Docket No.173738.02730 23T075WO TARGETING ALU RNA TO TREAT INFLAMATION AND RELATED INFLAMMATORY DISEASES AND CONDITIONS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to U.S. Provisional Patent Application No. 63/487,911, filed March 2, 2023, U.S. Provisional Patent Application No.63/490,751, filed March 16, 2023, and U.S. Provisional Patent Application No.63/490,753, filed March 16, 2023, the entire contents of which are each hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under R01HL128411 awarded by National Institutes of Health and the National Heart, Lung, and Blood Institute. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING [0003] The contents of the electronic sequence listing that accompanies this application named “173738_02730_Sequence_Listing.xml” (Size: 45,391 bytes; and Date of Creation: March 4, 2024) is herein incorporated by reference in its entirety. BACKGROUND [0004] Today, several diseases and conditions, such as atherosclerosis (and related conditions such as cardiovascular disease (CVD) such as atherosclerotic cardiovascular diseases) are considered a non-resolving chronic inflammatory disease, thus introducing a new era of therapeutic strategies that target inflammation. However, many pre-clinical and clinical trials targeting inflammation were unsuccessful due to off-target effects, cross-reactivity, redundancy of inflammatory mediators, compromised host immune responses, or discrepancies between animal models and human diseases. Further, despite remarkable advances in the field, the molecular mechanisms that induce sustained non-resolving inflammation in atherosclerosis are still not fully understood. Additionally, viral infections, such as SARS-CoV-2, accelerate atherosclerotic plaque progression
Docket No.173738.02730 23T075WO and increase the incidence of myocardial infarction and strokes. Previous studies indicated that viral infections increase retrotransposon (RT) expression; however, their role in exacerbating the inflammatory state of atherosclerosis has not been explored. [0005] (CVDs) and specifically atherosclerotic cardiovascular diseases continue to be the leading cause of death worldwide. Elevated plasma lipids, hypertension, and high glucose are the major risk factors for developing atherosclerotic plaques. To date, most pharmacological therapies aim to control these risk factors. However, despite remarkable advances in these therapies and the success of revascularization interventions to restore blood flow after plaque formation, heart disease has remained the leading cause of death globally. [0006] Thus, there remains a need in the art for developing therapeutic strategies that properly address inflammation and the role of viral infections in CVD. SUMMARY [0007] In some aspects, the present disclosure provides a method of inhibiting or treating a disease or condition in a subject in need thereof, the method comprising administering an agent to the subject, wherein the agent inhibits an Alu RNA. In some aspects, the disease or condition is associated with inflammation, and, in some aspects the disease or condition is selected from the group comprising atherosclerosis, cardiovascular disease, myocardial infarction, asthma, COPD, intra-amniotic inflammation, sterile intra-amniotic inflammation, an autoimmune disease, type I diabetes, and Alzheimer’s disease. In some aspects, the agent is an antisense oligonucleotide or siRNA that targets Alu RNA. [0008] In some aspects, the method further comprises measuring the expression of the Alu RNA in a cell, tissue, or organ sample from the subject in need thereof prior to and/or after delivering the agent to the subject in need thereof. One method of measuring the expression of the Alu RNA comprises measuring the level of a small cytoplasmic Alu (sc-Alu) RNA in the sample, and, in some aspects, further comprising measuring the expression of full-length Alu (fl-Alu) RNA in the sample and comparing the expression of sc-Alu RNA to fl-Alu RNA. In some aspects, the expression of Alu RNA is performed by a technique selected from the group consisting of polymerase chain reaction (PCR), reverse-transcription PCR (RT-PCR), quantitative PCR (qPCR), RT-qPCR, and in situ hybridization. In some aspects, the sample is a cell, tissue, or organ sample
Docket No.173738.02730 23T075WO from placenta, heart, lung, blood, serum, plasma, vaginal discharge, urine, lymphatic fluids, umbilical blood or tissue, and amniotic fluid. BRIEF DESCRIPTION OF THE DRAWINGS [0009] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. [00010] FIG. 1 shows representative images of de-identified human hearts. A six-month old donor with Hib meningitis (A) and a 55-year-old donor with history of heart disease (9 infarcts, 3 stents and, CABG surgery) (B). [00011] FIG.2 shows IL-1β expression in hCA increases with age and in the presence of CVD risk factors. RT-qPCR for IL-1β normalized to GAPDH in hCA isolated from different donors ranging in age from 6 months to 72 years old. The 6-month-old donor (6m) had no CVD history, the 51- and 58-year-old had history of hypertension and diabetes mellitus, whereas the 59- 72-year- old donors had history of hypercholesterinemia, hypertension, and MI. [00012] FIG.3 shows SARS-CoV-2 induces inflammasome pathway in human lungs and hCA. RT-qPCR for the indicated genes normalized to GAPDH in a representative sample of COVID-19 positive (pos) and negative (neg) lungs (A) and hCA (B). Data represent the mean ± SEM. [00013] FIG. 4 shows SARS-CoV-2 induces Alu RNA in human lungs and hCA. (A) Representative images of in situ hybridization for Alu probe (purple) or scramble control of lung biopsy and hCA from COVID-19 positive and negative patients. Nuclei were counterstained with nuclear fast red. Original magnification 20X. (B-C) Representative agarose gel of Alu and GAPDH RT- PCR (B) and densitometric quantification (C) of fl-Alu normalized to GAPDH in COVID-19 positive lungs (n=2) and hCA (n=1) and COVID-19 negative patients, and control or HeLa cells exposed to heat shock. Control Alu PCR using total RNA (bottom of panel B). Data represent the mean ± SEM.
Docket No.173738.02730 23T075WO [00014] FIG. 5 shows chaetocin increase Alu RNA expression and induce the expression of pro- inflammatory genes. A-C, ECs treated with 100 nM chaetocin for 16 hrs. Representative agarose gel of Alu and GAPDH RT-PCR, and control Alu PCR using total RNA (bottom panel) (A), densitometric quantification of fl-Alu normalized to GAPDH (B), and RT-qPCR for the indicated genes normalized to GAPDH (C). Data represent the mean ± SEM of a representative experiment of at least 2 independent experiments. [00015] FIG.6 shows Alu RNA induce pro- and anti-inflammatory response. RT-qPCR for the indicated genes normalized to GAPDH in EC treated with 100 nM chaetocin for 16 hrs or transfected with IVT AluSz RNA for 20 hrs. Data represent the mean ± SEM of a representative experiment. [00016] FIG. 7 shows Alu RNA increased poly I:C induced inflammation. RT-qPCR for the indicated genes normalized to GAPDH in EC transfected with IVT AluSz RNA, 1 ug/ml poly I:C or combination of both for 16 hrs. Data represent the mean ± SEM of a representative experiment. Note the logarithmic scale. [00017] FIG. 8 shows nanoparticles localize to advanced atherosclerotic plaques regions. (A) Representative scanning electron microscopy image of nanoparticles loaded with niRFP mRNA on a polycarbonate membrane with 200 nm pores (dark circles). (B-C) Representative en- face confocal images 48 hours after injection of niRFP mRNA loaded nanoparticle of aortic arch plaques and regions of the thoracic aorta from ApoE-/- mice fed on high fat diet (B), wire-injured and contralateral uninjured mouse femoral arteries (C), VE-Cadherin (VECad) to stain endothelial cell coverage and DAPI for nuclei staining. Images were captured at 20x magnification, insets magnified to 80x. Scale bars represents 100 µm. White arrowheads indicate RFP expressing cells. (D) uninjured (left) or wire injured femoral artery (right) 2 weeks after 5 injections of niRFP mRNA-p5RHH nanoparticle immunostained for α-smooth muscle actin (aSMA) and Nuclei were counterstained with DAPI. No niRFP signal was observed in the uninjured regions. Images were captured at 20x magnification. Scale bars represents 50 µm. [00018] FIG.9 shows a study protocol to assess the role of Alu RNA on atherosclerosis. [00019] FIG. 10 shows targeting Alu RNA to reduce inflammation. RT-qPCR for the indicated genes normalized to GAPDH in EC transfected with ASO for 2 hrs and treated with
Docket No.173738.02730 23T075WO vehicle control or with 1 ug/ml poly I:C for 12 hrs. Data represent the mean ± SEM of a representative experiment. Mean values are indicated above each bar. [00020] FIG. 11 shows effective delivery of niRFP nanoparticle to endothelial denuded regions of human coronary arteries. (A) Representative en-face confocal images of coronary artery 48 hours after one ex-vivo treatment with niRFP-p5RHH nanoparticles. No niRFP signal was observed in regions of healthy endothelium. Images were captured at 60x magnification. Scale bars represents 20 µm. (B) representative cross section preparations of uninjured or balloon injured coronary artery 4 days after 2 ex-vivo treatments (at day 0 and day 2) with niRFP-p5RHH nanoparticles. Arteries were immunostained for VE-Cadherin and nuclei were counterstained with DAPI. Arrowheads indicate ECs. Scale bars: 60 μm; original magnification, ×20 and ×60 (insets). [00021] FIG. 12 shows (A) Representative ISH images of miR-517a/b (purple), Alu (purple), or control scramble probes in term human chorioamniotic membranes (n = 3). Nuclei were counterstained with nuclear fast red. Scale bars: 100 µm; original magnification 40x. (B, C) E17.5 mouse chorioamniotic membranes derived from WT and C2MCΔ/Δ mice RT-qPCR of miR- 467a normalized to snoRNA202 (n=3, unpaired t test) (B), and a representative agarose gel for B1 and Gapdh RT-PCR (C). [00022] FIG. 13 shows (A-C) RT-qPCR of the indicated C19MC miRNAs normalized to snoRNA202 (unpaired t test) (A), representative ISH images of Alu (purple), or control scramble probes (B), and quantification of Alu ISH signal intensity (C) of human chorioamniotic membranes derived from PTB and term labor (n = 3). Nuclei were counterstained with nuclear fast red. Data represent the mean ± SEM. *p<0.05 and ****p<0.0001 vs term (A). Scale bars: 100 ^m; original magnification 40x. [00023] FIG. 14 shows (A) Representative agarose gel of Alu and GAPDH RT-PCR and control Alu PCR products using total RNA (bottom panel) and (B) densitometric quantification of fl-Alu to scAlu ratio normalized to GAPDH. [00024] FIG.15 shows representative in situ hybridization images of term human placentas with Alu (purple) or control scramble probes. Nuclei were counterstained with nuclear fast red. Scale bars: 50μm; original magnification, x40. [00025] FIG.16 shows IL1B expression in HUVECs can be inhibited with siRNA targeting Alu RNA.
