APOE antibodies, fusion proteins and uses thereof

Abstract

The invention relates to APOE antibodies, fusion proteins and uses thereof. Methods and compositions for preventing or treating cognitive decline and / or neurodegenerative disorders associated with dementia and / or mild cognitive impairment using antibodies, peptides, fusion proteins or genome editing systems that modulate the HSPG / heparin binding affinity of ApoE.

Description

[0001] This application is a divisional application of the patent application "APOE antibodies, fusion proteins and their uses" with an international application date of May 28, 2020 and application number 202080054661.6 (international application number PCT / US2020 / 034978).

[0002] Priority claim

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 853,676, filed May 28, 2019, and U.S. Provisional Application No. 62 / 873,019, filed July 11, 2019. The entire contents of the foregoing are incorporated herein by reference.

[0004] Federally sponsored research or development

[0005] This invention was made with U.S. government support under Grant No. OD019833 from the National Institutes of Health, Grant Nos. AG054671, AG031581 and AG19610 from the National Institute on Aging, and Grant Nos. NS100121 and NS110048 from the National Institute of Neurological Disorders and Stroke. The U.S. government has certain rights in this invention. Technical Field

[0006] Described herein are methods and compositions for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment by modulating the heparan sulfate proteoglycan (HSPG) / glycosaminoglycan (GAG) heparin binding affinity of apolipoprotein E (ApoE). Background Art

[0007] Alzheimer's disease (AD) is a chronic neurodegenerative disease that usually starts slowly and gradually worsens over time. It is the cause of 60-70% of dementia cases. The disease process is associated with plaques and neurofibrillary tangles in the brain. There is currently no treatment that can stop or reverse its progression, but some may temporarily improve symptoms. The accumulation, aggregation, and deposition of amyloid-β (Aβ) peptides in the brain are at the core of the pathogenesis of Alzheimer's disease (AD). There is growing evidence that ApoE strongly affects AD pathogenesis by controlling Aβ aggregation and metabolism (Fu et al., Mol Neurodegener 11:37, 2016). APOE affects the production, aggregation, and clearance of amyloid, is a component of amyloid plaques, and worsens tau-mediated neurodegeneration. ApoE is 299 amino acids long and is polymorphic, with three major alleles (ε2, ε3, and ε4) that differ by only one or two amino acids at positions 112 and 158: ApoE2 (cys112, cys158), ApoE3 (cys112, arg158), and ApoE4 (arg112, arg158). Therefore, there is a need for therapies that target and modulate the function of the ApoE protein for the treatment or prevention of AD and cognitive decline associated with dementia or mild cognitive impairment. Summary of the invention

[0008] In one aspect, the present disclosure features a separated monoclonal antibody that specifically binds to one or more (e.g., 1, 2, 3, or 4) HSPG binding sites or one or more (e.g., 1, 2, 3, or 4) HSPG binding allosteric regulatory sites of wild-type or mutant apolipoprotein E (ApoE). In some embodiments, the antibody binds to a polypeptide having an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to TEELRVRLASHLRK (SEQ ID NO: 3). In some embodiments, the antibody binds to a polypeptide having an amino acid sequence that is at least 95% (e.g., 96%, 97%, 98%, 99%, or 100%) identical to TEELRVSLASHLRK (SEQ ID NO: 2). In some embodiments, the antibody binds to one or more (e.g., 1, 2, 3, or 4) HSPG binding sites of wild-type or mutant ApoE2, ApoE3, or ApoE4.

[0009] In some embodiments, the antibody competes and / or binds to the same epitope as a reference anti-ApoE antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL of the reference antibody: (i) comprise the amino acid sequence set forth in SEQ ID NO: 13 and the amino acid sequence set forth in SEQ ID NO: 12, respectively; (ii) comprise the amino acid sequence set forth in SEQ ID NO: 23 and the amino acid sequence set forth in SEQ ID NO: 22, respectively; (iii) comprise the amino acid sequence set forth in SEQ ID NO: 33 and the amino acid sequence set forth in SEQ ID NO: 32, respectively; or (iv) comprise the amino acid sequence set forth in SEQ ID NO: 43 and the amino acid sequence set forth in SEQ ID NO: 42, respectively. In some embodiments of any of the antibodies described herein, the antibody competes and / or binds to the same epitope as a reference anti-ApoE antibody comprising a heavy chain and a light chain, wherein the heavy chain and the light chain of the reference antibody comprise the amino acid sequence set forth in SEQ ID NO: 53 and the amino acid sequence set forth in SEQ ID NO: 52.

[0010] On the other hand, provided herein is an anti-ApoE antibody comprising a VH comprising VHCDR1, VHCDR2 and VHCDR3 and a VL comprising VLCDR1, VLCDR2 and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3: (i) comprise SEQ ID No: 7, 8, 9, 4, 5, 6, respectively; (ii) comprise SEQ ID No: 17, 18, 19, 14, 15, 16, respectively; (iii) comprise SEQ ID No: 27, 28, 29, 24, 25, 26, respectively; (iv) comprise SEQ ID No: 37, 38, 39, 34, 35, 36, respectively; or (v) SEQ ID No: 47, 48, 49, 44, 45, 46, respectively. In some embodiments of any of the antibodies described herein, (i) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 13 and 12, respectively; (ii) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 23 and 22, respectively; (iii) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 33 and 32, respectively; or (iv) VH and VL comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 43 and 42, respectively. In some embodiments of any of the antibodies described herein, the antibody comprises a heavy chain and a light chain, and the heavy chain and the light chain respectively comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence recorded in SEQ ID No: 53 and 52. In some embodiments, the antibody comprises a mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3 heavy chain constant region. In some embodiments, the antibody comprises a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region. In some embodiments, the antibody comprises a human κ or human λ light chain constant region. In some embodiments, the antibody is a complete antibody, a single domain antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, Fv, scFv, sc(Fv)2, a diabody, a nanobody, Fab or F(ab')2. In some embodiments, the antibody further comprises a half-life extension portion. In some embodiments, the antibody further comprises a blood-brain barrier penetration portion. In some embodiments, the antibody further comprises a detectable label. In some embodiments, provided herein are pharmaceutical compositions comprising any of the antibodies described herein.In some embodiments, one or more polynucleotides encoding any of the antibodies described herein are provided herein. In some embodiments, one or more vectors comprising one or more polynucleotides described herein are provided herein. In some embodiments, a host cell comprising one or more polynucleotides described herein or one or more vectors described herein is provided herein. On the other hand, a method for preparing an anti-ApoE antibody is provided herein, the method comprising: (a) culturing any host cell described herein under conditions that allow expression of the antibody; and (b) isolating the antibody. In some embodiments, the method further comprises formulating the antibody into a sterile preparation suitable for administration to a human.

[0011] On the other hand, an Fc fusion protein is provided herein, comprising: a HSPG binding domain of wild-type ApoE or mutant ApoE, the HSPG binding domain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of: STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59) and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60). In some embodiments, the Fc fusion protein comprises the Fc region of a human antibody. In some embodiments, the human antibody is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 molecules. In some embodiments, a pharmaceutical composition comprising any Fc fusion protein described herein is provided herein. In some embodiments, one or more polynucleotides encoding the Fc fusion protein described herein are provided herein. In some embodiments, provided herein are one or more vectors comprising any of the one or more polynucleotides described herein. In some embodiments, provided herein are host cells comprising one or more polynucleotides described herein, or one or more vectors described herein.

[0012] On the other hand, provided herein is a pharmaceutical composition for eliciting an immune response, comprising: (i) a HSPG binding domain of wild-type ApoE or mutant ApoE, wherein the HSPG binding domain comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60); and (ii) a pharmaceutically acceptable adjuvant.

[0013] In another aspect, provided herein are pharmaceutical compositions comprising human cells expressing any of the antibodies described herein or any of the Fc fusion proteins described herein.

[0014] In another aspect, provided herein is a method for improving, slowing, delaying the onset, preventing or reversing cognitive decline and / or neurodegeneration associated with dementia and / or mild cognitive impairment in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any of the antibodies, Fc fusion proteins, or pharmaceutical compositions described herein.

[0015] On the other hand, the present invention provides a method for improving, slowing down, delaying the onset, preventing or reversing cognitive decline and / or neurodegenerative lesions associated with dementia and / or mild cognitive impairment in a human subject in need, the method comprising administering to the subject: (i) a viral vector comprising a nucleotide sequence encoding a gRNA molecule, the gRNA molecule comprising a targeting domain complementary to a target domain from an APOE gene; (ii) a viral vector comprising a nucleotide sequence encoding a Cas9 molecule; and (iii) a viral vector comprising a template nucleic acid, wherein the template nucleic acid comprises adenine to replace the cytosine at position 19: g.45412013C>A in the APOE gene, wherein the administration results in the production of one or more ApoE R136S alleles in one or more cells of the subject. In some embodiments, the targeting domain of the gRNA molecule comprises a sequence that is identical to or differs by no more than 3 nucleotides from a sequence from Table 5.

[0016] As used herein, "preventing" or "preventing" refers to reducing the risk of developing a condition.

[0017] In some embodiments, the human subject is diagnosed with Alzheimer's disease or is at risk of developing Alzheimer's disease. In some embodiments, the human subject carries one or more copies of the APOE4 allele. In some embodiments, the human subject carries one or more mutations in at least one gene selected from the group consisting of: APP, PSEN1 and PSEN2. In some embodiments, the human subject carries one or more mutations in other genes that cause autosomal dominant Alzheimer's disease (for example, in Bateman et al., Alzheimer's Research & Therapy 3 (1): 1, those described in 2011). In some embodiments, the human subject carries all or part of the third copy of chromosome 21. In some embodiments, the human subject is diagnosed with Alzheimer's disease by established biomarkers, such as those obtained by brain imaging or blood or CSF samples. In some embodiments, the human subject is more than (over) 50 years old (for example, more than 55, 60, 65, 70, 75, 80, 85, 90 or 95 years old).

[0018] In some embodiments, the human subject is diagnosed with or is at risk of developing a disorder selected from the group consisting of vascular cognitive impairment, vascular dementia, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Huntington's disease, neurodegenerative diseases, cerebrovascular diseases, brain injury, chronic traumatic encephalopathy, tauopathies, amyloidopathies, synucleinopathies, Creutzfeldt-Jakob disease, retinal degeneration, glaucoma, retinal damage, and aging.

[0019] On the other hand, a method for identifying a human subject who is less likely to develop an early-onset neurodegenerative disease is provided herein, the method comprising: obtaining a biological sample from a subject; detecting the presence of at least one APOE3 mutant allele or the presence of a mutant ApoE3 gene product in the biological sample; and based on the presence of the mutant ApoE3 allele or gene product in the biological sample, the subject is identified as less likely to develop an early-onset neurodegenerative disease. In some embodiments, the biological sample is blood, cerebrospinal fluid, saliva, urine, tears, vitreous humor, aqueous humor, or a tissue specimen. In some embodiments, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, retinal degeneration, or glaucoma. In some embodiments, retinal degeneration is age-related macular degeneration. In some embodiments, detection includes determining the sequence of the APOE3 allele in the subject. In some embodiments, detection includes determining the presence or absence of an APOE3 sequence encoding an ApoE3 protein having a mutation at R136 compared to a wild-type ApoE3 protein. In some embodiments, the mutation at R136 is R136S, R136H, or R136C. In some embodiments of any of the methods described herein for identifying a human subject as less susceptible to developing an early-onset neurodegenerative disease, the method further comprises selecting the subject to include in a clinical trial, and optionally administering an experimental treatment, or excluding the subject from the clinical trial if the subject does not have a mutant APOE3 allele. In some embodiments of any of the methods described herein for identifying a human subject as less susceptible to developing an early-onset neurodegenerative disease, the method further comprises selecting the subject to include in a clinical trial, and optionally administering an experimental treatment, or excluding the subject from the clinical trial if the subject has a mutant APOE3 allele.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A model showing the structure of the wild-type APOE3 protein. The N-terminal (residues 1-191) and C-terminal (residues 201-299) domains are shown. The amino acid positions of the APOE4 (C112R), APOE3ch (R136S), and APOE2 (R158C) variants are shown.

[0023] Figure 2 Representative Sanger sequencing results for APOE from control, proband, and offspring samples are shown.

[0024] Figure 3 The pedigree of the subjects is depicted, with circles representing females, squares representing males, diamonds representing individuals whose gender is masked for privacy, arrows depicting proband individuals with MCI, and shading representing individuals with a history of dementia. Deceased individuals are marked with a crossed bar. Individual APOE and PSEN1 genotypes are indicated as needed to maintain anonymity.

[0025] Figure 4A and 4B Fundus photographs of the right and left eyes, respectively. Figure 4C Infrared image showing the right eye, which describes Figure 4D Cross-section of the retina (line) as seen in . Figure 4D Shown are the results of optical coherence tomography (OCT) of the right eye. Figure 4E An infrared image of the left eye is shown. Figure 4F Shown are the results of OCT imaging of the left eye.

[0026] Figure 5 Shown are brain imaging results showing amyloid plaque burden and PHF Tau burden in the brains of PSEN1 mutation carriers with late-onset MCI (mild cognitive impairment) and PSEN1 mutation carriers with MCI onset at the typical age for this mutation (44 years).

[0027] Figure 6 Shown are measurements of amyloid burden, tau burden, hippocampal volume, and glucose metabolism levels in PSEN1 E280A mutation carriers. Red dots represent measurements of carriers with two APOE3ch alleles and unusually late onset of MCI. Black dots represent PSEN1 E280A mutation carriers with MCI at a younger age than typical for relatives at the onset of MCI. Gray dots represent PSEN1 E280A mutation carriers who have not yet developed MCI.

[0028] Figure 7Shown is the rate of Aβ42 fibril formation in the presence of APOE3 wild-type APOE3ch or in the absence of APOE as detected by Thioflavin T fluorescence. Changes in relative fluorescence units (RFU) are plotted against time in minutes (min). (***P<0.001, ****P<0.0001)

[0029] Figure 8 Schematic diagram showing split-luciferase complementation triggered by amyloid oligomerization (upper) and the percentage of luminescence obtained by the split-luciferase complementation assay after 24 hours in culture medium from 293T cells transfected with ApoE3ch or ApoE3 wild-type.

[0030] Fig. 9 ELISA results showing heparin binding affinity for APOE2 and APOE4.

[0031] Fig.10 Immunoblot analysis showing the heparin binding affinity of ApoE2, ApoE3, ApoE4 and ApoE3ch.

[0032] Fig.11A and 11B ELISA results showing the heparin binding affinity of ApoE2, ApoE3, ApoE4, and ApoE3ch.

[0033] Fig. 12A is a schematic diagram showing the experimental setup used to test the specificity of monoclonal ApoE3 antibodies in blocking ApoE3 / heparin binding. Fig. 12B and 12C is a schematic diagram showing the process of passing the ApoE3 protein pre-incubated with the monoclonal antibody through a heparin binding column, followed by washing and elution.

[0034] Figures 13A-13B Shown are the results of a BCA assay performed on various fractions from the heparin-binding column. Fig.13A The amounts of ApoE3 in various fractions in the absence of ApoE3 antibodies are shown. Fig. 13B The amount of ApoE3 in each fraction in the presence of ApoE3 antibody is shown.

[0035] Fig.14 Immunoblotting results are shown, showing the amount of ApoE3 in various fractions pre-incubated with or without monoclonal ApoE3 antibody.

[0036] Figures 15A-15G ELISA analysis of 19G10-2, 23B2, 2H79-1, 30E1-2, 16H8, 25F1-2 and 29G10-1 antibodies are shown, respectively.

[0037] Fig.16 Immunoblot analysis showing the heparin binding affinity of ApoE3 treated with wild-type ApoE3 peptide and ApoE3ch mutant peptide.

[0038] Figures 17A-17D Modeling showing the interaction of ApoE fragments with heparin. Fig.17A A model of the wild-type ApoE fragment containing amino acids 129-157 that interact with heparin is shown. Fig. 17B A model of the ApoE R136S fragment containing amino acids 129-157 that interact with heparin is shown. Fig. 17C A model of the wild-type ApoE fragment containing amino acids 114-144 that interact with heparin is shown. Fig.17D A model of the ApoE R136S fragment containing amino acids 114-144 that interact with heparin is shown.

[0039] Figures 18A-18B Heparin affinity chromatography and immunoblot analysis of antibody 1H4 are shown.

[0040] Fig.19 A-19D shows the ELISA results of 1H4-2 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), and ApoE3ch peptide (D).

[0041] Fig. 20 Shown are the ELISA results of 1H4-2 serum tested with ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, and ApoE3ch peptide.

[0042] Fig.21 Representative ELISA profiles of serial dilutions of antibody 1H4 incubated with human recombinant ApoE3 or mouse recombinant ApoE3 are shown.

[0043] Fig. 22 ELISA results of monoclonal 1H4 antibodies purified from cloned hybridomas are shown.

[0044] Fig.23A and 23B Heparin affinity chromatography and immunoblot analysis of antibody 7C11 are shown.

[0045] Fig.24 A-24D shows the ELISA results of 7C11-1 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), or ApoE3ch peptide (D).

[0046] Fig.25 Shown are ELISA results from testing 7C11-1 sera with ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, or ApoE3ch peptide.

[0047] Fig.26 ELISA results of the monoclonal 7C11-1 antibody purified from the cloned hybridoma are shown.

[0048] Fig. 27 Shown are the results of ELISA screening of the 19G10-2 antibody against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins.

[0049] Fig.28A and 28B Heparin affinity chromatography and immunoblot analysis of antibody 19G10-2 are shown.

[0050] Fig.29 Immunoblot of ApoE3 WT incubated with 19G10-2 serum antibody is shown.

[0051] Fig.30 Representative ELISA showing the difference in binding of both serum and monoclonal antibody hybridoma supernatant 19G10-2 to ApoE3WT or ApoE3ch.

[0052] Fig.31 yes Fig.30 Enlarged view of the Y axis.

[0053] Fig.32 ELISA results of the monoclonal 19G10-2 antibody purified from the cloned hybridoma are shown.

[0054] Fig.33 Shown are the results of ELISA screening of the 25F1-2 antibody against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins.

[0055] Fig.34A and 34B Heparin affinity chromatography and immunoblot analysis of antibody 25F1-2 are shown.

[0056] Fig.35 Immunoblot of ApoE3 WT incubated with 25F1-2 monoclonal antibody is shown.

[0057] Fig.36 Representative ELISA showing the difference in binding between 25F1-2 serum and monoclonal antibody hybridoma supernatant 25F1-2 to ApoE3WT or ApoE3ch.

[0058] Fig.37 yes Fig.36 A magnified view of the Y-axis.

[0059] Fig.38 ELISA results of the monoclonal 25F1-2 antibody purified from the cloned hybridoma are shown.

[0060] Fig.39 ELISA screening of 1343 antibodies against the heparin binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins is shown.

[0061] Fig.40 ELISA screening of 1343 antibodies against the heparin binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins is shown.

[0062] Fig.41A Shown is an immunoblot analysis of ApoE in protein fractions eluted from a heparin column in the presence or absence of 1343 antibody. Fig.41B ELISA analysis of fractions is shown.

[0063] Fig.42A An exemplary experimental outline showing an intraocular model of inducible APOE-dependent Tau hyperphosphorylation. Fig.42B PHF tau in control retinas injected with PBS is shown. Fig.42C A retina injected with recombinant human APOE3 is shown.

[0064] Figures 43A-43I Shown are PHF tau in control retinas compared to retinas injected with mouse 1H4-2 antibody or humanized 1343Ah antibody.

