Antisense oligonucleotide targeting MIR-34A-5P and method of use thereof

Abstract

This invention provides antisense oligonucleotides (ASOs) and compositions thereof. The ASOs provided herein are effective in regulating the activity or expression of miR-34a-5p, and / or enhancing the expression or activity of SIRT1, PGC-1α, and telomerase. Methods of using the ASOs are also provided herein, including methods for protecting tissues from senescence, mitochondrial dysfunction, and telomerase shortening in order to delay the aging process.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 522,377, filed on June 21, 2023, under 35 U.S.C. § 119(e). The disclosure of the prior application is considered a part of the disclosure of this application and is hereby incorporated by reference in its entirety.

[0002] Incorporation of Sequence Listing The accompanying sequence listing materials are hereby incorporated by reference into this application. The accompanying sequence listing xml file, named MDA1190 - 1WO_SL.xml, was created on May 30, 2024, and is 27 kb.

[0003] Field of the Invention The present invention generally relates to antisense oligonucleotides (ASOs), and more specifically to the use of ASOs targeting microRNAs for the treatment of aging - related diseases.

Background Art

[0004] Background Information The processes of cellular senescence and aging are known to be driven by complex metabolic networks that respond to damage such as environmental and endogenous stress factors. In one extreme of cellular senescence, cells completely stop proliferating and cannot grow. Mitochondrial activity decreases over time, affecting cells and tissues and thus the health of tissues. Damage to DNA due to radiation, stress, and translational errors particularly causes aging - related diseases. One of the symptoms of cellular senescence is the shortening of telomeres over time, which ultimately stops cell division. New and innovative therapeutic interventions are needed for the treatment of aging - related diseases.

[0005] Mitochondria play a crucial role in cellular health because they produce most of the adenosine triphosphate (ATP) used as the body's energy source. Many physiological pathways, including those related to cellular apoptosis and aging, interact with mitochondria. Mitochondrial-related diseases include skeletal muscle and motor disorders, metabolic disorders, cancer, neurodegenerative diseases, and aging. Pharmacological interventions targeting ion channels in mitochondria, the tricarboxylic acid cycle, and apoptotic proteins have beneficial effects. Many diseases associated with mitochondrial dysfunction have limited treatment options, and ongoing research aims to develop new therapies for these challenging conditions.

[0006] Mitochondrial biomass and activity are involved in diseases such as Duchenne muscular dystrophy, diabetes, Alzheimer's disease, and cancer. A decrease in mitochondrial biomass occurs before more obvious symptoms of the disease, such as muscle dysfunction, heart disease, or neurological problems. Restoring mitochondrial health is often achieved through the use of vitamins, minerals, resistance and strength training, physiotherapy, and other interventions.

[0007] DNA damage occurs throughout a cell's lifespan, but exposure to radiation and reactive oxygen species also causes DNA strand breaks. DNA damage is repaired by cellular pathways and enzymes that can patch or replace damaged base pairs or regions. DNA damage has long-term consequences if the body is unable to repair the damage and restore the function of the affected cells.

[0008] Cancer is one of the most studied diseases associated with DNA damage, but other diseases, including several neurodegenerative diseases, are also linked. Microcephaly, premature aging, and photosensitivity may occur in addition to immunodeficiency and growth retardation. Therapeutic interventions include the application of DNA damage inhibitors that slow apoptosis, or targeted enzymes that facilitate repair.

[0009] Damage to DNA resulting from normal cellular processes such as cell growth and division leads to telomere shortening. Telomere length is maintained by the activity of telomere enzymes, which lengthen shortened telomeres during cell replication. Mutations in genes related to telomere enzymes can cause diseases such as bone marrow failure, immunodeficiency, and lung disease. Currently, there is no cure for short telomeres, and therapeutic interventions rely on supportive interventions to maintain function. [Overview of the project]

[0010] This invention relates to antisense oligonucleotides (ASOs) and their use in methods for treating or slowing the progression of age-related diseases.

[0011] In one embodiment, the present invention relates to an antisense oligonucleotide (ASO) complementary to at least eight consecutive nucleotides of SEQ ID NO: 1. In one embodiment, the ASO is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In one embodiment, the ASO sequence is selected from SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0012] In one embodiment, ASO comprises at least one unnaturally occurring nucleotide. In one embodiment, the at least one unnaturally occurring nucleotide is a cytoskeletal modification, a nucleic acid base modification, a ribose modification, a 2'-ribose substitution, a locked nucleic acid, a morpholino oligonucleotide, a peptide nucleic acid, a phosphorothioate, a tricyclodeoxyribonucleic acid, 5-methylcytidine, 5-methyluridine, 5-ribothymidine, a debasalized nucleoside, a nucleotide lacking a 5'-terminal phosphate group, a nucleotide having a 5'-(E)-vinylphosphonate modification at the 5'-terminus, a terminally inverted debasalized ribonucleotide, a 3'-terminal nucleotide modified at the 2' position of a ribose sugar, a nucleotide having a 2'-O-methyl modification, a nucleotide having a 2'-O-methoxyethyl modification, a nucleotide having a 2'-fluoro modification, a morpholino oligonucleotide, a gapmer, an optionally selected uridine nucleotide, at least one thymine nucleotide, and any combination thereof. In one embodiment, at least one cytoskeletal modification of ASO is a phosphorothioate bond. In one embodiment, ASO is a morpholino oligonucleotide. In one embodiment, ASO is a gapmer. In one embodiment, at least one thymine nucleotide of ASO is optionally a uridine nucleotide. In one embodiment, ASO is covalently bonded to at least one non-nucleotide moiety.

[0013] In one embodiment, the present invention relates to a pharmaceutical composition comprising at least one antisense oligonucleotide (ASO) as described herein and a pharmaceutically acceptable carrier.

[0014] In one embodiment, the present invention relates to a method for treating or delaying the progression of an aging-related disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an ASO described herein, or at least one ASO, and a pharmaceutically acceptable carrier.

[0015] In one embodiment, the ASO or pharmaceutical composition inhibits or reduces the expression of miR-34a-5p.

[0016] In one embodiment, the ASO or pharmaceutical composition enhances or increases the level of SIRT1 activity. In another embodiment, the ASO or pharmaceutical composition enhances or increases the level of PGC-1α activity. In another embodiment, the ASO or pharmaceutical composition enhances or increases the level of telomerase activity or expression.

[0017] In one embodiment, age-related diseases include immunosenility, vascular senility, musculoskeletal dysfunction, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, type II diabetes, radiation-induced fibrosis, aging induced by radiation exposure from space, radiation-induced pulmonary fibrosis, mitochondrial dysfunction, or telomerase shortening. In one embodiment, age-related diseases include pulmonary fibrosis. In one embodiment, pulmonary fibrosis is induced by radiation, mitochondrial dysfunction, or telomerase shortening. [Brief explanation of the drawing]

[0018] The drawings of this disclosure provide exemplary examples of the present invention, and the whole thereof is incorporated herein by reference.

