Methods of treating, ameliorating and / or preventing vascular inflammation and diseases / disorders associated with the same

Abstract

Described herein is a method of treating, ameliorating and / or preventing vascular inflammation or diseases and disorders associated therewith in a subject. The method includes administering to the subject a compound for downregulating a Pcdhg cluster member / Pcdhga9 activity and / or expression level, such as in a vascular endothelial cell of the subject. Also described is a kit for performing a method described herein.

Description

[0001] METHODS OF TREATING, AMELIORATING AND / OR PREVENTING VASCULAR INFLAMMATION AND DISEASES / DISORDERS ASSOCIATED WITH THE SAME

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 621,466, filed January 16, 2024, which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] The XML file named " 047162-7494WOl(02519)_Seq Listing.xml " created on January’ 13, 2025, comprising 27,604 bytes, is hereby incorporated by reference in its entirety’.

[0006] BACKGROUND

[0007] Vascular inflammation is controlled mainly by the endothelial cells that line all blood vessels. Vascular inflammation is the mam cause of coronary, peripheral and cerebral artery disease, and a major contributing factor to pulmonary arterial hypertension, lung and kidney injury’ and intravascular coagulation due to sepsis, diabetic vasculopathy, and transplant rejection, among others.

[0008] While multiple methods exist to suppress inflammation, no methods are available to specifically target vascular inflammation. Vascular inflammation is sometimes treated by potent anti-inflammatory’ therapies, which unfortunately often limit immune defense against pathogens and increase risk of serious infections.

[0009] Therefore, there is a need for therapies that treat, ameliorate, prevent, reduce, and / or eliminate vascular inflammation, as well as for treating, ameliorating, and / or preventing the various disease or disorders caused by’ or involving vascular inflammation. The present invention addresses this need.

[0010] SUMMARY

[0011] In some aspects, the present invention is directed to the following non-limiting embodiments.

[0012] Methods of treating, ameliorating and / or preventing vascular inflammation

[0013] In some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing vascular inflammation in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of a compound that downregulates a Pcdhg cluster member / Pcdhga9.

[0014] In some embodiments, the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

[0015] In some embodiments, the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

[0016] In some embodiments, the method downregulates Pcdhga9 in a vascular endothelial cell of the subj ect.

[0017] In some embodiments, the vascular inflammation causes or contributes to an atherosclerotic cardiovascular disease, a coronary artery disease, a peripheral artery' disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, or a transplant rejection in the subject.

[0018] In some embodiments, the compound comprises a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9.

[0019] In some embodiments, the compound comprises a protein inhibitor of the Pcdhg cluster member / Pcdhga9.

[0020] In some embodiments, the compound comprises a nucleic acid that dow nregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference.

[0021] In some embodiments, the compound comprises a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme.

[0022] In some embodiments, the compound comprises an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity and / or expression level by CRISPR knockout or CRISPR knockdown.

[0023] In some embodiments, the compound comprises a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

[0024] In some embodiments, the compound that downregulates the Pcdhg cluster member / Pcdhga9 is an antibody against the Pcdhg cluster member / Pcdhga9.

[0025] Methods of treating, ameliorating, and / or preventing a disease or disorder caused by or involving vascular inflammation

[0026] In some aspects, the present invention is directed to a method of treating, ameliorating, and / or preventing a disease or disorder caused by or involving vascular inflammation in a subject in need thereof.

[0027] In some embodiments, the method comprises administering to the subject an effective amount of a compound that downregulates a Pcdhg cluster member.

[0028] In some embodiments, the method comprises administering to the subject an effective amount of a compound that downregulates Pcdhga9.

[0029] In some embodiments, the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

[0030] In some embodiments, the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

[0031] In some embodiments, the disease or disorder is at least one selected from the group consisting of an atherosclerotic cardiovascular disease, a coronary' artery disease, a peripheral artery’ disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, and a transplant rejection in the subject.

[0032] In some embodiments, the compound comprises a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9.

[0033] In some embodiments, the compound comprises a protein inhibitor of the Pcdhg cluster member / Pcdhga9.

[0034] In some embodiments, the compound comprises a nucleic acid that doyvnregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference.

[0035] In some embodiments, the compound comprises a ribozyme that doyvnregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme.

[0036] In some embodiments, the compound comprises an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity and / or expression level by CRISPR knockout or CRISPR knockdown.

[0037] In some embodiments, the compound comprises a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

[0038] In some embodiments, the compound is an antibody against the Pcdhg cluster member / Pcdhga9.

[0039] Kits

[0040] In some aspects, the present invention is directed to a kit.

[0041] In some embodiments, the kit is a kit for treating, ameliorating and / or preventing vascular inflammation or diseases or disorders associated therewith in a subject in need thereof.

[0042] In some embodiments, the kit comprises a compound for downregulating the Pcdhg cluster member / Pcdhga9 in the subject; and a manual instructing that the compound is to be administered to the subject in an effective amount.

[0043] In some embodiments, the compound down regulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

[0044] In some embodiments, the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

[0045] In some embodiments, the disease or disorder associated with the vascular inflammation is at least one selected from the group consisting of an atherosclerotic cardiovascular disease, a coronary' artery' disease, a peripheral artery' disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, and a transplant rejection.

[0046] In some embodiments, the compound comprises a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9.

[0047] In some embodiments, the compound comprises a protein inhibitor of the Pcdhg cluster member / Pcdhga9.

[0048] In some embodiments, the compound comprises a nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference.

[0049] In some embodiments, the compound comprises a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme.

[0050] In some embodiments, the compound comprises an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity and / or expression level by CRISPR knockout or CRISPR knockdown.

[0051] In some embodiments, the compound comprises a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

[0052] In some embodiments, the compound comprises the compound that downreg ulates the Pcdhg cluster member / Pcdhga9 comprises an antibody against the Pcdhg cluster member / Pcdhga9.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the present disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0055] Figs. 1A-1G illustrates certain aspects of a genome-wide CRISPR screen that identified KLF2 suppressors, in accordance with some embodiments. Fig. 1A: Schematic of the Klf2 reporter stably expressed in MAECs (A7 / 2:GFP reporter). Short-lived enhanced GFP is driven by the Klf2 promoter while mCherry is driven by a constitutive PGK promoter as an internal control. Fig. IB: Schematic of the genome-w ide CRISPR screen to identify modifiers (activators and suppressors) of KLF2 induction under LSS (15 dyn cm2LSS for 18 h). Fig. 1C: Functional categorization of the candidate KLF2 suppressors (cumulative z-score greater than 4, 160 hits) using ingenuity pathway analysis (QIAGEN). Fig. ID: Candidate suppressors intersecting with ‘cell-surface exposed proteins on outer plasma membrane’ Gene Ontology' term to identify candidates amenable to function neutralization by blocking antibodies (red box: validated hits). Fig. IE; A7 / 2:GFP reporter MAECs were transduced with Cas9 plus control sgRNA, PCDHGA9 sgRNA or with a human PCDHGA9 (Hs Pcdhga9) vector. Cells were exposed to static, LSS or OSS for 16 h and immunoblotted for 7 / 2:GFP (n = 3 independent experiments). Graph: quantitation of 7 / 2:GFP normalized to tubulin loading control. Fig. IF: Immunoblot for the inflammatory marker VCAM1 in control or PCDHGA9-depleted MAECs exposed to Static. LSS or OSS (n = 3 independent experiments). Graph: quantitation of VCAM1 normalized to tubulin loading control. Fig. 1G: THP1 monocyte binding to control siRNA or PCDHGA9 siRNA HUVECs exposed to static or OSS for 16 h (n = 4 independent experiments, mean value per experiment). Graph: quantitation of bound THP1 monocytes per field (THP1 binding index). Values are shown as the mean ± s.e.m. Statistical analysis was carried out using a one-way analysis of variance (ANOVA). Scale bar, 100 pm.

[0056] Figs. 2A-2H demonstrate that Pcdhg gene cluster suppresses KLF2 and KLF4, in accordance with some embodiments. Fig. 2A: Top, schematic of Pcdhg gene cluster organization consisting of 22 genes with their unique and variable first exon (and individual promoter, not shown) and three common and constant 3' exons that form the common cytoplasmic domain. Bottom, strategy for silencing (knocking down) the entire Pcdhg gene cluster with an siRNA targeting the common region (shaded bar and dashed lines). Fig. 2B: Validation of siRNA-mediated knockdown of the entire Pcdhg gene cluster (22 genes) in HUVECs by immunoblot with an antibody to the common cytoplasmic domain (n = 3 independent experiments). Right, quantitation of PCDHG levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control. Figs. 2C-2D: Quantitative PCR with reverse transcription (RT-qPCR) for Klf2 (Fig. 2C) and Klf4 (Fig. 2D) in control and PCDHG-depleted HUVECs exposed to static, LSS and OSS for 16 h (n = 3 independent experiments). Fig. 2E: THP1 monocyte binding to control siRNA or Pcdhg siRNA HUVECs exposed to static or OSS for 16 h (n = 3 independent experiments, mean value per experiment). Right, quantitation of THP1 monocytes per field per condition (THP1 binding index). Fig. 2F: Left, immunoblot for KLF4 in control siRNA or PCDHG siRNA HUVECs, treated with lovastatin (0. 0. 1 or 1.0 pM) for 16 h (n = 2 independent experiments). Right, quantitation of KLF4 normalized to GAPDH loading control. Fig. 2G: Top, immunoblot for VCAM1 and KLF4 in control siRNA or PCDHG siRNA HUVECs, untreated or treated with TNF (1 ng mF1) for 16 h (n = 3 independent experiments). Bottom left and bottom right, quantitation of VC AMI and KLF4 normalized to GAPDH loading control with or without TNF. Fig. 2H: THP1 monocyte binding to control siRNA or PCDHG siRNA HUVECs untreated or treated with TNF (1 ng ml ') for 16 h (n = 7 images across three independent experiments). Bottom, quantitation of THP1 monocytes per field per condition. Values are shown as the mean ± s.e.m. Statistical analysis was carried out using a one-way ANOVA. Scale bars. 100 pm (Fig. 2E), 250 pm (Fig. 2H).

[0057] Figs. 3 A-3Q demonstrate that Pcdhg endothelial KO protects against atherosclerosis, in accordance with some embodiments. Fig. 3A: Schematic showing the PcdhgloxP,con3mouse line and generation of ECKO by crossing Pcdhg^oxP con3with Cdh5-Cre. The third constant exon was tagged with GFP and floxed. Cre-mediated recombination excises this exon, creating a premature stop codon leading to nonsense-mediated decay. Fig. 3B: Analysis of progeny genotype showing Pcdhg ECKO at the expected Mendelian ratio. Fig. 3C: Aortic arch from control or Pcdhg ECKO adult mice immunostained for KLF4 and examined en face (mean fluorescence intensity (MFI) per nucleus from n = 3 animals). Greater curvature denotes the region of laminar shear while lesser curvature denotes a region of low, disturbed shear. Right, quantitation of the MFI per cell. Fig. 3D: Schematic of the atherosclerosis study: 8-10-week-old control or Pcdhg ECKO mice were injected with AAV8-PCSK9 and maintained on an HFD for 16 weeks, starved overnight and analyzed for blood lipids and vessel histology. Figs. 3E-3F: Whole aortas were stained with Oil Red O to mark lipid-rich regions and imaged en face (n = 6 animals for each sex). Bottom, quantitation of percentage Oil Red O+area for males (Fig. 3E) and females (Fig. 3F). Fig. 3G: Sections from aortic roots were stained with H&E (n = 5 animals) or Oil Red O (n = 5 animals). Quantitation of atherosclerotic plaque area (per animal), NC area (per plaque) and FC thickness (per plaque). Fig. 3H: Atherosclerotic plaques (dotted area) in aortic root sections were stained for the macrophage and monocyte marker CD68 (n = 3). Right, quantitation of CD68 MFI. Figs. 31- 3N: Immune function in control or Pcdhg ECKO tested according to infection with LCMV clone 13 and measuring spleen cell readouts and function (n = 6 animals). Percentage of live CD8a+cells on day 7 after infection (Fig. 31), GP33 tetramer+CD8a cells (Fig. 3 J), granzyme B+CD8a cells (Fig. 3K), IFNy+TNF+(Fig. 3L) or IFNy TNF (Fig. 3M) CD8a cells after restimulation with LCMV-specific GP33 peptide and viral loads in kidneys (Fig. 3N). Figs. 3O-3Q: Response in control or Pcdhg ECKO to intraperitoneally injected E. coli by measuring CFUs (Fig. 30), neutrophil numbers (percentage of CD1 l b+) (Fig. 3P) and phagocytic function (Fig. 3Q) in the peritoneal exudates at the indicated times after infection (n = 3-4 animals). Values are shown as the mean ± s.e.m. Statistical analysis was carried out using an unpaired two-tailed Student's Etest (Figs. 3E-3G (middle and right graphs) and Figs. 3H, 3I-3N), one-way ANOVA (Figs. 3C and 3G, left graph) or two-way ANOVA with Bonferroni’s multiple comparisons test (Figs. 3O-3Q). Scale bars, 50 pm (Fig. 3C), 500 pm (Figs. 3E and 3F), 200 pm (Figs. 3G and 3H).