[0065] Fig.44 A representative binding assay of 1H4 at increasing concentrations (nM) of ApoE3 protein on a Protein A biosensor is shown. DETAILED DESCRIPTION

[0066] The present disclosure discloses that homozygosity of APOE3ch (having two copies of APOE3 Christchurch (R136S) mutation) is associated with strong resistance to clinical onset of Alzheimer's disease, and the R136S mutation significantly reduces the ability of ApoE to bind to heparan sulfate proteoglycan (HSPG) / heparin. Therefore, the present disclosure relates to antibodies (e.g., antibodies that block the interaction between ApoE and HSPG / GAG / heparin and / or reduce its binding) that bind to wild-type ApoE and / or ApoE isoforms (one or more) containing R136S mutations. Fusion proteins comprising peptide fragments (e.g., HSPG / GAG / heparin binding domains) of wild-type and mutant ApoE containing R136S mutations are also contemplated. These proteins can be administered by human cells expressing such compositions. The present disclosure also relates to small molecules that block the interaction between ApoE and HSPG / heparin, and methods for screening the same small molecules. Compositions and methods for editing the ApoE locus with a genome editing system are also provided. The antibodies, fusion proteins, small molecules and genome editing systems described herein can be used to treat or prevent cognitive decline and / or neurodegenerative diseases associated with dementia and / or mild cognitive impairment (MCI), such as Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease. The antibodies, fusion proteins, small molecules and genome editing systems described herein can also be used to treat or prevent neurodegenerative diseases, cerebrovascular conditions, brain damage, retinal degeneration, optic nerve degeneration or retinal damage.

[0067] Apolipoprotein E (ApoE)

[0068] Apolipoprotein E variants are the main genetic modifiers of AD, promoting susceptibility to late-onset AD. The APOE4 allele causes the cysteine ​​at position 112 to change to arginine, and is associated with a 5-fold increase in AD risk in monoallele carriers, reaching a 20-fold increase in homozygous carriers. APOE2 causes an amino acid change from arginine to cysteine ​​at position 158, and has a protective effect on AD, while the APOE3 allele is considered neutral (Corder et al., Nat Genet (7) 180-184, 1994; Hauser et al., Cure Alzheimer Res (10); 808-817, 2013). APOE affects the production, aggregation and removal of amyloid protein, is a component of amyloid plaques, and worsens tau-mediated neurodegenerative lesions. APOE alleles also regulate lipid metabolism and cardiovascular risk. Approximately 5-10% of APOE2 homozygous individuals develop type III hyperlipoproteinemia (HLP III), while other rare APOE variants are associated with autosomal dominant HLP III. HLP III is characterized by increased plasma cholesterol and triglyceride levels, and the presence of nodular or rhabdomyomas on the palms. The mechanisms by which APOE alleles alter AD risk and cause HLP III are not fully understood. Significant APOE properties affected by specific mutations include differences in: 1) binding affinity for lipids and the LDL receptor; 2) the nature of the interdomain interactions between its N-terminus (amino acids 1 to 199) and C-terminal domains (216 to 299); and, 3) the ability to form homo-oligomers mediated by the C-terminal domain (Frieden et al., PNAS (109): 8913-8918, 2012; Georgiadou et al., PLoS One (6) e27037, 2011; Lalazar et al., J Biol Chem (263) 3542-3545, 1988).

[0069] Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues, which exists in the form of proteoglycans (HSPGs), in which two or three HS chains are attached to cell surface or extracellular matrix proteins in close proximity. HSPGs are partially present in hundreds of proteins located in the plasma membrane and extracellular matrix. Protein-protein interactions mediated by HSPGs play a key role in numerous processes associated with the pathology of Alzheimer's disease, including amyloid and tau pathology. Heparan sulfate is a member of the glycosaminoglycan family of carbohydrates and is closely related to heparin in structure. Both are composed of variable sulfated repeating disaccharide units. Heparan sulfate binds to a large number of extracellular proteins. These are generally collectively referred to as "heparin interactome" or "heparin binding proteins" because they are separated by affinity chromatography of the related polysaccharide heparin.

[0070] An exemplary amino acid sequence of human ApoE3 protein (Uniprot Accession No. P02649) is shown below:

[0071]

[0072] At least two HSPG / heparin binding domains have been identified in human ApoE, one located in the N-terminal domain and one located in the C-terminal domain (Weisgraber et al. J Biol Chem, 261(5):2068-76, 1986; Saito et al., J Biol Chem, 278(17):14782-7, 2003). The HSPG / heparin binding domain near arginine 136 (R136) (N-terminal HSPG / heparin binding domain) functions in full-length lipidated and defatted ApoE, while the HSPG / heparin binding domain in the C-terminal domain only functions in the absence of the N-terminal domain and in defatted ApoE. The N-terminal HSPG / heparin binding domain is well characterized and comprises amino acid residues 142 to 147 (bold) of SEQ ID NO: 1. The C-terminal HSPG / heparin binding domain is less well characterized and comprises a lysine (K) at position 233 and other charged amino acids nearby, including amino acid residues 211 to 218 and 243 to 272 of SEQ ID NO:1. The inventors show that the arginine at position 136 of ApoE plays a key role in the heparin binding of ApoE. Without wishing to be bound by theory, the underlying mechanism is the allosteric regulation of heparin binding mediated by the arginine at position 136. Allosteric regulation as used herein is related to the regulation of ligand binding by an allosteric modulator at one or more allosteric regulatory sites, which are different from the binding site (one or more) of the ligand. In some embodiments, one or more allosteric regulatory sites for ApoE and HSPG / heparin binding comprise the arginine at position 136 of ApoE as shown in SEQ ID NO:1. "HSPG / heparin binding domain (one or more)", "HSPG / heparin binding site (one or more)", "HSPG-binding domain (one or more)" and "HSPG-binding site (one or more)" are used interchangeably herein.

[0073] Anti-ApoE antibodies

[0074] Anti-ApoE antibodies that bind to wild-type or mutant ApoE proteins (e.g., human ApoE proteins) are provided. In some cases, the antibodies described herein bind to wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4), but do not bind to mutant ApoE proteins (e.g., ApoEch). In some cases, the antibodies described herein bind to mutant ApoE proteins (e.g., ApoEch), but do not bind to wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4). In some cases, the antibodies described herein bind to mutant ApoE proteins (e.g., ApoEch) and wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4).

[0075] In some cases, provided herein is an antibody that blocks the interaction between wild-type ApoE protein (e.g., ApoE2, ApoE3 or ApoE4) and HSPG. Provided herein is an antibody that can reduce or regulate the binding affinity of ApoE protein (e.g., ApoE2, ApoE3 or ApoE4) to HSPG. In some cases, provided herein is an antibody that is bound to the HSPG binding domain of wild-type ApoE protein. In some cases, provided herein is an antibody that is bound to the allosteric regulatory site (e.g., amino acid position 136 of ApoE) that one or more HSPG / ApoE are combined. In some cases, antibodies as described herein reduce fibril formation and / or amyloid oligomerization.

[0076] In some cases, the antibodies provided herein bind to amino acid sequences in wild-type or mutant ApoE comprising TEELRVSLASHLRK (SEQ ID NO: 2) or consisting thereof. In some cases, the antibodies provided herein bind to amino acid sequences in wild-type or mutant ApoE comprising TEELRVRLASHLRK (SEQ ID NO: 3) or consisting thereof. In some cases, the amino acid sequence TEELRVSLASHLRK (SEQ ID NO: 2) comprises or consists of an epitope for an antibody provided herein. In some cases, the amino acid sequence TEELRVRLASHLRK (SEQ ID NO: 3) comprises or consists of an epitope for an antibody provided herein. Variants of these sequences can also be used, for example, those having at least 80%, 85%, 90% or 95% identity to these sequences.

[0077] The calculation of "identity" between two sequences can be performed as follows. Align the sequences for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second nucleic acid sequences for optimal comparison, and non-identical sequences can be ignored for comparison purposes). The length of the sequence aligned for comparison purposes is at least 70% (e.g., at least 80%, 90%, or 100%) of the length of the reference sequence. The nucleotides at the corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules at that position are identical. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, and considering the number of spaces and the length of each space, these spaces need to be introduced to achieve the optimal comparison of the two sequences.

[0078] The comparison of sequence and the determination of the percentage identity between two sequences can be completed using a mathematical algorithm. In some embodiments, the percentage identity between two nucleotide sequences is determined using the Needleman and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm of the GAP program incorporated into the GCG software package (available on gcg.com), using the Blossum 62 matrix, the PAM250 matrix, the NWSgapdna.CMP matrix. In some embodiments, the percentage identity between two amino acids or nucleotide sequences can be determined using the E.Meyers and W.Miller ((1989) CABIOS, 4:11-17) algorithm incorporated into the ALIGN program (version 2.0), using the PAM120 weight residual table, the gap length penalty of 12 and the gap penalty of 4.

[0079] The use of the term "antibody" in the present disclosure is intended to encompass whole antibodies (as opposed to minibodies, nanobodies, or antibody fragments), bispecific antibodies, tetravalent antibodies, multispecific antibodies, minibodies, nanobodies, and antibody fragments. In some cases, the anti-ApoE antibodies of the present disclosure are whole antibodies. In some cases, the anti-ApoE antibodies of the present disclosure are chimeric antibodies, human antibodies, or humanized antibodies. In some cases, the heavy chain constant region of the anti-ApoE antibody is a human IgG1, human IgG2, human IgG3, or human IgG4 constant region. In some cases, the light chain constant region is a human κ constant region. In other cases, the light chain constant region is a human λ constant region. In some cases, the antibodies of the present disclosure are designed to have low effector functionality (e.g., by Fc modifications such as N297Q, T299A, etc. See also, Wang, X., Mathieu, M. & Brezski, RJ Protein Cell (2018) 9:63.doi.org / 10.1007 / s13238-017-0473-8 (incorporated herein by reference)). In some cases, the Fc portion of the antibody is hIgG1 Fc, a hIgG2 Fc, a hIgG3 Fc, a hIgG4 Fc, a hIgG1agly Fc, a hIgG2 SAAFc, a hIgG4(S228P)Fc or a hIgG4(S228P) / G1 agly Fc (in this format - to minimize effector function - the CH1 and CH2 domains are IgG4 with a "fixed" hinge (S228P), and are aglycosylated. The CH3 domain is hIgG1, or hIgG4(S228P)agly Fc). In one instance, the antibody has one of three scaffolds with reduced effector function: hIgG1 agly(N297Q); hIgG2 SAA (see, Vafa et al. Methods, 65(1): 114-26 (2014); and hIgG4P / G1 agly (see, US 2012 / 0100140A1).

[0080] In some embodiments, the antibodies or ApoE-binding fragments thereof described herein exhibit binding characteristics and / or biological properties as outlined for antibodies 1H4-2, 7C11-1, 19G10-2, 23B2(1343), 2H79-1, 30E1-2, 16H8, 25F1-2, and 29G10-1 as described in the Examples section below.

[0081] In some embodiments, the present disclosure provides antibodies that bind to wild-type human ApoE or a portion thereof and have one or more of the following properties: (i) bind to wild-type human ApoE with high affinity with KD ≤ 20 nM; (ii) compete with wild-type human ApoE for binding to heparin; and (iii) reduce the formation of paired helical fibrils (PHF) Tau in retinal cells.

[0082] In some embodiments, the present disclosure provides antibodies that bind to mutant human ApoE (e.g., those having a mutation at amino acid position 136 of human ApoE, such as ApoEch) or a portion thereof, and have one or more of the following properties: (i) binds to mutant human ApoE (e.g., a mutation at amino acid position 136 of human ApoE, such as ApoEch) with a high affinity of KD ≤ 20 nM; (ii) competes with wild-type human ApoE for binding to heparin; (iii) reduces the formation of paired helical fibers (PHF) Tau in retinal cells.

[0083] Any of the anti-ApoE antibodies described herein can be used to treat or prevent a condition associated with dementia or mild cognitive impairment (MCI) (e.g., Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, or Huntington's disease), a neurodegenerative disease, a cerebrovascular disease, a brain injury, a retinal degeneration, or a retinal injury.

[0084] Exemplary Antibody 1H4-2

[0085] Antibody 1H4-2 was generated by immunization with APOE:KLH-CTEELRVRLASHLRK-CONH2. The amino acid sequences of the complementarity determining regions (CDRs) and heavy and light chain variable regions of 1H4-2 are provided below.

[0086]

[0087] Variable light chain:

[0088] Nucleotide sequence:

[0089] signal sequence

[0090]

[0091] Amino Acid Sequence:

[0092] Signal peptide

[0093]

[0094] Variable heavy chain:

[0095] Nucleotide sequence

[0096] signal sequence

[0097]

[0098] Amino Acid Sequence:

[0099] Signal peptide

[0100]

[0101] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 1H4-2 and a VL comprising three VL CDRs of antibody 1H4-2. The six CDRs can be based on any definition known in the art, such as, but not limited to, Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs can be determined, for example, by using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0102] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) a VH comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO:8, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO:9; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.

[0103] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 13. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 12. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 13 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 12. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 13 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 12.

[0104] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO:13 and a VL with the amino acid sequence set forth in SEQ ID NO:12.

[0105] Exemplary Antibody 7C11-1

[0106] Antibody 7C11-1 was generated by immunization with APOE:KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54). The amino acid sequences of the complementarity determining regions (CDRs) and heavy and light chain variable regions of 7C11-1 are provided below.

[0107]

[0108] Variable heavy chain:

[0109] Nucleotide sequence

[0110] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0111]

[0112] Amino Acid Sequence:

[0113] Signal peptide-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0114]

[0115] Variable light chain:

[0116] Nucleotide sequence:

[0117] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0118]

[0119] Amino Acid Sequence:

[0120] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0121]

[0122] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 7C11-1 and a VL comprising three VL CDRs of antibody 7C11-1. The six CDRs may be based on any definition known in the art, such as, but not limited to, Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs may be determined, for example, by using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0123] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) VH, which includes VHCDR1 comprising the amino acid sequence recorded in SEQ ID NO: 17, VHCDR2 comprising the amino acid sequence recorded in SEQ ID NO: 18, and VHCDR3 comprising the amino acid sequence recorded in SEQ ID NO: 19; and (ii) VL, which includes VLCDR1 comprising the amino acid sequence recorded in SEQ ID NO: 14, VLCDR2 comprising the amino acid sequence recorded in SEQ ID NO: 15, and VLCDR3 comprising the amino acid sequence recorded in SEQ ID NO: 16.

[0124] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 23. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 22. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 23 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 22. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 23 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 23 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 22.

[0125] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO:23 and a VL with the amino acid sequence set forth in SEQ ID NO:22.

[0126] Exemplary Antibody 19G10-2

[0127] Antibody 19G10-2 was generated by immunization with KLH-CTEELRVSLASHLRK-CONH2. The following article provides the amino acid sequences of the complementarity determining regions (CDRs) and the heavy and light chain variable regions of 19G10-2.

[0128]

[0129] Variable light chain:

[0130] Nucleotide sequence:

[0131] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0132]

[0133] Amino Acid Sequence:

[0134] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0135]

[0136] Variable heavy chain analysis:

[0137] Nucleotide sequence

[0138] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0139]

[0140] Amino Acid Sequence:

[0141] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0142]

[0143] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 19G10-2 and a VL comprising three VL CDRs of antibody 19G10-2. The six CDRs may be based on any definition known in the art, such as, but not limited to, Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs may be determined, for example, by using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0144] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) a VH comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO:27, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO:28, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO:29; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO:24, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO:25, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO:26.

[0145] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 33. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 32. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 33 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 32. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 33 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 32. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 33 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 32.

[0146] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO:33 and a VL with the amino acid sequence set forth in SEQ ID NO:32.

[0147] Exemplary Antibody 25F1-2

[0148] Antibody 25F1-2 was generated by immunization with KLH-CTEELRVSLASHLRK-CONH2. The amino acid sequences of the complementarity determining regions (CDRs) and heavy and light chain variable regions of 25F1-2 are provided below.

[0149]

[0150] Variable light chain analysis:

[0151] Nucleotide sequence:

[0152] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0153]

[0154] Amino Acid Sequence:

[0155] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0156]

[0157] Variable heavy chain analysis:

[0158] Nucleotide sequence

[0159] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0160]

[0161] Amino Acid Sequence:

[0162] Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0163]

[0164] In some cases, the anti-ApoE antibody comprises a VH comprising three VH CDRs of antibody 25F1-2 and a VL comprising three VL CDRs of antibody 25F1-2. The six CDRs may be based on any definition known in the art, such as, but not limited to, Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs may be determined, for example, by using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0165] In one instance, the anti-ApoE antibody of the present disclosure comprises (i) a VH comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO:37, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO:38, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO:39; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO:34, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO:35, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO:36.

[0166] In some cases, the anti-ApoE antibody comprises a VH that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 43. In some cases, the anti-ApoE antibody comprises a VL that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 42. In one case, the anti-ApoE antibody comprises a VH that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 43 and a VL that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 42. In another instance, the anti-ApoE antibody comprises a VH that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 43 and a VL that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 42. In still another instance, the anti-ApoE antibody comprises a VH that is identical to the amino acid sequence set forth in SEQ ID NO: 43 and a VL that is identical to the amino acid sequence set forth in SEQ ID NO: 42.

[0167] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a VH with the amino acid sequence set forth in SEQ ID NO:43 and a VL with the amino acid sequence set forth in SEQ ID NO:42.

[0168] Exemplary Antibody 1343ab

[0169] Antibody 1343ab was generated by immunization with KLH-CTEELRVSLASHLRK-CONH2. The amino acid sequences of the complementarity determining regions (CDRs) and full length heavy and light chains are provided below.

[0170]

[0171] Full length heavy chain

[0172]

[0173] Full length light chain

[0174]

[0175] EC

[0176] N-linked glycosylation was detected on the heavy chain constant region N at 292. Loss of the C-terminal lysine was observed on the heavy chain.

[0177] In some cases, the anti-ApoE antibody comprises a VH containing three VH CDRs of antibody 1343ab and a VL containing three VL CDRs of antibody 1343ab. The six CDRs can be based on any definition known in the art, such as, but not limited to, Kabat, Chothia, enhanced Chothia, contact, IMGT, or Honegger definitions. These CDRs can be determined, for example, by using the AbYsis database (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi).

[0178] In one instance, the anti-ApoE antibody of the present disclosure comprises: (i) a VL comprising a VHCDR1 comprising the amino acid sequence set forth in SEQ ID NO:47, a VHCDR2 comprising the amino acid sequence set forth in SEQ ID NO:48, and a VHCDR3 comprising the amino acid sequence set forth in SEQ ID NO:49; and (ii) a VL comprising a VLCDR1 comprising the amino acid sequence set forth in SEQ ID NO:44, a VLCDR2 comprising the amino acid sequence set forth in SEQ ID NO:45, and a VLCDR3 comprising the amino acid sequence set forth in SEQ ID NO:46.

[0179] In some cases, the anti-ApoE antibody comprises a heavy chain that is at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 53. In some cases, the anti-ApoE antibody comprises a light chain that is at least 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 52. In one instance, the anti-ApoE antibody comprises a heavy chain that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 52. In another instance, the anti-ApoE antibody comprises a heavy chain that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 52. In still another instance, the anti-ApoE antibody comprises a heavy chain identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain identical to the amino acid sequence set forth in SEQ ID NO: 52.

[0180] In certain cases, an antibody of the disclosure that binds to ApoE is one that competes with or binds to the same epitope as a reference antibody having a heavy chain having the amino acid sequence set forth in SEQ ID NO:53 and a light chain having the amino acid sequence set forth in SEQ ID NO:52.

[0181] Chimeric antibodies, human antibodies, or humanized antibodies having the CDR sequences of any of the above antibodies can be produced based on the methods described herein.

[0182] Antibody fragments

[0183] Antibody fragments (e.g., Fab, Fab', F(ab')2, Facb and Fv) can be prepared by proteolytic digestion of intact antibodies. For example, antibody fragments can be obtained by treating whole antibodies with enzymes such as papain, pepsin or plasmin. Papain digestion of whole antibodies produces F(ab)2 or Fab fragments; pepsin digestion of whole antibodies produces F(ab')2 or Fab'; plasmin digestion of whole antibodies produces Facb fragments.

