notch signaling, such as immunosuppression, gastrointestinal bleeding and diarrhea, and cancerous skin lesions. The ApoE construct provided by the present invention does not affect gamma-cleavage of Notch, and therefore the ApoE construct provided by the present invention and the associated method may provide a new effective and safe treatment method.

[0030] The present application further discovered that the amino acid sequence at positions 221-274 of SEQ ID NO:12 in the C-terminal fragment of apolipoprotein E (MGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQR; SEQ ID NO:78; "ApoE CT ND5CD25") is an amino acid sequence necessary for the C-terminal fragment to exert a biological function (e.g., inhibiting γ-secretase-mediated APP γ-cleavage). In some embodiments, the shortest functional fragment of the C-terminal fragment of apolipoprotein E is the amino acid sequence at positions 220-274 of SEQ ID NO:12 (EMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQR; SEQ ID NO:87). In some embodiments, the shortest functional fragment of the C-terminal fragment of ApoE is the amino acid sequence 220-279 of SEQ ID NO:12, EMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGL (SEQ ID NO:34).

[0031] 1.Definition Unless otherwise specified, the practice of the present invention employs conventional methods in chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, cell biology, stem cell protocols, cell culture and transgenic biology within the ordinary skill of those skilled in the art, many of which are described below for illustrative purposes. Such techniques are fully explained in the literature. All publications, patents and patent applications cited herein are incorporated by reference in their entirety.

[0032] Unless otherwise explained, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined to be consistent with their meaning commonly understood in the art.

[0033] As used herein, the terms "treating" and "treatment" mean to delay the onset of, slow or reverse the progression of, reduce the severity of, alleviate or prevent the disease or condition to which the term applies or one or more symptoms of such disease or condition.

[0034] The term "effective amount" or "pharmacologically effective amount" refers to the amount and / or dosage and / or dosing regimen of one or more agents required to produce a desired result, such as an amount sufficient to reduce APPγ cleavage, or to treat one or more symptoms associated with a neurodegenerative disease (e.g., AD), or to reduce the severity or delay the progression of a disease characterized by amyloid deposits in the brain of a mammal (e.g., a therapeutically effective amount), or to reduce the risk of or delay the onset and / or ultimate severity of a disease characterized by amyloid deposits in the brain of a mammal (e.g., a prophylactically effective amount).

[0035] The term "ameliorating" refers to reducing or eliminating one or more symptoms of the condition or disease and / or reducing the rate of onset or severity of one or more symptoms of the condition or disease, and / or delaying the onset or severity of one or more symptoms of the condition or disease, and / or preventing said condition or disease. In some embodiments, 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 characteristic of a pathology or disease (e.g., an increase in the levels of one or more of the following: total-Tau (tTau), phosphorylated-Tau (pTau), APPneo, soluble Aβ40, Aβ42, pTau / Aβ42 ratio, tTau / Aβ42 ratio, and / or CSF Aβ42 / Aβ40 ratio, Aβ42 / Aβ38 ratio, sAPPα, sAPPα / sAPPβ ratio, sAPPα / Aβ40 ratio, sAPPα / Aβ42 ratio, etc.) and / or an increase in non-amyloid metabolites (e.g., α-CTF), and / or a reduction, stabilization, or reversal of one or more diagnostic criteria (e.g., Clinical Dementia Rating (CDR)).

[0036] An "individual" or "test subject" includes, but is not limited to, mammals, birds, frogs, fish, fruit flies, nematodes, etc. Mammals include, but are not limited to, primates (e.g., humans or non-human primates, e.g., monkeys), rodents (e.g., mice, rats, hamsters, gerbils, squirrels, guinea pigs, rabbits), farm animals (e.g., pigs, cows, sheep, horses, donkeys), and pets (e.g., cats, dogs, rabbits, hamsters, etc.). In some embodiments, the individual is a human.

[0037] The term "formulation" or "pharmaceutical formulation" or "dosage form" or "pharmaceutical formulation" as used herein refers to a composition that contains at least one therapeutic or pharmaceutical agent for delivery to a subject.

[0038] "Sequence identity" between two polypeptide or nucleic acid sequences refers to 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 based on the type of mutation. The types of mutations include, but are not limited to, insertions (extensions) at either or both ends of the sequence, deletions (truncations) at either or both ends of the sequence, substitutions / replacements of one or more amino acids / nucleotides, internal insertions in the sequence, and internal deletions in the sequence. Taking polypeptides as an example (and the same for nucleotides), the types of mutations can be one or more selected from substitutions / replacements of one or more amino acids / nucleotides, internal insertions in the sequence, and internal deletions in the sequence, and the total number of residues is calculated as the larger of the molecules being compared. Further types of mutations include insertions (lengthening) at either or both ends of the sequence or deletions (truncation) at either or both ends of the sequence, and the number of amino acids inserted or deleted at either or both ends (e.g., the number of insertions or deletions at both ends is less than 20) is not included in the total number of residues. When calculating percent identity, the sequences to be compared are aligned to maximize the match between the sequences, and gaps in the alignment (if any) are resolved by a specific algorithm.

[0039] The term "fragment" as used herein refers to a polypeptide and is defined as any isolated portion of a particular 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. Preferably, the retained active portion is at least 10% of the activity of the full-length polypeptide. More preferably, the retained 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 retained active portion is at least 95%, 96%, 97%, 98%, or 99% of the activity of the full-length polypeptide. Most preferably, the retained active portion is 100% of the activity of the full-length polypeptide. The term as used herein also refers to any portion of a given polypeptide that contains at least one specified sequence element of the full-length polypeptide. Preferably, 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.

[0040] The term "polypeptide" as used herein refers to a chain of consecutive amino acids linked by peptide bonds. The term can refer to any length of amino acid chain, but those skilled in the art will recognize that the term can be used to refer not only to long chains, but also to the shortest chain that contains two amino acids connected by a peptide bond. The term "protein" as used herein refers to one or more polypeptides that function as separate units. The terms "polypeptide" and "protein" are used interchangeably herein when a single polypeptide is a separate functional unit and does not require persistent physical association with other polypeptides to form its independent functional unit. When a separate functional unit consists of multiple polypeptides that are physically associated with each other, the term "protein" as used herein refers to multiple polypeptides that are physically associated and function together as a separate unit.

[0041] "Recombinantly expressed polypeptide," "recombinant polypeptide," and "modified polypeptide" (or protein) refer to a polypeptide expressed from a host cell that has been genetically engineered to express that polypeptide. A recombinantly expressed polypeptide may be the same as or similar to a polypeptide normally expressed in a host cell (e.g., a mammalian cell). A recombinantly expressed polypeptide may also be foreign to the host cell, e.g., a heterologous polypeptide to a polypeptide normally expressed in a mammalian host cell. Alternatively, a recombinantly expressed polypeptide is a chimeric polypeptide, e.g., a portion of the polypeptide contains the same or similar amino acid sequence as a polypeptide normally expressed in a mammalian host cell, and another portion is exogenous to the host cell.

[0042] The term "amino acid" refers to the 20 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).

[0043] Non-critical amino acids can be conservatively substituted without affecting the normal function of the protein. Conservative substitution means replacing an amino acid with a chemically or functionally similar amino acid. It is well known in the art to provide conservative substitution tables of similar amino acids. For example, in some embodiments, the following amino acids provided below are considered to be conservative substitutions for each other:

[0044] In some embodiments, amino acids that are considered conservative substitutions for one another are: [Table 3]

[0045] In some embodiments, another group of amino acids that are considered conservative substitutions for one another:

[0046] [Table 4]

[0047] In some embodiments, another group of amino acids that are considered conservative substitutions for one another:

[0048] [Table 5]

[0049] As used herein, the term "about" or "approximately" refers to an amount, level, value, quantity, frequency, percentage, scale, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference amount, level, value, amount, frequency, percentage, scale, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of an amount, level, value, amount, frequency, percentage, scale, size, amount, weight, or length around ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the reference amount, level, value, amount, frequency, percentage, scale, size, amount, weight, or length.

[0050] As used herein, the term "substantially / essentially" refers to an extent, amount, level, value, quantity, frequency, percentage, scale, size, quantity, weight, or length of about 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% or 99% or more compared to an extent, reference amount, level, value, amount, frequency, percentage, scale, size, amount, weight, or length.

[0051] Numeric ranges include the endpoints of the range and each specific value within the range; for example, "16 to 100 amino acids" includes 16 and 100 and each specific value between 16 and 100.

[0052] Throughout this specification, unless specifically limited, the terms "comprise," "containing," "having," and "having" should be understood to implicitly mean the inclusion of a stated step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. In certain embodiments, the terms "comprise," "containing," "having," and "having" are used interchangeably.

[0053] "Consisting of" is meant to include, and is limited to, everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required, and that no other elements may be present.

[0054] "Consisting essentially of" means including any of the elements listed after the phrase "consisting essentially of," and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required, but that the other elements are not optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

[0055] The articles "a / an" and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article's word. For example, "an element" means one element or more than one element.

[0056] The use of the alternative (eg, "or") should be understood to mean either, both, or any combination of the alternatives.

[0057] The term "and / or" should be understood to mean either or both of the alternatives.

[0058] Throughout this specification, references to "one embodiment," "an embodiment," "some embodiments," "related embodiments," "particular embodiments," "another embodiment," or "further embodiment," or combinations thereof, mean that the particular features, structures, or characteristics described in connection with an embodiment are included in at least one embodiment of the invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0059] 2. ApoE Constructs and ApoE Polypeptides In one aspect, the present application provides an isolated ApoE construct, comprising an ApoE polypeptide, the ApoE polypeptide comprising: (1) a fragment derived from the amino acid sequence set forth in SEQ ID NO:12, wherein the first amino acid residue at the N-terminus of the fragment (or the ApoE polypeptide) is any one of amino acid residues 215-221 of SEQ ID NO:12, and the final amino acid residue at the C-terminus of the fragment (or the ApoE polypeptide) is any one of amino acid residues 215-221 of SEQ ID NO:12. or (2) the amino acid sequence has at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with the fragment described in (1) above and retains at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5-fold, 2-fold, 5-fold, 10-fold or more) inhibition of gamma-secretase cleavage activity by the fragment described in (1). It should be understood that a "fragment" as used herein refers to a contiguous amino acid sequence from the corresponding protein.

[0060] In some embodiments, the ApoE polypeptide comprises a fragment derived from the amino acid sequence set forth in SEQ ID NO:12, wherein the first amino acid residue at the N-terminus of the ApoE polypeptide is any one of amino acid residues 215-221 of SEQ ID NO:12, and the final amino acid residue at the C-terminus of the ApoE polypeptide is any one of amino acid residues 274-299 (e.g., 279-299) of SEQ ID NO:12. In some embodiments, the ApoE construct comprises an ApoE polypeptide, wherein the ApoE polypeptide comprises a fragment derived from the amino acid sequence set forth in SEQ ID NO:12, wherein the first amino acid residue at the N-terminus of the ApoE polypeptide is any one of amino acid residues 215-221 of SEQ ID NO:12, and the final amino acid residue at the C-terminus of the ApoE polypeptide is any one of amino acid residues 274-299 (e.g., 279-299) of SEQ ID NO:12.

[0061] In some embodiments, the ApoE polypeptide has at least about 70% identity to a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 and retains at least about 50% of the inhibition of gamma-secretase cleavage activity by the fragment, provided that the first amino acid residue at the N-terminus of the fragment is any one of amino acid residues 215-221 of SEQ ID NO:12 and the most C-terminus of the fragment is any one of amino acid residues 274-299 (e.g., positions 279-299) of SEQ ID NO:12.

[0062] In some embodiments, the isolated ApoE construct consists or consists essentially of the ApoE polypeptide (e.g., an isolated ApoE polypeptide). Thus, in some embodiments, the present application further provides an ApoE polypeptide (e.g., an isolated ApoE polypeptide).

[0063] In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) consists of (1) a fragment derived from the amino acid sequence set forth in SEQ ID NO:12, or consists essentially of (1) a fragment derived from the amino acid sequence set forth in SEQ ID NO:12.

[0064] In some embodiments, the ApoE construct is not naturally occurring, for example, in some embodiments, the ApoE construct or the ApoE polypeptide is not ApoE2, not ApoE3, and / or not a full-length ApoE4 protein.

[0065] In some embodiments, the ApoE construct or the ApoE polypeptide is a full-length ApoE2 or ApoE3 protein (eg, a human full-length ApoE2 or ApoE3 protein), for example when the ApoE construct is used to express neural properties.

[0066] In some embodiments, the start point (or the first amino acid residue at the N-terminus) of the amino acid sequence of the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above is an amino acid residue at any one of positions 215-221 (e.g., positions 215, 216, 217, 218, 219, 220 or 221) of the ApoE SEQ ID NO:12 sequence, such as any one of the amino acid residues at positions 215, 216, 219, 220 or 221. In some embodiments, the end of the amino acid sequence of the ApoE polypeptide or the fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above (or the most C-terminal amino acid residue) is an amino acid residue at any one of positions 274 to 299 of the ApoE SEQ ID NO:12 sequence (e.g., positions 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, or 299), for example, an amino acid residue at any one of positions 279-299 (including the number), for example, any one of positions 274, 279, or 299.

[0067] In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above comprises (or consists of, or consists essentially of) any one of the following sequences: positions 215-299, 216-299, 217-299, 218-299, 219-299, 220-299, 221-299, 215-274, 216-274, 217-274, 218-274, 219-274, 220-274, 221-274, 215-279, 216-279, 217-279, 218-274, 219-274, 220-274, 221-274, 9th place, 218-279th place, 219-279th place, 220-279th place, 221-279th place, 215-294th place, 216-294th place, 217-294th place, 218-294th place, 219-29th place 4th place, 220-294th place, 221-294th place, 215-289th place, 216-289th place, 217-289th place, 218-289th place, 219-289th place, 220-289th place, 221-289th place 215-288, 216-288, 217-288, 218-288, 219-288, 220-288, 221-288, 215-287, 216-287 217-287, 218-287, 219-287, 220-287, 221-287, 215-286, 216-286, 217-286, 218-286 , amino acid residues at positions 219-286, 220-286, 221-286, 215-285, 216-285, 217-285, 218-285, 219-285, 220-285, 221-285, 215-284, 216-284, 217-284, 218-284, 219-284, 220-284, or 221-284.

[0068] In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above comprises (or consists of, or consists essentially of) any one of the following sequences: (i) the amino acid sequence of amino acid residues 215-299 of SEQ ID NO:12; (ii) the amino acid sequence of amino acid residues 216-299 of SEQ ID NO:12; (iii) the amino acid sequence of amino acid residues 217-299 of SEQ ID NO:12; (iv) the amino acid sequence of amino acid residues 218-299 of SEQ ID NO:12; (v) the amino acid sequence of amino acid residues 219-299 of SEQ ID NO:12; (vi) the amino acid sequence of amino acid residues 220-299 of SEQ ID NO:12; (vii) the amino acid sequence of amino acid residues 221-299 of SEQ ID NO:12; (viii) the amino acid sequence of amino acid residues 222-299 of SEQ ID NO:12; (ix) the amino acid sequence of amino acid residues at positions 216-294 of SEQ ID NO:12; (ix) the amino acid sequence of amino acid residues at positions 216-289 of SEQ ID NO:12; (x) the amino acid sequence of amino acid residues at positions 219-289 of SEQ ID NO:12; (xi) the amino acid sequence of amino acid residues at positions 219-288 of SEQ ID NO:12; (xii) the amino acid sequence of amino acid residues at positions 219-287 of SEQ ID NO:12; (xiii) the amino acid sequence of amino acid residues at positions 219-286 of SEQ ID NO:12; (xiv) the amino acid sequence of amino acid residues at positions 219-285 of SEQ ID NO:12; (xv) the amino acid sequence of amino acid residues at positions 219-284 of SEQ ID NO:12; (xvi) the amino acid sequence of amino acid residues at positions 219-279 of SEQ ID NO:12; (xvii) the amino acid sequence of amino acid residues at positions 219-279 of SEQ ID NO:12 (xviii) the amino acid sequence of amino acid residues 220-274 of SEQ ID NO:12; (xix) the amino acid sequence of amino acid residues 220-279 of SEQ ID NO:12; or (xx) the amino acid sequence of amino acid residues 221-274 of SEQ ID NO:12.

[0069] In some embodiments, the ApoE construct or ApoE polypeptide does not include one or more of the amino acid sequences at positions 211-214 of SEQ ID NO: 12. In some embodiments, the ApoE construct or ApoE polypeptide does not include one or more of the following amino acid fragments (or does not include any one of the following): positions 212-214, 213-214, 211-214, 211-215, 211-216, 211-217, 211-218, 211-219, 211-220, 211-221, 211-222, 212-214, 212-215, 212-216, 212-217, 212-218, 212-219, 212-220, 212-221, 212-222, 212-214, 212-215, 212-216, 212-217, 212-218, 212-219, 212-220, 212-223, 212-224, 212-225, 212-226, 212-227, 212-228, 212-229, 212-220, 212-221, 212-2 ... -221st place, 212-222nd place, 213-214th place, 213-215th place, 213-216th place, 213-217th place, 213-218th place, 213-219th place, 213-220th place, 213-221st place, 213-222nd place, 214-215th place, 214-216th place, 214-217th place, 214-218th place, 214-219th place, 214-220th place, 214-221st place, or 214-222nd place.

[0070] In some embodiments, the ApoE construct or ApoE polypeptide comprises the amino acid sequence of positions 211-214 of SEQ ID NO:12, but the C-terminus of the amino acid sequence of positions 211-214 of SEQ ID NO:12 is not directly connected to the N-terminus of the ApoE polypeptide, e.g., at least one non-ApoE-derived sequence (e.g., a FLAG sequence) is inserted therebetween. In some embodiments, the 3XFLAG sequence shown in SEQ ID NO:77 is inserted between the C-terminus of the amino acid sequence of positions 211-214 of SEQ ID NO:12 and the N-terminus of the ApoE polypeptide.

[0071] In some embodiments, the ApoE construct or the ApoE polypeptide may further comprise one or more non-ApoE-derived amino acid residues at the N-terminus and / or C-terminus of the fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above. In some embodiments, the ApoE construct or the ApoE polypeptide may further comprise one or more amino acid residues at the N-terminus and / or C-terminus of the fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above, which, when inserted at the N-terminus or C-terminus of the fragment (1), differs from the amino acid residue at the corresponding site in SEQ ID NO:12. For example, the first amino acid residue at the N-terminus of the fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above is amino acid residue 215 of SEQ ID NO:12, and the ApoE construct or ApoE polypeptide may further contain one or more amino acid residues at the N-terminus of amino acid residue 215 of SEQ ID NO:12, but the one or more amino acid residues are different from the amino acid residues at positions 214, 213, 212, 211, etc. of SEQ ID NO:12.

[0072] In some embodiments, the ApoE construct may further comprise one or more non-ApoE derived amino acid residues at the N-terminus and / or C-terminus of the ApoE polypeptide (e.g., a fragment derived from the amino acid sequence shown in SEQ ID NO:12 of (1), or an ApoE mutant). In some embodiments, the ApoE construct may further comprise one or more amino acid residues at the N-terminus and / or C-terminus of the ApoE polypeptide, which differ from the amino acid residue at the corresponding position in SEQ ID NO:12 when inserted at the N-terminus or C-terminus of the ApoE polypeptide. For example, the first amino acid residue at the N-terminus of the ApoE polypeptide is amino acid residue 215 of SEQ ID NO:12, and the ApoE construct may further include one or more amino acid residues N-terminal to amino acid residue 215 of SEQ ID NO:12, which amino acid residue or residues are different from amino acid residues at positions 214, 213, 212, 211, etc., of SEQ ID NO:12.

[0073] In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above includes at least the amino acid sequence 221-274 of SEQ ID NO:12 (SEQ ID NO:78). For example, the ApoE polypeptide or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above may further include 1, 2, 3, 4, 5, or 6 amino acid residues at the N-terminus and / or 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 at the C-terminus of the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above consists of, or consists essentially of, the amino acid sequence shown in SEQ ID NO:78. In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above includes at least the amino acid sequence 220-279 of SEQ ID NO:12 (SEQ ID NO:34). For example, the ApoE polypeptide or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above may further contain 1, 2, 3, 4, or 5 amino acid residues at the N-terminus of the amino acid sequence shown in SEQ ID NO:34, and / or may further contain 1, 2, 3, 4, 5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues at the C-terminus.In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above consists of, or consists essentially of, the amino acid sequence shown in SEQ ID NO:34. In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above includes at least the amino acid sequence 219-274 of SEQ ID NO:12 (SEQ ID NO:32). For example, the ApoE polypeptide or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above may further comprise 1, 2, 3, or 4 amino acid residues at the N-terminus and / or 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 at the C-terminus of the amino acid sequence shown in SEQ ID NO:32. In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above consists of, or consists essentially of, the amino acid sequence shown in SEQ ID NO:32. In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above includes at least the amino acid sequence of positions 219-279 of SEQ ID NO:12 (SEQ ID NO:31).For example, the ApoE polypeptide or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above may further comprise 1, 2, 3, or 4 amino acid residues at the N-terminus and / or 1, 2, 3, 4, 5, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues at the C-terminus of the amino acid sequence shown in SEQ ID NO:31. In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above consists of, or consists essentially of, the amino acid sequence shown in SEQ ID NO:31.

[0074] In some embodiments, the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide), or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above comprises any one of the amino acid sequences 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). In some embodiments, the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide), or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above consists of, or consists essentially of, the amino acid sequence of any one 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).

[0075] The ApoE constructs and the ApoE polypeptides described herein include mutants thereof (also referred to herein as "ApoE mutants"). For example, compared to a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) above (e.g., any one of SEQ ID NOs: 13, 18-34, 78, and 87), the mutants have one or more amino acid mutations (e.g., insertions, deletions, or substitutions), including, but not limited to, "conservative modifications that inhibit enzymatic activity". For example, the mutants have at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) above (e.g., any one of SEQ ID NOs: 13, 18-34, 78, and 87). In some embodiments, 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-fold, 2-fold, 5-fold, 10-fold or more) inhibition of gamma secretase cleavage activity by a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above (e.g., any one of SEQ ID NOs:13, 18-34, 78 and 87). In some embodiments, 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-fold, 2-fold, 5-fold, 10-fold or more) inhibition of gamma secretase cleavage activity by ApoE2 (SEQ ID NO:12) or ApoE3 (SEQ ID NO:89) or an ApoE fragment shown in SEQ ID NO:33.

[0076] As used herein, "conservative modifications that inhibit enzymatic activity" refers to amino acid modifications that do not significantly affect or alter (e.g., reduce by no more than about 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) the inhibition of γ-secretase cleavage (e.g., γ-cleavage of APP) by a protein construct or polypeptide. Such conservative modifications that inhibit enzymatic activity include amino acid substitutions, insertions and / or deletions. Modifications can be introduced into the protein constructs or polypeptides of the invention by standard techniques known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. In some embodiments, the ApoE mutant has one or more (e.g., up to 40, up to 30, up to 20, up to 10, up to 8, up to 5, up to 3, up to 2 or 1) amino acid insertions, substitutions and / or deletions (insertions and / or deletions at the N-terminus and / or C-terminus) and at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5-fold, 2-fold, 5-fold, 10-fold or more) of the amino acid sequence of SEQ ID NO:12 in (1) above (e.g., any one of SEQ ID NOs:13, 18-34, 78 and 87). The ApoE polypeptide (e.g., an isolated ApoE polypeptide) retains biological activity that inhibits gamma-secretase cleavage (e.g., gamma-cleavage on APP) by a fragment derived from the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, 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-fold, 2-fold, 5-fold, 10-fold or more) inhibition of gamma-secretase cleavage activity by ApoE2 (SEQ ID NO: 12) or ApoE3 (SEQ ID NO: 89) or an ApoE fragment set forth in SEQ ID NO: 33.In some embodiments, the ApoE polypeptide (e.g., an isolated ApoE polypeptide) has a higher (e.g., at least about 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold or more) biological activity that inhibits gamma-secretase cleavage (e.g., gamma-cleavage of APP) compared to a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) (e.g., any one of SEQ ID NOs:13, 18-34, 78 and 87, e.g., SEQ ID NO:33).

[0077] In some embodiments, the ApoE polypeptide comprises a mutation (e.g., an insertion, deletion, or substitution) at one or more amino acid sites in any of the ApoE polypeptides or ApoE constructs of Example 1. The invention provides any of the ApoE polypeptides or ApoE constructs (e.g., isolated) in Examples 1 and 2.

[0078] In some embodiments, the ApoE polypeptide comprises a mutation (e.g., an insertion, deletion, or substitution) at one or more amino acid sites relative to the amino acid sequence set forth 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, P2 93, 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+A247+Q248+Q249+I250+R251+L252+Q253+A254. In some embodiments, the ApoE polypeptide comprises a mutation at one or more amino acid sites of the amino acid sequence set forth 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. In some embodiments, the mutation comprises a substitution at said one or more sites with A, S or T, such as a substitution with A. In some embodiments, the mutation comprises a deletion of an amino acid residue at said one or more sites.