Docket No.173738.02730 23T075WO DETAILED DESCRIPTION [00026] Disclosed are agents, compositions, and methods for use for preventing, inhibiting, or treating a disease or condition in a subject, such as inflammation in a subject. Also disclosed are methods using such agents, compositions for preventing, inhibiting, or treating conditions or diseases associated with inflammation. [00027] It is to be understood that the invention is not limited to the particular embodiments described. It is also understood that the terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting. The scope of the present invention will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise. [00028] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10. Further, as used herein, ranges that are between two particular values should be understood to expressly include those two particular values. For example, “between 0 and 1” means “from 0 to 1” and expressly includes 0 and 1 and anything falling inside these values. Also, as used herein “about” means ±20% of the stated value, and includes more specifically values of ±10%, ±5%, ±2%, ±1%, and ±0.5% of the stated value. [00029] In one aspect, the present disclosure provides methods of inhibiting or treating a disease or condition in a subject. The methods comprise delivering or administering an agent to a subject that prevents, inhibits, or alleviates a disease or condition in the subject. In embodiments,
Docket No.173738.02730 23T075WO the agent targets or inhibits an Alu RNA or Alu dsRNA. Further aspects comprise agents or compositions for use in inhibiting or treating disease or condition in a subject, and uses of agents or compositions in medicaments or pharmaceutical compositions for inhibiting or treating inflammation in a subject. In embodiments, the disease or condition is inflammation or an inflammatory-related disease or condition. [00030] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising delivering or administering an agent to the subject in need thereof. In embodiments, the agent prevents, inhibits, or alleviates inflammation in the subject. In embodiments, the agent inhibits Alu RNA. "Treating" and grammatical variations thereof as used herein may refer to the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treating may include the delivery or administration of an agent or composition, including those disclosed herein, to prevent the onset of the symptoms or complications, to alleviate the symptoms or complications, or to eliminate the disease, condition, or disorder. "Disease" as used herein may include disease states or symptoms of a disease and may be used interchangeably with "condition" or "disorder". [00031] The present disclosure describes the role of Alu in promoting the expression of inflammatory genes in disease or condition that may be associated with inflammation (i.e., inflammatory diseases or conditions), including, but are not limited to, atherosclerosis, cardiovascular disease, myocardial infarction, asthma, COPD, intra-amniotic inflammation, sterile intra-amniotic inflammation, an autoimmune disease, type I diabetes, and Alzheimer’s disease. Therefore, by way of example, but not by way of limitation, diseases or conditions that may be detected, diagnosed, and/or treated by the disclosed compositions and methods include, but are not limited to atherosclerosis, cardiovascular disease, myocardial infarction, asthma, COPD, intra- amniotic inflammation, sterile intra-amniotic inflammation, an autoimmune disease, type I diabetes, and Alzheimer’s disease. In some embodiments, the condition is sterile intra-amniotic inflammation, and in some embodiments the subject in need thereof is a pregnant female and/or fetus, at a higher risk of preterm birth. [00032] As used herein, the terms “pre-term birth” pre-term labor, preterm birth, preterm labor, PTB, and/or PTL may be used interchangeably and refer to birth before about 37 weeks of gestation.
Docket No.173738.02730 23T075WO [00033] "Administering" and grammatical variations thereof as used herein refer to the introduction of a substance into a subject's body. Administration may be systemic or local, for example. Administration of a nucleotide, for example, may be via transfection, injection, and/or administered in formulations comprising an appropriate delivery vehicle such as, but not limited to liposomes, nanoparticles, viral vectors, cells. Such delivery vehicles for RNA, and methods of administration are well known in the art. The nucleotide or agent to be administered may also be naked RNA. By way of example, but not by way of limitation, methods of delivery or administration include oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, intradermal administration, intrathecal administration, and subcutaneous administration. Administration can be continuous or intermittent. [00034] Alu SINEs are specific to human and non-human primates. Therefore, "subject" as used herein may refer to human and non-human primates without being confined to any particular sex, age, and/or species. A subject which is "in need thereof" will be readily apparent to a practitioner based on the teachings of this disclosure and may be determined by condition or disease state (for example, a subject with atherosclerosis) and/or gene expression profiles (for example, a subject with increased expression levels of CASP1, IL1B, IL6, NLRP3, and TNF, and/or increased levels of Alu RNA circulating in the blood, for example). For example, the subject in need thereof may have elevated expression levels of at least one inflammation-associated gene, and/or had previously contracted an infectious agent. The present disclosure further discloses that viral infection increases expression of Alu which in turn elevates expression levels of inflammation-associated genes. In particular, the disclosure describes the relationship between Alu expression and expression of inflammation-associated genes CASP1, IL1B, IL6, NLRP3, and TNF. [00035] "Inflammation-associated gene" (which may be used interchangeably with “inflammasome-associated genes”) as used herein refer to the nucleic acid sequences encoding gene products (including but not limited to RNA and proteins) that contribute to inflammation, for
Docket No.173738.02730 23T075WO example inflammation involved in the development of atherosclerosis. Inflammation-associated genes may include but are not limited to CASP1, IL1B, IL18, IL6, NLRP3, and TNF. Therefore, in some aspects, the subject in need thereof has elevated expression levels of at least one inflammation-associated gene selected from the group comprising CASP1, IL1B, IL6, NLRP3, and TNF. [00036] "Elevated expression levels" as used herein refer to increased levels of RNA and/or protein. Expression levels may be determined by methods known in the art and/or those methods disclosed herein. [00037] "At least one" refers to one or more. [00038] In some aspects, the agent decreases expression levels of the least one inflammation-associated gene. Elevated expression levels may be relative to baseline (or "healthy" or "control") expression levels prior to infection or disease state or may be relative to the increased expression levels following the establishment of the disease or infection. "Decreasing expression levels" may be relative to baseline (or "healthy" or "control") expression levels prior to infection or disease state or may be relative to the increased expression levels following the establishment of the disease or infection. In some aspects, expression levels of the least one inflammation- associated gene are decreased for at least about 48 hours. [00039] "Agent" as used herein refers to a substance or composition that brings about a chemical, biological, or physical effect or causes a chemical, biological, or physical reaction. Suitable agents for use with the present invention include, without limitation, small molecule, oligonucleotide, antisense oligonucleotide, polynucleotide, peptide, polypeptide, and enzyme. Exemplary antisense oligonucleotides are described herein and, without limitation, may include antisense oligonucleotides comprising or consisting of CCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCACGAGTAGCTGGGACTACAGGCGC CCGACACCACTCCCGGCTAATTTTTTGTATTTTT (SEQ ID NO: 1). The antisense oligonucleotide sequence may have at least 99%, 98%, 97%, 96%, or 95% sequence identity to SEQ ID NO:1. [00040] In embodiments, the agent is a small interfering RNA (siRNA) which may include but is not limited to siRNA that targets Alu RNA. Exemplary siRNA targeting Alu RNA includes, without limitation, siRNA comprising the passenger (sense) strand comprising or consisting of
Docket No.173738.02730 23T075WO GCCACUGCACUCCAGCCUG-UU (SEQ ID NO: 41) and guide (antisense) strand comprising or consisting of UU-CGGUGACGUGAGGUCGGAC (SEQ ID NO: 42). [00041] "Inhibits" and grammatical variations thereof as used herein may refer to the ability of an agent to control, prevent, restrain, arrest, block, and/or negatively regulate the activity for function of a target. For example, an antisense oligonucleotide may be able to block the function of Alu RNA. [00042] The present disclosure further describes the use of an agent for targeting or inhibiting Alu RNA, for example in vitro, in vivo, and ex vivo. Transfection of an antisense oligonucleotide (ASO), for example, did not induce cytotoxicity or inflammatory response. Therefore, in some aspects, the agent does not induce a cytotoxic response and/or an inflammatory response. [00043] "Cytotoxic response" as used herein refers to a reaction in the subject that results in the killing of cells. [00044] "Inflammatory response" as used herein may refer to a reaction in the subject that results in increased immune response including, but not limited to, inflammation which may include the recruitment of cytokines, antibodies, immune cells, or other immune agents to the area of inflammation. [00045] The present disclosure describes an agent, such as an antisense oligonucleotide (ASO), that is capable of inhibiting Alu RNA. In some aspects, the agent inhibits Alu RNA. In some aspects, the agent is an antisense oligonucleotide. In some aspects, the antisense oligonucleotide comprises SEQ ID NO: 1. [00046] "Antisense oligonucleotide" as used herein refers to small pieces of nucleic acids, including DNA and RNA, that can bind to specific molecules of RNA, thus blocking the ability of the RNA to make a protein or function in other ways. In some aspects, the agent is encapsulated in a nanoparticle. [00047] "Encapsulation" (or "nanoencapsulation") and grammatical variants thereof as used herein may refer to a technique based on enclosing an agent in liquid, solid, or gaseous states within a matrix or inert material for preserving the agent inside of the matrix or inert material. [00048] "Nanoparticle" as used herein may refer to particles that are measured at the level of nanometers and is commonly known in the art. One example of a nanoparticle that may be used
Docket No.173738.02730 23T075WO in the described methods is the cationic amphipathic cell-penetrating peptide (p5RHH) that may self-assemble into compacted, endonuclease resistant nanoparticles. Therefore, in some aspects, the nanoparticle is endonuclease-resistant, and, in some aspects, the nanoparticle is less than about 200 nm in size. In some aspects, the nanoparticle targets delivery of the antisense oligonucleotide to the heart, and, in some aspects, delivered to atherosclerotic plaques and endothelial denuded regions. Methods of targeting delivery of a nanoparticle and its encapsulants to the heart, atherosclerotic plaques, and endothelial denuded regions are described in the prior art and may include, but is not limited to, p5RHH-niRFP nanoparticles selectively targeting atherosclerotic plaque regions as described in WO2023/164708. [00049] The present disclosure further describes viral infections that accelerate inflammatory conditions, such as atherosclerotic plaque progression, and increase the incidence of inflammation or inflammatory conditions, such as myocardial infarction and stroke. Therefore, in some aspects, the subject in need thereof has previously contracted an infectious agent. "Infectious agent" as used herein may refer to any organism or agent that can produce disease and may include, but is not limited to, pathogens, bacteria, viruses, fungi, protozoa, and helminths. The infectious agent may include, but is not limited to Porphyromonas gingivalis, Helicobacter pylori, Cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, herpes simplex virus, and coronavirus (CoV), such as severe acute respiratory syndrome (SARS)-CoV-2. Therefore, in some aspects, the infectious agent is selected from the group comprising Porphyromonas gingivalis, Helicobacter pylori, Cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, herpes simplex virus, and coronavirus (CoV). "Previously contracted" and grammatical variants thereof as used herein may refer to the transmission of an infectious agent to a subject. In some aspects, the subject in need thereof has previously contracted an infectious agent less than about one week to less than about a year prior to treatment. [00050] In some aspects, the method further comprises administering at least one anti- inflammatory agent. "Anti-inflammatory agent" as used herein may refer to an agent or composition that aids in reducing inflammation and may include, but is not limited to, NSAIDs (including, but not limited to aspirin, naproxen, ibuprofen, diclofenac, and COX-2 inhibitors such as celecoxib and meloxicam).