[0184] Alternatively, antibody fragments can be produced recombinantly. For example, a nucleic acid encoding an antibody fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host cell. See, e.g., Co, MS et al., J. Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, AH, Methods in Enzymology, 178:476-496 (1989); Pluckthun, A. and Skerra, A., Methods in Enzymology, 178:476-496 (1989); Lamoyi, E., Methods in Enzymology, 121:652-663 (1989); Rousseaux, J. et al., Methods in Enzymology, (1989) 121:663-669 (1989); and Bird, RE et al., TIBTECH, 9:132-137 (1991)). Antibody fragments can be expressed in E. coli and secreted therefrom, thus allowing for easy production of large amounts of these fragments. Antibody fragments can be separated from antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab)2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046.

[0185] Conjugated Antibodies

[0186] The antibodies disclosed herein may be conjugated antibodies bound to various molecules, including macromolecular substances such as polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI) modified with PEG (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive substances (e.g. 90 Y. 131 I), fluorescent substances, luminescent substances, haptens, enzymes, metal chelates and drugs.

[0187] In some embodiments, the antibodies described herein are modified by moieties that improve their stability and / or retention in the circulation, such as in blood, serum or other tissues, including the brain, for example, by at least 1.5, 2, 5, 10, 15, 20, 25, 30, 40 or 50 times. For example, the antibodies described herein can be combined with (e.g., conjugated to) polymers, such as substantially non-antigenic polymers, such as polyalkylene oxide or polyethylene oxide. Suitable polymers will be significantly different in weight. A polymer having a number average molecular weight of about 200 to about 35,000 daltons (or about 1,000 to about 15,000 and 2,000 to about 12,500) can be used. For example, the antibodies described herein can be conjugated to water-soluble polymers, such as hydrophilic polyethylene polymers, such as polyvinyl alcohol or polyvinyl pyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymer is maintained. Other useful polymers include polyoxyalkylenes, such as block copolymers of polyoxyethylene, polyoxypropylene and polyoxyethylene and polyoxypropylene; polymethacrylate; carbomer; and branched or unbranched polysaccharides. In some embodiments, the antibodies described herein are modified with portions that improve their penetration of the blood-brain barrier (e.g., those described in Pardridge, J Cereb Blood Flow Metab 32 (11): 1959-1972, 2012). Exemplary blood-brain barrier penetrating moieties include, but are not limited to, type 1 glucose transporter (GLUT1), type 1 cationic amino acid transporter (CAT1), type 1 monocarboxylate transporter (MCT1), type 2 concentrating nucleoside transporter (CNT2), active efflux transporters (AETs) (e.g., p-glycoprotein, and those described in Pardridge, J Cereb Blood Flow Metab 32(11):1959-1972, 2012), and additional blood-brain barrier penetrating moieties are known in the art.

[0188] The above-mentioned conjugated antibodies can be prepared by chemically modifying the antibodies described herein or their low molecular weight forms. Methods for modifying antibodies are well known in the art (eg, US 5,057,313 and US 5,156,840).

[0189] The anti-ApoE antibody can be in the form of a full-length (or whole) antibody, or a low molecular weight form of an anti-ApoE antibody (e.g., a biologically active antibody fragment or mini-antibody), such as Fab, Fab', F(ab') 2, Fv, Fd, dAb, scFv and sc(Fv)2. Other anti-ApoE antibodies encompassed by the present disclosure include single domain antibodies (sdAbs) containing a single variable chain, such as VH or VL, or a biologically active fragment thereof. See, for example, Moller et al., J. Biol. Chem., 285(49):38348-38361 (2010); Harmsen et al., Appl. Microbiol. Biotechnol., 77(1):13-22 (2007); US2005 / 0079574 and Davies et al. (1996) Protein Eng., 9(6):531-7. Like whole antibodies, sdAbs are capable of selectively binding to a specific antigen (e.g., ApoE2, ApoE3, ApoE4 or ApoEch). The molecular weight of sdAb is only 12-15 kDa, which is much smaller than ordinary antibodies, and even smaller than Fab fragments and single-chain variable fragments.

[0190] In certain embodiments, an anti-ApoE antibody or an antigen-binding fragment thereof or a low molecular weight antibody thereof specifically binds to the HSPG / heparin binding domain of ApoE and reduces the severity of symptoms when administered to a human patient or animal model suffering from one or more of the following diseases: dementia and / or mild cognitive impairment (MCI) (e.g., those associated with Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, Huntington's disease, or neurodegenerative diseases). In certain embodiments, an anti-ApoE antibody or an antigen-binding fragment thereof or a low molecular weight antibody thereof specifically binds to the HSPG / heparin binding domain of ApoE and reduces the severity of symptoms when administered to a human patient or animal model suffering from one or more of the following diseases: neurodegenerative diseases, cerebrovascular diseases (e.g., stroke, carotid stenosis, spinal stenosis, or aneurysm), brain injury (e.g., traumatic brain injury, acquired brain injury), retinal degeneration, glaucoma, or retinal injury. These characteristics of anti-ApoE antibodies or low molecular weight antibodies thereof can be measured according to methods known in the art.

[0191] Nucleic acids, vectors, host cells

[0192] The present disclosure is also characterized by nucleic acids encoding antibodies disclosed herein. Nucleic acids encoding VH CDR1, VH CDR2, and VH CDR3 of anti-ApoE antibodies described herein are provided herein. Nucleic acids encoding VL CDR1, VLCDR2, and VL CDR3 of anti-ApoE antibodies described herein are also provided. Nucleic acids encoding VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of anti-ApoE antibodies described herein are provided herein. Nucleic acids encoding the heavy chain variable region (VH) of anti-ApoE antibodies described herein, and / or nucleic acids encoding the light chain variable region (VL) of anti-ApoE antibodies described herein are also provided. In some cases, nucleic acids encoding VH and / or VL of anti-ApoE antibodies described herein are provided herein, respectively connected to human heavy chain constant regions and / or human light chain constant regions. Nucleic acids encoding both VH and VL of anti-ApoE antibodies described herein are also provided herein. In some cases, nucleic acids described herein include nucleic acids encoding Fc regions of human antibodies (e.g., human IgG1, IgG2, IgG3, or IgG4). In certain cases, nucleic acids include nucleic acids encoding Fc regions of human antibodies that have been modified to reduce or eliminate effector functions (e.g., N297Q or T299A substitutions in human IgG1 Fc regions (numbered according to EU numbering)). In some cases, nucleic acids include nucleic acids encoding Fc portions that are hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1agly Fc, hIgG2 SAAFc, hIgG4 (S228P) Fc, or hIgG4 (S228P) / G1 agly Fc.

[0193] Also disclosed herein are vectors (eg, expression vectors) comprising any of the above-described nucleic acids.

[0194] Furthermore, the present disclosure relates to host cells (eg, bacterial cells, yeast cells, insect cells, or mammalian cells) containing the above-mentioned vector(s) or nucleic acid(s).

[0195] Methods for obtaining anti-ApoE antibodies

[0196] Also provided herein is a method for preparing an anti-ApoE antibody for use in the present method. General methods for preparing antibodies such as monospecific, polyclonal or monoclonal antibodies are known in the art. For monoclonal antibodies, the process involves obtaining immune cells (lymphocytes) that secrete antibodies from the spleen of a mammal (e.g., a mouse) that has been previously immunized in vivo or in vitro with an antigen of interest (e.g., a peptide antigen as described herein). The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells that can replicate indefinitely in cell culture to produce immortal, immunoglobulin-secreting cell lines. The resulting fused cells or hybridomas are cultured, and the resulting colonies are screened to produce the desired monoclonal antibodies. Colonies that produce such antibodies are cloned and grown in vivo or in vitro to produce large quantities of antibodies. A description of the theoretical basis and practical methods for fusing such cells is described in Kohler and Milstein, Nature 256:495 (1975).

[0197] Mammalian lymphocytes can be immunized in vivo by immunizing an animal (e.g., mouse) with a peptide antigen that is, for example, identical to KLH-CTEELRVRLASHLRK-CONH 2 (SEQ ID NO:54) or KLH-CTEELRVSLASHLRK-CONH 2 (SEQ ID NO:55) is a peptide antigen that is at least 80%, 85%, 90% or 95% identical to (SEQ ID NO:55), optionally, for example, up to 20% of the residues have one or more substitutions or deletions. For example, the method can include immunizing the animal with a peptide comprising a sequence that is at least 80% identical to at least 10 consecutive amino acids from the following: the heparin binding domain of APOE, such as a peptide comprising TEELRVRLASHLRK (SEQ ID NO:3) or TEELRVSLASHLRK (SEQ ID NO:2). Such immunizations are repeated at intervals of up to several weeks as needed to obtain sufficient antibody titers. After the last antigen boost, the animal is sacrificed and spleen cells are removed.

[0198] Fusion with mammalian myeloma cells or other fusion partners capable of indefinite replication in cell culture is accomplished by known techniques, such as the use of polyethylene glycol ("PEG") or other fusing agents (see Milstein and Kohler, Eur. J. Immunol. 6:511 (1976), which is incorporated herein by reference). The immortalized cell line, preferably murine, is selected to lack enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capacity, but may also be derived from other mammalian species, including, but not limited to, rat and human. Many such cell lines are known to those skilled in the art, and others are regularly described.

[0199] The procedure for producing polyclonal antibodies is also known. Generally, such antibodies can be produced by subcutaneously administering the protein or polypeptide of the present invention to New Zealand white rabbits, which are first bled to obtain pre-immune serum. For example, antigens can be injected at six different sites with a total volume of 100 μl per site. Each injection material will contain a synthetic surfactant adjuvant polyether polyol (pluronic polyol), or a crushed acrylamide gel containing protein or polypeptide after SDS-polyacrylamide gel electrophoresis. Then, blood is taken from the rabbit two weeks after the first injection, and the same antigen is regularly used to strengthen three times every six weeks. Then serum samples are collected 10 days after each strengthening. Polyclonal antibodies are then recovered from serum by affinity chromatography using the corresponding antigen to capture antibodies. Finally, for example, rabbits are euthanized IV with pentobarbital 150mg / Kg. E. Harlow, et al., editors, Antibodies: A Laboratory Manual (1988) disclose this process and other processes for other production of polyclonal antibodies.

[0200] The methods described herein include any of the steps (one or more) of producing chimeric antibodies, humanized antibodies, single-chain antibodies, Fab fragments, bispecific antibodies, fusion antibodies, labeled antibodies, or analogs of any of them. The corresponding methods are known to those skilled in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor (1988). When derivatives of the antibodies are obtained by phage display technology, surface plasmon resonance used in the BIAcore system can be used to improve the efficiency of phage antibodies that bind to the same epitope as any of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). For example, the production of chimeric antibodies is described in International Application WO89 / 09622. Methods for producing humanized antibodies are described in, for example, European application EP-A1 0 239 400 and international application WO 90 / 07861. Other sources of antibodies used according to the present invention are so-called xenoantibodies. The general principles of producing xenoantibodies, such as human-like antibodies, in mice are described in, for example, international applications WO 91 / 10741, WO 94 / 02602, WO 96 / 34096 and WO 96 / 33735. As described above, the antibodies described herein can also exist in a variety of forms in addition to complete antibodies; including, for example, Fv, Fab and F(ab) 2, as well as single chains; see, for example, international application WO 88 / 09344.

[0201] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombination, and phage display technology, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd Edition. (1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier, NY, 563-681 (1981), which are incorporated by reference in their entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone including any eukaryotic, prokaryotic or phage clone, rather than a method for producing it. Therefore, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology.

[0202] In the known hybridoma method (Kohler et al., Nature 256 (1975), 495), relatively short-lived or non-immortal lymphocytes from mammals, such as B cells derived from a murine subject as described herein, are fused with an immortal tumor cell line (e.g., a myeloma cell line), thereby producing hybrid cells or "hybridomas" that are immortal and capable of producing antibodies encoded by the genes of the B cells. By selection, dilution and regrowth, the resulting hybrids are separated into individual genetic strains, each of which contains specific genes for forming a single antibody. They produce antibodies that are homogeneous against the desired antigen and are called "monoclonal" based on their pure genetic lineage.

[0203] The hybridoma cells thus prepared are inoculated and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. It will be appreciated by those skilled in the art that reagents, cell lines and culture media for the formation, selection and growth of hybridomas are commercially available from many sources and standardized protocols are well established. Typically, the culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies against the desired antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) as described herein. After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, the clones can be subcloned by a limiting dilution process and grown by standard methods; see, for example, Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp 59-103 (1986). It will further be appreciated that the monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0204] In another embodiment, lymphocytes can be selected and variable genes can be separated by micromanipulation. For example, peripheral blood mononuclear cells can be separated from immune or naturally immune mammals such as humans and cultured in vitro for about 7 days. The culture can be screened for specific immunoglobulins that meet the screening criteria. Cells from positive wells can be separated. Single B cells that produce Ig can be separated by FACS or identified in complement-mediated hemolytic plaque assays. B cells that produce Ig can be micromanipulated into tubes, and VH and VL genes can be amplified using, for example, RT-PCR. VH and VL genes can be cloned into antibody expression vectors and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.

[0205] Alternatively, cell lines producing antibodies can be selected and cultured using techniques well known to the skilled artisan. These techniques are described in various laboratory manuals and major publications. In this regard, techniques suitable for the invention as described below are described in Current Protocols in Immunology, Coligan et al., eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety including supplements.

[0206] Antibodies such as those described above can be prepared, for example, by preparing and expressing synthetic genes encoding the amino acid sequence. Methods for producing variants of any anti-ApoE antibody (e.g., comprising amino acid substitutions) are well known in the art. These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of DNA molecules prepared by encoding the antibody or any portion thereof (e.g., framework region, CDR, constant region). Site-directed mutagenesis is well known in the art (see, e.g., Carter et al., Nucl. Acids Res., 13: 4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82: 488 (1987)). PCR mutagenesis is also applicable to the preparation of amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nucl. Acids Res. 17:723-733 (1989). Another method for preparing sequence variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34:315-323 (1985).

[0207] Antibodies can be produced in bacterial cells or eukaryotic cells. Some antibodies, such as Fab, can be produced in bacterial cells such as Escherichia coli cells. Antibodies can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS, Hela). In addition, antibodies (e.g., scFv) can be expressed in yeast cells such as Pichia (Pichia) (see, e.g., Powers et al., J Immunol Methods. 251: 123-35 (2001)), Hanseula or Saccharomyces cerevisiae. In order to produce an antibody of interest or its antigen-binding fragment, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in a suitable host cell. Standard molecular biology techniques are used to prepare recombinant expression vectors, transfect host cells, select transformants, culture host cells and recover antibodies.

[0208] If the antibody is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have features that allow the vector to be amplified in bacterial cells. In addition, when using E. coli such as JM109, DH5α, HB101 or XL1-Blue as a host, the vector must have a promoter, such as the lacZ promoter (Ward et al., 341: 544-546 (1989), the araB promoter (Better et al., Science, 240: 1041-1043 (1988)), or a T7 promoter that allows efficient expression in E. coli. Examples of such vectors include, for example, M13 series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP and pET (when using this expression vector, the host is preferably a T7 promoter that expresses the T7 promoter). The expression vector may contain a signal sequence for antibody secretion. For production in the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987)) may be used as a signal sequence for antibody secretion. For bacterial expression, the expression vector may be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0209] If the antibody is to be expressed in animal cells such as CHO, COS and NIH3T3 cells, the expression vector includes a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18: 5322 (1990)) or CMV promoter. In addition to the nucleic acid sequence encoding the immunoglobulin or its domain, the recombinant expression vector can carry additional sequences, such as sequences that regulate the replication of the vector in the host cell (e.g., replication origin) and a selective marker gene. The selective marker gene helps to select the host cell into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017). For example, the selective marker gene usually confers resistance to drugs such as G418, hygromycin or methotrexate to the host cell into which the vector has been introduced. Examples of vectors having a selectable marker include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0210] In one embodiment, the antibody is produced in a mammalian cell. Exemplary mammalian host cells for expressing the antibody include Chinese hamster ovary (CHO cells) (including dhfr - CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, are used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621), human embryonic kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte cell lines, e.g., NS0 myeloma cells and SP2 cells, and cells from transgenic animals, e.g., transgenic mammals.

[0211] The antibodies disclosed herein can be separated and purified from the inside or outside (e.g., culture medium) of the host cell as substantially pure and homogeneous antibodies. The separation and purification methods commonly used for antibody purification can be used for the separation and purification of antibodies, without limitation to any specific method. Antibodies can be separated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be performed using liquid chromatography such as HPLC and FPLC. The column used for affinity chromatography includes protein A column and protein G column. The example of the column using protein A column includes Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present invention also includes antibodies that are highly purified using these purification methods.

[0212] Characteristics of antibodies

[0213] The ApoE binding properties of the antibodies described herein can be determined by any standard method, such as one or more of the following methods: Surface plasmon resonance (SPR), BIACORE TMAnalysis, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and fluorescence resonance energy transfer (FRET).

[0214] Methods for using SPR are described in, for example, U.S. Pat. No. 5,641,640; Raether (1988) Surface Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and online resources provided by BIAcore International AB. Information from SPR can be used to provide equilibrium dissociation constants (Kd) and K values ​​for binding of a biomolecule to a target. on and K off Accurate and quantitative determination of kinetic parameters.

[0215] The ability of different antibodies to compete with each other for binding to wild-type ApoE or spiked ApoE (e.g., ApoEch) can also be evaluated by using BIACORE chromatography technology (Pharmacia BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May 1994); see also Johne et al. (1993) J. Immunol. Methods, 160:191-198).

[0216] When using enzyme immunoassay, a sample containing the antibody, such as culture supernatant of antibody-producing cells or purified antibody, is added to an antigen-coated plate. A secondary antibody labeled with an enzyme such as alkaline phosphatase is added, the plate is incubated, and after washing, an enzyme substrate such as p-nitrophenyl phosphate is added, and the absorbance is measured to evaluate the antigen binding activity.

[0217] Additional general guidelines for evaluating antibodies, e.g., immunoblotting and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, ed. by Harlow and Lane, Cold Spring Harbor press (1988)).

[0218] Mutant ApoE protein, peptide and fusion protein thereof

[0219] The present disclosure provides a mutant ApoE protein or a fragment thereof containing an amino acid substitution at one or more positions in the HSPG binding domain compared to the wild-type ApoE protein. In some embodiments, the mutant ApoE protein or its fragment comprises an amino acid other than arginine at position 136. In some embodiments, the mutant ApoE protein or its fragment comprises serine, histidine or cysteine ​​at position 136. Also provided are nucleic acid (e.g., DNA or RNA) sequences encoding mutant ApoE proteins or their fragments, and vectors comprising the nucleic acid sequences. Mutated ApoE proteins or their fragments, nucleic acids encoding such proteins or fragments, and vectors comprising nucleic acid sequences can be used to treat or prevent conditions (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease) associated with dementia or mild cognitive impairment (MCI), neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration or retinal damage.

[0220] In some embodiments, the mutant ApoE protein is an ApoEch protein (e.g., ApoE2ch, ApoE3ch, or ApoE4ch protein). Also contemplated herein are fragments of the ApoEch protein including amino acid position 136. An exemplary sequence of the full-length ApoE3ch protein is shown below. The mutation from arginine to serine is marked in bold and double underlined.

[0221]

[0222] In some embodiments, the methods disclosed herein allow mutant ApoE proteins or fragments thereof to cross the blood-brain barrier. Mutant ApoE proteins or fragments thereof can be delivered using nanocarriers, including but not limited to polymer nanoparticles, lipid-based nanoparticles, liposomes, micelles, dendrimers, human cells expressing proteins, and nanotubes (see, Dominguez et al. J Nanosci nanotechnol. 14(1):766-79, 2014). In some embodiments, the mutant ApoE protein or fragment thereof is delivered intracarotidally or transmucosally (e.g., intracarotid infusion of a hypertonic solution (arabinose or mannitol); see, Sanchez-Covarrubias et al., Curr Pharm Des. 20(10): 1422-49, 2014 and Miyake et al., World J Otorhinolaryngol Head Neck Surg. 1(1): 11-16, 2015), or intranasally by using chlorotoxin (see, McCall et al., Tissue Barriers 2(4): e944449, 2014). Cryogenic techniques, receptor-mediated transport, cell-penetrating peptides, and cell-mediated delivery can also be used to promote ApoE3ch protein across the blood-brain barrier (see, Pandey et al., Tissue Barriers 4(1): e1129476, 2016). For example, immune cells and stem cells (e.g., neural stem cells, induced pluripotent cells, and mesenchymal stem cells) can be used to carry therapeutic loads across the BBB. Mesenchymal stem cells loaded with nanoparticles can be used for this purpose (see, for example, Roger et al. Biomaterials 31: 8393-401, 2010). Genetically modified stem cells (e.g., genetically modified mesenchymal stem cells) can also be used (see, for example, Ebrahimi and Lalvand Hygeia. JDMed. Vol. 5 (1): 90-104, 2013). Chemical drug delivery systems (CDDS) can also be used, such as those described in He et al., Cells, 7 (4): 24, 2018. Other methods of transporting proteins across the blood-brain barrier are known in the art.