[0079] In some embodiments, the conservative modification that inhibits the enzyme activity is a conservative substitution. A conservative substitution is a substitution in which an amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with the following side chains: basic side chains (such as lysine, arginine, histidine), acidic side chains (such as aspartic acid, glutamic acid), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (such as threonine, valine, isoleucine), aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0080] In some embodiments, the ApoE polypeptide comprises a mutation at one or more amino acid sites relative to the amino acid sequence set forth 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+P295A+S296A+D297A+N298A+H299A, R226A+D227A+R228A+L229A+D230A, E238A+V239A+R240A+K242A, or E244del+E245del+Q246del+A247del+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del. Contains one or more amino acid site mutations relative to the amino acid sequence shown in 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+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del, or R251S+T289A+A291T.

[0081] In some embodiments, the ApoE polypeptide or the ApoE mutant has at least about 70% sequence identity, such as at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more (but not 100%) identity to a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) (e.g., any one of SEQ ID NOs:13, 18-34, 78 and 87, e.g., SEQ ID NO:33). In some embodiments, the ApoE mutant inhibits gamma secretase cleavage at the same level as a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) above (e.g., any one of SEQ ID NOs: 13, 18-34, 78 and 87, e.g., SEQ ID NO: 33), or retains at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5-fold, 2-fold, 5-fold, 10-fold or more) inhibition of gamma secretase cleavage activity by a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) above. In some embodiments, the ApoE mutant, like the fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1), is derived from a human. In some embodiments, the ApoE polypeptide comprises or is derived from a sequence of non-human origin. The sequence identity between a mutant and a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above can be measured by methods well known in the art, for example, by BLASTP.

[0082] In some embodiments, the ApoE polypeptide comprises or is derived from a sequence of non-human origin, and the amino acid sequence has at least about 70% sequence identity with a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) above (e.g., any one of SEQ ID NOs: 13, 18-34, 78, and 87, e.g., SEQ ID NO: 33), and retains at least about 50% inhibition of gamma secretase cleavage activity by a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) above. In some embodiments, the ApoE polypeptide comprises or is derived from an ApoE sequence of any one of the following species: mammals, birds, frogs, fish, fruit flies, nematodes, etc. In some embodiments, mammals include, but are not limited to, primates (e.g., humans, or non-human primates, e.g., monkeys), rodents (e.g., mice, rats, hamsters, gerbils, squirrels, guinea pigs, rabbits), farm animals (e.g., pigs, cows, sheep, horses, donkeys), and pets (e.g., cats, dogs, rabbits, hamsters, etc.). In some embodiments, the ApoE polypeptide comprises non-naturally occurring modifications, e.g., further amino acid modifications to the monkey ApoE sequence.

[0083] In some embodiments, the ApoE polypeptide or the ApoE mutant comprises a sequence highly homologous and conserved to a non-human primate sequence, e.g., a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above (e.g., any one of SEQ ID NOs:13, 18-34, 78, and 87, e.g., SEQ ID NO:33). In some embodiments, the ApoE polypeptide comprises a monkey-derived ApoE C-terminal amino acid sequence set forth in SEQ ID NO:62. In some embodiments, the ApoE polypeptide consists of, or consists essentially of, the amino acid sequence set forth in SEQ ID NO:62.

[0084] In some embodiments, the ApoE polypeptide includes one or more mutations (e.g., insertions, deletions, or substitutions) based on the amino acid sequence set forth in SEQ ID NO:33, e.g., mutations at one or more sites described above, or has at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the ApoE polypeptide comprises one or more mutations (e.g., insertions, deletions, or substitutions) based on the amino acid sequence set forth in SEQ ID NO:33, e.g., mutations at any one or more of the above sites, or has at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO:33, and retains at least about 50% (e.g., at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5-fold, 2-fold, 5-fold, 10-fold or more) inhibition of gamma secretase cleavage activity by the amino acid sequence set forth in SEQ ID NO:33.

[0085] In some embodiments, the ApoE construct or the ApoE polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 46-52, 57-59, 62, and 76. In some embodiments, the ApoE construct or the ApoE polypeptide consists of, or consists essentially of, the amino acid sequence of any one of SEQ ID NOs: 46-52, 57-59, 62, and 76.

[0086] In some embodiments, the inhibitory effect of the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) (e.g., any one of SEQ ID NOs:13, 18-34, 78, and 87) on γ-secretase-mediated cleavage (e.g., γ-cleavage of APP) comprises one or more of the following properties: (i) inhibition of γ-secretase-mediated cleavage of APP (ii) inhibiting γ-cleavage (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); (iii) reducing amyloid metabolism of APP (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); (iv) reducing the production of Aβ (e.g., Aβ40 and / or Aβ42). (iv) reducing γ-cleavage to β-CTFs (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); (v) improving non-amyloid metabolism of APP (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). (vi) increasing production of α-CTFs (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more); (vii) not inhibiting (or inhibiting only about 30%, 20%, 10%, 5% or less) γ-cleavage against non-APP proteins (e.g., APLP1 or Notch) or fragments thereof;(viii) not increasing (or increasing by only about 30%, 20%, 10%, 5% or less) α-CTFs produced by γ-cleavage of non-APP proteins (e.g., APLP1, Notch, ErbB4, E-cadherin, N-cadherin, ephrin-B2 or CD44); (ix) reducing (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70% or less) amyloid plaques in the brain of individuals suffering from AD. %, 80%, 90%, 95%, or 100%), e.g., reducing the average plaque size, total area, and / or density of amyloid plaques (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); (x) reducing intraneuronal Aβ (e.g., Aβ40 and / or Aβ42) production in an individual with AD (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). , 50%, 60%, 70%, 80%, 90%, 95%, or 100%); (xi) reducing pathological processes in individuals with AD (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); (xii) improving survival and / or lifespan in individuals with AD (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, and / or (xiii) have no effect (or affect only about 30%, 20%, 10%, 5% or less) on mRNA and / or protein expression of α-secretase, β-secretase, and / or γ-secretase (or subunits thereof) (e.g., ADAM10, BACE1, PS1 and / or PS2);

[0087] In some embodiments, the ability of the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above to inhibit γ-secretase cleavage (e.g., γ-cleavage of APP) is greater than the ability to inhibit γ-secretase cleavage by the protein set forth in SEQ ID NO:12 or 33 or 89 (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more greater). In some embodiments, the ability of the ApoE construct, the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above to inhibit γ-secretase cleavage (e.g., γ-cleavage of APP) is the same as or similar to the ability to inhibit γ-secretase cleavage by the protein shown in SEQ ID NO:12 or 33 or 89 (e.g., the difference between them is not more than about 10%). In some embodiments, the ability of the ApoE construct, the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) to inhibit γ-secretase cleavage (e.g., γ-cleavage of APP) is up to about 50% lower (e.g., 40%, 30%, 20%, 10%, 5%, 1% or less lower) than the ability to inhibit γ-secretase cleavage by the protein shown in SEQ ID NO:12 or 33 or 89.

[0088] In some embodiments, the inhibitory activity of gamma-secretase-mediated cleavage (e.g., gamma-cleavage of APP) by the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) (e.g., any one of SEQ ID NOs:13, 18-34, 78, and 87) is the same as or similar to that of a gamma-secretase inhibitor (e.g., PF03084014) (e.g., the difference between them is not more than about 10%, 5%, or 1%). In some embodiments, the activity of the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above, in inhibiting gamma-secretase-mediated cleavage (e.g., gamma-cleavage of APP) is higher (e.g., at least about 10%, 20%, 50%, 100%, 1.5-fold, 2-fold, 5-fold or more higher) than that of a gamma-secretase inhibitor (e.g., PF03084014). In some embodiments, the activity of the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) above in inhibiting gamma-secretase-mediated cleavage (e.g., gamma-cleavage of APP) is lower (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more lower) than that of a gamma-secretase inhibitor (e.g., PF03084014).

[0089] In some embodiments, inhibition of gamma-cleavage of APP by the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence set forth in SEQ ID NO:12 in (1) (e.g., any one of SEQ ID NOs:13, 18-34, 78, and 87) is higher (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more higher) than inhibition of gamma-cleavage of non-APP proteins (e.g., APLP1, Notch). In some embodiments, the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence shown in SEQ ID NO:12 in (1) above, only inhibits γ-cleavage of APP, and does not or barely inhibits γ-cleavage of non-APP proteins (e.g., APLP1, Notch).

[0090] In some embodiments, the α-amylase inhibitor (e.g., GI254023X) and / or β-amylase inhibitor (e.g., AZD3839) does not affect the inhibition of γ-secretase cleavage (e.g., γ-cleavage of APP) activity by the ApoE construct (e.g., an isolated ApoE construct), the ApoE polypeptide (e.g., an isolated ApoE polypeptide, or an ApoE mutant), or a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1), or affects it by about 50% or less (e.g., about 40%, 30%, 20%, 10%, 5%, 1% or less). For example, in the presence of an α-amylase inhibitor or β-amylase inhibitor, the ApoE construct or ApoE polypeptide can still increase the α-CTF level produced from APP and reduce the production of Aβ.

[0091] The influence on gamma-secretase cleavage (e.g., gamma-cleavage on APP) activity may be studied in any suitable manner in the art, for example by studying the enzymatic activity of gamma-secretase itself, or by studying the properties or amount of the gamma-secretase cleavage products (e.g., Aβ, such as Aβ40 or Aβ42), or by studying the properties or amount (e.g., whether they are decreased by gamma-cleavage) of gamma-secretase substrates (e.g., APP, α-CTF, β-CTF, APLP1, Notch, ErbB4, E-cadherin, N-cadherin, ephrin-B2 or CD44). These methods of study include, but are not limited to, qRT-PCR, BCA, flow cytometry, proteomics, metabolomics, ELISA, Western blot, co-immunoprecipitation, protein fluorescent labeling, immunostaining, etc., and can be in vivo or in vitro (cell experiments or non-cell experiments). Any gamma secretase activity detection kit can be used here, such as MyBioSource's #MBS704513 kit, or R&D Systems' #FP003 kit, also see the research methods described in Examples 1 and 2.

[0092] In some embodiments, the ApoE construct further comprises an N-terminal domain sequence or a portion thereof of an ApoE2 protein (e.g., the amino acid sequence of positions 1-167 of SEQ ID NO:12 or a portion thereof; SEQ ID NO:79), and / or a hinge region sequence or a portion thereof (e.g., the amino acid sequence of positions 168-205 of SEQ ID NO:12 or a portion thereof; SEQ ID NO:80) at the N-terminus of any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87). NO:80), or a sequence having at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identity to the N-terminal domain sequence (or a portion of the sequence) and / or the hinge region sequence (or a portion of the sequence) of the ApoE2 protein (e.g., a non-human primate (e.g., monkey) ApoE homologous sequence). In some embodiments, the ApoE construct further comprises, at the N-terminus of any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87), an N-terminal domain sequence or a portion thereof (e.g., amino acid sequence of positions 1-167 or a portion thereof; SEQ ID NO:90) and / or a hinge region sequence or a portion thereof (e.g., amino acid sequence of positions 168-205 or a portion thereof; SEQ ID NO:80), or a sequence having at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identity to the N-terminal domain sequence (or a portion thereof) and / or the hinge region sequence (or a portion thereof) of an ApoE3 protein (e.g., a non-human primate (e.g., monkey) ApoE homologous sequence).In some embodiments, the ApoE construct further comprises, at the N-terminus of the ApoE polypeptide, a portion of a sequence (e.g., a portion of the N-terminus) derived from the ApoE C-terminal domain (positions 206-299 of SEQ ID NO:12), such as the sequence or a portion of amino acids 206-214 of SEQ ID NO:12, or a sequence having at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) identity to said portion of the ApoE C-terminal domain. In some embodiments, the C-terminus of the N-terminal domain sequence or a portion thereof, or the C-terminus of the (N-terminal domain + hinge region) sequence or a portion thereof, or the C-terminus of the (N-terminal domain + hinge region + N-terminal sequence of a portion of the C-terminal domain) sequence of the ApoE2 or ApoE3 protein (or a non-human homologous protein) is connected to the N-terminus of the ApoE polypeptide by a peptide bond or a linker sequence (e.g., a protein linker sequence). In some embodiments, the ApoE protein sequence homologous to the N-terminal domain (or a portion thereof) and / or the hinge region (or a portion thereof) and / or the C-terminal domain (or a portion thereof) of the human ApoE2 or ApoE3 protein is a sequence derived from any of the following species: mammals (e.g., non-human primates such as monkeys, rodents, livestock, pets), birds, frogs, fish, fruit flies, nematodes, etc.

[0093] In some embodiments, the ApoE construct further comprises at the N-terminus of any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87), an amino acid sequence of amino acid residues 1-167 (SEQ ID NO:79) or 1-205 (SEQ ID NO:81) or 1-214 of SEQ ID NO:12, or an amino acid sequence having at least about 70% identity to amino acid residues 1-167, 1-205, or 1-214 of SEQ ID NO:12. In some embodiments, the ApoE construct comprises an amino acid sequence set forth in SEQ ID NO:60 or 61. In some embodiments, the ApoE construct consists of, or consists essentially of, the amino acid sequence set forth in SEQ ID NO:60 or 61.

[0094] In some embodiments, the ApoE construct further comprises at the N-terminus of any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87), an amino acid sequence of amino acid residues 1-167 (SEQ ID NO:90) or 1-205 or 1-214 of ApoE3 (SEQ ID NO:89), or an amino acid sequence having at least about 70% identity to amino acid residues 1-167, 1-205, or 1-214 of ApoE3.

[0095] In some embodiments, 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).

[0096] In some embodiments, the ApoE construct further comprises a membrane-permeable peptide at the N-terminus or C-terminus of any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, or the full-length ApoE2 or ApoE3 sequences). In some embodiments, the membrane-permeable peptide comprises (or consists of, or consists essentially of) any one of the amino acid sequences in SEQ ID NOs: 35-45, 63-65, and 84-86, e.g., the amino acid sequence set forth in SEQ ID NO: 63 or 65. In some embodiments, the membrane penetrating peptide (e.g., at the C-terminus) is connected by a peptide bond or a linker sequence (e.g., a protein linker sequence) to the ApoE polypeptide (e.g., at the N-terminus), or to an amino acid sequence (e.g., at the N-terminus) that includes amino acid residues 1-167 (or a fragment thereof) or 1-205 (or a fragment thereof) or 1-214 (or a fragment thereof) of SEQ ID NO:12 or 89, or to a sequence (e.g., at the N-terminus) that has at least about 70% identity to amino acid residues 1-167 (or a fragment thereof) or 1-205 (or a fragment thereof) or 1-214 (or a fragment thereof) of SEQ ID NO:12 or 89. In some embodiments, the N-terminus of the membrane penetrating peptide is further connected by a peptide bond or a linker sequence to the C-terminus of a signal peptide. In some embodiments, the membrane penetrating peptide is located at the most N-terminus of the ApoE construct. In some embodiments, the membrane penetrating peptide is located at the most C-terminus of the ApoE construct.

[0097] In some embodiments, the present invention provides an ApoE construct (or fusion protein), the fusion protein comprising the following elements (1) and (2): (1) a membrane-permeable peptide, and (2) the full-length sequence of ApoE2 (e.g., SEQ ID NO: 12), or the full-length sequence of ApoE3 (e.g., SEQ ID NO: 89), or a fragment of ApoE2 comprising at least amino acid residues 221-274 of SEQ ID NO: 12. The elements (1) and (2) may be directly connected by a peptide bond or via a linker sequence.

[0098] Membrane-penetrating peptides or cell-membrane-penetrating peptides refer to a type of short peptide that can deliver macromolecules into cells. The fusion proteins of the present invention may be constructed with membrane-penetrating peptides that are well known in the art. For exemplary membrane-penetrating peptides, see Siegmund Reissmann, Cell penetration: scope and limitations by the application of cell-penetrating peptides, J. Pept. Sci. 2014;20:760-784, the entire contents of which are incorporated herein by reference. Membrane-penetrating peptides suitable for the present invention include RQIKIWFQNRRMKWKK (SEQ ID NO:35); YGRKKRRQRRR (SEQ ID NO:36); KQAIPVAK (SEQ ID NO:37); RRRRNRTRRNRRRVR (SEQ ID NO:38); oligoarginine (i.e., a fragment of 9-12 arginine residues in length; R9 (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);GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO:43);AGYLLGKINLKALAALAKKIL(SEQ ID NO:44);YTAIAWVKAFIRKLRK(SEQ ID NO:45);and KSVRTWNEIIPSKGCLRVGGRCHPHVNGGGRRRRRRRRR(SEQ ID NO:65).

[0099] In some embodiments, the fragment of ApoE2 described in element (2) in the fusion protein comprising at least amino acid residues 221-274 of SEQ ID NO:12 may be an isolated ApoE polypeptide described in any one of the embodiments herein, and a fragment containing the N-terminus and hinge region (or fragment thereof) and at least amino acid residues 221-274 of the C-terminus of ApoE2 or ApoE3. In some embodiments, the ApoE2 polypeptide may comprise any one of the following sequences: amino acid residues 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, 1-291, 1-292, 1-293, 1-294, 1-295, 1-296, 1-297, 1-298, or 1-299 of SEQ ID NO:12.

[0100] In some embodiments, element (2) in the fusion protein is selected from the amino acid sequence of any one of SEQ ID NOs: 12, 13, 18-34, 60, 78, 87, and 89.

[0101] In some embodiments, the linker is a non-protein linker. In some embodiments, the linker is a protein linker. In some embodiments, the linker is a peptide containing 3-25 residues, such as a peptide containing 3-15, 5-15, 10-20, 20-25, or 3-10 residues. Suitable examples of peptide linkers are known in the art. Typically, the linker contains one or more repeated motifs, which usually contain Gly and / or Ser. For example, the motif may 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). In some embodiments, the motifs are adjacent in the linker sequence, with no amino acid residues inserted between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motifs. In one embodiment, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is usually 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. Besides glycine and serine, the linker may contain other known amino acid residues, such as alanine, leucine, threonine, glutamic acid, phenylalanine, arginine, glutamine, etc. In some embodiments, the linker sequence is as set forth in SEQ ID NO:88.

[0102] The ApoE constructs or fusion proteins of the invention may also include amino acid residues or sequences introduced into the fusion protein for purposes such as gene cloning manipulation, constructing the fusion protein, facilitating expression of the recombinant protein, obtaining a recombinant protein that is automatically secreted from a host cell, or facilitating detection and / or purification of the recombinant protein. In some embodiments, the ApoE constructs or fusion proteins of the invention further include a signal peptide, a leader peptide, a terminal extension, a tag protein, and the like.

[0103] In some embodiments, the ApoE construct further comprises a signal peptide at the N-terminus of the ApoE polypeptide, particularly if it is to be expressed in a cell. In some embodiments, the C-terminus of the signal peptide is connected by a peptide bond or a linker sequence (e.g., a protein linker sequence) to the N-terminus of the ApoE polypeptide (e.g., any one of SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, or a full-length ApoE2 or ApoE3 sequence), or to the N-terminus of an amino acid sequence comprising amino acid residues 1-167 (or a fragment thereof), or 1-205 (or a fragment thereof), or 1-214 (or a fragment thereof) of SEQ ID NO: 12 or 89, or to the N-terminus of a sequence having at least about 70% identity to amino acid residues 1-167 (or a fragment thereof), or 1-205 (or a fragment thereof), or 1-214 (or a fragment thereof) of SEQ ID NO: 12 or 89, or to the N-terminus of a membrane-permeable peptide. In some embodiments, the signal peptide is located at the most N-terminus of the ApoE construct. In some embodiments, the signal peptide is further inserted at the N-terminus of the amino acid sequence shown in SEQ ID NO:60 or 61. In some embodiments, the signal peptide is further inserted at the N-terminus of the full-length sequence of ApoE2 (e.g., SEQ ID NO:12) or ApoE3 (e.g., SEQ ID NO:89). In some embodiments, the C-terminus of the signal peptide is further connected to the N-terminus of the membrane-permeable peptide by a peptide bond or a linker sequence (e.g., a protein linker sequence). Any well-known signal peptide may be used herein. In some embodiments, the signal peptide is homologous to the ApoE construct or the ApoE polypeptide, for example, a signal peptide derived from the ApoE2 (SEQ ID NO:12) or ApoE3 (SEQ ID NO:89) protein itself.In some embodiments, the signal peptide is heterologous to the ApoE construct or the ApoE polypeptide, e.g., a signal peptide derived from ApoA1 or ApoJ. In some embodiments, the signal peptide comprises (or consists of, or consists essentially of) any one of the amino acid sequences set forth in SEQ ID NOs:66-68.

[0104] In some embodiments, the ApoE construct comprises (or consists of, or consists essentially of) the following sequence from N-terminus to C-terminus: signal peptide-optional linker 1-membrane permeabilizing peptide (e.g., the amino acid sequence shown in SEQ ID NO:63 or 65)-optional linker 2-ApoE2 protein (e.g., SEQ ID NO:12) or ApoE3 protein (e.g., SEQ ID NO:89); signal peptide-optional linker-ApoE2 protein (e.g., SEQ ID NO:12) or ApoE3 protein (e.g., SEQ ID NO:89); ApoE2 protein (e.g., SEQ ID NO:12) or ApoE3 protein (e.g., SEQ ID NO:89); a membrane-permeable peptide (e.g., the amino acid sequence shown in SEQ ID NO:63 or 65)-optional linker-ApoE2 protein (e.g., SEQ ID NO:12) or ApoE3 protein (e.g., SEQ ID NO:89); a signal peptide - optional linker 1 - a membrane-permeable peptide (e.g., the amino acid sequence shown in SEQ ID NO: 63 or 65) - optional linker 2 - any one of the ApoE polypeptides (e.g., any one of the sequences of SEQ ID NO: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO: 33); a membrane-permeable peptide (e.g., the amino acid sequence shown in SEQ ID NO:63 or 65) - an optional linker - any one of the ApoE polypeptides (e.g., any one of the sequences of SEQ ID NO:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO:33); a signal peptide - optional linker - any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO:33); Any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO:33); a signal peptide - optional linker 1 - a membrane-permeable peptide (e.g., the amino acid sequence set forth in SEQ ID NO:63 or 65) - optional linker 2 - an ApoE2 or ApoE3 N-terminal domain and / or hinge region and / or (N-terminal to the C-terminal domain) sequence or fragment (e.g., the amino acid sequence set forth in any one of SEQ ID NOs:79-81 and 90 or a fragment thereof) - optional linker 3 - any one of the ApoE polypeptides (e.g., any one of the sequences of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, e.g., SEQ ID NO:33); a membrane-permeable peptide (e.g., the amino acid sequence set forth 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 the C-terminal domain) sequence or fragment (e.g., the amino acid sequence set forth in any one of SEQ ID NOs:79-81 and 90 or a fragment thereof) - optional linker 2 - any one of the ApoE polypeptides (e.g., any one of the sequences of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, e.g., SEQ ID NO:33); signal peptide - optional linker 1 - ApoE2 or ApoE3 N-terminal domain and / or hinge region and / or (N-terminal to the C-terminal domain) sequence or fragment (e.g., an amino acid sequence set forth in any one of SEQ ID NOs:79-81 and 90, or a fragment thereof) - optional linker 2 - any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, e.g., SEQ ID NO:33); ApoE2 or ApoE3 N-terminal domain and / or hinge region and / or (N-terminal to the C-terminal domain) sequence or fragment (e.g., an amino acid sequence set forth in any one of SEQ ID NOs:79-81 and 90, or a fragment thereof) - an optional linker - any one of the ApoE polypeptides (e.g., any one of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, e.g., SEQ ID NO:33); signal peptide-optional linker 1-membrane permeabilizing peptide (e.g., the amino acid sequence shown in SEQ ID NO: 63 or 65)-optional linker 2-(the amino acid sequence shown in SEQ ID NO: 60 or 61); a membrane-permeable peptide (e.g., the amino acid sequence shown in SEQ ID NO:63 or 65)-optional linker-(the amino acid sequence shown in SEQ ID NO:60 or 61); The amino acid sequence shown in SEQ ID NO:60 or 61; or Signal peptide - optional linker - (amino acid sequence shown in SEQ ID NO:60 or 61).

[0105] In some embodiments, the ApoE construct further comprises a tag protein at the N-terminus and / or C-terminus of the ApoE polypeptide. In some embodiments, the tag protein is located at the most N-terminus of the ApoE construct. In some embodiments, the tag protein is located at the most C-terminus of the ApoE construct. In some embodiments, the tag protein (e.g., the 3XFLAG sequence shown in SEQ ID NO:77) is located internally in the ApoE construct but at the N-terminus of the ApoE polypeptide, e.g., directly linked to the N-terminus of the ApoE polypeptide. The tag protein may be an expression purification tag (e.g., His-tag, FLAG-tag, GST-tag, c-Myc-tag, V5-tag, HA-tag) or a detection tag (e.g., GFP, RFP, V5, HA). In some embodiments, the purification tag may be used for detection, e.g., immunoblot or antibody detection. In some embodiments, the tag protein is located at the most N-terminus of the ApoE construct (e.g., ApoE2 or ApoE3, or an ApoE polypeptide comprising the sequence SEQ ID NO:33). In some embodiments, the tag protein is located at the most C-terminus of the ApoE construct. In some embodiments, the tag protein is a 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).

