Compositions and methods for using casein in alzheimer's disease

Abstract

Described herein are methods and compositions for the treatment of Alzheimer's Disease (AD), as well as compositions comprising winder-derived peptides and methods of use thereof.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 379,393, filed October 13, 2022; U.S. Provisional Patent Application Serial No. 63 / 419,574, filed October 26, 2022; and U.S. Provisional Patent Application Serial No. 63 / 502,038, filed May 12, 2023. The entire contents of the foregoing are incorporated herein by reference.

[0003] Federally funded research or development

[0004] This invention was made with U.S. Government support under Grant Nos. OD019833, AG054671, NS100121, and NS110048 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

[0005] Described herein are methods and compositions for treating Alzheimer's disease (AD), as well as compositions comprising reelin-derived peptides or nucleic acids encoding the same and methods of use thereof. Background Art

[0006] Alzheimer's disease (AD) is a progressive neurodegenerative disorder that currently affects approximately 6.2 million people in the U.S. Currently, there is no effective treatment to prevent or reverse disease progression. Summary of the Invention

[0007] Provided herein are methods for treating or preventing (as used herein, "prevention" 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 of treating or preventing a neurodegenerative disease. Preferably, the reelin protein comprises an H3447R or H3447K mutation and / or an R3454A mutation, optionally in combination with an H3447R or H3447K and R3454A mutation. In some embodiments, the subject is a mammal, such as a human or non-human veterinary subject. In addition to Alzheimer's disease, the present method can be used to treat other neurodegenerative diseases, disorders or conditions, including frontotemporal dementia, various types of memory loss, including but not limited to cognitive impairment of mild cognitive impairment (MCI), or other conditions associated with accumulation of beta-amyloid or accumulation of tau or other protein conformational diseases such as frontotemporal dementia, or amyotrophic lateral sclerosis (ALS), or cognitive decline associated with aging. Neurodegenerative diseases can also include eye diseases such as 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 infarcts and leukoencephalopathy (CADASIL) and abnormal angiogenesis such as wet age-related macular degeneration. Other applications include conditions associated with genetic or epigenetic loss-of-function of RELN, including lateral temporal lobe epilepsy, autism, schizophrenia, and bipolar disorder, as well as defects in cortical lamination, abnormal neuronal migration, and cerebellar hypotrophy.

[0008] In some embodiments, the resilin protein comprises full-length resilin (or a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to human resilin) or a mini-resilin comprising: A) a signal peptide; (B) an oligomerization (e.g., dimerization) domain, optionally a resilin CR-50 domain; (C) a receptor binding domain, optionally resilin domains (repeat) 5 and 6 (R5-6); and (D) a GAG binding domain, optionally from the C-terminus of resilin (CTR), e.g., comprising a signal peptide, a CR-50 domain, resilin domains 5 and 6 (R5-6), and the C-terminus of resilin.

[0009] In some embodiments, the method comprises administering a nucleic acid encoding a reelin protein, wherein the nucleic acid is naked mRNA or DNA encoding reelin, or is in a viral vector, such as an AAV vector.

[0010] In some embodiments, a resilin protein or a nucleic acid encoding a resilin protein is preferably administered in or around the entorhinal cortex of the brain, for example, to increase efficacy and / or reduce side effects.

[0011] Also provided herein are compositions comprising a resilin protein or a nucleic acid encoding a resilin protein, preferably wherein the resilin protein comprises an H3447R or H3447K mutation and / or an R3454A mutation, optionally a combination of H3447R or H3447K and R3454A mutations, as described herein.

[0012] In some embodiments, the resilin protein comprises a full-length resilin or a mini-resilin comprising: (a) a mini-resilin comprising: (A) a signal peptide; (B) an oligomerization (e.g., dimerization) domain, such as the resilin CR-50 domain, an Fc fragment of IgG, or FKBP; (C) an APOER2 / VLDLR binding domain, such as resilin domains (repeat) 5 and 6 (R5-6), or a receptor binding domain of APOE, RAP, urokinase-type fibroblasts; (D) glycosaminoglycan (GAG) binding domains, such as the C-terminus (CTR) from reelin, TAT peptide, or P21, e.g., as described herein, e.g., in Table A. Exemplary signal peptides for secretion include IL2 signal peptide, human albumin signal peptide, human α1-antitrypsin signal peptide, or human factor VIII signal peptide. Exemplary constructs include those in Table 5.

[0013] In some embodiments, the composition includes a nucleic acid encoding a reelin protein, optionally wherein the nucleic acid is naked mRNA or DNA encoding reelin, optionally with a human codon-optimized sequence, or is in a viral vector, such as an AAV vector.

[0014] In addition, provided herein are compositions comprising or consisting of a C-terminal region of a skein protein (CTR) and optionally a carrier, preferably wherein the CTR comprises an H3447R or H3447K mutation and / or an R3454A mutation, optionally a combination of H3447R or H3447K and R3454A mutations, optionally comprising or consisting of a sequence as shown in Table 1. In some embodiments, the composition further comprises a non-skein protein nucleic acid, such as an mRNA, optionally wherein the mRNA encodes a therapeutic peptide. In some embodiments, the composition further comprises an isolated non-skein protein, for example, complexed with or fused to a CTR. Also provided herein are methods of delivering a nucleic acid or protein to a cell, comprising administering to the cell an effective amount of a composition comprising a CTR.

[0015] Also provided herein are methods for treating or preventing a neurodegenerative disease (e.g., Alzheimer's disease) in a subject, the methods 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 the methylated promoter is: (i) a fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain and a guide RNA, the fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain, the guide RNA guiding the fusion protein to demethylate cytosine in the RELN promoter, optionally administered as an RNP; or (ii) a nucleic acid encoding the fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain and a guide RNA, the fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain, the guide RNA guiding the fusion protein to demethylate cytosine in the RELN promoter, optionally administered as mRNA or in one or more vectors, optionally viral vectors, optionally adeno-associated virus (AAV) vectors.

[0016] In addition, provided herein are methods for treating or preventing a neurodegenerative disease (e.g., Alzheimer's disease) in a subject. The method comprises administering to the subject an effective amount of: (i) a CRISPR / Cas protein, a guide RNA, and an ssODN, wherein the guide RNA guides the Cas protein to a region of the RELN allele comprising H3447, the ssODN comprising a sequence comprising an H3447R or H3447K mutation and / or an R3454A mutation, optionally a combination of H3447R or H3447K and R3454A mutations, or one or more of the ssODNs comprising a sequence comprising a combination of H3447R or H3447K and R3454A mutations, for insertion into the RELN allele, optionally as an R NP administration; or (ii) nucleic acid encoding a CRISPR / Cas protein, a guide RNA and an ssODN, wherein the guide RNA guides the Cas protein to a region of the RELN allele comprising H3447 and / or R3454, one or more of the ssODNs comprising a sequence comprising an H3447R or H3447K mutation and / or an R3454A mutation, optionally a combination of H3447R or H3447K and R3454A mutations, for insertion into the RELN allele, optionally administered as mRNA or in one or more vectors, optionally a viral vector, optionally an adeno-associated virus AAV vector.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) will control.

[0018] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A-CPET imaging of RELN-COLBOS(H3447R) carriers. (A) Comparison of representative PiB PET amyloid (upper left row) and Fluortaucipir tau PET (lower left row) images of a (PSEN1E280A; RELN-COLBOS) case (left panel) and a PSEN1E280A mutation carrier with MCI of typical age (right panel). For both measurements, specific binding of the tracer is represented using a scale with the lowest (0.8 DVR or SUVR) and highest (2.00 DVR or SUVR) binding. The right side of panel A shows representative images of a (PSEN1E280A; RELN-COLBOS) case (left panel) and a PSEN1E280A mutation carrier with MCI of typical age (right panel). 18 Comparison of F-fluorodeoxyglucose (FDG) PET precuneus cerebral glucose metabolic rate (CMRgI). The binding affinity of the dye is represented using a scale with the lowest (0.5SUVR) and highest (2.1SUVR) binding. (B) Dot plot analysis of amyloid burden, tau burden, glucose metabolism, and hippocampal volume measured by brain imaging shown in Figure A. Comparison of brain imaging measures of (PSEN1E280A; RELN-COLBOS) cases with previously published (PSEN1E280A; APOEch) homozygous cases, intact PSEN1E280A carriers (n=13-18), and younger MCIPSEN1E280A carriers (n=7-11) (2). Some previously published data points are included in the figures presented here because they were the only data available for comparison (2). The mean cortical to cerebellar distribution volume ratio (DVR) 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 cohorts. Data are presented as individual values with mean ± sem. (C) Anatomical details of tau burden in the temporal cortex. Planar representation of regions of interest in the right hemisphere temporal cortex (top left, PPC = posterior parahippocampal cortex, ERC = entorhinal cortex), superimposed with tauPET (Fluortaucipir, FTP) from four cases. The asymptomatic PSEN1E280A mutation carrier was 38 years old. The PSEN1E280A mutation carrier with typical MCI was 44 years old. PSEN1 carriers with the RELN-COLBOS mutation were distinguished by relatively lower tau burden in the medial temporal regions (ERC and PPC) compared with asymptomatic PSEN1E280A mutation carriers and PSEN1E280A mutation carriers with MCI at a typical age of onset.

[0020] Figure 2A-M. The RELN H3448R variant enhances Dab1 signaling and the affinity of the C-terminal region of RELN (CTR-RELN) for heparin in mice, reduces tau hyperphosphorylation, and preserves motor function. (A) Representative Western blots of pDAB1 levels (top) and total protein staining (bottom) in primary mouse cortical neurons treated with 4 μg / ml of full-length RELN WT or RELN H3448R (mouse ortholog of H3447R RELN) for 5 minutes at 37°C. (MOCK; P < 0.0029), RELN WT (P = 0.0246). Data are presented as mean ± sem and analyzed by Kruskal-Wallis (Dunn post hoc analysis for multiple comparisons of n = 4 independent experiments). (B) Spectral analysis of heparin chromatography fractions of CTR-RELN WT and CTR-RELN H3447R variant eluted in an increasing gradient of NaCl (0.05M NaCl step gradient) in 20mM Tris HCl buffer and detected via UV absorbance at 280nm. Data are expressed as a percentage of input versus NaCl gradient fractions from 0.4M to 5M NaCl. The data show that 0.55M NaCl can displace the binding of CTR-RELN WT to the heparin column. In the presence of the H3447R mutation, the affinity of CTR-RELN for heparin increases, as indicated by a shift in the peak with the greatest height of the eluted fraction from 0.55M NaCl to 0.7M NaCl. N = 3 independent experiments. Error bars represent sem. (C) Representative sensorgrams of binding analysis between a chip sensor coated with heparin and increasing concentrations of CTR-RELN variants ranging from 0 to 25 nM. Data are expressed as response units versus time in seconds. The equilibrium dissociation constant (K) for each SPR analysis D ) are shown in the interior of the figure and support the differences in affinity binding between heparin and CTR-RELN variants: H3447R (right panel, K D =3.75e-9M-1s-1)>H3347(left, K D =6.53e-9M-1s-1). Figure 7A-BSensorgrams of CTR-RELN and H3447K and H3447D control variants are reported for comparison. (D) Representative Western blotting of pDab1 levels (top blot) and GAPDH levels (bottom blot) detected in the cerebellum of both females (left) and males (right) mutated to wild type (RELN WT / WT), heterozygous (RELN WT / H3448R), or homozygous (RELN H3448R / H3448R) for the mRELN H3448R mutation. Levels were detected in 6-12 month old mice. (E, F) Quantification of pDab1 levels normalized to GAPDH and expressed as fold change of RELN WT, showing a genotype effect in pDab1 levels in male mice (F. WT / WT vs. H3448R / H3448R, p = 0.0284; WT / H3448R vs. H3448R / H3448R, p = 0.0037, one-way ANOVA), but not in female mice (E). G, Isothermal calorimetry measurements of the short variants CTR-RELN WT (left panel) and CTR-RELN H3447R (right panel) titrated with 5 μM heparin. Affinity calculations are reported at the top of each panel. H, Binding analysis between Fc-fused CTR-RELN WT or H3447R and heparin-coated biosensors via BLI. Association constant (k a ) and dissociation constant (k d ) is used to calculate the equilibrium dissociation constant (K D). I, Docking of CTR-RELN WT (purple) with a representative heparin molecule (cyan). AA in RELN CTR that makes polar contacts 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 a ptau T205 antibody. hTau tg / WT mice showed neurofibrillary tangles and neuropil filaments in the CA1 and dentate gyrus, while hTau tg / RELN H3448R showed a lesser degree of tau pathology (cell bodies of affected neurons depicted with dashed lines). Bar = 100 μm. 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 fewer signal intensity values. p = 0.022*, two-sided Student's T test. Error bars represent standard deviations from the mean. L, Representative phenotypes and relative scores observed during the tail elevation test (0 = severely impaired, 1: 50% impaired; 2 = normal). M, Tail suspension scores recorded for RELNWT / Tau-P301L (n = 13 male mice) and RLN-H3448RH3448R / Tau-P301L hybrid male mice (n = 11 male mice) showed that the presence of the RLN-H3448R variant significantly improved the tail suspension score compared to Tau-P301L mice expressing RLN WT (*p = 0.0305, two-tailed unpaired T test, t = 2.313, df = 22). Box plots are represented as minimum to maximum values around the mean.

[0021] Figure 3A-E. Neuropathological characterization of (PSEN1E280A; RELN H3447R) cases. (A) Amyloid beta (Aβ) pathology and hyperphosphorylated tau (ptau) pathology in the CA1 and EC are reported. Both pathologies are widespread and intense, with Aβ pathology showing diffuse plaques of varying distribution and size in the two structures (figure and inset). Ptau pathology shows neurofibrillary tangles of varying densities and diffuse tau pathology. Scale bar = 500 mm. (B) Representative images of neurons stained with Kluver-Barrera in the CA1 and EC of PSEN1E280ARELN-COLBOS (RELN-COLBOS) cases, PSEN1E280A / APOE Christchurch (APOEch) cases, average onset PSEN1E280A familial Alzheimer's disease (FAD) cases, and sporadic Alzheimer's (SAD) cases are shown. Scale bar = 125 mm. (C) 3D scatter plots of Aβ, ptau, and neuronal density in EC and CA1 from RELN-COLBOS, APOEch, FAD, and SAD cases. EC in RELN-COLBOS cases showed the highest neuronal density while exhibiting low Aβ and ptau pathology. In contrast, CA1 in the same case showed low neuronal density and high Aβ and ptau pathology levels. For all other cases, neuronal density in both regions was low while exhibiting different Aβ or ptau pathology levels. (D) Representative images of RELN-CT and ApoE staining in EC and CA1 for RELN-COLBOS, APOEch, FAD, and SAD cases. RELN-COLBOS cases showed a strong background signal in both structures, while the intraneuronal signal for RELN-CT in EC was low. Similarly, APOEch cases showed a lower intraneuronal signal in EC using RELN-CT antibodies, and very low intraneuronal signal in both structures using ApoE antibodies (enlarged right figure). Finally, ApoE staining showed obvious plaque-like and tangle-like signals in two structures (EC and CA1) in FAD and SAD cases. Scale bars in the enlarged panels = 100 mm and 25 mm. (E) Representative images of Kluver-Barrera staining of whole hippocampal and parahippocampal sections in RELN-COLBOS, APOEch, FAD and SAD cases (top) and representative enlarged images of parahippocampal subcortical white matter stained with RELN-CT antibody. RELN-COLBOS cases showed increased white matter luxol fast blue signal intensity, while RELN-COLBOS and SAD cases showed increased intracellular RELN-CT signals in the white matter.Scale bar for upper panel = 2.5 mm, scale bar for lower panel = 25 μm.

[0022] Figure 4A-D. ADAD pathology modifies a putative model of APOEch and RELN H3447R PS1E280A cases. Under normal conditions (A), RELN and ApoE can bind to the VLDLr / APOER2 receptor complex with the help of GAGs; regulation of this signaling pathway maintains physiological Dab1 activation (phosphorylation indicated by circles), which maintains basal phosphorylation levels of GSK3β and tau phosphorylation (left panel). In APOE Christchurch cases, ApoE binding to GAGs is reduced, allowing RELN to increase its 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, RELN binding to GAGs is increased, leading to enhanced Dab1 activation and subsequent inhibition of GSK3β and tau phosphorylation (right panel C). (D) A model of a protective state in which increased resilin signaling and / or reduced ApoE signaling have a beneficial effect.

[0023] Figure 5A-B . Pedigree of the subject and Sanger validation of the C-terminal RELN H3447R variant. (A) Pedigree of the subject. Circles represent females, squares represent males, and diamonds represent individuals whose sex has been masked for privacy. Arrows depict proband cases. Deceased individuals are marked with a slash. Black shapes indicate affected individuals, and white shapes indicate unaffected individuals. (B) Representative DNA Sanger sequencing of amplicons in the RELN gene from a carrier without the mutation (RELN / RELN, top panel) compared to an individual with the variant (RELN / RELN H3447R, bottom panel). The region of the H3447R mutation is highlighted by the black circle.

[0024] Figure 6A-B 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 vehicle, RELN H3448 wild-type (RELN WT), or RELN H3448R for one hour. (B) Quantification of normalized intensities from n=3 independent experiments shows a significant increase in the ptau / tau ratio in neurons treated with RELN H3448R compared to vehicle (p=0.03, unpaired T-test).

[0025] Figure 7A-BMutations at position 3447 in the CTR domain of RELN affect heparin binding. (A, B) Representative sensorgrams of binding assays between a chip sensor coated with heparin and increasing concentrations of CTR-reelin variants ranging from 0 to 25 nM. Sensorgrams of RELN H3447K (A) and RELN H3447D (B). Data are expressed as response units versus time in seconds. The equilibrium dissociation constant (k) for each SPR assay is shown in Table 1. D ) are shown in the interior of the figure and support the differential binding between heparin and the resilin variants, when compared with the data presented in Figure 2, the order is H3447R ( Figure 2C , K D =3.75e-9M-1s-1)>WT( Figure 2C , K D =6.53e-9M-1s-1)>H3447K(A,K D =7.33e-9M-1s-1)>>>H3447D(B,K D =1.64e-7M-1s-1).

[0026] Figure 8 A consensus C-terminus of reelin in 128 mammalian species. Analysis of reelin sequences across mammalian species reveals that the CTR is highly conserved. The basic AA may play a role in binding to GAG or lipoprotein receptors.

[0027] Figure 9 . Orientation of selected basic amino acids in the heparin binding motif. Analysis of reticulin sequences in mammalian species shows that CTR is highly conserved. Basic AAs may play a role in binding to GAGs or lipoprotein receptors. Position 3447 (arrow) is oriented in the same direction as most other arginines. The arginines at positions 3446 and 3453 can also interact with heparin as part of the heparin binding motif, but are oriented differently from most basic AAs. R3452 and R3457 are part of the heparin binding motif, but are unlikely to contribute to heparin interactions because they are each oriented differently from other basic amino acids in the potential binding site.

[0028] Figure 10 Twenty lowest energy structures of reticulin CTR generated by 2D NMR. The structures reveal the presence of a region of flexibility towards the c-terminus of the peptide, which includes the H3447R mutation.

[0029] Figure 11A-D. Representative HPLC chromatograms of sclerotin peptide variants. (A) Zero (R3446H), one (WT), or two (H3447R) basic amino acids in positions 3446-3447 show increased interaction with heparin, indicated by later peak retention times in isocratic 1 M KCl elution. (B) This pattern applies to both short and long peptides. n = 2 replicates, within < 0.5 min of the representative peak. (C) Compared to the long sclerotin variant (D), the short sclerotin variant has an earlier peak retention time; however, both short and long peptides are similarly affected by the AA substitution at position 3447. H3447D has an earlier peak retention time compared to WT. The basic substitutions H3447K and H3447R have increased later peak retention times, thus indicating increased interaction with heparin. n = 2 replicates, within < 0.5 min of the representative peak.

[0030] Figure 12A-B Surface plasmon resonance analysis of the binding kinetics of chorionin CTR to heparin. Binding of H3447 (A) or H3447R (B) to heparin-coated metal films. Association constant (k a ) and dissociation constant (k d ) is used to calculate the equilibrium dissociation constant (K D ).

[0031] Figure 13 BLI of the heparin-reflexin interaction showed that H3447R had a ~2-fold greater interaction compared to WT reflexin. Binding of WT reflexin (A) to the heparin-coated biosensor. Association constant (k a ) and dissociation constant (k d ) is used to calculate the equilibrium dissociation constant (K D ).

