US20170002362A1 - Composition for reducing cellular senescence level including activity inhibitor inhibiting dcun1d3 activity or expression inhibitor inhibiting expression of dcun1d3-encoding gene and use thereof - Google Patents

Composition for reducing cellular senescence level including activity inhibitor inhibiting dcun1d3 activity or expression inhibitor inhibiting expression of dcun1d3-encoding gene and use thereof Download PDF

Info

Publication number
US20170002362A1
US20170002362A1 US15/200,937 US201615200937A US2017002362A1 US 20170002362 A1 US20170002362 A1 US 20170002362A1 US 201615200937 A US201615200937 A US 201615200937A US 2017002362 A1 US2017002362 A1 US 2017002362A1
Authority
US
United States
Prior art keywords
dcun1d3
disease
seq
shrna
nucleotide sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/200,937
Other languages
English (en)
Inventor
Myoungsoon Kim
Yongsub Kim
Young-sam Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Samsung Electronics Co Ltd
Original Assignee
Samsung Electronics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020160008900A external-priority patent/KR20170004821A/ko
Application filed by Samsung Electronics Co Ltd filed Critical Samsung Electronics Co Ltd
Assigned to SAMSUNG ELECTRONICS CO., LTD. reassignment SAMSUNG ELECTRONICS CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KIM, MYOUNGSOON, KIM, YONGSUB, LEE, YOUNG-SAM
Publication of US20170002362A1 publication Critical patent/US20170002362A1/en
Priority to US15/681,262 priority Critical patent/US10329569B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • 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
    • 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
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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 N.A.
    • 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 N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications

Definitions

  • the present disclosure relates to a composition for reducing a cellular senescence level, a method of reducing a cellular senescence level in a mammal, and a method of treating a disease or a disease symptom associated with an increased cellular senescence level in a mammal.
  • Senescence may be defined as a permanent halt in cell division. Replicative senescence or cellular senescence is observed as a model for aging at a cellular level. When cells are consecutively cultured, the cells are divided a number of times, but the cells no longer divide once senescence is reached. Senescent cells often have resistance to programmed cell death, and, in some cases, the senescent cells are maintained in a non-dividing state for years.
  • DCUN1D3 was found during the process of high throughput screening of novel human genes associated with serum response element (SRE) pathway activation.
  • the DCUN1D3 gene is highly conserved among vertebrates.
  • Human DCUN1D3 complementary DNA (cDNA) encodes 304 amino acids with an apparent molecular weight of 34 kDa.
  • An amino acid sequence of DCUN1D3 and a nucleotide sequencing encoding the same may be those described in Genbank Accession Nos. NP_775746.1 and NM_173475.2, respectively.
  • DCUN1D3 is broadly expressed in several tumor tissues and cultured cell lines. UV irradiation significantly increases DCUN1D3 expression levels in cancer cell lines.
  • An aspect provides a composition for reducing a cellular senescence level.
  • Another aspect provides a method of reducing a cellular senescence level in a mammal.
  • Still another aspect provides a method of treating a disease or a disease symptom associated with an increased cellular senescence level in a mammal.
  • FIG. 1 is a microscopic image of cultured senescent cells transfected with DCUN1D3 shRNA-containing lentivirus
  • FIG. 2A is an X-gal staining image of senescent cells transfected with DCUN1D3 shRNA-containing lentivirus (panel A) or scrambled shRNA-containing lentivirus (panel B);
  • FIG. 2B is a percentage of the cells stained blue in the X-gal staining image of FIG. 2A ;
  • FIG. 3 shows the amount of lipofuscin according to fluorescence intensity in senescent cells transfected with DCUN1D3 shRNA or scrambled shRNA-containing lentivirus;
  • FIG. 4 shows relative cell size of senescent cells (% of control) transfected with DCUN1D3 shRNA-containing lentivirus or scrambled shRNA-containing lentivirus;
  • FIG. 5 shows the proportion of senescent cells introduced with DCUN1D3 siRNA or control siRNA in GO/G1, S, or G2/M phase of the cell cycle
  • FIG. 6 shows the relative number (A) and DNA content of cultured senescent cells transfected with DCUN1D3 shRNA or SEAT1 shRNA-containing lentivirus;
  • FIG. 7 shows an X-gal staining image of senescent cells transfected with scramble shRNA(A) or SEAT1 shRNA(B) or DCUN1D3 shRNA(C)-containing lentivirus, and a percentage (%) of the cells stained blue;
  • FIG. 8 shows results of measuring lipofuscin in senescent cells transfected with DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus
  • FIG. 9 shows results of measuring cell size (A) and cell granularity (B) of senescent cells transfected with DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus;
  • FIG. 10 shows results of measuring cell cycle of senescent cells transfected with SEAT1 siRNA or DCUN1D3 siRNA or control siRNA;
  • FIG. 11 shows expression levels of DCUN1D3 gene and SEAT1 gene in human cells (A) and mouse cells (B) introduced with siSEAT1;
  • FIG. 12 shows effects of SEAT1 shRNA and/or DCUN1D3 shRNA on intracellular DNA damage
  • FIG. 13 shows effects of SEAT1 or DCUN1D3 gene expression inhibition on hIL-6 expression
  • FIG. 14 shows effects of miR-20b on DCUN1D3 gene expression.
  • compositions for reducing a cellular senescence level including an activity inhibitor inhibiting activity of one or more of DCN1, defective in cullin neddylation 1, domain containing 3 (DCUNID3) protein and a polynucleotide having a nucleotide sequence of SEQ ID NO: 5, or an expression inhibitor inhibiting expression of one or more of a gene encoding DCUN1D3 and a gene encoding the nucleotide sequence of SEQ ID NO: 5 as an active ingredient.
  • DCUN1D3 was found during the process of high throughput screening of novel human genes associated with serum response element (SRE) pathway activation.
  • the DCUN1D3 gene is highly conserved among vertebrates.
  • Human DCUN1D3 complementary DNA (cDNA) encodes 304 amino acids with an apparent molecular weight of 34 kDa.
  • An amino acid sequence of DCUN1D3 and a nucleotide sequence encoding the same may be those described in Genbank Accession Nos. NP_775746.1 (SEQ ID NO: 3) and NM_173475.2 (SEQ ID NO: 4).
  • DCUN1D3 is broadly expressed in several tumor tissues and cultured cell lines. UV irradiation significantly increases a DCUN1D3 expression level in cancer cell lines.
  • the activity inhibitor inhibiting DCUN1D3 activity may be a substance inhibiting neddylation activity of DCUN1D3.
  • Neddylation may be a process by which the ubiquitin-like protein NEDD8 is conjugated to its target protein.
  • the nucleic acid with the nucleotide sequence of SEQ ID NO: 5 may be a gene encoding a long non-coding ribonucleic acid (IncRNA).
  • the non-coding RNA is a functional RNA molecule which is not translated into a protein.
  • the ncRNA may be small nucleolar RNA (snoRNA), microRNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), extracellular RNA (exRNA), Piwi-interacting RNA (piRNA), or long ncRNA (IncRNA).
  • the IncRNA is a transcript, which is not translated into a protein or has a low protein-coding potential, and has a length of about 50 nucleotides or more, about 100 nucleotides or more, about 200 nucleotides or more, or about 500 nucleotides or more.
  • the nucleic acid of the nucleotide sequence of SEQ ID NO: 5 may be a nucleic acid from a nucleic acid located at p-arm of human chromosome 16.
  • the nucleic acid of the nucleotide sequence of SEQ ID NO: 5 may be a nucleic acid from a nucleic acid located between an ERI1 Exoribonuclease Family Member 2 (ERI2) gene of human chromosome 16 and a DCN1, Defective In Cullin Neddylation 1, Domain Containing 3 (DCUN1D3) gene.
  • the nucleic acid of the nucleotide sequence of SEQ ID NO: 5 may have a sequence of GenBank Accession No. AK027199.
  • An activity inhibitor inhibiting SEAT1 RNA activity may be small interference RNA (siRNA) against SEAT1 RNA, small hairpin RNA (shRNA), antisense oligonucleotide, miRNA, or a combination thereof.
  • the siRNA be short double-stranded RNA(dsRNA) with phosphorylated 5′ ends and hydroxylated 3′ ends with two overhaning nucleotides.
  • the siRNA may have 20 to 24 nucleotides in length.
  • the siRNA may have a complementary nucleotide sequence to the mRNA of the target gene.
  • the siRNA against SEAT1 RNA may include a polynucleotide of SEQ ID NO: 6 and a polynucleotide of SEQ ID NO: 7 (SEAT1 siRNA #1); a polynucleotide of SEQ ID NO: 8 and a polynucleotide of SEQ ID NO: 9 (SEAT1 siRNA #2); or a polynucleotide of SEQ ID NO: 10 and a polynucleotide of SEQ ID NO: 11 (SEAT1 siRNA #3).
  • the activity inhibitor inhibiting SEAT1 RNA activity may be also an expression inhibitor inhibiting DCUN1D3 expression.
  • the gene encoding SEAT1 RNA may be located at p-arm of human chromosome 16.
  • the gene may be AK027199 gene, namely, a polynucleotide of SEQ ID NO: 5.
  • the gene encoding SEAT1 RNA may be located between ERI2 gene of human chromosome 16 and DCUN1D3 gene.
  • the expression inhibitor inhibiting expression of genes encoding one or more of DCUN1D3 and SEAT1 RNA may be any expression inhibitor, as long as it reduces expression of genes encoding one or more of DCUN1D3 and SEAT1 RNA in cells.
  • the expression inhibitor may be siRNA, shRNA, miRNA, antisense oligonucleotide, or a combination thereof.
  • the siRNA against the DCUN1D3 gene may include a nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2 (DCUN1D3 siRNA #1).
  • the siRNA against SEAT1 RNA may include a polynucleotide of SEQ ID NO: 6 and a polynucleotide of SEQ ID NO: 7 (SEAT1 siRNA #1); a polynucleotide of SEQ ID NO: 8 and a polynucleotide of SEQ ID NO: 9 (SEAT1 siRNA #2); or a polynucleotide of SEQ ID NO: 10 and a polynucleotide of SEQ ID NO: 11 (SEAT1 siRNA #3).
  • the expression inhibitor inhibiting expression of the SEAT1 RNA-encoding gene may be also an expression inhibitor inhibiting expression of DCUN1D3-encoding gene.
  • the expression inhibitor inhibiting expression of the DCUNID3-encoding gene may be any expression inhibitor, as long as it reduces expression of the DCUN1D3 gene in cells.
  • the expression inhibitor may be small interference RNA (siRNA), small hairpin RNA (shRNA), antisense oligonucleotide, or a combination thereof.
  • the siRNA may include a nucleotide sequence of SEQ ID NO: 1 and a nucleotide sequence of SEQ ID NO: 2.
  • the expression inhibitor may be miR-20b.
  • a reduction in the cellular senescence level may refer to delay or prevention of cellular senescence, or reversion of a senescent cell to a younger cell state (e.g., non-senescent cell).