[0106] Amyloid-beta precursor protein (APP) and hydrolysis APP is a protein embedded in the cell membrane and is mainly concentrated in the synapses of neurons. It contains cleavage sites for alpha, beta, and gamma secretases. APP is expressed in many different species and has high homology. The APP described herein may be derived from any species, such as mammals, birds, frogs, fish, fruit flies, nematodes, etc. Mammals include, but are not limited to, primates (e.g., humans or non-human primates, such as monkeys), rodents (e.g., mice, rats, hamsters, gerbils, squirrels, guinea pigs, rabbits), farm animals (e.g., pigs, cows, sheep, horses, donkeys), and pets (e.g., cats, dogs, rabbits, hamsters, etc.). In some embodiments, the APP is a human APP.

[0107] Amyloid metabolism of APP produces short β-amyloid polypeptides (Aβ). Aβ polypeptides are prepared by cleavage of APP by β-secretase activity near the N-terminal position of Aβ and by gamma-secretase activity near the C-terminal position of Aβ. APP is also cleaved by α-secretase activity to generate secreted nonamyloid fragments of soluble APPα. β-site APP cleaving enzyme ("BACE-1") is thought to be the major aspartyl protease involved in the production of Aβ by β-secretase activity. BACE-1 inhibition inhibits the production of Aβ. BACE1 is a type I transmembrane protein with a luminal active site that cleaves APP and releases the extracellular domain (sAPPβ) into the extracellular space. The remaining C-terminal fragment (CTF) is further cleaved by γ-secretase, resulting in the release of Aβ and the APP intracellular C-terminal domain (AICD). Presenilins are the main enzymatic components of γ-secretase, which inaccurately cleaves APP to produce consecutive Aβ polypeptides that differ in length by a few amino acids at the C-terminus. Most Aβ normally terminates at amino acid 40 (Aβ40), but the 42 amino acid variant (Aβ42) has been shown to be prone to aggregation and is hypothesized to nucleate senile plaque formation. Modulation of γ-secretase may also lead to an increase in the 38 amino acid variant (Aβ38). The competing α-secretase pathway is the result of sequential cleavage by α-secretase and γ-secretase. ADAM-10 is the α-secretase for APP cleavage, which releases the extracellular domain (sAPPα) that exhibits neuroprotective functions (Lammich et al., (1999) Proc. Natl. Acad. Sci. USA, 96:3922-3927). The 83 amino acid CTF (C83) is then cleaved, releasing p3 (nonamyloidogenic) and AICD (Furukawa et al., (1996) J. Neurochem., 67:1882-1896).

[0108] Aβ is the major component of β-amyloid fibrils and plaques, which are believed to be involved in increasing pathology. Examples of such conditions include, but are not limited to, neurodegenerative diseases such as 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 disorder (including that associated with Alzheimer's disease, Parkinson's disease, and Down's syndrome), dementia (including presenile dementia, senile dementia, and dementia associated with Alzheimer's disease (AD), Parkinson's disease, and Down's syndrome), progressive supranuclear palsy, corticobasal degeneration, neurodegeneration, olfactory disorders (including those associated with Alzheimer's disease, Parkinson's disease, and Down's syndrome), beta amyloid cerebrovascular disease (including cerebral amyloid cerebrovascular disease), hereditary cerebral hemorrhage, mild cognitive impairment ("MCI"), glaucoma, amyloidosis, type II diabetes mellitus, hemodialysis (beta-2 microglobulin and its complications), scrapie, bovine spongiform encephalitis, Creutzfeldt-Jakob disease, and traumatic brain injury.

[0109] In some embodiments, 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 mutant. APP mutations have been identified in 121 families. The most common missense mutation is Val 717 The residue was replaced with Ile (London mutation), Phe, or Gly residues, and Glu 693 to a Gln residue (Dutch mutation) or a Gly residue (Arctic mutation), or to Lys 670 and Met 671 to Asn and Leu residues (double mutation is Swedish mutation APP SW It is called APP 770 In some embodiments, the APP is a human APP and includes one or more of the following mutations: K670N, M671L, Swedish (K670N+M671L), Florida (I716V), London (V717I), APP M596V(beta cleavage is infeasible), a fAD mutation close to the gamma cleavage site (e.g., T714I, V717F, L723P), a fAD mutation close to the beta or alpha cleavage site (e.g., KM670 / 671NL, E693K, E693Δ), or a protective Iceland mutation (A673T). In some embodiments, the APP does not contain a mutation that affects gamma-cleavage, such as a mutation close to the gamma site.

[0110] In some embodiments, the ApoE construct or ApoE polypeptide can inhibit γ-cleavage of any one of the APP (e.g., inhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%). In some embodiments, the ability of the ApoE construct or ApoE polypeptide to inhibit γ-cleavage of any one of the APP mutants is at least about 30% (e.g., at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5-fold, 2-fold, 5-fold, 10-fold or more) of its ability to inhibit γ-cleavage of wild-type APP. In some embodiments, the ApoE construct or ApoE polypeptide has a lower ability to inhibit gamma-cleavage of APP containing mutations that affect gamma-cleavage (e.g., T714I, V717F, L723P) than it has an ability to inhibit gamma-cleavage of wild-type APP (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% lower).

[0111] Gamma-secretase The γ-secretase complex consists of four single proteins: PSEN1 (presenilin-1, PS1), nicastrin, APH-1 (anterior pharynx-defective 1), and PEN-2 (presenilin enhancer 2). Recent evidence suggests that the fifth protein in the complex, CD147, is a nonessential regulator and that its absence increases γ-secretase activity. The gamma-secretase described herein may be derived from any species, such as mammals, birds, frogs, fish, fruit flies, nematodes, etc. In some embodiments, the gamma-secretase is derived from a human or a non-human primate, such as a monkey.

[0112] In some embodiments, the gamma-secretase described herein is a wild-type gamma-secretase or a naturally occurring subtype. In some embodiments, the gamma-secretase comprises one or more mutations, such as mutations that enhance or reduce its cleavage activity. In some embodiments, the gamma-secretase has one or two mutations in PS1: M146L, L286V. In some embodiments, the PS1 and / or PS2 activity of the gamma-secretase is reduced or absent.

[0113] In some embodiments, the ApoE construct or ApoE polypeptide can inhibit any one gamma-secretase enzymatic activity (e.g., inhibit at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%). In some embodiments, the ability of the ApoE construct or ApoE polypeptide to inhibit any one gamma-secretase mutant enzymatic activity is at least about 30% (e.g., at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 1.5-fold, 2-fold, 5-fold, 10-fold, or more) of its ability to inhibit wild-type gamma-secretase enzymatic activity.

[0114] 3. Methods for producing ApoE constructs The present invention further provides isolated nucleic acids and vectors (e.g., cloning vectors or expression vectors) encoding any one of the above isolated ApoE constructs (e.g., any one of the amino acid sequences of SEQ ID NOs:12, 60, 61, and 89), fusion proteins, or protein portions of ApoE polypeptides (e.g., any one of the amino acid sequences of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO:33), and host cells expressing or containing any one of the above isolated ApoE constructs. In some embodiments, the vector is a recombinant AAV or lentivirus expression vector. In some embodiments, the vector comprises a neuron-specific promoter at the 5' end of the isolated nucleic acid. In some embodiments, the neuron-specific promoter is selected from a synapsin promoter (e.g., hSYN), a thy-1 promoter, and a calmodulin-dependent protein kinase II-alpha promoter. In some embodiments, the host cell is a neural cell, e.g., a neuron (e.g., a human neuron, a non-human primate neuron, or a rodent neuron). See Examples 1 and 2 for vectors, cells, and methods of manufacture.

[0115] In some embodiments, the present invention provides a nucleic acid or vector that mediates specific expression of any one of the ApoE constructs, fusion proteins, or ApoE polypeptides in neuronal cells. In some embodiments, the present invention provides a vector or isolated nucleic acid that includes the following 5' to 3' sequences: a neuron-specific promoter (e.g., hSYN)-a nucleotide sequence encoding a signal peptide-a nucleotide sequence encoding a membrane-permeable peptide (e.g., SEQ ID NO:63 or 65)-a nucleotide sequence encoding any one of the ApoE constructs (e.g., SEQ ID NO:12 or 89) / fusion protein / ApoE polypeptide (e.g., SEQ ID NO:33). In some embodiments, the present invention provides a vector or isolated nucleic acid that includes the following 5' to 3' sequences: a neuron-specific promoter (e.g., hSYN)-a nucleotide sequence encoding a membrane-permeable peptide (e.g., SEQ ID NO:63 or 65)-a nucleotide sequence encoding any one of the ApoE constructs (e.g., SEQ ID NO:12 or 89) / fusion protein / ApoE polypeptide (e.g., SEQ ID NO:33). In some embodiments, the invention provides a vector or isolated nucleic acid, comprising the following 5' to 3' sequences: a neuron-specific promoter (e.g., hSYN)-a nucleotide sequence encoding a signal peptide-a nucleotide sequence encoding any one of said ApoE constructs (e.g., SEQ ID NO:12 or 89) / fusion protein / ApoE polypeptide (e.g., SEQ ID NO:33). In some embodiments, said ApoE construct comprises (or consists of, or consists essentially of) the full length sequence of ApoE2 or ApoE3. In some embodiments, said ApoE construct comprises (or consists of, or consists essentially of) the amino acid sequence of SEQ ID NO:60 or 61.In some embodiments, the ApoE polypeptide comprises (or consists of, or consists essentially of) any one of the amino acid sequences of SEQ ID NO:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO:33.

[0116] The present application also includes nucleic acid molecules encoding any one of the above-described isolated ApoE constructs, fusion proteins, or ApoE polypeptides, and complementary sequences thereof. The present invention includes all forms of the nucleic acid molecules of the present invention, including RNA and DNA. A complementary sequence as described herein refers to a complementary sequence that is essentially identical in length to the nucleic acid molecule (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homologous in length), e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary.

[0117] The present invention also includes a nucleic acid construct containing said nucleic acid molecule or a complementary sequence thereof. The nucleic acid construct may be an expression cassette containing a promoter, said nucleic acid molecule, and a transcription termination sequence. The expression cassette may optionally contain an enhancer required for expression. Promoters, enhancers, transcription termination sequences, selectable markers, and signal sequences are well known in the art, and the skilled artisan can select them appropriately according to the host cell selected for expression of said polypeptide. These elements in the expression cassette are operably linked to each other so that they can perform their intended functions. For example, operably linking a promoter sequence to a nucleotide sequence of interest refers to connecting the promoter sequence and the nucleotide sequence of interest such that the promoter sequence can direct the transcription of the nucleotide sequence of interest and / or the synthesis of a polypeptide encoded by the nucleotide sequence of interest in the target cell. The selectable marker serves to select host cells into which the vector has been inserted from host cells into which the vector has not been inserted, for example, the selectable marker may be a gene that confers antibiotic resistance. A signal sequence is selected so that the expressed polypeptide can be transported out of the host cell.

[0118] Genetic control elements may be used to control gene expression of a polypeptide or protein. Such genetic control elements are selected to be active in the relevant host cell. The control elements may be constitutively active or inducible under certain conditions. Inducible control elements are particularly useful when expressing proteins that are toxic or have other adverse effects on cell growth and / or viability. In such cases, the expression of the polypeptide or protein may be controlled by the inducible control element to improve cell viability, cell density, and / or the total yield of the expressed polypeptide or protein. Numerous control elements that can be used to practice the present invention are known and available in the art.

[0119] Representative constitutive mammalian promoters useful in the present invention include, but are not limited to, the hypoxanthine phosphoribosyltransferase (HPTR) promoter, the adenosine deaminase promoter, the pyruvate kinase promoter, the β+actin promoter, and other constitutive promoters known to those skilled in the art. In addition, viral promoters capable of driving constitutive expression of coding sequences in eukaryotic cells include simian virus promoters, herpes simplex virus promoters, papillomavirus promoters, adenovirus promoters, human immunodeficiency virus (HIV) promoters, Rous sarcoma virus promoters, cytomegalovirus (CMV) promoters, Moloney murine leukemia virus, other retroviral long terminal repeats (LTRs), herpes simplex virus thymidine kinase promoters, and other viral promoters known to those skilled in the art.

[0120] Inducible promoters are capable of driving expression of an operatively linked coding sequence when an inducing agent is present, or can be used in accordance with the present invention. For example, in mammalian cells, the metallothionein promoter can induce transcription of a downstream coding sequence in the presence of certain metal ions. Other inducible promoters will be recognized and / or known to those of skill in the art.

[0121] In some embodiments, the gene expression control element suitable for the present invention is a neuron-specific control element, including promoter and enhancer.As used herein, the term "neuron-specific" control element refers to the control element, such as promoter and enhancer, for the specific expression of the nucleic acid molecule of interest in neuron.In some embodiments, the neuron-specific promoter is the promoter for the expression of transgene in a specific subtype of neuron (e.g., dopaminergic neuron, excitatory neuron, lateral prefrontal cortex neuron, etc.).

[0122] Neuron-specific promoters and other control elements (such as enhancers) are known in the art. Suitable neuron-specific promoters include, but are not limited to, 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-α promoter, CMV enhancer / platelet-derived growth factor-β promoter. In some embodiments, the neuron-specific promoter is selected from synapsin promoter (e.g., hSYN), thy-1 promoter, and calmodulin-dependent protein kinase II-α promoter.

[0123] Generally, gene expression sequences include 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, such as the TATA box, cap sequence, and CAAT sequence, respectively. Enhancer elements can be optionally used to enhance the expression levels of the expressed polypeptide or protein. Examples of enhancer elements that function in mammalian cells include the SV40 early gene enhancer (described in Dijkema et al., EMB0J. (1985) 4:761), the enhancer / promoter from Rous sarcoma virus (RSV) (described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777) and the human cytomegalovirus (described in Boshart et al., Cell (1985) 41:521) long terminal repeat.

[0124] Systems for connecting control elements to coding sequences are well known in the art (see, for example, general molecular biology and recombinant DNA techniques, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, incorporated herein by reference). Commercially available vectors suitable for inserting a desired coding sequence for expression in a variety of mammalian cells under a variety of growth and induction conditions are also known in the art.

[0125] In some embodiments, the nucleic acid construct is a vector, including expression vectors and cloning vectors. Expression vectors are vectors suitable for expressing foreign genes, such as nucleic acid molecules encoding the polypeptides described in the present invention (e.g., the full-length sequence of ApoE2 or ApoE3, or any ApoE constructs, ApoE polypeptides, or fusion proteins described herein), in host cells, including prokaryotic and eukaryotic expression vectors. Eukaryotic expression systems include yeast expression systems, mammalian expression systems, insect expression systems, and the like. Cloning vectors are used to amplify target genes (e.g., nucleic acid molecules encoding the polypeptides of the present invention) in host cells. Cloning vectors include plasmid vectors, phage vectors, viral vectors, and vectors fused to each other or to other genomic DNA. For molecular cloning, the most commonly used host cell is E. coli.

[0126] In some embodiments, the vector is a viral vector, including but not limited to lentivirus vector, herpes simplex virus vector, adenovirus vector, and adeno-associated virus (AAV) vector.Such vectors can be prepared using standard methods in the art.In some embodiments, the vector is a lentivirus or lentiviral vector.

[0127] In some embodiments, the vector is a recombinant AAV vector.AAV vector can be prepared using standard methods in the art.Any serotype of adeno-associated virus vector can be used, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16.

[0128] In some embodiments, the present invention provides a vector (e.g., AAV or lentiviral vector), comprising an expression cassette comprising a neuron-specific promoter (e.g., hSyn) operably linked to a nucleic acid molecule of interest, the nucleic acid molecule of interest being selected from a nucleic acid molecule encoding any one of the ApoE C-terminal fragments of the present invention, a nucleic acid molecule encoding an ApoE2 or ApoE3 full-length protein, a nucleic acid molecule encoding any one of the ApoE2 constructs or fusion proteins of the present invention, or a nucleic acid molecule encoding any one of the ApoE2 polypeptides of the present invention. Preferably, the vector is a recombinant AAV vector. Preferably, neuron-specific promoters suitable herein include synapsin promoter, thy-1 promoter, and calmodulin-dependent protein kinase II-α promoter. In some embodiments, the nucleic acid molecule of interest encodes an amino acid sequence set forth in any one of SEQ ID NOs: 12, 13, 18-34, 46-52, 57-59, 60-62, 76, 78, 87, and 89. In some embodiments, the nucleic acid molecule of interest further encodes an N-terminal signal peptide (e.g., any one of the amino acid sequences in SEQ ID NOs:66-68). In some embodiments, the nucleic acid molecule of interest further encodes an N-terminal and / or C-terminal membrane-permeable peptide (e.g., any one of the amino acid sequences in SEQ ID NOs:35-45, 63-65, and 84-86). In some embodiments, the nucleic acid molecule of interest further encodes an N-terminal and / or C-terminal tag protein (e.g., V5, EGFP).

[0129] The present invention further provides an expression system comprising the nucleic acid molecule, nucleic acid construct or vector disclosed herein. The expression system may be, for example, a Gram-positive or Gram-negative prokaryotic host cell system, such as Escherichia coli, modified to express the polypeptide of the present invention. In some embodiments, the expression system is a eukaryotic host cell system, such as a yeast, such as Pichia pastoris or Saccharomyces cerevisiae. In some embodiments, the host cell is a mammalian host cell, such as, for example, a HEK 293T, N2A, CHO cell. In some embodiments, the host cell is a neural cell, including, but not limited to, a glial cell and a neuron. Higher eukaryotic cells, particularly cells derived from multicellular organisms, can be used for the expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells, as well as vertebrate cells.

[0130] Suitable methods for introducing sufficient amounts of nucleic acid required for expression of a polypeptide or protein of interest into a host cell (e.g., a mammalian host cell) are well known in the art.See, for example, 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 of which are incorporated herein by reference.Vector may be introduced into a host cell using any suitable method known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitation, cationic lipid mediated delivery, liposome-mediated transfection, electroporation, microparticle bombardment, receptor-mediated gene delivery, polylysine, histone, chitosan, and peptide mediated delivery. For mammalian cells, the preferred transformation method is the calcium phosphate precipitation method described by Graham and van der Erb in Virology, 52:456-457 (1978) or the lipofectamine method described by Hawley-Nelson in Focus 15:73 (1193). TM(Gibco BRL) methods. General aspects of transformation of mammalian cell host systems are described in U.S. Patent No. 4,399,216, filed Aug. 16, 1983 by Axel. A variety of 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 expressing polypeptides or proteins in mammalian cells include P(3)NAl;p(3) (Okayama et al., (1985) Mol. Cell Biol. 5:1136-1142), pMClneoPoly-A (Thomas et al., (1987) Cell 51:503-512), baculovirus vectors (e.g., PAC373 or pAC610).

[0131] In some embodiments, the polypeptides or proteins according to the invention are stably transfected into the host cells, however, one of skill in the art should recognize that the present invention can be used to transiently or stably transfect the host cells.

[0132] There are many ways to introduce a target protein or polypeptide into a target cell, including using liposomes to deliver DNA (e.g., plasmids) or RNA encoding the target protein, or infecting cells with lentiviruses or adeno-associated viruses encoding the target protein.

[0133] Any mammalian cell or cell type amenable to cell culture and polypeptide expression can be used in the present invention. Non-limiting examples of mammalian cells useful in the present invention include BALB / c mouse myeloma cell line (NSO / 1, ECACC No: 85110503), human retinoblastoma cells (PER.C6 (CruCelI, Leiden, The Netherlands)), SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells + / - DHFR (CH0, Urlaub and Chasin, Proc. Natl. Acad. Sc1. USA, 77:42 16 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)), monkey kidney cells (CVI ATCC CCL70), African green monkey kidney cells (VER0-76, ATCC CRL-1587), human cervical carcinoma cells (HeLa, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad.Sc1., 383:44-68 (1982)), MRC 5 cells, FS4 cells and human hepatoma cell line (Hep G2), mouse neuronal cells N2A.

[0134] The isolated host cells are cultured under conditions that allow the expression of the isolated nucleic acid inserted into the vector. Suitable conditions for the expression of polynucleotides include, but are not limited to, an appropriate medium, an appropriate density of host cells in the medium, the presence of necessary nutrients, the presence of cofactors, appropriate temperature and humidity, and the absence of microbial contaminants. Those skilled in the art can select appropriate conditions for expression purposes and appropriately modify the steps and compositions of the invention to optimize cell growth and / or production of any given expressed polypeptide or protein.

[0135] Various methods of preparing host cells (e.g., mammalian cells) are known in the art. For example, sufficient nucleic acid for expression (generally a vector containing a gene encoding a polypeptide or protein of interest and any operably linked genetic control elements) may be introduced into a host cell line by any number of well-known techniques. Generally, the cells are screened to determine which host cells have actually acquired the vector and expressed the polypeptide or protein of interest. Routine methods for detecting a particular polypeptide or protein of interest expressed by a host cell include, but are 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. Those skilled in the art will know other suitable techniques for detecting expressed polypeptides or proteins. If multiple host cells express the polypeptide or protein of interest, some or all of the techniques can be used to determine which cell expresses the highest level of the polypeptide or protein.

[0136] The expressed polypeptide(s) and / or polypeptide complex(es) may be harvested using any suitable method. The expressed polypeptide(s) and / or polypeptide complex(es) may be expressed intracellularly, in the periplasmic space, or secreted into the extracellular medium. If the polypeptide(s) and / or polypeptide complex(es) are expressed intracellularly, the host cells containing the polypeptide(s) and / or polypeptide complex(es) may be lysed, and the polypeptide(s) and / or polypeptide complex(es) may be isolated from the lysate by centrifugation or ultrafiltration to remove undesired debris. If the polypeptide(s) and / or polypeptide complex(es) are secreted into the periplasmic space of E. coli, the cell paste may be thawed for about 30 minutes in the presence of agents such as sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF), and the cell debris may be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)). If the polypeptides and / or polypeptide complexes are secreted into the medium, the cell culture supernatant can be collected and concentrated using a commercially available protein concentration filter, such as an Amincon or Millipore Pellicon ultrafiltration device. Protease inhibitors and / or antibiotics may be included in the collection and concentration steps to inhibit protein degradation and / or the growth of contaminating microorganisms.

[0137] Polypeptides or proteins can be isolated and purified by standard methods, including but not limited to chromatography, gel filtration, centrifugation or differential solubility, ethanol precipitation, or other techniques available for protein purification (see Scopes, Protein Purification Principles and Practice, 2nd ed., Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), Protein Expression: A Practical Approach, Oxford University Press, 1999; and Deutscher, MP, Simon, M.1., Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997, all incorporated herein by reference). Specifically, in the case of immunoaffinity chromatography, proteins can be separated by binding them to an affinity column containing antibodies raised against the protein and immobilized on a solid support. Alternatively, affinity tags such as influenza coat sequence, polyhistidine, glutathione-S-transferase, etc., can be attached to the protein using standard recombinant techniques, allowing for easy purification by appropriate affinity columns. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepsin inhibitor, aprotinin, etc., can be added at any or all stages to reduce or eliminate peptide or protein degradation during purification. Protease inhibitors are particularly required when cells need to be lysed to isolate and purify the expressed polypeptide or protein. Those skilled in the art should recognize that a particular purification technique will vary depending on the characteristics of the polypeptide or protein being purified, the characteristics of the cells expressing the polypeptide or protein, and the composition of the medium for cell growth.

[0138] 4. Pharmaceutical Compositions and Kits The present invention further provides pharmaceutical compositions, which contain (i) any one of the isolated ApoE constructs described herein (e.g., any one of the amino acid sequences of SEQ ID NOs:12, 60, 61, and 89), fusion proteins, isolated ApoE polypeptides (e.g., any one of the amino acid sequences of SEQ ID NOs:13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO:33), isolated nucleic acids, vectors (e.g., an ApoE construct (e.g., full length ApoE2 or ApoE3) or a vector that neurospecifically expresses an ApoE polypeptide), or host cell, and (ii) a pharmaceutically acceptable carrier.

[0139] The actual dosage level of the active ingredient (i.e., the ApoE construct, ApoE polypeptide (e.g., ApoE C-terminal polypeptide), fusion protein or expression vector of the present invention) in the pharmaceutical composition may be varied to obtain an amount of active ingredient effective to achieve the following effect: achieve the desired therapeutic response for a particular patient, composition, and method of administration without causing toxicity to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the polypeptide or expression vector used in the present invention, the route of administration, the time of administration, the duration of treatment, other drugs, compounds and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0140] As used herein, pharma- ceutically acceptable carriers include diluents, adjuvants, excipients or vehicles, including, but not limited to, water, animal oils, vegetable oils, starch, glucose, lactose, sucrose, gelatin, sodium stearate, glyceryl monostearate, sodium chloride, skim milk powder, glycerin, propylene, propylene glycol, water, ethanol, etc. If necessary, the composition can also contain minor amounts of wetting or emulsifying agents or pH buffering agents. Typically, the physiologically acceptable carrier is an aqueous pH buffered solution. Acceptable carriers, excipients or stabilizers are non-toxic to exposed cells at the dosages and concentrations used and include the following: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol; hydrocarbyl p-hydroxybenzoates such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum proteins, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, lysine; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose, dextrins; chelating agents such as EDTA; tonicity adjusters such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or TWEEN (登録商標) , PLURONICS (登録商標) or non-ionic surfactants such as polyethylene glycol (PEG).

[0141] In some embodiments, 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 the pH ranges of about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0. In some embodiments, the pharmaceutical composition can also be made isotonic with blood by adding a suitable tonicity adjuster, such as glycerol.