[0223] Nucleic acids (e.g., DNA or mRNA) encoding mutant ApoE proteins (e.g., any mutant ApoE proteins described herein, such as ApoEch) or fragments thereof are contemplated herein. In some embodiments, the mRNA encoding the ApoEch protein can be modified to increase stability (e.g., those described in Zangi et al., Nat Biotechnol. 31(10):898-907, 2013 and developed by Moderna, Inc.; and those described in Alberer et al., Lancet 390(10101):1511-1520, 2017 and developed by Curevac and BioNTech).

[0224] Viral vectors containing DNA sequences encoding mutant ApoE or fragments thereof are contemplated herein. An exemplary cDNA sequence encoding a full-length ApoE3ch protein (including a signal peptide region) is shown below. Mutations from cytosine to adenine are marked in bold and double underlined.

[0225]

[0226] Suitable vectors are known in the art. In some embodiments, the viral vector is an AAV vector (e.g., those described in Rosenberg et al., Hum Gene Ther Clin Dev 29(1):24-47, 2018). Also included is a cDNA sequence encoding an ApoE protein containing a mutation at the R136 position except R136S. In some embodiments, the mutation is R136H or R136C.

[0227] Peptides and fusion proteins

[0228] In some embodiments, provided herein is a HSPG / heparin binding domain comprising wild-type or mutant ApoE (e.g., any mutant ApoE protein described herein) or a peptide consisting thereof. In some cases, the amino acid sequence of the peptide provided herein comprises or consists of a sequence selected from the group consisting of: STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59) and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60). It is also possible to use a variant with a sequence identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more with these sequences. Fusion proteins comprising the peptide provided above are also disclosed. In some embodiments, the fusion protein also includes the Fc region of a human antibody (e.g., human IgG1, IgG2, IgG3, or IgG4). In some cases, the fusion protein includes the Fc region of a human antibody at the C-terminus of the HSPG / heparin binding domain of wild-type or mutant ApoE. In some cases, the fusion protein includes the Fc region of a human antibody at the N-terminus of the HSPG / heparin binding domain of wild-type or mutant ApoE.

[0229] In some cases, peptides and fusion proteins provided herein compete with wild-type ApoE proteins for binding to HSPG / heparin. In some cases, peptides and fusion proteins provided herein reduce or regulate the combination between wild-type ApoE proteins and HSPG / heparin. In certain embodiments, peptides and fusion proteins provided herein inhibit and / or reduce the HSPG / heparin combination of wild-type ApoE proteins, and reduce the severity of symptoms when applied to one or more of the following human patients or the following animal models: diseases (such as Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, Huntington's disease) related to dementia or mild cognitive impairment (MCI), neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration or retinal damage. These features of peptides and fusion proteins provided herein can be determined according to methods known in the art.

[0230] Also provided herein are anti-ApoE vaccines that can be used to elicit a protective immune response against ApoE. In some embodiments, the anti-ApoE vaccine includes one or more ApoE peptides provided herein (e.g., and a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable adjuvants are known in the art.

[0231] Pharmaceutical compositions and methods of administration

[0232] The methods described herein include the use of pharmaceutical compositions comprising any of the antibodies, peptides, or fusion proteins described herein as an active ingredient.

[0233] Pharmaceutical compositions generally include a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration.

[0234] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g., intracranial, intranasal, intracarotid, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), and transmucosal.

[0235] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting tension, such as sodium chloride or glucose. The pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0236] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be present in a fluid that is easy to inject. It should be stable under manufacturing and storage conditions, and must prevent the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using a surfactant. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, sodium chloride. The extended absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0237] Sterile injection solutions can be prepared by mixing the required amount of active compound into a suitable solvent for one or more of the above-listed components, as needed, and then filtering and sterilizing. In general, dispersions are prepared by mixing the active compound into a sterile carrier, which contains a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired components from its previously sterile filtered solution.

[0238] Oral compositions typically include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be mixed with an excipient and used in the form of tablets, lozenges or capsules such as gelatin capsules. Oral compositions can also be prepared using fluid carriers for use as mouthwashes. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose, disintegrants, such as alginic acid, Primogel, corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate or orange flavoring.

[0239] For administration by inhalation, the compounds may be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer. Such methods include those described in US Pat. No. 6,468,798.

[0240] Systemic administration of therapeutic compounds as described herein can also be performed transmucosally. For transmucosal administration, penetrants suitable for the barrier to be penetrated are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories or injections.

[0241] In one embodiment, the active compound is prepared with a carrier that will protect the therapeutic compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such preparations can be prepared using standard techniques, or commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies to cell antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0242] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0243] CRISPR / Cas9-mediated gene editing of APOE

[0244] Included herein is a method for treating or preventing the following by editing the APOE gene using a genome editing system: a condition associated with dementia and / or mild cognitive impairment (MCI) (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease), neurodegenerative disease, cerebrovascular disease, brain injury, retinal degeneration, or retinal injury. Typically, the method includes administering a therapeutically effective amount of a genome editing system as described herein to a subject in need of such treatment, or a subject in need of such treatment that has been determined. The term "genome editing system" refers to any system with RNA-guided DNA editing activity. The genome editing system disclosed herein includes at least two components adapted from a naturally occurring CRISPR system: gRNA and RNA-guided nucleases. The two components form a complex that can bind to a specific nucleic acid sequence in a cell and, for example, edit the DNA in or around the nucleic acid sequence by producing one or more single-strand breaks (SSBs or nicks), double-strand breaks (DSBs), and / or base substitutions. For a complete description of the genome editing system, see, for example, WO2018 / 026976. In certain aspects, the present disclosure provides AAV vectors encoding the CRISPR / Cas9 genome editing system, and the use of such vectors in treating or preventing disorders as described herein.

[0245] RNA-guided nuclease / Cas9

[0246] Various RNA-guided nucleases can be used in the present method, for example, as described in WO 2018 / 026976. In some embodiments, the RNA-guided nuclease and composition used in the present method is Staphylococcus aureus (S. aureus) Cas9 or Streptococcus pyogenes (S. pyogenes) Cas9. Exemplary Cas9 proteins of the present disclosure may be isolated or derived from any species, including but not limited to bacteria or archaea. In some embodiments of the present disclosure, the Cas9 sequence is modified to include two nuclear localization sequences (NLS) at the C-terminus and N-terminus of the Cas9 protein (e.g., PKKKRKV (SEQ ID NO:61), and a mini-polyadenylation signal (or Poly-A sequence). Exemplary NLSs are the SV40 large T antigen NLS (PKKKRRV (SEQ ID NO:62)) and the nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO:63)). Other NLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov 15; 1(5):411-415; Freitas and Cunha, Curr Genomics. 2009 Dec; 10(8):550-557. Exemplary polyadenylation signals are

[0247] TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGGCGCG (SEQ ID NO: 64). In some embodiments, the RNA-guided nuclease is a nuclease-dead Cas protein (e.g., dCas9).

[0248] Guide RNA

[0249] Provided herein is a guide RNA (gRNA) designed to target one or more sites in the HSPG binding domain of wild-type ApoE. In some embodiments, gRNA is designed to introduce a mutation that causes a mutation at amino acid position 136 in wild-type ApoE. In some embodiments, provided herein is a guide RNA designed to introduce an R136S mutation in a wild-type APOE gene (e.g., APOE2, APOE3, or APOE4), wherein exemplary guide RNAs can be found in Table 5. In some embodiments, a template for repairing double-strand breaks and introducing an R136S mutation is also provided. Exemplary template sequences are as follows:

[0250]

[0251] The silent mutation that eliminates the PAM motif is double underlined, the codon corresponding to the R136S mutation is in bold, and the silent mutation that creates a Sad site for cleavage of the PCR product from clones receiving the template is in italics.

[0252] In some embodiments, the guide RNA provided herein is designed to target exon 3 (amino acids 1-61) of a wild-type APOE gene or a variant present in a subject (thus the method may include determining the sequence of the APOE gene in the subject and using the sequence to determine the sequence of a suitable guide RNA for targeting exon 3 in the subject). In some embodiments, double-strand break repair by non-homologous end joining (NHEJ) causes short insertions or deletions that lead to ApoE knockout. Exemplary guide RNA sequences for ApoE knockout are shown in Table 6.

[0253] Base editing

[0254] In some embodiments, the APOE gene is edited using base editing techniques (e.g., those described in Rees and Liu, Nature Reviews Genetics 19, 770-788, 2018; Komor et al., Nature 533, 420-424). In some embodiments, a guide RNA is designed using base editing to introduce an R136H mutation in a wild-type APOE gene (e.g., APOE2, APOE3, or APOE4), where exemplary guide RNAs can be found in Table 6. Base editors that convert C / G to A / T and adenine base editors that convert A / T to G / C can be used to introduce point mutations. Exemplary base editors include those described in Komor et al., Nature 533, 420-424 and Gaudelli et al., Nature 551, 464-471).

[0255] AAV delivery system

[0256] Method includes delivering a CRISPR / Cas9 genome editing system including a Cas9 nuclease and one or two guide RNAs to a subject in need. Delivery methods may include, for example, viral delivery, for example, preferably using an adeno-associated virus (AAV) vector comprising a sequence encoding Cas9 and guide RNA (one or more). Adeno-associated virus is a naturally occurring defective virus that requires another virus such as adenovirus or herpes virus as a helper virus to achieve efficient replication and productive life cycle. (Reviewed in Muzyczka et al., Curr. Topics in Micro and Immunol. 158: 97-129 (1992)). AAV vectors can effectively transduce various cell types and can produce long-term expressed transgenes in vivo. AAV vectors have been widely used for gene augmentation or replacement and have shown therapeutic effects in a range of animal models as well as in the clinic; see, e.g., Mingozzi and High, Nature Reviews Genetics 12, 341-355 (2011); Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-447; Asokan et al., Mol Ther. 2012 Apr; 20(4): 699-708. AAV vectors containing as little as 300 AAV base pairs can be packaged and can produce recombinant protein expression. For example, AAV2, AAV5, AAV2 / 5, AAV2 / 8, and AAV2 / 7 vectors have been used to introduce DNA into photoreceptor cells (see, e.g., Pang et al., Vision Research 2008, 48(3):377-385; Khani et al., Invest Ophthalmol Vis Sci. 2007 Sep;48(9):3954-61; Allocca et al., J. Virol. 2007 81(20):11372-11380). In some embodiments, the AAV vector can include an AAV capsid polypeptide described in PCT / US2014 / 060163 (or include a sequence encoding an AAV capsid polypeptide described in PCT / US2014 / 060163); for example, a viral particle comprising an AAV capsid polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 and 17 of PCT / US2014 / 060163, and a Cas9 sequence and a guide RNA sequence as described herein. In some embodiments, the AAV capsid polypeptide is an Anc80 polypeptide, such as Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; or Anc80L44.In some embodiments, AAV incorporates inverted terminal repeats (ITRs) derived from the AAV2 serotype. Exemplary left and right ITRs are shown in Table 6 of WO 2018 / 026976. However, it should be noted that many modified forms of the AAV2 ITRs are used in the field, and the ITR sequences shown below are exemplary and not intended to be limiting. Modifications of these sequences are known in the art or are obvious to the skilled person and are therefore included within the scope of the present disclosure.

[0257] Cas9 expression is driven by promoters known in the art. In some embodiments, expression is driven by one of three promoters: cytomegalovirus (CMV), elongation factor-1 (EFS) or human g-protein receptor coupled kinase-1 (hGRK1), which is specifically expressed in retinal photoreceptor cells. The nucleotide sequences of each of these promoters are provided in Table 5 of WO 2018 / 026976. In certain applications, modification of these sequences is possible or desirable, and such modifications are within the scope of the present disclosure.

[0258] The expression of gRNA in AAV vector is driven by a promoter known in the art. In some embodiments, polymerase III promoter, such as human U6 promoter. An exemplary U6 ​​promoter sequence is shown below:

[0259]

[0260] In some embodiments, the nucleic acid or AAV vector shares at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with one of the nucleic acids or AAV vectors listed above.

[0261] The above-mentioned AAV genome can be packaged into an AAV capsid (e.g., an AAV5 capsid), which can be included in a composition (e.g., a pharmaceutical composition) and / or administered to a subject. An exemplary pharmaceutical composition comprising an AAV capsid according to the present disclosure can include a pharmaceutically acceptable carrier, such as a balanced saline solution (BSS) and one or more surfactants (e.g., Tween 20) and / or a thermosensitive or reverse-thermosensitive polymer (e.g., Pluronic). Other pharmaceutical formulation ingredients known in the art may also be applicable to the compositions described herein.

[0262] Compositions comprising AAV vectors according to the present disclosure can be administered to a subject by any suitable means, including but not limited to injection (e.g., intracranial injection) and intranasal delivery. The concentration of the AAV vector in the composition is selected to ensure that a sufficient AAV dose is administered to the subject's brain, among other things, taking into account the dead volume within the injection device and the relatively limited volume that can be safely administered. Suitable doses may include, for example, 1×10 11 Viral genomes (vg) / mL, 2x10 11 Viral genomes (vg) / mL, 3x10 11 Viral genomes (vg) / mL, 4x10 11 Viral genomes (vg) / mL, 5x10 11 Viral genomes (vg) / mL, 6x10 11 Viral genomes (vg) / mL, 7x10 11 Viral genomes (vg) / mL, 8x10 11 Viral genomes (vg) / mL, 9x10 11 Viral genomes (vg) / mL, 1x10 12 vg / mL, 2x10 12 Viral genomes (vg) / mL, 3x10 12 Viral genomes (vg) / mL, 4x10 12 Viral genomes (vg) / mL, 5x10 12 Viral genomes (vg) / mL, 6x10 12 Viral genomes (vg) / mL, 7x10 12 Viral genomes (vg) / mL, 8x10 12 Viral genomes (vg) / mL, 9x10 12 Viral genomes (vg) / mL, lx10 13 vg / mL, 2x10 13 Viral genomes (vg) / mL, 3x10 13 Viral genomes (vg) / mL, 4x10 13 Viral genomes (vg) / mL, 5x10 13 Viral genomes (vg) / mL, 6x10 13 Viral genomes (vg) / mL, 7x10 13 Viral genomes (vg) / mL, 8x10 13 Viral genomes (vg) / mL or 9x10 13The composition can be delivered to the subretinal or cochlear space in any suitable volume. In some cases, the volume is selected to form a bleb in the subretinal space, such as 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, etc.

[0263] Explants are particularly useful for studying the expression of gRNA and / or Cas9 after viral transduction, and for studying genome editing in relatively short time intervals. These models also allow higher throughput than possible in animal models, and can predict expression and genome editing in animal models and subjects. Small (mice, rats) and large animal models (e.g., rabbits, pigs, non-human primates) can be used for pharmacology and / or toxicology studies, and for testing the systems, nucleotides, vectors and compositions of the present disclosure under conditions and volumes similar to those to be used in the clinic. Since the model system is selected to summarize the relevant aspects of human anatomy and / or physiology, the data obtained in these systems will generally (although not necessarily) predict the behavior of the AAV vectors and compositions according to the present disclosure in humans and animal subjects.

[0264] Screening method (test compound)

[0265] Included herein are methods for screening test compounds such as polypeptides, polynucleotides, inorganic or organic macromolecules or small molecule test compounds to identify agents for treating or preventing the following: disorders associated with dementia and / or mild cognitive impairment (e.g., Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease), neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration or retinal damage. In some embodiments, the test compound regulates the HSPG / heparin binding properties of ApoE protein (e.g., wild-type ApoE protein). In some embodiments, the test compound reduces the HSPG / heparin binding properties of ApoE protein (e.g., wild-type ApoE protein).

[0266] As used herein, "small molecule" refers to an organic or inorganic small molecule with a molecular weight lower than about 3,000 Daltons. Typically, the small molecule that can be used for the present invention has a molecular weight less than 3,000 Daltons (Da). Small molecule can be, for example, at least about 100Da to about 3,000Da (e.g., about 100 to about 3,000Da, about 100 to about 2500Da, about 100 to about 2,000Da, about 100 to about 1,750Da, about 100 to about 1,500Da, about 100 to about 1,250Da, about 100 to about 1,000Da, about 100 to about 750Da, about 100 to about 500Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000Da or about 100 to about 250Da).

[0267] The test compound can be, for example, a member of a natural product or a combinatorial chemical library. Various functions, such as charge, aromaticity, hydrogen bond, flexibility, size, length of side chain, hydrophobicity and rigidity, should be covered using groups of different molecules. Combinatorial techniques suitable for synthesizing small molecules are known in the art, for example, as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as "separation and merging" or "parallel" synthesis techniques, solid and liquid phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1: 60-6 (1997)). In addition, many small molecule libraries are commercially available. Many suitable small molecule test compounds are listed in U.S. Patent No. 6,503,713, which is incorporated herein by reference in its entirety.

[0268] The library screened using the method of the present invention can include various types of test compounds. A given library can include groups of structurally related or unrelated test compounds. In some embodiments, the test compound is a peptide or peptide mimetic molecule. In some embodiments, the test compound is a nucleic acid.

[0269] In some embodiments, for example, using methods known in the art or described herein, and correlating the structure with the resulting biological activity, such as performing structure-activity relationship studies, test compounds and libraries thereof can be obtained by systematically varying the structure of a first test compound, such as a first test compound that is structurally similar to a known natural binding partner of a target polypeptide, or a first small molecule identified as being capable of binding to a target polypeptide. Those skilled in the art will appreciate that there are various standard methods for generating such structure-activity relationships. Thus, in some cases, the work may be primarily empirical, while in other cases, the three-dimensional structure of an endogenous polypeptide or portion thereof may be used as a starting point for the rational design of one or more small molecule compounds. For example, in one embodiment, a universal library of small molecules is screened, such as using the methods described herein.

[0270] In some embodiments, the test compound is applied to a test sample, for example, a sample containing one or more ApoE proteins, and one or more effects of the test compound (e.g., HSPG / heparin binding affinity of ApoE protein (one or more)) are evaluated. For example, the ability of the test compound to modify the HSPG / heparin binding affinity of ApoE protein (one or more) can be evaluated using a heparin agarose column as described herein or an antibody that specifically recognizes the HSPG binding domain of ApoE. In some embodiments, the method for screening a test compound as described herein includes evaluating the ability of the test compound to modify (e.g., inhibit or reduce) the binding of an antibody to one or more HSPG binding sites or one or more HSPG-bound allosteric regulatory sites as described herein that bind to wild-type or mutant ApoE. In some embodiments, the test compound competes with an antibody as described herein for ApoE binding.

[0271] In some embodiments, the test sample is or is derived from (eg, taken from) an in vivo model of a disorder described herein. For example, an animal model, such as a rodent such as a rat, can be used.

[0272] The test compound that has been screened and determined to reduce or modify ApoE and HSPG / heparin binding by the methods described herein can be considered as a candidate compound. Screened in an in vivo model of, for example, a disease, such as dementia and / or mild cognitive impairment (such as those associated with Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease), neurodegenerative disease, cerebrovascular disease, brain injury, retinal degeneration or retinal injury, and determining that the disease, for example, one or more symptoms of the disease have a desired effect on the candidate compound, can be considered as a candidate therapeutic agent. Once screened in a clinical setting, the candidate therapeutic agent is a therapeutic agent. Candidate compounds, candidate therapeutic agents and therapeutic agents can be optionally optimized and / or derivatized, and are formulated with a physiologically acceptable excipient to form a pharmaceutical composition.

