Compositions and methods of using reelin in Alzheimer's disease

Abstract

Described herein are methods and compositions for treating Alzheimer's disease (AD), as well as compositions comprising reelin-derived peptides and methods of using the same.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of U.S. Provisional Patent Application Nos. 63 / 379,393, filed October 13, 2022, 63 / 419,574, filed October 26, 2022, and 63 / 502,038, filed May 12, 2023, the entire contents of which are incorporated herein by reference.

[0002] Federally Sponsored Research and Development This invention was made with government support under grant numbers OD019833, AG054671, NS100121 and NS110048 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field Described herein are methods and compositions for treating Alzheimer's disease (AD), as well as compositions comprising reelin-derived peptides or nucleic acids encoding same and methods of use thereof. [Background technology]

[0004] Alzheimer's disease (AD) is a progressive neurodegenerative disorder that currently affects approximately 6.2 million people in the U.S. Currently, there are no effective treatments to prevent or reverse disease progression. Summary of the Invention

[0005] Provided herein are methods for treating or preventing (as used herein, "preventing" means reducing the risk of developing) a neurodegenerative disease, such as Alzheimer's disease, in a subject. The method comprises administering to the subject an effective amount of a reelin protein or a nucleic acid encoding a reelin protein. Also provided herein are reelin proteins for use in methods for treating or preventing neurodegenerative diseases. Preferably, the reelin protein comprises an H3447R or H3447K mutation and / or an R3454A mutation, optionally an H3447R or H3447K mutation in combination with R3454A. In some embodiments, the subject is a mammal, such as a human or a non-human veterinary subject. In addition to Alzheimer's disease, the present methods can be used to treat other neurodegenerative diseases, disorders, or conditions, including frontotemporal dementia, various types of memory loss, cognitive dysfunction, including but not limited to mild cognitive impairment (MCI), or other conditions associated with beta-amyloid accumulation or tau accumulation, or other proteopathies such as holotemporal dementia, or amyotrophic lateral sclerosis (ALS), or age-related cognitive decline. Neurodegenerative diseases can also include ocular diseases such as age-related macular degeneration, glaucoma, diabetic retinopathy, or inherited retinal degeneration, stroke, brain trauma or concussion, retinal trauma, small vessel diseases such as cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), and abnormal angiogenesis such as wet age-related macular degeneration. Other applications include temporal lobe epilepsy, autism, schizophrenia and bipolar disorder, as well as conditions genetically or epigenetically associated with loss of RELN function, including cortical lamination defects, abnormal neuronal migration and cerebellar dysgenesis.

[0006] In some embodiments, the reelin protein comprises full-length reelin (or a sequence at least 80%, 85%, 90%, 95%, or 99% identical to human reelin), or a mini-reelin comprising A) a signal peptide, (B) an oligomerization domain, e.g., a dimerization domain, optionally a reelin CR-50 domain, (C) a receptor binding domain, optionally reelin domains (repeats) 5 and 6 (R5-6), and (D) a GAG binding domain, optionally including the C-terminus of reelin (CTR), e.g., a mini-reelin comprising the signal peptide, the CR-50 domain, reelin domains 5 and 6 (R5-6), and the C-terminus of reelin.

[0007] In some embodiments, the method comprises administering a nucleic acid encoding a reelin protein, where the nucleic acid is naked mRNA or DNA encoding reelin, or is in a viral vector, such as an AAV vector.

[0008] In some embodiments, the reelin protein or a nucleic acid encoding the reelin protein is preferentially administered in or around the entorhinal cortex of the brain, eg, to increase efficacy and / or reduce side effects.

[0009] Also provided herein are compositions comprising a reelin protein or a nucleic acid encoding a reelin protein, preferably wherein the reelin protein comprises the H3447R or H3447K mutation and / or the R3454A mutation described herein, optionally the H3447R or H3447K mutation in combination with R3454A.

[0010] In some embodiments, the reelin protein comprises a full-length reelin or a mini-reelin comprising, for example, as described herein, e.g., in Table A ((A) a signal peptide; (B) an oligomerization domain, e.g., a dimerization domain, e.g., a reelin CR-50 domain, an Fc fragment of IgG, or FKBP; (C) an APOER2 / VLDLR-binding domain, e.g., reelin domains (repeats) 5 and 6 (R5-6), or a receptor-binding domain of APOE, RAP, urokinase-type plasminogen activator (uPA), thrombospondin, f-spondin, or SEPP1; and (D) a glycosaminoglycan (GAG)-binding domain, e.g., a mini-reelin comprising the C-terminus (CTR) of reelin, a TAT peptide, or P21). Exemplary signal peptides for secretion include the IL2 signal peptide, human albumin signal peptide, human alpha1-antitrypsin signal peptide, or human factor VIII signal peptide. Exemplary constructs include those in Table 5.

[0011] In some embodiments, the composition comprises a nucleic acid encoding a reelin protein, and optionally the nucleic acid is naked mRNA or DNA encoding reelin, optionally with a human codon-optimized sequence, or is in a viral vector, e.g., an AAV vector.

[0012] Additionally, provided herein is a composition comprising or consisting of a reelin C-terminal region (CTR) and, optionally, a carrier, preferably wherein the CTR comprises an H3447R or H3447K mutation and / or an R3454A mutation, optionally an H3447R or H3447K mutation in combination with R3454A, and optionally comprises or consists of a sequence set forth in Table 1. In some embodiments, the composition further comprises a non-reelin nucleic acid, e.g., mRNA, optionally wherein the mRNA encodes a therapeutic peptide. In some embodiments, the composition further comprises an isolated non-reelin protein, e.g., complexed with or fused to the CTR. Also provided herein is a method for delivering a nucleic acid or protein to a cell, comprising administering to the cell an effective amount of a composition comprising the CTR.

[0013] Also provided herein are methods of treating or preventing a neurodegenerative disease, e.g., Alzheimer's disease, in a subject, the method comprising administering to the subject an effective amount of an agent that reduces methylation of the RELN promoter in an amount sufficient to increase RELN expression in the subject, wherein the agent that reduces methylation promoter is (i) a fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain and a guide RNA that directs the fusion protein to demethylate cytosines in the RELN promoter, optionally administered as an RNP, or (ii) a nucleic acid encoding a fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain and a guide RNA that directs the fusion protein to demethylate cytosines in the RELN promoter, optionally administered as an mRNA or in one or more vectors, optionally viral vectors, optionally adeno-associated virus AAV vectors.

[0014] Further provided herein is a method for treating or preventing a neurodegenerative disease, such as Alzheimer's disease, in a subject. The method comprises administering to the subject an effective amount of (i) a CRISPR / Cas protein, optionally administered as an RNP; a guide RNA that directs the Cas protein to a region of the RELN allele that includes H3447; and one or more ssODNs for insertion into the RELN allele, the ssODN comprising a sequence that includes the H3447R or H3447K mutation and / or the R3454A mutation, optionally the H3447R or H3447K mutation in combination with R3454A, or the H3447R or H3447K mutation in combination with R3454A; or (ii) optionally an mRNA. a nucleic acid encoding a CRISPR / Cas protein, administered as a vector or in one or more vectors, optionally a viral vector, optionally an adeno-associated virus (AAV) vector; a guide RNA that directs the Cas protein to a region of the RELN allele that includes H3447 and / or R3454; and an ssODN or multiple ssODNs that include a sequence that includes an H3447R or H3447K mutation and / or an R3454A mutation, optionally in combination with R3454A, for insertion into the RELN allele.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0016] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0017] [Figure 1]Figures 1A-C show PET imaging of RELN-COLBOS (H3447R) carriers. (A) Representative PiB PET amyloid (upper left column) and flortaucipir-tau PET (lower left column) imaging of a case (PSEN1 E280A; RELN-COLBOS) (left panel) compared with a PSEN1 E280A mutation carrier with MCI at typical ages (right panel). For both measurements, specific binding of the tracer is expressed using a scale with lowest (0.8 DVR or SUVR) and highest (2.00 DVR or SUVR) binding degrees. The right side of panel A shows representative 18F-fludeoxyglucose (FDG) PET precuneus glucose metabolic rate (CMRgI) of a case (PSEN1 E280A; RELN-COLBOS) (left panel) compared with a PSEN1 E280A mutation carrier with MCI at typical ages (right panel). Dye binding affinity is expressed using a scale with lowest (0.5 SUVR) and highest (2.1 SUVR) binding. (B) Dot plot analysis of the brain imaging measurements shown in panel A for amyloid burden, tau burden, glucose metabolism, and hippocampal volume. Brain imaging measurements of a case (PSEN1 E280A; RELN-COLBOS) compared with previously published data points from a homozygous case (PSEN1 E280A; APOEch), unaffected PSEN1 E280A carriers (n = 13–18), and younger MCI PSEN1 E280A carriers (n = 7–11). Some previously published data points are included in the figures shown here because they are the only data available for comparison (2). The mean distribution volume ratio (DVR) of the cortex to the cerebellum was used to quantify amyloid burden between individuals; the entorhinal to cerebellar SUVR was used to quantify tau burden; the hippocampal to whole-brain volume ratio was used to compare changes in hippocampal volume; and the precuneus to whole-brain CMRgl ratio was used to compare changes in glucose metabolism between different cohorts. Data are presented as individual values ​​and mean ± sem. (C) Anatomical details of tau burden in the temporal cortex.Planar map views of the right hemisphere temporal cortex of interest (upper left, PPC = posterior parahippocampal cortex, ERC = entorhinal cortex) for four cases are shown with tau PET (flourtaucipitin, FTP) overlay. The asymptomatic PSEN1 E280A mutation carrier was 38 years old. The PSEN1 E280A mutation carrier with typical MCI was 44 years old. It was notable that PSEN1 carriers with RELN-COLBOS mutations had relatively low tau burden in the medial temporal regions (ERC and PPC) compared with asymptomatic PSEN1 E280A mutation carriers and PSEN1 E280A mutation carriers with typical age-onset MCI. [Figure 2-1]Figures 2A-M show that the RELN H3448R variant improved Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin, reduced tau hyperphosphorylation, and preserved motor function in mice. (A) Representative Western blotting bands for pDAB1 levels (top) and total protein staining (bottom) in primary mouse cortical neuron cells treated with 4 μg / ml of full-length RELN WT or RELN H3448R, the mouse ortholog of H3447R RELN, at 37°C for 5 min. (Sham; P<0.0029) and RELN WT (P=0.0246). Data are presented as mean ± s.e.m. and analyzed by Kruskal-Wallis test (Dunn's post hoc analysis for multiple comparisons of n=4 independent experiments). (B) Spectroscopic analysis of heparin chromatography fractions of CTR-RELN WT and CTR-RELN H3447R variants eluted with an increasing gradient of NaCl (0.05 M NaCl step gradient) in 20 mM Tris-HCl buffer and detected by UV absorbance at 280 nm. Data are expressed as a percentage of input for NaCl gradient fractions from 0.4 M to 5 M NaCl. The data show that 0.55 M NaCl can displace CTR-RELN WT from the heparin column. The affinity of CTR-RELN for heparin increases in the presence of the H3447R mutation, as suggested by a peak shift, with the highest fraction eluting between 0.55 M and 0.7 M NaCl. N = 3 independent experiments. Error bars represent s.e.m. [Figure 2-2]Figure 2A-M shows that the RELN H3448R variant improved Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin, reduced tau hyperphosphorylation, and preserved motor function in mice. (C) Representative sensorgrams of binding assays between heparin-coated chip sensors and increasing concentrations of the CTR-RELN variants ranging from 0 to 25 nM. Data are expressed as response units versus time in seconds. The equilibrium dissociation constants (KD) for each SPR assay are shown within the graphs and support the difference in affinity binding between heparin and the CTR-RELN variants: H3447R (right plot, KD = 3.75e-9 M-1s-1) > H3347 (left plot, KD = 6.53e-9 M-1s-1). Sensorgrams of CTR-RELN with H3447K and H3447D control variants are reported in Figures 7A-7B for comparison. [Figure 2-3] Figure 2A-M shows that the RELN H3448R variant improved Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin, reduced tau hyperphosphorylation, and preserved motor function in mice. (D) Representative WB images of pDab1 levels (upper blot) and GAPDH levels (lower blot) detected in the cerebellum of female (left) and male (right) mice either wild-type (RELN WT / WT), heterozygous (RELN WT / H3448R), or homozygous (RELN H3448R / H3448R) for the mRELN H3448R mutation. Levels were detected in 6- to 12-month-old mice. (E, F) Quantification of pDab1 levels normalized to GAPDH and expressed as fold change of RELN WT showing a genotype effect on pDab1 levels in male mice (F. p = 0.0284 for WT / WT vs. H3448R / H3448R; p = 0.0037 for WT / H3448R vs. H3448R / H3448R, one-way ANOVA), and no effect in female mice (E). [Figure 2-4]Figure 2A-M shows that the RELN H3448R variant improved Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin, reduced tau hyperphosphorylation, and preserved motor function in mice. G, Isothermal calorimetry of the short variants CTR-RELN WT (left plot) and CTR-RELN H3447R (right plot) titrated with 5 μM heparin. Affinity calculations are reported at the top of each plot. H, Binding analysis by BLI between Fc-fused CTR-RELN WT or H3447R and a heparin-coated biosensor. The association constant (ka) and dissociation constant (kd) were used to calculate the equilibrium dissociation constant (KD) shown in the plot. [Figure 2-5] Figure 2A-M shows that the RELN H3448R variant improved Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin, reduced tau hyperphosphorylation, and preserved motor function in mice. I, Docked CTR-RELN WT (purple) with a representative heparin molecule (cyan). The AA of RELN CTR that make polar contact with heparin are highlighted in magenta. J, Representative IHC images of the hippocampus from WT / WT, WT / RELN H3448R, hTau tg / WT, and hTau tg / RELN H3448R mice stained with the pTau T205 antibody. hTau tg / WT mice exhibited neurofibrillary tangles and thread-like structures in the CA1 and dentate gyrus, while hTau tg / RELN H3448R mice exhibited milder tau pathology (cell bodies of affected neurons indicated by dotted lines). Bar = 100 μm. [Figure 2-6]Figures 2A-M show that the RELN H3448R variant improved Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin, reduced tau hyperphosphorylation, and preserved motor function in mice. K, Bar graph of pTau T205 signal intensity values ​​in hTau tg / WT (n = 3 mice) and hTau tg / RELN H3448R mice (n = 3 mice). The latter showed significantly lower signal intensity values. p = 0.022*, two-tailed Student's T-test. Error bars represent standard deviation from the mean. L, Representative phenotypes observed during the tail lift test and relative scores (0 = severe impairment, 1 = 50% impairment; 2 = normal). [Figure 2-7] Figure 2A-M shows that the RELN H3448R variant improved Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin, reduced tau hyperphosphorylation, and preserved motor function in mice. (M) Tail lift scores recorded for RELNWT / Tau-P301L (n = 13 male mice) and RLN-H3448R H3448R / Tau-P301L crossbred male mice (n = 11 male mice) show significantly improved tail lift scores in the presence of the RLN-H3448R variant compared to RLN WT-expressing Tau-P301L mice (*p = 0.0305, unpaired two-tailed t-test, t = 2.313, df = 22). Box plots are expressed as minimum to maximum values ​​around the mean. [Figure 3-1] Figures 3A-E show the neuropathological characterization of a case (PSEN1 E280A; RELN H3447R). (A) Reported amyloid beta (Aβ) and hyperphosphorylated tau (p-tau) pathology in the CA1 and EC. Both pathologies show broad distribution and intensity, with Aβ pathology showing diffuse plaques of varying distribution and size in both structures (panels and insets). p-tau pathology shows neurofibrillary tangles of varying density and diffuse tau pathology. Scale bar = 500 mm. [Figure 3-2]Figure 3A-E shows the neuropathological characterization of the case (PSEN1 E280A; RELN H3447R). (B) shows representative images of neurons stained with Klüver-Barrera in the CA1 and EC of case PSEN1 E280A RELN-COLBOS (RELN-COLBOS), case PSEN1 E280A / APOE Christchurch (APOEch), a PSEN1 E280A familial Alzheimer's disease (FAD) case with average onset, and a sporadic Alzheimer's disease (SAD) case. Scale bar = 125 mm. [Figure 3-3] Figures 3A-E show the neuropathological characterization of a case (PSEN1 E280A; RELN H3447R). (C) 3D scatter plot graphs of Aβ, p-tau, and neuronal density for the EC and CA1 of the RELN-COLBOS, APOEch, FAD, and SAD cases. The EC of the RELN-COLBOS case shows the highest neuronal density, while exhibiting low Aβ and p-tau pathology. In contrast, the CA1 of the same case shows high levels of Aβ and p-tau pathology along with low neuronal density. For all other cases, both regions have low neuronal density, while exhibiting varying levels of Aβ or p-tau pathology. [Figure 3-4]Figures 3A-E show the neuropathological characterization of the case (PSEN1 E280A; RELN H3447R). (D) Representative images of RELN-CT and ApoE staining in the EC and CA1 of RELN-COLBOS, APOEch, FAD, and SAD cases. The RELN-COLBOS case shows a stronger background signal in both structures, with less intraneuronal signal for RELN-CT in the EC. Similarly, the APOEch case shows a lower intraneuronal signal with the RELN-CT antibody in the EC and very low intraneuronal signal with the ApoE antibody in both structures (zoomed right panel). Finally, ApoE staining shows notable plaque-like and neurofibrillary tangle-like signals in both the EC and CA1 structures of the FAD and SAD cases. Scale bar = 100 mm, and 25 mm in the zoomed panel. (E) Representative images of Klüver-Barrera staining of whole hippocampal and parahippocampal sections (above) and representative magnified images of subcortical white matter stained with RELN-CT antibody in RELN-COLBOS, APOEch, FAD, and SAD cases. RELN-COLBOS cases showed high intensity Luxol Fast Blue signal in the white matter, while RELN-COLBOS and SAD cases showed high intracellular RELN-CT signal in the white matter. Scale bar = 2.5 mm for the upper panel and 25 μm for the lower panel. [Figure 4-1]Figures 4A-D show a putative model for the ADAD pathology-altered APOEch and RELN H3447R PS1E280A cases. Under normal conditions (A), RELN and ApoE can bind to the VLDLr / APOER2 receptor complex, assisted by GAGs; modulation of this signaling pathway maintains physiological Dab1 activation (phosphorylation indicated by circles), which maintains basal phosphorylation levels of GSK3β and tau (left panel). In APOE Christchurch cases, reduced binding of ApoE to GAGs allows increased RELN binding to the receptor complex, increasing Dab1 activation and inhibiting GSK3β and tau phosphorylation (middle panel B). In the presence of the RELN H3447R variant, increased RELN binding to GAGs leads to enhanced Dab1 activation and subsequent inhibition of GSK3β and tau phosphorylation (right panel C). [Figure 4-2] 4A-D show a putative model for ADAD pathology-altered APOEch and RELN H3447R PS1E280A cases. (D) Model of a protective state in which increased Reelin signaling and / or reduced ApoE signaling have beneficial effects. [Figure 5] Figures 5A-B show the subject's pedigree and Sanger confirmation of the C-terminal RELN H3447R variant. (A) Pedigree of the subject. Circles represent females, squares represent males, and diamonds represent individuals whose gender has not been disclosed for privacy reasons. Arrows indicate index cases. Deceased individuals are crossed out. Black marks indicate affected individuals, and white marks indicate unaffected individuals. (B) Representative DNA Sanger sequencing of amplicons in the RELN gene from a non-mutation carrier (RELN / RELN, top panel) compared with an individual with the variant (RELN / RELN H3447R, bottom panel). The region of the H3447R mutation is highlighted by a black circle. [Figure 6]Figure 6A - Treatment with RELN H3448R reduces tau phosphorylation in vitro. (A) Representative Western blots of pTau (Ser396, top blot), tau (Tau5, middle blot), and GAPDH (bottom blot) detected in primary mouse cortical neurons treated with either vehicle, RELN H3448 wild-type (RELN WT), or RELN H3448R for 1 hour. (B) Quantification of normalized intensity from n=3 independent experiments showing a significant increase in the pTau / tau ratio in neurons treated with RELN H3448R compared to vehicle (p=0.03, unpaired t-test). [Figure 7] Figures 7A-B show that mutations at position 3447 in the CTR domain of RELN affect heparin binding. (A, B) Representative sensorgrams of binding analysis between heparin-coated chip sensors and increasing concentrations of CTR-reelin variants ranging from 0 to 25 nM. Sensorgrams for RELN H3447K (A) and RELN H3447D (B). Data are expressed as response units over time in seconds. The equilibrium dissociation constants (kD) for each SPR analysis are shown in the graphs and support the difference in binding between heparin and the reelin variants when compared to the data shown in Figure 2, in the following order: H3447R (Figure 2C, KD = 3.75e-9 M-1s-1) > WT (Figure 2C, KD = 6.53e-9 M-1s-1) > H3447K (A, KD = 7.33e-9 M-1s-1) >>> H3447D (B, KD = 1.64e-7 M-1s-1). [Figure 8] Figure 1 shows the Reelin C-terminal consensus across 128 mammalian species. Analysis of Reelin sequences across mammalian species indicates that the CTR is highly conserved. Basic AA may play a role in binding GAG or lipoprotein receptors. [Figure 9]This figure shows the orientation of selected basic amino acids in the heparin-binding motif. Analysis of Reelin sequences across mammalian species indicates that CTRs are highly conserved. Basic AA may play a role in binding GAG or lipoprotein receptors. Position 3447 (arrow) faces the same direction as the majority of other arginines. Arginines at positions 3446 and 3453 may also interact with heparin as part of the heparin-binding motif, but they face a different direction than most basic AA. Although R3452 and R3457 are part of the heparin-binding motif, they face a different direction from the other basic amino acids in the potential binding site, and therefore are unlikely to contribute to heparin interaction. [Figure 10] Figure 1 shows the 20 lowest energy structures of Reelin CTR generated by 2D NMR. The structures show the presence of a flexible region towards the c-terminus of the peptide, including the H3447R mutation. [Figure 11] Figures 11A-D show representative HPLC chromatograms of Reelin-peptide variants. (A) Inclusion of zero (R3446H), one (WT), or two (H3447R) basic amino acids at positions 3446-3447 shows increased interaction with heparin, as indicated by a later retention time peak in isocratic 1 M KCl elution. (B) This pattern holds for short and long peptides. n = 2 replicates, within less than 0.5 min of a representative peak. (C) The short reelin variant had an earlier retention time peak compared to the long reelin variant (D); however, both the short and long peptides are similarly affected by the AA substitution at position 3447. H3447D had an earlier retention time peak compared to WT. The basic substitutions H3447K and H3447R had an increased later retention time peak, therefore indicating increased interaction with heparin. n=2 replicates, within <0.5 min of representative peak. [Figure 12]12A-B show surface plasmon resonance assays for Reelin CTR-heparin binding kinetics. Binding between H3447 (A) or H3447R (B) and heparin-coated metal films. The association constant (ka) and dissociation constant (kd) were used to calculate the equilibrium dissociation constant (KD). [Figure 13] BLI of heparin-Reelin interaction shows that H3447R has approximately two-fold greater interaction compared to WT Reelin. Binding between WT Reelin (A) and a heparin-coated biosensor. The association constant (ka) and dissociation constant (kd) are used to calculate the equilibrium dissociation constant (KD). [Figure 14] Figures 14A-B show RELN modulation of Aβ aggregation. (A) Thioflavin T (ThT) assay of Aβ aggregation with RELN CTR WT and H3447R long (left) and short (right) variants, showing that RELN CTR reduces Aβ aggregation. (B) ThT assay of Aβ aggregation alone or in the presence of RELN 3431HH-R3446H-3451HH long or RELN 3431HH-R3446H short peptides, showing that the anti-aggregation effect is significantly reduced in the presence of the RELN 3431HH-R3446H short variant alone. For both panels D and E, data are expressed as a percentage of the maximum ThT release of Aβ at 120 min of aggregation kinetics. Comparisons between the aggregation kinetics of Aβ alone or in the presence of RELN variants were performed using two-way ANOVA followed by Tukey's test for multiple comparisons. P values ​​were calculated for 30-minute and 40-45-minute samples (*p<0.05; **p<0.001; ***p<0.0001; ****p<0.00001). [Figure 15]Figure 1. RELN CTR modulation of Aβ aggregation. Thioflavin T (ThT) assay of Aβ aggregation with RELN CTR H3447K and H3447D long (left) and short (right) variants demonstrates that RELN CTR reduces Aβ aggregation. Data are expressed as a percentage of maximum ThT release of Aβ at 120 min of aggregation kinetics. Comparisons between the aggregation kinetics of Aβ alone or in the presence of RELN variants were performed using two-way ANOVA followed by Tukey's test for multiple comparisons. P values ​​were calculated for the 30 min and 40-45 min samples (*p<0.05; **p<0.001; ***p<0.0001; ****p<0.00001). [Figure 16] Figure 1 shows that RELN CTR promotes cellular uptake of mRNA cargo. Green fluorescent protein (GFP) expression in human retinal endothelial cells treated with 5 μg of encoding mRNA. The mRNA was conjugated to cationic peptides at three different ratios. The ratios are expressed as mRNA:peptide mass ratios. [Figure 17-1] 17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-2]17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-3] 17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-4]17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-5] 17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-6]17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-7] 17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-8]17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-9] 17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 17-10]17A-J show in vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Panels show representative brightfield acquisitions using 10X magnification of HREC cells untreated or treated with lipofectamine or mini-RELN constructs 225Q and 225Xf (17A), 225T and 225S (17B), 225F and 225SW (19C), 225ZZ and 233C (19D), 22WT and 225Z (E), 225SV (F), 225RR and 225D (G), 225E (H), 225SU and 233F (I), or 233A (J). Acquisitions were performed either before transfection (Pr. t., top row), 5 hours after transfection (5H pt., middle row) and 24 hours after transfection (24H pt., bottom row). Scale bar = 500 μm. [Figure 18-1] Figures 18A-E show in vitro screening of mini-RELN constructs in HRECs. Representative Western blots of HREC lysates upon 24-hour overexpression of 225Q, 225Xf, 225T, and 225S (A); 225Yf (n=2), 225SW (n=2), 225ZZ (n=2), and 233C (n=2) (B); 225SW (n=2), 225Z (n=2), and 225SV (n=2) (C); 225RR (n=2), 233D (n=2), and 233E (n=2) (D); or 225SU (n=2), 233F (n=2), and 233A (n=2) (E) plasmids using Lipofectamine as the transfection agent. As controls, untreated and Lipofectamine-treated cells were also tested. WB was used to detect total DAB1 and β-actin, and DAB1 levels were normalized. [Figure 18-2]Figures 18A-E show in vitro screening of mini-RELN constructs in HRECs. Representative Western blots of HREC lysates upon 24-hour overexpression of 225Q, 225Xf, 225T, and 225S (A); 225Yf (n=2), 225SW (n=2), 225ZZ (n=2), and 233C (n=2) (B); 225SW (n=2), 225Z (n=2), and 225SV (n=2) (C); 225RR (n=2), 233D (n=2), and 233E (n=2) (D); or 225SU (n=2), 233F (n=2), and 233A (n=2) (E) plasmids using Lipofectamine as the transfection agent. As controls, untreated and Lipofectamine-treated cells were also tested. WB was used to detect total DAB1 and β-actin, and DAB1 levels were normalized. [Figure 19-1] Figures 19A-E show quantification of total DAB1 levels, expressed as intensity normalized to β-actin and control (lipofectamine-treated, Lp), demonstrating that DAB1 levels were reduced in the presence of the mini-RELN construct compared to lipofectamine (Lp). [Figure 19-2] Figures 19A-E show quantification of total DAB1 levels, expressed as intensity normalized to β-actin and control (lipofectamine-treated, Lp), demonstrating that DAB1 levels were reduced in the presence of the mini-RELN construct compared to lipofectamine (Lp). [Figure 19-3] Figures 19A-E show quantification of total DAB1 levels, expressed as intensity normalized to β-actin and control (lipofectamine-treated, Lp), demonstrating that DAB1 levels were reduced in the presence of the mini-RELN construct compared to lipofectamine (Lp). [Figure 20-1]Figures 20A-F show in vitro screening of constructs on HREC cells. Representative Western blotting of HREC lysates upon 5-minute treatment with culture medium containing various mini-RELN constructs: 225Q, 225R (A); 225S, 225T (B); 225S, 225T, 225Z (C); 225FX, 225FY, 225SU, 225SV, 225SW, FL-RELN WT, FL-RELN Mut, and 225Z (D); 225ZZ, 225Z (E); or Fc-RELN WT (184I), Fc-RELN H3447R (184J), and RELN (F). Constructs were obtained from Innovagen or Creative Bio. Recombinant mouse reelin protein was obtained from R&D systems at 4 μg / mL. Phosphorylated DAB1 (pDAB1, 20A-F) and total DAB1 (20F) levels were measured. [Figure 20-2] Figures 20A-F show in vitro screening of constructs on HREC cells. Representative Western blotting of HREC lysates upon 5-minute treatment with culture medium containing various mini-RELN constructs: 225Q, 225R (A); 225S, 225T (B); 225S, 225T, 225Z (C); 225FX, 225FY, 225SU, 225SV, 225SW, FL-RELN WT, FL-RELN Mut, and 225Z (D); 225ZZ, 225Z (E); or Fc-RELN WT (184I), Fc-RELN H3447R (184J), and RELN (F). Constructs were obtained from Innovagen or Creative Bio. Recombinant mouse reelin protein was obtained from R&D systems at 4 μg / mL. Phosphorylated DAB1 (pDAB1, 20A-F) and total DAB1 (20F) levels were measured. [Figure 20-3]Figures 20A-F show in vitro screening of constructs on HREC cells. Representative Western blotting of HREC lysates upon 5-minute treatment with culture medium containing various mini-RELN constructs: 225Q, 225R (A); 225S, 225T (B); 225S, 225T, 225Z (C); 225FX, 225FY, 225SU, 225SV, 225SW, FL-RELN WT, FL-RELN Mut, and 225Z (D); 225ZZ, 225Z (E); or Fc-RELN WT (184I), Fc-RELN H3447R (184J), and RELN (F). Constructs were obtained from Innovagen or Creative Bio. Recombinant mouse reelin protein was obtained from R&D systems at 4 μg / mL. Phosphorylated DAB1 (pDAB1, 20A-F) and total DAB1 (20F) levels were measured. [Figure 20-4] Figures 20A-F show in vitro screening of constructs on HREC cells. Representative Western blotting of HREC lysates upon 5-minute treatment with culture medium containing various mini-RELN constructs: 225Q, 225R (A); 225S, 225T (B); 225S, 225T, 225Z (C); 225FX, 225FY, 225SU, 225SV, 225SW, FL-RELN WT, FL-RELN Mut, and 225Z (D); 225ZZ, 225Z (E); or Fc-RELN WT (184I), Fc-RELN H3447R (184J), and RELN (F). Constructs were obtained from Innovagen or Creative Bio. Recombinant mouse reelin protein was obtained from R&D systems at 4 μg / mL. Phosphorylated DAB1 (pDAB1, 20A-F) and total DAB1 (20F) levels were measured. [Figure 21-1] 21A-F show quantification of pDAB1 levels expressed as intensity normalized to GAPDH and control (medium). [Figure 21-2] 21A-F show quantification of pDAB1 levels expressed as intensity normalized to GAPDH and control (medium). [Figure 21-3] 21A-F show quantification of pDAB1 levels expressed as intensity normalized to GAPDH and control (medium). [Figure 21-4] 21A-F show quantification of pDAB1 levels expressed as intensity normalized to GAPDH and control (medium). [Figure 22] FIG. 1 shows quantification of total DAB levels, expressed as intensity normalized to GAPDH and control, upon 5 min overexpression of various mini-RELN constructs Fc-RELN WT(184I), Fc-RELN H3447R(184J), and Innovagen's RELN. [Figure 23] FIG. 1 shows the reduction of cytotoxicity induced by oligomeric tau in the presence of 184I mini-RELN peptide. [Figure 24-1] Figures 24A-C show that the C-terminal RELN domain modulates Aβ aggregation. A, B, Thioflavin T (ThT) assay of Aβ aggregation alone or in the presence of C-terminal RELN WT (either uncleaved or long, or furin-cleaved or short), panel A, or C-terminal RELN H3447R (either uncleaved or long, or furin-cleaved or short), panel B, or in the presence of vehicle or morin, used as a positive control for aggregation inhibition. Aggregation kinetics performed for up to 40 minutes showing that mini-RELN reduces Aβ aggregation. C. Analysis of the change in ThT fluorescence percentage of Aβ at various time points and under various treatment conditions, showing significant inhibition of Aβ aggregation. p-values ​​were calculated for the 30- and 40-minute samples. [Figure 24-2]Figures 24A-C show that the C-terminal RELN domain modulates Aβ aggregation. A, B, Thioflavin T (ThT) assay of Aβ aggregation alone or in the presence of C-terminal RELN WT (either uncleaved or long, or furin-cleaved or short), panel A, or C-terminal RELN H3447R (either uncleaved or long, or furin-cleaved or short), panel B, or in the presence of vehicle or morin, used as a positive control for aggregation inhibition. Aggregation kinetics performed for up to 40 minutes showing that mini-RELN reduces Aβ aggregation. C. Analysis of the change in ThT fluorescence percentage of Aβ at various time points and under various treatment conditions, showing significant inhibition of Aβ aggregation. p-values ​​were calculated for the 30- and 40-minute samples. [Figure 25-1] 25A-B show Western blotting confirmation of mini-RELN overexpression in HEK cells. Representative Western blots of both cell lysates and culture media of HEK cells transfected to overexpress a mini-RELN construct compared to controls (UT, untreated, and LP, lipofectamine) showing anti-Fc tag positive bands when the Fc-tagged mini-RELN construct was overexpressed. [Figure 25-2] 25A-B show Western blotting confirmation of mini-RELN overexpression in HEK cells. Representative Western blots of both cell lysates and culture media of HEK cells transfected to overexpress a mini-RELN construct compared to controls (UT, untreated, and LP, lipofectamine) showing anti-Fc tag positive bands when the Fc-tagged mini-RELN construct was overexpressed. [Figure 26-1]Figures 26A-E show in vitro screening of constructs on HREC cells. A. Representative Western blotting of HREC lysates upon treatment for 5 minutes with culture medium containing various concentrations of the 225Xf and 225Yf (A); 225ZZ and 233C (B); 225SU and 225SV (C); 225SW and 233F (D); or 225RR and 233A (E) mini-RELN constructs or controls, as indicated. [Figure 26-2] Figures 26A-E show in vitro screening of constructs on HREC cells. A. Representative Western blotting of HREC lysates upon treatment for 5 minutes with culture medium containing various concentrations of the 225Xf and 225Yf (A); 225ZZ and 233C (B); 225SU and 225SV (C); 225SW and 233F (D); or 225RR and 233A (E) mini-RELN constructs or controls, as indicated. [Figure 27-1] 27A-E show quantification of pDAB1 levels from FIGS. 26A-26E, expressed as intensity normalized to b-actin and control (lipofectamine-treated, Lp). [Figure 27-2] 27A-E show quantification of pDAB1 levels from FIGS. 26A-26E, expressed as intensity normalized to b-actin and control (lipofectamine-treated, Lp). [Figure 27-3] 27A-E show quantification of pDAB1 levels from FIGS. 26A-26E, expressed as intensity normalized to b-actin and control (lipofectamine-treated, Lp). [Figure 28-1]Figures 28A-K show in vivo drug delivery and activity of mini-RELN peptides 225S (A-F), 225T, and 225Z (G-K). A. Anti-human IgG-Fc ELISA showing that optical density levels in the hippocampus 24 and 72 hours after injection tended to be increased in the hippocampus 72 hours after injection of mini-RELN-Fc tagged peptides compared to PBS vehicle. B. Representative WB of pDAB1 levels in the hippocampus 24 and 72 hours after intranasal drug delivery. Quantification of CB. [Figure 28-2] Figures 28A-K show in vivo drug delivery and activity of mini-RELN peptides 225S (A-F), 225T, and 225Z (G-K). D. Representative WB of pDAB1 levels in the hippocampus and entorhinal cortex of Tau P301S mice 72 hours after intranasal drug delivery. GAPDH was detected as a loading control (bottom blot). E, F. Quantification of pDAB1-positive bands normalized to GAPDH and PBS-treated controls in the hippocampus (E) and entorhinal cortex (F), showing that pDAB1 levels increased in the entorhinal cortex 72 hours after injection. [Figure 28-3] Figures 28A-K show in vivo drug delivery and activity of mini-RELN peptides 225S (A-F), 225T, and 225Z (G-K). G. Representative WB of pDAB1 levels in the hippocampus 72 hours after intranasal drug delivery of mini-RELN peptides 225Z and 225T in WT male mice (upper blot). We used β-actin as a loading control (lower blot). H, I. Representative WB of pDAB1 levels in the hippocampus 72 hours after drug delivery of 225T (H) and 225Z (I), showing that pDAB1 levels were increased in the hippocampus. [Figure 28-4]Figures 28A-K show in vivo drug delivery and activity of mini-RELN peptides 225S (A-F), 225T, and 225Z (G-K). J. Representative WB of pDAB1 levels in the midbrain 72 hours after intranasal drug delivery of mini-RELN peptides 225Z and 225T in WT male mice (top blot). We used β-actin as a loading control (bottom blot). K. Representative WB of pDAB1 levels in the midbrain 72 hours after drug delivery of 225T and 225Z, showing that pDAB1 levels were increased in the midbrain. [Figure 29] Figures 29A-B show in vivo characterization of pDab1 expression in wild-type animals treated with vehicle (Pluronic) or mini-Reelin 225S via intranasal brain delivery. A. Entorhinal-hippocampal analysis of pDAB1 expression 24 hours after mini-Reelin administration. Positive pDab1 cells are outlined with a white dashed line. Mini-Reelin was detected using an FC antibody. DAPI is also shown. B. Segmentation was performed manually to show areas of positive pDab1 staining for mini-Reelin compared to Pluronic. Scale bar: 25 μm. Data demonstrate increased levels of RELN signaling, as measured by pDab1, in animals treated with mini-RELN. A direct correlation between the mini-RELN peptide signal and increased pDab1 signal is shown. [Figure 30]