[0058] Figs. 4A-4M demonstrate that a physical and functional PCDHG-Notch axis regulates KLF2 and KLF4, in accordance with some embodiments. Fig. 4A: Gene organization of the Pcdhg cluster with the three 3' exons (blue) coding for the CCD shared by all 22 members, and mutant construct design. Figs. 4B-4C: HUVECs transfected with the indicated constructs (GFP tagged for Fig. 4B and Flag tagged for Fig. 4C) and treated with LSS for 16 h. Left, immunoblotting confirmed expression of mutants and the effects on endogenous KLF4 (n = 2-3 independent experiments). Right, quantitation of KLF4 normalized to GAPDH loading control. The mutants were expressed at comparable levels (GFP, FLAG immunoblots). Full, Full length; ACCD. deleted CCD; ICD, VCD + CCD; ANLS, deleted NLS. Fig. 4D: Schematic of bulk RNA-seq analysis of control siRNA, Pcdhg siRNA and Pcdhg + Klf2 and Klf4 triple siRNA HUVECs to identify / W / ig-dependent and Klf2 and ^ / -independent DEGs (green shaded area). Fig. 4E: Immunoblot validation of Pcdhg and Klf4 depletion (n = 3 independent experiments). Fig. 4F : Upstream regulatory pathway and process analysis from the DEGs using Enrichr. Fig. 4G: Heatmap of upregulated Notch target genes in Pcdhg siRNA and Pcdhg siRNA + Klf2 and Klf4 siRNA compared to control siRNA from the RNA-seq analysis. Fig. 4H: Top, control siRNA or Pcdhg siRNA HUVECs were exposed to static or LSS for 2 h and immunoblotted for the cleaved NICD (Vall744) (n = 3 independent experiments). Bottom, quantitation of NICD Vall744 normalized to GAPDH loading control. Fig. 41: NICD-dependent transcription was blocked with RIN1, a pharmacological inhibitor of Notch-RBPJ interaction (RBPJi) or with RBPJ siRNA. Top, HUVECs were exposed to LSS for 16 h and immunoblotted for KLF4 (n = 3 independent experiments). Bottom, quantitation of KLF4 normalized to GAPDH loading control. Fig. 4J: Schematic showing Notchl. The asterisks show the NICD peptides identified in the mass spectrometry analysis of the IPs of full but not the ACCD Pcdhg mutant. Figs. 4K and 4L: Co-immunoprecipitation of Pcdhg and NICD Vall744. Inputs were used as loading controls for IPs. Fig. 4K. HUVECs expressing full or ACCD Pcdhg (C terminus, GFP- tagged) were immunoprecipitated with GFP nanobody beads and immunoblotted for NICD Vail 744 (n = 3). Vector alone was used as negative control for the immunoprecipitation. Fig. 4L, HUVECs expressing CCD or ANLS-CCD mutant (C terminus, FLAG-tagged) were immunoprecipitated with FLAG antibody beads and immunoblotted for NICD Vai 1744 (n = 3). Fig. 4M: Top, Notch transcriptional reporter in cells cotransfected with NICD-V5 and PICD-FLAG, as indicated. Bottom, quantitation of Notch activity reporter (GFP) normalized to GAPDH loading control (n = 3 independent experiments). Values are shown as the mean ± s.e.m. Statistical analysis was carried out using a one-way ANOVA. *P < 0.05, **P < 0.01, *** < 0.001, **** / > < 0.0001.

[0059] Figs. 5A-5I illustrate some results of the PCDHGA9 blocking antibody in experimental atherosclerosis, in accordance with some embodiments. Fig. 5A: Schematic of homophilic adhesion assay and function-blocking mAb generation from PCDHGA9 ECD. Fig. 5B: PCDHGA9 ECD was immobilized in 96 wells and the percentage of MAECs that adhered was measured (n = 3 independent experiments, mean value per experiment). Fig. 5C: Top, adhesion of MAECs to ECD in the presence of isotype control or function-blocking mAb A9 (n = 8 images from four independent experiments). Bottom, quantitation of percentage total cells adhered to the ECD. Figs. 5D-5E: 7 / 2:GFP reporter MAECs with isotype control or mAb A9 tested for Fig. 5D. 7 / 2:GFP expression after 16-h LSS (n = 12 images across three independent experiments) or Fig. 5E immunostained for VC AMI after 16-h OSS (n = 7-8 images across three independent experiments). Quantitation of A7 / AGFP and VCAM1 levels normalized to the mCherry internal control. Fig. 5F: Schematic of the partial carotid artery (PCA) ligation model of accelerated atherosclerosis. EC A, external carotid artery; ICA, internal carotid artery; LSA, left subclavian artery; RSA, right subclavian artery'; STA, superficial temporal artery Fig. 5G: Experimental design. Antibodies were administered via intraperitoneal injection into in 4 / ?oc mice. Fig. 5H: Whole-mount brightfield image showing the branching of the LCA and RCA from the aorta, with atherosclerotic plaque (yellow brackets) visible in the ligated LCA. Fig. 51: Left, LCA and RCA sections were stained with Oil Red O to identify lipid-rich atherosclerotic plaques (n = 6). Right, quantitation of Oil Red O+area fraction. Values are show n as the mean ± s.e.m. Statistical analysis was carried out using a one-way ANOVA (Fig. 5B) or an unpaired two-tailed Student’s / -test (Figs. 5C-5E and 51). Scale bars, 100 pm (Figs. 5C and 51), 1 mm (Fig. 6H). * < 0.05, ** < 0.01, ***P < 0.001, **** / > < 0.0001.

[0060] Figs. 6A-6B illustrate certain aspects of PCDHG in human atherosclerosis, in accordance with some embodiments. Fig. 6A: Top, artery sections from patients with CVD and healthy controls stained for PCDHG and for eRG to mark ECs (n = 8). Bottom, quantitation of PCDHG level in ECs. Fig. 6B: Top, human coronary artery sections from older donors stained for PCDHG levels, comparing the plaque region to segments of the same artery without evident plaque (normal region). Sections were stained for Erg to mark ECs (n = 3). Bottom, quantitation of PCDHG staining intensity in ECs. Values are shown as the mean ± s.e.m. Statistical analysis used an unpaired two-tailed Student’s t-test. Scale bars, 50 pm. *P < 0.05, **P < 0.01, ***P < 0.001, NS (not significant) > 0.05.

[0061] Figs. 7A-7F illustrate certain aspects of the validation of Klf2 reporter, in accordance with some embodiments. Fig. 7A: Immunoblot validation of LSS-mediated induction of A7 / 2:GFP in reporter MAECs; reporter is also sensitive to statin treatment, another potent inducer of Klf2 used as a positive control (N = 3 independent experiments). Fig. 7B: Rank- ordered candidate suppressors from the CRISPR screen to identify Klf2 modifiers (z < 4 gray, z > 4 blue), w ith validated cell-surface exposed candidates which are amenable to function neutralization marked in red (mid-gray), and known positive controls, Ccm2 and PdcdlO, marked in black. Fig. 7C: qRT-PCR validation of siRNA mediated knockdown of Pcdhga9 in MAECs (Pcdhga9 si) (N = 3 independent experiments). Fig. 7D: Immunoblot validation of human Pcdhga9-FLAG overexpression (Hs Pcdhga9 OE) in MAECs (N = 3 independent experiments). Fig. 7E: Control si and Pcdhga9 si Kip reporter MAECs were exposed to static (St), LSS, or oscillatory shear stress (OSS) for 16 h and immunoblotted for Klp.GYP (N = 3 independent experiments). Fig. 7F: qRT-PCR for endogenous Kip levels in Control si and Pcdhga9 si in Human Umbilical Vein Endothelial Cells (HUVECs) exposed to St or OSS for 16 h (N = 3 independent experiments). Values are means ± SEM. Statistical analysis used one-way AN OVA.

[0062] Figs. 8A-8C demonstrate that Protocadherin gamma (Pcdhg) gene cluster promotes inflammatory signaling, in accordance with some embodiments. Fig. 8A: Validation of siRNA mediated knockdown of Pcdhg gene cluster in HUVECs using two different siRNAs targeting the common region in the 3' end, by qRT-PCR for Pcdhgc3, the highest expressed Pcdhg member in HUVECs (N = 3 independent experiments). Fig. 8B: qRT-PCR for the OSS induced pro-inflammatory marker E-selectin (Sele) in control and Pcdhg depleted HUVECs exposed to St, LSS and OSS for 16 h (N = 3 independent experiments). Fig. 8C: Immunoblot for VCAM1 in Control si or Pcdhg si HUVECs. after treatment with indicated doses of TNFa for 16 h (N = 3 independent experiments). Graph: quantitation of VC AMI normalized to Tubulin loading control. Values are means ± SEM. Statistical analysis used one-way ANOVA

[0063] Figs. 9A-9G illustrate certain aspects of the validation and blood lipid analysis of Pcdhg ECKO mouse, in accordance with some embodiments. Fig. 9 A: Generation of Pcdhg endothelial knockout (ECKO) by crossing Pcdhg^con3with Cdh5Cre, and confirmation by genotyping PCR. wt: wild type for Pcdhg allele; fl ox: Pcdhg floxed, Cre: Cdh5Cre (N > 10 animals). Figs. 9B-9C: Analysis of progeny genotype showing no significant deviation from Mendelian ratio as tested by Chi-squared analysis. Figs. 9D-9G: Plasma triglycerides, cholesterol, HDL-C, and body w eights of male and female Control or Pcdhg ECKO mice injected with pCSK9-Adeno Associated Virus 8 (AAV8) and maintained on High Fat Diet (HFD) for 16 weeks, starved overnight before analysis (N = 5). Values are means ± SEM. Statistical analysis was carried out using one-way ANOVA.

[0064] Figs. 10A-10L demonstrate that Pcdhg ECKO does not affect immune function, in accordance with some embodiments. Figs. 10A-10C: Mice were infected with Lymphocytic Choriomeningitis Virus (LCMV) Clone 13 and spleen myeloid cell frequency measured on Day 7 post infection (N = 6 animals). Graphs: (Fig. 10A) percentages of live CD1 lb+ or CD11c + CD1 lb- non T / B-cells; (Fig. 10B) monocyte (Ly6c + Ly6g-) and neutrophil / granulocyte (Ly6c + Ly6g + ) population of the CD1 lb+ cells; (Fig. IOC) subsets of CD1 lb+ cells, as indicated. (Figs. 10D-10I) Immune response in Control or Pcdhg ECKO to intraperitoneally injected E. coli by measuring CFUs (Fig. 10D) and representative LB agar plates showing CFUs in males (Fig. 10E), CFUs in blood (Fig. 10F), flow cytometry quantification of neutrophils (CD45+CD11 b+F4 / 80 Ly6C Ly6G ) and monocytes (CD45+CD1 1 b F4 / 80 Ly6G Ly6C ). and macrophages (CD45+CD1 lb+F4 / 80+) numbers (Figs. 10G and 10H) and cell numbers in pentoneal exudate (Fig. 101) (N = 3). (Figs. 10J and 10K) Phagocytic function in the peritoneal exudates at 12 h post infection (N = 3). Fig. 10L: Phagocytic function of bone marrow neutrophils from uninfected Control or Pcdhg ECKO (Fig. 10J) (N = 4). Values are means ± SEM. Statistical analysis was carried out using unpaired two-tailed Student’s t-test (Figs. 10A-10C, 10J and 10L) or two-way ANOVA (Figs. 10D, 101 and 10H).

[0065] Figs. 11 A-l 1H demonstrate that conserved nuclear ICD region of Pcdhg associates with Notch ICD and is necessary and sufficient for function, in accordance with some embodiments. Fig. 11 A: Pcdhg mutants were expressed in HUVECs which were treated with Static, LSS or OSS and immunoblotted for GFP, Klf4 and GAPDH (N = 3 independent experiments). Fig. 1 IB: GFP was imaged in HUVECs expressing the above mutants (N = 3 independent experiments). Fig. 11C: HUVECs expressing additional mutants (C-terminal FLAG-tagged) were immunostained for FLAG and counterstained with DAPI to mark nuclei (N = 3 independent experiments). Figs. 1 ID-1 IE: Multiple sequence alignment to test domain homology domains, using Pcdhga9 as an example, showing near-complete conservation in CCDs (highlighted in yellow). Percent conservation shown in the box. Fig. 1 IF: RBPJ-Notch DNA-binding consensus motif. Fig. 11G: RBPJ-Notch binding motifs in mouse and human Klf2 and Klf4 promoters. Fig. 11H: NICD peptides detected in proteomic analysis of the IPs of full but not the ACCD Pcdhg mutant. Scale bar: (Figs. 1 IB and 11C) 20 pm.

[0066] The sequences show in Figs. 1 ID-1 IE are listed below: The sequences shown in Fig. 11H are also listed below:

[0067] Figs. 12A-12L illustrate certain aspects of Pcdhga9 blocking antibody generation and validation, in accordance with some embodiments. Fig. 12A: Pcdhga9 ECD (ECD-FLAG- TEV-GST) protein run on a 10% Polyacrylamide SDS gel and visualized using Imperial Protein stain (Thermo Scientific) (N = 3 independent experiments). Fig. 12B: ELISA for the 24 selected high affinity monoclonal antibody (mAb) from clones labeled as Al -12 and Bl- 12 using ECD alone (GST cleaved off using TEV protease). Fig. 12C: Adhesion of MAECs to ECD in the presence of Isoty pe control or mAbs A9, Bl and B4 (N = 8 images across 4 independent experiments). Graph: quantitation of percent total cells adhered to ECD. Fig. 12D: Immunoblot with mAbs A9, Bl. B4 shows detection of both ECD-FLAG-TEV-GST and ECD-FLAG (N = 3 independent experiments). Figs. 12E-12F: X7 / 2:GFP reporter MAECs tested for A7 / 2:GFP reporter expression after 16 h LSS (N = 12 images across 3 independent experiments) (Fig. 1 IE) or immunostained for VCAM1 when exposed to OSS for 16 h OSS (N = 7-8 images across 3 independent experiments) (Fig. 12F), in the presence of Isotype control or mAbs A9, Bl and B4 as indicated. Graphs: quantitation of Klf2 GFP and VCAM1 levels normalized to mCherry internal control. Fig. 12G: Immunofluorescence with mAbs A9. Bl, B4 showing highest signal from A9. Fig. 12H: Immunoblot of Pcdhg knockdown cells with mAb A9. Purified ECD used as positive control. Fig. 121: Antibody half-life in vivo. A single dose of 1 pg of Isotype control or mAb A9 antibody was administered IP, and plasma levels of antibody were measured using an ELISA as described in Methods. mAb levels at 3 h post injection were considered 100%. Fig. 12J: LCA and RCA sections were stained for smooth muscle specific Acta2 (SMA) for marking plaque neointima (N = 6). Graph: quantitation of the LCA to RCA inner diameter. Figs. 12K-12L: Plasma triglycerides and cholesterol in Isotype control or mAh A9 injected male and female Apoe mice on HFD from Figs. 5H and 51 (N = 6). Values are means ± SEM. Statistical analysis was carried out using unpaired two-tailed Student's t-test (Fig. 12J) or one-way ANOVA (Figs. 12C. 12E, 12F, 12K and 12L). Scale bar: (Fig. 12C) 100 pm, (Fig. 12G) 20 pm. (Fig. 12J) 100 pm.