[0019] [Figure 1A] Images of adenocarcinomatous human alveolar basal epithelial cells (A549) reverse-transfected with premicroRNA miR-34a or scrambled oligonucleotide (scr) as a control, and stained with MitoTracker after 48 hours are shown. [Figure 1B] The graph shows the mitochondrial DNA-to-nuclear DNA ratio (mtDNA / nDNA) measured in A549 and H1299 cells transfected with either synthetic miR-34a-5p double-stranded DNA (miR-34a mimetic) or, as a control, scrambled siRNA oligonucleotides. [Figure 1C] The image shows PGC-1α protein extracted from A549 and H1299 cells after transfection with a synthetic mimic of miR-34a-5p. [Figure 1D]Graph showing adenosine triphosphate (ATP) detected in cells transfected with either scrambled oligonucleotides or synthetic miR-34a duplex (mimic of miR-34a-5p). [Figure 2A] Exemplary schematic showing the relative abundance of DNA damage and repair genes regulated by miR-34a-5p. Red cells indicate high gene expression and blue cells indicate low gene expression. [Figure 2B] Graph showing the mRNA expression levels of genes RAD51, CHEK1, CHEK2, PRKDC, USP1, ATF1, BARD1, PIAS1, PML, DDB2, BLM, and CCNA2. [Figure 2C] Graph showing the PGC1 protein concentration in A549, H1299, and H1944 cells transfected with either scrambled oligonucleotides or pre-microRNA miR-34a. [Figure 2D] Images showing PGC-1α protein isolated from reverse-transfected A549, H1299, and H1944 cells transfected with either scrambled oligonucleotides or mimics of miR-34a-5p. [Figure 3A] Images showing SA-beta-Gal activity in A549 cells reverse-transfected with either scrambled oligonucleotides or pre-microRNA miR-34a. [Figure 3B] Set of images showing SA-beta-Gal activity in H1299 cells reverse-transfected with either pre-microRNA miR-34a or scrambled oligonucleotides. [Figure 3C] Images of Western blots of SIRT1, p21, and actin expression from human lung fibroblasts after treatment with antisense oligonucleotides. [Figure 3D] Images of Western blots of hTERT and actin expression after treatment with antisense oligonucleotides. [Figure 4A] A graph showing an antisense oligonucleotide targeting miR-34a-5p in mouse cells is shown. [Figure 4B] It is a graph showing an antisense oligonucleotide targeting miR-34a-5p in human cells. [Figure 5] An image of a Western blot of p21 expression from human lung fibroblasts after treatment with an antisense oligonucleotide targeting miR34a is shown. [Figure 6] An image of a Western blot of SIRT1 expression from human lung fibroblasts after treatment with an antisense oligonucleotide targeting miR34a is shown. [Figure 7] A graph showing the average mitochondrial DNA copy number of lung fibroblasts incubated with an antisense oligonucleotide (ASO) and treated with 4Gy XRT is shown. [Figure 8A] An exemplary schematic diagram of the treatment of young and old mice treated with an antisense oligonucleotide targeting miR-34a is shown. [Figure 8B] It is a graph showing SIRT1, PGC1-α, and telomerase expression in young and old mice treated with an antisense oligonucleotide targeting miR-34a. [Figure 8C] It is a graph showing the protein expression of p16, p21, telomerase PGC-1α, SIRT1, IL1b, and TNFα in mice treated with an antisense oligonucleotide targeting miR-34a. [Figure 9A] A graph showing the decrease in senescence marker p16 in mice treated with ASO SEQ ID NO: 3 is shown. [Figure 9B] A graph showing the decrease in senescence marker p21 in mice treated with ASO SEQ ID NO: 3 is shown.

Mode for Carrying Out the Invention

[0020] Detailed Description of the Invention This disclosure is based on the finding that inhibition of miR-34a-5p expression by antisense oligonucleotides (ASOs) correlates with increased expression of genes associated with DNA damage and repair in certain cancer cells. Based on this groundbreaking finding, it is understood that inhibition of miR-34a-5p may be associated with treating or slowing age-related diseases.

[0021] The need to slow down the aging process and protect specific tissues from senescence, mitochondrial dysfunction, and telomerase shortening remains unmet. Currently, anti-aging companies focus on only one of these areas, while the compounds and methods presented herein can simultaneously target different areas through extensive regulation of miRNAs in many biological processes, including the aging pathway.

[0022] The ASOs described herein can be used to prevent miR-34a-induced senility and pulmonary fibrosis, which are induced by radiation, mitochondrial dysfunction, and telomerase shortening, in immune cells, hepatocytes, vascular systems, the brain, and in diabetic patients.

[0023] The present invention is not limited to the specific compositions, methods, and experimental conditions described, and such compositions, methods, and conditions may vary. In addition, the technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, as the scope is limited only by the appended claims.

[0024] Unless otherwise defined, all other scientific and technical terms used herein, in the drawings and claims have their ordinary meanings as generally understood by those skilled in the art. Similar or equivalent methods and materials may be used in the implementation or testing of the nucleic acids, host cells, compositions, methods and uses disclosed herein, but preferred methods and materials are listed below. The materials, methods and examples presented herein are illustrative and not intended to be limiting.

[0025] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference.

[0026] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Therefore, it will be obvious to a person skilled in the art reading this disclosure that, for example, “the method” includes one or more methods and / or steps of the type described herein.

[0027] Where used herein, the term “approximately” accompanying a number means that it includes any additional number that is reasonably close to the given number. For example, depending on the context, the value may be 5-10% higher or lower. For instance, a value of approximately 100 means 90-110 (or any value between 90 and 110).

[0028] Terms such as “comprising,” “including,” “containing,” and “having” should be read expansively, unrestricted, and non-restrictively. Singular forms such as “a,” “an,” or “the” include plural references unless the context explicitly indicates otherwise. Thus, for example, a reference to “antisense oligonucleotide” includes any single antisense oligonucleotide of the same, e.g., the same sequence, or different, and multiple antisense oligonucleotides. Similarly, a reference to “cell” includes a single cell and multiple cells. Unless otherwise specified, the term “at least” preceding a set of elements should be understood to refer to all elements in the set. The terms “at least one” and “at least one of ~” include, for example, one, two, three, four, or five or more elements. Furthermore, it is understood that slight variations above and below the stated range can achieve substantially the same results as values ​​within the range. Also, unless otherwise specified, a range disclosure is intended as a continuous range including all values ​​between the minimum and maximum values.

[0029] In one embodiment, the present invention provides an antisense oligonucleotide (ASO) complementary to at least about eight consecutive nucleotides of SEQ ID NO: 1.

[0030] The terms “antisense oligonucleotide” and “antisense oligo” are used interchangeably and refer to compounds having a sequence of nucleotide bases and a subunit-subunit skeleton that allows the antisense oligomer to hybridize to a target nucleic acid molecule via hydrogen bonding. Antisense compounds are typically single-stranded nucleic acid molecules with a length of about 6 to 30, for example, about 15 to 30 nucleic acid bases. In one embodiment, the antisense compound may be a microRNA or mimetic RNA. In one embodiment, the antisense compound has a nucleic acid base sequence that, when written in the 5' to 3' direction, contains the reverse complement of the target segment of the target nucleic acid molecule it is targeted at.

[0031] As used herein, the terms “nucleic acid” or “oligonucleotide” refer to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinant and chemically synthesized molecules such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, conjugated nucleic acids, and oligonucleotides. According to the present invention, nucleic acids may exist as single-stranded or double-stranded molecules and linearly or covalently cyclically closed molecules. Nucleic acids can be isolated. The term “isolated nucleic acid” means that the nucleic acid has been (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) produced by recombinant cloning, (iii) purified, for example by separation by cleavage and gel electrophoresis, (iv) synthesized, for example by chemical synthesis, or (vi) extracted from a sample. The nucleus, in particular in the form of RNA which can be prepared by in vitro transcription from a DNA template, can be used for introduction into cells, i.e., cell transfection. Furthermore, the RNA can be modified before application by stabilizing the sequence, capping, and polyadenylation.