Docket No.173738.02730 23T075WO [00051] Most eukaryotic genomes contain large numbers of repetitive sequences known as transposable elements (TEs). In humans, retrotransposons (RTs) are the largest class of transposable elements of which the short interspersed nucleic elements (SINEs) account for ~13% of the human genome. Most of the human SINEs belong to a single family known as Alu repeats (Alu), which are specific to human and non-human primates. Alu repeats most often consist of two similar but not identical monomers with a short adenine-rich linker between the two monomers and a longer and more variable A-rich region at the 3'-end. Various dimeric Alu subfamilies have been identified. AluJ are the most ancient subfamilies, AluS represents the major burst of Alu elements, and AluY is the youngest subfamily, which continue to RT and cause polymorphism in the population. Therefore, in some aspects, the Alu is selected from the group comprising AluJ, AluS, and AluY. In some instances, Alu RNA can be double-stranded RNA. Therefore, in some aspects, the Alu RNA is dsRNA. It is known in the art that dsRNA is a target for antibody binding (for example, the J2 antibody as described in Schonborn, et al, Nucleic Acids Res, 1991). Therefore, in some aspects, the agent is an antibody. Alu are often described as "junk", "parasitic" or even "selfish" DNA. [00052] In recent years, increasing evidence has shown that Alu are implicated in aging, age-related diseases, and poor prognosis. The human genome sequence data have revealed numerous genetic variations caused by Alu repeat insertions in the germline and are implicated in several human genetic disease, including α-thalassaemia. Alu SINE insertions continue to contribute to genome evolution providing functional elements such as promoters, enhancers, novel gene isoforms and splice variants. Alu may comprise regulatory elements (that is, portions of DNA or RNA that regulate the expression of genes), therefore, in some aspects, the Alu comprises a promoter or an enhancer. [00053] Further, in some aspects, the Alu comprises a novel gene isoform, and, in some aspects, the Alu comprises a splice variant. Alu may splice variants possess a variety of ranges. For example, full-length (fl-Alu) Alu RNA is about 300 bp. However, shorter Alu or fragments of the Alu element can also be used including fragments at least 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 141, 142, 143, 144, 145, 156, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,
Docket No.173738.02730 23T075WO 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, or 300 bp in length. Shorter cytoplasmic Alu (sc-Alu) RNA is about 100 bp. In some aspects, the Alu comprises an insertion or deletion mutation. In some aspects, the Alu is about 100 base pairs (bp) to about 350 bp. [00054] In another aspect, the present disclosure provides a composition that can contain sense strands, antisense strands, or a combination thereof. The composition can also comprise combinations of different Alu elements in the same or different orientations. [00055] Embodiment include a composition comprising an antisense oligonucleotide that inhibits Alu. In some aspects, the antisense oligonucleotide comprises SEQ ID NO: 1. [00056] In some aspects, a composition comprising an antisense oligonucleotide that inhibits Alu may also include a carrier. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. In some embodiments, the carrier is a nanoparticle. In some embodiments, the antisense oligonucleotide is encapsulated in a nanoparticle. In some aspects, the nanoparticle is endonuclease resistant. In some aspects, the nanoparticle is less than about 200 nm in size. In some aspects, the nanoparticle targets delivery of the antisense oligonucleotide to a tissue or organ, such as a heart. In some aspects, the composition is for use in methods of treating a disease or condition in a subject in need thereof, wherein the method comprises administering the composition to the subject in need thereof. [00057] In some aspects, the composition further comprises a pharmaceutical carrier. "Pharmaceutical carrier", "pharmaceutically acceptable carrier", and grammatical variants thereof as used herein may refer to any carrier, diluent, or excipient that is compatible with the other ingredients of a formulation and is not deleterious to a recipient to which it is administered. Pharmaceutically acceptable carriers are known in the art and include, but are not limited to, diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), solubilizing agents (e.g., glycerol, polyethylene glycerol), emulsifiers, liposomes,
Docket No.173738.02730 23T075WO nanoparticles, and adjuvants. Pharmaceutically acceptable carriers may be aqueous or non- aqueous solutions, suspensions, or emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include isotonic solutions, alcoholic/aqueous solutions, emulsions, and suspensions, including saline and buffered media. The compositions of the present invention may further include additives, such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), antioxidants (e.g., ascorbic acid, sodium metabisulfite), bulking substances or tonicity modifiers (e.g., lactose, mannitol). Components of the compositions may be covalently attached to polymers (e.g., polyethylene glycol), complexed with metal ions, or incorporated into or onto particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels, etc.) or onto liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. The compositions may also be formulated in lipophilic depots (e.g., fatty acids, waxes, oils) for controlled or sustained release. Assays, systems, kits, platforms, and methods [00058] In yet another aspect, disclosed herein are methods, systems, kits and platforms useful for measuring the level of the Alu RNA in a sample from the subject in need thereof prior to and/or after delivering or administering the agent to the subject in need thereof. In the disclosed embodiments in this disclosure, a sample may be from a cell, tissue, or organ. Measuring the level of the Alu RNA may comprise obtaining a sample from the subject in need thereof and measuring the level (amount or expression) of the Alu RNA in the sample. Methods of measuring the level of Alu RNA may include, without limitation, a technique selected from the group consisting of polymerase chain reaction (PCR), reverse-transcription PCR (RT-PCR), quantitative PCR (qPCR), RT-qPCR, and in situ hybridization. In some aspects, measuring the level of the Alu RNA comprises measuring the level (e.g. expression) of a small cytoplasmic Alu (sc-Alu) RNA in the sample, and, in some aspects, the method further comprises measuring the level of full-length Alu (fl-Alu) RNA in the sample and comparing the level of sc-Alu RNA to fl-Alu RNA. In some aspects, the sample is a cell, tissue, or organ sample. In embodiments, by way of example, and not by way of limitation, the cell, tissue, or organ sample is from, placenta (including chorioamniotic membranes), heart, lung, blood, serum, plasma, vaginal discharge, urine, lymphatic fluids,
Docket No.173738.02730 23T075WO umbilical blood or tissue, and amniotic fluid. In some aspects, the cell, tissue, or organ sample is from placenta and measuring the expression of the Alu RNA comprises detecting the level (amount or expression) of C19MC Alu RNA in the placenta sample. In some aspects, measuring the level of the Alu RNA comprises contacting the sample with a probe configured to recognize the Alu RNA in the sample and detecting binding of the probe to the sample. Detecting Alu RNA may, for example, be performed by in situ hybridization, and, in some aspects of the present disclosure, the probe comprises or consists of SEQ ID NO: 40. [00059] These methods of measuring the level of the Alu RNA in a sample (which may interchangeably be referred to as “detection methods”) may include methods of measuring a level (an amount or expression) of Alu RNA in tissue. The methods of detecting Alu RNA in a tissue sample include obtaining a cell, tissue, or ogran sample from a subject and measuring the level of Alu RNA in the sample. Detecting Alu RNA may be performed by various methods known in the art, including techniques selected from polymerase chain reaction (PCR), reverse-transcription PCR (RT-PCR), including competitive RT-PCR, quantitative PCR (qPCR), and RT-qPCR. Detecting Alu RNA may also be performed by techniques for visualizing Alu RNA in tissue, such as by in-situ hybridization. [00060] Competitive RT-PCR is a method for quantifying RNA. The technique involves co-amplification from test RNA with an internal standard using common primers in a single reaction. In the present disclosure competitive RT-PCR is used to measure the level or quantify the expression level of Alu RNA. In embodiments, the method comprises a primer set that recognizes both the fl-Alu and sc-Alu (5’ CCGGGTGCGGTGGCACACGCT (SEQ ID NO: 2), and 5’-GCAATCTCCTTCTCACGGGTT (SEQ ID NO: 3)) and will amplify the most abundant form of Alu. The resulting RT-PCR products are analyzed, for example by gel electrophoresis, and the ratio of fl-Alu to sc-Alu is determined, for example by densitometry. Total RNA isolated from cells following heat shock, which has been shown to increase fl-Alu can be used as a positive control. Additionally, to exclude amplification of genomic Alu elements Alu PCR using total RNA can be used. In embodiments, methods of detecting an amount or expression of Alu RNA include measuring the amount or expression of a small cytoplasmic Alu (sc-Alu) RNA in the sample. Further, embodiments include measuring the amount to full length Alu (fl-Alu) RNA in the sample and comparing the amount of sc-Alu RNA to fl-Alu RNA.
Docket No.173738.02730 23T075WO [00061] In situ hybridization can be used to visualize Alu RNA in tissue. In situ hybridization (ISH) is a technique that allows the detection and localization of viral nucleic acid (DNA or RNA) in tissue sections or cytological specimens using labelled nucleic acid probes with complementary sequences to the target viral nucleic acid. To visualized Alu RNA in tissues, in situ hybridization can be performed using a locked nucleic acid (LNA) probe (5’CACTGCACTCCAGCCTG) (SEQ ID NO: 40) designed to recognize Alu RNA transcripts, or a scrambled control. To confirm that the Alu probe recognizes Alu RNA and not Alu elements in the genomic DNA, tissue sections can be pre-treated with either RNase A or DNase I. [00062] In situ hybridization probes can be modified for different methods of detection, including fluorescent detection, and labelled with biotin or digoxigenin. [00063] Kits, systems, platforms, and methods disclosed herein comprise one or more of the above-noted oligonucleotides, for e.g., amplification of the target ALU sequence and/or for labeling the target ALU sequence. Buffers, controls, and instructions for use may also be included. [00064] In yet another aspect, the present disclosure provides a method of treating a disease or condition in a subject in need thereof, the method comprising administering an agent to the subject in need thereof, wherein the disease is atherosclerosis, wherein the subject in need thereof has previously contracted an infectious agent selected from the group comprising Porphyromonas gingivalis, Helicobacter pylori, Cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, herpes simplex virus, and coronavirus (CoV), for example SARS-CoV-2; and has elevated expression levels of at least one inflammation-associated gene selected from the group comprising CASP1, IL1B, IL6, NLRP3, and TNF, and wherein the agent inhibits Alu RNA and decreases expression levels of the least one inflammation-associated gene. In some aspects, the agent is an antisense oligonucleotide, and, in some aspects, the antisense oligonucleotide comprises SEQ ID NO: 1. [00065] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited
Docket No.173738.02730 23T075WO herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and/or description found in the cited references. [00066] Non-limiting examples of embodiments. [00067] Embodiment 1. A method of inhibiting or treating a disease or condition in a subject in need thereof, the method comprising administering an agent to the subject, wherein the agent inhibits an Alu RNA. [00068] Embodiment 2. The method of embodiment 1, wherein the subject has elevated expression levels of at least one inflammation-associated gene selected from the group comprising CASP1, IL1B, IL6, IL18, NLRP3, and TNF. [00069] Embodiment 3. The method of embodiment 2, wherein the agent decreases expression level of the least one inflammation-associated gene. [00070] Embodiment 4. The method of embodiment 3, wherein the agent prevents, reduces, or alleviates inflammation in the subject. [00071] Embodiment 5. The method of any one of the preceding embodiments, wherein the disease or condition is associated with inflammation. [00072] Embodiment 6. The method of embodiment 5, wherein the disease or condition is selected from the group comprising atherosclerosis, cardiovascular disease, myocardial infarction, asthma, COPD, intra-amniotic inflammation, sterile intra-amniotic inflammation, an autoimmune disease, type I diabetes, and Alzheimer’s disease. [00073] Embodiment 7. The method of embodiment 6, wherein the disease is atherosclerosis. [00074] Embodiment 8. The method of embodiment 6, wherein the condition is sterile intra- amniotic inflammation. [00075] Embodiment 9. The method of embodiment 8, wherein the subject in need thereof is at a higher risk of preterm birth.
Docket No.173738.02730 23T075WO [00076] Embodiment 10. The method of any one of the preceding embodiments, wherein the agent does not induce a cytotoxic response and/or an inflammatory response. [00077] Embodiment 11. The method of any one of the preceding embodiments, wherein the agent inhibits production of inflammatory cytokines. [00078] Embodiment 12. The method of any one of the preceding embodiments, wherein the agent is an antisense oligonucleotide or siRNA that targets Alu RNA. [00079] Embodiment 13. The method of embodiment 12, wherein the agent is an antisense oligonucleotide that targets Alu RNA. [00080] Embodiment 14. The method of embodiment 13, wherein the antisense oligonucleotide comprises SEQ ID NO: 1. [00081] Embodiment 15. The method of embodiment 12, wherein the agent is an siRNA that targets Alu RNA. [00082] Embodiment 16. The method of embodiment 15, further comprising treating the subject in need thereof with chaetocin. [00083] Embodiment 17. The method of any one of the preceding embodiments, wherein administration comprises delivering the agent to a cell, tissue, or organ in the subject. [00084] Embodiment 18. The method of embodiment 17, wherein the agent is in a composition comprising a carrier. [00085] Embodiment 19. The method of embodiment 18, wherein the carrier is selected from one or more of nanoparticles, liposomes, viral vectors, cells. [00086] Embodiment 20. The method of embodiment 17, wherein the carrier targets delivery of the antisense oligonucleotide to a cell, tissue or organ in the subject. [00087] Embodiment 21. The method of any one of the preceding embodiments, further comprising administering at least one anti-inflammatory agent.
