Compositions and methods for treating Alzheimer's disease

Abstract

New compositions and methods are provided that are useful for the treatment, prevention, and potential cure of Alzheimer's disease by disrupting rs1921622 and / or 574 other sST2-associated genomic sites and / or the genomic locus of the IL1LR1 gene, including the 3'-UTR of the sST2 gene / transcript.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 226,165, filed July 27, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Brain diseases, such as neurodegenerative diseases and neuroinflammatory disorders, are devastating conditions that affect a large subset of the population. Many such diseases are incurable, highly debilitating, and often result in progressive deterioration of brain structure and function over time. Due to the growing aging population worldwide, disease prevalence is also increasing rapidly, as older adults are at higher risk of developing these conditions. Currently, many neurodegenerative diseases and neuroinflammatory disorders are difficult to diagnose due to limited understanding of the pathophysiology of these diseases. Meanwhile, current treatments are ineffective and do not meet market demand, which is increasing significantly every year due to the increasing aging of the population. For example, Alzheimer's disease (AD) is characterized by a gradual but progressive decline in learning and memory, and the leading cause of death in the elderly. The increasing prevalence of AD is driving the need and demand for better and earlier diagnosis. According to Alzheimer's Disease International, the disease currently affects 46.8 million people worldwide, but the number of cases is expected to triple over the next 30 years. One of the countries with the fastest growing elderly population is China. Based on population projections, by 2030, one in four people is expected to be over 60 years old, a large proportion of whom will be at risk of developing AD. In fact, the number of AD cases in China doubled from 3.7 million to 9.2 million cases between 1990 and 2010, and is projected to reach 22.5 million cases by 2050. Hong Kong's population is also rapidly aging. Elderly people aged 65 years and older are estimated to account for 24% of the population by 2025 and 39.3% of the population by 2050. The number of AD cases is projected to increase to 332,688 cases by 2039. Therefore, there is an urgent need to develop new methods to effectively treat AD patients suffering from this devastating condition. The present invention meets this and other related needs by disclosing novel compositions and methods useful for the effective treatment of disease, and potentially providing a cure. Summary of the Invention

[0003] This application discloses for the first time a genetic locus comprising rs1921622 and / or other 574 sST2-associated genetic variants and / or the 3'-end (3') untranslated region of sST2, which, when disrupted, results in reduced expression of soluble ST2 (sST2) protein, as a target for therapeutic intervention of Alzheimer's disease by genetic engineering. Thus, from this discovery, new compositions and methods are devised for treating Alzheimer's disease by suppressing or eliminating the effect of rs1921622, other 574 sST2-associated genetic variants, or genomic sequences encompassing the 3'-end (3') untranslated region of sST2.

[0004] Thus, in a first aspect, the present invention provides a method of treating Alzheimer's disease in an individual or reducing the risk of an individual developing Alzheimer's disease at a later date. The claimed method comprises administering to the individual an effective amount of a composition that disrupts (i) rs1921622, and / or (ii) any one or more of the other 574 sST2-associated genetic variants listed in Tables 4 and 5, and / or (iii) a genomic sequence encompassing the 3'-untranslated region (UTR) of the sST2 gene coding sequence or transcript.

[0005] In some embodiments, the claimed method includes sequencing at least a portion of the individual's genome prior to the administering step. In some embodiments, the individual is considered an APOE-ε4 carrier, either a heterozygote carrier or a homozygote carrier. In other cases, the individual is a non-APOE-ε4 carrier. In some cases, the individual is a female. In some cases, the individual is a male. In some embodiments, the individual has an A allele at rs1921622. In some embodiments, the individual has at least one allele at a particular locus shown in Table 4. In some embodiments, the individual has been diagnosed with AD. In some embodiments, the individual has not yet been diagnosed with AD, but has known risk factors for AD, such as a family history of AD, or carries one or more genetic alleles known to increase AD risk, for example as a female APOE-ε4 carrier. In some embodiments, the genomic sequence encompassing rs1921622 (or another locus named in Table 4 or Table 5) comprises about 300 base pairs upstream and downstream of rs1921622 (or another locus in Table 4 or Table 5), preferably about 250, 200, 150, 100, 50, 30, or 20 base pairs upstream and downstream of rs1921622 (or another locus in Table 4 or Table 5). In some embodiments, the composition comprises an siRNA, microRNA, miniRNA, lncRNA, or antisense oligonucleotide targeted to a genomic sequence encompassing rs1921622 (or another locus in Table 4 or Table 5) or the 3'-UTR of the sST2 gene. In some embodiments, the composition comprises one or more vectors encoding a small guide RNA (sgRNA)-guided endonuclease and two sgRNAs that target two locations within a genomic sequence encompassing rs1921622 (or another locus listed in Table 4 or Table 5) or the 3'-UTR of the sST2 gene. In some embodiments, the composition comprises a vector encoding a Cas9 nuclease and two sgRNAs.In some embodiments, the one or more vectors are one or more viral vectors. In some embodiments, the composition is administered by subcutaneous, intramuscular, intravenous, intraperitoneal, or intracranial injection, or by oral or nasal administration. In some embodiments, the composition is administered in the form of a solution, suspension, powder, paste, tablet, or capsule.

[0006] In a second aspect, the present invention provides compositions comprising an effective amount of one or more agents that disrupt genomic sequences including (i) rs1921622, and / or (ii) any one or more of the other 574 sST2-associated genetic variants listed in Tables 4 and 5, and / or (iii) the 3'-untranslated region (UTR) of the sST2 gene coding sequence or transcript, together with one or more physiologically acceptable excipients.

[0007] In some embodiments, the genomic sequence encompassing rs1921622 (or another locus named in Table 4 or Table 5) comprises about 300 bps upstream and downstream of rs1921622 (or another locus in Table 4 or Table 5), preferably about 250 bps, 200 bps, 150 bps, 100 bps, 50 bps, 30 bps, or 20 bps upstream and downstream of rs1921622 (or another locus in Table 4 or Table 5). In some embodiments, the composition comprises an siRNA, microRNA, miniRNA, lncRNA, or antisense oligonucleotide targeted to a genomic sequence encompassing rs1921622 (or another locus in Table 4 or Table 5) or the 3'-UTR of the sST2 gene. In some embodiments, the composition comprises one or more vectors encoding a small guide RNA (sgRNA)-guided endonuclease and two sgRNAs targeting two locations in a genomic sequence encompassing rs1921622 (or another locus listed in Table 4 or Table 5) or the 3'-UTR of the sST2 gene. In some embodiments, the composition comprises one vector encoding a Cas9 nuclease and two sgRNAs. In some embodiments, the one or more vectors are one or more viral vectors. In some embodiments, the composition is administered by subcutaneous, intramuscular, intravenous, intraperitoneal, or intracranial injection, or by oral or nasal administration. In some embodiments, the composition is administered in the form of a solution, suspension, powder, paste, tablet, or capsule.

[0008] In a third aspect, the invention provides a kit for treating Alzheimer's disease in an individual, or reducing an individual's risk of subsequently developing Alzheimer's disease, the kit comprising a first container containing a composition that disrupts rs1921622 (or another locus in Table 4 or Table 5) or a genomic sequence encompassing the 3'-UTR of the sST2 gene.

[0009] In some embodiments, the composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or intracranial injection, or is formulated for oral or nasal administration. In some embodiments, the composition comprises an siRNA, microRNA, miniRNA, lncRNA, or antisense oligonucleotide that targets a genomic sequence that encompasses the 3'-UTR of rs1921622 (or another locus in Table 4 or Table 5) or sST2 gene. In some embodiments, the composition comprises one or more vectors encoding (1) an endonuclease guided by a small guide RNA (sgRNA) and (2) two sgRNAs that target two positions in a genomic sequence that encompasses the 3'-UTR of rs1921622 (or another locus in Table 4 or Table 5) or sST2 gene. In some embodiments, the kit contains a composition that comprises a Cas9 nuclease and one vector encoding two sgRNAs. In some embodiments, the kit further comprises a second container containing an agent for sequencing at least a portion of an individual's genome (e.g., the APOE-ε4 gene). Optionally, the kit also further comprises instructions for administration of the composition.

[0010] In relation to this aspect of the invention, there is further provided in accordance with the disclosure herein the use of one or more agents that disrupt rs1921622, and / or the other 574 sST2-associated genetic variants, and / or genomic sequences encompassing the 3'-untranslated region of sST2, for the manufacture of (1) a pharmaceutical for treating Alzheimer's disease; and / or (2) a kit containing a pharmaceutical for treating Alzheimer's disease. [Brief description of the drawings]