[0068] Figs. 13A-13B demonstrate that Pcdhg level correlates with atherosclerosis, in accordance with some embodiments. Fig. 13 A: Mouse carotids from the Partial Carotid Artery (PCA) Ligation model of accelerated atherosclerosis stained for Pcdhg and counter stained with DAPI to mark nuclei (N = 3 animals). RCA: Right Carotid Artery (control), LCA: Left Carotid Artery (atherosclerotic plaque. Graph: quantitation of Pcdhg staining intensity. Fig. 13B: Commercial Pcdhg antibody and mAb A9 staining of retinal vasculature from Control and Pcdhg ECKO mouse (N = 3 animals). mAb A9 is specific to mouse Pcdhga9 and Pcdhg antibody targets the conserved region in 22 Pcdhg genes, also conserved between mouse and human. Values are means ± SEM. Statistical analysis was carried out using unpaired two-tailed Student’s t-test. Scale bar: (Fig. 13A) 100 pm, (Fig. 13B) 10 pm.

[0069] Figs. 14-16 listed siRNAs, antibodies and primers used in the study described in the Example section, in accordance with some embodiments.

[0070] The sequences shown in Fig. 14 are listed below:

[0071] The sequences shown in Fig. 16 is listed below:

[0072] DETAILED DESCRIPTION

[0073] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subj ect matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed. Targeting the vascular endothelium offers a specific means to limit vascular inflammation without compromising immune defense and thus safely and effectively treat the wide range of diseases where vascular inflammation plays a key role.

[0074] Klf2 / 4 are anti-inflammatory, anti-thrombotic transcription factors that confer resilience against a ver7wide range of insults and stresses. In the study described herein ("the present study” or ‘“this study”), a whole genome CRISPR-Cas9 screen was performed in an attempt to identify regulators of Klf2 / 4. The screening revealed Pcdhg gene cluster, as well as one specific gene of the cluster, protocadherin gamma subfamily A, 9 (Pcdhga9), as strong “gain-of-function” hit whose knockout increased Klf2 expression and decreased inflammatory gene expression.

[0075] Any Pcdhg gene cluster member, such as Pcdhga9, can be downregulated by various means, at the DNA level, the mRNA level, and / or at the protein level. It is also w orth noting that all the proteins expressed by the Pcdhg gene cluster, including Pcdhga9, are cell surface proteins, which means these proteins can be targeted by antibodies. Indeed, the preset study raised blocking antibodies and found that these also increase Klf2 / 4 levels and decrease inflammatory genes. When injected into mice, these antibodies greatly decrease experimental atherosclerosis.

[0076] Accordingly, in some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing vascular inflammation in a subject.

[0077] In some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing a disease or a disorder caused by or involving vascular inflammation in a subject. Such diseases or disorders include atherosclerotic cardiovascular disease, coronary artery disease, peripheral artery disease, cerebral vascular disease, pulmonary arterial hypertension, lung or kidney injury or intravascular coagulation caused by sepsis, diabetic vasculopathy, or transplant rejection.

[0078] Definitions

[0079] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein and the laboratory7procedures in animal pharmacology, pharmaceutical science, peptide chemistry, and organic chemistry are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0080] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components and can be selected from a group consisting of two or more of the recited elements or components.

[0081] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0082] In this document, the terms "a." "an." or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B."

[0083] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, in certain embodiments ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0084] A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0085] A "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0086] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0087] In one aspect, the terms "co-administered" and "co-administration" as relating to a subject refer to administering to the subject a compound and / or composition of the disclosure along with a compound and / or composition that may also treat or prevent a disease or disorder contemplated herein. In certain embodiments, the co-administered compounds and / or compositions are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound and / or composition may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.

[0088] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical administration.

[0089] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, z.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0090] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in earn ing or transporting a compound useful within the disclosure within or to the patient such that it may perform its intended function. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the disclosure, and not injurious to the patient. Some examples of materials that may sen e as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the disclosure, and are physiologically acceptable to the patient. The "pharmaceutically acceptable carrier" may further include a pharmaceutically acceptable salt of the compound useful within the disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0091] As used herein, the language "pharmaceutically acceptable salt" refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0092] As used herein, a "pharmaceutically effective amount," "therapeutically effective amount," or "effective amount" of a compound is that amount of compound that is sufficient to provide a beneficial effect to the subject to which the compound is administered.

[0093] As used herein, the term "prevent" or "prevention" means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

[0094] As used herein, the terms "subject" and "individual" and "patient" can be used interchangeably and may refer to a human or non-human mammal or a bird. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

[0095] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound useful within the disclosure (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder and / or a symptom of a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder and / or the symptoms of the disease or disorder. Such treatments may be specifically tailored or modified, based on knowdedge obtained from the field of pharmacogenomics.

[0096] Method of Treating, Ameliorating, and / or Preventing Vascular Inflammation

[0097] In some aspects, the present invention is directed to a method of treating, ameliorating and / or preventing vascular inflammation in a subject in need thereof.

[0098] In some embodiments, the subject is a mammal, such as a human.

[0099] In some embodiments, the method includes down-regulating a level (such as an expression level, a protein level, an mRNA level, a genomic DNA level / copy number, etc.) or an activity of a Pcdhg gene cluster member, such as protocadherin gamma subfamily A, 9 (Pcdhga9) in the subject. In some embodiments, the level or activity of Pcdhg gene cluster member, such as Pcdhga9, is down-regulated in a vascular endothelial cell of the subject

[0100] In some embodiments, the Pcdhga9 is the human protein having the sequence set forth in SEQ ID NO: 1.

[0101] In some embodiments, the Pcdhga9 is a gene that produces the protein of SEQ ID NO: 1. In some embodiments, the Pcdhga9 is a gene that occupies the same allele of the gene that produces the protein of SEQ ID NO: 1, mRNA transcribed from the gene, and / or protein products thereof. In some embodiments, the Pcdhga9 is a non-human (such as non-human mammalian animal) ortholog of the gene, mRNA or protein in this paragraph.

[0102] In some embodiments, the Pcdhg cluster member includes the Pcdhga9 gene herein (such as those described in the previous paragraph). In some embodiments, the Pcdhg cluster includes all the mRNAs expressed by the gene cluster. In some embodiments, the Pcdhg cluster includes all the proteins expressed by the gene cluster.

[0103] In some embodiments, when the vascular inflammation is treated, ameliorated, and / or prevented in the subject, one or more diseases or disorders associated with the vascular inflammation is also treated, ameliorated and / or prevented. Such diseases or disorders include atherosclerotic cardiovascular disease, coronary' artery' disease, peripheral artery' disease, cerebral vascular disease, pulmonary arterial hypertension, lung or kidney injury or intravascular coagulation caused by sepsis, diabetic vasculopathy, or transplant rejection.

[0104] In some embodiments, the method includes administering to the subject an effective amount of a compound that dow n-reg ulates an activity7and / or expression level of the Pcdhg gene cluster member, such as Pcdhga9.

[0105] In some embodiments, the compound that downregulates the activity and / or expression level acts at the genomic level. For example, the expression level of Pcdhga9 (or any Pcdhga cluster member) can be down-regulated by gene knockout, such as CRISPR knockout and other knockout techniques.

[0106] In some embodiments, the compound that downregulates the activity and / or expression level acts at the transcriptional level or the translational level. For example, the expression level of Pcdhga9 (or any Pcdhga cluster member) can be down-regulated by gene knockdown, such as by RNA interference technique, ribozy me knockdown, or CRISPR knockdown.

[0107] In some embodiments, the compound that downregulates the activity and / or expression level acts at the post-translational level. For example, the expression level of Pcdhga9 (or any Pcdhga cluster member) can be down-regulated by targeted protein degradation, such as proteolysis-targeting chimera (PROTAC) and other protein degradation strategies. Using Pcdhga 9 as an example, the activity of Pcdhga9 can be down-regulated by small molecules inhibitors of Pcdhga9, antibodies that neutralizes Pcdhga9, trans-dominant negative mutant of Pcdhga9, and the like.

[0108] In some embodiments, the compound that downregulates the activity and / or expression level includes a protein inhibitor of Pcdhga9 (or any other protein(s) expressed by the Pcdhg gene cluster), or a compound that downregulates the expression level and / or activity of Pcdhga9 (or any other protein(s) expressed by the Pcdhg gene cluster) by RNA interference, by ribozyme, by CRISPR knockout / knockdown. or by producing a trans- dominant negative mutant, and so forth.

[0109] In some embodiments, the compound contemplated herein can be delivered by a vector, such as a plasmid or a viral vector. One of ordinary skill in the art would understand that such vectors can be used to deliver compounds in the form of nucleic acids, such as RNA or DNA. Such vectors are described herein below.

[0110] Downregulating Pcdhg cluster member / Pcdhga9 by small molecule inhibitors

[0111] In some embodiments, the compound that downregulates the activity and / or expression level includes a small molecule that inhibits the activity of Pcdhga9 (or any other protein(s) expressed by the Pcdhg cluster). As used herein, the term “small molecule7’ refers to a molecule having a size of less than 2000, 1800, 1600, 1400, 1200, 1000, 800, or 600 daltons.

[0112] In some embodiments, the small molecule inhibitor comprises a PROTAC or a Proteolysis Targeting Chimeric Molecule. PROTACs are heterobifunctional nanomolecules that can target any protein for ubiquitination and degradation. In certain embodiments, the proteins contemplated in the present invention comprises a group that is recognized by the E3 ubiquitin ligase and a group that is recognized by Pcdhga9 or any other protein(s) expressed by the Pcdhg cluster. The PROTAC is able to simultaneously bind to the protein herein and the E3 ligase. Formation of such trimeric complex formation leads to the transfer of ubiquitins to the proteins herein, marking it for degradation. PROTAC molecules possess good tissue distribution and the ability' to target intracellular proteins, thus can be directly applied to cells or injected into animals without the use of vectors. PROTACS useful w ithin the invention can be prepared using any known compound that binds to and / or recognizes and / or inhibits Pcdhga9 or any other protein(s) expressed by the Pcdhg cluster, w hich is linked through a linker to an E3 ubiquitin ligase, such as but not limited to those described in WO 2013 / 106643, WO 2013 / 106646, and WO 2019 / 148055.

[0113] Downregulating Pcdhg cluster member / Pcdhga9 by protein inhibitors of Pcdhg cluster member / Pcdhga9

[0114] In some embodiments, the compound that downregulates the activity and / or expression level includes a protein that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

[0115] Since Pcdhg cluster members, including Pcdhga9, are proteins located on the surface of a cell, they can be targeted directed using antibodies.

[0116] The term “antibody” or “Ab” or “immunoglobulin” are terms of art and can be used interchangeably and refer to a protein, or polypeptide sequence which is or is derived from an immunoglobulin molecule having at least one antigen binding site which specifically binds to a specific epitope on an antigen (See, e.g., Harlow et al., 1998, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY: Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor. New York; Houston et al.. 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain-antibody heavy chain pair, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv). nanobodies, intracellular antibodies, intrabodies, camelized antibodies, camelid antibodies. IgNAR antibodies, affybodies. Fab fragments. F(ab') fragments, F(ab)2, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above. Antibodies can be of any ty pe (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2). or any subclass (e.g.. IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgGl or IgG4) or subclass thereof. Full-length antibodies are sometimes tetramers comprising two heavy chain and two light chain immunoglobulin molecules.

[0117] Antibodies against Pcdhg cluster members are available commercially. Using Pcdhga9 as an example, antibodies that target Pcdhga9 include, e.g., H00056107-M01 commercially available from Novus Biologicals (Centennial, CO, USA), ABIN565983 commercially available from antibodies-online Inc. (Pottstown, PA, USA), 1G10 (Catalog # MA5-22248) commercially available from Invitrogen (Waltham, MA, USA) and BS-11150R from Bioss (Wobum, MA, USA) and any humanized derivatives thereof.

[0118] In some embodiments, the protein that down regulates the expression level and / or activity of Pcdhg cluster members / Pcdhga9 is administered in form of a protein. In some embodiments, the protein that downregulates the expression level and / or activity of Pcdhg cluster members / Pcdhga9 is administered in form of a nucleic acid that expresses the protein, such as an expression vector. The expression vector is described in the '‘Vector” section elsewhere in the present disclosure.

[0119] Downregulating Pcdhg cluster member / Pcdhga9 by RNA Interference

[0120] In some embodiments, the compound that downregulates the activity and / or expression level of Pcdhg cluster member / Pcdhga9 includes a nucleic acid that downregulates the activity and / or expression level of Pcdhg cluster member / Pcdhga9 by the means of RNA interreference.

[0121] It is worth noting that, since all Pcdhg cluster mRNAs share the same 3’-end region, which is transcribed from the same exon (see e.g., Fig. 3A), RNA interference components can be designed to target the entire Pcdhg cluster without the need to specifically targeting multiple nucleotide sequences.

[0122] In some embodiments, the nucleic acid that downregulates the expression level of Pcdhg cluster member / Pcdhga9 by the means of RNA interreference includes an isolated nucleic acid. In other embodiments, the modulator is an RNAi molecule (such as but not limited to siRNA and / or shRNA and / or miRNAs) or antisense molecule, which inhibits Pcdhg cluster member / Pcdhga9 expression and / or activity. In yet other embodiments, the nucleic acid comprises a promoter / regulatory sequence, such that the nucleic acid is preferably capable of directing expression of the nucleic acid. Thus, the present disclosure provides expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

[0123] In certain embodiments, siRNA is used to decrease the level of Pcdhg cluster member / Pcdhga9. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391 (19):306- 311; Timmons et al., 1998, Nature 395:854; Montgomery' et al., 1998, TIG 14 (7):255-258; Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describes a chemical modification to siRNAs that aids in intravenous systemic delivery'. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz el al.. 2003, Cell. 115: 199-208 and Khvorova et al., 2003. Cell 115:209-216. Therefore, the present disclosure also includes methods of decreasing levels of Pcdhg cluster member / Pcdhga9 using RNAi technology.