[0023] Figure 1 shows quantification of pTau Ser396 expression in MAPT P301S mice injected intraperitoneally with either PBS or mini-RELN peptide 225T, as assessed using immunofluorescence microscopy. pTau S396 fluorescence intensity was used as a feature of tau pathology. The data demonstrated decreased levels of pTau S396 signaling, indicating reduced tau pathology in the hippocampus and entorhinal regions when treated systemically with mini-RELN. DETAILED DESCRIPTION OF THE INVENTION

[0018] Efforts to develop treatments for Alzheimer's disease (AD) have focused on removing amyloid, the neuropathological hallmark. We have characterized over 5,000 individuals of Colombian descent with autosomal dominant AD (ADAD) due to the E280A mutation in presenilin-1 (PSEN1), of which approximately 1,200 are mutation carriers. PSEN1-E280A carriers typically develop cognitive dysfunction in their 40s. E280A carriers develop mild cognitive impairment (MCI) by a median age of 44 years (95% CI, 43-45) and dementia by 49 years (95% CI, 49-50) (1), with rare exceptions (2). We reported a female PSEN1 E280A carrier with two copies of the APOE3 Christchurch (APOEch, R136S) mutation who remained cognitively intact nearly 30 years after the predicted age of clinical onset. (2) This disclosure is based, in part, on the clinical, in vivo neuroimaging, genetic, and neuropathological features of a male case from the same cohort with the PSEN1 E280A mutation, who also displayed an extreme phenotype with a delayed age of clinical ADAD onset.

[0019] We characterized a man heterozygous for the RELN-COLBOS variant who was resilient to cognitive dysfunction associated with the PSEN1 E280A mutation until age 67. Compared with other AD cases, we observed lower tau pathology and increased neuronal density in the entorhinal cortex, suggesting the involvement of this brain region in RELN-mediated mechanisms associated with protection against AD (Table 2 and Figures 3A–3E). A female sibling carrier of the RELN-COLBOS and PSEN1 E280A variants showed less optimal protection compared to her older brother, but a delayed age at onset of cognitive decline and prolonged end-stage disease. RELN-specific sexual dimorphism may have contributed to her distinct features. We cannot exclude the possibility that other factors contributed to the AD-resilient phenotype in RELN-COLBOS carriers. Other researchers have identified RELN as a candidate gene associated with AD pathology in cognitively healthy individuals ( 26 ), and DAB1 variants are associated with AD risk in APOE4 homozygotes, further linking the RELN / DAB1 pathway to Alzheimer's disease ( 27 ).

[0020] We previously reported the case of a woman homozygous for APOE3 Christchurch who was resistant to ADAD-related dementia and had extensive amyloid pathology and low tau pathology in the entorhinal cortex. (2) Tauopathy was more extensive in the RELN-COLBOS case compared with the APOE3 Christchurch homozygote, except for the entorhinal cortex, which was largely spared in both cases, suggesting resilience in the RELN-COLBOS case.

[0021] The hypermorph effect of RELN is mild. This is the first known report of a RELN hypermorph; stronger effects may not support the proper development of this important signaling process. Experimental evidence of a gain-of-function mechanism for the RELN-COLBOS variant, and the fact that patients with extreme protection against ADAD possess it, establishes theoretical evidence for genetic involvement in the observed phenotype. Without being bound by theory, it is hypothesized that RELN-COLBOS, but not neutral variants, likely contribute to the subject's resilience phenotype.

[0022] The APOE Christchurch mutation impairs ApoE binding to GAGs and the ApoE receptor (2, 28). Conversely, as shown herein, the RELN-COLBOS variant enhances RELN binding to GAGs and NRP1 (neurophilin 1), likely conferring a competitive advantage to RELN for binding to its receptor (4). RELN-COLBOS binding to GAGs or heparan sulfate proteoglycans may increase the local concentration of RELN, resulting in enhanced signaling. Our analysis of RELN-COLBOS cases reveals convergent mechanisms potentially linking ApoE and RELN interactions via GAGs or other receptors to protection against AD. RELN-COLBOS is a gain-of-function variant that exhibits enhanced ability to activate its canonical protein target, Dab1, and reduce human tau phosphorylation in knock-in mice. Modulation of this APOE-RELN defensive pathway, particularly in the entorhinal cortex, may have profound therapeutic effects on resistance to tau pathology and neurodegeneration, as well as resilience to cognitive decline and dementia in Alzheimer's disease.

[0023] Measurement of glucose metabolic rates in the precuneus relative to the whole brain using fluorodeoxyglucose (FDG) PET showed slightly elevated levels of glucose metabolism compared with the mean levels in younger relatives of typical MCI carriers (Figures 1a-1b).

[0024] Methods for treating and reducing the risk of cognitive decline and dementia Provided herein is a method for treating cognitive decline and dementia, or reducing the risk of its onset or worsening.The method can include administering RELN protein, such as the full-length RELN recombinant protein or mini-RELN recombinant protein described herein or their variants (see, for example, Table A), optionally comprising the variants described herein, for example, H3447R or H3447K mutation and / or R3454A mutation, optionally comprising H3447R or H3447K mutation combined with R3454A, or the nucleic acid encoding RELN protein.R3455A provides resistance to furin cleavage of the CTR of Reelin (Kohno et al., J Neurosci. 2015 Mar 18; 35(11): 4776-4787). Alternatively, or in addition, the method may include administering a gene editing agent that modifies at least one allele in a cell to include an H3447R or H3447K mutation and / or an R3454A mutation, optionally an H3447R or H3447K mutation in combination with an R3454A mutation, to an agent that increases the expression or activity of reelin, for example, an agent that reduces methylation of reelin (e.g., a crispr / cas-demethylase fusion). In some embodiments, the method includes directly administering the protein (and optionally a guide RNA / ssODN); administering a nucleic acid encoding the protein (and optionally a guide RNA / ssODN), for example, as naked DNA or mRNA, or in an expression vector, such as a viral vector; or administering a cell that expresses the protein (and optionally a guide RNA / ssODN). For example, naked DNA can be administered without a vector using an electroporation device (e.g., to the ciliary body of the eye).

[0025] Alzheimer's disease The methods described herein can be used to treat or reduce the risk of developing subjects with all types of Alzheimer's disease, including, but not limited to, familial and sporadic Alzheimer's disease, early-onset or late-onset Alzheimer's disease. In some embodiments, the methods can be used to treat or reduce the risk of developing early-onset familial Alzheimer's disease (AD) or age-related cognitive decline.

[0026] Typically, amnesia or mild, increasing confusion is an early symptom of Alzheimer's disease. Gradually, the cognitive dysfunction associated with Alzheimer's disease leads to memory loss, especially recent memory loss; disorientation and spatial errors; difficulty speaking, writing, thinking, and reasoning; changes in personality and behavior, resulting in depression, anxiety, social withdrawal, mood swings, distrust of others, irritability and aggression, changes in sleep habits, wandering, loss of reserve, delusions, and ultimately death.

[0027] Other neurodegenerative diseases, disorders or conditions In addition to Alzheimer's disease, the present methods can be used to treat other neurodegenerative diseases, disorders, or conditions, including frontotemporal dementia, various types of memory loss, cognitive dysfunction, including but not limited to mild cognitive impairment (MCI), or other conditions associated with beta-amyloid accumulation or tau accumulation, or other proteopathies such as frontotemporal dementia, or amyotrophic lateral sclerosis (ALS), or age-related cognitive decline. Neurodegenerative diseases can also include ocular diseases such as age-related macular degeneration, glaucoma, diabetic retinopathy, or inherited retinal degenerations, stroke, brain trauma or concussion, retinal trauma, inherited retinal degenerations, small vessel diseases such as cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), or abnormal angiogenesis. Other applications include conditions genetically associated with loss of RELN function, including temporal lobe epilepsy, autism, attention deficit hyperactivity disorder (ADHD), schizophrenia, and bipolar disorder, as well as cortical lamination defects, abnormal neuronal migration, and cerebellar hypoplasia. See, e.g., Shifman et al., 2008; Ober et al., 2008; Goes et al., 2010; Seripa et al., 2008; Feher et al., 2015; Kelemenova et al., 2009; and Abo El Fotoh et al., 2020.

[0028] Reelin' and Mini-Reelin' Reelin useful in the methods and compositions described herein may include full-length wild-type reelin, as well as truncated and deletion variants thereof that retain the function of the full-length protein, i.e., the ability to bind to HSPGs and / or to bind to receptors such as APOER2 or VLDLR, and / or to result in activation of downstream targets such as Dab1 or to promote resilience of neurons or glial cells, and / or to bind to NRP1.

[0029] Exemplary full-length human reelin sequences include:

[0030] [Table 1] In some embodiments, the methods and compositions described herein use a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to human reelin.

[0031] In some embodiments, mini-RELN (minimal RELN) is used. Exemplary mini-RELNs may include the sequences described herein; mini-RELN constructs may optionally include a protein linker between these domains, such as a Gly-Ser linker, which includes an amino acid between the domains that does not affect function; many others are known in the art; see, e.g., Chen et al., 2012. In some embodiments, mini-RELN includes A) a signal peptide, (B) an oligomerization domain, e.g., a dimerization domain, e.g., a CR-50 domain, (C) an APOER2 / VLDLR binding domain, e.g., reelin domains 5 and 6 (R5-6), and (D) a GAG-binding domain / cell-penetrating peptide, e.g., the C-terminus of reelin, e.g., as described herein, e.g., in Table A.