[0011] [Figure 1] Diagram illustrating IL-33 / ST2 signaling. (a) Schematic showing soluble ST2 (sST2) and full-length ST2 (ST2L) transcripts. (b) IL-33 / ST2 signaling pathway. IL-33, interleukin 33. [Diagram 2]Circulating sST2 levels are associated with Alzheimer's disease and its pathological changes. (a) Individual plasma soluble ST2 (sST2) levels stratified according to disease phenotype (n=336 healthy controls [HC], n=277 AD patients from Chinese_cohort_1; linear regression test, T=3.241, **P<0.01). Data are presented as box plots with maximum, 75th percentile, median, 25th percentile, and minimum values. Plus signs (+) represent the corresponding mean values. (b) Correlation between plasma sST2 levels and intracranial volume (ICV)-normalized gray matter volume (n=192 participants, from Chinese_cohort_1); r, Pearson's correlation coefficient. (c) Correlation between plasma sST2 and P-tau181 levels (n=271 participants, from Chinese_cohort_1). (d) Correlation between plasma sST2 and NfL (neurofilament light polypeptide) levels (n=251 participants, from Chinese_cohort_1). (e) Correlation between cerebrospinal fluid (CSF) and plasma levels of sST2 (n=107 participants, from the Stanford Alzheimer's Disease Research Center [ADRC] cohort). (f) Individual CSF sST2 levels stratified according to disease phenotype (n=11 HC, n=75 AD patients from the UKBBN cohort; linear regression test, T=3.355, **P<0.01). (g,h) Correlation between amyloid beta (Aβ) staining in postmortem frontal cortex and CSF sST2 levels in AD patients (n=51 participants, from the UKBBN cohort). Participants were stratified into two groups according to CSF ​​sST2 levels: low, ≦3.6 ng / mL; high, >3.6 ng / mL. The vertical dashed line in (h) indicates the CSF sST2 level (3.6 ng / mL), which has the largest Youden's index value for discriminating HC from AD patients. Representative images of Aβ staining in AD patients with low and high CSF sST2 levels (g) and the results of the correlation analysis (h). Scale bar, 100 μm. [Diagram 3]Plasma sST2 levels were elevated in Alzheimer's disease patients without a history of cardiovascular disease in Chinese_cohort_1. (a) Individual plasma soluble ST2 (sST2) levels stratified by cardiovascular disease (CVD) phenotype (n=97 cognitively normal participants with no history of CVD [non-CVD], n=30 cognitively normal participants with heart disease [HD], n=201 cognitively normal participants with hypertension [HT], n=72 cognitively normal participants with diabetes mellitus [DM], n=98 cognitively normal participants with hyperlipidemia [HL] from Chinese_cohort_1; linear regression test, T=2.206, 0.296, -1.493, and -0.461 for tests on heart disease, hypertension, diabetes mellitus, and hyperlipidemia, respectively; *P<0.05). Data are presented as box plots with maximum, 75th percentile, median, 25th percentile, and minimum values. Plus signs (+) represent the corresponding mean values. (b-e) Plasma sST2 levels are associated with Alzheimer's disease (AD) and related endophenotypes. (b) Individual plasma sST2 levels stratified by disease phenotype (n=97 healthy controls [HC], n=70 AD patients from Chinese_cohort_1 without history of CVD [non-CVD cohort], respectively; linear regression test, T=3.151, **P<0.01). (c-e) Correlation of plasma sST2 levels with endophenotypes in AD patients. (c) Gray matter volume normalized to intracranial volume (ICV) (n=48 participants, from the non-CVD cohort). (d) Plasma P-tau181 levels (n=77 participants, from the non-CVD cohort). (e) Plasma NfL (neurofilament light polypeptide) levels (n=60 participants, from the non-CVD cohort). r, Pearson's correlation coefficient. [Figure 4]Elevated brain sST2 levels exacerbate Aβ accumulation and impair microglial Aβ clearance capacity. (a) Schematic of intracerebroventricular (icv) delivery of soluble ST2 (sST2) to 3-month-old 5XFAD mice. (b-e) Amyloid beta (Aβ) deposits in the cortex of 4-month-old 5XFAD mice after icv delivery of sST2-Fc or Fc as control. (b, c) DAB staining of Aβ. (b) Representative image; scale bar, 1 mm. (c) Quantification of Aβ plaques (% of total cortical area) (control: n=6 mice, sST2: n=7 mice; unpaired Student's t test, T=2.758, *P<0.05). Data in bar graphs are mean+SEM. (d) Confocal images of X-34 stained Aβ deposits (blue) and 4G8 labeled Aβ (red). Fibrillar (open arrow) and compact (closed arrow) Aβ plaques are labeled. Scale bar, 100 μm. (e) Quantification of fibrillar, compact, and inactive Aβ plaques (control: n=6 mice, sST2: n=7 mice; unpaired Student's t test, T=3.325, 2.274, and −0.770, respectively; *P<0.05, **P<0.01). (f, g) Colocalization of microglia and Aβ plaques in the cortex of 4-month-old 5XFAD mice after icv delivery of sST2-Fc or Fc as control. (f) Representative image showing co-staining of 4G8-labeled Aβ plaques (white) and Iba-1+ microglia (green; red arrows point to microglial cell bodies) using merged confocal Z-stack images with orthogonal XZ and YZ views. Scale bar, 10 μm. (g) Quantification of microglial coverage of Aβ plaques (control: n = 6 mice, sST2: n = 7 mice; unpaired Student's t test, T = -2.298; *P < 0.05). (h, i) Microglial Aβ uptake activity in the cortex of 4-month-old 5XFAD mice after icv delivery of sST2-Fc or Fc as control. Representative scatter plot (h) and quantification (i) show the percentage of CD11b+ cells containing methoxy-X04-labeled Aβ (control: n=7 mice, sST2: n=7 mice; unpaired Student's t-test, T=-3.620, **P<0.01).(h) Scatter plot of wild-type (WT) mice was used to gate on MeX04+ microglia (i.e., MeX04+CD11b+ cells). [Diagram 5] Representative images of fibrillar, compact, and inactive Aβ plaques in 5XFAD mice. Confocal images of X-34 stained amyloid beta (Aβ) deposits (blue) and 4G8 labeled Aβ (red) in the cortex of a 4-month-old 5XFAD mouse. Scale bar, 10 μm. [Figure 6] Elevation of brain sST2 leads to increased microglial numbers in 5XFAD mice. (a, b) Brain soluble ST2 (sST2) increases microglial numbers in 5XFAD mice. Representative images (a) and quantification (b) of Iba-1+ microglia in the cortex of 4-month-old 5XFAD mice after intraventricular delivery of sST2-Fc or Fc as control (control: n=6 mice, sST2: n=7 mice; unpaired Student's t-test, T=4.491, ***P<0.001). Scale bar, 50 μm. Bar graph data are mean+SEM. (c) Quantification of microglia around small (i.e., radius ≦8 μm) and large (i.e., radius >8 μm) amyloid beta (Aβ) plaques (control: n = 6 mice, sST2: n = 7 mice, respectively; unpaired Student's t test, T = 2.661 and 2.551, respectively, *P < 0.05). [Figure 7] Gating strategy for amyloid beta (Aβ)+ microglia. Cells were gated by forward (FSC=size) and side scatter (SSC=internal structure). We used FSC and trigger pulse width to distinguish single cells from cell doublets or aggregates. We used unstained controls to identify the CD11b+ cell population. We used samples from C57BL6J mice to identify the methoxy-X04+ cell population. [Figure 8]Effect of gender and age on sST2 levels. (a) Individual plasma soluble ST2 (sST2) levels stratified by gender and disease phenotype (n=132 male healthy controls [HC], n=84 male patients with Alzheimer's disease [AD], n=204 female HC, n=193 female AD patients from Chinese_cohort_1, respectively; linear regression test, test for effect of gender: T=-4.918 and -2.407 in HC and AD patients, respectively, #P<0.05, ###P<0.001; test for effect of AD: T=1.534 and 3.008 in males and females, respectively, **P<0.01). Data are presented as box plots with maximum, 75th percentile, median, 25th percentile, and minimum values. Plus signs (+) represent the corresponding mean values. (b) Correlation between plasma sST2 levels and age in men and women (n=216 and 397: men and women from Chinese_cohort_1, respectively; men: Pearson's r2=0.0010, P=0.6410; women: Pearson's r2=0.0366, P<0.0001). (c) Correlation of plasma sST2 levels with age in men and women from the INTERVAL and LonGenity cohorts 20 (n=1,685 and 1,616 men and women from the INTERVAL cohort, n=432 and 530 men and women from the LonGenity cohort, respectively; correlation test in the INTERVAL cohort [age 18-76 years], men: Pearson's r2=0.0169, P<0.0001; women: Pearson's r2=0.0001, P=0.7715; correlation test in the LonGenity cohort [age 65-94 years], men: Pearson's r2=0.0100, P=0.0093; women: Pearson's r2=0.0196, P=0.0001). (d) Correlation of cerebrospinal fluid (CSF) sST2 levels with age in men and women from the Japanese cohort (n=68 and 65 men and women, respectively; men: Pearson's r2=0.0441, P=0.0812; women: Pearson's r2=0.1521, P=0.0014). (e, f) Contribution of age and sex to the variance of sST2 levels. Numbers represent the proportion of plasma sST2 (e) and CSF sST2 (f) variance explained by age, sex, and other factors in the Chinese_cohort_1 and Japanese cohorts, respectively. [Figure 9] The rs1921622 A allele is associated with lower circulating sST2 levels. (a) Manhattan plot showing genetic variants at the IL1RL1 locus associated with plasma soluble ST2 (sST2) levels identified by a genome-wide association study of plasma sST2 levels in Chinese_cohort_1. Horizontal lines indicate suggestive thresholds (P=1E-5, blue) and genome-wide thresholds (P=5E-8, red). (b) Regional association plot of genetic variants at the IL1RL1 locus and plasma sST2 levels. Purple diamonds indicate the sentinel variant rs1921622. The color scale indicates the linkage disequilibrium (measured as r2) between rs1921622 and its neighboring variants. (c, d) Plasma (c) and cerebrospinal fluid (CSF) (d) sST2 levels in individuals stratified according to rs1921622 genotype (plasma sST2 levels: n=107, 206, and 114 G / G, G / A, and A / A carriers, respectively, from Chinese_cohort_1; linear regression test, T=-11.207, ***P<0.001; CSF sST2 levels: n=20, 44, and 22 G / G, G / A, and A / A carriers, respectively, from UKBBN cohort; linear regression test, T=-2.615, *P<0.05). Data in box plots include maximum, 75th percentile, median, 25th percentile, and minimum; plus sign (+) represents the corresponding mean value. [Figure 10]Fine-mapping analysis of sST2-associated genetic variants in the IL1RL1 gene. (a) Quantile-quantile (QQ) plot showing p-value distribution of genome-wide association study results. Genomic inflation factor (λ) is indicated. (b) Haplotype analysis of 79 genetic variants associated with rs1921622 (r2>0.7). Each numbered column represents one of the 79 variants; red and blue indicate minor and major alleles, respectively. Each row represents a specific haplotype defined by a specific combination of major and minor alleles in a haplotype block, where haplotype frequency is indicated on the right and gene locus (±10 kbp) is indicated on the top. Triangle indicates the location of rs1921622. (c) Fine-mapping plot showing association of IL1RL1 genetic variants with plasma soluble ST2 (sST2) levels. The color intensity of the dots indicates the effect size of each individual variant on plasma sST2 levels, and the size of the dot indicates the probability that the variant exerts its causal effect. [Figure 11]Targeted deletion at the rs1921622 locus reduces sST2 expression and secretion in brain endothelial cells. (a) Effect of rs1921622 A allele on transcript levels of soluble ST2 (sST2) (ENST00000311734.2) in human tissues from the GTEx dataset. The effect size of rs1921622 A allele and 95% confidence intervals for each tissue (see Table 1 for details) are indicated by boxes and lines, respectively. Red and blue colors indicate significant (P<0.05) and non-significant (P>0.05) associations between rs1921622 genotype and sST2 transcript levels, respectively. (b-d) Single-nucleus RNA sequencing analysis revealed the association between rs1921622 and sST2 transcript levels in human brain endothelial cells. (b) Uniform manifold approximation and projection (UMAP) plot showing cell types in human frontal cortex (n=169,496 cells from 21 participants from the UKBBN cohort): Excitatory, excitatory neuron; Inhibitory, inhibitory neuron; Astro, astrocyte; Micro, microglia; Endo, endothelial cell; Oligo, oligodendrocyte; OPC, oligodendrocyte precursor cell. (c) Expression profile of sST2 transcripts (top) and CLDN5 transcripts (bottom) in human frontal cortex. (d) Dot plot showing expression levels of sST2 (top) and CLDN5 (bottom), stratified according to rs1921622 genotype in endothelial cells from human frontal cortex. Normalization experiment, normalized expression. (e-h) CRISPR / Cas9 genome editing reveals that the rs1921622 locus is important for sST2 expression. (e) Diagram showing the location of two sgRNA pairs (i.e., sgRNA-1 and sgRNA-4 for the 67 bp deletion [Δ67bp]; sgRNA-2 and sgRNA-3 for the 38 bp deletion [Δ38bp]) targeting the region harboring the rs1921622 locus (red). (f) ChIP-qPCR analysis of H3K27ac changes at the rs1921622 locus after 24 h of IL-33 treatment in hCMEC / D3 cells (n=3 per group; unpaired Student's t-test, T=4.593, *P<0.05).Data in bar graphs are mean + SEM. (g, h) Deletion of the rs1921622 locus reduces sST2 transcript levels and protein secretion in hCMEC / D3 cells. (g) sST2 transcript levels (n=6, 8, and 10 isogenic control clones, clones with the 38bp deletion, and clones with the 67bp deletion, respectively; unpaired Student's t test, T=-5.444 and -7.612 for Δ38bp vs. control and Δ67bp vs. control, respectively, ***P<0.001). (h) Secreted sST2 protein levels in conditioned medium (CM) (n=3, 3, and 4 for the isogenic control line, the line with the 38 bp deletion, and the line with the 67 bp deletion, respectively; unpaired Student's t test, T=-13.450 and -16.030 for Δ38bp vs. control and Δ67bp vs. control, respectively, ***P<0.001). [Figure 12] IL-33 induces the expression and secretion of sST2 in brain endothelial cells. (a, b) IL-33 administration enhances the expression (n=4 per group) (a) and secretion (n=3 per group) (b) of soluble ST2 (sST2) in hCMEC / D3 cells (unpaired Student's t-test, T=6.484 and 28.89 for sST2 transcript and protein levels, respectively, ***P<0.001). Data in bar graphs are mean+SEM. CM, conditioned medium; Control, control group. (c) ChIP-qPCR analysis of H3K4me3 changes in the sST2 promoter region after 24 h of IL-33 administration in hCMEC / D3 cells (n=3 per group; unpaired Student's t-test, T=29.69, ***P<0.001). [Figure 13]Validation of CRISPR / Cas9-based targeted deletion at the rs1921622-containing region in hCMEC / D3 cells. (a) Diagram showing the location of two sgRNA pairs (i.e., sgRNA-1 and sgRNA-4 for the 67-bp deletion [Δ67bp], and sgRNA-2 and sgRNA-3 for the 38-bp deletion [Δ38bp]) targeting the rs1921622-containing region (red). (b) Gel images of single clones from the isogenic control line (control), the 38-bp deletion line (Δ38bp), and the 67-bp deletion line (Δ67bp). L, DNA ladder. (c) Sanger validation of single clones. [Figure 14]The rs1921622 A allele exerts a protective effect against Alzheimer's disease in APOE-ε4 carriers. (a, b) Forest plots showing meta-analysis results of the rs1921622 A allele in APOE-ε4 carriers overall (a) and in female APOE-ε4 carriers (b) from six Alzheimer's disease (AD) datasets (N=5,436 healthy controls, N=5,556 AD patients). Effect sizes (log odds ratios) from the independent datasets and meta-analysis are displayed as rectangles and diamonds, respectively. For the independent datasets, horizontal lines indicate the 95% confidence intervals and the size of the rectangle is proportional to the weights used in the meta-analysis. RE2, Han and Eskin random effects model. (c) Survival plot of cumulative dementia-free probability in female APOE-ε4 carriers with AD stratified according to rs1921622 genotype (n=58, 108, and 66 G / G, G / A, and A / A carriers, respectively, from the LOAD cohort). HR, hazard ratio. (d) Individual cognitive performance (as determined by Mini-Mental State Examination [MMSE] score) in female APOE-ε4 carriers with AD stratified according to rs1921622 genotype (n=80, 97, and 41 G / G, G / A, and A / A carriers, respectively, from Chinese_cohort_2; linear regression test, T=2.644 and 2.207 for A / A vs. G / G and A / A vs. G / A, respectively, *P<0.05, **P<0.01). Data in box plots include maximum, 75th percentile, median, 25th percentile, and minimum values. Plus signs (+) represent the corresponding mean values. (e) Effect size of rs1921622 A allele on brain region volume in female APOE-ε4 carriers with cognitive impairment (n=29, 72, and 46 G / G, G / A, and A / A carriers, respectively, from the ADNI cohort). For each brain region, the effect size and 95% confidence interval of rs1921622 A allele are indicated by boxes and lines, respectively. Red and blue colors indicate significant (*P<0.05) and non-significant (P≧0.05) associations between rs1921622 genotype and brain region volume, respectively. Fusiform, fusiform gyrus; middle temporal, middle temporal gyrus.(f) Individual cognitive scores in amyloid beta (Aβ)+APOE-ε4 carriers stratified according to rs1921622 genotype (n=133 G carriers [G / -], n=57 A / A carriers from the AIBL cohort; Wilcoxon rank sum test, testing for the effect of rs1921622 genotype on AIBLPACC score, attention processing score, episodic recall score, and recognition score: W=4336.0, 1970.5, 1610.0, and 1523.5, respectively; *P<0.05, **P<0.01). (g) Longitudinal gray matter volumes in Aβ+APOE-ε4 carriers stratified according to rs1921622 genotype (n=281 and 112 data points from 128 G carriers [G / -] and 55 A / A carriers from the AIBL cohort, respectively; linear mixed model test, β=-0.046, F=4.839, *P<0.05). [Figure 15] Alzheimer's disease risk is not associated with rs1921622 genotype in APOE-ε4 non-carriers. Forest plot showing a meta-analysis of rs1921622 in APOE-ε4 non-carriers from six Alzheimer's disease datasets. Effect size (log odds ratio) values ​​from the independent datasets and meta-analysis results are displayed as rectangles and diamonds, respectively. For the independent datasets, horizontal lines indicate the range of the 95% confidence intervals, and the size of the rectangle is proportional to the weights used in the meta-analysis. RE2, Han, and Eskin random-effects models. [Figure 16]The rs1921622 A allele is associated with delayed age of onset of dementia in female APOE-ε4 carriers with Alzheimer's disease in the LOAD and ADC cohorts. (a, b) Survival plots of cumulative dementia-free probability in all APOE-ε4 carriers (n=82, 164, and 99 G / G, G / A, and 99 A / A carriers, respectively) (a) and in female APOE-ε4 carriers (n=197, 477, and 276 G / G, G / A, and A / A carriers, respectively) (b) in the Alzheimer's disease (AD) group from the LOAD cohort stratified according to rs1921622 genotype. (c, d) Survival plots of cumulative dementia-free probability in all APOE-ε4 carriers (n=438,959, and 543 G / G, G / A, and A / A carriers, respectively) (c) and female APOE-ε4 carriers (n=197,477, and 276 G / G, G / A, and A / A carriers, respectively) in the AD group from the ADC cohort stratified according to rs1921622 genotype. Cox regression test. HR, hazard ratio. [Figure 17]The rs1921622 A allele protects against entorhinal cortex atrophy in female APOE-ε4 carriers with cognitive impairment in the ADNI cohort. (a) Effect of the rs1921622 A allele on brain region volumes in APOE-ε4 carriers with cognitive impairment (n=374 participants from the ADNI cohort). For each brain region, the effect size and 95% confidence intervals of the rs1921622 A allele are indicated by boxes and lines, respectively. (b) Individual intracranial volume (ICV)-normalized entorhinal cortex volumes in APOE-ε4 carriers with cognitive impairment stratified according to rs1921622 genotype (n=80, 180, and 114 G / G, G / A, and A / A carriers from the ADNI cohort; linear regression test, T=0.330). Data in box plots include maximum, 75th percentile, median, 25th percentile, and minimum values. Plus signs (+) represent the corresponding mean values. (c) Effect of rs1921622 A allele on brain region volumes in female APOE-ε4 carriers with cognitive impairment (n=147 participants from the ADNI cohort). (d) Individual ICV-normalized entorhinal cortex volumes in female APOE-ε4 carriers with cognitive impairment stratified according to rs1921622 genotype (n=29, 72, and 46 G / G, G / A, and A / A carriers, respectively, from the ADNI cohort; linear regression test, T=2.285, *P<0.05). Fusiform, fusiform gyrus; middle temporal, middle temporal gyrus. [Figure 18]The rs1921622 A allele protects against neurodegeneration in female APOE-ε4 carriers with Alzheimer's disease from Chinese_cohort_1. (a, b) Individual plasma levels of P-tau181 (n=14, 32, and 11 G / G, G / A, and A / A carriers, respectively) (a) and NfL (neurofilament light polypeptide) (n=14, 33, and 12 G / G, G / A, and A / A carriers, respectively) (b) in APOE-ε4 carriers with Alzheimer's disease (AD) from Chinese_cohort_1 stratified according to rs1921622 genotype (linear regression test, T=-0.419 and -1.622 for plasma P-tau181 and NfL, respectively). Data in box plots include maximum, 75th percentile, median, 25th percentile, and minimum. Plus signs (+) represent the corresponding mean values. (c, d) Individual plasma levels of P-tau181 (n=13, 23, and 4 G / G, G / A, and A / A carriers, respectively) (c) and NfL (n=13, 23, and 5 G / G, G / A, and A / A carriers, respectively) in female APOE-ε4 carriers with AD from Chinese_cohort_1 stratified according to rs1921622 genotype (linear regression test, T=-2.065 and -2.498 for plasma P-tau181 and NfL, respectively; *P<0.05). [Figure 19]The rs1921622 A allele protects against cognitive decline and gray matter atrophy in female APOE-ε4 carriers in the AIBL Aβ+ cohort. (a, b) Individual cognitive scores and baseline gray matter volumes stratified according to rs1921622 genotype among amyloid beta (Aβ)+APOE-ε4 carriers in the AIBL cohort (n=45, 88, and 57 G / G, G / A, and A / A carriers, respectively; Wilcoxon rank sum test for rs1921622(A / A) on AIBLPACC score, attention processing, episodic recall score, and recognition score: W=4,336, 1,970.5, 1,610, and 1,523.5, respectively, *P<0.05, **P<0.01). (c, d) Individual cognitive scores and baseline gray matter volumes stratified according to rs1921622 genotype in female Aβ+APOE-ε4 carriers in the AIBL cohort (n=23, 45, and 33 G / G, G / A, and A / A carriers, respectively; Wilcoxon rank sum test, tests for rs1921622 (A / A) on AIBLPACC score, attention processing, episodic recall score, recognition score, and baseline gray matter volume: W=641, 692.5, 584,572, and 819, respectively; *P<0.05, **P<0.01). Data are presented as box plots with maximum, 75th percentile, median, 25th percentile, and minimum values. Plus signs (+) represent the corresponding mean values. [Figure 20]Co-harboring the rs1921622 A allele restores impaired microglial activity to Aβ in female APOE-ε4 carriers with Alzheimer's disease. (a, b) APOE-ε4 is associated with increased amyloid beta (Aβ) deposition in female patients with Alzheimer's disease (AD). (a) Representative image of Aβ plaque staining in the frontal cortex. Scale bar, 200 μm. (b) Quantification of Aβ plaque burden in the frontal cortex stratified according to gender and APOE-ε4 genotype (n=14 male APOE-ε4 non-carriers [non-ε4], n=22 male APOE-ε4 carriers [ε4], n=19 female non-APOE-ε4 carriers, n=23 female APOE-ε4 carriers from the UKBBN cohort; linear regression test, test for effect of gender: T=2.507 and 5.022 in non-APOE-ε4 and APOE-ε4, respectively, #P<0.05, ###P<0.001; test for effect of APOE-ε4: T=-0.802 and 2.062 in males and females, respectively, *P<0.05). Data in box plots include maximum, 75th percentile, median, 25th percentile, and minimum. Plus signs (+) represent the corresponding mean values. (c) Representative image of Aβ plaque staining in the frontal cortex of female patients with AD stratified according to APOE-ε4 and rs1921622 genotype. Scale bar, 5 mm. (d, e) The rs1921622 variant is associated with increased microglial coverage of Aβ in APOE-ε4 carriers but not in non-carriers. (d) Representative image of co-staining of Aβ plaques (brown) and Iba-1+ microglia (purple) in the frontal cortex of female APOE-ε4 non-carriers stratified according to rs1921622 genotype. Scale bar, 100 μm.(e) Quantification of the area of ​​Aβ plaques colocalized with microglia stratified according to APOE-ε4 genotype and rs1921622 genotype (n=4 and 15 G / G and A carriers [A / -], respectively, among APOE-ε4 non-carriers [non-ε4], and n=5 and 18 G / G and A / - carriers, respectively, among APOE-ε4 carriers [ε4], from the UKBBN cohort; linear regression test, testing the effect of APOE-ε4: T=-2.991 and -1.553 for G / G and A / -, respectively, #P<0.05; testing the effect of rs1921622: T=0.229 and 2.409 for non-ε4 and ε4, respectively, *P<0.05). [Figure 21]The rs1921622 A allele enhances microglial activity against Aβ in female APOE-ε4 carriers with Alzheimer's disease. (a, b) The rs1921622 A allele is associated with reduced amyloid beta (Aβ) deposition in female APOE-ε4 carriers with Alzheimer's disease (AD) (n=4 and 15 G / G carriers, A carriers [A / -] among APOE-ε4 noncarriers [non-ε4]; n=5 and 18 G / G carriers, A / - carriers among APOE-ε4 carriers [ε4] from the UKBBN cohort, respectively). (a) Representative images showing Aβ plaque staining in the frontal cortex. Scale bar, 200 μm. (b) Quantification of Aβ plaque area in the frontal cortex stratified according to APOE-ε4 and rs1921622 genotype (linear regression test; effect of APOE-ε4: T=2.912 and 0.956 in G / G and A / - carriers, respectively, #P<0.05; effect of rs1921622: T=-0.833 and -3.706 in non-APOE-ε4 and APOE-ε4 carriers, respectively, *P<0.05). Data in box plots include maximum, 75th percentile, median, 25th percentile, and minimum values. Plus sign (+) denotes the corresponding mean value. (c, d) The rs1921622 A allele is associated with increased microglial colocalization with Aβ in female APOE-ε4 carriers with AD (n=5 and 18 G / G and A / - carriers among APOE-ε4 carriers, respectively, from the UKBBN cohort). (c) Representative images showing co-staining of Aβ plaques (brown) and Iba-1+ microglia (purple) in the frontal cortex. Scale bar, 100 μm. (d) Quantification of Aβ plaque area and colocalization with microglia stratified according to rs1921622 genotype (linear regression test, T=2.409, *P<0.05). (e) Volcano plot showing association of rs1921622 genotype with genes expressed by microglia in the frontal cortex of female APOE-ε4 carriers with AD (n=2,636 microglia from 8 participants from the UKBBN cohort). Blue and red dots indicate microglial genes negatively and positively associated with the rs1921622 A allele, respectively.Dot size is proportional to the false discovery rate (FDR, log10 scale). The top 5 negatively and positively associated microglial genes are labeled. (f) Representative Gene Ontology (GO) terms enriched for rs1921622-associated microglial genes. GO terms enriched for down- and up-regulated microglial genes in rs1921622 A allele carriers are indicated in blue and red, respectively. (g) Dot plots showing expression levels of microglial activation and homeostasis genes stratified according to rs1921622 genotype. [Figure 22] The rs1921622 A allele and cerebrospinal fluid sST2 levels have opposite effects on microglial transcripts in female APOE-ε4 carriers with Alzheimer's disease. Scatter plot showing the normalized effect size (β) of cerebrospinal fluid (CSF) soluble ST2 (sST2) levels on microglial gene expression in the frontal cortex of female APOE-ε4 carriers with Alzheimer's disease (AD) and the correlation of the rs1921622 A allele (n=2,636 microglia from 8 individuals from the UKBBN cohort). [Figure 23]Deletion of the 3'-untranslated region of sST2 reduces sST2 levels and alleviates amyloid-related pathology in 5XFAD mice. (a, b) Deletion of the 3'-untranslated region of sST2 reduces serum sST2 levels. (a) Schematic of the deletion of the 3'-untranslated region of sST2. sST2 and ST2L share most of the coding sequence, whereas the 3'-untranslated region of sST2 is unique. (b) Quantitative analysis of serum sST2 levels by ELISA. Values ​​are means ± sem (***P<0.001, unpaired two-tailed t-test). (e-h) Deletion of the 3'-untranslated region of sST2 alleviates amyloid-related pathology in 5XFAD mice. (c, d) Quantitative analysis of soluble and insoluble Aβx-40 and Aβx-42 by ELISA. Values ​​are means ± sem (*P<0.05, unpaired two-tailed t-test). (e,f) DAB staining of Aβ. (e) Representative image. (f) Quantification of Aβ plaques (% of total cortical area). Values ​​are means ± sem (*P<0.05, unpaired two-tailed t-test). (g,h) X-34 stained Aβ deposits. (g) Representative image. (h) Quantification of X34 positive Aβ plaques (% of total cortical area). Values ​​are means ± sem (*P<0.05, unpaired two-tailed t-test). [Figure 24] Antisense oligonucleotides targeting the 3'-untranslated region of sST2 reduce sST2 levels and alleviate amyloid pathology. (a) Schematic of antisense oligonucleotides (ASOs) targeting the 3'-untranslated region of sST2. (b, c) Systematic screening of mouse sST2-ASOs in mouse fibroblast NIH-3T3 cells. (b) sST2 transcript levels in all mouse sST2 ASOs (c) sST2 transcript levels in the top 13 efficient ASOs. Values ​​are means ± sem. (d) Systemic delivery of sST2-ASOs reduces serum sST2 levels in vivo. Values ​​are means ± sem. (e, f) X-34 stained Aβ deposits. (e) Representative images. (f) Quantification of X34 positive Aβ plaques (% of total cortical area). [Figure 25]Antisense oligonucleotides targeting the 3'-untranslated region of sST2 reduce sST2 protein and transcript levels in human cells. (a) Schematic of antisense oligonucleotides (ASOs) targeting the 3'-untranslated region of target sST2. (b, c) Systematic screening of human sST2-ASOs in human umbilical vein endothelial cells (HUVECs). (b) Levels of secreted sST2 in the medium. Values ​​are normalized to sST2 levels in HUVECs transfected with a negative control ASO. (c) sST2 transcript levels in the top 15 efficient ASOs. Values ​​are means ± sem. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Table 1. Association of rs1921622 A allele with soluble ST2 and full-length ST2 transcript levels in human tissues. Linear regression tests adjusted for age, sex, RNA integrity, and population structure. β, effect size; SE, standard error; sST2, soluble ST2; ST2L, full-length ST2. Bold and red text indicates statistical significance with a cutoff of P<0.05. a Only tissues with >50% of individuals that exhibited expression (reads per kilobase mapped [RPKM] >0) were included to calculate mean expression of the candidate genes and association tests between candidate gene levels and rs1921622 genotypes. b Average expression of the candidate gene among individuals in each tissue. Table 2. Demographic characteristics of Chinese_cohort_1. CVD, cardiovascular disease; ICV, intracranial volume; MoCA, Montreal Cognitive Assessment; MRI, magnetic resonance imaging; NfL, neurofilament light polypeptide; SD, standard deviation; sST2, soluble ST2; WGS, whole genome sequencing. Table 3. Demographic characteristics of the UK Brain Bank Network cohort. SD, standard deviation; PMD, postmortem period; CSF, cerebrospinal fluid; sST2, soluble ST2. Table 4. Candidate gene variants associated with plasma sST2 levels in Chinese_cohort_1 (P<1E-5). Linear regression tests adjusted for age, sex, AD diagnosis, and population structure. Table 5. Candidate genetic variants in the IL1RL1 gene associated with plasma soluble ST2 levels after fine mapping (causal likelihood >0.001). Linear regression tests adjusted for age, sex, AD diagnosis, and population structure from fine mapping analysis. β, effect size; SE, standard error; SNP, single nucleotide polymorphism; sST2, soluble ST2. a Probability of a putative causative variant in plasma sST2 levels. Table 6. Demographic characteristics of the five Alzheimer's disease datasets for the meta-analysis. MMSE, Mini-Mental State Examination; SD, standard deviation. Table 7. Minor allele frequency of rs1921622 A allele in all participants, APOE-ε4 carriers, and APOE-ε4 non-carriers in six Alzheimer's disease datasets. AD, Alzheimer's disease; HC, healthy controls; OR, odds ratio. Bold letters indicate statistical significance at a cutoff of P<0.05. Table 8. Minor allele frequency of rs1921622 A allele in male APOE-ε4 carriers and female APOE-ε4 carriers in six Alzheimer's disease datasets. AD, Alzheimer's disease; HC, healthy controls; OR, odds ratio. Bold letters indicate statistical significance with a cutoff of P<0.05. Table 9: Oligonucleotide sequences.