[0124] In certain embodiments, the present disclosure provides a vector comprising an siRNA or antisense polynucleotide. In other embodiments, the siRNA or antisense polynucleotide inhibits the expression of Pcdhg cluster member / Pcdhga9. The incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art.

[0125] In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) inhibitor. shRNA inhibitors are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzy mes (e.g, dicer) that cleaves the shRNA to form siRNA.

[0126] The siRNA, shRNA, or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis.

[0127] In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected using a viral vector. In certain embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neomycin resistance and the like.

[0128] Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, in some embodiments, the siRNA polynucleotide is further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrwal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et u / ., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

[0129] Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and / or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and / or the inclusion of nontraditional bases such as inosine, queosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0130] In certain embodiments, an antisense nucleic acid sequence expressed by a plasmid vector is used to inhibit Pcdhg cluster member / Pcdhga9 protein expression. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of Pcdhg cluster member / Pcdhga9.

[0131] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g, Cohen, 1989. In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0132] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0133] Alternatively, antisense molecules of the present disclosure may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the present disclosure include oligonucleotide derivatives known in the art which have improved biological activity7compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0134] Downregulating Pcdhg cluster member / Pcdhga9 by ribozyme

[0135] In some embodiments, the compound that downregulates the activity or expression level of Pcdhg cluster member / Pcdhga9 includes a ribosome that inhibits Pcdhg cluster member / Pcdhga9 protein expression.

[0136] A ribozyme is used to inhibit Pcdhg cluster member / Pcdhga9 protein expression. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary7, for example, to the mRNA sequence encoding Pcdhg cluster member / Pcdhga9. Ribozy mes are antisense RNAs which have a catalytic site capable of specifically cleaving complementary7RNAs. Therefore, ribozymes having sequence complementary to Pcdhg cluster member / Pcdhga9 mRNA sequences are capable of downregulating the expression of Pcdhg cluster member / Pcdhga9 by reduces the level of Pcdhg cluster member / Pcdhga9 mRNA. Ribozymes targeting Pcdhg cluster member / Pcdhga9, may be synthesized using commercially available reagents (Applied Biosystems, Inc.. Foster City, CA) or they may be genetically expressed from DNA encoding them. In some embodiments, the DNA encoding the ribozymes are incorporated in a vector, which is described in the “Vector” section elsewhere in the present disclosure.

[0137] Downregulating Pcdhg cluster member / Pcdhga9 by CRISPR knockout / knockdown and other knockouts / knockdown techniques

[0138] In some embodiments, the compound that downregulates the activity or expression level of Pcdhg cluster member / Pcdhga9 comprises a nucleic acid that downregulates the expression level of Pcdhg cluster member / Pcdhga9 by the means of CRISPR knockout.

[0139] Again, as noted elsewhere herein, since Pcdhg cluster genes share the same 3’-end, the entire cluster can be targeted by CRISPR knockout / knockdown by targeting the exon shared by the cluster (or mRNA portion expressed from the shared exon).

[0140] In some embodiments, the compound downregulates the activity or expression level of Pcdhg cluster member / Pcdhga9 comprises a CRISPR / Cas9 system for knocking out Pcdhg cluster member / Pcdhga9.

[0141] The CRISPR / Cas9 system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a "seed" sequence within the guide RNA (gRNA) and a conserved di -nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The CRISPR / Cas9 system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and CAR T cells. The CRISPR / Cas9 system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes.

[0142] The Cas9 protein and guide RNA form a complex that identifies and cleaves target sequences. Cas9 is comprised of six domains: REC I, REC II, Bridge Helix, PAM interacting, HNH, and RuvC. The Reel domain binds the guide RNA, while the Bridge helix binds to target DNA. The HNH and RuvC domains are nuclease domains. Guide RNA is engineered to have a 5' end that is complementary to the target DNA sequence. Upon binding of the guide RNA to the Cas9 protein, a conformational change occurs activating the protein. Once activated, Cas9 searches for target DNA by binding to sequences that match its protospacer adjacent motif (PAM) sequence. A PAM is a two or three nucleotide base sequence within one nucleotide downstream of the region complementary to the guide RNA. In one nonlimiting example, the PAM sequence is 5’-NGG-3'. When the Cas9 protein finds its target sequence with the appropriate PAM, it melts the bases upstream of the PAM and pairs them with the complementary region on the guide RNA. Then the RuvC and HNH nuclease domains cut the target DNA after the third nucleotide base upstream of the PAM.

[0143] One non-limiting example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Patent Appl. Publ. No. US2014 / 0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.

[0144] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. In certain embodiments, the CRISPR / Cas system comprises an expression vector, such as, but not limited to, an pAd5F35-CRISPR vector. In other embodiments, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combinations thereof.

[0145] In certain embodiments, inducing the Cas expression vector comprises exposing the cell to an agent that activates an inducible promoter in the Cas expression vector. In such embodiments, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

[0146] The guide RNA is specific for a genomic region of interest and targets that region for Cas endonuclease-induced double strand breaks. The target sequence of the guide RNA sequence may be within a loci of a gene or within anon-coding region of the genome. In certain embodiments, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

[0147] Guide RNA (gRNA), also referred to as "short guide RNA" or "sgRNA". provides both targeting specificity and scaffolding / binding ability for the Cas9 nuclease. The gRNA can be a synthetic RNA composed of a targeting sequence and scaffold sequence derived from endogenous bacterial crRNA and tracrRNA. gRNA is used to target Cas9 to a specific genomic locus in genome engineering experiments. Guide RNAs can be designed using standard tools well known in the art. In the context of formation of a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence is designed to have some complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In certain embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In other embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or nucleus. Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As with the target sequence, it is believed that complete complementarity is not needed, provided this is sufficient to be functional.

[0148] In certain embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell, such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to ("upstream" of) or 3' with respect to ("downstream" of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In certain embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e g., each in a different intron, two or more in at least one intron, or all in a single intron).

[0149] In certain embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3. 4, 5, 6, 7. 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in U.S. Patent Appl. Publ. No. US20110059502, incorporated herein by reference. In certain embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.

[0150] Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian and non-mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery' systems include DNA plasmids, RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery' systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery' to the cell (Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy 1 : 13-26).

[0151] In certain embodiments, the CRISPR / Cas is derived from a type II CRISPR / Cas system. In other embodiments, the CRISPR / Cas system is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species.

[0152] In general, Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with the guiding RNA. Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. The Cas proteins can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In certain embodiments, the Cas-like protein of the fusion protein can be derived from a wild type Cas9 protein or fragment thereof. In other embodiments, the Cas can be derived from modified Cas9 protein. For example, the amino acid sequence of the Cas9 protein can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, and so forth) of the protein. Alternatively, domains of the Cas9 protein not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas9 protein is smaller than the wild type Cas9 protein. In general, a Cas9 protein comprises at least two nuclease (i.e., DNase) domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC and HNH domains work together to cut single strands to make a double-stranded break in DNA. (Jinek, et al., 2012. Science, 337:816-821). In certain embodiments, the Cas9-derived protein can be modified to contain only one functional nuclease domain (either a RuvC-like or a HNH-like nuclease domain). For example, the Cas9-derived protein can be modified such that one of the nuclease domains is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent). In some embodiments in which one of the nuclease domains is inactive, the Cas9-derived protein is able to introduce a nick into a doublestranded nucleic acid (such protein is termed a "nickase"), but not cleave the double-stranded DNA. In any of the above-described embodiments, any or all of the nuclease domains can be inactivated by one or more deletion mutations, insertion mutations, and / or substitution mutations using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.

[0153] In one non-limiting embodiment, a vector drives the expression of the CRISPR system. The art is replete with suitable vectors that are useful in the present disclosure. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. The vectors of the present disclosure may also be used for nucleic acid standard gene delivery protocols. Methods for gene deliver}' are known in the art (U.S. Patent Nos. 5,399,346. 5,580,859 & 5.589,466, incorporated by reference herein in their entireties).

[0154] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New' York, 2012), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326.193).

[0155] In some embodiments, the compound that downregulates the activity or expression level of Pcdhg cluster member / Pcdhga9 comprises a nucleic acid that downregulates the expression level of Pcdhg cluster member / Pcdhga9 by the means of CRISPR knockdown. CRISPR knockdown includes, but not limited to, CRISPRCasl3 knockdown. (See e.g., Mendez-Mancilla et al., Cell Chemical Biology 29, 1-7, 2021 Jul 27, and Kushawah et al., Dev Cell. 2020 Sep 28;54(6):805-817. The entireties of which are incorporated herein by reference).

[0156] In some embodiments, the present invention includes any other methods for effecting gene knockdown and / editing, which allow for deletion and / or inactivation of PDL3, such as but not limited to those described in WO 2018 / 236840 (which is incorporated herein in its entirety by reference).

[0157] Downregulating Pcdhg cluster member / Pcdhga9 by inactivating and / or sequestering

[0158] In some embodiments, the compound that downregulates the activity or expression level of Pcdhg cluster member / Pcdhga9 includes a protein that downregulates the activity7of Pcdhg cluster member / Pcdhga9 by inactivating and / or sequestering Pcdhg cluster member / Pcdhga9. In some embodiment, the compound includes a nucleic acid that express the protein that downregulates the activity of Pcdhg cluster member / Pcdhga9 by inactivating and / or sequestering Pcdhg cluster member / Pcdhga9. In some embodiments, the compound includes an expression vector that express the protein that downregulates the activity of Pcdhg cluster member / Pcdhga9 by inactivating and / or sequestering Pcdhg cluster member / Pcdhga9 (see '‘Vector” section for descriptions on vectors).

[0159] In some embodiments, the compound that downregulates the expression level of Pcdhg cluster member / Pcdhga9 is a trans-dominant negative mutant of Pcdhg cluster member / Pcdhga9, and / or a nucleic acid or a vector expressing the trans-dominant negative mutant of Pcdhg cluster member / Pcdhga9.

[0160] Method of Treating, Ameliorating and / or Preventing Vascular Inflammation-Associated Diseases / Disorders

[0161] In some embodiments, the present disclosure is directed to a method of treating, ameliorating, and / or preventing a disease or a disorder in a subject in need thereof. In some embodiments, the disease or a disorder is caused by or involving vascular inflammation. In some embodiments, the subject is a mammal, such as a human.

[0162] Non-limiting examples of diseases or disorders caused by or involving vascular inflammation include atherosclerotic cardiovascular disease, coronary artery disease, peripheral disease, cerebral artery disease, pulmonary arterial hypertension, lung or kidney injury’ or intravascular coagulation caused by sepsis, diabetic vasculopathy, or transplant rejection.

[0163] In some embodiments, the method includes administering to the subject an effective amount of a compound that down-rcgulates Pcdhg cluster member / Pcdhga9, such as a level or an activity thereof. In some embodiments, the compound that down-regulates Pcdhg cluster member / Pcdhga9 is the same as or similar to those described elsewhere herein, such as in the “Method of Treating, Ameliorating, and / or Preventing Vascular Inflammation” section.

[0164] Vectors

[0165] Vectors can increase the stability’ of the nucleic acids, make the delivery easier, or allow the expression of the nucleic acids or protein products thereof in the cells.

[0166] Therefore, in some embodiments, the protein inhibitors or the nucleic acids that that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level is incorporated into a vector.

[0167] In some embodiments, the present disclosure relates to a vector, including the nucleic acid sequence of the present disclosure or the construct of the present disclosure. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In certain embodiments, the vector of the present disclosure is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In certain embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokary ote- and / or eukaryote-vector based systems can be employed for use with the present disclosure to produce polynucleotide, or their cognate polypeptides. Many such systems are commercially and widely available.

[0168] In some embodiments, the vector is a viral vector. Viral vector technology is well known in the art and is described, for example, in virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.

[0169] (See, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193.

[0170] In some embodiments, the viral vector is a suitable adeno-associated virus (AAV), such as the AAV1-AAV8 family of adeno-associated viruses. In some embodiments, the viral vector is a viral vector that can infect a human. The desired nucleic acid sequence, such as the nucleic acids that downregulates a Pcdhg cluster member / Pcdhga9 described above, can be inserted between the inverted terminal repeats (ITRs) in the AAV. In various embodiments, the viral vector is an AAV2 or an AAV8. The promoter can be a thyroxine binding globulin (TBG) promoter. In various embodiments, the promoter is a human promoter sequence that enables the desired nucleic acid expression in the vascular endothelial cells. In some embodiments, the promoter is a vascular endothelium-selective promoter. The AAV can be a recombinant AAV, in which the capsid comes from one AAV serotype and the ITRs come from another AAV serotype. In various embodiments, the AAV capsid is selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and a AAV8 capsid. In various embodiments, the ITR in the AAV is at least one ITR selected from the group consisting of a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and an AAV8 ITR. In various embodiments, the present disclosure contemplates an AAV8 viral vector (recombinant or non-recombinant) containing a desired nucleic acid expression sequence and at least one promoter sequence that, when administered to a subject, causes elevated systemic expression of the desired nucleic acid. In some embodiments, the viral vector is a recombinant or non-recombinant AAV2 or AAV5 containing any of the desired nucleic acid expression sequences described herein.

[0171] In some embodiments, the vector in which the nucleic acid sequence is introduced is a plasmid that is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the present disclosure or the gene construct of the present disclosure can be inserted include a tet- on inducible vector for expression in eukaryote cells.

[0172] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In certain embodiments, the vector is a vector useful for transforming animal cells.

[0173] In certain embodiments, the recombinant expression vectors may also contain nucleic acid molecules which encode a peptide or peptidomimetic inhibitor of the present disclosure, described elsewhere herein.