  • the reduction in the cellular senescence level may include one or more of an increase in proliferation of a cell, an increase in activity of autophagy, a reduction in accumulation of lipofuscin, a reduction in activity of ⁇ -galactosidase, a reduction in the number of mitochondrial reactive oxygen species, and an increase in mitochondrial membrane potential.
  • the cell may be a muscle cell including myoblast, fibroblast, early senescent cell, or nerve cell.
  • the early senescent cell may be a cell derived (obtained) from a patient with progeria. Progeria may be Hutchinson-Gilford progeria or Werner syndrome.
  • the composition may be used to treat a disease or a disease symptom associated with increased cellular senescence level.
  • the disease or disease symptom associated with increased cellular senescence level may include skin wrinkles, wound healing declines, sarcopenia, muscular dystrophy, early senescent symptom (e.g., Hutchinson-Gilford progeria syndrome), or a combination thereof.
  • the disease or disease symptom associated with increased cellular senescence level may include a disease or a disease symptom associated with lipofuscin accumulation.
  • the disease or disease symptom associated with lipofuscin accumulation may be neuronal ceroid lipofuscinoses (NCL), age-related macular degeneration, neurofibrillary tangles, brown atrophy of the heart, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), acromegaly, denervation atrophy, lipid myopathy, or chronic obstructive pulmonary disease (COPD).
  • NCL neuronal ceroid lipofuscinoses
  • age-related macular degeneration neurofibrillary tangles
  • brown atrophy of the heart Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), acromegaly, denervation atrophy, lipid myopathy, or chronic obstructive pulmonary disease (COPD).
  • the disease or disease symptom associated with increased cellular senescence level may be a disease caused by mitochondrial damage such as an increase in mitochondrial ROS, reduction in mitochondrial membrane potential,
  • the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5 may be prepared as a pharmaceutically acceptable salt.
  • the cells may be cells of a mammal including a human.
  • the mammal may have a disease associated with increased cellular senescence level.
  • the cells may exist in vitro or in vivo.
  • the cells may be fibroblasts or nerve cells.
  • the composition may further include a pharmaceutically acceptable carrier.
  • the “pharmaceutically acceptable carrier” generally refers to an inert material, i.e., a material used in combination with an active ingredient to assist the application of the active ingredient.
  • the carrier may include a pharmaceutically acceptable excipient, additive, or diluent generally used.
  • the carrier may include one or more selected from, for example, a filler, a binder, a disintegrant, a buffer, a preservative, an antioxidant, a lubricant, a flavoring agent, a thickener, a coloring agent, an emulsifier, a suspending agent, a stabilizer, and an isotonic agent.
  • the carrier may include a vehicle for delivering nucleotide to a subject for example, a mammal.
  • vehicle may include nanoparticle, DNA templates encoding siRNA sequences, cationic liposomes, cholesterol conjugates, antibody conjugates cationic lipids, such as Lipofectamine, a positively charged peptide or protein, and/or peptide-mediated delivery systems.
  • a number of approaches for delivering siRNA to a specific target cell's cytoplasm may be used, ranging in complexity from simple naked siRNAs to complicated nanoparticle-based delivery vehicles.
  • Examples include, but are not limited to, DNA templates encoding siRNA sequences may be delivered to cells that can be transcribed to express siRNAs; the use of cationic liposomes, cholesterol conjugates, antibody conjugates, electroporation, direct injection, hydrodynamic transfection, electrical pulsing or any other suitable method of direct delivery; cationic lipids, such as Lipofectamine, as a transfection reagent may be used to deliver siRNA in vitro; siRNA may be delivered systemically using cholesterol conjugates, liposomes, and polymer-based nanoparticle sized delivery vehicles; and siRNA may be delivered via peptide-mediated delivery systems. Plasmid or viral vectors for the delivery may be used.
  • a positively charged peptide or protein may be used to produce a protein-siRNA complex that can be used to deliver siRNAs.
  • the phosphate backbone of siRNAs is negatively charged and allows complex formation with cationic peptides and proteins regardless of its sequence.
  • the protein-siRNA complex can include a non-specific cell-penetrating peptide (e.g. Tat) to deliver the siRNa to cells.
  • the protein-siRNA complex can include a targeting moiety, such as a receptor-binding peptide or antibody for specific delivery.
  • the composition may include the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5, the pharmaceutically acceptable salt thereof, or the solvate in a “therapeutically effective amount”.
  • the “therapeutically effective amount” refers to an amount that is sufficient enough to indicate a therapeutic effect when administered to a subject in need of treatment.
  • treatment refers to a practice of treating a disease or a medical symptom, e.g., a disease associated with cellular senescence, in a subject such as a mammal including a human, and examples of the treatment are as follows: (a) prevention of the occurrence of a disease or a medical symptom, that is, prophylactic treatment of a patient; (b) alleviation of a disease or a medical symptom, that is, involvement of removal or recovery of a disease or a medical symptom in a patient; (c) inhibition of a disease or a medical symptom, that is, involvement of delaying or stopping a disease or a medical symptom in a subject; or (d) reduction of a disease or a medical symptom in a subject.
  • the “effective amount” may be appropriately selected by one of ordinary skill in the art.
  • the “effective amount” may be in a range from about 0.01 mg to about 10,000 mg, about 0.1 mg to about 1,000 mg, about 1 mg to about 100 mg, about 0.01 mg to about 1,000 mg, about 0.01 mg to about 100 mg, about 0.01 mg to about 10 mg, or about 0.01 mg to about 1 mg per day.
  • the composition may be administered orally to a subject, or parenterally to a subject in a way of intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. Therefore, the composition may be formulated in various forms including tablets, capsules, aqueous solutions, or suspensions.
  • an excipient such as lactose or corn starch, and a lubricant such as magnesium stearate, may be generally added to the composition.
  • lactose and/or dry corn starch may be used as a diluent.
  • an active ingredient may be used in combination with an emulsifier and/or a suspending agent.
  • a particular sweetening agent and/or a flavoring agent may be added.
  • a sterile solution of an active ingredient is generally prepared, thereby appropriately adjusting and buffering the pH of the solution.
  • the total concentration of solutes is adjusted to render the formulation isotonicity.
  • the composition may be prepared as an aqueous solution containing a pharmaceutically acceptable carrier having a pH of 7.4 as of salt water.
  • the aqueous solution may be introduced into muscle or nerve blood flow of a patient by local bolus injection.
  • cellular senescence refers to, as compared with a reference cell, one or more of reduction in proliferation of a cell, reduction in activity of autophagy, accumulation of lipofuscin, increase in ⁇ -galactosidase activity, increase in the number of mitochondrial ROS, and reduction in mitochondrial membrane potential, or to a process causing the phenomena above.
  • the reference cell may be a known non-senescent cell of the same type.
  • the term “young cell” refers to, as compared with a reference cell, a cell with one or more of increased proliferation of a cell, increased activity of autophagy, decreased accumulation of lipofuscin, decreased activity of ⁇ -galactosidase, decreased number of mitochondrial ROS, and increased mitochondrial membrane potential.
  • the reference cell may be a known senescent cell of the same type.
  • a non-senescent cell as the reference cell may be a cell, for example, a fibroblast or a nerve cell derived from a person aged about 18 to about 25, about 18 to about 23, or about 18 to about 20 who are normal and healthy.
  • the composition may be used in combination with one or more additional therapeutic agents to treat a disease associated with increased cellular senescence level.
  • the composition may be free of other active ingredients to treat a disease associated with increased cellular senescence level other than the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5, the pharmaceutically acceptable salt thereof, or the solvate thereof.
  • compositions for reducing accumulation of lipofuscin in cells including the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5 as an active ingredient.
  • the composition may be used to treat a disease or a disease symptom associated with accumulation of lipofuscin in cells.