[0142] Pharmaceutical compositions intended for in vivo administration are usually formulated as sterile, substantially isotonic pharmaceutical compositions, and are in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration. Sterilization can be easily achieved by filtration through a sterile filter membrane. In some embodiments, the composition is pathogen-free. For injection, the pharmaceutical composition may be in the form of a solution in a physiologically compatible buffer, such as Hank's solution or Ringer's solution. In addition, the pharmaceutical composition is in solid form and can be redissolved or suspended immediately before use. Lyophilized compositions are also included.

[0143] As used herein, the pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release formulation, etc. The polypeptide of the present invention can be prepared into a suitable dosage form depending on the specific mode and route of administration.

[0144] The pharmaceutical composition can be adapted to various modes of administration described herein, including, for example, systemic administration or local administration.In some embodiments, the pharmaceutical composition is formulated for intravenous administration or subcutaneous injection.In some embodiments, the pharmaceutical composition is formulated for local administration, such as local administration (e.g., intratumoral injection) to tumor site, or local administration (e.g., injection) to brain (e.g., cortex or hippocampus).

[0145] In some embodiments, the pharmaceutical composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous, intraperitoneal, or intracerebral injection. Typically, the injectable composition is a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizing agent and a local anesthetic (such as lignocaine) to reduce pain at the site of injection. Typically, the ingredients are supplied 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 ampule or sachet indicating the quantity of active agent. If administered by infusion, the composition can be dispensed into an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, an ampule containing sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0146] In some embodiments, the pharmaceutical composition is suitable for administration to humans. In some embodiments, the pharmaceutical composition is suitable for administration to rodents (e.g., mice, rats, rabbits) or non-human primates (e.g., 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 multi-use vial. In some embodiments, the pharmaceutical composition is contained in a dispersed state within a container. In some embodiments, the pharmaceutical composition is cryopreserved.

[0147] The present invention further provides a unit dosage form comprising any one of the above-mentioned isolated ApoE constructs, fusion proteins, isolated ApoE polypeptides, isolated nucleic acids, vectors, host cells, or compositions thereof (e.g., pharmaceutical compositions) described herein. The term "unit dosage form" refers to a physically discrete unit suitable as a unit dosage form for administration to an individual, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect, together with a suitable pharmaceutical carrier, diluent, or excipient. These unit dosage forms can be stored in suitable packaging, in single or multiple unit doses, and can be further sterilized and sealed.

[0148] The present application also provides articles of manufacture comprising the compositions (such as pharmaceutical compositions) described herein in suitable packaging. Suitable packaging for the compositions (such as pharmaceutical compositions) described herein is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (such as sealed Mylar or plastic bags), and the like. These articles can be further sterilized and / or sealed.

[0149] The present invention further provides a kit comprising any one of the isolated ApoE constructs, fusion proteins, isolated ApoE polypeptides, isolated nucleic acids, vectors, host cells, or pharmaceutical compositions described herein. In some embodiments, the kit further comprises one or more instructions on how to use 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 useful for carrying out any of the methods described herein.

[0150] 5. Treatment of diseases The present invention further provides the use of an isolated ApoE construct (e.g., any one of the amino acid sequences of SEQ ID NOs: 12, 60, 61 and 89), fusion protein, isolated ApoE polypeptide (e.g., any one of the amino acid sequences of SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78 and 87, e.g., SEQ ID NO: 33), isolated nucleic acid, vector (e.g., ApoE construct (e.g., full length ApoE2 or ApoE3) or vector expressing ApoE polypeptide in a neurospecific manner), host cell or pharmaceutical composition according to any one of the embodiments in the preparation of a medicament for treating or preventing a disease associated with the cleavage activity of gamma-secretase (e.g., gamma-cleavage activity against APP) in an individual (e.g., a human). In some embodiments, the medicament inhibits the cleavage activity of gamma-secretase (e.g., gamma-cleavage activity against APP) and treats or prevents a disease associated with the cleavage activity of gamma-secretase. In some embodiments, the present invention provides a method for treating or preventing a disease associated with the cleavage activity of gamma secretase (e.g., gamma cleavage activity against APP) in an individual (e.g., a human), comprising administering to the individual an effective dosage of any one of the isolated ApoE constructs (e.g., any one of the amino acid sequences in SEQ ID NOs: 12, 60, 61, and 89), fusion proteins, isolated ApoE polypeptides (e.g., any one of the amino acid sequences in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO: 33), isolated nucleic acids, vectors (e.g., ApoE constructs (e.g., full-length ApoE2 or ApoE3) or vectors expressing ApoE polypeptides in a neurospecific manner), host cells, or pharmaceutical compositions described herein. In some embodiments, the method inhibits the cleavage activity of gamma secretase (e.g., gamma cleavage activity against APP), and treats or prevents a disease associated with the cleavage activity of gamma secretase. In some embodiments, the disease associated with the cleavage activity of gamma-secretase is selected from a degenerative disease of the nervous system, a tumor or cancer, an inflammatory disease, and a kidney disease.In some embodiments, the disease is selected from Alzheimer's disease and related diseases, amyloid cerebrovascular disease, Down's syndrome, other age-related neurodegenerative diseases (such as frontotemporal dementia), head and neck cancer (e.g., 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 (e.g., non-small cell lung cancer, lung adenocarcinoma and small cell lung cancer), glioma (e.g., malignant glioma), fibroma, lymphoma (e.g., B cell lymphoma), osteosarcoma, gastric cancer, bladder cancer, asthma (e.g., allergic asthma), pneumonia, airway inflammation, acute kidney injury, clear cell renal cell carcinoma, renal fibrosis, and obstructive nephropathy. In some embodiments, the disease is AD and related diseases, such as sporadic Alzheimer's disease (sAD).

[0151] The pharmaceuticals, therapeutic or prophylactic methods provided herein are suitable for any individual, including but not limited to mammals, birds, frogs, fish, fruit flies, nematodes, etc. Mammals include but are not limited to primates (e.g., humans, or non-human primates, e.g., monkeys), rodents (e.g., mice, rats, hamsters, gerbils, squirrels, guinea pigs, rabbits), farm animals (e.g., pigs, cows, sheep, horses, donkeys), and pets (e.g., cats, dogs, rabbits, hamsters, etc.). In some embodiments, the individual is a mouse or a monkey. In some embodiments, the individual is a human.

[0152] As used herein, suitable modes and routes of administration are well known in the art and include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration such as injection or perfusion. "Parenteral administration" refers to a method of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, nasal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and perfusion. The terms "systemic administration" and "systemically administered" refer to a method in which an agent or composition described herein is administered to an individual (e.g., a mammal) and the agent or composition is delivered via the circulatory system to a site in the body, including the target site of drug action. Systemic administration includes, but is not limited to, oral, intranasal, rectal, and parenteral (eg, outside the digestive tract, such as intramuscular, intravenous, intraarterial, transdermal, and subcutaneous) administration.

[0153] In some embodiments, any one of the isolated ApoE constructs (e.g., any one of the amino acid sequences in SEQ ID NOs: 12, 60, 61, and 89), fusion proteins, isolated ApoE polypeptides (e.g., any one of the amino acid sequences in SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO: 33), isolated nucleic acids, vectors, or pharmaceutical compositions of the invention are introduced into the central nervous system via any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be assisted, for example, by an intraventricular catheter connected to a reservoir (e.g., an Ommaya reservoir).

[0154] In some embodiments, nucleic acid molecules encoding the polypeptides of the present invention, such as coding sequences of the polypeptides of the present invention, including RNA and DNA sequences, can be delivered using techniques well known in the art. In some embodiments, the coding sequences of the polypeptides of the present invention are delivered to a subject (e.g., a human) in need thereof in the form of an expression vector (e.g., using a viral vector such as an AAV or lentiviral vector). In some embodiments, liposomes are used to deliver the DNA or RNA molecules described herein that can encode the polypeptides of the present invention. Thus, in these embodiments, the pharmaceutical compositions of the present invention include liposomes, DNA or RNA molecules encoding the polypeptides of the present invention described herein, and their expression vectors. Liposomes suitable for use in the present invention are well known in the art and may be liposomes suitable for administration to humans.

[0155] The ApoE constructs (e.g., full-length ApoE2 or ApoE3), fusion proteins, or ApoE polypeptides provided herein are inhibitors of γ-secretase (e.g., specifically inhibit γ-cleavage of APP), and thus the polypeptides of the present invention or pharmaceutical compositions thereof may be used to treat or prevent diseases that benefit from the inhibition of γ-secretase or diseases in which targeting the inhibition of γ-secretase is effective in treating or preventing (also referred to herein as γ-secretase-mediated diseases or diseases associated with the enzymatic cleavage activity of γ-secretase). Several diseases are known to be mediated by γ-secretase, including neurodegenerative diseases such as Alzheimer's disease and related diseases and amyloid cerebrovascular disease (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 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));Head and neck squamous cell carcinoma and oral squamous cell carcinoma (Liang Mao et al., γ-Secretase inhibitor reduces immunosuppressive cells and enhances tumour immunity in head and neck squamous cell carcinoma, Int. J. Cancer:142, 999-1009(2018)), breast cancer and hepatocellular carcinoma (Hui Jia et al., γ-Secretase inhibitors for breast cancer and hepatocellular carcinoma:From mechanism to treatment, Life Sciences 268(2021)119007), pancreatic cancer (including metastatic pancreatic cancer) (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. 2014 August; 32(4):739-745), ovarian cancer (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), Lung cancer (non-small cell lung cancer, lung adenocarcinoma, small cell lung cancer, etc.) (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 et al., Gamma secretase inhibition by BMS-906024 enhances efficacy of paclitaxel in lung adenocarcinoma, Mol Cancer Ther. 2017 December; 16(12):2759-2769), and gliomas such as malignant gliomas (Edward Pan et al., Phase I Study of RO4929097 with Bevacizumab in Patients with Recurrent Malignant Glioma, Neurooncol. 2016 December;130(3):571-579), fibroma (Shivaani Kummar et al., 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), lymphomas such as B-cell lymphoma (Shuji Tohda, Establishment of a novel B-cell lymphoma cell line with suppressed growth by gamma-secretase inhibitors, Leukemia Research 30(2006)1385-1390), gastric cancer (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 tumors or cancers, including bladder cancer (YIBING WANG et al., γ-secretase inhibitor inhibits bladder cancer cell drug resistance and invasion by reducing epithelial-mesenchymal transition, MOLECULAR MEDICINE REPORTS 12:2821-2827, 2015), and osteosarcoma (M Tanaka et al., Inhibition of Notch pathway prevents osteosarcoma growth by cell cycle regulation, British Journal of Cancer (2009) 100, 1957 - 1965);Asthma, such as allergic asthma (Weixi Zhang et al., 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), airway inflammation, such as allergic airway inflammation (Weixi Zhang et al., 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), pneumonia (Jin Hyun Kang et al., γ-Secretase Inhibitor Reduces Allergic Pulmonary Inflammation by Modulating Th1 and Th2 Responses, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 179, 2009); renal diseases such as acute kidney injury (Jean-Christophe Wyss et al., Targeted γ-secretase inhibition of Notch signaling activation in acute renal injury, Am J Physiol Renal Physiol 314:F736-F746, 2018);

[0156] In some embodiments, the disease associated with the cleavage activity of gamma secretase (e.g., gamma cleavage activity against APP) is Alzheimer's disease (AD), dementia with Lewy bodies (DLB), mild cognitive impairment (MCI), frontotemporal dementia (FTD), cerebral amyloid angiopathy (CAA), CAA-associated cerebral hemorrhage, and other neurodegenerative diseases, vascular cognitive impairment, Parkinson's disease (PD), multiple sclerosis (MS), traumatic brain injury (TBI), or fragile X syndrome-associated tremor / ataxia syndrome. CAA is a condition characterized by the deposition of proteins (mainly amyloid beta) in the walls of cerebral blood vessels. CAA is common in AD patients, but can occur without evidence of AD.

[0157] In some embodiments, the disease associated with the cleavage activity of gamma-secretase (e.g., gamma-cleavage activity against APP) is "amyloid protein β-related disease", which refers to any condition characterized by abnormally high levels of amyloid protein β peptide production, accumulation, and / or aggregation, particularly in the brain, for example in the form of amyloid plaques.Amyloid protein β-related diseases include AD, Down syndrome, fragile X syndrome, CAA, CAA-associated cerebral hemorrhage, and sporadic inclusion body myositis.

[0158] AD is the most common neurodegenerative disease in developed countries. Histopathologically, AD is characterized by the accumulation of amyloid protein plaques containing Aβ peptides and NFTs derived from tau protein. Clinically, AD is associated with progressive cognitive impairment characterized by loss of memory, function, language ability, judgment, and executive function. AD often causes severe behavioral symptoms in its later stages.

[0159] Approximately half of cases of early-onset Alzheimer's disease (diagnosed before age 65) are familial Alzheimer's disease (FAD), which is inherited in an autosomal dominant manner. FAD is caused by at least one mutation in three genes: APP mutations, PSENI mutations, or PSEN2 mutations. Amyloid precursor protein (APP) mutations have been identified in 121 families. The most common missense mutation is Val 717The residue was replaced with Ile (London mutation), Phe, or Gly residues, and Glu 693 to a Gln residue (Dutch mutation) or a Gly residue (Arctic mutation), or to Lys 670 and Met 671 to Asn and Leu residues (the double mutation is called the Swedish mutation and is 770 The 219 presenilin 1 (PSEN1) variants have been identified in 480 families, which show attenuated γ-secretase cleavage, increased oxidative stress in some mutations, and increased neuronal susceptibility to DNA damage-induced death. The 13 presenilin 2 (PSEN2) variants have been identified in 34 families, which show attenuated γ-secretase cleavage, increased oxidative stress in some mutations, and increased neuronal susceptibility to DNA damage-induced death.

[0160] In some embodiments, any one of the isolated ApoE constructs (e.g., any one of the amino acid sequences of SEQ ID NOs: 12, 60, 61, and 89), fusion proteins, isolated ApoE polypeptides (e.g., any one of the amino acid sequences of SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO: 33), isolated nucleic acids, vectors (e.g., ApoE constructs (e.g., full length ApoE2 or ApoE3) or vectors that neurospecifically express ApoE polypeptides), host cells, or pharmaceutical compositions provided herein are used to treat any disease associated with APP γ-cleavage (e.g., AD), such as any disease having an APP mutation (e.g., AD), by inhibiting γ-secretase enzymatic activity. In some embodiments, the APP is human APP and comprises one or more of the following mutations: K670N, M671L, Swedish (K670N+M671L), Florida (I716V), London (V717I), APP M596V(beta cleavage is not feasible), fAD mutations close to the gamma cleavage site (e.g., T714I, V717F, L723P), fAD mutations close to the beta or alpha cleavage site (e.g., KM670 / 671NL, E693K, E693Δ), or the protective Iceland mutation (A673T).

[0161] Several behavioral and histopathological tests are known in the art for assessing Alzheimer's disease phenotypes, characterizing therapeutic agents, and evaluating treatments. Histological analysis is usually performed postmortem. Histological analysis of Aβ levels is performed using thioflavin S. Congo red or anti-Aβ staining (e.g., 4G8, 10D5, or 6E10 antibodies) visualizes Aβ deposits 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). An in vivo method for visualizing Aβ deposits in genetically modified mice is also described. BSB ((trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene) and the PET tracer 11C-labeled Pittsburgh compound B (PIB) bind to Aβ plaques (see, e.g., Skovronsky et al., 2000, Proc. Natl. Acad. Sci. USA 97:7609-7614; Klunk et al., 2004, Ann. Neurol. 55:306-319). 19 The F-containing amyloidophilic Congo red-type compound FSB ((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene) allows visualization of Aβ plaques by MRI (see, e.g., Higuchi et al., 2005, Ature Neurosci. 8:527-533). Radioactively marked putrescine-modified amyloid β peptides also mark amyloid deposits in vivo in mouse models of Alzheimer's disease (see, e.g., Wengenack et al., 2000, Nat. Biotechnol. 18:868-872).

[0162] Standard diagnostic methods known in the art for Alzheimer's disease can be used to identify test subjects suffering from Alzheimer's disease. Typically, a diagnosis of Alzheimer's disease is made based on the patient's symptoms (e.g., progressive decline in memory function, progressive abandonment and frustration of normal activities, apathy, restlessness or irritability, aggression, anxiety, sleep disorders, agitation, abnormal motor behavior, disinhibition, social withdrawal, loss of appetite, hallucinations, dementia), medical history, neuropsychological testing, neurological and / or physical examinations. Multiple proteins associated with Alzheimer's disease can also be detected in cerebrospinal fluid, including tau, amyloid beta peptide and AD7C-NTP. Genetic testing may also be used for early-onset familial Alzheimer's disease (eFAD), an autosomal dominant inherited disorder. Clinical genetic testing is available for individuals with AD symptoms or for family members at risk for early-onset disease. In the United States, PS2 mutations and APP can be tested for in clinical laboratories or federally approved clinical laboratories under the Clinical Testing Improvement Amendments. A commercial test for PS1 mutations is also available (Elan Pharmaceuticals).

[0163] The therapeutic effect of the present invention can be evaluated by any of the methods or indicators described above. In some embodiments, the ApoE constructs, ApoE polypeptides, vectors, compositions, or treatment / prevention methods of the invention can achieve one or more of the following effects: (i) control cleavage of APP in neurons (in vivo or ex vivo) in a cell-autonomous manner; (ii) inhibit (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the enzymatic activity of γ-secretase, e.g., inhibiting γ-secretase-mediated γ-cleavage of APP, e.g., similar to the effect of inhibiting APP γ-cleavage by γ-secretase inhibitors (e.g., PF03084014); (iii) reduce (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the amyloid metabolism of APP, e.g., reducing amyloid protein (iv) reduce (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the production of β (Aβ) (e.g., Aβ40 and / or Aβ42) or reduce γ-cleavage to C-terminal fragment β (β-CTF) (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); (v) improve (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more) the non-amyloid metabolism of APP, e.g., improve (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more) the production of C-terminal fragment β (Aβ) (e.g., Aβ40 and / or Aβ42); (v) increase the production of α (α-CTF) (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more); (v) do not affect (or affect only about 30%, 20%, 10%, 5% or less) the mRNA and / or protein expression of α-secretase, β-secretase, and / or γ-secretase (or subunits thereof) (e.g., ADAM10, BACE1, PS1 and / or PS2);(vi) does not affect (or affects only about 30%, 20%, 10%, 5% or less) APP β-cleavage mediated by β-secretase and / or α-secretase mediated APP α-cleavage; (vii) does not inhibit (or inhibits only about 30%, 20%, 10%, 5% or less) γ-cleavage of non-APP proteins (e.g., APLP1 (APP like protein 1) or Notch) or fragments thereof, and does not increase (or increases only about 30%, 20%, 10%, 5% or less) α-CTFs produced by γ-cleavage of non-APP proteins; (viii) reduces amyloid plaques in the brain (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction) or prevention, e.g., reducing the average plaque size, total area, and / or density of amyloid plaques (e.g., reducing by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), reducing the production of Aβ (e.g., Aβ40 and / or Aβ42) in neurons (e.g., reducing by at least about 10%, 20%, 30%, 40%, , 50%, 60%, 70%, 80%, 90%, 95%, or 100%) or prevent AD; slow (e.g., 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 processes; improve (e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 2-fold, 5-fold, 10-fold or more) individual survival rate and / or lifespan; , 90%, 95%, 100%, 2-fold, 5-fold, 10-fold, 20-fold or more); (ix) the ApoE constructs described herein expressed locally in neurons (e.g., expressed in the cortex, dentate gyrus (DG), hippocampal CA3) migrate from distant sites to the subiculum and accumulate around amyloid plaques, thereby locally inhibiting γ-secretase enzymatic activity in these amyloidogenic hotspots;or (x) reduce (e.g., reduce by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), eliminate, or prevent (including delay in time of onset) one or more symptoms of AD (e.g., progressive decline in memory function, progressive abandonment of usual activities and frustration, apathy, restlessness or irritability, aggression, anxiety, sleep disorders, agitation, abnormal motor behavior, disinhibition, social withdrawal, loss of appetite, hallucinations, dementia);

[0164] In some embodiments, the cancer associated with the cleavage activity of gamma-secretase includes one or more of the following: head and neck cancer (e.g., 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 (e.g., non-small cell lung cancer, lung adenocarcinoma and small cell lung cancer), glioma (e.g., malignant glioma), fibroma, lymphoma (e.g., B cell lymphoma), osteosarcoma, gastric cancer, and bladder cancer.

[0165] In some embodiments, the medicaments or methods of treating cancer associated with gamma-secretase cleavage activity described herein have one or more of the following biological activities: (1) killing cancer cells, e.g., at least about any of 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more tumor cell death rates; (2) inhibiting cancer cell proliferation, e.g., inhibiting proliferation by at least about 10% (e.g., at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%); (3) reducing tumor size, e.g., at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100% reduction in tumor size; (4) alleviating one or more symptoms in a cancer patient; (5) inhibiting tumor metastasis (e.g., metastasis to lymph nodes), e.g., inhibiting tumor metastasis by at least about 10% (including, e.g., at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%); (6) prolonging survival, e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months or more; (7) preventing cancer or prolonging the time to cancer progression, e.g., prolonging the time to cancer progression by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more; and (8) preventing, inhibiting, or reducing cancer recurrence.

[0166] In some embodiments, the disease associated with the enzymatic cleavage activity of gamma-secretase is an inflammatory disease, including, but not limited to, asthma (such as allergic asthma), pneumonia, and airway inflammation. In some embodiments, the pharmaceutical or therapeutic / prophylactic methods described herein reduce (e.g., reduce by at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%) the secretion and / or activity of cytokines that cause inflammation, and / or reduce (e.g., reduce by at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%), eliminate or prevent (including delay in onset) one or more disease symptoms.

[0167] In some embodiments, the disease associated with the cleavage activity of gamma-secretase is a kidney disease, which refers to any disease, disorder or condition that affects the kidney or renal system. Examples of kidney-related diseases or conditions include, but are not limited to, acute kidney disease (or renal failure), clear cell renal cell carcinoma, renal fibrosis, or obstructive nephropathy. Conventional markers for evaluating and diagnosing kidney disease include GFR, creatinine, and albumin, and any marker or related method can be used to evaluate the therapeutic effect of the present invention. In some embodiments, the pharmaceutical or therapeutic / prophylactic method described herein can (i) reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%), eliminate, or prevent (including delay the onset time) one or more kidney disease indicators; and / or (ii) protect or improve kidney function.

[0168] The present invention provides a method for treating or preventing a gamma-secretase mediated disease in a subject (e.g., a human), comprising administering to the subject a therapeutically or prophylactically effective amount of any one of the ApoE constructs, fusion proteins, ApoE polypeptides, nucleic acid molecules, vectors, or pharmaceutical compositions of the present invention. The effective amount can be determined by one of skill in the art based on factors such as the subject's age, sex, weight, condition, general health, and previous medical history. The disease preferably includes the nervous system degenerative diseases, tumors or cancers, inflammatory diseases, and kidney diseases.

[0169] Some embodiments of the present invention provide a method of inhibiting amyloid metabolism of APP and reducing Aβ production by inhibiting γ-secretase cleavage, the method comprising producing ApoE2 (e.g., ApoE2 having a membrane-permeable peptide at the N-terminus) or an ApoE construct (e.g., a C-terminal fragment of ApoE2, or full-length ApoE2 or ApoE3) as described in any one of the embodiments herein, a fusion protein, or an ApoE polypeptide (e.g., any one of the amino acid sequences of SEQ ID NOs: 13, 18-34, 46-52, 57-59, 62, 76, 78, and 87, e.g., SEQ ID NO: 33) in a cell (e.g., a neuron) expressing γ-secretase. In some embodiments, the cell is a neuron. In some embodiments, the methods are used to delay (e.g., at least about any of 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%), prevent or treat the onset and progression of AD and amyloid cerebrovascular disease. As described herein, producing includes expressing a polypeptide and delivering the polypeptide from outside the cell to inside the cell. In some embodiments, the ApoE construct (e.g., ApoE2 and / or ApoE3 and / or C-terminal fragments thereof) or ApoE polypeptide-encoding DNA or RNA is delivered to the cell by liposomes to express and produce the ApoE construct or ApoE polypeptide in the cell; or the ApoE construct or ApoE polypeptide is produced in the cell by infecting the cell using lentivirus and adeno-associated virus; or the ApoE construct or ApoE polypeptide is entered into the cell by cell-penetrating peptides or enhanced cellular endocytosis.

[0170] In some embodiments, the polypeptides referred to in the ApoE constructs or fusion proteins, uses and methods described in any one of the embodiments herein include, in particular, the polypeptides set forth in any one of SEQ ID NOs: 12, 13, 18-33 and 89. EXAMPLES

[0171] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely illustrative of the present invention and do not limit the scope of the present invention. It should be understood that based on the disclosure of the present invention, a person skilled in the art may make various changes or modifications to the present invention, and these equivalent forms are also included in the scope defined in the claims appended to this application.

[0172] In the examples, data are analyzed using GraphPad Prism software, and all data are presented as mean±SEM. The following data analysis uses unpaired, paired or one sample t-test. Significant differences are indicated with an asterisk (*), where one asterisk (*) represents a P value of less than 0.05, two asterisks (**) represent a P value of less than 0.01, three asterisks (***) represent a P value of less than 0.001, and four asterisks (****) represent a P value of less than 0.0001.

