WO2025160114A1

WO2025160114A1 - Compositions and methods of treating arteriosclerosis

Info

Publication number: WO2025160114A1
Application number: PCT/US2025/012510
Authority: WO
Inventors: Mark W. Feinberg
Original assignee: Brigham and Womens Hospital Inc
Current assignee: Brigham and Womens Hospital Inc
Priority date: 2024-01-22
Filing date: 2025-01-22
Publication date: 2025-07-31
Anticipated expiration: 2026-07-22
Also published as: WO2025160114A8

Abstract

Disclosed herein are inhibitory nucleic acid molecules (e.g., a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), an anti-sense oligonucleotide (ASO), a microRNA (miRNA), or a short hairpin RNA (shRNA)), or a gapmeR) for reducing expression of the long noncoding RNA (IncRNA) epigenetically induced MYC interacting IncRNAt (EPIC1). Also disclosed herein are methods for treating arteriosclerosis (e.g., atherosclerosis, e.g., diabetes-associated atherosclerosis) in a subject using the EPIC1 inhibitory nucleic acid molecules.

Description

COMPOSITIONS AND METHODS OF TREATING ARTERIOSCLEROSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 63/623,528, filed on January 22, 2024, which is incorporated herein by reference in its entirety for any purpose.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 17, 2025 is named “51665-003WO2_Sequence_Listing_1_17_25” and is 90,969 bytes in size.

TECHNICAL FIELD

This disclosure relates to inhibitory nucleic acid molecules useful for targeting long non-coding RNAs, and methods of using such inhibitory nucleic acid molecules for the treatment of arteriosclerosis.

BACKGROUND

The increased prevalence of diabetes mellitus in recent years has triggered a global health crisis, particularly by contributing to accelerated atherosclerosis and associated cardiovascular morbidity and mortality. Type 2 diabetes mellitus, a complex and multifactorial metabolic disease, is characterized by the cardinal features of hyperglycemia, hyperinsulinemia, dyslipidemia, and chronic inflammation. Prolonged exposure to these metabolic alterations is now recognized as a major factor in the pathogenesis of arteriosclerosis in diabetes. There remains a need in the field to identify therapeutic targets for treating arteriosclerosis.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an inhibitory nucleic acid molecule including sufficient complementarity to a target nucleic acid molecule, wherein (i) the inhibitory nucleic acid molecule is at least 15 nucleotides in length, and (ii) the target nucleic acid molecule includes a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 70, or a splice variant thereof.

In some embodiments, the target nucleic acid molecule includes a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 70, or a splice variant thereof.

In some embodiments, the target nucleic acid molecule includes the nucleotide sequence of SEQ ID NO: 70, or a splice variant thereof.

In some embodiments, the inhibitory nucleic acid molecule is 15 to 1 ,815 nucleotides in length (e.g., 50 to 1 ,815 nucleotides in length, 100 to 1 ,815 nucleotides in length, 250 to 1 ,815 nucleotides in length, 500 to 1 ,815 nucleotides in length, 750 to 1 ,815 nucleotides in length, 1 ,000 to 1 ,815 nucleotides in length, or 1 ,500 to 1 ,815 nucleotides in length). In some embodiments, the inhibitory nucleic acid molecule is 15 to 49 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42,

43, 44, 45, 46, 47, 48, or 49 nucleotides in length), 50 to 99 nucleotides in length (e.g., 50, 51 , 52, 53, 54,

55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82,

83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 nucleotides in length), or 100 to 1 ,815 nucleotides in length (e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1 ,025, 1 ,050, 1 ,075, 1 ,100, 1 ,125, 1 ,150, 1 ,175, 1 ,200, 1 ,225, 1 ,250, 1 ,275, 1 ,300, 1 ,325, 1 ,350, 1 ,375, 1 ,400, 1 ,425, 1 ,450, 1 ,475, 1 ,500, 1 ,525, 1 ,550, 1 ,575, 1 ,600, 1 ,625, 1 ,650, 1 ,675, 1 ,700, 1 ,725, 1 ,750, 1 ,775, 1 ,800, or 1 ,815 nucleotides in length).

In some embodiments, the inhibitory nucleic acid molecule is 18 to 25 nucleotides in length (e.g., 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length).

In some embodiments, the inhibitory nucleic acid molecule is 18 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 19 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 20 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 21 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 22 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 23 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 24 nucleotides in length. In some embodiments, the inhibitory nucleic acid molecule is 25 nucleotides in length.

In some embodiments, the inhibitory nucleic acid molecule includes at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementarity to the target nucleic acid molecule, or splice variant thereof.

In some embodiments, the inhibitory nucleic acid molecule includes at least 90% (e.g., 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementarity to the target nucleic acid molecule, or splice variant thereof.

In some embodiments, the inhibitory nucleic acid molecule includes at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) complementarity to the target nucleic acid molecule, or splice variant thereof.

In some embodiments, the inhibitory nucleic acid molecule is complementary to the target nucleic acid molecule, or splice variant thereof.

In some embodiments, the inhibitory nucleic acid molecule further includes a modification.

In some embodiments, the modification includes: (a) a non-natural or modified nucleoside or nucleotide; and/or (b) a covalently or non-covalently conjugated moiety.

In some embodiments: (a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LNA), a 2’-O-methyl (2’-O-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2’-fiuoro (2’-F) modified nucleoside; and/or (b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

In some embodiments, the targeting moiety is vascular cell adhesion protein 1 (VCAM1). In some embodiments, the inhibitory nucleic acid molecule is selected from the group consisting of: a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), and a gapmeR.

In some embodiments, the inhibitory nucleic acid molecule is a dsRNA.

In some embodiments, the inhibitory nucleic acid molecule is a miRNA.

In some embodiments, the inhibitory nucleic acid molecule is an shRNA.

In some embodiments, the inhibitory nucleic acid molecule is an ASO.

In some embodiments, the inhibitory nucleic acid molecule is a gapmeR.

In some embodiments, the inhibitory nucleic acid molecule is an siRNA.

In some embodiments, the siRNA includes an antisense strand including at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -23.

In some embodiments, the antisense strand includes at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -23.

In some embodiments, the antisense strand includes at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1 -23.

In some embodiments, the antisense strand includes the nucleotide sequence of any one of SEQ ID NOs: 1 -23.

In some embodiments, the siRNA further includes a sense strand including at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 24-46.

In some embodiments, the siRNA includes: (a) the antisense strand of SEQ ID NO: 1 and the sense strand of SEQ ID NO: 24; (b) the antisense strand of SEQ ID NO: 2 and the sense strand of SEQ ID NO: 25; (c) the antisense strand of SEQ ID NO: 3 and the sense strand of SEQ ID NO: 26; (d) the antisense strand of SEQ ID NO: 4 and the sense strand of SEQ ID NO: 27; (e) the antisense strand of SEQ ID NO: 5 and the sense strand of SEQ ID NO: 28; (f) the antisense strand of SEQ ID NO: 6 and the sense strand of SEQ ID NO: 29; (g) the antisense strand of SEQ ID NO: 7 and the sense strand of SEQ ID NO: 30; (h) the antisense strand of SEQ ID NO: 8 and the sense strand of SEQ ID NO: 31 ; (i) the antisense strand of SEQ ID NO: 9 and the sense strand of SEQ ID NO: 32; (j) the antisense strand of SEQ ID NO: 10 and the sense strand of SEQ ID NO: 33; (k) the antisense strand of SEQ ID NO: 11 and the sense strand of SEQ ID NO: 34; (I) the antisense strand of SEQ ID NO: 12 and the sense strand of SEQ ID NO: 35; (m) the antisense strand of SEQ ID NO: 13 and the sense strand of SEQ ID NO: 36; (n) the antisense strand of SEQ ID NO: 14 and the sense strand of SEQ ID NO: 37; (o) the antisense strand of SEQ ID NO: 15 and the sense strand of SEQ ID NO: 38; (p) the antisense strand of SEQ ID NO: 70 or SEQ ID NO: 71 and the sense strand of SEQ ID NO: 39; (q) the antisense strand of SEQ ID NO: 17 and the sense strand of SEQ ID NO: 40; (r) the antisense strand of SEQ ID NO: 18 and the sense strand of SEQ ID NO: 41 ; (s) the antisense strand of SEQ ID NO: 19 and the sense strand of SEQ ID NO: 42; (t) the antisense strand of SEQ ID NO: 20 and the sense strand of SEQ ID NO: 43; (u) the antisense strand of SEQ ID NO: 21 and the sense strand of SEQ ID NO: 44; (v) the antisense strand of SEQ ID NO: 22 and the sense strand of SEQ ID NO: 45; or (w) the antisense strand of SEQ ID NO: 23 and the sense strand of SEQ ID NO: 46.

In some embodiments, the siRNA contains 3’ overhangs selected from the group consisting of: (i) a single uracil overhang at one or more 3’ ends of the siRNA; (ii) a double uracil overhang at one or more 3’ ends of the siRNA; (iii) a single thymine overhang at one or more 3’ ends of the siRNA; (iv) a double thymine overhang at one or more 3’ ends of the siRNA; or (v) a single cytosine and single thymine overhang at one or more 3’ ends of the siRNA.

In some embodiments, the siRNA targets the nucleotide sequence of any one of SEQ ID NOs: 47-69.

In a second aspect, the invention provides an inhibitory nucleic acid molecule including sufficient complementarity to a target nucleic acid molecule, wherein (i) the inhibitory nucleic acid molecule is at least 15 nucleotides in length, and (ii) the target nucleic acid molecule includes a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 71 , or a splice variant thereof.

In some embodiments, the target nucleic acid molecule includes a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 71 , or a splice variant thereof.

In some embodiments, the target nucleic acid molecule includes the nucleotide sequence of SEQ ID NO: 71 , or a splice variant thereof.

In some embodiments, the inhibitory nucleic acid molecule is 15 to 1 ,408 nucleotides in length (e.g., 50 to 1 ,408 nucleotides in length, 100 to 1 ,408 nucleotides in length, 250 to 1 ,408 nucleotides in length, 500 to 1 ,408 nucleotides in length, 750 to 1 ,408 nucleotides in length, or 1 ,000 to 1 ,408 nucleotides in length).

In some embodiments, the inhibitory nucleic acid molecule is 15 to 49 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42,

83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 nucleotides in length), or 100 to 1 ,408 nucleotides in length (e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1 ,025, 1 ,050, 1 ,075, 1 ,100, 1 ,125, 1 ,150, 1 ,175, 1 ,200, 1 ,225, 1 ,250, 1 ,275, 1 ,300, 1 ,325, 1 ,350, 1 ,375, 1 ,400, or 1 ,408 nucleotides in length).

In some embodiments: (a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a LNA, a 2'-O-Me modified nucleoside, a PS bond between nucleosides, and a 2'- F modified nucleoside: and/or (b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

In some embodiments, the targeting moiety is VCAM1 .

In some embodiments, the inhibitory nucleic acid molecule is selected from the group consisting of: a siRNA, a dsRNA, a miRNA, an shRNA, an ASO, and a gapmeR.

In some embodiments, the inhibitory nucleic acid molecule is a dsRNA.

In some embodiments, the inhibitory nucleic acid molecule is a miRNA.

In some embodiments, the inhibitory nucleic acid molecule is an shRNA.

In some embodiments, the inhibitory nucleic acid molecule is an ASO.

In some embodiments, the inhibitory nucleic acid molecule is a gapmeR.

In some embodiments, the inhibitory nucleic acid molecule is an siRNA.

In some embodiments of any of the foregoing aspects, the inhibitory nucleic acid molecule is formulated in a delivery vehicle.

In some embodiments, the delivery vehicle is selected from the group consisting of: a vector, a plasmid, a micelle, a liposome, an exosome, and a lipid nano particle (LNP).

In some embodiments, the vector is a viral vector.

In some embodiments, the viral vector is an adeno-associated viral (AAV) vector.

In some embodiments, the AAV vector is selected from the group consisting of: AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , and AAV12.

In some embodiments of any of the foregoing aspects, the inhibitory nucleic acid molecule is formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable excipient, diluent, and/or carrier.

In a third aspect, the invention provides a method of treating arteriosclerosis in a subject, the method including administering the inhibitory nucleic acid molecule of any of the foregoing aspects.

In some embodiments, the arteriosclerosis is atherosclerosis.

In some embodiments, the atherosclerosis is diabetes-associated atherosclerosis.

In some embodiments, the subject has a metabolic disorder, or the subject is at risk of developing the metabolic disorder.

In some embodiments, the metabolic disorder is diabetes. some embodiments, the subject at risk of developing diabetes is prediabetic and/or has one or more of the following: (a) hyperglycemia; (b) glucose resistance; (c) insulin resistance; (d) hyperlipidemia; and (e) has a family history of diabetes.

In some embodiments, the inhibitory nucleic acid molecule is administered to the subject intravenously, intraperitoneally, subcutaneously, intraarticularly, or intramuscularly.

In some embodiments, the inhibitory nucleic acid molecule is delivered to the aortic intima.

In some embodiments, the inhibitory nucleic acid molecule is delivered to a macrophage.

In some embodiments, the macrophage is an activated peritoneal macrophage.

In some embodiments, administration of the inhibitory nucleic acid molecule reduces proinflammatory macrophage recruitment.

In some embodiments, administration of the inhibitory nucleic acid molecule reduces lesion formation in the subject.

In some embodiments, the method further includes administering an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a statin, PCSK9 inhibitor, or ezetimibe.

In a fourth aspect, the invention provides a method of treating a subject who has a disease associated with excessive inflammation, the method including: administering to the subject a therapeutically effective dose of a pharmaceutical composition that decreases the expression of MERRICAL (SEQ ID NO: 71 ) or EPIC1 (SEQ ID NO 70) long non-coding RNAs in a cell of the subject in need thereof.

In some embodiments, the pharmaceutical composition includes a nucleic acid molecule, antisense oligonucleotide, or small molecule that targets all or part of the MERRICAL or EPIC1 long noncoding RNA sequences.

In a fifth aspect, the invention provides for all compositions, articles of manufacture, methods, and uses disclosed and/or described herein.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to illustrate embodiments of the disclosure and further an understanding of its implementations. FIG. 1A- FIG. 1F demonstrate that the long non-coding RNA (IncRNA), macrophage-enriched IncRNA regulates inflammation, chemotaxis, and atherosclerosis (MERRICAL), is coordinately regulated with a group of chemokine genes during high-fat, high-sucrose-containing (HFSC) diet-induced atherosclerosis in mice. FIG. 1 A is a schematic of an animal study in which RNA was derived from the aortic intima of Ldlr^/~ mice (n = 4, each sample represents RNA pooled from two mice) that were placed on a HFSC diet for 0 weeks (group 1 (“G1 ”)), 2 weeks (group 2 (“G2”)), or 12 weeks (group 3 (“G3”)). FIG. 1B illustrates the workflow of a genome-wide, RNA-seq profiling analysis used for the identification of differentially expressed (DE) IncRNAs (Iog2 fold change (FC) > 1 .5, FDR < 0.05). FIG. 1C is a radial heatmap showing the expression of the top 50 significantly regulated IncRNAs during the progression of atherosclerosis, as induced by the HFSC diet in Ldlr^/~ mice. FIG. 1D is a graph showing normalized read counts from the RNA-seq analysis of MERRICAL in the aortic intima during the progression of atherosclerosis. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 1E is a circos plot of DE mRNA in G3 vs. G1 paired with DE IncRNA in G3 vs. G1 . FIG. 1F is a heatmap showing the expression pattern of MERRICAL and its neighboring genes in the progression phase of diabetes-associated atherosclerosis.

FIG. 2A- FIG. 2L demonstrate the identification of the IncRNA MERRICAL in lesional intima. FIG. 2A is a graph of a quantitative real-time PCR (qRT-PCR) expression analysis for the IncRNA MERRICAL in the aortic intima and media in Ldlr'’- mice on the HFSC diet for 12 weeks (n = 3). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. ***p < 0.001 . FIG. 2B is a graph of a qRT- PCR expression analysis of the IncRNA MERRICAL in different body organs of 12-week-old C57BL/6 mice (n = 4). Values are mean ± SD. FIG. 2C is a graph of a qRT-PCR expression analysis of the IncRNA MERRICAL in different aortic cell types isolated by magnetic beads (cluster of differentiation (CD)-45⁺ leukocytes, CD31⁺ endothelial cells, CD90.2+ fibroblasts, and anti-smooth muscle actin (aSMA)⁺ smooth muscle cells (SMCs)) from C57BL/6 mice (n = 3). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *”*p < 0.001 . FIG. 2D is a graph of a MERRICAL expression analysis in different types of immune cells isolated from C57BL/5 mice. P.M, peritoneal macrophages; B.M, bone marrow-derived macrophages (BMDMs); NKT, natural killer T cells; CD19⁺, B cells; CD3⁺, total T cells; CD4⁺, CD4⁺ T cells; CD8⁺, cytotoxic T cells; (n = 3). Values are mean ± SD. FIG. 2E is a graph of IncRNA MERRICAL expression kinetics in macrophages differentiated from BM isolated from C57BL/6 mice (n = 3). Values are mean ± SD. FIG. 2F is a uniform manifold approximation and projection (UMAP) of expression of the IncRNA MERRICAL in different aortic cell types after aortic single-cell RNA-seq. FIG. 2G is a graph of the coding probability, as determined by coding potential assessment tool (CPAT), which predicted very low coding potential for the IncRNA MERRICAL. FIG. 2H is an immunoblot analysis testing the coding potential of the MERRICAL locus. Briefly, the MERRICAL sequence was cloned upstream of the 3xFLAG tag cassette, transfected in HEK293T cells, and immunoblotted for FLAG antibody. A positive control was provided with the kit (n = 3 experiments). FIG. 21 is an RNA in situ hybridization (RNA-ISH) for negative control and MERRICAL probes on paraformaldehyde (PFA)-fixed BMDMs. Scale bars, 200 pm. FIG. 2J is a graph of a qRT-PCR analysis onRNA derived from BMDMs, separated into cytoplasmic and nuclear fractions, and normalized to the cytoplasmic fraction (n = 3). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 2K is a representative image of MERRICAL (cyan dots) colocalized with F4/80+ macrophages (see internal box) in mouse aortic lesions of Ldlr- mice fed the HFSC diet. Scale bars, 200 pm. FIG. 2L is a heatmap showing MERRICAL (darker grey) and Cd68 (lighter grey) co-expression in the single-cell transcriptome analysis of RNA from the descending aorta of Ldlr^/~ mice fed the HFSC diet.

FIG. 3A- FIG. 3F demonstrate that MERRICAL-det\c\ent macrophages inhibit pro-inflammatory responses and expression of C-C motif chemokine ligand (CCL)-3 and CCL4 in vitro. FIG. 3A is a schematic summarizing a differential expression analysis using DEseq2 to compare non-specific (NS) control gapmeR-treated BMDMs (M1 phase) with the MERRICAL gapmeR knockdown (KD) BMDMs (M1 phase). FIG. 3B is an Ingenuity Pathway Analysis (IPA) of canonical pathways from differentially expressed genes (DEGs) of MERRICAL KD BMDMs compared with NS controls. FIG. 3C is a GOChord plot showing the significantly regulated genes (Iog2 FC > 3, FDR < 0.05) involved in the top 7 enriched pathways when comparing MERRICAL KD BMDMs with NS controls. Chemokine signaling was an enriched pathway and chemokines CCL3 and CCL4 (see boxes within FIG. 3C) were identified as significantly down regulated. FIG. 3D is a heatmap showing significantly regulated genes in the chemokine signaling pathway. CCL3 and CCL4, which were identified in FIG. 3C, are boxed to show emphasis. FIG. 3E is a Venn diagram showing the overlap of significantly downregulated genes (from comparison of MERRICAL KD BMDMs with NS controls) with significantly upregulated genes (from aortic intimal RNA-seq of Ldlr'’- mice fed the HFSC diet, G3 progression versus control). As emphasized by the central box, 173 genes were overlapped between significantly downregulated genes (from comparison of MERRICAL KD BMDMs with NS controls) with significantly upregulated genes (from aortic intimal RNA- seq of Ldlr^/~ mice fed the HFSC diet, G3 progression versus control). FIG. 3F is a GOChord plot showing overlapping genes in FIG. 3E (Iog2 FC > 0.58, FDR < 0.05) involved in the top 7 enriched pathways. CCL3 and CCL4, which were identified in FIG. 3C, are boxed to show emphasis.