Docket No.173738.02730 23T075WO [00088] Embodiment 22. The method of any one of the preceding embodiments, wherein the subject has previously contracted, or is currently infected with an infectious agent. [00089] Embodiment 23. The method of embodiment 22, wherein the infectious agent is selected from the group comprising pathogens, bacteria, viruses, fungi, protozoa, and helminths. [00090] Embodiment 24. The method of embodiment 23, wherein the infectious agent comprises one or more of Porphyromonas gingivalis, Helicobacter pylori, Cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, herpes simplex virus, and coronavirus (CoV). [00091] Embodiment 25. The method of any one of the preceding embodiments, wherein the Alu RNA is selected from the group comprising AluJ, AluS, and AluY. [00092] Embodiment 26. The method of any one of the preceding embodiments, wherein the Alu RNA is dsRNA. [00093] Embodiment 27. The method of embodiment 26, wherein the agent is an antibody specific to Alu dsRNA. [00094] Embodiment 28. The method of any one of the preceding embodiments, wherein the Alu RNA is about 100 bp to about 350 bp. [00095] Embodiment 29. The method of any one of the preceding embodiments, further comprising measuring the expression of the Alu RNA in a cell, tissue, or organ sample from the subject in need thereof prior to and/or after delivering the agent to the subject in need thereof, wherein measuring the expression of the Alu RNA comprises obtaining a sample from the subject and measuring a level of the Alu RNA in the sample. [00096] Embodiment 30. The method of embodiment 29, wherein measuring the expression of the Alu RNA comprises measuring the level of a small cytoplasmic Alu (sc-Alu) RNA in the sample. [00097] Embodiment 31. The method of 30, further comprising measuring the expression of full-length Alu (fl-Alu) RNA in the sample and comparing the expression of sc-Alu RNA to fl-
Docket No.173738.02730 23T075WO Alu RNA. [00098] Embodiment 32. The method of embodiment 31, wherein the fl-Alu RNA and sc- Alu RNA are detected with primers comprising SEQ ID NO: 2 and SEQ ID NO: 3. [00099] Embodiment 33. The method of embodiment 29, wherein measuring the expression of Alu RNA is performed by a technique selected from the group consisting of polymerase chain reaction (PCR), reverse-transcription PCR (RT-PCR), quantitative PCR (qPCR), RT-qPCR, and in situ hybridization. [000100] Embodiment 34. The method of embodiment 29, wherein the sample is a cell, tissue, or organ sample from placenta, heart, lung, blood, serum, plasma, vaginal discharge, urine, lymphatic fluids, umbilical blood or tissue, and amniotic fluid. [000101] Embodiment 35. The method of embodiment 34, wherein the cell, tissue, or organ sample is from placenta and measuring the level of the Alu RNA comprises detecting a level of C19MC Alu RNA in the placenta sample. [000102] Embodiment 36. The method of embodiment 29, wherein measuring the level of the Alu RNA comprises contacting the sample with a probe configured to recognize the Alu RNA in the sample and detecting binding of the probe to the sample. [000103] Embodiment 37. The method of embodiment 36, wherein detecting Alu RNA is performed by in situ hybridization. [000104] Embodiment 38. The method of embodiment 36, wherein the probe comprises SEQ ID NO: 40. EXAMPLES [000105] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims. [000106] Background [000107] Atherosclerotic cardiovascular disease (CVD) is the leading cause of death globally. In 2019, myocardial infarction (MI) and stroke were the world’s top killers, responsible
Docket No.173738.02730 23T075WO for approximately 16% and 11% of total deaths, respectively. The rapid increase in the aging population has transformed the demographics of many countries around the world. In the US, it is predicted that nearly one in four Americans will be 65 years or older by 2060. Aging increases the risk of CVD. Moreover, the elderly population has greater susceptibility to infections and develops more severe complications, highlighted by the COVID-19 pandemic. [000108] Elevated plasma lipids, hypertension, and high glucose are the major risk factors for developing atherosclerotic plaques. Most pharmacological therapies aim to control these risk factors
7. However, despite remarkable advances in these therapies and the success of revascularization interventions to restore blood flow after plaque formation, heart disease has remained the leading cause of death globally
7, 8. [000109] Viral infections, such as SARS-CoV-2, accelerate atherosclerotic plaque progression and increase the incidence of myocardial infarction and strokes. Previous studies indicated that viral infections increase retrotransposon (RT) expression; however, their role in exacerbating the inflammatory state of atherosclerosis had previously not been explored. The results here show that human lungs and human coronary arteries (hCA) isolated from SARS-CoV- 2 positive patients exhibited increased expression of Alu RNA as well as inflammasome- associated genes and pro- inflammatory cytokines. Thus, increases in RT expression by viral infections may exacerbate the inflammatory state of atherosclerosis and increase the risk of plaque rupture. [000110] Molecular mechanisms of sustained non-resolving inflammation in atherosclerosis [000111] The role of innate and adaptive immunity in atherogenesis has become prominent. Atherosclerosis is considered a non-resolving chronic inflammatory disease that develops in the medium to large arteries of the arterial tree at branching points with disturbed blood flow
1. Unstable atherosclerotic lesions are characterized by chronic inflammation with constant inflammatory cell recruitment, unsuccessful dead cell clearance from the site of inflammation, and failed switching of macrophages from pro-inflammatory to pro- resolving phenotypes
9. This revolution in understanding of the pathophysiology of atherosclerosis has started a new era of therapeutic strategies that target inflammation. However, many pre-clinical and clinical trials were unsuccessful due to off-target effects, cross-reactivity, redundancy of inflammatory mediators, compromised host immune responses, or discrepancies between animal models and human
Docket No.173738.02730 23T075WO diseases. The CANTOS trial was the first to show reduction in recurrent cardiovascular event rates in patients who received systemic administration of canakinumab, a monoclonal antibody that targets IL-1β. Although systemic immune suppression of IL-1β increased risk of fatal infections, the CANTOS trial established the inflammation hypothesis of atherosclerosis. However, despite remarkable advances in the field, the molecular mechanisms that induce sustained non- resolving inflammation in atherosclerosis are still not fully understood. [000112] Establish the role of the short interspersed nuclear elements (SINEs) in atherosclerosis [000113] Most eukaryotic genomes contain large numbers of repetitive sequences known as transposable elements. In humans, retrotransposons are the largest class of transposable elements and comprise ~45% of the genomic sequence, of which the short interspersed nucleic elements (SINEs) account for ~13% of the human genome. Most of the human SINEs belong to a single family known as Alu repeats (Alu), which are specific to human and non-human primates. Alu repeats consist of two similar but not identical monomers with a short adenine-rich linker between the two monomers and a longer and more variable A-rich region at the 3’-end. Various dimeric Alu subfamilies have been identified. AluJ are the most ancient subfamilies, AluS represents the major burst of Alu elements, and AluY is the youngest subfamily, which continue to retrotranspose and cause polymorphism in the population
10. Alu are often described as “junk”, “parasitic” or even “selfish” DNA. However, in recent years, increasing evidence has shown that Alu are implicated in aging, age-related diseases, and poor prognosis
4. The human genome sequence data have revealed numerous genetic variations caused by Alu repeat insertions in the germline and are implicated in several human genetic disease, including α-thalassaemia
11. Alu SINE insertions continue to contribute to genome evolution providing functional elements such as promoters, enhancers, novel gene isoforms and splice variants. In fact, most of the research related to Alu SINEs and atherosclerosis have focused on Alu-Alu recombination or insertions causing polymorphisms, such as the LDL receptor gene
12 and the antisense noncoding RNA in the INK4 locus (ANRIL)
13. [000114] Other studies have shown that inverted Alu repeats can form double strand RNA (dsRNA), which is detected by pattern recognition receptors (PRRs) such as MDA5 to induce an IFN-I response
14. To reduce the levels of Alu dsRNA, Alu RNA undergo post-transcriptional
Docket No.173738.02730 23T075WO modification that converts adenosines to inosines by ADAR. In fact, ADAR-dependent RNA editing has been shown to regulate the pro-inflammatory long noncoding RNA NEAT1
15, and cathepsin S
16 gene expression in human atherosclerotic vascular diseases. [000115] However, the human genome contains ~1 million copies of Alu repeats embedded near to or within coding and non-coding gene-rich regions that can be transcribed by RNA polymerase II. Additionally, Alu repeats contain an internal RNA polymerase III promoter and can be transcribed independently. To protect genomic integrity from deleterious insertions of Alu repeats, healthy somatic cells acquired multiple epigenetic mechanisms to strictly regulate their expression
17. During aging, profound alterations in DNA methylation patterns occurs. Since ~23% of all CpG sites in the human genome are in Alu repeats, global decrease in DNA methylation increases Alu RNA levels, which contribute to several age-related diseases. A classic example is age-related macular degeneration, in which increases in Alu RNA levels induce sterile inflammation characterized by activated NLRP3 inflammasomes that trigger the release of IL-1β and IL-18 in retinal pigmented epithelial cells causing cytotoxicity and degeneration
18, 19. Moreover, reverse-transcribed Alu RNA and its cDNA in the cytoplasm activated cGAS–STING signaling, type I interferon (IFN-I) and inflammatory responses, which were mitigated by treatment with nucleoside reverse transcriptase inhibitors (NRTIs)
20 21. Yet, the effect of global increase in Alu RNA expression in atherosclerosis has not been investigated. [000116] Infections, Alu RNA, and atherosclerosis [000117] Since the early 20th century, the association between seasonal influenza activity and CVD mortality was first noted. Since then, numerous clinical studies have shown that acute infections accelerate atherosclerotic plaque progression and increase the risk of MI and stroke
2. A recent study showed that the incidence of acute MI was six times as high during the 7 days after laboratory confirmation of influenza infection as during the control interval. Moreover, four- and three-times higher risk of MI was found a week after laboratory-confirmed infection with respiratory syncytial virus and other respiratory viruses, respectively
22. Other studies showed that infections with acute bacterial pneumonia increase the risk of MI by 48-fold during the first 15 days after hospitalization and correlated with the peaks at the onset of infection and with its severity
23-26. To date, a series of infectious agents has been shown to increase the short-term risk of MI including Porphyromonas gingivalis
27, Helicobacter pylori
28, Cytomegalovirus (CMV)
29,
Docket No.173738.02730 23T075WO Epstein-Barr virus (EBV)
30 and human immunodeficiency virus (HIV)
31. Although the risk of MI associated with acute infections decreased with time to baseline, in more severe infections it persisted for months and years after infection
32. In fact, the recent coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV- 2) also increased the risk of thrombosis, acute MI, ischemic stroke
33, and post-acute sequelae of COVID-19 in patients with severe infection
34. Moreover, SARS-CoV-2 was also associated with a serious and life-threatening multisystem inflammatory syndrome in children (MIS-C)
35, that shares some features with Kawasaki disease (KD)
36. Several clinical trials have shown that anti- infective therapy is ineffective in reducing atherosclerotic cardiovascular events
37. Thus, determining the right target pathway to reduce the incidence of adverse cardiac events in infected high-risk patients is vitally important
37. [000118] It has been shown that under stressful conditions such as viral infections, Alu SINEs regain transcriptional activation and become the largest class of virus-inducible noncoding RNA
5, 6. Viruses that upregulate Alu RNA expression include the herpes simplex virus
38 and SARS- CoV-2
39. In fact, the results disclosed here show that Alu RNA are highly expressed in human coronary arteries (hCA) and lungs isolated from COVID-19 positive patients compared to non- infected individuals (FIG. 4). While much progress has been made in the field of antiviral immunity, the effects and consequences of increased Alu RNA expression during viral infections on atherosclerosis had not been investigated. [000119] This study aims to unveil the fundamental role of the primate-specific Alu RNA in inducing sustained non-resolving inflammation in atherosclerosis, especially during infections, which will pave the way to novel therapeutic strategies that target Alu RNA, and will have a major impact on the CVD field. [000120] Methods of Investigation [000121] In this disclosure, several cutting-edge innovations are used that will greatly advance our understanding of the role of Alu RNA in non-resolving inflammation in atherosclerosis and during viral infection. [000122] First, there is a focus on Alu SINEs that constitute 11% of the human genome, which undergo transcriptional activation during viral infections and become the largest class of virus-inducible noncoding RNA
5, 6. Since Alu RNA is implicated in inducing sterile inflammation
Docket No.173738.02730 23T075WO and activation of NLRP3 inflammasomes and the release of IL-1β, which are implicated in the non-resolving inflammation in atherosclerosis, a thorough investigation into the effects of the global increase in their expression during infections on atherosclerosis is warranted. [000123] Second, Alu SINEs are specific to human and non-human primates. In humans, Alu repeats are ~300 nucleotides in length and consist of a dimer of two 7SL RNA-derived monomer units connected by an A-rich linker. Other species have different SINEs. Murine have two families of SINEs; the B1 SINEs are rodent specific, 135 nt long, and derived from 7SL RNA, whereas the B2 SINEs are ~209 nt long and originated from a tRNA gene. The species differences in SINEs may influence the RNA sensing by PRRs and consequently the immune response. Thus, the activation of the inflammasome and Caspase-1 by Alu RNA in humans may be difficult to replicate in mouse or other non-primate animal models. This fact may explain why many preclinical studies that used animal models have failed to translate clinically. [000124] Third, following the focus on primate specific Alu RNA, primary human EC, VSMC, and macrophages as well as peripheral blood mononuclear cells (PBMCs) and fresh hCA isolated from de-identified, viable and intact human hearts donated through the LifeLink Foundation (FIG. 1) are used. These hearts are acquired from brain-dead donors. Using this valuable resource allows for testing the role of Alu RNA in the innate immune response locally, in healthy and in advanced atherosclerotic plaques ex vivo, in the absence or presence of viral infections, while eliminating the effects of circulating and systemic immunity. [000125] Fourth, Alu RNA is recognized by PRRs and their downstream signaling, which are conserved in mouse and human. To investigate the role of the primate-specific Alu RNA in- vivo, a new “humanized” mouse model was developed in which Alu RNA is delivered to the atherosclerotic plaques using our cell-penetrating peptides that self-assemble into compacted, endonuclease resistant nanoparticles < 200 nm in size. Importantly, systemic administration of these nanoparticles in ApoE
-/- or wire injury mouse models specifically targeted atherosclerotic plaques and endothelial denuded regions, respectively, and not the liver, spleen, or kidneys (FIG.8). This approach will allow us to deliver Alu RNA to the atherosclerotic plaque itself and simultaneously treat the mice with poly I:C to mimic viral infections and investigate its role in atherogenesis in vivo.