[0013] definition The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), in either single-stranded or double-stranded form, and polymers thereof. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0014] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain. The segment may include regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons).

[0015] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., compounds that have an α carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0016] A variety of methods known in the art allow for the incorporation of unnatural amino acid derivatives or analogs into a polypeptide chain in a site-specific manner (see, e.g., WO 02 / 086075).

[0017] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0018] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the term encompasses amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0019] As used herein, "a composition that disrupts a genomic sequence that encompasses rs1921622" refers to any composition that includes one or more agents that can suppress or eliminate the transcription or translation of a genomic sequence, which can be achieved by direct deletion or modification of at least a portion of the genomic sequence (e.g., by genome editing techniques such as the clustered regularly interspaced short palindromic repeat (CRISPR) system) or by reduction or elimination of mRNA transcribed from the genomic sequence through the action of small inhibitory DNA or RNA molecules or other enzymes (e.g., antisense oligonucleotides, small inhibitory RNAs such as siRNA or shRNA, and ribozymes). Terms and phrases used in this disclosure and expressed similarly with respect to other loci (e.g., "other sST2-associated genetic variants listed in Tables 4 and 5") are defined functionally the same or similarly.

[0020] The term "targeted," when used in the context of describing an inhibitory oligonucleotide (such as a small inhibitory RNA or antisense oligonucleotide) or sgRNA in relation to a genomic sequence that the inhibitory oligonucleotide or gene editing system is used to negatively regulate, refers to sufficient sequence complementarity, e.g., at least 80%, 85%, 90%, 95%, or more percentage of nucleotide sequence complementarity based on Watson-Crick base pairing principles, between at least a portion of the oligonucleotide or sgRNA and the genomic sequence to allow specific hybridization of the sgRNA or oligonucleotide with the genomic sequence or its mRNA transcript, which subsequently results in cleavage of the genomic sequence at a predetermined location, or destruction of its mRNA transcript.

[0021] The term "recombinant," when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0022] A "promoter" is defined as an array of nucleic acid control sequences that direct the transcription of a polynucleotide sequence. As used herein, a promoter includes necessary polynucleotide sequences near the start site of transcription, such as, for example, a TATA element in the case of a polymerase II type promoter. A promoter also includes, optionally, distal enhancer or repressor elements, which may be located as far as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to the functional linkage of a polynucleotide expression control sequence (such as a promoter or an array of transcription factor binding sites) with a second polynucleotide sequence, where the expression control sequence directs the transcription of the polynucleotide sequence corresponding to the second sequence.

[0023] An "expression cassette" is a recombinantly or synthetically produced nucleic acid construct that has a series of specified polynucleotide elements that allow for transcription of a particular polynucleotide sequence in a host cell. An expression cassette can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette contains a polynucleotide that is to be transcribed and operably linked to a promoter.

[0024] The term "heterologous" used in the context of describing the relative positions of two elements refers to two elements, such as polynucleotide sequences (e.g., a promoter and an mRNA coding sequence or a protein / polypeptide coding sequence), or polypeptide sequences (e.g., two peptides as fusion partners in a fusion protein), that are not naturally found in the same relative positions. Thus, a "heterologous promoter" of a coding sequence refers to a promoter that is not naturally operably linked to that coding sequence. Similarly, a "heterologous polypeptide" or "heterologous polynucleotide" to a particular protein or its coding sequence is one that originates from a different source than that particular protein, or that originates from the same source but is not naturally linked to that particular protein or its coding sequence in the same manner. The fusion of one polypeptide (or its coding sequence) with a heterologous polypeptide (or polynucleotide sequence) does not result in a longer polypeptide or polynucleotide sequence that can be found in nature.

[0025] The phrase "specifically hybridizes" refers to the binding, duplexing, or hybridization of one polynucleotide sequence to another based on Watson-Crick nucleotide base pairing under stringent hybridization conditions when the polynucleotide sequences are present in a complex mixture (e.g., total cell or library DNA or library RNA). The phrase "stringent hybridization conditions" refers to conditions under which a nucleic acid (e.g., a polynucleotide probe) will hybridize to its target nucleotide sequence, typically in a complex mixture of nucleic acids, but not to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to nucleic acid hybridization can be found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are determined by the thermal melting point (T m ) is chosen to be about 5 to 10°C lower than T m is when 50% of the probes complementary to the target are at equilibrium (the target sequence is in excess, so T mHigh stringency hybridization is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which the probe hybridizes to the target sequence at equilibrium (at which 50% of the probes are occupied at equilibrium). Stringent conditions are conditions in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. In high stringency hybridization, a positive signal is at least 2 times, preferably 10 times, the background hybridization. Exemplary high stringency hybridization conditions include 50% formamide, 5xSSC, and 1% SDS incubated at 42°C, or 5xSSC and 1% SDS incubated at 65°C, and a wash in 0.2xSSC and 0.1% SDS at 65°C.

[0026] "Host cell" refers to a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells can be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast, insect, amphibian, or mammalian cells, such as CHO, HeLa, including cultured cells, explants, and in vivo cells.

[0027] The term "inhibiting" or "inhibition" as used herein refers to any detectable negative effect that an inhibitor has on a target biological process, such as expression of soluble ST2 (sST2) protein, formation of amyloid beta (Aβ) plaques in the brains of AD patients, cognitive decline in AD patients, protein phosphorylation, cell signaling, protein synthesis, cell proliferation, tumorigenicity, and metastatic potential. Typically, inhibition is reflected by at least a 10%, 20%, 30%, 40%, or 50% decrease in the target process (e.g., sST2 protein expression or Aβ plaque accumulation) or a decrease in any one of the downstream parameters mentioned above, when compared to a control not exposed to the inhibitor. Similarly, the terms "increasing" or "increase" are used to describe any detectable positive effect that an enhancer has on a target biological process, such as at least a 25%, 50%, 75%, 100%, or a 2-fold, 3-fold, 4-fold, 5-fold, or up to a 10-fold or 20-fold positive change when compared to a control in the absence of the enhancer. Conversely, the term "substantially unchanged" describes a condition in which the positive or negative change is 10%, 5%, 2%, 1%, or less.

[0028] The term "effective amount" as used herein refers to an amount sufficient to produce the intended effect for which the substance is administered. The effect may include a desired change in a biological process (e.g., a detectable decrease in sST2 expression, a reduction in Aβ plaque formation, or a delay in cognitive decline in AD patients), as well as prevention, correction, or inhibition to any detectable extent of the progression of symptoms of a disease / condition and associated complications. The exact amount that is "effective" to achieve the desired effect depends on the nature of the therapeutic agent, the mode of administration, and the purpose of the treatment, and can be ascertained by those skilled in the art using known techniques (e.g., Lieberman, "Pharmaceutical Dosage Forms" (Vol. 1-3, 1992); Lloyd, "The Art, Science and Technology of Pharmaceutical Compounding" (1999); and Pickar, "Dosage Calculations" (1999)).

[0029] As used herein, the term "treatment" or "treating" includes both therapeutic and preventative measures taken to address the presence of a disease or condition, or the risk of later developing such a disease or condition. This encompasses therapeutic or preventative measures to alleviate ongoing symptoms, inhibit or slow the progression of the disease, delay the onset of symptoms, or eliminate or reduce side effects caused by such disease or condition. Preventative measures in this context and its variations do not require 100% elimination of the occurrence of an event, rather they refer to inhibiting or reducing the likelihood or severity of such occurrence, or delaying such occurrence.

[0030] A "pharmacologically acceptable" or "pharmacologically acceptable" additive is a substance that is not biologically harmful or otherwise undesirable, i.e., the additive may be administered to an individual together with a bioactive agent without causing any undesirable biological effects. The additive does not interact in a deleterious manner with any of the components of the composition in which it is contained.

[0031] The term "additives" refers to any essentially accessory substance that may be present in the finished dosage form of the composition of the present invention. For example, the term "additives" includes vehicles, binders, disintegrants, fillers (diluents), lubricants, glidants (flow enhancers), compression aids, colorants, sweeteners, preservatives, suspending / dispersing agents, film formers / coating agents, flavors, and printing inks.

[0032] The term "consisting essentially of", when used in the context of describing a composition containing one or more active ingredients, refers to the fact that the composition does not contain other ingredients that possess any similar or related biological activity of the active ingredient(s) or that can enhance or suppress the activity, while one or more inactive ingredients, such as physiologically or pharma- ceutical acceptable additives, may be present in the composition. For example, a composition that consists essentially of an active agent(s) effective for disrupting rs1921622-containing genomic sequence in a subject or for suppressing mRNA transcribed from the genomic sequence is a composition that does not contain any other agent that may have any detectable positive or negative effect on the same target process or that may increase or decrease the occurrence or symptoms of disease in a subject to any measurable extent.

[0033] The term "about" refers to a range of + / - 10% of a given value. For example, "about 10" defines a range of 90% to 110% of 10, or 9 to 11.

[0034] I. Introduction In those earlier studies, the inventors discovered an anti-AD protective effect of the A allele of rs1921622, as shown by a correlation between increased soluble ST2 (sST2) protein serum levels and increased amyloid beta (Aβ) plaque formation in the brains of Alzheimer's disease (AD) patients, as well as a significant reduction in serum sST2 levels and Aβ plaque accumulation, particularly within certain segments of the general population (e.g., among female APOE-ε4 carriers). See, e.g., WO 2017 / 009750 and WO 2021 / 037027. The inventors further discovered that disruption of genomic sequences encompassing rs1921622 and / or other sST2-related gene variants listed in Tables 4 and 5 can serve as an effective means of directly suppressing sST2 protein expression and secretion in brain endothelial cells. Such disruption of the rs1921622 locus is therefore demonstrated to provide a therapeutic benefit in the treatment of patients suffering from Alzheimer's disease, as well as a prophylactic benefit in preventing or reducing the risk of Alzheimer's disease in individuals who have not yet been diagnosed with the disease.

[0035] II. General Recombinant Technology Basic texts disclosing general methods and techniques in the field of recombinant genetics include Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994).

[0036] For nucleic acids, sizes are given in either kilobases (kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

[0037] Oligonucleotides that are not commercially available can be chemically synthesized using an automated synthesizer, for example, according to the solid-phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett., 22:1859-1862 (1981), as described in Van Devanter et al., Nucleic Acids Res., 12:6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, for example, native acrylamide gel electrophoresis or anion-exchange HPLC, as described in Pearson and Reanier, J. Chrom., 255:137-149 (1983).

[0038] The sequences of genes of interest, polynucleotides encoding polypeptides of interest, and synthetic oligonucleotides can be verified after cloning or subcloning, for example, using the chain termination method of sequencing double-stranded templates as described in Wallace et al., Gene 16:21-26 (1981).

[0039] II. Compositions that disrupt genomic sequences Previous studies by the present inventors have illustrated the involvement of sST2 protein and the rs1921622 locus in the development of Alzheimer's disease. Their latest findings reveal that disrupting the genomic sequence encompassing the rs1921622 locus and other genomic sites listed in Tables 4 and 5 can reduce sST2 expression and Aβ plaque accumulation in the brain. This discovery has led to therapeutic and prophylactic uses of compositions that disrupt this genomic sequence, which may act at the level of the intact genomic sequence at or immediately surrounding the locus of rs1921622 and / or other candidate genomic sites listed in Tables 4 and 5, or may act at the level of the mRNA transcribed from the genomic sequence, to treat Alzheimer's disease in patients already diagnosed with the disease and who have a family history or known genetic background (e.g., carriers of one or two APOE ε4 alleles, point mutations in the genomic sequence encoding amyloid precursor protein (APP) on chromosome 21, point mutations in the genomic sequence encoding Presenilin 1 (PSEN1) on chromosome 14, and Presenilin 2 (PSEN2) on chromosome 1). 2:PSEN2) in individuals who have not yet been diagnosed but are at high risk for the disease. Various categories of possible agents acting via different mechanisms (e.g., by genome editing or mRNA suppression) are useful in formulating such compositions for the disruption of rs1921622-containing genomic sequences and are discussed below.

[0040] A. Antisense Oligonucleotides In some embodiments, the agent is an antisense oligonucleotide. Antisense oligonucleotides are relatively short nucleic acids that are complementary (or antisense) to the coding strand (sense strand) of RNA transcribed from a genomic sequence that includes the sST2 gene, e.g., rs1921622 or the 3'-untranslated region (3'-UTR) of the sST2 gene (Chr2:102, 959, 893-102, 961, 182). Antisense oligonucleotides are typically RNA-based, but may also be DNA-based. Antisense oligonucleotides are also often modified to increase stability.

[0041] Without being bound by theory, it is believed that binding of these relatively short oligonucleotides to mRNA induces stretches of double-stranded RNA that cause degradation of the message by endogenous ribonucleases. In addition, oligonucleotides may be specifically designed to bind near the promoter of a coding sequence; under these circumstances, the antisense oligonucleotides may additionally interfere with translation of the mRNA. Regardless of the specific mechanism by which antisense oligonucleotides function, their administration to cells or tissues allows for degradation of RNA transcribed from genomic sequences encompassing the sST2 gene, e.g., rs1921622 or the 3'UTR of the sST2 gene (Chr2:102, 959, 893-102, 961, 182). Thus, antisense oligonucleotides reduce the expression and / or activity of the product encoded from the genomic sequence.

[0042] The oligonucleotides can be DNA or RNA, or chimeric mixtures or derivatives, or modified versions thereof, single-stranded or double-stranded. The oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, the stability, hybridization, etc. of the molecule. Oligonucleotides may be tagged with other appended groups, such as peptides (e.g., for targeting host cell receptors), or agents that promote transport across cell membranes (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; WO 88 / 09810) or the blood-brain barrier (see, e.g., WO 89 / 10134), hybridization-triggered cleavage agents (see, e.g., Krol et al., 1988, BioTechniques 6:958-976), or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule.

[0043] The oligonucleotides of the present invention can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (e.g., commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res., vol. 16: 3209), methylphosphonate oligonucleotides can be prepared by using controlled pore glass polymer support (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA, vol. 85: 7448-7451), etc.

[0044] Several methods have been developed for delivering antisense DNA or RNA to cells: for example, antisense molecules can be directly injected into the target anatomical site, or modified antisense molecules designed to target the desired cells (e.g., antisense linked to a peptide or antibody that specifically binds to a receptor or antigen expressed on the target cell surface) can be administered systemically.

[0045] In certain cases, it may be difficult to achieve intracellular concentrations of antisense sufficient to suppress translation on endogenous mRNA. Therefore, another approach utilizes recombinant DNA constructs in which antisense oligonucleotides are placed under the control of a strong pol III or pol II promoter. For example, vectors can be introduced in vivo so that they are taken up by cells and direct the transcription of antisense RNA. Such vectors can remain episomal or be integrated into chromosomes, so long as they can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or other known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian cells, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), and the like. Any type of plasmid, cosmid, YAC, or viral vector can be used to prepare recombinant DNA constructs that can be directly introduced into the target tissue site. Alternatively, viral vectors can be used which selectively infect the desired tissue, in which case administration can be accomplished by another route (eg, systemically).

[0046] B. Small interfering RNA In some embodiments, the agent is a small interfering RNA (siRNA or RNAi) molecule. The RNAi construct comprises a double-stranded RNA that can specifically block expression of a target gene. "RNA interference" or "RNAi" is a term originally applied to the phenomenon in which double-stranded RNA (dsRNA) blocks gene expression in a specific and post-transcriptional manner. RNAi provides a useful method of inhibiting gene expression in vitro or in vivo. The RNAi construct can comprise small interfering RNA (siRNA), short hairpin RNA (shRNA), and other RNA species that can be cleaved in vivo to form siRNA. The RNAi construct herein also includes an expression vector ("RNAi expression vector") capable of generating transcripts that form dsRNA or hairpin RNA in cells and / or transcripts that can generate siRNA in vivo.