FIG. 4A- FIG. 41 show a loss of function and gain of function analysis of the IncRNA MERRICAL in regulating chemotaxis and pro-inflammatory response in vitro. FIG. 4A is a qRT-PCR analysis of KD efficiency using gapmeR targeting the IncRNA MERRICAL in BMDMs. Significance was determined by two-way ANOVA. *p < 0.05. FIG. 4B is a representative image of an RNA-ISH analysis that confirms MERRICAL KD in BMDMs. FIG. 4C is a qRT-PCR analysis confirming CCL3 expression in NS control and MERRICAL KD BMDMs. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05. FIG. 4D is a qRT-PCR analysis confirming CCL4 expression in NS control and MERRICAL KD BMDMs. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05. FIG. 4E is a radial heatmap showing cytokine array in supernatant from NS control and MERRICAL BMDMs. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05. FIG. 4F is an immunofluorescence image of representative fields of a peripheral blood mononuclear cell (PBMC) adhesion assay on the bEnd.3 cell monolayer cultured for 24 hours with supernatant from NS control or MERRICAL KD BMDMs. Shown to the right is a graph quantifying the number of PBMCs per area. Scale bars, 100 pm. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05. FIG. 4G is an immunofluorescence image of representative fields of BMDMs that migrated through a transwell filter with the supernatant of NS control or MERRICAL KD BMDMs, supplemented with the neutralization antibody anti-CCL3 and CCL4 loaded in the lower chamber. Shown to the right is a graph quantifying the number of migrated macrophages per area. The bars for NS and for MERRICAL KD are, left to right, IgG control, anti-CCL3, anto-CCL4, and anti- CCL3/CCL4. Values are mean ± SD. Significance was determined by two-way ANOVA. *p < 0.05, **p < 0.01 , ***p < 0.001 , ns = not significant. FIG. 4H is a graph of CCL3 mRNA expression in control vector- or MERRICAL overexpression vector-treated BMDMs. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. **p < 0.01 . FIG. 41 is a graph of CCL4 mRNA expression in control vector- or MERRICAL overexpression vector-treated BMDMs. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05.

FIG. 5A- FIG. 5M demonstrate that the IncRNA MERRICAL regulates CCL3 and CCL4 transcription through interaction with H3K4me3. FIG. 5A is a graph of a qRT-PCR analysis of CCL3 expression in BMDMs transfected with NS gapmeR (NS) and MERRICAL gapmeR (KD) in a timedependent manner (n = 3). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5B is a graph of a qRT-PCR analysis of CCL4 expression in BMDMs transfected with NS gapmeR (NS) and MERRICAL gapmeR (KD) in a time-dependent manner (n = 3). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5C is a graph of a qRT-PCR analysis of CCL3 expression in BMDMs transfected with pcDNA3.1 -vector (vector) and pcDNA3.1 -MERRICAL overexpression {MERRICAL) in a timedependent manner (n = 3). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5D is a graph of a qRT-PCR analysis of CCL4 expression in BMDMs transfected with pcDNA3.1 -vector (vector) and pcDNA3.1 -MERRICAL overexpression {MERRICAL) in a time-dependent manner (n = 3). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5E is a blot of a biotinylated IncRNA pull-down assay using wild-type (lacZ) or MERRICAL in BMDM nuclear lysates from control or lipopolysaccharide (LPS) treatment (1 pg/mL, 2 hours) (n = 3). FIG. 5F is a graph of an RNA immunoprecipitation (RIP) assay followed by qRT-PCR analysis of co-purified RNAs in BMDMs. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5G is a genomic snapshot showing CUT&RUN signals for H3K4me3 and IgG for CCL3, CCL4, WAP four-disulfide core domain (WFDC)-17, WFDC18, CCL5, and CCL12 from both NS and MERRICAL KD groups with or without LPS treatment (2 hours). FIG. 5H is a graph of a chromatin immunoprecipitation-quantitative PCR (ChlP-qPCR) analysis of H3K4me3 in BMDMs transfected with NS gapmeR (NS) or MERRICAL gapmeR (KD). Values are mean ± SD. Significance was determined by two- tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 51 is a graph of a ChlP-qPCR analysis of H3K4me3 in BMDMs transfected with NS gapmeR (NS) or MERRICAL gapmeR (KD). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5J is a graph of a ChlP-qPCR analysis of H3K4me3 in BMDMs transfected with pcDNA3.1 -vector (vector) or pcDNA3.1 - MERRICAL overexpression {MERRICAL). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5K is a graph of a ChlP- qPCR analysis of H3K4me3 in BMDMs transfected with pcDNA3.1 -vector (vector) or pcDNA3.1 - MERRICAL overexpression {MERRICAL). Values are mean ± SD. Significance was determined by two- tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5L is a graph quantifying relative CCL3 enrichment in a DNA enrichment in chromatin isolation by RNA purification (ChIRP) analysis with control (LacZ) and MERRICAL probes determined by quantitative-PCR (qPCR) and calculated as percentage of input with the indicated primer sets. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 . FIG. 5M is a graph quantifying relative CCL4 enrichment in a ChIRP analysis with control (LacZ) and MERRICAL probes determined by qPCR and calculated as percentage of input with the indicated primer sets. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , "*p < 0.001 .

FIG. 6A- FIG. 6F demonstrate that MERRICAL interacts with the WD repeat domain 5 protein- mixed lineage leukemia (WDR5-MLL1 ) complex and facilitates H4K4me3 modification at the promoter region of CCL3 and CCL4. FIG. 6A is a bar plot showing H3K4 methylation mediators, including lysine methyltransferase (KMT) (MLL) families, and apoptosis signal-regulating kinase 1 (ASHK1 ), SET domain containing 1A (SETD1A), and SET domain containing 1 B (SETD1 B) regulation in response to LPS- treated BMDMs and in response to MERRICAL KD in BMDMs from the RNA-seq analysis. Values are mean ± SD. Significance was determined by two-tailed Student’s t test for. *p < 0.05, **p < 0.01 , *"p < 0.001 . FIG. 6B is a qRT-PCR analysis of CCL3 expression in BMDMs treated with MM102 (10 pM) with LPS stimulation (1 pM; 0, 4, and 16 hours). Values are mean ± SD. Significance was determined by two- tailed Student’s t test. ***p < 0.001 , ns = not significant. FIG. 6C is a qRT-PCR analysis of CCL4 expression in BMDMs treated with MM102 (10 pM) with LPS stimulation (1 pM; 0, 4, and 16 hours). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. ***p < 0.001 , ns = not significant. FIG. 6D is a Western blot following an immunoprecipitation (IP) assay of WDR5 in both NS control and MERRICAL KD BMDMs treated with LPS (4 hours), which shows interaction between WDR5 and MLL1 under NS control and MERRICAL KD conditions in BMDMs. FIG. 6E is a graph of an RIP assay by IgG or WDR5, followed by qRT-PCR analysis of co-purified RNAs in BMDMs. Values are mean ± SD. Significance was determined by two-tailed Student’s t test for. *p < 0.05, **p < 0.01 . FIG. 6F is an illustration showing MERRICAL binding with the WDR5-MLL1 complex and facilitating H3K4me3 deposition on the promoter region of CCL3 and CCL4. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , "*p < 0.001 .

FIGS. 7A-7J demonstrate that MERRICAL KD inhibits pro-inflammatory responses and atherosclerotic lesion formation in vivo. FIG. 7A is a schematic of Ldlr^/~ mice on the HFSC diet and injected retro-orbitally (r.o.) with NS control gapmeR or MERRICAL gapmeR once per week (20 mg kg-¹ per injection per mouse) for 12 weeks. FIG. 7B is a graph of a qRT-PCR analysis of MERRICAL expression in the aortic intima from the NS control and MERRICAL KD groups (n = 5). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , *"p < 0.001 , *"*p < 0.0001 . FIG. 7C is a graph of a qRT-PCR analysis of CCL3 expression in the aortic intima from the NS control and MERRICAL KD groups (n = 5). Values are mean ± SD. Significance was determined by two- tailed Student’s t test. *p < 0.05, **p < 0.01 , ”’p < 0.001 , ”’*p < 0.0001 . FIG. 7D is a graph of a qRT-PCR analysis of CCL4 expression in the aortic intima from the NS control and MERRICAL KD groups (n = 5). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , *"p < 0.001 , *’**p < 0.0001 . FIG. 7E shows representative images and quantifications (right) for oil red O (ORO) staining in the in the aortic sinus from control and MERRICAL KD mice (n = 10). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , *"p < 0.001 , *"*p < 0.0001 . FIG. 7F shows representative images and quantifications (left) of plaque volume using 3D light sheet imaging in the descending aorta from control and MERRICAL KD mice (n = 3). Scale bar, 500 pm. FIG. 7G is a representative image of CD68 and a-SMA staining in aortic roots and quantification of CD68+ cells (n = 3). Scale bar: 100 pm. Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , ***p < 0.001 , *’**p < 0.0001 . FIG. 7H is a flow cytometry analysis of aortic cells (ascending aorta to diaphragm) from control and MERRICAL KD mice on the HFSC diet for 12 weeks (n = 4). Values are mean ± SD. Significance was determined by two-tailed Student’s t test. *p < 0.05, **p < 0.01 , *"p < 0.001 , *"*p < 0.0001 . FIG. 71 is a volcano plot showing the mouse cytokine array in plasma from control and MERRICAL KD mice on the HFSC diet for 12 weeks. FIG. 7J is a heatmap showing significantly regulated cytokines in plasma from control and MERRICAL KD mice on the HFSC diet for 12 weeks (n = 6).

FIG. 8 is a schematic of the proposed mechanism for IncRNA MERRICAL regulation of CCL3 and CCL4, and the effects on macrophage pro-inflammatory response and diabetes-associated- atherosclerosis. Increased MERRICAL expression in diabetic atherosclerotic progression in-cis regulates its neighboring chemokines CCL3 and CCL4 by interacting with the WDR5-MLL1 complex and facilitating the histone-modifying enzyme H3K4me3 deposition at the promoter regions of CCL3 and CCL4. Deficiency of MERRICAL abrogates pro-inflammatory macrophage recruitment into the vessel wall, inflammatory-associated responses, and diabetes-associated atherosclerotic lesion progression.

FIG. 9 is a graph showing the relative expression of epigenetically induced MYC interacting IncRNAI (EPIC1 ). The IncRNA EPIC1 is an ortholog of IncRNA MERRICAL and is also highly expressed in leukocytes (e.g., peripheral blood mononuclear cells), whereas it is not expressed in endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)) or smooth muscle cells (e.g., coronary artery smooth muscle cells (CASMCs)) as quantified by real time qPCR.

FIG. 10A- FIG. 10C demonstrate the homology between MERRICAL and human EPIC1 . FIG. 10A is a protein residue index showing that EPIC1 is a putative MERRICAL human homologue and its tertiary structure is highly predicted to interact with WDR5 in an analogous manner. FIG. 10B is a protein residue index showing that MERRICAL’s tertiary structure is also highly predicted to interact with WDR5 in an analogous manner (CatRapidv2.0). FIG. 10C is a Western blot of a IncRNA pulldown assay using biotin-labeled transcripts for EPIC1 or LacZ control in nuclear lysates of PBMC-derived human primary macrophages; these data show the interaction of human EPIC1 with WDR5.

DEFINTIONS

Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of "or" means "and/or" unless stated otherwise. The use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. As used herein, the term "about," as applied to one or more values of interest, refers to a value that falls within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

As used herein, “administration” refers to providing or giving a subject a therapeutic agent by any effective route. Exemplary routes of administration are described herein below.

As used herein, the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.

As used herein, the term "auxiliary moiety" refers to any moiety, including, but not limited to, a small molecule, a peptide, a carbohydrate, a neutral organic polymer, a positively charged polymer, a therapeutic agent, a targeting moiety, an endosomal escape moiety, and any combination thereof, which can be conjugated to a nucleic acid molecule. In some embodiments, an "auxiliary moiety" is linked to an inhibitory nucleic acid molecule disclosed herein by forming one or more covalent or non-covalent bonds with one or more conjugating groups attached to a phosphate linkage, a phosphorothioate linkage, a 5' positions of a nucleotide sugar, or any portion of a nucleobase. One skilled in the art will readily understand appropriate points of attachment of a particular auxiliary moiety to a nucleic acid molecule.

As used herein, “delivery vehicle” refers to any substance (e.g., molecule, peptide, conjugate, and construct) that facilitates, at least in part, the in vivo delivery of a nucleic acid molecule to targeted cells.

As used herein, the terms “effective amount,” “therapeutically effective amount,” and a “sufficient amount” of a composition described herein refer to a quantity sufficient to, when administered to the subject, effect beneficial or desired results; as such, an “effective amount” or synonym thereto depends upon the context in which it is being applied. For example, in the context of decreasing macrophage- enriched IncRNA regulates inflammation, chemotaxis, and atherosclerosis (MERRICAL), or in the context of decreasing epigenetically induced MYC interacting IncRNAI (EPIC1 ), it is an amount of the composition sufficient to achieve a treatment response as compared to the response obtained without administration of the composition. The amount of a given composition described herein that will correspond to such an amount will vary depending upon various factors, such as the given agent, the pharmaceutical compositions, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, weight) or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

As used herein, a “formulation” includes at least an inhibitory nucleic acid molecule and a delivery vehicle.

As used herein, the term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe). As used herein, the term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

As used herein, the term “inhibitory nucleic acid molecule” refers to a nucleic acid molecule that has sufficient complementarity to bind to a target nucleic acid molecule to inhibit expression of a product (e.g., a IncRNA) encoded by the target nucleic acid molecule. Exemplary inhibitory nucleic acid molecules are anti-sense oligonucleotides (ASOs), small interfering RNA (siRNAs), short hairpin RNA (shRNAs), double stranded RNAs (dsRNAs), and microRNA (miRNAs). Inhibitory nucleic acid molecules may reduce the target’s expression by 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more). In one embodiment, the target nucleic acid molecule is EPIC1 , or a splice variant thereof. In another embodiment, the target nucleic acid molecule is MERRICAL.

As used herein the term “modified” refers to a changed state or structure of a nucleic acid molecule described herein. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, the inhibitory nucleic acid molecules of the present invention are modified by the introduction of non-natural nucleosides and/or nucleotides. In other embodiments, the inhibitory nucleic acid molecules of the present invention are modified by conjugation of an auxiliary moiety.

As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and/or carriers, to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject.

As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

“Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

As used herein, an inhibitory nucleic acid molecule (e.g., an siRNA, a dsRNA, a miRNA, a shRNA, an ASO, or a gapmeR) having “sufficient complementarity” to a target nucleic acid molecule (e.g., a target IncRNA) means that the inhibitory nucleic acid molecule includes a nucleotide sequence capable of hybridizing to, and triggering the destruction of, the target nucleic acid molecule (e.g., by RISC- mediated cleavage or Rnase H-mediated cleavage of the target nucleic acid molecule). The inhibitory nucleic acid molecule can be designed such that every nucleotide is complementary to a nucleotide in the target nucleic acid molecule. Alternatively, mismatched nucleotides may be introduced so long as there remains hybridization and destruction of the target nucleic acid molecule (e.g., a target IncRNA).

As used herein, the term “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.

As used herein, “treatment” and “treating” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

As used herein, the term “vector” is considered a replicon, such as plasmid, phage, viral construct or cosmid, to which another nucleic acid (e.g., DNA or RNA) segment may be attached. Vectors are used to transduce and express the nucleic acid segment in cells.

DETAILED DESCRIPTION

Described herein are compositions (e.g., an inhibitory nucleic acid molecule) for reducing expression of a long non-coding RNA (IncRNA) and methods thereof for treating arteriosclerosis (e.g., diabetes-associated atherosclerosis) in a subject.

The IncRNA may be (i) macrophage-enriched IncRNA regulates inflammation, chemotaxis, and atherosclerosis (MERRICAL), and/or (ii) epigenetically induced MYC interacting IncRNAI (EPIC1 ). Inhibitors to these IncRNAs have the potential for treating inflammatory disease states, such as in diabetes and atherosclerosis, with implications for wide range of both acute and chronic inflammatory diseases. The inhibitory nucleic acid molecule may be a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), an anti-sense oligonucleotide (ASO), a microRNA (miRNA), or a short hairpin RNA (shRNA)), or a gapmeR described herein, or a composition (e.g., pharmaceutical composition) thereof. Furthermore, the inhibitory nucleic acid molecule may be used in methods for treating arteriosclerosis (e.g., diabetes-associated atherosclerosis) in a subject.

Whilst the invention has been disclosed in particular embodiments, it will be understood by those skilled in the art that certain substitutions, alterations and/or omissions may be made to the embodiments without departing from the spirit of the invention. Accordingly, the description is meant to be exemplary only and should not limit the scope of the invention. All references, scientific articles, patent publications, and any other documents cited herein or in the accompanying documents filed herewith are hereby incorporated by reference for the substance of their disclosure. For example, Chen, Jingshu et al., Cell Reports, 43 (3), 113815 (2024), is incorporated herein by reference in its entirety.

Inhibitory Nucleic Acid Molecules

Exemplary inhibitory nucleic acid molecules of the disclosure are siRNAs, dsRNAs, ASOs, miRNAs, gapmeRs, and shRNAs; however, any nucleic acid molecule capable of reducing EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof, is envisioned for use of the methods described herein. In some instances, the inhibitory nucleic acid molecules of the disclosure may be referred to as RNA inhibitory (RNAi) molecules.

For any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, gapmeR, or other inhibitory nucleic acid molecules capable of reducing expression of a target nucleic acid) the inhibitory nucleic acid molecule contains at least some sequence complementarity to the nucleotide sequence of SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 15 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 16 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 18 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 19 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 20 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 21 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 22 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 23 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 24 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 25 contiguous nucleotides set forth within SEQ ID NOs: 16. In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 26 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 27 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 28 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 29 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 30 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3).

For any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, ASO, miRNA, shRNA, gapmeR or other inhibitory nucleic acid molecules capable of reducing expression of a target nucleic acid) the inhibitory nucleic acid molecule comprises or consists of a sequence complementary to at least 15 to 30 contiguous nucleotides (e.g., 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 36 to 30, 27 to 30, 28 to 30, or 29 to 30 contiguous nucleotides) set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3).

In some embodiments, the inhibitory nucleic acid is an siRNA targeting EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof. In some embodiments, the inhibitory nucleic acid is an dsRNA targeting EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof. In some embodiments, the inhibitory nucleic acid is an ASO targeting EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof. In some embodiments, the inhibitory nucleic acid is an gapmeR targeting EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof. In some embodiments, the inhibitory nucleic acid is a miRNA targeting EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof. In some embodiments, the inhibitory nucleic acid is an shRNA targeting EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof. Each of these modalities is described further below. small interfering RNA (siRNA) siRNAs of the disclosure are single-stranded (ss) or double-stranded (ds) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target IncRNA of interest. Once an siRNA molecule enters a cell, it is incorporated into an RNA-induced silencing complex (RISC). Upon siRNA hybridization to a target IncRNA, the RISC complex will cleave the target IncRNA, thereby inactivating it and reducing the level of the IncRNA in the cell.

In some embodiments, siRNAs of the disclosure may include a nucleotide sequence of about 10 to about 30 nucleotides in length (e.g., 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or 31 nucleotides in length).

In some embodiments, siRNAs of the disclosure may include a nucleotide sequence of 10 to 30 nucleotides in length (e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed for the current invention.

In some embodiments, the siRNA contains an antisense strand. In some embodiments, lengths for an antisense strand of the siRNA molecules of the present disclosure is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 and 23 nucleotides

(e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 17 nucleotides. In some embodiments, the antisense strand is 18 nucleotides. In some embodiments, the antisense strand is 19 nucleotides. In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

In some embodiments, the siRNA contains a sense strand. In some embodiments, the sense strand of the siRNA molecules of the present disclosure is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides. In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.

In some embodiments, the sense and antisense strands of an siRNA molecule of the disclosure are completely complementary. In some embodiments, the sense and antisense strands of an siRNA molecule of the disclosure are completely complementary to the extent that their lengths overlap with one another. Depending on the sequence of the first and second strand, complementarity need not be complete or perfect, which means that the first and second strand are not 100% base-paired due to mismatches. One or more mismatches may be present within the ds siRNA without impacting the siRNA’s ability to reduced expression of a target IncRNA of interest.

The nucleotide sequence of an siRNA of the disclosure may contain sufficient complementarity to a portion of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof, e.g., see Table 3) such that the siRNA can hybridize with the target IncRNA of interest. In some embodiments, the siRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof, e.g., see Table 3), or a portion thereof. In some embodiments, the siRNA is complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof, e.g., see Table 3), or a portion thereof.

In some embodiments, the siRNAs described herein have 0-7 nucleotide 3’ overhangs or 0-4 nucleotide 5’ overhangs. In some embodiments, the siRNA molecule has a single uracil (e.g., U) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a double uracil (e.g., UU) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a single thymine (e.g., T) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a double thymine (e.g., TT) overhang at each 3’ end of the siRNA. In some embodiments, the siRNA molecule has a cytosine and thymine (e.g., CT) overhang at each 3’ end of the siRNA.

Different siRNAs can be combined for decreasing IcRNA expression of EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof(e.g., see Table 3). A combination of two siRNAs may be used in a method of the invention, such as two different siRNAs, three different siRNAs, four different siRNAs, or five different siRNAs targeting the same IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof). In some embodiments, the siRNA sequence may contain at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one or more of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA sequence may contain the sequence of any one or more of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 15 contiguous nucleotides set forth within any one of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 16 contiguous nucleotides set forth within any one of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 17 contiguous nucleotides set forth within any one of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 18 contiguous nucleotides set forth within any one of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 19 contiguous nucleotides set forth within any one of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA contains at least 20 contiguous nucleotides set forth within any one of SEQ ID NOs: 1 -23 (e.g., see Table 1 ). In some embodiments, the siRNA contains 21 contiguous nucleotides set forth within any one of SEQ ID NOs: 1 -23 (e.g., see Table 1 ).