[0174] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either dow nstream or upstream of that sequence. Alternatively, certain advantages will be gained bypositioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated w ith a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory- regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may- be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0175] It will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how- to use promoters, enhancers, and cell type combinations for protein expression. The promoters employed may be constitutive, tissue-specific, inducible, and / or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0176] The recombinant expression vectors may also contain a selectable marker gene which facilitates the selection of transformed or transfected host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG. The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0177] Combination Therapies

[0178] In some embodiments, the method of treating, ameliorating, and / or preventing the vascular inflammation or the associated diseases or disorders includes administering to the subject the effective amount of at least one compound and / or composition contemplated within the disclosure.

[0179] In some embodiments, the composition for treating vascular inflammation and associated diseases / disorders includes at least one compound and / or composition contemplated within the disclosure.

[0180] In some embodiments, the subject is further administered at least one additional agent that treats, ameliorates, and / or prevents a disease and / or disorder contemplated herein. In other embodiments, the compound and the at least one additional agent are co-administered to the subject. In yet other embodiments, the compound and the at least one additional agent are co-formulated.

[0181] The compounds contemplated within the disclosure are intended to be useful in combination with one or more additional compounds. These additional compounds may comprise compounds of the present disclosure and / or at least one additional agent for treating one or more diseases or disorders contemplated herein.

[0182] A synergistic effect may be calculated, for example, using suitable methods such as, for example, the Sigmoid-Emax equation (Holford & Scheiner. 1981. Clin. Pharmacokinet. 6:429-453), the equation of Loewe additivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol. 114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Each equation referred to above may be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

[0183] Administration / Dosage / Formulations

[0184] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations contemplated within the disclosure may be administered to the subject either prior to or after the onset of a disease and / or disorder contemplated herein. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations contemplated within the disclosure may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0185] Administration of the compositions contemplated within the disclosure to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures. at dosages and for periods of time effective to treat a disease and / or disorder contemplated herein in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound contemplated within the disclosure to treat a disease and / or disorder contemplated herein in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A nonlimiting example of an effective dose range for a therapeutic compound contemplated within the disclosure is from about 1 and 5,000 mg / kg of body weight / per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0186] Actual dosage levels of the active ingredients in the pharmaceutical compositions contemplated within the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0187] In particular, the selected dosage level depends upon a variety' of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex. weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

[0188] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds contemplated within the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0189] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated: each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms contemplated within the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic compound for the treatment of a disease and / or disorder contemplated herein.

[0190] In certain embodiments, the compositions of the disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the disclosure comprise a therapeutically effective amount of a compound of the disclosure and a pharmaceutically acceptable carrier.

[0191] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0192] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In another embodiment, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every' two, days, every' three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

[0193] Compounds of the disclosure for administration may be in the range of from about 1 pg to about 10,000 mg, about 20 pg to about 9,500 mg, about 40 pg to about 9,000 mg, about 75 pg to about 8.500 mg, about 150 pg to about 7,500 mg, about 200 pg to about 7,000 mg, about 3050 pg to about 6,000 mg, about 500 pg to about 5,000 mg, about 750 pg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1.000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

[0194] In some embodiments, the dose of a compound of the disclosure is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the disclosure used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg. or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg. or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0195] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of vascular inflammation and associated diseases / disorders in a patient.

[0196] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intracranially, intrathecal , oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

[0197] Routes of administration of any of the compositions of the disclosure include oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the disclosure may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal. (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0198] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry' powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0199] Oral Administration

[0200] For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

[0201] For oral administration, the compounds of the disclosure may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g.. polyvinylpyrrolidone, hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY -P Type, Aqueous Enteric OY -A Type, OY -PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g.. methyl or propyl p-hydroxy benzoates or sorbic acid).

[0202] The present disclosure also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the disclosure, and a further layer providing for the immediate release of another medication. Losing a wax / pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

[0203] Parenteral Administration

[0204] For parenteral administration, the compounds of the disclosure may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and / or dispersing agents may be used.

[0205] Additional Administration Forms

[0206] Additional dosage forms of this disclosure include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488.962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of this disclosure also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of this disclosure also include dosage forms as described in PCT Applications Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0207] Controlled Release Formulations and Drug Delivery Systems

[0208] In certain embodiments, the formulations of the present disclosure may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

[0209] The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

[0210] For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the disclosure may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

[0211] In certain embodiments of the disclosure, the compounds of the disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0212] The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

[0213] The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

[0214] The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

[0215] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.

[0216] As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

[0217] Dosing

[0218] The therapeutically effective amount or dose of a compound of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of the vascular inflammation or diseases / disorders associated therewith in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

[0219] A suitable dose of a compound of the present disclosure may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

[0220] It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every' other day, every 2 days, every 3 days, every' 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

[0221] In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the modulator of the disclosure is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%. 40%. 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0222] Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the patient's condition, to a level at which the improved disease is retained. In certain embodiments, patients require intermittent treatment on a longterm basis upon any recurrence of symptoms and / or infection.

[0223] The compounds for use in the method of the disclosure may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0224] Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Capsid assembly modulators exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such capsid assembly modulators lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

[0225] Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this disclosure and covered by the claims appended hereto. For example, it should be understood, that modifications in assay and / or reaction conditions, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present disclosure.

[0226] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present disclosure. Moreover, all values that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the present disclosure.

[0227] Examples

[0228] The present disclosure further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the present disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Example 1: Endothelial y-protocadherins inhibit KLF2 and KLF4 to promote atherosclerosis

[0229] Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide. Laminar shear stress from blood flow, sensed by vascular endothelial cells, protects from ASCVD by upregulating the transcription factors KLF2 and KLF4, which induces an anti-inflammatory program that promotes vascular resilience. Here the present study identify clustered y-protocadherins as therapeutically targetable, potent KLF2 and KLF4 suppressors whose upregulation contributes to ASCVD. Mechanistic studies show that y-protocadherin cleavage results in translocation of the conserved intracellular domain to the nucleus where it physically associates with and suppresses signaling by the Notch intracellular domain. y-Protocadherins are elevated in human ASCVD endothelium; their genetic deletion or antibody blockade protects from ASCVD in mice without detectably compromising host defense against bacterial or viral infection. These results elucidate a fundamental mechanism of vascular inflammation and reveal a method to target the endothelium rather than the immune system as a protective strategy in ASCVD.

[0230] Atherosclerotic cardiovascular disease (ASCVD), which is characterized by fatty plaques within arterial walls, results from converging metabolic, inflammatory' and biomechanical factors, including hypertension, hyperlipidemia, smoking and age. Atherosclerotic plaques form preferentially at curved or branched regions of arteries experiencing low, multidirectional shear stress from blood flow, termed disturbed shear stress (DSS). DSS is also the most reliable predictor of plaque erosion and plaque vulnerability' to rupture. Conversely, straight regions with high unidirectional laminar shear stress (LSS) suppress plaque formation mainly by upregulating the Kruppel-like 2 (KLF2) and 4 (KLF4) transcription factors in endothelial cells (ECs). The two genes, KLF2 and KLF4, which are generally7co-regulated in ECs and have partially redundant gene targets and functions, govern approximately 70% of LSS-induced anti-inflammatory' and antithrombotic protective genes. Extensive studies in mice, using both EC-specific knockout (ECKO) and transgenic overexpression, identified KLF2 and KLF4 as potent mediators of resilience against a multiplicity' of vascular conditions, including atherosclerosis, pulmonary hypertension and coronavirus disease 2019-mediated vascular dysfunction. Reduced endothelial KLF2 and KLF4 expression is similarly associated with worsened cardiovascular outcomes in humans. Therefore, restoring high KLF2 and KLF4 expression may protect against a range of inflammatory cardiovascular diseases (CVDs). However, the fundamental mechanisms regulating KLF2 and KLF4 expression are not fully understood.

[0231] Toward this goal, the present study carried out a genome-wide CRISPR knockout (KO) screen using a green fluorescent protein (GFP) reporter driven by the human KLF2 promoter, which identified approximately 300 genes required for shear stress induction of KLF2. Systematic analysis of the genes whose CRISPR KO reduced KLF2 (activators) identified a contribution from mitochondrial metabolism that synergizes with the established mechanism, the MEKK2 and MEKK3-MEK5-ERK5 kinase cascade. Unexpectedly, this screen uncovered an additional 160 genes whose KO increased KLF2 (suppressors). These genes are of great interest both to gain mechanistic insight into KLF2 regulation and as candidate therapeutic targets in vascular inflammatory diseases.

[0232] The present study selected protocadherin y subfamily A, 9 (PCDHGA9) for further study. This gene is a member of the 22-gene PCDHG subfamily among the clustered protocadherin (cPCDH) family of homophilic adhesion receptors. cPCDHs mediate a wide range of functions in the central nervous system, including adhesion, signaling and cell sorting. PCDHG has an essential role in neurons and its misexpression is associated with neuronal disorders; however, little is known about PCDHG in other cell types. The present study describes a role for the PCDHG cluster in vasculature inflammation and ASCVD.

[0233] Example 2: CRISPR screen identifies suppressors of KLF2

[0234] To identify the mechanisms underlying the mechanoregulation of KLF2, a genomewide CRISPR screen was performed using a A7 / 2:GFP reporter expressed in mouse aortic ECs (MAECs) and stimulated with LSS for 18 h (Figs. 1A-1B and 7A). In addition to the approximately 300 genes whose CRISPR KO decreased KLF2, this screen also identified approximately 160 genes whose KO increased the / 2:GFP reporter levels (Figs. 7B). Network analysis showed enrichment of pathways and processes that affect vascular and endothelial functions (Fig. 1C), consistent with a central role for KLF2 and KLF4 in ECs.

[0235] Reasoning that transmembrane proteins would be readily accessible for therapeutic intervention, the present study focused on cell surface candidates (Fig. ID, box). Of these, the top eight candidates were functionally verified using independent CRISPR single-guide RNAs (sgRNAs) by assaying for A7 / AGFP induction on LSS and oscillatory shear stress (OSS), commonly used to model in vivo DSS (Fig. ID, box). The top candidate, PCDHGA9, was chosen for further study. Examining induction of the 7 / 2:GFP reporter in the MAEC cell line under both LSS and OSS showed that CRISPR KO of Pcdhga9 increased A7 / 2:GFP in cells under LSS and did so even more strongly under OSS, where expression is normally low (Fig. IE). Conversely, overexpression of human PCDHGA9 suppressed reporter expression. Knockdown of Pcdhga9 using small interfering RNA (siRNA) similarly increased 7 / AGFP. which was reversed by overexpression of human protein (Figs. 7C-7E). Examining endogenous Klf2 in primary human umbilical vein ECs (HUVECs) replicated this behavior, demonstrating conservation across species and EC types (Fig. 7F). In ECs under pro-inflammatory OSS, PCDHGA9 depletion also suppressed the induction of the leukocyte adhesion receptor VCAM1 (Fig. IF) and the adhesion of THP1 monocytes (Fig. 1G). PCDPIGA9 thus restrains LSS-dependent induction of anti-inflammatory KLF2 and potentiates OSS-dependent pro-inflammatory activation.

[0236] Example 3: Protocadherin y gene cluster suppresses KLF2 and KLF4

[0237] PCDHGA9 is a member of the clustered protocadherin family, which belongs to the cadherin superfamily of cell-cell adhesion receptors. The clustered protocadherin genes, classified into alpha (a or a), beta (b or P) and gamma (g or y) clusters, are organized in tandem in the genome. The mouse Pcdhg gene cluster includes 22 genes (Fig. 2A). several of which are expressed in ECs (as shown in this study). Exon 1 is unique, while exons 2-4 are shared by all 22 members, allowing for targeting of the entire Pcdhg cluster by siRNA- mediated silencing (Fig. 2A, region enclosed between the dashed lines). Silencing the Pcdhg gene cluster in HUVECs (Fig. 2B and 8A) strongly increased endogenous Klf2 and Klf4 mRNA levels and suppressed pro-inflammatory E-selectin (Sele) even under OSS (Figs. 2C- 2D and 8B). Depletion of the Pcdhg gene cluster suppressed adhesion of THP1 monocytes to ECs under pro-inflammatory OSS to the same extent as Pcdhga9 depletion (Fig. 2E). Targeting Pcdhga9 or the entire cluster thus gives indistinguishable results.

[0238] In addition to LSS, KLF2 and KLF4 are also induced by statins, the cholesterol- lowering drugs used as first-line therapy for patients at risk of ASCVD, an effect believed to contribute to their therapeutic benefits. To test for interactions, control or / / ig-depleted HUVECs were treated with lovastatin for 16 h. Pcdhg depletion greatly amplified statin induction of KLF4 (Fig. 2F), showing clear synergy. Conversely, pro-inflammatory tumor necrosis factor (TNF) suppressed KLF4 and induced pro-inflammatory genes, including the leukocyte adhesion receptor VCAM1. Pcdhg depletion alleviated KLF4 suppression by TNF, suppressed TNF-induced VCAM1 expression and subsequent adhesion of THP1 monocytes (Figs. 2G-2H). Pcdhg depletion significantly reduced the induction of VCAM1 even at the highest doses of TNF tested (Fig. 8C). Targeting Pcdhg thus suppressed EC inflammatory activation in multiple contexts. Example 4: Pcdhg endothelial KO protects against atherosclerosis

[0239] EC KLF2 and KLF4 are essential for vascular resilience against inflammatory CVDs, including atherosclerosis. Importantly, elevating KLF4 in ECs protects against ASCVD, demonstrating its sufficiency for atheroprotection. To test if the effects of Pcdhg seen in culture are retained in vivo, PcdhgloxP con3(loxP- targeted Pcdhg constant exon 3, tagged with GFP) mice were crossed with constitutive Cdh5-Cre mice to delete the entire Pcdhg cluster in ECs (ECKO) (Fig. 3A). Homozygous Pcdhg ECKO pups were obtained at the expected Mendelian frequency and were phenotypically normal, showing that Pcdhg expression in ECs, unlike in neurons, is not essential for development (Figs. 3B and 9A-9C). These mice were thus further analyzed.