[0032] RNA types include messenger RNA (mRNA), microRNA (miRNA), and small interfering RNA (siRNA). This disclosure relates to miRNA. miRNA production begins in the nucleus with the transcription of primary microRNA from DNA. First, a strand of primary microRNA with a stem-loop and tail structure is produced. In the nucleus, the primary microRNA is processed into premicroRNA with a stem-loop structure. The premicroRNA is exported from the nucleus and further processed in the cytoplasm to produce double-stranded microRNA without the loop associated with the premicroRNA. When the double-stranded microRNA is unwound, single-stranded microRNA (miRNA) plays a role in regulating post-transcriptional gene expression. The strand of miRNA is very short, only 10 to 30 base pairs or nucleotides long. The structure of miRNA allows it to hybridize with RNA or DNA at specific sites, which can slow or halt protein expression with countless downstream effects on physiological pathways.

[0033] miRNA mimetics, such as miR-34a or miR-34a-5p mimes, are synthetic miR-34a double-stranded molecules. Anti-miRNAs, such as anti-miR-34a or anti-miR-34a-5p, are oligonucleotides complementary to the miR-34a sequence. Antisense oligonucleotides, which are miRNA mimetics, are double-stranded miRNA molecules with a stem-loop structure.

[0034] The terms “expression” and “expression” in relation to polypeptides are intended to be understood in the ordinary sense used in the art. Polypeptides are expressed by cells via transcription of nucleic acids into mRNA, which is then translated into initial polypeptides, which are folded and, in some cases, further processed into mature polypeptides. Thus, a statement that a cell or organism expresses such polypeptides indicates that the polypeptides are found intracellularly or on the cell, and in the cells of the organism, respectively, and that the polypeptides were synthesized by the expression mechanisms of the respective cells.

[0035] In relation to each biological process itself, the terms “expression,” “gene expression,” or “expression” refer to the entire regulatory pathway that translates the information encoded in the nucleic acid sequence of a gene first into messenger RNA (mRNA) and then into polypeptides. Thus, gene expression includes transcription into primary preRNA, processing of this preRNA into mature RNA, and translation of the mRNA sequence into the corresponding amino acid sequence of polypeptides. In this context, it should be noted that the term “gene product” refers not only to the mature polypeptide encoded by that gene (including its splice variants) and, where applicable, the polypeptide containing its respective precursor protein, but also to each mRNA that may be considered the “first gene product” during the process of gene expression.

[0036] In certain embodiments, the antisense oligomers and antisense oligomer conjugates described herein may contain basic functional groups such as amino or alkylamino compounds, and thus can form pharmaceutically acceptable acids and pharmaceutically acceptable salts. In this regard, the term “pharmaceutically acceptable salt” refers to inorganic and organic acid addition salts of the antisense oligomers or antisense oligomer conjugates of this disclosure.

[0037] The antisense compounds disclosed herein are specifically hybridizable to the indicated target sequences. Compounds such as the antisense compounds disclosed herein are specifically hybridizable if they have sufficient complementarity to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under the conditions under which the assay is performed in the case of in vitro assays.

[0038] In one embodiment, ASO is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In one embodiment, ASO is selected from SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0039] As used herein, the term “homology” refers to the degree of sequence identity, which can be determined using any computer program known in the art, such as BLAST 2.2.2 or FASTA version 3.0t78, and associated parameters, for example, with default parameters. Protein and / or nucleic acid sequence identity (homology) can be assessed using any of the various sequence comparison algorithms and programs known in the art. The terms “essentially identical” or “substantially identical” refer to sequences that have at least 85% sequence identity with the sequence being referred to. For example, the sequence of a nucleic acid molecule is substantially identical to a given nucleic acid sequence if it has 90% or more sequence identity with that sequence. As a further example, the sequence of a peptide or protein is substantially identical to a given amino acid sequence if it has 90% or more sequence identity with that sequence. A sequence that is essentially identical or substantially identical to another sequence may also be a sequence that has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence referred to.

[0040] The terms “sequence identity” and “identity percentage” are used interchangeably herein. To determine the identity percentage of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first polypeptide or polynucleotide for optimal alignment with the second polypeptide or polynucleotide sequence). The amino acids or nucleotides are then compared at their corresponding amino acid or nucleotide positions. If a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The identity percentage between two sequences is a function of the number of identical positions shared by the sequences (i.e., identity % = number of identical positions / total number of positions (i.e., overlapping positions) × 100%). In some embodiments, the length of the reference sequence aligned for comparison purposes (e.g., SEQ ID NOs. 1–4) is at least 80% of the length of the comparison sequence, and in some embodiments, it is at least 90% or 100%. In embodiments, the two sequences are of the same length.

[0041] The desired range of sequence identity is approximately 80% to 100%, and is an integer value in between. The percentage of identity between the disclosed sequence and the claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exact match exhibits 100% identity over the length of the reference sequence (e.g., sequence numbers 1-4).

[0042] Polynucleotides that are approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or more identical to the polynucleotides described herein are materialized within this disclosure. For example, polynucleotides can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% or more identity with SEQ ID NOs: 1-4.

[0043] The term “isolated” indicates that a cell or group of cells or a peptide(or nucleic acid))(or nucleic acid)(or nucleic acid)(or nucleic acid))(or nucleic acid)(or nucleic acid)(or nucleic acid))(or nucleic acid)(or nucleic acid))(or nucleic acid)(or nucleic acid))(or nucleic acid))(or nucleic acid)))(or nucleic acid)))(or nucleic acid)))(or nucleic acid)))(or nucleic acid)))(or nucleic acid))))))))))))))) or()( or nucleic acid)))))))( or nucleic acid)( or nucleic acid)( or nucleic acid)(e.g., 2 or more amino acids( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides) are bound together( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides)( or nucleotides) are more likely to be isolated from natural sources or synthesized. The term "isolated" does not mean that the sequence is the only amino acid or nucleotide chain in existence, but that it is essentially free from naturally associated substances, such as non-amino acid substances and / or non-nucleic acid substances, and is, for example, more than 90-95% pure.

[0044] The present invention generally relates to the field of aging. In particular, the present invention relates to the treatment of aging-related diseases, including immunosenility, vascular senility, musculoskeletal dysfunction, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, type II diabetes mellitus, and radiation-induced fibrosis, as well as aging caused by radiation exposure (space travel), by administering agents that modulate the activity or expression of microRNAs (miRNAs) by inhibiting the expression or activity of miR-34a-5p and / or enhancing the expression or activity of SIRT1, PGC-1α, and telomerase.

[0045] Examples of ASOs identified herein include those listed in Table 1, including SEQ ID NOs: 2, 3, and 4. ASOs may contain modified nucleotides that are known in the art or that can be used to enhance the benefits of the ASOs referred to herein. ASOs can be conjugated to portions such as lipids, peptides, aptamers, antibodies, and sugars.

[0046] [Table 1] Legend: * = phosphorothioate bond +=Affinity Plus (locked nucleic acid bases)

[0047] In one embodiment, the ASO contains at least one unnaturally occurring nucleotide.