[0047] The RNAi expression vector expresses (transcribes) an RNA that produces an siRNA portion in the cell in which the construct is expressed. Such a vector comprises a transcription unit that includes an assembly of (1) a genetic element(s) that has a regulatory role in gene expression (e.g., an assembly of a promoter, an operator, or an enhancer), (2) a "coding" sequence that is transcribed to produce a double-stranded RNA (two RNA portions that anneal to form siRNA in the cell, or a single hairpin RNA that can be processed into siRNA), and (3) an appropriate transcription start sequence and transcription stop sequence. The choice of promoter and other regulatory elements generally varies according to the intended host cell.

[0048] The RNAi construct contains a nucleotide sequence that hybridizes to at least a portion of the nucleotide sequence of the mRNA transcript of the gene to be inhibited (i.e., the RNA transcribed from the genomic sequence including the 3'UTR of the rs1921622 or sST2 gene) under physiological conditions of the cell. The double-stranded RNA only needs to be sufficiently similar to natural RNA to have the ability to mediate RNAi. In this way, the present invention has the advantage of being able to tolerate sequence mutations that may be expected due to genetic mutations, strain polymorphism, or evolutionary divergence. The number of nucleotide mismatches tolerated between the target sequence and the RNAi construct sequence is 1 in 5 base pairs or less, or 1 in 10 base pairs or less, or 1 in 20 base pairs or less, or 1 in 50 base pairs or less. Mismatches in the center of the siRNA duplex are most critical and can essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3' end of the siRNA strand that is complementary to the target RNA do not significantly contribute to the specificity of target recognition.

[0049] The production of RNAi constructs can be performed by chemical synthesis methods or recombinant nucleic acid technology. Endogenous RNA polymerase of treated cells can mediate transcription in vivo or cloned RNA polymerase can be used for in vitro transcription. RNAi constructs can include modifications to either the phosphate-sugar backbone or nucleosides, for example, to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and / or alter other pharmacokinetic properties. For example, the phosphodiester linkages of natural RNA can be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications of the RNA structure can be tailored to allow specific gene inhibition while avoiding a general response to dsRNA. Similarly, bases can be modified to block the activity of adenosine deaminase. RNAi constructs can be produced enzymatically or by partial / total organic synthesis, and any modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis.

[0050] In certain embodiments, the subject RNAi constructs are "small interfering RNAs" or "siRNAs." These nucleic acids are approximately 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length, e.g., lengths corresponding to fragments produced by nuclease "dicing" of longer double-stranded RNA. The siRNA is understood to recruit a nuclease complex and guide the complex to the target mRNA by pairing with a specific sequence. The target mRNA is then degraded by the nuclease in the protein complex. In certain embodiments, the 21-23 nucleotide siRNA molecule comprises a 3' hydroxyl group.

[0051] In certain embodiments, the RNAi construct is in the form of a short hairpin structure (called shRNA). shRNA can be exogenously synthesized or formed by transcription from an RNA polymerase III promoter in vivo. Examples of the creation and use of such hairpin RNA for gene silencing in mammalian cells are described, for example, in Paddison et al., Genes Dev, 2002, vol. 16:948-58; McCaffrey et al., Nature, 2002, vol. 418:38-9; Yu et al., Proc Natl Acad Sci USA, 2002, vol. 99:6047-52). Often, such shRNAs are engineered in cells or animals to ensure continuous and stable suppression of the desired gene. It is known in the art that siRNAs can be generated by processing hairpin RNAs in cells.

[0052] Plasmids can be used to deliver double-stranded RNA, for example as transcription products.In such embodiments, plasmids are designed to contain "coding sequences" on each of the sense and antisense strands of the RNAi construct.The coding sequences can be the same sequence, for example adjacent to an inverted promoter, or can be two separate sequences, each under the transcriptional control of a separate promoter.After the coding sequence is transcribed, the complementary RNA is transcribed base-paired to form double-stranded RNA.

[0053] C. Ribozyme In some embodiments, the agent is a ribozyme. Ribozyme molecules designed to catalytically cleave mRNA transcripts are also used to destroy and prevent downstream effects of mRNA (see, e.g., WO 90 / 11364; Sarver et al., 1990, Science, vol. 247:1222-1225, and U.S. Pat. No. 5,093,246). Ribozymes that cleave mRNAs at site-specific recognition sequences can be used to destroy specific mRNAs, while the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at positions dictated by adjacent regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has a sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art and is described in further detail in Haseloff and Gerlach, 1988, Nature 334:585-591.

[0054] Ribozymes for use in the present invention may also include RNA endoribonucleases (hereinafter "Cech-type ribozymes"), such as those naturally occurring in Tetrahymena thermophila (IVS, or known as L-19 IVS RNA), and have been described extensively by Thomas Cech and coworkers (Zaug et al., 1984, Science 224:574-578; Zaug and Cech, 1986, Science 231:470-475; Zaug et al., 1986, Nature 324:429-433; WO 88 / 04300; Been and Cech, 1986, Cell 47:207-216). Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.The present invention encompasses Cech-type ribozymes which target eight base pair active site sequences.

[0055] As with antisense approaches, ribozymes can be constructed from modified oligonucleotides (e.g., for improved stability, targeting, etc.) and delivered to cells in vitro or in vivo. A preferred delivery method involves using DNA constructs "encoding" ribozymes under the control of a strong constitutive pol III or pol II promoter, so that the transfected cell produces sufficient quantities of ribozyme to destroy the target mRNA and inhibit its effect. Ribozymes, unlike antisense molecules, are catalytic and therefore require lower intracellular concentrations for efficiency.

[0056] Currently, there are two basic types of DNA enzymes, both of which were identified by Santoro and Joyce (see, e.g., US Pat. No. 6,110,462).10-23 DNA enzymes contain a loop structure that connects two arms. The two arms provide specificity by recognizing a particular target nucleic acid sequence, while the loop structure provides catalytic function under physiological conditions.

[0057] Briefly, to design an ideal DNA enzyme that specifically recognizes and cleaves a target nucleic acid, one skilled in the art must first identify a unique target sequence. This can be done using the same approach outlined for antisense oligonucleotides. Preferably, the unique or substantial sequence is G / C rich, approximately 18-22 nucleotides. High G / C content helps ensure a stronger interaction between the DNA enzyme and the target sequence.

[0058] When synthesizing a DNA enzyme, the specific antisense recognition sequence that targets the enzyme to a message is split so that it comprises two arms of the DNA enzyme, and the DNA enzyme loop is positioned between the two specific arms.

[0059] Methods for making and administering DNA enzymes can be found, for example, in U.S. Patent No. 6,110,462. Similarly, methods for delivering DNA ribozymes in vitro or in vivo include methods for delivering RNA ribozymes, as outlined in detail above. In addition, those skilled in the art will recognize that, like antisense oligonucleotides, DNA enzymes can be modified as desired to improve stability and improve resistance to degradation.

[0060] D. Genome Editing Inhibition of sST2 protein expression can be achieved by disruption of the gene locus rs1921622 and / or other genomic sites listed in Tables 4 and 5, or gene sequences encompassing the 3'-UTR of the sST2 gene / transcript. One effective means of targeted gene cleavage is the CRISPR system.

[0061] The term CRISPR, an abbreviation for clustered regularly interspaced short palindromic repeats, was first coined in reference to segments of prokaryotic DNA that contain short repeated base sequences first found in bacteria and archaea. In palindromic repeats, the sequence of nucleotides is the same in both directions. Each repeat is followed by a short segment of spacer DNA from a previous exposure to foreign DNA (e.g., viral DNA). Small clusters of Cas (CRISPR-associated) genes are located next to the CRISPR sequence. Later, it was recognized that the CRISPR / Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements, especially those of viral origin, thereby providing a form of adaptive immunity. RNA carrying the spacer sequence helps the Cas (CRISPR-associated) protein recognize and cleave the exogenous DNA. Other RNA-guided Cas proteins cleave foreign RNA. CRISPR is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaeal genomes, and recently the CRISPR / Cas system has been adapted for use in targeted gene editing in eukaryotic cells. See, e.g., Ledford (2016) Nature 531(7593):156-9.

[0062] A simple version of the CRISPR / Cas system, CRISPR / Cas9, has been modified to edit genomes. By delivering Cas9 nuclease complexed with one or more synthetic guide RNAs (gRNAs) to a cell, typically by transfecting the cell with one or more expression vectors encoding Cas9 nuclease and gRNA(s), the genome of the cell can be cleaved at one or more preselected locations, allowing the target gene (e.g., the genomic sequence carrying rs1921622) to be removed and / or replaced with a new sequence.

[0063] In this case, an expression vector (e.g., a viral vector) carrying a coding sequence for one or more gRNAs specific for the rs1921622-containing genomic sequence and / or other genomic sequences containing the sites listed in Tables 4 and 5 can be introduced into a cell (e.g., an endothelial cell or an endothelial progenitor cell) in which the endogenous rs1921622-containing genomic sequence and / or other genomic sequences containing the sites listed in Tables 4 and 5 are to be knocked out. The same expression vector also optionally carries a coding sequence for a CRISPR / Cas9 nuclease or equivalent. Alternatively, a separate expression vector can be used to introduce a CRISPR / Cas9 nuclease coding sequence for its expression in the target cell. In some cases, two or more (e.g., two) different gRNAs are used to ensure the removal and / or replacement of the target genomic sequence (e.g., one that includes the rs1921622 locus and / or other genomic sites listed in Tables 4 and 5).

[0064] Additional gene editing systems that can be used to practice the invention include TALENs (transcription activator-like effector nucleases), ZFNs (zinc finger nucleases), and base editing, as well as newly developed technologies such as homing endonucleases and meganucleases (MegN) (which target and cleave DNA sequences) and prime editing (which produces RNA templates for genetic modification).

[0065] III. Pharmaceutical Compositions and Administration The present invention also provides pharmaceutical or physiological compositions comprising an effective amount of one or more agents useful in the methods of the present invention, both in prophylactic and therapeutic applications. Such pharmaceutical or physiological compositions also comprise one or more pharma- ceutical or physiologically acceptable additives or carriers. By way of example, one exemplary composition of the present invention comprises or consists essentially of one or more expression vectors encoding a CRISPR system (e.g., Cas9 nuclease or equivalent, and one or two sgRNAs) and one or more physiologically acceptable additives or carriers. Another exemplary composition of the present invention comprises or consists essentially of one or more expression vectors encoding one or more inhibitory oligonucleotides (e.g., small inhibitory RNA molecules, or antisense DNA or RNA oligonucleotides) and one or more physiologically acceptable additives or carriers. The pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. Suitable formulations for use in the present invention can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Company, Philadelphia, PA, 17th Edition (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0066] The pharmaceutical composition of the present invention can be administered by various routes, for example, oral, subcutaneous, transdermal, intramuscular, intravenous or intracranial.The preferred route of administering pharmaceutical composition is local delivery to the relevant organ or tissue for the target disease in recipient with a predetermined daily dose.The appropriate dose can be administered in a single dose per day, or as divided doses presented at appropriate intervals, for example, as divided doses twice, three times, four times or more times per day.

[0067] Inert and pharmaceutically acceptable carriers are also used to prepare pharmaceutical compositions containing one or more active agents of the present invention.Typically, pharmaceutical carriers can be either solid or liquid.Solid preparations include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories.Solid carriers can be one or more substances that can also act as diluents, flavorings, solubilizers, lubricants, suspending agents, binders, or tablets, disintegrating agents, and they can also be encapsulating materials.

[0068] In powders, the carrier is typically a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0069] For preparing pharmaceutical compositions in the form of suppositories, a low melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and solidify.

[0070] The powders and tablets preferably contain about 5% to about 70% by weight of the active ingredient. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like.

[0071] The pharmaceutical composition may include a formulation of active agent(s) with an encapsulating material as a carrier to provide a capsule in which one or more agents are surrounded by the carrier (with or without other carriers), such that the carrier is thus associated with the agent(s). Similarly, cachets may also be included. Tablets, powders, cachets, and capsules may be used as solid dosage forms suitable for oral administration.

[0072] Liquid pharmaceutical compositions include, for example, solutions suitable for oral or parenteral administration, suspensions, and emulsions suitable for oral administration. Sterile aqueous solutions of active ingredient(s) or active ingredient(s) in solvents including water, buffered water, saline, PBS, ethanol, or propylene glycol are examples of liquid compositions suitable for parenteral administration. The compositions may contain pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, isotonicity adjusting agents, wetting agents, detergents, etc., as required to approximate physiological conditions.

[0073] Sterile solutions can be prepared by dissolving the active ingredient(s) in a desired solvent system and then sterilizing the resulting solution through a membrane filter, or alternatively by dissolving the sterile component(s) in a previously sterilized solvent under sterile conditions. The resulting aqueous solution can be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier before administration. The pH of the preparation is typically 3-11, more preferably 5-9, most preferably 7-8.

[0074] Pharmaceutical compositions containing one or more active agents can be administered for preventive and / or therapeutic treatment. In therapeutic applications, the compositions are administered to patients already suffering from Alzheimer's disease in an amount sufficient to prevent, cure, reverse, or at least partially slow or stop the symptoms of the disease and its complications, such as the onset, progression, duration, and severity of the disease. An amount adequate to achieve this is defined as a "therapeutically effective dose". Amounts effective for this use depend on the severity of the disease, the weight and general condition of the patient, and the nature of the active agent(s).

[0075] In prophylactic applications, pharmaceutical compositions containing one or more active agents are administered to patients susceptible to or otherwise at risk of developing Alzheimer's disease in an amount sufficient to delay or prevent the onset of symptoms. Such an amount is defined as a "prophylactically effective dose." In this use, the exact amount of active agent(s) again depends on the patient's health and weight, as well as the nature of the active agent(s).

[0076] Single or multiple administrations of the composition can be administered, with the dose level and pattern being selected by the treating physician. In any case, the pharmaceutical formulation should provide a sufficient quantity of the agent(s) to effectively suppress serum levels of sST2 protein and Aβ plaque formation in the patient, either therapeutically or prophylactically.

[0077] IV. Therapeutic Applications of Nucleic Acids A variety of conditions can be treated by therapeutic approaches that include introducing into a cell a nucleic acid encoding one or more agents that disrupt the rs1921622-containing genomic sequence and / or other genomic sequences containing the sites listed in Tables 4 and 5, or inhibiting the mRNAs encoded by the genomic sequences (such as antisense or miRNAs or Cas9 nucleases and sgRNAs), such that the coding sequence is transcribed and a polypeptide or oligonucleotide agent is produced within the cell. For a discussion of the application of gene therapy to the treatment of inherited and acquired diseases, see Miller, Nature 357:455-460 (1992); and Mulligan, Science 260:926-932 (1993).

[0078] A. Vectors for Gene Delivery For delivery to cells or organisms, polynucleotides encoding one or more active agents can be incorporated into vectors. Examples of vectors used for such purposes include expression plasmids capable of directing the expression of nucleic acids in target cells. In another example, the vector is a viral vector system in which the polynucleotide is integrated into a viral genome capable of transfecting target cells. In one embodiment, the encoding polynucleotide can be operably linked to expression and control sequences capable of directing the expression of a polypeptide or oligonucleotide in a desired target host cell. In this way, expression of the polypeptide inhibitor or oligonucleotide inhibitor can be achieved under appropriate conditions in the target cell.

[0079] B. Gene Delivery Systems Viral vector systems useful for expressing polypeptides or oligonucleotides that disrupt genomic sequences that include rs1921622 and / or other genomic sites listed in Tables 4 and 5 include, for example, naturally occurring or recombinant viral vector systems. Depending on the particular application, suitable viral vectors include replication-competent, replication-deficient, and conditionally replicating viral vectors. For example, viral vectors can be derived from the genomes of human or bovine adenoviruses, vaccinia viruses, herpes viruses, adeno-associated viruses, minute virus of mice (MVM), HIV, Sindbis viruses, and retroviruses (including, but not limited to, Rous sarcoma virus and lentivirus), and MoMLV. Typically, a coding sequence of interest (e.g., encoding a polypeptide or oligonucleotide active agent of the present invention) is inserted into such a vector, typically packaging the genetic construct with accompanying viral DNA, followed by infection of susceptible host cells to express the coding sequence of interest.

[0080] As used herein, "gene delivery system" refers to any means for delivering a polynucleotide sequence of interest to a target cell. In some embodiments of the invention, the nucleic acid is conjugated to a cell receptor ligand for promoting uptake (e.g., invagination of coated pits and internalization of endosomes) via a suitable linking moiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92 / 06180) or by an ultrasound-microbubble delivery system (Lan HY et al., J. Am Soc. Nephrol. 14:1535-1548). For example, the nucleic acid can be linked via a polylysine moiety to asialo-oromucosid, a ligand for the asialoglycoprotein receptor of hepatocytes.

[0081] Similarly, the viral envelope used to package the genetic construct containing the nucleic acid of interest can be modified by adding receptor-specific receptor ligands or antibodies to allow receptor-mediated endocytosis into certain cells (see, e.g., WO 93 / 20221, WO 93 / 14188, and WO 94 / 06923). In some embodiments of the invention, the DNA construct of the invention is linked to a viral protein, such as an adenovirus particle, to drive endocytosis (Curiel et al., Proc. Natl. Acad. Sci. USA, 88:8850-8854 (1991)). In other embodiments, active agents of the invention may include microtubule inhibitors (WO / 9406922), synthetic peptides that mimic influenza virus hemagglutinin (Plank et al., J. Biol. Chem. 269:12918-12924 (1994)), and nuclear localization signals such as SV40 T antigen (WO93 / 19768).

[0082] Retroviral vectors may also be useful for introducing the coding sequence of the polypeptide or oligonucleotide active agents of the invention into target cells or tissues. Retroviral vectors are generated by genetically engineering retroviruses. The viral genome of retroviruses is RNA. Upon infection, this genomic RNA is reverse transcribed into a DNA copy that is integrated into the chromosomal DNA of the transduced cell with high stability and efficiency. The integrated DNA copy is called a provirus and is inherited by daughter cells like any other gene. Wild-type retroviral genomes and proviral DNA are flanked by two long terminal repeat (LTR) sequences and contain three genes: the gag gene, the pol gene, and the env gene. The gag gene encodes the internal structural (nucleocapsid) protein; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase); and the env gene encodes the viral envelope glycoprotein. The 5'LTR and 3'LTR serve to facilitate transcription and polyadenylation of the virion RNA. Adjacent to the 5'LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site) (see Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed.), pp. 155-173 (1983); Mann et al., Cell 33: 153-159 (1983); Cone and Mulligan, Proceedings of the National Academy of Sciences, USA 81: 6349-6353 (1984)).

[0083] The design of retroviral vectors is well known to those skilled in the art. Briefly, when sequences required for encapsidation (or packaging of retroviral RNA into infectious virions) are deleted from the viral genome, the result is a cis-acting defect that prevents encapsidation of genomic RNA. However, the resulting mutant is still capable of directing the synthesis of all virion proteins. Retroviral genomes with deleted sequences and cell lines containing mutant genomes stably integrated into chromosomes are well known in the art and are used to construct retroviral vectors. The preparation and use of retroviral vectors are described in, for example, European Patent Application Publication No. EPA 0178220; U.S. Pat. No. 4,405,712; Gilboa, Biotechniques, vol. 4:504-512 (1986); Mann et al., Cell, vol. 33:153-159 (1983); Cone and Mulligan, Proc. Natl. Acad. Sci. USA, vol. 8, pp. 1111-1115 (1983); 1:6349-6353 (1984); Eglitis et al., Biotechniques, vol. 6:608-614 (1988); Miller et al., Biotechniques, vol. 7:981-990 (1989); Miller (1992), supra; Mulligan (1993), supra; and WO 92 / 07943.