[0240] The aortic arch contains the athero-resistant greater curvature that is under LSS and the nearby athero-susceptible lesser curvature that is under DSS, offering a well-established model. Examination en face revealed modestly increased KLF4 expression in Pcdhg ECKO ECs in the greater curvature and a markedly larger increase in the lesser curvature compared to controls (Fig. 3C). The present study next induced hyperlipidemia by injecting adeno- associated virus 8 (AAV8)-PCSK9 and feeding them a high-fat diet (HFD) for 16 weeks (Fig. 3D). Pcdhg ECKO mice showed reduced atherosclerotic plaques as observed using Oil Red O staining in aortas from both males and females (n = 6, each sex) (Figs. 3E-3F). Examination of the aortic root using hematoxylin & eosin (H&E) and Oil Red O staining showed smaller atherosclerotic plaques with drastically smaller necrotic cores (NCs), thicker fibrous caps (FCs) (Fig. 3G) and greatly reduced macrophage and monocyte content in Pcdhg ECKO mice (Fig. 3H). No differences were observed in blood lipids (triglycerides, cholesterol, high-density lipoprotein cholesterol (HDL-C)) (Figs. 9D-9F) or body weight (Fig. 9G).

[0241] Example 5: Immune host-pathogen defense

[0242] Limiting EC inflammatory gene expression could conceivably suppress immune responses via effects on leukocyte trafficking or other mechanisms. To assess immune function, control and Pcdhg ECKO mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13, which triggers T cell activation. Seven days after infection, spleen cell readouts and function were measured using fluorescence-activated cell sorting (FACS). Control and Pcdhg ECKO mice displayed similar frequency of CD8a+T cells (Fig. 31) and similar functional capacity after restimulation with an LCMV -specific peptide (GP33) as observed by unaltered GP33 tetramer4, granzyme B+, interferon-y (IFNy)+TNF+and IFNy TNF populations (Figs. 3J-3M). The viral loads, measured as plaque-forming units (PFUs) from kidneys were similar in control and Pcdhg ECKO mice (Fig. 3N). Myeloid compartment (CD1 lb+) cells were similarly identical in frequency and activation status (Figs. 10A-10C). Thus, Pcdhg ECKO had no detectable effect on host defense against this virus.

[0243] The present study next examined responses to bacterial infection using a self-limited Escherichia coli serotype O6:K2:H1 peritonitis model that allows for assessment of the initiation, peak and resolution phases of inflammation. Pcdhg ECKO mice showed no deficit in bacterial defense; instead and suprisingly, they showed faster bacterial clearance compared to controls, as evidenced by reduced bacterial colony -forming units (CFUs) from peritoneal exudate 12 h after infection, in both males and females (Figs. 30 and 10D-10E); 12-24 h after infection marked the peak of inflammation in this study (Figs. 30 and 10D). No CFUs were detected in the blood from either control or Pcdhg ECKO mice, demonstrating no systemic bacterial dissemination (Fig. 10F). Bacteria trigger acute inflammation that mobilizes neutrophils from bone marrow (BM) reservoirs to the site of infection, which, owing to their short half-life (approximately 6 h), resolves within several hours. Accordingly, the peak of neutrophil recruitment and the percentage (of CD 1 lb+F4 / 80 cells) in the peritoneal exudate were lower in Pcdhg ECKO mice compared to controls (Figs. 3P and 10G-10H). The total number of leukocytes followed a similar but nonsignificant trend (Fig. 101). Although Pcdhg ECKO mice showed fewer neutrophils accumulating, peritoneal neutrophils collected at 12 h showed increased per-cell phagocytic clearance of bacteria compared to controls (Fig. 3Q and 10J-10K). BM neutrophils from uninfected mice showed no difference in phagocytic capacity, ruling out an inherent difference in neutrophil function (Fig. 10L). Thus. Pcdhg ECKO mice showed somewhat slower neutrophil recruitment in response to infection, but faster clearance of bacteria associated with greater phagocytosis per neutrophil. The combined data from viral and bacterial challenges showed that Pcdhg ECKO does not detectably limit host immune responses.

[0244] Example 6: Nuclear PCDHG intracellular domain suppresses KLF2 and KLF4

[0245] To investigate the molecular mechanism by which PCDGH suppresses KLF2 and KLF4, the present study performed a structure-function analysis using the alternative splicing of Pcdhg cluster members to define domain boundaries. Each PCDGH protein is organized into an extracellular domain (ECD) containing six cadherin domains involved in homophilic or heterophilic cis and trans interactions, a transmembrane (TM) domain governing its membrane localization and an intracellular domain (ICD) involved in intracellular trafficking, surface delivery, localization and signaling. The ICD is subdivided into a variable C-terminal domain (VCD) and a constant C-terminal domain (CCD). The conserved CCD is encoded by the three 3' exons shared by all 22 members (Fig. 4A).

[0246] Overexpression of Pcdhga9 suppressed KLF2 and KLF4 (Figs. IE and 7E). Therefore, the present study used this assay to identify functional domains. Full-length, ECD + TM, TM + ICD and CCD-lacking (ACCD) mutants (Figs. 4A-4B) were expressed in HUVECs, which were treated with LSS and OSS and assayed for KLF4 protein. The mutants were expressed at comparable levels and localization was analyzed using GFP fluorescence (Figs. 4B and 11 A-l IB). While the full-length mutant localized to both cell-cell junctions and intracellular vesicles, ECD + TM and ACCD were more junctional, as expected (owing to reduced internalization in the absence of the CCD), while TM + ICD was a mixture of punctate and distributed evenly (Fig. 1 IB). KLF4 immunoblotting revealed that the CCD is essential for KLF2 and KLF4 suppression, whereas the ECD is dispensable (Figs. 4B and 11 A). Further experiments thus focused on the CCD.

[0247] Unlike classical cadherins. PCDHG members are processed by proteolytic cleavage; this releases the ICD, which due to its nuclear localization signal (NLS) translocates to the nucleus (Fig. 4A). To test the role of cleavage and nuclear translocation, FLAG-tagged versions of the CCD and an NLS-deleted CCD mutant (ANLS-CCD) were expressed in HUVECs, stimulated with LSS and the KLF4 levels assayed (Fig. 4A). Mutants were expressed at comparable levels and localized as expected, with the full-length mutant present in the secretory’ system and plasma membrane, the CCD mainly in the nucleus and the ANLS- CCD in the cytoplasm (Fig. 11 C). The CCD suppressed KLF4 whereas the ANLS mutant was ineffective (Fig. 4C). Interestingly, the CCD is very highly conserved across species, supporting its critical role (Figs. 1 ID-1 IE). Thus, PCDHG signals via cleavage and nuclear translocation of its common cytoplasmic regions.

[0248] Example 7: Pcdhg regulates Klf2 and Klf4 via the Notch pathway

[0249] To assess the genes and processes regulated by PCDHG in ECs, the present study- performed bulk RNA sequencing (RNA-seq) analysis of control siRNA and Pcdhg siRNA HUVECs under OSS (where PCDHG had the greatest effect). The present study included Pcdhg + Klf2 and Klf4 triple siRNA to identify- Klf2 and V / fy-independent effects (Figs. 4D- 4E). Upstream regulatory pathway analysis of these T ig-dependent and Klf2 and Klf4- independent differentially expressed genes (DEGs) identified Notch as the most overrepresented pathway (Fig. 4F), confirmed by strong upregulation of known Notch target genes (Fig. 4G). Notchl-4 family transmembrane receptors are critical for EC functions, including determination of arterial identity and vascular stability, with Notchl being the main isoform. Binding of Notch ligands such as DLL4 triggers stepwise proteolysis of the receptors by members of the ADAM metalloprotease family followed by y-secretase, releasing the Notch intracellular domain (NICD), which translocates to the nucleus and binds the transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBPJ) to induce target genes. Interestingly, LSS both activates Notch and induces KLF2 and KLF4, both of which stabilize and protect vessels against inflammation and atherosclerosis, although the effects of Notch and KLF2 and KLF4 have not been linked. The present study found that Pcdhg depletion increased the levels of the cleaved, activated NICD (Vai 1744) (Fig. 4H). Notably, the promoters for both human KLF2 and mouse Klf2 and Klf4 contain previously unappreciated canonical Notch-RBPJ binding sites (Figs. 5F-5G). To test the role of Notch in KLF2 and KLF4 induction by LSS, RBPJ was blocked by the smallmolecule inhibitor RBPJ Inhibitor- 1 (RIN1) or by RBPJ siRNA, with or without Pcdhg depletion. ECs under LSS were then examined. The increase in KLF4 levels upon Pcdhg siRNA was completely prevented by inhibition of the Notch pathway (Fig. 41). The present study concludes that Pcdhg regulates Klf4 via Notch.

[0250] To identify proteins that interact with the Pcdhg CCD, the present study did a proteomic analysis of immunoprecipitates (IPs) from full-length Pcdhga9 and the ACCD mutant, looking for binding partners that required the CCD. Notchl peptides were detected in the IPs of full-length Pcdhga9 but not the ACCD mutant or vector alone. Sequence analysis showed that these Notchl peptides came from the NICD region (Figs. 4J and 11H), which is consistent with functional effects. The immunoprecipitation and immunoblot analyses showed that in addition to full-length Pcdhg, the CCD fragment but not ANLS-CCD physically associated with the NICD (Figs. 4K and 4L). Finally, a Notch transcriptional activity reporter showed that Pcdhg ICD (PICD) harboring the conserved CCD suppressed NICD transcriptional activity in a dose-dependent manner (Fig. 4M). Together, these results show that PICD physically associates with NICD to suppress Notch signaling and its downstream target Klf2 and Klf4.

[0251] Example 8: PCDHGA9 blocking antibody in experimental atherosclerosis

[0252] PCDHG was chosen as a target in part because its cell surface localization makes it amenable to inhibition by antibodies or other cell-impermeant reagents. Toward this goal, the present study purified murine PCDHGA9 ECD protein, which was used both for generating monoclonal antibodies (mAbs) and for developing a cell adhesion assay (Figs. 5 A and 12A). Cells were plated in 96-well microplates coated with PCDHGA9 ECD adhered over time; adhesion to uncoated wells was minimal (Fig. 5B), demonstrating specificity.

[0253] Rat hybridomas against PCDHGA9 ECD (Figs. 12A-12B) were purified and tested in the homophilic adhesion assay. Antibodies A9, Bl and B4 strongly blocked adhesion (Figs. 5C and 12C). Antibody specificity was verified by immunoblotting using purified ECD-GST or ECD alone (Fig. 12D). Next, these function-blocking mAbs were tested for their effect on KLF2 and KLF4 upon LSS, and VCAM1 upon OSS. Blocking antibodies increased flow induction of the / 2. GFP reporter and decreased VCAM1, normalized to the internal mCherry control (Figs. 5D-5E and 12E-12F). mAb A9 showed the strongest effect on adhesion to ECD, KLF2 induction upon LSS, VCAM1 induction upon OSS and the highest signal -to-noise ratio in the immunofluorescence (IF) assay (Fig. 12G). Hence, mAb A9 was studied further.

[0254] A9 was highly specific, as shown by immunoblotting of cell lysates from control versus Pcdhg or Pcdhga9 knockdown MAECs (Fig. 121). with purified ECD as a positive control. Therefore, the present study examined its efficacy in a model of experimental atherosclerosis in mice. Because A9 is a rat monoclonal antibody, the present study avoided immune recognition and clearance of the rat IgG by using the PCA ligation model of accelerated atherosclerosis in Apoe ' mice where lesions develop within 1 week (Fig. 5F). Mice were maintained on an HFD for a total of 3 weeks (1 week before ligation and 2 weeks after ligation). The A9 half-life, determined by intraperitoneal injection of 2 pg of isotype control or mAb A9 per mouse and measuring blood plasma levels, was approximately 8 days (Fig. 121). Hence, Apoe mice on an HFD were subjected to surgery and A9 versus control IgG was injected once per week for 2 weeks as described in the Example 1 1 (Fig. 5G). The operated left carotid artery (LCA) was then compared to the unligated right carotid artery (RCA) as an internal control. Treatment with mAb A9 strongly reduced plaque in this acute model of ASCVD, as seen by whole-mount carotid preparations (Fig. 5H, brackets) and verified by staining carotid sections with Oil Red O to detect hpid-rich plaques (Fig. 51). Reduction in lumen diameter was also prevented by A9 (Fig. 12J). No difference was observed in blood lipids (triglycerides and cholesterol) (Figs. 12K and 12L). Taken together, these results identify PCDHG as a new therapeutic target in atherosclerosis.

[0255] Example 9: PCDHG expression in atherosclerosis Lastly, the present study analyzed the levels of PCDHG in human arteries. Staining for PCDHG using an antibody against the CCD showed approximately three times higher expression of PCDHG in the endothelium (marked by eRG) from CVD donors compared to healthy, age-matched donors (Fig. 6A). Atherosclerotic mouse arteries also showed elevated PCDHG staining (Fig. 13 A). Coronary arteries from three asymptomatic older donors stained for PCDHG showed approximately a four times higher signal in the regions of plaque compared to regions of the same artery without evident plaque (Fig. 12B). Antibody specificity was verified with immunoblotting using control and PCDHG siRNA HUVECs (Fig. 2B) and staining of control versus PCDHG ECKO retinas (Fig. 13B). PCDHG is thus upregulated in the endothelium from individuals with ASCVD.

[0256] Example 10:

[0257] ECs have a pivotal role in vascular physiology7and pathology7via functions ranging from regulating vessel diameter to immune responses to nutrient transport. Cell-cell adhesions are a locus of EC signaling and function, including solute transport, leukocyte trafficking, growth control and shear stress signaling. This study is based on a genome-wide CRISPR KO screen that identified approximately 160 genes that suppress KLF2. KLF2 and KLF4 are generally co-regulated in ECs and show a high degree of functional redundancy; indeed, they recognize the identical consensus sequence in gene promoters and enhancers. The results obtained for the two were essentially identical in this study. The present study selected the strongest cell-surface-localized KLF2 suppressor, PCDHGA9, a member of the PCDHG family of adhesion receptors, for further study. In this study, depletion of PCDHGA9 or the whole PCDHG cluster showed essentially identical results. The present study found that PCDHG is a pro-inflammatory gene that suppresses KLF2 and KLF4 through a pathway that involves the release and translocation of the intracellular domain of its protein product to the nucleus where it binds the Notchl ICD to suppress transcription of Notch target genes. EC deletion of Pcdhg in mice increases KLF2 and KLF4 levels and protects against ASCVD without apparent effects on development or viability. Antibodies that block homophilic PCDHG adhesion also increase KLF2 and KLF4 and limit experimental atherosclerosis. Within the vasculature, Pcdhg is expressed primarily in ECs, with marked increases in atherosclerotic regions and low expression in other cell types.