[0032] Figure 14A-B.RELN regulation of Aβ aggregation. (A) Thioflavin T assay (ThT) of Aβ aggregation using RELN CTR WT and H3447R long (left) and short (right) showed that RELN CTR reduced Aβ aggregation. (B) ThT assay of Aβ aggregation alone or in the presence of RELN3431HH-R3446H-3451HH long peptide or RELN 3431HH-R3446H short peptide showed that the anti-aggregation effect was significantly reduced in the presence of RELN 3431HH-R3446H short variant. For both Figures D and E, data are expressed as the percentage of the maximum ThT emission of Aβ under 120 minutes of aggregation kinetics. Comparisons were made between the aggregation kinetics of Aβ alone or in the presence of RELN variants using a two-way ANOVA followed by a Tukey test for multiple comparisons. P values were calculated for samples at 30 min and 40-45 min (*p<0.05; **p<0.001; ***p<0.0001; ****p<0.00001).

[0033] Figure 15 .RELN CTR regulation of Aβ aggregation. Thioflavin T assay (ThT) of Aβ aggregation using RELN CTR H3447K and H3447D long (left) and short (right) showed that RELN CTR reduced Aβ aggregation. Data are expressed as the percentage of the maximum ThT emission of Aβ under 120 minutes of kinetic aggregation. The aggregation kinetics of Aβ alone or in the presence of RELN variants were compared using a two-way ANOVA followed by a Tukey test for multiple comparisons. P values for samples were calculated at 30 minutes and 40-45 minutes (*p<0.05; **p<0.001; ***p<0.0001; ****p<0.00001).

[0034] Figure 16 RELN CTR promotes cellular uptake of mRNA cargo. Green fluorescent protein (GFP) expression was demonstrated in human retinal endothelial cells treated with 5 μg of encoding-mRNA. The mRNA was conjugated to a cationic peptide at three different ratios. Ratios are expressed as mRNA:peptide mass ratio.

[0035] Figure 17A-J. In vitro screening of mini-RELN constructs on human retinal endothelial cells (HRECs). Shown are representative brightfield acquisitions (using 10X magnification) of HREC cells that were untreated or treated with Lipofectamine, 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 before transfection (Pr.t., top row), 5 hours after transfection (5H pt., middle row), and 24 hours after transfection (24H p.t., bottom row). Scale bar = 500 μm.

[0036] Figure 18A -E. In vitro screening of mini-RELN constructs on HRECs. A. Representative Western blots of HREC lysates after 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 a transfection agent. As controls, untreated and lipofectamine-treated cells were also tested. Western blot was used to detect total DAB1 and β-actin to normalize DAB1 levels.

[0037] Figure 19A -E. Quantification of total DAB1 levels expressed as normalized intensity to β-actin and control (lipofectamine treatment, Lp), showing reduced DAB1 levels in the presence of the mini-RELN construct compared to lipofectamine (Lp).

[0038] Figure 20A-F. In vitro screening of constructs on HREC cells. Representative Western blots of HREC lysates treated for 5 minutes with medium containing different 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 respin protein was obtained from R&D systems at 4 μg / mL. Phosphorylated DAB1 (pDAB1, 20A-F) and total DAB1 (20F) levels were measured.

[0039] Figure 21A -F. Quantification of pDAB1 levels expressed as normalized intensity to GAPDH and control (medium).

[0040] Figure 22 Quantification of total DAB levels in the case of 5 min overexpression of different mini-RELN constructs Fc-RELN WT (184I), Fc-RELNH3447R (184J) and RELN from Innovagen, expressed as normalized intensity to GAPDH and control.

[0041] Figure 23 Reduced oligomeric Tau-induced cytotoxicity in the presence of 184I mini-RELN peptide.

[0042] Figure 24A -CC-terminal RELN domain regulates Aβ aggregation. A, B. Thioflavin T (ThT) assay of Aβ aggregation alone or in the presence of C-terminal RELN WT (uncleaved or long, or furin-cleaved or short) (Figure A) or C-terminal RELN H3447R (uncleaved or long, or furin-cleaved or short) (Figure B), or in the presence of vehicle or morin as positive controls for aggregation inhibition. Aggregation kinetics for up to 40 minutes showed that mini-RELN reduced Aβ aggregation. C. Analysis of the change in the percentage of ThT fluorescence of Aβ at different time points and under different treatment conditions showed significant inhibition of Aβ aggregation. P values were calculated for samples at 30 and 40 minutes.

[0043] Figure 25A-B. Western blot validation of mini-RELN overexpression in HEK cells. Representative Western blots of both cell lysates and culture medium from HEK cells transfected to overexpress mini-RELN constructs compared to controls (UT, untreated and LP, lipofectamine), showing anti-Fc tag-positive bands when overexpressing Fc-tagged mini-RELN constructs.

[0044] Figure 26A -E. In vitro screening of constructs on HREC cells. A. Representative Western blots of HREC lysates treated for 5 minutes with medium containing 225Xf and 225Yf (A); 225ZZ and 233C (B); 225SU and 225SV (C); 225SW and 233F (D); or 225RR and 233A (E) mini-RELN constructs at different concentrations as indicated, or controls.

[0045] Figure 27A -E. Expressed as normalized intensity to b-actin and control (lipofectamine treated, Lp) from Figure 26A Quantification of pDAB1 levels in E.

[0046] Figure 28A-K. In vivo drug delivery and activity of mini-RELN peptides 225S (AF), 225T, and 225Z (GK). A. Anti-human IgG-Fc ELISA showed that the optical density levels in the hippocampus at both 24 and 72 hours post-injection tended to increase in the hippocampus 72 hours post-injection of mini-RELN-Fc labeled peptides compared to PBS vehicle. B. Representative WB of pDAB1 levels in the hippocampus 24 and 72 hours after nasal drug delivery. Quantification of CB. D. Representative WB of pDAB1 levels in the hippocampus and entorhinal cortex of TauP301S mice 72 hours after nasal drug delivery. GAPDH was detected as a loading control (bottom blot). E, F. Quantification of pDAB1-positive bands in the hippocampus (E) and entorhinal cortex (F) normalized to GAPDH and PBS-treated controls showed that the level of pDAB1 increased in the entorhinal cortex 72 hours post-injection. G. Representative WB of pDAB1 levels in the hippocampus 72 hours after nasal drug delivery of mini-RELN peptides 225Z and 225T to WT male mice (top blot). We used β-actin as a loading control (bottom blot). H, I. Representative WB of pDAB1 levels in the hippocampus 72 hours after drug delivery of 225T (H) and 225Z (I), showing that the level of pDAB1 increased in the hippocampus. J. Representative WB of pDAB1 levels in the midbrain 72 hours after nasal drug delivery of mini-RELN peptides 225Z and 225T to 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 shows that the level of pDAB1 increased in the midbrain.

[0047] Figure 29A-B In vivo characterization of pDab1 expression in wild-type animals treated with vehicle (pluronic) or mini-RELN 225S via transnasal brain delivery. A. Entorhinal-hippocampal analysis of pDAB1 expression 24 hours after mini-RELN administration. Positive pDab1 cells are surrounded by white dashed lines. Mini-RELN was detected using an FC antibody. DAPI is also shown. B. Manual segmentation was performed to show the area of positive pDab1 staining for mini-RELN compared to pluronic. Scale bar: 25 μm. Data show increased RELN signaling levels measured via pDab1 in animals treated with mini-RELN. A direct correlation between the signal of the mini-RELN peptide and the increased pDab1 signal is shown.

[0048] Figure 30Quantification of pTau Ser396 expression in MAPTP301S mice injected intraperitoneally with PBS or mini-RELN peptide 225T, assessed using immunofluorescence microscopy. pTau S396 fluorescence intensity was used as a hallmark of tau pathology. The data showed reduced pTau S396 signaling levels, indicating reduced tau pathology in the hippocampus and entorhinal region when treated with the mini-RELN system. DETAILED DESCRIPTION

[0049] Efforts to develop treatments for Alzheimer's disease (AD) have focused on removing amyloid, a hallmark of the neuropathology. The present inventors have characterized over 5,000 individuals from Colombian families with autosomal dominant AD (ADAD) due to the E280A mutation in presenilin-1 (PSEN1), of which approximately 1,200 were mutation carriers. PSEN1-E280A carriers typically develop cognitive impairment in their 40s. E280A carriers develop mild cognitive impairment (MCI) at a median age of 44 years (95% CI, 43-45) and dementia at 49 years (95% CI, 49-50), (1) with rare exceptions (2). We report a female PSEN1 E280A carrier with two copies of the APOE3 Christchurch (APOEch, R136S) mutation who remained cognitively intact nearly three decades after the expected age of clinical onset (2). The present disclosure is based in part on the clinical, in vivo neuroimaging, genetic, and neuropathological features of male cases from the same cohort with the PSEN1 E280A mutation, who also exhibited the extreme phenotype of delayed age of clinical onset of ADAD.

[0050] We characterized males heterozygous for the RELN-COLBOS variant who were resilient to cognitive impairment associated with the PSEN1E280A mutation until age 67. The observation of low tau pathology and increased neuronal density in the entorhinal cortex compared with other AD cases suggests that this brain region is involved in a RELN-mediated mechanism associated with protection against AD (Tables 2 and Figure 3A-E). A female sister carrier of the RELN-COLBOS and PSEN1E280A variants also exhibited a delayed age of onset of cognitive decline (although not optimally protected compared with her brother) and prolonged terminal disease. RELN-specific sex dimorphism may contribute to her unique features. We cannot rule out the possibility that other factors may contribute to the AD resilience phenotype in RELN-COLBOS carriers. Others have identified RELN as a candidate gene associated with AD pathology in cognitively healthy individuals (26), and DAB1 variants have been associated with AD risk in APOE4 homozygotes, further linking the RELN / DAB1 pathway to Alzheimer's disease (27).

[0051] The present inventors previously reported a female case of APOE3 Christchurch homozygotes 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 RELN-COLBOS cases compared to APOE3 Christchurch homozygotes, with the exception of the entorhinal cortex, which was largely spared in both, suggesting resiliency in RELN-COLBOS cases.

[0052] The hypervariable allele effect of RELN is mild. This is the first known report of a RELN hypervariable allele, and the stronger effect may not support the normal development of this key signaling process. The experimental evidence for a gain-of-function mechanism of the RELN-COLBOS variant and the fact that patients with extreme protection against ADAD have it provide a theoretical basis for the genetic significance of the observed phenotype. Without wishing to be bound by theory, it is hypothesized that RELN-COLBOS is not a neutral variant and may contribute to the restorative phenotype of the subjects.

[0053] 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 (neurophilin 1) NRP1, potentially conferring a competitive advantage for binding to its receptor (4). Binding of RELN-COLBOS to GAGs or heparan sulfate proteoglycans may increase the local concentration of RELN, leading to enhanced signaling. The current RELN-COLBOS case analysis reveals a convergent mechanism linking ApoE and RELN interaction to protection against AD, possibly via GAGs or other receptors. RELN-COLBOS is a gain-of-function variant that exhibits a robust ability to activate its canonical protein target, Dab1, and reduce human tau phosphorylation in knock-in mice. Modulation of this APOE-RELN protective pathway, particularly in the entorhinal cortex, may have profound therapeutic implications for combating tau pathology and neurodegeneration, as well as for resilience to cognitive decline and dementia in Alzheimer's disease.

[0054] Measurement of glucose metabolic rate using fluorodeoxyglucose (FDG) PET in the precuneus and whole-brain regions showed slightly higher levels of glucose metabolism compared with the average level in younger-aged typical MCI carriers from the family (Figures 1a-b).

[0055] Methods for treating and reducing the risk of cognitive decline and dementia

[0056] Provided herein are methods for treating cognitive decline and dementia or reducing the risk of cognitive decline and dementia or worsening cognitive decline and dementia. Methods may include administering RELN protein, for example, full-length RELN recombinant protein or mini-RELN recombinant protein as described herein or its variant (see, e.g., Table A), optionally wherein protein includes variant as described herein, such as H3447R or H3447K mutation and / or R3454A mutation, optionally a combination of H3447R or H3447K and R3454A mutation, or nucleic acid encoding RELN protein. R3455A provides resistance to furin cleavage of the CTR of filamentous protein (Kohno et al., J Neurosci. March 18, 2015; 35(11): 4776–4787). Alternatively or additionally, the method can include administering a gene editing agent that alters at least one allele in a cell to include an H3447R or H3447K mutation and / or an R3454A mutation, optionally a combination of H3447R or H3447K and R3454A mutations, to an agent that increases the expression or activity of reelin, such as an agent that reduces the methylation of reelin (e.g., a CRISPR / Cas-demethylase fusion). In some embodiments, the method includes directly administering the protein (and guide RNA / ssODN as needed); administering a nucleic acid encoding the protein (and guide RNA / ssODN as needed), for example as naked DNA or mRNA or in an expression vector such as a viral vector, or administering cells that express the protein (and guide RNA / ssODN as needed). For example, naked DNA can be administered without a vector (e.g., in the ciliary body of the eye) using an electroporation device.

[0057] Alzheimer's disease

[0058] The methods described herein can be used to treat subjects with all types of Alzheimer's disease or reduce the risk of subjects developing 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 method can be used to treat early-onset familial forms of Alzheimer's disease (AD) or cognitive decline associated with aging, or reduce the risk of developing early-onset familial forms of Alzheimer's disease (AD) or cognitive decline associated with aging.

[0059] Typically, increasing forgetfulness or mild confusion are early symptoms of Alzheimer's disease. Gradually, the cognitive impairment associated with Alzheimer's disease leads to memory loss, especially recent memory, disorientation and distorted spatial relationships, difficulty speaking, writing, thinking, and reasoning, personality and behavioral changes leading to depression, anxiety, social withdrawal, mood swings, distrust of others, irritability and aggression, changes in sleep habits, confusion, loss of inhibitions, delusions, and ultimately death.

[0060] Other neurodegenerative diseases, disorders or conditions

[0061] In addition to Alzheimer's disease, the present method can be used to treat other neurodegenerative diseases, disorders or conditions, including frontotemporal dementia, various types of memory loss, including but not limited to the cognitive impairment of mild cognitive impairment (MCI), or other conditions associated with the accumulation of beta-amyloid or the accumulation of tau or other protein conformational diseases such as frontotemporal dementia, or amyotrophic lateral sclerosis (ALS), or cognitive decline associated with aging. Neurodegenerative diseases can also include eye diseases such as age-related macular degeneration, glaucoma, diabetic retinopathy or hereditary retinal degeneration, stroke, brain trauma or concussion, retinal trauma, hereditary retinal degeneration, small vessel disease such as cerebral autosomal dominant arteriopathy (CADASIL) or abnormal angiogenesis with subcortical infarcts and leukoencephalopathy. Other applications include conditions genetically associated with RELN loss of function, including lateral temporal lobe epilepsy, autism, attention deficit hyperactivity disorder (ADHD), schizophrenia and bipolar disease as well as cortical lamination defects, abnormal neuronal migration and cerebellar atrophy). See, e.g., Shifman et al., 2008; Ober et al., 2008; Goes et al., 2010; Seripa et al., 2008; Fehér et al., 2015; Kelemenova et al., 2009; and Abo El Fotoh et al., 2020.

[0062] Reelin and mini-reelin

[0063] Reelin proteins useful in the methods and compositions described herein can include full-length wild-type Reelin proteins, 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 bind to receptors such as APOER2 or VLDLR, and / or result in activation of downstream targets such as Dab1 or promote neuronal or glial cell resilience, and / or bind to NRP1.

[0064] Exemplary full-length human striolarin sequences include the following:

[0065] Nucleic Acids protein Variants / isotypes NM_005045.4 NP_005036.2 Variant 1 / Reelin isoform a precursor* NM_173054.3 NP_774959.1 Variant 2 / Reelin isoform b precursor**

[0066] *Variant 1 represents the longer transcript and encodes the longer isoform a.

[0067] ** Variant 2 lacks an in-frame 6 nt microexon in the coding region compared to variant 1, resulting in isoform b being 2 aa shorter than isoform a.

[0068] In some embodiments, the methods and compositions described herein utilize sequences that are at least 80%, 85%, 90%, 95%, or 99% identical to human resilin.

[0069] In some embodiments, mini-RELNs (minimum RELNs) are used. Exemplary mini-RELNs can include sequences as described herein; mini-RELN constructs can optionally include protein linkers between these domains, for example, including amino acids such as Gly-Ser linkers that do not affect function between these domains; many other linkers are known in the art, see, for example, Chen et al., 2012. In some embodiments, the mini-RELN includes: A) a signal peptide, (B) an oligomerization domain, for example a dimerization domain, for example a CR-50 domain, (C) an APOER2 / VLDLR binding domain, for example, reticulin domains 5 and 6 (R5-6), and (D) a GAG binding domain / cell penetrating peptide, for example, from the C-terminus of reticulin, for example, as described herein, for example, in Table A.

[0070] Table A – Components of the Mini RELN

[0071]

[0072] Additional sequences, such as sequences from RELN, may also be included, but mini-RELN is not identical to full-length RELN. Mini-RELN may be preferred because it is easier to administer, cheaper to produce, and can be used for delivery for expression in standard AAV vectors or via mRNA or as a recombinant protein. Preferably, when used, the RELN sequence is from human RELN.

[0073] In some embodiments, the mini-RELN may comprise: a signal peptide, RAP, an IgG Fc, and a C-terminal RELN comprising a H3447R or H3447K mutation and / or a R3454A mutation, optionally in combination with H3447R or H3447K and R3454A mutations; a signal peptide, an IgG Fc, RAP, and a C-terminal RELN comprising a H3447R or H3447K mutation and / or a R3454A mutation, optionally in combination with H3447R or H3447K and R3454A mutations; or a signal peptide, R3-6, and a C-terminal RELN comprising a H3447R or H3447K mutation and / or a R3454A mutation, optionally in combination with H3447R or H3447K and R3454A mutations. In some embodiments, a C-terminal RELN comprising an H3447R or H3447K mutation and / or an R3454A mutation, optionally in combination with an H3447R or H3447K and R3454A mutation, can be longer than 30 amino acids (e.g., R8 to CTR - amino acids 3051-3460) but less than full-length resilin, e.g., at most 100, 105, 110, 120, 130, 150, 200, 300, 400, or 410 amino acids.

[0074] In some embodiments, at the N-terminus, the mini-RELN includes a signal peptide from RELN, or a signal peptide from another protein (e.g., an IL2 signal peptide, a human albumin signal peptide, a human α1-antitrypsin signal peptide, or a human Factor VIII signal peptide, not shown in SEQ ID NO: 1 below); a CR-50 domain (e.g., amino acids 229 to 345 of full-length RELN, shown below in lowercase); a linker (e.g., shown in lowercase italics); R5-6 of RELN (e.g., amino acids 1918 to 2664); and a C-terminus (e.g., amino acids 3429 to 3460, shown in italics, with the position of the H3447R variant indicated in bold). SEQ ID NO: 1 is an exemplary 225Z construct; other constructs, including those shown herein, can also be used.

