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WO2024182813A2 - Ciblage d'arn alu pour traiter l'inflammation et des maladies et états pathologiques inflammatoires associés - Google Patents

Ciblage d'arn alu pour traiter l'inflammation et des maladies et états pathologiques inflammatoires associés Download PDF

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Publication number
WO2024182813A2
WO2024182813A2 PCT/US2024/018437 US2024018437W WO2024182813A2 WO 2024182813 A2 WO2024182813 A2 WO 2024182813A2 US 2024018437 W US2024018437 W US 2024018437W WO 2024182813 A2 WO2024182813 A2 WO 2024182813A2
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alu
rna
alu rna
expression
agent
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WO2024182813A3 (fr
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Hana Totary-Jain
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University of South Florida
University of South Florida St Petersburg
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University of South Florida
University of South Florida St Petersburg
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • Atherosclerosis and related conditions such as cardiovascular disease (CVD) such as atherosclerotic cardiovascular diseases
  • CVD cardiovascular disease
  • atherosclerotic cardiovascular diseases are considered a non-resolving chronic inflammatory disease, thus introducing a new era of therapeutic strategies that target inflammation.
  • CVD cardiovascular disease
  • 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.
  • the molecular mechanisms that induce sustained non-resolving inflammation in atherosclerosis are still not fully understood.
  • RT retrotransposon
  • 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.
  • 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.
  • 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.
  • the agent is an antisense oligonucleotide or siRNA that targets Alu RNA.
  • 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.
  • 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.
  • 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.
  • FIG. 1 shows representative images of de-identified human hearts.
  • A six-month old donor with Hib meningitis
  • B a 55-year-old donor with history of heart disease (9 infarcts, 3 stents and, CABG surgery)
  • FIG.2 shows IL-1 ⁇ expression in hCA increases with age and in the presence of CVD risk factors.
  • FIG.3 shows SARS-CoV-2 induces inflammasome pathway in human lungs and hCA.
  • FIG. 4 shows SARS-CoV-2 induces Alu RNA in human lungs and hCA.
  • 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.
  • Data represent the mean ⁇ SEM of a representative experiment of at least 2 independent experiments.
  • FIG.6 shows Alu RNA induce pro- and anti-inflammatory response.
  • FIG. 7 shows Alu RNA increased poly I:C induced inflammation.
  • 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.
  • FIG.9 shows a study protocol to assess the role of Alu RNA on atherosclerosis.
  • FIG. 10 shows targeting Alu RNA to reduce inflammation.
  • 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.
  • FIG. 1 Images were captured at 60x magnification. Scale bars represents 20 ⁇ m.
  • FIG. 1 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 ⁇ m; original magnification, ⁇ 20 and ⁇ 60 (insets). [00021] FIG.
  • 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.
  • 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: 50 ⁇ m; original magnification, x40.
  • FIG.16 shows IL1B expression in HUVECs can be inhibited with siRNA targeting Alu RNA.
  • ranges that are between two particular values should be understood to expressly include those two particular values.
  • “between 0 and 1” means “from 0 to 1” and expressly includes 0 and 1 and anything falling inside these values.
  • “about” means ⁇ 20% of the stated value, and includes more specifically values of ⁇ 10%, ⁇ 5%, ⁇ 2%, ⁇ 1%, and ⁇ 0.5% of the stated value.
  • 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.
  • the agent prevents, inhibits, or alleviates inflammation in the subject.
  • the agent inhibits Alu RNA.
  • 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”.
  • 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.
  • 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.
  • the condition is sterile intra-amniotic inflammation
  • the subject in need thereof is a pregnant female and/or fetus, at a higher risk of preterm birth.
  • pre-term birth pre-term labor
  • preterm birth pre-term labor
  • preterm birth preterm birth
  • preterm labor preterm labor
  • PTB preterm labor
  • PTL may be used interchangeably and refer to birth before about 37 weeks of gestation.
  • 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.
  • RNA Ribonucleic acid
  • 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.
  • Alu SINEs are specific to human and non-human primates.
  • 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).
  • condition or disease state for example, a subject with atherosclerosis
  • 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.
  • 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.
  • the disclosure describes the relationship between Alu expression and expression of inflammation-associated genes CASP1, IL1B, IL6, NLRP3, and TNF.
  • "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.
  • Elevated expression levels 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.
  • At least one refers to one or more.
  • 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.
  • Agent 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.
  • the agent is a small interfering RNA (siRNA) which may include but is not limited to siRNA that targets Alu RNA.
  • 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).
  • 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.
  • an antisense oligonucleotide may be able to block the function of Alu RNA.
  • 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.
  • ASO antisense oligonucleotide
  • Cytotoxic response refers to a reaction in the subject that results in the killing of cells.
  • Inflammatory response 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.
  • the present disclosure describes an agent, such as an antisense oligonucleotide (ASO), that is capable of inhibiting Alu RNA.
  • the agent inhibits Alu RNA.
  • the agent is an antisense oligonucleotide.
  • the antisense oligonucleotide comprises SEQ ID NO: 1.
  • Antisense oligonucleotide 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.
  • the agent is encapsulated in a nanoparticle.
  • 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.
  • Nanoparticle as used herein may refer to particles that are measured at the level of nanometers and is commonly known in the art.
  • 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.
  • 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.
  • infectious agent 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.
  • 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.
  • 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).
  • TEs transposable elements
  • RTs retrotransposons
  • SINEs short interspersed nucleic elements
  • Alu repeats Alu repeats
  • 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.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • 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
  • Shorter cytoplasmic Alu (sc-Alu) RNA is about 100 bp.
  • the Alu comprises an insertion or deletion mutation.
  • the Alu is about 100 base pairs (bp) to about 350 bp.
  • 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.
  • Embodiment include a composition comprising an antisense oligonucleotide that inhibits Alu.
  • the antisense oligonucleotide comprises SEQ ID NO: 1.
  • a composition comprising an antisense oligonucleotide that inhibits Alu may also include a carrier.
  • 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.
  • the carrier is a nanoparticle.
  • the antisense oligonucleotide is encapsulated in a nanoparticle.
  • the nanoparticle is endonuclease resistant.
  • the nanoparticle is less than about 200 nm in size.
  • the nanoparticle targets delivery of the antisense oligonucleotide to a tissue or organ, such as a heart.
  • 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.
  • 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 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.
  • nonaqueous solvents examples include 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.
  • 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).
  • 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).
  • 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.
  • polymeric compounds e.g., polylactic acid, polyglycolic acid, hydrogels, etc.
  • liposomes e.g., microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • lipophilic depots e.g., fatty acids, waxes, oils
  • 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.
  • 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.
  • These methods of measuring the level of the Alu RNA in a sample 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.
  • PCR polymerase chain reaction
  • RT-PCR reverse-transcription PCR
  • qPCR quantitative PCR
  • RT-qPCR quantitative PCR
  • Detecting Alu RNA may also be performed by techniques for visualizing Alu RNA in tissue, such as by in-situ hybridization.
  • 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.
  • 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.
  • 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.
  • ISH In situ hybridization
  • DNA or RNA viral nucleic acid
  • tissue sections or cytological specimens using labelled nucleic acid probes with complementary sequences to the target viral nucleic acid.
  • LNA locked nucleic acid
  • SEQ ID NO: 40 locked nucleic acid
  • 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.
  • 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.
  • an infectious agent selected from the group comprising Porphyromonas gingivalis, Helicobacter pylori, Cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, herpes simplex
  • the agent is an antisense oligonucleotide, and, in some aspects, the antisense oligonucleotide comprises SEQ ID NO: 1.
  • 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.
  • 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.
  • Embodiment 5 The method of any one of the preceding embodiments, wherein the disease or condition is associated with inflammation.
  • 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.
  • Embodiment 6 wherein the disease is atherosclerosis.
  • Embodiment 8. The method of embodiment 6, wherein the condition is sterile intra- amniotic inflammation.
  • 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
  • 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.
  • Embodiment 11 The method of any one of the preceding embodiments, wherein the agent inhibits production of inflammatory cytokines.
  • Embodiment 13 The method of embodiment 12, wherein the agent is an antisense oligonucleotide that targets Alu RNA.
  • Embodiment 14 The method of embodiment 13, wherein the antisense oligonucleotide comprises SEQ ID NO: 1.
  • Embodiment 15 The method of embodiment 12, wherein the agent is an siRNA that targets Alu RNA.
  • Embodiment 16 The method of embodiment 15, further comprising treating the subject in need thereof with chaetocin. [00083] Embodiment 17.
  • Embodiment 18 The method of embodiment 17, wherein the agent is in a composition comprising a carrier.
  • Embodiment 19 The method of embodiment 18, wherein the carrier is selected from one or more of nanoparticles, liposomes, viral vectors, cells.
  • 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.
  • Embodiment 21 The method of any one of the preceding embodiments, further comprising administering at least one anti-inflammatory agent.
  • 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.
  • Embodiment 23 The method of embodiment 22, wherein the infectious agent is selected from the group comprising pathogens, bacteria, viruses, fungi, protozoa, and helminths.
  • 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).
  • 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.
  • Embodiment 26 The method of any one of the preceding embodiments, wherein the Alu RNA is dsRNA.
  • Embodiment 27 The method of embodiment 26, wherein the agent is an antibody specific to Alu dsRNA.
  • Embodiment 28 The method of any one of the preceding embodiments, wherein the Alu RNA is about 100 bp to about 350 bp.
  • Embodiment 29 Embodiment 29.
  • 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.
  • Embodiment 31 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.
  • 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.
  • 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.
  • Embodiment 35 Embodiment 35.
  • 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.
  • 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.
  • Embodiment 37 The method of embodiment 36, wherein detecting Alu RNA is performed by in situ hybridization.
  • Embodiment 38 The method of embodiment 36, wherein the probe comprises SEQ ID NO: 40.
  • Atherosclerotic cardiovascular disease is the leading cause of death globally.
  • MI myocardial infarction
  • 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.
  • Atherosclerotic plaques 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 .
  • Viral infections such as SARS-CoV-2, accelerate atherosclerotic plaque progression and increase the incidence of myocardial infarction and strokes.
  • 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.
  • SINEs short interspersed nuclear elements
  • 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.
  • AluJ are the most ancient subfamilies
  • AluS represents the major burst of Alu elements
  • 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.
  • 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 ⁇ -thalassaemia 11 .
  • Alu SINE insertions continue to contribute to genome evolution providing functional elements such as promoters, enhancers, novel gene isoforms and splice variants.
  • functional elements such as promoters, enhancers, novel gene isoforms and splice variants.
  • 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 .
  • ANRIL INK4 locus
  • Alu RNA undergo post-transcriptional Docket No.173738.02730 23T075WO modification that converts adenosines to inosines by ADAR.
  • 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.
  • 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.
  • Alu repeats contain an internal RNA polymerase III promoter and can be transcribed independently.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus-2
  • MI acute respiratory syndrome coronavirus-2
  • KD Kawasaki disease
  • 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-1 ⁇ , 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.
  • 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.
  • 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.
  • Alu RNA is recognized by PRRs and their downstream signaling, which are conserved in mouse and human.
  • PRRs nuclear-binding receptors
  • 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.
  • 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).
  • 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).
  • fl-Alu full-length Alu RNA of ⁇ 300 bp
  • sc-Alu shorter cytoplasmic Alu RNA of ⁇ 100 bp
  • a histone methyltransferase inhibitor was used to release the epigenetic suppression of Alu SINEs and increase their RNA levels (FIG.5).
  • 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.
  • NLRP3 NOD [nucleotide oligomerization domain]-, LRR [leucine-rich repeat]-, and PYD [pyrin domain]-containing protein 3
  • DAMP danger-associated molecular pattern
  • Activated inflammasomes convert pro- caspase 1 into active caspase-1, which then cleaves pro-interleukin-1 ⁇ (IL-1 ⁇ ) 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-1 ⁇ is actively produced by inflammasomes in ECs, VSMCs, and macrophages in the atheroma, their activation driven by cholesterol crystals and disturbed flow.
  • IL-1 ⁇ 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 .
  • ICM-1 intercellular adhesion molecule-1
  • VCAM-1 vascular cell adhesion molecule-1
  • CCL-2 chemokines
  • IL-1 ⁇ 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 .
  • TNF tumor necrosis factor
  • IL6 interleukin-6
  • Alu SINEs 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.
  • 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 .
  • SUV39H1 SUV39 methyltransferases
  • H3K9 histone H3 on lysine 9
  • 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.
  • 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 .
  • inflammasome activation requires a priming signal to increase the expression of NLRP3/pro–IL-1 ⁇ and an activation signal, which promotes inflammasome assembly
  • Western blot will be performed to evaluate NLRP3-mediated pro–IL-1 ⁇ 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).
  • ATP adenosine triphosphate
  • Enzyme-linked immunosorbent assay (ELISA) analysis of IL-1 ⁇ , 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.
  • ELISA Enzyme-linked immunosorbent assay
  • 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.
  • oxLDL oxidized low-density lipoprotein
  • 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-1 ⁇ posttranslational processing and cytokine release as described above.
  • 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).
  • 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.
  • PBMCs Peripheral blood mononuclear cells
  • RNA may be determined by in- situ hybridization and RT-PCR in hCA and in PMBCs as shown in FIG.4.
  • RT-qPCR 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.
  • 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.
  • 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-1 ⁇ , IL-6, and TNF into the supernatant.
  • 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.
  • 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.
  • 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.
  • niRFP near-infrared fluorescent protein
  • FIG. 8B wire injury
  • ApoE -/- FIG. 8C
  • mouse models specifically targeted atherosclerotic plaques and endothelial denuded regions respectively, and not the liver, spleen, or kidneys 52 .
  • 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).
  • mice 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.
  • collagen may be detected by Masson's trichrome and sirius red staining, while elastin may be detected by Verhoeff staining.
  • hs-CRP high-sensitivity C-reactive protein
  • the levels of circulating IL-1 ⁇ 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.
  • RNA nanoparticles and poly I:C may exacerbate atherosclerotic plaques and increase infiltration of macrophages and the expression of inflammatory cytokines including IL-1 ⁇ , mimicking the conditions of acute infections in humans that accelerate disease progression and increase the risk of stroke and MI.
  • 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.
  • Example 3 Test the efficacy of novel anti-atherosclerotic therapies targeting Alu RNA.
  • 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-1 ⁇ 18,19 46 , targeting Alu RNA may hold great therapeutic potential.
  • ASO antisense oligo
  • ECs were transfected with ASO and after 2 hr, cells were treated with 1 ug/mL poly I:C and collected after 12 hours.
  • 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-1 ⁇ 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.
  • 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.
  • RNA may be extracted and the expression levels of NLRP3 inflammasome components and pro-inflammatory genes may be determined by RT-qPCR.
  • 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).
  • 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).
  • Cy5 labeled ASO may be used.
  • 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.
  • 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.
  • 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.
  • ANOVA analysis of variance
  • Example 4 Alu RNA associated with pre-term birth
  • PTB Preterm birth
  • PTB is a global health challenge, being the leading cause of neonatal mortality and associated with long-term physical, intellectual, and mental disabilities.
  • 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.
  • PTB Preterm birth
  • 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.
  • 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.
  • 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-1 ⁇ and pro-IL-18.
  • This priming process occurs through transcriptional regulation mediated by NF- ⁇ B in response to a primary 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-1 ⁇ and pro-IL-18 into their mature forms.
  • caspase-1 cleaves gasdermin D (GSDMD), resulting in the formation of membrane pores and cell death, a process known as pyroptosis.
  • GDMD gasdermin D
  • 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-1 ⁇ , suggesting inflammasome-mediated pyroptosis in the intra-amniotic space during SIAI.
  • rigorous studies have subjected approved anti-inflammatory drugs for use during pregnancy to scrutiny.
  • ISH in-situ hybridization
  • Example 5 Methods of detecting for measuring and visualizing Alu RNA
  • 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.
  • fl-Alu full- length Alu transcript which are processed into a stable small cytoplasmic Alu RNA.
  • sc-Alu small cytoplasmic
  • 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.
  • Example 6 siRNA targeting Alu RNA induces IL1B expression
  • 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.
  • Panning B Smiley JR. Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6. Mol Cell Biol. 1993;13(6):3231-44. Epub 1993/06/01. doi: 10.1128/mcb.13.6.3231. PubMed PMID: 7684492; PMCID: PMC359768. [000231] 6.
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Abstract

La divulgation concerne des agents, des compositions et des méthodes destinés à être utilisés pour prévenir, inhiber ou traiter une inflammation chez un sujet. Les méthodes comprennent l'utilisation de tels agents, des compositions pour prévenir, inhiber ou traiter des états pathologiques ou des maladies associés à une inflammation.
PCT/US2024/018437 2023-03-02 2024-03-04 Ciblage d'arn alu pour traiter l'inflammation et des maladies et états pathologiques inflammatoires associés Pending WO2024182813A2 (fr)

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US8809517B2 (en) * 2010-06-01 2014-08-19 University Of Kentucky Research Foundation Method of inhibiting Alu RNA and therapeutic uses thereof
US11219623B2 (en) * 2015-02-26 2022-01-11 University Of Kentucky Research Foundation Inflammasome inhibition for the treatment of Parkinson's disease, Alzheimer's disease and multiple sclerosis
US20210348164A1 (en) * 2018-10-09 2021-11-11 University Of Virginia Patent Foundation Endogenous cytoplasmic alu complementary dna in age-related macular degeneration
EP4114928A4 (fr) * 2020-03-05 2024-10-16 Flagship Pioneering Innovations VI, LLC Procédés de suppression de la défense de l'hôte et compositions pour la modulation d'un génome

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