[0032] [Table 2] Additional sequences, such as additional sequences from RELN, may also be included, although mini-RELN is not the same as full-length RELN. Mini-RELN may be preferred because it is easier to administer, cheaper to produce, and feasible to deliver in standard AAV vectors, or via mRNA, or for expression as a recombinant protein. Preferably, the RELN sequence, if used, is that of human RELN.

[0033] In some embodiments, mini-RELN can comprise a signal peptide, RAP, IgG Fc, and a C-terminal RELN comprising a H3447R or H3447K mutation and / or a R3454A mutation, optionally in combination with R3454A; a signal peptide, IgG Fc, RAP, and a C-terminal RELN comprising a H3447R or H3447K mutation and / or a R3454A mutation, optionally in combination with R3454A; or a C-terminal RELN comprising a signal peptide, R3-6, and a H3447R or H3447K mutation and / or a R3454A mutation, optionally in combination with R3454A. In some embodiments, the C-terminal RELN comprising the H3447R or H3447K mutation and / or the R3454A mutation, optionally in combination with R3454A, may be longer than 30 amino acids (e.g., R8 to CTR--amino acids 3051 to 3460), but less than full-length reelin, e.g., up to 100, 105, 110, 120, 130, 150, 200, 300, 400, or 410 amino acids.

[0034] In some embodiments, at the N-terminus, mini-RELN comprises the signal peptide of RELN, or the signal peptide of another protein (e.g., the IL2 signal peptide, human albumin signal peptide, human alpha 1-antitrypsin signal peptide, or human factor VIII signal peptide, not shown in SEQ ID NO: 1 below); a CR-50 domain (e.g., amino acids 229-345 of full-length RELN, shown in lowercase letters below); a linker (e.g., shown in lowercase italics); R5-6 of RELN (e.g., amino acids 1918-2664); and a C-terminus (e.g., amino acids 3429-3460, shown in italics, with bold indicating the position of the H3447R variant). SEQ ID NO: 1 is an exemplary 225Z construct; other constructs, including those shown herein, can also be used.

[0035] [ka]

[0036] Additional exemplary sequences of mini-RELN constructs include the following (the font and style key below indicates the order of the different modules): 184I pfcn-huIgG2-C-term H3447WT (nucleotide sequence) IL2SS-igg2-fc-linker-c-term reln h3447WT

[0037] [ka] 184I pfcn-huIgG2-C-term H3447WT (amino acid sequence - translation of domain-by-domain sequence) IL2SS-igg2-fc-linker-c-term reln h3447WT

[0038] [ka] 184J pfcn-huIgG2-C-term H3447H3447R (nucleotide sequence) IL2SS-igg2-fc-linker-c-term reln h3447r

[0039] [ka] 184J pfcn-huIgG2-C-term H3447R (amino acid sequence - translation of domain-by-domain sequence) IL2SS-igg2-fc-linker-c-term reln h3447r

[0040] [ka] 225Q pFUSEN-hG2Fc-R5~6-C-term H3447R (nucleotide sequence) IL2SS-igg2fc-linker-reln r5-6-c-term reln h3447r

[0041] [ka] 225Q pFUSEN-hG2Fc-R5~6-C-term H3447R (amino acid sequence - translation of each domain sequence) IL2SS-igg2fc-linker-reln r5-6-c-term reln h3447r

[0042] [ka] 225R pFUSEN-flag-CR-50-R5~6-C terminus H3447R (nucleotide sequence) IL2SS-linker-flag tag-linker-reln cr50-linker-RELN R5-6-c-terminus reln H3447R

[0043] [ka]

[0044] [ka] 225R pFUSEN-flag-CR-50-R5~6-C-term H3447R (amino acid sequence - translation of each domain sequence) IL2SS-linker-flag tag-linker-reln cr50-linker-RELN R5-6-c-terminus reln H3447R

[0045] [ka] 225S pFUSEN-FcIgG-RAP-CTRH3447R (nucleotide sequence) IL2SS-igg2-fc-linker-rap-linker-c-term reln H3447R

[0046] [ka] 225S pFUSEN-FcIgG-RAP-CTRH3447R (amino acid sequence - translation of domain sequence) IL2SS-igg2-fc-linker-rap-linker-c-term reln H3447R

[0047] [ka] 225T pFUSEN-RAP-FcIgG-CTRH3447R (nucleotide sequence) IL2SS-rap-linker-igg2-fc-linker-c-term reln H3447R

[0048] [ka] 225T pFUSEN-RAP-FcIgG-CTRH3447R (amino acid sequence - translation of domain sequence) IL2SS-rap-linker-igg2-fc-linker-c-term reln H3447R

[0049] [ka] 225Xf pFUSEN-IL2ss-FcigG-APOE-R154S-CTRH3447R (nucleotide sequence) IL2SS-igg2-fc-linker-apoe r154s Christchurch-linker-c-term reln H3447R

[0050] [ka] 225Xf pFUSEN-IL2ss-FcigG-APOE-R154S-CTRH3447R (amino acid sequence - translation of domain-by-domain sequence) IL2SS-igg2-fc-linker-apoe r154s Christchurch-linker-c-term reln H3447R

[0051] [ka] 225Yf pFUSEN-IL2ss-FcigG-APOE-CTRH3447R (nucleotide sequence) IL2SS-igg2-fc-linker-apoe-linker-c-term reln H3447R

[0052] [ka] 225Yf pFUSEN-IL2ss-FcigG-APOE-CTRH3447R (Amino acid sequence - Translation of sequences by domain) IL2SS-igg2-fc-linker-apoe-linker-c-term reln H3447R

[0053] [ka] 225Z pFUSEN-6xHISCR-50-R5~6-C terminus H3447R (nucleotide sequence) IL2SS-linker-6XHis tag-reln cr50-linker-RELN R5-6-c-term reln H3447R

[0054] [ka]

[0055] [ka] 225Z pFUSEN-6xHISCR-50-R5~6-C terminus H3447R (amino acid sequence - translation of domain-by-domain sequence) IL2SS-linker-6XHis tag-reln cr50-linker-RELN R5-6-c-term reln H3447R

[0056] [ka] 225SU pFUSEN-IL2 signal peptide-6XHis tag-hReelin (AALeu1220 to Ile2660) C-terminus-REELIN H3447R (nucleotide sequence) IL2SS-linker-6Xhistag-linker-Ile1120 to Ile2660 hreln-linker-c-term reln H3447R

[0057] [ka]

[0058] [ka] 225SU pFUSEN-IL2 signal peptide-6XHis tag-hReelin (AALeu1220 to Ile2660) C-terminus-REELIN H3447R (amino acid sequence - translation of each domain) IL2SS-linker-6Xhistag-linker-Ile1120 to Ile2660 hreln-linker-c-term reln H3447R

[0059] [ka] 225SV pFUSEN-IL2 signal peptide-6XHis tag-hReelin (AALeu1220 to Ile2660) C-terminus-REELINWT (nucleotide sequence) IL2SS-linker-6Xhistag-linker-Ile1120 to Ile2660 hreln-linker-c-term reln wt

[0060] [ka]

[0061] [ka] 225SV pFUSEN-IL2 signal peptide-6XHis tag-hReelin (AALeu1220 to Ile2660) C-terminus-REELINWT (amino acid sequence - translation of each domain) IL2SS-linker-6Xhistag-linker-Ile1120 to Ile2660 hreln-linker-c-term reln wt

[0062] [ka] 225ZZ pFUSEN-Il2ss6xHISmini-RELN-FspCR50-R5~6-C-term H3447R (nucleotide sequence) IL2SS-linker-6Xhistag-linker-reln fspcr50-linker-RELN R5-6-c-terminus reln h3447r

[0063] [ka]

[0064] [ka] 225ZZ pFUSEN-Il2ss6xHISmini-RELN-FspCR50-R5~6-C-term H3447R (Amino acid sequence - Translation of sequences by domain) IL2SS-linker-6Xhistag-linker-reln fspcr50-linker-RELN R5-6-c-terminus reln h3447r

[0065] [ka] 225SW pFUSEN-IL2ss-6xHis-hReelin R3~R6 CT-H3447R (nucleotide sequence) IL2SS-linker-6Xhistag-linker-reln r3-linker-RELN RELN r6-linker-c-term reln wt

[0066] [ka] 225SW pFUSEN-IL2ss-6xHis-hReelin R3~R6 CT-H3447R (amino acid sequence - translation of each domain sequence) IL2SS-linker-6Xhistag-linker-reln r3-linker-RELN r6-linker-c-term reln wt

[0067] [ka] 233A pFUSEN-IL2ss-6xHis-hReelinR3~R6CT-WT (nucleotide sequence) IL2SS-linker-6Xhistag-linker-reln r3-linker-RELN r6-linker-c-term reln H3447R

[0068] [ka] 233A pFUSEN-IL2ss-6xHis-hReelinR3~R6CT-WT (amino acid sequence - translation of each domain sequence) IL2SS-linker-6Xhistag-linker-reln r3-linker-RELN r6-linker-c-term reln H3447R

[0069] [ka] 233C pFUSEN-Il2ss-6xHISR5~6C-term H3447R (nucleotide sequence) IL2SS-linker-6Xhis tag-linker-reln r5~6-c-terminus reln H3447R

[0070] [ka] 233C pFUSEN-Il2ss-6xHISR5~6C-term H3447R (amino acid sequence - translation of domain-by-domain sequence) IL2SS-linker-6Xhis tag-linker-reln r5~6-c-terminus reln H3447R

[0071] [ka] 233D pFUSEN-IL2ss-6xHis-hReelinR6~R6CT-H3447R (nucleotide sequence) IL2SS-linker-6Xhistag-linker-reln r6-linker-reln r6-linker-c-term reln H3447R

[0072] [ka] 233D pFUSEN-IL2ss-6xHis-hReelinR6~R6CT-H3447R (amino acid sequence - translation of domain-by-domain sequence) IL2SS-linker-6Xhistag-linker-reln r6-linker-reln r6-linker-c-term reln H3447R

[0073] [ka] 233E -pFUSEN-Il2ss-6xHIS-R5~6-FspCR50-C-term H3447R (nucleotide sequence) IL2SS-linker-6Xhistag-linker-reln r5~6-linker-reln fspcr50-linker-c-term reln H3447R

[0074] [ka] 233E pFUSEN-Il2ss-6xHIS-R5~6-FspCR50-C-term H3447R (amino acid sequence - translation of domain-by-domain sequence) IL2SS-linker-6Xhistag-linker-reln r5~6-linker-reln fspcr50-linker-c-term reln H3447R

[0075] [ka] 233F pFUSEN-IL2ss-6xHis-FspCR50-R6~R6C-term RELN-H3447R (nucleotide sequence) IL2SS-linker-6Xhistag-linker-reln fspcr50-linker-reln r6-linker-reln r6-linker-c-term reln H3447R

[0076] [ka] 233F pFUSEN-IL2ss-6xHis-FspCR50-R6~R6C-term RELN-H3447R (amino acid sequence - translation of each domain sequence) IL2SS-linker-6Xhistag-linker-reln fspcr50-linker-reln r6-linker-reln r6-linker-c-term reln H3447R

[0077] [ka] 225RR pFUSEN-Il2ss-flag mini-RELNFspCR50R5-6 C-terminus H3447R (nucleotide sequence) IL2SS-linker-flag tag-linker-reln fspcr50-linker-RELN R5-6-c-terminus reln H3447R

[0078] [ka]

[0079] [ka] 225RR pFUSEN-Il2ss-flag mini-RELNFspCR50R5-6 C-terminus H3447R (amino acid sequence - translation of domain-by-domain sequence) IL2SS-linker-flag tag-linker-reln fspcr50-linker-RELN R5-6-c-terminus reln H3447R

[0080] [ka]

[0081] In some embodiments, the proteins and nucleic acids used herein are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequences provided herein, so long as they retain the desired function of the parent sequence. In some embodiments, a protein may comprise a sequence provided herein with at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 altered amino acids.

[0082] Residues that can be changed without disrupting function can be identified, for example, by aligning similar sequences and making conservative substitutions in non-conserved regions (see, for example, the alignments provided herein). To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or 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 gaps that need to be introduced for optimal alignment of the two sequences and the number of identical positions shared by the sequences, taking into account the length of each gap.

[0083] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two amino acid sequences can be determined using the Needleman-Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program in the GCG software package (available on the World Wide Web at gcg.com) using default parameters, such as a Blossum 62 scoring matrix and a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0084] Increased RELN signaling can also be achieved by increasing the activity or expression level of DAB1 using various techniques, including increasing DAB1 phosphorylation, decreasing dephosphorylation, or increasing dimerization or oligomerization. Exemplary sequences of DAB1 are included herein. DAB1 expression can be achieved by administering mRNA. DAB1 dimerization can be achieved by expressing DAB1 fusion to FKBP, and the activity of the protein fusion can be controlled by administering rapamycin. An exemplary Dab1 dimerization construct protein sequence (Dab1 is shown in bold, followed by FKBP) is as follows:

[0085] [ka]

[0086] Exemplary sequences encoding Dab1 dimerization constructs are shown in the Examples below.

[0087] RAP clustering may also be used to increase reelin signaling; see, e.g., Strasser et al., Mol Cell Biol. 2004 Feb;24(3):1378-86. An exemplary sequence for a protein fusion of RAP to IgG2 is (RAP in uppercase bold, linker in uppercase italics, igG2 in lowercase, linker in uppercase, RELN CTR with H3447R variant in lowercase bold) (which corresponds to the 225T exemplary sequence):

[0088] [ka] The HNEL sequence can be removed to improve secretion.

[0089] Also provided herein are nucleic acids encoding the proteins described herein (e.g., mini-reelin and variants described in Table A) and cells that express the proteins, and nucleic acids encoding the variants, including vectors, such as viral vectors.

[0090] Gene editing Also, CRISPR-based method can be used to increase the level or activity of reelin.For example, CRISPR-Cas9 can be used to introduce H3447R or H3447K mutation and / or R3454A mutation, optionally H3447R or H3447K mutation combined with R3454A into the reelin gene of a subject having one or more wild-type reelin alleles.For example, prime editing (see, for example, Anzalone et al., Nature volume 576, pages 149-157 (2019)) can be used to introduce the change, and optionally the following sequence:

[0091] [ka] and single guide RNA (sgRNA), or tracrRNA and crNA, containing the following sequence: TGAATTTTTCACGACAACAT The spacer region comprises:

[0092] In some embodiments, the ssODN contains two mismatches to the wild-type RELN gene, the first mismatch restoring the mutation H3447R (CAT to CGT) and the second optional mismatch disrupting the PAM (NGG) from GGG to GGA, as shown below.

[0093] [ka] Similarly, ssODNs can be designed to restore the H3447R or H3447K mutation and / or the R3454A mutation, optionally the H3447R or H3447K mutation in combination with R3454A.

[0094] The Cas9 nuclease of Streptococcus pyogenes (S. pyogenes) can be guided by simple base pair complementarity between an engineered guide RNA (gRNA) of 17-20 nucleotides, e.g., a single guide RNA or a crRNA / tracrRNA pair, and the complementary strand of a target genomic DNA sequence of interest next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence NGG or NAG (Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang et al., Nat Biotechnol 31, 233-239 (2013); Jinek et al., Elife 2, e00471 (2013); Hwang et al., Nat Biotechnol 31, 227-229 (2013); Cong et al., Science 339, 819-823 (2013);Mali et al., Science 339, 823-826 (2013c);Cho et al., Nat Biotechnol 31, 230-232 (2013);Jinek et al., Science 337, 816-821 (2012)). Engineered CRISPR1 (Cpf1, also known as Cas12a) nucleases from Prevotella and Francisella can also be used, as described, for example, in Zetsche et al., Cell 163, 759-771 (2015); Schunder et al., Int J Med Microbiol 303, 51-60 (2013); Makarova et al., Nat Rev Microbiol 13, 722-736 (2015); Fagerlund et al., Genome Biol 16, 251 (2015). Unlike SpCas9, Cpf1 / Cas12a requires only a single 42-nt crRNA, which has 23 nt at its 3' end complementary to the protospacer of the target DNA sequence (Zetsche et al., 2015).Furthermore, SpCas9 recognizes the NGG PAM sequence 3′ of the protospacer, while AsCpf1 and LbCp1 recognize the TTTN PAM found 5′ of the protospacer (Id.).

[0095] In some embodiments, the system utilizes wild-type or variant Cas9 proteins from S. pyogenes or Staphylococcus aureus, or wild-type or variant Cpf1 proteins from Acidaminococcus sp. BV3L6 or Lachnospiraceae bacterium ND2006 that are bacterially encoded or codon-optimized for expression in mammalian cells and / or have improved PAM recognition specificity and / or genome-wide specificity.Several variants have been described, for example, in WO 2016 / 141224, PCT / US2016 / 049147, Kleinstiver et al., Nat Biotechnol. 2016 Aug;34(8):869-74; Tsai and Joung, Nat Rev Genet. 2016 May;17(5):300-12; Kleinstiver et al., Nature. 2016 Jan 28;529(7587):490-5; Shmakov et al., Mol Cell. 2015 Nov 5;60(3):385-97; Kleinstiver et al., Nat Biotechnol. 2015 Dec;33(12):1293-1298; Dahlman et al., Nat Biotechnol. 2015 Nov;33(11):1159-61;Kleinstiver et al., Nature. 2015 Jul 23;523(7561):481-5;Wyvekens et al., Hum Gene Ther. 2015 Jul;26(7):425-31;Hwang et al., Methods Mol Biol. 2015;1311:317-34;Osborn et al., Hum Gene Ther. 2015 Feb;26(2):114-26;Konermann et al., Nature. 2015 Jan 29;517(7536):583-8;Fu et al., Methods Enzymol. 2014;546:21-45; and Tsai et al., Nat Biotechnol. 2014 See Jun;32(6):569-76.

[0096] In some embodiments, TrueCut Cas9 Protein v2 is used. Cas9 and analogs are shown in Table B, and engineered protospacer adjacent motifs (PAMs) or high-fidelity variants are shown in Table C.

[0097] [Table 3]

[0098] [Table 4-1]

[0099] [Table 4-2]

[0100] [Table 4-3]

[0101] In some embodiments, the RGN sequence is modified to include a nuclear localization sequence (NLS) and a mini-polyadenylation signal (or poly-A sequence), for example, at the C- and / or N-terminus of the RGN protein. Exemplary NLSs include the SV40 large T antigen NLS (PKKKRRV); PKKKRKV; KRTADGSEFES)PKKKRKV; and the nucleoplasmin NLS (KRPAATKKAGQAKKKK). 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.

[0102] A guide RNA appropriate for the RGN should be used; in some embodiments, the gRNAs used in this disclosure may be unimolecular or modular, as known in the art.

[0103] In some embodiments, a CRISPR / Cas9 construct, ssODN, and sgRNA, e.g., a ribonucleoprotein complex (RNP) encoding the above-described ssODN and sgRNA, or a gene therapy vector or mRNA, is administered to introduce the H3447R or H3447K mutation and / or the R3454A mutation in RELN, optionally the H3447R or H3447K mutation in combination with R3454A, throughout the brain or specifically into the entorhinal cortex, e.g., using stereotactic injection as described herein. In some embodiments, when the Cas9 protein is administered in a viral vector, the sequence may be split between two vectors (see, e.g., Truong et al., Nucleic Acids Res. 2015 Jul 27;43(13):6450-8).

[0104] RELN promoter demethylation Methylation of the RELN promoter reduces expression of the reelin protein (Chen et al., Nucleic Acids Res. 2002 Jul 1;30(13):2930-9). Therefore, the method can include inducing demethylation of the RELN promoter. As an example, a CRISPR / Cas-based demethylase, in which the demethylase (e.g., Tet1 catalytic domain (Tet-CD)) is linked to a catalytically inactive (dead) Cas protein (Xu et al., Cell Discovery (2016) 2, 16009), can be used to induce demethylation of the RELN promoter sequence, derepressing and thus increasing expression of reelin (57). The promoter sequence of the RELN gene is listed below, along with suggested gRNAs for use with dCas9 targeted to modulate methylation. RELN promoter sequence features: 346~365 misc_feature:sgRNA1-ggccaaaggggctggccttc 383~384 misc_features:-458 Lintas et al.(56) 430~431 misc_features:-411 Lintas et al.(56) 439~440 misc_features:-402 Lintas et al.(56) 394~413 misc_feature:sgRNA2-ctctgcggggctttgacgtc 441~460 misc_feature:sgRNA3-gtcctcgacagcgtccccgt 1001~1003 misc_features:orf start RELN promoter sequence:

[0105] [ka]

[0106] In some embodiments, a ribonucleoprotein complex (RNP), gene therapy vector, or mRNA encoding a CRISPR / Cas9 construct and the ssODN and sgRNA described above is administered to introduce the H3447R or H3447K and / or R3454A mutation in RELN, optionally the H3447R or H3447K mutation in combination with R3454A, throughout the brain or specifically to the entorhinal cortex, e.g., using stereotactic injection as described herein.

[0107] Other compounds can also be used, including administration of hsa_circRNA_102049, which acts as a sponge for hsa-miR-214-3p, to reduce RELN promoter methylation and thus increase reelin gene expression (Wang et al., Bioengineered. 2022 Feb;13(2):2272-2284).

[0108] Delivery Vector Nucleic acids encoding Reelin or CRISPR / cas polypeptides (e.g., wild-type, variant, peptide, or fragments thereof) can be incorporated into genetic constructs to be used as part of gene therapy protocols. For example, targeted expression vectors for in vivo delivery and expression of polynucleotides encoding Reelin polypeptides or active fragments thereof in specific cell types, particularly cerebral cortical neuronal cells, are described herein. Such component expression constructs can be administered in any effective carrier, e.g., any formulation or composition capable of effectively delivering the component genes to cells in vivo. Techniques include inserting genes into viral vectors, preferably adeno-associated viruses. Viral vectors typically transduce cells directly.

[0109] Viral vectors capable of highly efficient transduction of CNS neurons may be used, including rAAV vectors of any serotype (e.g., AAV1-AAV12), recombinant or chimeric AAV vectors, and lentivirus or other suitable viral vectors. In some embodiments, the polynucleotide encoding Reelin is operably linked to a promoter suitable for expression in the CNS. For example, a neuronal subtype-specific promoter, such as the alpha-calcium / calmodulin kinase 2A promoter, may be used to target excitatory neurons. Alternatively, a general neuronal promoter, such as the synapsin I promoter, may be used to drive Reelin expression. Other exemplary promoters include, but are not limited to, the cytomegalovirus (CMV) early enhancer / promoter, the hybrid CMV enhancer / chicken β-actin (CBA) promoter, a promoter containing the CMV early enhancer element, the first exon and first intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin gene (commonly referred to as the "CAG promoter"); or a 1.6 kb hybrid promoter consisting of the CMV immediate early enhancer and CBA intron 1 / exon 1 (commonly referred to as the CAGGS promoter; Niwa et al., Gene, 108:193-199 (1991)). The CAGGS promoter (Niwa et al., 1991) has been shown to provide ubiquitous and long-term expression in the brain (Klein et al., Exp. Neurol. 176:66-74 (2002)). A typical method for in vivo introduction of nucleic acid into cells is by using a viral vector containing a nucleic acid, for example, a cDNA encoding Reelin.Another advantage of infecting cells with a viral vector is that most of the targeted cells can receive the nucleic acid.In addition, the molecule encoded in the viral vector, for example, by the cDNA contained in the viral vector, is efficiently expressed in cells that have taken up the viral vector nucleic acid.