[0075]

[0076]

[0077] Additional exemplary sequences of mini-RELN constructs include the following (keys to the font and style below indicate the order of the various modules):

[0078] 184I pfcn-huIgG2-C-termH3447WT (nucleotide sequence)

[0079] IL2SS-IgG2-Fc-Linker-C-terminal reln h3447WT

[0080]

[0081] 184I pfcn-huIgG2-C-termH3447WT (amino acid sequence – translation of the sequence by domain)

[0082] IL2SS-IgG2-Fc-Linker-C-terminal reln h3447WT

[0083]

[0084] 184J pfcn-huIgG2-C-termH3447H3447R (nucleotide sequence)

[0085] IL2SS-IgG2-Fc-Linker-C-Terminus Reln H3447R

[0086]

[0087]

[0088] 184J pfcn-huIgG2-C-termH3447R (amino acid sequence – translation of the domain sequence)

[0089] IL2SS-IgG2-Fc-Linker-C-Terminus Reln H3447R

[0090]

[0091] 225Q pFUSEN-hG2Fc-R5-6-C-termH3447R (nucleotide sequence)

[0092] IL2SS-IgG2Fc-Linker-Reln R5-6-C-terminal Reln H3447R

[0093]

[0094]

[0095] 225Q pFUSEN-hG2Fc-R5-6-C-termH3447R (amino acid sequence – translation of the sequence by domain)

[0096] IL2SS-IgG2Fc-Linker-Reln R5-6-C-terminal Reln H3447R

[0097]

[0098]

[0099] 225R pFUSEN-flag-CR-50-R5-6-CtermH3447R (nucleotide sequence)

[0100] IL2SS-Linker-flag tag-Linker-reln cr50-Linker- -C-terminal RelN H3447R

[0101]

[0102]

[0103] 225R pFUSEN-flag-CR-50-R5-6-CtermH3447R (amino acid sequence – translation of the sequence by domain)

[0104] IL2SS-Linker-flag tag-Linker-reln cr50-Linker- -C-terminal RelN H3447R

[0105]

[0106]

[0107] 225S pFUSEN-FcIgG-RAP-CTRH3447R (nucleotide sequence)

[0108] IL2SS-IgG2-Fc-Linker-Rap-Linker-C-Terminus Reln H3447R

[0109]

[0110]

[0111] 225S pFUSEN-FcIgG-RAP-CTRH3447R (amino acid sequence – translation of the sequence by domain)

[0112] IL2SS-IgG2-Fc-Linker-Rap-Linker-C-Terminus Reln H3447R

[0113]

[0114] 225T pFUSEN-RAP-FcIgG-CTRH3447R (nucleotide sequence)

[0115] IL2SS-rap-linker-IgG2-fc-linker-C-terminal reln H3447R

[0116]

[0117]

[0118] 225T pFUSEN-RAP-FcIgG-CTRH3447R (amino acid sequence – translation of the sequence by domain)

[0119] IL2SS-rap-linker-IgG2-fc-linker-C-terminal reln H3447R

[0120]

[0121] 225Xf pFUSEN-IL2ss-FcigG-APOE-R154S-CTRH3447R (nucleotide sequence) IL2SS-IgG2-Fc-LINKER-APOE R154S Christchurch-LINKER-C-terminal reln H3447R

[0122]

[0123]

[0124] 225Xf pFUSEN-IL2ss-FcigG-APOE-R154S-CTRH3447R (amino acid sequence – translation of the sequence by domain)

[0125] IL2SS-IgG2-Fc-Linker-Apoe R154s Christchurch-Linker-C-Terminus Reln H3447R

[0126]

[0127] 225Yf pFUSEN-IL2ss-FcigG-APOE-CTRH3447R (nucleotide sequence)

[0128] IL2SS-IgG2-Fc-Linker-Apoe-Linker-C-Terminus Reln H3447R

[0129]

[0130] 225Yf pFUSEN-IL2ss-FcigG-APOE-CTRH3447R

[0131] (Amino acid sequence – translation of sequence by domain)

[0132] IL2SS-IgG2-Fc-Linker-Apoe-Linker-C-Terminus Reln H3447R

[0133]

[0134]

[0135] 225Z pFUSEN-6xHISCR-50-R5-6-CtermH3447R (nucleotide sequence)

[0136] IL2SS-Linker-6Xhis tag-reln cr50-Linker- -C-terminal RelN H3447R

[0137]

[0138]

[0139] 225Z pFUSEN-6xHISCR-50-R5-6-CtermH3447R (amino acid sequence – translation of the sequence by domain)

[0140] IL2SS-Linker-6Xhis tag-reln cr50-Linker- -C-terminal RelN H3447R

[0141]

[0142]

[0143] 225SU pFUSEN-IL2signalpeptide-6XHistag-hReelin(AALeu1220-Ile2660)C-term-REELIN H3447R (nucleotide sequence)

[0144] IL2SS-LINKER-6Xhis tag-LINKER-l1120-Ile2660hreln-LINKER-c-terminal reln H3447R

[0145]

[0146]

[0147] 225SU pFUSEN-IL2signalpeptide-6XHistag-hReelin(AALeu1220-Ile2660)C-term-REELIN H3447R (amino acid sequence – translation of the sequence by domain)

[0148] IL2SS-LINKER-6Xhis tag-LINKER-l1120-Ile2660hreln-LINKER-c-terminal reln H3447R

[0149]

[0150] 225SV pFUSEN-IL2signalpeptide-6XHistag-hReelin(AALeu1220-Ile2660)Cterm-REELINWT (nucleotide sequence)

[0151] IL2SS-LINKER-6Xhis tag-LINKER-l1120-Ile2660hreln-LINKER-c-terminal reln wt

[0152]

[0153]

[0154]

[0155] 225SV pFUSEN-IL2 signal peptide-6XHistag-hReelin (AALeu1220-Ile2660) Cterm-REELINWT (amino acid sequence – translation of the sequence by domain)

[0156] IL2SS-LINKER-6Xhis tag-LINKER-l1120-Ile2660hreln-LINKER-c-terminal reln wt

[0157]

[0158] 225ZZ pFUSEN-Il2ss6xHISMini-RELN-FspCR50-R5-6-CtermH3447R (nucleotide sequence)

[0159] IL2SS-Linker-6Xhis Tag-Linker-reln's fspcr50-Linker- -C-terminal RelN H3447R

[0160]

[0161]

[0162] 225ZZ pFUSEN-Il2ss6xHISMini-RELN-FspCR50-R5-6-CtermH3447R (amino acid sequence – translation of the sequence by domain)

[0163] IL2SS-Linker-6Xhis Tag-Linker-reln's fspcr50-Linker- -C-terminal RelN H3447R

[0164]

[0165]

[0166] 225SW pFUSEN-IL2ss-6xHis-hReelin R3-R6CT-H3447R (nucleotide sequence) IL2SS-linker-6xhis tag-linker-reln r3-linker- -LINKER-c-terminal reln wt

[0167]

[0168]

[0169] 225SW pFUSEN-IL2ss-6xHis-hReelin R3-R6CT-H3447R (amino acid sequence – translation of the sequence by domain)

[0170] IL2SS-Linker-6Xhis Tag-Linker-reln's r3-Linker- LINKER-c-terminal reln wt

[0171]

[0172] 233ApFUSEN-IL2ss-6xHis-hReelinR3-R6CT-WT (nucleotide sequence)

[0173] IL2SS-Linker-6Xhis Tag-Linker-reln's r3-Linker- LINKER-c-terminal reln H3447R

[0174]

[0175]

[0176] 233ApFUSEN-IL2ss-6xHis-hReelinR3-R6CT-WT (amino acid sequence – translation of the sequence by domain)

[0177] IL2SS-Linker-6Xhis Tag-Linker-reln's r3-Linker- LINKER-c-terminal reln H3447R

[0178]

[0179]

[0180] 233C pFUSEN-Il2ss-6xHISR5-6CtermH3447R (nucleotide sequence)

[0181] IL2SS-Linker-6Xhis tag-Linker-reln r5-6-c-terminus relnH3447R

[0182]

[0183]

[0184] 233C pFUSEN-Il2ss-6xHISR5-6CtermH3447R (amino acid sequence – translation of the sequence by domain)

[0185] IL2SS-Linker-6Xhis tag-Linker-reln r5-6-c-terminus relnH3447R

[0186]

[0187] 233D pFUSEN-IL2ss-6xHis-hReelinR6-R6CT-H3447R (nucleotide sequence) IL2SS-Linker-6xHis tag-Linker-reln r6-Linker-reln r6-Linker-C-terminal reln H3447R

[0188]

[0189]

[0190] 233D pFUSEN-IL2ss-6xHis-hReelinR6-R6CT-H3447R (amino acid sequence – translation of the sequence by domain)

[0191] IL2SS-Linker-6Xhis Tag-Linker-Reln R6-Linker-Reln R6-Linker-C-Terminus Reln H3447R

[0192]

[0193] 233E-pFUSEN-Il2ss-6xHIS-R5-6-FspCR50-CtermH3447R (nucleotide sequence) IL2SS-Linker-6Xhis tag-Linker-reln r5-6-Linker- -LINKER-c-terminal reln H3447R

[0194]

[0195]

[0196]

[0197] 233E pFUSEN-Il2ss-6xHIS-R5-6-FspCR50-CtermH3447R (amino acid sequence – translation of the sequence by domain)

[0198] IL2SS-Linker-6Xhis Tag-Linker-reln r5-6-Linker- -LINKER-c-terminal reln H3447R

[0199]

[0200] 233F pFUSEN-IL2ss-6xHis-FspCR50-R6-R6Cterm RELN-H3447R (nucleotide sequence)

[0201] IL2SS-LINKER-6Xhis tag-LINKER- Linker-reln r6-Linker-reln r6-Linker-reln C-terminal reln H3447R

[0202]

[0203]

[0204] 233F pFUSEN-IL2ss-6xHis-FspCR50-R6-R6Cterm RELN-H3447R (amino acid sequence – translation of the sequence by domain)

[0205] IL2SS-LINKER-6Xhis tag-LINKER- Linker-reln r6-Linker-reln r6-Linker-reln C-terminal reln H3447R

[0206]

[0207] 225RR pFUSEN-Il2ss-flagMini-RELNFspCR50R5-6CtermH3447R (nucleotide sequence)

[0208] IL2SS-Linker-flag tag-Linker-reln's fspcr50-Linker- -C-terminal RelN H3447R

[0209]

[0210]

[0211]

[0212] 225RR pFUSEN-Il2ss-flagMini-RELNFspCR50R5-6CtermH3447R (amino acid sequence – translation of the sequence by domain)

[0213] IL2SS-Linker-flag tag-Linker-reln's fspcr50-Linker- -C-terminal RelN H3447R

[0214]

[0215] 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 functionality of the parent sequence. In some embodiments, the proteins may include sequences provided herein with exactly, at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 altered amino acids.

[0216] Residues that can be changed without destroying function can be identified, for example, by comparing similar sequences and making conservative substitutions in non-conserved regions (see, for example, the comparison provided herein). In order to determine the percentage identity of two amino acid sequences or two nucleic acid sequences, sequences are compared for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal comparison, and non-homologous sequences can be disregarded for the purpose of comparison). In some embodiments, the length of the reference sequence compared for the purpose of comparison 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 the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for the optimal comparison of the two sequences.

[0217] The comparison of sequences and the determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available on the World Wide Web at gcg.com), using default parameters (e.g., the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5).

[0218] Increased RELN signaling can also be achieved by increasing the activity or expression level of DAB1 using various approaches, including increasing its phosphorylation, decreasing dephosphorylation, or increasing dimerization or oligomerization. An exemplary sequence of DAB1 is included herein. DAB1 expression can be achieved via mRNA administration. Dimerization of DAB1 can be achieved by expressing a fusion of DAB1 and 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:

[0219]

[0220]

[0221] Exemplary sequences encoding Dabl dimerization constructs are provided below in the Examples.

[0222] RAP clustering can also be used to increase reelin signaling; see, for example, Strasser et al., Mol Cell Biol. 2004 Feb; 24(3): 1378-86. An exemplary sequence of a protein fusion of RAP and IgG2 is as follows (RAP in bold capital letters, linker in capital italics, IgG2 in lowercase, linker in capital letters, RELN CTR with H3447R variant in lowercase bold) (this corresponds to the 225T exemplary sequence):

[0223]

[0224] The HNEL sequence can be removed to enhance secretion.

[0225] Also provided herein are nucleic acids encoding the proteins described herein (eg, the mini-reel proteins and variants described in Table A), as well as cells expressing the proteins, and nucleic acids encoding variants, including vectors such as viral vectors.

[0226] gene editing

[0227] CRISPR-based methods can also be used to increase the level or activity of resilin. For example, CRISPR-Cas9 can be used to introduce H3447R or H3447K mutations and / or R3454A mutations, optionally a combination of H3447R or H3447K and R3454A mutations, into the resilin gene of a subject having one or more wild-type resilin alleles. For example, lead editing (see, e.g., Anzalone et al., Nature 576, 149–157 (2019)) can be used to introduce changes, optionally using an ssODN comprising the following sequence:

[0228] and a single guide RNA (sgRNA), or tracrRNA and crNA, comprising a spacer having the following sequence:

[0229] TGAATTTTTCACGACAACAT.

[0230] In some embodiments, the ssODN includes two mismatches to the wild-type RELN gene; the first mismatch restores the mutation H3447R (CAT to CGT), and the second optional mismatch disrupts the PAM (NGG) (from GGG to GGA), as shown below.

[0231]

[0232] Similarly, ssODNs can be designed to restore the H3447R or H3447K mutation and / or the R3454A mutation, optionally in combination with the H3447R or H3447K and R3454A mutations.

[0233] The Cas9 nuclease from Streptococcus pyogenes (S. pyogenes) can be guided via simple base pair complementarity between 17-20 nucleotides of an engineered guide RNA (gRNA) (e.g., a single guide RNA or a crRNA / tracrRNA pair) and the complementary strand of a target genomic DNA sequence of interest immediately adjacent to a protospacer adjacent motif (PAM), such as 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 CRISPR 1 (Cpf1, also known as Cas12a) nucleases from Prevotella and Francisella can also be used, for example, as described 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 with 23nt complementary to the protospacer sequence of the target DNA sequence at its 3' end (Zetsche et al., 2015). Furthermore, while SpCas9 recognizes the NGG PAM sequence 3' to the protospacer sequence, AsCpf1 and LbCp1 recognize the TTTN PAM present 5' to the protospacer sequence (ibid.).

[0234] In some embodiments, the present system utilizes a wild-type or variant Cas9 protein from Streptococcus pyogenes or Staphylococcus aureus, or a wild-type or variant Cpf1 protein from Acidaminococcus sp. BV3L6 or Lachnospiraceae bacterium ND2006, which is encoded in bacteria or codon-optimized for expression in mammalian cells and / or modified in its PAM recognition specificity and / or its genome-wide specificity. Many variants have been described; see, e.g., 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 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 Jun;32(6):569-76.

[0235] 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.

[0236] Table B: List of exemplary Cas9 or Cas12a orthologs

[0237]

[0238] Table C: List of exemplary high-fidelity and / or PAM-relaxed RGN orthologs

[0239]

[0240]

[0241]

[0242] *Prediction based on UniRule annotations on the UniProt database.

[0243] In some embodiments, the RGN sequence is modified to include a nuclear localization sequence (NLS) (e.g., at the C-terminus and / or N-terminus of the RGN protein) and a mini-polyadenylation signal (or Poly-A sequence). Exemplary NLSs include the SV40 large T antigen NLS (PKKKRRV); PKKKRKV; KRTADGSEFES)PKKKRKV; and nucleoplasmin NLS (KRPAATKKAGQAKKKK). Other NLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov 15; 1(5):411–415; Freitas and Cunha, Curr Genomics. 2009 Dec; 10(8):550–557. An exemplary polyadenylation signal is TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTGATCAGGCGCG.

[0244] A guide RNA suitable for RGNs should be used; in some embodiments, the gRNAs used in the present disclosure can be unimolecular or modular, as known in the art.

[0245] In some embodiments, a ribonucleoprotein complex (RNP), or a gene therapy vector or mRNA encoding a CRISPR / Cas9 construct, ssODN, and sgRNA (e.g., the ssODN and sgRNA described above) is administered to introduce H3447R or H3447K mutations and / or R3454A mutations in RELN, optionally a combination of H3447R or H3447K and R3454A mutations, into the whole brain or more specifically into the entorhinal cortex, e.g., using stereotactic injection as described herein. In some embodiments, where the Cas9 protein is administered in a viral vector, the sequence can be split into two vectors (see, e.g., Truong et al., Nucleic Acids Res. 2015 Jul 27; 43(13): 6450-8).

[0246] Demethylation of the RELN promoter

[0247] Methylation of the RELN promoter reduces expression of the resilin protein (Chen et al., Nucleic Acids Res. 2002 Jul 1;30(13):2930-9. Thus, the present method may comprise inducing demethylation of the RELN promoter. As an example, a CRISPR / Cas-based demethylase, in which a 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 trigger demethylation of the promoter sequence of RELN to derepress and thereby increase expression of resilin (57). The promoter sequence of the RELN gene is listed below, with gRNAs proposed for use with dCas9 targeting to regulate methylation.

[0248] RELN promoter sequence characteristics:

[0249] 346-365misc_feature:

[0250] 383-384 misc_feature: -458 Lintas et al. (56)

[0251] 430-431 misc_feature:-411 Lintas et al. (56)

[0252] 439-440 misc_feature:-402 Lintas et al. (56)

[0253] 394-413misc_feature:

[0254] 441-460misc_feature:

[0255] 1001-1003 misc_feature: orf start

[0256] RELN promoter sequence:

[0257]

[0258] In some embodiments, ribonucleoprotein complexes (RNPs), gene therapy, or mRNA encoding a CRISPR / Cas9 construct and the above-described ssODNs and sgRNAs are administered to introduce the H3447R or H3447K mutation and / or the R3454A mutation in RELN, optionally in combination with the H3447R or H3447K and R3454A mutations, into the whole brain or more specifically into the entorhinal cortex, e.g., using stereotactic injection as described herein.

[0259] Other compounds can also be used to reduce methylation of the RELN promoter and thereby increase expression of the resilin gene, including administration of hsa_circRNA_102049, which acts as a sponge for hsa-miR-214-3p (Wang et al., Bioengineered. 2022 Feb;13(2):2272-2284).

[0260] Delivery vehicle

[0261] Nucleic acids encoding reticulin or CRISPR / cas polypeptides (e.g., wild type, variants, peptides, or fragments thereof) can be incorporated into gene constructs for use as part of a gene therapy regimen. For example, described herein are targeted expression vectors for in vivo delivery and expression of polynucleotides encoding reticulin polypeptides or active fragments thereof in specific cell types, particularly cerebral cortical neuronal cells. The expression constructs of such components can be administered in any effective carrier, such as any formulation or composition that can effectively deliver the component genes to cells in vivo. The method includes inserting the gene into a viral vector, preferably an adeno-associated virus. Viral vectors typically directly transduce cells.

[0262] Viral vectors capable of efficiently transducing CNS neurons can be used, including rAAV (e.g., AAV1-AAV12) vectors of any serotype, recombinant or chimeric AAV vectors, and lentivirus or other suitable viral vectors. In some embodiments, the polynucleotide encoding the reelin protein is operably linked to a promoter suitable for expression in the CNS. For example, neuronal subtype-specific promoters such as the α-calcium / calmodulin kinase 2A promoter can be used to target excitatory neurons. Alternatively, pan-neuronal promoters such as the synapsin I promoter can be used to drive reelin protein 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 comprising 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 introducing nucleic acids into cells in vivo is by using a viral vector containing a nucleic acid (e.g., a cDNA encoding reelin). Among other things, infecting cells with viral vectors has the advantage that a large portion of the targeted cells can receive the nucleic acid. In addition, molecules encoded within the viral vector, for example by cDNA contained in the viral vector, are efficiently expressed in cells that have taken up the viral vector nucleic acid.

[0263] A particularly useful viral vector system for the delivery of nucleic acids is adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires other viruses such as adenovirus or herpes virus as helper viruses for efficient replication and productive life cycle. (For review, see Muzyczka et al., Curr. Topics in Micro and Immunol. 158: 97-129 (1992)). AAV vectors effectively transduce various cell types and can produce long-term expression of transgenes in vivo. Although the AAV vector genome can be maintained within the cell as an episome, vector integration has also been observed (see, e.g., Deyle and Russell, Curr Opin Mol Ther. 2009 Aug;11(4):442–447; Asokan et al., Mol Ther. 2012 Apr;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 the clinic; 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 Apr; 20(4): 699–708. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression. Protocols for producing recombinant retroviruses and for infecting cells with such viruses in vitro or in vivo 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 has been 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.

[0264] Thus, in some embodiments, the nucleic acid encoding the reelin protein 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, AAVrh10, AAV11, and AAV12.

[0265] The vectors described herein can be pseudotyped vectors. Pseudotyping provides a mechanism for regulating the target cell population of the vector. For example, pseudotyped AAV vectors can be used in the various methods described herein. Pseudotyped vectors are those that include the genome of a vector (e.g., a genome of an AAV serotype) in the capsid of a second vector (e.g., a second AAV serotype). Pseudotyping methods are well known in the art. For example, the vector can be pseudotyped with an envelope glycoprotein derived from the following: Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana strain and Chandipura strain), rabies virus (e.g., various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)), Lyssavirus Mokola virus (a rabies-related virus), vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein type B (FuG-B), variant of FuG-B (FuG-B2), or Moloney murine leukemia virus (MuLV). The virus can be pseudotyped for transduction of one or more neurons or cell groups. Additionally, the capsid can be altered 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, 1361–1368(2019); Nonnenmacher et al., Mol. Ther. Methods Clin. Dev. 20, 366–378(2021).

[0266] Without limitation, illustrative examples of pseudotyped vectors include recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV9, AAVrh10, AAV11, and AAV12 serotype vectors. It is known in the art that such vectors can be engineered to include transgenes encoding human or other proteins. In certain instances, the present disclosure may include pseudotyped AAV9 or AAVrh10 viral vectors containing nucleic acids as disclosed herein. See Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003.

[0267] In some cases, a particular AAV serotype vector may be selected based on its intended use, such as based on its intended route of administration.