Docket No.173738.02730 23T075WO [000126] Fifth, investigating Alu RNA expression is technically challenging because the human genome has ~one million homologous copies. To overcome this technical challenge that hinders detection and quantification of Alu RNA, an Alu-RNA specific probe is used to determine in situ Alu RNA expression in human tissues (FIG.4A). A primer set was also designed for RT- PCR that recognize the full-length (fl-Alu) Alu RNA of ~300 bp and the shorter cytoplasmic Alu (sc-Alu) RNA of ~100 bp (FIG.4B-C). Moreover, to directly test the effect of overexpression of Alu RNA on EC, VSMC, and macrophages in vitro, in hCA ex vivo and in the humanized mouse in vivo, a histone methyltransferase inhibitor was used to release the epigenetic suppression of Alu SINEs and increase their RNA levels (FIG.5). To directly test the effects of Alu RNA cells were transfected with in vitro transcribed (IVT) Alu RNA using 100% pseudouridine substitution to reduce their immunogenicity
40 (FIG.6). [000127] Sixth, the role of Alu RNA during viral infection was investigated. The COVID-19 pandemic caused by SARS-CoV-2 highlighted the alarming increase in adverse cardiac events in infected patients. Our data provide evidence of the increase in Alu RNA in COVID-19 positive tissues and its role in exacerbating inflammation, which has not been thoroughly investigated. [000128] Seventh, targeting the inflammatory pathways has been recently proven by the CANTOS trial to be beneficial in reducing cardiac events. Yet, many pre-clinical and clinical trials were unsuccessful mostly due to discrepancies between animal models and human diseases. We can investigate the role of the primate-specific Alu RNA in the non-resolving in atherosclerosis and during viral infection and assess the efficacy of a novel Alu RNA-specific antisense oligo (ASO) in reducing inflammation, both in vitro and ex vivo. [000129] Disclosed are studies showing that human lungs and coronary arteries (hCA) isolated from SARS-CoV-2 positive patients exhibited increased expression of Alu RNA as well as inflammasome-associated genes and pro-inflammatory cytokines, revealing the role of Alu RTs in inducing atherosclerosis-related inflammatory genes and lay the foundation for a novel anti- atherosclerotic therapeutic strategy that targets the inflammatory pathway. [000130] Example 1: Investigate the role of Alu RNA in regulating vessel inflammation during aging and infections. [000131] Background
Docket No.173738.02730 23T075WO [000132] Atherosclerosis is classified as a non-resolving inflammatory disease that progresses with age and is aggravated by infections. Numerous mechanistic studies have investigated the drivers of chronic inflammation in atherosclerosis and during infections. However, the role of stress induced Alu RNA, that constitutes 13% of the human genome, has not been investigated in atherosclerosis. [000133] Previous studies have shown that the NLRP3 (NOD [nucleotide oligomerization domain]-, LRR [leucine-rich repeat]-, and PYD [pyrin domain]-containing protein 3) inflammasome signaling complex is a key player in the innate immunity and vascular inflammation during atherosclerotic plaque initiation, progression, and rupture
41, 42. Several danger-associated molecular pattern (DAMP) stimuli have been shown to activate NLRP3 inflammasomes, including oxidized LDL
43, cholesterol crystals
42 and disturbed blood flow
44, as well as pathogen- associated molecular patterns (PAMPs). Activated inflammasomes convert pro- caspase 1 into active caspase-1, which then cleaves pro-interleukin-1β (IL-1β) and pro-interleukin- 18 (IL-18) into their active forms. Concurrently, caspase-1 cleaves gasdermin D (GSDMD) and its N-terminal domain forms pores in cell membranes through which the pro-inflammatory cytokines escape
45. IL-1β is actively produced by inflammasomes in ECs, VSMCs, and macrophages in the atheroma, their activation driven by cholesterol crystals and disturbed flow. In addition to the induction of its own gene expression, IL-1β increases the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and chemokines such as MCP-1 (CCL-2) that recruit leukocytes and monocytes to the atherosclerotic legion
46. Thus, IL-1β is a potent pro- inflammatory cytokine that stimulates the production of other cytokines, including tumor necrosis factor (TNF) and interleukin-6 (IL6) in ECs, VSMCs, and macrophages, which contribute to atherogenesis
47. In fact, the results show that hCAs isolated from donors of various ages exhibited increased IL-1β transcript levels with increasing age and the presence of CVD risk factors (FIG.2). [000134] Since viral infections increase the incidence of MI and stroke, the expression levels of NLRP3, CASP1, IL1B, and other pro-inflammatory cytokines such as IL6 and TNF in lungs and hCA isolated from COVID-19 positive donors compared to non-infected controls by RT- qPCR was assessed. A notable increase in inflammasome components and pro-inflammatory cytokines in the COVID-19 positive lungs and hCAs was found (FIG.3).
Docket No.173738.02730 23T075WO [000135] Since viral infections including SARS-CoV-2 were reported to induce Alu RNA expression, in-situ hybridization of COVID-19 positive lungs and hCA sections was performed using probes designed to recognize Alu RNA transcripts, or a scrambled control. Results show that Alu RNA are highly expressed in the COVID-19 positive lungs and hCA compared to uninfected donors (FIG.4A). [000136] To confirm that the Alu probe recognizes Alu RNA and not genomic Alu SINEs, the sections were pre-treated with either RNase A or DNase I and the Alu signal was abolished when sections were pre-treated with RNase A and not when pre-treated with DNase I (data not shown). Since Alu transcripts exist in a full-length (fl-Alu) RNA of ~300 bp that can be processed into a shorter cytoplasmic Alu (sc-Alu) RNA of ~100 bp, RT-PCR was performed using a primer set that recognizes both. As a positive control, total RNA isolated from HeLa cells after heat shock recovery was used, which has been shown to increase fl-Alu
48. To exclude amplification of the genomic Alu SINEs, Alu PCR was also performed using total RNA. Results show that lungs and hCA from COVID-19 positive patients exhibited 3- and 2-fold increase, respectively, in fl-Alu compared to non-infected patients, similar to HeLa cells after heat shock recovery, whereas total RNA showed fl-Alu PCR product only in one of the positive lungs (FIG. 4B-C). Fl-Alu PCR product of HeLa cells after heat shock recovery was cloned and sequenced to verify that indeed the RT-PCR products are all derived from Alu RNA. These results demonstrate that IL1B expression increases with age, CVD risk factors, viral infection, and correlates with an increase in Alu RNA levels. [000137] Under physiological conditions, ~99% of the one million Alu SINE promoters, which are transcribed by RNA polymerase III, are epigenetically silenced and packaged into chromatin structures that deny access of transcription factors. Although Alu SINEs are rich in CpG sites, which are heavily methylated, their transcription is not enhanced by DNA demethylation, but rather by inhibition of SUV39 methyltransferases (SUV39H1) that methylates histone H3 on lysine 9 (H3K9)
49. Importantly, during aging, profound alterations in DNA methylation patterns occur including decreases in SUV39H1 expression in both humans and mice, increasing the transcription of Alu SINEs
50. In fact, pol III loading and expression of Alu RNA increases significantly when cells are treated with the histone methyl-transferase inhibitor chaetocin
49.
Docket No.173738.02730 23T075WO [000138] To investigate the effects of the histone methyltransferase inhibitor on Alu RNA expression in vitro, primary human ECs were treated with 100 nM of chaetocin for 16 hrs and ~ 2.5-fold increase in the fl-Alu RNA expression occurred, whereas the sc-Alu were not detected (FIG. 5A-B). Intriguingly, chaetocin treatment also induced IL1B and TNF (FIG. 5C). These results indicate that increased Alu RNA by chaetocin treatment increases the expression of pro- inflammatory genes. [000139] To directly test the effect of Alu RNA on the expression of pro-inflammatory genes, and eliminate the non-specific effects of chaetocin, a member of the most abundant Alu subfamilies, AluSz was in vitro transcribed (IVT), in the forward direction, to mimic the RNA polymerase III stress-induced Alu RNA expression, using 100% pseudouridine substitution to reduce their immunogenicity
40. Like the chaetocin treatment, EC transfected with 125 nM AluSz RNA also showed increased expression of pro-inflammatory genes including CASP1, IL1B, and TNF (FIG.6). [000140] Taken together, these results support the approach and demonstrate that increases in Alu RNA, by either chaetocin treatment or by direct transfection of Alu RNA, increases pro- inflammatory cytokines. Therefore, the effects and consequences of Alu RNA expression, especially during viral infection, on atherosclerosis must be investigated. [000141] Research [000142] Establish the role of Alu RNA in inducing the pro-inflammatory response in-vitro. [000143] A first step will be to determine the role of SUV39H1 histone lysine methyltransferase in the induction of Alu RNA and pro- inflammatory gene expression. To mimic the age-associated decrease in SUV39H1 expression
50, which releases the epigenetic suppression of Alu SINEs and increases the expression of Alu RNA, primary human ECs, VSMCs, and THP- 1 monocytes differentiated into macrophages will be treated with chaetocin and the expression of Alu RNA will be determined by RT-PCR, as well as inflammasome components and pro- inflammatory genes by RT-qPCR. Dose response and time course experiments will be performed as well as cell viability assays using the RealTime-Glo™ MT Cell Viability Assay (Promega) on each cell type to determine the optimal response. The fl-Alu RT-PCR products will also be cloned, sequenced, and BLAT to confirm that they are indeed derived from Alu RNA.
