WO2022135486A1 - 鉴定和/或调节衰老的方法 - Google Patents

鉴定和/或调节衰老的方法 Download PDF

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WO2022135486A1
WO2022135486A1 PCT/CN2021/140550 CN2021140550W WO2022135486A1 WO 2022135486 A1 WO2022135486 A1 WO 2022135486A1 CN 2021140550 W CN2021140550 W CN 2021140550W WO 2022135486 A1 WO2022135486 A1 WO 2022135486A1
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erv
hervk
cells
inhibitor
locus
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PCT/CN2021/140550
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French (fr)
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刘光慧
曲静
张维绮
刘晓倩
刘尊鹏
武泽明
王思
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中国科学院动物研究所
北京干细胞与再生医学研究院
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Priority claimed from CN202011527970.9A external-priority patent/CN114657281A/zh
Application filed by 中国科学院动物研究所, 北京干细胞与再生医学研究院 filed Critical 中国科学院动物研究所
Priority to CN202180048695.9A priority Critical patent/CN116157529A/zh
Priority to EP21909481.0A priority patent/EP4163379A4/en
Publication of WO2022135486A1 publication Critical patent/WO2022135486A1/zh
Priority to US18/150,917 priority patent/US20240060131A1/en

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Definitions

  • the present invention relates to the field of biomedicine.
  • the present invention provides methods of identifying and/or modulating aging. More specifically, the present invention relates to identifying the degree of senescence of a cell, tissue, organ or object by detecting the level of endogenous retrovirus activation, or providing for the modulation of a cell, tissue, organ or object by inhibiting or promoting the level of endogenous retrovirus activation. A method for the degree of aging of the subject.
  • geriatric medicine covers a wide area, not only for the old age, but for the whole life cycle, with the goal of prolonging the healthy time of people in a state of younger physical function. Therefore, the discovery of biological markers that can identify the degree of aging of an individual or its cells, tissues or organs, and therapeutic targets for preventing or delaying the aging of an individual or its cells, tissues or organs is of great interest to geriatrics and the field of medicine as a whole. very important.
  • telomere shortening due to DNA end duplication, DNA damage, altered activity of tumor suppressor genes and oncogenes, oxidative stress, inflammation, chemotherapeutic agents, and exposure to ultraviolet radiation and ionizing radiation Wait.
  • the secretome is one of the hallmarks of cellular senescence, which is a phenotype with significantly increased expression and secretion of different kinds of cytokines, and is also the most important environmental effect produced by senescent cells, known as the senescence-associated secretory phenotype. associated secretory phenotype, SASP). These secreted factors facilitate communication with neighboring cells and the immune system, ultimately affecting the fate of senescent cells.
  • SASP can promote tumor cell development by secreting factors that promote angiogenesis, extracellular matrix remodeling, or epithelial-mesenchymal transition (EMT).
  • EMT epithelial-mesenchymal transition
  • aging-induced chronic inflammation can cause systemic immune dysregulation, leading to aging-related tissue damage and degeneration, and the occurrence of aging-related diseases.
  • Retroelements include long terminal repeats (LTRs) and non-LTR retrotransposons.
  • LTR retrotransposons include endogenous retroviruses (ERVs), which are relics of ancient retroviruses that infected humans and integrated into the human genome, where they were fixed during evolution.
  • Human endogenous retroviruses (HERVs) comprise approximately 8% of the human genome.
  • the present invention provides a method for identifying the degree of aging of a subject or its cells, tissues or organs or assessing the physiological age of a subject, or diagnosing diseases associated with aging, such as progeria and frailty, or assessing the use of isolated animal cells or cell populations.
  • methods of aging including
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof.
  • the reference value is obtained by detecting a reference subject or reference cell. In some embodiments, the reference value is obtained by detecting a population of reference subjects or a population of cells.
  • the level of activation of the ERV includes ERV locus methylation levels, ERV transcript levels, ERV protein levels, presence, localization and/or release of ERV particles in cells, and/or ERV The transposition probability of the locus.
  • the activation level of the ERV includes ERV locus methylation levels, ERV transcript levels, ERV protein levels, and/or the presence and/or release of ERV particles in cells.
  • the activation level of the ERV includes the transposition probability of the ERV locus.
  • the level of activation of the ERV is manifested by the localization of ERV particles in the cell.
  • the subject or animal is a mammal. In some embodiments, the subject or animal is a non-human mammal.