[0268] Various methods for applying AAV vector constructs in gene therapy are known in the art, including methods for modification, purification, and preparation for administration to human subjects (see, e.g., Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). In addition, AAV-based gene therapy targeting cells of the CNS has been described (see, e.g., U.S. Patents 6,180,613 and 6,503,888). High-titer AAV preparations can be produced using techniques known in the art, such as those described in U.S. Patent No. 5,658,776.

[0269] Vector construct refers to a polynucleotide molecule comprising all or part of a viral genome and a transgene. In some cases, gene transfer can be mediated by DNA viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV). Other vectors useful in gene therapy methods are known in the art. For example, constructs as disclosed herein may comprise alphaviruses, herpes viruses, retroviruses, lentiviruses, or vaccinia viruses.

[0270] Adenovirus is a relatively well-characterized group of viruses, including more than 50 serotypes (see, e.g., WO 95 / 27071, which is incorporated herein by reference). Adenovirus is easily tractable by applying molecular biological 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 or generation of wild-type viruses (see, e.g., International Patent Publications WO 95 / 00655 and WO 95 / 11984, which are incorporated herein by reference). Wild-type AAV is highly infective and can be integrated into the host genome with a high degree of 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).

[0271] Non-native regulatory sequences, gene control sequences, promoters, non-coding sequences, introns or coding sequences may be included in nucleic acids as disclosed herein. Further contemplated herein are nucleic acids that include nucleic acid tags or signaling sequences, or that encode protein tags or protein signaling sequences. Typically, the coding region is operably linked to one or more regulatory nucleic acid components.

[0272] Promoters included in nucleic acids as disclosed herein can be tissue-specific promoters or cell-type-specific promoters, promoters specific for multiple tissues or cell types, organ-specific promoters, promoters specific for multiple organs, systemic or widespread promoters, or nearly systemic or widespread promoters. Promoters with stochastic expression, inducible expression, conditional expression, or otherwise intermittent, variable, or unpredictable expression are also included within the scope of this disclosure. Promoters can include any of the above characteristics or other promoter characteristics known in the art.

[0273] In clinical settings, the gene delivery system for therapeutic genes can be introduced into the subject by any of many methods, and each method is familiar in the art.For example, the pharmaceutical preparation of gene delivery system can be systematically introduced, for example, by intravenous injection, and the specific transduction of the protein in the target cell will occur by the specificity of the transfection provided by the gene delivery vector, due to the cell type or tissue type expression or its combination caused by the transcriptional regulatory sequence of the expression of the control receptor gene.In other embodiments, the initial delivery of recombinant genes is more limited, and it is quite local to introduce into the subject.For example, gene delivery vectors can be introduced by conduit (referring to United States Patent (USP) 5,328,470) or by stereotactic injection such as optionally injected into the cerebellomedullary cistern, cerebral ventricle, lumbar intrathecal space, directly injected into hippocampus (such as Chen et al., PNAS USA91:3054-3057 (1994)) and / or entorhinal cortex.In some embodiments, the delivery method of the virus expressing skeletal protein includes intravenous, intrathecal, intraventricular, intracisternal and stereotactic intraparenchymal administration.

[0274] The pharmaceutical preparation of gene therapy construct can be basically made up of the gene delivery system in an acceptable diluent, or can comprise a slow-release matrix in which the gene delivery vehicle is embedded. Alternatively, in the case where a complete gene delivery system can be completely produced from recombinant cells such as retroviral vectors, the pharmaceutical preparation can comprise one or more cells that produce the gene delivery system.

[0275] Reticulin preparations and pharmaceutical compositions

[0276] In some embodiments, a vesicle protein polynucleotide as disclosed herein is encapsulated by or associated with a nanoparticle for in vivo delivery to a target tissue. 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-labelled AZT bound to nanoparticles inrats determined by radioluminography. J Drug Target 5:171.1998); Sakuma SR et al. (Mucoadhesion of polystyrene nanoparticles having surface hydrophilicpolymeric chains in the gastrointestinal tract. Int JPharm 177:161.1999); Virovic L et al. Novel delivery methods for treatment of viral hepatitis:anupdate.Expert Opin Drug Deliv 2:707.2005); and Zimmermann E et al. Electrolyte-andpH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions inartificial 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, supra, pp. 317-327; see generally as described above). In some embodiments, lipid-based nanoparticles (LNPs) are used; see, e.g., Robinson et al., Mol Ther. 2018 Aug 1; 26(8): 2034-2046; US9956271B2.

[0277] The present methods and compositions may include microvesicles or their preparations containing one or more therapeutic molecules as described herein, such as polynucleotides or RNA. As used herein, the term "microvesicle" refers to membrane-derived microvesicles, which include 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, EP2010663B1. The methods and compositions described herein can be applied to microvesicles of all sizes; in some embodiments, 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 can also be more broadly applied to all extracellular vesicles (which is a term covering exosomes, shed microvesicles, oncosomes, ectosomes, and retrovirus-like particles). Such microvesicles or preparations are 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 are produced, for example, in vitro by culturing cells expressing the nucleic acid molecules of the invention 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 Aug; 11(8): 230-43) and 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 / fractionation and differential centrifugation, and can be adapted for use in the methods or compositions described herein. See, for example, EP2010663B1.

[0278] In some embodiments, the microvesicles are isolated by gentle centrifugation of the culture medium of the donor cells (e.g., at about 300 g) for a period of time sufficient to separate the cells from the culture medium (e.g., about 15 minutes). This leaves the microvesicles in the supernatant, thereby producing a microvesicle preparation. In some embodiments, the culture medium or supernatant from the gentle centrifugation is centrifuged more vigorously (e.g., at about 16,000 g) for a period of time sufficient to pellet the cell debris (e.g., about 30 minutes). This leaves the microvesicles in the supernatant, thereby producing a microvesicle preparation. In some embodiments, the culture medium, the gently centrifuged preparation, or the vigorously centrifuged preparation is subjected to filtration (e.g., through a 0.22 μm filter or a 0.8 μm filter, thereby passing the microvesicles through the filter. In some embodiments, the filtrate is subjected to a final ultracentrifugation (e.g., at about 110,000 g) for a period of time (e.g., about 80 minutes) that will sufficiently pellet the microvesicles. The resulting pellet contains the microvesicles and can be resuspended in a volume of buffer that produces a concentration useful for further use, thereby producing a microvesicle preparation. In some embodiments, the microvesicle preparation is produced by sucrose concentration gradient purification. In some embodiments, the microvesicles are further treated with DNAse (e.g., DNAse I) and / or RNAse and / or protease to eliminate any contaminating DNA, RNA, or protein from the outside, respectively. In some embodiments, the microvesicle preparation comprises one or more RNAse inhibitors.

[0279] The molecules contained in the microvesicle preparation will include therapeutic molecules. Typically, the microvesicles in the preparation will be a heterogeneous population, and each microvesicle will include a complement of molecules that may be different or identical with the molecules of other microvesicles in the preparation. The content of the therapeutic molecules in the microvesicle preparation can be quantitatively or qualitatively represented. A type of such method is to express content as the percentage of the total molecules in the microvesicle preparation. As an example, if the therapeutic molecule is mRNA, content can be expressed as the percentage of the total RNA content of the microvesicle preparation or alternatively as the percentage of the total mRNA content of the microvesicle preparation. Similarly, if the therapeutic molecule is a protein, content can be expressed as the percentage of total protein in the microvesicle. In some embodiments, compared with the microvesicles obtained from control cells (cells obtained from the same source that have not undergone scientific manipulation to increase the expression of therapeutic molecules), the therapeutic microvesicles produced by the methods described herein or its preparations include a detectable statistically significantly increased amount of therapeutic molecules. In some embodiments, the therapeutic molecule is present in an amount that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% greater than in microvesicles obtained from control cells. Higher levels of enrichment can also be achieved. In some embodiments, the therapeutic molecule is present in the microvesicles or their preparations at least 2-fold greater than in the microvesicles of control cells. Higher fold enrichment (e.g., 3, 4, 5, 6, 7, 8, 9 or 10-fold) can also be achieved.

[0280] In some embodiments, a relatively high percentage of the microvesicle contents are therapeutic molecules (e.g., by overexpression or specific targeting of molecules to microvesicles). In some embodiments, the microvesicle content of the therapeutic molecule 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 microvesicles). Higher levels of enrichment can also be achieved. In some embodiments, the therapeutic molecule is present in the microvesicles or their preparations at least 2 times more than all other such (similar) molecules. Higher times of enrichment (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 times) can also be obtained.

[0281] Reticulin CTR

[0282] Preliminary evaluation of the resilin CTR sequence indicates that it is highly conserved across mammalian species, suggesting that the previously uncharacterized resilin CTR may play an important neurobiological role (11). We show that the AA at position 3447 is aligned on the same side as other arginines in known heparin binding motifs. Therefore, a basic AA at this position has the potential to contribute to HSPG interactions ( Figure 9Based on this initial result, we used NMR to elucidate the previously unknown structure of the reticulin CTR ( Figure 10 We found that the reticulin CTR is primarily an α-helical structure, with the last 16 amino acids exhibiting increased flexibility. Increased flexibility in the secondary structure could indicate a potential binding site.

[0283] In addition, as observed by HPLC, the basic AA at position 3447 is conducive to heparin interaction. In two different experimental designs, H3447R tracing protein has a significantly later peak retention time compared to R3446H or WT peptide. We believe that the significant difference in peak retention time is at least greater than 30 seconds. Compared with neutral (WT) or basic AA (H3447R, H3447K), acidic AA (H3447D) has significantly less interaction. In short, tracing protein CTR has charge-based interactions with heparin, and position 3447 plays a role in these interactions.

[0284] To gain further insight into the kinetics of this interaction, SPR was used to assess the heparin-Restorin CTR peptide interaction. H3447R Restorin had ~2-fold more interaction with heparin compared to Restorin WT peptide ( Figure 12A-B ); To verify this small difference, we additionally evaluated this interaction using BLI and found the same difference as for the fc-fusion peptide ( Figure 13 ). In this case, the fc-fusion peptide is likely short due to the production method. Therefore, using two different methods, we found that H3447R had 2-fold more interactions compared to WT resilin.

[0285] We then used ITC to further understand the thermodynamics of this interaction and found that H3447R favors the interaction, especially in vivo. However, we also found that for long peptides, K a, ΔH, and ΔG, and ΔS are similar. This can be partially explained because this method evaluates binding in solution, while the kinetic and HPLC methods evaluate binding to heparin attached to the surface; therefore, the structure of the sclerotin peptide and the potential binding site exposure can be affected. Significantly, compared with the sclerotin WT short variant, the short peptide shows a more positive entropy and a more negative Gibbs free energy. Because sclerotin CTR is usually cleaved by furin, the short peptide is the most representative in vivo variant. Therefore, in vivo, compared with WT sclerotin, the H3447R sclerotin-HSPG interaction may be thermodynamically favorable. In addition, the negative control of ITC shows that the basic amino acids at positions 3446-3447 play an important role in the heparin interaction, and in the absence of at least one basic amino acid in the binding site, there is a ~100x less affinity.

[0286] We also found that H3447R interacted with NRP1 10-fold more than the WT fc-fusion peptide, suggesting that reticulin CTR may have additional interactions on the cell surface.

[0287] Overall, these data suggest that H3447R has increased interaction with heparin compared to WT. Without wishing to be bound by theory, chorionin first binds to HSPG and then to LDL receptors or other receptors, with chorionin CTR interacting with HSPG and chorionin middle domain interacting with LDL receptors. These cell surface interactions may ultimately regulate downstream NFT formation, as dysregulation of chorionin has previously been shown to lead to tau hyperphosphorylation (43, 44).

[0288] Significantly, RELN CTR 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 higher reduction. The patients described herein who were resilient to ADAD had high levels of amyloid plaque burden. These results suggest that the flexibility of the N-terminal region may play a role in the seeding of Aβ, as H is less flexible than R or K amino acids (45). In addition, the relative flexibility of the α-GAG binding site may help to resist the proliferation of Aβ plaques ( Figure 14A-B In contrast, our results show that the β-GAG site interacts with both GAG and NRP1, and that the H3447R mutation optimizes these interactions ( Figure 11A-D , Figure 12A-B , Figure 13 ).

[0289] The RELN CTR can have multiple interacting partners, including GAGs, NRP1, amyloid, and potentially others. The RELN H3447R mutation found in AD-protected cases is clearly not neutral and may contribute to the AD-protective phenotype through multiple mechanisms. We show that the RELN CTR–GAG interaction is reproducible across multiple methods and that the H3447R mutation enhances the binding site, potentially affecting interactions with multiple other molecules implicated in neurodegenerative diseases.

[0290] Provided herein are compositions comprising RELN CTR peptides. 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 comprise one or more optional or additional mutations as described herein (e.g., as shown in Table 1) and / or a mutation at G3444 (e.g., G3444H or G3444P).

[0291] Table 1. Properties of exemplary reticulin CTR peptides

[0292]

[0293] In some embodiments, the resilin 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.

[0294] In some embodiments, a peptide may include a sequence provided herein with exactly, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 altered amino acids.

[0295] Compositions comprising a CTR peptide are also provided, optionally in a pharmaceutically acceptable carrier, and optionally admixed with a non-reeling protein or nucleic acid. The nucleic acid may be, for example, an mRNA encoding a therapeutic or prophylactic agent, such as an antigen for a vaccine. The CTR peptide may also be incorporated into a fusion protein with a non-reeling protein sequence, for example, where the CTR peptide sequence is inserted internally at the N-terminus, the C-terminus, or at a position that does not affect the function of the non-reeling protein sequence.

[0296] Pharmaceutical compositions and methods of administration

[0297] The methods described herein include the use of pharmaceutical compositions comprising or consisting of a therapeutic agent described herein, such as a resilin protein or a fragment thereof, such as a mini-resilin; or a nucleic acid encoding the same, as described herein, as an active ingredient.

[0298] Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with drug administration. Supplementary active compounds may also be incorporated into the composition, such as mRNA encoding a therapeutic or diagnostic protein.

[0299] Pharmaceutical composition is usually formulated to be compatible with its expected route of administration.The example of route of administration includes parenteral such as intravenous, intradermal, subcutaneous, oral (such as inhalation), transdermal (topical), transmucosal and rectal administration.In some embodiments, composition is delivered to brain, for example, by being administered to cerebellomedullary cistern, cerebral ventricle, lumbar vertebra intrathecal space, directly injected into hippocampus (such as Chen et al., PNAS USA91:3054-3057 (1994)).In some embodiments, the delivery method of the virus expressing filament protein includes intravenous, intrathecal, intraventricular, intracisternal and stereotactic intraparenchymal administration.In some embodiments, composition is applied in or around the entorhinal cortex of brain.

[0300] Methods for preparing 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 for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for adjusting tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0301] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that is easy to inject. It should be stable under the conditions of manufacture and storage and must be protected against the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. Suitable fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the required particle size (in the case of dispersions) and by using a surfactant. The prevention of the effect of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid and thimerosal, etc. In many cases, it will be preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. The extended absorption of the injectable composition can be achieved by including an agent such as aluminum monostearate and gelatin that delays absorption in the composition.

[0302] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound into an appropriate solvent with one or a combination of the components listed above, as needed, followed by sterilization filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0303] Oral compositions typically include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be combined 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 adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients, or compounds of similar properties: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as mint, methyl salicylate, or orange flavoring.

[0304] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant (e.g., a gas such as carbon dioxide), or from a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0305] Systemic administration of the therapeutic compounds described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel, or cream as generally known in the art.

[0306] The pharmaceutical compositions may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0307] Therapeutic compounds that are or include nucleic acids 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 needle-free methods, such as the microparticle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and the percutaneous needle-free vaccination of mammals using a powdered vaccine as disclosed in U.S. Patent No. 6,168,587. In addition, intranasal delivery is possible, for example, as described, among others, in Hamajima et al., Clin. Immunol. Immunopathol., 88(2), 205-10 (1998). Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microcapsules can also be used. Biodegradable targetable microparticle delivery systems (e.g., as described in U.S. Patent No. 6,471,996) can also be used.

[0308] In one embodiment, the therapeutic compound is prepared with a carrier that will protect the therapeutic compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques, or, for example, commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies directed against cellular antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0309] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0310] Ocular administration can be achieved via intravitreal or subretinal injection of biologics (including proteins, nucleic acids, or vectors including viruses) or via electroporation of DNA plasmids in the ciliary body.

[0311] Methods for determining the risk of developing AD

[0312] The present invention includes a method for determining the risk of identifying AD in a subject, such as a human subject. The method relies on the detection of the H3447R variant of the RELN gene in the subject's DNA. The method includes obtaining a sample comprising genomic DNA from the subject and evaluating the sample for the presence of the H3447R variant.

[0313] As used herein, the term "sample," when referring to a material to be tested for the presence of the H3447R variant, includes, among other things, tissue (including buccal cells from a swab) or blood. Various methods for identifying and / or isolating and / or purifying genomic DNA from a sample are well known in the art. For example, the nucleic acids contained in the sample can first be isolated according to standard methods, such as using a lytic enzyme, a chemical solution, or by a nucleic acid-binding resin according to the manufacturer's instructions.

[0314] The presence and / or level of H3447R variant nucleic acid can be assessed using methods known in the art, for example, using 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, Magnetics) Diehl (2006) Nat Methods 3:551-559); RNAse protection assays; Northern blots; various types of nucleic acid sequencing (Sanger, pyrosequencing, next generation sequencing); or gene arrays / chips) (Lehninger Biochemistry (Worth Publishers, Inc., latest edition; Sambrook, et al., Molecular Cloning: A Laboratory Manual (3. Sup. rd 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 known in the art (see, e.g., Griffiths et al., ed. Modern genetic Analysis, 1999, Chapter 12 Genomics in 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) can be used to detect the presence of the H3447R variant. In some embodiments, techniques suitable for detecting changes in the structure or sequence of nucleic acids (such as the presence of deletions, amplifications, or substitutions) can be used to detect the H3447R variant.

[0315] Gene arrays can be prepared by selecting probes comprising the H3447R variant polynucleotide sequence and then fixing such probes to a solid support or surface. For example, the probes can include a DNA sequence, an RNA sequence, a copolymer sequence of DNA and RNA, a DNA and / or RNA analog, or a combination thereof. The probe sequence can be enzymatically synthesized in vivo, enzymatically synthesized in vitro (e.g., by PCR), or non-enzymatically synthesized in vitro.

[0316] Subjects having the H3447R variant in their genome can be identified as having a lower risk of developing AD compared to subjects who do not have the H3447R variant.In some embodiments, the subject has or is also identified as having an APOE4 variant allele.

[0317] Example

[0318] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0319] Example 1. Resilience to Autosomal Dominant Alzheimer's Disease in Carriers of the Reelin-COLBOS (H3447R) Variant

[0320] method

[0321] The following methods and materials were used in this Example 1.

[0322] Clinical evaluation

[0323] Patients were evaluated after providing written informed consent approved by the Institutional Review Boards of the University of Antioquia and Massachusetts General Hospital (MGH).

[0324] Brain imaging

[0325] We imaged brain Aβ and tau burden in vivo using Pittsburgh Compound B (PiB) and Flortaucipir (FTP) positron emission tomography (PET), respectively. Structural magnetic resonance imaging (MRI) and both PET scans were performed at MGH. 18 F-fluorodeoxyglucose PET was performed at the University of Antioquia, and the procedure and data analysis were performed as described previously ( 2 ).

[0326] Genetic and molecular studies

[0327] We performed whole-exome and whole-genome sequencing (WGS) and Genomizer analysis (v10.10) of the subjects to obtain a ranking of potential risk factors associated with AD, as previously shown (2) and described in detail below.

[0328] Mouse model

[0329] We generated a knock-in mouse model carrying the RELN-COLBOS variant via homologous recombination as a fee-for-service (Cyagen). Additional details about the mouse model and in vivo analysis are described in the Methods section in the Supplementary Appendix.

[0330] Neuropathology

[0331] Postmortem interval time was 210 minutes after death, and brain weight was 745.4 g. After fixation in 4% paraformaldehyde for 5 days and sample preparation in paraffin, 3 μm thick sections were cut from the indicated brain regions, dewaxed and stained with hematoxylin and eosin, or processed for immunohistochemical staining using antibodies as described in the additional neuropathological characterization section.

[0332] Additional Plasma Nfl Assay Details

[0333] Plasma neurofilament light chain (NfL) analysis was performed as previously reported by Sahlgrenska University Hospital ( The data were collected from 12 clinical neurochemistry laboratories in Sweden (1). The experimenters were blinded to the clinical or genetic data of the probands to avoid any bias.