[0258] Analysis of / W / ig-dependent genes identified the Notch pathway as a major downstream target. Notchl limits angiogenesis in development and promotes arterial identity and decreases atherosclerosis in adults; thus, it was further investigated. Mechanistic data in this study showed Notch as a direct inducer of KLF2 and KLF4 via consensus sites in their promoters and enhancers, which is suppressed by PCDHG, and which correlates with physical association of the PICD and NICD.

[0259] Example 11: Materials and Methods

[0260] Primary cells, cell lines and cell culture reagents

[0261] The PyMT-immortalized MAECs that express the Klf2 promoter reporter (A7 / 2:GFP MAEC) were described in Coon et al. (J. Cell Biol. 221, e202109144 (2022)). MAECs were maintained in complete EC medium (cat. no. Ml 166, Cell Biologies). HUVECs obtained from Yale Vascular Biology and Therapeutics Core were pooled from three donors. They were screened for the absence of pathogens, maintained in EGM2 endothelial cell growth medium (cat. no. CC-3162, Lonza) and used at passages 2-5. All cells were routinely screened for Mycoplasma. siRNA transfection was performed with Lipofectamine RNAiMAX (cat. no. 13778150, Thermo Fisher Scientific) in Opti-MEM medium (cat. no. 31985070, Thermo Fisher Scientific) using ON-TARGETplus SMARTpool siRNAs from Horizon Discovery. For NICD transcriptional activity reporting, 12><CSL / RBPJ-dlEGFP (cat. no. 47684, Addgene) was used. For lentiviral transduction, HEK 293T cells were transfected with lentiviral vectors along with pVSV-G (cat. no. 138479, Addgene) and psPAX2 (cat. no. 12260, Addgene) packaging plasmids using Lipofectamine 2000 (cat. no. 11668019, Thermo Fisher Scientific) according to the manufacturer’s instructions. Supernatants were collected 48-96 h after transfection and filtered through a 0.45-pm low- protein binding filter. Primary HUVECs were infected with lentivirus for 24 h, then the medium was replaced with EGM2 medium. For the monocyte adhesion assays. THP1 labeled with CellTracker Deep Red (cat. no. C34565, Thermo Fisher Scientific) were resuspended in Hanks’ Balanced Salt Solution (HBSS) supplemented with 1 mM Ca2+, 0.5 mM Mg2+and 0.5% BSA, added to the slides with HUVECs, incubated for 20 min at 37 °C, washed three times in HBSS and fixed with 3.7% formaldehyde. Cells were counterstained with 4', 6- diamidino-2-phenylindole (DAPI) before fluorescence imaging. Drugs used were lovastatin (cat. no. 438185. Sigma-Aldrich), TNF (cat. no. 300-01A, PeproTech) and RIN1 (cat. no. SS3376, Selleckchem). All antibodies were validated in knockdown and KO depletion using IF or immunoblotting.

[0262] CRISPR library screen and KLF2 suppressor (gam-of-function) phenotype determination

[0263] CRISPR library screening, library preparation and next-generation sequencing (NGS) were described in Coon et al. (J. Cell Biol. 221, e202109144 (2022)). Briefly, immortalized Klf2 GFP reporter MAECs were infected with a genome-wide CRISPR library of approximately 160,000 CRISPR sgRNAs, treated with 15 dyn cm2LSS for 18 h, FACS- sorted based on Klf2:GFP reporter levels, with subsequent analysis of sgRNAs in the high 7 / 2:GFP gate by NGS on an HiSeq 2500 system (Illumina). The KLF2 suppressor (gain-of- function) phenotype was determined as done previously for the loss-of function phenotype ranking genes based on the cumulative z-score from the three highest scoring unique sgRNAs.

[0264] Shear stress stimulation

[0265] All shear stress experiments, unless otherwise indicated, were performed in parallel plate flow chambers perfused within a pump and environmental control system as described in Conway et al. (Curr. Biol. 27, 2219-2225 (2017); erratum 27, 2727 (2017)). Briefly, cells were seeded at 70-90% confluency on 10 pg mF1fibronectin-coated glass slides for 48-72 h in complete medium. For shear stress stimulation, slides were mounted in custom-made 25 x 55-mm parallel plate shear chambers with 0.5-mm-thick silicone gaskets and stimulated with 15 dyn cm2for LSS or 1 ± 4 dyn cm2for OSS in complete medium. The medium was maintained at 37 °C and 5% CO2 with a heat gun and humidified bubbler, respectively. An orbital shaker was used for mAh functional testing in vitro (Figs. 6D-6E). The well radius was divided into three equal parts, with the outermost part representing the pulsatile LSS region (to measure Klf2:GFP levels) and the innermost part representing the disturbed shear region (to measure VCAM1 levels).

[0266] Animals and tissue preparation

[0267] Mice were maintained in a light-controlled and temperature-controlled environment with free access to food and water; all efforts were made to minimize animal suffering. PcdhgloxP / con3mice and Cdh5-Cre mice were used. Pcdhg,oxP / co”3mice were a gift from J. Lefebvre, University of Toronto, Canada. All mice in this study were on the C57BL / 6J background. PcdhgloxP / con3and Cdh5-Cre mice were maintained and bred as heterozygotes. Euthanasia was performed using an overdose of isoflurane inhalation and death was confirmed by subsequent cervical dislocation or by removing vital organs or opening the chest canty to verily the absence of cardiovascular function. Mice were perfused through the left ventricle with PBS and then 3.7% formaldehyde followed by tissue collection, as described. The heart and spinal column, with the aorta and carotids attached, were removed and fixed under gentle agitation for an additional 24 h at 4 °C, washed three times with PBS and taken for further analysis. For the whole-aorta en face preparation, the isolated aortas were bisected along the lesser curvature; the aortic arch was also bisected through the greater curvature. For the aortic arch segment preparation, the aortic arch was bisected through the greater curvature. Hearts (containing aortic roots) and carotids were allowed to sink in 30% sucrose in PBS overnight at 4 °C, embedded in optimal cutting temperature (O.C.T.) compound (cat. no. 4583, Sakura) and frozen on dry ice for sectioning. Tissue blocks were cut into 8-10-pm sections using a cryostat (Leica); sections were stored at -80 °C until use.

[0268] Atherosclerosis and blood lipid analysis

[0269] To induce atherosclerosis, murine AAV8-PCSK9 (pAAV / D377Y-mPCSK9; 2 x 1011PFUs) produced by the Gene Therapy Program Vector Core at the University of Pennsylvania School of Medicine was injected intraperitoneally. Mice were maintained on an HFD (Clinton / Cybulsky high-fat rodent diet with regular casein and 1.25% added cholesterol; cat. no. D12108c, Research Diet) for 16 weeks. Blood samples were collected from mice starved overnight, centrifuged at 8,000g at 4 °C for 10 min; the supernatant (plasma) was separated, and HDL-C was isolated by precipitation of non-HDL-C (Wako Pure Chemicals). Both HDL-C fractions and total plasma were stored at -80 °C. Total plasma cholesterol and triglycerides were measured using kits according to the manufacturer’s instructions (Wako Pure Chemicals).

[0270] Tissue analysis

[0271] For IF, O.C.T. tissue sections were thawed and washed three times with PBS to remove O.C.T. Cells were fixed with 3.7% formaldehyde in PBS for 15 min at ambient temperature, washed with and stored in PBS. De-identified human specimens were deparaffinized in Histo-Clear (cat. no. HS-200, National Diagnostics). Sections were progressively rehydrated before antigen retrieval for 30 min at 95 °C in 1 x antigen retrieval buffer (cat. no. 51699, Dako). Samples were incubated in perm-block buffer (5% donkey serum, 0.2% BSA. 0.3% Triton X-100 in PBS) for 1 h at room temperature, incubated with primary antibodies in perm-block overnight at 4 °C, washed three times in perm-block and then incubated with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) at 1 : 1,000 dilution in perm-block for 1 h at room temperature. Slides were washed three times in perm-block and three times in PBS before mounting in DAPI Fluoromount G (cat. no. 0100-20, Southern Biotech). Images were acquired on a Leica SP8 confocal microscope with the Leica Application Suite software. Confocal stacks were flattened by maximum-intensity z-proj ection in Image! After background subtraction, the MFI or nuclear intensity (with DAPI mask) was recorded. For aorta Oil Red O staining, the whole aorta was opened longitudinally on a soft-bottomed silica dish, incubated with Oil Red O solution (0.6% Oil Red O in 60% isopropanol) with gentle rocking for 1 h at ambient temperature, washed in 60% isopropanol for 20 min. washed in distilled H2O three times and mounted on slides with the endothelium side up in O.C.T. compound. Images were acquired with a digital microscopic camera (Leica DFC295, Leica Microsystems). Oil Red O staining on O.C.T. tissue sections was done similarly. Quantitation of Oil Red O+area was done in Image! H&E staining of O.C.T. tissue sections was done by the Yale Research Histology Core using standard techniques. Plaque morphometric and vulnerability analysis was performed as described in Seimon et al. (J. Clin. Invest. 119, 886-898 (2009)). Plaque area was determined by Oil Red O+staining. For each plaque, the NC area was defined as a clear area in the plaque that was H&E-free; FC thickness was quantified by selecting the largest NC and measuring the thinnest part of the cap.

[0272] LCMV infection, immune function analysis and FACS analysis

[0273] Control or Pcdhg ECKO mice were infected with 4 x 106PFU LCMV clone 13 by intravenous injection, euthanized on day 7 after infection and examined for immune function. Spleens were collected for flow cytometry analysis. For the cytotoxic T lymphocyte assay (restimulation assay), CD8a T cells from the spleen were pulsed with LCMV-specific peptide (GP33) or an irrelevant control peptide (SIINFEKL) and assayed for IFNy and TNF levels. Kidneys were collected for viral load assessment.

[0274] E. coli peritonitis

[0275] Methods were performed as described in Libreros et al. (Blood 142, 589-606 (2023)). Briefly, peritonitis was induced in male and female control or Pcdhg ECKO mice byinjecting intraperitoneally with live E. coli serotype O6:K2:H1 at 105CFUs per mouse, in sterile saline. Mice were euthanized and exudates (peritoneal lavage) were collected for flow cytometry at the designated times. For bacterial titers, serially diluted exudates and blood were plated onto lysogeny broth (LB) agar plates and incubated overnight at 37 °C. The next day, LB plates were imaged and CFUs counted using Image! Leukocyte populations were determined by flow cytometry. Peritoneal exudates were stained with surface antibodies: antimouse PerCP / Cy5.5 CD45; anti-mouse APC F4 / 80; anti-mouse APC / Cy7 Ly6G; anti-mouse FITC Ly6C; and anti-mouse PE / Cy7 CD1 lb for the identification of neutrophils (CD45 CD1 1 b F4 / 80 Ly6C Ly6G ). monocytes (CD45 CD 1 l b F4 / 8O Ly6G Ly6C ) and macrophages (CD45+CDl lb+F4 / 80+). For in vivo exudate E. coli phagocytosis, peritoneal leukocytes were collected at 12 h after infection and stained with surface antibodies: antimouse PerCP / Cy5.5 CD45; anti-mouse APC F4 / 80; anti-mouse APC / Cy7 Ly6G; anti-mouse BV421 Ly6C; and anti-mouse PE / Cy7 CDl lb for the identification of neutrophils, monocytes and macrophages. Exudates were then permeabilized using the BD Cytoperm Permeabilization Buffer (BD Biosciences) for 15 min according to the manufacturer’s protocol. Exudates were washed twice with BD Perm / Wash buffer (BD Biosciences). Fc receptor-mediated, nonspecific antibody binding was blocked using CD16 / CD32 Fc block antibody (cat. no. MA5-29707, Thermo Fisher Scientific) and stained for intracellular E. coli using an FITC-conjugated anti-E. coli antibody (1:50 dilution) (cat. no. GTX40856;

[0276] GeneTex). Cells were then analyzed using flow cytometry to assess the percentage intracellular E. coli (FITC+) in macrophages (CD45 CD I I b LyOG Ly6C F4 / 80 ). neutrophils (CD45 CD I l b F4 / 80 Ly6C Ly6G ) and monocytes

[0277] (CD45 CD 1 1 b Ly6G F4 / 80 Ly6C ). For the in situ E. coli phagocytosis assay, BM cells were collected from uninfected mice by flushing with PBS. BM neutrophils were isolated using the EasySep mouse neutrophil enrichment kit (STEMCELL Technologies). BM neutrophils were incubated with BacLight green (cat. no. B35000, Thermo Fisher Scientific) labeled E. coli (1 :25 ratio) at 37 °C for 45 min for phagocytosis, washed, fixed and analyzed using flow cytometry. The BD FACS Diva v.9.0 was used for data acquisition and FlowJo v. 10. 10.0 for analysis.

[0278] Cloning and purification of Pcdhga9 ECD

[0279] Human Pcdhga9 mutants were generated by cloning PCR-amplified fragments into the pBob-GFP vector (Addgene). For GFP-tagged constructs, fragments were cloned upstream and in-frame with the GFP open reading frame. For FL AG-tagged constructs, GFP was excised and the fragments were cloned with an added C-terminal FLAG-tag using PCR. Mouse Pcdhga9 ECD was cloned in the pCDNA3. 1 vector (Invitrogen). Secreted Pcdhga9 ECD-FLAG-GST was purified from the cell culture supernatant from transfected HEK 293T cells by collecting medium at 48 h and 96 h after transfection. Supernatant was spun at 6,000g. at 4 °C for 15 min to remove debris, incubated with 200 pl washed glutathione beads per 40 ml medium O / N at 4 °C with tumbling, washed three times with 0.1% Triton X-100 in PBS, and eluted by adding excess reduced glutathione solution. The concentration of ECD was estimated using Coomassie brilliant blue staining compared to BSA standards.