[0110] A particularly useful viral vector system for delivering nucleic acids is the adeno-associated virus (AAV). The adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a 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 a variety of cell types and produce long-term expression of transgenes in vivo. Although AAV vector genomes can persist in cells as episomes, vector integration has been observed (see, for example, Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-447; Asokan et al., Mol Ther. 2012 April; 20(4): 699-708; Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7: 349-356 (1992); Samulski et al., J. Virol. 63: 3822-3828 (1989); and McLaughlin et al., J. Virol. 62: 1963-1973 (1989)). AAV vectors, such as AAV2, have been widely used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models and in clinical settings. See, for example, 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 as little as 300 base pairs of AAV can be packaged and are capable of producing recombinant protein expression.Protocols for producing recombinant retroviruses and infecting cells in vitro or in vivo with such viruses are known in the art and can be found, for example, in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals.The use of AAV vectors to deliver constructs for expression in the brain is described, for example, in Iwata et al., Sci Rep. 2013;3:1472; Hester et al., Curr Gene Ther. 2009 Oct;9(5):428-33; Doll et al., Gene Therapy 1996,3(5):437-447; and Foley et al., J Control Release. 2014 Dec 28;196:71-8.

[0111] Therefore, in some embodiments, the Reelin encoding nucleic acid is present in a vector for gene therapy, such as an AAV vector.In some cases, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11 and AAV12.

[0112] The vector described herein can be a pseudotype vector.Pseudotype provides a mechanism for modulating the target cell population of vector.For example, pseudotyped AAV vector can be used in various methods described herein.Pseudotyped vector is a vector that contains the genome of one vector, for example, the genome of one AAV serotype, in the capsid of a second vector, for example, the second AAV serotype.Pseudotyped methods are well known in the art. For example, vectors can be pseudotyped with envelope glycoproteins derived from the rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains), rabies virus (e.g., various ERA (Evelyn-Rokitnicki-Abelseth) strains and challenge virus standard (CVS)), the lyssavirus Mokola virus, rabies-associated virus, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein type B (FuG-B), a variant of FuG-B (FuG-B2), or Moloney murine leukemia virus (MuLV). Viruses can be pseudotyped for transduction of one or more neurons or groups of cells. Additionally, the capsid may be modified to include one or more peptides that increase expression in the CNS; see, e.g., Yao et al., Nat Biomed Eng. 2022 Oct 10; Chatterjee et al., Gene Ther. 2022 Jun;29(6):390-397; Meng et al., Mol Ther Methods Clin Dev. 2021 Feb 27;21:28-41; Zhang et al., Biomaterials. 2022 Feb;281:121340; Gray, Cell Gene Ther. Insights 5, 13611368 (2019); Nonnenmacher et al., Mol. Ther. Methods Clin. Dev. 20, 366-378 (2021).

[0113] Illustrative examples of pseudotyped vectors include, but are not limited to, recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV9, AAVrhlO, AAV11, and AAV12 serotype vectors. It is known in the art that such vectors can be engineered to contain transgenes encoding human or other proteins. In certain cases, the present disclosure may include pseudotyped AAV9 or AAVrhlO viral vectors containing the nucleic acids disclosed herein. See Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003.

[0114] In some cases, a particular AAV serotype vector may be selected based on the intended use, for example, based on the intended route of administration.

[0115] Various methods for the application of AAV vector constructs in gene therapy are known in the art, including methods of modification, purification and preparation for administration to human subjects (see, for example, Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003).In addition, AAV-based gene therapy targeted to cells of the CNS has been described (see, for example, U.S. Patent Nos. 6,180,613 and 6,503,888).High-titer AAV preparations can be produced using techniques known in the art, for example, as described in U.S. Patent No. 5,658,776.

[0116] Vector construct refers to the polynucleotide molecule that contains all or part of viral genome and transgene.In some cases, gene transfer can be mediated by DNA virus vector, such as adenovirus (Ad) or adeno-associated virus (AAV).Other vectors that are useful in gene therapy methods are known in the art.For example, the construct disclosed herein can comprise alphavirus, herpesvirus, retrovirus, lentivirus or vaccinia virus.

[0117] Adenoviruses are a relatively well-characterized group of viruses, including more than 50 serotypes (see, e.g., International Publication No. WO 95 / 27071, which is incorporated herein by reference). Adenoviruses are amenable to application of molecular biology techniques and may not require integration into the host cell genome. Recombinant Ad-derived vectors have been constructed, including vectors that reduce the potential for recombination and generation of wild-type virus (see, e.g., International Publication Nos. WO 95 / 00655 and WO 95 / 11984, which are incorporated herein by reference). Wild-type AAV has high infectivity and the ability to integrate into the host genome with high specificity (see, e.g., Hermonat and Muzyczka 1984 Proc. Natl. Acad. Sci., USA 81:6466-6470 and Lebkowski et al. 1988 Mol. Cell. Biol. 8:3988-3996).

[0118] As disclosed herein, non-native regulatory sequence, gene regulatory sequence, promoter, non-coding sequence, intron or coding sequence can be included in nucleic acid.The inclusion of nucleic acid that codes nucleic acid tag or signaling sequence, or protein tag or protein signaling sequence is also contemplated herein.Typically, coding region is operably linked with one or more regulatory nucleic acid components.

[0119] The promoter contained in the nucleic acid disclosed herein may be a tissue- or cell-type-specific promoter, a multi-tissue- or multi-cell-type-specific promoter, an organ-specific promoter, a multi-organ-specific promoter, a systemic or ubiquitous promoter, or a near-systemic or ubiquitous promoter.Promoters with stochastic, inducible, conditional, or otherwise discontinuous, variable, or unpredictable expression are also included within the scope of the present disclosure.The promoter may include any of the above characteristics or other promoter characteristics known in the art.

[0120] In a clinical setting, a gene delivery system for a therapeutic gene may be introduced into a subject by any of several methods, each of which is well known in the art. For example, a pharmaceutical preparation of the gene delivery system may be introduced systemically, e.g., by intravenous injection, with specific transduction of the protein in target cells occurring predominantly due to the transfection specificity provided by the gene delivery vehicle, cell-type or tissue-type expression due to transcriptional regulatory sequences controlling receptor gene expression, or a combination thereof. In other embodiments, the initial delivery of the recombinant gene is more limited, and introduction into the subject is fairly localized. For example, the gene delivery vehicle may be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection, for example, optionally into the cisterna magna, ventricles, lumbar intrathecal space, hippocampus (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)), and / or entorhinal cortex. In some embodiments, methods of delivery of the reelin-expressing virus include intravenous, intrathecal, intracerebroventricular, intracisternal, and stereotactic intraparenchymal administration.

[0121] The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0122] Reelin formulations and pharmaceutical compositions In some embodiments, the reelin polynucleotides disclosed herein for delivery to target tissues in vivo are encapsulated in or associated with nanoparticles.Methods for nanoparticle packaging are well known in the art and are described, for example, in Bose S, et al. (Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells. J. Virol. 78:8146. 2004); Dong Y et al. Poly(d,l-lactide-co-glycolide) / montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 26:6068. 2005); Lobenberg R. et al. (Improved body distribution of 14C-labeled AZT bound to nanoparticles in rats determined by radioluminography. J. Drug Target 5:171. 1998); Sakuma SR et al. (Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int. J. Pharm 177:161. 1999); Virovic L et al. Novel delivery methods for treatment of viral hepatitis: an update. Expert Opin Drug Deliv 2:707. 2005); and Zimmermann E et al, Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media. Eur J Pharm Biopharm 52:203. 2001).In some embodiments, one or more polynucleotides are delivered to a target tissue in vivo in a vesicle, such as a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see the entire text). In some embodiments, lipid-based nanoparticles (LNPs) are used; see, e.g., Robinson et al., Mol Ther. 2018 Aug. 1;26(8):2034-2046; U.S. Pat. No. 9,956,271.

[0123] The methods and compositions may include microvesicles or preparations thereof containing one or more therapeutic molecules, such as the polynucleotides or RNAs described herein. "Microvesicles," as used herein, refer to membrane-derived microvesicles, which encompass a range of extracellular vesicles, including exosomes, microparticles, and shed microvesicles secreted by many cell types under both normal physiological and pathological conditions. See, for example, European Patent No. 2010663. The methods and compositions described herein may be applied to microvesicles of all sizes. In some embodiments, sizes range from 30 to 200 nm, in some embodiments, 30 to 800 nm, and in some embodiments, up to 2 μm. The methods and compositions described herein may also be applied more broadly to all extracellular vesicles, a term that encompasses exosomes, shed microvesicles, oncosomes, ectosomes, and retrovirus-like particles. Such microvesicles or preparations may be produced by the methods described herein. As used herein, a microvesicle preparation refers to a population of microvesicles obtained / prepared from the same cell source. Such preparations can be produced, for example, by culturing cells expressing the nucleic acid molecules of the present invention in vitro and isolating the microvesicles produced by the cells. Methods for isolating such microvesicles are known in the art (Thery et al., Isolation and characterization of exosomes from cell culture supernatants and biological fluids, in Current Protocols Cell Biology, Chapter 3, 322, (John Wiley, 2006); Palmisano et al., (Mol Cell Proteomics. 2012 August; 11(8):230-43) and Waldenstrom et al., ((2012) PLoS ONE 7(4): e34653)), some examples of which are described herein.Such techniques for isolating microvesicles from cells in culture include, but are not limited to, sucrose gradient purification / separation and differential centrifugation, and may be adapted for use in the methods or compositions described herein. See, e.g., EP 2010663.

[0124] In some embodiments, microvesicles are isolated by gentle centrifugation (e.g., about 300 g) of donor cell culture medium for a period sufficient to separate the cells from the medium (e.g., about 15 minutes). This leaves the microvesicles in the supernatant, thereby yielding a microvesicle preparation. In some embodiments, the culture medium or the supernatant from the gentle centrifugation is centrifuged more vigorously (e.g., about 16,000 g) for a period sufficient to precipitate cellular debris (e.g., about 30 minutes). This leaves the microvesicles in the supernatant, thereby yielding a microvesicle preparation. In some embodiments, the culture medium, the gently centrifuged preparation, or the strongly centrifuged preparation is filtered (e.g., through a 0.22 μm or 0.8 μm filter, allowing the microvesicles to pass through the filter). In some embodiments, the filtrate is subjected to a final ultracentrifugation (e.g., about 110,000 g) for a period sufficient to precipitate the microvesicles (e.g., about 80 minutes). The resulting pellet contains microvesicles and may be resuspended in a volume of buffer to produce a concentration useful for further use, thereby producing a microvesicle preparation. In some embodiments, the microvesicle preparation is produced by sucrose density gradient purification. In some embodiments, the microvesicles are further treated with DNase (e.g., DNase I) and / or RNase and / or proteinase to remove any external contaminating DNA, RNA, or protein. In some embodiments, the microvesicle preparation contains one or more RNase inhibitors.

[0125] The molecules contained in the microvesicle preparation include therapeutic molecules. Typically, the microvesicles in the preparation are a heterogeneous population, with each microvesicle containing a complement of molecules that may or may not differ from the complements of molecules in other microvesicles in the preparation. The content of therapeutic molecules in a microvesicle preparation can be expressed quantitatively or qualitatively. One such method is to express the content as a percentage of the total molecules in the microvesicle preparation. For example, if the therapeutic molecule is mRNA, the content can be expressed as a percentage of the total RNA content of the microvesicle preparation, or alternatively as a percentage of the total mRNA content. Similarly, if the therapeutic molecule is a protein, the content can be expressed as a percentage of the total protein in the microvesicles. In some embodiments, therapeutic microvesicles or preparations thereof produced by the methods described herein contain a detectable, statistically significant increased amount of therapeutic molecules compared to microvesicles obtained from control cells (cells obtained from the same source that have not been subjected to chemical manipulations to increase the expression of therapeutic molecules). In some embodiments, the therapeutic molecule is present in an amount at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater than the amount in microvesicles obtained from control cells. Higher levels of enrichment may also be achieved. In some embodiments, the therapeutic molecule is present in microvesicles or preparations thereof at least 2-fold greater than in control cell microvesicles. Higher fold enrichment may also be achieved (e.g., 3, 4, 5, 6, 7, 8, 9, or 10-fold).

[0126] In some embodiments, a relatively high percentage of the microvesicle content is a therapeutic molecule (e.g., achieved through overexpression of the molecule or specific targeting of the molecule to the microvesicle). In some embodiments, the therapeutic molecule content of the microvesicle is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total (similar) molecule content (e.g., the therapeutic molecule is mRNA and is about 10% of the total mRNA content of the microvesicle). Higher levels of enrichment may also be achieved. In some embodiments, the therapeutic molecule is present in the microvesicle or preparation thereof at least 2-fold more than all other such (similar) molecules. Higher fold enrichment may also be obtained (e.g., 3, 4, 5, 6, 7, 8, 9, or 10-fold).

[0127] Reelin CTR Initial evaluation of the Reelin CTR sequence showed that it is highly conserved across mammalian species, suggesting that the previously uncharacterized Reelin CTR may play an important neurobiological role (11). We show that the AA at position 3447 aligns with other arginines within the known heparin-binding motif. Therefore, a basic AA at this position has the potential to contribute to HSPG interactions (Figure 9). Building on this initial result, we used NMR to solve the previously unknown structure of Reelin CTR (Figure 10). We find that Reelin CTR is primarily alpha-helical, with the last 16 AA residues exhibiting high flexibility. Here, high flexibility in the secondary structure may indicate potential binding sites.

[0128] Additionally, the basic AA at position 3447 favored heparin interactions as observed by HPLC. H3447R Reelin has a significantly later retention time peak compared to the R3446H or WT peptide across two different experimental designs. We considered significant differences in retention time peaks to be at least >30 seconds. The acidic AA (H3447D) had significantly fewer interactions compared to the neutral (WT) or basic AA (H3447R, H3447K). In summary, Reelin CTR has charge-based interactions with heparin, and position 3447 plays a role in these interactions.

[0129] To gain further insight into the dynamics of this interaction, the heparin-Reelin CTR peptide interaction was assessed using SPR. H3447R Reelin had approximately two-fold more interactions with heparin compared to the Reelin WT peptide (Figures 12A-B); to confirm this small difference, we further assessed this interaction using BLI and found the same difference with the fc-fusion peptide (Figure 13). In this case, the fc-fusion peptide is likely shortened due to the production method. Therefore, using two different methods, we find that H3447R has two-fold more interactions compared to WT Reelin.

[0130] We then used ITC to further understand the thermodynamic properties of this interaction and found that H3447R is particularly favored for interaction in vivo. Nevertheless, we also found that for long peptides, the K aWe found that the ΔH, ΔG, and ΔS were similar. This may be partially explained by the fact that this method assesses binding in solution, whereas the kinetic and HPLC methods assess binding to surface-bound heparin, thus affecting the configuration of the Reelin peptide and potential binding site exposure. Significantly, the shorter peptides exhibited more positive entropy and more negative Gibbs free energy compared to the Reelin WT short variants. Because the Reelin CTR is typically cleaved by furin, the shorter peptides are the most representative in vivo variants. Therefore, the H3447R Reelin-HSPG interaction may be thermodynamically favored in vivo compared to WT Reelin. Additionally, the ITC negative control indicated that the basic amino acids at positions 3446–3447 play a prominent role in heparin interaction, and that the lack of at least one basic amino acid in the binding site resulted in approximately 100-fold lower affinity.

[0131] We also found that H3447R interacted 10-fold more strongly with NRP1 than the WT fc-fusion peptide, suggesting that Reelin CTR may have additional interactions on the cell surface.

[0132] Overall, these data indicate that H3447R has increased interaction with heparin compared to WT. Without being bound by theory, Reelin first binds to HSPGs and then to LDL receptors or other receptors, where the Reelin CTR interacts with HSPGs and the Reelin middomain interacts with LDL receptors. Because it has previously been shown that dysregulation of Reelin can cause tau hyperphosphorylation (43, 44), these cell surface interactions may ultimately modulate downstream NFT formation.

[0133] RELN CTR significantly reduced amyloid aggregation, suggesting a role in the progression of AD pathology (Figure 14A). However, mutations at position 3447, including the Reelin-COLBOS variant H3447R, did not significantly affect Aβ aggregation compared to WT, although there was a trend toward a greater reduction. The patients described herein, who were resilient to AD, had high levels of amyloid plaque burden. These results suggest that flexibility in the N-terminal region may play a role in Aβ seeding, as H is less flexible than R or K amino acids (45). Furthermore, the relative flexibility of the alpha-GAG binding site may aid in the prevention of Aβ plaque propagation (Figures 14A-14B). In contrast, our results indicate that the beta-GAG site interacts with GAGs and NRP1, and the H3447R mutation optimizes these interactions (Figures 11A-11D, Figures 12A-12B, Figure 13).

[0134] The RELN CTR may have multiple interaction partners, including GAGs, NRP1, amyloids, and potentially others. The RELN H3447R mutation found in AD-protected cases is not clearly neutral and may contribute to the AD-protective phenotype through multiple mechanisms. We show that the RELN CTR-GAG interaction is reproducible across multiple pathways, and that the H3447R mutation enhances a binding site that may affect interactions with numerous other molecules associated with neurodegenerative diseases.

[0135] Compositions comprising RELN CTR peptides are provided herein. These peptides may comprise or consist of RELN-CTR wild-type (WT) or RELN-CTR H3447R / K variant peptides (preferably H3447R:RKQNYMMNFSRQHGLRRFYNRRRRSLRRYP). In some embodiments, the CTR peptide is a variant that may include one or more alternative or additional mutations described herein, for example, as shown in Table 1, and / or a mutation at G3444, for example, G3444H or G3444P.

[0136] [Table 5]

[0137] In some embodiments, the Reelin CTR peptides are at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the sequences provided herein, so long as they retain the desired functionality of the parent sequence.

[0138] In some embodiments, the peptides may include just the sequences provided herein with at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 altered amino acids.

[0139] Also provided are compositions comprising a CTR peptide, optionally mixed with a non-reelin protein or nucleic acid, optionally in a pharmaceutically acceptable carrier. The nucleic acid can be, for example, an mRNA encoding a therapeutic or prophylactic agent, such as an antigen for a vaccine. The CTR peptide can also be in a fusion protein with a non-reelin sequence, for example, where the CTR peptide sequence is at the N-terminus, C-terminus, or internally inserted at a position that does not affect the function of the non-reelin sequence.

[0140] Pharmaceutical Compositions and Methods of Administration The methods described herein include the use of a pharmaceutical composition comprising or consisting of, as an active ingredient, a therapeutic agent described herein, such as a Reelin protein or a fragment thereof, such as mini-Reelin; or a nucleic acid encoding same, as described herein.

[0141] Pharmaceutical compositions typically contain pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carriers" includes physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. In addition, additional active compounds, such as mRNAs encoding therapeutic or diagnostic proteins, can be incorporated into the composition.

[0142] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. In some embodiments, the composition is delivered to the brain, for example, by administration into the cisterna magna, ventricles, lumbar intrathecal space, or direct administration to the hippocampus (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)). In some embodiments, methods for delivering reelin-expressing viruses include intravenous, intrathecal, intraventricular, intracisternal, and stereotaxic intraparenchymal administration. In some embodiments, the composition is administered into or around the entorhinal cortex of the brain.

[0143] 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 application can contain the following components: sterile diluents, such as water for injection, physiological saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for adjusting isotonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0144] Pharmaceutical compositions suitable for injectable use 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 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 the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, a sugar, a polyalcohol, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0145] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the components listed above as necessary, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into sterile medium that contains basic dispersion medium and other necessary components from the components listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which produces powder of active ingredient and any additional desired components from the solution that has been previously sterilized and filtered.

[0146] Oral compositions generally contain an inert diluent or an edible carrier. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or sterols; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0147] 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. Such methods include those described in U.S. Patent No. 6,468,798.

[0148] In addition, the systemic administration of the therapeutic compounds described herein can be carried out by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration.Transmucosal administration can be achieved through the use of nasal drops or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels or creams generally known in the art.

[0149] Pharmaceutical compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0150] The therapeutic compound that is or contains nucleic acid can be administered by any method suitable for administering nucleic acid agents, such as DNA vaccines.These methods include gene guns, bioinjectors and skin patches, as well as needleless methods, such as the microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389 and the mammalian transdermal needleless vaccination with powder-form vaccines disclosed in U.S. Patent No. 6,168,587.In addition, intranasal delivery is possible, as described in, among others, Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998).Liposomes (for example, as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used.Biodegradable targetable microparticle delivery systems can also be used (for example, as described in U.S. Patent No. 6,471,996).

[0151] In one embodiment, the therapeutic compound is prepared with a carrier that protects the therapeutic compound against rapid elimination from the body, for example, a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Such formulations may be prepared using standard techniques or may be commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells by monoclonal antibodies against cellular antigens) may also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0152] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0153] Ocular administration can be achieved by intravitreal or subretinal injection of biologics, including protein, nucleic acid, or virus-containing vectors, or by electroporation of DNA plasmids into the ciliary body.

[0154] Methods for determining risk of developing AD The present invention includes a method for determining the risk of identifying AD in a subject (for example, a human subject).The method relies on detecting the H3447R variant of RELN gene in the DNA of the subject.The method includes obtaining a sample comprising genomic DNA from the subject, and evaluating the existence of H3447R variant in the sample.

[0155] As used herein, the term "sample" refers to the material that is tested for the presence of H3447R variant, and includes, among others, tissue (including oral cells collected by swab) or blood.For identifying and / or isolating and / or purifying genomic DNA from sample, various methods are well known in the art.For example, the nucleic acid contained in sample can first be isolated according to standard methods, for example, by using lytic enzymes, chemical solutions, or by nucleic acid binding resin according to manufacturer's instructions.

[0156] The presence and / or levels of H3447R variant nucleic acids can be assessed using methods known in the art, such as polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT-PCR, digital PCR, i.e., BEAMing ((beads, emulsion, amplification, magnetic) Diehl (2006) Nat Methods 3:551-559); RNase protection assay; Northern blot; various types of nucleic acid sequencing (Sanger, pyrosequencing, next generation sequencing); or gene arrays / chips (Lehninger Biochemistry (Worth Publishers, Inc., current addition; Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Bernard (2002) Clin Chem 48(8): 1178-1185; Miranda (2010) Kidney International 78:191-199;Bianchi (2011) EMBO Mol Med 3:495-503;Taylor (2013) Front. Genet. 4:142;Yang (2014) PLOS One 9(11):e110641);Nordstrom (2000) Biotechnol. Appl. Biochem. 31(2):107-112; Ahmadian (2000) Anal Biochem 280:103-110).In some embodiments, high-throughput methods, such as protein or gene chips, can be used to detect the presence of H3447R variants, as known in the art (see, for example, Chapter 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999, WH Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485):1760-1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002; Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003).In some embodiments, suitable techniques for detecting the presence of alterations in nucleic acid structure or sequence, such as deletion, amplification or substitution, can be used to detect H3447R variants.

[0157] Gene arrays can be prepared by selecting probes that contain H3447R variant polynucleotide sequences, and then immobilizing these probes on solid support or surface.For example, probes can include DNA sequences, RNA sequences, DNA and RNA copolymer sequences, DNA and / or RNA analogs, or combinations thereof.Probe sequences can be synthesized in vivo by enzymes, in vitro by enzymes (for example, by PCR), or in vitro by non-enzymatic synthesis.

[0158] A subject with the H3447R variant in their genome can be identified as having a lower risk of developing AD compared to a subject without the H3447R variant. In some embodiments, the subject has or is identified as having an APOE4 variant allele. [Example]

[0159] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0160] [Example 1] Resilience against autosomal dominant Alzheimer's disease in Reelin-COLBOS (H3447R) variant carriers method The following methods and materials were used in this Example 1.