[0334] Additional genome sequencing details

[0335] Whole genome sequencing analysis called 44 million variants from the genomes of the cases. The variants were subjected to multiple filters using Genomiser: population allele frequency <= 2%, variant effect filters excluding intergenic, untranslated regions (UTRs) and non-coding intronic regions, regulatory feature filters to remove all non-regulatory, non-coding variants over 20 Kb from known genes, and hiPhive sorter phenotype gene priority scores >= 0.401. 6,779 variants passed the filter and were ranked according to the composite Exomiser score from the gene-phenotype score and the variant pathogenicity score and compatibility with the inheritance pattern. The pathogenicity of the top three variants was manually reviewed. 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, SORL1CACNA1G, HFE, MPO, NOS3, and PLAU. Gene variants were extracted from WGS and annotated in the Alissa Interpret platform for independent pathogenicity interpretation (Agilent, Santa Clara, CA). We used combined annotation-dependent depletion (CADD) (2, 3) to calculate a "C score" to compare the pathogenicity of human-derived variants with simulated variants. The Regulatory Mendelian Mutation (ReMM) framework uses machine learning techniques to train a classifier to predict the likelihood of causing a Mendelian disease when a mutation occurs at any position in the noncoding genome (4).

[0336] Additional Single-Cell RNA Sequencing Details

[0337] We isolated peripheral blood mononuclear cells using Ficoll gradients and analyzed these cells by scRNA sequencing using a previously published protocol (1).

[0338] Additional details on RELN genotyping by Sanger DNA sequencing

[0339] DNA was first extracted using the Gentra Puregene kit (Qiagen) using the following protocol using 10 μM primers (forward: 5′-GTCCCAGCCTTTAGTTCCT-3′;

[0340] Reverse: 3'-CAACTTTCACGGACACATCAA-5') in 1 μL, 50 μL of the obtained sample (both RELN H3447R and RELN H3447 carriers) was amplified via polymerase chain reaction (PCR): 3 minutes at 94°C for initial denaturation, 33 cycles of 30 seconds at 94°C, 35 seconds at 62°C for annealing, 35 seconds at 72°C for elongation, and 5 minutes at 72°C for final extension. Horizontal electrophoresis was performed at 100 V using a 1.5% agarose gel in TAE buffer (Tris-acetate EDTA, T8280-1L, Sigma Aldrich) and mixed with GelRed (41003-T, Biotium). Fluorescence of positive bands was detected via a Bio-RAD molecular imager GelDoc XR+ and acquired via Image Lab software (version 6.0.1, Biorad). Amplified DNA was purified using the QIAquick gel extraction kit (Qiagen) and sequenced by MGH CCIB DNA core using a 3730xl sequencer (Applied Biosystems) as previously published (1).

[0341] Additional Cell Culture Details

[0342] The plasmid encoding full-length mouse recombinant RELN was a gift from Dr. Tom Curran via Addgene (plasmid #122444 (5)). The plasmid was subsequently mutagenized to obtain the homologous H3448R mutation of human RELN H3447R as a fee-based service from CustomDNAConstructs (New York, USA). We generated WT and RELN-COLBOS via transient transfection in Flp-In T-Rex293 mammalian cells (R78007, Thermo Fisher Scientific) and used them for receptor binding assays via ELISA. The plasmid encoding the CTR-RELN-Fc fusion peptide was obtained from CustomDNAConstructs as a fee-based service. All constructs were overexpressed in Flp-In T-Rex 293 mammalian cells (R78007, Thermo Fisher Scientific) using lipofectamine 2000 via transient transfection according to the manufacturer (11668030, Thermo Fisher). 5 hours after transfection, conditioned medium was collected using Opti-MEM. Cells were incubated for 24 hours and collected and cleared from cell debris by centrifugation at 1,800 rcf for 3 minutes at room temperature (RT). Primary CD1 cortical mouse neurons (M-CX-400, Lonza) were cultured in neurobasal media (Gibco) supplemented with B-27 (Thermo Fisher), glutamax (Gibco) and normocin (Invivogen). Cells were plated on poly-L-lysine (Sigma)-coated wells and processed on the 6th day after liquid nitrogen recovery. The treatment with recombinant RELN (RELN WT or RELN H3448R, 4 μg / mL) was incubated at 37°C, 5% CO2 for 5 minutes or 1 hour in the presence of 10 μM Mg-132 (ab141003, Abcam). Cells were washed in ice-cold dPBS (Gibco) and lysed in RIPA (9806, Cell Signaling) supplemented with 10 μM Mg-132, Triton-X100 (Sigma Aldrich), a protease inhibitor cocktail (4693159001, Millipore) and phosphatase inhibitors (4906837001, Sigma Aldrich and P0044, Millipore). Protein concentration was determined using a Pierce Bicinchonic acid (BCA) protein assay kit (23227, Thermo Fisher) 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 minutes.

[0343] Additional Western Blot Details

[0344] 20 μg of total 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 denatured by heating at 90°C for 5 minutes. Samples were separated by electrophoresis at 90V for 1 hour using a 4-20% precast gradient gel (Mini-PROTEAN TGX, Bio-Rad) and SDS-Tris-glycine buffer (Bio-Rad). Proteins were transferred to a 0.45 μm nitrocellulose membrane in ice-cold 20% methanolic Tris-glycine buffer (Bio-Rad) at 90V for 1 hour. To detect pDAB1 levels, proteins were transferred to a PVDF membrane using the iBlot2 Dry Blotting System (IB21002S, Thermo Fisher). Total protein levels were detected using Licor membranes blocked with Odyssey blocking buffer (LI-COR Biosciences) for one hour or with 5% milk powder (M17200-100.0, RPI) for two hours and, when blocking for anti-pDAB1 Western blots, with both protease and phosphatase inhibitor cocktails. β-tubulin (ms; 1:2,000; 86298S, Cell Signaling), anti-phospho-Dab1 (Rb; 1:7,500; MBS8511213, MyBiorsorce), total tau (ms, 1:1,000, ab80579, Abcam), phosphorylated tau (Ser396, rb, 1:1,000, 44-752G, Thermo Fisher), and anti-RELN antibody (ms, 1:1,000, clone CR-50, D223-3, MBL) were used as primary antibodies and incubated in blocking buffer for 2 h at room temperature or 18 h at 4°C. After washing the blot three times with TBS-T buffer (Pierce, Thermo Fisher), the secondary antibody was incubated at room temperature for 1 hour or 45 minutes (IRDye 800CW donkey anti-mouse, 925-32212, or IRDye 680CW donkey anti-rabbit; 1:10,000, 925-68073, Li-COR). Immunoreactive bands were detected using the Odyssey infrared imaging system and visualized on Image Studio software (version 2.1, LI-COR Biosciences). Dab1 was detected using an anti-rabbit-HRP conjugated antibody (HAF008, R&D Systems) followed by Western blot with pico Super Signal TMThe fluorescence was acquired by incubating with West Pico PLUS chemiluminescent substrate for five minutes and acquiring on a Syngene G:Box Digital ECL detection system.

[0345] Additional Heparin-Sepharose Affinity Chromatography Details

[0346] We chromatographically tested the changes in binding of RELN variants to heparin 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 5 volumes of degassed 20 mM TRIS-HCl buffer (pH 7.5). Recombinant C-terminal RELN peptides were produced and purified by Innovagen (Sweden): (WT)

[0347] RKQNYMMNFSRQHGLRHFYNRRRRSLRRYP, and (H3447R)

[0348] RKQNYMMNFSRQHGLRRFYNRRRRSLRRYP. 1 mL of 50 μg / mL peptide (H3447 or WT, and H3447R) was recycled through the column five times, and the final flow-through collected was used for further analysis. The column was washed five times with the same buffer and the protein was eluted using a 0.05M step gradient of NaCl (0-1M, 1 mL per fraction) in 20 mM Tris-HCl. To ensure complete release of the protein, the column was washed with 5 M NaCl 20 mM Tris-HCl. Three independent experiments were performed on C-terminal RELN WT and H3447R. All eluted fractions were subjected to spectral analysis by reading the absorbance at 280 nm using a Nanodrop 2000 spectrophotometer. GraphPad Prism 8 was subsequently used to analyze the blank correction fractions.

[0349] Additional ELISA Details

[0350] Enzyme-linked immunosorbent assay (ELISA) was used to quantify changes in the binding of RELN variants (full-length culture-derived or CTR-RELN) to VLDLr or ApoEr2 according to an optimized version of a previously published protocol (6). 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 CaCl2 (pH 7.4) (TBS-C buffer). After incubation at 4°C for 18 hours, the plates were blocked with 3% BSA (22070008-6, Bioworld), 0.05% Tween-20 (Sigma Aldrich) in TBS-C buffer for 1 hour. We evaluated binding by incubating with 100 μL / well 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) as the primary antibody for 1 hour at room temperature, and (1:10,000; 100 μL / well, donkey anti-goat IgG H&L, HRP, ab6885, Abcam) as the secondary antibody for 30 minutes at room temperature. Between each previous step, the plate was washed four times with TBS-C buffer (200 μL / well). The plate was washed three times before starting the colorimetric reaction (DY008, R&D Systems). After 5 minutes of incubation, stop solution (50 μL / well, DY008, R&D Systems) was added 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 Gen51.11 software and GraphPad Prism, respectively.

[0351] Additional SPR Assay Details

[0352] Surface plasmon resonance (SPR) was used to perform binding kinetics using a Biacore 3000 instrument (GE Healthcare) at 25°C as a paid service of Precision Antibody (Maryland, USA) according to a previously published protocol (7 24). Biotinylated heparin (B9806, Sigma Aldrich) was covalently linked 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 passed through the chip at a flow rate of 30 μL / min. The binding of the antigen to the ligand was monitored in real time to obtain the association (on) (ka) and dissociation (off) (kd) rates. The equilibrium constant (K) was calculated from the observed ka and kd. D The accuracy of the SPR analysis was determined by chi-square (χ2) analysis as described in the statistical analysis section. The WT and H3447R peptides we included in the analysis, as well as peptides with the hypothetical H3447D change (RKQNYMMNFSRQHGLRDFYNRRRRSLRRYP) and H3447K change (RKQNYMMNFSRQHGLRKFYNRRRRSLRRYP), were also produced and purified by Innovagen (Sweden).

[0353] Additional mouse model and in vivo analysis details

[0354] We generated the RELN H3448R-Tg knock-in (KI) mouse model in TACONIC by introducing the H3448R (CAC to CGT) mutation into exon 64 in the 3' homology arm of the RELN gene. Gene targeting was achieved using C57BL / 6 ES cells. KI mice were generated by injecting targeted ES cells into blastocysts (which were introduced into foster mothers for the generation of mouse crosses). Mice were euthanized using a chamber saturated with CO2. The cerebellum was removed and stored at -80°C with cervical dislocation, and the postmortem interval was ensured to be less than 3 minutes. All procedures were performed using protocols approved by the Institutional Animal Care Committee of the Massachusetts Eye and Ear Infirmary. Brain homogenates from the removed cerebellum were obtained in a modified RIPA buffer (Cellsignaling) supplemented with proteases (Roche) and phosphatase inhibitors (Sigma) using a tissue homogenizer (two 15" pulses). The homogenized tissue was then vortexed 20" every 10 minutes for 1 hour and centrifuged at 10,000 rpm at 4°C for 10 minutes. The soluble protein fraction was then analyzed using the BCA assay (Pierce).

[0355] We measured the levels of RELN (clone CR-50, D223-3, MBL), Dab1 (clone G-5, sc-271136, Santa Cruz), phospho-Dab1 (Tyr232, MBS8511213, My Biosource) in the cerebellum of adult male and female mice (6 to 12 months of age, n=3-4 per genotype) that were wild type, heterozygous, or homozygous for the RELN H3448R mutation by western blot.

[0356] Additional neuropathological features details

[0357] The postmortem interval of the brain tissue was 210 minutes after death. The brain showed significant atrophy of the frontal lobe; the brain and related structures weighed 745.4 g, and the interuncal 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 (CN), 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 processed for amyloid β (Aβ, 1:100; BAM-10, Mob410; DBS Emergo Europe, The Hague, The Netherlands), hyperphosphorylated tau (ptau, 1:100; AT8, MN1020, Thermo Fisher Scientific, The Hague, The Netherlands), and morphological changes of the cerebral cortex (Central South University, Cen- t ... Immunohistochemical (IHC) staining of the retinal tissues was performed using antibodies against 1% B cells (ApoE) and 2% C-terminal reelin (RELN-CT, 1:200; E-5, sc-25346, Santa Cruz Biotechnology Inc., Heidelberg, DE) with specific secondary antibodies against mouse and rabbit (P0260 and P0447, respectively, DAKO GmbH, Jena, DE), neuronal nuclei (NeuN; 1:100; MAB377; Merck / Millipore, Darmstadt, Germany). Visualization was achieved using 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 the 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 staining and Klüver-Barrera (KV) staining. Cresyl violet staining was used for neuronal perikarya.Neuropathology examination was performed by an experienced morphologist who was not aware of the source of the sample (MG and DS-F.). The slices were scanned using a Hamamatsu NanoZoomer automatic digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan), and images and regions of interest (cortex for cortical areas and whole stained sections for non-cortical areas) were obtained with a resolution of at least one pixel per μm. By using ImageJ software (version 1.52p, NIH, Bethesda, MA, USA), after color deconvolution and thresholding, signal intensity and particles and total area (8) were assessed in the brown (DAB) color channel. Neuron counts were performed manually and normalized by the area of the selected region of interest in the hippocampus and parahippocampal structures. Information about statistical analysis is reported in a dedicated section.

[0358] Additional Immunoprecipitation Details

[0359] Mouse frontal cortex tissue was homogenized by two 15" pulses in ice-cold M-PER protein extraction reagent (78503, Thermo Fisher) supplemented with phosphatase and protease inhibitors using a homogenizer as described in the previous paragraph. 100 μg of total protein was pulled down using anti-phosphotyrosine magnetic beads (clone 4G10, 16-282, Millipore), anti-total Dab1 agarose beads (clone G-5, sc-271136AC, Santa Cruz), and anti-normal mouse IgG isotype control agarose beads (SC-2343, Santa Cruz). Before the samples were immunoprecipitated overnight at 4°C, 10% was removed and analyzed as input. The beads were washed in PBS while collecting the unbound fraction and then boiled in 4X reducing sample buffer for 6 minutes to allow release of the immunoprecipitated proteins.

[0360] Additional sequencing analysis by mass spectrometry

[0361] 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). Excised gel bands were analyzed (as a fee-based service) at the Taplin Biomass Spectrometry Facility (Harvard Medical School, Boston, MA). The gel bands were subsequently dehydrated in acetonitrile and then concentrated in vacuo (speed-vac). Rehydration of the bands was obtained at 4°C in a solution of 50mM ammonium bicarbonate supplemented with 12.5ng / μL modified sequencing grade trypsin (Promega, Madison, WI). The samples were washed with 50mM ammonium bicarbonate solution. Protein extraction was obtained by removing the ammonium bicarbonate solution after overnight incubation at 37°C. Proteins were washed in 50% acetonitrile and 1% formic acid (9). Reconstituted samples were sequenced via electrospray ionization-LTQ Orbitrap Velos Pro ion trap mass spectrometer (ThermoFisher Scientific, Waltham, MA) using a nanoscale reversed-phase HPLC capillary column with a gradient elution of acetonitrile and formic acid. Protein-specific fragment ion sequences were analyzed using Sequest (10) (Thermo Fisher Scientific, Waltham, MA) using known peptide sequences.

[0362] Additional statistical analysis details

[0363] All data presented are expressed as mean values, and errors are expressed as standard error of the mean (sem) or standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, La Jolla California USA, graphpad.com). P values less than 0.05 and α of 0.05 were considered statistically significant. We used Kruskal-Wallis (Dunn post hoc analysis for multiple comparisons of 4 independent experiments) to compare changes between primary cortical neurons treated with MOCK, RELN WT, or RELN H3448R, and data are presented as mean ± sem. For SPR data ( Figure 2C and Figure 7A-B), we verified the accuracy of the results via chi-square test (χ2) analysis, and compared the sensorgrams obtained experimentally (colored lines) with the sensorgrams (black lines) generated by BIAnalysis software arithmetic. Values ranging from 1 to 2 were interpreted as significant (accurate), and those below 1 were interpreted as highly significant (highly accurate). The Western blot analysis presented in Figure 2E-F was performed using GraphPad Prism9, using one-way analysis of variance followed by Fisher's LSD test for multiple comparisons. Neuropathological data (Figure 3) were analyzed and graphs were generated using GraphPadPrism 6 (GraphPad Software, Inc., La Jolla, CA, USA) and R statistical software (R Foundation for Statistical Computing, Vienna, Austria; R-project.org.). Analyses including distribution analysis and correlation analysis were performed using Spearman's Rho test. Brain color maps were created using the R package cerebroViz. Statistical significance for all analyses was determined by *p≤0.05, **p≤0.01, and ***p≤0.001.

[0364] Example 1.1 - Case Report

[0365] We identified a male carrier of the PSEN1E280A mutation who remained cognitively intact until age 67. He completed five years of formal education in his native Colombia and worked until his retirement at age 64. He is married with two children. A first assessment at age 67 revealed limited verbal learning skills and language difficulties despite functional independence. The patient was diagnosed with MCI, characterized by decreased short-term memory and verbal fluency, at age 70.

[0366] At age 72, his speech deteriorated further. He progressed to mild dementia at age 72 (Table 2). An episode of septic shock associated with a urinary tract infection preceded cognitive decline. At age 73, he required assistance with basic and instrumental activities of daily living, meeting criteria for moderate dementia. He died of aspiration pneumonia at age 74, and his relatives consented to brain donation for neuropathology.

[0367] This subject's sister, who carried the PSEN1E280A mutation, had severe dementia when she was first evaluated at age 64, which progressed to end-stage dementia at age 72 (see pedigree in Figure 5A). According to her family, she developed depression, hypothyroidism, hypertension, and cognitive decline at age 58, and dementia developed at age 61. Prior to dementia, she suffered ocular trauma and a tibia fracture following a fall, which required surgery under general anesthesia. She died of pulmonary sepsis at age 73.

[0368] Table 2. Test Scores and Percentiles by Age and Education Neuropsychological test scores adjusted for age and education of the subjects revealed a pattern of significant global and progressive cognitive decline over the 5-year assessment period.

[0369]

[0370] NOTE. MMSE, Mini-Mental State Examination; CERAD, Consortium to Establish a Registry for the Test Battery of Alzheimer's Disease; GDS, Geriatric Depression Scale; EDG, Global Decline Scale. Percentiles were calculated using age- and education-based norms from the Colombian population aged 65 years and older and are presented as means and standard deviations (SD); for a complete description of neuropsychological measures and normative data, see Torres, Vila-Castelar et al. 2019 (11). The following guidelines can be used for the qualitative interpretation of his performance: 90-75 percentile = above average; 74-25 percentile = average; 24-9 percentile = below average; 8-2 percentile = borderline; 1 = very low. ***

[0371] Example 1.2 - Identification of RELN-COLBOS mutations

[0372] The patient participated in the Columbia-Boston Biomarker Study (COLBOS) and underwent neuroimaging at MGH when he was 73 years old. Amyloid PET, measured by cortical and cerebellar Pittsburgh compound B (PiB), revealed a higher cortical Aβ plaque burden in this subject (distribution volume ratio, DVR = 1.77) than in younger MCI carriers from this family with a typical age of onset (DVRs 1.49–1.60, Figure 1). Tau tangle burden in the inferior temporal lobe, measured by flortaucipir (FTP), was similar to that observed in younger PSEN1E280 MCI carriers with a typical age of onset (SUVR 1.78). However, he had relatively limited tau pathology in the entorhinal cortex (EC = 1.34 SUVR, Figure 1A, C) and in other neocortical regions such as the posterior cingulate cortex and precuneus (PCC = 1.51; precuneus = 1.49 SUVR, Figure 1A), which typically show higher levels of tau pathology in PSEN1E280A carriers who develop MCI and dementia at typical ages (3) (Figure 1). Sparing the entorhinal cortex from tau pathology is a prominent feature of RELN-COLBOS cases that may be crucial for the protective phenotype.

[0373] Measurement of glucose metabolic rate in the precuneus and whole brain regions using fluorodeoxyglucose PET showed relatively preserved metabolism (Figure 1). He had brain atrophy similar to that of typical MCI carriers as measured by MRI-based hippocampal and whole brain volumes. These imaging findings suggest that in this patient and in APOE3 Christchurch homozygous cases, (2) protection against ADAD dementia may be mediated by mechanisms that limit regional tau pathology, even in the face of high amyloid burden and substantial tau pathology in brain regions other than the medial temporal lobe (Figure 1).