  • the subject or animal is a mouse, and optionally, the ERV is from the MMTV family; or the subject or animal is a non-human primate, and optionally, the ERV is from the ERVW family.
  • the subject or animal is a human, and optionally, the ERV is from the HERVK family.
  • the sample is a body fluid sample, including but not limited to urine, saliva, and blood.
  • the blood includes, but is not limited to, whole blood, plasma, or serum.
  • the sample comprises tissue from the subject or animal.
  • the tissue includes, but is not limited to, skin, lung, liver tissue.
  • the sample comprises cells from the subject or animal.
  • the cells include, but are not limited to, fibroblasts and mesenchymal stem cells.
  • the present invention provides a method of treating premature aging in a subject or cells, tissues or organs thereof, or preventing or delaying aging in a subject or cells, tissues or organs thereof.
  • the method comprises administering to the subject an inhibitor of ERV locus activation or an inhibitor of the ERV.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof.
  • the ERV locus activation inhibitor increases the methylation level of the ERV locus, reduces the level of the ERV locus transcript, reduces the level of the ERV locus protein, reduces the level of the ERV locus in the ERV locus present in and/or released from cells.
  • the subject or animal is a mammal. In some embodiments, the subject or animal is a non-human mammal.
  • the subject or animal is a mouse, and optionally, the ERV is from the MMTV family; or the subject or animal is a non-human primate, and optionally, the ERV is from the ERVW family.
  • the subject or animal is a human, and optionally, the ERV is from the HERVK family.
  • the method comprises administering to the subject a retroviral inhibitor, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • a retroviral inhibitor eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the present invention provides a method of modulating cellular senescence comprising contacting the cell with an ERV locus activation modulator.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof.
  • the modulator is an ERV locus activation inhibitor or an inhibitor of the ERV.
  • the ERV locus activation inhibitor increases the methylation level of the ERV locus, reduces the level of the ERV locus transcript, reduces the level of the ERV locus protein, reduces the level of the ERV locus in the ERV locus present in and/or released from cells.
  • the modulator is an ERV locus activation promoter of the cell.
  • the ERV locus activation promoter reduces the methylation level of the ERV locus, increases the transcript level of the ERV locus, increases the protein level of the ERV locus, increases the level of the ERV locus at the ERV locus present in and/or released from cells.
  • the subject or animal is a mammal. In some embodiments, the subject or animal is a non-human mammal.
  • the subject or animal is a mouse, and optionally, the ERV is from the MMTV family; or the subject or animal is a non-human primate, and optionally, the ERV is from the ERVW family.
  • the subject or animal is a human, and optionally, the ERV is from the HERVK family.
  • the present invention provides a method of culturing isolated animal cells or cell populations or preventing or delaying cellular senescence.
  • the method comprises contacting the cell with an inhibitor of ERV locus activation of the cell or an inhibitor of the ERV.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof.
  • the ERV locus activation inhibitor increases the ERV locus methylation level, reduces the ERV locus transcript level, reduces the ERV locus protein levels, decrease in the presence of and/or release from cells of the ERV particles.
  • the subject or animal is a mammal. In some embodiments, the subject or animal is a non-human mammal.
  • the subject or animal is a mouse, and optionally, the ERV is from the MMTV family; or the subject or animal is a non-human primate, and optionally, the ERV is from the ERVW family.
  • the subject or animal is a human, and optionally, the ERV is from the HERVK family.
  • the method comprises contacting the cell with a retroviral inhibitor.
  • the retroviral inhibitor comprises a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the present invention provides a method of promoting cellular senescence.
  • the method comprises contacting the cell with an ERV locus activation promoter of the cell or the ERV.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof.
  • the modulator is an ERV locus activation promoter of the cell.
  • the ERV locus activation promoter reduces the methylation level of the ERV locus, increases the transcript level of the ERV locus, increases the protein level of the ERV locus, increases the level of the ERV locus at the ERV locus present in and/or released from cells.
  • the cells are mammalian cells.
  • the cells are non-human mammalian cells.
  • the cell is a mouse cell, and optionally, the ERV is from the MMTV family; or the cell is a non-human primate cell, and optionally, the ERV is from the ERVW family.
  • the cells are human cells, and optionally, the ERV is from the HERVK family.
  • the method comprises contacting the cell with a DNA methyltransferase inhibitor, such as decitabine, Lomeguatrib, SGI-1027, or 5-azacytidine.