Docket No.173738.02730 23T075WO [000144] The role of IVT Alu RNA on pro-inflammatory cytokine expression will be investigated. Similarly, the effect of Alu RNA on the expression of inflammasome components and pro-inflammatory cytokines will also be directly tested. primary human ECs, VSMCs, and THP-1 derived macrophages may be transfected with increasing concentrations of IVT Alu RNA of the different Alu subfamilies, including the J and the S, or control GFP mRNA in the presence of 100% pseudouridine substitution
40. Cell viability and the expression of inflammasome genes and pro-inflammatory cytokines may be assessed at different time points after transfection as described above. [000145] The effects of chaetocin and IVT Alu RNA on inflammasome activation, posttranslational processing, and secretion of IL-1β and other pro-inflammatory cytokines will be investigated. Since inflammasome activation requires a priming signal to increase the expression of NLRP3/pro–IL-1β and an activation signal, which promotes inflammasome assembly, Western blot will be performed to evaluate NLRP3-mediated pro–IL-1β cleavage in EC, VSMC, and THP- 1 derived macrophages treated with chaetocin or transfected with IVT Alu RNA in the absence or presence of the inflammasome activator adenosine triphosphate (ATP). Enzyme-linked immunosorbent assay (ELISA) analysis of IL-1β, IL-6, and TNF will be performed on the supernatants of ECs, VSMC, and THP-1 derived macrophages treated with chaetocin or transfected with IVT Alu RNA in the presence or absence of ATP compared to vehicle control. [000146] The effects of Alu RNA on the function of EC, VSMC, and macrophages in culture will be evaluated. the proliferation and migration properties of EC and VSMC treated with chaetocin or transfected with IVT Alu RNA compared to control GFP mRNA will be determined. The expression levels of ICAM-1, VCAM-1, and CCL-2 by RT-qPCR may also be determined by Western blotting. the ability of THP-1 derived macrophages treated with chaetocin or transfected with IVT Alu RNA to uptake oxidized low-density lipoprotein (oxLDL) will be evaluate and the expression levels of cholesterol trafficking genes such as scavenger receptors SR-A and CD36 and the cholesterol efflux transporters ABCA1 and ABCG1 assessed. [000147] Establish the role of Alu RNA during viral infection in vitro. [000148] Alu RNA expression in response to viral infection will be determined. To mimic viral infection, EC, VSMC, and THP-1 derived macrophages will be treated with increasing concentrations of poly I:C, a dsRNA TLR3 agonist that mimics viral infections, and the cells will
Docket No.173738.02730 23T075WO be infected with increasing MOI of SARS-CoV-2 and the expression of Alu RNA will be determined by RT-PCR at different time points after treatment or infection. [000149] We will also investigate the role of Alu RNA during viral infection. Several studies have shown that viral infections increase RNA polymerase III transcription of Alu RNA. However, the role of Alu RNA activation during SARS-CoV-2 infection are not fully understood. To determine whether Alu RNAs play an active role in the host defense against the virus EC, VSMC, and THP-1 derived macrophages may be pretreated with either chaetocin or transfected with IVT Alu RNA, then cells may be treated with poly I:C or infected with SARS- CoV-2. The expression of the NLRP3 inflammasome and pro-inflammatory genes may be determined by RT- qPCR. We may also examine IL-1β posttranslational processing and cytokine release as described above. [000150] Results show that EC treated with poly I:C or transfected with IVT Alu RNA for 16 hrs increased the expression of NLRP3 and pro-inflammatory genes and combination of both poly I:C and IVT Alu RNA exhibited much higher increase in their expression (FIG.7). [000151] Establish the role of Alu RNA in hCA and PBMCs ex vivo. [000152] The expression levels of Alu RNA in hCA and PBMCs during aging, the presence of CVD risk factors, and infection history ex-vivo will be assessed. Upon arrival to the lab, plasma from peripheral blood may be collected for analysis of pro-inflammatory and specialized pro- resolving mediators (SPMs) including the main polyunsaturated fatty acids, arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) and bioactive lipid mediators with major emphasis on SPMs and may be quantified by liquid chromatography–mass spectrometry as previously described
51. [000153] Peripheral blood mononuclear cells (PBMCs) may be isolated and total RNA will be extracted. The hearts will be maintained in cold Organ Recovery Systems solution (ORS, ViaSpan solution) and the left coronary, circumflex, anterior intraventricular, right coronary, and marginal arteries may be dissected. After careful removal of the surrounding fat and connective tissue, hCAs may be cut into several pieces to be used for total RNA extractions and for fixation and paraffin embedding. The expression of Alu RNA may be determined by in- situ hybridization and RT-PCR in hCA and in PMBCs as shown in FIG.4. We may also determine the expression levels of SUV39H1, NLRP3 inflammasome components, and pro-inflammatory cytokines in PBMCs and hCAs by RT-qPCR. Donor age, sex, CVD risk factors including cholesterol levels,
Docket No.173738.02730 23T075WO blood pressure, body mass index, diabetes, and smoking as well as infections and other health history may be considered when analyzing the data. [000154] The effects of viral infection on Alu RNA and pro-inflammatory cytokine expression in hCA and PBMCs ex-vivo will be assessed. PBMCs and hCA cut into 1 cm long pieces from each donor described elsewhere may be treated or perfused, respectively, with: 1. Poly I:C, 2. Chaetocin, 3. chaetocin and poly I:C, 4. infected with SARS-CoV-2, or 5. control DMEM medium supplemented with 10% FBS and 2% penicillin/ streptomycin. [000155] After incubation for time periods optimized in the previous experiments, total RNA may be extracted and the expression levels of Alu RNA, inflammasome, and pro-inflammatory genes may be determined by RT-qPCR. [000156] Results [000157] Based on results (FIGs.5 and 6), chaetocin treatment and IVT Alu RNA transfected ECs, VSMCs, and THP-1 differentiated into macrophages are expected to have increased expression of Alu RNA and pro-inflammatory genes. Chaetocin treatment and IVT Alu RNA transfection are expected to increase the expression of pro–IL-1b, increase its cleavage by ATP induced inflammasome activation and increase the levels of secreted IL-1β, IL-6, and TNF into the supernatant. Moreover, chaetocin treatment and IVT Alu RNA transfections increase the expression of ICAM-1, VCAM-1, and CCL-2 in EC and increase the expression of SR-A, CD36, ABCA1 and ABCG1 as well as oxLDL uptake in macrophages. Based on results (FIG. 4), Alu RNA expression increases with increasing poly I:C concentrations. Additionally, infection with SARS-CoV-2 and chaetocin treatment increase poly I:C induced pro-inflammatory cytokine production and release. Based on the literature
49-51 and the disclosed results (FIGs. 2 and 3), increased levels of pro-inflammatory genes and a reduction in SPMs are expected with age of the donor and with the presence of CVD risk factors and infections. Finally, poly I:C and SARS-CoV- 2 infected hCA and PBMCs is expected to exhibit higher expression levels of Alu RNA, inflammasome genes, and pro-inflammatory cytokines, especially in older donors with CVD risk factors. RNAseq analysis will be performed for EC, VSMC, and THP-1 derived macrophages treated with vehicle control, chaetocin, poly I:C, or transfected with either IVT Alu RNA or control GFP mRNA in the absence or presence of poly I:C. Since active caspase-1 from the NLRP3
Docket No.173738.02730 23T075WO inflammasome cleaves IL-18 as well as GSDMD that facilitates pro-inflammatory cytokines escape45, the role of Alu RNA in IL-18 and GSDMD expression will also be investigated. [000158] Example 2: Determine the effects of Alu RNA on atherosclerotic plaque inflammation in “humanized” mice in vivo. [000159] Background [000160] Although mouse models have contributed to understanding of CVD and identified therapeutic approaches, very few have reached clinical practice. Atherosclerotic mouse models, including ApoE
-/- which is the most widely used, do not display the same characteristics as human lesions such as plaque rupture. [000161] Alu SINEs are specific to human and non-human primates and our results show that Alu RNA, which is induced by viral infection, is a major player in regulating the inflammasome and pro-inflammatory cytokines. Although mice have the B1 and B2 SINEs, which are also transcribed by RNA polymerase III during viral infection, their sensing by PRR may be different than the human Alu RNA. Therefore, activation of the inflammasome and Caspase-1 by Alu RNA in humans may be difficult to replicate in mouse or other non-primate animal models. [000162] To overcome this challenge, we established self-assembled nanoparticles to deliver Alu RNA specifically to atherosclerotic plaques in ApoE
-/- mice in vivo. We have previously shown that in the presence of IVT RNA, the cationic amphipathic cell-penetrating peptide (p5RHH), self-assembled into compacted, endonuclease resistant nanoparticles < 200 nM in size (FIG. 8A)
52. These nanoparticles are rapidly taken up by cells and trafficked through the endosomes that require acidification for its intrinsic endosomolytic activity and release to the cytosol, without inducing cytotoxicity or apoptosis of the transfected cells. Importantly, systemic administration of near-infrared fluorescent protein (niRFP) mRNA nanoparticles in wire injury (FIG. 8B), or ApoE
-/- (FIG. 8C), mouse models specifically targeted atherosclerotic plaques and endothelial denuded regions respectively, and not the liver, spleen, or kidneys
52. [000163] The inventors recently showed that wire injured mice that received repeated retro- orbital venous injections of p5RHH-niRFP mRNA nanoparticles every 3 days for 2 weeks (total of 5 doses) exhibited strong niRFP expression only in regions of neointimal hyperplasia and restenosis (FIG. 8D right), but niRFP expression was not detected in the contralateral uninjured
Docket No.173738.02730 23T075WO femoral artery (FIG. 8D left). niRFP was also not detected either by confocal microscopy or by RT-qPCR in the different organs
52. Therefore, this novel approach allows targeting of Alu RNA to the atherosclerotic plaque itself and investigation of its role in-vivo. [000164] Research [000165] Investigate the role of Alu RNA in atherosclerotic plaque progression in “humanized” ApoE
-/- mice. [000166] ApoE
−/− mice fed a standard chow diet start having lesions as early as 10 weeks of age that progress into intermediate lesions containing foam cells and smooth muscle cells by 15 weeks, and fibrous plaques by 20 weeks of age
53. Therefore, we can treat 12-week-old ApoE-/- mice fed a standard chow diet with p5RHH complexed with: 1. control niRFP mRNA, 2. IVT Alu RNA, 3. niRFP mRNA and treated with poly I:C, or 4. IVT Alu RNA and treated with poly I:C The p5RHH nanoparticle may be delivered via retro-orbital venous injections every 3 days for 4 weeks for a total of 8 doses and poly I:C (250 ug in PBS) may be given intraperitoneally on alternate days (FIG.9). After 4 weeks of treatment, mice may be euthanized and the effects of Alu RNA on atherogenesis may be determined as follows: [000167] Atherosclerotic plaque burden will be assessed. After perfusion- fixation and careful removal of the surrounding fat and connective tissue, the aorta may be excised and further fixed overnight. The aorta may be longitudinally dissected, stained with Oil Red O solution, and pinned onto a flat surface for imaging. Quantitative morphometric analysis of digital images may be performed using ImageJ software to determine the extent of atherosclerosis. For each animal, the arterial area stained with Oil Red O may be reported as a percentage of the total investigated area. Advanced stages of atherosclerosis may be defined by the presence of cholesterol clefts in cross sections stained with Oil Red O and may be reported as an overall incidence for each treatment. Cross-sections may also be H&E stained and the neointima/media ratio for each section may be determined as previously described
52, 54, 55. Sections may also be immunostained with anti a-smooth muscle actin to visualize VSMCs. In addition, collagen may be detected by Masson's trichrome and sirius red staining, while elastin may be detected by Verhoeff staining. [000168] Assessment of inflammation. The levels of circulating IL-1β and high-sensitivity C-reactive protein (hs-CRP) may be determined by ELISA kits as well as levels of bioactive lipids and SPM in aortas and in plasma collected from mice at the end of the treatments as described
Docket No.173738.02730 23T075WO above. Immunostaining for anti-CD45 and anti-CD68 will also be performed to visualize infiltration of immune cells and macrophages, respectively, in aortic cross sections. [000169] The global delivery of Alu RNA will be assessed. At the end of the 4 week treatment, mouse organs (liver, lungs, kidneys, and spleen) and aortas will be collected and snap frozen (for total RNA and protein extraction). Total RNA may be extracted and the expression of Alu RNA and pro-inflammatory genes normalized to GAPDH may be determined by RT-PCR and RT-qPCR, respectively. [000170] Results [000171] Based on prior results (FIG. 8), Alu RNA-nanoparticles are expected to be delivered to atherosclerotic plaques only and may increase the expression of inflammasome and pro-inflammatory genes, similar to the niRFP-nanoparticles. Compared to the niRFP-treated mice, Alu RNA nanoparticles and poly I:C may exacerbate atherosclerotic plaques and increase infiltration of macrophages and the expression of inflammatory cytokines including IL-1β, mimicking the conditions of acute infections in humans that accelerate disease progression and increase the risk of stroke and MI. [000172] Since mice have their own B1 and B2 SINEs that can be induced by poly I:C treatment, it may confound the effects of Alu RNA. Therefore, since bacterial infections are also implicated in atherosclerotic plaque progression, poly I:C may be replaced with LPS treatment to mimic bacterial infection. [000173] Example 3: Test the efficacy of novel anti-atherosclerotic therapies targeting Alu RNA. [000174] Background [000175] It is established today that inflammation is an important driver of atherosclerosis, with the CANTOS trial providing the first evidence that targeting inflammation can reduce adverse cardiac events. However, many pre-clinical and clinical trials have been unsuccessful due to discrepancies between animal models and human diseases. Despite recent advances, there is still a tremendous gap between our understanding of the molecular mechanisms involved in atherosclerosis and the development of effective therapies that reduce adverse cardiovascular events, especially during acute infections.