[0374] Our genetic analysis confirmed that the subject was a heterozygous carrier of the PSEN1E280A mutation (confirmed by single-cell RNA sequencing), ruled out the presence of the Christchurch mutation (the subject was APOE3 / APOE3 and had a normal lipid profile), and identified a heterozygous variant in RELN (H3447R, Figure 5B), which we named "RELN-COLBOS," as the most promising missense variant that could contribute to the phenotype in the protected subject. The RELN-COLBOS variant was found only in the subject and his sister (also APOE3 / APOE3), who also had late-onset cognitive decline. In brief, we focused on the RELN-COLBOS variant because it ranked among the top three candidate genes in the Genomizer prioritization score analysis and because RELN is functionally closely related to APOE, a gene mutated in other cases with extreme protection against ADAD (2).

[0375] Example 1.3 - Molecular analysis of RELN-COLBOS variants

[0376] RELN binding triggers clustering and activation of VLDLr and APOER2, leading to a signaling cascade that regulates disabled 1 (Dab1) activity, thereby causing reduced tau phosphorylation. (4-7) In primary cultured mouse cortical neurons, RELN-COLBOS was approximately two times more effective than wild-type (WT) RELN in triggering Dab1 phosphorylation (Figure 2A, p=0.0246) and reduced tau phosphorylation at position 396, an early marker of tauopathy (8-10) ( Figure 6A-B In cell-free binding assays, RELN COLBOS did not directly affect RELN binding to VLDLr or APOER2.

[0377] The C-terminal region of RELN (CTR-RELN), where the H3447R variant is located, indirectly regulates signaling via interactions with previously unidentified co-receptors on the cell membrane. (11) CTR-RELN has many basic amino acids that are well conserved across species (11), which we hypothesized could mediate interactions with glycosaminoglycans (GAGs). Interaction with GAGs is the rate-limiting step in the interaction of ApoE with some receptors (12), and the role of GAGs in RELN activity has not been fully resolved (11, 13). We used affinity chromatography to examine heparin (a type of GAG) binding of recombinant CTR RELN peptides. CTR RELN WT and CTR RELN H3447R bound to heparin. CTR RELN H3447R required a higher NaCl salt concentration for release from the heparin column, indicating increased binding affinity (Figure 2B). Surface plasmon resonance (SPR) measurements of kinetic constants showed that the affinity of CTR RELN H3447R was approximately twice that observed in WT ( Figure 2C ).

[0378] Substitution of histidine at position 3447 with aspartic acid, a highly acidic amino acid (H3447D), reduced heparin interaction, whereas substitution of lysine, a basic amino acid (H3447K), had minimal effect, further supporting the critical role of position 3447 in GAG binding. Surface plasmon resonance (SPR) measurements of kinetic constants showed that the affinity of CTRRELN H3447R was approximately twice that observed in WT: H3447R>WT>H3447K>>>H3447D ( Figure 2C , Figure 7A, B). RELN H3447D could not be produced in sufficient quantities for signaling analysis. This limitation is consistent with previous reports of specific mutations in the C-terminus that limit secretion of RELN (52).

[0379] We used ELISA to evaluate the direct binding of recombinant RELN H3448R to the classical RELN receptors VLDLr and APOER2 (53) to further examine the molecular mechanisms leading to the observed gain of function. We used experimental conditions with RELN H3448R, RELN WT, or an equimolar mixture of WT and variant proteins, because RELN oligomerization is a key property that regulates binding to the receptor (53). Under 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 region of 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 5th and 6th reticulin repeats (R5-6) of RELN are essential for mediating binding to the receptor (53). We conclude that the gain-of-function of the RELN H3447R variant is unlikely to be explained by direct changes in binding affinity to the VLDLr or APOER2 receptors.

[0380] Example 1.4 - Disease-Modifying Effects of RELN-COLBOS Mutations

[0381] We generated a knock-in mouse model carrying an equivalent of the RELN-COLBOS variant (H3448R or mRELN-H3448R (11)) to further support genetic imputation of causality, a common practice for studying rare variants. This mouse model is viable, fertile, and lacks the obvious structural and phenotypic brain abnormalities of RELN loss-of-function variants (e.g., cortical lamination defects, abnormal neuronal migration, and cerebellar atrophy) (14, 15). Analysis of the cerebellum from mice with mRELN-COLBOS confirmed the observation of gain-of-function of RELN-H3448R, as determined by enhanced phosphorylation of Dab1 in males (Figure 2D, F, p = 0.0284), and revealed a propensity for the formation of higher molecular weight protein oligomers of RELN-COLBOS, a feature that may be critical for enhanced activity (16). The cerebellum is a target of RELN phenotypes in mice and humans (14, 15). Morphological analysis revealed a mild but statistically significant increase in cerebellar neuron number in mice harboring the RELN-COLBOS variant, supporting the hypothesis of a gain-of-function mechanism, although no neuronal density phenotype was observed in other brain regions.

[0382] This mouse model allowed us to examine the sexually dimorphic effects of the RELN-COLBOS variant, a feature that has been described for conditions associated with genetic variation in RELN, including schizophrenia, bipolar disorder, autism, and Alzheimer's disease (17-22). Increased Dab1 phosphorylation and enhanced oligomerization of RELN were observed only in male mice (Figure 2D). This finding is consistent with the best association of RELN-COLBOS with protection against ADAD that we observed in male and female cases. Homozygosity was required to detect changes in Dab1 activity and GSK3β activity (another downstream target of RELN signaling) associated with the RELN-COLBOS variant. In summary, these data suggest that RELN H3447R is a gain-of-function (super allele) variant.

[0383] To attempt to correlate the phenotypes of RELN-COLBOS in mice and humans, we employed a cross-breeding strategy using our knock-in mouse models and tauopathy mouse models, specifically the STOCK Tg(Prnp-MAPT*P301L)JNPL3Hlmc mouse from Dr. Huton's laboratory, distributed by Taconic. This mouse model expresses a mutation in the tau gene, leading to the accumulation of tau tangles and neuronal loss in specific brain regions, and is commonly used to study tauopathies. 23 The decision to use this mouse model was based on the known effects of RELN signaling on tau phosphorylation. 24 , and our clinical observations of relatively reduced tauopathy in certain brain regions of postmortem human brain samples from protected cases. Our study found that compared with controls ( Figure 2J ) compared to male P301L mice expressing the RELN-COLBOS allele in the hippocampus ( Figure 2J -K) and medulla oblongata with significantly reduced human tau phosphorylation (ptau205). We also observed a significant rescue of the abnormal limb grasping response (a common consequence of tauopathies in mice) in RELN-COLBOS mice harboring a tau transgene ( Figure 2L -M). Although additional studies of this model are necessary, our findings strongly support our hypothesis that RELN-COLBOS is a gain-of-function mutation and is likely to be associated with the restoration of tau disease. This analysis also shows direct evidence of rescue in a mouse model with the MAPT mutation associated with frontotemporal dementia.

[0384] Postmortem examination of this case showed neuropathological evidence of severe AD (classified as CERADC, Braak stage VI and Thal stage 5) with extensive amyloid and tau pathology. Figure 3A). Recently, we reported the neuropathological profile of a homozygous PSEN1E280A carrier of the APOE Christchurch mutation. This case showed a unique pathological phenotype among PSEN1E280A cases, with significantly lower ptau pathology in most brain regions except the primary visual cortex (23). In contrast, a side-by-side comparison showed that RELN-COLBOS cases had more ptau pathology relative to APOEch cases, except in specific regions. Both cases showed widespread Aβ pathology in all evaluated regions, although there was some individual variability.

[0385] We focused our analysis on the hippocampus and related cortices because these structures are known to be affected early in AD (24). Neurons within layer II of the entorhinal cortex and entorhinal cortical neurons are generally particularly vulnerable to aging and AD (25). We measured neuronal density in the hippocampus and parahippocampal regions of RELN-COLBOS cases, AD-resistant APOEch cases, typical PSEN1E280A 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 association was not evident in other subregions such as CA1 ( Figure 3C ; S17 and S18). RELN-COLBOS and APOEch cases showed significantly lower intraneuronal ApoE signaling compared with FAD and sporadic AD controls ( Figure 3D ), while RELN-COLBOS showed higher intracellular signal of reelin in the white matter ( Figure 3D ).

[0386] Neuropathological findings are consistent with our in vivo neuroimaging observations and confirm the potential role of entorhinal cortex integrity as a target for RELN-mediated mechanisms critical for resilience to ADAD.

[0387] Example 2. Computer simulation of filamentous protein

[0388] To understand the potential importance of the reelin CTR, computational analysis revealed that the RELN CTR, which includes basic AAs that likely form charge-based binding sites, is highly conserved across mammalian species. We also showed that the reelin CTR is likely intrinsically disordered. Intrinsically disordered regions (IDRs) within proteins lack a well-defined tertiary structure and may be important for several biological functions, including protein interactions. Furthermore, IDRs may play a role in interactions within plaque deposits in individuals with neurodegenerative diseases, including AD.

[0389] Previous work has shown that reticulin CTR is highly conserved across vertebrate species (11). This work also showed that reticulin is not required for secretion but may play a role in downstream signaling.

[0390] method

[0391] Phylogenetic trees were created using BEAST, which uses Bayesian Markov Chain Monte Carlo (MCMC) to create trees based on the filament protein CTR sequences. Used to evaluate intrinsically disordered regions of RELN. Specifically, VL-XT (various long disordered regions and X-ray characterized terminal disordered regions) is a combination of three feedforward neural networks. Individual neural networks are trained on long disordered regions (greater than 39 AA), N-terminal disorder, and C-terminal disorder.

[0392] result

[0393] The reelin CTR is highly conserved

[0394] The reelin CTR is also highly conserved among mammalian species ( Figure 8 ). Therefore, it is hypothesized that the C-terminal region of reelin is important for the binding of reelin to HSPG and the subsequent regulation of downstream pathways. Among the species available in the National Center for Biotechnology Information (NCBI) database, there is a high level of conservation at position 3447 in most mammalian species, except in horses (Equus caballus, Equus asinus, Przewalskii).

[0395] Reticulin CTR contains a GAG-binding motif

[0396] Initial structural data suggest that there may be two binding sites in the C-terminal region ( Figure 9 ). The key AA positions for interaction 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 AAs with acidic GAGs is primarily driven by electrostatic forces (30). Therefore, it was hypothesized that these basic amino acids may be particularly important for GAG interactions and that the C-terminal region may contain binding sites for interaction with GAGs.

[0397] H3447 is oriented in the same direction as other basic amino acids

[0398] Bioinformatics analysis shows that the RELN CTR is highly conserved across mammalian species, including position H3447R (purple), a mutation found in recovered AD cases. Many of the conserved amino acids are highly basic residues, suggesting potential binding to GAGs. The basic AAs shown in magenta, including position 3447, are oriented on the same side, suggesting a primary role in the interaction with heparin. Here, the orientation of the basic amino acids may contribute to the creation of the binding site.

[0399] in conclusion

[0400] The filamentous protein CTR is highly conserved across a wide variety of species. The basic AAs hypothesized to contribute to GAG interactions are >90% conserved. The only species with a different AAs at position 3447 is Equidae (Q3447), which is apparently uncharged and should not produce significant differences compared to the weakly basic histidine. Furthermore, these basic AAs, including position H3447R, are oriented on the same side of the α-helix, which has previously been shown to be an important aspect of GAG interactions.

[0401] Example 3: Kinetics of the Resin-Heparin Interaction

[0402] To further understand the kinetics of the RELN CTR-heparin interaction, surface plasmon resonance (SPR) was used to add additional support to the proposed neuroprotective mechanism of the H3447R variant. D ) showed that H3447R had ~2x the binding affinity of H3447. In addition, biolayer interferometry was used to further confirm these results.

[0403] method

[0404] surface plasmon resonance

[0405] Surface plasmon resonance (SPR) was used for binding kinetic studies using a Biacore 3000 instrument (GE Healthcare) at 25°C as a paid service from Precision Antibody (Maryland, USA) according to a previously published protocol. Heparin was covalently linked to a streptavidin-coated chip at a concentration of 13 response units (RU), and unoccupied sites were blocked with biocytin. A range of single analyte concentrations prepared in DPBS buffer was used and the antigen was flowed through the chip at a flow rate of 30 μL / min. The binding of the antigen to the ligand was monitored in real time to obtain the association (ka) and dissociation (kd) rates. The equilibrium constant (KD) was calculated from the observed ka and kd. The accuracy of the SPR analysis was determined by chi-square test (2), as described in the statistical analysis section.

[0406] Biolayer Interferometry (BLI)

[0407] We used the Fc-fusion chorionin CTR peptide because the peptide alone would be below the detection limit in this experimental design. To evaluate heparin-Fc-chorionin interactions, the octet system (biolayer interferometry) was used to assess heparin-protein dynamics. 50 μg / mL biotinylated heparin was immobilized on the biosensor tip surface of the pretreated biosensor for 300 seconds. This was followed by quenching with 50 μg / mL biocytin, baseline buffer diluent for 120 seconds, analyte (Fc-fusion protein) at 200 nM for 120 seconds, and dissociation in assay buffer for 120 seconds.

[0408] NRP1 protein dynamics were additionally assessed using biolayer interferometry (BLI) at 30°C and 1000 rpm agitation. 1 mg / mL NRP1 (R&D 3870-N1-025) was biotinylated, desalted, and immobilized on the surface of a SA biosensor tip (Pall ForteBio) at a 1:2 molar ratio. This was followed by: 1) 180 seconds of baseline buffer dilution, 2) loading of the ligand (NRP1), 3) 180 seconds of baseline buffer dilution, 4) 240 seconds of binding (analyte), and 5) 300 seconds of dissociation in assay buffer. Assay buffer: SD buffer (pH 7.4 PBS, 0.05% tween 20, 0.01% BSA).

[0409] The experimental data were fitted with a 1:1 binding model and analyzed using global fitting using Octet data analysis software to calculate K D .

[0410] result

[0411] We recently showed using surface plasmon resonance (SPR; submitted to NEJM) that the RELNCTR H3447R peptide has a 2-fold higher interaction with heparin relative to RELN WT. These kinetics were evaluated using synthetic peptides (AA3431 to AA3460). We designed Fc-fused RELN CTR proteins to confirm this finding in proteins produced in mammalian cells. The interaction of Fc-fused RELN WT and H3447R with heparin was evaluated using biolayer interferometry (BLI; Figure 1C). These proteins were cleaved by furin during production in mammalian cells, resulting in a short RELN CTR variant lacking the last 6 amino acids. In this construct, RELN H3447R also had approximately a 2-fold higher affinity for heparin compared to WT. We found that K a It's K DThe difference in the H3447R values is the main contributing factor to the difference, suggesting that the variant requires less energy to interact with heparin or that the variant saturates the heparin substrate twice as fast as the WT. Therefore, in the presence of the H3447R mutation, the CTR sequence upstream of furin cleavage may contribute to the CTR-heparin affinity.

[0412] in conclusion

[0413] The kinetic data show that RELN H3447R has a ~2-fold increased interaction with heparin compared to RELN WT. Because the difference is relatively small, it is relevant that this difference is seen in two different experimental systems with both long and short RELN variants. These data suggest that RELN H3447R can increase the association of RELN and GAGs and, therefore, may have a competitive advantage over other molecules or proteins that interact with GAGs in vivo.

[0414] Example 4: Affinity data of chorionin-heparin interaction

[0415] High performance liquid chromatography (HPLC) was used to assess the interaction between HSPG and filamentous protein CTR. The delayed peak interaction in isotonic PBS supplemented with 1M KCl showed an increased peptide-heparin interaction. Our data show a delayed peak retention time between 0-2 arginines; this difference in peak retention time 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 peak retention times, there may be a second binding site in the last 6 AA of the long peptide sequence. We also show that the basic mutation at position 3447 has a later peak retention time compared to neutral (H) or acidic (D) AA.

[0416] method

[0417] Heparin column chromatography

[0418] To assess the resilin-heparin binding affinity, resilin variants were run on a heparin column in 20 mM Tris HCl over 20 1 mL column volumes in a 0.05 M step range of 0.05-1 M NaCl as described in Arboleda et al. 2019 (2). Sample fractions were eluted at increasing NaCl step sizes to determine the ionic strength required to disrupt the binding between resilin and heparin. Thus, the stronger the interaction, the higher the salt concentration required to disrupt the bond.

[0419] High performance liquid chromatography (HPLC)

[0420] HPLC gives a more accurate 50 μL of 0.3 μg / μL of uncut or cleaved WT silybum peptide in 0.15M KCl, 10mM PBS at 0.3 ml / min. 0-13.5 min: 0.15M KCl loading, 13.5-14.5 min: ramp to 0.5M KCl, 14.5-24.5 min: 0.5-1M KCl gradient (ramp), 24.5-45 min: 1M KCl (isocratic elution), 45-55 min: 1M KCl at 0.6 ml / min (wash), 55.0-56.0 min: ramp to 0.15M KCl at 0.3 ml / min, 56.0-59.0 min: 0.15M KCl (reset column). Based on the fluorescent 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 fluorescent properties of the peptides, 15 ul of -0.6 ug / mL of peptide was diluted in 500 uL of 0.15 M KCl, 10 mM PBS, and the peak excitation and emission of the peptide was calculated from the 2D plot data.

[0421] HPLC method development

[0422] The column chromatography method was adapted from previous studies in the 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, the high salt gradient is corrosive to the HPLC mechanical components. Therefore, KCl was considered because this salt has a higher ionic strength than NaCl. A lower molar concentration of KCl-containing buffer compared to NaCl can be used to disrupt the heparin-filament protein CTR interaction to help keep the HPLC pump free from residual salt and potential corrosion. Due to the use of a lower concentration of salt, the amount of time required to maintain the isotonic hold at 1 M KCl was extended until no peptide signal could be detected before the column was equilibrated for use with the next sample.

[0423] result

[0424] Heparin column chromatography showed that RELN H3447R could enhance the interaction with heparin

[0425] Eluted peptides from heparin column chromatography, quantified with respective 0.05 M NaCl steps, were evaluated using both nanodrop and ELISA. Overall, nanodrop measurements resulted in sharper, more defined peaks compared to ELISA, likely because ELISA relies on the affinity of the antibody for RELN CTR, whereas nanodrop measurements directly quantify the amount of peptide. The peak elution step was determined as the maximum ratio of each sample to the input.

[0426] For all RELN variants, the long (uncleaved) peptide had a later peak retention time compared to the short peptide, suggesting that the last six AAs may include the heparin binding site. In addition, the basic AAs (R, K) at position 3447 had a peak eluting at higher salt concentrations compared to WT or H3447D. Therefore, the charge of the AAs at position 3447 may be an important factor in the RELN CTR-GAG interaction.

[0427] While heparin column chromatography can quantify the RELN CTR-heparin interaction to some extent, HPLC can provide more accurate quantification of the interaction using a gradient. Representative normalized peaks show that for both long and short peptides, RELN H3447R has delayed peak times compared to RELN WT. Significant differences are characterized by average peaks with retention times greater than 30 seconds apart.

[0428] in conclusion

[0429] Chromatographic data show that the RELN H3447R mutation increases heparin interaction as a model for overall GAG interactions. The differences in retention times of the RELN peptide variants are due to the AA mutations, rather than changes in fluorescence properties or changes in the detectability of the peptides, as the peak excitation and emission spectra remain similar among the peptide variants. Additional mutations may include substitutions of other key arginine residues (R3451, R3454, and R3458) in the same orientation as H3447R to facilitate RELN CTR-GAG interactions.

[0430] Example 5: Other potential reticulin CTR interactions

[0431] Recent work has shown that the reticulin CTR also interacts with neuropilin (NRP1) (46). This study found that the last 6 AAs after the furin cleavage site are critical for NRP1 interaction. The uncleaved "long" CTR-RELN interacts with NRP1, and the cleaved "short" CTR-RELN does not interact with NRP1 (46). Therefore, BLI was used 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). Specifically, the C-terminal arginine is associated with binding to the NRP1b1-b2 domain (48). Therefore, based on sequence alone, RELN CTR may have additional interactions beyond GAGs. In particular, RELN H3447R may help optimize the NRP1 binding motif, especially after furin cleavage.

[0432] method

[0433] Docking

[0434] The NRP1 and RELN CTR structures were uploaded to ClusPro 2.0 (cluspro.org / ) to model these protein interactions. Briefly, the top 1000 rotatamers with the lowest scores among the 70,000 rotations were selected. The algorithm clustered these 1000 rotations, which required The positions with the most neighbors within the RMSD radius are ranked. The top position then becomes the center of the first cluster, and the process is repeated to rank up to 30 clusters based on size (i.e., the number of neighbors). Energy minimization then removes steric overlap. Thus, ClusPro considers the lowest energy structure of the largest cluster, not just a single energy minimization (49, 50).