[0161] Clinical evaluation Patients were evaluated after providing written informed consent approved by the Institutional Review Boards of Antioch University and Massachusetts General Hospital (MGH).

[0162] Brain imaging We used Pittsburgh Compound B (PiB) and flortaucipir (FTP) positron emission tomography (PET) to image cerebral Aβ and tau burden in vivo, respectively. Structural magnetic resonance imaging (MRI) and both PET scans were performed at MGH. 18 F-fludeoxyglucose PET was performed at the University of Antioch, and the procedure and data analysis were performed as previously described ( 2 ).

[0163] Genetic and molecular studies We performed whole-exome, whole-genome sequencing (WGS) and Genomizer analysis (v10.10) of subjects to obtain a ranking of potential AD-associated risk factors, as previously described (2) and described in detail below.

[0164] Mouse model We generated knock-in mouse models harboring the RELN-COLBOS variant by homologous recombination through a fee-for-service service (Cyagen). Additional details about the mouse model and in vivo analysis are described in the Methods section of the Supplementary Appendix.

[0165] Neuropathology The postmortem time at autopsy was 210 minutes postmortem, and the brain weight was 745.4 grams. After 5 days of fixation in 4% paraformaldehyde and sample preparation in paraffin, 3 μm thick sections were cut from identified brain regions, deparaffinized, and stained with hematoxylin and eosin or processed for immunohistochemical staining using antibodies as described in the Additional Neuropathological Characterization section.

[0166] Additional Plasma NFL Assay Details Plasma neurofilament light chain (NfL) analysis was performed by the Clinical Neurochemistry Laboratory at Sahlgrenska University Hospital (Mölndal, Sweden) as previously reported (1). Experimenters were blinded to the clinical or genetic data of the probands to avoid any bias.

[0167] Additional genome sequencing details Whole-genome sequencing analysis extracted 44 million variants from the case genomes. Using Genomizer, variants were subjected to multiple filters: population allele frequency ≤ 2%; a variant effect filter that excluded intergenic, untranslated regions (UTRs), and noncoding intronic regions; a regulatory feature filter that removed all nonregulatory, noncoding variants > 20 kb from known genes; and a hiPhive prioritizer phenotype gene priority score ≥ 0.401. 6,779 variants passed the filters and were ranked by a composite Exomiser score derived from gene-phenotype and variant pathogenicity scores, as well as compatibility with inheritance patterns. The top three ranked variants were manually reviewed for pathogenicity. We also manually searched a list of 23 genes previously associated with Alzheimer's disease, including AAGAB, ABCC8, AKT2, APOE, APP, BEAN1, GATA1, GCK, HMGA1, HNF1B, HNF4A, LDB3, PAX4, PSEN1, PSEN2, ABCA7, SORL1, CACNA1G, HFE, MPO, NOS3, and PLAU. Gene variants from WGS were extracted and annotated for independent pathogenicity interpretation using the Alissa Interpret platform (Agilent, Santa Clara, CA). We calculated a "C-score" using Combined Annotation Dependent Depletion (CADD) (2, 3) to compare the pathogenicity of human-derived variants against simulated variants. The Regulatory Mendelian Mutation (ReMM) framework uses machine learning techniques to train classifiers to predict the potential of any position in the noncoding genome to cause a Mendelian disease if mutated ( 4 ).

[0168] Additional single-cell RNA sequencing details We isolated peripheral blood mononuclear cells using a Ficoll gradient and analyzed these cells by scRNA sequencing using a previously published protocol ( 1 ).

[0169] Additional RELN genotyping details by Sanger DNA sequencing DNA was first extracted using the Gentra Puregene kit (Qiagen). Fifty microliters of the resulting samples (both RELN H3447R and RELN H3447 carrier) were amplified by polymerase chain reaction (PCR) using 1 μL of 10 μM primers (forward: 5'-GTCCCAGCCTTTAGTTCCT-3'; reverse: 3'-CAACTTTCACGGACACATCAA-5') premixed with PCR Master Mix 2X (K0171, Thermo Fisher Scientific) using the following protocol: 94°C for 3 minutes for initial denaturation, 33 cycles of 94°C for 30 seconds, 62°C for 35 seconds for annealing, 72°C for 35 seconds for extension, and 72°C for 5 minutes for final extension. Horizontal electrophoresis was performed at 100 V using 1.5% agarose gels mixed with GelRed (41003-T, Biotium) in TAE buffer (Tris-acetate-EDTA, T8280-1L, Sigma-Aldrich). Fluorescence of positive bands was detected with a Bio-RAD Molecular Imager Gel Doc XR+ and acquired with Image Lab software (version 6.0.1, Biorad). Amplified DNA was purified using a QIAquick gel extraction kit (Qiagen) and sequenced by the MGH CCIB DNA Core using a 3730xl sequencer (Applied Biosystems) as previously published (1).

[0170] Additional cell culture details A plasmid encoding full-length murine recombinant RELN was donated by Dr. Tom Curran via Addgene (plasmid no. 122444 (5)). Subsequently, the plasmid was mutagenized to obtain the H3448R mutation, homologous to human RELN H3447R, through a fee-for-service arrangement by CustomDNAConstructs (New York, USA). We generated WT and RELN-COLBOS in Flp-In T-Rex 293 mammalian cells (R78007, Thermo Fisher Scientific) by transient transfection and used them for receptor binding assays by ELISA. A plasmid encoding the CTR-RELN-Fc fusion peptide was obtained through a fee-for-service arrangement by CustomDNAConstructs. All constructs were overexpressed in Flp-In T-Rex 293 mammalian cells (R78007, Thermo Fisher Scientific) by transient transfection using Lipofectamine 2000 according to the manufacturer's instructions (Product ID: 11668030, Thermo Fisher Scientific). Five hours after transfection, conditioned medium was collected using Opti-MEM. Cells were incubated for 24 hours, and the supernatant was collected and cellular debris removed by centrifugation for 3 minutes at 1,800 rcf and room temperature (RT). Primary CD1 cortical mouse neurons (M-CX-400, Lonza) were cultured in neurobasal medium (Gibco) supplemented with B-27 (Thermo Fisher), glutamax (Gibco), and normocin (Invivogen). Cells were seeded onto wells coated with poly-L-lysine (Sigma) and processed on day 6 after liquid nitrogen recovery. Treatment with recombinant RELN (RELN WT or RELN H3448R, 4 μg / mL) was incubated for 5 minutes or 1 hour at 37°C, 5% CO2 in the presence of 10 μM Mg-132 (ab141003, Abcam).Cells were washed with ice-cold dPBS (Gibco) and lysed in RIPA (9806, Cell Signaling) supplemented with 10 μM Mg-132, Triton-X100 (Sigma-Aldrich), protease inhibitor cocktail (4693159001, Millipore), and phosphatase inhibitors (4906837001, Sigma-Aldrich and P0044, Millipore). Protein concentration was determined using a Pierce bicinchoninic acid (BCA) protein assay kit (23227, Thermo Fisher Scientific) according to the manufacturer's instructions. Samples containing 10 μL of Laemmli buffer (Boston Bioproducts) and 4 μL of 1 M DTT (Sigma-Aldrich) were prepared, diluted with water to a final volume of 40 μL, and denatured at 90°C for 5 min.

[0171] Additional Western Blotting Details Twenty micrograms of whole cell lysate was prepared in 4 μL of 1 M 1,4-dithiothreitol (DTT; Sigma-Aldrich) and 10 μL of Laemmli buffer (Boston Bioproducts) to a final volume of 40 μL and heat-denatured at 90°C for 5 minutes. Samples were separated by electrophoresis using a 4-20% precast gradient gel (Mini-PROTEAN TGX, Bio-Rad) and SDS-Tris-glycine buffer (Bio-Rad) at 90 V for 1 hour. Proteins were transferred to a 0.45 μm nitrocellulose membrane in ice-cold 20% methanol-Tris-glycine buffer (Bio-Rad) at 90 V for 1 hour. To detect pDAB1 levels, proteins were transferred to a PVDF membrane using the iBlot2 dry transfer system (IB21002S, Thermo Fisher Scientific). Total protein levels were detected using Licor Membranes blocked with both protease and phosphatase inhibitor cocktails for anti-pDAB1 Western blotting with Odyssey blocking buffer (LI-COR Biosciences) for 1 h or 5% milk powder (M17200-100.0, RPI) for 2 h. The primary antibodies used were β-tubulin (ms; 1:2,000; 86298S, Cell Signaling), anti-phospho-Dab1 (Rb; 1:7,500; MBS8511213, MyBiorsource), total tau (ms; 1:1,000; ab80579, Abcam), phosphorylated tau (Ser396, rb; 1:1,000; 44-752G, Thermo Fisher Scientific), and anti-RELN (ms; 1:1,000; clone CR-50, D223-3, MBL) and were incubated in blocking buffer for 2 h at room temperature or 18 h at 4°C. Blots were washed three times with TBS-T buffer (Pierce, Thermo Fisher) and then incubated with secondary antibodies for 1 h or 45 min at room temperature (IRDye 800CW donkey anti-mouse, 925-32212 or IRDye 680CW donkey anti-rb; 1:10,000, 925-68073, Li-COR).Immunoreactive bands were detected using an Odyssey Infrared Imaging System and visualized with Image Studio software (version 2.1, LI-COR Biosciences). Detection of Dab1 was achieved by incubation with an anti-rb-HRP conjugated antibody (HAF008, R&D Systems) followed by a 5-minute incubation with West pico Super Signal™ West Pico PLUS chemiluminescent substrate and acquisition on a SyngeneG:Box Digital ECL detection system.

[0172] Additional Heparin-Sepharose Affinity Chromatography Details We examined the changes in heparin binding of RELN variants by chromatography using an optimized version of a protocol previously published by our laboratory (1). Briefly, after equilibration of a heparin column (BioVision 6554-1) at room temperature, the column was washed with five volumes of degassed 20 mM Tris-HCl buffer (pH 7.5). Recombinant C-terminal RELN peptides were produced and purified by Innovagen (Sweden): (WT) RKQNYMMNFSRQHGLRHFYNRRRRSLRRYP and (H3447R) RKQNYMMNFSRQHGLRRFYNRRRRSLRRYP. One mL of 50 μg / mL peptide (H3447 or WT, and H3447R) was recycled through the column five times, and the final flow-through was collected for further analysis. The column was washed five times with the same buffer, and the protein was eluted using a 0.05 M gradient of NaCl in 20 mM Tris-HCl (0 to 1 M, 1 mL per fraction). To ensure complete release of the protein, the column was washed with 5 M NaCl, 20 mM Tris-HCl. Three independent experiments were performed for C-terminal RELN WT and H3447R. All eluted fractions were analyzed spectrophotometrically by reading the absorbance at 280 nm using a Nanodrop 2000 spectrophotometer. Blank-corrected fractions were then analyzed using GraphPad Prism 8.

[0173] Additional ELISA details Enzyme-linked immunosorbent assays (ELISAs) were used to quantify changes in binding of RELN variants (media-derived full-length or CTR-RELN) to either VLDLr or ApoEr2, according to an optimized version of a previously published protocol. Briefly, ELISA strips (DY008, R&D Systems) were coated with 1 ng / μL (100 μL / well) of VLDLr (8444-VL, R&D) or ApoEr2 (TP320903, OriGene) receptor diluted in 25 mM Tris-HCl, 140 mM NaCl, 27 mM KCl, 2 mM CaCl, pH 7.4 (TBS-C buffer). After 18 hours of incubation at 4°C, plates were blocked for 1 hour with 3% BSA (22070008-6, Bioworld), 0.05% Tween-20 (Sigma-Aldrich) TBS-C buffer. We assessed binding by incubation with 100 μL / well of serial dilutions of recombinant RELN protein (Ser1221-Gln2666, 8546-MR-050, R&D) or RELN variants in TBS-C buffer for 1 hour at room temperature. For detection, we used goat anti-mouse RELN primary antibody (LS-C793521-100, LS-Bio, 1:2,000, 100 μL / well) for 1 hour at room temperature and donkey anti-goat IgG H&L, HRP, ab6885, Abcam, for 30 minutes at room temperature. Between each step, the plate was washed four times with TBS-C buffer (200 μL / well). The plate was washed three times, after which the colorimetric reaction (DY008, R&D Systems) was initiated. Stop solution (50 μL / well, DY008, R&D Systems) was added after 5 minutes of incubation, and the absorbance of the samples was measured at 450 nm using a Synergy 2 microplate reader (BioTek Instruments). Data processing and analysis were performed using Gen5 1.11 software and GraphPad Prism, respectively.

[0174] Additional SPR Assay Details Surface plasmon resonance assays (SPR) were used to perform binding kinetics according to a previously published protocol (7 24) using a Biacore 3000 instrument (GE Healthcare) at 25 °C, as a fee-for-service service provided by Precision Antibody (Maryland, USA). Biotin-labeled heparin (B9806, Sigma-Aldrich) was covalently coupled to a streptavidin-coated chip, and unoccupied sites were blocked with biocytin. A range of single analyte concentrations prepared in DPBS buffer was used, and the antigen was flowed over the chip using a flow rate of 30 μL / min. Binding of the antigen to the ligand was monitored in real time to obtain the association rate (ka) and dissociation rate (kd). The equilibrium constant (K D ) was calculated from the observed k and k. The accuracy of the SPR analysis was determined by chi-squared (χ) analysis as described in the statistical analysis section. We included in the analysis the WT and H3447R peptides, as well as peptides with the hypothetical H3447D change (RKQNYMMNFSRQHGLRDFYNRRRRSLRRYP) and H3447K change (RKQNYMMNFSRQHGLRKFYNRRRRSLRRYP), also produced and purified by Innovagen (Sweden).

[0175] Additional mouse model and in vivo analysis details We generated the RELNH3448R-Tg knock-in (KI) mouse model by introducing the H3448R mutation (CAC to CGT) into exon 64 of the 3' homology arm of the RELN gene in TACONIC mice. Gene targeting was achieved using C57BL / 6 ES cells. The targeted ES cells were injected into blastocysts, which were then transferred to foster mice and crossed with other mice to generate KI mice. Mice were euthanized using a CO2-saturated chamber. The cerebellum was removed by cervical dislocation and stored at -80°C, ensuring a postmortem time of less than 3 minutes for autopsy. All procedures were performed using protocols approved by the Institutional Animal Care and Massachusetts Eye and Otolaryngology Review Board. Brain homogenates from dissected cerebella were obtained using a tissue homogenizer (two 15-second pulses) in modified RIPA buffer (Cell Signaling) supplemented with protease (Roche) and phosphatase inhibitors (Sigma). The homogenized tissue was then vortexed for 20 seconds every 10 minutes for 1 hour and centrifuged at 10,000 rpm for 10 minutes at 4°C. The soluble protein fraction was then analyzed using the BCA assay (Pierce).

[0176] We measured Western blotting levels of RELN (clone CR-50, D223-3, MBL), Dab1 (clone G-5, sc-271136, Santa Cruz), and phospho-Dab1 (Tyr232, MBS8511213, My Biosource) in the cerebellum of adult male and female mice (6–12 months old, n = 3–4 per genotype) that were either wild-type, heterozygous for the RELN H3448R mutation, or homozygous.

[0177] Additional neuropathological characterization details The postmortem interval for the autopsy of brain tissue was 210 minutes. The brain showed predominantly frontal lobe atrophy; the brain and associated structures weighed 745.4 g, and the interuncular distance was 2.3 cm. After 5 days of fixation in 4% paraformaldehyde and sample preparation, 3 μm-thick sections were cut from the medial frontal gyrus (MFG), superior temporal gyrus (STG), medial temporal gyrus (MTG), inferior temporal gyrus (ITG), hippocampus / collateral sulcus (HP-C), hippocampus / uncus (HP-Uncus), amygdala (Amy), insula (Ins), inferior parietal lobule (IPL), occipital lobe (OL), cingulate gyrus (GC), lentiform nucleus (LN), caudate nucleus (CN), thalamus / hypothalamus (TH), cerebellum (CB), midbrain / pons (MP), and medulla oblongata (MO), deparaffinized, and stained with hematoxylin and eosin (HE) or amyloid beta (Aβ, 1:100; BAM-10, Mob410; DBS Emergo Europe, The Netherlands). The Hague, The Netherlands), hyperphosphorylated tau (pTau, 1:100; AT8, MN1020, Thermo Fisher, Dreieich, DE), ionized calcium-binding adaptor molecule 1 (Iba1, 1:500; 019-19741; Wako, Neuss, Germany), glial fibrillary acidic protein (GFAP, 1:200; M0761, DAKO GmbH, Jena, DE), C-terminal Reelin (RELN-CT, 1:200; E-5, sc-25346, Santa Cruz Biotechnology Inc., Heidelberg, DE) and apolipoprotein E (ApoE, 1:100; goat polyclonal, AB947, Merck Millipore, Darmstadt, DE), as well as specific secondary antibodies anti-mouse and anti-rabbit (P0260 and P0447, respectively, DAKO The tissue was then processed for immunohistochemistry (IHC) staining for neuronal nuclei (NeuN; 1:100; MAB377; Merck / Millipore, Darmstadt, Germany) and neuronal nuclei (NeuN; 1:100; MAB377; Merck / Millipore, Darmstadt, Germany). Visualization was achieved with 3,3'-diaminobenzidine (DAB, Ventana, Roche AG, Basel, Switzerland) and the Ultraview Universal Detection kit (Roche AG, Basel, Switzerland) according to the manufacturer's instructions.Automated immunostaining was performed using a Ventana Benchmark XT system (Roche AG, Basel, Switzerland) according to the manufacturer's instructions. Selected brain regions were also stained with Luxol Fast Blue (LFB) for myelin and Klüver-Barrera (KV) staining. Cresyl violet staining was used for the perineuronal area. Diagnosis was made by neuropathological workup by experienced morphologists blinded to sample origin (M.G. and D.S.F.). Sections were scanned using a Hamamatsu NanoZoomer automated digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan), and images and regions of interest (cortex for cortical areas, whole stained sections for noncortical areas) were acquired at a resolution of at least 1 pixel per μm. Signal intensity, along with grains and total area, was assessed after color deconvolution and thresholding in the brown (DAB) channel using ImageJ software (version 1.52p, NIH, Bethesda, MA, USA) (8). Neuronal counts were performed manually in selected regions of interest in the hippocampus and parahippocampal structures and normalized by region. Information on statistical analysis is reported in a dedicated section.

[0178] Additional immunoprecipitation details Mouse frontal cortex tissue was homogenized in ice-cold M-PER Protein Extraction Reagent (78503, Thermo Fisher Scientific) supplemented with phosphatase and protease inhibitors using the homogenizer described in the previous paragraph with two 15-second pulses. 100 μg of total protein was precipitated using either anti-phosphotyrosine magnetic beads (clone 4G10, 16-282, Millipore), anti-total Dab1 agarose beads (clone G-5, sc-271136 AC, Santa Cruz), or anti-normal mouse IgG isotype control agarose beads (SC-2343, Santa Cruz). Before immunoprecipitation, 10% of the sample was removed and analyzed as input. After collecting the unbound fraction, the beads were washed in PBS and then boiled in four volumes of sample reducing buffer for 6 minutes to release the immunoprecipitated proteins.

[0179] Additional sequencing analysis by mass spectrometry Immunoprecipitated fractions obtained using anti-Dab1-conjugated beads were separated by electrophoresis on 10% acrylamide precast gels (Biorad) and stained with Coomassie Blue (Thermo Fisher). The excised gel bands were analyzed by the Taplin Biological Mass Spectrometry Facility (Harvard Medical School, Boston, MA) through a fee-for-service service. The gel bands were then dehydrated in acetonitrile followed by speed-vac. The bands were rehydrated at 4°C in a 50 mM solution of ammonium bicarbonate supplemented with 12.5 ng / μL of modified sequencing-grade trypsin (Promega, Madison, WI). The samples were washed with 50 mM ammonium bicarbonate. Proteins were extracted by overnight incubation at 37°C and removal of the ammonium bicarbonate solution. Proteins were washed in 50% acetonitrile and 1% formic acid (9). The reconstituted samples were sequenced by electrospray ionization on a LTQ Orbitrap Velos Pro ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using a nanoscale reversed-phase HPLC capillary column after gradient elution with acetonitrile and formic acid. Protein-specific fragment ion sequences were analyzed using Sequest (10) (Thermo Fisher Scientific, Waltham, MA) with known peptide sequences.

[0180] Additional statistical analysis details All data shown are expressed as means and errors expressed as either standard error of the mean (s.e.m.) or standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, La Jolla, California, USA; graphpad.com). A P value of less than 0.05 and an α of 0.05 were considered statistically significant. We compared changes between primary cortical neurons treated with either mock, RELN WT, or RELN H3448R using Kruskal-Wallis analysis (Dunn's post hoc analysis for multiple comparisons of four independent experiments). Data are expressed as means ± s.e.m. For SPR data (Figure 2C and Figures 7A-7B), we confirmed the accuracy of the results by chi-squared (χ2) analysis, comparing the experimental sensorgram (colored line) with the mathematically generated sensorgram (black line) using BIAnalysis software. Values ​​between 1 and 2 were interpreted as significant (exact), and values ​​less than 1 were interpreted as highly significant (high precision). Western blotting analysis shown in Figures 2E–2F was performed using GraphPad Prism 9 with one-way analysis of variance and Fisher's LSD test for multiple comparisons. Neuropathology data (Figure 3) were analyzed and graphs were generated using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) and R statistical software (R Foundation for Statistical Computing, Vienna, Austria; R-project.org). Analyses included distribution analysis, and correlation analysis was performed using Spearman's rho test. Brain color maps were generated using the cerebroViz package for R. Statistical significance for all analyses was determined by *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

[0181] Example 1.1 Case report We identified a male carrier of the PSEN1 E280A mutation whose cognitive function remained intact until age 67. He completed five years of schooling in his home country (Colombia) and worked until retirement at age 64. He was married and had two children. An initial evaluation at age 67 revealed limited language learning ability and language difficulties related to functional independence. At age 70, the patient was diagnosed with mild cognitive impairment (MCI), characterized by declines in short-term memory and verbal fluency.

[0182] At age 72, his language skills further deteriorated. He progressed to mild dementia at age 72 (Table 2). The cognitive decline was preceded by an episode of urinary tract infection-associated septic shock. At age 73, he required assistance with basic and instrumental activities of daily living, meeting the criteria for moderate dementia. He died at age 74 from aspiration pneumonia, and his relatives consented to brain donation for neuropathology.

[0183] The subject's sister carried the PSEN1 E280A mutation and had severe dementia when she was first evaluated at age 64, progressing to end-stage dementia at age 72 (see pedigree in Figure 5A). According to family history, she had depression, hypothyroidism, hypertension, and cognitive decline at age 58 and developed dementia at age 61. Prior to the onset of dementia, she had suffered an eye injury and tibia fracture from a fall, which required surgery under general anesthesia. She died at age 73 from sepsis of pulmonary origin.