[0048] Many unnaturally occurring nucleotides are known and can be used in the methods described herein. Unnaturally occurring nucleotides include, for example, nucleotides containing modifications to the ribose sugar, nucleic acid base, or nucleotide backbone. For example, the in vivo stability of RNA can be improved by substituting the 2'-ribose sugar with a 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl residue. The ribose sugar of a nucleotide can be modified in several ways, including but not limited to the addition of substituents and the cross-linking of the 2' and 4' carbon atoms to form locked nucleic acids (LNA, nucleic acids with 2'-O, 4'-C methylene cross-links), bicyclic nucleic acids (BNA), or 2'-O, 4'-C-ethylene cross-linked nucleic acids (ENA). Modifications at the nucleic acid base may include modifications of adenine, thiamine, cytosine, guanine, or uracil, including pyrimidine methylation such as 5-methylcytidine and 5-methyluridine / ribothymidine. Nucleic acid base modifications include debasalized nucleotides, which are nucleotides lacking a nucleic acid base. Skeletal modifications include phosphorothioate or boranophosphate bonds, which may include additional sulfur atoms to introduce stereochemistry. Non-native nucleotides can be substantially different from native nucleotides. For example, phosphorodiamidoic acid morpholino oligonucleotides (PMOs) can be incorporated into ASOs, and peptide nucleic acids (PNAs) can be used to assist RNA delivery. TricycloDNA (tcDNA) can also be used as a substitute for native nucleotides in ASOs.

[0049] A "nucleic acid base" refers to a heterocyclic portion that can pair with a base from another nucleic acid molecule. The term "sequential nucleotide" refers to nucleic acid bases that are directly adjacent to each other.

[0050] In one embodiment, at least one unnaturally occurring nucleotide of ASO is selected from the group consisting of skeletal modifications, nucleic acid base modifications, ribose modifications, 2'-ribose substitutions, locked nucleic acids (bases), morpholinonucleotides, peptide nucleic acids, phosphorothioates, tricyclodeoxyribonucleic acids, 5-methylcytidine, 5-methyluridine, 5-ribothymidine, debasalized nucleosides, nucleotides lacking a 5'-terminal phosphate group, nucleotides having a 5'-vinylphosphonate modification at the 5' end, terminally inverted debasalized ribonucleotides, nucleotides with a 3' terminal modified at the 2' position of a ribose sugar, nucleotides having a 2'-O-methyl modification, nucleotides having a 2'-O-methoxyethyl modification, nucleotides having a 2'-fluoro modification, and any combination thereof.

[0051] In one embodiment, at least one of the ASO's skeletons is a phosphorothioate bond.

[0052] In one embodiment, ASO is morpholino.

[0053] The term "morpholino" refers to a single-stranded nucleic acid analog consisting of 15 to 30 bases, typically around 25 bases. In morpholino oligomers, the bases are covalently bonded to a methylmorpholine ring skeleton linked via phosphorus-containing groups, typically phosphorodiamidate groups.

[0054] In one aspect, ASO is a gapmer.

[0055] As used herein, the term “gapmer” refers to a short DNA antisense oligonucleotide structure having RNA-like segments on both sides of the sequence. These linear fragments of genetic information are designed to hybridize to a target fragment of RNA and silence the gene by inducing RNase H cleavage. Gapmer binding to its target has higher affinity due to the modified RNA facies region, as well as resistance to degradation by nucleases. A gapmer consists of a short DNA strand facies a strand of RNA mimetic. The mimetic is typically composed of locked nucleic acid (LNA), 2'-OMe, or 2'-F modified bases. The LNA sequence is an RNA analog “fixed” to an ideal Watson-Crick base pair. Gapmers often utilize nucleotides modified with phosphorothioate (PS) groups. The mechanism of therapeutic gene silencing relies on degradation by the action of RNase H. Almost all organisms utilize this family of enzymes to degrade DNA-RNA hybrids as a defense against viral infection. In protein synthesis, DNA is first transcribed into mRNA, and then translated into an amino acid sequence. Gapmers utilize this biological pathway by binding to mRNA targets. In humans, gapmer DNA-mRNA double strands are degraded by RNase H1. mRNA degradation interferes with protein synthesis.

[0056] In one embodiment, at least one thymine nucleotide of the ASO is optionally a uridine nucleotide.

[0057] In one embodiment, the ASO is covalently bonded to at least one non-nucleotide portion.

[0058] In one embodiment, the present invention relates to a pharmaceutical composition comprising at least one antisense oligonucleotide (ASO) as described herein and a pharmaceutically acceptable carrier.

[0059] As used herein, “pharmaceutical composition” refers to a formulation comprising an active ingredient and, optionally, a pharmaceutically acceptable carrier, diluent, or excipient. The term “active ingredient” may interchangeably refer to “active ingredient” and means any agent capable of inducing the desired effect upon administration. In one embodiment, the active ingredient comprises a biologically active molecule. As used herein, the phrase “biologically active molecule” refers to a molecule that has a biological effect within a cell. In certain embodiments, the active molecule may be an inorganic molecule, an organic molecule, a small organic molecule, a drug compound, a polypeptide such as a peptide, an enzyme, or a transcription factor, an antibody, an antibody fragment, a peptide mimetic, a lipid, a nucleic acid such as DNA or RNA molecules, a ribozyme, a hairpin RNA, siRNA (small interfering RNA) of various chemicals, a miRNA, an siRNA-protein conjugate, an siRNA-peptide conjugate, and an siRNA-antibody conjugate, antagomyl, PNA (peptide nucleic acid), LNA (locked nucleic acid), or morpholino. In certain exemplary embodiments, the activator is an ASO described herein.

[0060] "Pharmacologically acceptable" means that a carrier, diluent, or excipient must be compatible with the other components of the formulation and must not be detrimental to its recipient or to the activity of the active ingredient of the formulation. Pharmaceutically acceptable carriers, excipients, or stabilizers are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmacochemically acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used, and these include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin This may include proteins such as methyl esters, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.Examples of excipients include, but are not limited to, anti-adhesive agents such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as gelatin and synthetic polymers, lubricants such as talc and silica, and antioxidants, vitamins A, E, C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil, and dimethyl sulfoxide (DMSO).

[0061] Pharmaceutical compositions containing ASOs as identified herein can be formulated as powders, liquids, solutions, or suspensions. Formulations may include drenches, tablets, boluses, granules, creams, ointments, or sprays. Possible routes of administration include topical, ocular, sublingual, nasal, intrathecal, intravenous, intravitreous, oral, or subcutaneous.

[0062] ASOs can also be delivered with pharmaceutically acceptable excipients and in combination with other pharmaceutically active compounds such as steroids or antihistamines.

[0063] Suitable pharmaceutical carriers for use in ASO include, in particular, sugars, starches, cellulose, oils, waxes, agars, buffers, coatings, preservatives, and antioxidants.

[0064] ASO or pharmaceutically acceptable salts of ASO may be formulated with pharmaceutically acceptable excipients as pharmaceutical compositions for therapeutic use.

[0065] The pharmaceutical compositions described herein can be administered in doses of approximately 0.1–20 mg / kg, approximately 1–10 mg / kg, approximately 1–5 mg / kg, and within ranges and values ​​between the recommended ranges. For example, the dose may be 10 mg / kg. For example, the dose may be administered daily, every other day, weekly, or monthly.

[0066] In one embodiment, the present invention relates to a method for treating or delaying the progression of an aging-related disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an ASO as described herein or a pharmaceutical composition comprising an ASO as described herein.

[0067] As used herein, the term “subject” refers to any individual or patient on whom the method of the present invention is performed. Generally, the subject is human, as will be understood to those skilled in the art, but the subject may be a non-human animal. As used herein, the terms “subject,” “patient,” or “subjects” refer to humans or other mammals, including ungulates, rodents, or primates, such as horses, cattle, sheep, pigs, goats, llamas, camels, dogs, cats, birds, ferrets, rabbits, squirrels, mice, opossums, lemurs, or rats. In one embodiment, the subject is a human subject.

[0068] The term "effective dose" refers to the amount of compound, composition, or formulation that is sufficient to treat a condition or disease, to produce a desired effect or outcome, or to reduce, alleviate, or stop the symptoms of a condition or disease. "Effective dose" is used interchangeably with the term "therapeutic effective dose."

[0069] As used herein, the term “treatment” means an approach or regimen designed to improve or alleviate the symptoms of a disease, illness, or debilitation. Treatment may lead to pain reduction or an improvement in quality of life. As used herein, the terms “treatment” and “treating” mean preventive or deterrent measures that have a therapeutic effect and slow down, or at least partially alleviate or inhibit, an undesirable condition in the organism in question. Conditions requiring treatment include those that already have a condition, those that are prone to having a condition, or those that should be prevented (prevented). Generally, treatment reduces, stabilizes, or inhibits the progression of symptoms associated with the presence and / or progression of an undesirable condition.

[0070] As used herein, the term “beneficial” means improvement of the symptoms of a disease, reduction of adverse symptoms, improvement of clinical test results, reduction of pain, improvement of quality of life, or other desirable outcome.

[0071] The term "therapeutic effect" refers to the inhibition or activation of factors that cause or contribute to an undesirable condition. A therapeutic effect alleviates, to some extent, one or more symptoms of an undesirable condition or disease. The term "undesirable condition" refers to a function in the cells or tissues of an organism that deviates from the optimal function of that organism. Abnormal conditions can, among other things, relate to cell proliferation, cell differentiation, or cell survival.

[0072] The terms “administering of ~” and / or “administering” should be understood to mean providing a therapeutically effective amount of the pharmaceutical composition to a subject in need of treatment. The route of administration may be intestinal, topical, or parenteral. Therefore, routes of administration, though not limited to these, include, but are: intradermal, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal; oral, sublingual, buccal, rectal, vaginal, nasal, and ocular administration; as well as infusion, inhalation, and spraying. As used herein, the phrases “parenteral administration” and “administered parenterally” mean modes of administration other than intestinal and topical administration. As used herein, the term “administer” refers to any method by which treatment is given to a patient or subject. “Administer” refers to any method of transferring, delivering, introducing, or transporting a substance to a subject, such as a compound, for example, a pharmaceutical compound, or other drug such as an antisense oligonucleotide. Administration can be achieved by local, intravenous, intramuscular, systemic, oral, or parenteral methods.

[0073] ASOs can be delivered through various methods, including chemical modification, covalent attachment to a portion that improves cell delivery, or delivery in nanoparticles. ASOs can also be delivered to target tissues using exosome loading, globular nucleic acids, nanotechnology, lipid nanoparticles, DNA cages, or other delivery methods.

[0074] In one embodiment described herein, the compound or formulation is administered by parenteral or by various routes including systemic, oral, topical, intravenous, intramuscular, subcutaneous, or intratumor routes.

[0075] In one embodiment, the ASO or pharmaceutical composition enhances or increases the level of SIRT1 activity.

[0076] SIRT1 encodes an enzyme involved in DNA repair, such as recombination repair of DNA damage.

[0077] Sirtuin 1 (SIRT1), also known as NAD-dependent deacetylase sirtuin-1, is a protein encoded by the SIRT1 gene in humans. SIRT1 is an abbreviation for Sirtuin 1 (Silent Mating Information Regulation 2 homolog). SIRT1 is an enzyme mainly located in the cell nucleus that deacetylates transcription factors contributing to cell regulation (response to stressors, longevity). Sirtuin 1 is a member of the sirtuin family of proteins, which are homologs of the Sir2 gene in S. cerevisiae. Members of the sirtuin family are characterized by their sirtuin core domain and are classified into four classes. Although the function of human sirtuins has not yet been determined, yeast sirtuin proteins are known to regulate epigenetic gene silencing and suppress rDNA recombination. The protein encoded by this gene belongs to class I of the sirtuin family. Sirtuin 1 is downregulated in cells with high insulin resistance. Furthermore, SIRT1 has been shown to deacetylate and affect the activity of both members of the PGC1-alpha / ERR-alpha complex, an essential metabolic regulatory transcription factor. In vitro, SIRT1 has been shown to deacetylate and thereby inactivate the p53 protein, potentially playing a role in activating T helper 17 cells.

[0078] In one embodiment, the ASO or pharmaceutical composition enhances or increases the level of PGC-1α activity.

[0079] PGC-1α is a transcriptional coactivator crucial for mitochondrial health and involved in energy metabolism. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a protein encoded in humans by the PPARGC1A gene. PPARGC1A is also known as human accelerating region 20 (HAR20). PGC-1α is a major regulator of mitochondrial biogenesis. PGC-1α is also a major regulator of hepatic gluconeogenesis, inducing increased gene expression for gluconeogenesis. PGC-1α is a gene containing two promoters and four alternative splicings. PGC-1α is a transcriptional coactivator that regulates genes involved in energy metabolism. It is a major regulator of mitochondrial biogenesis. This protein interacts with the nuclear receptor PPAR-γ, enabling interaction between this protein and multiple transcription factors. PGC-1α provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis and is a major factor in causing slow-twitch muscle fibers rather than fast-twitch muscle fibers.

[0080] In one embodiment, the ASO or pharmaceutical composition enhances or increases the level of telomerase activity.

[0081] Telomerase, also known as terminal transferase, is a ribonucleoprotein that adds species-dependent telomere repeat sequences to the 3' end of telomeres. Telomeres are regions of repeat sequences at each end of chromosomes in most eukaryotes. Telomeres protect the ends of chromosomes from DNA damage or fusion with adjacent chromosomes. Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule (for example, the sequence 3'-CCCAAUCCC-5' in Trypanosoma brucei), which is used as a template when elongating telomeres. Telomerase is active in gametes and most cancer cells, but is normally absent in most somatic cells.

[0082] In one embodiment, age-related diseases include immunosenility, vascular senility, musculoskeletal dysfunction, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, type II diabetes, radiation-induced fibrosis, aging induced by radiation exposure from being in space, radiation-induced pulmonary fibrosis, mitochondrial dysfunction, or telomerase shortening.

[0083] In one aspect, age-related diseases include pulmonary fibrosis.

[0084] Pulmonary fibrosis, or lung fibrosis, is a condition in which the lungs scar over time. Symptoms include shortness of breath, dry cough, fatigue, weight loss, and watch-eye nails. Complications may include pulmonary hypertension, respiratory failure, pneumothorax, and lung cancer. Causes include environmental pollution, certain drugs, connective tissue diseases, infections, and interstitial lung diseases. However, in most cases, the cause is unknown and is called idiopathic pulmonary fibrosis. Diagnosis can be made based on symptoms, medical imaging, lung biopsy, and pulmonary function tests. There is no cure, and the available treatment options are limited. Treatment is directed towards efforts to improve symptoms and may include oxygen therapy and lung rehabilitation. Certain drugs may be used to slow the progression of scarring. Occasionally, lung transplantation may be an option. Life expectancy is generally less than 5 years. Methods provided herein may extend life expectancy.

[0085] In one embodiment, pulmonary fibrosis is induced by radiation, mitochondrial dysfunction, or telomerase shortening.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but it should be understood that modifications and variations are included within the spirit and scope of this disclosure. Methods and materials are described herein.

[0087] The following embodiments are provided to further illustrate embodiments of the present invention, but are not intended to limit the scope of the invention. They are typical of those that may be used, but alternatively, other procedures, methods, or techniques known to those skilled in the art may be used. [Examples]

[0088] Example 1 Upregulation of miR-34a reduces mitochondrial health and biomass. To investigate the effects of miR-34a on intracellular mitochondria, adenocarcinoma human alveolar basal epithelial cells from the A549 cell line and human non-small cell lung cancer cells from the H1299 cell line were reverse-transfected with premicroRNA miR-34a and scrambled oligonucleotides (Life Technologies) as negative controls using Lipofectamine® 2000 (Life Technologies). The premicroRNAs for miR-34a and scrambled oligonucleotides were purchased from Thermo Fisher (mirVana miRNA mimetic, catalog number 4464066) and used at a final concentration of 100 nmol / L. Cells were stained with MitoTracker Red CMXRos (catalog number M7512), and mitochondrial health was measured after 48 hours. Cells transfected with miR-34a showed a decrease in mitochondrial health levels compared to control cells (Figure 1A).

[0089] Mitochondrial health can also be observed using DNA analysis. The ratio of mitochondrial DNA to nuclear DNA (mtDNA / nDNA) was measured using the NovaQUANT Mitochondrial to Nuclear DNA Ratio Kit (catalog no. 72620-1KIT), which contains genome and mitochondrial-specific primers, in A549 and H1299 cells transfected with scrambled oligonucleotides (control) and a mimetic of miR-34a-5p. The mtDNA / nDNA ratio was reduced in both A549 and H1299 lung cancer cell lines transfected with miR-34a premicroRNA compared to cells treated with the control oligonucleotide (Figure 1B).

[0090] PGC-1α expression is positively correlated with mitochondrial biomass. Therefore, to further investigate the role of miR-34a in mitochondria, A549 and H1299 lung cancer cell lines were transfected with scrambled oligonucleotides (control) and mimics of miR-34a-5p, and PGC-1α protein expression was evaluated after 72 hours of incubation. Total protein was extracted from A549 and H1299 cells, degraded on a denaturing gel, transferred to a membrane, and the membrane was probed with anti-PGC-1α and anti-actin antibodies (Sigma, St. Louis, MO). The membrane was then incubated with a secondary antibody labeled with horseradish peroxidase (Amersham GE Healthcare, Cleveland, OH), and the labeled protein was visualized using the chemiluminescent reagent Pierce ECL kit (Thermo Fisher Scientific, Waltham, MA). PGC-1α expression was reduced in cells transfected with miR-34a-5p compared to control cells in both A549 and H1299 cell lines (Figure 1C). Therefore, mitochondrial biomass was reduced by transfection with a mimic of miR-34a-5p.

[0091] Mitochondrial imaging, DNA analysis, and protein expression all showed lower mitochondrial health and activity in cells transfected with miR-34a. Adenosine triphosphate (ATP) is produced by mitochondria and is directly correlated with mitochondrial health and function. The amount of ATP produced by mitochondria in cells was measured using Cell Titer-GLO® Luminescent Cell Viability Assay (Promega) after transfecting A549 cells with scrambled oligonucleotides (control) and a mimetic of miR-34a-5p. Luminescence intensity was used to measure ATP production in transfected cells, and cells transfected with miR-34a-5p showed decreased ATP production (Figure 1D). These results indicate that transfection with miR-34a-5p resulted in decreased mitochondrial function.

[0092] Example 2 miR-34a suppresses DNA damage and repairs proteins. The expression of DNA damage and repair genes regulated by miR-34a-5p was measured using PCR array analysis. PremicroRNAs of miR-34a, anti-miR-34a, and their respective negative controls (scrambled oligonucleotides) (Life Technologies) were reverse-transfected into A549, H460 (large cell lung cancer), H1299 (non-small cell lung cancer), and H1944 cells with Lipofectamine® 2000 (Life Technologies) at a final concentration of 100 nmol / L. Total RNA was isolated from the cells using triazole (Life Technologies) for miRNA analysis according to the manufacturer's protocol. To analyze the expression of mature microRNA, total RNA was reverse-transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies) according to the manufacturer's protocol, followed by QPCR using the Taqman MicroRNA assay. The expression of DNA damage and repair genes was analyzed using a custom 384-well panel quantitative polymerase chain reaction (QPCR) plate (58 genes). Using the comparative Ct method, the relative abundances of miRNA and mRNA were calculated compared to the expression of U6 and beta-actin (ACTB), respectively (Figure 2A).

[0093] miR-34 expression was inversely correlated with the expression of genes associated with DNA damage and repair in non-small cell lung cancer tumors. To identify genes involved in DNA damage and repair affected by miR-34a expression, gene expression data from The Cancer Genome Atlas (TGCA) were analyzed for 532 patients with non-small cell lung cancer (NSCLC). Expression levels of DNA damage and repair pathways, as well as 12 mRNAs associated with miR-34a, were obtained from the TCGA portal (https: / / tcga-data.nci.nih.gov / tcga / dataAccessMatrix.htm). RNA-Seq and miRNA data from tumors isolated from patients contributing to TGCA were downloaded and used for subsequent analyses. Spearman-Roe correlation and statistical significance statistical analyses were performed using R: Language and Environment for Statistical Computation (https: / / www.R-project.org / ). All reported statistical significance p-values ​​were two-sided, and P ≤ 0.05 was considered statistically significant for all analyses. hsa-miR-34a expression was inversely correlated with several genes associated with DNA damage and repair, including RAD51 (Spearman-Low = -0.184, P = 0.0051), USP1 (Spearman-Low = -0.307, P = 2.37e-06), ATF1 (Spearman-Low = -0.329, P = 3.75e-07), BARD1 (Spearman-Low = -0.179, P = 0.0067), and CCNA2 (Spearman-Low = -0.201, P = 0.0022) (Figure 2B). Increased miR-34a expression was significantly correlated with decreased expression of several genes involved in DNA damage and repair.

[0094] PremicroRNAs of miR-34a and a negative control (scrambled oligonucleotide) (Life Technologies) were reverse-transfected into A549 and H1299 cells. Anti-miR-34a and scrambled oligonucleotides were transfected into H1944 cells as a control. Transfection was performed using Lipofectamine® 2000 (Life Technologies) at a final concentration of 100 nmol / L. Total protein was extracted, denatured on a denaturing gel, transferred to a membrane, and the membrane was probed with anti-PGC-1α and anti-actin antibodies (Sigma, St. Louis, MO). The membrane was then incubated with a secondary antibody labeled with horseradish peroxidase (Amersham GE Healthcare, Cleveland, OH) and visualized using the Pierce® Enhanced Chemiluminescence (ECL) Western Blotting Substrate kit (Thermo Fisher Scientific, Waltham, MA). Cells transfected with miR-34a premicroRNA or the miR-34a inhibitor (alpha-miR-34a) showed lower expression of the RAD51, MYC, CHEK1, CHEK2, and PIAS1 genes compared to cells transfected with control premicroRNA or anti-miR-34a oligonucleotides (Figures 2C and 2D). miR-34a levels in transfected cells were measured using the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies) according to the manufacturer's protocol, followed by qPCR using the Taqman MicroRNA assay.

[0095] Example 3 miR-34a regulates senescence and telomerase. The potential effects of miR-34a on the cellular senescence process were tested by measuring senescence-related beta-galactosidase (SA-β-Gal), an enzyme expressed only in senescent cells. SA-β-Gal is not expressed in pre-senescent, quiescent, or immortal cells. PremicroRNAs of miR-34a and a negative control (scrambled oligonucleotide) (Life Technologies) were reverse-transfected into A549 and H1299 cells with Lipofectamine® 2000 (Life Technologies) at a final concentration of 100 nmol / L. Senescence was measured 72 hours post-transfection using the Senescence Detection Kit (ab65351, Abcam). This assay is designed to histochemically detect SA-β-Gal activity in cultured cells and tissue sections, a known characteristic of senescent cells. Cultured A549 and H1299 cells transfected with miR-34a showed higher SA-beta-Gal activity than cells transfected with scrambled oligonucleotide-controlled premicroRNA (Figures 3A and 3B).

[0096] Genes involved in telomerase function include SIRT1 and p21. SIRT1 is involved in telomere dynamics, and p21 is a cyclin-dependent kinase inhibitor involved in the p53-p21 cellular senescence pathway. The role of miR-34a in these pathways is unclear. PremicroRNAs of miR-34a, anti-miR-34a, and their respective negative controls (scrambled oligonucleotides) (Life Technologies) were reverse-transfected into A549 and H1299 cells with Lipofectamine® 2000 (Life Technologies) at a final concentration of 100 nmol / L. Total protein was extracted, denatured on a denatured gel, transferred to a membrane, and the membrane was probed with anti-SIRT1, anti-p21, and anti-actin antibodies (Sigma, St. Louis, MO). Next, the membranes were incubated with a secondary antibody labeled with horseradish peroxidase (Amersham GE Healthcare, Cleveland, OH), and the labeled proteins were visualized using the Pierce® Enhanced Chemiluminescence (ECL) Western Blotting Substrate kit (Thermo Fisher Scientific, Waltham, MA). In A549 and H1299 cells transfected with premicroRNA miR-34a, SIRT1 expression was decreased, but p21 expression was increased compared to the scrambled oligonucleotide control (Figure 3C).

[0097] Telomerase function is influenced by the telomerase reverse transcriptase (hTERT) gene, but no identified functional relationship exists between hTERT and miR-34a. To measure the effect of miR-34a on hTERT expression, premicroRNAs of miR-34a, anti-miR-34a, and their respective negative controls (scrambled oligonucleotides) (Life Technologies) were reverse-transfected into A549 cells with Lipofectamine® 2000 (Life Technologies) at a final concentration of 100 nmol / L. Total protein was extracted from cells 72 and 96 hours after transfection, degraded on a denaturing gel, transferred to a membrane, and the membrane was probed with anti-hTERT and anti-actin antibodies (Sigma, St. Louis, MO). Next, the membranes were incubated with a secondary antibody labeled with horseradish peroxidase (Amersham GE Healthcare, Cleveland, OH) and visualized using the Pierce® Enhanced Chemiluminescence (ECL) Western Blotting Substrate kit (Thermo Fisher Scientific, Waltham, MA). Cells transfected with miR-34a showed low hTERT expression even 96 hours after transfection, demonstrating a remarkable correlation between miR-34A and hTERT expression, and therefore between miR-34a and telomerase function (Figure 3D).

[0098] Example 4 Antisense oligonucleotides designed to target miR-34a-5p Five sequences of ASOs targeting miR-34a-5p were tested for efficacy in mouse cells (lung cancer cells 344SQ) (Figure 4A) and validated in human cells using a sea urchin luciferase assay (Figure 4B). This assay allows for rapid and accurate measurement of ASO regulation by miR-34a-5p. 344SQ or A549 cells were used, 4 × 10⁶ 4Cells were plated in 96-well dishes at a cell / well rate. Cells were treated with an ASO (100 nmol / L) targeting miR-34a. 48 hours after transfection, cells were incubated for 15 minutes with 20 μl / well of 1× Passive Lysis Buffer (Promega, Madison, WI). Firefly and sea urchin luciferase activity was measured sequentially using a dual luciferase assay (Promega) with a Fluostar Optima plate reader (BMG Lab Technologies GmbH, Durham, NC). The ASOs tested included 9003001MIR, 9003002MIR, 9003003MIR, 9003004MIR, and 9003005MIR. Three sequences, 9003003MIR (SEQ ID NO: 2), 9003004MIR (SEQ ID NO: 3), and 9003005MIR (SEQ ID NO: 4), exhibited remarkably effective reductions in miR-34a expression in both mouse and human cells at normalized luminescence velocities detected at less than 0.5 relative light units (RLU) (Figures 4A and 4B).

[0099] Example 5 In human lung fibroblasts, antisense oligonucleotides targeting miR-34a-5p reduce the senescence marker p21 after radiation treatment. We analyzed the protein marker p21 of cellular senescence to detect the potential mitigating effect of miR-34a-targeting antisense oligonucleotides against irradiation-induced cytotoxicity. Antisense oligonucleotides targeting miR-34a-5p (ASO-miR-34a-1(9003001MIR)), ASO-miR-34a-2 (9003002MIR), ASO-miR-34a-3 (SEQ ID NO: 2), ASO-miR-34a-4 (SEQ ID NO: 3), and ASO-miR-34a-5 (SEQ ID NO: 4) were added to the culture medium of human lung fibroblasts (MRC-5). Lung fibroblasts were plated at 300,000 cells / mL. The following day, cells were pretreated with ASO-miR-34a-1, ASO-miR-34a-2, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 at both 125 nM and 2000 nM concentrations. Five days after treatment, cells were irradiated with a dose of 4 Gy (XRAD320 X-Ray System), and total protein was extracted from the cells 72 hours after irradiation. Western blotting was performed to analyze p21 expression, a senescence marker. For this purpose, total protein was extracted from cells, degraded on a denatured gel, transferred to a membrane, and the membrane was probed with anti-p21 and anti-actin antibodies (Sigma, St. Louis, MO). The membrane was incubated with a secondary antibody labeled with horseradish peroxidase (Amersham GE Healthcare, Cleveland, OH) and visualized using the Pierce® Enhanced Chemiluminescence (ECL) Western Blotting Substrate kit (Thermo Fisher Scientific, Waltham, MA). Surprisingly, we were able to identify two ASOs, SEQ ID NOs. 3 and 4, which exhibit a significant gene expression repression effect on the p21 protein after cell irradiation (Figure 5).

[0100] Example 6 Antisense oligonucleotides targeting miR-34a-5p inhibit the reduction of SIRT1 protein expression caused by miR-34a. Antisense oligonucleotides targeting miR-34a-5p (ASO-miR-34a-1, ASO-miR-34a-2, SEQ ID NO: 2, SEQ ID NO: 3, and ASO-miR-34a-5) were added to the culture medium of human lung fibroblasts (MRC-5). Human lung fibroblasts were plated at 300,000 cells / mL. The cells were then pretreated with ASO-miR-34a-1, ASO-miR-34a-2, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 at 12 nM and 2000 nM for 5 days, and total protein was collected 72 hours after pretreatment. Western blotting was performed to analyze SIRT1, a protein involved in the aging process. Total protein was extracted from the cells, degraded on a denatured gel, transferred to a membrane, and the membrane was probed with anti-SIRT1 and anti-actin antibodies (Sigma, St. Louis, MO). The membranes were incubated with a secondary antibody labeled with horseradish peroxidase (Amersham GE Healthcare, Cleveland, OH) and visualized using the Pierce® Enhanced Chemiluminescence (ECL) Western Blotting Substrate kit (Thermo Fisher Scientific, Waltham, MA). SIRT1 targeting by miR-34a was remarkably mitigated in cells incubated with three of the tested ASOs: SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 (Figure 6).