[0173] Example 1 1. Main drugs and materials The HEK 293T cell line was cultured in DMEM medium (HyClone) containing 10% fetal bovine serum (FBS) (Life) and 1% penicillin / streptomycin and grown in a sterile cell culture incubator (ThermoFisher) at 37°C with 5% CO2.

[0174] Primary cultured neurons were derived from the cortex or hippocampus of 16-day-old mouse embryos. Mouse cortex or hippocampus was harvested under a stereoscope; digested with 20 U / ml papain (Worthington) for 10 min at 37°C; after digestion, neurons present in the form of single cells were plated at 100,000 neurons per well (24-well plate); neurons were cultured in neurobasal medium (ThermoFisher) containing 2% B27 (ThermoFisher) and 1% penicillin / streptomycin; fresh medium was replaced once a week.

[0175] C57 wild-type mice and AD model mice aged 0–7 months (5×FAD mice [Tg6799], mice expressing APP and PS1 containing pathogenic mutations; expressed APP contains three mutations K670N / M671L, I716V, and V717I, while PS1 contains two mutations M146L, L286V) were used. Mice were provided with adequate food and water, housed at room temperature on a 12 h / 12 ​​h light / dark cycle, and the housing environment met the SPF (specific pathogen-free) level criteria.

[0176] Enzyme-linked immunosorbent assay kits (ELISA) were purchased from Invitrogen; human Aβ40 (KHB3481), human Aβ42 (KHB3441), and mouse Aβ40 (KMB3481).

[0177] The transfection reagents are lipofectamine2000 from ThermoFisher and Polyjet from SignaGen.

[0178] Quantitect SYBR-Green qRT-PCR kit for qRT-PCR was purchased from Qiagen company.

[0179] [Table 6]

[0180] 2. Experimental Method 2.1 Protein sample preparation and immunoblotting (Western Blot) a) A plasmid is constructed to express a protein of interest in mammalian cells, and the protein expression promoter is CAG.

[0181] b) The target protein was expressed in the HEK 293T cell line. One day before transfection, the cells were passaged, and transfection was performed when the cell adhesion rate reached 70% to 80%. The transfection reagent was lipofectamine2000 (ThermoFisher).

[0182] c) 4–6 hours after transfection, the cell culture medium was replaced with fresh medium.

[0183] d) 36–48 hours after transfection, cells were lysed (10% 1M tris-HCl (pH=6.8), 4% SDS, 20% glycerol, 0.01% bromophenol blue) and protein samples were collected and used for immunoblotting experiments or frozen in a -80°C refrigerator.

[0184] e) Polyacrylamide gel electrophoresis. Proteins were boiled at 98°C for 10 min before loading and then centrifuged at 12,000 rpm for 10 min. Electrophoresis: top stacking gel 80V; bottom separating gel 120V.

[0185] f) Transfer onto membrane. Before use, the filter paper was immersed in an electric transfer solution, and the PVDF membrane was hydrophilized in methanol for 10 seconds. After electrophoresis, the gel was removed from the electrophoresis tank, and the filter paper, PVDF membrane, gel, and filter paper were placed in this order from bottom to top, and the gel was placed in a membrane transfer device, where the membrane was transferred at a constant flow rate of 0.1 A for 70 minutes.

[0186] g) Blocking and antibody incubation: The transferred PVDF membrane was placed in 5% BSA / TBST blocking solution and blocked for 1 hour at room temperature on a shaker. Primary antibodies were incubated overnight (12-16 hours) at 4°C on a shaker; washed 3 times with 1x TBST, 10 minutes each; secondary antibodies were incubated 1-2 hours at room temperature on a shaker; washed 3 times with 1x TBST, 10 minutes each.

[0187] h) Development and imaging: Developed with ECL chemiluminescent ultrasensitive color reagent.

[0188] 2.2 Brain slice preparation and immunohistochemistry a) Perfusion and brain harvest. Mice were perfused transcardially, first flushing the blood with 20 ml of PBS, and then perfused with 20 ml of 4% PFA.

[0189] b) Fixation and dehydration. First, the tissue was fixed in 4% PFA at 4°C for 24 hours, and then dehydrated in 30% sucrose at 4°C.

[0190] c) Slicing of mouse brains. After dehydration for 24 h, mouse brains were cut into 40 μm thick brain slices and either stored in freezing buffer or used for subsequent immunohistochemistry.

[0191] d) THS staining. Washed 3 times with ddH2O for 5 min each time; soaked in 1% THS aqueous solution for 10 min; washed twice with 80% ethanol for 3 min each time; washed once with 95% ethanol for 3 min. Subsequent immunohistochemistry was performed. If THS staining was not performed, proceeded directly to the next immunohistochemistry.

[0192] e) Immunohistochemistry. Washed 3 times with 1x PBS for 10 min each time; disrupted cell membrane with 0.1% Triton-X100; blocked with 5% donkey or goat serum; then incubated with primary antibody overnight (12-16 h) at 4°C on a shaker; washed 3 times with 1x PBS for 10 min each time; incubated with secondary antibody for 1-2 h at room temperature on a shaker; washed 3 times with 1x PBS for 10 min each time.

[0193] f) Patching and sealing.

[0194] g) Developed 24 hours after sealing.

[0195] h) Images were processed and signal density analyzed using ImageJ.

[0196] 2.3 Enzyme-linked immunosorbent assay (ELISA) The experiment was carried out according to the standard procedure of the ELISA analysis kit of Invitrogen company.

[0197] a) The target protein was expressed in HEK 293T cell line. One day before transfection, the cells were passaged, and transfection was performed when the cell adhesion rate reached 70%-80%. The transfection reagent was lipofectamine2000 (ThermoFisher).

[0198] b) 4–6 hours after transfection, the medium was replaced with fresh medium.

[0199] c) 36-48 hours after transfection, the cell culture fluid was collected and either subjected to ELISA process or frozen in a -80℃ refrigerator.

[0200] d) 50 μl of standard and experimental samples were added to each well of a 96-well plate.

[0201] e) 50 μl of human Aβ40 or Aβ42 or mouse Aβ40 detection antibody was added to each well of a 96-well plate.

[0202] f) Shake gently and leave at room temperature for 3 hours.

[0203] g) The liquid was removed from the wells and washed 4 times with 1× wash buffer.

[0204] h) 100 μl of 1× anti-rabbit IgG HRP in buffer was added.

[0205] i) Shake gently and leave at room temperature for 30 minutes.

[0206] j) The liquid was removed from the wells and washed 4 times with 1× wash buffer.

[0207] k) 100 μl of stabilized chromogen was added to each well and placed in the dark at room temperature for 30 minutes.

[0208] l) 100 μl of Stop Solution was added to each well.

[0209] m) The absorbance at 450 nm was measured using a microplate reader.

[0210] 2.4 qRT-PCR TRIzol (商標) RNA was extracted from the treated cells according to the instructions of the reagent (ThermoFisher).

[0211] a) The target protein was expressed in HEK 293T cell line. One day before transfection, the cells were passaged, and transfection was performed when the cell adhesion rate reached 70%-80%. The transfection reagent was lipofectamine2000 (ThermoFisher).

[0212] b) Six hours after transfection, the medium was replaced with fresh medium.

[0213] c) 48 hours after transfection, RNA was extracted.

[0214] d) 500 μl of Trizol (ThermoFisher Company) was added to each well of the 24-well plate, shaken and incubated at room temperature for 20 min.

[0215] e) The cell suspension was collected into a centrifuge tube.

[0216] f) 250 μl of chloroform was added, mixed by inversion, and allowed to stand at room temperature for 3 minutes.

[0217] g) Centrifuge at 12000g for 15 minutes at 4°C. After centrifugation, the solution appeared to be clearly layered, with chloroform containing phenol (pink color) at the bottom, DNA and protein in the middle, and aqueous phase containing RNA (clear) at the top. The supernatant was transferred to a new centrifuge tube.

[0218] h) An equal volume (supernatant) of isopropyl alcohol was added, mixed by inversion, and allowed to stand at room temperature for 20 minutes.

[0219] i) Centrifuge at 12000g for 15 minutes at 4°C. The supernatant was removed and a small amount of white precipitate (RNA) was visible at the bottom.

[0220] j) 400 μl of 75% ethanol was added. Centrifugation was performed at 4° C. and 12000 g for 5 minutes.

[0221] k) Remove as much ethanol as possible and dry thoroughly in a fume hood, but avoid over-drying. Add an appropriate amount of DEPC water.

[0222] l) After removing the DNA, the mixture was stored at -80°C for the next experiment.

[0223] The relative abundance of mRNA was quantitatively analyzed with the Quantitect SYBR-Green qRT-PCR kit from Qiagen. The qRT-PCR primers are listed in Table 2.

[0224] [Table 7]

[0225] 2.5 Construction of PS1 KO, PS2 KO and PS1 / 2 dKO cell lines In this study, we selected HEK 293T cells and used CRISPR / CAS9 technology to construct PS1 KO, PS2 KO and PS1 / 2 dKO cell lines according to the standard procedures published by Zhang Feng et al., 2013 ( Ran et al., 2013 ). As shown in Table 3 , sgRNAs that specifically knocked out PS1 and PS2 were designed using the https: / / zlab.bio / guide-design-resources website.

[0226] a) PS2 sgRNA and control empty plasmid were expressed in HEK 293T cells. One day before transfection, cells were passaged and transfection was performed when the cell adhesion reached 70%-80%. The transfection reagent was lipofectamine2000 (ThermoFisher).

[0227] b) Six hours after transfection, the medium was replaced with fresh medium.

[0228] c) 18 hours after transfection, the medium was replaced with fresh medium containing 3 μg / ml puromycin (Select Company).

[0229] d) After all the HEK 293T cell lines transfected with empty plasmid were killed under the action of 3 μg / ml puromycin, the HEK 293T cells transfected with PS2 sgRNA were collected.

[0230] e) HEK 293T cells transfected with PS2 sgRNA were passaged at a ratio of 1:400 onto a 10 cm cell culture dish, and it was confirmed that the cells proliferated in a monoclonal manner.

[0231] f) After monoclonal expanded cells grew into cell clusters, single cell clusters were collected and passaged into 12-well plates.

[0232] g) After the cell mass covered the whole 12-well plate, the cells were harvested. Half of the cells were used to extract DNA and protein to detect the DNA sequence and protein expression of PS2. Another part of the cells was used for passage.

[0233] h) Cells in which the PS2 DNA sequence has been edited so that they no longer express the PS2 protein are called PS2 knockout cell lines - PS2 KO cell lines.

[0234] i) The present invention constructed a PS1 / PS2 dKO cell line based on the PS2 KO cell line. Furthermore, the PS1 sgRNA or control empty plasmid was expressed in the PS2 KO cell line; steps a-g were carried out to detect the DNA sequence and protein expression of PS1.

[0235] j) The PS2 KO cell line, in which the PS1 DNA sequence has been edited and no longer expresses the PS1 protein, is a PS1 and PS2 double knockout cell line--PS1 / 2 KO cell line.

[0236] [Table 8]

[0237] 2.6 Virus preparation and injection. 2.6.1 Preparation of lentivirus a) We constructed lentiviral expression plasmids, lentiviral GFP and lentiviral ApoE CT, which can be specifically expressed in neurons.

[0238] b) The lentivirus expression vector was co-transfected with the capsid protein vector (PSPAX2 and PMD2G) into HEK 293T cell line. One day before transfection, the cells were passaged and transfection was performed when the cell adhesion reached 70%-80%. The transfection reagent was Polyjet (SignaGen).

[0239] c) 36-48 hours after transfection, cell culture fluid was collected and filtered through a 0.22 μm filter.

[0240] d) The filtered cell culture fluid was ultracentrifuged at 20,000 rpm for 2 hours and 5 minutes at 4°C.

[0241] e) After centrifugation, the supernatant was removed and the pellet was resuspended in 50 μl of PBS per three 10 cm cell culture medium.

[0242] f) The resuspended virus was individually packaged and stored in a -80°C refrigerator for further experiments.

[0243] 2.6.2 Preparation of adeno-associated virus Adeno-associated viruses AAV-CT (AAV2 / 9-hSyn-ApoE CT) and AAV-GFP (AAV2 / 9-hSyn-EGFP-WPRE-pA) expressed in neurons were purchased from Teltu Biotechnology company.

[0244] 2.6.3 Virus injection Using a stereotaxic syringe, 1 μl of the adeno-associated virus prepared above was injected into the cerebral cortex (x, y, z, + / -1.6, 0, -1) and hippocampus (x, y, z, + / -1.5, -2, -1.5) of 1-month-old mice.

[0245] 2.7 Protein purification 2.7.1 Purification of γ-secretase For the expression vector of human γ-secretase, see Lu et al., 2014.

[0246] a) Human γ-secretase was expressed in the HEK 293T cell line (four proteins, human γ-secretase, NCT, PS1, PLAG-PEN2, and APH, were simultaneously expressed using the same expression plasmid). One day before transfection, the cells were passaged, and transfection was performed when the cell adhesion reached 70%-80%. The transfection reagent was lipofectamine2000 (ThermoFisher).

[0247] b) 4–6 hours after transfection, the cell culture medium was replaced with fresh medium.

[0248] c) 48 hours after transfection, the cells were washed once with 1x ice-cold PBS, scraped off with a cell scraper, and collected by centrifugation at 800g for 5 minutes.

[0249] d) Resuspended cells were lysed in lysis buffer [25 mM HEPES, pH 7.4; 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.05% (v / v) Tween-20, 1x cocktail].

[0250] e) Lysed cells were sonicated for 20 cycles (3 seconds on, 3 seconds off) at 45% power.

[0251] f) Centrifuge at 3000 g for 10 minutes at 4°C and collect the supernatant in a new centrifuge tube.

[0252] g) Centrifuge at 15,000 g and 4° C. for 1 hour and discard the supernatant.

[0253] h) The precipitate was resuspended in 1 ml of dissolution buffer, and after resuspension, 1% CHAPSO was added and incubated at 4° C. for 2 hours. The mixture was centrifuged at 15,000 g for 30 minutes, and the supernatant was collected in a new centrifuge tube.

[0254] i) 100 μl of FLAG beads (SIGMA) was added to the supernatant and incubated at 4° C. for 2 hours.

[0255] j) Wash three times with the following buffer: 25 mM HEPES, pH 7.4; 150 mM NaCl, 0.1% digitonin.

[0256] k) Human γ-secretase was eluted with the following buffer: 25 mM HEPES, pH 7.4; 150 mM NaCl, 0.1% digitonin and 500 μg / ml 3xFLAG peptide (SIGMA).

[0257] l) Purified human γ-secretase was detected by Western blot and stored at -80°C.

[0258] 2.7.2 Purification of ApoE a) ApoE2 and ApoE2 NT were expressed in HEK 293T cell line. ApoE2 protein and ApoE2NT protein contain fusion-expressed FLAG. One day before transfection, cells were passaged, and transfection was performed when the cell adhesion reached 70%-80%. The transfection reagent was lipofectamine2000 (ThermoFisher).

[0259] b) 4–6 hours after transfection, the cell culture medium was replaced with fresh medium.

[0260] c) 48 hours after transfection, the cells were washed once with 1x ice-cold PBS, scraped off with a cell scraper, and collected by centrifugation at 800g for 5 minutes.

[0261] d) The resuspended cells were lysed with lysis buffer [25 mM HEPES, pH 7.4; 150 mM NaCl, 1× cocktail].

[0262] e) Lysed cells were sonicated for 20 cycles (3 seconds on, 3 seconds off) at 45% power.

[0263] f) To obtain the supernatant containing the protein, the mixture was centrifuged at 12000g for 15 minutes at 4°C, and the supernatant was collected in a new centrifuge tube.

[0264] g) The protein-containing supernatant was incubated with anti-FLAG M2 affinity resin (SIGMA) for 90 minutes at 4° C. After incubation, ApoE2 and ApoE2 NT were transferred to the anti-FLAG M2 affinity resin.

[0265] h) The anti-FLAG M2 affinity resin containing ApoE2 and ApoE2 NT was washed three times with the following buffer: 25 mM HEPES, pH 7.4; 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.05% (v / v) Tween-20.

[0266] i) ApoE2 and ApoE2 NT were eluted with the following buffer: 25 mM HEPES, pH 7.4; 150 mM NaCl and 500 μg / ml 3xFLAG peptide (SIGMA).

[0267] j) Purified ApoE2 and ApoE2 NT were detected by Western blot and stored at -80°C.

[0268] 2.7.3 Purification of β-CTF a) β-CTF was expressed in E. coli and the expression plasmid was pET22 β-CTF-Myc-6xHis.

[0269] b) After induction with 0.5 mM IPTG and expression at 37°C for 4 hours, E. coli cells were lysed in the following lysis buffer: 30 mM Tris-HCl, pH 7.5; 150 mM NaCl, 1 mM EDTA.

[0270] c) Centrifuge at 25,000 g for 10 minutes at room temperature. The supernatant was removed.

[0271] d) The precipitate was resuspended in the following buffer and mixed slowly at room temperature: 30 mM Tris-HCl, pH 8.0; 300 mM NaCl, 1 mM EDTA, 6 M urea, 1% TritonX-100, 1 mM CaCl2, 1× cocktail.

[0272] e) The mixture was centrifuged at 40,000 g for 30 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.45 mM filter membrane.

[0273] f) The filtered supernatant was incubated with Ni-NTA beads for 2 hours at room temperature, then the incubated Ni-NTA beads and the supernatant were simultaneously loaded into an empty chromatography column and allowed to drain by gravity. The flow-through liquid was collected and once again combined with the beads.

[0274] g) Wash the Ni-NTA beads with 10x the Ni-NTA bead column volume as follows: 30 mM Tris-HCl, pH 8.0; 300 mM NaCl, 1 mM EDTA, 6 M urea, 1% TritonX-100, 1 mM CaCl2, 1× cocktail.

[0275] h) Wash the Ni-NTA beads with 20 Ni-NTA bead column volumes as follows: 30 mM Tris-HCl, pH 8.0; 300 mM NaCl, 0.1% Trion X-100.

[0276] i) Wash the Ni-NTA beads with 20 Ni-NTA bead column volumes as follows: 50 mM Tris-HCl, pH 8.0; 300 mM NaCl, 0.1% CHAPSO.

[0277] j) β-CTF was eluted with the following buffer: 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% CHAPSO, 300 mM imidazole.

[0278] k) Purified β-CTF was detected by Western blot and stored at -80°C.

[0279] 2.7.4 Purification of GFP The purification method for GFP was the same as that for ApoE in 2.7.2.

[0280] 3. Experimental Results 3.1 Effects of the three ApoE isoforms and various ApoE protein fragments on APP metabolism The present invention co-expressed human APP with ApoE2, ApoE3, and ApoE4 in HEK 293T cells, respectively, used GFP as a control group, and measured the production of α-CTF using Western blot. The results of the immunoblot experiment (Figure 1, panels A-B) showed that ApoE2 and ApoE3 co-expressed with APP could significantly increase the non-amyloid metabolite α-CTF of APP, but ApoE4 co-expressed with APP could not significantly change α-CTF (compared with α-CTF in the GFP control group). The present invention further investigated the effect of ApoE on the APP amyloid metabolic pathway. Aβ is a product of amyloid metabolism, and the less Aβ produced, the weaker the amyloid metabolism of APP. In the present invention, human wild-type APP and β-secretase were co-expressed with ApoE2, ApoE3 or ApoE4, respectively, in HEK 293T cells, and GFP was used as a control group to detect the effect of ApoE on Aβ production. ELISA results (Figure 1, panel C) showed that expression of ApoE2 significantly reduced the production of Aβ40, while ApoE3 and ApoE4 did not have this effect. Expression of ApoE2 also significantly reduced Aβ40 compared with ApoE4. These results indicated that ApoE2 expressed in the same cells as APP attenuated the amyloid metabolism of APP and reduced Aβ40, but increased α-CTF, a non-amyloid metabolite of APP. ApoE4 does not have this function.

[0281] The present invention further detected which fragment of ApoE exerts the function of attenuating APP amyloid metabolism. ApoE2, ApoE3, and ApoE4 only differ in two amino acid residues in the N-terminal domain, and have completely identical amino acid sequences in the C-terminal domain. Therefore, the present invention constructed expression plasmids for the N-terminal fragment and the C-terminal fragment of human ApoE, and named the plasmids ApoE2 NT, ApoE3 NT, ApoE4 NT, or ApoE CT protein, respectively, for subsequent experiments. When expressed in cells, the N-terminus of all constructed proteins was further connected 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 the immunoblot experiment (Figure 1, panels D-E) showed that only when ApoE CT was co-expressed, α-CTF was significantly increased by more than 5-fold over the original level. Co-expression of either ApoE2 NT or ApoE4 NT did not change α-CTF. At the same time, ELISA experiment results showed that ApoE CT significantly reduced Aβ40 to about 25% of the GFP control, while none of the three ApoE NTs reduced Aβ40 (Figure 1, panel F); similarly, ApoE CT significantly reduced Aβ42 to about 26% of the GFP control (Figure 1, panel G).

[0282] AD is a neurological disease, and in the brain, Aβ is mainly produced by cleavage of APP expressed in neurons. The present invention constructed a lentivirus expressing ApoE CT in neurons. The ELISA results (Figure 1, panel H) showed that overexpression of ApoE CT in neurons could reduce Aβ40 endogenously produced in primary cultured mouse hippocampal or cortical neurons to about 71% and about 73% of the control group, respectively. Similarly (Figure 1, panel I), overexpression of ApoE CT in neurons induced by human pluripotent stem cells also reduced Aβ40 endogenously produced in human neurons to about 54% of the control group.

[0283] These results indicate that ApoE2 can increase the non-amyloid metabolite α-CTF of APP via its C-terminal fragment in a cell-autonomous manner in various cells, and simultaneously attenuate the amyloid metabolic pathway and reduce Aβ.

[0284] The ApoE CT amino acid sequence used in each experiment of the present invention is as follows:

[0285] ARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH(SEQ ID NO:33).

[0286] 3.2 ApoE2 and ApoE CT secreted into the culture medium do not affect APP cleavage HEK 293T cells expressing ApoE2 or ApoE CT were cultured, and the culture medium was obtained. The results showed that the culture medium contained secreted ApoE2 or ApoE CT (Figure 2, panel A). HEK 293T cells expressing APP were co-incubated with this culture medium containing ApoE2 or ApoE CT. HEK293T cells expressing APP were co-incubated with culture medium containing GFP as a negative control. The results showed that ApoE2 and ApoE CT in the co-incubated culture medium did not significantly change α-CTF levels compared with the GFP control (Figure 2, panels B-C). The immunofluorescence results (Figure 2, panels D-E) showed that even after long-term co-incubation, ApoE2 and ApoE CT in the culture medium mainly adhered to the cell surface and hardly entered into the cells. ApoE2 and ApoE CT expressed in HEK 293T cells (Fig. , panel D) or primary cultured mouse neurons (Fig. 2, panel E) were abundantly present intracellularly.

[0287] The results showed that ApoE2 and ApoE CT in cell culture could not alter the production of α-CTF and Aβ. ApoE2 and ApoE CT must be cell-autonomous, i.e., present in the same cells as APP, to regulate APP cleavage, thereby increasing α-CTF and decreasing Aβ production. Among them, there are many ways to make ApoE2 and ApoE CT enter target cells, specifically including: liposomes deliver DNA or RNA encoding such proteins, and unless otherwise specified, in the present invention, liposomes are used to transfect cells with plasmids encoding the corresponding proteins to express ApoE and ApoE CT; ApoE2 and ApoE CT can be expressed in APP-expressing cells by infecting cells with lentivirus or adeno-associated virus, and the present invention uses lentivirus or adeno-associated virus expression system to express ApoE2 or ApoE CT in neurons; cell-penetrating peptides, enhanced cell endocytosis and other methods can allow ApoE2 and ApoE CT proteins to enter target cells expressing APP; the present invention will discuss the role of cell-penetrating peptides in detail later.

[0288] 3.3 ApoE CT attenuates the core sequence of APP amyloid metabolism In order to find out which amino acid on ApoE CT is the core sequence that changes APP metabolism, 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 plasmids expressing CT NA1, CT NA2, CT NA3, CT NA4 and CT NA5; deletes 1, 2, 3, 4 or 5 amino acids at the N-terminus of ApoE CT to construct plasmids expressing CT ND1, CT ND2, CT ND3, CT ND4 and CT ND5; deletes 5 and 10 original amino acids of ApoE from the C-terminus of ApoE CT to construct plasmids expressing CT CD5 and CT CD10. The amino acid sequences of CTNA1, CTNA2, CTNA3, CTNA4, CTNA5, CTND1, CTND2, CTND3, CTND4, CTND5, CTCD5 and CTCD10 are shown in SEQ ID NO: 13-24, respectively. When expressed in cells, the N-terminus of all constructed proteins was further connected to the signal peptide of ApoE itself (SEQ ID NO: 66).