Docket No.173738.02730 23T075WO [000176] Since Alu RNA are induced by viral infection and are implicated in sterile inflammation and activation of NLRP3 inflammasomes and downstream pro-inflammatory cytokines including IL-1β
18,19 46, targeting Alu RNA may hold great therapeutic potential. [000177] We designed an antisense oligo (ASO; SEQ ID NO: 1) [Inventors do you have the sequence of this oligo?] that targets Alu RNA. To test its efficacy in reducing poly I:C induced inflammation, ECs were transfected with ASO and after 2 hr, cells were treated with 1 ug/mL poly I:C and collected after 12 hours. ASO transfection did not induce any cytotoxicity or inflammatory response but rather reduced poly I:C induced NLRP3, CASP1, IL1B, and TNF expression (FIG.10). These exciting findings highlight the feasibility of these studies and lay the foundation for a novel anti-atherosclerotic therapeutic strategy that targets the inflammatory pathway in atherosclerosis. [000178] Research [000179] Assess the efficacy of ASO in inhibiting the pro-inflammatory response in vitro. [000180] During aging, decreases in SUV39H1 expression
50 may be responsible for the increase in Alu RNA levels, which may contribute to the non-resolving inflammation characteristic of atherosclerosis. To assess the efficacy of ASO in inhibiting pro- inflammatory responses, we will transfect primary human ECs, VSMCs, and THP-1 monocytes differentiated into macrophages with increasing concentrations of ASO and after 2 hrs, cells will be treated with the optimal concertation of chaetocin (determined above) to induce Alu RNA. After 12 hr, the expression of NLRP3 and other pro-inflammatory genes will be determined by RT-qPCR. The expression levels of pro-IL-1β and cleaved IL-1β as well as the secretion of the pro-inflammatory cytokines to the supernatant will also be determined by Western blotting and ELISA, respectively as described above. Cell viability, proliferation and migration properties of EC and VSMC will also be assessed to assure the safety of the ASO treatment as described above. [000181] Assess the efficacy of ASO in reducing poly I:C- and SARS-CoV-2-induced inflammation in vitro. [000182] We have established that viral infections accelerate atherosclerotic plaque progression and increase the expression of Alu RNA. To assess the efficacy of ASO in reducing inflammation during viral infection, EC, VSMC, and THP-1 derived macrophages will be transfected with increasing concentrations of ASO and after 2 hr, cells will be treated with poly
Docket No.173738.02730 23T075WO I:C or will be infected with SARS-CoV-2, and the expression of the NLRP3 inflammasome components and pro-inflammatory genes will be determined by RT-qPCR. IL-1β posttranslational processing and cytokine release as described above will also be examined. [000183] Assess the efficacy of ASO in inhibiting the pro-inflammatory response in hCA atherosclerotic plaques ex vivo. [000184] To deliver the ASO to the atherosclerotic plaque in hCA, the same self-assembled nanoparticles described in FIG. 8 that was previously established in a wire injury mouse model may be used
52 will be used. hCA from donors with CVD history will be cut into 1 cm long pieces and perfused with DMEM supplemented with 10% FBS and 2% penicillin/ streptomycin containing: 1. control niRFP-nanoparticle, 2. control niRFP-nanoparticle and poly I:C, 3. control niRFP-nanoparticle and SARS-CoV-2, 4. ASO-nanoparticle, 5. ASO-nanoparticle and poly I:C, or 6. ASO-nanoparticle and SARS-CoV-2. After 48 hrs, total RNA may be extracted and the expression levels of NLRP3 inflammasome components and pro-inflammatory genes may be determined by RT-qPCR. [000185] To assess the feasibly of this approach and to confirm the delivery of the nanoparticle to regions of endothelial cell denudation, hCA was isolated and perfused with p5RHH-niRFP mRNA nanoparticles for 48 hours. En-face confocal imaging of the longitudinally dissected arteries immunostained with VE-cadherin showed clear niRFP expression in regions of disrupted endothelial cell barriers (FIG. 11A). To further confirm that the mRNA-p5RHH nanoparticle delivers its cargo to regions of endothelial cell denudation, a sham or balloon injury was performed on the same human coronary artery using embolectomy catheter followed by p5RHH-niRFP mRNA nanoparticle treatment every 2 days for 4 days (total 2 treatments). Confocal images of arterial cross sections showed a large neointima/atherosclerotic lesion in both the injured and uninjured regions (FIG.11B). Importantly, balloon injured arteries which showed no VE-Cadherin positive staining, exhibited clear niRFP expression across the different layers of the artery, whereas the uninjured artery that exhibited VE-Cadherin positive staining showed very low niRFP expression after 48 hours (FIG.11B). These results are consistent with the wire injury and ApoE
-/- mice models (FIG. 8) and provide strong proof of the feasibility of the disclosed therapeutic approach. [000186] Results
Docket No.173738.02730 23T075WO [000187] Based on results (FIGs.10 and 11), ASO is expected to reduce chaetocin-, poly I:C- and SARS-Cov-2-induced inflammation and release of pro-inflammatory cytokines in-vitro and ex-vivo. Also, ASO may not affect cell viability, proliferation, and migration. [000188] To increase the efficacy of the ASO in reducing inflammation ex-vivo, hCA may be treated with ASO nanoparticles every other day for at least 8 days, which may allow evaluation of the anti- inflammatory properties for prolonged periods of time. It may also be possible to assess apoptosis and cytotoxicity ex-vivo using Tunnel assay. To visualize the efficacy of the ASO delivery, Cy5 labeled ASO may be used. [000189] METHODS [000190] Reproducibility [000191] To overcome the challenge of working with Alu RNA because Alu SINEs are extremely abundant (~ 13% of the human genome) and the subfamilies are highly homologous, in this application the RT-PCR products will be confirmed to be derived from Alu RNA by cloning, sequencing, and BLAT to confirm that they aligned with Alu SINEs. To exclude amplification of genomic Alu SINEs the PCR will also be performed using total RNA in each experiment. The in- vivo and ex-vivo experiments will be performed blindly to assure transparency and will include appropriate controls. All the in vivo and ex-vivo experiments will be performed in males and females and the health of the donors will be carefully considered (see sample size). Rigorous statistical analysis will be performed to assure reproducibility. [000192] Statistical Analysis [000193] For comparisons of two groups, unpaired, two-tailed Student’s t-test will be used. For comparison of 3 or more groups, One-way ANOVA will be used followed by Tukey post hoc test. In the event of non-normal distributions, the non-parametric Mann Whitney U test will be used. A p<0.05 will be considered significant. All the statistical analysis will be performed using Prism 9 Software. [000194] Sample Sizes [000195] Demographic data will be summarized using descriptive statistics such as mean/SD, median/IQR, and frequency/rate.
Docket No.173738.02730 23T075WO [000196] For Example 1, a linear regression will be performed to determine if the treatment group influences the expression level of IL1B and adjust for any potential covariates. Potential covariates include age, sex, and the presence of CVD risk factors. To define the relationship between outcome and treatment groups, the regression coefficients and 95% confidence intervals will be reported. A sample size of 50, or 10 per treatment group, achieves 72% power to detect a medium effect size in this analysis design. [000197] For Example 2, there is no concern with any potential covariates and a one-way analysis of variance (ANOVA) will be used to determine if there is a significant difference in the IL1B expression between groups if the data is normally distributed. If the data is not normally distributed, a Kruskal Wallis H test will be used instead. If there is a significant difference observed between groups, then post hoc tests will be done to determine where the difference exists. A sample size of 9 per group, or 36 total, achieves 65% power to detect a large effect size due to group. [000198] For Example 3, a linear regression will be perform to determine if the treatment group influences the expression level of IL1B while adjusting for any potential covariates including age, sex, and presence of CVD risk factors. To define the relationship between outcome and treatment groups, the regression coefficients and 95% confidence intervals will be reported. A sample size of 60, or 10 per treatment group, achieves a 78% power to detect a medium effect size. [000199] Example 4: Alu RNA associated with pre-term birth [000200] Preterm birth (PTB), commonly defined as birth occurring before 37 weeks of gestation, is a global health challenge, being the leading cause of neonatal mortality and associated with long-term physical, intellectual, and mental disabilities. Despite the efforts made during the past decade to improve maternal and fetal health, PTB continues to affect 13.5 million newborns, making up to 10% of all live births worldwide. Intra-amniotic inflammation is the most well- characterized cause of PTB, presenting in two distinct contexts: inflammation arising from microbial invasion of the amniotic cavity and sterile intra-amniotic inflammation (SIAI), which occurs without microbial presence. SIAI is characterized by an increase in endogenous mediators activating the innate immune system (Gomez-Lopez et al, Reproduction, 2022). However, the mechanism leading to SIAI and PTB remains not fully understood.