[0435] The output of the ClusPro algorithm is a list of the most likely conformations. One model for each ApoE peptide was considered preliminary data for further analysis 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 by the PyMol software using the 'measure mode' function. Internal contact.

[0436] Biolayer Interferometry (BLI)

[0437] Biolayer interferometry (BLI) was used to assess NRP1 protein dynamics at 30°C and 1000 rpm stirring using previously published methods. 1 mg / mL NRP1 (R&D 3870-N1-025) was biotinylated, desalted, and immobilized on the surface of a SA biosensor tip (Pall ForteBio) at a 1:2 molar ratio. This was followed by: 1) 180 seconds of baseline buffer dilution, 2) loading of the ligand (NRP1), 3) 180 seconds of baseline buffer dilution, 4) 240 seconds of association (analyte), and 5) 300 seconds of dissociation in assay buffer. Assay buffer: SD buffer (pH 7.4 PBS, 0.05% tween 20, 0.01% BSA).

[0438] The experimental data were fitted with a 1:1 binding model and analyzed using global fitting using Octet data analysis software to calculate K D .

[0439] result

[0440] Molecular docking indicated that the α- and β-GAG binding sites overlap with the NRP1 binding site. However, the N-terminal AA of RELN CTR may also contribute to the NRP1 interaction.

[0441] 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 expected to be cleaved by furin. Previously published data concluded that WT CTR did not bind to NRP1 after removal of the last 6 amino acids by furin.

[0442] We used isothermal titration calorimetry (ITC) Figure 2G ) and biolayer interferometry (BLI, Figure 2H ) confirmed the enhanced interaction of CTR-RELN H3447R with heparin. a It's K d The COLBOS mutation was the main contributor to the difference in the values of , and the mutant CTR-RELN was found to have a more negative Gibbs free energy compared to the WT, suggesting that the COLBOS mutation enabled the spontaneous CTR-RELN reaction with heparin. Our NMR ( Figure 2I , Figure 9 ) studies revealed that CTR-RELN may have an α-helical structure (including a flexible region with a domain we named "Flexibility Vertex") in the presence of trifluoroethanol, although it may be unstructured under native conditions, as revealed by circular dichroism (Table 3).

[0443] Table 3. Secondary structure analysis of circular dichroism spectroscopy data of CTR-RELN WT alone or CTR-RELN WT with 50% TFE

[0444]

[0445] Heparin binding analysis of a library of mutant CTR-RELN peptides revealed two GAG binding sites, which we named the "α-GAG binding site" and the "β-GAG binding site" ( Figure 9). The α-GAG binding site is located in the last 6 amino acids and overlaps with the previously identified binding site for neuropilin 1 (NRP1), which is released by furin. The β-GAG binding site is located upstream of the furin cleavage site and spans amino acids 3446 to 3451. Our studies also found that due to the optimization of the β-GAG binding site, CTR-RELN COLBOS has a 10-fold higher affinity for NRP-1 compared to the wild-type version of CTR-RELN (Table 4). We analyzed the binding of CTR-RELN COLBOS by HPLC (Figure 11-D), BLI ( Figure 2H ) and NMR structures ( Figure 2I , 9 ) performed extensive studies of the interaction of mutant CTR-RELN with heparin to support these assertions.

[0446] Table 4. Analysis of binding parameters of CTR-RELN-NRP-1 interaction

[0447] Analytes KD ka kd Fc / / / Fc-WT 6.10E-07M <![CDATA[9.40E3M -1 ·s -1 ]]> <![CDATA[5.73E-3s -1 ]]> Fc-H3447R 7.93E-08M <![CDATA[1.29E4M -1 ·s -1 ]]> <![CDATA[1.02E-3s -1 ]]> VEGF 5.53E-09M <![CDATA[2.72E5M -1 ·s -1 ]]> <![CDATA[1.50E-3s -1 ]]>

[0448] Our data show that RELN CTR peptides without the α-GAG binding site located in the last 6 amino acids of RELN CTR have an affinity for NRP1 that is 100-fold lower than the affinity of VEGF for NRP1, consistent with previously published data. However, for the H3447R mutation, the affinity was 10-fold higher compared to WT, suggesting that H3447R forms a new binding site or significantly increases the affinity for NRP1 via the β-GAG site. In summary, the RELN H3447R peptide has a ~10-fold increased affinity for NRP1 compared to the WT RELN peptide, which is closer to the affinity range of VEGF and therefore has a gain-of-function.

[0449] in conclusion

[0450] Previously published data showed that NRP1 interaction is lost in the WT short RELN peptide. The data here add more context to the story, with WT short having significantly less interaction with NRP1, especially compared to VEGF. However, these data support the hypothesis that RELN H3447R adds a new β-GAG binding site or significantly optimizes the β-GAG binding site, given that the mutation rescues the RELN CTR-NRP1 interaction ∼10-fold. Therefore, RELN gain-of-function could be achieved by introducing the H3447R variant and / or by reducing furin cleavage of RELN-CTR by introducing R3454A.

[0451] Example 6. Structural Analysis of Reelin

[0452] Circular dichroism (CD) was used to further understand the unstructured nature of RELN CTR. Modeling results showed that RELN CTR may have high fluctuations in the most disordered regions of the RELN protein. In addition, recently published data showed a high-resolution structure of CR8 within the C-terminus of RELN, but could not characterize RELN CTR, possibly due to the flexibility of the domain (32). NMR helped to obtain high-resolution structural conditions by using circular dichroism (CD) and found that 50% TFE (2,2,2-trifluoroethanol) was required to stabilize the WT RELN peptide structure. Both methods provided insight into the unstructured regions of RELN CTR. Computer modeling methods added additional support, showing that the flexible regions can be predicted by combining the RELN CTR sequence with known disordered regions of other proteins.

[0453] method

[0454] Normal Mode Analysis (NMA)

[0455] NMA is a principal component analysis (PCA)-based method that considers the harmonic potential of peptides, in which the force field is simplified to a ball and spring elastic network model. This model treats the harmonic potential of the Ca atom of each AA as a node connected by springs. Here, the energy function is minimized by diagonalizing the Hessian matrix consisting of the second-order derivatives of the potential energy (33).

[0456] Predictors of Naturally Disordered Regions (PONDR) VL-XT Disorder Analysis

[0457] This algorithm uses a feedforward neural network to predict disordered regions based on AA sequences. VL-XT combines three feedforward neural networks trained on: 1) regions of varying length (VL), 2) X-ray characterized N-termini, and 3) X-ray characterized C-termini (XT). Compared to NMA, VL-XT can predict disordered regions using known disordered regions of other proteins.

[0458] Circular dichroism

[0459] The CD signal was recorded at a wavelength ranging from 190 to 260 nm with a quartz cuvette of 1 mm path length at 0.1 nm intervals. The sample was scanned at 50 nm / min using a 1 nm bandwidth and a 2 second integration time. The data were plotted as the average of four spectra. The data were deconvoluted using the online deconvolution software BeStSel (bestsel.elte.hu / ).

[0460] 2D nuclear magnetic resonance (NMR) structure

[0461] NMR structures were performed as a paid service. The final NMR sample consisted of 50% (v / v) H2O, 50% (v / v) 98% pure 2,2,2-trifluoroethanol-d2 (CF3CD2OH), 0.25 mM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid, dissolved in H2O) as an internal standard, and ~3.3 mM silylin peptide. 1 HTOCSY (80ms spin lock time), NOESY (200ms mixing time), 1 H- 15 N HSQC, 1 H- 13 C HSQC experiments were performed at 25°C on an Agilent 600 MHz NMR spectrometer (DD2). Data were analyzed using NMRPipe and CCPNmr 2.4.2; structures were calculated using Cns 1.2.1 and aria 2.3.2. A total of 200 structures were calculated, and the 20 lowest energy structures were selected.

[0462] result

[0463] Structural characterization of the RELN CTR reveals an alpha helix with flexible arms

[0464] We used circular dichroism (CD) to determine that the RELN CTR is highly unstructured under native conditions. Addition of 50% TFE (2,2,2-trifluoroethanol) stabilized the unstructured CTR and enabled characterization of the structural domains. With 50% TFE, the peptide secondary structure was closer to an α-helix, with characteristic negative minima at 208 nm and 222 nm. Even in the presence of TFE, we cannot rule out the presence of unstructured regions within the RELN CTR. The structure of the disordered peptide could explain the "antiparallel β-sheets" or "other classes" (34, 35) found as a greater percentage of structure in the peptide alone compared to the peptide with 50% TFE.

[0465] The CD results informed the experimental conditions for NMR to resolve the CTR of the filamentous protein at atomic resolution. NMR is advantageous for smaller proteins because it can capture dynamics and does not require crystallization (51). The 20 lowest energy structures were used to create the The final NMR structure of the WT RELN CTR at a resolution of approximately 100 nm was obtained. Using this technique, we demonstrated that the C-terminal domain of RELN in the presence of TFE forms an α-helix with a highly flexible arm towards the C-terminal region. The apex of the flexible region spans from the glycine residue at position 3444 to the histidine 3447 that was mutated in the protected case. We named this region the flexible apex domain (FVD). The consensus motif for GAG binding includes basic amino acids on the same side of the α-helix and basic amino acids on consecutive turns separated by ∼3.5 amino acids to allow for strong bond formation (36). In addition, the 20 lowest energy structures showed that the helix containing position 3447 is unwound, potentially exposing H3447R for GAG interactions.

[0466] The CTR-GAG interaction of resilin depends on the basic AA near H3447R

[0467] Our structural data combined with our functional analyses suggest that the α-GAG binding site is likely dependent on R3454, R3457, and R3458. As we concluded earlier from our functional studies, R3446 may also mediate GAG interactions together with R3451 comprising the β-GAG binding side. It is clear from the structure that the arginine introduced by the mutation at position 3447 found in the protected case is in the correct orientation for GAG interactions. Importantly, the new β-GAG binding site is not cleaved by furin, becoming a constitutive active site for the interaction of RELN with GAGs, NRP1, and potentially other receptors. Polar contacts between WT RELN CTR and heparin suggest that this interaction is likely due to the presence of basic amino acids ( Figure 9 ).

[0468] The C-terminal peptide of sibiricin has more atomic perturbations in the α- and β-GAG binding sites

[0469] Glycine residues are labeled to highlight the structured region found in the NMR spectrum toward the N-terminal region of the peptide. The data indicate that the last six aa of the H3447R long peptide have more perturbations compared to the WT long peptide. The WT and H3447R short peptides are comparable; overall, the short peptides have reduced perturbations compared to the long peptides. Atomic perturbations are measured as the root mean square deviations in all normal modes.

[0470] Modeling reveals increased disorder in the reticulin CTR

[0471] To assess the flexibility of this region, normal mode analysis (NMA) was first used to assess the harmonic potential of each amino acid (37). NMA showed that long peptides had increased flexibility, and RELN H3447R long had increased flexibility compared to WT long. The short filamentous protein peptides showed similar flexibility. NMA generally showed that long peptides had increased flexibility compared to short peptides; however, since all other peptides except WT long were modeled peptides, these results may not be completely accurate. In contrast, the predictor of naturally disordered regions (PONDR) analysis used only the AA sequence to score the relative disorder of RELN variants. Compared to H3447R long, which had a score of 0.9225 in AA 3444-3460, WT long had a score of 0.9032 in AA 3444-3460. Therefore, the H3447R mutation increases disorder in the RELN CTR and may contribute to the increased ability to bind GAGs or inhibit AB plaque formation. On the contrary, R3446H reduced the disordered region to 3447-3460, with an average score of 0.8583. Therefore, the arginine in the β-GAG binding site may contribute to the increase in disorder in the reelin CTR. VL-XT (a neural network-based method) more closely predicted the disordered region of RELN CTR found in the experimental data. Here, the predicted disordered region at the RELN C-terminus begins at G3444, which is also shown in the 20 lowest energy NMR structures. Because RELN CTR is extremely well conserved among species, and because it is also highly disordered, it is obvious that its function requires highly disordered properties.

[0472] in conclusion

[0473] The RELN CTR has an unstructured region beginning at the FVD that also contains α- and β-GAG binding sites. The RELN H3447R mutation creates a new β-GAG binding site at a position that aligns with other basic amino acids within the GAG binding motif, thereby facilitating GAG interactions. Furthermore, computational analysis confirmed that the region from G3444 to P3460 is highly disordered. Disordered regions may contribute to increased cellular interactions with GAGs or other proteins. NMA and VL-XT (a machine learning-based approach using known disordered regions) were used to estimate potential flexibility within the RELN CTR.

[0474] Example 7. Resiliin CTR regulates Aβ aggregation

[0475] Although previous experiments suggested a protective mechanism based on RELN–GAG interactions, it was important to assess whether RELNH3447R directly modulates known hallmarks of Alzheimer's disease. Previous studies have suggested that disordered regions of the molecule may regulate Aβ aggregation (38); purified RELN has also been reported to reduce amyloid beta (Aβ) aggregation in vitro (39). Therefore, we examined whether this effect was attributable to CTR-RELN.

[0476] method

[0477] Thioflavin T (ThT) binds to amyloid and has also been shown to be linearly correlated with amyloid concentration. Therefore, the ThT assay can be used to quantify AB aggregation using a previously developed method (40).

[0478] We incubated CTR-RELN with Aβ and monitored aggregation using thioflavin T fluorescence assay, normalized to the maximum ThT emission of Aβ alone. We also assayed Aβ in the presence of morin, a known inhibitor (41), as a negative control. Long and short WT and H3447R RELN CTR peptides were included to better understand the potential role of the H3447R mutation in amyloid pathology (Figures 14 and 15). Negative controls included long and short RELN CTR peptides with a neutrally charged AA (histidine) in place of the basic AA in the α- and β-GAG binding sites.

[0479] result

[0480] RELN peptide alone did not trigger ThT fluorescence ( Figure 15 We found that RELN CTR significantly reduced amyloid aggregation and that the long peptide was as effective as the short peptide ( Figure 14A-B , Figure 15 For both long and short variants, the RELN H3447R variant peptide was similarly effective as the WT in inhibiting ThT aggregation ( Figure 14A-B). Interestingly, when R3431, K3432, and R3446 were replaced with histidine in the short peptide variant, the anti-aggregation effect of CTR-RELN was significantly reduced, thus suggesting that the N-terminal amino acid may play a major role in the interaction with the amyloid seed (Figure 14B). H3447D long and H3447K long similarly reduced aggregation, but H3447D short had ~3-fold higher aggregation than H3447K short. Therefore, the charge of the β-GAG binding site may not be the only contributing factor affecting the interaction with Aβ plaques. This hypothesis is further supported by a control (Figure 14B) in which several arginines in RELN CTR were replaced with histidine, including in the α-GAG binding site. In this case, there was significantly higher Aβ aggregation in samples with the short peptide compared to the long peptide. The negative control showed minimal ThT fluorescence with the peptide alone without Aβ.

[0481] in conclusion

[0482] RELN CTR can play a role in AB aggregation, but the mechanism may rely on the overall flexibility of the peptide rather than the charge of the interaction, considering that the amyloidogenicity of AB is enhanced by the sequence enrichment of hydrophobic AA residues (42). It has been previously shown that intrinsically disordered proteins (IDPs) such as certain cleaved APP variants can form seeds for amyloid aggregation. Proteins with IDPs can potentially interact with amyloid aggregates 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 disordered regions. Therefore, replacing histidine with arginine can have some contribution to increased aggregation.

[0483] RELN peptides with more flexible AA(H) in key positions at the N-terminus, α-, and β-GAG sites reduced ThT fluorescence to levels similar to the WT long peptide. However, with a similar short peptide with the β-GAG site removed, AB aggregation increased ~4.5-fold.

[0484] Additional peptides may include substitution of the most flexible AA (G3444) in the RELN CTR to a less flexible AA such as histidine, which has moderate flexibility, or proline, which would significantly reduce flexibility.

[0485] Example 8. Reticulin CTR Enhances Cell Membrane Permeability

[0486] The highly basic structure of the C-terminal domain of RELN and its ability to interact with membranes may make this domain suitable for inducing cell membrane penetration of other proteins (either in protein fusions or as mixtures) or nucleic acids (such as mRNA).

[0487] To evaluate this, HMREC were cultured in 24-well plates until 80% confluence in complete endothelial growth medium 2 (EGM-2, Lonza, Switzerland). For treatment, 5 μg of mRNA was added to all formulations. We prepared mRNA / peptide conjugates by mixing each amount of peptide (RKQNYMMNFSRQHGLRHFYNRRRR) and mRNA before dilution in EGM-2. HMREC were cultured with treatment medium for 24 hours and then fluorescence was measured in a microplate reader Synergy H1 (Biotek, VT) with excitation at 482 nm and emission recorded at 520 nm.

[0488] Figure 16 The results shown in show that expression was increased when mRNA was mixed with CTR.

[0489] Example 9. Exemplary sequences encoding Dab1 dimerization constructs

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496] The following is the codon-optimized sequence encoding the Dab1-FKBP fusion protein:

[0497]

[0498]

[0499] Example 10. Analysis of mini-RELN constructs in HREC cells

[0500] Both in vitro overexpression and recombinant protein were used to evaluate mini-RELN constructs in human retinal endothelial cells (HRECs). The constructs used in Examples 10-12 are shown in Table 5.

[0501] Table 5. Exemplary constructs

[0502]

[0503]

[0504] 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 tested experimentally at passage 7. 24 hours before the experiment, cells were plated on 6-well plates at 500,000 cells / mL. Cell transfection of the plasmid was completed with Lipofectamine 2000 (5 μL, Life technologies) according to the manufacturer. In detail, 1 day before transfection, cells were seeded to 70% confluence at the time of transfection. On the day of transfection, lipofectamine was diluted and incubated at room temperature for 10 minutes to allow the formation of lipophilic complexes. Subsequently, each plasmid (1 μg, Invivogen) was added to the lipid complex of Lipofectamine and incubated for 10 minutes. We used OPTIMEM medium (Gibco) to block transfection after 5 hours to allow cells to recover and promote replication. Cells were imaged using a light microscope (Olympus CKX53) at 10X magnification before transfection and 5 and 24 hours after transfection. Figure 17A The results shown in -J demonstrate the integrity of mammalian cells expressing all mini-RELN constructs.

[0505] 24 hours after transfection, cells were harvested for western blot analysis. Total protein levels were quantified via BCA assay. For western blotting, 7 μg of protein homogenate was prepared under reducing and denaturing conditions using Laemmli buffer enriched with 10 mM DTT and a 5-minute boil. Samples were separated by electrophoresis in 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 with TBS blocking buffer for 2 hours. We detected β-actin (1:5000, mouse, #66009-1-Ig, Proteintech) and total DAB1 (1:100, SC-red tube; sc-271136Dab1 (G-5), Santa Cruz). (TBS) blocking buffer was diluted and incubated at 4 ° C overnight under gentle shaking. Before the secondary antibody incubation, the membrane was washed twice with TBS-T for 10 minutes. As the secondary antibody, we used IR Dye 800nCW goat anti-rabbit (925-32211, Licor) and IR Dye 680CW goat anti-mouse (925-68070, Licor) diluted 1: 10000 in blocking buffer and HRP (m-IgG1BP-HRP-SC-525408, Santa Cruz) diluted 1: 1000 in blocking buffer, under gentle shaking at room temperature for 1 hour. Before using Odyssey imager for IR detection, the membrane was washed three times with TBS-T. In order 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. Figure 18A -E shows a decrease in total Dab1, presumably due to increased Dab1 signaling activity triggered by the mini-RELN construct. The decrease in total Dab1 was quantified as Figure 19A - E. These experiments show that the mini-RELN constructs can function directly in the cells in which they are expressed. Figure 18A The results shown in Figure 5-E demonstrate that expression of the mini-RELN construct triggers a significant turnover of total DAB1 and a sustained activation of RELN signaling as measured by reduced DAB1 levels in the presence of the mini-RELN construct compared to lipofectamine (Lp) for all constructs. These findings allow us to conclude that all mini-RELN constructs tested are active.

[0506] All constructs tested triggered significant activation, with 225Z being the most active. 225Z includes the CR50 domain of RELN to induce oligomerization, R5-6 to bind the receptor, and the RELN C-terminus with a COLBOS variant to bind HSPG.

[0507] Our analysis showed that the modular design described herein is also effective in activating RELN signaling when it includes RAP to bind the receptor, Fc to induce oligomers, and a RELN C-terminus with a COLBOS variant to bind HSPG. In this design, Fc replaces the CR50 of RELN and RAP replaces the receptor binding domain. In this design, the mini-RELN construct was shown to be effective when produced by cells such as HRECs and to signal in an autocrine manner in the same cell.