[0289] [Table 9]

[0290] SEQ ID NO:18(CTND1): RMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH; SEQ ID NO:19(CT ND2): MEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH; SEQ ID NO:20(CT ND3): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH; SEQ ID NO:21(CT ND4): EMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH; SEQ ID NO:22(CT ND5): MGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH; SEQ ID NO:23(CT CD5): ARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPV; SEQ ID NO:24(CT CD10): ARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGT。

[0291] Human APP and β-secretase were co-expressed with each of the plasmids in HEK 293T cells, GFP was used as a control group, and ELISA was used to measure the effect of the proteins on Aβ40 production. The ELISA results (Figure 3, panels A-B) show that even after adding one of the original amino acid residues to the N-terminus of ApoE CT, CT NA1 still reduced Aβ40 production, but after adding two or more amino acid residues, CT NA2, CT NA3, CT NA4, and CT NA5 completely lost the ability to reduce Aβ. Even after deleting 1 to 5 amino acid residues from the N-terminus of ApoE CT, ApoE ND1, ApoE ND2, ApoE ND3, ApoE ND4, and ApoE ND5 could still reduce Aβ40. Even after deleting 5 or 10 amino acid residues from the C-terminus of ApoE CT, CT CD5 and CT CD10 still reduced Aβ production. These results indicate that the N-terminus of ApoE CT is crucial and that even changes of just one or two amino acids can affect the activity of ApoE CT in inhibiting Aβ40 production, although the effect on activity of the C-terminal residues is relatively weak.

[0292] According to the above results, the present invention is based on the deletion of three amino acid residues (CT ND3) at the N-terminus of ApoE CT, and deletes 10, 11, 12, 13, 14, 15, 20 or 25 amino acid residues at the C-terminus of ApoE CT, and constructs plasmids expressing CT ND3CD10, CT ND3CD11, CT ND3CD12, CT ND3CD13, CT ND3CD14, CT ND3CD15, CT ND3CD20 and CT ND3CD25, whose amino acid sequences are shown in SEQ ID NO: 25-32. When expressed in cells, the N-terminus of all constructed proteins was further connected to the signal peptide of ApoE itself (SEQ ID NO: 66).

[0293] SEQ ID NO:25(CTND3CD10): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGT; SEQ ID NO:26(CT ND3CD11): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVG; SEQ ID NO:27(CT ND3CD12): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAV; SEQ ID NO:28(CT ND3CD13): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAA; SEQ ID NO:29(CT ND3CD14): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQA; SEQ ID NO:30(CT ND3CD15): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQ; SEQ ID NO:31(CT ND3CD20): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGL; SEQ ID NO:32(CT ND3CD25): EEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQR。

[0294] Human APP, β-secretase were co-expressed with each of the plasmids in HEK 293T cells, GFP was the control group, and ELISA was used to measure the effect of the proteins on Aβ40 production in cells. The ELISA results (Figure 3, Panel C) show that CT ND3CD10, CT ND3CD11, CT ND3CD12, CT ND3CD13, CT ND3CD14, CT ND3CD15, CT ND3CD20, or CT ND3CD25 can still significantly reduce Aβ40 after deleting 3 amino acid residues from the N-terminus of ApoE CT and 10, 11, 12, 13, 14, 15, 20, or 25 amino acids from the C-terminus. These results indicate that the terminal position of the core sequence of ApoE CT that inhibits Aβ production is from residue 274 to the last amino acid residue of ApoE protein.

[0295] 3.4 ApoE CT affects APP cleavage via γ-cleavage To study the mechanism by which ApoE CT affects APP cleavage, the present invention detected whether ApoE CT changes the expression levels of key secretory enzymes of APP. The present invention used qRT-PCR and Western Blot techniques to detect the mRNA and protein expression levels of ADAM10 (major α-secretase), BACE1 (β-secretase), PS1 and PS2 (γ-secretase active subunits), respectively. The results of qRT-PCR (Figure 4, Panel A) and Western Blot (Figure 4, Panel B) showed that compared with the GFP control, overexpression of ApoE CT in cells did not change the expression of ADAM10, BACE1, PS1 and PS2 mRNA and protein, indicating that ApoE CT does not affect APP cleavage by changing the expression levels of APP metabolic enzymes.

[0296] α-CTF is a substrate for γ-secretase, and Aβ is a cleavage product of γ-secretase. Therefore, when the activity of γ-secretase is changed, the production of α-CTF and Aβ changes significantly, and ApoE CT changes the production of α-CTF and Aβ. Therefore, ApoE CT may change the activity of γ-secretase. To verify this speculation, the present invention co-expressed APP and PS1, which is the active center of γ-secretase, in HEK 293T cells. The immunoblot results (Figure 5, panels A-B) showed that overexpression of PS1 (which can enhance γ-secretase activity) reduced α-CTF. Expression of ApoE CT or treatment with 1 μM of the γ-secretase inhibitor PF03084014 both significantly increased α-CTF (Figure 5, panels A-B). This result suggested that the effect of intracellularly expressed ApoE CT on APP cleavage was similar to that of γ-secretase inhibition. β-CTF is a product of APP digested by β-secretase and is also a substrate for γ-secretase, and Aβ can be produced by a single step of γ-secretase cleavage. The ELISA results (Figure 5, panels C-D) showed that expression of ApoE CT not only reduced Aβ40 produced by β-CTF to about 80% of the original level, but also reduced Aβ42 to about 8% of the original level, indicating that the ability of ApoE CT to reduce Aβ may be achieved by controlling γ-cleavage. The present invention further demonstrated that expression of APP with the APP intracellular C-terminal domain deleted (APP) in HEK293T cells was significantly improved by inhibiting γ-secretase inhibition. ΔAICD ) was co-expressed with BACE1 (β-secretase) and ApoE CT. ΔAICD The ELISA results (Figure 5, Panel E) showed that ApoECT expression was significantly increased in APP ΔAICD These results indicate that ApoE CT no longer reduces Aβ produced from ApoE CT, indicating that ApoE CT does not affect β cleavage.

[0297] These results indicate that intracellularly expressed ApoE CT requires gamma cleavage to reduce Aβ, and overexpressed ApoE CT simulates the effect of gamma-secretase inhibitors, suggesting that ApoE CT may inhibit gamma cleavage of APP, thereby increasing α-CTF and reducing Aβ.

[0298] 3.5 Regulation of APP cleavage by ApoE CT requires the involvement of γ-secretase The active center of γ-secretase is PS1 or PS2. When PS1 and PS2 are knocked out (PS1 / 2 dKO), α-CTF is no longer metabolized by γ-secretase and accumulates in large amounts. Immunoblot results (Figure 6, panels A and D) showed that expressed ApoE CT significantly increased α-CTF in wild-type HEK 293T cell line, and α-CTF content was significantly increased in PS1 / 2 dKO cells expressing GFP compared to wild-type HEK 293T cell line. Expression of ApoE CT in PS1 / 2 dKO cells did not further increase α-CTF, indicating that ApoE CT can no longer increase α-CTF in the absence of γ-secretase. ApoE CT expressed in PS1 / 2 dKO cells supplemented with PS1 or PS2 expression significantly increased α-CTF (Figure 6, panels B-D), indicating that the increase in α-CTF by ApoE CT is dependent on PS1 or PS2. Similarly, overexpression of ApoE CT reduced Aβ40 in PS1 / 2 dKO cell lines supplemented with PS2 expression (Figure 6, panel E). These results indicated that ApoE CT can regulate APP metabolism only in the presence of γ-secretase.

[0299] 3.6 ApoE directly inhibits β-CTF cleavage by γ-secretase in a purified system To further verify whether ApoE directly inhibits γ-secretase, the present invention purified β-CTF, ApoE2, ApoE2 NT and γ-secretase, and examined these purified proteins by immunoblotting (Figure 7, Panel A). Among them, PS1, NCT and PEN2 are constituent proteins of γ-secretase. The ELISA results (Figure 7, Panel B) showed that co-incubation of γ-secretase and β-CTF can produce Aβ, and in this case, the addition of 10 μM of γ-secretase inhibitor (PF03084014) can inhibit the production of Aβ. Purified ApoE2 was added to this reaction system, which inhibited the production of Aβ40 in a concentration-dependent manner, with a half maximal inhibitory concentration (IC50) of about 1.2 μM (Figure 7, Panel C). Furthermore, under the same concentration conditions (2.5 μM), ApoE2 could significantly inhibit the production of Aβ, but ApoE2 NT could not inhibit the production of Aβ40 (Figure 7, panel D). These results suggested that ApoE2 could directly inhibit the cleavage of β-CTF by γ-secretase and that ApoE CT is required for the said activity of ApoE, suggesting that ApoE CT is an important fragment of ApoE2 that inhibits the cleavage of APP by γ-secretase.

[0300] 3.7 ApoE CT does not inhibit gamma cleavage of APLP1 APLP1 (APP like Protein 1) is a protein with a structure similar to APP and is one of the substrates of α- and γ-secretase. Immunoblot results (Figure 8) showed that PF03084014, a γ-secretase inhibitor, can inhibit γ-cleavage of APLP1 in HEK 293T cells and significantly increase α-like CTF, but co-expressed ApoE CT did not increase α-like CTF of APLP1, and ApoE CT did not inhibit γ-cleavage of APLP1. This result indicated that ApoE CT inhibits the cleavage activity of γ-secretase with a certain degree of substrate selectivity.

[0301] 3.8 Neuronally expressed ApoE CT reduces amyloid plaques in the brains of AD model mice Adeno-associated virus capable of expressing ApoE CT in neurons was injected into the cortex and hippocampus of 4-week-old AD model mice (5×FAD). Six months after expressing ApoE CT (or GFP as a negative control) in neurons, its effect on amyloid plaques was detected by staining the amyloid plaques with ApoE CT, GFP, and GFP. The results showed that the amyloid plaque area in the brain of 5×FAD mice expressing ApoE CT (the N-terminus of which is connected to the signal peptide of human ApoE itself (SEQ ID NO:66)) in neurons was smaller, the fluorescence intensity was weaker, the percentage of the total amyloid plaque area in the brain was smaller, and the density of amyloid plaques was also reduced (Figure 9, panels A-E). These results indicated that ApoE CT expressed in neurons inhibited the deposition of amyloid plaques in the brain of 5×FAD mice and attenuated the pathological progression of AD in mice.

[0302] 3.9 ApoE2 expressed in neurons can inhibit endogenously produced Aβ40 in mice The above experiments demonstrated that ApoE CT is an important functional domain of ApoE2 in inhibiting γ-secretase activity, and ApoE CT can inhibit the production of Aβ40 in mouse neurons. Next, we examined whether ApoE2 also inhibits the production of endogenous Aβ40 in neurons (Figure 10). GFP or ApoE2 was expressed in primary cultured mouse neurons using a lentivirus expression system and a neuron-specific promoter, respectively. The N-terminus of ApoE2 was further connected to the signal peptide of human ApoE itself (SEQ ID NO:66). The ELISA results (Figure 10, panel A) demonstrated that ApoE2 overexpressed in neurons can reduce endogenously produced Aβ40 compared to the GFP control. Furthermore, the use of an AAV expression system allowed ApoE2 to be expressed in neurons in the brains of wild-type mice (WT mice). ELISA results (Figure 10, panel B) showed that ApoE2 expressed in neurons of mouse brain also reduced the production of endogenous Aβ40 compared to the GFP control. These results indicated that ApoE2, like ApoE CT, can inhibit the production of endogenous Aβ40 in neurons.

[0303] 3.10 ApoE with tagged proteins at both ends still has the activity to regulate APP cleavage To investigate whether the tag protein added to ApoE destroys the activity of ApoE, the present invention added a tag protein V5 (SEQ ID NO: 82) to the C-terminus and N-terminus of ApoE2 and ApoE CT, respectively, and named them ApoE2-V5 (V5 is at the C-terminus), V5-ApoE2 (V5 is at the N-terminus), ApoE CT-V5, and V5-ApoE CT, respectively. The N-terminus of all constructed proteins was further connected to the signal peptide (SEQ ID NO: 66) of human ApoE itself. Then, these expression plasmids and APP were co-expressed in HEK 293T cells. Immunoblot results showed that ApoE2-V5 and V5-ApoE2 (Figure 11, panels A-B) and ApoE CT-V5 and V5-ApoE CT (Figure 11, panels C-D) both significantly increased α-CTF (Figure 11), and their activity was similar to that of ApoE2 or ApoE CT. This indicated that ApoE2 and ApoE CT with tag proteins inserted at both ends still had activity to regulate APP cleavage.

[0304] 3.11 ApoE CT mutations can still regulate APP cleavage To investigate whether mutations in ApoE CT alter the activity of ApoE CT in controlling APP cleavage, the present invention constructed and analyzed a series of ApoE CT mutants, 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 positions of the mutation sites are relative to ApoE CT (SEQ ID NO:33). Their amino acid sequences are shown in SEQ ID NO:46-59. The N-terminus of all constructed proteins was further connected to the signal peptide of human ApoE itself (SEQ ID NO:66).

[0305] [Table 10]

[0306] [Table 11]

[0307] [Table 12]

[0308] Human APP was co-expressed with each of the above plasmids in HEK 293T cells, GFP was used as a negative control, and ApoE CT was used as a positive control. Immunoblot results showed that among the single-site and double-site mutations of the constructed ApoE CT (Figure 12, panels A-B), CT A20, CT A40, CT A60, CT A65, CT A72, CT A80, and CT A36 / 74 all significantly increased α-CTF, and compared with ApoE CT, the degree of α-CTF increase by these mutations was not reduced (CT A72, CT A80, and CT A36 / 74 further significantly increased α-CTF). Although the multiple site mutations CT E5, CT E10, CT E20 and CT F5 were unable to increase α-CTF, CT R10, CT LF5 and CT LM5 still significantly increased α-CTF, and these mutations increased α-CTF to levels similar to ApoE CT (Figure 12, panels C-D). These data indicated that ApoE CT mutants (e.g., CT A20, CT A40, CT A60, CT A65, CT A72, CT A80, CT A36 / 74, CT R10, CT LF5 and CT LM5) also had similar activity to the original ApoE CT in controlling APP cleavage.

[0309] 3.12 Insertion or deletion of amino acid residues in ApoE2 can still regulate APP metabolism This experiment explored whether the insertion or deletion of amino acid residues in ApoE2 affects the activity of ApoE. The present invention first inserts a tag protein 3XFLAG between the amino acid residues at positions 215 and 216 of ApoE2 (SEQ ID NO:12), and names it ApoE2-M3F (SEQ ID NO:60; bold indicates 3XFLAG sequence; underline indicates the sequence of SEQ ID NO:33), and this modification does not affect the ApoE CT sequence (SEQ ID NO:33). The present invention also constructs ApoE2 MD10 (SEQ ID NO:61), which is obtained by deleting amino acid residues 244-254 of ApoE2, and these deleted amino acids are located in the C-terminal region where ApoE2 controls APP metabolism, i.e., the sequence of SEQ ID NO:33. When expressed intracellularly, the N-terminus of all constructed proteins was further connected to the signal peptide of human ApoE itself (SEQ ID NO:66).

[0310] [Table 13]

[0311] [Table 14]

[0312] Human APP was co-expressed with ApoE2-M3F and ApoE2 MD10 in HEK 293T cells, and GFP was used as a negative control group. Immunoblot results (Figure 13) showed that both ApoE2-M3F, which inserts an amino acid residue in the middle of ApoE2 (without affecting the ApoE CT sequence), and ApoE2 MD10, which deletes an amino acid residue in the middle of the C-terminal functional region, significantly increased α-CTF. This result indicated that ApoE2 can still regulate the metabolism of APP even if an amino acid sequence is inserted or deleted in ApoE2.

[0313] 3.13: Non-human ApoE CT also regulates APP cleavage To further study whether non-human ApoE has the same activity, this experiment analyzed ApoE CT from non-human primates (monkey CT), which has a very similar amino acid sequence to human ApoE CT, with only three amino acid substitutions: R36S+T74A+A76T (Figure 14, Panel C). The monkey ApoE CT amino acid sequence is shown below.

[0314] [Table 15]

[0315] 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)) were co-expressed in HEK 293T cells, and GFP was used as a negative control group, and human ApoE CT (N-terminus is further connected to the signal peptide of human ApoE itself (SEQ ID NO: 66)) was used as a positive control. Immunoblot results (Figure 14, panels A and B) showed that both non-human primate ApoE CT (monkey CT) and human ApoE CT increased α-CTF. This result indicated that non-human primate ApoE CT also has the activity of regulating APP cleavage.

[0316] 3.14: ApoE inserting a membrane-permeable peptide has the activity of regulating APP cleavage To examine whether the membrane-permeable peptides change the activity of ApoE, the present invention added the membrane-permeable peptides to ApoE. The selected membrane-permeable peptide sequences were as follows:

[0317] [Table 16]

[0318] 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 N-terminus of all protein constructs was further connected to the signal peptide of human ApoE itself (SEQ ID NO:66). R9-ApoE2, FGF4-ApoE2, and RDP-ApoE2 were co-expressed with human APP in HEK 293T cells, respectively, and GFP was used as the negative control group. The immunoblot results (Figure 15) showed that FGF4-ApoE2 did not increase α-CTF, but both R9-ApoE2 and RDP-ApoE2 significantly increased α-CTF. This result indicated that ApoE2 bound to the membrane-permeable peptide could still control the cleavage of APP.

[0319] 3.15 The signal peptide is required for ApoECT activity in regulating APP cleavage In the above experiments to study the function of ApoE expressed in cells, all the amino acid sequences used to express the corresponding peptides contain the signal peptide of human ApoE itself (ApoE SP; SEQ ID NO: 66), which is located at the N-terminus of ApoE or ApoE CT and each mutant of ApoE or ApoE CT described above.To study whether signal peptide affects the activity of ApoE CT, in the present invention, the signal peptide of ApoE CT is replaced by the signal peptide of ApoA1 and ApoJ, respectively (ApoA1 SP and ApoJ SP; SEQ ID NO: 67 and SEQ ID NO: 68).The activity of ApoE CT that is not connected to signal peptide is also analyzed.

[0320] [Table 17]

[0321] The ApoE CT mutant substituted with ApoE A1 signal peptide was named ASP CT, the ApoE CT mutant substituted with ApoE J signal peptide was named JSP CT, and the ApoE CT mutant without signal peptide was named NSP CT. Human APP was co-expressed with each of the ApoE CT mutants in HEK 293T cells, and GFP was used as a negative control and ApoE CT was used as a positive control. Immunoblot results (Figure 16) showed that ASP CT and JSP CT still increased α-CTF, but not as much as ApoE CT with ApoE's own signal peptide; after the signal peptide was deleted, NSP CT no longer increased α-CTF. This result suggested that the CT of ApoE expressed in cells must be bound to a signal peptide to control the cleavage of APP by γ-secretase, but this signal peptide can be a different signal peptide and is not limited to the original ApoE signal peptide.

[0322] 4. Experimental Results The present invention is the first to reveal that ApoE2 can control APP cleavage 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 α-CTF, a non-amyloid metabolite of APP, but also attenuates the amyloid metabolism of APP and reduces Aβ. It has also been found that ApoE2 and its C-terminal fragment control APP metabolism not by indirectly affecting the cleavage of APP in cells via ApoE protein secreted outside the cells, but by directly inhibiting γ-cleavage of APP coexisting with APP in the same cells, which is a cell-autonomous mode of action. It has also been found that ApoE2 directly inhibits γ-secretase cleavage of β-CTF in experiments using purified protein, and ApoE NT, which lacks the C-terminus, does not inhibit γ-secretase cleavage of β-CTF. Furthermore, the C-terminal fragment of ApoE did not inhibit the gamma cleavage of APLP1, suggesting that the inhibition of gamma cleavage by the C-terminal fragment of ApoE is substrate selective to some extent. The present invention also found that the C-terminal fragment of ApoE expressed specifically in neurons significantly reduced the size and density of amyloid plaques in the brain of AD model mice. ApoE expressed specifically in mouse neurons suppresses endogenous Aβ40 production. In addition, the insertion of tag proteins at the N- or C-terminus did not affect the APPγ cleavage inhibitory activity of ApoE2 and ApoE C-terminal fragment. The present invention also constructed a series of mutants of the internal sequence of ApoE C-terminal fragment and ApoE2, and found that they had the same APPγ cleavage inhibitory activity as the original ApoE CT or ApoE2. The present invention also found that the insertion of a membrane-permeable peptide at the N-terminus of ApoE did not affect the activity of inhibiting APPγ cleavage, but that the absence of a signal peptide at the N-terminus caused the loss of APPγ cleavage inhibitory activity of ApoE. The findings of the present invention are not limited to human-derived ApoE C-terminal fragments, but ApoE C-terminal fragments from non-human primates also have similar inhibitory activity against APPγ cleavage.

[0323] These results suggest that ApoE2 and its C-terminal fragments (or mutants based on ApoE2 or the C-terminal fragments) may be used as medicines to prevent or treat AD. Such potential medicines should act in a cell-autonomous manner in neurons. Liposome delivery of DNA or RNA, lentivirus and adeno-associated virus infection, etc. can express ApoE2 and / or its C-terminal fragments in target cells; cell-penetrating peptides, enhanced phagocytosis, etc. can also make ApoE2 and / or its C-terminal fragments abundant in target cells; and ApoE2 and / or its C-terminal fragments can inhibit the cleavage activity of γ-secretase in a cell-autonomous manner, reduce Aβ, and suppress amyloid metabolism of APP.

[0324] The above experiment is merely an example of AD disease to illustrate that ApoE2 and / or its C-terminal fragment inhibits the cleavage activity of gamma-secretase in a cell-autonomous manner. Furthermore, diseases such as amyloid cerebrovascular disease are associated with gamma-secretase dysfunction. Down's syndrome is also prone to progress to AD. Therefore, ApoE2 and / or its C-terminal fragment may be applicable to the treatment and prevention of these APP or gamma-secretase-related diseases.

[0325] Example 2: Precise inhibition of gamma-secretase by ApoE leads to prevention of Alzheimer's disease 1. Overview Abnormally reduced gamma-secretase activity is associated with most progeroid mutations that cause familial Alzheimer's disease (fAD). However, the role of gamma-secretase in the more common sporadic AD (sAD) remains unresolved. Human apolipoprotein E (ApoE) is the most important genetic risk factor for sAD, and the present invention reports that ApoE interacts with gamma-secretase and inhibits gamma-secretase activity in a substrate-specific and cell-autonomous manner through its conserved C-terminal region (CT). This ApoE CT-mediated inhibitory activity is differentially impaired by different ApoE subtypes, resulting in a potency that is inversely proportional to AD risk, in the order ApoE2>ApoE3>ApoE4. Interestingly, in AD mouse models, ApoE CT expressed in neurons can migrate from other regions to amyloid plaques and delay their formation. In conclusion, the data from this experiment revealed that ApoE has substrate-specific γ-secretase inhibitory activity and strongly suggested that precise inhibition of γ-secretase by ApoE prevents the risk of sAD.

[0326] 2. Introduction Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting nearly 50 million people worldwide (Prince, 2015). The β-amyloid (Aβ) hypothesis proposes that AD is caused by the excessive aggregation of Aβ, a peptide produced by sequential cleavage of the amyloid precursor protein (APP) by the β-secretase and γ-secretase enzymes (De Strooper and Karran, 2016; De Strooper et al., 1998; Haass and Selkoe, 1993; Hardy and Selkoe, 2002; Wolfe et al., 1999). Mutations in presenilin 1 (PS1) and presenilin 2 (PS2), the catalytic centers of γ-secretase, cause the rare familial form of AD (fAD). These lines of evidence suggest that γ-secretase is a potential drug target for AD. Unfortunately, treatment with conventional γ-secretase inhibitors can cause severe side effects (Panza et al., 2019; Xia, 2019). Recent studies have found that progerin mutations associated with fAD are usually associated with a global decrease in γ-secretase activity (Shen and Kelleher, 2007; Sun et al., 2017; Walker et al., 2005; Xia et al., 2015), which is inconsistent with the predicted role of γ-secretase based on the Aβ hypothesis. Given that Aβ-based drug development efforts have been unsatisfactory (Karran and De Strooper, 2016), there is an urgent need to clarify the role that γ-secretase plays in the more generalized sporadic AD (sAD).

[0327] Although there is no causative gene for sAD, the risk of developing it is influenced by many genetic factors. Among them, apolipoprotein E4 (ApoE4) is one of the most influential (Corder et al., 1993; Rhinn et al., 2013). The ApoE gene encodes three isoforms, namely ApoE2, 3, and 4, with a single amino acid difference only in its N-terminal region (NT) (Kanekiyo et al., 2014). ApoE4 not only increases the risk of AD, but also accelerates the age of onset and aggravates the severity of AD (Belloy et al., 2019; Li et al., 2020). Conversely, ApoE2 reduced AD risk. In rodent brains, under normal physiological conditions, ApoE is usually detected only in glial cells (Kanekiyo et al., 2014), whereas Aβ is produced in neurons. However, ApoE is also expressed 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). A recent study using neurons generated from human induced pluripotent stem cells (hiPSCs) with different ApoE subtypes showed increased Aβ production in ApoE4 positive neurons, suggesting that ApoE in neurons regulates APP metabolism in a cell-autonomous manner (Wang et al., 2018). This evidence suggests that ApoE in neurons has a crucial physiological role, which has not been thoroughly investigated so far.