[0273] Thus, test compounds identified as "hits" in a first screen (e.g., test compounds with a desired effect on a condition) can be selected and systematically altered, for example, using rational design to optimize binding affinity, avidity, specificity, or other parameters. Such optimizations can also be screened using the methods described herein. Thus, in one embodiment, the invention includes screening a first library of compounds using methods known in the art and / or described herein, identifying one or more hits in the library, systematically structurally altering the hits to generate a second library of compounds structurally related to the hits, and screening the second library using the methods described herein.

[0274] Test compounds identified as hits can be considered candidate therapeutic compounds for treating, preventing or delaying the development and progression of conditions associated with dementia and / or mild cognitive impairment as described herein, such as Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease, and for treating, preventing or delaying the development or progression of neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration or retinal damage. Various techniques that can be used to determine the structure of a "hit" can be used in the methods described herein, such as NMR, mass spectrometry, gas chromatography equipped with an electron capture detector, fluorescence and absorption spectroscopy. Therefore, the present invention also includes compounds identified as "hits" by the methods described herein, as well as methods of their administration and methods for treating, preventing or delaying the development or progression of conditions described herein.

[0275] Test compounds identified as candidate therapeutic compounds can be further screened by administration to animal models of disorders associated with any disorder as described herein. The animal can be monitored for changes in the disorder, such as improvements in a parameter of the disorder, such as a parameter associated with a clinical outcome.

[0276] Treatment

[0277] Methods described herein include methods for treating, preventing or delaying the development or progression of conditions, neurodegenerative diseases, cerebrovascular diseases, brain damage, retinal degeneration or retinal damage associated with dementia and / or mild cognitive impairment. In some embodiments, the conditions associated with dementia and / or mild cognitive impairment are Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease or Huntington's disease. Other non-limiting examples of neurodegenerative diseases include prion disease, motor neuron disease and amyotrophic lateral sclerosis (ALS). Non-limiting examples of cerebrovascular diseases include stroke, carotid stenosis, spinal stenosis and aneurysm. Non-limiting examples of brain damage include traumatic brain injury and acquired brain injury. Retinal degeneration, such as glaucoma, age-related macular degeneration, can involve amyloid β and neurofibrillary tangles toxicity, establishing a link between retinal degeneration and neurodegeneration (e.g., Alzheimer's disease) (see, e.g., McKinnon, Frontiers in Bioscience 8, s1140-1156, 2003; Johnson et al. PNAS 99 (18) 11830-11835, 2002; and Sivak, Investigative Ophthalmology & Visual Science, 54 (1) 871-880, 2013). Therefore, treatments for neurodegenerative diseases can be used to treat retinal or optic nerve degeneration.

[0278] The method comprises administering to a subject in need or determined to be in need of such treatment a therapeutically effective amount of any antibody, peptide, fusion protein, or genome editing system as described herein.

[0279] Methods described herein can also be used for subjects at risk of developing any disease described herein. Subjects at risk of developing Alzheimer's disease can include subjects homozygous or heterozygous for APOE4 alleles, carriers of autosomal dominant Alzheimer's disease pathogenic mutations (e.g., amyloid beta precursor (APP) gene, PSEN1 gene or PSEN2 gene mutations), trisomy 21 (e.g., subjects whose cognitive impairment is only developmental). Subjects at risk of developing Alzheimer's disease can also include subjects with polygenic risk scores associated with the increased risk of developing the disease, and subjects with brain imaging or other biomarkers (e.g., biomarkers in body fluids) evidence of Alzheimer's disease. Methods for preventing or delaying the development of diseases described herein can also be used for subjects who are not at risk of developing the above-mentioned diseases, such as subjects over 50 years old (e.g., 55 years old, 60 years old, 65 years old, 70 years old, 75 years old, 80 years old, 85 years old, 90 years old or 95 years old).

[0280] As used in this context, "treating" refers to ameliorating at least one symptom of a disorder associated with a disorder described herein. Typically, Alzheimer's disease leads to fibril formation, amyloid aggregation, and cognitive decline; thus, treatment may result in a reduction in fibril formation and / or amyloid aggregation in the brain, a reduction in the formation of tangled tau proteins, improved brain metabolism, improved neurocognitive function, and / or improved cognitive ability.

[0281] Example

[0282] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. In terms of the specific materials mentioned, they are only for illustrative purposes and are not intended to limit the present invention. Those skilled in the art may develop equivalent means or reactants without exercising inventive ability and without departing from the scope of the present invention.

[0283] Example 1: Identification of the APOE3 Christchurch R136S mutation in PSEN1 mutation carriers Materials and methods:

[0284] Clinical evaluation: The institutional review boards of the University of Antioquia, Massachusetts General Hospital, and the Schepens Eye Research Institute of Massachusetts Eye and Ear approved this study. As with all study participants, the proband case provided her written informed consent. Clinical ratings and neuropsychological testing were performed as shown in Table 1. PSEN1 E280A genotyping was performed as previously described. 1 .

[0285] All clinical measures were performed at the University of Antioquia (Medellín, Colombia) and were conducted in Spanish by physicians and psychologists trained in the assessment. Neurocognitive testing included a comprehensive multidomain assessment. Some of the tests performed were the Spanish version of the Mini-Mental State Examination (MMSE), the Clinical Dementia Rating (CDR), and the Consortium to Establish a Registry for Alzheimer's disease battery, which have been adapted to this Colombian population. 2 Other tests include the Yesavage Geriatric Depression Scale3 and functional assessment staging test 4 These tests were completed within six months of the brain imaging.

[0286] A detailed ophthalmological evaluation was performed. It included visual acuity assessment, slit lamp and indirect ophthalmoscopy. Ultra-wide field fundus and fundus autofluorescence images using the Optos Panoramic 200Tx imaging system (Optos PLC, Dunfermline, Scotland, UK) were obtained. In addition, OCT angiography using Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany) and Cirrus HD-OCT with AngioPlex (Carl Zeiss Meditec, Dublin, CA) was performed.

[0287] Additional studies were performed after the discovery that PSEN1 E280A mutation carriers had two copies of the APOE3ch variant. Fasting serum lipid testing was performed to explore the possibility of type III hyperlipoproteinemia, a condition found in 5-10% of people who are homozygous for the relatively AD-protective APOE2 allele and in most but not all APOE3ch carriers. 5 .

[0288] Finally, analysis of data from clinically and neuropathologically validated AD cases and controls from the AD Genetics Consortium was used to elucidate whether homozygosity for the APOE2 allele is associated with a very low risk of late-onset AD dementia.

[0289] Whole exome sequencing: Whole exome capture and sequencing were performed using Illumina chemistry for variant discovery; rare variants with frequencies below 1% in genes previously associated with AD were considered for the search for candidate risk modifiers. Specifically, rare DNA variants (minor allele frequency <1%) within exonic regions and splice site junctions (5 bp into introns) of genes were identified using bioinformatics tools. Whole exome libraries were constructed and sequenced on an Illumina HiSeq 4000 sequencer using 151 bp paired-end reads. A previously described protocol modified as follows was used. 6Library construction was performed. The genomic DNA input in 10 μL solution was reduced from 3 μg to 50 ng and enzymatic shearing was performed. The dual-index Illumina double-end adapter was replaced with a palindromic forked adapter, in which a unique 8-base index sequence was embedded and added to each end for adapter connection. In-solution hybridization selection was performed using an Illumina Rapid Capture Exome Enrichment Kit with a 38Mb target region (29Mb bait). The target region includes 98.3% of the intervals in the Refseq exome database. Prior to hybridization, the dual-index libraries were merged into groups of up to 96 samples. After elution from the streptavidin beads, the enriched library pools were quantified by PicoGreen and then standardized. For cluster amplification and sequencing, quantitative PCR (KAPA Biosystems) was used to quantify the library prepared using bifurcated, indexed joints, standardized to 2 nM using the Hamilton Starlet liquid handling system, and equal volumes were merged using the Hamilton Starlet liquid handling system. The pool was then denatured in 0.1 N NaOH. The denatured sample was diluted into a strip tube using the Hamilton Starlet liquid handling system. Cluster amplification of templates was performed using Illumina cBot according to the manufacturer's protocol (Illumina). Flow cell (flowcell) was sequenced on a HiSeq 4000 synthetic sequencing kit and then analyzed using RTA2.7.3.

[0290] Exome sequencing data were processed and analyzed using the bioinformatics analysis pipeline of the Center for Personalized Medicine (CPM) Clinical Genomics Laboratory and the Central Clinical Exome Sequencing of the Translational Genomics Institute. In brief, the Dragen Genome Pipeline of EdicoGenome with default parameters was used for sequence alignment and variant calling. The open source software samtools and bcftools (samtools.github.io / ) were used with a set of custom scripts to determine coverage and initial variant filtering based on ExAC (Exome Aggregation Consortium, exac.broadinstitute.org / ) allele frequencies. 7 . Sequence alignment was performed against the human hs37d5 bait genome. 8To identify potential modifying variants, a master gene list of 15 genes was generated based on two HPO terms: HP:0002511, Alzheimer's disease; HP:0003584, late onset. These genes are AAGAB, ABCC8, AKT2, APOE, APP, BEAN1, GATA1, GCK, HMGA1, HNF1B, HNF4A, LDB3, PAX4, PSEN1, and PSEN2. 9 Rare DNA variants (minor allele frequency <1%) in exonic regions and splice site junctions (5 bp into introns) of these genes were further annotated and analyzed using a commercial tool (Cartagenia v5.0). Sequence alterations were reported according to the Human Genome Variation Society (HGVS v2.0) nomenclature guidelines.

[0291] Whole-genome sequencing: Whole-genome sequencing (WGS) and Genomizer analysis (v 10.1.0) were used to perform comprehensive and unbiased sequencing of other potential genetic risk modifiers, including those associated with a lower risk of Alzheimer's disease, helping to rule out other potential protective genetic factors. 10 To process the WGS data, the same dragen analysis pipeline as above was used. The data was aligned to the GRCh37 bait genome (hs37d5). Variants detected at a depth of <10X were filtered out and then annotated using the Variant Effect Predictor (VEP) tool from Ensembl. The VEP version used was v93. The filtered and annotated variant set was then compiled for Genomizer analysis.

[0292] APOE structure display: The image was created using NGL Viewer 12 From the previously published structure of PDB 2L7B 11 The data were obtained and modified from RCSBPDB (rcsb.org).

[0293] APOE genotyping by Sanger sequencing: The reaction mixture for the amplification process was carried out in a 50 μL volume and included the following components: 1x PfuUltra II Hostart Master Mix, 1 μL of each primer (10 μmol / L) (forward primer: 5'- A GCCCTTCTCCCCGCCTCCCACTGT-3' (SEQ ID NO: 67) and reverse primer: 5'-CTCCGCCACCTGCTCCTTCACCTCG-3' (SEQ ID NO: 68)), 5% DMSO and 1 μL of genomic DNA (100 ng / μL). 13PCR cycles were run as follows: initial denaturation at 95° C. for 2 min, followed by 35 cycles of denaturation at 95° C. for 20 s, annealing at 60° C. for 30 s, extension at 72° C. for 40 s, and a final extension at 72° C. for 5 min. PCR products were purified using the QIAquick gel extraction kit from Qiagen and sequenced by the MGHCCIB DNA core using a 3730xl sequencer from Applied Biosystems.

[0294] MRI and PET Imaging: Pittsburgh Compound B (PiB), flortaucipir (FTP) positron emission tomography (PET), and structural magnetic resonance imaging (MRI) measurements were obtained at Massachusetts General Hospital as described previously and analyzed at Massachusetts General Hospital and the Banner Alzheimer's Disease Institute. 14 Fluorodeoxyglucose PET images were acquired at the University of Antioquia, Colombia, and analyzed as previously described. 15 Imaging data from cases were compared with data from younger PSEN1 E280A mutation carriers who developed MCI at the expected age of clinical onset in relatives and from cognitively intact mutation carriers.

[0295] As mentioned before 16-19 MRI was performed on a 3T Tim Trio (Siemens) and included magnetization prepared rapid gradient echo (MPRAGE) processed with Freesurfer (FS) to identify gray and white matter and pial surfaces to allow segmentation of regions of interest (ROIs) as follows: cerebellar gray matter, hippocampus, and the following Braak stage-related cortices: entorhinal cortex, parahippocampal gyrus, inferior temporal gyrus, fusiform gyrus, posterior cingulate gyrus.

[0296] 18F-Flortaucipir (FTP) was prepared at MGH with a radiochemical yield of 14 ± 3% and a specific activity of 216 ± 60 GBq / μmol at the end of the synthesis (60 min) and has been validated for use in humans (Shoup et al., 2013). 11C-Pittsburgh compound B was prepared and PET images were obtained as described previously. 16All PET images were acquired using a Siemens / CTI (Knoxville, TN) ECAT HR+ scanner (3D mode; 63 image planes; 15.2-cm axial field of view; 5.6-mm lateral resolution, and 2.4-mm slice interval. 11C PiB PET was acquired using a bolus of 8.5 to 15 mCi followed by 60-min dynamic acquisition of 69 frames (12 x 15 sec, 57 x 60 sec). 18F FTP was acquired at 4 x 5-min frames over 80–100 min after a bolus of 9.0 to 11.0 mCi. PET images were reconstructed and attenuation corrected, and individual frames were evaluated to verify adequate counting statistics and the absence of head motion.

[0297] 18F FTP-specific binding in the FS ROI is expressed as the standardized uptake value ratio (SUVR) to the cerebellum, similar to previous reports. 19 , using the FS cerebellar gray matter ROI as reference. For voxelwise analysis, each subject's MPRAGE was registered to the template MR in SPM8 (SPM), and the spatially transformed SUVR PET data were smoothed using an 8 mm Gaussian kernel to account for individual anatomical differences. 20 To account for possible off-target binding of 18F FTP in the choroid plexus (which could confound hippocampal signaling), we used linear regression to regress the choroid plexus as previously reported. 21 .

[0298] 11C PiB PET data are expressed as distribution volume ratio (DVR) with cerebellar gray matter as reference tissue; regional time-activity curves were used to calculate regional DVR for each ROI using the Logan graphical method applied to data from 40 to 60 minutes after injection 16,22 11C PiB preservation was assessed using a large cortical ROI aggregate that included the previously described frontal, lateral temporal, and retrosplenial cortices (FLR) 23,24

[0299] 8F-fluorodeoxyglucose PET was performed on a 64-section PET / computed tomography imaging system (Biograph mCT; Siemens) at rest in a dark room after a 30-minute radiotracer uptake period, using intravenous administration of 5 mCi (185 million Bq) of 18F-fluorodeoxyglucose, followed by 30 minutes of dynamic emission scanning (six 5-minute frames). Images were reconstructed using computed tomography attenuation correction. The ratio of the precuneus to the whole brain cerebral glucose metabolic rate (CMRgl) was characterized from bilateral regions of interest (ROIs) in each participant's 18F-fluorodeoxyglucose PET images using an automated brain mapping algorithm (SPM8; fil.ion.ucl.ac.uk / spm / software / spm8). The hippocampus to total intracranial volume ratio was characterized from bilateral ROIs in each participant's T1-weighted MR images using FreeSurfer (surfer.nmr.mgh.harvard.edu). All images were visually inspected to verify ROI characterization.

[0300] Amyloid aggregation studies: Human ApoE3 protein fragments (including the carboxyl-terminal domain and histidine tag) with and without the Christchurch variant were synthesized in bacteria, purified (Innovagen), and used for the analysis of amyloid aggregation using Thioflavin T ( ThTβ-amyloid protein (1-42) aggregation kit, catalog number AS-72214) in vitro evaluation of these proteins against Aβ 42 For this assay, 55 μM Aβ 42 Add to a solution of 10 μM wild-type apoE3 protein or mutant 136Arg→Ser ApoE3 protein in a clear, non-binding 96-well plate. Samples were then mixed with 2 mM Thioflavin T dye and fluorescence was read at Ex / Em=440 / 484 at intermittent intervals over 2 hours. Plates were kept at 37°C with 15 seconds of shaking between readings.

[0301] Full-length ApoE3 proteins with and without the Christchurch mutation were also transiently transfected in the Flp-In TM T-REx TM 293 (Thermo Fisher Scientific) mammalian cells to confirm the effects of these proteins on Aβ using a previously published split-luciferase complementation assay. 42 The effect of aggregation. 26 The latter analysis was performed using plasmid #87086 from Addgene 27) was expressed as WT or APOE3 introduced into the Christchurch variant by site-directed mutagenesis. Luciferase assay reagents were purchased from Promega.

[0302] result

[0303] Approximately 1,200 Columbia presenilin 1 (PSEN1) E280A mutation carriers and 4,600 non-carriers were identified, who together comprise the world's largest known autosomal dominant Alzheimer's disease (ADAD) family. 28,29 Mutation carriers typically develop mild cognitive impairment (MCI) and dementia at a median age of 44 (95% CI, 43-45) and 49 (95% CI, 49-50) years, respectively. 30,31 Studying autosomal dominant AD (ADAD) mutation carriers who remain cognitively intact until older age may help identify risk-reducing genetic variants. 32 Characterizing AD biomarkers in these individuals will help understand the potentially targetable mechanisms by which these genes exert their relative protective effects.We identified a PSEN1 E280A mutation carrier who did not develop MCI until her 70s, nearly 30 years after the median age of onset.

[0304] This study was conducted in accordance with the guidelines of the Institutional Review Board with the written informed consent of the participant (her exact age and other identifying information have been omitted to protect her anonymity and confidentiality). 42 (Aβ 42 )-overproducing PSEN1 E280A mutation, family informant reports confirmed that she was cognitively intact until age 70, then consistent with MCI at annual evaluations at 24-month intervals 33 . She remained fully independent in basic and instrumental activities of daily living, with no overt signs of deterioration in her ability to perform these activities. At the intake evaluation, her memory deficits were limited to recent events, and her neurologic examination was normal. Her age- and education-adjusted neuropsychological test scores indicated preferential impairments in recall memory, relatively preserved recognition memory, initial learning, naming, visuospatial abilities, and verbal fluency, as well as relatively stable cognitive performance over the 24-month evaluation period (Table 1).

[0305] Table 1: Cognitive test scores and percentiles

[0306]

[0307] MMSE: Mini-Mental State Examination

[0308] CERAD: Establishing an Alzheimer's Disease Registry Alliance

[0309] GDS: Geriatric Depression Scale

[0310] EDG: Full Decline Scale

[0311] *Due to her limited literacy, the MMSE subtests requiring reading and writing skills were not administered. Her maximum possible score was 23 (not 30).

[0312] **Percentiles are calculated using the norm for this Colombian population.

[0313] A percentile between the 25th and 75th places her performance within the average range for her age and education.

[0314] A percentile between 9 and 25 would classify her performance as below average. A percentile between 2 and 8 would classify her performance as low. A percentile below 1 would classify her performance as very low.

[0315] #The brain imaging described in this report was obtained three months after initial cognitive testing.

[0316] Whole exome sequencing confirmed her PSEN1 E280A mutation and found that she also had two copies of the rare APOE3 Christchurch R136S (APOEch) mutation. Sanger sequencing confirmed the latter finding. Whole genome sequencing and Genomizer analysis were used to comprehensively identify and rank all potentially important rare and common variants. 34 Using this approach, the PSEN1 E280A mutation was confirmed to be the participant's major risk factor, and APOE3ch homozygosity was confirmed to be her major resistance factor.

[0317] APOE, the major susceptibility gene for late-onset AD, has three common alleles (APOE2, 3, and 4). Compared with the most common APOE3 / 3 genotype, APOE2 is associated with a lower risk of AD and a greater age of dementia onset. 35 And each extra copy of APOE4 was associated with higher risk and a younger age of onset. 36,37 APOEch variant, arginine-to-serine substitution at amino acid 136 (136Arg→Ser), corresponding to codon 154, 38 Can be located on any common APOE allele, 39 This included the participant's two APOE3 alleles. Figure 1The structural model of the wild-type ApoE3 protein is shown. The N-terminal (residues 1-191) and C-terminal (residues 201-299) domains are highlighted. The amino acid positions of the APOE4 (C112R), APOE3ch (R136S), and APOE2 (R158C) variants are shown.