  • the disease or disease symptom associated with accumulation of lipofuscin may be NCL, age-related macular degeneration, neurofibrillary tangles, brown atrophy of the heart, Alzheimer's disease, Parkinson's disease, ALS, acromegaly, denervation atrophy, lipid myopathy, or COPD.
  • the terms used in the ‘composition for reducing accumulation of lipofuscin in cells’ are the same as those in the ‘composition for reducing a cellular senescence level’, unless otherwise mentioned herein.
  • compositions for increasing proliferation of a cell including the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5 as an active ingredient.
  • the composition may be used to treat a disease or a disease symptom associated with reduced proliferation of a cell.
  • the disease or disease symptom associated with reduced proliferation of a cell may be skin wrinkles, wound healing declines, sarcopenia, muscular dystrophy, early senescent symptom (e.g., Hutchinson-Gilford progeria syndrome), or a combination thereof.
  • the terms used in the ‘composition for increasing proliferation of a cell’ are the same as those in the ‘composition for reducing a cellular senescence level’, unless otherwise mentioned herein.
  • Still another aspect provides a method of reducing a cellular senescence level in a mammal, the method including administering an effective amount of the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5 to the mammal to reduce a cellular senescence level.
  • the effective amount refers to an amount sufficient enough to reduce a cellular senescence level” when administered to a subject.
  • the administration refers to administration of the above-described composition including the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5, the pharmaceutically acceptable salt thereof, or the solvate thereof”.
  • Still another aspect provides a method of treating a disease or a disease symptom associated with increased cellular senescence level in a mammal, the method including administering a therapeutically effective amount of the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5 to a mammal to treat the disease or disease symptom associated with increased cellular senescence level.
  • the effective amount refers to “an amount sufficient enough to treat the disease or disease symptom associated with increased cellular senescence level” when administered to a subject having the disease or disease symptom associated with increased cellular senescence level.
  • the administration refers to administration of the above-described composition including the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5, the pharmaceutically acceptable salt thereof, or the solvate thereof”.
  • the administration may be oral, parenteral, or topical administration.
  • the administration may be topically applied to tissue consisting of or including senescent cells.
  • the administration may be topically applied to skin tissue, muscle tissue, or nerve tissue.
  • the “therapeutically effective amount” is an amount that is effective enough to treat the disease or disease symptom associated with increased cellular senescence level in a mammal.
  • the administration amount may vary, as described above, according to a variety of factors, such as a patient's condition, an administration route, or physician's determination.
  • the effective administration amount may be estimated by a dose-response curve obtained in vitro or from an animal model test.
  • the ratio or concentration of the compound of the present invention may be determined according to chemical properties, the route of administration, or therapeutic amounts.
  • the administration amount may be effective in a subject when administered in a range from about 1 ⁇ g/kg to about 1 g/kg per day, or about 0.1 mg/kg to about 500 mg/kg weight per day.
  • the administration amount may vary according to a subject's age, weight, susceptibility, or symptoms.
  • Still another aspect provides a method of reducing a lipofuscin level in a mammalian cell, the method including administering a therapeutically effective amount of the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5 to a mammal to reduce the lipofuscin level in the cell.
  • the method may be used to treat a disease or a disease symptom associated with increased lipofuscin level in a mammalian cell.
  • the disease or disease symptom associated with increased lipofuscin level may be NCL, age-related macular degeneration, neurofibrillary tangles, brown atrophy of the heart, Alzheimer's disease, Parkinson's disease, ALS, acromegaly, denervation atrophy, lipid myopathy, or COPD.
  • the terms used in the ‘method of reducing a lipofuscin level in a mammalian cell’ are the same as those in the ‘method of treating a disease or a disease symptom associated with increased cellular senescence level in a mammal’, unless otherwise mentioned herein.
  • Still another aspect provides a method of increasing proliferation of a mammalian cell, the method including administering an effective amount of the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5 to a mammal to increase proliferation of the cell.
  • the method may be used to treat a disease or a disease symptom associated with decreased proliferation of a mammalian cell.
  • the disease or disease symptom associated with decreased proliferation of a cell may be skin wrinkles, wound healing declines, sarcopenia, muscular dystrophy, early senescent symptom (e.g., Hutchinson-Gilford progeria syndrome), or a combination thereof.
  • the terms used in the ‘method of increasing proliferation of a mammalian cell’ are the same as those in the ‘method of treating a disease or a disease symptom associated with increased cellular senescence level in a mammal’, unless otherwise mentioned herein.
  • composition according to an aspect may be used to reduce a cellular senescence level, the composition including the activity inhibitor inhibiting activity of one or more of the DCUN1D3 protein and the polynucleotide having the nucleotide sequence of SEQ ID NO: 5, or the expression inhibitor inhibiting expression of one or more of the gene encoding DCUN1D3 and the gene encoding the nucleotide sequence of SEQ ID NO: 5, the pharmaceutically acceptable salt thereof, or the solvate thereof.
  • the method of reducing a cellular senescence level in a mammal according to another aspect may be used to efficiently reduce the cellular senescence in the mammal.
  • the method of treating a disease or a disease symptom associated with increased cellular senescence level in a mammal may be used to efficiently treat the disease or disease symptom associated with increased cellular senescence level in the mammal.
  • DCUN1D3 shRNA was transduced into senescent cells to examine its effect on proliferation of senescent cells.
  • Senescent cells to be used were prepared by subculturing human dermal fibroblast (HDF) M4 cells obtained from a 4-year-old boy (derived from the foreskin of a 4-year-old donor, Seoul National University) in DMEM (hereinafter, referred to as ‘DMEM’) (HyClone, Logan, Utah, USA:Cat no.
  • HDF human dermal fibroblast
  • SH30243 containing high concentration of glucose, glutamine, and pyruvate and being supplemented with 10% (v/v) fetal bovine serum (FBS), and 1 ⁇ penicillin/streptomycin (100 U/ml peninicillin, 100 ug/ml streptomycin) under conditions of 37° C. and 5% CO 2 .
  • FBS fetal bovine serum
  • penicillin/streptomycin 100 U/ml peninicillin, 100 ug/ml streptomycin
  • DCUN1D3 shRNA was introduced into lentivirus to prepare a DCUN1D3 shRNA-containing lentivirus, which was transduced into the senescent cells.
  • the DCUN1D3 shRNA-containing lentiviral pLKO.1 puro vector (OriGene, USA) was prepared by synthesizing nucleotide sequences of SEQ ID NOS: 1 and 2, respectively to prepare a double stranded shRNA construct (IDT, Korea), and ligating the shRNA construct into pLKO.1 puro vector digested with restriction enzymes AgeI and EcoRI.
  • the prepared DCUN1D3 shRNA-containing lentivirus was transduced into 293FT human embryonic kidney cells (#R700-07: Invitrogen) using Lipofectamine 2000, together with packaging vectors, VSV-G and PAX2 (OriGene, USA).
  • VSV-G and PAX2 packaging vectors
  • 6 ug/ml polybrene was added to the growth medium for kidney cells to increase the transduction efficiency.
  • a lentiviral supernatant was collected and concentrated using Lenti-X (Clontech, cat. no.631231).
  • the DCUN1D3 shRNA-containing lentivirus thus prepared was transfected into the senescent cells and subcultured in DMEM (hereinafter, referred to as ‘DMEM’) (HyClone, cat.no. SH30243, USA) containing high concentration of glucose, glutamine, and pyruvate and being supplemented with 10% (v/v) fetal bovine serum (FBS), and 1 ⁇ penicillin/streptomycin (100 U/ml peninicillin, 100 ug/ml streptomycin) for 4 weeks under conditions of 37° C. and 5% CO 2 .
  • DMEM DMEM
  • FBS fetal bovine serum
  • penicillin/streptomycin 100 U/ml peninicillin, 100 ug/ml streptomycin
  • the nucleotide sequence of the DCUN1D3 shRNA was a nucleotide sequence of SEQ ID NO: 1 (sense) and SEQ ID NO: 2 (antisense). Scrambled RNA (Bioneer, Korea) provided by the manufacturer was used as a control group.