[0084] Retroviral vector particles are prepared by recombinantly inserting a desired nucleotide sequence into a retroviral vector and packaging the vector with retroviral capsid proteins using a packaging cell line. The resulting retroviral vector particles are unable to replicate in the host cell, but can integrate into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, the patient can produce, for example, a polypeptide or polynucleotide active agent useful in the methods of the present invention, thus restoring target cells (e.g., brain endothelial cells) to normal phenotype.

[0085] Packaging cell lines used to prepare retroviral vector particles are typically recombinant mammalian tissue culture cell lines that produce the necessary viral structural proteins required for packaging but are incapable of producing infectious virions. On the other hand, the defective retroviral vectors used lack these structural genes but encode the remaining proteins required for packaging. To prepare packaging cell lines, an infectious clone of the desired retrovirus can be constructed in which the packaging site has been deleted. Cells containing this construct express all structural viral proteins but are incapable of packaging the introduced DNA. Alternatively, packaging cell lines can be generated by transforming the cell line with one or more expression plasmids encoding the appropriate core and envelope proteins. In these cells, the gag, pol, and env genes can be derived from the same or different retroviruses.

[0086] Several packaging cell lines suitable for the present invention are also available in the prior art. Examples of these cell lines include Crip, GPE86, PA317 and PG13 (see Miller et al., J. Virol. 65:2220-2224 (1991)). Other examples of packaging cell lines are described in Cone and Mulligan, Proceedings of the National Academy of Sciences, USA 81:6349-6353 (1984); Danos and Mulligan, Proceedings of the National Academy of Sciences, USA 85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller (1990), supra.

[0087] Packaging cell lines capable of producing retroviral vector particles with chimeric envelope proteins may be used, or amphotropic or xenotropic envelope proteins, such as those produced by the PA317 and GPX packaging cell lines, may be used to package retroviral vectors.

[0088] C. Pharmaceutical Preparations When used for pharmaceutical purposes, nucleic acids encoding polypeptide or oligonucleotide active agents will generally be formulated in a suitable buffer, which can be any pharma- ceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of skill in the art, such as those described in Good et al., Biochemistry, 5:467 (1966).

[0089] The composition may additionally comprise a stabilizer, enhancer, or other pharma- ceutically acceptable carrier or vehicle. A pharma-ceutically acceptable carrier may contain, for example, a physiologically acceptable compound that acts to stabilize the nucleic acid of the present invention and any related vector. Physiologically acceptable compounds may include carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or additives. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Company, Philadelphia, PA, 17th Edition (1985).

[0090] D. Administration of Formulations Formulations containing polynucleotide sequences encoding polypeptide or oligonucleotide active agents can be delivered to target tissues or organs using any delivery method known to those of skill in the art. In some embodiments of the invention, the encoding polynucleotide sequences are formulated for subcutaneous, intramuscular, intravenous, or intraperitoneal injection, or for oral ingestion or topical application.

[0091] Formulations containing the nucleic acid of interest are typically administered directly to cells. Cells may be provided as part of a tissue, such as red blood cells as part of the circulatory system, or as isolated cells, such as in tissue culture. Cells may be provided in vivo, ex vivo, or in vitro.

[0092] The formulation can be introduced into the tissue of interest in vivo or ex vivo by various methods. In some embodiments of the present invention, the nucleic acid of interest is introduced into cells by methods such as microinjection, calcium phosphate precipitation, liposome fusion, ultrasound, electroporation, or biolistics. In further embodiments, for example, when the target cell is brain endothelial cell intracranial injection, the nucleic acid is suitably taken up directly by the target tissue or organ associated with the disease or condition being treated.

[0093] In some embodiments of the invention, the nucleic acid of interest is administered ex vivo to cells or tissues explanted from the patient and then returned to the patient. Examples of ex vivo administration of therapeutic gene constructs include Nolta et al., Proc. Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23(1):46-65 (1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg. 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1):402-6 (1996).

[0094] The effective dosage of the formulation will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, and other drugs administered. Thus, the therapeutic dosage must be titrated to optimize safety and efficacy. In determining the effective amount of vector to be administered, the physician should evaluate the particular nucleic acid used, the disease state being diagnosed; the age, weight, and general condition of the patient, circulating plasma levels, vector toxicity, disease progression, and the generation of anti-vector antibodies. The size of the administration may also be determined by the presence, nature, and extent of any adverse side effects associated with the administration of a particular vector. For example, an amount of 1-1000 mg, 10-200 mg, or 20-100 mg of antisense oligonucleotide may be delivered to a patient by intravenous injection at a frequency of weekly, biweekly, or monthly administrations for a period of at least 1-3 months or longer. As another example, 5×10 5Cells (e.g., hCMEC / D3 cells) are transfected with 0.5 µg-50 µg; 1-20 µg; or 2-10 µg of vector carrying a gene encoding Cas9 together with a pair of sgRNAs. For CRISPR editing targeting the sST2 genomic region in human patients, doses of lipid nanoparticles carrying sgRNA and mRNA encoding Cas9 range from 0.01-2 mg / kg body weight; 0.02-1.0 mg / kg body weight; 0.05-0.5 mg / kg body weight; or 0.10-0.30 mg / kg body weight, delivered by 1-3 iv injections over 1-4 weeks.

[0095] V.Kit The present invention also provides kits for treating Alzheimer's disease or reducing the risk of Alzheimer's disease in an individual in need thereof according to the methods of the present invention. The kits typically include a container containing (1) a pharmaceutical composition having an effective amount of one or more active agents capable of disrupting a genomic sequence encompassing rs1921622 and / or other genomic sites listed in Tables 4 and 5 and / or suppressing mRNA transcribed from the genomic sequence, and (2) informational material including instructions on how to dispense the pharmaceutical composition, including descriptions of the types of patients that may be treated (e.g., human patients suffering from or at high risk for Alzheimer's disease), schedules (e.g., dose and frequency), and routes of administration. In some cases, the kit includes two or more containers to provide a plurality of pharmaceutical compositions, each including an effective amount of at least one active agent, such as a vector or vector encoding a component of the CRISPR system (e.g., Cas9 nuclease, or equivalent, and one or more sgRNAs) or encoding an siRNA, microRNA, miniRNA, lncRNA, or antisense oligonucleotide that targets a genomic sequence that includes rs1921622, including the 3'-UTR of the sST2 gene / transcript. Optionally, the kit may further include one or more additional containers, each containing at least one agent useful for sequencing at least a portion of the individual's genome, particularly a genomic sequence that includes the locus rs1921622, and / or other genomic sites listed in Tables 4 and 5. EXAMPLES

[0096] The following examples are offered by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results.

[0097] overview Altered levels of circulating proteins are associated with Alzheimer's disease (AD), while their pathogenic role in AD is unclear. Here, we report that soluble ST2 (sST2), a soluble decoy receptor for IL-33 / ST2 signaling, is involved in AD pathogenesis. We found that elevated sST2 levels are associated with more severe neurodegeneration and Aβ pathological lesions in AD patients, and can lead to exacerbated Aβ accumulation and reduced Aβ-microglia colocalization in amyloidosis mouse models. Our genome-wide association study identified genetic variants in IL1RL1 (the gene encoding sST2) that are associated with reduced sST2 levels. Deletion of the rs1921622 variant by CRISPR / Cas9 genome editing, typically located in the enhancer element of IL1RL1, reduces the expression and secretion of sST2 in brain endothelial cells; that reduced sST2 levels are associated with a reduced risk of developing AD and less severe AD-related endophenotypes in female APOE-ε4 carriers. Immunohistochemical and single-nucleus transcriptome analysis of AD brains further suggests that rs1921622 / lower sST2 levels exert a protective effect by reducing Aβ accumulation by enhancing microglial activation and their colocalization with Aβ in female APOE-ε4 carriers. Furthermore, disruption of the 3'-untranslated region (UTR) of the sST2 transcript, either by deleting the genomic region of the sST2 3'-UTR or by administering an antisense oligomer (ASO) targeting the sST2 3'-UTR, also reduced sST2 gene and protein levels and ameliorated AD-related pathological changes in amyloidosis mouse models. Collectively, these findings demonstrate that sST2 is a novel disease-causing factor in AD and that reducing sST2 levels is a potential intervention strategy against the disease.

[0098] Introduction Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of death in the elderly. 1It is characterized by memory decline and cognitive impairment. Its pathological hallmarks include extracellular accumulation of amyloid beta (Aβ) peptides forming Aβ plaques, and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein (P-tau). 2 In AD, Aβ acts as a risk factor-associated molecular pattern that triggers the activation of microglia, stimulating them to migrate toward and clear Aβ plaques via phagocytosis. 3、4 Although the pathophysiological mechanisms underlying AD remain unclear, large-scale genome-wide association studies (GWAS) have revealed over 40 AD-related genes that are associated with microglial function (e.g., APOE, TREM2, BIN1, and CD33), suggesting that microglia play an important role in the pathogenesis of AD. 5~7 Of note, the strongest known risk factor for sporadic AD, APOE-ε4, 5 , which has been suggested to have a major impact on Aβ accumulation in AD. 8、9 The APOE gene contributes to Aβ clearance through distinct cellular mechanisms; specifically, it promotes the formation of microglial clusters around Aβ and subsequently degrades Aβ plaques. 6、10、11 Postmortem single-cell / nuclear transcriptome analysis of AD brains revealed a microglial subpopulation with increased APOE expression in AD 12、13 Moreover, in a mouse model of amyloidosis, APOE is required to induce specific microglial subpopulations that interact with Aβ plaques. 6 Thus, microglial dysfunction is an essential causative factor in AD.

[0099] In addition to genetic factors, changes in the brain environment and secreted signaling proteins in the circulation may disrupt microglial activity and contribute to AD pathogenesis. 14、15For example, levels of soluble TREM2 (sTREM2), a proteolytic cleavage product of the extracellular domain-containing microglial receptor TREM2, are increased in the cerebrospinal fluid (CSF) of AD patients. 16~18 In a transgenic mouse model of amyloidosis, injection of sTREM2 attenuates Aβ accumulation and rescues memory deficits by enhancing microglial interactions with Aβ and therefore subsequent phagocytosis of Aβ. 19、20 Furthermore, expression of VCAM1 (vascular cell adhesion molecule 1), a soluble form cleaved from the full-length protein on endothelial cells, is elevated in the plasma and CSF of AD patients. 21、22 This suggests that microglia mediate reduced hippocampal neurogenesis and proinflammatory responses during aging. 23 .

[0100] Other secreted soluble receptors that may contribute to AD pathogenesis include soluble cytokine receptors that contain membrane-bound cytokine receptor ectodomains that function as decoy receptors and attenuate cytokine-mediated signaling. 24、25 In particular, soluble ST2 (sST2) is a secreted isoform of the receptor for the cytokine IL-33 (i.e., ST2L [full-length ST2]) that is produced by alternative promoter activation (Figure 1). 26 ST2L is expressed by brain microglia. 27 Activation of IL-33 / ST2 signaling reduces Aβ accumulation and increases microglial Aβ clearance in a transgenic mouse model of amyloidosis 28、29 On the other hand, sST2 can act as a decoy receptor for IL-33 and effectively inhibit IL-33 / ST2 signaling. 30、31 Altered plasma levels of sST2 have been observed in several inflammatory diseases, cancer, and cardiovascular diseases and are a promising biomarker for these diseases in the periphery. 32~35 Of note, recent evidence also indicates that sST2 levels are elevated in the blood of patients with mild cognitive impairment or AD. 28、36Nevertheless, the regulatory mechanisms underlying the dysregulation of sST2 and whether sST2 has a pathological role in AD remain unclear.

[0101] Therefore, in this study, we investigated the role of sST2 in AD pathogenesis and the regulation of sST2 expression and AD-related pathological changes by genetic factors. We showed that elevated sST2 levels impair microglial function and exacerbate Aβ accumulation in a mouse model of amyloidosis. Furthermore, we identified single nucleotide polymorphisms (SNPs) in IL1RL1 (the gene encoding sST2 and ST2L) that were associated with lower plasma and CSF sST2 levels. Typically, genotype-expression association analysis and CRISPR / Cas9-based genome editing demonstrated that one of the sST2-related SNPs, rs1921622, downregulates gene expression and secretion of sST2 in human endothelial cells, and female APOE-ε4 carriers carrying this variant have a lower risk of AD and a lower severity of AD-related endophenotypes. Subsequent single-nucleus transcriptome profiling revealed that both the presence of rs1921622 and reduced sST2 levels were associated with enhanced microglial activation in female patients with AD carrying APOE-ε4, indicating that sST2 levels regulate the microglial activation state in AD. Consistently, reduction of sST2 levels by disruption of the sST2 3'-untranslated region (UTR) using antisense oligomers (ASOs) or genome editing attenuated AD-related pathological changes in amyloidosis mouse models. Collectively, our findings indicate that sST2 is a soluble factor in the brain environment that plays a disease-causing role in the pathogenesis of AD, which may be a novel target for AD therapy, and that reduction of sST2 levels may be a potential intervention strategy for the disease.

[0102] result sST2 levels are associated with Alzheimer's disease and its pathological changes To explore how sST2 is involved in the pathogenesis of AD, we investigated the association of sST2 levels with AD and its associated endophenotypes. Specifically, we measured plasma sST2 levels in Chinese patients with AD and HC recruited in Hong Kong (hereafter "Chinese_cohort_1"; n=345 HC and n=345 AD patients; Table 2). We then performed linear regression analysis of plasma sST2 levels with AD, adjusting for age, sex, and population structure. The results show that plasma sST2 levels were significantly higher in AD patients than in HCs (P<0.01; Fig. 2a). Furthermore, it has been shown that sST2 is a well-known biomarker for cardiovascular disease (CVD), and 35、37 Given that plasma sST2 levels were elevated in patients with cardiac disease in our cohort (P<0.05; Fig. 3a), we investigated changes in plasma sST2 levels in AD in a subcohort of participants without a clinical history of CVD (referred to as the "non-CVD cohort"). Consistent with our findings in the overall cohort, patients with AD also had significantly higher plasma sST2 levels than HCs (P<0.01; Fig. 3b), suggesting that increased plasma sST2 levels in AD are independent of CVD status. In addition, plasma sST2 levels correlated with the AD-related endophenotypes examined, namely gray matter atrophy, as well as neurodegeneration and AD (i.e., P-tau181 and [NfL] neurofilament light polypeptide) in the overall cohort (Fig. 2b-d) and non-CVD cohorts (Fig. 3c-e). 38、39 ) were positively correlated with the levels of plasma biomarkers corresponding to AD. These observations collectively suggest that plasma sST2 levels are associated with AD and related endophenotypes.

[0103] Furthermore, we showed that plasma and CSF sST2 levels were positively correlated within the same individual (P<0.0001; n=107 participants from the ADRC cohort). 40; Fig. 2e). Therefore, we subsequently investigated the association of CSF sST2 levels with AD. Consistent with the regulation of plasma sST2 levels in AD (Fig. 2a), in the UK Brain Bank Network cohort (hereafter "UKBBN cohort"; n=11 HCs and n=75 AD patients; Table 3), CSF sST2 levels were significantly higher in AD patients than in HCs (P<0.01; Fig. 2e). Notably, in AD patients, CSF sST2 levels were positively correlated with Aβ plaque burden in the frontal cortex (P<0.05; Fig. 2g, Fig. 2h). These findings indicate that circulating sST2 levels in both blood and CSF are increased in AD and associated with disease progression.

[0104] Increased brain sST2 levels exacerbate Aβ accumulation and impair microglial Aβ clearance To investigate the causal relationship between increased sST2 levels and AD progression, we investigated the effects of increased sST2 levels on AD-related pathological changes in 5XFAD mice, a transgenic mouse model of amyloidosis. We found that activation of IL-33 / ST2 signaling attenuated the pathological features of AD, particularly Aβ accumulation. 28、29 Consistent with our previous findings showing that continuous intracerebroventricular administration of recombinant murine sST2 to 3-month-old 5XFAD mice for 28 days (Fig. 4a) significantly increased Aβ plaque burden in the cortical region compared to mice treated with vehicle control (P<0.05; Fig. 4b-c). In particular, the fibrillar morphology of Aβ plaques (i.e., X-34 without a dense core) was significantly increased in the cortical region compared to mice treated with vehicle control (P<0.05; Fig. 4b-c). + Diffusion fibrils 41 ) and compact morphology of Aβ plaques (i.e., X-34 with 4G8 halo + The amount of both Aβ plaques (i.e., dense cores) was increased in sST2-treated mice (P<0.05; Fig. 4d, Fig. 4e, and Fig. 5). However, sST2 administration reduced the amount of less toxic inactive Aβ plaques (i.e., X-34 without 4G8 labeling) in 5XFAD mice. + The load on the high density core was not affected.

[0105] Considering that microglia play an important role in Aβ clearance, we next investigated how sST2 regulates the interaction of microglia with Aβ plaques. In 5XFAD mice, sST2 injection increased the total number of microglia and the clustering of microglia around Aβ plaques in the cortical region (P<0.05; Fig. 6). However, the coverage of Aβ plaques by microglia in the cortical region was lower in sST2-injected 5XFAD mice than in vehicle control-injected 5XFAD mice (Fig. 4f, g), suggesting that sST2 reduces the formation of a microglial barrier around Aβ plaques. Furthermore, flow cytometry analysis showed that sST2 reduces the formation of a microglial barrier around Aβ plaques. + We showed that sST2 administration reduced the Aβ phagocytosis capacity of microglia in 5XFAD mice, as indicated by a decrease in the percentage of microglia (i.e., 23.57% of CD11b in the sST2-treated group). + and Methoxy-X04 + (32.79% in cells vs. control; P < 0.01; Fig. 4h, Fig. 4I, and Fig. 7). Thus, these findings collectively suggest that increasing sST2 levels in the brain exacerbates Aβ accumulation and impairs microglial Aβ clearance capacity.

[0106] sST2 levels are associated with genetic variants in IL1RL1 To understand how sST2 is regulated during the development of AD, we investigated how various factors contribute to changes in sST2 levels. Consistent with previous studies, we found that plasma sST2 levels were significantly associated with both age and sex in Chinese and European ancestry populations. 42 This was significantly lower in women than in men (P<0.001 in Chinese_Cohort_1, INTERVAL cohort, and LonGenity cohort). 42), and increased significantly with age (P<0.001 in both cohorts; Fig. 8a-c). Furthermore, CSF sST2 levels were also associated with both age and sex in the Japanese cohort (both P<0.01; Fig. 8d). 43 However, when we examined the contribution of age, sex, and other factors to the variance in sST2 levels, age and sex together accounted for only 6.92% and 13.01% of the variance in plasma sST2 levels in the Chinese_cohort_1 and CSF sST2 levels in the Japanese cohort, respectively (Fig. 8e, f), suggesting that other factors modulate such changes.