[0280] Generation and validation of mAbs and PCDHGA9 ECD- -cell adhesion assay

[0281] A total of 1 mg purified PCDHGA9 ECD protein was used for mAb generation in rats (BiCell Scientific). mAbs were purified and concentrated from serum-free hybridoma cultures (BiCell Scientific). Low-adhesion 96-well plates were coated with ECD (10 pg mE1) for 1 h at ambient temperature, washed three times with 0.1% Triton X-100 in PBS, and blocked with 1% heat-denatured BSA in PBS for 1 h at ambient temperature. These plates were used for testing IgG specificity and cell adhesion to ECD, as described below. For specificity, affinity and the amount of IgG, mAbs were added to the plates for 1 h at ambient temperature with shaking, washed three times with 0. 1% Triton X-100 in PBS. incubated with secondary horseradish peroxidase (HRP) antibody (1:5,000 dilution) for 1 h at ambient temperature with shaking, washed three times with 0.1% Triton X-100 in PBS and three times with PBS, follow ed by the addition of 100 pl 3,3 ',5,5 '-tetramethylbenzidine incubated for 15-30 min and absorbance measured at 605 nm, followed by the addition of 100 pl 0. 1 N HC1 and absorbance measured at 450 nm.

[0282] For the cell adhesion assays, wells were washed three times with complete EC medium followed by the addition of 7 / 2:GFP MAECs in 100 pl total EC medium (with isotype control or test mAbs), incubated at 37 °C in the CO2 incubator for the indicated time and fixed by adding 33 pl of 16% paraformaldehyde directly to the wells. Unadhered cells were removed by turning the plate upside down in a water bath and cells counted based on mCherry fluorescence.

[0283] PCA ligation model of accelerated experimental atherosclerosis

[0284] Apoe mice aged 8-10 weeks maintained on an HFD for 1 week w ere anesthetized with ketamine and xylazine; surgery was performed as described in Budatha et al. (J. Am. Heart Assoc. 10, e021160 (2021)). Briefly, three out of four branches of the left common carotid artery (left external carotid, internal carotid and occipital artery) were ligated with sutures, with the superior thyroid artery left intact. Then, 2 pg mAb A9 or isotype control antibody was injected intraperitoneally once every week for 2 weeks. Mice were euthanized with an overdose of isoflurane and perfused through the left ventricle with PBS and then 3.7% formaldehyde. Aortas with carotid arteries were isolated and imaged whole. Carotid arteries were embedded in O.C.T.. sectioned at 10 pm, and immunohistochemistry or IF performed as described. The LCA / RCA inner diameter ratio was calculated by measuring the perimeter to avoid interference from changes in vessel morphology during mounting and handling. For testing mAh retention in vivo (half-life), 1 pg of mAbs (100 pl of 0.25 mg mF1in saline for a 20-g mouse) were injected intraperitoneally in C57BL / 6 mice; 100 pl of blood was collected via the retro-orbital route at the indicated times, centrifuged at 13,000g at 4 °C for 15 min and the plasma was removed and immediately stored at -80 °C. The mAb concentration was determined using a sandwich enzyme-linked immunosorbent assay with immobilized anti-rat IgG (100 ng) as trap and secondary anti-rat IgG HRP antibody for detection, as described above.

[0285] Immunoblotting and immunoprecipitation

[0286] Cells were collected and lysed in radioimmunoprecipitation assay buffer (Roche) containing l x Halt Protease Inhibitor Cocktail (cat. no. 78429, Thermo Fisher Scientific) and 1 x PhosStop (cat. no. 4906837001, Roche) for 30 min on ice, clarified at 13,000g at 4 °C for 15 min, the supernatant was transferred to new 1.5-ml tubes, 4x loading buffer (250 mM Tris- HC1, pH 6.8, 8% SDS, 40% glycerol, 20% p-mercaptoethanol, 0.008% bromophenol blue) added and the samples were heated to 95 °C for 5 min. Cell lysates were resolved by a 4-15% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to 0.2-pm nitrocellulose membranes, which w ere blocked w ith 5% nonfat skimmed milk for 1 h at ambient temperature and incubated with the desired antibodies diluted in 5% BSA using a standard immunoblotting procedure and detection using electrochemiluminescence (Merck Millipore). Images w ere quantified with ImageJ using densitometry’ and normalized to GAPDH or tubulin loading controls. For immunoprecipitation, GFP-trap (cat. no. GTA-20, Chromotech) or FLAG M2 (cat. no. A2220, Sigma-Aldrich) agarose was used. Lysates were collected in 25 mM Tris, pH 7.4, 150 mM NaCl, 1% TritonX-100. lx Halt Protease Inhibitor Cocktail and 1 x PhosStop, clarified at 13,000g at 4 °C for 15 min, incubated with antibody -bound beads at 4 °C for 2 h to overnight. Beads were washed three times with lysis buffer at 4 °C and eluted with 2x protein sample buffer (for GFP-trap) or 3x FLAG peptide competition (for FLAG M2) and subjected to SDS-PAGE or mass spectrometry’. Uncropped gels, blots and scans provided.

[0287] RNA isolation, sequencing and quantitative PCR

[0288] Total RNA was extracted from cells with the RNeasy Plus Mini Kit (cat. no. 74136, QIAGEN) according to the manufacturer’s instructions. RNA was quantified using NanoDrop; RNA integrity was measured with an Agilent Bioanalyzer. Samples were subjected to RNA-seq using an Illumina NovaSeq 6000 (HiSeq paired-end. 100 bp). The base calling data from the sequencer were transferred into FASTQ files, using the bcl2fastq2 conversion software v.2.20 (Illumina). PartekFlow (a start-to-finish software analysis solution for NGS data applications) was used to determine DEGs. For the quantitative PCR with reverse transcription (RT-qPCR) analysis, reverse transcription was performed with the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad Laboratories). RT-qPCR was performed with the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories). The expression of target genes was normalized to GAPDH. The RT-qPCR primers are listed in Fig. 16.

[0289] Quantification, statistics and reproducibility /

[0290] ImageJ v.1.51 (National Institutes of Health) was used for morphometric analysis. Graph preparation and statistical analysis was performed using Prism 10.0 (GraphPad Software). Unless otherwise indicated, all experiments were repeated at least three times, as described in the figure legends. Data were considered normally distributed and statistical significance was performed using a two-tailed Student / -test for two-group comparisons, a one-way ANOVA with Tukey post hoc analysis or a tw o-w ay ANOVA with Bonferroni correction for multiple comparisons, as described in the figure legends. Data are presented as the mean± s.e.m.

[0291] Enumerated Embodiments:

[0292] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0293] Embodiment 1: A method of treating, ameliorating and / or preventing vascular inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound that downregulates a Pcdhg cluster member, optionally Pcdhga9.

[0294] Embodiment 2: The method of Embodiment 1, wherein the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

[0295] Embodiment 3 : The method of any one of Embodiments 1 -2, wherein the method dow nregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

[0296] Embodiment 4: The method of any one of Embodiments 1-3, wherein the vascular inflammation causes or contributes to an atherosclerotic cardiovascular disease, a coronary artery disease, a peripheral artery' disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, or a transplant rejection in the subject.

[0297] Embodiment 5 : The method of any one of Embodiments 1 -4, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises: a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9, a protein inhibitor of the Pcdhg cluster member / Pcdhga9, a nucleic acid that ownregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme, an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity and / or expression level by CRISPR knockout or CRISPR knockdown, or a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

[0298] Embodiment 6: The method of any one of Embodiments 1-5, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises an antibody against the Pcdhg cluster member / Pcdhga9.

[0299] Embodiment 7: A method of treating, ameliorating, and / or preventing a disease or disorder caused by or involving vascular inflammation in a subject in need thereof, the method comprises administering to the subject an effective amount of a compound that downregulates a Pcdhg cluster member, optionally Pcdhga9.

[0300] Embodiment 8: The method of Embodiment 7, wherein the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

[0301] Embodiment 9: The method of any one of Embodiments 7-8, wherein the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

[0302] Embodiment 10: The method of any one of Embodiments 7-9, wherein the disease or disorder is at least one selected from the group consisting of an atherosclerotic cardiovascular disease, a coronary artery disease, a peripheral artery disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury' or intravascular coagulation caused by sepsis, a diabetic vasculopathy, and a transplant rejection in the subject.

[0303] Embodiment 11 : The method of any one of Embodiments 7-10, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises: a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9, a protein inhibitor of the Pcdhg cluster member / Pcdhga9, a nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity' and / or expression level by RNA interference, a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme, an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity and / or expression level by CRISPR knockout or CRISPR knockdown, or a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

[0304] Embodiment 12: The method of any one of Embodiments 7-11, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises an antibody against the Pcdhg cluster member / Pcdhga9.

[0305] Embodiment 13: A kit for treating, ameliorating and / or preventing vascular inflammation or diseases or disorders associated therewith in a subject in need thereof, the kit comprising: a compound for downregulating the Pcdhg cluster member / Pcdhga9 in the subject; and a manual instructing that the compound is to be administered to the subject in an effective amount.

[0306] Embodiment 14: The kit of Embodiment 13. wherein the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

[0307] Embodiment 15: The kit of any one of Embodiments 13-14, wherein the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

[0308] Embodiment 16: The kit of any one of Embodiments 13-15, wherein the disease or disorder associated with the vascular inflammation is at least one selected from the group consisting of an atherosclerotic cardiovascular disease, a coronary artery disease, a peripheral artery disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, and a transplant rejection.

[0309] Embodiment 17: The kit of any one of Embodiments 13-16. wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises: a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9, a protein inhibitor of the Pcdhg cluster member / Pcdhga9, a nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme, an expression vector compnsing an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity7and / or expression level by CRISPR knockout or CRISPR knockdown, or a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

[0310] Embodiment 18: The kit of any one of Embodiments 13-17, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises an antibody against the Pcdhg cluster member / Pcdhga9.

[0311] The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and / or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they' may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

CLAIMSWhat is claimed is:

1. A method of treating, ameliorating and / or preventing vascular inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound that downregulates a Pcdhg cluster member, optionally Pcdhga9.

2. The method of claim 1, wherein the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

3. The method of any one of claims 1-2, wherein the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

4. The method of any one of claims 1-3, wherein the vascular inflammation causes or contributes to an atherosclerotic cardiovascular disease, a coronary artery' disease, a peripheral artery disease, a cerebral vascular disease, a pulmonary' arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, or a transplant rejection in the subject.

5. The method of any one of claims 1-4, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises: a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9. a protein inhibitor of the Pcdhg cluster member / Pcdhga9, a nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity' and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity’ and / or expression level by RNA interference, a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity’ and / or expression level, and / or an expression vector expressing the ribozyme, an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity7and / or expression level by7CRISPR knockout or CRISPR knockdown. or a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9,and / or an expression vector that expresses the trans -dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

6. The method of any one of claims 1-5, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises an antibody against the Pcdhg cluster member / Pcdhga9.

7. A method of treating, ameliorating, and / or preventing a disease or disorder caused by or involving vascular inflammation in a subject in need thereof, the method comprises administering to the subject an effective amount of a compound that downregulates a Pcdhg cluster member, optionally Pcdhga9.

8. The method of claim 7, wherein the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

9. The method of any one of claims 7-8, wherein the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

10. The method of any one of claims 7-9, wherein the disease or disorder is at least one selected from the group consisting of an atherosclerotic cardiovascular disease, a coronary artery disease, a peripheral artery disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, and a transplant rejection in the subject.

11. The method of any one of claims 7-10, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises: a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9, a protein inhibitor of the Pcdhg cluster member / Pcdhga9, a nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme,an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that downregulate a Pcdhg cluster member / Pcdhga9 activity and / or expression level by CRISPR knockout or CRISPR knockdown, or a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

12. The method of any one of claims 7-11, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises an antibody against the Pcdhg cluster member / Pcdhga9.

13. A kit for treating, ameliorating and / or preventing vascular inflammation or diseases or disorders associated therewith in a subject in need thereof, the kit comprising: a compound for down regulating the Pcdhg cluster member / Pcdhga9 in the subject; and a manual instructing that the compound is to be administered to the subject in an effective amount.

14. The kit of claim 13, wherein the compound downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level.

15. The kit of any one of claims 13-14, wherein the method downregulates the Pcdhg cluster member / Pcdhga9 in a vascular endothelial cell of the subject.

16. The kit of any one of claims 13-15, wherein the disease or disorder associated with the vascular inflammation is at least one selected from the group consisting of an atherosclerotic cardiovascular disease, a coronary artery disease, a peripheral artery disease, a cerebral vascular disease, a pulmonary arterial hypertension, a lung or kidney injury or intravascular coagulation caused by sepsis, a diabetic vasculopathy, and a transplant rejection.

17. The kit of any one of claims 13-16, wherein the compound that downregulates the Pcdhg cluster member / Pcdhga9 comprises: a small molecule inhibitor of the Pcdhg cluster member / Pcdhga9,a protein inhibitor of the Pcdhg cluster member / Pcdhga9, a nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, and / or an expression vector expressing the nucleic acid that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level by RNA interference, a ribozyme that downregulates a Pcdhg cluster member / Pcdhga9 activity and / or expression level, and / or an expression vector expressing the ribozyme, an expression vector comprising an expression cassette, wherein the expression cassette expresses CRISPR components that down reg ulate a Pcdhg cluster member / Pcdhga9 activity and / or expression level by CRISPR knockout or CRISPR knockdown, or a trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9, and / or an expression vector that expresses the trans-dominant negative mutant protein of the Pcdhg cluster member / Pcdhga9.

18. The kit of any one of claims 13-17, wherein the compound that dow nregulates the Pcdhg cluster member / Pcdhga9 comprises an antibody against the Pcdhg cluster member / Pcdhga9.

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