  • SEQ ID NO: 1 sense: GUCACUGCAUCGGGAAAUA(dTdT)
  • SEQ ID NO: 2 antisense: UAUUUCCCGAUGCAGUGAC(dTdT)
  • FIG. 1 is a microscopic image of the cultured senescent cells transfected with DCUN1D3 shRNA-containing lentivirus.
  • control shRNA indicates senescent cells transfected with scrambled shRNA-containing lentivirus
  • DCUN1D3 shRNA indicates senescent cells transfected with DCUN1D3 shRNA-containing lentivirus.
  • the senescent cells transfected with DCUN1D3 shRNA-containing lentivirus showed an increase in the number of cells and similar morphology to young cells, compared to senescent cells transfected with scrambled shRNA-containing lentivirus, that is, a negative control group, indicating that DCUN1D3 shRNA induces proliferation of senescent cells, after introduced into the senescent cells.
  • Senescent cells were stained blue with X-gal due to increased activity of SA ⁇ -gal. As explained in (1), senescent cells were transfected with DCUN1D3 shRNA or scrambled shRNA-containing lentivirus, and SA ⁇ -gal activity in the cultured senescent cells was measured. Senescent cells that were transfected with none of DCUN1D3 shRNA or scrambled shRNA-containing lentivirus were used as a negative control group.
  • FIG. 2A is an X-gal staining image (A) of senescent cells transfected with DCUN1D3 shRNA (right) or scrambled shRNA-containing lentivirus (left) and FIG. 2B is a percentage (B) of the cells stained blue in the X-gal staining image of FIG. 2A .
  • senescent cells show a reduction in autophagy activity, accumulation of lipofuscin, and mitochondrial damage.
  • senescent cells were transfected with DCUN1D3 shRNA or scrambled shRNA-containing lentivirus, and the amount of lipofuscin was measured in the cultured senescent cells.
  • Senescent cells that were transfected with none of DCUN1D3 shRNA or scrambled shRNA-containing lentivirus were used as a negative control group. Further, the size of the cell was measured.
  • FIG. 3 shows results of measuring the amount of lipofuscin in the senescent cells transfected with DCUN1D3 shRNA or scrambled shRNA-containing lentivirus.
  • an autofluorescence level of the senescent cells transfected with DCUN1D3 shRNA-containing lentivirus was about 66%, compared to that of the senescent cells transfected with scrambled shRNA-containing lentivirus. Therefore, it is confirmed that lipofuscin accumulation was reduced in senescent cells by transfection of the cells with DCUN1D3 shRNA-containing lentivirus, indicating that a cellular senescence level is reduced by transfection of senescent cells with DCUN1D3 shRNA-containing lentivirus.
  • FIG. 4 shows results of measuring the size of the senescent cells transfected with DCUN1D3 shRNA or scrambled shRNA-containing lentivirus. As shown in FIG. 4 , the size of the senescent cells transfected with DCUN1D3 shRNA-containing lentivirus was smaller than that of the senescent cells transfected with scrambled shRNA-containing lentivirus.
  • FIG. 4 the size, granularity, and autofluorescence of the cells were estimated by flow cytometry.
  • trypsin-treated cells were collected in PBS, and analyzed by FACS Caliber instrument (Becton Dickson, USA). The size and granularity of 100,000 cells were evaluated by previous forward and side scatter. Autofluorescence was measured using a 488-nm laser for excitation and a 530/30 bandpass filter for detection.
  • FSC and SSC in the vertical axis of A and B of FIG. 4 represent forward scatter (cell size) and side scatter (cell granularity), respectively.
  • siRNA and control siRNA were introduced into senescent cells by introducing it into senescent cells using lipofectamine without packaging shRNA or scrambled RNA in lentivirus, and the senescent cells were cultured and then cell cycle was examined.
  • DCUN1D3 shRNA was introduced, and therefore, DCUN1D3 siRNA was allowed to produce within the cells.
  • FIG. 5 shows results of measuring the cell cycle of the senescent cells introduced with DCUN1D3 siRNA or control siRNA.
  • the experiment was performed in triplicate, and culture was performed for 3 days.
  • the senescent cells introduced with DCUN1D3 siRNA showed increased cell percentages in the G1/G1 phase and increased cell percentages in the S phase and G2/M phase, compared to the senescent cells introduced with control siRNA.
  • the unit of the vertical axis of FIG. 5 is %, which is a percentage when the result of control siRNA is taken as 100.
  • DCUN1D3 shRNA or SEAT1 (senescence-associated transcript1) shRNA was transduced into senescent cells to examine their effects on proliferation of senescent cells.
  • Senescent cells to be used were prepared by subculturing human dermal fibroblast (HDF) M4 cells obtained from a 4-year-old boy (derived from the foreskin of a 4-year-old donor, Seoul National University) in DMEM (hereinafter, referred to as ‘DMEM’) (HyClone, Logan, Utah, USA:Cat no.
  • SH30243 containing high concentration of glucose, glutamine, and pyruvate and being supplemented with 10% (v/v) fetal bovine serum (FBS), and 1 ⁇ penicillin/streptomycin (100 U/ml peninicillin, 100 ug/ml streptomycin) under conditions of 37° C. and 5% CO 2 .
  • FBS fetal bovine serum
  • penicillin/streptomycin 100 U/ml peninicillin, 100 ug/ml streptomycin
  • DCUN1D3 shRNA or SEAT1 shRNA was introduced into lentivirus to prepare a DCUN1D3 shRNA or SEAT1 shRNA-containing lentivirus, which was transduced into the senescent cells.
  • the DCUN1D3 shRNA or SEAT1 shRNA-containing lentiviral pLKO.1 puro vector (OriGene, USA) was prepared by synthesizing nucleotide sequences of SEQ ID NOS: 1 and 2, respectively to prepare a double stranded shRNA construct (IDT, Korea), and ligating the shRNA construct into pLKO.1 puro vector digested with restriction enzymes AgeI and EcoRI.
  • the prepared DCUN1D3 shRNA or SEAT1 shRNA-containing lentivirus was transduced into 293FT human embryonic kidney cells (#R700-07: Invitrogen) using Lipofectamine 2000, together with packaging vectors, VSV-G and PAX2 (OriGene, USA).
  • VSV-G and PAX2 packaging vectors
  • 6 ug/ml polybrene was added to the growth medium for kidney cells to increase the transduction efficiency.
  • a lentiviral supernatant was collected and concentrated using Lenti-X (Clontech, cat. no.631231).
  • the DCUN1D3 shRNA or SEAT1 shRNA-containing lentivirus thus prepared was transfected into the senescent cells and subcultured in DMEM (hereinafter, referred to as ‘DMEM’) (HyClone, cat.no. SH30243, USA) containing high concentration of glucose, glutamine, and pyruvate and being supplemented with 10% (v/v) fetal bovine serum (FBS), and 1 ⁇ penicillin/streptomycin (100 U/ml peninicillin, 100 ug/ml streptomycin) for 4 weeks under conditions of 37° C. and 5% CO 2 .
  • DMEM DMEM
  • FBS fetal bovine serum
  • penicillin/streptomycin 100 U/ml peninicillin, 100 ug/ml streptomycin
  • the nucleotide sequence of the used DCUN1D3 shRNA was a nucleotide sequence of SEQ ID NO: 1 (sense) and SEQ ID NO: 2 (antisense).
  • the nucleotide sequence of the used SEAT1 shRNA was a nucleotide sequence of SEQ ID NO: 6 and a polynucleotide of SEQ ID NO: 7 (SEAT1 siRNA #1); a nucleotide sequence of SEQ ID NO: 8 and a polynucleotide of SEQ ID NO: 9 (SEAT1 siRNA #2); or a nucleotide sequence of SEQ ID NO: 10 and a polynucleotide of SEQ ID NO: 11 (SEAT1 siRNA #3).
  • Scrambled RNA (Bioneer, Korea) used as a control group was provided by the manufacturer.
  • SEQ ID NO: 1 sense: GUCACUGCAUCGGGAAAUA(dTdT)
  • SEQ ID NO: 2 antisense: UAUUUCCCGAUGCAGUGAC(dTdT)
  • SEAT1 siRNA #1 polynucleotide of SEQ ID NO: 6 (sense) and SEQ ID NO: 7 (antisense)
  • SEAT1 siRNA #2 polynucleotide of SEQ ID NO: 8 (sense) and SEQ ID NO: 9 (antisense)
  • SEAT1 siRNA #3 polynucleotide of SEQ ID NO: 10 (sense) and SEQ ID NO: 11 (antisense)
  • FIG. 6 shows the relative number (A) and DNA content of cultured senescent cells transfected with DCUN1D3 shRNA or SEAT1 shRNA-containing lentivirus.
  • control shRNA represents senescent cells transfected with scramble shRNA-containing lentivirus
  • DCUN1D3 shRNA and SEAT1 shRNA represent senescent cells transfected with DCUN1D3 shRNA (SEQ ID NOs: 1 and 2) or SEAT1 shRNA (SEQ ID NOs: 6 and 7)-containing lentivirus, respectively.
  • the senescent cells transfected with DCUN1D3 shRNA or SEAT1 shRNA-containing lentivirus showed increases in the number of cells and DNA content, compared to senescent cells transfected with scramble shRNA-containing lentivirus, namely, a negative control group. Further, their cell morphology was the same as that of young cells. Therefore, it can be seen that introduction of DCUN1D3 shRNA or SEAT1 shRNA into senescent cells induces proliferation of senescent cells.
  • the DNA content was measured as follows. The cells were seeded in a flat-bottomed 6-well microplate at a density of 20,000 cells/well. The number of cells was determined by measuring SYBR Gold (Invitrogen) fluorescence intensity in a fluorstar microplate reader (BMG Labtec, Cary, N.C., USA) at a predetermined time.