[0107] Association studies in populations of European ancestry have identified various genetic variants in the IL1RL1 gene that are associated with plasma sST2 levels. 44 , suggesting that genetic factors contribute to the regulation of sST2 levels. Nevertheless, it is possible that these identified SNPs may form a haplotype structure in this gene region. 45 Considering the above, these SNPs may simply be inherited together with the causal variant. Therefore, to identify the key genetic regulator(s) of sST2, we performed a GWAS of plasma sST2 levels using our whole genome sequencing dataset of Chinese_cohort_1. 46 Accordingly, we identified 575 genetic variants that were significantly associated with plasma sST2 levels (P<1E-5) and found that these variants accounted for 54.86% of the variants (Figure 9a, Figure 10a, and Table 4). Specifically, among these 575 variants, 79 genetic variants in or near IL1RL1 forming a haplotype were most strongly associated with sST2 levels (Figure 9b and Figure 10b). Subsequent fine mapping analysis by CAVIAR 47In the Chinese cohort, the sentinel variant rs1921622(G / A) was identified as a putative causal variant (99.9% probability) modulating plasma sST2 levels (Fig. 10c and Table 5). In Chinese_cohort_1, the rs1921622 A allele was associated with a 20% decrease in plasma sST2 levels in an allele-dose-dependent manner (P<0.001; Fig. 9c). Furthermore, CSF sST2 levels were consistently lower in carriers of the rs1921622 A allele than in noncarriers in the UKBBN cohort (P<0.05; Fig. 9d). Notably, rs1921622 alone accounted for 18.04% and 18.29% of the variance in plasma and CSF sST2 levels, respectively, which is much larger than the contributions of age and sex. Thus, our fine mapping analysis using whole genome sequencing data identified rs1921622 as a key genetic factor regulating plasma and CSF levels of sST2.

[0108] The rs1921622 locus regulates sST2 expression in human brain endothelial cells Because rs1921622 is a non-coding variant located in an intronic region of ST2L downstream of the region encoding sST2, we investigated whether it regulates the expression of sST2 and ST2L transcripts. Genotype-Expression Association Analysis Using the Genotype-Tissue Expression (GTEx) Dataset 48、49 showed that compared with non-carriers, individuals carrying the rs1921622 A allele exhibited significantly lower transcript levels of sST2, but not ST2L, in multiple brain regions (e.g., nucleus accumbens, amygdala, hippocampus, and frontal cortex; P < 0.05; Fig. ​ Fig.11a andTable1). Furthermore, analysis of our previously published human frontal cortex single nucleus RNA sequencing (snRNA-seq) dataset from the UKBBN cohort revealed that rs1921622 A allele-carriers exhibited significantly lower transcript levels of sST2, but not ST2L, in multiple brain regions (e.g., nucleus accumbens, amygdala, hippocampus, and frontal cortex; P < 0.05; Fig.11a and Table 1). 50revealed that sST2 is exclusively expressed by endothelial cells (i.e., CLDN5-expressing cells) in the frontal cortex (Fig. 11b, Fig. 11c). In addition, cell type-specific genotype-expression association analysis showed that compared with individuals carrying the major allele, individuals carrying the rs1921622 A allele had lower endothelial cell sST2 transcript levels (P<0.01) and fewer sST2-expressing endothelial cells in an allele dose-dependent manner (P<0.05; Fig. 11d). These results collectively indicate that the rs1921622 variant is associated with reduced sST2 expression in human brain endothelial cells.

[0109] To investigate the role of rs1921622 in the decreased sST2 expression in endothelial cells, we investigated whether rs1921622 and the surrounding genomic region (Figure 11e) regulate sST2 transcription. Considering that non-coding variants generally regulate gene expression by functioning as enhancer elements, 51 We first examined enhancer activity at the rs1921622 locus in hCMEC / D3 cells, a human brain microvascular endothelial cell line. Administration of the cytokine IL-33 (a ligand for ST2L) increased the expression and secretion of sST2 in hCMEC / D3 cells (both P<0.001; Fig. 12a, b). Furthermore, chromatin immunoprecipitation (ChIP) assays showed that these IL-33-treated hCMEC / D3 cells exhibited increased occupancy of active enhancer histone marks (i.e., H3K27ac) at the rs1921622 locus, accompanied by high levels of H3K4me3 histone modification (indicating an active promoter region) at the sST2 promoter region (both P<0.05; Fig. 11f and c). Therefore, these results indicate that rs1921622 is located in a potential enhancer element of the sST2 gene.

[0110] To further demonstrate that the region carrying rs1921622 contributes to the regulation of sST2 expression, we used a CRISPR / Cas9-based approach to delete the genomic region carrying the rs1921622 locus in hCMEC / D3 cells. Thus, we generated two different hCMEC / D3 cell lines with biallelic 38bp and 67bp deletions (designated Δ38bp and Δ67bp, respectively) encompassing the rs1921622 locus (FIG. 11e and FIG. 13). Notably, loss of 38bp or 67bp adjacent to the rs1921622 locus significantly reduced sST2 transcription levels in hCMEC / D3 cells (P<0.001; FIG. 11g), and concomitantly abolished sST2 protein secretion (P<0.001; FIG. 11h). Collectively, these results indicate that the rs1921622-containing region has a putative regulatory role as an enhancer element that controls sST2 expression in endothelial cells.

[0111] The rs1921622 A allele protects against Alzheimer's disease in APOE-ε4 carriers Given our findings that circulating sST2 levels are associated with AD risk and related endophenotypes, we investigated whether the rs1921622 A allele is associated with reduced AD risk. Therefore, we used the following six independent AD datasets as discovery cohorts: Chinese_cohort_1 dataset; Chinese_cohort_2 WGS and array datasets. 52 as well as three public datasets from populations of European ancestry (i.e., Late-Onset Alzheimer's Disease (LOAD) 53 , Alzheimer's Disease Center [ADC] 54、55, and Alzheimer's Disease Neuroimaging Initiative (ADNI) datasets (N = 5,436 HCs and N = 5,556 AD patients; Tables 2 and 6). Genotype-phenotype association analysis showed that the rs1921622 A allele was weakly associated with AD risk in all subjects (odds ratio [OR] = 0.945, Han and Eskin random-effects model [RE2] = 8.90E-2; Table 7). Nevertheless, we observed a significant genetic interaction between the rs1921622 A allele and APOE-ε4 (P=0.0046): the rs1921622 A allele exerted a significant protective effect against AD risk in APOE-ε4 carriers (OR=0.860, RE2=3.18E-2; Fig. 14a and Table 7), and this protective effect was absent in APOE-ε4 non-carriers (OR=0.964, RE2=5.22E-1; Fig. 15). Specifically, the rs1921622 A allele had a more pronounced protective effect against AD risk in female APOE-ε4 carriers than in male APOE-ε4 carriers (OR=0.717, RE2=1.77E-4; Fig. 14b and Table 8). Furthermore, with regard to AD-associated endophenotypes, the protective effect of the rs1921622 variant was not evident in APOE-ε4 carriers with AD overall, but was significant in female APOE-ε4 carriers with AD, including delayed onset of dementia, better cognitive scores, less atrophy of the entorhinal cortex, and less neurodegeneration (as indicated by plasma levels of P-tau181 and NfL) (all P<0.05; Figures 14c-e and 16-18).

[0112] To confirm the protective effect of the rs1921622 A allele against AD, we conducted an independent replication cohort (i.e., participants with low Aβ deposition in the brain (i.e., Aβ + We investigated AD-related endophenotypes in the Australian Imaging, Biomarkers and Lifestyle cohort (AIBL; n = 190) with positron emission tomography-confirmed AD.56 Aβ carrying the APOE-ε4 allele + Among subjects, consistent with findings from the discovery cohort, the presence of the rs1921622 A allele was associated with improved cognitive performance as indicated by AIBL Preclinical Alzheimer Cognitive Composite (AIBLPACC) scores and cognitive subprocesses including episodic recall and recognition (all P<0.05; Fig. S14f). Furthermore, the rs1921622 A allele was associated with improved overall Aβ function as indicated by associations of the rs1921622 A allele with endophenotypes including attention processing ability and gray matter volume. + Women with Aβ were more likely to be APOE-ε4 carriers than + APOE-ε4 carriers had a strong protective effect against AD (both P<0.05; Figure 19). Importantly, Aβ levels in 183 individuals from this cohort who were followed for 10 years for gray matter volume were significantly higher in the control group than in the control group. + In the subgroup of APOE-ε4 carriers, progression of gray matter atrophy was slower in carriers of the rs1921622 A allele than in noncarriers (P < 0.05; Fig. S14g). Thus, these results support the independent role of Aβ + To examine the protective effect of the rs1921622 A allele against cognitive decline and gray matter atrophy among APOE-ε4 carriers in our cohort.

[0113] Carriage of the rs1921622 A allele in female APOE-ε4 carriers with Alzheimer's disease is associated with reduced Aβ deposition and increased microglial activation Given our findings that the rs1921622 A allele exerts an AD protective effect in APOE-ε4 carriers, specifically in women, we subsequently investigated whether this protective variant modulates Aβ deposition in postmortem human brain. Among AD patients, female but not male APOE-ε4 carriers exhibited greater Aβ deposition in the frontal cortex than APOE-ε4 noncarriers (P<0.05; Fig. 20a, Fig. 20b). However, when further stratifying according to rs1921622 genotype, female APOE-ε4 carriers carrying the rs1921622 A allele exhibited significantly less Aβ deposition than those without this allele (P<0.05; Fig. 21a, Fig. 21b, and Fig. 20c), suggesting that the rs1921622 A allele attenuates the effect of APOE-ε4 on Aβ-related pathological changes. Immunohistochemical analysis revealed that female APOE-ε4 carriers with AD had a lower microglial coverage of Aβ plaques (i.e., Iba-1) than APOE-ε4 noncarriers. + We found that those carrying the rs1921622 A allele exhibited a decreased proportion of Aβ colocalized with microglia (P<0.05; Fig. 20d, e), whereas those carrying the rs1921622 A allele exhibited a decreased Iba-1 + There was a significant increase in colocalization of microglia with Aβ plaques (P<0.05; Fig. S21c, d). Thus, these results suggest that the rs1921622 A allele is associated with enhanced microglia-Aβ interactions and reduced Aβ pathology in female APOE-ε4 carriers with AD.

[0114] Next, to investigate the regulatory effect of the rs1921622 variant on microglial activity at the molecular level, we investigated the frontal cortex of female APOE-ε4 carriers with AD from the UKBBN cohort. 50We performed an association analysis with a microglial snRNA-seq dataset in . There was a strong negative correlation between the effect of the rs1921622 A allele and the effect of CSF sST2 levels on microglial gene expression (P<0.0001; Figure 22), supporting the idea that the protective rs1921622 variant exerts its regulatory effect on microglia via regulating CSF sST2 levels. Specifically, we identified 1,696 microglial genes that were significantly associated with rs1921622 genotype: there were 445 and 1,251 genes whose expression was up-regulated and down-regulated, respectively, in patients who carried the rs1921622 A allele compared to patients who did not carry the allele (false discovery rate [FDR] adjusted P<0.05; Figure 21e). Furthermore, gene ontology (GO) analysis showed that among the microglial genes associated with the rs1921622 A allele, those with upregulated expression were associated with leukocyte migration (FDR-adjusted P = 1.2E-4) and innate immune responses (FDR-adjusted P = 5.4E-3), while those with downregulated expression were mainly involved in protein refolding (FDR-adjusted P = 8.1E-3) or mRNA splicing (FDR-adjusted P = 7.7E-2) (Figure 21f).

[0115] Recent studies of microglia in mouse and human brains have revealed a subset of “microglial activation genes,” including CD74, APOE, and TREM2, whose expression levels are upregulated in association with AD. 12、13 and is involved in Aβ phagocytosis by microglia 6、57、58 Therefore, we investigated whether these genes are associated with the rs1921622 A allele. Interestingly, among female APOE-ε4 carriers with AD, the rs1921622 A allele was associated with increased expression of these microglial activation genes in an allele-dosage-dependent manner, specifically increased transcript levels of CD74, APOE, and TREM2 in microglia, as well as TMEM163. +The rs1921622 A allele was associated with an increased proportion of microglia (Figure 21g). In contrast, the rs1921622 A allele was also associated with decreased expression of homeostatic genes, including SRGAP2, TMEM119, and P2RY12, which generally points to a less responsive microglial state. 6、57 (FIG. 21g). Thus, these results collectively indicate that the rs1921622 A allele promotes the transition of microglia to a more activated state in female APOE-ε4 carriers with AD.

[0116] Disruption of the 3'-untranslated region of sST2 reduces sST2 levels and alleviates amyloid-related pathology in 5XFAD mice To establish a more generalized method for reducing sST2 levels and improving AD-related genotypes, we explored alternative solutions to manipulate sST2 levels without affecting the expression of ST2L. sST2 and ST2L share most of the coding sequence, while the 3'-untranslated region (UTR) of the sST2 transcript is unique (Fig. 23a). Therefore, the 3'UTR region of sST2 may be a perfect target for manipulating sST2 levels. By generating a mouse model with deletion of sST2, we found that deletion of the 3'UTR region of sST2 significantly reduced serum sST2 levels (Fig. 23b). Furthermore, deletion of sST2 3'UTR also reduced cortical Aβ levels of both soluble and insoluble content in 6-month-old amyloidogenic mouse model, 5XFAD mice (Fig. 23c, d). Deletion of the 3'UTR of sST2 also led to a reduction in amyloid plaque burden (Figures 23e-h). In summary, the 3'UTR region may be a good target for reducing sST2 levels and AD-related pathology.

[0117] Antisense oligonucleotides could target the 3'UTR region, cause RNase H-mediated degradation of mRNA, and finally reduce the mRNA level of the target gene (Fig. 24a), so they could serve as a good translational tool to manipulate sST2 levels. We performed a systematic screening of all potential ASOs that could specifically target the sST2 3'UTR region in mouse fibroblasts NIH3-3 and human umbilical vein endothelial cells (HUVEC) (Fig. 24b, Fig. 25a, Table 9). We performed a second round of screening with the top 13 msST2-ASOs and the top 15 efficient hsST2-ASOs to examine sST2 transcript levels. Most ASOs showed more than 50% reduction in sST2 transcripts (Fig. 24c, Fig. 25b). Furthermore, intravenous injection of mouse sST2 ASO reduced serum sST2 levels in C57 mice (Fig. 24d) and also reduced Aβ burden in 5XFAD mice (Fig. 24e, f), providing a possible hint for future clinical translation.

[0118] Consideration GWAS studies suggest that most AD risk genes are enriched in microglia and that changes in their expression regulate microglial phagocytic function. 6 Nevertheless, emerging studies suggest that soluble factors in the brain environment also regulate microglial activity and disease-associated pathological changes. 20、23Here, we report that expression of the endothelial gene IL1RL1, encoding sST2 (a secreted decoy receptor for IL-33 / ST2 signaling), is regulated by the genetic variant rs1921622; this variant exerts a protective effect against AD via modulation of plasma and CSF sST2 levels, which in turn regulates microglial phenotype and Aβ accumulation in AD. Elevated sST2 levels are associated with higher Aβ deposition in AD patients, exacerbate Aβ plaque formation and impair microglial clearance of Aβ in a mouse model of amyloidosis, whereas we show that rs1921622 downregulates sST2 expression; this variant protects against neurodegeneration, cognitive decline, and Aβ accumulation in female APOE-ε4 carriers, who tend to be at higher risk for AD and more severe AD-related pathological changes. 59 Analysis of postmortem human brain suggests that the protective effect of rs1921622 is mediated by modulation of microglia-Aβ plaque interactions. Thus, our results collectively indicate that sST2, a soluble protein secreted by endothelial cells, regulates microglial activity and that alterations in sST2 levels in the brain environment impair the microglial ability to clear Aβ, thus modulating the risk and pathological changes of AD associated with APOE-ε4.

[0119] sST2 contains only the extracellular domain of ST2L and is transcribed independently. 26 , an effective decoy receptor for IL-33 / ST2 signaling. Considering that IL-33 / ST2 signaling has an essential regulatory role for microglial activity involved in tissue repair, Aβ clearance, and synapse phagocytosis, 28、29、60、61 Increased brain sST2 levels likely affect microglial function and AD-related pathological changes by blocking the binding of IL-33 to ST2L on microglia. In AD, Aβ accumulation induces microglia to migrate toward Aβ plaques, prolonging the process of forming a barrier surrounding them and initiating phagocytic clearance.62 As AD progresses, these microglial functions become impaired, leading to the accumulation of Aβ plaques. 63 In this study, sST2 injection in a mouse model of amyloidosis disrupted the interaction of microglia with Aβ and the subsequent phagocytosis of Aβ (Figure 4). This finding suggests that in these mice, IL-33 administration activates IL-33 / ST2 signaling and initiates microglial chemotaxis to Aβ plaques, subsequently enhancing Aβ phagocytosis. 28、29 This is consistent with our previous observation that, among patients with AD carrying APOE-ε4, those carrying rs1921622 (those with lower sST2 levels in the brain environment) exhibited enhanced microglia-Aβ plaque interactions and smaller Aβ plaque areas than non-carriers, further supporting the pathological role of sST2 in microglia in AD. Thus, perturbation of endogenous IL-33 / ST2 signaling by sST2 may result in impaired microglial chemotaxis, barrier formation, and Aβ uptake, all of which subsequently contribute to the pathogenesis of AD.

[0120] What is the regulatory mechanism of sST2? Our analysis in Chinese and European ancestry populations confirms previous studies that sST2 levels are associated with age, sex, and genetic variants. 42、44, further revealing that genetic components play a dominant role in regulating sST2 levels, accounting for 54.86% of the variance in sST2 levels. Notably, our GWAS and fine mapping analysis identified rs1921622 as a putative causal variant associated with sST2 (causal probability = 99.99%); our ChIP assay and CRISPR / Cas9 editing experiments verified that the rs1921622-containing region is an enhancer element regulating sST2 expression in endothelial cells. These results collectively suggest that rs1921622 is an important genetic regulator of sST2. Therefore, future investigations into epigenetic events at the rs1921622 locus may help elucidate the regulatory mechanism of sST2. Indeed, recent studies have demonstrated that activation of TNFα-mediated NF-κB signaling can induce the expression and release of sST2 from endothelial cells. 64、65 , and suggests that inhibition of NF-κB signaling abolishes sST2 production. 66 Considering that NF-κB is an essential transcription factor regulating gene expression in endothelial cells, 67 It will be interesting to investigate whether NF-κB is a candidate transcription factor involved in the rs1921622-mediated regulation of sST2 expression in endothelial cells.

[0121] In this study, single-nucleus transcriptome profiling of postmortem human brain shows that sST2 is mainly expressed in brain endothelial cells (Figure 11). Dysregulation of sST2 expression in the brain exacerbates AD-associated pathological changes, particularly impairing the phagocytic capacity of microglia. Thus, our results demonstrate a novel pathogenic role of cerebrovascular structures in AD, which mediate microglial activity in the brain via sST2, a secreted soluble factor. Indeed, apart from Aβ- and tau-associated pathological changes, neurovascular dysfunction occurs early in AD and is involved in its pathogenesis. 68 Brain transcriptome profiling revealed numerous dysregulated genes in AD endothelial cells associated with angiogenesis and antigen presentation. 50Furthermore, APOE-ε4 leads to leakage of blood-borne proteins such as thrombin and plasmin. 69、70 , leading to synapse loss 71、72 , which have recently been shown to exacerbate the breakdown of the blood-brain barrier. The exact pathological function of the cerebral vasculature that causes AD remains unclear, whereas in the peripheral system, vasculature-secreted soluble cytokines and chemokines (e.g., CXCL1) commonly regulate the activation and migration of immune cells, thereby mediating immune responses in tissues. 73 Similarly, emerging research suggests that the cerebral vasculature not only provides nutrients and removes metabolic products, but also serves as an important source of soluble inflammatory proteins, such as IL-1β, IL-6, IL-8, TNFα, TGFβ, and MCP-1. 74、75 Thus, in addition to sST2, other soluble factor-based crosstalk between the vasculature and other cell types may occur in the brain. Therefore, identifying those components and mediators of such crosstalk may expand our understanding of the role of the cerebral vasculature and provide insight into novel pathological mechanisms of AD.