[0318] The APOE3ch variant is not present in the AlzAD or ExAC databases reporting approximately 180,000 exomes. R136S was previously identified in APOE2 individuals with HLP III, but its potential impact in the progression of AD has not been previously reported. 40 We sequenced DNA samples from two other PSEN1 E280A carriers with delayed age of onset by whole genome sequencing, at ages 62 and 70. Neither of these individuals had the APOE3 R136S variant nor APOE2, which had previously been shown to delay disease onset in this family. 41

[0319] To confirm the potential association between the APOE3 R136S mutation and delayed age of AD onset, we performed whole-genome sequencing and neurological and neuropsychological testing in four offspring of the proband case who were older than 50 years and predicted to carry APOE3 R136S. Figure 2 Representative Sanger sequencing results for APOE from control, proband, and offspring samples are shown. Top row: C112 homozygous sequences shown in all cases. Middle row: R136 homozygous sequences from control individuals are shown in the left panel. The middle panel shows the homozygous changes that lead to the R136S mutation. The right panel shows an example of the R136S heterozygous mutation in the offspring of the proband. Bottom row: R158 homozygous sequences shown in all cases. Figure 3 The pedigree of the proband is shown, with circles representing females, squares representing males, diamonds representing individuals whose sex is masked for privacy, arrows depicting proband individuals with MCI, and shading representing individuals with a history of dementia. Deceased individuals are marked with slashes. Individual APOE and PSEN1 genotypes are shown as appropriate to preserve anonymity. Although other unknown genetic or epigenetic factors may contribute to the late age of onset of cognitive impairment in these two related PSEN1 E280A carriers, we propose that the APOE3 R136S variant modifies the AD phenotype by buffering the effects of amyloid β accumulation in the brain and subsequently delays the onset of tau pathology, neurodegeneration (i.e., brain atrophy), and symptom onset.

[0320] Carriers of APOEch and other rare mutations in the low-density lipoprotein receptor (LDLR) binding region of APOE typically have type III hyperlipoproteinemia (HLP-III), similar to that observed in 5-10% of APOE2 homozygotes. 43,44 Participants in this report demonstrated HLP-III, including APOEch and elevated triglyceride and total cholesterol levels (see Table 2).

[0321] Table 2: Dyslipidemia examination

[0322]

[0323] HDL-C: high-density lipoprotein cholesterol, VLDL-C: very low-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol. *Normal range according to Merck Manual and laboratory values.

[0324] Detailed laboratory examinations showed abnormal lipid profiles (lipid profile) in three of our proband individuals and four offspring carrying APOE3 R136S (Table 3). The total cholesterol and triglyceride levels of these four subjects were high. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in two offspring were higher, and could not be determined in the proband individual and one offspring, and their triglyceride levels were higher than 400mg / dL (accuracy threshold of indirect method of lipid analysis) (Table 3). Further analysis using direct enzymatic testing showed that the LDL of these two individuals was higher than normal LDL. Despite carrying APOE3R136S and APOE4, the lipid profile of one offspring was within the normal range. Incomplete penetrance of HLP III in APOE2 and R136S mutation carriers has been reported previously. 45 We excluded secondary causes of lipid disorders in these subjects, such as diabetes, obesity, alcoholism, renal disorders, or thyroid disease. None of the mutation carrier individuals had xanthomas, which are diagnostic of HLP III, or cardiovascular disease. The combination of abnormal lipid profiles and the APOE3 R136S mutation in these subjects is consistent with a diagnosis of familial HLP III.

[0325] Table 3: Dementia and dyslipidemia examinations in the study population

[0326]

[0327] A detailed ophthalmologic evaluation was performed in a PSEN1 E280A mutation carrier with two APOE3ch alleles. The carrier had visual acuity of 20 / 70 in the right eye and 20 / 40 in the left eye. Anterior segment examination noted a dense posterior capsule opacity in the posterior chamber intraocular lens in the right eye. A nuclear sclerotic cataract ( Figure 4A and 4B Posterior segment examination was normal in both eyes, with clear vitreous cavities, normal optic nerve, macula, and peripheral retina. Further testing by optical coherence tomography (OCT) of the right eye was normal, except for a small area of ​​hyper-reflectivity covering the fovea ( Figure 4D ). Figure 4C Infrared image showing the right eye, depicting Figure 4D In addition, OCT imaging of the left eye showed a degenerative lamellar hole (caused by Figure 4F * in the figure), with small defects in the external membrane and ellipsoidal layer ( Figure 4F ).

[0328] Although several mechanisms have been proposed to explain the effects of APOE variants on AD risk, most studies have focused on their effects on Aβ 42 Differential effects of aggregation and plaque burden (APOE2<3<4). 47 In this study, neuroimaging measures were used to elucidate whether participants' resistance to clinical onset of AD was associated with: a) despite more than seven decades of Aβ 42 overproduction of tau but relatively small Aβ plaque burden or b) relatively high Aβ plaque burden but limited paired helical fibril (PHF) tau (neurofibrillary tangle burden) and downstream assays of neurodegeneration.

[0329] Neuroimaging findings among participants Figure 5 Figure 2. Positron emission tomography (PET) images superimposed on the medial and lateral surfaces of the left hemisphere. The upper row shows PET measurements of amyloid plaque burden (PiB DVR). The lower row shows PET measurements of paired helical fibril (PHF) tau (i.e., neurofibrillary tangles) burden. The individual with late-onset MCI was in his 70s, compared with a typical age of onset of MCI of 44 years.

[0330] like Figure 5As shown, a person with late-onset MCI had abnormally high PET measurements of Aβ plaque burden, as indicated by a higher mean cortical to cerebellar Pittsburgh compound B (PiB) distribution volume ratio (DVR = 1.96) compared to PSEN1 E280A carriers who developed MCI in their 40s (DVR of 1.49-1.60). Despite her high Aβ plaque burden, the magnitude and / or spatial extent of her PHFtau burden and neurodegeneration was relatively limited: her flortaucipir (tau) PET measurements were limited to the medial temporal region and the occipital region that is typically less affected, and relatively few other regions that are characteristically affected in the clinical stage of AD ( Figure 5 ). Her fluorodeoxyglucose PET measurements of brain glucose metabolic rate were preserved in brain regions known to be preferentially affected by AD, including precuneus-to-whole-brain measures that were higher than those of PSEN1 E280A mutation carriers who developed MCI at a younger age and many younger, cognitively unimpaired mutation carriers.

[0331] Figure 6 Show average cortical amyloid plaque load, entorhinal cortex PHF tau load, hippocampal volume, precuneus glucose metabolism measured value.These measured values ​​are based on brain imaging results, obtained from PSEN1 E280A mutation carriers (red dots) with two APOE3ch alleles and abnormal late-onset MCI, PSEN1 E280A mutation carriers (black dots) suffering from MCI under the typical MCI onset age of relatives are younger, and PSEN1 E280A mutation carriers (grey dots) not yet developed MCI are obtained.Amyloid plaque load is expressed as average cortex-to-cerebellum distribution volume ratio (DVR).Paired spiral-like fiber (PHF) tau load is expressed as entorhinal cortex-to-cerebellum flortaucipir (FTP) standard uptake value ratio (SUVR).The hippocampal volume that can be reduced due to hippocampal atrophy is expressed as hippocampus-to-whole brain volume ratio. Reduced cerebral glucose metabolism in AD-affected brain regions with synaptic dysfunction and loss is reflected in the precuneus-to-whole brain cerebral glucose metabolic rate (CMRgl) ratio. Figure 6As shown, although the PSEN1 E280A mutation carrier with two APOE3ch alleles had the highest amyloid plaque burden by far, she did not have a relatively severe PHF tau burden or hippocampal atrophy, and she had no evidence of precuneus glucose hypometabolism. Her MRI-based hippocampal-to-whole brain volume (a measure of hippocampal atrophy that can be affected by AD and / or normal aging) was within the range of mutation carriers who develop MCI in their 40s. Without wishing to be bound by theory, these results suggest that the resistance of this APOE3ch homozygote to the clinical onset of AD is mediated by a mechanism that limits tau pathology and neurodegeneration even in the face of high Aβ plaque burden.

[0332] To investigate the functional consequences of the APOE3ch variants, Aβ was expressed in the presence of wild-type human ApoE3 protein from bacteria, in the presence of mutant ApoE3ch protein, or in the absence of any ApoE protein. 42 In vitro aggregation was compared. Aβ was detected by Thioflavin T fluorescence 42 The rate of fibril formation. 42 Aggregation was highest in the presence of wild-type human ApoE3 protein (C-terminal domain) and lower in the presence of human ApoE3ch (similar to that observed in the presence of ApoE2 48 ), and lowest in the absence of any ApoE ( Figure 7 ).

[0333] This finding was confirmed using a sensitive split-luciferase complementation assay, in which the luciferase signal is reestablished once the amyloid protein forms oligomers. 48 Oligomers are some of the most toxic species of amyloid. 49 Full-length ApoE3ch expression in mammalian cells elicits significantly less Aβ compared to wild-type ApoE3 42 Oligomerization, as luciferase luminescence via oligomer formation was significantly reduced in ApoE3ch compared to wild-type ApoE3 ( Figure 8 These results provide validation for the genetic analysis and suggest that the protective effects of ApoEch may be due, at least in part, to its ability to promote Aβ 42 Limited capacity to aggregate. It remains possible that the study participant could have had larger Aβ plaque deposits, allowing her to survive into her 70s without the APOEch / 3ch genotype, and that the ApoE3ch protein altered the morphology of Aβ aggregation in a way that limited downstream neuroinflammation, tau pathology, neurodegeneration, and cognitive decline.

[0334] A small number of Colombian relatives were found to carry one copy of the APOE3ch mutation. 50Four PSEN1 E280A mutation carriers were included, who developed MCI at a median age of 45 years. Therefore, it is speculated that APOE3ch homozygosity may be required to significantly reduce risk and delay the clinical onset of autosomal dominant AD. Due to the small sample size, APOEch heterozygous individuals may still have partial protection against cognitive decline associated with autosomal dominant AD and substantial protection against sporadic late-onset AD and / or neurodegeneration.

[0335] These results suggest that APOE variants differ in the extent of their pathogenic function (APOEch and APOE2<3<4), and that APOE3ch / 3ch and APOE2 / 2 are associated with the greatest loss of function. Interventions that safely and adequately edit APOE, reduce its expression, or inhibit its pathogenic function could have profound implications for the treatment and prevention of AD. Interestingly, inhibition of APOE expression in the brain using antisense oligonucleotides in mice with Aβ overproduction altered Aβ plaque morphology and reduced dystrophic neurites. 51 This approach may be feasible because loss of APOE expression is tolerated in middle-aged men who are homozygous for the frameshift variant. 52 Moreover, the availability of statins to treat HPL-III supports the potential tolerability of ApoE-lowering therapy. See, e.g., Reiman et al. Nat Commun 1191):667, 2020.

[0336] Without wishing to be bound by theory, these results further suggest that homozygosity for APOE3ch and APOE2 is associated with strong resistance to clinical onset of AD; these genotypes exert their beneficial effects by directly or indirectly limiting downstream tau pathology and neurodegeneration; these effects are not based solely on the size of Aβ plaque load, despite the relative reduction in Aβ aggregation mediated by ApoE. These findings suggest a role for APOE in the understanding, treatment, and prevention of AD, and may stimulate interest in developing APOE-modifying genes and drug therapies for the condition.

[0337] Example 2: Heparin Binding Properties of APOE3ch Mutant Protein

[0338] Materials and methods

[0339] Heparin column protocol: A 1 ml heparin column (BioVision-6554-1) was used to compare the heparin binding affinity of ApoE2, ApoE3, ApoE3ch and ApoE4 protein isoforms. The column was adapted to room temperature for 1 hour before use. The column was washed with 5 mL of 20 mM TRIS-HCL (pH 7.5). Then a 1 mL sample containing 50 μg / mL of APOE recombinant protein in 20 mM TRIS-HCL (pH 7.5) was circulated through the column 5 times. The column was then washed 5 times with 20 mM TRIS-HCL (pH 7.5). An increased NaCl gradient (0.025-1 M) in 20 mM TRIS-HCL was passed through the column, and 1 mL fractions were collected and subsequently prepared for immunoblotting.

[0340] Immunoblotting: Immunoblotting confirmed the elution of ApoE isoforms in fractions collected from the heparin-bound column. Fractions were diluted to a final volume of 40 μl in 10 μl RIPA buffer (Cell Signaling Technology), 4 μl DTT (1 M) and 10 μl Laemmli buffer. TGX TM Samples were separated on precast protein gels (Bio-Rad), transferred to nitrocellulose membranes (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-CORBiosciences, Lincoln, NE), and probed with mouse anti-his tag (Novus biologicals.) and IRDye800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using the Odyssey infrared imaging system and visualized on Image Studio version 2.1 (LI-COR Biosciences). Individual gels were combined to generate Fig.10 .

[0341] Heparin plate ELISA protocol: ELISA was performed using heparin microplates (Bioworld; 50-197-531). They were blocked for one hour using sample preparation reagent (DY008). The heparin plates were incubated for 2 hours with 0.1 μg / well of each recombinant ApoE protein isotype (ApoE2, ApoE3, ApoE3ch, and ApoE4), then the plates were washed five times in PBS containing a NaCl gradient (0-0.5M), and then washed three times in wash buffer (DY008). The anti-His tag antibody was incubated overnight at 1:10,000 (Novusbiologicals; NBP2-61482). The plates were then washed five times to ensure removal of unbound primary antibodies, incubated for 45 minutes with donkey anti-rabbit-HRP (1:10000), and then washed five times to ensure removal of secondary antibodies. Sulfuric acid from the ELISA kit (DY008) was heated to 37°C before adding 100 μl of tetramethylbenzidine (Millipore) to start the detection phase of the reaction. After incubation for 5 minutes, sulfuric acid was added to terminate the reaction. The plate was then read using SPECTRAmax plus 384 (Molecular Devices). The reading wavelength was 450 nm. To calculate the amount of antigen present in the sample, a standard curve was drawn based on serially diluted recombinant Notch3 protein using Prism 6 (GraphPad Software).

[0342] result

[0343] The heparan sulfate proteoglycan (HSPG) fraction is a glycosaminoglycan present in hundreds of proteins in the plasma membrane and extracellular matrix. Protein-protein interactions mediated by HSPGs play a key role in numerous processes associated with Alzheimer's disease pathology, including amyloid and tau pathology, as well as neurodegenerative disorders. The ability of various ApoE isoforms, including ApoEch, to bind heparin, a glycosaminoglycan commonly used to mimic HSPG-protein interactions, was investigated. Briefly, fractions containing the ApoE isoforms ApoE2 and ApoE4 eluted from a heparin column under an increasing NaCl gradient (0-0.65 M) were analyzed using ELISA. As Fig. 9 As shown, the ApoE variant ApoE4, which is associated with a higher risk of Alzheimer's disease, has a higher affinity for heparin than the ApoE2 variant, which is known to be protective. Next, fractions containing His-tagged ApoE2, ApoE3, ApoE4, and ApoE3ch eluted from the heparin column under increasing NaCl gradients (0-0.65M) were analyzed using immunoblotting. Fig.10As shown, heparin binding of ApoE3ch was impaired and much lower than that of ApoE2. The affinity of the ApoE isoforms for heparin was also analyzed using the heparin plate ELISA protocol as described in Materials and Methods. Figures 11A-11B As shown, ApoE3ch exhibited significantly lower levels of heparin binding, as ApoE3ch was released from the heparin column at much lower NaCl concentrations than those required for ApoE4 release.

[0344] Example 3: Generation of antibodies against wild-type ApoE and ApoEch mutant proteins

[0345] Materials and methods

[0346] Antibody competition assay: The antibody was incubated with ApoE3 recombinant protein (50ug / ml in 20mM Tris-HCL) at a ratio of 1:10 and incubated for 3 hours at room temperature. A negative control containing only culture medium and a positive control containing only recombinant protein ApoE3 were used. The antibody / ApoE3 recombinant protein solution and control were passed through a heparin column and exposed to an increasing NaCl gradient (as described in Example 2 above). Fractions were collected and evaluated by ELISA and immunoblotting.

[0347] BCA assay: First, the fractions collected from the heparin column were screened using the bicinchoninic acid assay (BCA assay) (Pierce BCA protein assay kit). The assay was performed using 200 μl of a mixture of reagents A and B and 25 μl of each fraction. The 96-well plate was incubated at 37°C for 30 minutes and read at 562 nm. The plate was read using a Synery2 microplate reader (BioTek Instrument. Inc) and Gen5 version 1.11 software).

[0348] Immunoblotting: Immunoblotting confirmed the elution of ApoE3 recombinant protein in the fractions collected from the heparin-bound column. 10 μl of the fractions were diluted to a final volume of 40 μl using RIPA buffer (Cell Signaling Technology), 10x (DTT 1M) and 4x Laemmli buffer. TGX TMSamples were separated on precast protein gels (Bio-Rad), transferred to nitrocellulose membranes (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-CORBiosciences, Lincoln, NE), and probed with mouse anti-his tag (Novus bios) and IRDye 800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using the Odyssey infrared imaging system and visualized on Image Studio version 2.1 (LI-COR Biosciences).

[0349] ELISA: The affinity of antibodies designed against the heparin binding domain of ApoE to ApoE3 and ApoEch mutant recombinant proteins was tested using ELISA. Wash the Ni-NTA HisSorb plate (Qiagen) 3 times with wash buffer 1 (DY008). Suspend the ApoE recombinant protein in buffer (DY008) to produce a final concentration of 0.5ug / ml. Incubate the plate with 200μl of ApoE recombinant protein for 2 hours and wash 5 times with 1x wash buffer (DY008). The plate was then incubated overnight at 4°C with serially diluted antibodies from 1:1,000 to 1:32,000. The plate was then washed 5 times in 1X wash buffer (DY008) and incubated with anti-mouse HRP (Abcam; ab97046, 1:10,000) for 45 minutes, followed by 5 washes with 1x wash buffer to ensure complete removal of unbound secondary antibodies. Sulfuric acid from an ELISA kit (DY008) was heated to 37°C. 100 μl of tetramethylbenzidine (Millipore) was added to initiate the detection phase of the reaction. After 5 minutes of incubation, sulphuric acid was added to terminate the reaction. The plates were then read using a Synery2 microplate reader (BioTek Instrument. Inc) and Gen5 version 1.11 software.

[0350] result

[0351] Monoclonal antibodies against amino acids 130 to 143 of ApoE were generated and tested for their effect on binding between full-length ApoE3 protein and heparin. Briefly, full-length wild-type ApoE3 protein or those pre-incubated with monoclonal antibodies were passed through a heparin column and cycled five times to ensure maximum ApoE3 binding. The column was then washed five times with 20 mM Tris-HCl (pH = 7.5) and exposed to an increasing NaCl (0 to 1 M) gradient in 20 mM Tris-HCl (pH = 7.5). The eluates from the Tris-HCl washes and from different NaCl concentrations ( Figures 12A-12C ).

[0352] The fractions collected from the column were first screened by the bicinchoninic acid (BCA) assay. Fig.13A As shown, the protein signal of wild-type ApoE3 was detected in the fraction on the right side of the curve, indicating the strong binding of ApoE3 to heparin. In contrast, when ApoE3 was pre-incubated with a monoclonal antibody, a strong signal was observed in the early fraction with low ionic strength ( Fig. 13B To verify the results, the column washes and NaCl gradient fractions collected from the heparin column were analyzed using immunoblotting. Fig.14 As shown, pre-incubation of wild-type ApoE3 with a monoclonal antibody (A3Ab) (the antibody is named 1343 in Arboleda-Velasquez et al., Nature Medicine, 25, pages 1680-1683 (2019)) reduced its ability to bind heparin to a level similar to that of the ApoE3ch mutant protein. The individual gels were combined to generate Fig.14 These results suggest that antibodies could be used to alter the binding properties of ApoE to heparin, thereby preventing or treating Alzheimer's disease or related dementias or neurodegenerative disorders.