  • Senescent cells were stained blue with X-gal due to increased activity of SA ⁇ -gal. As explained in (1), senescent cells were transfected with DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus, and SA ⁇ -gal activity in the cultured senescent cells was measured. Senescent cells that were transfected with none of DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus were used as a negative control group.
  • FIG. 7 shows an X-gal staining image of senescent cells transfected with scramble shRNA(A) or SEAT1 shRNA(B) or DCUN1D3 shRNA(C)-containing lentivirus, and a percentage (%) of the cells stained blue.
  • senescent cells show a reduction in autophagy activity, accumulation of lipofuscin, and mitochondrial damage.
  • senescent cells were transfected with DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus, and the amount of lipofuscin was measured in the cultured senescent cells.
  • Senescent cells that were transfected with none of DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus were used as a negative control group. Further, the size of the cell was measured.
  • FIG. 8 shows results of measuring the amount of lipofuscin in the senescent cells transfected with DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus.
  • an autofluorescence level of the senescent cells transfected with SEAT1 shRNA or DCUN1D3 shRNA-containing lentivirus was about 10% and about 66%, respectively, compared to that of the senescent cells transfected with scrambled shRNA-containing lentivirus. Therefore, it is confirmed that lipofuscin accumulation was reduced in senescent cells by transfection of the cells with SEAT1 shRNA or DCUN1D3 shRNA-containing lentivirus, indicating that a cellular senescence level is reduced by transfection of senescent cells with SEAT1 shRNA or DCUN1D3 shRNA-containing lentivirus.
  • FIG. 9 shows results of measuring cell size (A) and cell granularity (B) of senescent cells transfected with DCUN1D3 shRNA or SEAT1 shRNA or scramble shRNA-containing lentivirus.
  • A cell size
  • B cell granularity
  • FIGS. 8 and 9 the size, granularity, and autofluorescence of the cells were estimated by flow cytometry.
  • trypsin-treated cells were collected in PBS, and analyzed by FACS Caliber instrument (Becton Dickson, USA). The size and granularity of 100,000 cells were evaluated by previous forward and side scatter. Autofluorescence was measured using a 488-nm laser for excitation and a 530/30 bandpass filter for detection.
  • FSC and SSC in the vertical axis of A and B of FIG. 9 represent forward scatter (cell size) and side scatter (cell granularity), respectively.
  • siRNA and control siRNA were introduced into senescent cells by introducing it into senescent cells using lipofectamine without packaging shRNA or scrambled RNA in lentivirus, and the senescent cells were cultured and then cell cycle was examined.
  • SEAT1 siRNA and DCUN1D3 siRNA were introduced, respectively and therefore, SEAT1 siRNA and DCUN1D3 siRNA were allowed to produce within the cells.
  • FIG. 10 shows results of measuring the cell cycle of the senescent cells introduced with SEAT1 siRNA or DCUN1D3 siRNA or control siRNA.
  • the experiment was performed in triplicate, and culture was performed for 3 days.
  • the senescent cells introduced with SEAT1 siRNA or DCUN1D3 siRNA showed decreased cell percentages in the G0/G1 phase and increased cell percentages in the S phase and G2/M phase, compared to the senescent cells introduced with control siRNA.
  • the unit of the vertical axis of FIG. 10 is %, which is a percentage when the result of control siRNA is taken as 100.
  • SEAT1 shRNA #2 is as follows.
  • SEAT1 siRNA #2 polynucleotide of SEQ ID NO: 8 (sense) and SEQ ID NO: 9 (antisense)
  • DCUN1D3 gene is a target of an Lnc RNA, SEAT1.
  • FIG. 11 shows expression levels of DCUN1D3 gene and SEAT1 gene in human cells (A) and mouse cells (B) introduced with siSEAT1.
  • SEAT1 siRNA was introduced, expression of DCUN1D3 gene as well as expression of SEAT1 gene was reduced, indicating that expression of DCUN1D3 gene is strongly associated with expression of SEAT1 gene. That is, DCUN1D3 gene is a target of SEAT1.
  • each of SEAT1 shRNA and DCUN1D3 shRNA was introduced into senescent HDF M4 cells described in (1) according to the method described in (1), and cultured. Then, monoclonal antibodies specific to DNA damage markers, vH2AX and 53BP1 were used to examine vH2AX and/or 53BP1-positive cells. In detail, cells were grown on a poly-L-lysine-coated cover slip (#08-774-383; Corning Inc., Corning N.Y., USA) or in a 35-mm dish (#81156: Corning Inc.).
  • Cells were fixed with 2% paraformaldehyde in fresh PBS at room temperature for 10 to 20 minutes, and then permeabilized with 0.5% Triton X-100 and 2 ug/ml Hoechst 33342(Sigma-Aldrich) in PBS for 5 minutes. Blocking was performed at room temperature for 30 minutes using 2% BSA in PBS. The sample was incubated with primary antibodies diluted with 2% BSA/PBS-d at room temperature for 1 to 2 hr or overnight, and washed with PBS. Then, the sample was incubated with secondary antibodies (Jackson ImmunoResearch) for 30 minutes.
  • FIG. 12 shows effects of SEAT1 shRNA and/or DCUN1D3 shRNA on intracellular DNA damage.
  • SEAT1 shRNA or DCUN1D3 shRNA when SEAT1 shRNA or DCUN1D3 shRNA is introduced into senescent cells, DNA damage was remarkably reduced, indicating that SEAT1 shRNA or DCUN1D3 shRNA converts senescent cells into young cells, or protects senescent cells, and also indicating that SEAT1 shRNA or DCUN1D3 shRNA may prevent or treat a senescence-associated disease or symptom.
  • “Proliferating” represents HDF M4 cells.
  • the vertical axis in FIG. 12 represents a percentage of vH2AX and 53BP1-double positive cells. As this value is higher, DNA damage is higher.
  • IL-6 interleukin-6
  • IL-6 is a pro-inflammatory cytokine, and expressed by IL6 gene in human. It is known that senescent cells show a higher IL-6 level than young cells. A reduction in the intracellular IL-6 level may be an index of an ability to protect senescent cells or to convert senescent cells into young cells.
  • each of SEAT1 siRNA and DCUN1D3 siRNA was introduced into senescent HDF M4 cells described in (1) according to the method described in (1), and cultured. Then, monoclonal antibodies specific to hIL-6 were used to perform ELISA, and hIL-6 levels in the culture supernatant were measured by Luminex. The results are given in FIG. 13 .
  • FIG. 13 shows effects of SEAT1 or DCUN1D3 gene expression inhibition on hIL-6 expression.
  • siSEAT1 #3, DCUN1D3 #2, and #3 represent siRNAs formed from shRNAs having the following sequences.
  • siSEAT1 #1,2,3 and siDCUN1 D3 #1,2,3 represent co-introduction of three siRNAs, respectively.
  • miR-20b (SEQ ID NO: 16) involved in the regulation of DCUN1D3 gene expression was identified by comparing the sequences of SEAT1 and DCUN1D3 genes with the known miRNA sequences.
  • miR-20b was introduced into senescent HDF M4 cells described in (1) according to the method described in (1), and cultured. Then, monoclonal antibodies specific to DCUN1D3 were used to perform ELISA, and DCUN1D3 levels in the cells were measured. Specifically, miR-20b was transfected into senescent HDF M4 cells according to the following method, and cultured for 3 days. The cells were harvested and RNA was extracted therefrom.
  • the extracted RNA was used as a template to perform RT-PCR, and DCUN1D3 mRNA levels were measured:
  • the transfection of HDF M4 cells was performed using 50 nM sRNA mixed with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. 3 days after transfection, cells were harvested and RNA was extracted therefrom.
  • a miR-20b gain-of-function study was performed using miR-20b mimics (50 nM) and its negative control (50 nM) on the HDF M4 cells.
  • the loss-of-function study was performed with miR-20b inhibitor (75 nM) and its negative control (75 nM) on the HDF M4 cells. The results are given in FIG. 14 .
  • FIG. 14 shows effects of miR-20b on DCUN1D3 gene expression.
  • the vertical axis represents relative DCUN1D3 levels.
  • FIG. 14 when miR-20b was introduced into senescent cells, DCUN1D3 expression was remarkably reduced.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Neurosurgery (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Dermatology (AREA)
  • Psychology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Psychiatry (AREA)
US15/200,937 2015-07-01 2016-07-01 Composition for reducing cellular senescence level including activity inhibitor inhibiting dcun1d3 activity or expression inhibitor inhibiting expression of dcun1d3-encoding gene and use thereof Abandoned US20170002362A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US15/681,262 US10329569B2 (en) 2015-07-01 2017-08-18 Composition for reducing cellular senescence level including activity inhibitor inhibiting DCUN1D3 activity or expression inhibitor inhibiting expression of DCUN1D3-encoding gene and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2015-0094272 2015-07-01
KR20150094272 2015-07-01
KR1020160008900A KR20170004821A (ko) 2015-07-01 2016-01-25 Dcun1d3의 활성을 저해하는 활성 저해제 또는 dcun1d3을 코딩하는 유전자의 발현을 저해하는 발현 저해제를 포함하는 세포의 노화 수준을 감소시키기 위한 조성물 및 그의 용도
KR10-2016-0008900 2016-01-25