[0184] [Table 6]

[0185] Example 1.2 Identification of RELN-COLBOS mutations When the patient was 73 years old, he enrolled in the Columbia-Boston Biomarker Research Study (COLBOS) and underwent neuroimaging at MGH. Cortical-cerebellar amyloid PET scans, measured with Pittsburgh compound B (PiB), revealed a higher level of cortical Aβ plaque burden (distribution volume ratio (DVR) = 1.77) compared with that of younger relatives with typical ages of onset of MCI (1.49-1.60; Figure 1). Tau tangle burden in the inferior temporal lobe, measured with flortaucipitin (FTP), was similar to that seen in younger PSEN1 E280A MCI carriers with typical ages of onset (1.78 SUVR). However, he had relatively limited tau pathology in other neocortical regions, such as the entorhinal cortex (EC = 1.34 SUVR, Figure 1A, C), as well as the posterior cingulate cortex (PCC) and precuneus (PCC = 1.51; precuneus = 1.49 SUVR, Figure 1A), which typically show higher levels of tau pathology in PSEN1 E280A carriers, who develop MCI and dementia at typical ages (3) (Figure 1). The lack of tau pathology in the entorhinal cortex is a distinctive feature of RELN-COLBOS cases that may be important for their protective phenotype.

[0186] Measurement of glucose metabolic rate in the precuneus-to-whole brain region using fluorodeoxyglucose PET showed relatively preserved metabolism (Figure 1). He had brain atrophy, as measured by MRI-based hippocampal-to-whole brain volumes, similar to typical MCI carriers. These imaging findings suggest that protection against ADAD dementia in this patient and in the APOE3 Christchurch homozygous case (2) may be mediated through mechanisms that limit regional tau pathology despite high amyloid burden and substantial tau pathology in brain regions other than the medial temporal lobe (Figure 1).

[0187] Our genetic analysis confirmed that the subject was a heterozygous carrier of the PSEN1 E280A mutation (confirmed by single-cell RNA sequencing), excluded the presence of the Christchurch mutation (the subject was APOE3 / APOE3 and had a normal blood lipid profile), and identified a heterozygous variant in RELN (H3447R, Figure 5B), which we named "RELN-COLBOS," as the most likely missense variant potentially contributing to the phenotype in the protected subject. The RELN-COLBOS variant was found only in the subject and his sister (who was also APOE3 / APOE3), and she also had a later onset of cognitive decline. Briefly, we focused on the RELN-COLBOS variant because it ranked among the top three candidate genes in the Genomizer Priority Score analysis and because RELN is closely functionally related to APOE, and the gene mutated in other cases confers extreme protection against ADAD (2).

[0188] Example 1.3 Molecular analysis of RELN-COLBOS variants RELN binding induces clustering and activation of VLDLr and APOER2, triggering Dab1 (Dab1) signaling cascade activation and thus reducing tau phosphorylation (4-7). RELN-COLBOS was approximately twice as effective at inducing Dab1 phosphorylation as wild-type (WT) RELN (Figure 2A, p = 0.0246) and reduced tau phosphorylation at position 396 (8-10), an early marker of tauopathy, in primary cultured mouse cortical neurons (Figures 6A-6B). RELN-COLBOS did not directly affect RELN binding to VLDLr or APOER2 in cell-free binding assays.

[0189] The C-terminal region of RELN (CTR-RELN), where the H3447R variant is located, indirectly modulates signaling through interactions with a previously unidentified coreceptor on the cell membrane (11). CTR-RELN contains many basic amino acids that are highly conserved across species (11), and we hypothesized that these may mediate interactions with glycosaminoglycans (GAGs). Interaction with GAGs is a rate-limiting step in the interaction of ApoE with some receptors (12), while the role of GAGs in RELN activity has not been fully elucidated (11, 13). We used affinity chromatography to examine heparin (a type of GAG) binding of recombinant CTR-RELN peptides. Both CTR-RELN WT and CTR-RELN H3447R bound to heparin. CTR-RELN H3447R required a higher NaCl concentration to be released from the heparin column, suggesting a higher binding affinity (Figure 2B). Measurement of the kinetic constants by surface plasmon resonance (SPR) showed that the affinity of CTR RELN H3447R was approximately twice that observed in the WT (Fig. 2C).

[0190] Substitution of histidine at position 3447 with aspartic acid, a highly acidic amino acid (H3447D), reduced heparin interaction, whereas substitution with lysine, a basic amino acid (H3447K), had minimal effect, further supporting the important role of position 3447 in GAG binding. Measurement of kinetic constants by surface plasmon resonance (SPR) indicated that the affinity of CTR RELN H3447R was approximately twice that observed in WT: H3447R>WT>H3447K>>>H3447D (Figure 2C, and Figures 7A and 7B). RELN H3447D could not be produced in sufficient quantities for signal transduction analysis. This limitation is consistent with a previous report of specific mutations in the C-terminus that limit RELN secretion (52).

[0191] We further investigated the molecular mechanism underlying the observed gain of function by using ELISA to assess the direct binding of recombinant RELN H3448R to the canonical RELN receptors, VLDLr and APOER2 (53). Because RELN oligomerization is a key property that modulates receptor binding (53), we used experimental conditions with RELN H3448R, RELN WT, or an equimolar mixture of WT and variant proteins. In all conditions, full-length RELN H3448R and RELN WT did not differ in their ability to bind to VLDLr or APOER2. We further determined that the C-terminal regions of the RELN(CTR-RELN)WT or CTR-RELN H3447R recombinant peptides were unable to directly bind to VLDLr or APOER2. This finding confirms previous reports demonstrating that the fifth and sixth reelin repeats (R5-6) of RELN are required to mediate receptor binding (53). We conclude that the gain of function of the RELN H3447R variant is unlikely to be explained by a direct change in binding affinity for the VLDLr or APOER2 receptors.

[0192] Example 1.4 Disease-modifying effects of RELN-COLBOS mutations To further support genetic imputation of causality, a common practice in studying rare variants, we generated knock-in mouse models harboring the RELN-COLBOS variant equivalent (H3448R or mRELN-H3448R (11)). These mouse models were viable and fertile and lacked the overt structural and phenotypic brain abnormalities of RELN loss-of-function variants (e.g., cortical lamination defects, abnormal neuronal migration, and cerebellar hypoplasia) (14, 15). Analysis of the cerebellum of mice carrying mRELN-COLBOS confirmed the gain-of-function observation for RELN-H3448R, as determined by hyperphosphorylation of Dab1 in males (Figure 2D, Figure 2F; p = 0.0284), and revealed a propensity for the formation of higher molecular weight protein oligomers in RELN-COLBOS, a feature that may be important for its high activity (16). The cerebellum is a target of the RELN phenotype in mice and humans. (14, 15) Morphological analysis revealed a modest but statistically significant increase in the number of cerebellar neurons in mice carrying the RELN-COLBOS variant, supporting the hypothesis of a gain-of-function mechanism; however, the neuronal density phenotype was not observed in other brain regions.

[0193] This mouse model allowed us to examine the sexually dimorphic effects of the RELN-COLBOS variant, a feature described for conditions associated with genetic mutations in RELN, including schizophrenia, bipolar disorder, autism, and Alzheimer's disease (17-22). Increased Dab1 phosphorylation and enhanced RELN oligomerization were observed only in male mice (Figure 2D). This finding was consistent with our observation of an optimal association of RELN-COLBOS with protection against ADAD in males versus females. Homozygosity was required to detect changes in Dab1 activity and GSK3β activity (another downstream target of RELN signaling) associated with the RELN-COLBOS variant. Together, these data indicate that RELN H3447R is a gain-of-function (hypermorph) variant.

[0194] To attempt to correlate the RELN-COLBOS phenotype in mice and humans, we used crossbreeding techniques with our knock-in mouse model and a tauopathy mouse model, specifically the STOCK Tg(Prnp-MAPT*P301L)JNPL3Hlmc mouse from Dr. Hutton's laboratory, distributed by Taconic. This mouse model expresses a mutation in the tau gene, which is commonly used to study tauopathies. 23 The decision to use this mouse model was based on the known effect of RELN signaling on tau phosphorylation, which results in the accumulation of tau neurofibrillary tangles and neuronal loss in specific brain regions. 24 Based on our clinical observations, postmortem human brain samples from protected cases showed a relative reduction in tauopathy in certain brain regions. Our study found that male P301L mice expressing the RELN-COLBOS allele had a substantial reduction in human tau phosphorylation (pTau205) in the hippocampus (Figures 2J-2K) and medulla oblongata compared with controls (Figure 2J). We also observed that abnormal limb grip responses, a common consequence of tauopathy in mice, were significantly rescued in RELN-COLBOS mice carrying a tau transgene (Figures 2L-2M). Although additional studies in this model are warranted, our findings strongly support our hypothesis that RELN-COLBOS is a gain-of-function mutation, which appears to be genetically linked to resilience to tauopathy. This analysis also provides direct evidence of rescue in a mouse model carrying a MAPT mutation associated with frontotemporal dementia.

[0195] Postmortem examination of the case revealed neuropathological evidence of severe AD (classified as CERAD C, Braak stage VI, and Thal phase 5) with widespread amyloid and tau pathology (Figure 3A). Recently, we reported the neuropathological profile of a PSEN1 E280A carrier homozygous for the APOE Christchurch mutation. This case exhibited a unique pathological phenotype among PSEN1 E280A cases, with significantly less p-tau pathology in most brain regions, except for the primary visual cortex (23). In contrast, side-by-side comparisons showed that RELN-COLBOS cases had more p-tau pathology than APOEch cases, with the exception of certain regions. Both cases showed widespread Aβ pathology in all assessed regions, although with some individual variation.

[0196] Because the hippocampus and associated cortex are known to be affected early in AD (24), we focused our analysis on these structures. Neurons within layer II of the entorhinal cortex, and entorhinal cortex neurons in general, are particularly susceptible to aging and AD (25). We measured neuronal density in the hippocampus and parahippocampal gyrus regions of RELN-COLBOS cases, AD-resistant APOEch cases, typical PSEN1 E280A cases, and typical sporadic AD cases (Figure 3B). We found that lower AD pathology was associated with higher neuronal density in the entorhinal cortex of RELN-COLBOS cases compared with APOEch cases or FAD and sporadic AD controls (Figure 3C). This relationship was not evident in other subregions, such as the CA1 (Figure 3C; S17 and S18). RELN-COLBOS and APOEch cases showed characteristically lower intraneuronal ApoE signals compared with FAD and sporadic AD controls ( Fig. 3<em>D ), whereas RELN-COLBOS showed higher Reelin intracellular signals in the white matter ( Fig. 3<em>D ).

[0197] The neuropathological findings are consistent with our in vivo neuroimaging observations and confirm the potential role of entorhinal cortex integrity as a target of RELN-mediated mechanisms important for resilience to ADAD.

[0198] [Example 2] In silico modeling of Reelin To understand the potential importance of Reelin CTR, we used computational analysis to show that the Reelin CTR, which contains basic AA residues that likely form a charge-based binding site, is highly conserved across mammalian species. We also show that Reelin CTR appears to be prone to intrinsic unfolding. Intrinsically disordered regions (IDRs) within proteins lack defined tertiary structure and may be important for several biological functions, including protein interactions. In addition, IDRs may play a role in interactions within plaque deposits in individuals with neurodegenerative diseases, including AD. Previous studies have shown that Reelin CTR is highly conserved across vertebrate species (11). This study also showed that Reelin is not required for secretion but may play a role in downstream signaling.

[0199] method Phylogenetic trees were generated using BEAST, which uses Bayesian Markov Chain Monte Carlo (MCMC) to generate phylogenetic trees based on the Reelin CTR sequence. PONDR® was used to access intrinsically disordered regions of RELN. Specifically, VL-XT (Variously Long Disordered Regions and X-ray Characterized Terminal Disordered Regions) is a combination of three feedforward neural networks. Individual neural networks were trained for long disordered regions (>39 AA), n-terminal disorders, and c-terminal disorders.

[0200] result Reelin CTR is highly conserved Again, the Reelin CTR is highly conserved among mammalian species (Figure 8). Therefore, it is hypothesized that the C-terminal region of Reelin is important for Reelin binding to HSPGs and subsequent regulation of downstream pathways. Among species available in the National Center for Biotechnology Information (NCBI) database, there is a high level of conservation at position 3447 among most mammalian species, except for horses (Equus caballus, donkeys (Equus asinus), and Przewalskii)).

[0201] Reelin CTR contains a GAG-binding motif Initial structural data suggest that there may be two binding sites in the c-terminal region (Figure 9). The location of the critical AA that interacts with GAGs can be predicted based on the orientation of arginines in the known heparin-binding motif ((B)Bxx(x / B)BxxB(B)), where B represents a positively charged AA and (x) represents a non-consensus AA (29). The interaction of basic AA with acidic GAGs is primarily driven by electrostatic forces (30). Therefore, it is hypothesized that these basic amino acids may be particularly important for GAG interaction and that the c-terminal region may contain binding sites that interact with GAGs.

[0202] H3447 is oriented in the same direction as other basic amino acids Bioinformatics analysis showed that the RELN CTR, including the H3447R position (purple), which was found to be mutated in cases with resilience to AD, is highly conserved across mammalian species. Many of the conserved amino acids are highly basic residues, suggesting potential binding to GAGs. The basic AA at position 3447 (shown in magenta) face the same direction, which may indicate a key role in the interaction with heparin. Here, the orientation of the basic amino acids may contribute to creating a binding site.

[0203] conclusion The Reelin CTR is highly conserved across diverse species. The basic AA hypothesized to contribute to GAG interaction is over 90% conserved. The only species with a different AA at position 3447 is the equid species (Q3447), which is clearly uncharged and should not pose a significant difference compared to the weakly basic histidine. Furthermore, these basic AA, including position H3447R, face the same side of the alpha-helix, which has previously been shown to be an important face for GAG interaction.

[0204] [Example 3] Kinetics of Reelin-heparin interaction To further understand the dynamics of the RELN CTR-heparin interaction, surface plasmon resonance (SPR) adds additional support to the proposed neuroprotective mechanism of the H3447R variant. The equilibrium dissociation constant (K D ) indicates that H3447R has approximately twice the binding affinity of H3447. Furthermore, biolayer interferometry was used to further confirm these results.

[0205] method Surface plasmon resonance Surface plasmon resonance (SPR) assays were performed to measure binding kinetics using a Biacore 3000 instrument (GE Healthcare) at 25°C according to a previously published protocol (34, 24) via a fee-for-service service provided by Precision Antibody (Maryland, USA). Heparin was covalently coupled to a streptavidin-coated chip at a concentration of 13 response units (RU), and unoccupied sites were blocked with biocytin. Antigen was flowed over the chip at a flow rate of 30 μL / min using a range of single analyte concentrations prepared in DPBS buffer. Binding of the antigen to the ligand was monitored in real time to obtain the association rate (ka) and dissociation rate (kd). The equilibrium constant (KD) was calculated from the observed ka and kd. The accuracy of the SPR analysis was determined by chi-squared (2) analysis, as described in the statistical analysis section.

[0206] Biolayer Interferometry (BLI) Because the peptide alone may be below the detection limit in this experimental design, we used the Fc-fused Reelin CTR peptide. To assess heparin-Fc-Reelin interactions, we used an octet system (biolayer interferometry) to evaluate heparin-protein kinetics. Biotinylated heparin (50 μg / mL) was immobilized on the biosensor tip surface for 300 seconds on pretreated biosensors. This was followed by quenching with 50 μg / mL biocytin, 120 seconds in baseline buffer diluent, 120 seconds with 200 nM analyte (Fc-fusion protein), and 120 seconds of dissociation in assay buffer.

[0207] Biolayer interferometry (BLI) was further used to evaluate NRP1-protein dynamics at 30 °C and 1000 rpm agitation. NRP1 (R&D 3870-N1-025) at 1 mg / mL was biotinylated at a 1:2 molar ratio and immobilized on the tip of an SA biosensor (Pall ForteBio). This was followed by 1) 180 s of baseline buffer dilution, 2) ligand (NRP1) loading, 3) 180 s of baseline buffer dilution, 4) 240 s of association (analyte), and 5) 300 s of dissociation in assay buffer. Assay buffer: SD buffer (pH 7.4 PBS, 0.05% Tween 20, 0.01% BSA).

[0208] The experimental data were fitted to a 1:1 binding model and analyzed by global fitting using octet data analysis software to obtain the K D was calculated.

[0209] result We recently demonstrated by surface plasmon resonance (SPR; published in NEJM) that the RELN CTR H3447R peptide has a two-fold higher affinity for heparin compared to RELN WT. These kinetics were assessed with synthetic peptides (AA3431–AA3460). To confirm this finding in proteins produced in mammalian cells, we engineered Fc-fused RELN CTR proteins. The interaction of Fc-fused RELN WT and H3447R with heparin was assessed using biolayer interferometry (BLI; Figure 1C). These proteins are cleaved by furin during production in mammalian cells, resulting in a short RELN CTR variant lacking the last six amino acids. In this configuration, RELN H3447R also has approximately two-fold higher affinity for heparin compared to WT. We observed that K a K D We found that the H3447R mutation contributes significantly to the difference in CTR-heparin affinity, indicating that the variant requires less energy to interact with heparin or that it saturates the heparin substrate twofold faster than the WT. Therefore, the CTR sequence upstream of furin cleavage may contribute to the CTR-heparin affinity in the presence of the H3447R mutation.

[0210] conclusion The kinetic data indicate that RELN H3447R has approximately two-fold higher interaction with heparin compared to RELN WT. The relatively small difference is relevant given that this difference was seen across two different experimental systems with both the long and short RELN variants. These data indicate that RELN H3447R may increase the association of RELN with GAGs and therefore may have a competitive advantage over the interaction of other molecules or proteins with GAGs in vivo.

[0211] [Example 4] Affinity data for Reelin-heparin interactions We used high-performance liquid chromatography (HPLC) to evaluate the interaction between HSPG and reelin CTR. A delayed interaction peak in isotonic PBS supplemented with 1 M KCl indicates a high peptide-heparin interaction. Our data show a delayed retention time peak with zero to two arginines; this difference in retention time peak was observed in both long and short peptides. Therefore, the basic amino acids at positions 3446–3447 are important for heparin interaction. Since all short peptides have earlier retention time peaks, a second binding site may exist in the last six AA of the long peptide sequence. We also show that the basic mutation at position 3447 has a later retention time peak compared to neutral (H) or acidic (D) AA.

[0212] method Heparin column chromatography To assess Reelin-heparin binding affinity, Reelin variants were loaded onto a heparin column in 20 mM Tris-HCl over 20 1 mL column volumes, ranging from 0.05 to 1 M NaCl in 0.05 M increments, as described in Arboleda et al. (2019). Sample fractions were eluted with increasing NaCl steps to determine the ionic strength required to disrupt the bond between Reelin and heparin. Therefore, the stronger the interaction, the higher the salt concentration required to disrupt the bond.

[0213] High-performance liquid chromatography (HPLC) HPLC provided more accurate results. 50 μL of 0.3 μg / μL uncleaved or cleaved WT Reelin peptide was used in 0.15 M KCl, 10 mM PBS at 0.3 mL / min. Samples were loaded at 0.15 M KCl from 0 to 13.5 min, ramped to 0.5 M KCl from 13.5 to 14.5 min, ramped to 0.5 M KCl from 14.5 to 24.5 min, eluted with a 0.5 to 1 M KCl gradient (stamp), eluted with 1 M KCl from 24.5 to 45 min (isocratic elution), washed with 1 M KCl at 0.6 mL / min from 45 to 55 min, ramped to 0.15 M KCl at 0.3 mL / min from 55.0 to 56.0 min, and reset to 0.15 M KCl from 56.0 to 59.0 min. Based on the fluorescence properties of aromatic amino acids, fluorescence intensity was measured at an excitation wavelength of 260 nm and an emission wavelength of 290 nm. To evaluate the fluorescence properties of the peptides, 15 μl of approximately 0.6 μg / mL of peptide was diluted in 500 μL of 0.15 M KCl, 10 mM PBS, and the excitation and emission peaks of the peptides were calculated from the 2D graph data.

[0214] Method development for HPLC The column chromatography method was adapted from previous work in our laboratory (2). Based on the column chromatography data, the initial protocol used a 0-1 M NaCl gradient and a 5 M salt wash to reset the HPLC column. However, high salt gradients were corrosive to HPLC instrument components. Therefore, KCl was considered because it has a higher ionic strength than NaCl. Using a lower molarity buffer with KCl compared to NaCl helped keep the HPLC pump free of residual salt and avoid potential corrosion, which could disrupt the heparin-Reelin CTR interaction. Because a lower concentration of salt was used, the time required for the 1 M KCl isotonic hold was extended until the peptide signal was no longer detectable, after which the column was equilibrated for the next sample.

[0215] result Heparin column chromatography shows that RELN H3447R allows increased interaction with heparin Peptides eluted from heparin column chromatography were quantified at each 0.05M NaCl step and evaluated by both Nanodrop and ELISA. Overall, Nanodrop measurements have sharper, more defined peaks compared to ELISA, likely because ELISA relies on the affinity of the antibody for RELN CTR, while Nanodrop directly quantifies the amount of peptide. The elution step peak is determined as the maximum ratio to the input per sample.

[0216] For all RELN variants, the long (uncleaved) peptides had a later retention time peak compared to the short peptides, suggesting that the last six AAs may contain the heparin-binding site. Furthermore, the basic AA (R, K) at position 3447 has an elution peak at a higher salt concentration compared to WT or H3447D. Therefore, the charge of the AA at position 3447 may be an important factor in the RELN CTR-GAG interaction.

[0217] While heparin column chromatography can quantify the RELN CTR-heparin interaction to some extent, HPLC can provide more accurate quantification of this interaction using a gradient. Representative normalized peaks show that RELN H3447R has delayed peak times compared to RELN WT for both long and short peptides. Significant differences are characterized when the average peaks have more than 30 seconds between each retention time.

[0218] conclusion Chromatography data showed that the RELN H3447R mutation increased heparin interaction as a model for global GAG interactions. Because the excitation and emission spectral peaks are similar between peptide variants, the difference in retention time of the RELN peptide variants is due to the AA mutation, not a change in fluorescence properties or peptide detectability. Additional mutations, along with H3447R, may include substitutions at other key arginine residues (R3451, R3454, and R3458) that are directed to contribute to the RELN CTR-GAG interaction.

[0219] [Example 5] Other potential Reelin CTR interactions Recent studies have shown that the reelin CTR also interacts with neuropilin (NRP1) (46). This study found that the last six AA residues after the furin cleavage site are important for NRP1 interaction. Uncleaved "long" CTR-RELN interacts with NRP1, whereas cleaved "short" CTR-RELN does not (46). Therefore, we used BLI to further evaluate whether the H3447R mutation affects the NRP1-RELN CTR interaction. All known NRP1-binding domains have been shown to have a C-terminal arginine with a motif called CendR (R / KXXR / K) (47). In particular, the C-terminal arginine is involved in binding to the NRP1 b1-b2 domain (48). Therefore, based on sequence alone, the RELN CTR may have additional interactions in addition to GAG. Notably, the RELN H3447R mutation may contribute to optimizing the NRP1-binding motif, especially after furin cleavage.

[0220] method docking The NRP1 and RELN CTR structures were uploaded to ClusPro 2.0 (cluspro.org / ) to model these protein interactions. Briefly, the top 1,000 rotatamers with the lowest scores among 70,000 rotations were selected. The algorithm clustered these 1,000 rotations, ranking them with their nearest neighbors within a 9 Å RMSD radius. The top-ranked one then became the center of the first cluster, and this process was repeated to rank up to 30 clusters based on size (i.e., number of neighbors). Next, energy minimization was used to remove steric overlaps. Therefore, ClusPro considers the lowest-energy structures in the largest clusters, rather than simply minimizing energy (49, 50).

[0221] The output of the ClusPro algorithm is a list of the most likely conformations. One model for each ApoE peptide was selected as preliminary data and further analyzed using PyMol version 2.3.3 (pymol.org / ). In the future, the top three lowest energy models ranked by ClusPro will be evaluated. Polar contacts between peptides were defined as contacts within 3 Å using the "Measurement Mode" function in the PyMol software.