[0353] To generate monoclonal antibodies against the ApoE heparin-binding domain, the wild-type ApoE peptide was used: KLH-CTEELRVRLASHLRK-CONH 2 (SEQ ID NO: 54) and ApoEch peptide: KLH-CTEELRVSLASHLRK-CONH 2 (SEQ ID NO: 55) was used to immunize mice. A cysteine ​​residue was added at the N-terminus to promote binding of the peptide. Cell fusions were obtained from positive clones and cell suspensions tested for activity against wild-type and mutant peptides and proteins. Seven antibodies generated were analyzed by ELISA as examples as described in the Materials and Methods section. The 19G10-2 antibody serum showed specificity for both the full length and C-terminus of the APOE3ch mutant protein and some interactions with the wild-type APOE3 protein ( Fig.15A ). The 23B2 antibody showed reactivity to both the wild-type ApoE3 and ApoE3ch mutant C-terminal and full-length recombinant proteins ( Fig. 15B ). The 2H79-1 antibody showed nonspecific binding to bovine serum albumin (BSA) and showed no affinity for wild-type ApoE3 or ApoE3ch mutant ( Fig. 15C Both 30E1-2 and 16H8 antibodies showed reactivity to full-length and C-terminal ApoE3ch mutant proteins and the C-terminal form of wild-type ApoE3, but did not react with full-length wild-type APOE3 protein ( Fig.15D and 15E). The 25F1-2 antibody serum showed high affinity for both full-length and C-terminal ApoE3ch mutant proteins, appeared to have variable binding to the C-terminus of wild-type ApoE3 protein, and had some interaction with full-length wild-type ApoE3 ( Fig.15F ). The 29G10-2 antibody showed high affinity to the full-length and C-terminal of the ApoE3ch mutant protein, and also showed reactivity to the C-terminal wild-type ApoE3 and BSA. Finally, the 29G10-2 antibody did not interact with the full-length wild-type ApoE3 protein ( Figure 15G ).

[0354] Described herein are the variable heavy chain (VH), variable light chain (VL), and complementarity determining region (CDR) sequences of 25F1-2 and 19G10-2.

[0355] In addition, the following parental clones specific for wild-type ApoE were generated and tested for specificity to wild-type ApoE peptide (WT peptide), wild-type ApoE protein (WT protein), mutant ApoE peptide (ApoEch; Mut peptide) and mutant ApoE3ch protein (Mut protein) as shown in Table 4. These values ​​represent the absorbance levels detected by ELISA. The bold clones show specificity to wild-type ApoE peptide (KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54) and wild-type ApoE protein).

[0356] Table 4

[0357] WT peptide WT protein Mut protein Mut protein 1D5 1.742 1.086 0.060 0.062 1H4 2.578 2.113 0.070 0.056 3A6 2.412 0.733 0.059 0.056 7C3 1.698 1.245 0.064 0.051 7C4 2.097 0.586 0.057 0.056 7C11 1.282 0.689 0.058 0.055 16G6 0.739 0.449 0.076 0.067 Positive Control 1.980 1.171 0.254 0.144 Negative control 0.068 0.051 0.056 0.048

[0358] Example 4: Production of fusion proteins containing the APOE heparin binding domain

[0359] Materials and methods

[0360] Peptide competition assay: Wild-type ApoE3 and ApoE3ch mutant peptides (50ug / ml) were incubated with ApoE3 recombinant protein (50ug / ml, prepared in 20mM Tris-HCL) at room temperature for 3 hours. The peptide / ApoE3 recombinant protein solution was then passed through a heparin column and exposed to an increasing NaCl gradient (as described in Examples 2 and 3 above). Fractions were collected and evaluated by immunoblotting.

[0361] Immunoblotting: Immunoblotting confirmed the elution of ApoE3 within the fractions collected from the heparin-bound column. Fractions were diluted to a final volume of 40 μl in 10 μl RIPA buffer (Cell Signaling Technology), 10X (DTT 1M) and 4X Laemmli buffer. TGXTM Samples were separated on precast protein gels (Bio-Rad), transferred to nitrocellulose membranes (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-CORBiosciences, Lincoln, NE), and probed with mouse anti-his tag (Novus biologicals) and IRDye800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using the Odyssey infrared imaging system and visualized on Image Studio version 2.1 (LI-COR Biosciences).

[0362] result

[0363] Peptides containing amino acids 130-143 of wild-type ApoE protein and ApoEch mutant protein, respectively, were generated. To examine the effect of these peptides on the binding between wild-type ApoE3 recombinant protein and heparin agarose, peptide competition assays were performed as described in Materials and Methods. Fig.16 As shown, the wild-type ApoE peptide caused a one-fraction shift in the binding of the wild-type ApoE3 recombinant protein, indicating that the peptide can compete with the wild-type full-length ApoE3 for binding to heparin. These results indicate that an ApoE fragment containing amino acids 130-143 of the wild-type ApoE protein can be used to alter the binding properties of ApoE to heparin, thereby preventing or treating Alzheimer's disease or related dementias or neurodegenerative diseases.

[0364] In order to increase the protein stability of the peptide, the backbone Fc IgG2 constructs pfuse-hfc1 and pfcn-hg2 (Invivogen) were used to generate C-terminal and N-terminal fusion proteins containing the heparin binding domain or the allosteric regulation site of the heparin binding domain of human ApoE (wild type and R136S mutant forms). The human ApoE fragment excludes the sites of APOE2 and APOE4 variants. The administration of ApoE fragments with R at position 136 can compete with endogenous ApoE for interactions with binding partners including HSPG, causing protection against neurodegenerative diseases. The administration of ApoE fragments with S at position 136 can bind to molecules that do not bind to wild-type ApoE, causing protection against neurodegenerative diseases. The amino acid sequences of fragments from wild-type and R136S mutant ApoE used to generate fusion proteins are shown below (the R136 position is marked with bold and double underline).

[0365] Downstream of R136 fragment

[0366] WT

[0367]

[0368] Mutants

[0369]

[0370] Upstream of R136 fragment

[0371] WT

[0372]

[0373] Mutants

[0374]

[0375] Figures 17A-17D Model showing the interaction of ApoE fragments with heparin. Fig.17A Model showing the wild-type ApoE fragment (downstream of the R136 fragment, helix) interacting with heparin. Fig. 17B A model showing the ApoE R136S fragment interacting with heparin (downstream of the R136 fragment, helix). Fig. 17C Model showing the wild-type ApoE fragment (upstream of the R136 fragment, helix) interacting with heparin. Fig.17D A model of the ApoE R136S fragment (upstream of the R136 fragment, helix) interacting with heparin is shown.

[0376] The nucleic acid and amino acid sequences of exemplary fusion protein constructs containing the downstream of the R136 fragment or the upstream of the R136 fragment are shown below.

[0377] 184Q pfcn-hg2 ApoE 114-144

[0378] Nucleic acid sequence (SEQ ID NO:81)

[0379]

[0380]

[0381] Amino acid sequence (SEQ ID NO: 82)

[0382]

[0383] 184R pfcn-hg2 ApoE 114-144R136S nucleotide sequence (SEQ ID NO:83)

[0384]

[0385]

[0386]

[0387] Amino acid sequence (SEQ ID NO:84)

[0388]

[0389] 197F pfuse hfc2 ApoE 114-144 nucleic acid sequence (SEQ ID NO:85)

[0390]

[0391]

[0392] Amino acid sequence (SEQ ID NO:86)

[0393]

[0394] 197G pfuse-hfc2 ApoE 114-144R136S nucleic acid sequence (SEQ ID NO:87)

[0395]

[0396] Amino acid sequence (SEQ ID NO:88)

[0397]

[0398] 184U pfcn hg2 ApoE 129-157 nucleic acid sequence (SEQ ID NO: 89)

[0399]

[0400]

[0401] Amino acid sequence (SEQ ID NO:90)

[0402]

[0403] 184V pfcn hg2 129-157R136S nucleic acid sequence (SEQ ID NO:91)

[0404]

[0405]

[0406]

[0407] Amino acid sequence (SEQ ID NO:92)

[0408]

[0409] 197H pfuse hfc2 ApoE 129-157 nucleic acid sequence (SEQ ID NO:93)

[0410]

[0411]

[0412] Amino acid sequence (SEQ ID NO:94)

[0413]

[0414] 197I pfuse hfc2 ApoE 129-157R136S nucleic acid sequence (SEQ ID NO:95)

[0415]

[0416]

[0417] Amino acid sequence (SEQ ID NO:96)

[0418]

[0419] Example 5: CRISPR-Cas9-mediated editing and base editing of APOE

[0420] In order to introduce the R136S mutation in APOE using CRISPR-Cas9, a gRNA sequence was designed (Table 5). In one embodiment, the gRNA sequence was cloned into lentiCRISPR v2 using 2 oligonucleotides to form a joint containing a 20-base sequence, which was cloned into the BsmB1 site downstream of the U6 promoter. To support repair, a template with 2 additional silent mutations was designed. The template has 50 bases on both sides of the mutation region. Exemplary template sequences are as follows:

[0421]

[0422] The silent mutation that eliminates the PAM motif is double underlined, the codon corresponding to the R136S mutation is in bold, and the silent mutation that generates a Sad site for cleavage of the PCR product in the clone receiving the template is in italics.

[0423] Table 5: gRNA design for introducing R136S mutation

[0424]

[0425] Without wishing to be bound by theory, the mechanism proposed for APOE3ch mutation is the loss of function (e.g., in combination with HSPG). Therefore, gRNA sequence is designed as "knockout" APOE (Table 6) using CRISPR-Cas9. Design gRNA to target exon 3 (amino acids 1-61) of ApoE. In one example, by using 2 oligonucleotides, gRNA sequence is cloned into lentiCRISPRv2 to form a joint containing 20 base sequences, which is cloned into the BsmB1 site downstream of U6 promoter. Repair is completed by non-homologous end joining (NHEJ), which is an error-prone process and usually results in short insertions or deletions, thereby causing APOE to be knocked out.

[0426] Table 6: gRNA design for APOE knockout

[0427]

[0428] The R136H mutation in APOE is expected to have a similar effect to the R136S mutation. Therefore, in order to introduce the R136H mutation in APOE using base editing technology, the following gRNA was designed (Table 7). The gRNA sequence GAGGCGCACCCGCAGCTCCT was cloned into pLenti sgRNA (addgene 71409), and 2 oligonucleotides were used to form a linker containing a 20-base sequence, which was cloned into the BsmB1 site downstream of the U6 promoter. Plasmid Addgene base editor plasmid pCMV-BE3 (#73021) was used to generate base editing.

[0429] Table 7: gRNA design for APOE base editing

[0430]

[0431] Example 6: High-throughput screening of molecules that modify ApoE and heparin binding

[0432] In order to screen the molecules that affect ApoE and heparin binding, ApoE protein is pre-incubated with candidate polypeptides, small molecules, nucleic acids, lipids or carbohydrates. The pre-incubated ApoE protein is introduced into a heparin-coated surface (such as a plate or column) so that it binds heparin / HSPG / GAG, and unbound ApoE and candidate molecules are washed off. Alternatively, before applying ApoE protein, the surface (such as a plate) coated with heparin can be pre-incubated with candidate molecules. The level of ApoE binding heparin is detected using antibodies, protein assays or fluorescence, and the influence of candidate molecules on ApoE / heparin binding is evaluated. The candidate molecules that reduce ApoE / heparin binding can represent a new therapy for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment in human subjects with such treatment needs. Examples of such molecules include EZ-482 (see, for example, Mondal et al. Biochemistry 55 (18): 2613-21, 2016. The structure of EZ-482 is shown below).

[0433]

[0434] Example 7: High-throughput screening of molecules that modify ApoE and bind to anti-ApoE antibodies

[0435] In order to screen the molecules that affect the binding of ApoE and anti-ApoE antibodies, the ApoE protein is pre-incubated with candidate polypeptides, small molecules, nucleic acids, lipids or carbohydrates. The pre-incubated ApoE protein is introduced into a surface (e.g., plate or column) coated with an antibody (such as any anti-ApoE antibody described herein) that binds to the HSPG binding site of ApoE, so that it binds the antibody, and unbound ApoE and candidate molecules are washed away. Alternatively, before applying the ApoE protein, a surface (such as a plate) coated with heparin can be pre-incubated with candidate molecules. Antibodies, protein assays or fluorescence detection of the level of anti-ApoE binding to anti-ApoE antibodies are used, and the effect of candidate molecules on binding is evaluated. Candidate molecules that increase or decrease ApoE / heparin binding can represent a new therapy for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment in human subjects with such treatment needs.

[0436] Example 8: Antibody Characterization

[0437] Antibodies binding to wild-type ApoE and ApoEch mutants were further evaluated using heparin affinity chromatography, immunoblotting, subclone analysis, monoclonal antibody screening, screening for selectivity between huApoE3 and msApoE, and in vivo subretinal injections.

[0438] method

[0439] Chromatographic experiments

[0440] At room temperature (RT), His-tagged recombinant ApoE peptide (50 μg / mL) was incubated for 3 hours with each antibody diluted 1:10 in 20mM Tris HCl buffer (pH7.5). Heparin binding of the samples was tested using a heparin agarose column. In short, the column was brought to room temperature and washed 5 times with 20mM Tris-HCL. After collecting 10 μL for WB experiments, the protein input was loaded onto the column. The input cycle passed through the column 5 times. The effluent was collected, the column was subsequently washed 5 times, and the recovered fractions were labeled "washings". For each 0.05M step of the NaCl salt gradient from 0 to 1M, 1mL fractions were recovered. The 5M fraction was also tested to ensure that the protein was completely released from the column. WB or ELISA was used to test the changes in ApoE binding to heparin.

[0441] Immunoblotting

[0442] Samples for WB analysis were prepared by diluting 10 μL of each fraction in 4X sample buffer (Laemmli's SDS-Sample buffer, BP-110R, Boston bioproducts), 4 μL DTT (Sigma Aldrich) and 16 μL 1XRIPA buffer. Electrophoresis was performed under denaturing conditions using a vertical electrophoresis chamber (Biorad). Bands were separated on a 4-20% precast gel (Biorad) using a constant voltage (15'70V, 1h 100V). Proteins were transferred on a nitrocellulose membrane (Millipore) for 1 hour at a constant voltage of 70V. Before incubation with primary antibody (Anti-His, rb, 1: 5000, Novus biological) and secondary antibody (Donkey anti-rb-800, 1: 10000, Licor), the membrane was blocked at room temperature for 1 hour using Odyssey blocking buffer (Licor) and the membrane was washed 3X 10' with TBS-0.05% Tween 20 (Thermo fisher). Image acquisition was completed using Odyssey scanner. Image J was used to analyze data. Data were standardized by input and expressed as standardized intensity relative to fraction number (0 = input, 0.05M NaCl step gradient increased in 1-27 = 20mM Tris HCl pH 7.5, 5M NaCl in 20mM Tris HCl pH 7.5).

[0443] ELISA

[0444] To test the selectivity of the antibodies for huApoE3WT, huApoE3ch, or msApoE, anti-his ELISA coated plates were incubated with 0.0025 μg / μL target protein for 2 hours at room temperature with slow shaking. The wells were washed 5 times with 1X wash buffer (R&D) and then incubated overnight at 4°C on a shaker (100 μL / well) with serial dilutions of the Innovagen antibody of interest. The next day, the wells were washed 5 times with sample buffer and incubated for 45 minutes with rabbit anti-mouse HRP coupling buffer (1:10,000; 100 μL / well; Abcam). After washing 5 more times, the plates were incubated 1:1 with chromogen A+B to initiate the colorimetric reaction (100 μL / well). The reaction was terminated with 2N sulfuric acid (R&D stop solution, 50 μL / well) and the absorbance at 450 nm was detected spectrally.

[0445] ELISA was used to test the affinity of antibodies designed for the heparin binding domain of APOE to APOE3 and APOEch mutant recombinant proteins. The Ni-NTA HisSorb Plates (Qiagen) plates were washed 3 times with wash buffer 1 (DY008), and the APOE recombinant protein was suspended in buffer (DY008) to produce a final concentration of 0.5ug / ml. The plate was incubated with 200ul for 2 hours. The plate was then washed 5 times with 1x wash buffer (DY008), and then incubated overnight at 4°C with antibodies in a serial dilution series from 1:1000 to 1:32000. The plate was then washed 5 times in 1X wash buffer (DY008). The plate was then incubated for 45 minutes with anti-mouse HRP (Abcam; ab97046) (1:10000). The plate was then washed 5 times in 1x wash buffer to ensure complete removal of unbound secondary antibodies. Sulfuric acid from the ELISA kit (DY008) was heated to 37°C before adding 100 μl of tetramethylbenzidine (Millipore) to start the detection phase of the reaction. After 5 minutes of incubation, sulphuric acid was added to stop the reaction. The plates were then read using a Synery 2 microplate reader (BioTek Instrument. Inc) and Gen5 version 1.11 software.

[0446] result

[0447] Antibody 1H4 was evaluated using heparin affinity chromatography and immunoblotting. ApoE3 was incubated with a negative control (vehicle, top blot) or 1H4 (bottom blot), and each fraction was subjected to heparin affinity chromatography and immunoblotting ( Fig.18A ). The ApoE3-positive band is indicated by an arrow and was detected using an antibody anti-his tag (rb, 1:5,000) that specifically detects the his tag of recombinant human APOE. Fig.18B Display by Fig.18A Quantification of WB blot bands detected by anti-his tag antibody shown in . Intensity normalized to input. These results indicate that ApoE3 binding to heparin is reduced in the presence of antibody and that antibody 1H4 competes with ApoE for heparin binding. N = 2 independent experiments.

[0448] Next, subclone analysis of 1H4 serum was performed. Fig.19 A-19D shows the ELISA results of 1H4-2 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C) and ApoE3ch peptide (D). The results are expressed as the optical density at 450nm versus the dilution factor of the serum tested. Fig. 20 It is a comparison Fig.19 A-19D shows a graph of the results.

[0449] Fig.21 Representative ELISA profiles of serial dilutions of antibody 1H4 incubated with human recombinant ApoE3 or mouse recombinant ApoE3 are shown. The results indicate that the antibody binds preferentially to human protein rather than mouse and that antibody 1H4 is selective for human ApoE. Results are shown as mean optical density detected at 450 nm ± sem (n = 2).

[0450] Next, monoclonal 1H4 antibodies were purified from cloned hybridomas and evaluated by ELISA. Fig. 22 Results of ELISA experiments are shown.

[0451] Antibody 7C11 was evaluated using heparin affinity chromatography, immunoblotting, quantitative ELISA of chromatography fractions, and competitive ELISA for binding analysis.

[0452] Heparin affinity chromatography fractions of ApoE3 were incubated with negative control (vehicle, top blot) or antibody 7C11 (bottom blot) and immunoblotted ( Fig.23A ). The ApoE3-positive band is indicated by an arrow and was detected using an antibody anti-his tag (rb, 1:5,000) that specifically detects the his tag of the recombinant peptide. Fig. 23B Display by Fig.23A Quantification of WB blot bands detected by anti-his tag antibody shown in . Intensity normalized to input. These results indicate that ApoE3 binding to heparin is reduced in the presence of antibody and that 7C11 competes with ApoE for heparin binding.

[0453] Next, subclone analysis of 7C11-1 serum was performed. Fig.24A-24D shows the ELISA results of 7C11-1 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C) or ApoE3ch peptide (D). The results are shown as optical density at 450nm versus the dilution factor of the serum tested. Fig.25 It is a comparison Fig.19 A-19D is a graph showing the results. Next, the monoclonal 7C11-1 antibody was purified from the cloned hybridoma and evaluated by ELISA. Fig.26 Results of ELISA experiments are shown.