[0122] Our genetic analysis demonstrated that rs1921622 exerted a protective effect against AD in APOE-ε4 carriers, suggesting a potential interaction between IL-33 / ST2 signaling and ApoE. ApoE, a major component of lipoproteins and Aβ plaques, has various functions including cholesterol transport, lipid metabolism, and Aβ clearance. 6、76 In the brain, ApoE is primarily produced by astrocytes, and its expression is upregulated in microglia under neuropathological conditions, including AD. 12、77 Single-cell RNA sequencing of amyloidosis mouse models reveals that microglial subpopulations transition from a homeostatic state to an activated state termed "disease-associated microglia" or "activation-responsive microglia" 6、57 This activated state is associated with pattern recognition, lipid metabolism, and lysosomal pathways, and is involved in the detection, phagocytosis, and degradation of lysosomal proteins.78~81 Microglial activation genes, including APOE, AXL, TREM2, and CD74, are key regulators of the phagocytic process. 12、13、57、58 In contrast, disruption of APOE and TREM2 function in the brain abolishes the induction of this microglial activation state, locking microglia in a homeostatic state and resulting in a lower Aβ phagocytosis capacity. 6、57 Thus, ApoE-mediated microglial activation may have a protective role against AD and may be required for Aβ clearance and brain homeostasis. In this study, snRNA-seq analysis of human postmortem brains revealed that in AD patients carrying APOE-ε4, the presence of rs1921622 regulates the transition of microglia from a homeostatic to an activated state, characterized by increased expression of the previously mentioned microglial activation genes (Figure 21). Thus, IL-33 / ST2 signaling and ApoE may converge to regulate the expression of these specific genes in microglia, thereby regulating the activation state and Aβ clearance capacity of microglia. Interestingly, in the periphery, IL-33 administration suppresses ApoE in atherosclerosis. - / - Improves the formation of macrophage foam cells (lipid-containing macrophages that cause atherosclerosis) and the development of atherosclerotic plaques in models 82 It will therefore be interesting to determine whether modulation of IL-33 / ST2 signaling reduces the detrimental effects of APOE-ε4 on Aβ accumulation through the regulation of lipid metabolism in AD. Indeed, ABCA1 (ATP-binding cassette transporter A1) and ABCG1 (ATP-binding cassette transporter G1), responsible for cholesterol efflux, are among the microglial genes whose expression is upregulated by rs1921622. 83 Furthermore, these two genes play essential roles in ApoE lipidation. 84、85 , which is involved in Aβ degradation 86 , impaired in APOE-ε4 carriers 87、88These two genes may be important components shared by both ApoE- and IL-33 / ST2-mediated signaling in AD, whereas how they are involved in the crosstalk between these two signaling pathways awaits further investigation.

[0123] Our findings collectively suggest that sST2 is a promising therapeutic target for AD. First, because sST2 is primarily expressed by endothelial cells, this may allow cell-type-specific manipulation of sST2 expression, which may not require crossing the blood-brain barrier. Second, sST2 levels are elevated in patients with mild cognitive impairment or early AD. 28、36 , suggesting the applicability of sST2 in early intervention strategies. Third, our findings that deletion of the rs1921622 locus can be performed with high efficacy in human brain endothelial cell lines may be a feasible method to specifically silence sST2 expression and secretion without disrupting ST2L activity; this suggests that the epigenetic and transcriptional regulation of sST2 is distinct from that of ST2L. 89 , because rs1921622 only regulates the expression of sST2, not ST2L. Fourth, as we have shown that rs1921622 is a common AD-associated variant, manipulation of sST2 targeting this genetic variant could be developed for specific subgroups of patients with high sST2 levels (e.g., female patients carrying APOE-ε4 but not rs1921622 A allele account for 6.2%-12.2% of AD patients), enabling patient stratification and precision medicine. In addition, sST2 also has a potential pathogenic role in atherosclerosis and sepsis. 82、90 Because it is a well-known biomarker for CVD 35、37 , Such genome-based manipulation targeting sST2 may also be beneficial for the treatment of such peripheral diseases.

[0124] Nevertheless, the function and regulation of sST2 in AD remains to be elucidated. First, we demonstrated that increased brain sST2 levels contribute to the pathogenesis of AD. 91 Whether plasma sST2, which constitutes the major pool of sST2 produced by peripheral endothelial cells, plays a pathological role in AD remains unclear. The positive correlation between plasma sST2 levels and CSF sST2 levels suggests that sST2 may be blood-brain barrier permeable. Therefore, further investigations are needed to confirm whether peripheral sST2 can penetrate the brain parenchyma and contribute to the pathological changes associated with the disease. Indeed, recent studies have shown that some angiotensin receptor blockers, such as valsartan, can reduce peripheral sST2 levels in patients with heart failure. 92 It is interesting to examine whether these drugs can also regulate peripheral sST2 levels in AD patients and ameliorate disease-related pathological changes. Second, our association analysis in multiple cohorts consistently demonstrated that rs1921622 protects against AD in APOE-ε4 carriers, especially in women. This variant regulates sST2 expression in both men and women, while the reason for this gender-specific protective effect in AD remains unclear. Notably, sex hormones, including estrogen and testosterone, have different regulatory effects on IL-33 / ST2 signaling. 93、94 It will therefore be interesting to examine whether such signaling has different activities and functions in men and women with AD. Finally, since IL-33, ST2L, and sST2 all contribute to IL-33 / ST2 signaling, in addition to increased sST2 levels, dysregulation of IL-33 and / or ST2L may also contribute to AD. Recent studies have shown that genetic variants in the IL33 gene are associated with AD risk. 95 , and that brain IL-33 transcript and protein levels are lower in AD than in physiological conditions. 36、95Therefore, future integrated studies of sST2, IL-33, and ST2L in AD at both genomic and genetic levels may further elucidate how impaired IL-33 / ST2 signaling contributes to AD pathogenesis.

[0125] In summary, we have discovered an alternative pathogenic mechanism of AD involving microglial dysfunction mediated by sST2, a soluble protein in the brain. Dysregulation of endothelial cell secreted sST2 leads to increased plasma and CSF levels of sST2, impairing Aβ clearance by microglia, thereby exacerbating Aβ accumulation in AD. Furthermore, we found that the AD-protective gene variant rs1921622, which downregulates sST2 expression, attenuates APOE-ε4-associated risk and pathological changes of AD through modulation of microglial signaling. Consistently, reduction of sST2 expression and protein levels by 3'-UTR-targeting ASO or genome editing ameliorated AD-associated pathological changes. Thus, a better understanding of how circulating levels of sST2, a novel biomarker and potential drug target for AD, are genetically regulated can aid in the design of intervention strategies and clinical trials.

[0126] Methods and Materials Recruiting participants for Chinese cohort 1 A total of 690 Hong Kong participants aged 60 years or older, including 345 AD patients and 345 HCs, who visited the Specialized Outpatient Department at the Prince of Wales Hospital of the Chinese University of Hong Kong from April 2013 to February 2018, were recruited. The clinical diagnosis of AD was established based on the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5). 96All participants underwent a medical history evaluation, clinical evaluation, cognitive and functional evaluation using the Montreal Cognitive Assessment (MoCA) test, and neuroimaging evaluation using MRI. 97、98 Participants with any significant neurological disease other than AD or psychiatric disorders were excluded. Age, sex, grade, medical history, and history of CVD (i.e., cardiac disease, hypertension, diabetes mellitus, and hyperlipidemia) were recorded. DNA and plasma samples were prepared from whole blood samples and stored at -80°C until use. Brain imaging data were retrieved from 192 participants (n=77 AD patients, n=115 HC) from Prince of Wales Hospital using T1-weighted MRI. Raw imaging files were anonymized and processed by AccuBrain® IV1.2 (BrainNow Medical Technology Ltd, Hong Kong, China) for analysis of gray matter volume. A subcohort consisting only of participants without a history of CVD was also selected from Chinese_cohort_1 ("non-CVD cohort"; n=86 AD patients, n=97 HC).

[0127] The study was approved by the Prince of Wales Hospital, the Chinese University of Hong Kong, and the Hong Kong University of Science and Technology. All participants provided written informed consent for both study enrollment and sample collection.

[0128] Extraction of DNA and plasma from human blood samples We collected whole blood samples (3 mL) from participants into K3EDTA tubes (VACUETTE). We centrifuged the samples at 2,000×g for 15 min to separate cell pellets and plasma. Plasma was collected, aliquoted, and stored at −80° C. until use. We sent the cell pellets to the Centre for PanorOmic Sciences (Genomics and Bioinformatics Cores, University of Hong Kong, Hong Kong, China) for genomic DNA extraction using the QIAsymphony DSP DNA Midi Kit (QIAGEN) on the QIAsymphony SP platform (QIAGEN). Genomic DNA was eluted with water or elution buffer ATE (QIAGEN) and stored at 4° C. We determined DNA concentration by BioDrop μLITE+ (BioDrop).

[0129] UKBBN Dataset The following samples were obtained from the MRC UKBBN (Bristol Brain Bank): CSF samples, frontal cortex sections, frozen frontal cortex tissue, and genomic DNA samples (Table 3). For initial sample selection from the UKBBN dataset, subjects with other neurodegenerative diseases, vascular diseases, toxic conditions, infectious diseases, prions, inflammatory diseases, structural brain disorders, metabolic / nutritional diseases, trauma, delirium, genetic disorders (e.g., Down's syndrome), or other systemic diseases were excluded. For CSF samples, samples with a postmortem period of 30 hours or less were selected, resulting in a total of 86 participants (n=75 AD patients, n=11 HC). In addition, snRNA-seq data from frozen frontal cortex samples from the UKBBN (n=12 AD patients, n=9 HC) were obtained from our previously published dataset. 50 .

[0130] Other cohorts and data for association studies The following data were obtained for the replication study: (i) genomic, demographic, and clinical data from Chinese_cohort_2, participants were recruited as previously described.52 (ii) genomic, demographic, and clinical data from the LOAD Family Study 53 (iii) genomic, demographic, and clinical data from the NIA ADC cohort 54、55 (iv) genomic, demographic, clinical and brain imaging data from the ADNI cohort; (v) genomic, demographic, and transcriptomic data from the GTEx dataset. 48、49 (vi) plasma biomarker data, CSF biomarker data, demographic data, and clinical data from the Stanford Alzheimer's Disease Research Center (ADRC) cohort. 40 (vii) Plasma biomarker data and demographic data of the INTERVAL and LonGenity cohorts retrieved from an online database (https: / / twc-stanford.shinyapps.io / aging_plasma_proteome / ). 42 (viii) CSF biomarkers and demographic data from the Japanese cohort 43 and (ix) genomic, demographic, clinical, and brain imaging data from the AIBL cohort. 56 .

[0131] Measurement of protein levels in human samples and cell lines Plasma levels of sST2 in 613 participants from Chinese_cohort_1 (n = 277 AD patients, n = 336 HCs), CSF levels of sST2 in 86 participants from the UKBBN cohort (n = 75 AD patients, n = 11 HCs), and levels of sST2 secreted by hCMEC / D3 cells were measured using the Human ST2 / IL-33 R Quantikine ELISA Kit (DST200; R&D Systems). Plasma levels of NfL (n = 135 AD patients, n = 116 HC) and P-tau181 (n = 145 AD patients, n = 126 HC) in participants from Chinese_Cohort_1 were measured by Quanterix Accelerator Lab (Boston, MA, USA) using the Quanterix NF-light SIMOA Assay Advantage Kit (103186) and P-Tau 181 Advantage V2 Kit (103714), respectively.

[0132] Whole-genome sequencing and SNP arrays for genotyping DNA samples from 427 participants from Chinese_cohort_1 (n=233 AD patients, n=194 HC) were submitted to Novogene (Shenzhen, China) for library construction and whole genome sequencing. Samples were sequenced on an Illumina HiSeq X platform (average depth: 5x) and individual genotypes were analyzed using the GotCloud pipeline. 46263 genomic DNA samples from Chinese_cohort_1 (n=112 AD patients, n=151 HCs) and 113 genomic DNA samples from UKBBN cohort (n=102 AD patients, n=11 HCs) were genotyped using SNP arrays for chr2:102966067 (GRCh37 / hg19), APOE-ε2, and APOE-ε4 using TaqMan Assays (rs1921622, C___1226146_10, Cat. No. 4351376; rs7412, C___904973_10, Cat. No. 4351376; and rs429358, C___3084793_20, Cat. No. 4351376, respectively; Thermo Fisher Scientific). We performed real-time quantitative PCR using the 7500 Fast and QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). We saved the results in EDS files and entered them into TaqMan Genotyper Software (Applied Biosystems) for collaborative genotyping of SNPs.

[0133] Immunohistochemical staining of postmortem human brain sections Formalin-fixed, paraffin-embedded postmortem frontal cortex sections from 78 AD patients were obtained from the UKBBN cohort. Sections were first deparaffinized and rehydrated with xylene and graded ethanol solutions. To stain Aβ, sections were first treated with formic acid for 5 min at room temperature. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide solution. Sections were then incubated with mouse anti-human Aβ antibody (1:500, clone NAB228, SC-32277, Santa Cruz Biotechnology) overnight at 4°C. After washing, sections were incubated with HRP-labeled anti-mouse IgG (QD 440-XAKE, RTU, BioGenex) and signals were developed with 3,3'-diaminobenzidine (DAB) substrate (QD 440-XAKE, BioGenex). To co-stain microglia and Aβ protein, double immunohistochemical staining was performed; after deparaffinization and rehydration, sections were treated with sodium citrate buffer (10 mM sodium citrate, pH 6.0) for 25 min, blocked, and then endogenous peroxidase activity was quenched with 3% hydrogen peroxide solution. Sections were then incubated overnight at 4°C with mouse anti-human Aβ antibody (SC-32277) and rabbit anti-human Iba-1 antibody (1:100, 019-19741, polyclonal, Fujifilm Wako Pure Chemical Corporation). After washing, sections were incubated with HRP-labeled anti-mouse Ig and AP-labeled anti-rabbit Ig (HK597-50K, double staining kit, BioGenex), followed by substrate development with DAB (QD440-XAKE, BioGenex) and Fast Red Substrate (HK182-5KE, BioGenex). Sections were then counterstained with Mayer's hematoxylin (HK100-9K, BioGenex) and coverslipped. The buffer used for washing was Tris Buffer Saline (TBS) with 0.01% Triton X-100, and primary antibodies were diluted in TBS. Images were captured with a ZEISS Axio Scan.Z1 scanner and processed with ZEN microscope software v3.2 (ZEISS).

[0134] To quantify Aβ plaques, we took 10 random images of each section. After background subtraction and threshold adjustment, we analyzed Aβ plaques using the Analyze Particles function in Fiji-ImageJ (v1.53c). We determined the total Aβ area, the number of Aβ plaques, and the median plaque size for each section. We calculated the total Aβ area as the total image area (10 mm 2 Aβ plaque burden (area%) was calculated by dividing the area by the total area of ​​Aβ. To quantify microglia-Aβ co-staining, we selected 20 random images of each section and processed them with the Colour Deconvolution function to separate the data into three color channels (i.e., DAB, Fast Red, and hematoxylin). After adjusting the threshold, we selected Aβ plaques and microglia using the Create Selection function and then analyzed them using the Analyze function. We determined the total Aβ area and Aβ area that colocalized with Iba-1 staining. We calculated the Aβ plaque area that colocalized with microglia (total Aβ%) by dividing the Aβ area that colocalized with Iba-1 staining by the total Aβ area. Two independent researchers performed section staining, image acquisition, and image quantification; they also randomly selected and quantified images in a blinded manner.

[0135] GWAS association analysis and data visualization Association analysis between SNPs and plasma sST2 levels at genome-wide level in Chinese_cohort_1 was performed using PLINK software (v1.9) adjusted for age, sex, AD diagnosis, and top 5 principal components as covariates with the following parameters: --keep-allele-order, --linear, --ci 0.95, --hwe 0.00001, and --maf 0.05. 99To visualize the data, Manhattan and quantile plots were generated using the manhattan() and qq() functions, respectively, of the R qqman package (v0.1.4). Region plots of the IL1RL1 locus were generated using LocusZoom. Fine mapping analysis of the effect of the IL1RL1 locus on plasma sST2 levels was performed using CAVIAR software (v2.2) with association test results and pairwise linkage disequilibrium information generated from PLINK software (v1.9) using the following parameters: --hwe 0.00001, --maf 0.05, --r, --matrix, --chr 2, --from-bp 102000000, and --to-bp 104000000. 47 Fine-mapped region plots were generated using the plot_ly() function in the R plotly package (v4.9.1). Linkage disequilibrium and haplotype structure were plotted using Haploview (v4.2). All independent sST2-associated variants (r 2 To identify variants with a P<1E-5 across the sST2 GWAS (<0.2), variants with a P<0.01E-5 across the sST2 GWAS were subjected to analysis with PLINK software (v1.9) (parameters: --hwe 0.00001, --maf 0.05, --clump-p1 0.00001, --clump-r2 0.2, --chr 2, and --clump-kb 2000), yielding 29 independent sST2-associated variants. The calc.relimp() function in the R relaimpo package (v2.2-3) was used. 100、101 was used to quantify the contribution of genetic factors (i.e., 29 independent sST2-associated variants) and non-genetic factors (i.e., age and sex) to sST2 level variance.

[0136] Association analysis of rs1921622 in transcriptome datasets at tissue and single-cell levels Human tissue sST2 and ST2L transcript levels from the GTEx dataset 48、49and rs1921622 genotype data were adjusted for age, sex, RNA integrity (i.e., RNA integrity number) and population structure (i.e., top four principal components) and used for genotype-expression association studies. Rank-based normalization of transcript levels was performed using the R rntransform() function in the GenABEL package (v1.8).

[0137] Transcription levels of sST2 and ST2L in the human frontal cortex at the single-cell level were obtained by realigning FASTQ files of our previously published snRNA-seq dataset with a modified reference genome. 50 Specifically, the IL1RL1 region (chr2:102, 311, 563 to 102, 352, 037) in the GTF file of the original GRCh38 / hg38 pre-mRNA reference genome was separated into three parts: the sST2-specific region (chr2:102, 343, 416 to 102, 346, 100), the ST2L-specific region (chr2:102, 311, 563 to 102, 337, 147, and 102, 346, 101 to 102, 352, 037), and the overlapping region (chr2:102, 337, 148 to 102, 343, 415). The modified reference genome was generated by Cell Ranger (v3.0.1) using the new GTF file and the original FASTA file. Subsequent quality control steps included gene level quantification and cell type identification, performed as described above. 50 For association analysis between genotype and candidate gene expression in each cell cluster, linear regression analysis was performed, adjusting for age, sex, AD diagnosis, and postmortem interval. The significance level was set at FDR-adjusted P<0.05. GO analysis of associated genes was performed using DAVID Bioinformatics Resources. 102、103 .