In any of the foregoing embodiments, the siRNA further contains the sequence of any one of SEQ ID NOs: 24-26. Table 1 below provides the antisense and sense strands of exemplary siRNA sequences of the invention. TABLE 1. EXEMPLARY siRNA SEQUENCES

A = adenine; C = cytosine; G = guanine; U = uracil.

In some embodiments, the siRNA of the disclosure may target a nucleotide sequence of any one of SEQ ID NOs: 47-71 (e.g., see Table 2), or a complementary sequence thereof, or variant thereof with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% thereto.

TABLE 2. TARGET SEQUENCES

In some embodiments, the siRNA comprises a sequence complementary to at least 15 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 16 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 17 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 18 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 19 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 20 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 21 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 22 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 23 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 24 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 25 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 26 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 27 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 28 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 29 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). In some embodiments, the siRNA comprises a sequence complementary to at least 30 contiguous nucleotides set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3).

In some embodiments, the siRNA includes a sequence complementary at least 15 to 30 contiguous nucleotides (e.g., 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 36 to 30, 27 to 30, 28 to 30, or 29 to 30 contiguous nucleotides) set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3). The nucleotide sequence of SEQ ID NO: 70 or SEQ ID NO: 71 is set forth in Table 2 while variants (e.g., splice variants) of SEQ ID NO: 70 are provided in Table 3.

TABLE 3. EPIC1 SPLICE VARIANTS

Double-stranded RNA (ds RNA) dsRNAs of the disclosure are ds nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target IncRNA of interest. Typically, dsRNAs are longer than an siRNA and are processed within a cell to form an siRNA molecule. The siRNA is then incorporated into an RNA-induced silencing complex (RISC). Upon siRNA hybridization to a target IncRNA, the RISC complex will cleave the target IncRNA, thereby inactivating it and reducing the level of the IncRNA in the cell.

In some embodiments, dsRNAs of the disclosure may include a sense strand and an antisense strand, each containing a nucleotide sequence of about 25 to about 1815 nucleotides in length, or longer (e.g., 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 1 10, about 1 15, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about

165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 210, about

220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about

310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 380, about

400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about

625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about

850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, or about 1815 nucleotides in length).

In some embodiments, dsRNAs of the disclosure may include a sense strand and an antisense strand, each containing a nucleotide sequence of 25 to 1815 nucleotides in length, or longer (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,

155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,

320, 330, 340, 350, 360, 370, 380, 380, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700,

725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,

1800, or 1815 nucleotides in length).

In some embodiments, the dsRNA includes a sequence complementary at least 25 to 1815 contiguous nucleotides (e.g., 25 to 1815, 30 to 1815, 35 to 1815, 40 to 1815, 45 to 1815, 50 to 1815, 55 to 1815, 60 to 1815, 65 to 1815, 70 to 1815, 75 to 1815, 80 to 1815, 85 to 1815, 90 to 1815, 95 to 1815, 100 to 1815, 110 to 1815, 120 to 1815, 130 to 1815, 140 to 1815, 150 to 1815, 160 to 1815, 170 to 1815,

180 to 1815, 190 to 1815, 200 to 1815, 210 to 1815, 220 to 1815, 230 to 1815, 240 to 1815, 250 to 1815,

275 to 1815, 300 to 1815, 325 to 1815, 350 to 1815, 375 to 1815, 400 to 1815, 425 to 1815, 450 to 1815,

475 to 1815, 500 to 1815, 550 to 1815, 600 to 1815, 650 to 1815, 700 to 1815, 750 to 1815, 800 to 1815,

850 to 1815, 900 to 1815, 950 to 1815, 1000 to 1815, 1100 to 1815, 1200 to 1815, 1300 to 1815, 1400 to 1815, 1500 to 1815, 1600 to 1815, 1700 to 1815, or 1800 to 1815 contiguous nucleotides) set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3).

In some embodiments, the sense and antisense strands of an dsRNA molecule of the disclosure are completely complementary. In some embodiments, the sense and antisense strands of an dsRNA molecule of the disclosure are completely complementary to the extent that their lengths overlap with one another. Depending on the sequence of the first and second strand, complementarity need not be complete or perfect, which means that the first and second strand are not 100% base-paired due to mismatches. One or more mismatches may be present within the ds dsRNA without impacting the dsRNA’s ability to reduced expression of a target IncRNA of interest.

The nucleotide sequence of an dsRNA of the disclosure may contain sufficient complementarity to a portion of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof) such that the dsRNA can hybridize with the target IncRNA of interest. In some embodiments, the dsRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof. In some embodiments, the dsRNA is complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof.

Different dsRNAs can be combined for decreasing the expression of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof). A combination of two dsRNAs may be used in a method of the invention, such as two different dsRNAs, three different dsRNAs, four different dsRNAs, or five different dsRNAs targeting the same IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof. micro RNA (miRNA) miRNAs of the disclosure are single stranded (ss) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target IncRNA of interest. Once a miRNA molecule enters a cell, it is incorporated into a RNA-induced silencing complex (RISC). Upon miRNA hybridization to a target IncRNA, the RISC complex will cleave the target IncRNA, thereby inactivating it, resulting in reduced IncRNA levels in the cell.

In some embodiments, miRNAs of the disclosure may include a nucleotide sequence of about 6 to about 30 nucleotides in length (e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length).

In some embodiments, miRNAs of the disclosure may include a nucleotide sequence of 6 to 30 nucleotides in length (e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

In some embodiments, the miRNA comprises a sequence complementary at least 6 to 30 contiguous nucleotides (e.g., 6 to 30, 7 to 30, 8 to 30, 9 to 30, 10 to 30, 11 to 30, 12 to 30, 13 to 30, 14 to 30, 15 to 30, 16 to 30, 17 to 30, 18 to 30, 19 to 30, 20 to 30, 21 to 30, 22 to 30, 23 to 30, 24 to 30, 25 to 30, 26 to 30, 27 to 30, 28 to 30, or 29 to 30 contiguous nucleotides) set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3).

The nucleotide sequence of the miRNA may contain sufficient complementarity to a portion of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof) such that the miRNA can hybridize with the target IncRNA of interest. In some embodiments, the miRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof. In some embodiments, the miRNA is complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof.

Different miRNAs can be combined for decreasing the expression of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof). A combination of two or more miRNAs may be used in a method of the invention, such as two different miRNAs, three different miRNAs, four different miRNAs, or five different miRNAs targeting the same IcRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof, e.g., see Table 3). short hairpin RNA (shRNA) shRNAs of the disclosure are ss or ds nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target IncRNA of interest. Once a shRNA molecule enters a cell, it is incorporated into a RNA-induced silencing complex (RISC). Upon shRNA hybridization to a target IncRNA, the RISC complex will cleave the target IncRNA, thereby inactivating it and reducing the level of the IncRNA in the cell.

In some embodiments, shRNAs of the disclosure may include a nucleotide sequence of about 50 to about 100 nucleotides in length (e.g., 45 to 105, 50 to 105, 55 to 105, 60 to 105, 65 to 105, 70 to 105, 75 to 105, 80 to 105, 85 to 105, 90 to 105, or 95 to 105 nucleotides in length, e.g., 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 nucleotides in length).

In some embodiments, shRNAs of the disclosure may include a nucleotide sequence of 50 to 100 nucleotides in length (e.g., 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, or 95 to 100 nucleotides in length, e.g., 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length).

It is within the scope of the disclosure that any length, known and previously unknown in the art, may be employed for the current invention. shRNAs of the disclosure contain a variable hairpin loop structure and a stem sequence. In some embodiments the stem sequence may be 10 to 50 nucleotides in length (e.g., 10 to 50, 11 to 50, 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50,

24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to 50, 32 to 50, 33 to 50, 34 to 50,

35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to 50, 43 to 50, 44 to 50, 45 to 50,

46 to 50, 47 to 50, 48 to 50, or 49 to 50 nucleotides in length, e.g., 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,

20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length). In some embodiments, the hairpin size is between 4 to 50 nucleotides in length (e.g., 4 to 50, 5 to 50, 6 to 50, 7 to 50, 8 to 50, 9 to 50, 10 to 50, 11 to 50, 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to

50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to

50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to

50, 48 to 50, or 49 to 50 nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19,

20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length), although the loop size may be larger without significantly affecting silencing activity. shRNA molecules of the disclosure may contain mismatches, for example G-U mismatches between two strands of the shRNA stem without decreasing potency. In some embodiments, shRNAs are designed to include one or several G-U pairings in the hairpin stem to stabilize hairpins during propagation in bacteria, for example.

In some embodiments, the shRNA includes a sequence (e.g., a stem sequence) complementary at least 10 to 50 contiguous nucleotides (e.g., 10 to 50, 11 to 50, 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50, 31 to 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50, 42 to 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to 50, 48 to 50, or 49 to 50 contiguous nucleotides) set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3).

The nucleotide sequence of the shRNA may contain sufficient complementarity to a portion of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof) such that the shRNA can hybridize with the target IncRNA of interest. In some embodiments, the shRNA is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof. In some embodiments, the shRNA is complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof.

Different shRNAs can be combined for decreasing the expression of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof). A combination of two or more shRNAs may be used in a method of the invention, such as two different shRNAs, three different shRNAs, four different shRNAs, or five different shRNAs targeting the same IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof).

Anti-Sense Oligonucleotide (ASO)

ASOs of the disclosure are single (ss) nucleic acid molecules made of DNA, RNA, or both DNA and RNA (e.g., a chimeric) that are complementary to a target IncRNA of interest. Upon hybridization to a target IncRNA in a cell, RNase H will degrade the IncRNA by hydrolyzation, resulting in reduced IncRNA levels in the cell.

In some embodiments, ASOs of the disclosure may include a nucleotide sequence of about 12 to about 50 nucleotides in length (e.g., 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 nucleotides in length).

In some embodiments, ASOs of the disclosure may include a nucleotide sequence of 12 to 50 nucleotides in length (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

In some embodiments, the ASO includes a sequence complementary at least 12 to 50 contiguous nucleotides (e.g., 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50,

31 to 50, 32 to 50, 33 to 50, 34 to 50, 35 to 50, 36 to 50, 37 to 50, 38 to 50, 39 to 50, 40 to 50, 41 to 50,

42 to 50, 43 to 50, 44 to 50, 45 to 50, 46 to 50, 47 to 50, 48 to 50, or 49 to 50 contiguous nucleotides) set forth within SEQ ID NO: 70 or SEQ ID NO: 71 (or a variant thereof, e.g., see Table 3).

The nucleotide sequence of the ASO may contain sufficient complementarity to a portion of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof) such that the ASO can hybridize with the target IncRNA of interest. In some embodiments, the ASO is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof. In some embodiments, the ASO is complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof.

Different ASOs can be combined for decreasing the expression of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof, e.g., see Table 3). A combination of two ASOs may be used in a method of the invention, such as two different ASOs, different three ASOs, four different ASOs, or five different ASOs targeting the same IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof, e.g., see Table 3).

GapmeR

GapmeRs of the disclosure are single (ss) nucleic acid molecules made of DNA and RNA with the central 8-10 nucleotide of the gapmeR being DNA that is complementary to a target IncRNA of interest. Upon hybridization to a target IncRNA in a cell, RNase H will degrade the IncRNA by hydrolyzation, resulting in reduced IncRNA levels in the cell.

In some embodiments, gapmeRs of the disclosure may include a nucleotide sequence of about 12 to about 50 nucleotides in length (e.g., 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51 nucleotides in length).

In some embodiments, gapmeRs of the disclosure may include a nucleotide sequence of 12 to 50 nucleotides in length (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length).

In some embodiments, gapmeRs of the disclosure may include a nucleotide sequence of 8, 9, or 10 internal DNA nucleotides.

In some embodiments, the gapmeR includes a sequence complementary at least 12 to 50 contiguous nucleotides (e.g., 12 to 50, 13 to 50, 14 to 50, 15 to 50, 16 to 50, 17 to 50, 18 to 50, 19 to 50, 20 to 50, 21 to 50, 22 to 50, 23 to 50, 24 to 50, 25 to 50, 26 to 50, 27 to 50, 28 to 50, 29 to 50, 30 to 50,

The nucleotide sequence of the gapmeR may contain sufficient complementarity to a portion of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof) such that the gapmeR can hybridize with the target IncRNA of interest. In some embodiments, the gapmeR is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof. In some embodiments, the gapmeR is complementary to the target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof), or a portion thereof.

Different gapmeRs can be combined for decreasing the expression of a target IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof). A combination of two gapmeRs may be used in a method of the invention, such as two different gapmeRs, different three gapmeRs, four different gapmeRs, or five different gapmeRs targeting the same IncRNA of interest (e.g., EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof).

Modifications to the Inhibitory Nucleic Acid Molecules

It is contemplated that any of the inhibitory nucleic acid molecules disclosed herein may be used in the methods disclosed herein in an unmodified or in a modified form. Unmodified inhibitory nucleic acid molecules contain nucleobases that include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid molecules are described in more detail below.

Modifications may be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences.

Modifications may be achieved by incorporating, for example, one or more alternative nucleosides, alternative 2’ sugar moieties, and/or alternative internucleoside linkages, which are described further below. Typically, these types of modifications are introduced to optimize the molecule’s efficacy or biophysical properties (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, reduce immunogenicity, and/or targeting to a particular location or cell type).

Modification may further be achieved by covalently or non-covalently conjugating a moiety (e.g., a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer) to the 5’ end and/or 3’ end of the inhibitory nucleic acid molecule, as described in more detail below.

Nucleoside Modifications

Modification of the inhibitory nucleic acid molecules described herein include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and/or 3-deazaguanine and 3-deazaadenine. The inhibitory nucleic acid molecules may also include nucleobases in which the purine or pyrimidine base is replaced with other heterocycles, for example 7- deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and/or 2-pyridone. Further modification of the inhibitory nucleic acid molecules described herein may include nucleobases disclosed in US 3,687,808; Kroschwitz, J. I., ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition 30:613, 1991 ; and Sanghvi, Y.S., Chapter 16, Antisense Research and Applications, CRC Press, Gait, M.J. ed., 1993, pp. 289-302.

Sugar Modifications

Modifications of the inhibitory nucleic acid molecules described herein may also include one or more of the following 2’ sugar modifications: 2’-O-methyl (2’-O-Me), 2'-methoxyethoxy (2'-O- CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e. , a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, and/or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2OCH2N(CH3)2. Other possible 2'-modifications that can modify the inhibitory nucleic acid molecules described herein include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (- OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the interfering RNA molecule, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

Internucleoside Linkage Modifications

Modifications of the inhibitory nucleic acid molecules described herein may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'- alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.

Conjugates

Any of the inhibitory nucleic acid molecules described herein may be modified via the addition of an auxiliary moiety, e.g., a cell penetrating peptide (CPP), a polymer, a hydrophobic moiety, or a targeting moiety. The auxiliary moiety may be present as a 5’ terminal modification (e.g., covalently bonded to a 5’- terminal nucleoside), a 3’ terminal modification (e.g., covalently bonded to a 3’-terminal nucleoside), or an internucleoside linkage (e.g., covalently bonded to phosphate or phosphorothioate in an internucleoside linkage). CPPs are known in the art (e.g., TAT or Arg8) (Snyder and Dowdy, 2005, Expert Opin. Drug Deliv. 2, 43-51 ). Specific examples of CPPs are provided in WO2011157713, which is incorporated herein by reference in its entirety.

Inhibitory nucleic acid molecules of the disclosure may include covalently attached neutral polymer-based auxiliary moieties. Neutral polymers include poly(C1 -6 alkylene oxide), e.g., polyethylene glycol) and polypropylene glycol) and copolymers thereof, e.g., di- and triblock copolymers.

An inhibitory nucleic acid molecule containing a hydrophobic moiety may exhibit superior cellular uptake, as compared to an inhibitory nucleic acid molecule lacking the hydrophobic moiety. A hydrophobic moiety is a monovalent group (e.g., a bile acid (e.g., cholic acid, taurocholic acid, deoxycholic acid, oleyl lithocholic acid, or oleoyl cholenic acid), glycolipid, phospholipid, sphingolipid, isoprenoid, vitamin, saturated fatty acid, unsaturated fatty acid, fatty acid ester, triglyceride, pyrene, porphyrine, texaphyrine, adamantine, acridine, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butydimethylsilyl, t-butyldiphenylsilyl, cyanine dye (e.g., Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen) covalently linked to the nucleic acid backbone (e.g., 5’- terminus) of the inhibitory nucleic acid molecule.

A targeting moiety is selected based on its ability to target oligonucleotides of the invention to a desired or selected cell population that expresses the corresponding binding partner (e.g., either the corresponding receptor or ligand) for the selected targeting moiety. For example, an oligonucleotide of the invention could be targeted to hepatocytes expressing asialoglycoprotein receptor (ASGP-R) by selecting a targeting moiety containing N-acetylgalactosamine (GalNAc).

In some embodiments, the targeting moiety is vascular cell adhesion protein 1 (VCAM1).

A targeting moiety may include one or more ligands (e.g., 1 to 9 ligands, 1 to 6 ligands, 1 to 3 ligands, 3 ligands, or 1 ligand). The ligand may target a cell expressing asialoglycoprotein receptor (ASGP-R), IgA receptor, HDL receptor, LDL receptor, or transferrin receptor. Non-limiting examples of the ligands include N-acetylgalactosamine (e.g., a triantennary N-acetylgalactosamine), glycyrrhetinic acid, glycyrrhizin, lactobionic acid, lactoferrin, IgA, or a bile acid (e.g., lithocholyltaurine or taurocholic acid).

The ligand may be a small molecule, e.g., a small molecule targeting a cell expressing asialoglycoprotein receptor (ASGP-R). A non-limiting example of a small molecule targeting an asialoglycoprotein receptor is N-acetylgalactosamine. Alternatively, the ligand can be an antibody or an antigen-binding fragment or an engineered derivative thereof (e.g., Fcab or a fusion protein (e.g., scFv)).

Preparation of Inhibitory Nucleic Acid Molecules

Inhibitory nucleic acid molecules of the disclosure may be prepared using techniques and methods known in the art for the oligonucleotide synthesis. For example, inhibitory nucleic acid molecules of the disclosure may be prepared using a phosphoramidite-based synthesis cycle. This synthesis cycle includes the steps of (1 ) de-blocking a 5’-protected nucleotide to produce a 5’-deblocked nucleotide, (2) coupling the 5’-deblocked nucleotide with a 5’-protected nucleoside phosphoramidite to produce nucleosides linked through a phosphite, (3) repeating steps (1 ) and (2) one or more times as needed, (4) capping the 5’-terminus, and (5) oxidation or sulfurization of internucleoside phosphites. The reagents and reaction conditions useful for the oligonucleotide synthesis are known in the art. The inhibitory nucleic acid molecules disclosed herein may be linked to solid support as a result of solid-phase synthesis. Cleavable solid supports that may be used are known in the art. Non-limiting examples of the solid support include, e.g., controlled pore glass or macroporous polystyrene bonded to a strand through a cleavable linker (e.g., succinate-based linker) known in the art (e.g., UnyLinkerTM). A nucleic acid linked to solid support may be removed from the solid support by cleaving the linker connecting a nucleic acid and solid support.

Compositions

The inhibitory nucleic acid molecules described herein may be formulated into various compositions (e.g., a pharmaceutical composition) for administration to a subject in a biologically compatible form suitable for administration in vivo. For example, the inhibitory nucleic acid molecules described herein (e.g., the siRNA molecules of SEQ ID NOs: 1 -46, or variants thereof) may be administered in a suitable diluent, carrier, or excipient, and may further contain a preservative, e.g., to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington, J.P. The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22^nd ed. And in The United States Pharmacopeial Convention, The National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates and mammals.

Compositions containing the inhibitory nucleic acids described herein may further include a second therapeutic agent (e.g., a nucleic acid molecule to be expressed within a cell, a polypeptide, or a drug). For example, a second therapeutic agent may be a blood pressure medication, an antiinflammatory medication (e.g., a steroid or colchicine), or immunosuppressive agent. In some embodiments, the second therapeutic agent is a statin. Non-limiting examples of second therapeutic agents are a statin (e.g., atorvastatin), a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor (e.g., an siRNA or monoclonal antibody targeting PCSK9), or ezetimibe (e.g., ZETIA™).