[0101] Example 7 Antisense oligonucleotides targeting miR-34a-5p increase mitochondrial DNA in human fibroblasts after radiation treatment. Mitochondrial copy number was analyzed using the NovaQUANT® Human Mitochondrial to Nuclear DNA Ratio Kit (EMD Millipore, Billerica, MA). ASOs targeting miR-34a-5p (SEQ ID NOs: 3 and 4) were added to the culture medium of human lung fibroblasts (MRC-5). Human lung fibroblasts were plated at 300,000 cells / mL. The cells were then pre-treated with SEQ ID NOs: 3 and 4 at 125 nM for 5 days. The cells were then irradiated with a dose of 4 Gy (XRAD320 cell irradiator), and DNA was collected 72 hours after irradiation. The NovaQUANT® Human Mitochondrial to Nuclear DNA Ratio Kit compares nuclear DNA (nDNA) levels to mitochondrial DNA (mtDNA) levels in human DNA samples. Real-time PCR provides a platform for quantitative assays to measure mtDNA copy number and compare it to nDNA copy number using a set of four optimized PCR primer pairs targeting two nuclear genes (BECN1 and NEB) and two mitochondrial genes (ND1 and ND6). The threshold cycle number (C) of the results obtained from the real-time PCR instrument t The expression levels of each gene were represented using ( ). Nuclear gene 1 (BECN1) was compared to mitochondrial gene 1 (ND1), and nuclear gene 2 (NEB) was compared to mitochondrial gene 2 (ND6). The ratio of mtDNA to nDNA was calculated and averaged to represent the number of mtDNA copies per cell. Sequence ID 3 had a significant mitigating effect against mitochondrial damage caused by cell irradiation (Figure 7).

[0102] Example 8 Antisense oligonucleotides targeting miR-34a-5p prevent immunosenility and hepatic senility in aged mice compared to young mice and untreated control mice. Mice were sourced from Jackson Laboratory. C57BL / 6J mice over 20 months of age were treated with SEQ ID NO: 3 (10 mg / kg on Mondays, Wednesdays, and Fridays for either 1 or 2 months), and gene expression levels were compared with untreated mice of approximately 3 months of age and untreated mice over 20 months of age using quantitative PCR. Mice were randomized into three treatment groups: (1) young (control, untreated), (2) aged (control, untreated), and (3) aged (treated with SEQ ID NO: 3, 10 mg / kg). Treated mice were injected with a solution containing SEQ ID NO: 3 at 10 mg / kg for 3 days a week, and peripheral blood mononuclear cell (PBMC) samples were collected 30 and 61 days after the start of treatment. Expression levels of genes associated with mitochondrial health, telomerase activity, and senescence-associated secretory phenotype (SASP) factors were measured using qPCR, including the genes SIRT1, PGC-1α, telomerase, p21, TNFα, and IL1b. Treatment of aged mice with SEQ ID NO: 3 improved the expression levels of PGC-1α, SIRT1, p21, and SASP factors TNFα and IL1b at 1 and 2 months post-treatment (Figures 8A, 8B, and 8C).

[0103] Example 9 Antisense oligonucleotides targeting miR-34a-5p reduce aging markers. Aged mice (20 months old, n=5 per group) of C57BL / 6 were treated with SEQ ID NO: 3 by intraperitoneal injection (IP) three times a week for one month. Immunohistochemical analysis of the senescence marker CDKN1A(p21) in the liver of the treated mice showed a significant decrease in CDKN1A(p21) expression (Figure 9A).

[0104] Aged mice (20 months old, n=6 per treatment group) of 129SvEv and C57BL / 6 were treated with SEQ ID NO: 3 by intraperitoneal injection (IP) three times a week for one month. Immunohistochemical analysis of the senescence marker CDKN2A(p16) in the livers of the treated mice showed a significant decrease in CDKN2A(p16) expression (Figure 9B).

[0105] Although the present invention has been described with reference to the above embodiments, it should be understood that modifications and variations are included in the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims.

[0106] Sequence List TIFF2026521620000003.tif65161 Legend: * = phosphorothioate bond +=Affinity Plus (locked nucleic acid bases)

Claims

1. An antisense oligonucleotide (ASO) complementary to at least eight consecutive nucleotides of SEQ ID NO:

1.

2. The ASO according to claim 1, wherein the ASO is at least 90% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO:

4.

3. The ASO according to claim 2, wherein the ASO is selected from sequence number 2, sequence number 3, or sequence number 4.

4. The ASO according to any one of claims 1 to 3, wherein the ASO comprises at least one unnaturally occurring nucleotide.

5. The ASO according to claim 4, wherein the at least one unnaturally occurring nucleotide is selected from the group consisting of a cytoskeletal modification, nucleic acid base modification, ribose modification, 2'-ribose substitution, locked nucleic acid base, morpholino oligonucleotide, peptide nucleic acid, phosphorothioate, tricyclodeoxyribonucleic acid, 5-methylcytidine, 5-methyluridine, 5-ribothymidine, debased nucleoside, nucleotide lacking a terminal phosphate group at the 5' end, nucleotide having a 5'-(E)-vinylphosphonate modification at the 5' end, terminal inverted debased ribonucleotide, nucleotide with a 3' end modified at the 2' position of a ribose sugar, nucleotide having a 2'-O-methyl modification, nucleotide having a 2'-O-methoxyethyl modification, nucleotide having a 2'-fluoro modification, morpholino oligonucleotide, gapmer, optionally uridine nucleotide, at least one thymine nucleotide, and any combination thereof.

6. The ASO according to claim 5, wherein at least one skeletal modification is a phosphorothioate bond.

7. The ASO according to claim 5, wherein the ASO is a morpholino oligonucleotide.

8. The ASO according to claim 5, wherein the ASO is a gapmer.

9. The ASO according to claim 5, wherein at least one thymine nucleotide is optionally a uridine nucleotide.

10. The ASO according to claim 4, wherein the ASO is covalently bonded to at least one non-nucleotide portion.

11. A pharmaceutical composition comprising at least one antisense oligonucleotide (ASO) according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

12. A method for treating or delaying the progression of an aging-related disease in a subject, comprising administering to the subject a therapeutically effective amount of an ASO according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11.

13. The method according to claim 12, wherein the ASO or pharmaceutical composition inhibits or reduces the expression of miR-34a-5p.

14. The method according to claim 14, wherein the ASO or pharmaceutical composition enhances or increases the level of SIRT1 activity.

15. The method according to claim 14, wherein the ASO or pharmaceutical composition enhances or increases the level of PGC-1α activity.

16. The method according to claim 14, wherein the at least one ASO or pharmaceutical composition enhances or increases the level of telomerase activity.

17. The method according to claim 13, wherein the aging-related disease is immunosenility, vascular senility, musculoskeletal dysfunction, non-alcoholic steatohepatitis (NASH), Alzheimer's disease, type II diabetes, radiation-induced fibrosis, aging induced by radiation exposure due to being in space, radiation-induced pulmonary fibrosis, mitochondrial dysfunction, or telomerase shortening.

18. The method according to claim 13, wherein the age-related disease is pulmonary fibrosis.

19. The method according to claim 18, wherein the pulmonary fibrosis is induced by radiation, mitochondrial dysfunction, or telomerase shortening.

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