[0328] This experiment reports that ApoE directly interacts with γ-secretase and specifically inhibits γ-cleavage of APP via its C-terminal region. Excitingly, such inhibitory activity was differentially impaired by ApoE subtypes and negatively correlated with the AD risk corresponding to ApoE subtype. Furthermore, ApoE CT expressed in neurons accumulates around amyloid plaques and may achieve localized γ-inhibition in this amyloidogenic hotspot region (Jorda-Siquier et al., 2022). In short, our data suggest that precise inhibition of γ-secretase by ApoE prevents the risk of sAD and reveal that ApoE CT is a novel γ-secretase inhibitor with unprecedented specificity.

[0329] 3. Experimental Results 3.1. Endogenous ApoE and APP are co-expressed in human cortical neurons and show overlapping subcellular distribution patterns ApoE is mainly expressed in glial cells in rodent brains, and Aβ is metabolized by APP expressed in neurons. Therefore, most studies focus on how ApoE secreted from glial cells affects the formation of extracellular amyloid plaques (Huang et al., 2017; Huynh et al., 2017; Liu et al., 2017). However, recent studies have shown that ApoE can control Aβ production in a cell-autonomous manner (Wang et al., 2018). Before exploring this possible mechanism, we first stained human brain samples with immunofluorescence to confirm that ApoE is indeed expressed in neurons. This experimental result is consistent with previously reported human brain data (Aoki et al., 2003; Belonwu et al., 2022; Han et al., 1994; Zalocusky et al., 2021) in which ApoE was detected in both neurons and glial cells in the human brain. Specifically, in brain samples from the young control group (Figure 17, panel A; Figure 25, panel B; and human brain donor information in Figure 32), age-matched non-AD control group (elderly, 60 years and older), and AD patients (AD, 60 years and older), 30-50% of cortical neurons were ApoE positive (Figure 25, panels A and B); whereas, in human cerebral cortex samples, 40-62% of ApoE positive cells were neurons (Figure 17, panel A; Figure 25, panels A and C). Furthermore, human cortical astrocytes also expressed ApoE (Figure 25, panel D). In monkey cerebral cortex, most of the detectable ApoE signal was located in neurons (Figure 17, panel A). In human AD patient brains, strong ApoE staining signals were detected in amyloid plaques, whereas no ApoE signal was detected in the same sites of non-AD control brain samples (Figure 25, panel E). Importantly, immunostaining demonstrated that endogenous ApoE and APP colocalized in human cortical neurons and exhibited overlapping intracellular signaling patterns (FIG. 17, panel B).

[0330] 3.2. ApoE2 increases α-CTF and reduces Aβ in a cell-autonomous manner To study whether ApoE can regulate APP metabolism in a cell-autonomous manner, we co-expressed APP with ApoE in a mouse neuronal line (N2A; Figure 17, Panel C) and HEK 293T cells (Figure 26, Panel C). α-CTF is a cleavage product of the nonamyloid metabolic pathway of APP, and its production is known to be highly dependent on ADAM10 (Figure 26, Panels A and B), an α-secretase (Esch et al., 1990; Haass et al., 1992; Mucke and Selkoe, 2012). In N2A and HEK 293T cells, ApoE2 or ApoE3 significantly increased the levels of α-CTF produced from co-expressed APP (Figure 17, Panels C and D, N2A cells; Figure 26, Panels C and D, HEK 293T cells). Furthermore, ApoE2 also significantly reduced the levels of Aβ40 produced from co-expressed APP (Figure 17, panel E). Interestingly, co-expression of ApoE4 and APP failed to alter α-CTF (Figure 17, panels C and D) and Aβ40 levels (Figure 17, panel E). These results suggest that ApoE2 increases α-CTF in a cell-autonomous manner.

[0331] 3.3. The C-terminal region of ApoE2 has a higher activity of increasing α-CTF concentration than full-length ApoE2 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 sequence differences between the three ApoE subtypes are only within their NT. Experiments with HEK 293T cells carrying truncated mutants of ApoE subtypes showed that NT from ApoE2, 3, and 4 did not significantly alter α-CTF produced from co-expressed APP (Figure 26, panels E and F). Conversely, co-expression of the conserved ApoE CT increased α-CTF levels by more than six-fold (Figure 26, panels G and H).

[0332] Surprisingly, the experimental results showed that co-expression of ApoE CT with APP reduced Aβ secreted into the cell culture medium. 40was reduced by approximately 75% (Figure 26, Panel I), and Aβ in the cell lysate was 40 was reduced by approximately 42% (Figure 26, panel J), and Aβ 42 was significantly reduced by approximately 74% (Figure 26, panel K), but still Aβ 42 / Aβ 40 The ratio of ApoE NT to β-CTF was unchanged (FIG. 26, panel K). However, co-expression of ApoE NT with APP and BACE1 did not change the β-CTF level (FIG. 26, panels L and M). 40 (Figure 26, Panel I). Similarly, in experiments with N2A cells, only co-expression of ApoE CT and APP, but not ApoE NT, significantly increased α-CTF (Figure 17, Panels F and G); moreover, ApoE CT did not significantly increase total Aβ 40 also significantly decreased (Figure 17, panel H).

[0333] Experiments showed that compared with ApoE2, ApoE CT significantly increased α-CTF levels (FIG. 17, panels I and J, N2A cells; FIG. 26, panels N and O, HEK 293T cells), indicating that ApoE CT is more active than full-length ApoE2 in regulating APP metabolism. These results suggest that ApoE CT, a conserved region of ApoE, increases α-CTF levels, and suggest that ApoE NT may somehow interfere with this activity of ApoE CT.

[0334] To investigate whether secreted ApoE also affects APP cleavage, we cultured HEK 293T cells expressing only APP in culture medium containing secreted ApoE or ApoE CT (Figure 27). The experiment showed that free ApoE2 or ApoE CT in the culture medium did not change the α-CTF produced by APP in cells (Figure 27, panels A, B, and C). Notably, ApoE2 was stable in the conditioned medium at 37°C for 48 hours (Figure 27, panel D). In HEK 293T cells and primary cultured rat neurons treated with conditioned medium, ApoE2 or ApoE CT was detected mainly on the outside of the cell membrane (Figure 27, panels E and F). In contrast, ApoE CT expressed intracellularly or in neurons was distributed within cells or neurons (Figure 27, panels E and F). Comparing the relative amounts of ApoE2 in cell lysates and secreted into the culture medium, both contained similar amounts of ApoE2 (Figure 27, Panel G). Furthermore, small amounts of ApoE in the conditioned medium were endocytosed by cells and neurons after co-incubation (Figure 27, Panels E and F). Thus, the regulation of APP metabolism by ApoE is more likely to be mediated by intracellular ApoE in a cell-autonomous manner than by secreted ApoE.

[0335] 3.4. ApoE CT expressed in neurons reduces endogenous Aβ In addition to human APP, ApoE CT also increased α-CTF levels in HEK 293T cells transiently expressing mouse APP (mAPP) (Figure 26, panels P and Q). Experiments with primary neurons showed that overexpression of ApoE CT (lentiviral infection) in mouse cortical and hippocampal neurons suppressed endogenous Aβ expression. 40 The amount of Aβ produced by human neurons was reduced by 30% compared to the GFP control (Figure 17, panel K). In hiPSC-derived neurons, ApoE CT (lentivirus infection) reduced the amount of Aβ produced by human neurons. 40was reduced by approximately 46% (Figure 17, panel L). Thus, these results suggest that ApoE CT slows endogenous Aβ in rodent and human neurons. Furthermore, expression of ApoE CT reduced Aβ expression in HEK 293T cells expressing the pathogenic Swedish APP mutant (APPSW) (Citron et al., 1992). 40 Slowed production: Aβ in culture media 40 was reduced by approximately 76% (Figure 26, panel R), and Aβ in the cell lysate was 40 was decreased by about 43% (FIG. 26, panel S), indicating that ApoE CT reduced Aβ produced from APPSW.

[0336] Apolipoprotein A1 (ApoA1), another apolipoprotein coexpressed with APP, failed to alter the levels of α-CTFs and Aβ (Figure 26, panels T, U, and V), suggesting that the activity of ApoE in regulating APP cleavage is not a nonspecific activity of any apolipoprotein.

[0337] 3.5. ApoE2 interacts with APP and γ-secretase via CT In N2A cells, ApoE2 or ApoE CT showed a perinuclear subcellular distribution pattern very similar to that of co-expressed APP-FLAG (Figure 18, Panel A). In cultured rat hippocampal neurons (Figure 18, Panel B) and hiPSC-derived neurons (Figure 18, Panel C), expression of ApoE2-V5 or ApoE CT-V5 showed a subcellular distribution pattern similar to that of endogenously expressed APP in neurons (V5 is the tag protein). These results suggested that ApoE and ApoE CT have a subcellular distribution pattern similar to that of APP.

[0338] To detect the potential interaction of ApoE with APP or APP secretase, the protein proximity-dependent labeling technique was used in this experiment (Hung et al., 2016). When coexpressed in HEK293T cells, ApoE2 and ApoE CT were biotinylated by APP-APEX2 (ascorbate peroxidase 2 attached to the C-terminus of APP; Figure 18, panels D and E). Also, FL-APP (full-length APP) and α-CTF were biotinylated by ApoE2-APEX2 in the same system (Figure 18, panel F). In primary cultured rat neurons, ApoE2-APEX2 biotinylated endogenously expressed APP (Figure 18, panel G). Furthermore, ApoE2-APEX2 biotinylated coexpressed ADAM10 (α-secretase), PS1, and PS2 (catalytic cleavage activity of γ-secretase), but not β-secretase BACE1 (Figure 18, panel H). Furthermore, when coexpressed in HEK 293T cells, ApoE2 and ApoE CT (but not ApoE2 NT) could immunoprecipitate with β-CTF-FLAG (Figure 18, panel I). Similarly, when coexpressed in HEK 293T cells, ApoE2 and ApoE CT (but not ApoE2NT) immunoprecipitated with components of γ-secretase (APH, NCT, PEN2) (Figure 18, panel J; immunoprecipitation with anti-FLAG-PEN2). Interestingly, endogenous γ-secretase (NCT, PS1, PS2, PEN2) was immunoprecipitated with endogenous ApoE in human brain samples (Figure 18, Panel K). These results indicate that only ApoE2 and ApoE CT (but not ApoE NT) can interact with APP, γ-secretase, and α-secretase, but not with BACE1, and that endogenous interaction between ApoE and γ-secretase exists in human brain.

[0339] 3.6. ApoE CT reduces γ-cleavage of APP To further explore the potential mechanism, this study further explored the interaction of AD-associated APP mutants with ApoE CT. ApoE CT increased α-CTF produced from all APP mutants tested (Figure 28). Interestingly, compared to wild-type APP (WT APP), the ability of ApoE CT to increase α-CTF levels was significantly reduced in cells expressing APP mutants with fAD mutations near the γ-cleavage site (T714I, V717F, L723P) (Kumar-Singh et al., 2000; Kwok et al., 2000; Murrell et al., 1991) (Figure 28). When studying WT APP and APP subtypes with fAD mutations near the β- or α-cleavage sites (KM670 / 671NL, E693K, E693Δ) (Bugiani et al., 2010; Citron et al., 1992; Tomiyama et al., 2008; Xia et al., 2021), no statistically significant effect of ApoE CT on α-CTF levels was detected (Figure 28); APP subtypes co-expressing the Icelandic mutation (A673T) (Xia et al., 2021), which is protective and reduces the probability of developing AD, also did not detect a significant effect of ApoE CT on α-CTF levels (Figure 28). Taken together, when mutations occur near the γ-site of APP, ApoE CT activity is disrupted, suggesting that γ-cleavage is involved in the activity of ApoE CT to control APP metabolism. Because α-CTF is degraded by γ-cleavage (De Strooper et al. 2012; De Strooper et al. 1998; Haass et al. 1992), these results suggest that ApoE CT may affect α-CTF degradation.

[0340] Next, this study investigated in detail the role of γ-secretase in APP metabolism. Consistent with previous reports on α-CTF cleavage by γ-secretase (De Strooper et al., 2012; De Strooper et al., 1998; Haass et al., 1992), overexpression of PS1 significantly reduced the level of α-CTF by 50% (Figure 19, panels A and B); when treated with the γ-secretase inhibitor PF03084014 (1 μM), overexpressed PS1 no longer reduced α-CTF, and PF03084014 also increased α-CTF; when treated with the γ-secretase inhibitor, the level of α-CTF was similar to that in HEK 293T cells co-expressing ApoE CT (Figure 19, panels A, B, C and D). Interestingly, in the presence of PF03084014, ApoE CT failed to further increase α-CTF (Figure 19, panels C and D), suggesting that ApoE CT activity can be blocked by inhibiting γ-secretase. Furthermore, with the ADAM10 inhibitor GI254023X (1 μM), α-CTF was reduced by approximately 45% (Figure 29, panels A and B). Even in the presence of GI254023X, ApoE CT increased α-CTF (approximately 4-fold; Figure 29, panels A and B), and Aβ 40 (Figure 29, panels C and D). Furthermore, even when β-secretase was inhibited by its inhibitor AZD3839 (5 ​​μM), ApoE CT increased α-CTF (Figure 29, panels E and F). ApoE CT also increased APP M596V (Figure 29, panels G and H), which enhances the α-CTF produced from APP M596V is an APP mutation that cannot undergo β-cleavage (Citron et al., 1995). Furthermore, ApoE CT is a β-CTF that is produced by Aβ 40 (Figure 19, Panel E) and Aβ 42 (FIG. 19, panel F) also significantly reduced Aβ production, suggesting that ApoECT reduces Aβ production by inhibiting γ-cleavage. ΔAICD is Aβ 40Interestingly, ApoECT is a ubiquitous β-cleavage enzyme that is required to produce APP. ΔAICD Aβ produced from 40 (FIG. 19, panels G and H). All of these results support the hypothesis that ApoECT inhibits γ-cleavage of APP but does not directly regulate α- or β-cleavage.

[0341] In addition, neither ApoE2 nor ApoE CT altered the mRNA or protein expression levels of ADAM10, BACE1, PS1, or PS2 (Figure 29, panels I and J), suggesting that there was no change in the expression level of APP secretase. In addition, ApoE CT did not alter the expression of APP on the cell surface (Figure 29, panel K).

[0342] 3.7. Regulation of APP metabolism by ApoE CT requires the involvement of γ-secretase To study whether the regulation of APP metabolism by ApoE CT requires γ-secretase, we constructed PS1 and / or PS2 knockout (KO) HEK 293T cell lines (Lu et al., 2014) (Figure 20, Panel A). α-CTF levels in PS1 KO cells were elevated, which could be reversed by compensatory expression of PS1 (Figure 20, Panel B). PF03084014 treatment further increased α-CTF levels in PS1 KO cells (Figure 20, Panel B). Co-expressed ApoE CT could also increase α-CTF in PS1 KO cells (Figure 20, Panels C and D) and PS2 KO cells (Figure 20, Panels E and F). Thus, knocking down PS1 or PS2 alone is not sufficient to completely abolish ApoE CT activity.

[0343] Compared to WT cells, α-CTF levels were also significantly increased in PS1 / 2 double knockout cells (PS1 / 2 dKO) (Figure 20, panel G); co-expression of ApoE CT (Figure 20, panels G and J) or ApoE2 (Figure 20, panels K and L) in PS1 / 2 dKO cells did not further increase α-CTF levels. Supplementing expression of PS1 or PS2 restored the activity of ApoE CT, and ApoE CT again significantly increased α-CTF (Figure 20, panels H, I, J). In PS1 / 2 dKO cells supplemented with expression of PS2, ApoE CT suppressed Aβ production from β-CTF. 40 (FIG. 20, panel M). These results suggest that γ-secretase is required for ApoE CT or ApoE2 to increase α-CTF.

[0344] 3.8. Endogenously expressed ApoE in mouse neurons regulates γ-cleavage of endogenous APP. To study the physiological activity of endogenous ApoE in neurons, shRNAs against mouse ApoE (shApoE_1, shApoE_2, and shScramble as control) were constructed. shApoE_1 and shApoE_2 expressed in mouse neurons using the lentiviral system successfully reduced the expression of endogenous ApoE protein in primary cultured mouse neurons (Figure 21, panel A). After treatment with γ-secretase inhibitor PF03084014, the ~14 kD CTF (C-terminal fragment, CTF) produced by APP metabolism in neurons was downregulated. 14kD ) was significantly increased, indicating that the CTF 14kD We also demonstrated that shApoE_1 and shApoE_2 are cleaved by γ-secretase in mouse neurons (Figure 21, Panel B). 14kD The basal levels of the band were significantly reduced (Figure 21, panels C and D), indicating that endogenous ApoE inhibits the CTF 14kDIn the presence of γ-secretase inhibitor PF03084014, these two shRNAs for ApoE no longer increased CTF activity. 14kD ApoE was unable to reduce Aβ levels in neurons (Figure 21, panel E), suggesting that this activity of ApoE requires the presence of active γ-secretase. Knockdown of ApoE also increased the levels of Aβ produced in neurons (Figure 21, panel F). These results suggest that endogenous ApoE in neurons regulates γ-cleavage of endogenous APP.

[0345] 3.9. ApoE CT inhibits γ-cleavage in a substrate-specific manner. The present invention further detected whether ApoE CT also inhibited the gamma-cleavage of NOTCH. NOTCH is a substrate of gamma-secretase (Xia, 2019; Yang et al., 2019). In HEK 293T cells, co-expressed gamma-secretase reduced N100 (NOTCH fragment), and treatment with PF03084014 reversed this reduction (Figure 30, panel A). Interestingly, co-expressed ApoE CT could not suppress this gamma-secretase-dependent reduction of N100 (Figure 30, panel A). However, when ApoE CT was co-expressed with gamma-secretase, ApoE CT still inhibited the gamma-cleavage of APP by gamma-secretase (Figure 30, panel B). APP-like protein 1 (APLP1) is another substrate for α- and γ-secretase (Eggert et al., 2004). Treatment with PF03084014 reduced the levels of APLP1, but increased its cleavage product (α-like CTF); however, α-like CTF was not altered by coexpression of ApoE CT and APLP1 (Figure 30, panel C). These results indicate that ApoE CT does not inhibit γ-cleavage of NOTCH and APLP1.

[0346] 3.10. ApoE2 directly inhibits γ-cleavage of APP To determine whether ApoE directly inhibits the gamma cleavage of β-CTF, the present invention purified β-CTF, ApoE2, ApoE2 NT, and gamma secretase (Lu et al., 2014) for further testing (Figure 22, Panel A; Figure 31). Incubation of purified gamma secretase with β-CTF inhibited the gamma cleavage of Aβ 40 was produced; in the absence of γ-secretase, β-CTFs were produced by Aβ 40 and Aβ production by γ-secretase incubated with β-CTF. 40 The production of Aβ40 was completely blocked by PF03084014 (Figure 22, panel B). Purified ApoE2 inhibited Aβ40 production in a dose-dependent manner with an IC50 of 1.2 μM (Figure 22, panel C). Conversely, ApoE2 NT failed to suppress Aβ40 production (Figure 22, panel D). These results suggest that ApoE2 directly inhibits γ-cleavage of β-CTF via its CT.

[0347] 3.11. Neuronally expressed ApoE CT trafficked to amyloid plaques and attenuated plaque burden in 5×FAD mice To study whether ApoE CT can delay the development of amyloid plaques in AD mouse models, we expressed ApoE CT via AAV vectors in neurons in the cerebral cortex and hippocampus of 4-week-old 5×FAD mice (Figure 23, Panel A). After 10 weeks, the average plaque size, percentage of total amyloid plaque area, and plaque density were significantly reduced in the subiculum brain region of 5×FAD mice expressing ApoE CT compared to GFP controls (Figure 23, Panels B, C, D, E).

[0348] Interestingly, in these 5xFAD mice, ApoE CT expression was restricted to neurons in the cerebral cortex, dentate gyrus (DG) and CA3 regions of the hippocampus, but migrated to the subiculum and accumulated around amyloid plaques (Figure 23, Panel B); in contrast, GFP expressed in neurons did not. Data reported by us (Figure 25, Panel E) and others (Metzger et al., 1996) suggest that ApoE CT expressed in neurons can migrate to amyloid plaques and locally regulate γ-secretase at hotspots of amyloid formation (Jorda-Siquier et al., 2022). When ApoE2 and ApoE CT were expressed in 5×FAD mice using lentiviral vectors, a similar distribution pattern was detected around amyloid plaques (Fig. 24, Panel A, arrows, ApoE in amyloid plaques; sessile arrows, ApoE in neurons); on the other hand, GFP and ApoE2 NT did not accumulate around plaques (Fig. 24, Panel A). In 1-month-old 5×FAD mice that did not produce amyloid plaques (Fig. 24, Panel B) and WT mice (Fig. 24, Panel C), ApoE CT was present only in neurons and colocalized with the intracellular distribution of expressed human APP (hAPP). These results suggest that CT is required for the accumulation of ApoE around amyloid plaques.

[0349] 4. Consideration The continued failure of efforts to target Aβ for AD treatment has called into question the role of Aβ in AD (Liu et al. 2019; Panza et al. 2019; Roberson and Mucke 2006). Inevitably, this failure also challenges much of our fundamental understanding of AD itself. For example, γ-secretase dysfunction is undoubtedly a causative factor in fAD, while human genetic evidence strongly links Aβ dysregulation to fAD (Selkoe, 2001). However, the evidence supporting the role of Aβ in sAD is much weaker. We cannot completely rule out the possibility that sAD is a separate disease that happens to share the same pathological features as fAD (Armstrong, 2013; De Strooper and Karran, 2016). Indeed, fAD and sAD differ in ways beyond the underlying genetic factors, including age at onset, detailed pathological abnormalities, and symptoms (Dorszewska et al. 2016; Roher et al. 2016).

[0350] ApoE is the most important genetic factor for sAD, and the data obtained from this experiment show for the first time that ApoE appears to reduce the risk of sAD by selectively inhibiting γ-cleavage of APP, a key step in amyloid formation. These results of the present invention explain why ApoE4 carriers have higher amyloid burden and less metabolites from non-amyloidogenic pathways compared to ApoE2 carriers (Corder et al., 1993; Hashimoto et al., 2012; Olsson et al., 2003; Schmechel et al., 1993). In other words, the data obtained from this experiment support that precise inhibition of γ-secretase prevents the risk of sAD, and therefore it is reasonable to treat sAD by inhibiting γ-secretase. Of note, recent studies have found that most of the progerin mutations in fAD result in reduced γ-secretase activity (Sun et al., 2017; Xia et al., 2015). This suggests that although γ-secretase dysfunction plays a central role in the pathogenesis of sAD and fAD, the changes in γ-secretase activity are opposite in these two different AD types. In this light, γ-secretase inhibition may not be applicable to fAD, which has loss-of-function progerin mutations. This raises the interesting questions of why both high and low γ-secretase activity lead to AD, and whether fAD should be classified into different groups based on the effect of progerin mutations on γ-secretase activity.

[0351] At the same time, considering the wide range of γ-substrates yet to be tested (Guner and Lichtenthaler, 2020), APP may not be the only substrate whose cleavage is inhibited by ApoE, and thus the effect of ApoE on γ-enzyme cleavage of APP may not be the only cause of sAD risk, but it is still plausible. Thus, inhibiting γ-secretase may not have the same effect as Aβ clearance. This is a particularly interesting question, as it may help explain why Aβ clearance strategies alone do not yield satisfactory clinical results. By analyzing which substrates of γ-secretase are affected by ApoE, the present study provides an opportunity to explore the role of γ-secretase substrates other than Aβ in the pathogenesis of AD.

[0352] In mouse models, ApoE is mainly expressed in glial cells (Xu et al., 2006). Because Aβ is produced in neurons, most studies have focused on how ApoE secreted from glial cells affects extracellular amyloid processes (Holtzman et al., 1999; Huang et al., 2017; Huynh et al., 2017; Liu et al., 2017; Yu et al., 2014). Indeed, many papers from different laboratories have independently shown that both ApoE mRNA and protein are detectable in human neurons (Aoki et al., 2003; Han et al., 1994; Metzger et al., 1996; Strittmatter et al., 1993; Xu et al., 1999). Recent single-nucleus RNA-seq data also suggest that ApoE is detectable in neurons, with up to 28% of neurons in AD patients expressing high levels of ApoE (Belonwu et al., 2022; Zalocusky et al., 2021), consistent with our observation that approximately 30–40% of neurons in the human cerebral cortex express ApoE. 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). It has also been reported that human neurons with the ApoE4 subtype produced more Aβ than those with other ApoE subtypes (Wang et al., 2018), strongly supporting the idea that endogenously expressed ApoE in human neurons controls endogenous APP metabolism. However, the role of ApoE in neurons remains poorly understood. The present invention found that the protective subtype ApoE2 directly inhibits γ-cleavage of APP in a cell-autonomous manner through its CT, whereas ApoE4 lacks this activity. Importantly, the present invention also found that endogenous ApoE in mouse neurons significantly inhibits γ-cleavage of endogenously expressed APP; and that there is an interaction between endogenous ApoE and endogenously expressed γ-secretase in human brain. Furthermore, a mouse model of AD harbors many of the major pathological features of AD, but does not harbor ApoE-induced amyloid deposition (Oakley et al., 2006).Surprisingly, we found that ApoE expressed in neurons of 5xFAD mice migrated to and accumulated around amyloid plaques, which is consistent with previous reports that ApoE is present in amyloid plaques in the brains of AD patients (Han et al., 1994; Strittmatter et al., 1993), suggesting that ApoE in plaques is of neuronal origin. These findings emphasize the crucial role of ApoE in neurons in AD and provide a new mechanistic explanation for the differential contribution of ApoE2 and ApoE4 to the pathogenesis of AD.