[0508] Experimental evidence showed that we could substitute modules within the mini-RELN domain based on their function (e.g., Fc with CR-50, RAP with R5-6) without compromising efficacy, demonstrating that our selected modules are sufficient to support RELN signaling.

[0509] For in vitro screening of constructs using recombinant mini-RELN on HREC, cells 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 plated at 500,000 cells / mL in 6-well plates. 2 hours prior to the experiment, cells were washed with PBS to remove residual growth medium and then incubated with starvation medium including EBM-2, 1% GlutaMax + 0.2% Normicin. Treatments were performed using recombinant mini-RELN constructs obtained from Innovagen and recombinant mouse reticulin protein (3820-MR-025 / CF) from R&D systems. Each treatment was incubated at a concentration of 4ug / mL for 5 minutes and then the cells were harvested in an applied lysis buffer consisting of RIPA (Cell signaling), protease inhibitors (cOmplete TM , Mini, EDTA-free protease inhibitor cocktail, Roche), phosphatase inhibitor (PhosSTOP, Roche), 1 uL of proteasome inhibitor MG-132 10 mM, 1% Triton X 100. The lysate was scraped and centrifuged at 15000 rpm for 10 minutes at 4 ° C. The total protein level was quantified by BCA assay. For Western blotting, 3.5ug protein homogenate was prepared under reducing and denaturing conditions using Laemmli buffer enriched with 10mM DTT and boiling for 5 minutes. The samples were separated by electrophoresis in a 4-20% gel at 90V, and the proteins were transferred to a PVDF membrane using the iBlotTM 2 dry blotting system. The membrane was blocked with TBS blocking buffer for 2 hours. We detected pDAB1 (Tyr232; 1:1000, rabbit, #3325 Cell signaling) and total protein staining (Licor). Antibodies used (TBS) blocking buffer is diluted and incubated overnight at 4 DEG C under gentle shaking.Before secondary antibody incubation, TBS-T is used to wash the membrane 2 times, and continues 10 minutes.As secondary antibody, we use the IR Dye 800n CW goat anti-rabbit (925-32211, Licor) diluted in 1:10000 in blocking buffer, under gentle shaking, at room temperature 1 hour.Before using Odyssey imager to carry out IR detection, use TBST-T washing membrane three times.In order to quantify the result, use ImageJ to quantify each band, and data are represented as the pDAB1 that is normalized to control: total protein ratio.

[0510] RELN triggers the phosphorylation and proteasomal degradation of Dab1. Figure 20A -F shows increased levels of phosphorylated DAB1 (pDAB1) after treatment with the mini-RELN purification construct. Figure 21A pDAB1 levels were quantified in - E. These experiments showed that the mini-RELN construct also functions in a non-cell autonomous manner.

[0511] like Figure 21A As seen in Figure 3, increased pDAB1 levels were observed with 225Q treatment. rmRELN (rmRELN), which lacks the C-terminus of RELN, was less effective than 225Q, which contains a C-terminal RELN with the COLBOS variant. This finding suggests that modules containing either the C-terminus or the GAG-binding domain of RELN are 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 was shown to work when produced by some cells, such as HEK, and to signal in other cells, such as HREC. Figure 21B Increased pDAB1 levels were demonstrated with treatment of the 225S and 225T mini-RELN constructs. 225T, which contains an Fc for oligomerization, a RAP for receptor binding, and the C-terminal domain of RELN with a COLBOS variant, was more effective than the control, which is larger and lacks the C-terminal RELN domain with a COLBOS variant (mouse and human rmRELN at much higher concentrations, including R3-6 of RELN). This confirms the superiority of constructs containing GAG binding domains, such as the C-terminal domain of RELN with a COLBOS variant. Figure 21CIncreased pDAB1 levels were demonstrated in the presence of various configurations of mini-RELN constructs compared to vehicle; those containing CR50, R5-6 of RELN, and 225Z at the C-terminus of RELN with the COLBOS variant were more effective than commercially available positive controls lacking the GAG binding domain and lacking the oligomerization domain. Figure 21D The data in Figure 2 show increased pDAB1 levels with treatment with 225SV, 225SW, and 225Z. This experiment shows that mini-RELN constructs, such as 225Z, containing only CR50, R5-6, and the C-terminus of RELN with a COLBOS variant are more effective at triggering signaling compared to full-length RELN WT and full-length RELN COLBOS. This data suggests that even when full-length RELN has a COLBOS variant, the mini-RELN construct is more effective than full-length RELN. Figure 21E , the data show increased pDAB1 levels in the presence of 225Z treatment. Figure 21F As shown, there was no detectable increase in RELN signaling, as expected for a construct containing only Fc for oligomerization and the C-terminus of RELN but lacking the receptor binding domain. Therefore, the presence of the receptor binding domain is necessary for activation of RELN signaling.

[0512] Together, these results provide evidence that the functional module we describe in Table A is necessary and sufficient to stimulate optimal RELN signaling with therapeutic interest.

[0513] Depending on the indication, such as whether more or less RELN signaling recruitment is desired, or depending on the particular route of administration, a particular mini-RELN may be more appropriate. For example, a smaller mini-RELN construct may be preferred for nasal delivery.

[0514] Lactate dehydrogenase assay was performed in SH-SY5Y cells treated with preformed oligomeric Tau (oTau) for 24 hours either alone or in the presence of C-terminal RELN WT (184I). Lactate dehydrogenase assay was performed as follows. 100k neuroblastoma (SH-SY5Y, ATCC) cells / well were plated in a 96-well TC-treated plate in growth medium (DMEM / F12, Gibco, 10% heat-inactivated FBS, R&D, 200ug normocin) and allowed to attach and grow overnight. The next day, the test compound was allowed to thaw on wet ice. Phenol-free and serum-free DMEM / F12 serum-free antibiotic-free culture medium were warmed, and 50mL aliquots were sterile filtered (0.22um) as test culture medium. In the TC cabinet, the thawed test compound was mixed via pipetting, and then added to the final concentration of each test condition in the filtered test culture medium and mixed by pipetting. Test condition (test condition) was allowed to pre-incubate for 15 minutes at room temperature in the TC cabinet. The wells were gently aspirated and gently washed with 200 uL of pre-warmed test medium, then gently aspirated and 100 uL of the test condition was added and allowed to incubate for 21 hours in a TC incubator under 5% CO2. LDH was performed using the Roche kit according to the manufacturer's protocol. Figure 23 The results shown in Figure 1 show a reduction in oTau-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 of 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 rather a direct effect mediated by binding of Cterm RELN to tau.

[0515] Synthetic thioflavin peptides were also used for thioflavin T (ThT) assays to assess the different effects of these proteins on Aβ42 aggregation in vitro using thioflavin T (SensoLyte ThTβ-amyloid protein (1-42) aggregation kit, catalog number AS-72214). Aβ42 was added to a final concentration of 55 μM in a solution of 10 μM thioflavin peptide variants in a transparent, unbound 96-well plate. The sample was then mixed with 2 mM thioflavin T dye and fluorescence was read at excitation wavelength / emission wavelength (Ex / Em) = 440 / 484 nm at intermittent intervals over 2 hours. The plates were kept at 37°C with 15 seconds of shaking between reads. Figure 24AThe results shown in Figure 5-C show that the Cterm domain of RELN with and without the COLBOS variant reduces amyloid aggregation. The presence of the COLBOS variant enhances this effect. In general, it appears that the RELN Cterm domain is highly unstructured. Interactions with tau and / or amyloid and / or HSPG may allow RELN to assume a more stable structure, resulting in aggregation resistance. These data suggest that as long as the mini-RELN construct contains the C-terminal domain of RELN, preferably with the COLBOS variant, it may have a protective effect. If the mini-RELN includes only the C-terminal domain without the receptor binding domain, protection will not involve signaling, but will still involve direct interaction with tau. If the mini-RELN includes both the C-terminal domain and the receptor binding domain, it will have at least two protective mechanisms, namely RELN signaling and direct interaction with tau.

[0516] Whenever 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 separated by electrophoresis in a 4-20% gel at 90 V, and proteins were transferred to nitrocellulose membranes using 20% methanol tris-glycine transfer buffer. The membrane was blocked with TBS blocking buffer for 2 hours. We detected the antibodies using anti-Fc-TAF (1:1,000, goat, Sigma Aldrich) and GAPDH (1:5000, mouse, Abcam). (TBS) blocking buffer was diluted and incubated overnight at 4 ° C under gentle shaking. Before the secondary antibody incubation, the membrane was washed twice with TBS-T for 10 minutes. As the secondary antibody, we used IRDye 800n CW goat anti-rabbit (925-32211, Licor), IR Dye 680CW goat anti-mouse (925-68070, Licor) diluted 1: 10000 in blocking buffer and HRP (m-IgG1BP-HRP-SC-525408, Santa Cruz) diluted 1: 1000 in blocking buffer, under gentle shaking at room temperature for 1 hour. Before using Odyssey imager for IR detection, the membrane was washed three times with TBS-T. In order to quantify the results, each band was quantified using ImageJ, and the data were expressed as the pDAB1: β-actin ratio normalized to the control.

[0517] Figure 25AThe results presented in Figure 25A-B 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 (25A-B). Mini-RELN constructs containing Fc from IgG are expected to form oligomers, while constructs containing CR50 and fspCR50 are expected to form higher molecular weight multimers.

[0518] The effects of in vitro treatment with RELN or mini-RELN proteins 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 before the experiment, cells were plated on 6-well plates at 500,000 cells / mL. Two hours before the experiment, cells were washed with PBS to remove residual growth medium and then incubated with starvation medium including EBM-2, 1% GlutaMax+0.2% Normicin. Treatment was performed using culture medium derived from HEK 293T cells transiently transfected with different mini-RELN constructs for 24 hours in the presence of Lipofectamine 2000. HEK-derived Lipofectamine 2000 culture medium was used as a negative control. HEK 293T-derived culture medium was tested at different ratios of 1:1, 1:4, and 1:14 (transfection medium: starvation medium). Each treatment was incubated for 5 minutes, and then cells were harvested in an applied lysis buffer consisting of RIPA (Cellsignaling), protease inhibitors (cOmplete TM , Mini, EDTA-free protease inhibitor cocktail, Roche), phosphatase inhibitor (PhosSTOP, Roche), 1 uL of proteasome inhibitor MG-132 10 mM, 1% Triton X 100. The lysate was scraped and centrifuged at 15000 rpm for 10 minutes at 4 ° C. The total protein level was quantified by BCA assay. For Western blotting, 7ug protein homogenate was prepared under reducing and denaturing conditions using Laemmli buffer enriched with 10mM DTT and boiling for 5 minutes. The samples were separated by electrophoresis in a 4-20% gel at 90V, and the proteins were transferred to a PVDF membrane using the iBlotTM 2 dry blotting system. The membrane was blocked with TBS blocking buffer for 2 hours. We detected pDAB1 (Tyr232; 1:100, rabbit, #3325 Cell signaling) and β-actin (1:5000, mouse, #66009-1-Ig, Proteintech). Antibodies were diluted in (TBS) blocking buffer and incubated overnight at 4°C under gentle shaking. Before incubation with the secondary antibody, the membrane was washed twice with TBS-T for 10 minutes. As secondary antibodies, IR Dye 800n CW goat anti-rabbit (925-32211, Licor) and IR Dye 680CW goat anti-mouse (925-68070, Licor) diluted 1:10000 in blocking buffer were used for 1 hour at room temperature under gentle shaking. Before IR detection using an Odyssey imager, the membrane was washed three times with TBS-T. To quantify the results, each band was quantified using ImageJ, and the data were expressed as pDAB1:β-actin ratios normalized to the control.

[0519] Transient overexpression of the mini-RELN construct in HEK 293T was obtained as follows. HEK T293 cells (ATCC) were cultured in DMEM culture medium supplemented with 10% fetal bovine serum (FBS, R&D) and 400uL / L normocine at 37°C and 5% CO2, and up to 10 generations were used in the experiment. 24 hours before the experiment, cells were plated on 6-well plates at 300,000 cells / mL. According to the manufacturer, cell transfection of the plasmid was completed with Lipofectamine 2000 (7mL, Life technologies). In detail, 1 day before transfection, cells were seeded to 70% confluence when transfected. On the day of transfection, lipofectamine was diluted and incubated at room temperature for 10 minutes to allow the formation of lipophilic complexes. Subsequently, each plasmid (3mg, Invivogen) was added to the lipid complex of Lipofectamine and incubated for 10 minutes. We use OPTIMEM culture medium (Gibco) to block transfection after 5 hours, to allow cell recovery and promote replication.After transfection 24 hours, harvested cells, by centrifugal culture medium is removed from cell, and store under the aseptic condition of-80 until use.Before transfection, after transfection 5 hours and 24 hours, use 10X magnification optical microscope (Olympus CKX53) to cell imaging.

[0520] Figure 26A and 27AIt was shown that the mini-RELN construct containing Fc that induces oligomerization, receptor-binding APOE, and HSPG-binding C-TermRELN was able to activate RELN signaling better than the commercially available construct containing only RELN R5-6. As expected, 225Yf, which includes WT APOE, which is expected to bind the receptor more effectively, was more effective and clearly dose-dependent. 225Xf, which includes APOE with the Christchurch mutation, was also effective in activating signaling. Figure 26B and 27B The results shown in may be related to the expected nature of the oligomerization of this construct. Figure 26C and 27C The efficacy of the 225SU and 225SV mini-RELN constructs was shown. Figure 26D and 27D Efficacy was demonstrated for the mini-RELN constructs 225SW and 233F; 233F comprises two RELN R6 domains in tandem, a non-naturally occurring construct. Figure 26E and 27E Activation of Dab1 signaling with the 225RR construct including the RELN Cterm with the COLBOS variant was shown to be more robust compared to the control and compared to 233A including the RELN Cterm WT.

[0521] Example 11. In vivo validation of purified recombinant mini-RELN constructs

[0522] We administered the mini-RELN construct into mice using MIND procure (nasal delivery using minimally invasive nasal reservoir (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 shown the importance of the entorhinal cortex in extreme protection against Alzheimer's disease. Using the MIND methodology provides a way to reach the entorhinal cortex with minimal systemic exposure. We show that it is possible to deliver mini-RELN to the hippocampal formation, including the entorhinal cortex (a brain area we show to be critical for extreme protection against Alzheimer's disease). C57Bl / 3 (Jackson Laboratory) were housed under a regular light / night cycle. All procedures were performed under approved IACUC animal protocols. Before incision, the nasal area was shaved and cleaned with betadine and 70% alcohol, and mice were subcutaneously injected with buprenorphine hydrochloride and meloxicam (5 mg / kg). The nasal cavity was then opened with a scalpel and the nasal mucosa was exposed using a microdrill. Once the mucosa was exposed, a gel dispersion of PBS or mini-RELN constructs (Innovagen) in 20% Pluronic (Sigma Aldrich) was delivered subcutaneously into the surgically generated bag. Mice were injected with meloxicam for 3 days and euthanized on day 4 to collect brain tissue for postmortem analysis.

[0523] Western blotting was performed as follows. 20 μg of protein homogenate was prepared under reducing and denaturing conditions using Laemmli buffer enriched with 10 mM DTT and boiled for 5 minutes. Samples were separated by electrophoresis in 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 with TBS blocking buffer for 2 hours. We detected pDAB1 (Tyr232; 1:1000, rabbit, #3325 Cell signaling) and β-actin (1:5000, mouse, #66009-1-Ig, Proteintech). (TBS) blocking buffer was diluted and incubated overnight at 4 ° C under gentle shaking. Before the secondary antibody incubation, the membrane was washed twice with TBS-T for 10 minutes. As the secondary antibody, we used IR Dye 800n CW goat anti-rabbit (925-32211, Licor) and IR Dye 680CW goat anti-mouse (925-68070, Licor) diluted 1: 10000 in blocking buffer, under gentle shaking at room temperature for 1 hour. Before using Odyssey imager for IR detection, the membrane was washed three times with TBS-T. In order to quantify the results, each band was quantified using ImageJ, and the data were expressed as pDAB1: β-actin ratio normalized to the control.

[0524] The data shown in Figure 28A-K demonstrate that the mini-RELN peptide is delivered to both the hippocampus and the entorhinal cortex and to the effective brain of the midbrain, resulting in increased downstream RELN pathway levels, as confirmed by the pDAB1 levels of increase. The mice processed with the mini-RELN construct have more RELN signaling in the hippocampus and the entorhinal cortex, as determined by the pDab1 of increase. The effect of mini-RELN processing was confirmed in WT mice and in mice with tau mutations that cause tauopathy. 225Z includes CR50 of RELN to induce oligomerization, includes R5-6 of RELN to mediate receptor binding, and includes Cterm RELN with COLBOS mutation to realize HSPG combination. This demonstrates that these RELN domains are enough to drive protective signaling.

[0525] Immunofluorescence staining of mouse brains was also used to demonstrate the delivery of mini-RELN using MIND. 24 or 72 hours after mini-RELN (225S) or vehicle administration, mice were intracardially perfused with 4% PFA in PBS, and brains were harvested and incubated in 4% PFA at 4°C for 24 hours. Sagittal sections were obtained at 1 mm thickness using a stainless steel brain mold. Entorhinal-hippocampal sections of each brain were selected for use according to the kit instructions. Tissue Clearing TM Clarity kit (HRTC-012) was used. Sections were incubated with IgG-Fc tag and pDAB1 primary antibody for 72 hours, followed by incubation with secondary antibodies: Alexa 488 and Alexa 647 and DAPI for 1 hour. Subsequently, sections were imaged using a confocal SP8 microscope for quantification. Results ( Figure 29A-B ) demonstrates the presence of the mini-RELN peptide in the brain, which co-localizes with brain cells with increased levels of pDab 1. This is evidence of efficient drug delivery, target engagement, and targeted RELN signaling activation in the hippocampal formation.

[0526] Example 12. Systemic delivery of mini-RELN reduces tau pathology in P301S Tau mice

[0527] The effects of systemic delivery of mini-RELN peptides were evaluated in vivo. Six-month-old female MAPT P301STau Tg mice were intraperitoneally injected with either 500 μL of vehicle (PBS) or mini-RELN peptide solution on day 1, and 250 μL of vehicle (PBS) or mini-RELN peptide solution 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.

[0528] Immunofluorescence staining of mouse brain was performed as follows. After fixation with 4% PFA at 4°C for 24 hours, sagittal brain sections were obtained at a thickness of 1 mm using a stainless steel brain mold. The entorhinal-hippocampal sections of each brain were selected for use according to the kit instructions. Tissue Clearing TM Clarity was performed using the HRTC-012 kit. Sections were incubated with the pTauS396 primary antibody for 72 hours, followed by incubation with the secondary antibodies: Alexa 647 and DAPI for 1 hour. Sections were then imaged using a confocal SP8 microscope for quantification. 14 images were taken at 63X magnification for each group. Matlab (2021a) was used to automatically quantify the fluorescence intensity of pTau. The Otsu thresholding method was used to obtain a mask for the image signal, and the average signal intensity was calculated.

[0529] Quantification of pTau S396 fluorescence intensity was used as a signature of tau pathology. Figure 30 The results shown in the , show that when mice were treated with the mini-RELN peptide, pTau S396 levels were significantly reduced. The data show that when systemically administered, the administration of the mini-RELN construct is effective, and the beneficial effects lead to a significant reduction in tau phosphorylation (a pathological feature of tauopathies such as frontotemporal dementia and Alzheimer's disease). This was shown in a mouse model of tau pathology (MAPT P301S).

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[0589] 59.Chen

[0590] Other implementation plans

[0591] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

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

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

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

4. A composition comprising a resilin protein or a nucleic acid encoding a resilin protein, preferably wherein the resilin protein comprises an H3447R or H3447K mutation and / or an R3454A mutation, optionally a combination of H3447R or H3447K and R3454A mutations.

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

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

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 protein, or is in a viral vector, optionally an AAV vector.

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

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

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

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

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

14. A method of treating or preventing a neurodegenerative 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 the methylated promoter is: (i) a fusion protein comprising a catalytically inactive CRISPR / Cas protein fused to a demethylation domain and a guide RNA, wherein the guide RNA directs the fusion protein to demethylate cytosine 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, wherein the guide RNA directs the fusion protein to demethylate cytosine in the RELN promoter, optionally administered as mRNA or in one or more vectors, optionally viral vectors, optionally adeno-associated virus (AAV) vectors.

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

16. The method of claims 14-15, wherein the administration is to the entorhinal cortex.

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

18. A method of assessing the risk of developing Alzheimer's disease in a subject, the method comprising determining the presence or absence of an H3447R variant allele in the RELN gene in the subject, wherein the presence of the H3447 variant indicates that the subject has a reduced risk of developing AD compared to a subject not having the H3447R variant.

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

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