[0138] Analysis of the association between rs1921622 and risk of Alzheimer's disease A meta-analysis was conducted to examine the effect of rs1921622 genotype on AD risk. Specifically, effect sizes (i.e., log odds ratios) and standard errors (SEs) for APOE-ε4 carriers and noncarriers from six AD datasets (i.e., Chinese_cohort_1 dataset, Chinese_cohort_2 WGS and array dataset, and LOAD, ADC, and ADNI datasets) were determined using logistic regression with age, sex, and top five principal components as covariates. Results were summarized and processed by METASOFT (v2.0.0) to determine the mean mean and mean variance. 104 The joint risk effects and significance levels under the Han and Eskin random effects model (RE2) were estimated. Results were then input into ForestPMPlot (v1.0.2) to generate forest plots for data visualization.

[0139] In vivo experiments in mice We housed all mice in the HKUST Animal and Plant Care Facility. All animal experiments were approved by the HKUST Animal Ethics Committee and performed in accordance with the guidelines of the Animal Care Facility at HKUST. We housed four mice of the same sex per cage at 22°C and 60% relative humidity, with a 12-h light / dark cycle and food and water available ad libitum. Wild-type (WT) C57BL6J mice were obtained from the Jackson Laboratory. 5XFAD mice were generated as previously described by overexpressing the K670N / M671L (Sweden), I716V (Florida), and V717I (London) mutations in human APP, and the M146L and L286V mutations in human PSEN1. 100We confirmed the genotype by PCR analysis of tail or ear biopsy specimens. sST2 3'UTR deletion mice were produced by GemPharmatech Co.,Ltd. sST2 3'UTR deletion mice were produced by CRISPR / Cas9-mediated deletion. The sequences of gRNA were 5'-GTCCCTTGTAGTCGGTACAA-3' and 5'-GACACTCTACTTGTACCTAG-3'. We confirmed the genotype by PCR analysis of tail or ear biopsy specimens. We performed all in vivo experiments in age-matched groups, and mice were randomly assigned to the experimental conditions. We chose the sample size mainly based on our experience with similar types of experiments. We performed all animal experiments during the light phase. Mouse recombinant sST2-Fc (1004-MR-050; R&D Systems) was delivered at 0.11 μL / h via miniosmotic pumps (model 1004; Alzet) to 5XFAD mice (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas / Mmjax). Specifically, pumps were implanted in the ventricles above the right hemisphere and loaded with mouse recombinant sST2-Fc protein (240 ng per pump; 10 μg / mL) or human IgG (as a control) in artificial cerebrospinal fluid (119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2·2H2O, 1 mM NaH2PO4·2H2O, 1.3 mM MgCl2·6H2O, 26.2 mM NaHCO3, and 11 mM d-glucose). 28 days after administration, mice were anesthetized with isoflurane, perfused transcardially with phosphate-buffered saline (PBS), and mouse brains were collected. 40 mg / kg of mouse sST2 ASO was intravenously injected into C57 and 5XFAD mice. Five days after administration, mice were anesthetized with isoflurane, perfused with PBS, and serum and brains were collected.

[0140] Immunohistochemical staining of mouse brain The left hemisphere of mouse brains was fixed in 4% paraformaldehyde for 24 hours at 4°C, transferred to 30% sucrose, and stored at 4°C until sectioning. Brains were cut coronally into 50 μm sections with a vibrating blade microtome (VT1000S, Leica) and stored in cryoprotectant solution (30% glycerol, 30% ethylene glycol, and PBS) at -20°C until use. For immunohistochemistry, sections were rinsed with PBST (i.e., 0.1% Triton X-100 in PBS) and then treated with formic acid for 5 minutes at room temperature for antigen retrieval, followed by 3% hydrogen peroxide solution for 10 minutes to quench endogenous peroxidase activity. Sections were blocked with 5% horse serum in PBST for 2 hours and then labeled with 4G8 antibody (1:1000, 800703, BioLegend) in blocking buffer overnight at 4°C. The next day, sections were incubated with biotin-conjugated anti-mouse secondary antibody (1:1000, BA 2000, Vector Laboratories) followed by avidin-biotin-HRP complex (PK-6100, Vector Laboratories) and signals were developed with DAB (SK-4100, Vector Laboratories). Image processing was performed using a Leica DM6000 B compound microscope. Cortical sections were analyzed for Aβ plaque area using the Analyze Particles function of Fiji-ImageJ (v1.53c). Specifically, four brain sections per mouse in the cortical region (approximately 200-300 µm apart) were analyzed, and the average percentage of cortical area occupied by Aβ plaques was calculated.

[0141] For immunofluorescence analysis, we washed the sections and incubated them in 1 μM X-34 for 10 min. We then washed them with X-34 buffer (40% EtOH in PBS) and then in PBS. We then blocked the sections for 2 h in blocking buffer (4% horse serum, 1% bovine serum albumin [BSA], and 0.3% Triton X-100 in PBS). Primary antibodies used in the experiments include mouse anti-Aβ antibody (1:1000, clone 4G8, 800703, BioLegend), rabbit anti-Iba-1 (1:1000, 019-19741, Wako), and rat anti-Ki67 (1:200, clone SolA15, 14-5698-80, eBioscience); we diluted these in blocking buffer and incubated the sections at 4°C overnight. Sections were then incubated with fluorophore-conjugated secondary antibodies against mouse, rabbit, and rat Ig (Alexa Fluor 488, 568, and 647; 1:1000, Life Technologies) in blocking buffer for 2 hours at room temperature; washed extensively with PBST; stained with SYTOX Green (1:300000, S7020, Life Technologies) or DAPI (1:5000, D3571, Life Technologies); and mounted with FluorSave™ reagent (345789, Millipore).

[0142] We performed image processing using a Leica TCS SP8 confocal microscope equipped with a Leica 40x oil immersion objective. We took five images totaling 40 μm from each mouse cortex with a step size of 1 μm, then merged them into a single image with a maximum intensity Z projection. We identified three distinct plaque morphologies using anti-Aβ immunolabeling (i.e., 4G8) and X-34 staining: (i) fibrillar plaques characterized by fibrillar X-34 and 4G8 labeling without a plaque core; (ii) compact plaques characterized by 4G8 amyloid fibrils protruding radially outward with an X-34-labeled core; and (iii) inactive plaques characterized by an X-34-labeled core without 4G8 labeling. We segmented individual Aβ plaques using a custom macro and manually identified each plaque type. For plaque-associated microglia, we used DAPI in the barrier surrounding the plaque and the processes in contact with the plaque. + The number of microglia surrounding small plaques (i.e., radius ≦8 μm) and large plaques (i.e., radius >8 μm), as defined by nuclear staining, was manually counted. +Microglia were defined as microglia with Ki67 signal in the nucleus. To quantify microglial coverage of Aβ plaques, we selected only compact plaques. We used 10 optical slices spaced 0.5 μm apart through the center of the plaque for analysis. We processed all images with a customized macro in Fiji-ImageJ (v1.53c). For each slice, after adjusting the threshold, we determined the plaque perimeter using a function of area for analysis particles and lines. We also determined the plaque perimeter and plaque perimeter arcs that colocalized with microglial staining. We calculated the percentage of plaque perimeter covered by microglia by summing the plaque perimeter arcs over a three-dimensional (3D) stack that were in close (within 1 μm) contact with Iba-1 immunolabeled cells (approximately 25 plaques per group). We performed 3D reconstruction of microglia-plaque interactions using Imaris v9.7.2 (Oxford Instruments).

[0143] Assessment of microglial Aβ phagocytosis Microglial Aβ phagocytic capacity was examined as previously described 29Briefly, 4-month-old 5XFAD or wild-type mice were intraperitoneally injected with methoxy-X04 (10 mg / kg) to label Aβ. Mice were anesthetized with isoflurane 3 h after methoxy-X04 injection, and the left ventricle was perfused with ice-cold PBS. Mouse forebrains were isolated, minced, and incubated for 30 min at 37°C in 5 U / mL papain (LS003126) and 35 U / mL DNase I (LS002140; Worthington Biochemical) for enzymatic digestion. After incubation, myelin debris was depleted by 30% isotonic Percoll (P1644; Sigma-Aldrich) gradient centrifugation, and mononuclear cell suspensions were obtained in DMEM / F12 medium with ice-cold 10% heat-inactivated FBS. For cell population identification, unstained controls were prepared from a mixture of different sample cell suspensions. To label microglia, cell suspensions were stained with Alexa Fluor 488-conjugated mouse CD11b antibody (1:200; 53-0112-82; eBioscience) for 45 min at 4 °C. The resulting labeled cell suspensions were analyzed using a BD Influx cell sorter-flow cytometer. Scatter plot data recorded for microglial cell populations were analyzed using FlowJo software v10.5.0 (TreeStar).

[0144] CRISPR / Cas9-mediated genomic deletion of the rs1921622-harboring region in a cell culture system Human brain microvascular endothelial cell line (hCMEC / D3) was purchased from Cedarlane and cultured as previously described. 101Briefly, we coated tissue culture plates with 100 μg / mL type I collagen (Millipore) for 1 h at 37° C. and 5% CO2. We then washed the plates with Dulbecco's phosphate-buffered saline (DPBS) and replaced with complete culture medium (endothelial cell growth medium-2 [EGM-2] [Lonza] supplemented with 5% FBS [HyClone], 1% chemically defined lipid concentrate [Gibco], 10 mM HEPES [Gibco], 5 μg / mL ascorbic acid [Sigma], 1.4 μM hydrocortisone [Sigma], 1 ng / mL bFGF [PeproTech], 10 U / mL penicillin, and 10 μg / mL streptomycin [Gibco]). Cultured cells were dissociated with 0.05% trypsin for 5 min and plated at 25,000 cells / cm2. 2 and returned to culture at 37°C in a 5% CO2 incubator. Three to four days after seeding, the cells reached confluence and could be passaged. We used cells at passages 27 to 35 for our experiments.

[0145] For ChIP quantitative PCR (ChIP-qPCR) experiments, we completely changed the medium of hCMEC / D3 cells 2 hours before treatment. We then treated the cultured cells with recombinant human IL-33 (BioLegend) or DPBS as a vehicle control for 24 hours.

[0146] To evaluate the efficiency of single guide RNA (sgRNA) editing in endothelial cells, we transfected 5 × 10 sgRNAs with a single CRISPR construct by nucleofection using the Human Umbilical Vein Endothelial Cell Nucleofector Kit (Lonza) with the Nucleofector 2b device (Lonza). 5hCMEC / D3 cells were transfected. One day after transfection, we changed the culture medium to complete culture medium with 1 μg / mL puromycin (Thermo Fisher Scientific). After 3 days of puromycin selection, we extracted genomic DNA using QuickExtract™ DNA Extraction Solution (Lucigen), followed by T7EI (NEB) editing efficiency test. All four sgRNAs exhibited high editing efficiency (data not shown).

[0147] A dual guide RNA-mediated knockout approach was used to delete the region carrying rs1921622 in hCMEC / D3 cells. Cells were genotyped from 300 bp upstream and downstream of rs1921622 by Sanger sequencing. Screening of potential Streptococcus pyogenes Cas9 (SpCas9) guide RNAs was performed using the CRISPR design tool (crispr.mit.edu) at 100 bp upstream and downstream of rs1921622. The following sgRNAs were used: sgRNA-1, 5'-TTATGGACAGAATTAAGAAG-3' (SEQ ID NO: 1); sgRNA-2, 5'-CTGTCCATAAGATTTGAAAG-3' (SEQ ID NO: 2); sgRNA-3, 5'-AATTTTGTTCTGGTAGCCAT-3' (SEQ ID NO: 3); and sgRNA-4, 5'-GGTATTTCAGCTAGTGCCTA-3' (SEQ ID NO: 4). The sgRNA was subcloned into PX459v2, which contains the sgRNA cassette, human codon-optimized SpCas9, and a puromycin resistance gene.

[0148] To generate dual gRNA-mediated deletion cell lines, hCMEC / D3 cells were transfected with plasmids containing sgRNA-1 / sgRNA-4 (targeted 67 bp deletion), or sgRNA-2 / sgRNA-3 (targeted 38 bp deletion), or PX459v2 as a no sgRNA control. After 3 days of puromycin selection (1 μg / mL) starting the day after transfection, puromycin-resistant cells were seeded into two 24-well plates. Medium was changed twice weekly. After 3 weeks when single colonies were observed, wells containing only one colony were passaged into 12-well plates. Each clone was genotyped, and clones that may have had targeted deletions were subjected to Sanger sequencing. This protocol produced six control lines, eight lines with 38 bp deletions, and ten lines with 67 bp deletions.

[0149] CRISPR / Cas9-mediated genome editing in human patients In a dual guide RNA-mediated genome editing system aimed at deleting the genomic sequence encompassing the rs1921622 locus, spanning approximately 100 bp upstream and downstream from the s1921622 locus, human patients diagnosed with Alzheimer's disease and genotyped to have genetic markers indicative of high risk for AD (e.g., female patients APOE-ε4 carriers) are administered intravenously with viral vectors (e.g., adenoviral or adeno-associated viral vectors) or lipid nanoparticles packaged from DNA or RNA encoding the CRISPR / Cas9 system or ribonucleoprotein (RNP) complexes encoding Streptococcus pyogenes Cas9 (SpCas9) nuclease and two sgRNAs. The sgRNA targeting and deletion strategy is the same as that used in the culture system (see last section). For CRISPR editing targeting the sST2 genomic region in human patients, lipid nanoparticles carrying sgRNA and mRNA encoding Cas9 are delivered to the patient at a dose ranging from 0.01-2 mg / kg; 0.02-1.0 mg / kg; 0.05-0.5 mg / kg; or 0.1-0.3 mg / kg of patient body weight. After 4 weeks, administration of 0.1 mg / kg of nanoparticles is expected to achieve approximately a 50% reduction in circulating levels of the target protein (e.g., sST2 protein), while administration of 0.3 mg / kg of nanoparticles is expected to achieve a greater than 80% reduction in target protein levels.

[0150] Screening of sST2 antisense oligonucleotides The specificity of sST2 antisense oligonucleotides (ASOs) was defined by the absence of identical sequences and one or two mismatches with the sequence in other transcripts in the mouse genome reference (GRCm38) and human genome reference (GRCh37). All ASOs were synthesized by Integrated DNA Technologies (IDT). To examine the effect of each ASO, mouse fibroblast NIH-3T3 cells (ATCC) and human umbilical vein endothelial cells (ATCC) were transfected with 500 nM ASO using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher). Cells were harvested one day after transfection. The protein amount of the cell lysates was measured to normalize the level of medium sST2 levels.

[0151] Other statistical analyses and data visualization For the remaining statistical analyses of human subjects, the significance of the association between AD-related endophenotypes and sST2 levels and rs1921622 genotypes was determined by linear regression analysis. CSF sST2 level cutoffs were determined according to the levels of CSF sST2 at the maximum value of the Youden index using the optimal.cutpoints() function in R and the Youden method in the OptimalCutpoints package (v1.1-4). 105To examine the association between age at onset of dementia and the rs1921622 A allele, Cox regression was performed with sex and the top 5 principal components as covariates using the coxph() function of the survival package in R (v1.3-24). The significance level was set at P<0.05. For data visualization, volcano plots were generated using the plot() function in R, and dot plots were generated using the ggplot() function of the ggplot2 package in R (v3.2.1). For data from mouse and cell culture system experiments, the significance of differences was assessed by unpaired Student's t-test, or one-way or two-way analysis of variance followed by Bonferroni post hoc tests as indicated. The significance level was set at P<0.05. All statistical plots were generated using GraphPad Prism v8.0 (GraphPad Software).

[0152] Data availability The consent forms given by individual participants from Chinese_Cohort_1 stated that the study contents would be kept confidential under the control of the hospital and research team. Therefore, phenotypic, genomic, and proteomic data of individual participants will be available and shared in a formal collaboration. Use for data sharing and project collaboration will be processed and reviewed by a review panel hosted at HKUST. Researchers may contact [sklneurosci@ust.hk] for details of data sharing and project collaboration related to this study.

[0153] Code Availability All requests for code used in data analysis and data visualization will be promptly reviewed by the corresponding author and a review panel hosted at HKUST to verify that the request is subject to any intellectual property, confidentiality, or other license obligations. In the absence of restrictions, the corresponding author will communicate with the requester to share the code.

[0154] All patents, patent applications, and other publications cited in this application, including any GenBank accession number or equivalent, are incorporated by reference in their entirety for all purposes.

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Table 9-7

Claims

1. 1. A method for treating Alzheimer's disease (AD) or reducing the risk of AD in an individual in need thereof, comprising administering to the individual an effective amount of a composition that disrupts the 3'-untranslated region (UTR) of the sST2 gene / transcript or a genomic sequence encompassing rs1921622.

2. The method of claim 1, further comprising sequencing at least a portion of the individual's genome prior to the administering step.

3. The method of claim 1, wherein the individual is an APOE-ε4 carrier or a non-APOE-ε4 carrier.

4. The method of claim 1 , wherein the individual is a female or a male.

5. The method described in claim 1, wherein the individual has an A allele or a G allele at rs1921622.

6. 10. The method of claim 1, wherein the individual has been diagnosed with AD, or the individual has not yet been diagnosed with AD but has known risk factors for AD.

7. The method of claim 1, wherein the genomic sequence encompassing rs1921622 comprises a sequence of approximately 300 base pairs upstream and downstream of rs1921622, preferably approximately 250 base pairs, 200 base pairs, 150 base pairs, 100 base pairs, 50 base pairs, 30 base pairs, or 20 base pairs upstream and downstream of rs1921622.

8. The method of claim 1, wherein the composition comprises an siRNA, microRNA, miniRNA, lncRNA, or antisense oligonucleotide that targets a genomic sequence comprising the 3'-UTR or rs1921622 of the sST2 gene.

9. The method of claim 1, wherein the composition comprises one or more vectors encoding an endonuclease guided by a small guide RNA (sgRNA) and two sgRNAs targeting two locations within the genomic sequence including rs1921622.

10. The method described in claim 9, wherein the composition comprises a vector encoding a Cas9 nuclease and two sgRNAs.

11. 11. The method of claim 9 or claim 10, wherein the one or more vectors are one or more viral vectors.

12. The method of claim 1, wherein the composition is administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, or intracranial injection, or by oral or nasal administration.

13. The method of claim 12, wherein the composition is administered in the form of a solution, suspension, powder, paste, tablet, or capsule.

14. A kit for treating Alzheimer's disease (AD) or reducing the risk of AD in an individual in need thereof, the kit comprising a container containing a composition that disrupts a genomic sequence encompassing the 3'-UTR or rs1921622 of the sST2 gene.

15. 15. The kit of claim 14, wherein the composition is formulated for subcutaneous, intramuscular, intravenous, intraperitoneal, or intracranial injection, or is formulated for oral or nasal administration.

16. 15. The kit of claim 14, wherein the composition comprises an siRNA, microRNA, miniRNA, lncRNA, or antisense oligonucleotide targeting the genomic sequence comprising the 3'-UTR or rs1921622 of the sST2 gene.

17. The kit of claim 14, wherein the composition comprises an endonuclease guided by a small guide RNA (sgRNA) and one or more vectors encoding two sgRNAs targeting two locations within the genome sequence including rs1921622.

18. 18. The kit of Claim 17, wherein the composition comprises a vector encoding a Cas9 nuclease and two sgRNAs.

19. 15. The kit of claim 14, further comprising a second container containing agents for sequencing at least a portion of the individual's genome.

20. 15. The kit of claim 14, further comprising instructions for administration of the composition.

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