[0353] γ-secretase is a potential drug target for AD. However, in past drug development, inhibition of γ-secretase activity was found to lead to severe side effects, possibly due to the low selectivity of γ-secretase inhibitors across different substrates (Panza et al., 2019; Roberson and Mucke 2006; Xia 2019). Broad and nonspecific inhibitors, when inhibiting γ-secretase and blocking the production of Aβ, also inhibit the normal processing of other substrates of γ-secretase (Panza et al., 2019; Xia, 2019), causing side effects. The development of γ-secretase modulators that selectively inhibit γ-cleavage of APP is a promising new strategy (Xia, 2019). The present invention found that ApoE CT inhibits γ-cleavage of APP but does not affect γ-cleavage of NOTCH and APLP1, indicating that the γ-cleavage inhibitory effect of ApoE CT is substrate specific. As previously reported, small molecule drugs inhibit γ-secretase activity by occupying the same position as the β-chain on PS1 that interacts with substrates (e.g., APP or Notch (Yang et al., 2021)). ApoE CT is a structurally distinct molecule from conventional small molecule inhibitors, and therefore may act on many altered sites, affecting the binding of only certain substrates to γ-secretase but not others, and thus achieving substrate selectivity. However, the exact mechanism has yet to be elucidated. Nevertheless, ApoE CT may serve as a new strategic therapeutic approach that may have fewer side effects than conventional γ-secretase inhibitors. In other words, the present invention not only provides a potential mechanism for understanding the role of neuronal ApoE subtypes in the pathogenesis of sAD, but also identifies ApoE CT as a potential therapeutic approach for AD by selectively inhibiting γ-cleavage of AD-associated γ-secretase substrates.

[0354] These results support the idea that precise inhibition of γ-secretase prevents the risk of sAD, as opposed to fAD, which is usually associated with reduced γ-secretase activity. The present invention also identifies ApoE CT as an effective inhibitor of γ-secretase, with rare substrate specificity and the ability to translocate from a distance and act locally at amyloidogenic hotspots around plaques.

[0355] 5. Materials and Methods animal Neurons from 5×FAD mice express human-derived APP and PS1 with five fAD mutations: 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 (lights on from 8 a.m. to 8 p.m.) and had free access to food and water. All mouse procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Chinese Academy of Sciences and the US National Institutes of Health.

[0356] Human and monkey brain tissue All human brain tissues were obtained from the Chinese Brain Bank Center (CBBC), Zhejiang University School of Medicine and the National Health And Disease Human Brain Tissue Resource Center, Hubei, China. Donor information is summarized in Figure 32. Human brain tissues were stored at -80°C before the experiment, fixed in 4% PFA overnight, and frozen in 30% sucrose overnight before sectioning. The following steps are the same as IHC using mouse brain sections.

[0357] Fixed brain slices from adult rhesus monkeys were obtained from the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China ( Wu et al., 2021 ).

[0358] Primary neuron culture Primary isolated neuronal cultures were prepared as previously described (Chen et al., 2014). Whole mouse (C57BL / 6) or rat (SD) hippocampi or cortices were removed from day 16 wild-type embryos and dissociated by treatment with papain (Worthington) for 10 min at 37°C. Isolated cells were seeded at 150,000 cells / well (24-well plates) in B27 neuronal growth medium. Neurons were cultured at 37°C in a 5% CO2 incubator and fresh medium was added once a week.

[0359] Construction of PS1 KO, PS2 KO and PS1 / 2 dKO cell lines HEK 293T cells were cultured in DMEM medium (Gibco) supplemented with 10% bovine serum albumin (Life Technologies) and 1% penicillin / streptomycin (Life Technologies). To prepare PS1, PS2 single knockout or PS1 / 2 double knockout HEK 293T cell lines, we used the CRISPR / Cas9 genome editing method (Ran et al., 2013). Guide RNA (gRNA) targeting PS1 exon 4, 5'-ATTATCTAATGGACGACCCC-3' (SEQ ID NO:91) and gRNA targeting PS2 exon 4, 5'-CAAATACGGAGCGAAGCACG-3' (SEQ ID NO:92), were subcloned into pSpCas9(BB)-2A-puro backbone, respectively (PS1gRNA, PS2gRNA). Plasmids containing gRNAs were transfected into HEK 293T cells using Lipofectamine2000 (Invitrogen Technology). 18 hours after transfection with PS1 gRNA or PS2 gRNA, HEK 293T cells were treated with 3 μg / ml puromycin (Selleck). When empty plasmid-transfected HEK 293T cells had completely died, PS1 gRNA- or PS2 gRNA-transfected HEK 293T cells were cultured in a 10 cm dish at a dilution rate of 1:400 to confirm that each cell was a single clone. After individual cells grew into cell clusters, genome-edited cell clusters were selected and verified by DNA sequencing and Western blot. To construct the PS1 / 2dKO cell line, PS1 gRNA was transfected into PS2 knockout cells and a second screening was performed.

[0360] Protein blotting (WB) Plasmids were transfected into HEK 293T or N2A cells for 36-48 h and lysed in cell lysis buffer [10% 1M Tris-HCl (pH = 6.8), 4% SDS, 20% glycerol, 0.01% bromophenol blue]. The lysates were shaken for 10 min at room temperature, boiled for 10 min, and centrifuged at 12,000 × g for 10 min. Proteins were subjected to 8% or 12% Tris-glycine SDS-PAGE and transferred to PVDF membranes (Life Technologies). Samples were incubated overnight at 4°C with primary antibodies diluted in 5% BSA buffer (1 × TBST). After washing three times with 1 × TBST, the cells were incubated with secondary antibodies diluted in 5% BSA buffer (1 × TBST) for 2 h at room temperature. Finally, after washing three times with 1× TBST, signals were detected with ECL protein blotting substrate (Solarbio, PE0010).

[0361] Brain tissue samples were lysed in RIPA buffer (Solarbio, R0010) using a TissueLyser II (QIAGEN, 85300). The lysate was boiled for 10 min and centrifuged at 12,000 × g for 10 min. Western blot procedures were the same as for cell samples.

[0362] Preparation of APEX-tagged proteins for protein blotting The method to target protein interactions in live cells by APEX2 was applied as previously described (Hung et al., 2016). Approximately 30 hours after transfection, HEK 293T cells were incubated with APEX2 plasmid through Lipofectamine 2000 (Invitrogen Technology) in 1 ml complete medium with or without 250 uM biotin phenol (BP) for 30 min at 37 °C. Cells were treated with 1 mM H2O2 for 1 min and washed three times with quencher solution (10 mM sodium ascorbate, 5 mM Trolox, 10 mM sodium azide in 1x DPBS). Cells were lysed with RIPA lysis buffer (1x protein inhibitor mix, 1 mM PMSF and 10 mM sodium ascorbate, 5 mM Trolox and 10 mM sodium azide). Lysates were sonicated on ice, centrifuged (12,000 × g, 4 °C, 10 min), and the supernatant was incubated with streptavidin magnetic beads overnight at 4 °C. Beads were collected on a magnetic rack and washed with a series of buffers (twice with RIPA buffer, once with 1 M KCl, once with 0.1 M Na2CO3, twice with 10 mM Tris-HCl (pH 8.0) in 2 M urea, and twice with RIPA lysis buffer). Biotinylated proteins were eluted from the beads by boiling each sample for 10 min in loading buffer containing 2 mM biotin and 20 mM DTT, and the eluate was collected. Samples were further subjected to protein blotting.

[0363] Co-immunoprecipitation (co-IP) HEK 293T cells were treated with Lipofectamine 2000 (Invitrogen Technology) together with the plasmid to be transfected for 48 hours, and the HEK 293T cells were lysed in 1% CHAPSO (SIGMA), 25 mM HEPES (pH 7.4) (Gibco), 150 mM NaCl (containing 1x protease inhibitor mixture), centrifuged at 14,000 x g for 20 minutes, and the supernatant was collected. The supernatant was incubated with anti-FLAG antibody (Sigma) or anti-IgG antibody (Santacruz) for 3 hours at 4°C, and pulled down with ProteinG Sepharose beads (Gen Script), then washed three times for 15 minutes with 0.1% digitalis saponin-HEPES buffer, and recovered with cell lysis buffer [10% 1M tris-HCl (pH = 6.8), 4% SDS, 20% glycerol, 0.01% bromophenol blue]. The samples were then subjected to protein blotting.

[0364] Immunofluorescence staining and immunohistochemistry (IHC) Neurons and HEK 293T cells cultured on coverslips in 24-well plates were washed with 1x PBS (2.5 mM MgCl2 and 0.5 mM CaCl2) and fixed with 4% PFA + 4% sucrose in 1x PBS for 10 min at room temperature. They were then permeabilized with 0.15% Triton-X in 1x PBS for 15 min and blocked with 5% BSA in 1x PBS for 30 min at room temperature. Samples were incubated with primary antibodies diluted in 5% BSA (in 1x PBS) buffer overnight at 4°C. Samples were then washed once with 0.5 M NaCl in 1x PBS and three times with 1x PBS. They were then incubated with secondary antibodies diluted in 5% BSA (in 1x PBS) buffer and left at room temperature for 1 h. After three final washes with 1x PBS, samples were mounted with Prolong Gold Antifade Mountant (Life Technology). All images were taken and analyzed in a blinded fashion.

[0365] 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. Frozen brain sections (40 μM) were obtained from fixed and cryoprotected mouse brains using a freezing microtome (Leica CM3050S). Sections were floated, washed three times with 1× PBS, and permeabilized for 30 min with 1× PBS supplemented with 0.5% Triton-X. After blocking with 5% BSA in 1× PBS for 1 h at room temperature, sections were incubated overnight at 4°C in 5% BSA (in 1× PBS) buffer containing primary antibodies. Sections were washed three times with 1× PBS and incubated with secondary antibodies diluted in 5% BSA (in 1× PBS) buffer for 1 h at room temperature. Samples were then washed three times with 1× PBS and mounted with Prolong Gold Antifade Mountant (LifeTechnology).

[0366] THS staining 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. Frozen brain sections (40 μM) were obtained from fixed and cryoprotected mouse brains using a freezing microtome (Leica, CM3050S). Sections were floated and washed three times with ddH2O, then incubated in 1% THS (SIGMA) in water for 5 min at room temperature. Sections were then washed twice with 80% ethanol and once with 95% ethanol for 3 min each. For IHC, samples were washed with ddH2O and three times with 1× PBS.

[0367] Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from HEK 293T cells using TRIzol Reagent (Invitrogen). Reverse transcription was performed using the QuantiTect SYBR-Green RT-PCR (reverse transcription PCR) kit (Qiagen). GAPDH was used as an internal control. For ADAM10 mRNA, forward primer 5'-AGAGCAACATCTGGGGACAAA-3' (SEQ ID NO:93) and reverse primer 5'-TCCACAAATAGGTTGGCCAGAT-3' (SEQ ID NO:94); for BACE1 mRNA, forward primer 5'-AACGAATTGGCTTTGCTGTC (SEQ ID NO:95) and reverse primer 5'-AGGCTATGGTCATGAGGGTTG-3' (SEQ ID NO:96); for PS1 mRNA, forward primer 5'-TGACTCTCTGCATGGTGGTG-3' (SEQ ID NO:97) and reverse primer 5'-TCAGAATTGAGTGCAGGGCT-3' (SEQ ID NO:98); for PS2 mRNA, forward primer 5'-TTTGAGCCTCCCTTGACTGG-3' (SEQ ID NO:99) and reverse primer 5'-GGTATTCCAGTCCCCGCTG-3' (SEQ ID NO:99). NO:100) was used for qRT-PCR. Quantitation was tested using comparative threshold cycling measurements of SYBR green fluorescent signal (Applied Biosystems, Thermo Fisher Scientific).

[0368] Enzyme-linked immunosorbent assay (ELISA) HEK293T and N2A cells were transfected for 48 h or neurons were infected with lentivirus 4.5 days later, and media and lysates were harvested. Aβ levels in media and cell lysates were measured by Aβ ELISA kit according to the protocol instructions. 40 and Aβ 42 (Invitrogen Technology; human Aβ40 was detected with KHB3481, and human Aβ 42 KHB3441 is a mouse Aβ 40 is detected by KMB3481).

[0369] Protein purification Human γ-secretase complex was purified as previously described ( Lu et al., 2014 ), and the cDNA and expression vector encoding human γ-secretase complex were also referenced in Lu et al., 2014 .

[0370] Purification of human ApoE protein. 48 hours after transfection of cells with human ApoE plasmids (ApoE2-3xFLAG or ApoE2NT-3xFLAG), the transfected cells were washed once with cold 1x PBS, harvested and centrifuged at 800g. The cell pellet was resuspended in 5 ml of lysis buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), 0.05% (v / v) Tween-20 and 1x protease inhibitor mixture. Cell lysis was performed by sonication and cell debris was separated by centrifugation (12,000xg, 4°C, 15 min). The supernatant was incubated with anti-FLAGM2 affinity gel (SIGMA) for 90 min at 4°C. The resin was then washed three times with a buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 0.05% (v / v) Tween-20. ApoE protein was eluted with a buffer containing 25 mM HEPES, pH 7.4, 150 mM NaCl, and 500 μg / ml 3×FLAG peptide (SIGMA). SDS-PAGE and protein blotting were used to demonstrate the purified protein.

[0371] Purification of β-CTF. Human β-CTF-Myc-6×His plasmid was transformed into E. coli cells, and when the OD260 reached 0.6-0.8, the E. coli cells were induced with 0.5 μM IPTG (Solarbio) at 37 °C for 4 h. The E. coli cells were lysed in the corresponding 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 min and dissolved in urea buffer (30 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 6 M urea, 1% TritonX-100, 1 mM CaCl2, 1× protease inhibitor mixture) and slowly rotated for 3 h. The resuspended lysate was centrifuged twice at 40,000 × g for 30 min, and the supernatant was incubated with Ni-NTA beads (GE Healthcare) for 2 h at room temperature. 10 column volumes (CV) of urea buffer 1 (30 mM Tris-HCl, pH 8.0; 300 mM NaCl, 1 mM EDTA, 6 M urea, 1% TritonX-100, 1 mM CaCl2, 1 × cocktail), 20 CV of wash buffer 2 (30 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% TritonX-100), and 20 CV of wash buffer 3 (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% Sharpsol) were used. Bound material was eluted with wash buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% CHAPSO) with a 0-300 mM imidazole gradient. SDS-PAGE and protein blotting were used to visualize purified proteins.

[0372] Three-dimensional stereotactic injection of viruses After deep anesthesia with isoflurane, mice (5 weeks old) were placed in a stereotaxic apparatus (RWD Instruments). The zero point (x,y,z,0,0,0) is the bregma. Lentivirus (lentivirus-GFP, lentivirus-ApoE and lentivirus-ApoE-IRES-GFP) and AAV (AAV2 / 9-hSyn-EGFP-WPRE-pA and AAV2 / 9-hSyn-ApoECT; purchased from Shanghai Taitou Biotechnology) were injected into the cerebral cortex (x,y,z,+ / -1.6,0,-1) or hippocampus (x,y,z,+ / -1.5,-2,-1.5), with 1ul of each injection site. After surgery, the mice were returned to their cages for rehabilitation.

[0373] Quantization and Statistical Analysis Data were analyzed using unpaired, paired or one sample t-tests with GraphPadPrism software (v6). 40 and Aβ 42 Levels were normalized to the corresponding GFP control. α-CTF levels were normalized to FL-APP and the corresponding GFP control. All data are expressed as mean ± SEM. Statistical significance was defined as p<0.05. One, two, three, and four stars (*) in the figures indicate p-values ​​<0.05, <0.01, <0.001, and <0.0001, respectively.

[0374] 6. References

[0375] [Table 18]

[0376] [Table 19]

[0377] [Table 20]

[0378]

Table 21

[0379]

Table 22

[0380]

Table 23

[0381]

Table 24

[0382]

Table 25

[0383]

Table 26

[0384]

Table 27

[0385]

Table 28

Claims

1. An isolated ApoE construct comprising an ApoE polypeptide. The ApoE polypeptide (1) A fragment derived from the amino acid sequence shown in SEQ ID NO: 12, in which the first amino acid residue at the N-terminus of the fragment is any one of amino acid residues 215-221 of SEQ ID NO: 12, and the most C-terminal amino acid residue of the fragment is any one of amino acid residues 274-299 (preferably positions 279-299) of SEQ ID NO: 12; or (2) The amino acid sequence has at least about 70% sequence identity with the fragment described in (1) and retains at least about 50% of the inhibition of gamma-secretase cleavage activity by the fragment described in (1).

2. The isolated ApoE construct according to claim 1, wherein the ApoE polypeptide or a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) comprises any one of the following sequences: (i) the amino acid sequence of amino acid residues 215-299 of SEQ ID NO:12; (ii) the amino acid sequence of amino acid residues 216-299 of SEQ ID NO:12; (iii) the amino acid sequence of amino acid residues 217-299 of SEQ ID NO:12; (iv) the amino acid sequence of amino acid residues 218-299 of SEQ ID NO:12; (v) the amino acid sequence of amino acid residues 219-299 of SEQ ID NO:12; (vi) the amino acid sequence of amino acid residues 220-299 of SEQ ID NO:12; (vii) the amino acid sequence of amino acid residues 221-299 of SEQ ID NO:12; (viii) the amino acid sequence of amino acid residues 216-294 of SEQ ID NO:12; (ix) the amino acid sequence of amino acid residues 216-289 of SEQ ID NO:12; (x) the amino acid sequence of amino acid residues 219-289 of SEQ ID NO: 12; (xi) the amino acid sequence of amino acid residues 219-288 of SEQ ID NO:12; (xii) the amino acid sequence of amino acid residues 219-287 of SEQ ID NO:12; (xiii) the amino acid sequence of amino acid residues 219-286 of SEQ ID NO: 12; (xiv) the amino acid sequence of amino acid residues 219-285 of SEQ ID NO:12; (xv) the amino acid sequence of amino acid residues 219-284 of SEQ ID NO:12; (xvi) the amino acid sequence of amino acid residues 219-279 of SEQ ID NO:12; (xvii) the amino acid sequence of amino acid residues 219-274 of SEQ ID NO:12; (xviii) the amino acid sequence of amino acid residues 220-274 of SEQ ID NO:12; (xix) the amino acid sequence of amino acid residues 220-279 of SEQ ID NO: 12; or (xx) the amino acid sequence of amino acid residues 221-274 of SEQ ID NO:

12.

3. The isolated ApoE construct according to claim 1 or 2, characterized in that the ApoE polypeptide or a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) comprises any one of the amino acid sequences in SEQ ID NO: 13, 18-34, 78 and 87.

4. The isolated ApoE construct described in any one of claims 1 to 3, characterized in that the ApoE polypeptide or a fragment derived from the amino acid sequence shown in SEQ ID NO: 12 in (1) comprises an amino acid sequence shown in any one of SEQ ID NO: 31-34, 78 and 87.

5. 5. The isolated ApoE construct of any one of claims 1 to 4, wherein the ApoE polypeptide comprises one or more amino acid mutations relative to the amino acid sequence set forth 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+E245+Q246+A247+Q248+Q249+I250+R251+L252+Q253+A254.

6. The isolated ApoE construct of any one of claims 1 to 5, characterized in that the ApoE polypeptide comprises one or more amino acid site mutations 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, A255A, A256A, A257A, A258A, A259A, A260A, A261A, A262A, A263A, A264A, A265A, A266A, A267A, A268A, A269A, A270A, A271A, A272A, A273A, A274A, A275A, A276A, A277A, A278A, A279A, A280A, A287A, A289A, A282A, A283A, A284A, A285A, A286A, A287A, A289A, A288A, A289A, A290A, A291A, A292A, A293A, A294A, A295A, A296A, A297A, A298A, A299A, A300A, A301A, A302 91T, P293A, V294A, P295A, S296A, D297A, N298A, H299A, R251A+T289A, R251S+T28 9A+A291T, S290A+P293A+V294A+P295A+S296A+D297A+N298A+H299A, R226A+D227 A+R228A+L229A+D230A, E238A+V239A+R240A+K242A, or E244del+E245del+Q246del+A247del+Q248del+Q249del+I250del+R251del+L252del+Q253del+A254del.

7. The isolated ApoE construct of any one of claims 1-6, wherein the ApoE polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 46-52, 57-59, 62 and 76.

8. The isolated ApoE construct of any one of claims 1 to 7, characterized in that the N-terminus of the ApoE polypeptide in the ApoE construct further comprises an amino acid sequence of amino acid residues 1-167, 1-205, or 1-214 of SEQ ID NO:12, or has a sequence that is at least about 70% identical to amino acid residues 1-167, 1-205, or 1-214 of SEQ ID NO:

12.

9. 9. The isolated ApoE construct of claim 8, wherein the ApoE construct comprises the amino acid sequence shown in SEQ ID NO: 60 or 61.

10. The isolated ApoE construct of any one of claims 1 to 9, characterized in that the ApoE construct further comprises a membrane-permeable peptide at the N-terminus or C-terminus of the ApoE polypeptide.

11. The isolated ApoE construct of claim 10, wherein the membrane-permeable peptide comprises any one of the amino acid sequences of SEQ ID NOs: 35-45, 63-65 and 84-86.

12. The isolated ApoE construct of claim 10 or 11, wherein the membrane-permeable peptide comprises the amino acid sequence shown in SEQ ID NO: 63 or 65.

13. The isolated ApoE construct according to any one of claims 10-12, characterized in that the membrane-permeable peptide is connected to the ApoE polypeptide, or to an amino acid sequence comprising amino acid residues 1-167, 1-205, or 1-214 of SEQ ID NO: 12, or to a sequence having at least about 70% identity to amino acid residues 1-167, 1-205, or 1-214 of SEQ ID NO: 12, via a linker sequence.

14. The isolated ApoE construct of any one of claims 1 to 13, wherein the ApoE construct further comprises a signal peptide at the N-terminus of the ApoE polypeptide.

15. The isolated ApoE construct of claim 14, wherein the signal peptide comprises any one of the amino acid sequences selected from SEQ ID NOs: 66-68.

16. An isolated nucleic acid encoding a protein portion of the isolated ApoE construct of any one of claims 1-15.

17. A vector comprising the isolated nucleic acid of claim 16.

18. 18. The vector of claim 17, wherein the vector contains a neuron-specific promoter at the 5' end of the isolated nucleic acid.

19. The vector according to claim 18, characterized in that the neuron-specific promoter is selected from the group consisting of a synapsin promoter, a thy-1 promoter and a calmodulin-dependent protein kinase II-α promoter.

20. The vector according to any one of claims 17 to 19, characterized in that the vector is a recombinant AAV or lentivirus expression vector.

21. A host cell characterized in that it expresses the isolated ApoE construct according to any one of claims 1-15, contains the isolated nucleic acid according to claim 16, or contains the vector according to any one of claims 17-20.

22. The host cell of claim 21 , wherein the host cell is a neuron.

23. 21. A pharmaceutical composition comprising: (i) an isolated ApoE construct according to any one of claims 1-15, an isolated nucleic acid according to claim 16, a vector according to any one of claims 17-20, or a host cell according to claim 21 or 22; and (ii) a pharma- ceutical acceptable carrier.

24. Use of an isolated ApoE construct according to any one of claims 1-15, an isolated nucleic acid according to claim 16, a vector according to any one of claims 17-20, a host cell according to claim 21 or 22, or a pharmaceutical composition according to claim 23 in the preparation of a medicament for treating or preventing a disease associated with the cleavage activity of gamma-secretase in an individual.

25. A method for treating or preventing a disease associated with the cleavage activity of gamma-secretase in an individual, the method comprising administering to said individual an effective dosage of an isolated ApoE construct according to any one of claims 1-15, an isolated nucleic acid according to claim 16, a vector according to any one of claims 17-20, a host cell according to claim 21 or 22, or a pharmaceutical composition according to claim 23.

26. The use or method according to claim 24, characterized in that the medicament or method inhibits the cleavage activity of gamma-secretase and treats or prevents a disease associated with the cleavage activity of gamma-secretase.

27. The use or method according to any one of claims 24 to 26, characterized in that the disease is selected from the group consisting of degenerative diseases of the nervous system, cancer, inflammatory diseases and kidney diseases.

28. (i) the nervous system degenerative disease is selected from Alzheimer's disease and related diseases, amyloid cerebrovascular disease, Down's syndrome, and other age-related neurodegenerative diseases (such as frontotemporal dementia); (ii) the cancer is selected from head and neck cancer, breast cancer, liver cancer, pancreatic cancer, ovarian cancer, lung cancer, glioma, fibroma, lymphoma, osteosarcoma, gastric cancer, and bladder cancer; (iii) the inflammatory disease is selected from asthma, pneumonia, and airway inflammation; and / or (iv) the kidney disease is selected from acute kidney injury, clear cell renal cell carcinoma, renal fibrosis, and obstructive nephropathy; 28. Use or method according to any one of claims 24 to 27.

29. The use or method according to any one of claims 24 to 28, characterized in that the individual is a human.

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