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/681,262 Division US10329569B2 (en) 2015-07-01 2017-08-18 Composition for reducing cellular senescence level including activity inhibitor inhibiting DCUN1D3 activity or expression inhibitor inhibiting expression of DCUN1D3-encoding gene and use thereof

Publications (1)

Publication Number Publication Date
US20170002362A1 true US20170002362A1 (en) 2017-01-05

Family

ID=56296680

Family Applications (2)

Application Number Title Priority Date Filing Date
US15/200,937 Abandoned US20170002362A1 (en) 2015-07-01 2016-07-01 Composition for reducing cellular senescence level including activity inhibitor inhibiting dcun1d3 activity or expression inhibitor inhibiting expression of dcun1d3-encoding gene and use thereof
US15/681,262 Active US10329569B2 (en) 2015-07-01 2017-08-18 Composition for reducing cellular senescence level including activity inhibitor inhibiting DCUN1D3 activity or expression inhibitor inhibiting expression of DCUN1D3-encoding gene and use thereof

Family Applications After (1)

Application Number Title Priority Date Filing Date
US15/681,262 Active US10329569B2 (en) 2015-07-01 2017-08-18 Composition for reducing cellular senescence level including activity inhibitor inhibiting DCUN1D3 activity or expression inhibitor inhibiting expression of DCUN1D3-encoding gene and use thereof

Country Status (3)

Country Link
US (2) US20170002362A1 (zh)
EP (1) EP3112466A1 (zh)
CN (1) CN106310275A (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4082554A1 (en) * 2021-04-27 2022-11-02 Amorepacific Corporation Composition for use in inhibiting lipofuscin accumulation or removing lipofuscin

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10208098B2 (en) * 2016-09-15 2019-02-19 Council Of Scientific & Industrial Research Recombinant protein-based method for the delivery of silencer RNA to target the brain
EP3784795A4 (en) * 2018-04-27 2022-01-19 The Regents Of The University Of California INHIBITION OF LIPOFUSCIN AGGREGATION BY MOLECULAR TWEEZERS
WO2020022499A1 (ja) * 2018-07-27 2020-01-30 国立大学法人大阪大学 老化の抑制、加齢性の疾患もしくは症状の予防、改善、もしくは治療、または寿命の延長のための組成物
JPWO2022259950A1 (zh) * 2021-06-10 2022-12-15
CN116041477B (zh) * 2022-10-13 2023-08-08 呈诺再生医学科技(北京)有限公司 Tdgf1基因在制备治疗衰老相关疾病或逆转细胞衰老的药物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009137912A1 (en) * 2008-05-15 2009-11-19 Topigen Pharmaceuticals Inc. Oligonucleotides for treating inflammation and neoplastic cell proliferation
US20120052487A9 (en) * 2002-11-14 2012-03-01 Dharmacon, Inc. Methods and compositions for selecting sirna of improved functionality