[0454] The 19G10-2 antibody was further evaluated using heparin affinity chromatography, immunoblotting, quantitative ELISA of chromatographic fractions, and ELISA for binding analysis. ELISA screening of the 19G10-2 antibody was performed against the heparin binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins. Fig. 27 As shown, the 19G10-2 antibody exhibited specificity for both the full-length and C-terminal domains of the APOE3ch (amino acids 125 to 299) mutant recombinant protein, and some interaction with APOE3 WT.

[0455] ELISA analysis was performed on heparin affinity chromatography fractions of ApoE3 incubated with negative control (vehicle, top blot) or antibody 19G10-2 (bottom blot) ( Fig.28A ). The ApoE3-positive band is indicated by an arrow and was detected using an antibody anti-his tag (rb, 1:5,000) that specifically detects the his tag of the recombinant peptide. Fig.28B Display by Fig.28A Quantification of WB blots detected by anti-his tag antibodies shown in . Intensity normalized to input. These results show that, although designed for ApoE3ch-HSPG domain, 19G10-2 competes with wild-type ApoE for heparin binding and causes ApoE3 to bind heparin to decrease. Without wishing to be bound by theory, antibody 19G10-2 can recognize and / or stabilize conformation-specific features of APOE (e.g., APOE polymers or aggregates) that are unlikely to bind heparin / HSPG / GAG.

[0456] Fig.29 Immunoblots of ApoE3 WT incubated with 19G10-2 serum antibody are shown. Top blot: The membrane was labeled with a secondary mouse probe to detect the 19G10-2 antibody. The bottom membrane was incubated with an anti-his tag to detect the ApoE3 positive fraction as described above. This analysis shows that the antibody-APOE complex (left blot; top and bottom) does not bind heparin, while free APOE binds heparin with high affinity (right blot; bottom).

[0457] Fig.30 Representative ELISA showing differential binding of both serum and monoclonal antibody hybridoma supernatant 19G10-2 to ApoE3WT or ApoE3ch. The data demonstrates the superior selectivity of this antibody for the ApoE3ch variant. Fig.31 is an enlarged graph of the Y axis, showing some limited binding fraction of antibody 19G10-2 in the presence of ApoE3WT (serum, gray fraction; monoclonal, black binding fraction). Next, monoclonal 19G10-2 antibodies were purified from cloned hybridomas and evaluated by ELISA. Fig.32 Results from ELISA experiments are shown. The signal of recognition of full-length WT APOE is higher than that of WT ApoE peptide indicating a conformation-specific feature of the binding.

[0458] The 25F1-2 antibody was further evaluated using heparin affinity chromatography, immunoblotting, quantitative ELISA of chromatography fractions, and ELISA for binding analysis. Fig.33 ELISA screening of 25F1-2 antibodies against the heparin binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins is shown. Fig.33 As shown, the 25F1-2 antibody showed high affinity for the APOE3 mutant full-length and C-terminal proteins, however, it did not appear to have strong binding to the C-terminus of the APOE3WT protein and showed limited interaction with the APOE3WT full-length protein. The results are shown as optical density at 450 nm versus the dilution factor of the serum tested.

[0459] Immunoblotting was performed on heparin affinity chromatography fractions of ApoE3 incubated with negative control (vehicle, top blot) or antibody 25F1-2 (bottom blot). Fig.34A ). The ApoE3-positive band is indicated by an arrow and was detected using an antibody anti-His tag (rb, 1:5,000) that specifically detects the his tag of the recombinant peptide. Fig.34B Display by Fig.34A Quantification of WB blot bands detected with anti-his tag antibody as shown in . Intensity normalized to input. These results indicate that, despite being designed against the ApoE3ch-HSPG domain, 25F1-2 competes with wild-type ApoE for heparin binding and results in reduced binding of ApoE3 to heparin.

[0460] Fig.35Immunoblots of ApoE3 WT incubated with 25F1-2 monoclonal antibody are shown. Top blot: membrane was labeled with secondary mouse probe to detect 25F1-2. Bottom membrane was incubated with anti-his tag to detect ApoE3 positive fraction as before. This analysis shows that antibody-APOE complexes (left blot; top and bottom) do not bind heparin, while free ApoE binds heparin with high affinity (right blot, bottom).

[0461] Fig.36 Representative ELISA showing the difference in binding of 25F1-2 serum and monoclonal antibody hybridoma supernatant 25F1-2 to ApoE3WT or ApoE3ch. These results demonstrate the superior selectivity of this antibody for the ApoE3ch variant. Fig.37 yes Fig.36 The Y-axis of the graph shows the binding profile of antibody 25F1-2 in the presence of ApoE3 WT. Next, the monoclonal 25F1-2 antibody was purified from the cloned hybridoma and evaluated by ELISA. Fig.38 Results from ELISA experiments are shown. The signals for some recognitions of full-length WT APOE were higher than those of WT ApoE peptide, indicating conformation-specific features of binding.

[0462] The mouse antibody 1343ab (renamed from 23B2) was evaluated using heparin affinity chromatography and immunoblotting, and quantitative ELISA of the chromatography fractions. Fig.39 and 40 ELISA screening of 1343 antibody against the heparin binding domain of APOE3 wild type (WT) and APOE3ch mutant recombinant proteins is shown. 1343 showed reactivity to both APOE3 WT and APOE3ch mutant C-termini and full-length recombinant APOE protein (mutant refers to Christchurch mutant).

[0463] Immunoblotting of ApoE in protein fractions eluted from a heparin column using an increasing NaCl gradient in the presence or absence of 1343 ( Fig.41A ). Individual blots were cropped between 25 and 50 kDa. Blank spaces separate individual blots. FT = flow through. ELISA was performed to quantify the differences in the NaCl elution patterns of different ApoE in the presence and absence of 1343 ( Fig.41B ). N=3 columns per isomer were analyzed twice side by side on different days in independent experiments to quantify differences. Error bars depict standard error of the mean.

[0464] It was demonstrated that CDR sequences from mouse antibodies 1H4-2, 7C11-1, 19G10-2, 25F1-2 and 1343ab were grafted into human IgG frameworks (IgG2 or IgG4) to generate humanized counterparts.

[0465] Example 9: In vivo validation of ApoE antibodies

[0466] An intraocular model of inducible APOE-dependent Tau hyperphosphorylation (paired helical fibril formation) was generated. This model was used to test the ability of ApoE antibodies to inhibit Tau pathology, a hallmark of neurodegeneration. Fig.42A An exemplary experiment summary is shown.

[0467] Briefly, his-tagged recombinant human APOE3 was injected into B6; C3-Tg (Prnp-MAPT * P301S) PS19Vle / J mice (Jackson Laboratory 008169) (Yoshiyama et al. 53 (3): 337-51, 2007). This mouse model contains the human tau P301S mutation and is a validated animal model of Alzheimer's disease and other tauopathies such as frontotemporal dementia (see, for example, Bugiani et al. 58 (6): 667-77, 1999). Mice injected with PBS were used as controls.

[0468] like Fig.42B As shown, in the control retina of 6-week-old mice, paired helical fibers of phosphorylated tau (PHF) are absent in ganglion cells and their axonal fibers (the signal in the blood vessels labeled with isolectin B4 is the background signal). In contrast, the administration of recombinant human APOE3 (his tag) triggered a strong formation of PHF detected with AT8 antibody. PHF is strong in ganglion cell axons (arrows) and ganglion cell bodies. Human APOE was detected around ganglion cell bodies using anti-His antibody.

[0469] Mouse 1H4-2 antibody and humanized 1343Ah antibody were injected intravitreally into the eyes of mice from the above mouse model (final volume 2 μL). The animals were sacrificed on day 3 after injection, and the retinas were dissected and immunolabeled. The retinas were stained with DAPI, isolectin, and AT8 (pTAU), which recognizes phosphorylated paired helical fibril tau (PHF tau). The retinas were imaged using an SP8 confocal microscope. Fig.43B , 43CAs shown in Figures 43F and 43G, APOE3 WT resulted in a significant increase in PHF tau, which was significantly reduced by 1H4-2 (***p<0.001). Similarly, administration of the humanized 1H4-2 IgG2 / κ recombinant monoclonal antibody effectively reduced the induction of APOE-dependent PHF tau pathology in vivo ( Figure 43H ; **p<0.01, ***p<0.001). Fig.43D and 43H As shown, humanized 1343Ah significantly reduced PHF Tau levels (**p<0.01, ***p<0.001).

[0470] The similarity of efficacy between the mouse monoclonal antibody and the corresponding humanized antibody confirmed the affinity of the binding domain for ApoE or ApoEch, and the binding properties were retained during the humanization process. This suggests that the binding properties of the CDR can be transferred from the original mouse IgG1 to other proteins including human IgG2.

[0471] The binding affinity between ApoE3 and monoclonal antibody 1H4 (mAb 1H4) was determined using the advanced kinetics module (Blitz Pro, FB-609928, version 1.3.1.3) of the BLItz system. In short, the protein A biosensor was loaded with mAb1H4, and the association constant and dissociation constant were determined at the concentration of the increased full-length ApoE3 protein (Innovagen) from 0 to 571.4nM. The following run settings were used: an initial baseline of 30s in the experimental medium, 120s loading of the ligand (mAb 1H4) on the biosensor, and a new baseline of 30s before the binding (120s) and dissociation (120s) steps. A total of 6 runs of 420s were performed at 2200rpm and room temperature to determine the binding parameters of ApoE3. Global fitting and step correction (step correction) were performed for the dissociation experiment using BLItz software (version 1.1.0.7). Fig.44 Representative binding measurements of ApoE3 protein with increasing concentrations (nM) and 1H4 on a protein A biosensor are shown. KD, Ka, and Kd were measured using the BLItz system (Table 8) and calculated. The upper graph represents the association step and the lower graph represents the dissociation step of the binding kinetics. The X-axis and Y-axis represent time in seconds and binding in nM, respectively.

[0472] Table 8

[0473]

[0474] Example 10: In vivo validation of APOE fragment fusion protein

[0475] Next, a fusion protein containing an APOE fragment that includes the HSPG binding domain and the Fc region of human IgG was tested using a similar in vivo model. Briefly, 0.78 μg of recombinant full-length APOE was used to induce tau pathology. 0.14 μg of the fusion protein was injected intravitreally into mice. Fig.43E and 43I As shown, the fusion protein reduced APOE-dependent tau pathology in neurons.

[0476] Example 11: Sequences of chimeric antibodies

[0477] The chimeric antibody sequences in which CDRs were transferred from mouse to human IgG2 or IgG4 are shown below: Normal font = vector

[0478] Italics = signal peptide

[0479] Underline = VL / VH

[0480] Double underline = constant part (human κ / IgG4 / IgG2) Sequence of the expression vector of mAb 1H4 IgG2 / κ:

[0481]

[0482]

[0483]

[0484]

[0485]

[0486] Sequence of the expression vector for mAb 1343Ah IgG2 / κ:

[0487]

[0488]

[0489]

[0490]

[0491]

[0492] Sequence of the expression vector for mAb 19G10 IgG4 / κ:

[0493]

[0494]

[0495]

[0496]

[0497]

[0498] Sequence of the expression vector for mAb 25F1 IgG4 / κ:

[0499]

[0500]

[0501]

[0502]

[0503]

[0504] Sequence of the expression vector for mAb 7C11.1 IgG2 / κ:

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511] References

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[0564] Other Implementations

[0565] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. An isolated monoclonal antibody that specifically binds to one or more HSPG binding sites or one or more allosteric regulatory sites of HSPG binding of wild-type or mutant apolipoprotein E (ApoE).

2. The antibody of claim 1, wherein the antibody binds to a polypeptide having an amino acid sequence that is at least 95% identical to TEELRVRLASHLRK (SEQ ID NO: 3).

3. The antibody of claim 1, wherein the antibody binds to a polypeptide having an amino acid sequence that is at least 95% identical to TEELRVSLASHLRK (SEQ ID NO: 2).

4. The antibody of any one of claims 1 to 3, wherein the antibody binds to one or more HSPG binding sites of wild-type or mutant ApoE2, ApoE3 or ApoE4.

5. The antibody according to any one of claims 1 to 4, wherein the antibody competes with and / or binds to the same epitope as a reference anti-ApoE antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL of the reference antibody: (i) comprising the amino acid sequence described in SEQ ID NO: 13 and the amino acid sequence described in SEQ ID NO: 12, respectively; (ii) comprising the amino acid sequence set forth in SEQ ID NO: 23 and the amino acid sequence set forth in SEQ ID NO: 22, respectively; (iii) comprising the amino acid sequence set forth in SEQ ID NO: 33 and the amino acid sequence set forth in SEQ ID NO: 32, respectively; or (iv) comprising the amino acid sequence described in SEQ ID NO: 43 and the amino acid sequence described in SEQ ID NO: 42, respectively.

6. The antibody according to any one of claims 1 to 4, wherein the antibody competes with and / or binds to the same epitope as a reference anti-ApoE antibody comprising a heavy chain and a light chain, wherein the heavy chain and the light chain of the reference antibody comprise the amino acid sequence recorded in SEQ ID NO:53 and the amino acid sequence recorded in SEQ ID NO:

52.

7. An anti-ApoE antibody comprising a VH comprising VHCDR1, VHCDR2 and VHCDR3 and a VL comprising VLCDR1, VLCDR2 and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3: (i) comprising SEQ ID No: 7, 8, 9, 4, 5, 6 respectively; (ii) comprising SEQ ID No: 17, 18, 19, 14, 15, 16, respectively; (iii) comprising SEQ ID No: 27, 28, 29, 24, 25, 26, respectively; (iv) comprising SEQ ID No: 37, 38, 39, 34, 35, 36, respectively; or (v) contain SEQ ID No: 47, 48, 49, 44, 45, 46 respectively.

8. The antibody according to claim 7, in: (i) the VH and the VL comprise amino acid sequences that are at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 13 and 12, respectively; (ii) the VH and the VL comprise amino acid sequences that are at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 23 and 22, respectively; (iii) the VH and the VL comprise amino acid sequences that are at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 33 and 32, respectively; or (iv) the VH and the VL comprise amino acid sequences that are at least 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 43 and 42, respectively.

9. The antibody according to claim 7, comprising a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the amino acid sequence recorded in SEQ ID No: 53 and 52.

10. The antibody of any one of claims 1 to 9, wherein the antibody comprises a mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3 heavy chain constant region.

11. The antibody of any one of claims 1 to 9, wherein the antibody comprises a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

12. The antibody of any one of claims 1 to 11, wherein the antibody comprises a human kappa or human lambda light chain constant region.

13. The antibody according to any one of claims 1 to 12, wherein the antibody is a whole antibody, a single domain antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, Fv, scFv, sc(Fv)2, a diabody, a nanobody, a Fab or a F(ab')2.

14. The antibody of any one of claims 1 to 13, further comprising a half-life extending moiety.

15. The antibody according to any one of claims 1 to 14, further comprising a blood-brain barrier penetrating portion.

16. The antibody of any one of claims 1 to 12, further comprising a detectable label.

17. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 16.

18. One or more polynucleotides encoding the antibody according to any one of claims 1 to 13.

19. One or more vectors comprising one or more polynucleotides according to claim 18.

20. A host cell comprising one or more polynucleotides according to claim 18, or one or more vectors according to claim 19.

21. A method for preparing an anti-ApoE antibody, the method include: (a) culturing the host cell according to claim 20 under conditions that allow expression of the antibody; and (b) isolating the antibody.

22. The method of claim 21, further comprising formulating the antibody into a sterile formulation suitable for administration to a human.

23. An Fc fusion protein comprising: a HSPG binding domain of wild-type ApoE or mutant ApoE, wherein the HSPG binding domain comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of: STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59) and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60). The Fc fusion protein according to claim 23 , comprising the Fc region of a human antibody.

25. The Fc fusion protein of claim 24, wherein the human antibody is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 molecules.

26. A pharmaceutical composition comprising the Fc fusion protein according to any one of claims 23 to 25.

27. One or more polynucleotides encoding an Fc fusion protein according to any one of claims 23 to 25.

28. One or more vectors comprising one or more polynucleotides according to claim 27.

29. A host cell comprising one or more polynucleotides according to claim 28, or one or more vectors according to claim 28.

30. A pharmaceutical composition for eliciting an immune response, comprising: (i) a HSPG binding domain of wild-type ApoE or mutant ApoE, the HSPG binding domain comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO:57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO:58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO:59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO:60); and (ii) a pharmaceutically acceptable adjuvant.

31. A method of improving, slowing, delaying the onset, preventing or reversing cognitive decline associated with dementia and / or mild cognitive impairment in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of an antibody according to any one of claims 1 to 16, an Fc fusion protein according to any one of claims 23 to 25, or a pharmaceutical composition according to claim 17 or 26.

32. A method of improving, slowing, delaying the onset, preventing or reversing cognitive decline associated with dementia and / or mild cognitive impairment in a human subject in need thereof, the method comprising administering to the subject: (i) a viral vector comprising a nucleotide sequence encoding a gRNA molecule, wherein the gRNA molecule comprises a targeting domain complementary to a target domain from an APOE gene; (ii) a viral vector comprising a nucleotide sequence encoding a Cas9 molecule; and (iii) a viral vector comprising a template nucleic acid, wherein the template nucleic acid comprises adenine to replace the cytosine at position 19: g.45412013C>A in the APOE gene, wherein said administering results in the production of one or more ApoE R136S alleles in one or more cells of said subject.

33. A method according to claim 32, wherein the targeting domain of the gRNA molecule comprises a sequence that is identical to or differs by no more than 3 nucleotides from a sequence from Table 7.

34. The method of claim 31 or 32, wherein the human subject is diagnosed with or is at risk of developing Alzheimer's disease.

35. The method of claim 34, wherein the human subject carries one or more copies of the APOE4 allele.

36. The method of claim 34, wherein the human subject carries one or more mutations in at least one gene selected from the group consisting of: APP, PSEN1, and PSEN2.

37. The method of claim 34, wherein the human subject carries all or part of a third copy of chromosome 21.

38. The method of claim 34, wherein the human subject is diagnosed with Alzheimer's disease by brain imaging.

39. The method of claim 34, wherein the human subject is over 50 years old.

40. The method of claim 31 or 32, wherein the human subject is diagnosed with or is at risk for developing a disorder selected from the group consisting of vascular cognitive impairment, vascular dementia, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Huntington's disease, neurodegenerative disease, cerebrovascular disease, brain injury, chronic traumatic encephalopathy, tauopathy, amyloid disease, synucleinopathy, Creutzfeldt-Jakob disease, retinal degeneration, glaucoma, retinal damage, and aging.

41. A method of identifying a human subject who is less susceptible to developing an early-onset neurodegenerative disease, the method include: Obtaining or having obtained a biological sample from a subject; detecting the presence of at least one APOE3 mutant allele, or the presence of a mutant ApoE3 gene product, in the biological sample; and Based on the presence of the mutant ApoE3 allele or gene product in the biological sample, the subject is identified as being less susceptible to developing an early onset neurodegenerative disease.

42. The method of claim 41, wherein the biological sample is blood, cerebrospinal fluid, saliva, urine, tears, vitreous humor, aqueous humor, or a tissue specimen.

43. The method of claim 41, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, retinal degeneration, or glaucoma.

44. The method of claim 43, wherein the retinal degeneration is age-related macular degeneration.

45. The method of claim 41, wherein the detecting comprises determining the sequence of the APOE3 allele in the subject.

46. ​​The method of claim 41, wherein the detecting comprises determining the presence or absence of an APOE3 sequence encoding an ApoE3 protein having a mutation at R136 compared to a wild-type ApoE3 protein.

47. The method of claim 46, wherein the mutation at R136 is R136S, R136H or R136C.

48. The method of any one of claims 41 to 47, further comprising selecting the subject for inclusion in the clinical trial, and optionally administering an experimental treatment, or excluding the subject from the clinical trial if the subject does not have a mutant APOE3 allele.

49. The method of any one of claims 41 to 47, further comprising selecting a subject for inclusion in a clinical trial, and optionally administering an experimental treatment, or excluding the subject from the clinical trial if the subject has a mutant APOE3 allele.

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