In some embodiments, the second therapeutic agent (e.g., statin) is administered in combination with an inhibitory nucleic acid molecule of the disclosure. In some embodiments, the subject is orally administered a statin. In some embodiments, the subject is administered a statin daily. Methods of Treatment

The disclosure provides methods of treating arteriosclerosis in a subject. In some embodiments, the method contains the steps of administering to a subject an inhibitory nucleic acid molecule described herein, wherein the inhibitory nucleic acid molecule targets EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof (e.g., see Table 3). In some embodiments, the method contains the steps of administering to a subject an siRNA molecule described herein (e.g., SEQ ID NOs: 1 -46, or a variant thereof), wherein the siRNA molecule targets EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof (e.g., see Table 3). In some embodiments, the method contains the steps of administering to a subject an dsRNA molecule described herein, wherein the dsRNA molecule targets EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof (e.g., see Table 3). In some embodiments, the method contains the steps of administering to a subject an ASO molecule described herein, wherein the ASO molecule targets EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof (e.g., see Table 3). In some embodiments, the method contains the steps of administering to a subject a gapmeR described herein, wherein the gapmeR targets EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof (e.g., see Table 3). In some embodiments, the method contains the steps of administering to a subject an miRNA molecule described herein, wherein the miRNA molecule targets EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof (e.g., see Table 3). In some embodiments, the method contains the steps of administering to a subject an shRNA molecule described herein, wherein the shRNA molecule targets EPIC1 (SEQ ID NO: 70) or MERRICAL (SEQ ID NO: 71 ), or a variant thereof (e.g., see Table 3).

In some embodiments, the arteriosclerosis is atherosclerosis. In some embodiments, the atherosclerosis is diabetes-associated atherosclerosis.

In some embodiments, an additional therapeutic agent (e.g., a statin, a PCSK9 inhibitor, or ezetimibe) can be administered prior to, subsequent to, or concurrently with an inhibitory nucleic acid described herein.

Any of the methods can administer a composition (e.g., a pharmaceutical composition) or delivery vehicle (e.g., a vector or nanoparticle) that contains or expresses any of the inhibitory nucleic acid molecules described herein (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, or gapmeR).

Delivery Vehicle

The inhibitory nucleic acid molecule of the disclosure may be delivered to a subject (e.g., a human) using any suitable delivery vehicle. For example, a delivery vehicle for any of the inhibitory nucleic acid molecules described herein may be a vector, plasmid, or nano particle, (e.g., a micelle, a liposome, an exosome, or a lipid nano particle (LNP)).

The inhibitory nucleic acid molecule of the disclosure and compositions thereof may be delivered to a subject via a vector (e.g., a viral vector). Any suitable viral vector system can be used including, e.g., adenoviruses (e.g., Ad2, Ad5, Ad9, Ad15, Ad17, Ad19, Ad20, Ad22, Ad26, Ad27, Ad28, Ad30, or Ad39), rhabdoviruses (e.g., vesicular stomatitis virus), retroviruses, adeno-associated vectors (AAV), poxviruses, herpes viral vectors, and Sindbis viral vectors. For example, the vector may be an AAV vAAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV12 vector. The inhibitory nucleic acid molecule of the disclosure and compositions thereof may be delivered to a subject via liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of the inhibitory nucleic acids described herein, and compositions thereof. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical composition. The liposomes encapsulating the inhibitory nucleic acid molecules may be administered, e.g., intravenously, subcutaneously, or intramuscularly.

The inhibitory nucleic acid molecule of the disclosure and compositions thereof may be delivered to a subject via exosomes. Exosomes produced from cells can be collected from cell culture medium by any suitable method. Typically, a preparation of exosomes can be prepared from cell culture or tissue supernatant by centrifugation, filtration or combinations of these methods. For example, using standard methods, exosomes can be prepared by differential centrifugation, that is low speed (<20000 g) centrifugation to pellet larger particles followed by high speed (>100000 g) centrifugation to pellet exosomes, size filtration with appropriate filters (for example, 0.22 micrometer filter), gradient ultracentrifugation (for example, with sucrose gradient) or a combination of these methods.

The inhibitory nucleic acid molecules of the disclosure, and compositions thereof, may be delivered to a subject via LNPs. For example, the inhibitory nucleic acid molecules (e.g., siRNA, dsRNA, miRNA, shRNA, ASO, or gapmeR) may be formulated in a lipid nanoparticle such as those described in International Publication No. WO2012170930, herein incorporated by reference in its entirety. As a nonlimiting example, LNP formulations may contain cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol, polyethylene glycol (PEG), R-3-[(w-methoxy polyethylene glycol)2000)carbamoyl)]-1 ,2- dimyristyloxl-propyl-3-amine (PEG-c-DOMG), distearoyl-rac-glycerol (DSG) and/or dimethylaminobutanoate (DMA). As a non-limiting example, 1 -5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. In another embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1 ,2-Distearoyl-sn-glycerol, methoxypoly ethylene glycol) or PEG-DPG (1 ,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, (6Z,9Z,28Z,31Z)- heptatriacont-6,9,28,31 -tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 1 ,2-dil inoleyloxy- n,n-dimethyl-3-aminopropane (DLin-DMA), C 12-200, and N,N-dimethyl-2,2-di-(9Z,12Z)-9,12- octadecadien-1 -yl-1 ,3-dioxolane-4-ethanamine (DLin-KC2-DMA).

Exemplary commercial reagents useful for lipid-based delivery of inhibitory nucleic acid molecules including, but not limited to, TransIT-TKO™ (Mirus, Catalog No. MIR 2150), Transmessenger™ (Qiagen, Catalog No. 301525), Oligofectamine™ and Lipofectamine™ (Invitrogen, Catalog No. MIR 12252-011 and Catalog No. 13778-075), siPORT™ (Ambion, Catalog No. 1631 ), and DharmaFECT™ (Fisher Scientific, Catalog No. T-2001 -01 ). Subject

The subject to be treated may have a metabolic disorder, or is at risk of developing a metabolic disorder, such as diabetes. Subjects at risk of developing diabetes may be prediabetic and/or have experienced one or more of the following risk factors: hyperglycemia, glucose resistance, insulin resistance, hyperlipidemia, or has a family history of diabetes.

Dosage

The actual dosage amount of a composition of the present disclosure administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage (e.g., mg/kg) and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Administration may occur any suitable number of times per day, and for as long as necessary. Subjects may be adult or pediatric humans, with or without comorbid diseases.

Routes of Administration

The compositions utilized in the methods described herein can be administered to a subject by any suitable route of administration. For example, a composition containing an inhibitory nucleic acid of the disclosure may be administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions.

In some embodiments, the compositions utilized in the methods described herein can be administered to the subject intravenously. In some embodiments, the compositions utilized in the methods described herein can be administered to the subject subcutaneously. In some embodiments, the compositions utilized in the methods described herein can be administered to the subject intraarticularly. In some embodiments, the compositions utilized in the methods described herein can be administered to the subject intramuscularly. EXAMPLES

The following examples are provided as a description of how the compositions and methods described herein may be used and evaluated and are intended to be purely exemplary of the invention and is not intended to limit the scope of what the inventors regard as their invention.

Example 1. Deficiency of IncRNA MERRICAL abrogates macrophage chemotaxis and diabetes- associated atherosclerosis

The present example describes the following discoveries: (1 ) macrophage-enriched IncRNA regulates inflammation, chemotaxis, and atherosclerosis (MERRICAL) is increased in diabetes- associated atherosclerosis; (2) MERRICAL in cis regulated C-C motif chemokine ligand (CCL)-3 and CCL4 by guiding the WD repeat domain 5 protein-mixed lineage leukemia (WDR5-MLL1 ) complex and H3K4me3; (3) MERRICAL knockdown (KD) in macrophages reduced CCL3 and CCL4 expression and chemotactic responses; (4) MERRICAL KD abrogated pro-inflammatory macrophage recruitment and lesion formation; and (5) epigenetically induced MYC interacting IncRNAI (EPIC1 ) is a human ortholog of MERRICAL.

Diabetes-associated atherosclerosis involves excessive immune cell recruitment and plaque formation. However, the mechanisms remain poorly understood. Transcriptomic analysis of the aortic intima in LdlH- mice on a high-fat, high-sucrose-containing (HFSC) diet identifies a macrophage-enriched nuclear long noncoding RNA (IncRNA), MERRICAL (macrophage-enriched IncRNA regulates inflammation, chemotaxis, and atherosclerosis). MERRICAL expression increases by 249% in intimal lesions during progression. IncRNA-mRNA pair genomic mapping reveals that MERRICAL positively correlates with the chemokines CCL3 and CCL4. MERRICAL-deficient macrophages exhibit lower CCL3 and CCL4 expression, chemotaxis, and inflammatory responses. Mechanistically, MERRICAL guides the WDR5-MLL1 complex to activate CCL3 and CCL4 transcription via H3K4me3 modification. MERRICAL deficiency in HFSC diet-fed Ldlr mice reduces lesion formation by 74% in the aortic sinus and 86% in the descending aorta by inhibiting leukocyte recruitment into the aortic wall and pro-inflammatory responses. These findings unveil a regulatory mechanism (FIG. 8) whereby a macrophage-enriched IncRNA potently inhibits chemotactic responses, alleviating lesion progression in diabetes.

The increased prevalence of diabetes mellitus in recent years has triggered a global health crisis, particularly by contributing to accelerated atherosclerosis and associated cardiovascular morbidity and mortality.¹ Type 2 diabetes mellitus, a complex and multifactorial metabolic disease, is characterized by the cardinal features of hyperglycemia, hyperinsulinemia, dyslipidemia, and chronic inflammation.² Prolonged exposure to these metabolic alterations is now recognized as a major factor in the pathogenesis of atherosclerosis in diabetes.³ Accumulating studies highlight that chronic inflammation coupled with dysregulation of macrophage effector functions may be a critical link between lesion progression and diabetes.⁴ Following recruitment and differentiation of circulating monocytes into the vessel wall, lesional macrophages phagocytose modified low-density lipoprotein particles and contribute to the growth and subsequent de-stabilization of plaques through secretion of pro-inflammatory mediators, such as chemokines and cytokines.⁴’⁵’⁶ However, significant gaps remain in the molecular underpinnings that regulate macrophage chemokine/cytokine secretion and the pro-inflammatory response in diabetes-associated atherosclerosis.

Advances in genome-wide transcriptome analysis demonstrated that only 2% of the mammalian genome is transcribed into protein-coding RNAs (mRNAs),⁸⁹ indicating that the majority of the mammalian genome is utilized for generating transcripts that lack protein-coding potential. These transcripts are defined as noncoding RNAs (ncRNAs).¹⁰’¹¹’¹² Currently, ncRNAs are broadly classified into two major groups based on size: (1 ) a variable class of small ncRNAs (<200 bp) and (2) transcripts that exceed 200 bp and are broadly defined as long ncRNA (IncRNA).¹³’¹⁴ An accumulating body of evidence indicates that IncRNAs play crucial roles in regulating key cellular processes and are dysregulated in diverse disease states, including atherosclerosis.¹⁵’¹⁶’¹⁷’¹⁸’¹⁹’²⁰’²¹’²²’²³’²⁴

Surprisingly, IncRNAs discovered to date in cardiovascular disease function through a variety of mechanisms, ranging from interactions between RNA, chromatin, DNA, and protein to cell signaling.¹⁰ While some IncRNAs have been reported to act in trans and pervasively regulate gene expression from a different genomic location, a growing proportion of IncRNAs have been reported to act in cis and function as local effectors to regulate the expression of neighboring mRNA transcripts.²⁵’²⁶²⁷ For example, several co-expressed neighboring mRNA-IncRNA pairs have been identified in endothelial cells with positive correlations in vascular disease models.¹⁹’²⁸²⁹ However, whether such cis-acting IncRNAs participate in the inflammatory and chemotactic response in macrophages in the development of diabetes-associated atherosclerosis remains to be investigated.

There are several established diabetic atherosclerosis mouse models, including a streptozotocin (STZ)-induced type 1 diabetes model³⁰’^{31 32} and a genetic diet-induced type 2 diabetic mouse model.³³ In this study, a diet-induced diabetic atherosclerosis mouse model was utilized by placing Ldlr mice on a high-fat, high-sucrose-containing (HFSC) diet in a similar manner as described previously.³⁴ Ldlr^/_ mice on this HFSC diet exhibited hyperglycemia, glucose and insulin resistance, and hyperlipidemia and developed atherosclerosis with accelerated lesion formation.³⁴ Transcriptomic analyses were performed on the aortic intima during lesion progression from Ldlr mice under HFSC diet conditions, which identified neighboring mRNA-IncRNA pairs differentially expressed with atherosclerosis progression. One IncRNA captured in this analysis showed a strong positive correlation with a well-studied group of chemokine genes in the CCL family. Functional experiments aimed at characterizing this IncRNA were performed; this IncRNA was termed macrophage-enriched IncRNA regulates inflammation, chemotaxis, and atherosclerosis (MERRICAL) due to its critical role in macrophage chemotaxis and inflammation during the development of diabetes-associated atherosclerosis in Ldlr^z- mice. Overall, these findings provide insights into the pathobiology of a broad range of chronic disease states associated with the maladaptive macrophage chemotactic response in diabetes.

Results

Discovery of MERRICAL, a coordinately regulated IncRNA with a group of chemokine genes in diabetes-associated atherosclerosis

Significantly regulated IncRNA candidates in the aortic intima during the progression phase of diabetes-associated atherosclerosis were identified. A diet-induced diabetic atherosclerosis mouse model was used by placing Ldlr^z- mice on an HFSC diet. After 12 weeks of HFSC diet feeding, Ldlr^z- mice exhibited hyperglycemia, glucose and insulin intolerance, and hyperlipidemia, as reported previously.³⁴ Transcriptomic analysis was performed on RNA samples derived from the aortic intima of Ldlr mice after a progression phase of 0, 2, and 12 weeks on the HFSC diet (groups 1-3) (FIG. 1 A). RNA sequencing (RNA-seq) profiling captured a total of 16,917 mRNAs and 2,807 IncRNAs (FIG. 1 B). DEseq2 analysis was performed to identify significantly differentially expressed transcripts. In total, 140 IncRNAs and 2,763 mRNAs were differentially expressed (false discovery rate [FDR] < 0.05) at the 12-week progression time point (G3). Subsequent analyses focused on the 12-week progression time point because phenotypic changes at this time point are magnified during the development of diabetic atherosclerosis.³⁴ The top dysregulated noncoding transcript was the IncRNA AI662270 (hereafter called MERRICAL), which is upregulated at 12 weeks of lesion progression (FIGS. 1 C and 1 D).

Given the potential for IncRNAs to regulate their neighboring genes,³⁵ nearby regulatory IncRNAs involved in the progression of atherosclerosis were sought. Compared with the physical distance of IncRNA and mRNA pairs, chromatin organization appears to play a more important role, as mRNA- IncRNA pairs localized within the same or adjacent topologically associating domain (TAD) showed the highest correlation, irrespective of the distance between them.²⁸ A differential expression (DE) analysis was performed with DE-mRNA/DE-IncRNA pairs being matched back to a genomic Hi-C database³⁶, which identified 75 mRNA-IncRNA pairs within a 500-kb distance (FIG. 1 E). Surprisingly, among those pairs, the IncRNA MERRICAL positively correlated with several mRNA transcripts in close proximity (FIGS. 1 E and 1 F) that comprised a family of chemokine genes known to be closely associated with chemotaxis and pro-inflammatory responses in the progression of atherosclerosis.³⁷ Interestingly, only the chemokine genes exhibited upregulated expression during the progression phase, whereas the Schlafen (SLFN) genes did not show any such change (FIG. 1 F). Because of its expression pattern and function, as detailed below, this IncRNA was termed MERRICAL.

Identification of the IncRNA MERRICAL in the intima of atherosclerotic lesions

The IncRNA MERRICAL has a total of four spliced variants. Statistical analysis of differential transcript usage for MERRICAL was performed and found that the transcript ENSMUST00000216842.2 (1 ,428 bp) is the dominant isoform and was significantly upregulated in the progression (G3) group.

Studies have shown that IncRNAs are enriched in a tissue- or cell-specific manner that can exert profound phenotypic effects.³⁸³⁹ The IncRNA MERRICAL was enriched in the aortic intima compared with the media isolated from Ldlr mice on the HFSC diet (FIG. 2A). Tissue profiling revealed that the IncRNA MERRICAL was highly enriched in the bone marrow (BM), spleen, thymus, and peripheral blood mononuclear cells (PBMCs) compared with the liver and lungs (FIG. 2B). The aortic intima was not only enriched in endothelial cells but also highly expressed several leukocyte markers in atherosclerotic mice.⁴⁰ Using magnetic bead-isolated aortic cells (CD45⁺ leukocytes, CD31 ⁺ endothelial cells, CD90.2+ fibroblasts, and aSMA⁺ smooth muscle cells), IncRNA MERRICAL expression was found to be uniquely enriched in CD45⁺ leukocytes (FIG. 2C). Consistent enrichment of the IncRNA MERRICAL was also observed in the uniform manifold approximation and projection (UMAP) of single-cell RNA-seq from mouse aortic cells (FIG. 2F). Among the macrophage and monocyte cluster, 81 .8% of MERRICAL’s expression is expressed in this cluster (FIG. 2L). Moreover, by comparing different types of immune cells, the IncRNA MERRICAL had the highest expression in macrophages, especially activated peritoneal macrophages (FIG. 2D). The expression of the IncRNA MERRICAL was exponentially upregulated with the differentiation of BM-derived macrophages (BMDMs) and in pro-inflammatory M1 -like macrophages (FIG. 2E).

MERRICAL is annotated as an IncRNA in both the Ensembl database and NCBI. This transcript’s coding potential was validated by first checking the coding probability using the in silico coding potential assessment tool (CPAT), which found that MERRICAL had a score similar to those of other well- described IncRNAs, such as MALAT1^{41 42} or CARMN²⁰⁴³ (FIG. 2G). Consequently, to further validate any peptide coding potential, the MERRICAL sequence was cloned upstream of the p3xFLAG-CMV plasmid, transfected into HEK293T cells, and immunoblotted for a FLAG tag. No peptide or protein from MERRICAL overexpression was detected (FIG. 2H). RNA in situ hybridization (RNA-ISH) demonstrated that MERRICAL localized to the nucleus of BMDMs (FIG. 2I), which was further validated by quantitative real-time polymerase chain reaction (PCR) of nuclear and cytoplasmic fractions (FIG. 2J). To further assess the cellular specificity in atherosclerotic lesions, RNA-ISH identified that MERRICAL (nuclear staining) was found in close proximity to the cytoplasmic macrophage marker F4/80 in atherosclerotic lesions, indicating high specificity for lesional macrophages (FIG. 2K).

MERRICAL-deficient macrophages inhibit pro-inflammatory responses and chemokine CCL3/CCL4 expression in vitro

To explore the role of the IncRNA MERRICAL in regulating macrophage functions, a transcriptomics analysis in MERRICAL knockdown (KD) BMDMs in a resting phase (control) and a pro- inflammatory phase (LPS (lipopolysaccharide)) was performed. MERRICAL gapmeR-mediated KD effectively reduced MERRICAL expression in both control and LPS-treated BMDMs (FIG. 4B). RNA-seq (NCBI GEO accession no. GSE235419) captured 222 DE genes (DEGs) in the MO control BMDMs and 5,827 DEGs in the M1 LPS-treated BMDMs (FDR < 0.05) (FIG. 3A). Ingenuity Pathway Analysis (IPA) of canonical pathways in combination with R package GOplot⁴⁴ were used on the set of DEGs (adjusted p < 0.05). Notably, several inflammatory pathways were shown in the top dysregulated pathways; i.e., “inflammasome pathway,” “IL-2 signaling,” “IL-3 signaling,” “STAT3 pathway,” “chemokine signaling,” and “IL-8 signaling” (FIGS. 3B and 3C). GOchord plots of the top DEGs associated with these enriched pathways revealed several downregulated pro-inflammatory transcripts, including CCL3 and CCL4, the two neighboring genes of MERRICAL (FIGS. 3C and 3D). Interestingly, 173 significantly regulated genes were found in common between the decreased DEGs from BMDM RNA-seq (MERRICAL KD vs. nonspecific (NS) control) and the upregulated DEGs in the athero-intimal RNA-seq (progression G3 vs. control G1 ) (FIG. 3E). IPA canonical pathway analysis of these overlapping 173 genes revealed that the top enriched pathways included “inflammatory response” and “chemotaxis.” CCL3 and CCL4 were identified as the top decreased DEGs in the MERRICAL KD BMDMs, and as the top upregulated DEGs during the progression of diabetic atherosclerosis (G3 versus G1 ) (FIG. 3F). Together, these results indicate that the IncRNA MERRICAL contributes to the regulation of inflammatory response and chemotaxis, potentially through in cis regulation of its neighboring genes CCL3 and CCL4. Loss and gain of function of the IncRNA MERRICAL regulate chemotaxis and pro-inflammatory response in vitro

The results from the RNA-seq indicated that MERRICAL plays a critical role in regulating immune responses in BMDMs under pro-inflammatory stimuli. To further investigate this possibility, loss and gain- of-function studies were performed by knocking down MERRICAL using gapmeR (loss of function) or overexpressing MERRICAL using an expression vector carrying the full-length sequence of MERRICAL (gain of function). Both quantitative real-time PCR and RNA-ISH confirmed the efficiency of KD (80%) of MERRICAL in BMDMs (FIGS. 4A and 4B). Cell viability and apoptosis were not affected after MERRICAL KD in BMDMs. Expression of CCL3 and CCL4 was decreased at both the gene and protein levels, as shown by quantitative real-time PCR and mouse cytokine multiplex assays (FIGS. 4C and 4E), while other chemokine neighbor genes of the IncRNA MERRICAL were not affected. BMDMs were treated with palmitic acid, a saturated fatty acid that is known to promote inflammation and insulin resistance.⁴⁵’⁴⁶ MERRICAL expression was potently induced in response to palmitic acid in BMDMs. Consistently, MERRICAL KD in BMDMs reduced the expression of CCL3 and CCL4 in the presence of palmitic acid treatment, as shown by quantitative real-time PCR. However, MERRICAL KD was found not to affect oxidized low-density lipoprotein (oxLDL) uptake or phagocytosis.