[0222] Biolayer Interferometry (BLI) Biolayer interferometry (BLI) was used to assess NRP1 protein dynamics at 30 °C and 1000 rpm agitation using a previously published method. NRP1 (R&D 3870-N1-025) at 1 mg / mL was biotinylated, desalted, and immobilized on an SA biosensor tip (Pall ForteBio) at a 1:2 molar ratio. This was followed by 1) 180 s of baseline buffer dilution, 2) ligand (NRP1) loading, 3) 180 s of baseline buffer dilution, 4) 240 s of association (analyte), and 5) 300 s of dissociation in assay buffer. Assay buffer: SD buffer (pH 7.4 PBS, 0.05% Tween 20, 0.01% BSA).

[0223] The experimental data were fitted with a 1:1 binding model and analyzed by global fitting using Octet Data Analysis software to obtain the K D was calculated.

[0224] result Molecular docking suggests that the alpha- and beta-GAG binding sites overlap with the NRP1 binding site. However, the n-terminal AA of the RELN CTR may also contribute to NRP1 interaction.

[0225] Vascular endothelial growth factor (VEGF) is known to interact with NRP1 and was therefore used as a positive control (31). As previously mentioned, our Fc-fusion protein was produced in mammalian cells and was therefore predicted to be cleaved by furin. Previously published data concluded that WT CTR does not bind to NRP1 after removal of the last six amino acids by furin.

[0226] We confirmed the enhanced interaction of CTR-RELN H3447R with heparin through the use of isothermal titration calorimetry (ITC, Figure 2G) and biolayer interferometry (BLI, Figure 2H). a is K dThe COLBOS mutations were found to be the major contributor to the difference in the β-helix values, and the mutant CTR-RELN was found to have a more negative Gibbs free energy compared to the WT, suggesting that the COLBOS mutations enable spontaneous CTR-RELN reaction with heparin. Our nuclear magnetic resonance (NMR, Figure 2I, Figure 9) studies revealed that in the presence of trifluoroethanol, CTR-RELN can have an alpha-helical structure containing a flexible region with a domain we termed the "Flexibility Vertex," which cannot be structured under native conditions, as revealed by circular dichroism (Table 3).

[0227] [Table 7]

[0228] Heparin-binding analysis of a library of mutant CTR-RELN peptides revealed two binding sites for GAGs, which we named the "alpha-GAG binding site" and "beta-GAG binding site" (Figure 9). The alpha-GAG binding site is located in the last six amino acids and overlaps with a previously identified binding site for neuropilin 1 (NRP1), which is released by furin. The beta-GAG binding site is located upstream of the furin cleavage site and spans amino acids 3446–3451. Our studies also found that CTR-RELN COLBOS possesses a 10-fold higher affinity for NRP-1 compared to the wild-type version of CTR-RELN due to the optimization of the beta-GAG binding site (Table 4). To support these claims, we conducted extensive studies of the interaction of mutant CTR-RELN with heparin by HPLC (Figure 11D), BLI (Figure 2H), and NMR structure (Figure 2I, Figure 9).

[0229] [Table 8]

[0230] Our data showed that the RELN CTR peptide, lacking the alpha-GAG binding site located in the last six amino acids of RELN CTR, had an affinity for NRP1 that was 100-fold lower than that for VEGF, consistent with previously published data. However, with the H3447R mutation, the affinity was 10-fold higher compared to WT, suggesting that H3447R either forms a new binding site or significantly increases affinity for NRP1 via the beta-GAG site. In summary, the RELN H3447R peptide had an approximately 10-fold increased affinity for NRP1 compared to the WT RELN peptide, which is much closer to the affinity range for VEGF and therefore represents a gain of function.

[0231] conclusion Previously published data indicated that NRP1 interaction is lost in the WT-short RELN peptide. Our data further support this theory, demonstrating that the WT-short has significantly less interaction with NRP1, particularly compared to VEGF. However, these data support the hypothesis that RELN H3447R adds or substantially optimizes a new beta-GAG binding site, given that the mutation rescues the RELN CTR-NRP1 interaction by approximately 10-fold. Therefore, RELN gain-of-function may be achieved by introducing the H3447R variant and / or by reducing furin cleavage of RELN-CTR by introducing R3454A.

[0232] [Example 6] Structural analysis of Reelin Circular dichroism (CD) was used to further understand the unstructured nature of Reelin CTR. Modeling results indicated that Reelin CTR may have higher fluctuations in the most unfolded regions of the Reelin protein. Furthermore, recently published data demonstrated a high-resolution structure of the C-terminal CR8 of Reelin but failed to characterize Reelin CTR, potentially due to the flexibility of the domain (32). NMR assisted the acquisition of high-resolution structural information using CD and found that 50% TFE (2,2,2-trifluoroethanol) was required to stabilize the WT Reelin peptide structure. Both techniques provided insight into the unstructured regions of RELN CTR. In silico techniques added additional support, demonstrating that flexible regions can be predicted through the combination of the RELN CTR sequence with known unfolded regions of other proteins.

[0233] method Normal Mode Analysis (NMA) NMA is a method based on principal component analysis (PCA) that considers the harmonic oscillator potential of peptides. Here, the force field is simplified to a ball-and-spring elastic network model. The model considers the harmonic oscillator potential of the Ca atom of each AA as nodes connected by springs. Here, the energy function is minimized by transforming the Hessian matrix, consisting of the second derivative of the potential energy, into a diagonal matrix (33).

[0234] Predictor of Natively Disordered Regions (PONDR) VL-XT Denaturation Analysis This algorithm uses a feed-forward neural network to predict denatured regions based on AA sequences. VL-XT combines three feed-forward neural networks trained on 1) long regions of various lengths (VL), 2) N-termini characterized by X-rays, and 3) C-termini characterized by X-rays (XT). Compared with NMA, VL-XT can predict denatured regions using known denatured regions of other proteins.

[0235] Circular dichroism CD signals were recorded at wavelengths ranging from 190 to 260 nm at 0.1 nm intervals using a quartz cuvette with a 1 mm path length. Samples were scanned at 50 nm / min with a 1 nm bandwidth and a 2 s integration time. Data were plotted as the average of four spectra. Data were deconvoluted using the online deconvolution software BeStSel (bestsel.elte.hu / ).

[0236] 2D nuclear magnetic resonance (NMR) structure The NMR structure was completed using a fee-for-service service. The final NMR sample consisted of 50% (V / V) HO, 50% (V / V) of 98% pure 2,2,2-trifluoroethanol-d2 (CF3CD2OH), 0.25 mM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid, dissolved in HO) as an internal standard, and approximately 3.3 mM reelin peptide. 1 H TOCSY (80 ms spin-lock time), NOESY (200 ms mixing time), 1 H- 15 N HSQC, 1 H- 13 C HSQC experiments were performed on an Agilent 600 MHz NMR spectrometer (DD2) at 25 °C. Data were analyzed with NMRPipe and CCPNmr 2.4.2; structures were calculated with Cns 1.2.1 and aria 2.3.2. A total of 200 structures were calculated, and the 20 lowest energy structures were selected.

[0237] result Structural characterization of RELN CTR reveals an alpha helix with flexible arms Using circular dichroism (CD), we determined that RELN CTR was not highly structured under native conditions. The addition of 50% TFE (2,2,2-trifluoroethanol) stabilized the unstructured CTR, allowing for domain characterization. With 50% TFE, the peptide secondary structure more closely resembled an alpha helix, with characteristic negative minima at 208 nm and 222 nm. We cannot exclude the presence of unstructured regions within RELN CTR, even in the presence of TFE. This structure of the denatured peptide may explain the greater percentage of "antiparallel β-sheet" or "other category" structures found in the peptide alone compared with the peptide with 50% TFE (34, 35).

[0238] The CD results informed the NMR experimental conditions for resolving the Reelin CTR at atomic resolution. NMR is advantageous for smaller proteins because it can capture dynamics and eliminates the need for crystallization (51). Using the 20 lowest-energy structures, we generated the final NMR structure of WT RELN CTR at approximately 2.8 Å resolution. Using this technique, we confirmed that the C-terminal domain of RELN in the presence of TFE forms an alpha helix with a highly flexible arm pointing toward the C-terminal region. The apex of the flexible region extends from the glycine residue at position 3444 to histidine 3447, which is mutated in the protected case. We named this region the flexible apex domain (FVD). The consensus motif for GAG binding contains basic amino acids on the same side of the alpha helix and in consecutive turns separated by approximately 3.5 amino acids, which allows for strong binding (36). In addition, the 20 lowest energy structures show that the helix containing position 3447 unwinds, potentially exposing H3447R for GAG interaction.

[0239] Reelin CTR-GAG interaction depends on a basic AA near H3447R Our structural data, combined with our functional analysis, indicate that the alpha-GAG binding site appears to depend on R3454, R3457, and R3458. As we concluded earlier from our functional studies, R3446 also mediates GAG interaction, likely in concert with R3451, which comprises the beta-GAG binding side. The structure reveals that the arginine introduced by mutation at position 3447, found in protected cases, is appropriately oriented for GAG interaction. Importantly, the new beta-GAG binding site is not cleaved by furin, making it a constitutively active site of RELN for interaction with GAGs, NRP1, and potentially other receptors. Polar contacts between WT RELN CTR and heparin suggest that this interaction is likely due to basic amino acids in the alpha- and beta-GAG binding sites (Figure 9).

[0240] The Reelin C-terminal peptide has more atomic fluctuations in the alpha- and beta-GAG binding sites It is noted that the glycine residues represent a structured region found in the NMR spectrum, toward the n-terminal region of the peptide. The data suggest that the last six AA of the H3447R long peptide have more fluctuation compared to the WT Reelin long peptide. The WT and H3447R Reelin short peptides were comparable; overall, the short peptide had less fluctuation compared to the long peptide. Atomic fluctuations were measured as the mean square deviation over all normal vibrations.

[0241] Modeling shows that Reelin CTR has more denaturation To assess the flexibility of the region, the harmonic potential of each amino acid was first evaluated using normal mode analysis (NMA) (37). NMA indicates that the long peptide has high flexibility, and the RELN H3447R length has higher flexibility compared to the WT length. The Reelin short peptide shows similar flexibility. Overall, NMA indicates that the long peptide has higher flexibility compared to the short peptide; however, because all other peptides except the WT length were modeled peptides, these results may not be completely accurate. In contrast, the Predictor of Intrinsically Disordered Regions (PONDR) analysis scores the relative degeneration of RELN variants using only the AA sequence. The WT length has a score of 0.9032 at AA3444-3460 compared to the H3447R length, which has a score of 0.9225 at AA3440-3460. Therefore, the H3447R mutation increases degeneration in the RELN CTR, which may contribute to its increased ability to bind to GAGs or inhibit AB plaque formation. In contrast, R3446H reduces the unfolded region to 3447-3460, with an average score of 0.8583. Therefore, arginine in the beta-GAG binding site may contribute to increased unfolding in Reelin CTR. The neural network-based method VL-XT more closely predicted the unfolded region of RELN CTR found in experimental data. Here, the predicted unfolded region in the RELN C-terminus begins at G3444, which was also represented in the 20 lowest-energy NMR structures. Because RELN CTR is highly conserved across species and is also highly unfolded, this supports the notion that its function requires highly unfolded properties.

[0242] conclusion The RELN CTR has an unstructured region beginning at the FVD that also contains alpha- and beta-GAG binding sites. The RELN H3447R mutation creates a new beta-GAG binding site at a position that aligns with other basic amino acids in the GAG-binding motif, contributing to GAG interaction. Furthermore, computational analysis confirms that the region from G3444 to P3460 is highly unstructured. Unstructured regions may contribute to enhanced cellular interactions with GAGs or other proteins. We estimated the potential flexibility within the RELN CTR using NMA and VL-XT, a machine-learning-based method using known unstructured regions.

[0243] [Example 7] Reelin CTR modulates Aβ aggregation Although previous experiments suggest a protective mechanism based on RELN-GAG interactions, it is important to assess whether RELN H3447R directly modulates known hallmarks of Alzheimer's disease. Previous studies have suggested that altered molecular regions can modulate Aβ aggregation (38); also, purified RELN has been reported to reduce amyloid-β (Aβ) aggregation in vitro (39). Therefore, we investigated whether this effect is due to CTR-RELN.

[0244] method Thioflavin T (ThT) binds to amyloid and has been shown to correlate linearly with amyloid concentration. Therefore, the ThT assay can be used to quantify AB aggregation using previously developed methods (40).

[0245] We incubated CTR-RELN with Aβ and monitored aggregation using a thioflavin T fluorescence assay, normalized to the maximum ThT release of Aβ alone. We also assayed Aβ in the presence of morin, a known inhibitor, as a negative control (41). To better understand the potential role of the H3447R mutation in amyloid pathology, we included long and short WT and H3447R RELN CTR peptides (Figures 14 and 15). Negative controls included long and short RELN CTR peptides with a neutrally charged AA (histidine) instead of a basic AA in the alpha- and beta-GAG binding sites.

[0246] result The RELN peptide alone does not induce ThT fluorescence (Figure 15). We found that RELN CTR significantly reduced amyloid aggregation, with the long peptide being as effective as the short peptide (Figures 14A-14B, Figure 15). The RELN H3447R variant peptide was as effective in inhibiting ThT aggregation as the WT for both the long and short variants (Figures 14A-14B). Interestingly, the anti-aggregation effect of CTR-RELN was significantly reduced when R3431, K3432, and R3446 were replaced by histidines in the short peptide variant, thus suggesting that the N-terminal amino acid may play a major role in interacting with amyloid seeds (Figure 14B). While the H3447D-long and H3447K-long variants similarly reduced aggregation, the H3447D-short variant had approximately three-fold higher aggregation compared to the H3447K-short variant. Therefore, the charge of the beta-GAG binding site may not be the only contributing factor affecting its interaction with Aβ plaques. This hypothesis is further supported by a control in which several arginines in the RELN CTR, including those in the alpha-GAG binding site, were replaced with histidines (Figure 14B). In this case, there was significantly higher Aβ aggregation in samples with short peptides compared to long peptides. The negative control, the peptide alone without Aβ, shows minimal ThT fluorescence.

[0247] conclusion The RELN CTR may play a role in AB aggregation, but given that the amyloidogenicity of AB is enhanced by enrichment of the sequence with hydrophobic AA residues (42), the mechanism may rely on the overall flexibility of the peptide rather than the charge of the interaction. It has previously been shown that intrinsically disordered proteins (IDPs), such as certain truncated APP variants, can form seeds for amyloid aggregation. Proteins with IDPs can potentially interact with amyloid aggregation in a sequence-independent manner (Ikeda et al., Sci Rep. 2020 Jul 23;10(1):12334). However, in general, net charge and hydrophobicity can increase the likelihood of unfolded regions. Therefore, substituting arginine with histidine may contribute somewhat to increased aggregation.

[0248] The RELN peptide, which has a more flexible AA(H) at the N-terminus, a critical position in the alpha- and beta-GAG sites, reduced ThT fluorescence to a level similar to that of the WT length. However, a similar short peptide in which the beta-GAG site was removed increased AB aggregation by approximately 4.5-fold.

[0249] Additional peptides may include substituting the most flexible AA in the RELN CTR (G3444) with a less flexible AA such as histidine, which has intermediate flexibility, or proline, which may significantly reduce flexibility.

[0250] [Example 8] Reelin CTR improves cell membrane penetration Due to the highly basic structure of the C-terminal domain of RELN and its ability to interact with membranes, this domain may be suitable for directing other proteins (as proteins in protein fusions or as mixtures) or nucleic acids (e.g., mRNA) through cell membranes.

[0251] To evaluate this, HMRECs were cultured in complete endothelial growth medium 2 (EGM-2, Lonza, Switzerland) in 24-well plates to 80% confluence. For treatment, 5 μg of mRNA was added to all formulations. We prepared mRNA / peptide conjugates by mixing pre-diluted amounts of peptide (RKQNYMMNFSRQHGLRHFYNRRRR) and mRNA in EGM-2. HMRECs were cultured in treatment medium for 24 hours, followed by fluorescence measurements in a microplate reader, Synergy H1 (Biotek, VT), with excitation at 482 nm and emission recorded at 520 nm.

[0252] The results shown in Figure 16 showed an increase in expression when the mRNA was mixed with CTR.

[0253] [Example 9] Exemplary sequences encoding Dab1 dimerization constructs

[0254] [ka]

[0255] [ka]

[0256] [ka]

[0257] [ka]

[0258] [ka]

[0259] [ka]

[0260] Below is the codon-optimized sequence encoding the Dab1-FKBP fusion protein.

[0261] [ka]

[0262] [Example 10] Analysis of mini-RELN constructs in HREC cells Mini-RELN constructs were evaluated in human retinal endothelial cells (HRECs) using both in vitro overexpression and recombinant protein. The constructs used in Examples 10-12 were as shown in Table 5.

[0263] [Table 9]

[0264] For in vitro overexpression, HRECs were cultured in EBM-2 medium enriched with EGM-2 Single Quots (Lonza) at 37°C and 5% CO2, and experiments were performed at passage 7. 24 hours before the experiment, cells were seeded at 500,000 cells / mL in 6-well plates. Plasmid transfection was performed using Lipofectamine 2000 (5 μL, Life Technologies) according to the manufacturer's instructions. Specifically, cells were seeded one day before transfection to achieve 70% confluence at the time of transfection. On the day of transfection, Lipofectamine was diluted and incubated at room temperature for 10 minutes to form lipophilic complexes. Subsequently, each plasmid (1 μg, Invivogen) was added to the Lipofectamine lipid complex and incubated for 10 minutes. We blocked the transfection after 5 hours using OPTIMEM medium (Gibco) to allow cells to recover and promote replication. Five and 24 hours after transfection, cells were imaged using a light microscope (Olympus CKX53) at 10X magnification. The results, shown in Figures 17A-17J, demonstrated the integrity of mammalian cells expressing all mini-RELN constructs.

[0265] Twenty-four hours after transfection, cells were harvested and subjected to Western blotting analysis. Total protein levels were quantified by BCA assay. For Western blotting, 7 μg of protein homogenate was prepared by boiling for 5 minutes in Laemmli buffer enriched with 10 mM DTT under reducing and denaturing conditions. Samples were electrophoretically separated on a 4-20% gel at 90 V, and proteins were transferred to a PVDF membrane using the iBlot™2 dry blotting system. The membrane was blocked for 2 hours using Intercept® (TBS) blocking buffer. We detected β-actin (1:5000, mouse, no. 66009-1-Ig, Proteintech) and total DAB1 (1:100, SC-red tube; sc-271136 Dab1(G-5), Santa Cruz). Antibodies were diluted in Intercept® (TBS) blocking buffer and incubated overnight at 4°C with gentle shaking. Prior to secondary antibody incubation, the membrane was washed twice for 10 minutes with TBS-T. For secondary antibodies, we used IR Dye 800n CW goat anti-rabbit (925-32211, Licor), IR Dye 680 CW goat anti-mouse (925-68070, Licor), both diluted 1:10,000 in blocking buffer, and HRP (m-IgG1 BP-HRP-SC-525408, Santa Cruz), also diluted 1:1,000 in blocking buffer, for 1 hour at room temperature with gentle shaking. The membrane was washed three times with TBS-T, followed by IR detection using an Odyssey imager. To quantify the results, each band was quantified using ImageJ, and the data were expressed as the DAB1:β-actin ratio normalized to the control. Figures 18A-18E show a reduction in total Dab1, likely as a result of increased Dab1 signaling activity induced by the mini-RELN construct. The reduction in total DAB1 was quantified as shown in Figures 19A-19E. These experiments demonstrated that mini-RELN constructs can function directly in cells expressing them.The results shown in Figures 18A-18E demonstrate that expression of mini-RELN constructs induces a significant turnover and sustained activation of RELN signaling, detected as a reduction in total DAB1. The data show a reduction in DAB1 levels in the presence of mini-RELN constructs compared to lipofectamine (Lp) for all constructs. These findings led us to conclude that all mini-RELN constructs tested were active.

[0266] All tested constructs induced significant activation, with 225Z being the most active. 225Z contains the CR50 domain of RELN, which induces oligomerization, R5-6, which binds to the receptor, and the RELN C-terminus with the COLBOS variant, which binds to HSPGs.

[0267] Our analysis showed that the modular design described herein, including the RELN C-terminus with RAP for receptor binding, Fc for oligomerization, and a COLBOS variant for HSPG binding, was also effective in activating RELN signaling. In this design, Fc replaced the CR50 domain of RELN, and RAP replaced the receptor-binding domain. With this design, the mini-RELN construct is effective when produced by cells such as HRECs, demonstrating that it signals in an autocrine manner in the same cells.

[0268] Experimental evidence showing that we can replace modules within the mini-RELN domain according to their function (e.g., Fc for CR-50 and RAP for R5-6) without compromising efficacy demonstrates that the modules we selected are sufficient to support RELN signaling.

[0269] For in vitro screening of constructs using recombinant mini-RELN in HRECs, cells were cultured in EBM-2 medium enriched with EGM-2 Single Quots (Lonza) at 37°C and 5% CO2, and experimental testing was performed at passage 7. 24 hours prior to the experiment, cells were seeded at 500,000 cells / mL in 6-well plates. Two hours prior to the experiment, cells were washed with PBS to remove residual growth medium and then incubated in starvation medium containing EBM-2, 1% GlutaMax, and 0.2% Normicin. Treatments were performed using recombinant mini-RELN constructs obtained from Innovagen and recombinant mouse Reelin protein (3820-MR-025 / CF) from R&D systems. Each treatment was incubated at 4 μg / mL for 5 minutes, followed by application of a lysis buffer consisting of RIPA (Cell signaling), protease inhibitors (cOmplete™ mini, EDTA-free protease inhibitor cocktail, Roche), phosphatase inhibitors (PhosSTOP, Roche), 1 μL of 10 mM protease inhibitor MG-132, and 1% Triton X-100. Cells were harvested in this lysis buffer. Lysates were scraped and centrifuged at 15,000 rpm for 10 minutes at 4°C. Total protein levels were quantified by BCA assay. For Western blotting, 3.5 μg of protein homogenate was prepared by boiling for 5 minutes using Laemmli buffer enriched with 10 mM DTT under reducing and denaturing conditions. Samples were electrophoretically separated on a 4-20% gel at 90 V, and proteins were transferred to a PVDF membrane using the iBlot™ dry blotting system. The membrane was blocked for 2 hours using Intercept® (TBS) blocking buffer. We detected pDAB1 (Tyr232; 1:1000, rabbit, #3325 Cell Signaling) and total protein stain (Licor). Antibodies were diluted using Intercept® (TBS) blocking buffer and incubated overnight at 4°C with gentle shaking.Prior to secondary antibody incubation, the membrane was washed twice for 10 minutes with TBS-T. For the secondary antibody, we used IR Dye 800n CW goat anti-rabbit (925-32211, Licor) diluted 1:10,000 in blocking buffer and incubated at room temperature for 1 hour with gentle shaking. The membrane was washed three times with TBS-T, followed by IR detection using an Odyssey imager. To quantify the results, each band was quantified using ImageJ, and the data were expressed as a pDAB1:total protein ratio normalized to the control.

[0270] RELN induced the phosphorylation and proteosomal degradation of Dab1. Figures 20A-20F show increased levels of phosphorylated DAB1 (pDAB1) after treatment with the mini-RELN purified construct. pDAB1 levels were quantified in Figures 21A-21E. These experiments indicate that the mini-RELN construct also acts in a non-cell-autonomous manner.