Docket No.173738.02730 23T075WO [000201] Recently, we uncovered an intriguing case of convergent evolution involving the primate-specific chromosome 19 miRNA cluster (C19MC) and the rodent-specific miRNA cluster on chromosome 2 (C2MC). These trophoblast-specific clusters, rich in short interspersed nuclear elements (SINEs), generate double-stranded (ds)RNA, triggering a viral mimicry immune response. This response activates interferon lambda (IFNL) signaling, providing constant antiviral protection to the immunologically underdeveloped fetus, independently of the miRNAs. However, the role of the dsRNA produced by C19MC and C2MC in SIAI and PTB remain unexplored. [000202] Introduction [000203] Preterm birth (PTB), defined as childbirth occurring before 37 weeks of gestation, remains a significant global health challenge, with an annual incidence exceeding 13 million pregnancies (Ohuma et al, Lancet 2023). PTB is a leading cause of neonatal morbidity and mortality, and its impact extends to long-term physical, intellectual, and mental health outcomes for affected individuals (Teune et al, Am J Obstet Gynecol 2011). While the precise etiology of PTB is multifaceted, a significant proportion is attributed to spontaneous preterm labor, a syndrome with diverse causal and associated factors. [000204] The spontaneous onset of preterm labor, whether with intact or ruptured membranes, accounts for two-thirds of PTB. This phenomenon is a key component of the great obstetrical syndromes, where presenting symptoms and signs signify the activation of the common pathway of parturition. This activation involves increased uterine contractility, cervical remodeling, and membrane/decidual activation. Various pathological factors can lead to the activation of this common pathway. Among these factors, inflammation of the amniotic cavity, referred to as intra-amniotic inflammation, stands out as a well-established cause of spontaneous PTB. [000205] Intra-amniotic inflammation manifests in two distinct contexts: intra-amniotic inflammation resulting from microbial invasion of the amniotic cavity and sterile intra-amniotic inflammation (SIAI), occurring in the absence of microbial presence. SIAI is marked by an increase in endogenous mediators that activate the innate immune system. Notably, SIAI is more prevalent than intra-amniotic infection and is associated with acute inflammatory lesions in the placenta, resembling pregnancy and neonatal outcomes observed in cases of intra-amniotic
Docket No.173738.02730 23T075WO infection. This highlights the clinical significance of this inflammatory state (Romero et al, Am J Reprod Immunol 2014). Therefore, SIAI has emerged as a distinct clinical entity and is more common than intra-amniotic infection in women with preterm labor with intact membranes, as well as in women with an asymptomatic sonographic short cervix or cervical insufficiency. However, the precise trigger(s) for SIAI in patients with PTB have not been identified. [000206] Sterile inflammation is initiated when pattern recognition receptors (PRRs) engage with damage-associated molecular patterns (DAMPs) or alarmins released during cellular stress, necrosis, or senescence. Classical alarmins include interleukin 1α (IL-1α), high mobility group box-1 (HMGB1), S100 calcium binding protein B (S100B) and heat-shock protein 70 (HSP70), which play a pivotal role in inflammatory responses. Importantly, elevated concentrations of these alarmins have been detected in the amniotic fluid of women with SIAI and PTB (Bhatti et al, J Perinat Med 2007). Furthermore, studies employing ultrasound-guided intra-amniotic administration of these alarmins have solidified the association between heightened alarmin levels and PTB with adverse neonatal outcomes. Moreover, exposure of chorioamniotic membranes, which are in direct contact with the amnionic fluid, to alarmins induced inflammatory responses that involved the activation of the NF-κB pathway and to the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome (Gomez-Lopez et al, Am J Reprod Immunol 2018). Inflammasome activation requires a priming step that involves the upregulation of inflammasome- associated gene expression and production of pro-inflammatory cytokines such as pro-IL-1β and pro-IL-18. This priming process occurs through transcriptional regulation mediated by NF-κB in response to a primary stimulus of DAMPs. Upon receiving a secondary stimulus of DAMPs, specific sensors within the inflammasome are activated, leading to the recruitment and assembly of the adaptor protein ASC, which in turn recruits and activates caspase-1 that cleaves pro-IL-1β and pro-IL-18 into their mature forms. Additionally, caspase-1 cleaves gasdermin D (GSDMD), resulting in the formation of membrane pores and cell death, a process known as pyroptosis. Importantly, GSDMD was detected in amniotic fluid and in chorioamniotic membranes of women with PTB and SIAI cases and correlated with increased protein expression of caspase-1 and IL-1β, suggesting inflammasome-mediated pyroptosis in the intra-amniotic space during SIAI. [000207] In the pursuit of potential SIAI therapies, rigorous studies have subjected approved anti-inflammatory drugs for use during pregnancy to scrutiny. Notably, corticosteroid
Docket No.173738.02730 23T075WO betamethasone and antibiotic clarithromycin, evaluated in an HMGB1-induced SIAI mouse model, not only exhibited effectiveness but demonstrated a compelling capacity to prevent PTB. Delving into the intricate pathways implicated in SIAI, the exploration of drugs inhibiting inflammasome activation has proven noteworthy. MCC950, for instance, exhibited promising outcomes in S100B-induced SIAI mouse models, underscoring its potential as a therapeutic candidate. However, it is imperative to note that the safety of MCC950 during pregnancy necessitates further rigorous investigation. Despite these strides, to date, there is no approved treatment for SIAI, emphasizing the urgent need for advancements in this critical area of reproductive health. [000208] Results [000209] miRNA and SINEs of C19MC and C2MC are expressed in human and mouse chorioamniotic membranes, respectively. [000210] Through in-situ hybridization (ISH) utilizing probes designed for miR-517a/b, Alu RNA transcripts, or a scrambled control on term chorioamniotic membrane sections, we found that both miR-517a/b and Alu RNA were unmistakably expressed solely in the trophoblast cell layer of the chorioamniotic membrane (FIG. 12A). Further confirmation of these findings involved assessing the expression of C2MC miRNA in mouse chorioamniotic membranes from both wild-type (WT) and C2MC
Δ/Δ mice through RT-qPCR. Our results indicated exclusive expression of miR-467a, a C2MC member (FIG. 12B), and B1 RNA (FIG. 12C) in the WT specimens. These results establish the expression of the C19MC and C2MC miRNAs and SINE RNA in human and mouse chorioamniotic membranes, respectively. [000211] Expression levels of C19MC miRNAs and Alu dsRNA are elevated in human chorioamniotic membranes from women diagnosed with SIAI and PTB [000212] Given that C19MC Alu and C2MC B1 SINEs form dsRNA capable of initiating a robust viral mimicry immune response even in the absence of an actual infection, our primary objective is to investigate their potential involvement in inducing SIAI within the chorioamniotic membranes, consequently triggering PTB. [000213] RT-qPCR evaluation of the expression levels of C19MC miRNA and Alu RNA in human chorioamniotic membranes isolated from term (n=3) and PTB (n=3) cases unveiled a significant increase in the expression levels of four randomly selected C19MC miRNAs in PTB
Docket No.173738.02730 23T075WO specimens compared to term cases (FIG.13A). Furthermore, in situ hybridization (ISH) for Alu RNA corroborated these findings, revealing a substantial upregulation of Alu RNA expression in chorioamniotic membranes isolated from PTB cases compared to those from term labor (FIGs. 13B and 13C). These results demonstrate the potential role of C19MC Alu RNA in the induction of SIAI and subsequent PTB and warrant further investigations. [000214] Example 5: Methods of detecting for measuring and visualizing Alu RNA [000215] RT-PCR [000216] Alu SINEs contain at least one internal RNA polymerase III promoter and under stressful conditions they are transcribed independently by pol III that produce short lived, full- length (fl-Alu) Alu transcript which are processed into a stable small cytoplasmic (sc-Alu) Alu RNA. [000217] To quantify the expression levels of Alu RNA, we performed competitive RT-PCR. This assay contains a limited amount of a primer set that recognizes both the fl-Alu and sc-Alu (5' CCGGGTGCGGTGGCACACGCT (SEQ ID NO: 2), and 5'-GCAATCTCCTTCTCACGGGTT (SEQ ID NO: 3)) and will amplify the most abundant form of Alu. The resulting RT-PCR products were analyzed by gel electrophoresis and the ratio of fl-Alu to sc-Alu was determined by densitometry. As a positive control, we used total RNA isolated from HeLa cells after heat shock recovery, which has been shown to increase fl-Alu. To exclude amplification of genomic Alu elements, we also performed Alu PCR using total RNA. [000218] Under steady state condition (HeLa at 37°C), most of the endogenous Alu RNA are processed into sc-Alu, thus the competitive Alu RT-PCR showed very low ratio of fl-Alu compared to sc-Alu (FIG.14). However, upon heat shock exposure which induce fl-Alu, most of the primers amplified the fl-Alu and thus increased the fl-Alu to sc-Alu ratio (FIG.14). To confirm that the RT-PCR products are derived from Alu RNA, the products were cloned and sequenced to find that all the clones aligned with Alu SINEs (Table 1). Table 1. Sequences and BLAT alignment of fl-Alu RT-PCR products in HeLa cells exposed to heat shock. Sequences and BLAT alignment (human GRCh38/hg38) of single colonies of fl-Alu RT-PCR products after gel purification and cloning into TOPO TA vectors obtained from HeLa cells subjected to heat shock (n=20). Each clone was sequenced in both directions using the T7 and T3 primers. Forward and reserve primers are indicated in single underline
Docket No.173738.02730 23T075WO (red) and double underline (blue),, respectively. Sequences that did not include EcoRI restriction sites or the PCR primer sequences are indicated as bad sequencing. “N” in the sequences is understood to be any nucleotide. Clone Se uencin with T3 rimer Se uencin with T7 rimer S an h 38/ an
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[000219] In Situ Hybridization [000220] To visualize Alu RNA in tissues, we performed in situ hybridization (ISH) of term human placental sections using LNA probe (5’CACTGCACTCCAGCCTG (SEQ ID NO: 40)) designed to recognize Alu RNA transcripts, or a scrambled control. To confirm that the Alu probe recognizes Alu RNA and not Alu elements in the genomic DNA, we pre-treated the sections with
Docket No.173738.02730 23T075WO either RNase A or DNase I. A strong Alu signal was observed in the STB layer, which was abolished when sections were pre-treated with RNase A and not when pre-treated with DNase I (FIG.15). [000221] Viral infections including SARS-CoV-2 have been reported to induce Alu RNA expression. We performed ISH of COVID-19 positive lungs and human coronary artery sections using the sane LNA Alu probe or a scrambled control. We found that Alu RNA are highly expressed in the COVID-19 positive lungs and coronary artery compared to uninfected donors (FIG.4A). [000222] Example 6: siRNA targeting Alu RNA induces IL1B expression [000223] RT-qPCR was performed to measure the expression of IL1B (normalized to GAPDH) in HUVECs (human umbilical vein endothelial cells) at passage 5, confirmed to be mycoplasma negative. These cells were initially plated in a 24-well plate at a density of 200,000 cells per well. After 24 hours, the cells underwent transfection with either control siRNA or Alu siRNA (15 pmol). The media was changed 4 hours post-siRNA transfection. Subsequently, after an additional 24 hours, the cells were treated with 100nM of chaetocin. Total RNA was then extracted 24 hours post-chaetocin treatment for the RT-qPCR analysis. [000224] Treatment of HUVECs with siRNA, including siRNA targeting Alu RNA, induce the expression of IL1B with and without chaetocin supplementation (FIG. 16). These results indicate that siRNA or Alu siRNA treatment is a potential medium for reversing the anti- inflammatory effects of Alu. [000225] References [000226] 1. Libby P, Ridker PM, Hansson GK, Leducq Transatlantic Network on A. Inflammation in atherosclerosis: from pathophysiology to practice. J Am Coll Cardiol. 2009;54(23):2129-38. doi: 10.1016/j.jacc.2009.09.009. PubMed PMID: 19942084; PMCID: PMC2834169.
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