MERRICAL KD BMDMs also exhibited a strong reduction in the adhesion of calcein-labeled mouse PBMCs to endothelial cell (bEnd3 cells) monolayers in the presence of supernatants from MERRICAL KD or control BMDMs (FIG. 4F). Consistent with this finding, there was a significant decrease in BMDM migration using supernatants of the MERRICAL KD BMDMs in the Transwell migration assay. To further investigate whether the migration difference in MERRICAL KD was associated with CCL3 and CCL4, Transwell migration assays were performed using neutralization antibodies to CCL3 and CCL4 (FIG. 4G). Migration was significantly decreased in the NS control group treated with neutralization antibodies to CCL3 and CCL4 compared with the immunoglobulin G (IgG) control. The decrease with these neutralization antibodies was markedly blunted in the MERRICAL KD group, highlighting the dependent nature of this migratory effect on CCL3 and CCL4.

Consistent results were further shown by profiling the cytokine antibody arrays of 42 cytokines related to immunity and inflammation from the culture supernatants of BMDMs (KD vs. NS control). In contrast, gain of function by overexpression of the IncRNA MERRICAL in BMDMs upregulated CCL3 and CCL4 expression but not other chemokines or cytokines in the presence or absence of pro-inflammatory stimuli by quantitative real-time PCR (FIGS. 4H and 41). These loss- and gain-of function results in BMDMs highlight that the IncRNA MERRICAL likely plays an important role in chemotaxis by regulating its neighboring chemokine genes CCL3 and CCL4.

The IncRNA MERRICAL regulates CCL3 and CCL4 transcription through interaction with H3K4me3

As the IncRNA MERRICAL exhibited predominant nuclear localization (FIGS. 21 and 2J), the possibility that the IncRNA MERRICAL controls CCL3 and CCL4 gene expression by regulating their transcription was explored next. MERRICAL expression was upregulated in a time-dependent manner in response to LPS. CCL3 and CCL4 expression was measured in MERRICAL KD and overexpressing BMDMs after stimulation with LPS. CCL3 and CCL4 mRNA expression was significantly reduced in KD BMDMs in a time-dependent manner (FIGS. 5A-5D). Chromatin immunoprecipitation (ChIP) was performed, followed by qPCR to monitor the recruitment of RNA polymerase II (RNA Pol II) to regulatory regions of CCL3 and CCL4 that were down-regulated with MERRICAL deficiency. RNA Pol II recruitment to the transcription start site (TSS) of CCL3 and CCL4 was observed in the LPS-stimulated BMDMs; however, these responses were ameliorated in the MERRICAL KD BMDMs. Consistent with these observations, the inducible recruitment of RNA Pol II at CCL3 and CCL4 TSS sites was further enhanced in the MERRICAL overexpression BMDMs. These observations strongly indicate that the IncRNA MERRICAL regulates the expression of CCL3 and CCL4 at the level of transcription.

Several chemokine gene promoters are enriched for H3 methylation dynamics. One well-known transcription activation marker is H3K4me3 (trimethylation of histone H3 at lycine 4).⁴⁹’⁵⁰’⁵¹ We examined whether IncRNA MERRICAL interacts with chromatin-modifying enzymes and, in turn, epigenetically regulate CCL3 and CCL4 expression. LncRNA pulldown assays were performed, which found that H3K4me3 showed specifically stronger binding to MERRICAL in LPS-stimulated BMDMs compared with the LacZ control (FIG. 5E). In contrast, there was no difference in H3K4me2 or H3K9me2 (histone H3 lysine 9 dimethylation) indicating specificity for H3K4me3. RNA immunoprecipitation (IP) studies showed consistent binding efficiency between H3K4me3 and MERRICAL (FIG. 5F).

The cleavage under targets and release using nuclease (CUT&RUN) assay⁵⁴ was performed to explore chromatin-associated interaction between H3K4me3 and DNA loci under various conditions, including MERRICAL KD and NS groups treated with PBS (control) or LPS (2 h). Notably, LPS treatment increased the presence of H3K4me3 in the genomic areas of CCL3 and CCL4. Conversely, in the MERRICAL KD LPS group, there was a reduction in H3K4me3 enrichment specifically within the genomic regions of CCL3 and CCL4 compared with the NS LPS group. I ntriguing ly, this diminished H3K4me3 enrichment was exclusive to the genome regions of CCL3 and CCL4, unlike neighboring genes, such as Wfdc17, Wfdc18, and CCL5, and other chemokine genes, such as CCL12 (FIG. 5G). In addition, ChlP- qPCR was performed to quantify H3K4me3 near the TSS of CCL3 and CCL4, which were transcriptionally regulated by MERRICAL. H3K4me3 levels on the TSS of CCL3 and CCL4 were decreased in LPS-stimulated MERRICAL-deficient BMDMs (FIGS. 5G and 5H). In contrast, H3K4me3 enrichment on the TSS of CCL3 and CCL4 was upregulated in BMDMs overexpressed with MERRICAL (FIGS. 5J and 5K).

Based on data demonstrating the regulatory role of MERRICAL in the H3K4me3 modification on the promoter region of CCL3 and CCL4, it was hypothesized that MERRICAL interacts physically with CCL3 and CCL4 at their respective promoter regions. To investigate this, a chromatin isolation by RNA purification (ChIRP) assay was performed, which displayed specific enrichment for MERRICAL at the promoter regions of CCL3 and CCL4 (FIGS. 5L and 5M). Conversely, no significant enrichment was observed at the classic pro-inflammatory gene interleukin-1 p (IL-1 p) promoter.

H3K4 methylation is mediated by several SET (Su(var)3-9, enhancer of zeste, trithorax) domaincontaining methyltransferases, including mixed-lineage leukemia 1 -5 (MLL1 -MLL5) and SET1 A/B.⁵⁵ RNA-seq data was utilized to trace whether any of these were inversely regulated in response to LPS in the NS control group compared with the MERRICAL KD group in the presence of LPS. Among all comparisons, MLL1 (KMT2A) was the top upregulated family member in LPS-treated vs. control in BMDMs and was also markedly downregulated in the LPS-treated MERRICAL KD group compared with LPS-treated NS controls (FIG. 6A). MLL1 in particular exerts its activity through interactions with a complex that includes WDR5. Mechanistically, at the gene promoter level, MLL1 engages with the subunit WDR5, an RNA-binding adapter protein that recognizes H3K4 methylation. This interaction typically leads to the activation of targeted genes. To investigate the functional importance of the MLL1 -WDR5 complex in gene regulation, BMDMs were treated with MM102, a small-molecule inhibitor specifically designed to block the interaction between WDR5 and MLL1 ,⁵⁶ As a result, a significant reduction in the transcription of the CCL3 and CCL4 genes was observed (FIGS. 6B and 6C). This finding highlights the critical role of the MLL1 -WDR5 complex in the activation of H3K4me3 at the promoters of the CCL3 and CCL4 genes, emphasizing the necessity of this complex for gene activation.

To investigate the potential role of the IncRNA MERRICAL in conjunction with WDR5, IP experiments targeting MLL1 and WDR5 were performed. These experiments were carried out under both NS control conditions and MERRICAL KD conditions in BMDMs stimulated with LPS. Remarkably, LPS treatment for 4 h resulted in an upregulated interaction between WDR5 and MLL1 , while the deficiency of MERRICAL completely abolished this interaction (FIG. 6D).

To further explore the potential binding between MERRICAL and WDR5, an RNA-protein interaction prediction method was employed.⁵⁷ This analysis indicated a predicted binding affinity of the nucleotides spanning 950-1 ,100 of MERRICAL to WDR5. The direct binding of the IncRNA MERRICAL to WDR5 was subsequently tested using RNA IP. The results demonstrated markedly higher binding efficiency between the IncRNA MERRICAL and WDR5 compared with the IgG control, an effect enhanced in the presence of LPS (FIG. 6E). In contrast, there was no MERRICAL binding to other reported atherosclerosis-associated IncRNAs, including CARMN²⁰ or SNHG12¹⁵. Taken together, these findings demonstrate that the IncRNA MERRICAL binds with WDR5, facilitating the WDR5-MLL1 interactions and promoting the H3K4me3 epigenetic modification at the promoter region of the CCL3 and CCL4 genes (FIG. 6F).

MERRICAL silencing inhibits pro-inflammatory responses and diabetes-associated atherosclerosis

To explore the role of the IncRNA MERRICAL in regulating the progression of diabetes- associated atherosclerosis, Ldlr^z- mice on the HFSC diet were injected retro-orbitally (r.o.) with NS control or MERRICAL gapmeRs over 12 weeks (FIG. 7A). After 12 weeks on the HFSC diet, gapmeR- mediated silencing of the IncRNA MERRICAL reduced its expression in the aortic intima by 80% (FIG. 7B). The silencing effect was also observed in PBMCs, the spleen, and BM but not in the lungs or liver. Mouse weight gain, glucose and insulin tolerance, and lipid metabolism, including cholesterol, triglyceride, and high-density lipoprotein (HDL) levels, were not affected by IncRNA MERRICAL KD (data not shown). Analysis of atherosclerotic lesion formation revealed a 74% decrease in lesion area by oil red O (ORO) staining (FIG. 7E) and an 86% decrease in lesion area of the arch and descending aorta by light sheet fluorescent microscopy⁵⁸ (FIG. 7F) after MERRICAL KD in mice. As shown by immunohistochemistry, MERRICAL-deficient lesions at the aortic sinus showed significantly decreased accumulation CD68+ macrophages by 76% as quantified by flow cytometry and by 88% as quantified by immunofluorescent staining, with no changes in aSMA⁺ (anti-alpha smooth muscle actin) smooth muscle cells (SMCs) after normalization to the lesion area (FIG. 7G). In addition to CD68+ macrophages, there was a significant decrease in the accumulation of CD4⁺ T cells by 87.5% and a non-significant trend in CD8⁺ T cells in the aortic root in MERRICAL KD mice compared with the control (data not shown). Further quantification of leukocyte subsets in the aorta by flow cytometry analysis showed that the percentages of CD45⁺ leukocytes, F4/80+ macrophages, and CD86+ M1 -like macrophages were significantly decreased in the MERRICAL KD mice, whereas the percentage of CD206+ M2-like macrophages was significantly upregulated in the MERRICAL KD group (FIG. 7H). There was no effect of MERRICAL KD on the BM cell CD45+ leukocyte, CD115⁺ monocyte, or Ly6C monocytic populations (data not shown). Based on the in vitro regulation of MERRICAL on chemokine and inflammatory markers, quantitative real-time PCR was performed and found that CCL3/CCL4 and 111 -p were significantly inhibited in the MERRICAL KD aortic intima (FIGS. 7C and 7D). Circulating cytokines in the plasma were profiled using mouse cytokine multiplex assays, and, interestingly, CCL3 and CCL4 were the top significantly decreased cytokines in plasma from the MERRICAL KD group (FIGS. 7I and 7J). Collectively, these data strongly support the contention that MERRICAL-deficient mice on an HFSC diet have decreased CCL3 and CCL4 expression, reduced chemokine recruitment of leukocytes into the blood vessel wall, and markedly alleviated progression of diabetes-associated atherosclerosis.

In this study, a diet-induced diabetic atherosclerosis mouse model was applied by placing Ldlr^z- mice on an HFSC diet. After 12 weeks HFSC diet, Ldlr^/_ mice exhibited hyperglycemia, glucose and insulin intolerance, and hyperlipidemia (data now shown). Changes in cholesterol levels were assessed during the progression and regression phases, showing that the HFSC diet was a more aggressive diet and had a delayed regression phenotype (data not shown) compared with the conventional high- cholesterol diet. Significantly regulated IncRNA candidates in the aortic intima during the progression or regression phases of diabetes-associated atherosclerosis were sought.

To date, projects such as ENCODE (the Encyclopedia of DNA Elements), LNCipedia, and the HUGO Gene Nomenclature Committee have distinguished IncRNAs based on expression levels and functions in processing of RNA, DNA, or protein.⁵⁹ However, there are still numerous unidentified IncRNAs with unknown functions in regulating gene expression and signaling pathways in metabolic diseases such as diabetes-associated atherosclerosis. This study aimed to identify functional IncRNA candidates that are involved in the robust chronic inflammation and accelerated lipid deposition manifest in the development of diabetes-associated atherosclerosis.

Among the identified IncRNAs pervasively transcribed in the mammalian genome, some IncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes.⁴⁷’⁵⁸’⁶⁰’⁶¹’⁶²⁶³ Thus, significantly correlated IncRNA-mRNA pairs were screened using intima RNA-seq data from the HFSC diet-induced atherosclerosis mouse model. Based on recent Hi-C genomic sequencing studies, the genome is compartmentalized into chromatin neighborhoods that have been referred to as TADs⁶⁴ that highlight that genomic distance alone does not explain the actual physical interactions between transcripts and that IncRNA-mRNA pairs can be dictated by these higher-order chromatin neighborhoods or TADs. LncRNA-mRNA pairs were mapped to the Hi-C genomic database, which defined a total of 75 IncRNA-mRNA pairs. Among these pairs, one of the IncRNAs that positively correlated with a group of chemokine genes implicated in inflammation and chemotaxis during the progression of atherosclerosis was chosen for further analysis. Based on cell and tissue profiling and the in vitro/in vivo functional assays, this IncRNA was termed MERRICAL. MERRICAL was the top-expressing IncRNA from differential expression analysis over 12 weeks of atherosclerotic progression on the HFSC diet, and the expression of the IncRNA MERRICAL significantly decreased in the regression phase (24-week time point, data not shown).

Several lines of evidence support a pro-inflammatory role of the IncRNA MERRICAL in macrophages. Based on the transcriptomic analysis of MERRICAL-deficient BMDMs in both MO (resting phase) and M1 (pro-inflammatory phase), surprisingly, about 5,800 DEGs were found in the M1 phase comparison but only 222 DEGs in the MO phase comparison. This huge spike of DEGs between MERRICAL KD and the NS control indicated that the IncRNA MERRICAL may play a causal role in regulating the pro-inflammatory response. The observed MERRICAL KD effect might also be more prominently displayed in the highly activated M1 state, further amplifying the differences in gene expression. Pathway enrichment of the DEGs identified from the M1 BMDM comparison showed a variety of inflammation-associated pathways, including chemotaxis and innate immune response. Among the DEGs, CCL3 and CCL4, which were earlier captured as neighboring transcripts of MERRICAL, were the top significantly decreased genes in MERRICAL KD BMDMs. In vitro loss and gain of function of MERRICAL consistently indicated that the IncRNA MERRICAL positively regulated its neighboring genes CCL3 and CCL4 in cis both under inflammatory stimuli such as LPS or diabetes-associated stimuli such as palmitic acid. The chemotaxis signaling pathway was among the top enriched pathways in response to MERRICAL KD.

The present study revealed a mechanistic mode of action for the IncRNAs MERRICAL and EPIC1 . The localization of IncRNAs can be closely related to its function in cells.⁶⁶ While the majority of IncRNAs are found to be nucleus enriched, the amount present in the cytoplasmic fraction is often variable. Using RNA-ISH and cellular fractions, the IncRNA MERRICAL was predominantly localized in the nucleus of BMDMs. Nucleus-enriched IncRNAs are often found to interact with transcription factors and histone modifiers.⁶⁶ Consistent with its nuclear localization, the IncRNA MERRICAL was found to control expression of CCL3 and CCL4 at the transcriptional level. An RNA pull-down assay showed that IncRNA MERRICAL possessed a particularly strong interaction with a histone activation marker, H3K4me3, compared with other histone modifiers. Several chemokine gene promoters are enriched for H3K4 mono-, di-, and trimethylation.⁴⁹’⁵⁰ After conducting a CUT&RUN assay,⁵⁴ the chromatin-associated interactions between H3K4me3 and DNA loci in response to LPS and MERRICAL KD was performed in macrophages. Under the proinflammatory stimulus of LPS, heightened enrichment peaks on the genomic regions of CCL3 and CCL4 were identified (FIG. 5G). This indicates that LPS stimulation triggers increased H3K4me3 enrichment on the genomic elements of CCL3 and CCL4, subsequently activating their expression. In contrast, MERRICAL KD diminished this enrichment of H3K4me3 in the presence of LPS compared with the NS group, both in global genomic regulation and in the CCL3 and CCL4 genomic region (data not shown). I ntrigui ngly , this diminished H3K4me3 enrichment was exclusive to the genome regions of CCL3 and CCL4, unlike neighboring genes, such as Wfdc17, Wfdc18, and CCL5, and other chemokine genes, such as CCL12 (FIG. 5G). MLL1 -WDR5 interaction has been found to be critical for facilitating H3K4me3 activation at the promoter region of targeted genes. Initially, treatment of macrophages with an MLL1 -WDR5 interaction inhibitor resulted in decreased activation of CCL3 and CCL4. This finding confirmed the dependency of chemokine activation, specifically CCL3 and CCL4, on the H3K4me3 modification mediated by the interaction between MLL1 and WDR5. Furthermore, IP experiments between WDR5 and MLL1 revealed the complete loss of their interaction under IncRNA MERRICAL deficiency. Collectively, these findings support a mechanistic role of MERRICAL as a scaffold, regulating the activation of H3K4me3 on CCL3 and CCL4 by facilitating the interaction between the MLL1 and WDR5 complex. The CUT&RUN assay demonstrates selectivity of the MERRICAL complex for the chemokines CCL3 and CCL4 in response to inflammatory stimuli in macrophages.

To further investigate the mechanism, RNA pull-down assays were performed, which found a particularly strong interaction between the IncRNA MERRICAL and the histone activation marker H3K4me3 compared with other histone modifiers. These results collectively support a mechanism whereby the IncRNA MERRICAL binds to WDR5, facilitating the interaction between WDR5 and MLL1 , orchestrating the deposition of H3K4me3 at the promoter region of CCL3 and CCL4. Consequently, this leads to the activation of CCL3 and CCL4 transcription under inflammatory conditions. While WDR5- MLL1 is not a cell-specific complex, by virtue that MERRICAL is highly expressed in macrophages and strongly associates with WDR5-MLL1 , this paradigm is likely to be primarily operative in macrophages and other immune cell types where MERRICAL is robustly expressed. Notably, a decrease in RNA Pol II enrichment was observed at the TSS of CCL3 and CCL4 under MERRICAL KD conditions, while an increase in RNA Pol II enrichment was observed under MERRICAL overexpression conditions (data not shown). Previous reports have indicated that loss of H3K4me3 results in a widespread decrease in transcriptional output, accompanied by RNA Pol II pausing and slower elongation.⁶⁷ Importantly, there exists an ortholog of MERRICAL; this orthologous IncRNA is termed epigenetically induced MYC interacting IncRNAI (EPIC1 ). EPIC1 showed remarkable conservation with MERRICAL, sharing 22 completely conserved sequences and overall 46% similarity. Moreover, an RNA-protein interaction tool⁶⁸ predicted a strong interaction between the IncRNA EPIC1 and human WDR5, further highlighting its functional relevance in the context of WDR5-mediated processes.

Besides its genomic local function on CCL3 and CCL4, numerous innate immune response signaling pathways were also observed to be regulated. These in vivo studies also showed that MERRICAL-deficient mice systemically inhibited inflammation with decreased shifting of pro-inflammatory Ly6chi monocytes in PBMCs (data not shown). Intimal RNA-seq and quantitative real-time PCR data showed that the IncRNA MERRICAL was induced in the intimal layer during atherosclerotic lesion progression (FIGS. 1 C and 1 D) and in response to inflammatory stimuli such as LPS or palmitic acid in BMDMs in a time-dependent manner (data not shown).

This in vivo study showed that deficiency of the IncRNA MERRICAL inhibited leukocyte recruitment into the vessel wall and lesion formation, ultimately alleviating the development of diabetes- associated atherosclerosis. However, MERRICAL deficiency did not affect glucose metabolism or the circulating lipid profile in mice or BMDM cell oxLDL uptake or the phagocytosis process in vitro. These observations support the theory that the underlying molecular mechanisms of the IncRNA MERRICAL in regulating atherosclerosis is primarily through controlling macrophage chemotaxis and inflammatory responses. These studies build on the known roles of the chemokines CCL3 and CCL4 in promoting lesion progression in atherosclerosis models.³⁷’⁷¹ In conclusion, this study has identified the IncRNA MERRICAL as a macrophage-enriched

IncRNA and a key regulator of chemotaxis and pro-inflammatory response in diabetes-associated atherosclerosis. The regulatory role of MERRICAL is in part through its in cis function by interacting with the WDR5-MLL1 complex to facilitate H3K4me3 and transcriptional activation at the CCL3 and CCL4 gene promoters. These findings establish mechanistic insights into macrophage-mediated chronic inflammation and chemotaxis in diabetic atherosclerosis and highlight targets (e.g., MERRICAL (e.g., SEQ ID NO: 71 ) and EPIC1 (e.g., SEQ ID NO: 70) for anti-inflammatory intervention.

Materials and Methods

TABLE 4. RESOURSES

TABLE 5. PRIMERS

Data and code availability

The NCBI GEO accession numbers is GSE235419 for the RNA-seq data.

Cell culture bEnd.3 cells (ATCC, CRL-2299) were cultured in Dulbecco’s Modified Eagle Medium/F12(1 :1 ) (DMEM; Gibco, 1 1320-033) supplemented with 10% fetal bovine serum (FBS) and 1 % Penicillinstreptomycin (P/S). HEK293T cells (ATCC, CRL-3216) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1 % P/S. For culturing of bone marrow-derived macrophages (BMDMs), bone marrow was isolated from the femur and tibia of mice and cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; Sigma, I3390) and 15% filtered L929 cell culture supernatant (containing monocyte-colony stimulating factor, M-CSF). The BMDMs were mature and ready for treatment after 7 days. For M1 activation, BMDMs were treated with 100 ng/mL LPS overnight; for M2 activation, BMDMs were treated with 10 ng/mL IL-4 overnight. Transfection was performed using Lipofectamine 2000 (Invitrogen) as described in the manufacturer’s protocol. Customized GapmeRs for MERRICAL (Qiagen) or nonspecific control #1 (Qiagen) were used for transfection at 50 nM in BMDMs. Peritoneal macrophages (PM) were isolated according to method previously described.⁴ Briefly, mice were intraperitoneally injected (i.p) with 1 mL 3.8% brewer’s thiog lycollate medium. After three days, treated mice were euthanized by C02. 5 mL of cold PBS was injected into the peritoneal cavity, and peritoneal fluid was aspirated by needle syringe. Peritoneal fluid was centrifuged for 10 min (400 g, 4°C). The cell pellet was resuspended in RPMI medium and allowed to sit for 2 h and then the non-adherent cells were washed away with warm PBS.

Isolation of mouse peripheral blood mononuclear cells

Peripheral blood was collected by cardiac puncture of anesthetized mice, and 1 :1 diluted with HBSS. PBMC were separated through density centrifugation (400 g at 20°C for 30 min) using histopaque- 1077 gradient. The interphase fraction containing PBMCs was aspirated and red blood cells were lysed with ammonium chloride solution. After centrifugation (200 g, 5 min), pelleted PBMC was resuspended in RPMI medium supplemented with 10% FBS.

Animal experiments

Eight-week-old Ldlr male mice were purchased from the Jackson Laboratory, and randomly divided into Control and MERRICAL knockdown group (n = 15 per group). All mice were continuously fed a high fat, high sucrose-containing diet (HFSC, Research Diets Inc., D09071704) for 12 weeks. Control group and MERRICAL knockdown mice were retro-orbitally (r.o.) injected with non-specific (NS) control or MERRICAL gapmeRs (20 mg/kg per mouse) once per week. C57BL/6J mice were purchased from Charles River. All animal experiments were approved by the Institutional Animal Care and Use Committee at Brigham and Women’s Hospital and Harvard Medical School, Boston, MA and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Intimal RNA isolation from aorta tissue

Isolation of medial and intimal RNA from aorta was conducted as previously described.^{1 2} Briefly, mouse aorta between the heart and diaphragm was exposed, and then the peri-adventitial tissues were removed carefully. The cleaned aorta was carefully flushed with 1X PBS, and then flushed with 300 pL Trizol using 1 mL insulin needle. The intima eluate was collected into 1 .5 mL Eppendorf tube and then intimal RNA was isolated as described above. The aorta leftover (media) was washed once with 1X PBS and the medial RNA was isolated by Trizol as described above.

Vector construction

Transcripts for MERRICAL (ENSMUST00000216842.2) were synthesized by Genewiz with 5' Kpnl and 3'Notl restriction sites. For overexpression, MERRICAL transcript was cloned into pcDNA3.1 using Kpnl and Notl. For RNA synthesis in vitro, pcDNA3.1 containing MERRICAL transcript was subcloned into pBluescript SK II + using Xbal and Hind III. pcDNA3.1 vector containing MERRICAL transcript was sub-cloned upstream of p3xFLAG-CMV-14 expression vector (Sigma, E7908) using Hind III and Notl restriction site. Protein-coding potential

In silico CPAT online algorithm was used for prediction of coding potential.³ In total, 293T cells were transfected with 500 ng of either positive control (p3XFLAG-CMV-7-BAP) or p3xFLAG-CMV-14 expression vector containing MERRICAL transcript using Lipofectamine 2000 (Invitrogen) and protein lysate was isolated 72 h post-transfection, followed by immunoblotting for FLAG Tag (Cell Signaling, 8146).

RNA-in situ hybridization (RNA-ISH)

Probe for MERRICAL was specially developed to detect mouse MERRICAL (Advanced Cell Diagnostics). Cells or tissues sections were fixed for 2 h in 4% paraformaldehyde and further prepared as described by the manufacturer. In situ hybridization was performed using RNAscope 2.5 HD Reagent Kit- Red (Advanced Cell Diagnostics) based on manufacturer’s protocol.

Cell fractionation

Cell fractionation was performed to separate cytoplasmic, nuclear, and chromatin components using a nuclear extract kit (Active Motif, 40010) following the manufacturer’s protocol. RNA was harvested as described previously and cleaned up using the RNeasy kit (Qiagen). Different protein or RNA contents in the fractions were then estimated by western blotting or RT-qPCR.

Preparation of mouse bone marrow derived macrophages

Bone marrow derived macrophages (BMDM) were obtained according to method described.⁵ The mice were euthanized by CO2. The femur and tibia bones were isolated with hair, skin, and most of muscle tissue removed. The bones were cut open, and bone marrow was flushed out with a 21 G needle and syringe into cold PBS with 2% FBS. The bone marrow was passed through a 70 pm cell strainer in order to remove bone fragments and other tissue. Ammonium chloride solution was used to lyse red blood cells. The collected bone marrow cells were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS and 15% filtered L929 cell culture supernatant (containing monocyte-colony stimulating factor, M-CSF). The BMDMs were mature and ready for treatment after 7 days. For M1 (LPS group) activation, BMDMs were treated with 100 ng/mL LPS overnight. To assess the impact of MM102 small molecule inhibitor on the interaction between MLL-WDR5 and subsequent cytokine expression in the presence or absence of LPS, BMDMs were treated with MM102 (S7265, 10uM, Selleck) for 16 h, then treated with 1 pM/ml LPS for 0, 4, and 16 h.

RNA isolation and real-time quantitative PCR

Total RNA was extracted by using Trizol reagent following the manufacturer’s protocol (15596- 026 Invitrogen, MA, USA). The concentration and quality control of RNA was examined using NanoDrop 2000 (Thermo Fisher, MA, USA). cDNA was produced using High-Capacity cDNA Reverse Transcription Kit (4368814, Thermofisher, MA, USA). mRNA expression levels were normalized to b-actin. GoTaq qPCR (A6001 , Promega) was used for quantitative real-time PCR analysis with Quantstudio 6 Pro (Thermofisher) following the manufacturer’s instructions. A list of primers is presented in Table 5.

Cytokine profiling in the plasma and supernatants

Mouse plasma blood samples were obtained by cardiac puncture during sacrifice, then centrifuged to separate the plasma. For the supernatants from primary CD4⁺ T cells, 1 *10⁶ isolated cells per well were plated in the 24-well plate with Mouse T-Activator CD3/CD28 for T cell expansion and activation (11452D, Gibco, MA, USA) in 1 mL RPMI Media 1640 with 10% FBS and 1%P/S according to the manufacturer’s instructions. After culture for 24h, the supernatants were collected and directly subjected for the further experiments. Plasma and supernatants from primary CD4⁺ T cells were then subjected to Mouse Cytokine/Chemokine 31 -Plex Discovery Assay Array (MD31 ), and TGFp 3-Plex Discovery Assay Multi Species Array (TGFpl -3) (Eve Technologies, AB, Canada).

Phagocytosis assay

Assessment of BMDM phagocytic capacity was performed using the Vybrant Phagoctyosis Assay Kit (V6694, Thermofisher). Briefly, BMDMs were transfected with GapmeRs for MERRICAL or NS control at 50mM, and seeded into 96-well plates. 100 pL of prepared E. co// Bio Particle suspension was added to each well, and cells were intubated for 2 h. Following intubation, 100 pL of trypan blue suspension was added to all wells and subsequently aspirated. Fluoroescence was measured using a microplate reader at -480 nm excitation, -520- mm emission. Net phagocytosis was subsequently calculated per manufacturer’s protocol.

Transwell-cell migration assay

BMDMs were serum starved for 12 h. Cells (1 x 10⁵) were seeded into the 8 pm pore size inserts and induced by supernatant from NS control or MERRICAL KD BMDM, and in the absence (IgG) or presence of neutralization antibodies against CCL3 and CCL4 for 16h. Cells were then fixed and stained with DAPI. The bottom sides of the inserts were imaged and quantified by Imaged software.

Endothelial cell monolayer adhesion assay bEnd3 cells were seeded in 12-well tissue culture plates. After the endothelial cells reached sub- confluency, they were incubated with supernatant from NS control or MERRICAL KD BMDM for 16h. Isolated mouse PBMCs were resuspended and labeled with Calcein AM (Molecular Probes, Eugene, OR, U.S.A.) at 37°C for 45 min. After washing once with Dulbecco’s phosphate buffered saline (DPBS), PBMC were added to the bEnd3 cell monolayers and co-cultured for 1 h. After washing once with DPBS, fluorescent images were taken using an inverted fluorescence microscope.

Bulk RNA-Seq analysis

RNA-Seq transcriptomic analysis was performed after ribodepletion and library construction by using Illumina performed after ribodepletion and standard library construction using Illumina HiSeq2500 V42x150 PE (Genewiz). All samples were processed by using a pipeline published in the bcbio-nextgen project (github.com/bcbio/bcbio-nextgen). Raw reads were filtered and examined for quality control through running FastQC (bioinformatics.babraham.ac.uk/projects/fastqc/) and filtered reads were used to generate library and further analysis. Trimmed reads were aligned to UCSC build mm10 of the mouse genome and augmented with transcript information from Ensembl releases 86 (H. sapiens) using STAR. Total gene hit counts and CPM values were calculated for each gene and downstream differential expression analysis between specified groups was performed using DESeq2 and an adapted DESeq2 algorithm that excludes overlapping reads. Genes with adjusted p value <0.05 and Iog2fold-change (>0.58) were identified as differentially expressed genes for each comparison. The mean quality score of all samples was 37.21 with a range of 27,963,821 -45,892,062 reads per sample. All samples had at least >90% of mapped fragments over total fragments.

Pathway enrichment analysis

Differentially expressed genes (DEGs) were identified as having at least 1 .5-fold changes and adjusted p values <0.05 (false discovery rate). The DEGs were visualized using hierarchical clustering plot. DEGs were subjected to gene set enrichment analyses by using Ingenuity Pathway Analysis (IPA) software functional annotation tool. The significant values for the canonical pathways were calculated by Fisher exact test. R package GOplot91 was used for visualization of pathway enrichment analysis on the set of DEGs (adjusted P-value < 0.05).

Immunofluorescence staining

For immunofluorescence staining, cells were fixed in 4% PFA (Boston Bio Products) for 24h and embedded in paraffin for sectioning. To characterize atherosclerotic lesions, the aortic root and aortic arch was embedded in optimal cutting temperature (OCT) compound (Cat#23730571 , Fisher Scientific, USA) after harvesting the hearts and aortas. About 7-pm frozen serial sections through the aortic sinus were prepared with all three valve leaflets visible, and the aortic arch with all three branches (left subclavian artery, left common carotid artery, and brachiocephalic artery) visible. For plaque area analysis in aorta, mouse aortas were stained with fresh Oil Red O solution for 2 h at room temperature. The stained aortas were destained with 70% ethanol several times. Serial aortic root sections were stained with oil red O or used for immunostaining to detect vascular smooth muscle cells (VSMCs) (anti- aSMA, 1 :500, Sigma-Aldrich, A5228) and macrophages (CD68 (1 :100, Abeam, AB201845)). Slide sections were blocked with 5% donkey serum (Jackson ImmunoResearch Lab) for 1 h and then incubated with primary antibodies overnight at 4°C. Slides were washed and incubated with conjugated secondary antibodies (Jackson ImmunoResearch Lab) Cy3 conjugated donkey anti-rat secondary antibody (1 :300, Cat#: 712-165-153), Alexa 647 conjugated donkey anti-rabbit secondary antibody (1 :300, Cat#: 711 -605-152) and Alexa 488 conjugated donkey anti-rabbit secondary antibody (1 :300, Cat#: 711 -545-152) for 90 min at room temperature. Cell nuclei were stained with 4',6-diamidino-2- phenylindole (DAPI). Immunofluorescence imaging was performed by BIDMC confocal imaging and IHC core facility. Images were acquired on a Carl Zeiss LSM 880 confocal microscope using Zen black software version 2.3 SP1 (BIDMC confocal imaging and IHC core facility). Objective lenses 10x 0.45 NA and 20x 0.8 NA were used for image acquisition. Lesion areas of aorta roots were defined by the internal elastic lamina to the luminal edge of the lesion. All the evaluations were carried out by two observers in a blind manner.

Optical clearing and 3D light sheet microscopy

Mouse aortas were isolated and fixed in 4% paraformaldehyde overnight at 4°C. iDISCO optical clearing protocol was followed as described elsewhere with modifications.⁶ Following the incubation with Alexa Fluor conjugated antibodies - CD31 -Alexa Fluor 488 (1 :100, Biolegend, 102406), aSMA-Alexa Fluor 594 (1 :500, Sigma-Aldrich, A5228) and CD68-Alexa Fluor 647(1 :100, Abeam, AB201845), aortas were embedded in 5% agarose in PBS and allowed to cool on ice. Excess agarose was trimmed, and the blocks were subjected to subsequent steps in the protocol until the final incubation and clearing in 100% Dibenzyl ether (DBE). An Ultramicroscope II (LaVision Biotec, Germany) light-sheet microscope at the Harvard Neurobiology Imaging Facility Core was used to image the cleared samples (2x objective lens, 1 x zoom; light-sheet thickness set to 3.89 pm, step size 5 pm). Images were analyzed in Imaris software (Oxford Instruments). Plaques were segmented using the manual surface creation method. Plaque volume was obtained from the surface statistics tab. CD68⁺ spots within the plaque were visualized by first masking the 647 nm channel using the plaque surface and then employing the spot detection algorithm.

Mononuclear cell preparation and flow cytometry

Mononuclear cells for flow cytometry were isolated from the aorta, PBMC and BMDM to detect the characterization of different cell populations. Mouse aortas were digested by using an optimized digestion enzyme mix recipe (Collagenase I 450U/mL, Collagenase XI 125U/mL, DNase I 60U/mL, Hyaluronidase 60U/mL, and Elastase 50 ng/ml). After that, samples were resuspended to obtain single cell suspensions.¹ BMDMs were digested with cell stripper (Corning 25-056-CI) to single cell suspensions. After preparing the single cell suspension according to the above methods, the samples from were sequentially filtered through 40pm strainers. Following the manufacturer’s instructions, appropriately fluorescently labeled antibodies were added at predetermined optimum concentrations and incubated on ice for 20 min in the dark for cell-surface staining. Cells were stained with LIVE/DEAD Cell Stain (Invitrogen), followed by staining for cell surface markers, and then fixed and permeabilized with the Cytofix/Cytoperm kit (554714BD, Biosciences) for intracellular staining. After washing with PBS, centrifuging at 350xg for 5 min, samples were resuspended for flow cytometric analysis (BD FACS Analyzer LSR, or BD FACS Analyzer Symphony). The antibodies for flow cytometry are listed in Table 4. All flow data were analyzed by FlowJo 10.7.1 .

Western blot

Cells were lysed in RIPA buffer (ThermoFisher Scientific, USA) containing 1% protease and phosphatase inhibitors and resolved by SDS-PAGE. The proteins were separated by gel electrophoresis and then transferred onto PVDF membranes (Bio-Rad, USA). The membranes were blocked with 5% non-fat milk in 1 X TBST at room temperature for 1 h and incubated overnight at 4°C with antibodies against p-actin (3700, 1 :2000. Cell Signaling), Histone H3 (tri methyl K4) (ab8580, 1 :1000, Abeam), Histone H3 (di methyl K9) (Ab32521 , 1 :1000, Abeam), and Histone H3K4me2 Polyclonal Antibody (39141 , 1 :1000, Thermofisher). Membranes were incubated with secondary antibody for 1 h at room temperature. Protein bands were detected by enzyme-linked chemiluminescence using a luminescent image analyzer (Bio-Rad, Chemidoc).

Co-immunoprecipitation ( Co- IP)

Co-IP assay of MLL and WDR5 was performed using the Universal Magnetic Co-IP Kit (#54002, Active Motif), according to the manufacturer’s protocol. Briefly, BMDMs were transfected with GapmeRs for MERRICAL or nonspecific control #1 at 50mM for 48 h, then subsequently treated with LPS (1 pM/ml, 4 h) and PBS (vehicle control). Cells were harvested and resuspended in Complete Whole-cell Lysis Buffer and incubated at 4°C for 30 min. Part of the cell lysates (10%) was saved as an input sample. The remaining lysate was combined with WDR5 (13105, 3 pg, Cell Signaling), or anti- IgG (2727, 3 pg, Cell Signaling) antibody, along with Co-IP/Wash Buffer at 4°C for 4 h. Protein G Magnetic beads were then added to the mixture, incubated at 4°C for 1 h, and washed with Co-IP/Wash Buffer four times. Each bead pellet was resuspended in 20 pL 2X loading buffer and subjected to western blot for MLL (61295, 1 :500, Active Motif) and WDR5 (13105, 1 :1000, Cell Signaling).

Chromatin immunoprecipitation (Ch IP)

ChIP assay was performed according to the manufacturer’s protocol from Upstate, using the ChIP assay kit (#9003, Cell signal) with modifications. Briefly, BMDMs were treated with LPS (1 pM/ml, 2 h) and PBS (vehicle control). Cells were cross-linked with 1% formaldehyde for 15 min at room temperature, and then the reaction was stopped by incubating in glycine with a final concentration of 0.125 M for 5 min. Cells were washed three times with cold PBS and harvested by scraping with cell scraper. Then the cells were lysed in the SDS lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-HCI, pH 8.1 ) on ice for 10 min. The samples were sonicated into DNA fragments of 0.2-1 kb (checked by agarose gel electrophoresis/ethidium bromide staining) and microcentrifuged at maximal speed for 10 min at 4°C. The supernatant was precleared by rotating with 60 pl of Salmon Sperm DNA/protein-agarose slurry for 30 min at 4°C and then aliquoted after centrifugation. 20 pl was saved as input and 200 pl (equal to one-fifth the number of cells from one 100% confluent 15-cm dish) was used for each antibody. Each 200 pl supernatant was diluted with 800 pl of ChIP dilution buffer (0.01% SDS, 1 .1% Triton X-100, 1 .2 mM EDTA, 16.7 mM Tris-HCI, pH 8.1 , and 167 mM NaCI) and incubated with the specific antibody (1g/sample) at 4°C overnight. A mock precipitation without antibody was used as negative control. The next day, 60 pl of salmon sperm DNA/protein-agarose slurry was added to each sample and incubated at 4°C for another 2-4 h. The beads were then washed for 3-5 min with 1 mL of each buffers listed: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris-HCI, pH 8.1 , 150 mM NaCI), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCI, pH 8.1 , 500 mM NaCI), and LiCI wash buffer (0.25 M LiCI, 1 % IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris-HCI, pH 8.1 ). After all washes, pellets were suspended by vortexing with 150 pl of freshly prepared elution buffer (0.1 M NaHCO3, 1% SDS) for 15 min, and then the supernatant was collected. This elution progress was repeated once again, and in total 300 pl elutes were collected. The one-tenth input was diluted with dilution buffer to a total volume of 300 pl. Elutes and diluted inputs were incubated in 0.3 M NaCI at 65°C for 4 h to reverse formaldehyde cross-linking. Then 10 L of 0.5 M EDTA, 20 pl of 1 M Tris- HCI, pH 6.5, and 20 g of proteinase K were added to the sample and incubated at 45°C for 1 h. DNA was extracted with phenol/chloroform and then incubated with 10 g of glycogen in 75% ethanol at 20°C overnight. After precipitation by centrifuging at 12,000 g for 30 min at 4°C, the recovered DNA pellets were dissolved in 30 pl of distilled water. Amplifications were performed using RT qPCR with SYBR Green Master Mix (GoTag PCR system, Promega M7122). The qPCR primers used are listed in Table 5.

RNA-in situ hybridization (RNA-ISH)

Probe for MERRICAL was specially developed to detect mouse MERRICAL (ENSMUST00000216842.2) (Advanced Cell Diagnostics). Cells or tissues sections were fixed for 2 h in RNAase-free 4% paraformaldehyde and further prepared as described by the manufacturer (Advanced Cell Diagnostics). In situ hybridization was performed using RNAscope 2.5 HD Reagent Kit-Red (Advanced Cell Diagnostics) based on manufacturer’s protocol.

RNA pull down

Biotin-labeled MERRICAL and LacZ were generated in vitro using T7 RNA Polymerase transcription kit (Promega) and Biotin RNA Labeling Mix (Roche). The Biotinylated RNA was incubated with 2 pL DNase I at 37°C for 15 min to remove DNA template and then purified using G-50 Sephadex columns (Roche). The nuclear protein was homogenized by 20 strokes using a Dounce homogenizer, followed by centrifuging at 15,000 x g for 15 min at 4°C. The nuclear lysate was pre-cleared by incubating with 60 pL streptavidin agarose beads (Thermo Scientific) at 4°C for 1 h with gentle rotation. The biotinylated RNA was heated to 90°C for 2 min and placed on ice for 2 min to allow proper RNA secondary structure formation. The folded RNA was then added to the pre-cleared nuclear lysate and incubated at 4°C for 2 h. Then 60 pL prewashed streptavidin agarose beads were added to the reaction and rotated at 4°C for 1 h. At the end of incubation, beads were collected by centrifugation at 12,000 x g for 1 min and washed with ice-cold NT2 buffer at 4°C five times. After washing, 40pL 2xLaemmli loading buffer were added to the beads and boiled for 10 min at 100°C. The supernatant was collected and analyzed by Western blot.

RNA immunoprecipitation (RIP)

RNA immunoprecipitation (RIP) assays were conducted to confirm MERR/CAL-H3K4me3 interactions using a Magna RIP Kit (Millipore, 17-700) according to the manufacturer’s instructions. Briefly, cells were collected for lysis using RIP lysis buffer containing RIP buffer, a protein inhibitor cocktail and an RNase inhibitor for 10 min on the ice. Part of the cell lysates (10%) was saved as an input sample. The remaining lysates (90%) were diluted with RIP immunoprecipitation buffer and incubated with magnetic beads conjugated to anti-Histone H3 (tri methyl K4) (ab8580, 1 :50, Abeam), or anti-IgG (Millipore, PP64B) antibody overnight at 4°C. The beads were washed with RIP wash buffer six times. After washing, 50 pL of the immunoprecipitate was saved for western blot analysis. Immunoprecipitated RNA was extracted by Trizol reagent (Invitrogen, 15596-026), and analyzed by RT-qPCR. RIP assays were also conducted to confirm MERRICAL-WDR5 interactions in the presence of absence of 1 pM/ml LPS for 4 h. Briefly, cells were collected for lysis using polysome lysis buffer, a protein inhibitor cocktail, and an RNA inhibitor and incubated overnight at -80°C. Part of the cell lysates (10%) was saved as an input sample. The remaining lysates (90%) were added to magnetic beads resuspended in NET-2 buffer conjugated to WDR5 (13105, 5 pg, Cell Signaling), or anti-IgG (2727, 5 pg, Cell Signaling) antibody overnight at 4°C, after they were washed in NT-2 buffer six times. The resulting immunoprecipitate was washed with NT-2 buffer six times, and resuspended in 150pL proteinase K buffer. Immunoprecipitated RNA was isolated and purified using phenol:chloroform:isoamyl alcohol (125:24:1 ), chloroform, 5M ammonium acetate, 7.5M LiCI, glycogen, and absolute ethanol, and analyzed by RT-qPCR.

Chromatin Isolation by RNA Purification (ChIRP)

BMDM cells were treated with LPS (1 ug/ml, 2h) to induce chemokine expression. The ChIRP assay was performed using crosslinked nuclear extract obtained from approximately 9 x 10⁷ cells, following a previously described protocol. {Chu, 2012 #228} Briefly, the cells were crosslinked with 1% formaldehyde and subsequently lysed using lysis Buffer (50 mM Tris HCI pH = 7, 10 mM EDTA, 1% SDS). The cell extract was then subjected to sonication using a Bioruptor device for 15 cycles (30 s ON/45 s OFF). After sonication, the extract was centrifuged at maximum speed to remove insoluble chromatin. A 1% fraction of the cleared extract was preserved as input, while the remaining material was diluted with Hybridization Buffer (containing 15% formamide, 500 mM NaCI, 1 mM EDTA, 0.5% SDS, and supplemented with protease and RNAse inhibitors). The diluted extract was incubated overnight at room temperature with specific probes, and the mixture was rotated to facilitate probe-target interactions. On the following day, 400 pL of Streptavidin magnetic beads (Dynabeads MyOne Streptavidin C1 - Thermo Fisher) were added to each pulldown condition. The pulldown reactions were then incubated for 4 h at room temperature with rotation. Subsequently, the beads were washed five times with Wash Buffer (composed of 2x saline sodium citrate [SSC] and 0.5% SDS). Elution of the bound material was performed using PK buffer (containing 100 mM NaCI, 1 mM EDTA, 0.5% SDS, and 10 mM Tris HCI [pH 7] or [pH 8] for RNA or DNA elution, respectively). To analyze RNA enrichment, 10% of the ChIRP samples was used for RT-qPCR analysis after standard RNA extraction. For DNA analysis, the remaining material was utilized to amplify promoter region of genes including CCL3, CCL4 and IL-1 p by qPCR. The ChIRP assay employed a pool of 20 different biotinylated oligonucleotides as probes to specifically pull down MERRICAL RNA. As independent controls, a probe set the negative control, LacZ gene were used. Each reaction utilized a total of 300 p.m. of probe (100 p.m. of each MERRICAL probe or 150 p.m. of LacZ biotinylated oligos). MERRICAL Biotinylated probes were obtained from LGC Biosearch Technologies and negative control LacZ probes were obtained from Sigma-Aldrich (Magna ChIRP Negative Control Probe Set).

CUT&RUN assay

CUT&RUN Assay (EpiCypher, 14-1408) and subsequent sequencing analyses were utilized to determine the specificity of MERRICAL’s targets, according to the manufacturer’s instructions. Briefly, BMDMs were transfected with NS control or MERRICAL gapmers and treated with LPS (1 ug/ml, 2h). 1 .5 million cells per replicate were harvested per condition and washed and mixed with concanavalin A beads for 10 min at room temperature. Then, the cell/bead conjugates were resuspended with antibody buffer containing 0.01 % digitonin and either 0.5 pg H3K4me3 antibody or IgG negative control antibody, and incubated overnight at 4°C on a nutator. After overnight incubation, the mixture was washed and incubated with CUTANA pA/G-MNase for 10 min at room temperature. The reaction was stopped, the released chromatin fragments were purified using SPRIselect beads, and the DNA was eluted into TE buffer. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7103), NEBNext Multiplex Oligos for Illumina - Index Primers Set 1 (NEB, #E75335S) and NEBNext Multiplex Oligos for Illumina - Index Primers Set 2 (NEB, #E7500S). Libraries were sent to the Massachusetts General Hospital NextGen Sequencing Core where the samples were quality-checked by TapeStation, pooled, and sequenced using the Illumina NextSeq 2000 50 PE P2 flow cell (Illumina).

CUT&RUN data was performed with the following analytical steps. The paired-end sequencing read FASTQ files for each sample were taken as the input. Trimmomatic v0.36 was used for adapter sequences trimming at the 3' ends of each read (Bolger et al., 2014) with a two-step trimming process to improve the quality (K-seq). The reads were aligned to the reference mouse mm10 assembly by Bowtie2 v2.5.1 with settings -end-to-end, -very-sensitive, -no-mixed, -no-discordant, -dovetail, -I 10 -X 700 considers mates that overlap with each other, usually when fragment length is less than read length, as a concordant alignment. After alignment, Samtools v1 .14 was used to do mapping quality filtering and file format conversion in preparation for further analysis. Sparse Enrichment Analysis for CUT&RUN (SEACR v1 .3), developed by the Henikoff Lab for the peak calling, was used. The bedGraph files from paired-end sequencing were used as input and peaks were defined as contiguous blocks of base pair coverage that did not overlap with blocks of background signal delineated in the IgG control. Since the fragment counts were normalized with the spike-in read count, the normalization option of SEACR was set to “non”. Finally, the peak calling results from each sample were displayed by the Integrative Genomic Viewer (IGV v2.16).

Study approval

All protocols concerning animal use were approved by the Institutional Animal Care and Use Committee at Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA and conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Animal studies were performed in male Ldlr^z- mice (The Jackson Laboratory).

Quantification and statistical analysis

Statistical analyses were performed using GraphPad Prism version 7.0 (GraphPad Software Inc). Student t test was used to determine statistical significance between two groups.⁷ ANOVA with Bonferroni’s test was used to determine differences between more than two groups. Data are expressed as mean ± SEM. In all figures: *, **, ***, and **** denote p < 0.05, 0.025, 0.01 , and 0.001 respectively, while no significant difference is abbreviated “ns.” REFERENCES

1 . Al-Lawati, J. A. (2017). Diabetes mellitus: a local and global public health emergency. Oman Med. J. 32, 177 -179.

2. Kaur, J. (2014). A comprehensive review on metabolic syndrome. Cardiol. Res. Pract. 2014.

3. Zhang, Y., Sun, X., Icli, B., and Feinberg, M.W. (2017). Emerging roles for microRNAs in diabetic microvascular disease: novel targets for therapy. Endocr. Rev. 38,145 -168.

4. Tabas, I., and Bornfeldt, K.E. (2016). Macrophage phenotype and function in different stages of atherosclerosis. Circ. Res. 1 18, 653 -667.

5. Moore, K.J., Sheedy, F.J., and Fisher, E.A. (2013). Macrophages in atherosclerosis: a dynamic balance. Nat. Rev. Immunol. 13, 709 -721 .

6. Moore, K.J., and Tabas, I. (201 1 ). Macrophages in the pathogenesis of atherosclerosis. Cell 145, 341 -355.

7. Hilgendorf, I., Swirski, F.K., and Robbins, C.S. (2015). Monocyte fate in atherosclerosis. Arterioscler. Thromb. Vase. Biol. 35, 272 -279.

8. Djebali, S., Davis, C.A., Merkel, A., Dobin, A., Lassmann, T., Mortazavi, A., Tanzer, A., Lagarde, J., Lin, W., Schlesinger, F., et al. (2012). Landscape of transcription in human cells. Nature 489, 101 -108.

9. ENCODE Project Consortium (2012). An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57 -74.

10. Wang, K.C., and Chang, H.Y. (201 1 ). Molecular mechanisms of long noncoding RNAs. Mol. Cell 43, 904 -914.

1 1 . Consortium, R.G.S.P. (2004). Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493.

12. Hangauer, M.J., Vaughn, I.W., and McManus, M.T. (2013). Pervasive transcription of the human genome produces thousands of previously unidentified long intergenic noncoding RNAs. PLoS Genet. 9, e1003569.

13. Mattick, J.S., and Rinn, J.L. (2015). Discovery and annotation of long noncoding RNAs. Nat. Struct. Mol. Biol. 22, 5 -7.

14. Rinn, J.L., and Chang, H.Y. (2012). Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81 , 145 -166.

15. Haemmig, S., Yang, D., Sun, X., Das, D., Ghaffari, S., Molinaro, R., Chen, L., Deng, Y., Freeman, D., Moullan, N., et al. (2020). Long noncoding RNA SNHG12 integrates a DNA- PK -mediated DNA damage response and vascular senescence. Sci. Transl. Med. 12, eaaw1868.

16. Simion, V., Zhou, H., Haemmig, S., Pierce, J.B., Mendes, S., Tesmenitsky, Y., Pe' rez- Cremades, D., Lee, J.F., Chen, A.F., Ronda, N., et al. (2020). A macrophage-specific IncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus. Nat. Commun. 1 1 , 6135 -6216.

17. Simion, V., Zhou, H., Pierce, J.B., Yang, D., Haemmig, S., Tesmenitsky, Y., Sukhova, G., Stone, P.H., Libby, P., and Feinberg, M.W. (2020). LncRNA VINAS regulates atherosclerosis by modulating NF-kB and MAPK signaling. JCI insight 5, e140627.

18. Shihabudeen Haider Ali, M.S., Cheng, X., Moran, M., Haemmig, S., Naldrett, M.J., Alvarez, S., Feinberg, M.W., and Sun, X. (2019). LncRNA Meg3 protects endothelial function by regulating the DNA damage response. Nucleic Acids Res. 47, 1505 -1522.

19. Zhou, H., Simion, V., Pierce, J.B., Haemmig, S., Chen, A.F., and Feinberg, M.W. (2021 ). LncRNA-MAP3K4 regulates vascular inflammation through the p38 MAPK signaling pathway and cis-modulation of MAP3K4. Faseb. J. 35, e21 133. 20. Ni, H., Haemmig, S., Deng, Y., Chen, J., Simion, V., Yang, D., Sukhova, G., Shvartz, E., Wara, A.K.M.K., Cheng, H.S., et al. (2021 ). A Smooth Muscle Cell -Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting With Serum Response Factor. Arterioscler. Thromb. Vase. Biol. 41 , 2399 -2416.

21 . Josefs, T., and Boon, R.A. (2020). The long non-coding road to atherosclerosis. Curr. Atheroscler. Rep. 22. 55-12.

22. Li, FL, Zhu, FL, and Ge, J. (2016). Long noncoding RNA: recent updates in atherosclerosis. Int. J. Biol. Sci. 12, 898 -910.

23. Ferna' ndez-Ruiz, I. (2018). A new role for IncRNAs in atherosclerosis. Nat. Rev. Cardiol. 15, 195.

24. Zhang, Z., Salisbury, D., and Sallam, T. (2018). Long noncoding RNAs in atherosclerosis: JACC review topic of the week. J. Am. Coll. Cardiol. 72, 2380 -2390.

25. Rinn, J.L., and Chang, H.Y. (2020). Long noncoding RNAs: molecular modalities to organismal functions. Annu. Rev. Biochem. 89, 283 -308.

26. Marchese, F.P., Raimondi, I., and Huarte, M. (2017). The multidimensional mechanisms of long noncoding RNA function. Genome Biol. 18, 206 -213.

27. Engreitz, J.M., Haines, J.E., Perez, E.M., Munson, G., Chen, J., Kane, M., McDonel, P.E., Guttman, M., and Lander, E.S. (2016). Local regulation of gene expression by IncRNA promoters, transcription and splicing. Nature 539, 452 -455.

28. Khyzha, N., Khor, M., DiStefano, P.V., Wang, L., Matic, L., Hedin, U., Wilson, M.D., Maegdefessel, L., and Fish, J.E. (2019). Regulation of CCL2 expression in human vascular endothelial cells by a neighboring divergently transcribed long noncoding RNA. Proc. Natl. Acad. Sci. USA 1 16, 16410 -16419.

29. Liu, S., Liu, J., Yang, X., Jiang, M., Wang, Q., Zhang, L., Ma, Y., Shen, Z., Tian, Z., and Cao, X. (2021 ). Cis-acting Inc-Cxcl2 restrains neutrophilmediated lung inflammation by inhibiting epithelial cell CXCL2 expression in virus infection. Proc. Natl. Acad. Sci. USA 1 18, e21082761 18.

30. Bornfeldt, K.E. (2022). 2021 George Lyman Duff Memorial Lecture: The Remnant Lipoprotein Hypothesis of Diabetes-Associated Cardiovascular Disease. Arterioscler. Thromb. Vase. Biol. 42, 819 -830.

31 . Goldberg, I.J., Isaacs, A., Sehayek, E., Breslow, J.L., and Huang, L.-S. (2004). Effects of streptozotocin-induced diabetes in apolipoprotein Al deficient mice. Atherosclerosis 172, 47 -53.

32. Srinivasan, K., Viswanad, B., Asrat, L., Kaul, C.L., and Ramarao, P. (2005). Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacol. Res. 52, 313 -320.

33. Collins, A.R., Meehan, W.P., Kintscher, U., Jackson, S., Wakino, S., Noh, G., Palinski, W., Hsueh, W.A., and Law, R.E. (2001 ). Troglitazone inhibits formation of early atherosclerotic lesions in diabetic and nondiabetic low density lipoprotein receptor -deficient mice. Arterioscler. Thromb. Vase. Biol. 21 , 365 -371 .

34. Neuhofer, A., Wernly, B., Leitner, L., Sarabi, A., Sommer, N.G., Staffler, G., Zeyda, M., and Stulnig, T.M. (2014). An accelerated mouse model for atherosclerosis and adipose tissue inflammation. Cardiovasc. Diabetol. 13. 23-12.

35. Statello, L., Guo, C.-J., Chen, L.-L., and Huarte, M. (2021 ). Gene regulation by long noncoding RNAs and its biological functions. Nat. Rev. Mol. Cell Biol. 22, 96 -118.

36. Liu, T., Porter, J., Zhao, C„ Zhu, H., Wang, N„ Sun, Z., Mo, Y.-Y., and Wang, Z. (2019). TADKB: Family classification and a knowledge base of topologically associating domains. BMC Genom. 20. 217-17. 37. Chang, T.-T., Yang, H.-Y., Chen, C., and Chen, J.-W. (2020). CCL4 inhibition in atherosclerosis: effects on plaque stability, endothelial cell adhesiveness, and macrophages activation. Int. J. Mol. Sci. 21 , 6567.

38. Bell, R.D., Long, X., Lin, M., Bergmann, J.H., Nanda, V., Cowan, S.L., Zhou, Q., Han, Y., Spector, D.L., Zheng, D., and Miano, J.M. (2014). Identification and initial functional characterization of a human vascular cell -enriched long noncoding RNA. Arterioscler. Thromb. Vase. Biol. 34, 1249 -1259.

39. Haemmig, S., and Feinberg, M.W. (2017). Targeting LncRNAs in cardiovascular disease: options and expeditions. Circ. Res. 120, 620 -623.

40. Chen, J., Zhuang, R., Cheng, H.S., Jamaiyar, A., Assa, C., McCoy, M., Rawal, S., Pe' rez- Cremades, D., and Feinberg, M.W. (2022). Isolation and culture of murine aortic cells and RNA isolation of aortic intima and media: Rapid and optimized approaches for atherosclerosis research. Atherosclerosis 347, 39 -46. 2022.03.01 1 .

41 . Arun, G., Aggarwal, D., and Spector, D.L. (2020). MALAT1 long non-coding RNA: Functional implications. Noncoding. RNA 6, 22.

42. Chen, J., Ke, S., Zhong, L., Wu, J., Tseng, A., Morpurgo, B., Golovko, A., Wang, G., Cai, J.J., Ma, X., et al. (2018). Long noncoding RNA MALAT1 regulates generation of reactive oxygen species and the insulin responses in male mice. Biochem. Pharmacol. 152, 94 - 103.

43. Dong, K., Shen, J., He, X., Hu, G., Wang, L., Osman, I., Bunting, K.M., Dixon-Melvin, R., Zheng, Z., Xin, H., et al. (2021 ). CARMN Is an Evolutionarily Conserved Smooth Muscle Cell -Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin. Circulation 144, 1856 -1875.

44. Walter, W., Sa' nchez-Cabo, F., and Ricote, M. (2015). GOplot: an R package for visually combining expression data with functional analysis. Bioinformatics 31 , 2912 -2914.

45. Sachithanandan, N., Graham, K.L., Galic, S., Honeyman, J.E., Fynch, S.L., Hewitt, K.A., Steinberg, G.R., and Kay, T.W. (201 1 ). Macrophage deletion of SOCS1 increases sensitivity to LPS and palmitic acid and results in systemic inflammation and hepatic insulin resistance. Diabetes 60, 2023 -2031 .

46. Saraswathi, V., Kumar, N., Gopal, T., Bhatt, S., Ai, W., Ma, C., Talmon, G.A., and Desouza, C. (2020). Lauric acid versus palmitic acid: effects on adipose tissue inflammation, insulin resistance, and non-alcoholic fatty liver disease in obesity. Biology 9, 346.

47. Wang, K.C., Yang, Y.W., Liu, B., Sanyal, A., Corces-Zimmerman, R., Chen, Y., Lajoie, B.R., Protacio, A., Flynn, R.A., Gupta, R.A., et al. (201 1 ). A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature 472, 120 -124.

48. Li, H„ Ilin, S., Wang, W„ Duncan, E.M., Wysocka, J., Allis, C.D., and Patel, D.J. (2006). Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF. Nature 442, 91 -95.

49. Chervona, Y., and Costa, M. (2012). The control of histone methylation and gene expression by oxidative stress, hypoxia, and metals. Free Radic. Biol. Med. 53, 1041 - 1047.

50. Russ, B.E., Olshanksy, M., Smallwood, H.S., Li, J., Denton, A.E., Prier, J.E., Stock, A.T., Croom, H.A., Cullen, J.G., Nguyen, M.L.T., et al. (2014). Mapping histone methylation dynamics during virus-specific CD8+ T cell differentiation in response to infection. Immunity 41 , 853 -865.

51 . Russ, B.E., Olshanksy, M., Smallwood, H.S., Li, J., Denton, A.E., Prier, J.E., Stock, A.T., Croom, H.A., Cullen, J.G., Nguyen, M.L.T., et al. (2014). Distinct epigenetic signatures delineate transcriptional programs during virus-specific CD8+ T cell differentiation. Immunity 41 , 853 -865. 52. Gomez, J. A., Wapinski, O.L., Yang, Y.W., Bureau, J.-F., Gopinath, S., Monack, D.M., Chang, H.Y., Brahic, M., and Kirkegaard, K. (2013). The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-g locus. Cell 152, 743 - 754.

53. Fanucchi, S., Fok, E.T., Dalia, E., Shibayama, Y., Bo" rner, K., Chang, E.Y., Stoychev, S., Imakaev, M., Grimm, D., Wang, K.C., et al. (2019). Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments. Nat. Genet. 51 , 138 -150.

54. Skene, P.J., and Henikoff, S. (2017). An efficient targeted nuclease strategy for high- resolution mapping of DNA binding sites. Elife 6, e21856.

55. Wang, X., Zhu, K., Li, S„ Liao, Y., Du, R., Zhang, X., Shu, H.-B., Guo, A.-Y., Li, L, and Wu, M. (2012). MLL1 , a H3K4 methyltransferase, regulates the TNFa-stimulated activation of genes downstream of NF-kB. J. Cell Sci. 125, 4058 -4066.

56. Cao, F., Townsend, E.C., Karatas, FL, Xu, J., Li, L., Lee, S., Liu, L., Chen, Y., Ouillette, P., Zhu, J., et al. (2014). Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia. Mol. Cell 53, 247 -261 .

57. Muppirala, U.K., Honavar, V.G., and Dobbs, D. (201 1 ). Predicting RNAprotein interactions using only sequence information. BMC Bioinf. 12, 489 -499.

58. MacRitchie, N., and Maffia, P. (2021 ). Light sheet fluorescence microscopy for quantitative three-dimensional imaging of vascular remodelling. Cardiovasc. Res. 1 17, 348 -350.

59. Palazzo, A.F., and Lee, E.S. (2015). Non-coding RNA: what is functional and what is junk? Front. Genet. 6, 2.

60. Nagano, T., Mitchell, J. A., Sanz, L.A., Pauler, F.M., Ferguson-Smith, A.C., Feil, R., and Fraser, P. (2008). The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science 322, 1717 -1720.

61 . 0rom, U.A., Derrien, T., Beringer, M., Gumireddy, K., Gardini, A., Bussotti, G., Lai, F., Zytnicki, M., Notredame, C., Huang, Q., et al. (2010). Long noncoding RNAs with enhancer-like function in human cells. Cell 143, 46 -58.

62. Guil, S., and Esteller, M. (2012). Cis-acting noncoding RNAs: friends and foes. Nat. Struct. Mol. Biol. 19, 1068 -1075.

63. Ebisuya, M., Yamamoto, T., Nakajima, M., and Nishida, E. (2008). Ripples from neighboring transcription. Nat. Cell Biol. 10, 1 106 -1 1 13.

64. Dixon, J.R., Selvaraj, S., Yue, F., Kim, A., Li, Y., Shen, Y., Hu, M., Liu, J.S., and Ren, B. (2012). Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376 -380.

65. Kopp, F., and Mendell, J.T. (2018). Functional classification and experimental dissection of long noncoding RNAs. Cell 172, 393 -407.

66. Bridges, M.C., Daulagala, A.C., and Kourtidis, A. (2021 ). LNCcation: IncRNA localization and function. J. Cell Biol. 220, e202009045.

67. Wang, H., Fan, Z., Shliaha, P.V., Miele, M., Hendrickson, R.C., Jiang, X., and Helin, K. (2023). H3K4me3 regulates RNA polymerase II promoterproximal pause-release. Nature 615, 339 -348.

68. Livi, C.M., Klus, P., Delli Ponti, R., and Tartaglia, G.G. (2016). catRAPID signature: identification of ribonucleoproteins and RNA-binding regions. Bioinformatics 32, 773 -775.

69. Hirota, K., Miyoshi, T., Kugou, K., Hoffman, C.S., Shibata, T., and Ohta, K. (2008). Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs. Nature 456, 130 -134. 70. Anderson, K.M., Anderson, D.M., McAnally, J.R., Shelton, J.M., Bassel-Duby, R., and Olson, E.N. (2016). Transcription of the non-coding RNA upperhand controls Hand2 expression and heart development. Nature 539, 433 -436.

71 . de Jager, S.C.A., Bot, I., Kraaijeveld, A.O., Korporaal, S.J.A., Bot, M., van Santbrink, P.J., van Berkel, T.J.C., Kuiper, J., and Biessen, E.A.L. (2013). Leukocyte-specific CCL3 deficiency inhibits atherosclerotic lesion development by affecting neutrophil accumulation. Arterioscler. Thromb.

Example 2. EPIC1 as a therapeutic target for treating arteriosclerosis

As described in Example 1 , a long non-coding RNA (IncRNA) termed epigenetically induced MYC interacting IncRNAI (EPIC1 ) was identified as an ortholog of macrophage-enriched IncRNA regulates inflammation, chemotaxis, and atherosclerosis (MERRICAL). These two IncRNA share 22 completely conserved sequences and 46% similarity. Moreover, an RNA-protein interaction tool predicted a strong interaction between the IncRNA EPIC and human WDR5, further highlighting its relevance in the context of WDR5-mediated processes (data not shown).

EPIC1 is predominantly expressed in leukocytes (e.g., peripheral blood mononuclear cells), and not expressed in endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)) or smooth muscle cells (e.g., coronary artery smooth muscle cells (CASMCs)) as quantified by real time qPCR (FIG. 9). Protein residue indexing revealed that EPIC1 ’s tertiary structure is highly predicted to interact with WDR5 in an analogous manner (FIG. 10A), similar to that of MERRICAL (FIG. 10B). Furthermore, a Western blot of a IncRNA pulldown assay using biotin-labeled transcripts for EPIC1 or LacZ control in nuclear lysates of PBMC-derived human primary macrophages confirmed the interaction of human EPIC1 with WDR5 (FIG. 10C). Such results illustrate that EPIC1 (and MERRICAL) are therapeutic targets for treating arteriosclerosis (e.g., by alleviating lesion progression in diabetes).

Numbered Embodiments

1 . An inhibitory nucleic acid molecule comprising sufficient complementarity to a target nucleic acid molecule, wherein (i) the inhibitory nucleic acid molecule is at least 15 nucleotides in length, and

(ii) the target nucleic acid molecule comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 70.

2. The inhibitory nucleic acid molecule of embodiment 1 , wherein the target nucleic acid molecule comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 70.

3. The inhibitory nucleic acid molecule of embodiment 1 or 2, wherein the target nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 70.

4. The inhibitory nucleic acid molecule of any one of embodiments 1 -3, wherein the inhibitory nucleic acid molecule is 15 to 1 ,815 nucleotides in length.

5. The inhibitory nucleic acid molecule of embodiment 4, wherein the inhibitory nucleic acid molecule is 15 to 49 nucleotides in length, 50 to 99 nucleotides in length, or 100 to 1 ,815 nucleotides in length. 6. The inhibitory nucleic acid molecule of embodiment 5, wherein the inhibitory nucleic acid molecule is 18 to 25 nucleotides in length.

7. The inhibitory nucleic acid molecule of embodiment 6, wherein the inhibitory nucleic acid molecule is 21 nucleotides in length.

8. The inhibitory nucleic acid molecule of any one of embodiments 1 -7, wherein the inhibitory nucleic acid molecule comprises at least 85% complementarity to the target nucleic acid molecule.

9. The inhibitory nucleic acid molecule of embodiment 8, wherein the inhibitory nucleic acid molecule comprises at least 90% complementarity to the target nucleic acid molecule.

10. The inhibitory nucleic acid molecule of embodiment 9, wherein the inhibitory nucleic acid molecule comprises at least 95% complementarity to the target nucleic acid molecule.

1 1 . The inhibitory nucleic acid molecule of embodiment 10, wherein the inhibitory nucleic acid molecule is complementary to the target nucleic acid molecule.

12. The inhibitory nucleic acid molecule of any one of embodiments 1 -1 1 , further comprising a modification.

13. The inhibitory nucleic acid molecule of embodiment 12, wherein the modification comprises:

(a) a non-natural or modified nucleoside or nucleotide; and/or

(b) a covalently or non-covalently conjugated moiety.

14. The inhibitory nucleic acid molecule of embodiment 13, wherein:

(a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LN A), a 2’-O-methyl (2’-O-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2'-F) modified nucleoside; and/or

(b) the covalently or non-covalently conjugated moiety is selected from the group consisting of: a targeting moiety, a hydrophobic moiety, a cell penetrating peptide, or a polymer.

15. The inhibitory nucleic acid molecule of any one of embodiments 1 -14, wherein the inhibitory nucleic acid molecule is selected from the group consisting of: a small interfering RNA (siRNA), a doublestranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), and a gapmeR.

16. The inhibitory nucleic acid molecule of embodiment 15, wherein the inhibitory nucleic acid molecule is an siRNA.

17. The inhibitory nucleic acid molecule of embodiment 16, wherein the siRNA comprises an antisense strand comprising at least 85% sequence identity to any one of SEQ ID NOs: 1 -23.

18. The inhibitory nucleic acid molecule of embodiment 17, wherein the antisense strand comprises at least 90% sequence identity to any one of SEQ ID NOs: 1 -23.

19. The inhibitory nucleic acid molecule of embodiment 18, wherein the antisense strand comprises at least 95% sequence identity to any one of SEQ ID NOs: 1 -23.

20. The inhibitory nucleic acid molecule of embodiment 19, wherein the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1 -23. 21 . The inhibitory nucleic acid molecule of any one of embodiments 17-20, wherein the siRNA further comprises a sense strand comprising at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 24-46.

22. The inhibitory nucleic acid molecule of embodiment 21 , wherein the siRNA comprises:

(a) the antisense strand of SEQ ID NO: 1 and the sense strand of SEQ ID NO: 24;

(b) the antisense strand of SEQ ID NO: 2 and the sense strand of SEQ ID NO: 25;

(c) the antisense strand of SEQ ID NO: 3 and the sense strand of SEQ ID NO: 26;

(d) the antisense strand of SEQ ID NO: 4 and the sense strand of SEQ ID NO: 27;

(e) the antisense strand of SEQ ID NO: 5 and the sense strand of SEQ ID NO: 28;

(f) the antisense strand of SEQ ID NO: 6 and the sense strand of SEQ ID NO: 29;

(g) the antisense strand of SEQ ID NO: 7 and the sense strand of SEQ ID NO: 30;

(h) the antisense strand of SEQ ID NO: 8 and the sense strand of SEQ ID NO: 31 ;

(i) the antisense strand of SEQ ID NO: 9 and the sense strand of SEQ ID NO: 32;

(j) the antisense strand of SEQ ID NO: 10 and the sense strand of SEQ ID NO: 33;

(k) the antisense strand of SEQ ID NO: 11 and the sense strand of SEQ ID NO: 34;

(l) the antisense strand of SEQ ID NO: 12 and the sense strand of SEQ ID NO: 35;

(m) the antisense strand of SEQ ID NO: 13 and the sense strand of SEQ ID NO: 36;

(n) the antisense strand of SEQ ID NO: 14 and the sense strand of SEQ ID NO: 37;

(o) the antisense strand of SEQ ID NO: 15 and the sense strand of SEQ ID NO: 38;

(p) the antisense strand of SEQ ID NO: 16 and the sense strand of SEQ ID NO: 39;

(q) the antisense strand of SEQ ID NO: 17 and the sense strand of SEQ ID NO: 40;

(r) the antisense strand of SEQ ID NO: 18 and the sense strand of SEQ ID NO: 41 ;

(s) the antisense strand of SEQ ID NO: 19 and the sense strand of SEQ ID NO: 42;

(t) the antisense strand of SEQ ID NO: 20 and the sense strand of SEQ ID NO: 43;

(u) the antisense strand of SEQ ID NO: 21 and the sense strand of SEQ ID NO: 44;

(v) the antisense strand of SEQ ID NO: 22 and the sense strand of SEQ ID NO: 45; or

(w) the antisense strand of SEQ ID NO: 23 and the sense strand of SEQ ID NO: 46.

23. The inhibitory nucleic acid molecule of any one of embodiments 15-22, wherein the siRNA contains 3’ overhangs selected from the group consisting of:

(i) a single uracil overhang at one or more 3’ ends of the siRNA;

(ii) a double uracil overhang at one or more 3’ ends of the siRNA;

(iii) a single thymine overhang at one or more 3’ ends of the siRNA;

(iv) a double thymine overhang at one or more 3’ ends of the siRNA; or

(v) a single cytosine and single thymine overhang at one or more 3’ ends of the siRNA.

24. The inhibitory nucleic acid molecule of any one of embodiments 15-23, wherein the siRNA targets the nucleotide sequence of any one of SEQ ID NOs: 47-69.

25. The inhibitory nucleic acid molecule of any one of embodiments 1 -24, wherein the inhibitory nucleic acid molecule is formulated in a delivery vehicle. 26. The inhibitory nucleic acid molecule of embodiment 25, wherein the delivery vehicle is selected from the group consisting of: a vector, a plasmid, a micelle, a liposome, an exosome, and a lipid nano particle (LNP).

27. The inhibitory nucleic acid molecule of embodiment 26, wherein the vector is a viral vector.

28. The inhibitory nucleic acid molecule of any one of embodiments 1 -27, wherein the inhibitory nucleic acid molecule is formulated as a pharmaceutical composition.

29. The inhibitory nucleic acid molecule of embodiment 28, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent, and/or carrier.

30. A method of treating arteriosclerosis in a subject, the method comprising administering the inhibitory nucleic acid molecule of any one of embodiments 1 -29.

31 . The method of embodiment 30, wherein the arteriosclerosis is atherosclerosis.

32. The method of embodiment 31 , wherein the atherosclerosis is diabetes-associated atherosclerosis.

33. The method of any one of embodiments 30-32, wherein the subject has a metabolic disorder, or the subject is at risk of developing the metabolic disorder.

34. The method of embodiment 33, wherein the metabolic disorder is diabetes.

35. The method of embodiment 34, wherein the subject at risk of developing diabetes is prediabetic and/or has one or more of the following:

(a) hyperglycemia;

(b) glucose resistance;

(c) insulin resistance;

(d) hyperlipidemia; and

(e) has a family history of diabetes.

36. The method of any one of embodiments 30-35, wherein the inhibitory nucleic acid molecule is delivered to the aortic intima.

37. The method of any one of embodiment 30-36, wherein the inhibitory nucleic acid molecule is delivered to a macrophage.

38. The method of embodiment 37, wherein the macrophage is an activated peritoneal macrophage.

39. The method of any one of embodiments 30-38, further comprising administering an additional therapeutic agent.

Other Embodiments

All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

Claims

2. The inhibitory nucleic acid molecule of claim 1 , wherein the target nucleic acid molecule comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 70.

3. The inhibitory nucleic acid molecule of claim 1 or 2, wherein the target nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 70.

4. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is 15 to 1 ,815 nucleotides in length.

5. The inhibitory nucleic acid molecule of claim 4, wherein the inhibitory nucleic acid molecule is 15 to 49 nucleotides in length, 50 to 99 nucleotides in length, or 100 to 1 ,815 nucleotides in length.

6. The inhibitory nucleic acid molecule of claim 5, wherein the inhibitory nucleic acid molecule is 18 to 25 nucleotides in length.

7. The inhibitory nucleic acid molecule of claim 6, wherein the inhibitory nucleic acid molecule is 21 nucleotides in length.

8. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule comprises at least 85% complementarity to the target nucleic acid molecule.

9. The inhibitory nucleic acid molecule of claim 8, wherein the inhibitory nucleic acid molecule comprises at least 90% complementarity to the target nucleic acid molecule.

10. The inhibitory nucleic acid molecule of claim 9, wherein the inhibitory nucleic acid molecule comprises at least 95% complementarity to the target nucleic acid molecule.

11 . The inhibitory nucleic acid molecule of claim 10, wherein the inhibitory nucleic acid molecule is complementary to the target nucleic acid molecule.

12. The inhibitory nucleic acid molecule of claim 1 , further comprising a modification.

13. The inhibitory nucleic acid molecule of claim 12, wherein the modification comprises:

(a) a non-natural or modified nucleoside or nucleotide; and/or

(b) a covalently or non-covalently conjugated moiety.

14. The inhibitory nucleic acid molecule of claim 13, wherein:

(a) the non-natural or modified nucleoside or nucleotide is selected from the group consisting of: a locked nucleic acid (LN A), a 2’-O-methyl (2’-0-Me) modified nucleoside, a phosphorothioate (PS) bond between nucleosides, and a 2'-fluoro (2’-F) modified nucleoside; and/or

15. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is selected from the group consisting of: a small interfering RNA (siRNA), a double-stranded RNA (dsRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), an anti-sense oligonucleotide (ASO), and a gapmeR.

16. The inhibitory nucleic acid molecule of claim 15, wherein the inhibitory nucleic acid molecule is an siRNA.

17. The inhibitory nucleic acid molecule of claim 16, wherein the siRNA comprises an antisense strand comprising at least 85% sequence identity to any one of SEQ ID NOs: 1 -23.

18. The inhibitory nucleic acid molecule of claim 17, wherein the antisense strand comprises at least 90% sequence identity to any one of SEQ ID NOs: 1 -23.

19. The inhibitory nucleic acid molecule of claim 18, wherein the antisense strand comprises at least 95% sequence identity to any one of SEQ ID NOs: 1 -23.

20. The inhibitory nucleic acid molecule of claim 19, wherein the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1 -23.

21 . The inhibitory nucleic acid molecule of claim 17, wherein the siRNA further comprises a sense strand comprising at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs: 24- 46.

22. The inhibitory nucleic acid molecule of claim 21 , wherein the siRNA comprises:

(a) the antisense strand of SEQ ID NO: 1 and the sense strand of SEQ ID NO: 24;

(b) the antisense strand of SEQ ID NO: 2 and the sense strand of SEQ ID NO: 25;

(c) the antisense strand of SEQ ID NO: 3 and the sense strand of SEQ ID NO: 26;

(d) the antisense strand of SEQ ID NO: 4 and the sense strand of SEQ ID NO: 27; (e) the antisense strand of SEQ ID NO: 5 and the sense strand of SEQ ID NO: 28;

(f) the antisense strand of SEQ ID NO: 6 and the sense strand of SEQ ID NO: 29;

(g) the antisense strand of SEQ ID NO: 7 and the sense strand of SEQ ID NO: 30;

(i) the antisense strand of SEQ ID NO: 9 and the sense strand of SEQ ID NO: 32;

23. The inhibitory nucleic acid molecule of claim 15, wherein the siRNA contains 3’ overhangs selected from the group consisting of:

(i) a single uracil overhang at one or more 3’ ends of the siRNA;

(ii) a double uracil overhang at one or more 3’ ends of the siRNA;

(iii) a single thymine overhang at one or more 3’ ends of the siRNA;

(iv) a double thymine overhang at one or more 3’ ends of the siRNA; or

24. The inhibitory nucleic acid molecule of claim 15, wherein the siRNA targets the nucleotide sequence of any one of SEQ ID NOs: 47-69.

25. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is formulated in a delivery vehicle.

26. The inhibitory nucleic acid molecule of claim 25, wherein the delivery vehicle is selected from the group consisting of: a vector, a plasmid, a micelle, a liposome, an exosome, and a lipid nano particle (LNP).

27. The inhibitory nucleic acid molecule of claim 26, wherein the vector is a viral vector.

28. The inhibitory nucleic acid molecule of claim 1 , wherein the inhibitory nucleic acid molecule is formulated as a pharmaceutical composition.

29. The inhibitory nucleic acid molecule of claim 28, wherein the pharmaceutical composition comprises a pharmaceutically acceptable excipient, diluent, and/or carrier.

30. A method of treating arteriosclerosis in a subject, the method comprising administering the inhibitory nucleic acid molecule of claim 1 .

31 . The method of claim 30, wherein the arteriosclerosis is atherosclerosis.

32. The method of claim 31 , wherein the atherosclerosis is diabetes-associated atherosclerosis.

33. The method of claim 30, wherein the subject has a metabolic disorder, or the subject is at risk of developing the metabolic disorder.

34. The method of claim 33, wherein the metabolic disorder is diabetes.

35. The method of claim 34, wherein the subject at risk of developing diabetes is prediabetic and/or has one or more of the following:

(a) hyperglycemia;

(b) glucose resistance;

(c) insulin resistance;

(d) hyperlipidemia; and

(e) has a family history of diabetes.

36. The method of claim 30, wherein the inhibitory nucleic acid molecule is delivered to the aortic intima.

37. The method of claim 30, wherein the inhibitory nucleic acid molecule is delivered to a macrophage.

38. The method of claim 37, wherein the macrophage is an activated peritoneal macrophage.

39. The method of claim 30, further comprising administering an additional therapeutic agent.