[0271] As seen in Figure 21A, pDAB1 levels increased upon 225Q treatment. rmRELN lacking the C-terminus of RELN (rmRELN) was less effective than 225Q, which contains the C-terminal RELN with COLBOS variant. This finding suggests that modules containing the C-terminus of RELN or the GAG-binding domain may be useful for optimal RELN signaling. Signaling by mini-RELN was measured by directly detecting phosphorylated Dab1 (pDab1) after short-term treatment. In this design, the mini-RELN peptide is shown to function when produced by some cells, such as HEK, and signal in other cells, such as HREC. Figure 21B shows increased pDAB1 levels upon treatment with the 225S and 225T mini-RELN constructs. 225T, which contains Fc for oligomerization, RAP for receptor binding, and the C-terminus of RELN with COLBOS variants, was larger and more effective than controls lacking the C-terminal RELN domain with COLBOS variants (higher concentrations of rmRELN mouse and human containing R3-6 of RELN). This confirms the conclusion that constructs containing GAG-binding domains, such as the C-terminal domain of RELN with COLBOS variants, are superior. Figure 21C shows the increase in pDAB1 levels in the presence of various mini-RELN constructs compared to vehicle; 225Z, which contains CR50, R5-6 of RELN, and the C-terminus of RELN with COLBOS variants, lacks the GAG-binding domain and was more effective than the commercially available positive control lacking the oligomerization domain. Data in Figure 21D show the increase in pDAB1 levels upon treatment with 225SV, 225SW, and 225Z. This experiment showed that mini-RELN constructs such as 225Z, which contain only the C-terminus of RELN with CR50, R5-6, and COLBOS variants, are more effective at inducing signaling compared to full-length RELN WT and full-length RELN COLBOS. This data demonstrates that mini-RELN constructs are more effective than full-length RELN, even when full-length RELN carries a COLBOS variant.The data in Figure 21E show an increase in pDAB1 levels upon treatment with 225Z. As shown in Figure 21F, there was no detectable increase in RELN signaling, as expected for a construct containing only the Fc and C-terminus of RELN for oligomerization but lacking the receptor binding domain. Therefore, the presence of the receptor binding domain is required to activate RELN signaling.

[0272] In summary, these results provide evidence that the functional modules we describe in Table A are necessary and sufficient to stimulate optimal RELN signaling with therapeutic intent.

[0273] Depending on the indication, e.g., whether more or less RELN signaling supplementation is required, or depending on the particular route of administration, a particular mini-RELN may be more appropriate, e.g., a smaller mini-RELN construct may be preferred for nasal delivery.

[0274] Lactate dehydrogenase assays were performed on SH-SY5Y cells treated with preformed oligomeric tau (o-tau) alone or in the presence of C-terminal RELN WT(184I) for 24 hours. Lactate dehydrogenase assays were performed as follows: Neuroblastoma (SH-SY5Y, ATCC) cells (100,000 cells / well) were seeded into 96-well TC-treated plates in growth medium (DMEM / F12, Gibco, 10% heat-inactivated FBS, R&D, 200 μg normocin) and allowed to adhere and grow overnight. The following day, test compounds were thawed on ice. Phenol-free, serum-free DMEM / F12 serum-free, antibiotic-free medium was warmed, and 50 mL aliquots were filter-sterilized (0.22 μm) as test medium. In a TC cabinet, the thawed test compound was mixed by pipetting and then added to the filtered test medium to the final concentration for each test condition and mixed by pipetting. The test conditions were pre-incubated for 15 minutes at room temperature in a TC cabinet. The wells were gently aspirated and gently washed with 200 μL of pre-warmed test medium, then gently aspirated and 100 μL of test condition was added and incubated for 21 hours in a TC incubator at 5% CO2. LDH was performed using a Roche kit according to the manufacturer's protocol. The results, seen in Figure 23, demonstrated a reduction in tau-derived cytotoxicity in the presence of 184I, thus suggesting a direct interaction with neurotoxic oligomeric tau aggregates. 184I did not exhibit any cytotoxicity. These results suggest a mechanism for direct protection against tau toxicity mediated by the mini-RELN construct. Without wishing to be bound by theory, it is proposed that this mechanism does not involve RELN signaling, but is a direct effect mediated by binding of C-terminal RELN and tau.

[0275] We also performed a thioflavin (ThT) assay using thioflavin T (SensoLyte ThT β-Amyloid (1-42) Aggregation Kit, catalog number AS-72214) and synthetic Reelin peptide to assess the differential effects of these proteins on Aβ42 aggregation in vitro. Aβ42 was added to a clear, non-binding 96-well plate to a final concentration of 55 μM in a 10 μM solution of the Reelin peptide variant. The samples were then mixed with 2 mM thioflavin T dye, and fluorescence was read at excitation / emission wavelengths (Ex / Em) of 440 / 484 nm at intermittent time intervals over a 2-hour period. The plate was maintained at 37°C with 15 seconds of shaking between readings. The results, shown in Figures 24A-24C, demonstrated that the C-terminal domain of RELN, with or without the COLBOS variant, reduced amyloid aggregation. The presence of the COLBOS variant enhanced this effect. In general, the RELN C-terminal domain appears to be highly unstructured. Interaction with tau and / or amyloid and / or HSPGs may allow RELN to adopt a more stable conformation, resulting in anti-aggregation properties. These data suggest that as long as a mini-RELN construct contains the RELN C-terminal domain, preferably the RELN C-terminal domain with a COLBOS variant, it may have a protective effect. When mini-RELN contains only the C-terminal domain without the receptor-binding domain, the protection does not involve signal transduction, but still involves direct interaction with tau. When mini-RELN contains the C-terminal domain and the receptor-binding domain, it has at least two protective mechanisms: RELN signal transduction and direct interaction with tau.

[0276] When antibodies were available, overexpression of HEK-derived constructs was tested using Western blotting as follows. 15 μg of protein homogenate was prepared using Laemmli. Samples were electrophoretically separated on a 4-20% gel at 90 V, and proteins were transferred to a nitrocellulose membrane using 20% ​​methanolic Tris-glycine transfer buffer. The membrane was blocked for 2 hours using Intercept® (TBS) blocking buffer. We detected antibodies against Fc-taf (1:1,000, goat, Sigma-Aldrich) and GAPDH (1:5,000, mouse, Abcam). Antibodies were diluted using Intercept® (TBS) blocking buffer and incubated overnight at 4°C with gentle shaking. Prior to secondary antibody incubation, the membrane was washed twice for 10 minutes using TBS-T. For secondary antibodies, we used IR Dye 800n CW goat anti-rabbit (925-32211, Licor), IR Dye 680 CW goat anti-mouse (925-68070, Licor), both diluted 1:10,000 in blocking buffer, and HRP (m-IgG1 BP-HRP-SC-525408, Santa Cruz), also diluted 1:1,000 in blocking buffer, for 1 hour at room temperature with gentle shaking. The membrane was washed three times with TBS-T, followed by IR detection using an Odyssey imager. To quantify the results, each band was quantified using ImageJ, and the data were expressed as a pDAB1:β-actin ratio normalized to the control.

[0277] The results shown in Figures 25A-25B provide evidence of protein expression. For 225R, which contains the CR50 domain of RELN, a higher molecular weight band representing mini-RELN oligomers was detected (Figures 25A-25B). Mini-RELN constructs containing the Fc from IgG are predicted to form oligomers, while constructs containing CR50 and fspCR50 are predicted to form higher molecular weight multimers.

[0278] The effects of in vitro treatment with RELN or mini-RELN protein were also evaluated. HRECs were cultured in EBM-2 medium enriched with EGM-2 Single Quots (Lonza) at 37°C and 5% CO2 and tested at passage 7. 24 hours prior to the experiment, cells were seeded at 500,000 cells / mL in 6-well plates. Two hours prior to the experiment, cells were washed with PBS to remove residual growth medium and then incubated in starvation medium containing EBM-2, 1% GlutaMax, and 0.2% Normicin. Treatments were performed using medium from HEK293T cells transiently transfected with various mini-RELN constructs for 24 hours in the presence of Lipofectamine 2000. HEK-derived Lipofectamine 2000 medium was used as a negative control. HEK 293T-derived medium was tested at various ratios (1:1, 1:4, and 1:14) (transfected medium:starvation medium). Each treatment was incubated for 5 minutes, followed by application of a lysis buffer consisting of RIPA (Cell signaling), protease inhibitors (cOmplete™ mini, EDTA-free protease inhibitor cocktail, Roche), phosphatase inhibitors (PhosSTOP, Roche), 1 μL of 10 mM protease inhibitor MG-132, and 1% Triton X-100. The lysate was scraped and centrifuged at 15,000 rpm for 10 minutes at 4°C. Total protein levels were quantified by BCA assay. For Western blotting, 7 μg of protein homogenate was prepared by boiling for 5 minutes using Laemmli buffer enriched with 10 mM DTT under reducing and denaturing conditions. Samples were electrophoretically separated on a 4-20% gel at 90 V, and proteins were transferred to a PVDF membrane using the iBlot™2 dry blotting system. The membrane was blocked for 2 hours using Intercept® (TBS) blocking buffer.We detected pDAB1 (Tyr232; 1:1000, rabbit, no. 3325 Cell Signaling) and beta-actin (1:5000, mouse, no. 66009-1-Ig, Proteintech). The antibodies were diluted using Intercept® (TBS) blocking buffer and incubated overnight at 4°C with gentle shaking. Prior to secondary antibody incubation, the membrane was washed twice for 10 minutes with TBS-T. As secondary antibodies, we used IR Dye 800n CW goat anti-rabbit (925-32211, Licor) and IR Dye 680 CW goat anti-mouse (925-68070, Licor), both diluted 1:10000 in blocking buffer, for 1 hour at room temperature with gentle shaking. The membrane was washed three times with TBS-T, followed by IR detection using an Odyssey imager. To quantify the results, each band was quantified using ImageJ and data were expressed as pDAB1:β-actin ratios normalized to the control.

[0279] Transient overexpression of the mini-RELN construct in HEK293T cells was achieved as follows. HEK T293 cells (ATCC) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, R&D) and 400 μL / L normocin at 37°C and 5% CO2 and used in experiments up to passage 10. 24 hours before the experiment, cells were seeded in 6-well plates at 300,000 cells / mL. Plasmid transfection into cells was performed using Lipofectamine 2000 (7 mL, Life Technologies) according to the manufacturer. Specifically, 1 day before transfection, cells were seeded to be 70% confluent at the time of transfection. On the day of transfection, Lipofectamine was diluted and incubated at room temperature for 10 minutes to form lipophilic complexes. Subsequently, each plasmid (3 mg, Invivogen) was added to the lipofectamine lipid complex and incubated for 10 minutes. We blocked the transfection after 5 hours using OPTIMEM medium (Gibco) to allow cells to recover and promote replication. 24 hours after transfection, cells were harvested, the medium was removed from the cells by centrifugation, and they were stored under sterile conditions at -80°C until use. Five and 24 hours after transfection, cells were imaged using a light microscope (Olympus CKX53) at 10X magnification.

[0280] Figures 26A and 27A show that mini-RELN constructs containing Fc, APOE, and HSPG-binding C-terminal RELN were more effective at activating RELN signaling than commercially available constructs containing only RELN R5-6. As expected, 225Yf, which contains WT APOE and is predicted to bind the receptor more efficiently, was more effective and clearly dose-dependent. 225Xf, which contains APOE with the Christchurch mutation, was also effective at activating signaling. The results shown in Figures 26B and 27B for treatment with 225ZZ may be related to the predicted oligomerization properties of this construct. Figures 26C and 27C show the efficacy of the 225SU and 225SV mini-RELN constructs. Figures 26D and 27D show the efficacy of mini-RELN constructs 225SW and 233F; 233F contains two RELN R6 domains in tandem, and this construct does not occur in nature. Figures 26E and 27E show Dab1 signaling activation by the 225RR construct, which contains the RELN C-terminus with the COLBOS variant, which was more robust compared to the control and compared to 233A, which contains the RELN C-terminus WT.

[0281] [Example 11] In vivo validation of purified recombinant mini-RELN constructs We administered the mini-RELN construct to mice using either intranasal delivery using the Minimally Invasive Nasal Depot (MIND) (Padmakumar et al., J Control Release. 2021 Mar 10;331:176-186) or intraperitoneal injection. MIND utilizes the olfactory nerve to bypass the blood-brain barrier. Our clinical studies have demonstrated the importance of the entorhinal cortex in extreme protection against Alzheimer's disease. Using the MIND method provides one way to reach the entorhinal cortex with minimal systemic exposure. We demonstrated that mini-RELN can be delivered to the hippocampal formation, including the entorhinal cortex, a brain region we have shown to be important for extreme protection against Alzheimer's disease. C57Bl / 3 mice (Jackson Laboratory) were housed under a regular light / dark cycle. All procedures were performed under approved IACUC animal protocols. Prior to incision, the nasal area was shaved and cleaned using betadine and 70% alcohol, and mice were injected subcutaneously with buprenorphine HCl and meloxicam (5 mg / kg). The nasal cavity was then opened using a scalpel, and the nasal mucosa was exposed using a microdrill. Once the mucosa was exposed, a gel dispersion of either PBS or the mini-RELN construct (Innovagen) in 20% Pluronic (Sigma-Aldrich) was delivered subcutaneously into the surgically created pocket. Mice were injected with meloxicam for 3 days, then euthanized on day 4, and brain tissue was collected for postmortem analysis.

[0282] Western blotting was performed as follows: 20 μg of protein homogenate was prepared by boiling for 5 minutes in Laemmli buffer enriched with 10 mM DTT under reducing and denaturing conditions. Samples were electrophoretically separated on a 4-20% gel at 90 V, and proteins were transferred to a PVDF membrane using the iBlot™2 dry blotting system. The membrane was blocked for 2 hours using Intercept® (TBS) blocking buffer. We detected pDAB1 (Tyr232; 1:1000, rabbit, no. 3325 Cell Signaling) and beta-actin (1:5000, mouse, no. 66009-1-Ig, Proteintech). Antibodies were diluted using Intercept® (TBS) blocking buffer and incubated overnight at 4°C with gentle shaking. Prior to secondary antibody incubation, the membrane was washed twice for 10 minutes with TBS-T. As secondary antibodies, we used IR Dye 800n CW goat anti-rabbit (925-32211, Licor) and IR Dye 680 CW goat anti-mouse (925-68070, Licor), both diluted 1:10,000 in blocking buffer, for 1 hour at room temperature with gentle shaking. The membrane was washed three times with TBS-T, followed by IR detection using an Odyssey imager. To quantify the results, each band was quantified using ImageJ, and the data were expressed as a pDAB1:β-actin ratio normalized to the control.

[0283] The data shown in Figures 28A-28K demonstrate effective brain delivery of the mini-RELN peptide to the hippocampus and entorhinal cortex, as well as the midbrain, resulting in increased levels of the downstream RELN pathway, as confirmed by increased pDAB1 levels. Mice treated with the mini-RELN construct had greater RELN signaling in the hippocampus and entorhinal cortex, as determined by increased pDab1 levels. The efficacy of mini-RELN treatment was confirmed in WT mice and mice with tau mutations that result in tauopathy. 225Z contains CR50 of RELN, which induces oligomerization, R5-6 of RELN, which mediate receptor binding, and the C-terminal RELN domain with a COLBOS mutation that achieves HSPG binding. This indicates that these RELN domains were sufficient to drive protective signaling.

[0284] Immunofluorescence staining of mouse brains was also used to demonstrate delivery of mini-RELN using MIND. 24 or 72 hours after administration of mini-Reelin (225S) or vehicle, mice were intracardially perfused with 4% PFA in PBS, and the brains were harvested and incubated in 4% PFA at 4°C for 24 hours. 1 mm-thick sagittal sections were obtained using a stainless steel brain matrix. Entorhinal-hippocampal sections from each brain were selected and cleared using the Binaree® Tissue Clearing™ Kit (HRTC-012) according to the kit's guidelines. The sections were incubated with IgG-Fc tag and pDAB1 primary antibodies for 72 hours, followed by secondary antibodies: Alexa 488 and Alexa 647, and DAPI for 1 hour. The sections were then imaged and quantified using a confocal SP8 microscope. The results (exemplary results for construct 225S shown in Figures 29A-29B) demonstrated the presence of the mini-RELN peptide in the brain, co-localizing with brain cells with increased pDab1 levels, evidence of effective drug delivery, target binding, and targeted activation of RELN signaling in the hippocampal formation.

[0285] [Example 12] Systemic delivery of mini-RELN reduced tau pathology in P301S tau mice. The effects of systemic delivery of mini-RELN peptide were evaluated in vivo. Six-month-old female MAPT P301S tau Tg mice were intraperitoneally injected with 500 μL of either vehicle (PBS) or mini-RELN peptide solution on day 1 and 250 μL on days 2–4. On day 5, mice were euthanized with saturated CO2 gas and perfused intracardially with 4% PFA. Brains were then harvested and used for histological analysis.

[0286] Immunofluorescence staining of mouse brains was performed as follows. After 24 hours of fixation with 4% PFA at 4°C, 1 mm-thick sagittal brain sections were obtained using a stainless steel brain matrix. Entorhinal-hippocampal sections from each brain were selected and cleared using the Binaree® Tissue Clearing™ Kit (HRTC-012) according to the kit's guidelines. The sections were incubated with p-tau S396 primary antibody for 72 hours, followed by secondary antibodies: Alexa 647 and DAPI for 1 hour. The sections were then imaged and quantified using a confocal SP8 microscope. 14 images per group were acquired at 63X magnification. The fluorescence intensity of p-tau was automatically quantified using Matlab (2021a). Image signal masks were obtained using Otsu's thresholding method, and then the signal intensity mean was calculated.

[0287] Quantification of p-tau S396 fluorescence intensity was used as a characteristic of tau pathology. The results shown in Figure 30 showed that when mice were treated with mini-RELN peptide, p-tau S396 levels were significantly reduced. This data indicates that the administration of mini-RELN constructs is effective when administered systemically, and that the beneficial effect results in a significant reduction in tau phosphorylation, which is a pathological characteristic of tauopathies such as frontotemporal dementia and Alzheimer's disease. This was demonstrated in a mouse model of tauopathy (MAPT P301S).

[0288] References

[0289] [Table 10-1]

[0290] [Table 10-2]

[0291] [Table 10-3]

[0292] [Table 10-4]

[0293] [Table 10-5]

[0294] [Table 10-6]

[0295] Other embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the above description is intended to be illustrative of the invention and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for treating or preventing a neurodegenerative disease in a subject, comprising administering to said subject an effective amount of a reelin protein or a nucleic acid encoding a reelin protein, preferably wherein said reelin protein comprises a H3447R or H3447K mutation and / or a R3454A mutation, optionally a H3447R or H3447K mutation in combination with R3454A.

2. The method of claim 1, wherein the reelin protein comprises full-length reelin or a mini-reelin comprising: (A) a signal peptide; (B) an oligomerization domain, optionally a reelin CR-50 domain; (C) a receptor binding domain, optionally reelin domains (repeats) 5 and 6 (R5-6); and (D) a GAG binding domain, optionally a C-terminus (CTR) of reelin.

3. The method of claim 1 or 2, comprising administering a nucleic acid encoding a reelin protein, wherein the nucleic acid is naked mRNA or DNA encoding the reelin, or is in a viral vector, optionally an AAV vector.

4. 1. A composition comprising a reelin protein or a nucleic acid encoding a reelin protein, preferably wherein said reelin protein comprises a H3447R or H3447K mutation and / or a R3454A mutation, optionally a H3447R or H3447K mutation in combination with R3454A.

5. The composition of claim 4 , wherein the reelin protein comprises a full-length reelin.

6. The composition of claim 4, wherein the reelin protein comprises: A) a signal peptide; (B) an oligomerization domain, optionally a reelin CR-50 domain; (C) a receptor binding domain, optionally reelin domains (repeats) 5 and 6 (R5-6); and (D) a GAG binding domain, optionally a mini-reelin comprising the C-terminus of reelin (CTR).

7. 7. The composition of any one of claims 4 to 6, comprising a nucleic acid encoding a reelin protein, optionally wherein the nucleic acid is naked mRNA or DNA encoding the reelin, or is in a viral vector, optionally an AAV vector.

8. 7. A composition according to any one of claims 4 to 6 for use in a method of treating or preventing a neurodegenerative disease in a subject.

9. 8. The method of claims 1 to 3, or the composition for use of claim 7, wherein the neurodegenerative disease is Alzheimer's disease, frontotemporal dementia, memory loss, cognitive dysfunction, amyotrophic lateral sclerosis (ALS), age-related cognitive decline, age-related macular degeneration, glaucoma, diabetic retinopathy or hereditary retinal degeneration, stroke, brain trauma or concussion, retinal trauma, small vessel disease such as cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), and wet age-related macular degeneration.

10. 1. A composition comprising or consisting of a reelin C-terminal region (CTR), and optionally a carrier, preferably wherein said CTR comprises a H3447R or H3447K mutation and / or a R3454A mutation, optionally a H3447R or H3447K mutation in combination with R3454A, and wherein said composition comprises or consists of a sequence as shown in Table 1.

11. 9. The composition of claim 8, further comprising a non-reelin nucleic acid, optionally an mRNA, optionally wherein the mRNA encodes a therapeutic peptide.

12. 10. The composition of claim 8, further comprising an isolated non-reelin protein, optionally complexed with or fused to a RELN CTR.

13. 11. A method for delivering a nucleic acid or protein to a cell, comprising administering to said cell an effective amount of a composition according to any one of claims 8 to 10.

14. 1. A method of treating or preventing a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of an agent that reduces methylation of the RELN promoter in an amount sufficient to increase RELN expression in the subject, wherein the agent that reduces promoter methylation: (i) a fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain, optionally administered as an RNP, and a guide RNA that directs the fusion protein to demethylate cytosines in the RELN promoter; or (ii) a nucleic acid encoding a fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain, optionally administered as mRNA or in one or more vectors, optionally viral vectors, optionally adeno-associated virus AAV vectors, and a guide RNA that directs the fusion protein to demethylate cytosines in the RELN promoter. That's the method.

15. 1. A method of treating or preventing a neurodegenerative disease in a subject, comprising administering to the subject an effective amount of: (i) a CRISPR / Cas protein, optionally administered as an RNP; a guide RNA that directs said Cas protein to a region of a RELN allele comprising H3447 or R3454; and a ssODN comprising a sequence comprising an H3447R or H3447K mutation and / or an R3454A mutation, for insertion into said RELN allele, one or more ssODNs comprising a sequence comprising an H3447R or H3447K mutation, optionally in combination with R3454A; or (ii) a nucleic acid encoding a CRISPR / Cas protein, optionally administered as naked DNA or mRNA and / or in one or more vectors, optionally viral vectors, optionally adeno-associated virus (AAV) vectors; a guide RNA that directs said Cas protein to a region of the RELN allele that comprises H3447; and a ssODN for insertion into said RELN allele, comprising a sequence comprising the H3447R or H3447K mutation and / or the R3454A mutation, optionally the H3447R or H3447K mutation in combination with R3454A. Administering

16. 16. The method of claim 14, wherein the administering is to the entorhinal cortex.

17. 16. The method of claims 14 to 15, wherein the disease is Alzheimer's disease, frontotemporal dementia, memory loss, cognitive dysfunction, amyotrophic lateral sclerosis (ALS), age-related cognitive decline, age-related macular degeneration, glaucoma, diabetic retinopathy or hereditary retinal degeneration, stroke, brain trauma or concussion, retinal trauma, small vessel disease such as cerebral autosomal dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL), and wet age-related macular degeneration.

18. 1. A method of assessing the risk of developing Alzheimer's disease in a subject, comprising determining the presence or absence of an H3447R variant allele in the RELN gene of said subject, wherein the presence of the H3447R variant indicates that said subject has a lower risk of developing AD compared to a subject who does not have the H3447R variant.

19. 19. The method of claim 18, wherein the subject has an APOE4 variant sequence.

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