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE321857T1 (de) 1999-09-29 2006-04-15 Diagnocure Inc Pca3 mrna in gutartigen und bösartigen prostatageweben
US8354384B2 (en) 2005-06-23 2013-01-15 Yale University Anti-aging micrornas
UY29774A1 (es) 2005-08-31 2007-03-30 Kudos Pharm Ltd Inhibidor de atm
EP2111863B1 (en) 2005-10-26 2012-03-28 Asahi Kasei Pharma Corporation Fasudil in combination with bosentan for the treament of pulmonary arterial hypertension
US20080279866A1 (en) 2006-08-25 2008-11-13 The Brigham And Women's Hospital, Inc. Induction of immunosuppression by inhibition of ATM
US20120010196A1 (en) 2010-04-02 2012-01-12 Qin Quingyu Methods of treating neurodegenerative disorders and diseases
US20120149645A1 (en) 2010-12-13 2012-06-14 Emory University Compositions and modulation of myocyte enhancer factor 2 (mef2)
WO2012158789A2 (en) 2011-05-17 2012-11-22 St. Jude Children's Research Hospital Methods and compositions for inhibiting neddylation of proteins
US20130183369A1 (en) 2011-07-12 2013-07-18 John Douglas Furber Means of inducing cellular exocytosis and uses thereof
KR20140093347A (ko) 2013-01-15 2014-07-28 삼성전자주식회사 항 ｃ-Ｍｅｔ 항체 처리에 의해 부작용을 유발하는 유전자 및 이를 이용한 약물 스크리닝 방법
US20160289763A1 (en) * 2013-11-13 2016-10-06 The Texas A&M University System Micro-rnas that modulate lymphangiogenesis and inflammatory pathways in lymphatic vessel cells
KR102276424B1 (ko) 2014-10-06 2021-07-12 삼성전자주식회사 로-키나제 저해제를 포함하는 세포의 노화를 감소시키기 위한 조성물 및 그의 용도
KR20160049435A (ko) 2014-10-27 2016-05-09 삼성전자주식회사 Atm 저해제를 포함하는 세포의 노화를 감소시키기 위한 조성물 및 그의 용도
KR20160063082A (ko) * 2014-11-26 2016-06-03 삼성전자주식회사 노화 바이오마커 및 그의 용도
KR20160146099A (ko) 2015-06-11 2016-12-21 삼성전자주식회사 Ｒｃｃ1 조절 물질을 포함하는 세포 또는 개체의 노화를 감소시키기 위한 조성물 및 이를 이용한 방법

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120052487A9 (en) * 2002-11-14 2012-03-01 Dharmacon, Inc. Methods and compositions for selecting sirna of improved functionality
WO2009137912A1 (en) * 2008-05-15 2009-11-19 Topigen Pharmaceuticals Inc. Oligonucleotides for treating inflammation and neoplastic cell proliferation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4082554A1 (en) * 2021-04-27 2022-11-02 Amorepacific Corporation Composition for use in inhibiting lipofuscin accumulation or removing lipofuscin
US11744865B2 (en) 2021-04-27 2023-09-05 Amorepacific Corporation Composition for inhibiting lipofuscin accumulation or removing lipofuscin

Also Published As

Publication number Publication date
CN106310275A (zh) 2017-01-11
US10329569B2 (en) 2019-06-25
US20180002703A1 (en) 2018-01-04
EP3112466A1 (en) 2017-01-04

Similar Documents

Publication Publication Date Title
US10329569B2 (en) Composition for reducing cellular senescence level including activity inhibitor inhibiting DCUN1D3 activity or expression inhibitor inhibiting expression of DCUN1D3-encoding gene and use thereof
US20180187199A1 (en) Multi-target modulation for treating fibrosis and inflammatory conditions
JPWO2009020119A1 (ja) ＨＩＦ−１αおよびＨＩＦ−２α発現阻害物質含有医薬
US20130323241A1 (en) Treatment of angiogenesis disorders
US10537591B2 (en) Method for promoting muscle regeneration
US20140343124A1 (en) Method for reducing expression of downregulated in renal cell carcinoma in malignant gliomas
KR101541974B1 (ko) miR29b를 포함하는 신경줄기세포의 신경세포로의 분화 촉진용 조성물 및 방법
US20160220599A1 (en) Novel lincrna and interfering nucleic acid molecules, compositions and methods and uses thereof for regulating angiogenesis and related conditions
JP5850702B2 (ja) 間葉系細胞の分化調節剤およびこれを用いた医薬、並びに間葉系細胞への分化調節作用を有する物質のスクリーニング方法
EP3220901A1 (en) Means and methods for treatment of early-onset parkinson's disease
US20230287427A1 (en) Inhibition of lncExACT1 to Treat Heart Disease
EP3406719A1 (en) Calcineurin b homologous protein 1 inhibitors and therapeutic and non-therapeutic uses thereof
KR102301572B1 (ko) 신규한 핵산 분자 전달용 조성물 및 그 용도
WO2021207854A1 (en) Compositions and methods for inhibiting tdp-43 and fus aggregation
KR102177130B1 (ko) 근육 질환 및 신경근육 질환 예방, 치료 또는 진단을 위한 miR-18b의 용도
US20220186228A1 (en) Synthetic microrna mimics
US8188264B2 (en) RNAi mediated knockdown of NuMA for cancer therapy
US9567583B2 (en) Method for treating glioma using Tarbp2 expression inhibitor
KR20170004821A (ko) Dcun1d3의 활성을 저해하는 활성 저해제 또는 dcun1d3을 코딩하는 유전자의 발현을 저해하는 발현 저해제를 포함하는 세포의 노화 수준을 감소시키기 위한 조성물 및 그의 용도
KR20140143880A (ko) miRNA의 발현 증가를 이용한 성체줄기세포의 노화 억제 조성물
US20150111950A1 (en) Inhibition of dna2 in fanconi anemia
Lim Development of Antisense Therapies for Facioscapulohumeral and Duchenne Muscular Dystrophy
US20120035241A1 (en) Use of inhibitors of plac8 activity for the modulation of adipogenesis
KR20230068278A (ko) Upf1 단백질 또는 upf1 단백질 유래 폴리펩티드의 암의 예방 또는 치료 용도
EP3145553A1 (en) Small interfering rna (sirna) for the therapy of type 2 (ado2) autosomal dominant osteopetrosis caused by clcn7 (ado2 clcn7-dependent) gene mutation

Legal Events

Date Code Title Description
AS Assignment

Owner name: SAMSUNG ELECTRONICS CO., LTD., KOREA, REPUBLIC OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KIM, MYOUNGSOON;KIM, YONGSUB;LEE, YOUNG-SAM;REEL/FRAME:039066/0952

Effective date: 20160630

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION