ApoE Antibodies, Fusion Proteins, and Uses Thereof

Abstract

Methods and compositions for preventing or treating cognitive decline and / or neurodegeneration 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

[Technical Field]

[0001] Priority claims This application claims the benefit of U.S. Provisional Patent Application No. 62 / 853,676, filed May 28, 2019, and U.S. Provisional Patent Application No. 62 / 873,019, filed July 11, 2019, the entire contents of which are incorporated herein by reference.

[0002] Federally sponsored research or development This invention was made with government support under Grant No. OD019833 awarded by the National Institutes of Health, Grant Nos. AG054671, AG031581, and AG19610 awarded by the National Institute on Aging, and Grant Nos. NS100121 and NS110048 awarded by the National Institute of Neurological Disorders and Stroke. The government has certain rights in this invention.

[0003] 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 technology]

[0004] Alzheimer's disease (AD) is a chronic neurodegenerative disease that typically begins slowly and worsens over time. It accounts for 60–70% of dementia cases. The disease process is associated with the formation of plaques and neurofibrillary tangles in the brain. While some treatments may temporarily improve symptoms, there are currently no treatments that can halt or reverse its progression. The accumulation, aggregation, and deposition of amyloid-β (Aβ) peptides in the brain are central to the pathogenesis of Alzheimer's disease (AD). Increasing evidence demonstrates that ApoE strongly influences AD pathogenesis by regulating Aβ aggregation and metabolism (Fu et al., Mol Neurodegener 11:37, 2016). ApoE influences amyloid production, aggregation, and clearance, is a component of amyloid plaques, and exacerbates tau-mediated neurodegeneration. ApoE is 299 amino acids long and is polymorphic with three major alleles (epsilon 2, epsilon 3, and epsilon 4): ApoE2 (cys112, cys158), ApoE3 (cys112, arg158), and ApoE4 (arg112, arg158), which differ from each other by only one or two amino acids at positions 112 and 158. Thus, there is a need for therapeutic agents that target and modulate the function of the ApoE protein to treat or prevent cognitive decline associated with AD and dementia or mild cognitive impairment. Summary of the Invention

[0005] In one aspect, this disclosure features an isolated 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) sites of allosteric regulation of HSPG binding of wild-type or mutant apolipoprotein E (ApoE). In some embodiments, the antibody binds to a polypeptide having an amino acid sequence 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 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 (eg, 1, 2, 3, or 4) HSPG binding sites of wild-type or mutant ApoE2, ApoE3, or ApoE4.

[0006] In some embodiments, 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 said reference antibody comprise (i) 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) 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) 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) 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 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 light chain of said 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.

[0007] In another aspect, 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 comprise (i) SEQ ID NOs: 7, 8, 9, 4, 5, 6, respectively; (ii) SEQ ID NOs: 17, 18, 19, 14, 15, 16, respectively; (iii) SEQ ID NOs: 27, 28, 29, 24, 25, 26, respectively; (iv) SEQ ID NOs: 37, 38, 39, 34, 35, 36, respectively; or (v) SEQ ID NOs: 47, 48, 49, 44, 45, 46, respectively. In some embodiments of any of the antibodies described herein, (i) the 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) the 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) the 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) the 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, respectively, comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 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 kappa or human lambda light chain constant region.In some embodiments, the antibody is a whole antibody, a single domain antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, an Fv, a scFv, a sc(Fv)2, a diabody, a nanobody, a Fab, or a F(ab')2. In some embodiments, the antibody further comprises a half-life-extending moiety. In some embodiments, the antibody further comprises a blood-brain barrier-permeable moiety. In some embodiments, the antibody further comprises a detectable label. In some embodiments, provided herein is a pharmaceutical composition comprising any of the antibodies described herein. In some embodiments, provided herein is a polynucleotide encoding any of the antibodies described herein. In some embodiments, provided herein is a vector comprising a polynucleotide described herein. In some embodiments, provided herein is a host cell comprising a polynucleotide described herein or a vector described herein. In another aspect, provided herein is a method of making an anti-ApoE antibody, the method comprising: (a) culturing any of the host cells described herein under conditions allowing expression of the antibody, and (b) isolating the antibody. In some embodiments, the method further comprises formulating the antibody as a sterile formulation suitable for human administration.

[0008] In another aspect, provided herein is an Fc fusion protein comprising an HSPG-binding domain of wild-type or mutant ApoE comprising an amino acid sequence 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 an 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, provided herein is a pharmaceutical composition comprising any of the Fc fusion proteins described herein. In some embodiments, provided herein is a polynucleotide encoding any of the Fc fusion proteins described herein. In some embodiments, provided herein is a vector comprising any of the polynucleotides described herein. In some embodiments, provided herein is a host cell comprising a polynucleotide described herein or a vector described herein.

[0009] In another aspect, provided herein is a pharmaceutical composition for eliciting an immune response, comprising: (i) an HSPG-binding domain of wild-type or mutant ApoE comprising an amino acid sequence 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.

[0010] In another aspect, provided herein is a pharmaceutical composition comprising a human cell expressing any of the antibodies described herein or any of the Fc fusion proteins described herein.

[0011] In another aspect, provided herein is a method of ameliorating, slowing, delaying the onset of, preventing, or reversing cognitive decline and / or neurodegeneration associated with dementia and / or mild cognitive impairment in a human subject in need thereof, comprising the step of administering to the human subject a therapeutically effective amount of any of the antibodies, Fc fusion proteins, or pharmaceutical compositions described herein.

[0012] In another aspect, provided herein is a method of ameliorating, slowing, delaying the onset of, 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 subject: (i) a viral vector comprising a nucleotide sequence encoding a gRNA molecule comprising a targeting domain complementary to a target domain from the APOE gene; (ii) a viral vector comprising a nucleotide sequence encoding a Cas9 molecule; and (iii) a viral vector comprising a template nucleic acid comprising an adenine instead of a cytosine at position 19 of the APOE gene: g.45412013C>A, wherein said administration results in the generation 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 identical to or differing from a sequence from Table 7 by no more than 3 nucleotides.

[0013] As used herein, "prevent" means reducing the risk of developing a disorder.

[0014] In some embodiments, the human subject has been diagnosed with or is at risk of developing Alzheimer's disease. In some embodiments, the human subject has one or more copies of the APOE4 allele. In some embodiments, the human subject has 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 has one or more mutations in additional genes that cause autosomal dominant Alzheimer's disease (e.g., those described in Bateman et al., Alzheimer's Research & Therapy 3(1): 1, 2011). In some embodiments, the human subject has all or a portion of a third copy of chromosome 21. In some embodiments, the human subject has been 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 over 50 years old (e.g., over 55, over 60, over 65, over 70, over 75, over 80, over 85, over 90, or over 95 years old).

[0015] In some embodiments, the human subject has been diagnosed with or is at risk of developing a disorder selected from the group consisting of: vascular cognitive impairment, vascular dementia, autosomal overt cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), autosomal recessive cerebral 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 disease, brain injury, chronic traumatic brain injury, tauopathy, amyloidopathy, synucleinopathy, Creutzfeldt-Jakob disease, retinal degeneration, glaucoma, retinal damage, optic nerve degeneration, and aging.

[0016] In yet another aspect, provided herein is a method for identifying a human subject who is less likely to develop an early-onset neurodegenerative disease, comprising the steps of obtaining a biological sample from the subject; detecting the presence of at least one variant allele of APOE3 or a variant ApoE3 gene product in the biological sample; and identifying the subject as less likely to develop an early-onset neurodegenerative disease based on the presence of the variant APOE3 allele or gene product in the biological sample. In some embodiments, the biological sample is blood, cerebrospinal fluid, saliva, urine, tears, vitreous fluid, 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, the retinal degeneration is age-related macular degeneration. In some embodiments, the detecting step comprises determining the sequence of an APOE3 allele in the subject. In some embodiments, the detecting step 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. In some embodiments, the mutation at R136 is R136S, R136H, or R136C. In some embodiments of any of the methods for identifying a human subject who is less likely to develop an early-onset neurodegenerative disease described herein, the method further comprises selecting the subject for inclusion 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 for identifying a human subject who is less likely to develop an early-onset neurodegenerative disease described herein, the method further comprises selecting the 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.

[0017] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can 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 by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0018] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [Brief explanation of the drawings]

[0019] [Figure 1]

[0023] Figure 1 shows a model of 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 for the APOE4 (C112R), APOE3ch (R136S), and APOE2 (R158C) variants are shown. [Figure 2] FIG. 1 shows representative Sanger sequencing results for APOE from control, proband, and offspring samples. [Figure 3] Figure 1 shows the subject's pedigree. Circles represent females, squares represent males, diamonds represent individuals whose gender has been concealed for privacy reasons, arrowheads depict proband individuals with MCI, and shading indicates individuals with a history of dementia. Deceased individuals are marked with crossed bars. Individual APOE and PSEN1 genotypes are appropriately labeled to protect anonymity. [Figure 4-1]Figures 4A and 4B show fundus photographs of the right and left eyes, respectively. Figure 4C shows an infrared image of the right eye depicting a cross section of the retina (line) seen in Figure 4D. Figure 4D shows optical coherence tomography (OCT) results for the right eye. Figure 4E shows an infrared image of the left eye. Figure 4F shows OCT imaging results for the left eye. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] FIG. 1 shows 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 a typical age (44 years) for this mutation. [Figure 6] 1 shows the measurement values ​​of amyloid burden, tau burden, hippocampal volume, and glucose metabolism in PSEN1 E280A mutation carriers.Red points represent the measurement values ​​for the carriers with two APOE3ch alleles and exceptionally late onset of MCI.Black points represent PSEN1 E280A mutation carriers with MCI who have a younger age than typical for their family at the time of MCI onset.Gray points represent PSEN1 E280A mutation carriers who have not yet developed MCI. [Figure 7] Figure 1 shows the rate of Aβ42 fibril formation in the presence of APOE3 wild-type, APOE3ch, or the absence of APOE, as detected by thioflavin T fluorescence. The change in relative fluorescence units (RFU) was plotted against time in minutes (min) (***P<0.001, ****P<0.0001). [Figure 8] A diagram showing a summary of split luciferase complementation caused by amyloid oligomerization (top) and the percentage of luminescence obtained by split luciferase complementation assay after 24 hours in medium from 293T cells transfected with ApoE3ch or ApoE3 wild type. [Figure 9] FIG. 1 shows ELISA results for heparin binding affinity of APOE2 and APOE4. [Figure 10]FIG. 1 shows Western blot analysis of heparin binding affinity of ApoE2, ApoE3, ApoE4, and ApoE3ch. [Figure 11-1] FIG. 1 shows ELISA results for heparin-binding affinity of ApoE2, ApoE3, ApoE4, and ApoE3ch. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12] Figure 12A is a schematic diagram showing the experimental setup for testing the specificity of a monoclonal ApoE3 antibody in blocking ApoE3 / heparin binding, and Figures 12B and 12C are schematic diagrams showing the process of passing ApoE3 protein preincubated with the monoclonal antibody through a heparin-binding column, followed by washing and elution. [Figure 13] Figure 13 shows the results from a BCA assay performed on various fractions from a heparin-binding column. Figure 13A shows the amount of ApoE3 in various fractions in the absence of ApoE3 antibody, and Figure 13B shows the amount of ApoE3 in various fractions in the presence of ApoE3 antibody. [Figure 14] FIG. 1 shows Western blot results showing the amount of ApoE3 in various fractions with or without preincubation with monoclonal ApoE3 antibody. [Figure 15-1] FIG. 1 shows ELISA analysis of 19G10-2, 23B2, 2H79-1, 30E1-2, 16H8, 25F1-2, and 29G10-1 antibodies, respectively. [Figure 15-2] This is a continuation of Figure 15-1. [Figure 15-3] This is a continuation of Figure 15-1. [Figure 15-4] This is a continuation of Figure 15-1. [Figure 15-5] This is a continuation of Figure 15-1. [Figure 15-6] This is a continuation of Figure 15-1. [Figure 15-7] This is a continuation of Figure 15-1. [Figure 16]FIG. 1 shows Western blot analysis of heparin binding affinity of ApoE3 treated with wild-type ApoE3 peptide and ApoE3ch mutant peptide. [Figure 17]

[0033] Figure 17A shows a model of a wild-type ApoE fragment containing amino acids 129-157 that interacts with heparin. Figure 17B shows a model of a fragment of ApoE R136S containing amino acids 129-157 that interacts with heparin. Figure 17C shows a model of a wild-type ApoE fragment containing amino acids 114-144 that interacts with heparin. Figure 17D shows a model of a fragment of ApoE R136S containing amino acids 114-144 that interacts with heparin. [Figure 18-1] FIG. 1 shows heparin affinity chromatography and Western blot analysis of antibody 1H4. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19] FIG. 1 shows ELISA results of 1H4-2 sera tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), and ApoE3ch peptide (D). [Figure 20] FIG. 1 shows ELISA results of 1H4-2 sera tested with ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, and ApoE3ch peptide. [Figure 21] FIG. 1 shows representative ELISA profiles of serial dilutions of antibody 1H4 incubated with either human or mouse recombinant ApoE3. [Figure 22] FIG. 1 shows ELISA results for monoclonal 1H4 antibody purified from a cloned hybridoma. [Figure 23-1] FIG. 1 shows heparin affinity chromatography and Western blot analysis of antibody 7C11. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 24] FIG. 1 shows ELISA results from testing 7C11-1 serum with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), or ApoE3ch peptide (D). [Figure 25] FIG. 1 shows ELISA results from testing 7C11-1 serum with ApoE3 WT full-length protein, ApoE3 WT peptide, ApoE3ch full-length protein, or ApoE3ch peptide. [Figure 26] FIG. 1 shows ELISA results for monoclonal 7C11-1 antibody purified from a cloned hybridoma. [Figure 27] FIG. 1 shows the results from an ELISA screening of the 19G10-2 antibody against the heparin-binding domains of ApoE3 wild-type (WT) and ApoE3ch mutant recombinant proteins. [Figure 28-1] FIG. 1 shows heparin affinity chromatography and Western blot analysis of antibody 19G10-2. [Figure 28-2] This is a continuation of Figure 28-1. [Figure 29] FIG. 1 shows Western blotting of ApoE3 WT incubated with 19G10-2 serum antibody. [Figure 30] FIG. 1 shows a representative ELISA demonstrating the difference in binding of both serum 19G10-2 and monoclonal antibody hybridoma supernatant 19G10-2 to ApoE3 WT or ApoE3ch. [Figure 31] FIG. 31 is a diagram showing an enlargement of the Y axis in FIG. 30. [Figure 32] FIG. 1 shows ELISA results for monoclonal 19G10-2 antibody purified from a cloned hybridoma. [Figure 33] FIG. 1 shows the results from ELISA screening of the 25F1-2 antibody against the heparin-binding domain of ApoE3 wild-type (WT) and ApoE3ch mutant recombinant proteins. [Figure 34-1]FIG. 1 shows heparin affinity chromatography and Western blot analysis of antibody 25F1-2. [Figure 34-2] This is a continuation of Figure 34-1. [Figure 35] FIG. 1 shows Western blotting of ApoE3 WT incubated with 25F1-2 monoclonal antibody. [Figure 36] FIG. 1 shows a representative ELISA demonstrating the difference in binding of both 25F1-2 serum and monoclonal antibody hybridoma supernatant 25F1-2 to ApoE3 WT or ApoE3ch. [Figure 37] FIG. 37 is an enlarged view of the Y axis in FIG. 36. [Figure 38] FIG. 1 shows ELISA results for monoclonal 25F1-2 antibody purified from a cloned hybridoma. [Figure 39] FIG. 1 shows ELISA screening of the 1343 antibody against the heparin-binding domain of ApoE3 wild-type (WT) and ApoE3ch mutant recombinant proteins. [Figure 40] FIG. 1 shows ELISA screening of the 1343 antibody against the heparin-binding domain of ApoE3 wild-type (WT) and ApoE3ch mutant recombinant proteins. [Figure 41-1] Figure 41A shows Western blot analysis of ApoE in protein fractions eluted from a heparin column in the presence or absence of antibody 1343. Figure 41B shows ELISA analysis of the fractions. [Figure 41-2] This is a continuation of Figure 41-1. [Figure 42] Figure 42A shows an exemplary experimental outline for an intraocular model of inducible ApoE-dependent tau hyperphosphorylation. Figure 42B shows PHF tau in control retinas injected with PBS. Figure 42C shows retinas injected with recombinant human ApoE3. [Figure 43-1]FIG. 1 shows PHF tau in control retinas compared to retinas injected with either murine 1H4-2 or humanized 1343Ah antibody. [Figure 43-2] This is a continuation of Figure 43-1. [Figure 43-3] This is a continuation of Figure 43-1. [Figure 44] FIG. 1 shows representative binding measurements of increasing concentrations (nM) of ApoE3 protein with 1H4 on a Protein A biosensor. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure has discovered that homozygosity for APOE3ch (having two copies of the APOE3 Christchurch (R136S) mutation) is associated with significant resistance to the clinical onset of Alzheimer's disease, and that the R136S mutation significantly attenuates ApoE's ability to bind heparan sulfate proteoglycans (HSPGs) / heparin. Accordingly, the present disclosure relates to antibodies that bind to wild-type ApoE and / or ApoE isoforms containing the R136S mutation (e.g., antibodies that block and / or reduce the interaction between ApoE and HSPGs / GAGs / heparin). Fusion proteins containing peptide fragments (e.g., HSPG / GAG / heparin-binding domains) of wild-type and mutant ApoE containing the R136S mutation are also contemplated. These proteins can be administered via human cells expressing such compositions. The present disclosure further relates to small molecules that block the interaction between ApoE and HSPGs / heparin, and methods of screening for such small molecules. Compositions and methods for editing the ApoE locus using a genome editing system are also provided. The antibodies, fusion proteins, small molecules, and genome editing systems described herein are useful in treating or preventing cognitive decline and / or neurodegeneration associated with dementia and / or mild cognitive impairment (MCI), such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, or Huntington's disease. The antibodies, fusion proteins, small molecules, and genome editing systems described herein are also useful in treating or preventing neurodegenerative diseases, cerebrovascular conditions, brain injuries, retinal degeneration, optic nerve degeneration, or retinal damage.

[0021] Apolipoprotein E (ApoE) Apolipoprotein E variants are major genetic modifiers of AD, contributing to susceptibility to late-onset AD. The APOE4 allele, resulting in a cysteine-to-arginine change at position 112, is associated with a five-fold increase in AD risk in single-allele carriers and a 20-fold increase in homozygous carriers. The APOE2 allele, resulting in an arginine-to-cysteine ​​amino acid change at position 158, is protective against AD, whereas 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 influences amyloid production, aggregation, and clearance, is a component of amyloid plaques, and exacerbates tau-mediated neurodegeneration. APOE alleles also regulate lipid metabolism and cardiovascular risk. Approximately 5-10% of APOE2 homozygous individuals develop hyperlipoproteinemia type III (HLP III), whereas other rare APOE variants have been associated with autosomal dominant HLP III. HLP III is characterized by elevated plasma cholesterol and triglyceride levels and the presence of nodular or linear palmar xanthomas. The mechanisms by which APOE alleles modify AD risk and cause HLP III are not fully understood. Notable APOE properties affected by specific mutations include differences in 1) binding affinity for lipid and LDL receptors; 2) the nature of the interdomain interactions between its N-terminal domain (amino acids 1-199) and C-terminal domain (216-299); 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).

[0022] Heparan sulfate (HS) is a linear polysaccharide found in all animal tissues and exists as proteoglycans (HSPGs), in which two or three HS chains are attached in close proximity to cell surface or extracellular matrix proteins. HSPG moieties are present in hundreds of proteins located in the plasma membrane and extracellular matrix. HSPG-mediated protein-protein interactions play a key role in numerous processes related to Alzheimer's disease pathology, including amyloid and tau pathology. Heparan sulfate is a member of the glycosaminoglycan family of carbohydrates and is structurally very closely related to heparin. Both consist of variably sulfated repeating disaccharide units. Heparan sulfate binds to numerous extracellular proteins, which are often collectively referred to as the "heparin interactome" or "heparin-binding proteins," because they are isolated by affinity chromatography on its related polysaccharide, heparin.

[0023] An exemplary amino acid sequence of the human ApoE3 protein (Uniprot accession number P02649) is shown below:

[0024] [ka]

[0025] 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) (the N-terminal HSPG / heparin-binding domain) can function in full-length, lipidated, and delipidated ApoE, whereas the HSPG / heparin-binding domain in the C-terminal domain can function in the absence of the N-terminal domain and only in delipidated ApoE. The N-terminal HSPG / heparin-binding domain has been well characterized and includes amino acid residues 142-147 (bold) of SEQ ID NO: 1. The C-terminal HSPG / heparin-binding domain is less well characterized and contains a lysine (K) at position 233 of SEQ ID NO:1, as well as other charged amino acids nearby, including amino acid residues 211-218 and 243-272. The inventors have shown that the arginine at position 136 of ApoE plays an important role in ApoE heparin binding. While not intending to be bound by theory, a possible mechanism is allosteric regulation of heparin binding mediated by the arginine at position 136. As used herein, allosteric regulation refers to the regulation of ligand binding through the binding of an allosteric regulator at one or more sites of allosteric regulation (which may be different from the binding site of the ligand). In some embodiments, the one or more sites of allosteric regulation for ApoE and HSPG / heparin binding, as set forth in SEQ ID NO:1, include the arginine at position 136 of ApoE. The terms "HSPG / heparin binding domain," "HSPG / heparin binding site," "HSPG binding domain," and "HSPG binding site" are used interchangeably herein.

[0026] Anti-ApoE antibody Anti-ApoE antibodies that bind to wild-type or mutant ApoE proteins (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 not to mutant ApoE proteins (e.g., ApoEch). In some cases, the antibodies described herein bind to mutant ApoE proteins (e.g., ApoEch) but not to wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4). In some cases, the antibodies described herein bind to both mutant ApoE proteins (e.g., ApoEch) and wild-type ApoE proteins (e.g., ApoE2, ApoE3, or ApoE4).

[0027] In some cases, the antibodies provided herein block the interaction between wild-type ApoE protein (e.g., ApoE2, ApoE3, or ApoE4) and HSPG. The antibodies provided herein may reduce or modulate the binding affinity of ApoE protein (e.g., ApoE2, ApoE3, or ApoE4) to HSPG. In some cases, the antibodies provided herein bind to the HSPG-binding domain of wild-type ApoE protein. In some cases, the antibodies provided herein bind to one or more sites of allosteric regulation of HSPG / ApoE binding (e.g., amino acid position 136 of ApoE). In some cases, the antibodies described herein reduce fibril formation and / or amyloid oligomerization.

[0028] In some cases, the antibodies provided herein bind to an amino acid sequence in wild-type or mutant ApoE that comprises or consists of TEELRVSLASHLRK (SEQ ID NO: 2). In some cases, the antibodies provided herein bind to an amino acid sequence in wild-type or mutant ApoE that comprises or consists of TEELRVRLASHLRK (SEQ ID NO: 3). 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, e.g., those that are at least 80%, 85%, 90%, or 95% identical to these sequences, can also be used.

[0029] The calculation of "identity" between two sequences can be carried out as follows: The sequences are aligned for optimal comparison (for example, gaps can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). The length of the sequence aligned for comparison purposes is at least 70% (for example, at least 80%, 90%, or 100%) of the length of the reference sequence. Then, the nucleotides at corresponding nucleotide positions are compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0030] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, percent identity between two nucleotide sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (as incorporated into the GAP program in the GCG software package (available at gcg.com)) using either the Blossum 62 matrix, the PAM250 matrix, or the NWSgapdna.CMP matrix. In some embodiments, percent identity between two amino acid or nucleotide sequences can be determined using the E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) algorithm (as incorporated into the ALIGN program (version 2.0)) using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4.

[0031] The use of the term "antibody" in this 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 this disclosure are whole antibodies. In some cases, the anti-ApoE antibodies of this disclosure are chimeric, human, 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 kappa constant region. In other cases, the light chain constant region is a human lambda constant region. In some cases, the antibodies of this disclosure are engineered to have reduced effector function (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 examples, the Fc portion of the antibody is hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1agly Fc, hIgG2 SAA Fc, hIgG4(S228P) Fc, or hIgG4(S228P) / G1 agly Fc (in this format (which minimizes 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 example, 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 U.S. Patent Application Publication No. 2012 / 0100140).

[0032] 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, exemplified in the Examples section below.

[0033] 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 a KD of ≦20 nM; (ii) compete with wild-type human ApoE for binding to heparin; and (iii) reduce paired helical filament (PHF) tau formation in retinal cells.

[0034] In some embodiments, the present disclosure provides antibodies that bind to mutant human ApoE (e.g., 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) bind to mutant human ApoE (e.g., having a mutation at amino acid position 136 of human ApoE, such as ApoEch) with high affinity, with a KD of ≦20 nM; (ii) compete with wild-type human ApoE for binding to heparin; and (iii) reduce paired helical filament (PHF) tau formation in retinal cells.

[0035] Any of the anti-ApoE antibodies described herein are useful for treating or preventing disorders 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), neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage.

[0036] Exemplary Antibody 1H4-2 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.

[0037] [Table 1]

[0038] Variable light chain: Nucleotide sequence: signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0039] [ka]

[0040] Amino acid sequence: signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0041] [ka]

[0042] Variable heavy chain: Nucleotide sequence signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0043] [ka]

[0044] Amino acid sequence: signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0045] [ka]

[0046] In some cases, the anti-ApoE antibody comprises a VH comprising the three VH CDRs of antibody 1H4-2 and a VL comprising the three VL CDRs. The six CDRs may be based on any definition known in the art, such as, but not limited to, the 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).

[0047] In one example, an anti-ApoE antibody of this disclosure comprises (i) a VH comprising a VHCDR1 having the amino acid sequence set forth in SEQ ID NO:7, a VHCDR2 having the amino acid sequence set forth in SEQ ID NO:8, and a VHCDR3 having the amino acid sequence set forth in SEQ ID NO:9; and (ii) a VL comprising a VLCDR1 having the amino acid sequence set forth in SEQ ID NO:4, a VLCDR2 having the amino acid sequence set forth in SEQ ID NO:5, and a VLCDR3 having the amino acid sequence set forth in SEQ ID NO:6.

[0048] 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 example, 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 example, 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 yet another example, 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.

[0049] In some cases, the antibody of this disclosure that binds to ApoE is an antibody that competes with or binds to the same epitope as a reference antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 13 and a VL having the amino acid sequence set forth in SEQ ID NO: 12.

[0050] Exemplary Antibody 7C11-1 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.

[0051] [Table 2]

[0052] Variable heavy chain: Nucleotide sequence Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0053] [ka]

[0054] Amino acid sequence: Signal peptide -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0055] [ka]

[0056] Variable light chain: Nucleotide sequence Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0057] [ka]

[0058] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0059] [ka]

[0060] In some cases, the anti-ApoE antibody comprises a VH comprising the three VH CDRs and a VL comprising the 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, the 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).

[0061] In one example, an anti-ApoE antibody of this disclosure comprises (i) a VH comprising a VHCDR1 having the amino acid sequence set forth in SEQ ID NO: 17, a VHCDR2 having the amino acid sequence set forth in SEQ ID NO: 18, and a VHCDR3 having the amino acid sequence set forth in SEQ ID NO: 19; and (ii) a VL comprising a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 14, a VLCDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and a VLCDR3 having the amino acid sequence set forth in SEQ ID NO: 16.

[0062] 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 example, 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 example, 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 yet another example, 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.

[0063] In some cases, the antibody of this disclosure that binds to ApoE is an antibody that competes with or binds to the same epitope as a reference antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 23 and a VL having the amino acid sequence set forth in SEQ ID NO: 22.

[0064] Exemplary Antibody 19G10-2 Antibody 19G10-2 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 19G10-2 are provided below.

[0065] [Table 3]

[0066] Variable light chain: Nucleotide sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0067] [ka]

[0068] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0069] [ka]

[0070] Variable Heavy Chain Analysis: Nucleotide sequence Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0071] [ka]

[0072] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0073] [ka]

[0074] In some cases, the anti-ApoE antibody comprises a VH comprising the three VH CDRs and a VL comprising the 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, the 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).

[0075] In one example, an anti-ApoE antibody of this disclosure comprises (i) a VH comprising a VHCDR1 having the amino acid sequence set forth in SEQ ID NO: 27, a VHCDR2 having the amino acid sequence set forth in SEQ ID NO: 28, and a VHCDR3 having the amino acid sequence set forth in SEQ ID NO: 29; and (ii) a VL comprising a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 24, a VLCDR2 having the amino acid sequence set forth in SEQ ID NO: 25, and a VLCDR3 having the amino acid sequence set forth in SEQ ID NO: 26.

[0076] 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 example, 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 example, 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 yet another example, 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.

[0077] In some cases, the antibody of this disclosure that binds to ApoE is an antibody that competes with or binds to the same epitope as a reference antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 33 and a VL having the amino acid sequence set forth in SEQ ID NO: 32.

[0078] Exemplary Antibody 25F1-2 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.

[0079] [Table 4]

[0080] Variable Light Chain Analysis: Nucleotide sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0081] [ka]

[0082] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0083] [ka]

[0084] Variable Heavy Chain Analysis: Nucleotide sequence Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0085] [ka]

[0086] Amino acid sequence: Signal sequence -FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4

[0087] [ka]

[0088] In some cases, the anti-ApoE antibody comprises a VH comprising the three VH CDRs and a VL comprising the 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, the 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).

[0089] In one example, an anti-ApoE antibody of this disclosure comprises (i) a VH comprising a VHCDR1 having the amino acid sequence set forth in SEQ ID NO: 37, a VHCDR2 having the amino acid sequence set forth in SEQ ID NO: 38, and a VHCDR3 having the amino acid sequence set forth in SEQ ID NO: 39; and (ii) a VL comprising a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 34, a VLCDR2 having the amino acid sequence set forth in SEQ ID NO: 35, and a VLCDR3 having the amino acid sequence set forth in SEQ ID NO: 36.

[0090] 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 example, 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 example, 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 yet another example, 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.

[0091] In some cases, the antibody of this disclosure that binds to ApoE is an antibody that competes with or binds to the same epitope as a reference antibody comprising a VH having the amino acid sequence set forth in SEQ ID NO: 43 and a VL having the amino acid sequence set forth in SEQ ID NO: 42.

[0092] Exemplary antibody 1343ab 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.

[0093] [Table 5]

[0094] Full-length heavy chain

[0095] [ka]

[0096] Full-length light chain

[0097] [ka]

[0098] EC N-linked glycosylation was detected on the heavy chain constant region N at 292. Loss of the C-terminal lysine was observed in the heavy chain.

[0099] In some cases, the anti-ApoE antibody comprises a VH comprising the three VH CDRs and a VL comprising the three VL CDRs of antibody 1343ab. The six CDRs may be based on any definition known in the art, such as, but not limited to, the 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).

[0100] In one example, an anti-ApoE antibody of this disclosure comprises (i) a VH comprising a VHCDR1 having the amino acid sequence set forth in SEQ ID NO: 47, a VHCDR2 having the amino acid sequence set forth in SEQ ID NO: 48, and a VHCDR3 having the amino acid sequence set forth in SEQ ID NO: 49; and (ii) a VL comprising a VLCDR1 having the amino acid sequence set forth in SEQ ID NO: 44, a VLCDR2 having the amino acid sequence set forth in SEQ ID NO: 45, and a VLCDR3 having the amino acid sequence set forth in SEQ ID NO: 46.

[0101] 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 example, 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 example, 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 yet another example, the anti-ApoE antibody comprises a heavy chain that is identical to the amino acid sequence set forth in SEQ ID NO: 53 and a light chain that is identical to the amino acid sequence set forth in SEQ ID NO: 52.

[0102] In some cases, the antibody of this disclosure that binds to ApoE is an antibody that competes with or binds to the same epitope as a reference antibody comprising 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.

[0103] Chimeric, human, or humanized antibodies having the CDR sequences of any of the above antibodies can be produced according to the methods described herein.

[0104] antibody fragment 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 yields F(ab)2 or Fab fragments; pepsin digestion of whole antibodies yields F(ab')2 or Fab'; and plasmin digestion of whole antibodies yields Facb fragments.

[0105] Alternatively, antibody fragments can be produced recombinantly: for example, a nucleic acid encoding the antibody fragment of interest can be constructed, introduced into an expression vector, and expressed in a suitable host cell. For example, 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 and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated 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)). By another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab)2 fragments containing salvage receptor-binding epitope residues and having increased in vivo half-lives are described in U.S. Pat. No. 5,869,046.

[0106] Conjugated antibodies The antibodies disclosed herein may be combined with polymers (e.g., polymeric substances such as polyethylene glycol (PEG), PEG-modified polyethyleneimine (PEI) (PEI-PEG), polyglutamic acid (PGA) (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer), hyaluronic acid, radioactive materials (e.g., 90 Y, 131 I) They may be conjugated antibodies bound to a variety of molecules including fluorescent substances, luminescent substances, haptens, enzymes, metal chelates, and drugs.

[0107] In some embodiments, an antibody described herein is modified with a moiety that improves its stabilization and / or circulatory retention, e.g., in blood, serum, or other tissues, including the brain, by at least 1.5-fold, 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, or 50-fold. For example, an antibody described herein can be associated (e.g., conjugated) with a polymer, e.g., a substantially non-antigenic polymer, e.g., polyalkylene oxide or polyethylene oxide. Suitable polymers vary substantially by weight. Polymers having molecular number-average weights ranging from about 200 daltons 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, an antibody described herein can be conjugated to a water-soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g., polyvinyl alcohol or polyvinylpyrrolidone. 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 the block copolymers remain water soluble). Additional useful polymers include polyoxyalkylenes, such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides. In some embodiments, the antibodies described herein are modified with a moiety that improves 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 permeable moieties include, but are not limited to, glucose transporter type 1 (GLUT1), cationic amino acid transporter type 1 (CAT1), monocarboxylic acid transporter type 1 (MCT1), concentrative nucleoside transporter type 2 (CNT2), activated efflux transporters (AETs) (e.g., p-glycoprotein and those described in Pardridge, J Cereb Blood Flow Metab 32(11):1959-1972, 2012). Additional blood-brain barrier permeable moieties are known in the art.

[0108] The conjugated antibodies can be prepared by chemically modifying the antibodies described herein or their lower molecular weight forms. Methods for modifying antibodies are well known in the art (e.g., U.S. Pat. Nos. 5,057,313 and 5,156,840).

[0109] Anti-ApoE antibodies can take the form of full-length (or whole) anti-ApoE antibodies, or smaller forms (e.g., biologically active antibody fragments or minibodies), such as Fab, Fab', F(ab')2, Fv, Fd, dAb, scFv, and sc(Fv)2. Other anti-ApoE antibodies encompassed by this disclosure include single domain antibodies (sdAbs) containing a single variable chain, such as VH or VL, or biologically active fragments thereof. See, e.g., Moller et al., J. Biol. Chem., 285(49): 38348-38361 (2010); Harmsen et al., Appl. Microbiol. Biotechnol., 77(1):13-22 (2007); U.S. Patent Application Publication No. 2005 / 0079574, and Davies et al. (1996) Protein Eng., 9(6):531-7. Like whole antibodies, sdAbs can selectively bind to specific antigens (e.g., ApoE2, ApoE3, ApoE4, or ApoEch). With a molecular weight of only 12-15 kDa, sdAbs are much smaller than typical antibodies, and even smaller than Fab fragments and single-chain variable fragments.

[0110] In certain embodiments, an anti-ApoE antibody, or antigen-binding fragment thereof, or low-molecular-weight antibody thereof, specifically binds to the HSPG / heparin-binding domain of ApoE and, when administered to a human patient with one or more of the following or an animal model, reduces the severity of symptoms of dementia and / or mild cognitive impairment (MCI) (e.g., Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, Huntington's disease, or those associated with neurodegeneration). In certain embodiments, an anti-ApoE antibody, or antigen-binding fragment thereof, or low-molecular-weight antibody thereof, specifically binds to the HSPG / heparin-binding domain of ApoE and, when administered to a human patient with one or more of the following or an animal model, reduces the severity of symptoms of neurodegenerative disease, cerebrovascular disease (e.g., stroke, carotid artery stenosis, vertebral artery stenosis, or aneurysm), brain injury (e.g., traumatic brain injury, acquired brain injury), retinal degeneration, glaucoma, or retinal injury. These characteristics of an anti-ApoE antibody or low molecular weight antibody thereof can be measured according to methods known in the art.

[0111] Nucleic acids, vectors, and host cells This disclosure also features nucleic acids encoding the antibodies disclosed herein. Nucleic acids encoding the VH CDR1, VH CDR2, and VH CDR3 of the anti-ApoE antibodies described herein are provided herein. Nucleic acids encoding the VL CDR1, VL CDR2, and VL CDR3 of the anti-ApoE antibodies described herein are also provided herein. Nucleic acids encoding the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of the anti-ApoE antibodies described herein are also provided herein. Nucleic acids encoding the heavy chain variable region (VH) of the anti-ApoE antibodies described herein and / or the light chain variable region (VL) of the anti-ApoE antibodies described herein are also provided herein. Nucleic acids encoding the VH and / or VL of the anti-ApoE antibodies described herein, optionally linked to a human heavy chain constant region and / or a human light chain constant region, respectively, are provided herein. Also provided herein are nucleic acids encoding both the VH and VL of the anti-ApoE antibodies described herein. In some cases, the nucleic acids described herein include nucleic acids encoding the Fc region of a human antibody (e.g., human IgG1, IgG2, IgG3, or IgG4). In some cases, the nucleic acids include nucleic acids encoding the Fc region of a human antibody that has been modified to reduce or eliminate effector function (e.g., an N297Q or T299A substitution (EU numbering) in a human IgG1 Fc region). In some examples, the nucleic acids include nucleic acids encoding an Fc portion that is hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1agly Fc, hIgG2 SAA Fc, hIgG4(S228P) Fc, or hIgG4(S228P) / G1 agly Fc.

[0112] Also disclosed herein are vectors (eg, expression vectors) containing any of the above nucleic acids.

[0113] Additionally, this disclosure relates to host cells (eg, bacterial, yeast, insect, or mammalian cells) containing the above-described vectors or nucleic acids.

[0114] How to obtain anti-ApoE antibodies Methods for producing anti-ApoE antibodies useful in the present method are also provided herein. General methods for producing antibodies, e.g., monospecific, polyclonal, or monoclonal antibodies, are known in the art. For monoclonal antibodies, the method involves obtaining antibody-secreting immune cells (lymphocytes) from the spleen of a mammal (e.g., a mouse) that has been previously immunized with an antigen of interest (e.g., a peptide antigen as described herein) either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells capable of replicating indefinitely in cell culture, thereby generating immortal immunoglobulin-secreting cell lines. The resulting fused cells or hybridomas are cultured, and the resulting colonies are screened for the production of the desired monoclonal antibody. Colonies producing such antibodies are cloned and grown either in vivo or in vitro to produce large amounts of the antibody. A description of the theoretical basis and practical methodology for fusing such cells is presented in Kohler and Milstein, Nature 256:495 (1975).

[0115] Mammalian lymphocytes can be immunized by in vivo immunization of animals (e.g., mice) with peptide antigens, such as peptide antigens at least 80%, 85%, 90%, or 95% identical to KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54) or KLH-CTEELRVSLASHLRK-CONH2 (SEQ ID NO: 55), optionally containing one or more substitutions or deletions, for example, up to 20% of the residues. For example, the method can include immunizing animals with a peptide containing a sequence at least 80% identical to at least 10 consecutive amino acids from a peptide containing the heparin-binding domain of APOE, such as TEELRVRLASHLRK (SEQ ID NO: 3) or TEELRVSLASHLRK (SEQ ID NO: 2). Such immunizations can be repeated at intervals of up to several weeks as needed to obtain sufficient antibody titers. After the final antigen boost, the animals are sacrificed and spleen cells are removed.

[0116] Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by known techniques, for example, using polyethylene glycol ("PEG") or other fusing agents (see Milstein and Kohler, Eur. J. Immunol. 6:511 (1976), incorporated herein by reference). The immortal cell line (preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rat and human) is selected to be deficient in enzymes required for the utilization of certain nutrients, to have the ability to grow rapidly, and to have good fusion potential. Many such cell lines are known to those of skill in the art, and others have been frequently described.

[0117] Procedures for producing polyclonal antibodies are also known. Typically, such antibodies can be produced by subcutaneously administering a protein or polypeptide of the present invention to New Zealand White rabbits that have been initially bled to obtain preimmune serum. The antigen can be injected, for example, at six different sites in a total volume of 100 μl per site. Each injection contains a synthetic surfactant adjuvant, pluronic polyol, or finely divided acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. Two weeks after the initial injection, the rabbits are then bled and periodically boosted with the same antigen three times every six weeks. Ten days after each boost, serum samples are then collected. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Finally, the rabbits are euthanized, for example, with pentobarbital 150 mg / kg IV. This and other procedures for producing polyclonal antibodies are disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988).

[0118] The methods described herein include any one of the steps of producing a chimeric antibody, a humanized antibody, a single-chain antibody, a Fab fragment, a bispecific antibody, a fusion antibody, a labeled antibody, or an analog thereof. 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 the antibody derivatives are obtained by phage display technology, surface plasmon resonance, such as that used in the BIAcore system, can be used to increase the efficiency of phage antibodies that bind to the same epitope as any one of the antibodies described herein (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in WO 89 / 09622. Methods for producing humanized antibodies are described, for example, in European Patent Application Publication No. 0239400 and International Publication No. WO 90 / 07861. Another source of antibodies that can be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principles for producing xenogenic antibodies, such as human-like antibodies in mice, are described, for example, in International Publication Nos. 91 / 10741, 94 / 02602, 96 / 34096, and 96 / 33735. As discussed above, the antibodies described herein can exist in various forms other than complete antibodies, including, for example, Fv, Fab, and F(ab)2, as well as single chains; see, for example, International Publication No. WO 88 / 09344.

[0119] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and taught in, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier, NY, 563-681 (1981) (these references are incorporated by reference in their entirety). As used herein, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology.

[0120] In the known hybridoma method (Kohler et al., Nature 256 (1975), 495), relatively short-lived or mortal lymphocytes from a mammal, e.g., B cells from a murine subject as described herein, are fused with an immortal tumor cell line (e.g., a myeloma cell line), thus producing hybrid cells or "hybridomas" that are immortal and capable of producing the genetically encoded antibodies of the B cells. The resulting hybrids are separated by selection, dilution, and regrowth into monogenic strains, each individual strain containing a specific gene for the formation of a single antibody. They produce antibodies that are homogeneous against the desired antigen and, in reference to their pure genetic lineage, are termed "monoclonal."

[0121] The hybridoma cells thus prepared are seeded and grown in a suitable medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. Those skilled in the art will recognize that reagents, cell lines, and media for hybridoma formation, selection, and growth are commercially available from several sources, and standardized protocols are well established. Generally, the culture medium in which the hybridoma cells are growing 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 (ELSA), as described herein. After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods; see, e.g., Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986). The monoclonal antibodies secreted by the subclones can be isolated 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.

[0122] In another embodiment, lymphocytes can be selected by micromanipulation and variable genes isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized or naturally immune mammal, such as a human, and cultured in vitro for about 7 days. The cultures are screened for specific immunoglobulins that meet the screening criteria. Cells from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be micromanipulated into tubes and the VH and VL genes can be amplified, for example, using RT-PCR. The VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g., eukaryotic or prokaryotic cells) for expression.

[0123] Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. Such techniques are described in a variety of laboratory manuals and major publications. In this regard, techniques suitable for use in the present invention, such as those 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 any appendices.

[0124] Antibodies such as the above antibodies can be produced, for example, by preparing and expressing a synthetic gene encoding the listed amino acid sequence. Methods for producing any variant of an anti-ApoE antibody (e.g., containing 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 a prepared DNA molecule encoding an antibody or any portion thereof (e.g., framework region, CDR, constant region). Site-directed mutagenesis is well known in the art (see, for example, 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 suitable for producing 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).

[0125] Antibodies can be produced in bacterial cells or eukaryotic cells. Some antibodies, such as Fabs, can be produced in bacterial cells, such as E. coli cells. Antibodies can also be produced in eukaryotic cells, such as transformed cell lines (e.g., CHO, 293E, COS, Hela). In addition, antibodies (scFv) can be expressed in yeast cells, such as Pichia (see, e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hansenula, or Saccharomyces. To produce an antibody or antigen-binding fragment of interest, 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 the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody.

[0126] If the antibody is to be expressed in bacterial cells (e.g., E. coli), the expression vector should have properties that allow the vector to be amplified in the bacterial cells. Additionally, when E. coli such as JM109, DH5α, HB101, or XL1-Blue is used as the host, the vector must have a promoter that can enable efficient expression in E. coli, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter. 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 this expression vector is used, the host is preferably BL21, which expresses T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. For production into 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 calcium chloride method or electroporation method may be used to introduce the expression vector into bacterial cells.

[0127] When antibodies are to be expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector contains a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter. In addition to the nucleic acid sequence encoding an immunoglobulin or a domain thereof, the recombinant expression vector may carry additional sequences such as sequences that control replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Examples of vectors having selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0128] In one embodiment, the antibody is produced in mammalian cells. Exemplary mammalian host cells for expressing the antibody include Chinese hamster ovary cells (CHO cells) (e.g., dhfr cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selectable marker, as described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621). - CHO cells), 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.

[0129] The antibodies of the present disclosure can be isolated from the inside or outside of host cells (such as the culture medium) and purified as substantially pure and homogeneous antibodies. Isolation and purification methods commonly used in antibody purification can be used for antibody isolation and purification, and are not limited to any particular method. Antibodies can be isolated 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, reversed-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-phase chromatography such as HPLC and FPLC. Columns used for affinity chromatography include Protein A and Protein G columns. Examples of columns that use Protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also includes antibodies that have been highly purified using these purification methods.

[0130] Antibody characterization The ApoE binding properties of the antibodies described herein can be measured by any standard method, for example, by one or more of the following methods: OCTET®, surface plasmon resonance (SPR), BIACORE™ analysis, enzyme-linked immunosorbent assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay), and fluorescence resonance energy transfer (FRET).

[0131] Methods for using SPR are described, for example, in U.S. Patent 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 (Uppsala, Sweden). Information from SPR can be used to determine the equilibrium dissociation constant (K) for binding of a biomolecule to a target. d ), and K on and K. off The present invention can be used to provide accurate and quantitative measurements of kinetic parameters, including:

[0132] Epitopes can also be mapped directly using BIACORE chromatography technology to assess the ability of different antibodies to compete with each other for binding to wild-type or mutant ApoE (e.g., ApoEch) (Pharmacia BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May 1994); see also Johne et al. (1993) J. Immunol. Methods, 160:191-198).

[0133] In enzyme immunoassays, an antibody-containing sample, such as a 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, washed, and then an enzyme substrate such as p-nitrophenyl phosphate is added. The absorbance is measured to assess antigen-binding activity.

[0134] Additional general guidance for evaluating antibodies, for example, Western blot and immunoprecipitation assays, can be found in Antibodies: A Laboratory Manual, ed. by Harlow and Lane, Cold Spring Harbor press (1988).

[0135] Mutant ApoE proteins, peptides, and fusion proteins thereof The present disclosure provides mutant ApoE proteins or fragments thereof containing amino acid substitutions at one or more positions in the HSPG-binding domain compared to wild-type ApoE proteins. In some embodiments, the mutant ApoE proteins or fragments thereof contain an amino acid other than arginine at position 136. In some embodiments, the mutant ApoE proteins or fragments thereof contain serine, histidine, or cysteine ​​at position 136. Nucleic acid (e.g., DNA or RNA) sequences encoding mutant ApoE proteins or fragments thereof, and vectors containing the nucleic acid sequences, are also provided. Mutant ApoE proteins or fragments thereof, nucleic acids encoding such proteins or fragments, and vectors containing the nucleic acid sequences are useful for treating or preventing disorders 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), neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage.

[0136] In some embodiments, the mutant ApoE protein is an ApoEch protein (e.g., an ApoE2ch, ApoE3ch, or ApoE4ch protein). Fragments of ApoEch proteins comprising amino acid position 136 are also contemplated herein. An exemplary sequence of a full-length ApoE3ch protein is shown below. The arginine to serine mutation is shown in bold and double underlined.

[0137] [ka]

[0138] In some embodiments, the methods disclosed herein enable a mutant ApoE protein or fragment thereof to cross the blood-brain barrier. The mutant ApoE protein or fragment thereof can be delivered using nanocarriers, including, but not limited to, polymeric nanoparticles, lipid-based nanoparticles, liposomes, micelles, dendrimers, human cells expressing the protein, 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 intranasally, by intracarotid or transmucosal delivery (e.g., intracarotid infusion of hypertonic solutions (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 by use of chlorotoxin (see McCall et al., Tissue Barriers 2(4):e944449, 2014). Hypothermia, receptor-mediated transport, cell-penetrating peptides, and cell-mediated delivery can also be used to facilitate crossing of the blood-brain barrier by ApoE3ch protein (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 payloads across the BBB. Nanoparticle-loaded mesenchymal stem cells can be used for this purpose (see, e.g., Roger et al. Biomaterials 31:8393-401, 2010). Genetically modified stem cells (e.g., genetically modified mesenchymal stem cells) can also be used (see, e.g., 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. Additional methods for transporting proteins across the blood-brain barrier are known in the art.

[0139] Nucleic acids (e.g., DNA or mRNA) encoding mutant ApoE proteins (e.g., any of the mutant ApoE proteins described herein, e.g., ApoEch) or fragments thereof are contemplated herein. In some embodiments, the mRNA encoding the ApoEch protein may be modified to increase stability (e.g., as described in Zangi et al., Nat Biotechnol. 31(10):898-907, 2013 and developed by Moderna, Inc.; and as described in Alberer et al., Lancet 390(10101):1511-1520, 2017 and developed by Curevac and BioNTech).

[0140] Viral vectors containing DNA sequences encoding mutant ApoE proteins or fragments thereof are contemplated herein. An exemplary cDNA sequence (including the signal peptide region) encoding the full-length ApoE3ch protein is shown below. The cytosine to adenine mutation is shown in bold and double underlined.

[0141] [ka]

[0142] Suitable vectors are known in the art. In some embodiments, the viral vector is an AAV vector (e.g., as described in Rosenberg et al., Hum Gene Ther Clin Dev 29(1):24-47, 2018). Also included are cDNA sequences encoding ApoE proteins containing mutations at R136 other than R136S. In some embodiments, the mutation is R136H or R136C.

[0143] Peptides and fusion proteins In some embodiments, provided herein are peptides comprising or consisting of the HSPG / heparin-binding domain of wild-type or mutant ApoE (e.g., any of the mutant ApoE proteins described herein). In some cases, the amino acid sequence of the peptides 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). Variants having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to these sequences can also be used. Fusion proteins comprising the peptides provided above are also disclosed. In some embodiments, the fusion protein further comprises an Fc region of a human antibody (e.g., human IgG1, IgG2, IgG3, or IgG4). In some cases, the fusion protein comprises an Fc region of a human antibody C-terminal to the HSPG / heparin-binding domain of wild-type or mutant ApoE. In some cases, the fusion protein comprises an Fc region of a human antibody N-terminal to the HSPG / heparin-binding domain of wild-type or mutant ApoE.

[0144] In some cases, the peptides and fusion proteins provided herein compete with wild-type ApoE protein for binding to HSPG / heparin. In some cases, the peptides and fusion proteins provided herein reduce or modulate the binding between wild-type ApoE protein and HSPG / heparin. In certain embodiments, the peptides and fusion proteins provided herein inhibit and / or reduce the HSPG / heparin binding of wild-type ApoE protein and reduce the severity of symptoms when administered to human patients with one or more of the following, or to the following animal models: dementia or mild cognitive impairment (MCI)-related disorders (e.g., Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, Huntington's disease), neurodegenerative diseases, cerebrovascular diseases, brain injury, retinal degeneration, or retinal damage. These characteristics of the peptides and fusion proteins provided herein can be measured according to methods known in the art.

[0145] Also provided herein is an anti-ApoE vaccine that can be used to induce a protective immune response against ApoE. In some embodiments, the anti-ApoE vaccine comprises one or more of the ApoE proteins provided herein, for example, and a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable adjuvants are known in the art.

[0146] Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of pharmaceutical compositions that contain, as an active ingredient, any of the antibodies, peptides, or fusion proteins described herein.

[0147] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0148] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration, which includes parenteral, e.g., intracranial, intranasal, intracarotid, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), and transmucosal.

[0149] Methods for formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0150] Pharmaceutical compositions suitable for injection can 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™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin in the composition.

[0151] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent that contains one or combination of the above-listed components, and then optionally sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into sterile medium, and this sterile medium contains basic dispersion medium and other components that are required from above-listed.For the sterile powder that is used to prepare sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying, which produces the powder of active ingredient and any additional desired components from its previously sterile-filtered solution.

[0152] Oral compositions generally include an inert diluent or an edible carrier. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a flowable carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. Tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, or cornstarch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

[0153] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer, including those described in U.S. Patent No. 6,468,798.

[0154] Systemic administration of the therapeutic compound as described herein can also be via transmucosal route.For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, surfactants, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved by using nasal sprays or suppositories or injections.

[0155] In one embodiment, therapeutic compounds are prepared with carriers that protect the therapeutic compound from rapid elimination from the body, such as sustained-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0156] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0157] CRISPR / Cas9-mediated gene editing of APOE Included herein are methods for treating or preventing disorders associated with dementia and / 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), neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage by editing the APOE gene using a genome editing system. Generally, the method includes administering a therapeutically effective amount of a genome editing system as described herein to a subject in need of, or determined to be in need of, such treatment. The term "genome editing system" refers to any system with RNA-guided DNA editing activity. The genome editing system of the present disclosure includes at least two components adapted from naturally occurring CRISPR systems: a gRNA and an RNA-guided nuclease. These two components associate with a specific nucleic acid sequence in a cell and form a complex capable of editing DNA within or surrounding the nucleic acid sequence, for example, by generating one or more single-strand breaks (SSBs or nicks), double-strand breaks (DSBs), and / or base substitutions. See, e.g., WO 2018 / 026976 for a complete description of genome editing systems. In certain embodiments, the present disclosure provides AAV vectors encoding CRISPR / Cas9 genome editing systems and uses of such vectors to treat or prevent disorders as described herein.

[0158] RNA-guided nuclease / Cas9 Various RNA-guided nucleases can be used in the present methods, for example, as described in International Publication No. WO 2018 / 026976. In some embodiments, the RNA-guided nuclease used in the present methods and compositions is Staphylococcus aureus (S. aureus) Cas9 or Streptococcus pyogenes (S. pyogenes) Cas9. Exemplary Cas9 proteins of the present disclosure can be isolated or derived from any species, including, but not limited to, bacteria or archaea. In some embodiments of this disclosure, the Cas9 sequence is modified to include two nuclear localization sequences (NLSs) (e.g., PKKKRKV (SEQ ID NO: 61)) and a mini-polyadenylation signal (or polyA sequence) at the C- and N-termini of the Cas9 protein. 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, for example, Cokol et al., EMBO Rep. 2000 Nov 15; 1(5):411-415; Freitas and Cunha, Curr Genomics. 2009 Dec; 10(8): 550-557. An exemplary polyadenylation signal is TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGGCGCG (SEQ ID NO: 64). In some embodiments, the RNA-guided nuclease is a nuclease-inactive Cas protein (e.g., dCas9).

[0159] guide RNA Provided herein are guide RNAs (gRNAs) designed to target one or more sites in the HSPG-binding domain of wild-type ApoE. In some embodiments, the gRNA is designed to introduce a mutation in wild-type ApoE, resulting in a mutation at amino acid position 136. In some embodiments, the guide RNA provided herein is designed to introduce an R136S mutation in a wild-type APOE gene (e.g., APOE2, APOE3, or APOE4), in which case exemplary guide RNAs can be found in Table 7. In some embodiments, a template for repairing double-strand breaks and introducing an R136S mutation is also provided. An exemplary template sequence is as follows:

[0160] [ka] However, silent mutations that could eliminate the PAM motif are double underlined, the codon corresponding to the R136S mutation is shown in bold, and silent mutations that could create a SacI site for cleaving the PCR product from clones that accepted the template are shown in italics.

[0161] In some embodiments, the guide RNAs provided herein are designed to target exon 3 (amino acids 1-61) of the wild-type APOE gene or of a variant present in a subject (thus, the method may include determining the sequence of the APOE gene in a subject and using the sequence to determine the sequence of an appropriate guide RNA for targeting exon 3 in the subject). In some embodiments, double-strand break repair through non-homologous end joining (NHEJ) generates a short insertion or deletion that results in an ApoE knockout. Exemplary guide RNA sequences for ApoE knockout are shown in Table 8.

[0162] BasesEdit In some embodiments, the APOE gene is edited using base editing technology (e.g., as 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 to introduce an R136H mutation in a wild-type APOE gene (e.g., APOE2, APOE3, or APOE4) using base editing, in which case exemplary guide RNAs can be found in Table 6. Base editors that change C / G to A / T and adenine base editors that change 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).

[0163] AAV delivery system The method comprises delivering a CRISPR / Cas9 genome editing system comprising Cas9 nuclease and one or two guide RNAs to a subject in need.Delivery methods can include, for example, viral delivery, preferably using an adeno-associated virus (AAV) vector comprising a Cas9 coding sequence and guide RNA.Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as adenovirus or herpesvirus, as a helper virus for efficient replication and productive life cycle (for a review, see Muzyczka et al., Curr. Topics in Micro and Immunol.158:97-129 (1992)).AAV vectors can efficiently transduce various cell types and cause long-term expression of transgenes in vivo. AAV vectors have been widely used for gene augmentation or replacement and have shown therapeutic efficacy in various animal models as well as in clinical settings; 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 April; 20(4): 699-708. AAV vectors containing at least 300 base pairs of AAV can be packaged and can result in 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 comprise (or can comprise a sequence encoding) an AAV capsid polypeptide described in International Application No. 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 NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 of International Application No. PCT / US2014 / 060163, a Cas9 sequence, and a guide RNA sequence as described herein. In some embodiments, the AAV capsid polypeptide is an Anc80 polypeptide, e.g., Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; or Anc80L44. In some embodiments, the AAV incorporates inverted terminal repeats (ITRs) derived from the AAV2 serotype. Exemplary left and right ITRs are provided in Table 6 of WO 2018 / 026976. However, it should be noted that numerous modified versions of AAV2 ITRs are used in the art, and the ITRs shown below are exemplary and not intended to be limiting. Modifications of these sequences are known in the art or would be apparent to one of ordinary skill in the art and are therefore within the scope of this disclosure.

[0164] 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-coupled receptor kinase-1 (hGRK1) (specifically expressed in retinal photoreceptor cells). The nucleotide sequences for each of these promoters are provided in Table 5 of WO 2018 / 026976. Modifications of these sequences may be possible or desirable in certain applications, and such modifications are within the scope of this disclosure.

[0165] Expression of the gRNA in the AAV vector is driven by a promoter known in the art. In some embodiments, a polymerase III promoter, such as the human U6 promoter. An exemplary U6 ​​promoter sequence is provided below:

[0166] [ka]

[0167] 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.

[0168] The AAV genomes described above can be packaged into AAV capsids (e.g., AAV5 capsids), and the capsids can be included in compositions (e.g., pharmaceutical compositions) and / or administered to a subject. Exemplary pharmaceutical compositions comprising AAV capsids according to this disclosure can include a pharmaceutically acceptable carrier, such as balanced salt solution (BSS), and one or more surfactants (e.g., Tween 20) and / or temperature-sensitive or reverse temperature-sensitive polymers (e.g., pluronics). Other pharmaceutical formulation elements known in the art can also be suitable for use in the compositions described herein.

[0169] Compositions comprising AAV vectors according to this 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, taking into account, among other things, the dead volume in the injection device and the relatively limited volume that can be safely administered. Suitable doses include, for example, 1 x 10 11 Viral genomes (vg) / mL, 2 × 10 11 Viral genome (vg) / mL, 3 x l0 11 Viral genomes (vg) / mL, 4 × 1011 Viral genome (vg) / mL, 5 × l0 11 Viral genomes (vg) / mL, 6 × 10 11 Viral genomes (vg) / mL, 7 × 10 11 Viral genome (vg) / mL, 8 x l0 11 Viral genomes (vg) / mL, 9 × 10 11 Viral genome (vg) / mL, 1 × 10 12 vg / mL, 2 x l0 12 Viral genome (vg) / mL, 3 x l0 12 Viral genome (vg) / mL, 4 x l0 12 Viral genome (vg) / mL, 5 × l0 12 Viral genome (vg) / mL, 6 x l0 12 Viral genome (vg) / mL, 7 x l0 12 Viral genome (vg) / mL, 8 x l0 12 Viral genome (vg) / mL, 9 x l0 12 Viral genome (vg) / mL, l x 10 13 vg / mL, 2 x l0 13 Viral genome (vg) / mL, 3 x l0 13 Viral genome (vg) / mL, 4 x l0 13 Viral genome (vg) / mL, 5 × l0 13 Viral genome (vg) / mL, 6 x l0 13 Viral genome (vg) / mL, 7 x l0 13 Viral genome (vg) / mL, 8 x l0 13 viral genomes (vg) / mL, or 9 x l0 13 The amount of viral genome (vg) / mL may be included. Any suitable volume of the composition can be delivered to the subretinal or cochlear space. 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.

[0170] Explants are particularly useful for studying gRNA and / or Cas9 expression after viral transduction and for studying genome editing over relatively short time intervals. These models also allow for higher throughput than may be possible in animal models and may be predictive of expression and genome editing in animal models and subjects. Small (mouse, rat) and large animal models (e.g., rabbits, pigs, non-human primates) can be used for pharmacological and / or toxicological studies and to test the systems, nucleotides, vectors, and compositions of this disclosure under conditions and volumes that approximate those used clinically. Because model systems are selected to reproduce relevant aspects of human anatomy and / or physiology, data obtained in these systems generally (but not necessarily) predict the behavior of AAV vectors and compositions of this disclosure in human and animal subjects.

[0171] Screening Method (Test Compound) Included herein are methods for screening test compounds, e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds, to identify agents useful in the treatment or prevention of disorders associated with dementia or mild cognitive impairment (e.g., Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, or Huntington's disease), neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. In some embodiments, the test compound modulates 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).

[0172] As used herein, "small molecule" refers to a small organic or inorganic molecule with a molecular weight below about 3,000 Daltons. Generally, small molecules useful in the present invention have a molecular weight of less than 3,000 Daltons (Da). Small molecules can be, for example, at least about 100 Da and up to about 3,000 Da (e.g., between about 100 and about 3,000 Da, about 100 and about 2,500 Da, about 100 and about 2,000 Da, about 100 and about 1,750 Da, about 100 and about 1,500 Da, about 100 and about 1,250 Da, about 100 and about 1,000 Da, about 100 and about 750 Da, about 100 and about 500 Da, about 200 and about 1,500, about 500 and about 1,000, about 300 and about 1,000 Da, or about 100 and about 250 Da).

[0173] The test compound can be, for example, a natural product or a member of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions, such as charge, aromaticity, hydrogen bonding, flexibility, size, side chain length, hydrophobicity, and rigidity. Suitable combinatorial techniques for synthesizing small molecules are known in the art, and include techniques such as "split and pool" or "parallel" synthesis techniques, solid phase and solution phase techniques, and encoding techniques, as exemplified, for example, in Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998) (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, some small molecule libraries are commercially available. Some suitable small molecule test compounds are listed in US Pat. No. 6,503,713, which is incorporated herein by reference in its entirety.

[0174] The library screened using the method of the present invention can contain various types of test compounds.A given library can contain a set of structurally related or unrelated test compounds.In some embodiments, the test compound is a peptide or peptidomimetic molecule.In some embodiments, the test compound is a nucleic acid.

[0175] In some embodiments, test compounds and libraries thereof can be obtained by systematically varying the structure of a first test compound, e.g., a first test compound structurally similar to a known natural binding partner of a target polypeptide, or a first small molecule identified as capable of binding to a target polypeptide, using, for example, methods known in the art or described herein, and correlating the resulting biological activity with its structure, e.g., structure-activity relationship studies. As those skilled in the art will appreciate, there are a variety of standard methods for generating such structure-activity relationships. Thus, in some cases, the study can be largely empirical, while in other cases, the three-dimensional structure of an endogenous polypeptide or a portion thereof can be used as a starting point for rational design of small molecule compounds. For example, in one embodiment, a general library of small molecules is screened, e.g., using the methods described herein.

[0176] In some embodiments, a test compound is applied to a test sample, e.g., a sample containing one or more ApoE proteins, and one or more effects of the test compound (e.g., the HSPG / heparin binding affinity of the ApoE protein) are evaluated. The ability of a test compound to alter the HSPG / heparin binding affinity of an ApoE protein can be evaluated, for example, using a heparin Sepharose column or an antibody that specifically recognizes the HSPG-binding domain of ApoE as described herein. In some embodiments, a method for screening test compounds as described herein includes evaluating the ability of the test compound to alter (e.g., inhibit or reduce) the binding of an antibody as described herein that binds to one or more HSPG-binding sites or one or more sites of allosteric regulation of HSPG binding of wild-type or mutant ApoE. In some embodiments, the test compound competes with an antibody as described herein for ApoE binding.

[0177] In some embodiments, the test sample is or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein. For example, an animal model, e.g., a rodent such as a rat, can be used.

[0178] A test compound that has been screened by the methods described herein and determined to reduce or alter the binding of ApoE to HSPG / heparin can be considered a candidate compound. For example, a candidate compound that has been screened in an in vivo model of a disorder, such as dementia and / or mild cognitive impairment (e.g., those associated with Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, or Huntington's disease), a neurodegenerative disease, a cerebrovascular disease, brain injury, retinal degeneration, or retinal damage, and determined to produce a desired effect on the disorder, e.g., one or more symptoms of the disorder, can be considered a candidate therapeutic agent. Once screened in a clinical setting, a candidate therapeutic agent is a therapeutic agent. Candidate compounds, candidate therapeutic agents, and therapeutic agents can be optionally optimized and / or derivatized and formulated with a physiologically acceptable excipient to form a pharmaceutical composition.

[0179] Thus, test compounds identified as "hits" in the first screen (e.g., test compounds that produce a desired effect on a disorder) can be selected and systematically varied, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameters. Such optimization 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, subjecting the hits to systematic structural variation to generate a second library of compounds structurally related to the hits, and screening the second library using the methods described herein.

[0180] Test compounds identified as hits can be considered candidate therapeutic compounds useful for treating, preventing, or delaying the onset or progression of disorders associated with dementia and / or mild cognitive impairment, such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies, frontotemporal dementia, Parkinson's disease, or Huntington's disease, or for treating, preventing, or delaying the onset or progression of neurodegenerative diseases, cerebrovascular diseases, brain injury, retinal degeneration, or retinal damage, as described herein. Various techniques useful for determining the structure of "hits," such as NMR, mass spectrometry, gas chromatography with electron capture detection, fluorescence, and absorption spectroscopy, can be used in the methods described herein. Thus, the present invention also includes compounds identified as "hits" by the methods described herein, as well as methods for their administration and use in treating, preventing, or delaying the onset or progression of the disorders described herein.

[0181] Test compounds identified as candidate therapeutic compounds can be further screened by administration to an animal model of a disorder associated with any of the disorders described herein. The animals can be monitored for changes in the disorder, e.g., improvements in parameters of the disorder, e.g., parameters associated with clinical outcome.

[0182] Treatment method The methods described herein include methods for treating, preventing, or delaying the onset or progression of dementia and / or mild cognitive impairment-related disorders, neurodegenerative diseases, cerebrovascular diseases, brain injuries, retinal degeneration, or retinal damage. In some embodiments, the dementia and / or mild cognitive impairment-related disorder is Alzheimer's disease, vascular dementia, Lewy body dementia, frontotemporal dementia, Parkinson's disease, or Huntington's disease. Additional non-limiting examples of neurodegenerative diseases include prion diseases, motor neuron diseases, and amyotrophic lateral sclerosis (ALS). Non-limiting examples of cerebrovascular diseases include stroke, carotid artery stenosis, vertebral artery stenosis, and aneurysm. Non-limiting examples of brain injuries include traumatic brain injury and acquired brain injury. Retinal degeneration such as glaucoma and age-related macular degeneration can involve amyloid beta and neurofibrillary tangle toxicity, and the relationship between retinal degeneration and neurodegeneration (e.g., Alzheimer's disease) has been established (see, for example, 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 neurodegeneration can be used to treat retinal or optic nerve degeneration.

[0183] The methods include administering a therapeutically effective amount of any of the antibodies, peptides, fusion proteins, or genome editing systems as described herein to a subject in need of, or determined to be in need of, such treatment.

[0184] The method described herein is also useful for the subject at risk of developing any of the disorders described herein.The subject at risk of developing Alzheimer's disease can include those who are homozygous or heterozygous for APOE4 allele, those who carry mutations that cause autosomal dominant Alzheimer's disease (for example, mutations in amyloid beta precursor (APP) gene, PSEN1 gene, or PSEN2 gene), and those who carry trisomy 21 (for example, subjects who only have developmental cognitive impairment).The subject at risk of developing Alzheimer's disease can also include those who have polygenic risk scores associated with increased risk of developing the disease, and those who have evidence of brain imaging or other biomarkers of Alzheimer's disease (for example, biomarkers in body fluids). The methods for preventing or delaying the onset of the disorders described herein may also be useful for subjects who are not at risk of developing the disorders, for example, any subject over the age of 50 (e.g., over the age of 55, 60, 65, 70, 75, 80, 85, 90, or 95).

[0185] In this context, " treating " refers to alleviating at least one symptom of the disorder related to the disorder as described herein.Alzheimer's disease often causes fibrillation, amyloid aggregation and cognitive decline; therefore, treatment can cause reduction in fibrillation and / or amyloid aggregation in the brain, reduction in the formation of tau tangles, improvement in cerebral metabolism, improvement in cognitive neuroscience function and / or cognitive decline. [Example]

[0186] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0187] [Example 1] Identification of the APOE3 Christchurch R136S mutation in a PSEN1 mutation carrier material and method: Clinical Evaluation: The institutional review boards of the University of Antioquia, Massachusetts General Hospital, and Schepens Eye Research Institute of Massachusetts Eye and Ear approved this study. The proband patient, as well as all study participants, provided written informed consent. Clinical evaluation and neuropsychological testing were performed as noted in Table 1. PSEN1 E280A genotyping was performed as previously described. 1 ,went.

[0188] All clinical measurements were conducted in Spanish by physicians and psychologists trained at the University of Antioquia (Medellin, Colombia). Neurocognitive testing included a comprehensive multidomain assessment. Some of the tests administered were the Mini-Mental State Examination (MMSE), the Clinical Dementia Rating (CDR), and the Spanish version of the Consortium to Establish a Registry for Alzheimer's Disease Battery, adapted for this Colombian population. 2 Additional testing was performed within 6 months of brain imaging, including the Yesavage Geriatric Depression Scale. 3 and Functional Assessment Staging test 4 It was made up of:

[0189] A detailed ophthalmologic evaluation was performed, including visual acuity assessment, slit-lamp, and indirect ophthalmoscopy. Ultra-wide-angle fundus and fundus autofluorescence images were obtained using an Optos Panoramic 200Tx imaging system (Optos PLC, Dunfermline, Scotland, UK). Additionally, optical coherence tomography (OCT) angiography was performed using a Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany) and a Cirrus HD-OCT with AngioPlex (Carl Zeiss Meditec, Dublin, CA).

[0190] After discovering that PSEN1 E280A mutation carriers have two copies of the APOE3ch variant, additional studies were performed to identify hyperlipoproteinemia type III, a condition found in 5–10% of individuals homozygous for the relatively AD-protective APOE2 allele and in most, but not all, APOE3ch carriers. 5 A fasting serum lipid panel was performed to explore the possibility of .

[0191] Finally, analysis of data from clinically and neuropathologically verified AD cases and controls from the AD Genetics Consortium was used to determine whether homozygosity for the APOE2 allele is associated with an exceptionally low risk of late-onset AD dementia.

[0192] Whole-exome sequencing: For variant discovery, whole-exome capture and sequencing were performed using Illumina chemistry; rare variants at frequencies less than 1% in genes previously associated with AD were considered candidate risk modifiers in this study. 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. Library construction was performed using a previously described protocol with the following modifications: 6Genomic DNA input was reduced from 3 μg to 50 ng in 10 μL of solution and enzymatically sheared. Dual-indexed Illumina paired-end adapters were replaced with palindromic forked adapters with an 8-base index sequence embedded within the adapter, added to each end for adapter ligation. In-solution hybrid selection was performed using the Illumina Rapid Capture Exome Enrichment Kit with a 38 Mb target territory (29 Mb bait). The targeted region encompassed 98.3% of the intervals in the Refseq exome database. Dual-indexed libraries were pooled into groups of up to 96 samples prior to hybridization. The enriched library pools were quantified by PicoGreen after elution from streptavidin beads and then normalized. For cluster amplification and sequencing, libraries prepared with forked indexing adapters were quantified using quantitative PCR (KAPA biosystems), normalized to 2 nM using a Hamilton Starlet liquid handling system, and pooled in equal volumes using a Hamilton Starlet liquid handling system. The pools were then denatured in 0.1 N NaOH. The denatured samples were diluted into strip tubes using a Hamilton Starlet liquid handling system. Cluster amplification of the templates was performed using an Illumina cBot according to the manufacturer's protocol (Illumina). Flow cells were sequenced using a HiSeq 4000 Sequencing-by-Synthesis kit and subsequently analyzed using RTA 2.7.3.

[0193] Exome sequencing data were processed and analyzed using the Center for Personalized Medicine (CPM) Clinical Genomics Laboratory and Translational Genomics Research Institute's clinical exome sequencing bioinformatics pipeline. Briefly, sequence alignment and variant calling were performed using Edico Genome's Dragen genome pipeline with default parameters. Coverage determination and initial variant filtering were performed based on ExAC (Exome Aggregation Consortium, exac.broadinstitute.org / ) allele frequencies using the open-source software samtools and bcftools (samtools.github.io / ) with a set of custom scripts. 7 Sequence alignment was performed against the human hs37d5 decoy genome. 8 To identify potential modifier variants, a core gene list of 15 genes was created based on two HPO terms: HP:0002511, Alzheimer's disease; HP:0003584, late-onset. These genes were AAGAB, ABCC8, AKT2, APOE, APP, BEAN1, GATA1, GCK, HMGA1, HNF1B, HNF4A, LDB3, PAX4, PSEN1, and PSEN2. 9 Rare DNA variants (minor allele frequency <1%) within exonic regions and splice site junctions (5 bp into introns) of these genes were further annotated and analyzed using a commercially available tool (Cartagenia v5.0). Sequence variations were reported according to the Human Genome Variation Society (HGVS v2.0) nomenclature guidelines.

[0194] Whole Genome Sequencing: Whole genome sequencing (WGS) and Genomizer analysis (v 10.1.0) were used to perform a comprehensive and unbiased ranking of other potential genetic risk modifiers, including those associated with a lower risk of Alzheimer's disease, to help rule out other potential protective genetic factors. 10 The same dragen pipeline described above was used to process the WGS data. Data were aligned to the GRCh37 decoy genome (hs37d5). Variants called at a depth of <10× were filtered out and then annotated using Ensembl's Variant Effect Predictor (VEP) tool. The version of VEP used was v93. The filtered and annotated set of variants was then compiled for Genomizer analysis.

[0195] APOE structure display: PDB 2L7B, previously published structure, from the RCSB PDB (rcsb.org) using NGL Viewer 11 Image obtained and modified from 12 .

[0196] APOE genotyping by Sanger sequencing: The reaction mix for amplification was performed in a 50 μL volume containing the following components: 13The PCR mixture contained: 1x PfuUltra II Hostart Master Mix, 1 µL of each primer (10 µmol / L) (forward primer F: 5'-AGCCCTTCTCCCCGCCTCCCACTGT-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). PCR cycling was performed with an initial denaturation at 95°C for 2 minutes, followed by 35 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 40 seconds, and a final extension at 72°C for 5 minutes. PCR products were purified using a Qiagen QIAquick gel extraction kit and sequenced by the MGH CCIB DNA core using an Applied Biosystems 3730xl sequencer.

[0197] 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 and as previously described. 14 Fluorodeoxyglucose PET images were acquired at the University of Antioquia, Colombia, and analyzed at the Massachusetts General Hospital and Banner Alzheimer's Institute, as previously described. 15 Imaging data from cases were compared with imaging data from younger PSEN1 E280A mutation carriers who developed MCI at the expected age for their families at clinical onset, and with imaging data from mutation carriers who remained cognitively intact.

[0198] MRI was performed on a 3T Tim Trio (Siemens) and as previously described. 16-19, included magnetization-prepared rapid gradient echo (MPRAGE) processed with Freesurfer (FS) to identify gray matter and pial surfaces and allow region of interest (ROI) segmentation as follows: cerebellar gray matter, hippocampus, and the following Braak stage-related cortices: entorhinal, parahippocampal, inferior temporal, fusiform, and posterior cingulate.

[0199] At MGH, 18F-flortaucipir (FTP) was prepared 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 was validated for human use (Shoup et al., 2013). 11C-Pittsburgh Compound B was prepared and analyzed as previously described. 16 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 axial resolution and 2.4 mm slice spacing). 11C PiB PET was acquired with an 8.5–15 mCi bolus injection followed immediately by a 60-minute dynamic acquisition (12 × 15 s, 57 × 60 s) of 69 frames. 18F FTP was acquired with a 9.0–11.0 mCi bolus injection in 4 × 5-minute frames for 80–100 minutes. PET images were reconstructed and attenuation-corrected, and each frame was evaluated to verify adequate counting statistics and the absence of head motion.

[0200] 18F-FTP specific binding was measured in the FS ROI, using the FS cerebellar gray matter ROI as a reference, in accordance with previous reports. 19 The PET data were expressed as the standardized uptake value ratio (SUVR) for the cerebellum. For voxel-based analysis, each subject's MPRAGE was registered to the MR matrix in SPM8 (SPM), and the spatially transformed SUVR PET data were smoothed with an 8 mm Gaussian kernel to account for individual anatomical differences. 20 To account for possible 18F FTP off-target binding in the choroid plexus, which may confound the hippocampal signal, we performed a 18F FTP assay, as previously reported.21 , Linear regression was used to regress the choroid plexus.

[0201] 11C PiB PET data were expressed as distribution volume ratio (DVR) using cerebellar gray matter as the reference tissue; local 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 retention was performed as previously described. 23,24 , was assessed using a large cortical ROI ensemble including the frontal, lateral temporal, and retrosplenial cortex (FLR).

[0202] 18F-fludeoxyglucose PET was performed on a 64-section PET / computed tomography imaging system (Biograph mCT; Siemens) using an intravenous administration of 5 mCi (185 million Bq) of 18F-fludeoxyglucose after a 30-minute radiotracer uptake period while resting in a darkened room, followed by a 30-minute dynamic emission scan (six frames at 5-minute intervals). Images were reconstructed with computed tomography attenuation correction. The precuneus to whole-brain cerebral metabolic rate for glucose (CMRgl) ratio was characterized using an automated brain mapping algorithm (SPM8; fil.ion.ucl.ac.uk / spm / software / spm8) from bilateral regions of interest (ROIs) on each participant's 18F-fludeoxyglucose PET images. The ratio of hippocampal volume to total intracranial volume was characterized using FreeSurfer (surfer.nmr.mgh.harvard.edu) from bilateral ROIs on T1-weighted MR images of each participant. All images were visually inspected to verify ROI characterization.

[0203] Amyloid aggregation studies: Human ApoE3 protein fragments (including the carboxyl-terminal domain and histidine tag) with and without the Christchurch variant were bacterially synthesized and purified (Innovagen), and the Aβ of these proteins was analyzed in vitro using Thioflavin T (SensoLyte® ThT β-Amyloid (1–42) Aggregation Kit, Cat. No. AS-72214). 42 For this assay, 55 μM Aβ was incubated in a clear, non-binding 96-well plate to assess the differential effects on aggregation. 42 was added to a solution of either 10 μM wild-type ApoE3 or mutant 136Arg→Ser ApoE3 protein. Samples were then mixed with 2 mM Thioflavin T dye, and fluorescence was read at intermittent time intervals over 2 hours at Ex / Em = 440 / 484. Plates were kept at 37°C and shaken for 15 seconds between readings.

[0204] Full-length ApoE3 proteins with and without the Christchurch mutation were also expressed by transient transfection in Flp-In™ T-REx™ 293 (Thermo Fisher Scientific) mammalian cells, and the effects of these proteins on Aβ42 aggregation were confirmed using a previously published split luciferase complementation assay. 26 The latter analysis was performed using the APOE3 vector (Addgene, plasmid no. 87086) as either WT or containing the Christchurch variant introduced by site-directed mutagenesis. 27 ) was used. Reagents for the luciferase assay were purchased from Promega.

[0205] result Approximately 1,200 Colombian presenilin 1 (PSEN1) E280A mutation carriers and approximately 4,600 non-carriers were identified, who together constitute the largest known kindred with autosomal dominant Alzheimer's disease (ADAD) in the world. 28,29Mutation carriers typically develop mild cognitive impairment (MCI) and dementia at a median age of 44 years (95% CI, 43-45) and 49 years (95% CI, 49-50), respectively. 30,31 Studying autosomal dominant AD (ADAD) mutation carriers who remain cognitively intact until older ages may help discover risk-reducing genetic variants. 32 Characterizing AD biomarkers in these individuals may help inform 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 at onset.

[0206] This study was conducted in accordance with Institutional Review Board guidelines and with the written informed consent of the participant (her exact age and other identifying information have been redacted to protect her anonymity and confidentiality). 42 (Aβ 42 ) confirmed to have the overproducing PSEN1 E280A mutation and confirmed by family informant reports that her cognitive abilities remained intact into her 70s, and thereafter met the criteria for MCI for 24 months of annual evaluations. 33 She met the criteria for cognitive impairment. She remained fully independent for basic and instrumental activities of daily living, with no obvious signs of deterioration in her ability to perform these activities. At the time of intake evaluation, her memory impairment was limited to recent events, and her neurological examination was normal. Neuropsychological test scores, adjusted for her age and education, showed preferential impairments in recall memory, relative preservation of recognition memory, initial learning ability, naming ability, visuospatial ability, and letter fluency skills, as well as relatively stable cognitive abilities over the 24-month evaluation period (Table 1).

[0207] [Table 6]

[0208] Whole-exome sequencing confirmed her PSEN1 E280A mutation and discovered 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 significant rare and common variants. 34 Using this approach, the PSEN1 E280A mutation was identified as the participant's primary risk factor, and APOE3ch homozygosity was identified as her primary resistance factor.

[0209] 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 has been associated with a lower risk of AD and an older age at dementia onset. 35 Each additional copy of APOE4 is associated with higher risk and a younger age at onset. 36,37 APOEch variant (arginine to serine substitution at amino acid 136 (136Arg→Ser)) corresponding to codon 154 38 could be present in any of the common APOE alleles, including this participant's two APOE3 alleles. 39 Figure 1 shows a model of the structure of the wild-type ApoE3 protein. The N-terminal (residues 1-191) and C-terminal (residues 201-299) domains are highlighted. The amino acid positions for the APOE4 (C112R), APOE3ch (R136S), and APOE2 (R158C) variants are indicated.

[0210] The APOE3ch variant was not present in the AlzAD or ExAC databases, which report approximately 180,000 exomes. R136S was previously identified in APOE2 individuals with HLP III, but its potential effect on AD progression has not been previously reported. 40 We sequenced DNA samples from two other PSEN1 E280A carriers with delayed age of onset (age of onset 62 and 70 years) by whole-genome sequencing. Neither of these individuals had the APOE3 R136S variant nor APOE2; the latter was previously shown to delay disease onset in this family. 41 .

[0211] To confirm the possible association between the APOE3 R136S mutation and delayed age at onset of AD, we performed whole-genome sequencing, neurological, and neuropsychological testing in four offspring of the proband patient, all of whom were older than 50 years and predicted to have APOE3 R136S. Figure 2 shows representative Sanger sequencing results for APOE from control, proband, and offspring samples. Top row: C112 homozygous sequences are shown in all cases. Middle row: R136S homozygous sequences are shown in the left panel from control individuals. The center panel shows the homozygous change resulting in the R136S mutation. The right panel shows an example of an R136S heterozygous mutation in a proband's offspring. Bottom row: R158 homozygous sequences are shown in all cases. Figure 3 shows the proband's pedigree, with circles representing females, squares representing males, diamonds representing individuals whose gender has been concealed for privacy reasons, arrowheads depicting proband individuals with MCI, and shading indicates individuals with a history of dementia. Deceased individuals are marked with crossed bars. The individuals' APOE and PSEN1 genotypes are appropriately indicated to protect anonymity. Although other unknown genetic or epigenetic factors may have contributed to the later 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 mitigating the effects of amyloid-β accumulation in the brain and subsequently delaying the appearance of tau pathology, neurodegeneration (i.e., brain atrophy), and symptom onset.

[0212] Carriers of APOEch and other rare mutations within the low-density lipoprotein receptor (LDLR) binding domain of APOE typically have hyperlipoproteinemia type III (HLP-III), similar to that observed in 5-10% of APOE2 homozygotes. 43,44 The participants in this report had HLP-III, including elevated APOEch and triglyceride and total cholesterol levels (see Table 2).

[0213] [Table 7]

[0214] Detailed laboratory workup revealed abnormal lipid profiles in our proband individual with APOE3 R136S and three of his four offspring (Table 3). These four subjects had high levels of total cholesterol and triglycerides. Very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were higher in two of the offspring and were unmeasurable in the proband individual and one of the offspring, who had triglyceride levels higher than 400 mg / dL (the threshold for accuracy of indirect methods of lipid profiling) (Table 3). Further analysis using direct enzymatic tests showed higher-than-normal LDL in these two individuals. One of the offspring had a lipid profile within the normal range despite having APOE3 R136S and APOE4. Incomplete penetrance of HLP III has previously been reported for APOE2 and R136S mutation carriers. 45 We excluded secondary causes of lipid disorders in these subjects, such as diabetes, obesity, alcoholism, renal impairment, or thyroid disease. None of the mutation carriers had xanthomas or cardiovascular disease, which are diagnostic for HLP III. The combination of abnormal lipid profiles and the APOE3 R136S mutation in these subjects is consistent with a diagnosis of familial HLP III.

[0215] [Table 8-1]

[0216] [Table 8-2]

[0217] A detailed ophthalmologic evaluation of a PSEN1 E280A mutation carrier with two APOE3ch alleles was performed. This carrier had 20 / 70 visual acuity in the right eye and 20 / 40 visual acuity in the left eye. Anterior segment examination was notable for a posterior chamber lens in the right eye with dense posterior capsule opacification. The anterior segment of the left eye was notable for a nuclear sclerosis cataract (Figures 4A and 4B). Posterior segment examination of both eyes was normal, with a clear vitreous cavity, normal-appearing optic nerve, macula, and peripheral retina. Further examination by optical coherence tomography (OCT) of the right eye was normal, except for a small area of ​​hyperreflectivity overlying the fovea (Figure 4D). Figure 4C shows an infrared image of the right eye depicting a cross-section of the retina (lines) seen in Figure 4D. Furthermore, OCT imaging of the left eye revealed degenerative lamellar lacunae (indicated by "*" in Figure 4F) along with small defects in the external limiting membrane and ellipsoid layer (arrows in Figure 4F).

[0218] Although several mechanisms have been proposed to explain the effect of APOE variants on AD risk, most studies have focused on Aβ 42 Focusing on their differential effects (APOE2<3<4) on aggregation and plaque burden 47 In this study, participants' resistance to clinical onset of AD was associated with a) longer than 70 years of Aβ 42 Neuroimaging measurements were used to determine whether a) Aβ overproduction is associated with relatively low Aβ plaque burden, despite its overproduction, or b) relatively high Aβ plaque burden, but with limited downstream measures of paired helical filament (PHF) tau (neurofibrillary tangle burden) and neurodegeneration.

[0219] The participants' neuroimaging findings are shown in Figure 5. Positron emission tomography (PET) images are overlaid on the medial and lateral surfaces of the left hemisphere. The top row shows PET measurements of amyloid plaque burden (PiB DVR). The bottom row shows PET measurements of paired helical filament (PHF) tau (i.e., neurofibrillary tangle) burden. The individual with late-onset MCI is in their 70s, while the individual with a typical age at MCI onset is 44 years old.

[0220] As shown in Figure 5, the individual with late-onset MCI had unusually high PET measurements of Aβ plaque burden, as indicated by a higher mean Pittsburgh Compound B (PiB) distribution volume ratio in the cortex to the cerebellum (DVR = 1.96) than in PSEN1 E280A carriers who developed MCI in their 40s (DVR = 1.49–1.60). Despite her high Aβ plaque burden, the size and / or spatial extent of her PHF tau burden and neurodegeneration were relatively limited; her flortaucipitin (tau) PET measurements were restricted to the medial temporal lobe and, to a lesser extent, affected the occipital lobe, and were relatively modest in other regions characteristically affected in the clinical stages of AD (Figure 5). Her fluorodeoxyglucose PET measurements of cerebral metabolic rate for glucose were preserved in brain regions known to be preferentially affected by AD, with higher whole-brain precuneus measurements in PSEN1 E280A mutation carriers who developed MCI at an earlier age and in many younger, cognitively intact mutation carriers.

[0221] Figure 6 shows measurements of mean cortical amyloid plaque burden, entorhinal cortex PHF tau burden, hippocampal volume, and precuneus glucose metabolism. These measurements were based on brain imaging results from a PSEN1 E280A mutation carrier with two APOE3ch alleles and exceptionally late onset of MCI (red dots), a PSEN1 E280A mutation carrier with MCI at a younger age than typical for their family (black dots), and a PSEN1 E280A mutation carrier who had not yet developed MCI (gray dots). Amyloid plaque burden is expressed as the mean cortical to cerebellum distribution volume ratio (DVR). Paired helical fiber (PHF) tau burden is expressed as the entorhinal cortex to cerebellum flortaucipitin (FTP) standard uptake value ratio (SUVR). Hippocampal volume, which may be reduced by hippocampal atrophy, is expressed as the hippocampal to whole brain volume ratio. Cerebral glucose metabolism, reflected as the ratio of the cerebral metabolic rate for glucose in the precuneus to the whole brain (CMRgl), is reduced in AD-affected brain regions with synaptic dysfunction and loss. As shown in Figure 6, the PSEN1 E280A mutation carrier with two APOE3ch alleles had by far the highest amyloid plaque burden, but she did not have comparable severe PHF tau burden or hippocampal atrophy, and she had no evidence of precuneus glucose hypometabolism. Her MRI-based hippocampal volume relative to the whole brain, a measure of hippocampal atrophy that can be affected by AD and / or normal aging, was within the range of mutation carriers who developed MCI in their 40s. Without wishing to be bound by theory, these results suggest that this APOE3ch homozygote's resistance to clinical AD onset is mediated through a mechanism that limits tau pathology and neurodegeneration, even in the face of high Aβ plaque burden.

[0222] To study the functional impact of APOE3ch variants, we cultured Aβ in vitro in the presence of bacterially derived wild-type human ApoE3 protein, mutant ApoE3ch proteins, or in the absence of any ApoE protein. 42 The aggregation of Aβ was compared. 42 The rate of fibril formation was detected by thioflavin T fluorescence. 42Aggregation was highest in the presence of wild-type human ApoE3 protein (C-terminal domain) and lower in the presence of human ApoE3ch (as observed in the presence of ApoE). 48 (similar to ) and was lowest in the absence of any ApoE (Figure 7).

[0223] This finding was confirmed using a sensitive split luciferase complementation assay, in which the luciferase signal was observed once amyloid was present, some of the most toxic amyloid species. 49 , and if oligomers are formed, they are reassembled. 48 As luciferase luminescence due to oligomerization was significantly reduced in ApoE3ch compared to wild-type ApoE3 (Figure 8), full-length ApoE3ch expression in mammalian cells resulted in significantly lower Aβ expression compared to wild-type ApoE3. 42 These results provide confirmation of the genetic analysis and suggest that the protective effect of ApoEch proteins is due, at least in part, to Aβ 42 It is still possible that the study participant may have had even higher Aβ plaque deposition if she had survived into her 70s without the APOEch / 3ch genotype, and that the ApoE3ch protein altered the morphology of Aβ aggregates in a manner that limited downstream neuroinflammation, tau pathology, neurodegeneration, and cognitive decline.

[0224] A small proportion of Colombian family members were found to carry one copy of the APOE3ch mutation, including four PSEN1 E280A mutation carriers who progressed to MCI at a median age of 45 years. 50 For this reason, it was speculated that APOE3ch homozygosity dramatically reduces the risk of autosomal dominant AD and delays its clinical onset. Because the sample size was small, it is still possible that APOEch heterozygous individuals may have partial protection against autosomal dominant AD-related cognitive decline and substantial protection against sporadic late-onset AD and / or neurodegeneration.

[0225] These results suggest that APOE variants vary in the extent of their pathogenic function (APOEch and APOE2<3<4), with APOE3ch / 3ch and APOE2 / 2 associated with the greatest loss of function. Interventions that safely and sufficiently edit APOE, reduce its expression, or inhibit its pathogenic function could have a major impact on the treatment and prevention of AD. Interestingly, suppression of APOE expression in the brain using antisense oligonucleotides in Aβ-overproducing mice resulted in altered Aβ plaque morphology and fewer degenerated neurites. 51 This approach may be feasible and tolerable in middle-aged men whose lack of APOE expression is homozygous for a frameshift variant. 52 The availability of statins to treat HPL-III supports the potential tolerability of ApoE-reducing treatments. See, e.g., Reiman et al. Nat Commun 1191):667, 2020.

[0226] Without wishing to be bound by theory, these results further suggest that homozygosity for APOE3ch and APOE2 is associated with increased resistance to the clinical onset of AD; that these genotypes exert their beneficial effects by directly or indirectly limiting downstream tau pathology and neurodegeneration; and that these effects are not solely based on the magnitude of Aβ plaque burden despite a relative reduction in ApoE-mediated Aβ aggregation. These findings have implications for the role of APOE in the understanding, treatment, and prevention of AD and may spur interest in developing APOE-modifying genetic and pharmacological therapies for this disorder.

[0227] [Example 2] Heparin-binding properties of APOE3ch mutant proteins material and method Heparin Column Protocol: The heparin-binding affinities of ApoE2, ApoE3, ApoE3ch, and ApoE4 protein isoforms were compared using a 1 ml heparin column (BioVision-6554-1). The column was acclimated to room temperature for 1 h before use. The column was washed with 5 ml of 20 mM TRIS-HCl (pH 7.5). A 1 ml sample containing 50 μg / ml APOE recombinant protein in 20 mM TRIS-HCl (pH 7.5) was then recirculated through the column five times. The column was then washed five times with 20 mM TRIS-HCl (pH 7.5). An increasing NaCl gradient (0.025 to 1 M) in 20 mM TRIS-HCl was passed through the column, and 1 ml fractions were collected and then prepared for Western blotting.

[0228] Western blotting: Western blotting confirmed the elution of ApoE isoforms within the fractions collected from the heparin-binding 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. Samples were separated on a 4-20% Mini-PROTEAN® TGX™ precast protein gel (Bio-Rad), transferred to a nitrocellulose membrane (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), and probed with mouse anti-His tag (Novus biologicals) and IRDye 800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using an Odyssey infrared imaging system and visualized on Image Studio version 2.1 (LI-COR Biosciences). Individual gels were combined to produce Figure 10.

[0229] Heparin Plate ELISA Protocol: ELISA was performed using heparin microplates (Bioworld; 50-197-531). These were blocked for 1 hour with sample preparation reagent (DY008). Heparin plates were incubated with 0.1 μg / well of each recombinant ApoE protein isoform (ApoE2, ApoE3, ApoE3ch, and ApoE4) for 2 hours. Afterwards, the plates were washed five times in PBS containing a gradient of NaCl (0-0.5 M), followed by three washes in wash buffer (DY008). Anti-His tag antibody (Novus biologicals; NBP2-61482) was incubated overnight at 1:10,000. The plate was then washed five times to ensure removal of unbound primary antibody, incubated with donkey anti-rabbit-HRP (1:10,000) for 45 minutes, and washed five times to ensure removal of the secondary antibody. Sulfuric acid from the ELISA reagent kit (DY008) was warmed to 37°C before the addition of 100 μl of tetramethylbenzidine (Millipore) to initiate the detection phase of the reaction. After a 5-minute incubation, sulfuric acid was added to terminate the reaction. The plate was then read using a 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 plotted using Prism 6 (GraphPad Software) based on serially diluted recombinant Notch3 protein.

[0230] result Heparin sulfate proteoglycan (HSPG) moieties are a type of glycosaminoglycan present in hundreds of proteins located in the plasma membrane and extracellular matrix. Protein-protein interactions mediated through HSPGs play an important role in numerous processes related to Alzheimer's disease pathology, including amyloid and tau pathology and neurodegeneration. We investigated the ability of various ApoE isoforms, including ApoEch, to bind heparin (a glycosaminoglycan commonly used to model HSPG-protein interactions). Briefly, fractions containing ApoE isoforms ApoE2 and ApoE4 eluted from a heparin column under an increasing NaCl gradient (0-0.65 M) were analyzed using ELISA. As shown in Figure 9, the ApoE variant associated with a higher risk of Alzheimer's disease, ApoE4, has a higher affinity for heparin compared to the variant ApoE2, which is known to be protective. Next, fractions containing His-tagged ApoE2, ApoE3, ApoE4, and ApoE3ch eluted from the heparin column under an increasing NaCl gradient (0-0.65 M) were analyzed by Western blot. As shown in Figure 10, ApoE3ch exhibited impaired heparin binding, much lower than that of ApoE2. The affinity of ApoE isoforms for heparin was also analyzed using a heparin plate ELISA protocol, as described in Materials and Methods. As shown in Figures 11A-11B, ApoE3ch exhibited significantly lower levels of heparin binding, as it was released from the heparin column at much lower NaCl concentrations compared to those required for ApoE4 release.

[0231] [Example 3] Generation of antibodies against wild-type ApoE and ApoEch mutant proteins material and method Antibody competition assay: Antibodies were incubated with ApoE3 recombinant protein (50 μg / ml in 20 mM Tris-HCl) at a 1:10 ratio and incubated at room temperature for 3 hours. A negative control containing medium alone and a positive control containing recombinant protein ApoE3 alone 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 Western blotting.

[0232] BCA Assay: Fractions collected from the heparin column were first screened using a bicinchoninic acid assay (BCA assay) (Pierce BCA Protein Assay Kit). The assay was performed using 200 μl of Reagent A and B mix 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 Synery 2 microplate reader (BioTek Instrument, Inc.) and Gen5 version 1.11 software.

[0233] Western blotting: Western blotting confirmed the elution of ApoE3 recombinant protein in fractions collected from the heparin-binding column. Fractions were diluted to 10 μl with RIPA buffer (Cell Signaling Technology), 10x (1M DTT), and 4x Laemmli buffer for a final volume of 40 μl. Samples were separated on a 4-20% Mini-PROTEAN® TGX™ precast protein gel (Bio-Rad), transferred to a nitrocellulose membrane (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), and probed with mouse anti-His tag (Novus biologicals) and IRDye 800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using an Odyssey infrared imaging system and visualized on Image Studio version 2.1 (LI-COR Biosciences).

[0234] ELISA: Antibodies designed against the heparin-binding domain of ApoE were tested for their affinity to ApoE3 and ApoEch mutant recombinant proteins using ELISA. Ni-NTA HisSorb plates (Qiagen) were washed three times with wash buffer 1 (DY008). ApoE recombinant proteins were suspended in buffer (DY008) to a final concentration of 0.5 μg / ml. The plates were incubated with 200 μl of ApoE recombinant proteins for 2 hours and washed five times with 1× wash buffer (DY008). The plates were then incubated overnight at 4°C with antibodies at serial dilutions from 1:1,000 to 1:32,000. The plate was then washed five times with 1x wash buffer (DY008) and incubated with anti-mouse HRP (Abcam; ab97046, 1:10,000) for 45 minutes, followed by five washes in 1x wash buffer to ensure complete removal of unbound secondary antibody. Sulfuric acid from the ELISA reagent kit (DY008) was warmed to 37°C. 100 μl of tetramethylbenzidine (Millipore) was added to initiate the detection phase of the reaction. After a 5-minute incubation, sulfuric acid was added to terminate the reaction. The plate was then read using a Synery 2 microplate reader (BioTek Instrument, Inc.) and Gen5 version 1.11 software.

[0235] result A monoclonal antibody (mAb) against amino acids 130-143 of ApoE was generated and its effect on the binding between full-length ApoE3 protein and heparin was tested. Briefly, full-length wild-type ApoE3 protein or its preincubation with the mAb was passed through a heparin column and recirculated 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 gradient of NaCl (0-1 M) in 20 mM Tris-HCl (pH = 7.5). The elution from the Tris-HCl wash and from various NaCl concentrations was collected (Figures 12A-12C).

[0236] Fractions collected from the column were first screened by bicinchoninic acid (BCA) assay. As shown in Figure 13A, for wild-type ApoE3 in the fractions, the protein signal was detected on the right side of the curve, indicating strong binding of ApoE3 to heparin. In contrast, when ApoE3 was preincubated with the monoclonal antibody, a strong signal was observed in the early fractions at low ionic strength (Figure 13B). To verify the results, Western blotting was used to analyze the column wash and NaCl gradient fractions collected from the heparin column. As shown in Figure 14, preincubation with a monoclonal antibody (A3Ab) against wild-type ApoE3 (this antibody was designated 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 produce Figure 14. These results suggest that antibodies may be used to alter the heparin-binding properties of ApoE and thereby prevent or treat Alzheimer's disease or related dementia or neurodegeneration.

[0237] To generate monoclonal antibodies against the heparin-binding domain of ApoE, mice were immunized with the wild-type ApoE peptide: KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54) and the ApoEch peptide: KLH-CTEELRVSLASHLRK-CONH2 (SEQ ID NO: 55). A cysteine ​​residue was added to the N-terminus to facilitate conjugation of the peptides. Cell fusions were obtained from positive clones, and cell supernatants were tested for activity against wild-type and mutant peptides and proteins. Seven antibodies generated were analyzed by ELISA, as described in the Materials and Methods section. The 19G10-2 antibody serum showed specificity for both the full-length and C-terminal ends of the APOE3ch mutant protein and some interaction with the wild-type APOE3 protein (Figure 15A). The 23B2 antibody showed reactivity to the C-terminal and full-length recombinant proteins of both wild-type ApoE3 and ApoE3ch mutants (Figure 15B). The 2H79-1 antibody showed nonspecific binding to bovine serum albumin (BSA) and no affinity for either wild-type ApoE3 or ApoE3ch mutants (Figure 15C). Both the 30E1-2 and 16H8 antibodies showed reactivity to full-length and C-terminal ApoE3ch mutant proteins, as well as the C-terminal form of wild-type ApoE3, but did not react with full-length wild-type APOE3 protein (Figures 15D and 15E). The 25F1-2 antibody serum showed high affinity for full-length and C-terminal ApoE3ch mutant proteins and appeared to have variable binding to the C-terminus of wild-type ApoE3 protein and some interaction with full-length wild-type ApoE3 protein (Figure 15F). The 29G10-2 antibody showed high affinity for both full-length and C-terminus ApoE3ch mutant proteins and also showed reactivity to C-terminal wild-type ApoE3 and BSA. Finally, the 29G10-2 antibody did not interact with the full-length wild-type ApoE3 protein (FIG. 15G).

[0238] The sequences of the variable heavy chain (VH), variable light chain (VL), and complementarity determining regions (CDRs) of 25F1-2 and 19G10-2 are described herein.

[0239] Additionally, the following parent clones specific for wild-type ApoE were generated and tested for specificity against 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 6. The values ​​indicate the absorbance level as detected by ELISA. The bolded clones showed specificity for the wild-type ApoE peptide (KLH-CTEELRVRLASHLRK-CONH2 (SEQ ID NO: 54) and wild-type ApoE protein).

[0240] [Table 9]

[0241] [Example 4] Generation of fusion proteins containing the heparin-binding domain of APOE material and method Peptide competition assay: Wild-type ApoE3 and ApoE3ch mutant peptides (50 μg / ml) were incubated with ApoE3 recombinant protein (50 μg / ml prepared in 20 mM 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 Western blotting.

[0242] Western blotting: Western blotting confirmed the elution of ApoE3 in fractions collected from the heparin-binding column. Fractions were diluted to a final volume of 40 μl in 10 μl RIPA buffer (Cell Signaling Technology), 10x (1M DTT), and 4x Laemmli buffer. Samples were separated on 4-20% Mini-PROTEAN® TGX™ precast protein gels (Bio-Rad), transferred to nitrocellulose membranes (VWR; 27376-991), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE), and probed with mouse anti-His tag (Novus biologicals) and IRDye 800CW donkey anti-rabbit (LI-COR Biosciences) antibodies. Immunoreactive bands were visualized using an Odyssey infrared imaging system and visualized in Image Studio version 2.1 (LI-COR Biosciences).

[0243] result Peptides containing amino acids 130-143 of wild-type ApoE protein and ApoEch mutant protein were generated, respectively. To examine the effect of these peptides on the binding between wild-type ApoE3 recombinant protein and heparin Sepharose, a peptide competition assay was performed as described in Materials and Methods. As shown in Figure 16, the wild-type ApoE peptide caused a one-fraction shift in wild-type ApoE3 recombinant protein binding, suggesting that this peptide can compete with wild-type full-length ApoE3 for heparin binding. These results suggest that an ApoE fragment containing amino acids 130-143 of wild-type ApoE protein alters the heparin-binding properties of ApoE and may thereby be used to prevent or treat Alzheimer's disease or related dementia or neurodegeneration.

[0244] To increase the protein stability of the peptides, C- and N-terminal fusion proteins containing the heparin-binding domain of human ApoE (wild-type and R136S mutant versions) or the site of allosteric modulation of the heparin-binding domain were generated using the backbone Fc IgG2 constructs pfuse-hfc1 and pfcn-hg2 (Invivogen). The human ApoE fragments excluded the sites for APOE2 and APOE4 variants. Administration of an ApoE fragment with an R at position 136 may compete with endogenous ApoE for interactions with binding partners, including HSPGs, providing protection from neurodegeneration. Administration of an ApoE fragment with an S at position 136 may bind molecules that do not bind wild-type ApoE, providing protection from neurodegeneration. The amino acid sequences for the fragments from wild-type and R136S mutant ApoE used to generate the fusion proteins are shown below (the R136 position is shown in bold and double underlined). Downstream of the R136 fragment WT STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57) Mutants STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58) Upstream of the R136 fragment WT RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59) Mutants RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60)

[0245] Figures 17A-17D show models of ApoE fragment interaction with heparin. Figure 17A shows a model of a wild-type ApoE fragment (downstream of the R136 fragment, helix) interacting with heparin. Figure 17B shows a model of an ApoE R136S fragment (downstream of the R136 fragment, helix) interacting with heparin. Figure 17C shows a model of a wild-type ApoE fragment (upstream of the R136 fragment, helix) interacting with heparin. Figure 17D shows a model of an ApoE R136S fragment (upstream of the R136 fragment, helix) interacting with heparin.

[0246] The nucleic acid and amino acid sequences for exemplary fusion protein constructs containing either the R136 fragment downstream or the R136 fragment upstream are shown below. 184Q pfcn-hg2 ApoE 114~144 Nucleic acid sequence (SEQ ID NO: 81)

[0247] [ka]

[0248] [ka] Amino acid sequence (SEQ ID NO: 82)

[0249] [ka] 184R pfcn-hg2 ApoE 114~144 R136S Nucleic acid sequence (SEQ ID NO: 83)

[0250] [ka]

[0251] [ka] Amino acid sequence (SEQ ID NO: 84)

[0252] [ka] 197F pfuse hfc2 ApoE 114~144 Nucleic acid sequence (SEQ ID NO: 85)

[0253] [ka]

[0254] [ka] Amino acid sequence (SEQ ID NO: 86)

[0255] [ka] 197G pfuse-hfc2 ApoE 114~144 R136S Nucleic acid sequence (SEQ ID NO: 87)

[0256] [ka]

[0257] [ka] Amino acid sequence (SEQ ID NO: 88)

[0258] [ka] 184U pfcn hg2 ApoE 129~157 Nucleic acid sequence (SEQ ID NO: 89)

[0259] [ka]

[0260] [ka] Amino acid sequence (SEQ ID NO: 90)

[0261] [ka] 184V pfcn hg2 129~157 R136S Nucleic acid sequence (SEQ ID NO: 91)

[0262] [ka]

[0263] [ka] Amino acid sequence (SEQ ID NO: 92)

[0264] [ka] 197H pfuse hfc2 ApoE 129~157 Nucleic acid sequence (SEQ ID NO: 93)

[0265] [ka]

[0266] [ka] Amino acid sequence (SEQ ID NO: 94)

[0267] [ka] 197I pfuse hfc2 ApoE 129~157 R136S Nucleic acid sequence (SEQ ID NO: 95)

[0268] [ka]

[0269] [ka] Amino acid sequence (SEQ ID NO: 96)

[0270] [ka]

[0271] [Example 5] CRISPR-Cas9-mediated editing and base editing of APOE To introduce the R136S mutation into APOE using CRISPR-Cas9, a gRNA sequence was designed (Table 7). In one example, the gRNA sequence was cloned into lentiCRISPR v2 using two oligos to form a linker containing a 20-base sequence that was cloned into the BsmB1 site downstream of the U6 promoter. To aid in repair, a template with two additional silent mutations was designed. The template has 50 bases flanking the area with the mutation. An exemplary template sequence is as follows:

[0272] [ka] However, the silent mutation to abolish the PAM motif is double underlined, the codon corresponding to the R136S mutation is shown in bold, and the silent mutation to generate a SacI site for cleaving PCR products from clones that accepted the template is shown in italics.

[0273] [Table 10]

[0274] While not intending to be bound by theory, the proposed mechanism for the APOE3ch mutation is loss of function (in binding to HSPGs). Therefore, gRNA sequences were designed to "knock out" APOE using CRISPR-Cas9 (Table 8). The gRNA is designed to target exon 3 (amino acids 1-61) of ApoE. In the example, the gRNA sequence is cloned into lentiCRISPR v2 by ordering two oligos to form a linker containing a 20-base sequence that is cloned into the BsmB1 site downstream of the U6 promoter. Repair is performed by non-homologous end joining (NHEJ), an error-prone method that often results in short insertions or deletions that result in APOE knockout.

[0275] [Table 11] The R136H mutation in APOE is predicted to have a similar effect to the R136S mutation. Therefore, the following gRNA was designed to introduce the R136H mutation into APOE using base editing technology (Table 9). The gRNA sequence GAGGCGCACCCGCAGCTCCT was cloned into pLenti sgRNA (addgene 71409) using two oligos to form a linker containing a 20-base sequence that was cloned into the BsmB1 site downstream of the U6 promoter. The Addgene base editor plasmid pCMV-BE3 (#73021) was used to generate the base edits.

[0276] [Table 12]

[0277] [Example 6] High-throughput screening of molecules that alter the binding of ApoE to heparin To screen for molecules that affect the binding of ApoE to heparin, ApoE protein is preincubated with a candidate polypeptide, small molecule, nucleic acid, lipid, or carbohydrate. The preincubated ApoE protein is introduced to a heparin-coated surface (such as a plate or column) and allowed to bind to heparin / HSPG / GAG, and unbound ApoE and the candidate molecule are washed away. Alternatively, the heparin-coated surface (such as a plate) can be preincubated with the candidate molecule before applying the ApoE protein. The level of heparin-bound ApoE is detected using antibodies, protein assays, or fluorescence to evaluate the effect of the candidate molecule on ApoE / heparin binding. Candidate molecules that reduce ApoE / heparin binding may represent novel therapeutic agents for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment in human subjects in need of such treatment. Examples of such molecules include EZ-482 (see, e.g., Mondal et al. Biochemistry 55(18):2613-21, 2016; the structure of EZ-482 is shown below).

[0278] [ka]

[0279] [Example 7] High-throughput screening of molecules that alter the binding of ApoE to anti-ApoE antibodies To screen for molecules that affect the binding of ApoE to anti-ApoE antibodies, ApoE protein is preincubated with a candidate polypeptide, small molecule, nucleic acid, lipid, or carbohydrate. The preincubated ApoE protein is introduced to a surface (such as a plate or column) coated with an antibody that binds to the HSPG-binding site of ApoE (any of the anti-ApoE antibodies described herein), allowing it to bind to the antibody, and unbound ApoE and candidate molecules are washed away. Alternatively, a heparin-coated surface (such as a plate) can be preincubated with the candidate molecule before applying the ApoE protein. The level of ApoE bound to the anti-ApoE antibody is detected using an antibody, protein assay, or fluorescence, and the effect of the candidate molecule on the binding is evaluated. Candidate molecules that increase or decrease ApoE / heparin binding may represent novel therapeutic agents for preventing or treating cognitive decline associated with dementia and / or mild cognitive impairment in human subjects in need of such treatment.

[0280] [Example 8] Antibody characterization Antibodies binding to wild-type ApoE and ApoEch variants were further evaluated using heparin affinity chromatography, Western blotting, subclone analysis, monoclonal antibody screening, screening for selectivity between huApoE3 and msApoE, and in vivo subretinal injection.

[0281] method Chromatography experiments His-tagged recombinant ApoE peptide (50 μg / mL) was incubated with each antibody at a 1:10 dilution in 20 mM Tris-HCl buffer (pH 7.5) for 3 hours at room temperature. Samples were tested for heparin binding using a heparin-Sepharose column. Briefly, the column was allowed to reach room temperature and washed five times with 20 mM Tris-HCl. Protein input was loaded onto the column, with 10 μL collected for WB experiments. The input was recirculated through the column five times. The flow-through fraction was collected, and then the column was washed five times; the collected fractions were labeled "wash." 1 mL fractions were collected at every 0.05 M step in a 0 to 1 M NaCl salt gradient. A 5 M fraction was also tested to ensure complete release of protein from the column. Changes in ApoE heparin binding were tested using WB or ELISA.

[0282] Western blotting Samples for WB analysis were prepared by diluting 10 μL of each fraction in 4× sample buffer (Laemmli's SDS sample buffer, BP-110R, Boston Bioproducts), 4 μL DTT (Sigma-Aldrich), and 16 μL 1× RIPA 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 (70 V for 15 min, 100 V for 1 h). Protein transfer was performed on a nitrocellulose membrane (Millipore) at a constant voltage of 70 V for 1 h. The membrane was blocked with Odyssey blocking buffer (Licor) for 1 h at room temperature, and then washed 3 × 10 min with TBS-0.05% Tween 20 (Thermo Fisher) before incubation with the primary antibody (anti-His, rb (rabbit), 1:5000, Novus Biological) and the secondary antibody (donkey anti-rb-800, 1:10000, Licor). Image acquisition was performed using an Odyssey scanner. Data were analyzed using Image J. Data were normalized by input and expressed as normalized intensity relative to fraction number (0 = input, 1–27 = increasing 0.05 M NaCl step gradient in 20 mM Tris HCl pH 7.5, 28 = 5 M NaCl in 20 mM Tris HCl pH 7.5).

[0283] ELISA To test the selectivity of antibodies for huApoE3WT, huApoE3ch, or msApoE, anti-his ELISA-coated plates were incubated with 0.0025 μg / μL of target protein for 2 hours at room temperature with gentle shaking. Wells were washed five times with 1x wash buffer (R&D) and then incubated overnight at 4°C on a shaker with serial dilutions of the Innovagen antibody of interest (100 μL / well). The following day, wells were washed five times with sample buffer and incubated for 45 minutes with rabbit anti-mouse HRP conjugate buffer (1:10,000; 100 μL / well; Abcam). After five additional washes, plates were incubated with chromogen A+B 1:1 to initiate the colorimetric reaction (100 μL / well). The reaction was stopped with 2N sulfuric acid (R&D stop solution, 50 μL / well) and absorbance was detected spectrophotometrically at 450 nm.

[0284] Antibodies designed against the heparin-binding domain of APOE were tested for affinity to recombinant proteins of APOE3 and APOEch variants using ELISA. Ni-NTA HisSorb plates (Qiagen) were washed three times with wash buffer 1 (DY008), and APOE recombinant protein was suspended in buffer (DY008) to a final concentration of 0.5 μg / ml. The plates were incubated with 200 μl of the antibody for 2 hours. The plates were then washed five times with 1× wash buffer (DY008). The plates were then incubated with serial dilutions of antibody from 1:1000 to 1:32000 and incubated overnight at 4°C. The plates were then washed five times with 1× wash buffer (DY008). The plates were then incubated with anti-mouse HRP (Abcam; ab97046) (1:10000) for 45 minutes. The plate was then washed five times in 1x wash buffer to ensure complete removal of unbound secondary antibody. Sulfuric acid from the ELISA reagent kit (DY008) was warmed to 37°C before the addition of 100 μl of tetramethylbenzidine (Millipore), which initiated the detection phase of the reaction. After a 5-minute incubation, sulfuric acid was added to terminate the reaction. The plate was then read using a Synery 2 microplate reader (BioTek Instrument, Inc.) and Gen5 version 1.11 software.

[0285] result Antibody 1H4 was evaluated using heparin affinity chromatography and Western blotting. ApoE3 was incubated with either a negative control (vehicle, upper blot) or 1H4 (lower blot), and each fraction was subjected to heparin affinity chromatography and Western blotting (Figure 18A). The ApoE3-positive band is indicated by an arrow and detected using the antibody anti-his tag (rb, 1:5,000), which specifically detects the his tag of recombinant human APOE. Figure 18B shows quantification of the WB blotting band detected with the antibody anti-his tag as shown in Figure 18A. Intensity was normalized to input. These results indicate that ApoE3 binding to heparin was reduced in the presence of the antibody and that antibody 1H4 competes with ApoE for heparin binding. N = 2 independent experiments.

[0286] Next, subclonal analysis of the 1H4 serum was performed. Figures 19A-19D show the ELISA results of the 1H4-2 serum tested with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), and ApoE3ch peptide (D). Results are expressed as optical density at 450 nm versus the dilution factor of the tested serum. Figure 20 compares the results shown in Figures 19A-19D.

[0287] Figure 21 shows representative ELISA profiles of serial dilutions of antibody 1H4 incubated with either human or mouse recombinant ApoE3. The results demonstrate that the antibody binds preferentially to the human protein, but not to the mouse, and that antibody 1H4 is selective for human ApoE. The results are presented as the mean optical density detected at 450 nm ± sem (n = 2).

[0288] The monoclonal 1H4 antibody was then purified from the cloned hybridoma and subjected to ELISA evaluation. Figure 22 shows the results from the ELISA experiment.

[0289] Antibody 7C11 was evaluated using heparin affinity chromatography, Western blotting, quantitative ELISA on chromatography fractions, and competitive ELISA for binding analysis.

[0290] Heparin affinity chromatography fractions of ApoE3 were incubated with either a negative control (vehicle, upper blot) or antibody 7C11 (lower blot) and subjected to Western blotting (Figure 23A). ApoE3-positive bands are indicated by arrows and detected using the antibody anti-his tag (rb, 1:5,000), which specifically detects the his tag of the recombinant peptide. Figure 23B shows quantification of the WB blotting bands detected with the antibody anti-his tag as shown in Figure 23A. Intensity was normalized to input. These results indicate that the binding of ApoE3 to heparin was reduced in the presence of the antibody and that antibody 7C11 competes with ApoE for heparin binding.

[0291] Subclonal analysis of the 7C11-1 serum was then performed. Figures 24A-24D show ELISA results from testing the 7C11-1 serum with ApoE3 WT full-length protein (A), ApoE3 WT peptide (B), ApoE3ch full-length protein (C), or ApoE3ch peptide (D). Results are expressed as optical density at 450 nm versus the dilution factor of the serum tested. Figure 25 compares the results shown in Figures 19A-19D. Monoclonal 7C11-1 antibody was then purified from the cloned hybridoma and subjected to ELISA evaluation. Figure 26 shows the results from the ELISA experiment.

[0292] Furthermore, the 19G10-2 antibody was evaluated using heparin affinity chromatography, Western blotting, quantitative ELISA on chromatography fractions, and ELISA for binding analysis. ELISA screening of the 19G10-2 antibody was performed against the heparin-binding domains of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins. As shown in Figure 27, the 19G10-2 antibody shows specificity for both the full-length and C-terminal domains (amino acids 125-299) of the APOE3ch mutant recombinant proteins, as well as some interaction with APOE3 WT.

[0293] Heparin affinity chromatography fractions of ApoE3 incubated with either the negative control (vehicle, upper blot) or antibody 19G10-2 (lower blot) were subjected to ELISA analysis (Figure 28A). ApoE3-positive bands are indicated by arrows and detected using the antibody anti-his tag (rb, 1:5,000), which specifically detects the his tag of the recombinant peptide. Figure 28B shows quantification of the WB blotting bands detected with the antibody anti-his tag as shown in Figure 28A. Intensities were normalized to input. These results indicate that, despite being designed against the ApoE3ch-HSPG domain, 19G10-2 competes with wild-type ApoE for heparin binding, resulting in decreased heparin binding of ApoE3. Without wishing to be bound by theory, it is possible that antibody 19G10-2 recognizes and / or stabilizes conformation-specific features of APOE (e.g., APOE polymers or aggregates) that are unlikely to bind heparin / HSPG / GAGs.

[0294] Figure 29 shows Western blotting of ApoE3 WT incubated with 19G10-2 serum antibody. Top blot: Membrane probed with secondary anti-mouse to detect 19G10-2 antibody. Bottom membrane was incubated with anti-His tag as previously described to detect ApoE3-positive fractions. This analysis demonstrates that the antibody-APOE complex (left side of blot; top and bottom) does not bind heparin, whereas free APOE (right side of blot; bottom) binds heparin with high affinity.

[0295] Figure 30 shows a representative ELISA demonstrating the difference in binding of both serum 19G10-2 and monoclonal antibody hybridoma supernatant 19G10-2 to ApoE3 WT or ApoE3ch. The data confirm the antibody's predominant selectivity for ApoE3ch variants. Figure 31 shows a magnified Y-axis showing several limited binding profiles of antibody 19G10-2 (serum, gray profile; monoclonal, black binding profile) in the presence of ApoE3 WT. The monoclonal 19G10-2 antibody was then purified from the cloned hybridoma and subjected to ELISA evaluation. Figure 32 shows the results from the ELISA experiment. The higher signal for recognition of full-length WT APOE compared to the WT ApoE peptide suggests conformation-specific binding features.

[0296] Furthermore, the 25F1-2 antibody was evaluated using heparin affinity chromatography, Western blotting, quantitative ELISA on chromatography fractions, and ELISA for binding analysis. Figure 33 shows the ELISA screening of the 25F1-2 antibody against the heparin-binding domain of APOE3 wild-type (WT) and APOE3ch mutant recombinant proteins. As shown in Figure 33, the 25F1-2 antibody exhibited high affinity for the APOE3 mutant full-length and C-terminal proteins, but did not appear to bind strongly to the C-terminus of the APOE3 WT protein, showing limited interaction with the APOE3 WT full-length protein. The results are shown as the optical density at 450 nm against the dilution factor of the tested serum.

[0297] Heparin affinity chromatography fractions of ApoE3 incubated with either the negative control (vehicle, upper blot) or antibody 25F1-2 (lower blot) were subjected to Western blotting (Figure 34A). ApoE3-positive bands are indicated by arrows and detected using the antibody anti-his tag (rb, 1:5,000), which specifically detects the his tag of the recombinant peptide. Figure 34B shows quantification of the WB blotting bands detected with the antibody anti-his tag as shown in Figure 34A. Intensities were 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, resulting in decreased heparin binding of ApoE3.

[0298] Figure 35 shows Western blotting of ApoE3 WT incubated with 25F1-2 monoclonal antibody. Top blot: Membrane probed with secondary anti-mouse to detect 25F1-2. The bottom membrane was incubated with anti-His tag as previously described to detect the ApoE3-positive fraction. This analysis demonstrates that the antibody-APOE complex (left blot; top and bottom) does not bind heparin, whereas free APOE binds heparin with high affinity (right blot; bottom).

[0299] Figure 36 shows a representative ELISA demonstrating the difference in binding of both 25F1-2 serum and monoclonal antibody hybridoma supernatant 25F1-2 to ApoE3 WT or ApoE3ch. These results confirm the antibody's superior selectivity for ApoE3ch variants. Figure 37 is an expansion of the Y-axis of Figure 36, showing the binding profile of antibody 25F1-2 in the presence of ApoE3 WT. Monoclonal 25F1-2 antibody was then purified from the cloned hybridoma and subjected to ELISA evaluation. Figure 38 shows the results from the ELISA experiment. The higher signal for some recognition of full-length WT APOE than for the WT ApoE peptide suggests the binding of conformation-specific features.

[0300] The mouse antibody 1343ab (renamed from 23B2) was evaluated using heparin affinity chromatography and Western blotting, as well as quantitative ELISA for the chromatography fractions. Figures 39 and 40 show ELISA screening of the 1343 antibody against the heparin-binding domain of APOE wild-type (WT) and APOE3ch mutant recombinant proteins. 1343 showed reactivity to both the C-terminus and full-length recombinant APOE proteins of APOE3 WT and APOE3ch mutant (mutant refers to Christchurch mutant).

[0301] ApoE in protein fractions eluted from a heparin column using an increasing NaCl gradient in the presence or absence of 1343 was subjected to Western blotting (Figure 41A). Individual blots were cut between 25 kDa and 50 kDa. Blank spaces separate individual blots. FT = flow-through. ELISA was performed to quantify differences in the NaCl elution patterns of different ApoEs in the presence and absence of 1343 (Figure 41B). N = 3 columns per isoform in independent experiments were analyzed side-by-side in duplicate on different days to quantify differences. Error bars indicate the standard error of the mean.

[0302] In validation, CDR sequences from murine antibodies 1H4-2, 7C11-1, 19G10-2, 25F1-2, and 1343ab were grafted onto a human IgG backbone (IgG2 or IgG4) to generate humanized counterparts.

[0303] [Example 9] In vivo validation of ApoE antibodies An intraocular model of inducible APOE-dependent tau hyperphosphorylation (versus helical fibril formation) was created. This model was used to test the ability of ApoE antibodies to inhibit tau pathology, a marker of neurodegeneration. Figure 42A shows an exemplary experimental outline.

[0304] Briefly, his-tagged recombinant human ApoE3 was transfected into B6;C3-Tg (Prnp-MAPT * P301S) PS19Vle / J mice (Jackson lab 008169) were injected intravitreally (Yoshiyama et al. 53(3):337-51, 2007). This mouse model contains the human tau P301S mutation and is a validated animal model for Alzheimer's disease and other tauopathies, such as frontotemporal dementia (see, e.g., Bugiani et al. 58(6):667-77, 1999). Mice injected with PBS served as controls.

[0305] As shown in Figure 42B, in the control retina of a 6-week-old mouse, phosphorylated tau paired helical filaments (PHFs) are absent from ganglion cells and their axon fibers (the signal inside blood vessels labeled with isolectin B4 is background signal). In contrast, administration of recombinant human ApoE3 (His-tagged) caused robust formation of PHFs, which were detected with the AT8 antibody. PHFs were robust in ganglion cell axons (arrows) and ganglion cell bodies. Human ApoE was detected around the ganglion cell bodies using an anti-His antibody.

[0306] The mouse 1H4-2 antibody and the humanized 1343Ah antibody were injected intravitreally into the eyes of mice from the above mouse model (final volume 2 μL). The animals were sacrificed 3 days after injection, and the retinas were dissected and immunolabeled. The retinas were stained with DAPI, isolectin, and AT8 (pTAU), which recognizes phosphorylated paired helical filament tau (PHF tau). The retinas were imaged using an SP8 confocal microscope. As shown in Figures 43B, 43C, 43F, and 43G, ApoE3 WT caused a significant increase in PHF tau, which was significantly reduced by 1H4-2 ( *** p<0.001). Similarly, administration of the humanized 1H4-2 IgG2 / kappa recombinant monoclonal antibody effectively reduced ApoE-dependent induction of PHF tau pathology in vivo (Figure 43H; ** p<0.01, *** p<0.001). As shown in Figures 43D and 43H, PHF tau levels were significantly reduced by humanized 1343Ah ( ** p<0.01, *** p<0.001).

[0307] The similarity in potency between the mouse monoclonal antibody and the corresponding humanized antibody confirms that the affinity and binding properties of the binding domain for ApoE or ApoEch are preserved during humanization, demonstrating that the binding properties of the CDRs can be transferred from the original mouse IgG1 to other proteins, including human IgG2.

[0308] The binding affinity between ApoE3 and monoclonal antibody 1H4 (mAb 1H4) was determined using the advanced kinetics module of the BLItz system (Blitz Pro, FB-609928, ver. 1.3.1.3). Briefly, a protein A biosensor was loaded with mAb 1H4, and both the association and dissociation constants were determined at increasing concentrations of full-length ApoE3 protein (Innovagen) from 0 nM to 571.4 nM. The following run settings were used: a 30-second initial baseline in experimental medium, 120 seconds of loading of the ligand (mAb 1H4) on the biosensor, and a 30-second new baseline before the association (120 seconds) and dissociation (120 seconds) steps. A total of six runs of 420 seconds each at 2200 rpm and room temperature were performed to determine the binding parameters of ApoE3. Global fitting and step correction of the dissociation experiments were performed using BLItz software (ver. 1.1.0.7). Figure 44 shows representative binding measurements of increasing concentrations (nM) of ApoE3 protein with 1H4 on a Protein A biosensor. KD, Ka, and Kd were measured (Table 10) and calculated using the BLItz system. The top panel represents the association step of the binding kinetics, and the bottom panel represents the dissociation step. The X-axis and Y-axis represent time in seconds and binding in nM, respectively.

[0309] [Table 13]

[0310] [Table 14]

[0311] [Example 10] In vivo validation of APOE fragment fusion proteins Next, a fusion protein containing an APOE fragment containing an 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 into the vitreous of mice. As shown in Figures 43E and 43I, the fusion protein reduced ApoE-dependent tau pathology in neurons.

[0312] [Example 11] Chimeric antibody sequence The sequences of chimeric antibodies in which the CDRs have been transferred from mouse to human IgG2 or IgG4 are shown below: Regular font = Vector Italics = signal peptide Underline=VL / VH Double underlined = constant region (human kappa / IgG4 / IgG2) Sequence of the expression vector for mAb 1H4 IgG2 / kappa: >p1H4.VL.hk

[0313] [ka]

[0314] [ka] >p1H4.VH.hIgG2

[0315] [ka]

[0316] [ka]

[0317] [ka] Sequence of the expression vector for mAb 1343Ah IgG2 / kappa: >p1343Ah.VL.hk

[0318] [ka]

[0319] [ka] >p1343Ah.VH.hIgG2

[0320] [ka]

[0321] [ka]

[0322] [ka] Sequence of the expression vector for mAb 19G10 IgG4 / kappa: >p19G10.VL.hk

[0323] [ka]

[0324] [ka] >p19G10.VH.hIgG4

[0325] [ka]

[0326] [ka]

[0327] [ka] Sequence of the expression vector for mAb 25F1 IgG4 / kappa: >p25F1.VL.hk

[0328] [ka]

[0329] [ka] >p20F1.VH.hIgG4

[0330] [ka]

[0331] [ka]

[0332] [ka] Sequence of the expression vector for mAb 7C11.1 IgG2 / kappa: >p7C11.1.VL.hk

[0333] [ka]

[0334] [ka] >p7C11.1.VH.hIgG2

[0335] [ka]

[0336] [ka]

[0337] [ka]

[0338] References:

[0339] [Table 15-1]

[0340] [Table 15-2]

[0341] [Table 15-3]

[0342] [Table 15-4]

[0343] [Table 15-5]

[0344] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to illustrate, but not to 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 following claims. Various embodiments of the present invention are described below. 1. An isolated monoclonal antibody that specifically binds to one or more HSPG binding sites of wild-type or mutant apolipoprotein E (ApoE), or one or more sites of allosteric regulation of HSPG binding. 2. The antibody according to 1 above, which binds to a polypeptide having an amino acid sequence at least 95% identical to TEELRVRLASHLRK (SEQ ID NO: 3). 3. The antibody according to 1 above, which binds to a polypeptide having an amino acid sequence at least 95% identical to TEELRVSLASHLRK (SEQ ID NO: 2). 4. An antibody according to any one of 1 to 3 above, which binds to one or more HSPG binding sites of wild-type or mutant ApoE2, ApoE3, or ApoE4. 5. 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 said reference antibody are (i) 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) 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) 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) the amino acid sequence set forth in SEQ ID NO: 43 and the amino acid sequence set forth in SEQ ID NO: 42, respectively; 5. The antibody according to any one of 1 to 4 above, comprising: 6. An antibody according to any one of 1 to 4 above, 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, and the heavy chain and light chain of the reference antibody comprise the amino acid sequence shown in SEQ ID NO: 53 and the amino acid sequence shown 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 are (i) SEQ ID NOs: 7, 8, 9, 4, 5, 6, respectively; (ii) SEQ ID NOs: 17, 18, 19, 14, 15, and 16, respectively; (iii) SEQ ID NOs: 27, 28, 29, 24, 25, and 26, respectively; (iv) SEQ ID NOs: 37, 38, 39, 34, 35, and 36, respectively; or (v) SEQ ID NOs: 47, 48, 49, 44, 45, and 46, respectively. an antibody, 8. (i) VH and 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) VH and VL comprise amino acid sequences at least 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NOs: 23 and 22, respectively; (iii) VH and 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) VH and 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; The antibody according to item 7 above. 9. The antibody described in 7 above, comprising a heavy chain and a light chain, each of which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the amino acid sequences set forth in SEQ ID NOs: 53 and 52. 10. The antibody according to any one of 1 to 9 above, which comprises a mouse IgG1, IgG2a, IgG2b, IgG2c, or IgG3 heavy chain constant region. 11. The antibody according to any one of 1 to 9 above, which comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. 12. The antibody according to any one of 1 to 11 above, which comprises a human kappa or human lambda light chain constant region. 13. The antibody according to any one of 1 to 12 above, which 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, Fab, or F(ab')2. 14. The antibody according to any one of 1 to 13 above, further comprising a half-life extending moiety. 15. The antibody according to any one of 1 to 14 above, further comprising a blood-brain barrier-permeable portion. 16. The antibody according to any one of 1 to 12 above, further comprising a detectable label. 17. A pharmaceutical composition comprising the antibody described in any one of 1 to 16 above. 18. A polynucleotide encoding the antibody according to any one of 1 to 13 above. 19. A vector comprising the polynucleotide described in 18 above. 20. A host cell comprising the polynucleotide according to 18 above or the vector according to 19 above. 21. A method for producing an anti-ApoE antibody, comprising: (a) culturing the host cell according to claim 20 under conditions that allow expression of the antibody; and (b) isolating the antibody A method comprising: 22. The method of claim 21, further comprising formulating the antibody as a sterile preparation suitable for administration to humans. 23. STEELRVRLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 57), STEELRVSLASHLRKLRKRLLRDADDLQK (SEQ ID NO: 58), RLVQYRGEVQAMLGQSTEELRVRLASHLRKL (SEQ ID NO: 59), and RLVQYRGEVQAMLGQSTEELRVSLASHLRKL (SEQ ID NO: 60) An Fc fusion protein comprising the 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: 24. An Fc fusion protein according to claim 23, comprising the Fc region of a human antibody. 25. The Fc fusion protein according to claim 24, wherein said 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 23 to 25 above. 27. A polynucleotide encoding the Fc fusion protein according to any one of 23 to 25 above. 28. A vector comprising the polynucleotide according to 27 above. 29. A host cell comprising a polynucleotide according to 28 above or a vector according to 28 above. 30. (i) an HSPG-binding domain of wild-type ApoE or mutant ApoE 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 10. A pharmaceutical composition for eliciting an immune response comprising: 31. A method for ameliorating, slowing, delaying the onset of, preventing, or reversing cognitive decline associated with dementia and / or mild cognitive impairment in a human subject in need thereof, comprising the step of administering to the human subject a therapeutically effective amount of an antibody described in any of 1 to 16 above, an Fc fusion protein described in any of 23 to 25 above, or a pharmaceutical composition described in any of 17 to 26 above. 32. A method of ameliorating, slowing, delaying the onset of, preventing, or reversing cognitive decline associated with dementia and / or mild cognitive impairment in a human subject in need thereof, said method comprising: (i) a viral vector containing a nucleotide sequence encoding a gRNA molecule containing a targeting domain complementary to a targeting domain derived from the APOE gene; (ii) a viral vector comprising a nucleotide sequence encoding a Cas9 molecule; and (iii) a viral vector containing a template nucleic acid containing an adenine instead of a cytosine at position 19 of the APOE gene: g.45412013C>A to the subject, wherein said administration results in production of one or more ApoE R136S alleles in one or more cells of the subject. 33. The method of claim 32, wherein the targeting domain of the gRNA molecule comprises a sequence that is the same as or differs from a sequence from Table 7 by no more than 3 nucleotides. 34. The method according to claim 31 or 32, wherein the human subject has been diagnosed with or is at risk of developing Alzheimer's disease. 35. The method of claim 34, wherein the human subject has one or more copies of the APOE4 allele. 36. The method of claim 34, wherein the human subject has 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 has all or part of a third copy of chromosome 21. 38. The method according to claim 34, wherein the human subject has been diagnosed with Alzheimer's disease by brain imaging. 39. The method according to claim 34, wherein the human subject is over 50 years of age. 40. The method according to claim 31 or 32, wherein the human subject has been diagnosed with or is at risk of developing a disorder selected from the group consisting of vascular cognitive impairment, vascular dementia, autosomal overt cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), autosomal recessive cerebral 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 brain injury, tauopathy, amyloidopathy, synucleinopathy, Creutzfeldt-Jakob disease, retinal degeneration, glaucoma, retinal damage, and aging. 41. A method for identifying a human subject who is less likely to develop an early-onset neurodegenerative disease, comprising the steps of: obtaining or obtaining a biological sample from said subject; detecting the presence of at least one mutant allele of APOE3 or the presence of a mutant ApoE3 gene product in said biological sample; and Identifying the subject as being less susceptible to developing an early-onset neurodegenerative disease based on the presence of a mutant ApoE3 allele or gene product in said biological sample. 42. The method according to claim 41, wherein the biological sample is blood, cerebrospinal fluid, saliva, urine, tears, vitreous fluid, aqueous humor, or a tissue specimen. 43. The method according to 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 according to claim 43, wherein the retinal degeneration is age-related macular degeneration. 45. The method of claim 41, wherein the detecting step comprises determining the sequence of an APOE3 allele in the subject. 46. ​​The method of claim 41, wherein the detecting step comprises determining the presence or absence of an APOE3 sequence encoding an ApoE3 protein having a mutation at R136, as compared to a wild-type ApoE3 protein. 47. The method according to claim 46, wherein the mutation at R136 is R136S, R136H, or R136C. 48. A method according to any one of claims 41 to 47, further comprising the steps of 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 does not have a mutant APOE3 allele. 49. A method according to any one of claims 41 to 47, further comprising the steps of 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.

Claims

1. 1. An isolated monoclonal antibody, comprising: (a) specifically binds to wild-type apolipoprotein E (ApoE) comprising the amino acid sequence TEELRVRLASHLRK (SEQ ID NO: 3); (b) does not specifically bind to a mutant ApoE protein E containing the amino acid sequence TEELRVSLASHLRK (SEQ ID NO: 2); and (c) binds to an epitope comprising or consisting of the amino acid sequence TEELRVRLASHLRK (SEQ ID NO: 3); the antibody comprises a heavy chain variable region (VH) comprising VHCDR1, VHCDR2, and VHCDR3, and a light chain variable region (VL) comprising VLCDR1, VLCDR2, and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 are (i) SEQ ID NOs: 7, 8, 9, 4, 5, and 6, respectively; or (ii) SEQ ID NOs: 17, 18, 19, 14, 15, and 16, respectively. an antibody,

2. The antibody of claim 1, which, when bound to ApoE, prevents or inhibits the binding of heparin or HSPG to ApoE.

3. an antibody that competes with and / or binds to the same epitope as a reference anti-ApoE antibody comprising a heavy chain variable region (VH) comprising VHCDR1, VHCDR2, and VHCDR3, and a light chain variable region (VL) comprising VLCDR1, VLCDR2, and VLCDR3, wherein VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 of said reference antibody are (i) SEQ ID NOs: 7, 8, 9, 4, 5, 6, or (ii) SEQ ID NOs: 17, 18, 19, 14, 15, and 16, respectively. The antibody of claim 1 or 2, comprising:

4. 4. The antibody of any one of claims 1 to 3, wherein the antibody competes with and / or binds to the same epitope as a reference anti-ApoE antibody according to an assay, wherein the heavy chain variable region (VH) and light chain variable region (VL) of the reference antibody comprise the amino acid sequences set forth in SEQ ID NOs: 22 and 23, respectively, and wherein the assay comprises incubating the reference antibody with 50 μg / ml ApoE3 recombinant protein in 20 mM Tris-HCL at a ratio of 1:10 for 3 hours at room temperature, followed by incubation with the antibody.

5. The VH and VL of the reference antibody are (i) the amino acid sequence set forth in SEQ ID NO: 13 and the amino acid sequence set forth in SEQ ID NO: 12, respectively; or (ii) the amino acid sequence set forth in SEQ ID NO: 22 and the amino acid sequence set forth in SEQ ID NO: 23, respectively; The antibody according to any one of claims 1 to 3, comprising:

6. an antibody that competes with and / or binds to the same epitope as a reference anti-ApoE antibody according to an assay, wherein the heavy chain variable region (VH) and light chain variable region (VL) of said reference antibody comprise the amino acid sequences set forth in SEQ ID NOs: 13 and 12, respectively, and said assay comprises incubating said reference antibody with 50 μg / ml ApoE3 recombinant protein in 20 mM Tris-HCL at a ratio of 1:10 for 3 hours at room temperature, followed by incubation with the antibody; The antibody according to any one of claims 1 to 3.

7. The antibody of any one of claims 1 to 6, wherein (i) the VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13, with the proviso that VHCDR1, VHCDR2, and VHCDR3 comprise SEQ ID NOs: 7, 8, and 9, respectively, and (ii) the VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 12, with the proviso that VLCDR1, VLCDR2, and VLCDR3 comprise SEQ ID NOs: 4, 5, and 6, respectively.

8. The antibody of any one of claims 1 to 6, wherein (i) the VH comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22, with the proviso that VHCDR1, VHCDR2, and VHCDR3 comprise SEQ ID NOs: 17, 18, and 19, respectively, and (ii) the VL comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 23, with the proviso that VLCDR1, VLCDR2, and VLCDR3 comprise SEQ ID NOs: 14, 15, and 16, respectively.

9. The antibody of any one of claims 1 to 8, comprising a murine IgG1, IgG2a, IgG2b, IgG2c, or IgG3 heavy chain constant region.

10. The antibody of any one of claims 1 to 8, comprising a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

11. The antibody of any one of claims 1 to 10, comprising a human kappa or human lambda light chain constant region.

12. The antibody of any one of claims 1 to 11, which is a whole antibody, a humanized antibody, a chimeric antibody, an Fv, a scFv, a sc(Fv)2, a diabody, a Fab, or an F(ab')2.

13. The antibody of any one of claims 1 to 12, further comprising a half-life extending moiety.

14. The antibody of any one of claims 1 to 13, further comprising a blood-brain barrier-permeable moiety.

15. The antibody of any one of claims 1 to 11, further comprising a detectable label.

16. A pharmaceutical composition comprising the antibody of any one of claims 1 to 15.

17. A polynucleotide encoding the antibody of any one of claims 1 to 12.

18. A vector comprising the polynucleotide of claim 17.

19. 19. A host cell comprising the polynucleotide of claim 17 or the vector of claim 18.

20. 1. A method for producing an anti-ApoE antibody, comprising: (a) culturing the host cell of claim 19 under conditions that allow expression of the antibody; and (b) isolating the antibody A method comprising:

21. 21. The method of claim 20, further comprising formulating the antibody as a sterile formulation suitable for administration to a human.

22. 17. The pharmaceutical composition of claim 16, for use in a method of ameliorating, slowing, delaying the onset of, preventing, or reversing cognitive decline associated with dementia and / or mild cognitive impairment in a human subject in need thereof.

23. 17. The pharmaceutical composition of claim 16 for use in a method of treating, delaying the onset of, or preventing Alzheimer's disease in a subject.

24. 24. The pharmaceutical composition of claim 22 or 23, wherein the APOE3 allele in the subject does not encode an ApoE3 protein having a mutation at R136 compared to the wild-type ApoE3 protein.

25. 24. The pharmaceutical composition of claim 22 or 23, wherein the subject is at risk or has been diagnosed as being at risk for developing Alzheimer's disease.

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