  • a DNA methyltransferase inhibitor such as decitabine, Lomeguatrib, SGI-1027, or 5-azacytidine.
  • the cells are mammalian cells, such as mouse cells, non-human primate cells, and human cells.
  • the present invention provides a method for preventing or delaying degeneration of tissues and organs, treating related diseases, and delaying body aging, such as methods for preventing or delaying skin aging, treating or preventing osteoarthritis and frailty. .
  • the method comprises administering a retroviral inhibitor to a subject in need thereof.
  • the retroviral inhibitor comprises a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the method comprises administering to a subject in need thereof an inhibitor of HERVK locus activation or a HERVK inhibitor.
  • said HERVK locus activation inhibitor said ERV locus activation inhibitor increases said HERVK locus methylation level, decreases said HERVK locus transcript level, decreases said HERVK locus protein levels, decrease in the presence of and/or release from cells of the HERVK particles.
  • the administration is topical, intradermal, or subcutaneous.
  • the present invention provides a method of treating progeria, such as Hutchinson-Gilford Progeria Syndrome (HGPS) or Werner Syndrome (WS).
  • HGPS Hutchinson-Gilford Progeria Syndrome
  • WS Werner Syndrome
  • the method comprises administering to a subject in need thereof an inhibitor of HERVK locus activation or a HERVK inhibitor.
  • said HERVK locus activation inhibitor said ERV locus activation inhibitor increases said HERVK locus methylation level, decreases said HERVK locus transcript level, decreases said HERVK locus protein levels, decrease in the presence of and/or release from cells of the HERVK particles.
  • the method comprises administering a retroviral inhibitor to a subject in need thereof.
  • the retroviral inhibitor comprises a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • Figure 1 The left panel is a schematic diagram of the progeria cell model; the right panel is a schematic diagram of HERVK activation (including transcription, translation, packaging and release).
  • Figure 2 Schematic illustration of the integration of the ERVW and ERVK families into the genome during primate evolution.
  • Figure 3A Left panel: doughnut plot showing the composition of shared up-regulated repeat elements (RE) and replicative aged (RS) hMPCs in premature senescence classified by each repeat element class. REs that were up-regulated in at least three comparisons were considered (enlarged REs are shown in Figure 3C).
  • Right panel Family enrichment analysis of premature senescence and shared up-regulated REs of LTR classes in RS hMPCs.
  • Figure 3B is a heatmap showing the relative expression levels (log2 (fold change)) of the RepeatMasker-annotated repeat elements in premature senescence and RS hMPCs.
  • the bar graph of Figure 3C shows an overlay of premature senescence and RepeatMasker-annotated repeat elements that are upregulated in RS hMPCs.
  • the heatmap of Figure 3D shows the relative expression levels of all proviral sequences of HERVK in prematurely senescent hMPCs, with the color key from blue to red representing low to high expression levels.
  • Figure 3E is a volcano plot showing differential expression of all proviral sequences of HERVK in prematurely senescent hMPCs.
  • Figure 3F shows that HERVK is upregulated at the RNA level at proviral HML2_1q22 in prematurely senescent hMPCs.
  • Figure 4A is a heatmap showing HERVK RNA levels detected by RT-qPCR
  • Figure 4B left panel is a representative image of HERVK RNA-FISH
  • right panel is a quantitative analysis of fluorescence signal intensity.
  • Figure 5 is a violin plot showing CpG DNA methylation levels of HERVK.
  • Figure 7A is the result of immunofluorescence staining for HERVK-Env in hMPC of WT, HGPS and WS;
  • Figure 7B is the result of western blotting of HERVK-Env, p16 INK4a and LAP2 in hMPC of WT, HGPS and WS.
  • Figure 7C is the result of immunofluorescence staining for HERVK-Env in hMPC of RS;
  • Figure 7D is the result of western blotting of HERVK-Env, p16 INK4a and LAP2 in hMPC of RS.
  • Figure 8 shows the RVLP in hMPC of WT, HGPS and WS and RS stained with heavy metals, the left panel is the TEM image, the right panel is the RVLP count result, the RVLP in hMPC of HGPS, WS and RS was significantly increased.
  • Figure 9 shows the results of TEM analysis of the immunogold label against HERVK-Env.
  • the left panel in Figure 9A is the TEM image of hMPC of WT, HGPS and WS, the scale is 200nm (left) and 100nm (right), the dotted line represents the cell membrane, E: extracellular, I: intracellular; the right panel is the result of statistical analysis.
  • the left panel is the control TEM without primary antibody
  • the scale bar is 200nm (upper) and 100nm (lower)
  • the dotted line represents the cell membrane, E: extracellular, I: intracellular
  • the middle panel is the TEM image of hMPC of RS
  • the scale bars are 200 nm (left) and 100 nm (right)
  • the dotted line represents the cell membrane, E: extracellular, I: intracellular
  • the right panel is the result of statistical analysis.
  • Figure 10 shows the results of the analysis of transposition probability for HERVK occurrence.
  • Figure 10A is a schematic diagram of constructing a vector for detecting HERVK transposition probability;
  • Figure 10B shows the transposition probability of HERVK in hMPCs of RS analyzed by flow cytometry, and the ratio of green fluorescence to red fluorescence represents the transposition probability;
  • Figure 10C shows HERVK copy number in the genome detected by single-molecule DNA-FISH in hMPCs of WT, HGPS and WS;
  • Figure 10D shows HERVK copy number in the genome detected by single-molecule DNA-FISH in hMPC of RS.
  • Figure 11 shows epigenetic de-repression of HERVK in RS fibroblasts.
  • Figure 12 shows that HERVK-Env protein level is increased after activation by the CRISPR-dCas system (12A); qPCR heat map (12B) shows that HERVK-Env expression is up-regulated, and it is related to the up-regulation of senescence positive marker p16 INK4a expression and negative marker LAP2 expression down-regulation; SA- ⁇ -gal staining (12C), Ki67 staining (12D) and clonal expansion ability (12E) showed cellular senescence.
  • Figure 13 shows that knockdown of HERVK with shRNA in hMPCs of HGPS and WS reduces the senescence phenotype.
  • Figure 14 shows that HERVK-Env protein levels are reduced (14A) after systematic inhibition by Cas9KRAB, and SA- ⁇ -gal staining (14B), Ki67 staining (14C) and clonal expansion ability (14D) show delayed cellular senescence.
  • Figure 15 shows that 5-AZA treatment results in decreased HERVK-LTR5HS methylation (14A), activation of HERVK (increased levels of HERVK RNA, 14B), and induction of senescence (14B-E), while knockdown of HERVK (14F) effectively reduces 5 - AZA-induced senescence phenotype (reduced SA- ⁇ -gal staining positive cells, 14G).
  • Figure 16 shows that cellular senescence is delayed with HERVK reverse transcriptase inhibitors and integrase inhibitors.
  • Figure 16A is a schematic diagram of the life cycle of HERVK, in which reverse transcriptase and integrase can be inhibited by small molecules; flow cytometry analysis of Figure 16B shows that the reverse transcriptase inhibitor Abacavir reduces the transposition efficiency of HERVK;
  • Figure 16C shows reverse transcription The enzyme inhibitor Abacavir decreased HERVK DNA copy number; the reverse transcriptase inhibitors Abacavir, Lamivadine, and integrase inhibition of Ratelgravir decreased the number of SA- ⁇ -gal staining positive cells ( Figure 16C) and increased the number of Ki67 positive cells ( Figure 16D).
  • Figure 17 is a schematic representation of the cGAS pathway that induces innate immune responses.
  • Figure 18 shows analysis of cGAS enrichment on cytoplasmic HERVK DNA by immunoprecipitation and qPCR.
  • Figure 19 shows cGAS pathway activation in prematurely senescent hMPCs, inducing an innate immune response.
  • Figure 19A shows ELISA analysis of 2'3'-cGAMP levels in WT, HGPS and WS hMPC;
  • Figure 19B shows ELISA analysis of IL-6 levels in culture medium of WT, HGPS and WS hMPC;
  • Figure 19C shows WT, HGPS and WS Western blot analysis of p-TBK1, p-IRF3 and p-RelA in hMPCs.
  • Figure 20 shows that knockdown of HERVK inhibits the cGAS pathway and innate immune responses in HGPS and WS hMPCs.
  • the results of knockdown of HERVK ( Figures 20A and B) showed a trend consistent with the results of knockdown of STING ( Figures 20C-E).
  • Figure 21A shows analysis of HERVK DNA copy number in WT, HGPS and WS hMPC by ddPCR
  • Figure 21B shows ELISA analysis of HERVK-Env levels in WT, HGPS and WS hMPC media
  • Figure 21C shows HERVK-Env of WT, HGPS and WS hMPC Immunogold-labeled TEM images (bars are 200 nm (left) and 100 nm (right), dotted lines indicate cell membranes, E: extracellular, I: intracellular).
  • Figure 22 shows that incubation with old CM induces senescence in young hMPCs.
  • Figure 23 shows that after removal of HERVK particles in old CMs, their ability to induce senescence is reduced.
  • Figure 24 shows the in vitro synthesis and purification of live HERVK virus bearing GFP.
  • 293T cells were transfected with GFP-bearing HERVK vector, and HERVK viral RNA, viral protein, and viral particles were all detected ( Figure 24B-E).
  • Figure 25 shows that hMPCs were infected with purified GFP-bearing live HERVK virus, HERVK virus could enter target cells (Figure 25B-G), activate innate immune pathways and up-regulated the expression of inflammatory factors (Figure 25H-J), resulting in hMPC senescence (Figure 25H-J) 25K-M).
  • Figure 26 shows that neutralization of live HERVK virus with HERVK antibody reduces innate immune response and expression of inflammatory factors (Figure 26E-F) and alleviates hMPC senescence ( Figure 26B-D).
  • Figure 27A shows RT-qPCR analysis of MMTV in liver tissue of young and old mice
  • Figure 27B shows Western blot analysis of MMTV-Env and p-RelA in lung tissue of young and old mice
  • 27C shows young and old mice, respectively Quantitative analysis of immunohistochemical staining for MMTV-Env in murine lung, skin and liver tissues.
  • Figure 28A shows Western blot analysis of ERVW-Env and p-RelA in HGPS and WT cynomolgus monkey lung tissues
  • Figure 22B shows immunohistochemical staining of ERVW-Env in HGPS and WT cynomolgus monkey lung, skin and liver tissues, respectively Quantitative analysis of ERVW-Env in young and old cynomolgus monkey lung tissues
  • Figure 22C shows the quantitative results of ERVW-Env western blot analysis in young and old cynomolgus monkey lung tissues
  • Figure 22D shows the expression of ERVW-Env in young and old cynomolgus monkey lung, skin and liver tissues Quantitative analysis of immunohistochemical staining.
  • Figure 29A shows RT-qPCR results of the HERVK gene in primary hPMCs from young and old humans
  • Figure 23B shows immunohistochemical staining for HERVK-Env in skin tissue from young and old humans, representative on the left Sex images, right panel results of statistical analysis
  • Figure 23C shows ELISA analysis of HERVK in sera from young and old humans.
  • Figure 30A shows a schematic diagram of the culture of young-derived primary hPMCs with young and old human sera and HERVK-depleted aged sera; RT-qPCR analysis of inflammatory factors in primary hPMCs derived from young adults cultured with human serum of SA- ⁇ -gal analysis in primary hPMCs.
  • Figure 31A shows the experimental flow chart of injecting MMTV-inhibiting lentivirus (sgRNA-encoding lentivirus, the sgRNA targets MMTV) into the joints of aged mice;
  • Figure 31B shows that MMTV protein levels are reduced after systemic inhibition by Cas9KRAB;
  • Figure 31C shows After 8 weeks of injection, the tension level of the limbs of the mice was tested;
  • Figure 31D shows that 10 weeks after injection, the bone mineral density of the joint was detected by micro-NMR;
  • Figure 31E shows the RT-qPCR analysis of inflammatory factors in the joint after 10 weeks of injection.
  • Figure 32A shows the experimental flow chart of injecting the reverse transcriptase inhibitor Abacavir into the joints of aged mice;
  • Figure 32B shows the test of the limb pulling force level of mice after 8 weeks of injection of the reverse transcriptase inhibitor Abacavir;
  • Figure 3C shows the injection of the reverse transcriptase inhibitor After 13 weeks of Abacavir, mice were tested for running ability;
  • Figure 32D shows that after 14 weeks of injection of the reverse transcriptase inhibitor Abacavir, the bone mineral density of the joint was detected by micro-NMR;
  • Figure 32D shows the RT of inflammatory factors in the joint after injection of the reverse transcriptase inhibitor Abacavir - qPCR analysis.
  • Figure 33A shows a schematic diagram of daily administration to aged mice by dissolving the reverse transcriptase inhibitor Abacavir in drinking water
  • Figure 33B shows the test of the limb tension level in mice after administration of the reverse transcriptase inhibitor Abacavir for 12 weeks
  • Figure 33C shows After 24 weeks of administration of the reverse transcriptase inhibitor Abacavir, the body weight, skin, hair, arched back, cataracts and mobility of the mice were tested
  • Figure 33D shows the survival curve after administration of the reverse transcriptase inhibitor to the aged mice.
  • Figure 34A shows a schematic diagram of administration of the integrase inhibitor Ratelgravir to aged mice
  • Figure 34B shows the test of the level of pulling force on the limbs of the mice, and the body weight, skin, hair, arched back of the mice 16 weeks after the administration of the reverse transcriptase inhibitor Abacavir , cataract and mobility tests.
  • Figure 35 shows the results of anti-MMTV antibodies for western blot analysis of MMTV-Env protein in mice ( Figure 35A) and immunohistochemical staining experiments for MMTV ( Figure 35B).
  • the term “and/or” covers all combinations of the items linked by the term, as if each combination had been individually listed herein.
  • “A and/or B” covers “A”, “A and B", and “B”.
  • “A, B and/or C” encompasses "A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and "A and B and C”.
  • subject refers to non-human animal subjects such as non-human mammals, birds, reptiles, fish, etc., including livestock, poultry, pets, racing animals, or human subjects.
  • Aging of a subject generally refers to the progressive reduction of an organism's physical and/or psychological adaptability to the environment, with a gradual progression toward death. Aging can be divided into physiological aging and pathological aging. Physiological aging refers to the physiological degeneration process after maturity, while pathological aging is the senile changes caused by various external factors (including various diseases). However, aging is the result of the combined action of many pathological, physiological and psychological processes, and in fact in most cases it is impossible to completely distinguish between physiological aging and pathological aging.
  • the “aging degree” of the subject is usually related to the "biological age”, which refers to the level reflected by the subject's physiology and its function when the subject exhibits a certain natural age, that is, the performance degree of the physiology and its function corresponding to a certain natural age. .
  • Premature aging of a subject refers to a subject whose biological age is significantly greater than its natural age, usually due to factors such as the environment, disease, overwork, and the like.
  • premature aging includes hereditary progeria, such as Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner Syndrome (WS).
  • HGPS Hutchinson-Gilford Progeria Syndrome
  • WS Werner Syndrome
  • senescence is considered to be an important cause of biological aging, usually referring to cells that maintain viability and metabolic activity but lose the ability to proliferate. Distinctive features of senescent cells include (i) growth arrest, (ii) enlarged and flattened cell morphology, (iii) foci of DNA damage in the nucleus, (iv) senescence-associated secretory phenotype (SASP), (v) senescence-associated The ⁇ -galactosidase (SA- ⁇ -gal) activity was increased, (vi) the expression of the tumor suppressor p16 INK4a was increased, and (vii) the number and size of PML nuclear bodies were increased.
  • the senescence phenotype pathways that lead to cells typically include replicative senescence (RS), premature senescence, and postdifferentiation senescence (SAD).
  • RS replicative senescence
  • SAD postdifferentiation senescence
  • "Replicative senescence” is the type of senescence that occurs after a large number of cell divisions. For example, when grown in culture, primary cells undergo cellular senescence after approximately 50 cell divisions. This barrier to further proliferation is thought to be due to shortening of the cell's telomeres with each successive cell division, causing the cell to reach a point at which a DNA damage response is triggered (the so-called "Hayflick limit”), ultimately leading to induction of proliferative arrest and senescence.
  • Premature senescence of cells refers to cellular senescence in the absence of telomere loss or dysfunction. Premature aging can be caused by a variety of stimuli including, for example, chemotherapy, radiation therapy, DNA damage, oxidative stress, inflammation, strong mitotic signaling, and ribosomal stress. In addition, genetic defects in cells can also contribute to premature cellular senescence, for example, the cells of HGPS or WS patients mentioned above can experience premature senescence.
  • SAD refers to a senescence-like phenotype (including SASP) exhibited by terminally differentiated post-mitotic cells that can be induced by a variety of stressors, including genotoxic, proteotoxic, oxidative, and ribosomal stressors. Studies have shown that SAD is observed in certain diseases.
  • the "degree of senescence" of a cell means that its senescence-related phenotype is comparable to the corresponding phenotype of a cell at a particular number of passages.
  • An "endogenous retrovirus (ERV)” is a retrovirus-derived genomic sequence generally considered to be a relic of a retrovirus integrated into germline chromosomes during evolution (eg, millions of years ago). Although most ERVs are defective and in most cases inactive, they may be activated under certain physiological and pathological conditions. For example, the ERVW family inserted into the primate germline chromosome about 25 million years ago, while the HERVK family inserted into the human germline chromosome about 6 to 7 million years ago. Studies have shown that, under certain conditions, MMTV remains active in rodents such as mice. The ERVW family remains active in non-human primates such as Old World monkeys, while the HERVK family remains active in humans. ERV loci typically include flanking LTR regions and coding sequences.
  • reference value is meant to include a representative of the morphology, function and metabolites of a normal subject or its cells, tissues or organs in a specific state (such as age or passage number), or normal isolated cells or cell populations Indicator constants for physiological and biochemical characteristics, also known as normal values.
  • the reference value may be a predetermined value, or may be a value obtained by detecting a reference object or a reference cell.
  • a "reference subject” or “reference cell” refers to a subject or cell having the above-mentioned representative characteristics.
  • a reference object in the present invention refers to a subject having representative characteristics such as a specific degree of aging or physiological age
  • a reference cell in the present invention refers to a cell having representative characteristics such as a cell that has been passaged for a specific number of times.
  • locus refers to a region on a chromosome that includes one or more genes, gene portions and/or regulatory sequences.
  • homologues of ERVs are ERVs of other species that are phylogenetically homologous to them or derived from homologs of the same virus family. For example, according to Stockinga & Kozak, Cell. Mol. Life Sci. 65 (2008) 3383-3398, HERVK is a homologue of MMTV in mice.
  • ERV locus activation refers to the transition of the ERV locus from a silent state to a state in which transcription, translation and even packaging and secretion of viral particles are possible.
  • Inhibitors/promoters of ERV locus activation encompass any substance capable of inhibiting/promoting (including specifically inhibiting/promoting and non-specifically inhibiting/promoting) ERV locus activation.
  • nonspecific retroviral inhibitor refers to a retroviral inhibitor that is not specific for a particular ERV locus.
  • nucleic acid molecule includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA produced using nucleotide analogs.
  • the nucleic acid molecule may be single-stranded or double-stranded, preferably double-stranded DNA.
  • the nucleic acid can be synthesized using nucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids with altered base-pairing ability or increased nuclease resistance.
  • the term "encode” refers to a polynucleotide that directly specifies the amino acid sequence of its protein product.
  • the boundaries of the coding sequences are generally defined by open reading frames, which typically begin with the ATG start codon or additional start codons such as GTG and TTG and end with stop codons such as TAA, TAG and TGA.
  • the coding sequence may be a DNA, cDNA or recombinant nucleotide sequence.
  • antisense nucleic acid refers to a nucleic acid molecule that has a complementary sequence to a target nucleic acid (eg, mRNA), and participates in the regulation of gene expression through base-pairing binding with the target nucleic acid.
  • Antisense nucleic acids include RNA or DNA molecules that are precisely complementary to a specific mRNA, specifically blocking its translation.
  • interfering nucleic acid refers to nucleic acid molecules encoding RNA molecules for RNA interference (RNAi), including siRNA, shRNA, miRNA, and the like.
  • RNAi RNA interference
  • protein refers to a biological macromolecule composed of one or more "polypeptide” chains.
  • Polypeptide refers to a chain containing ten or more amino acid residues linked by peptide bonds. All peptide and polypeptide formulas or sequences herein are written left to right, indicating the orientation from the amino terminus to the carboxy terminus.
  • amino acid In the context of peptides, the terms "amino acid”, “residue” and “amino acid residue” are used interchangeably and include both naturally occurring amino acids and unnatural amino acids in proteins.
  • amino acid residue The one-letter and three-letter names of the amino acids naturally occurring in proteins use the names commonly used in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
  • percent identity refers to comparing the amino acids of two polypeptides or the nucleotides of two nucleic acid molecules that, when optimally aligned, have approximately the same amino acid as specified or percentage of nucleotides.
  • 95% amino acid/nucleotide identity refers to comparing the amino acids of two polypeptides or the nucleotides of two nucleic acid molecules that, when optimally aligned, have 95% amino acid/nucleotide identity Nucleotides are the same.
  • an "isolated" cell refers to a cell that is separated from the organism from which it is derived, and generally refers to an ex vivo cultured cell, including but not limited to adherent cultured cells, suspension cultured cells, or 3D cultured cells.
  • the CRISPR/Cas system refers to clustered regularly interspaced short palindromic repeats and related systems, which can cut DNA strands at specific positions under the guidance of guide RNAs.
  • the CRISPR-dCas system involves the loss of the nuclease activity of Cas through modification, so that it cannot cut the DNA strand, and at the same time, it is linked to the transcriptional activation domain to achieve the effect of site-specific transcriptional activation.
  • the CRISPR-dCas system can also be used to modulate the methylation level of the region of interest to regulate transcription.
  • dCas can be linked to methylation-promoting sequences, such as amino acid sequences with methyltransferase activity (e.g., CRISPR-dCas-SunTag-DNMT3A), to increase methylation levels in the region of interest, thereby Reduce transcription of the region of interest.
  • dCas with sequences that inhibit methylation, such as the Tet (ten-eleven translocation) catalytic domain (CRISPR-dCas-Tet), to reduce methylation levels in the region of interest, thereby increasing the region of interest transcription.
  • the present invention provides a method for identifying the aging degree of a subject or its cells, tissues or organs, or assessing the physiological age of the subject, or diagnosing progeria, comprising:
  • ERVs eg, by epigenetics.
  • ERVs are able to bypass organismal surveillance such that their loci are activated due to changes in epibiological regulation (such as DNA methylation) so that retroviral elements begin transcription, retrotransposition, or even Produces and releases retrovirus-like particles (RVLPs) capable of infecting cells.
  • epibiological regulation such as DNA methylation
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the "activation level" of an ERV locus includes ERV locus methylation levels, ERV transcript levels, ERV protein levels, and/or ERV particles present in and/or released from cells. Lower methylation levels, higher transcript levels, higher protein levels, and/or higher ERV particles present in or released from cells represent higher levels of ERV locus activation.
  • the activation level of the ERV locus includes the transposition probability of the ERV locus. A higher probability of transposition represents a higher level of activation.
  • the reference value is obtained by detecting a reference object.
  • the reference subject refers to a subject at a specific biological age.
  • the desired reference object can be determined by detecting representative characteristics associated with biological age, such as characteristics selected from brain activity, skin elasticity, reflex action, balance, and the like. Due to individual differences, preferably, the reference value is obtained by detecting a population of reference subjects.
  • a statistically significant number may also be selected to be at a specific natural age (eg 20, 30, 40, 50, 60, 70, 80, 90 or higher) or natural age range Normal subjects (eg, 18-22 years old, 28-32 years old, 38-42 years old, 48-52 years old, 58-62 years old, 68-72 years old, 78-82 years old, 88-92 years old, etc.) are used as reference objects.
  • a specific natural age eg 20, 30, 40, 50, 60, 70, 80, 90 or higher
  • Normal subjects eg, 18-22 years old, 28-32 years old, 38-42 years old, 48-52 years old, 58-62 years old, 68-72 years old, 78-82 years old, 88-92 years old, etc.
  • the reference value can be expressed in the form of "mean ⁇ standard deviation" or in the form of a range. Where a population is tested to determine reference values, the highest and lowest parts of the measured values can also be removed, eg the highest 2.5% and/or the lowest 2.5%.
  • the detection result ie, the activation level of the ERV locus
  • the subject or the degree of aging of its cells, tissues or organs, or the physiological age of the subject is identified as being higher than the reference value.
  • the reference value is obtained by detecting a reference subject or population of reference subjects.
  • the aging degree of the object or its cells, tissues or organs or the physiological age of the object is identified as being higher than the reference object or the population of the reference object or its cells, tissues or the (average) degree of aging of the organ or the (average) physiological age of the reference subject or the reference subject; when the test result is lower than the reference value, the degree of aging of the subject or its cells, tissues or organs or The physiological age of the subject is identified as being below the (average) degree of aging of the reference subject or a population of the reference subjects or cells, tissues or organs thereof or the (average) physiological age of the reference subject or the reference subject age.
  • the reference value is a value or range that represents the activation level of the ERV locus for a reference subject or population of reference subjects, including a value or range of ERV locus methylation levels, ERV transcript levels A value or range of values, a value or range of ERV protein levels, a value or range of ERV particles present in and/or released from cells, and/or a value or range of transposition probability of an ERV locus.
  • the reference value is a value or range that represents the activation level of the ERV locus for a reference subject or population of reference subjects, including a value or range of ERV locus methylation levels, ERV transcript levels A value or range of values, a value or range of ERV protein levels and/or a value or range of amounts of ERV particles present in and/or released from cells.
  • the reference value is a value or range that represents the level of activation of the ERV locus in a reference subject or population of reference subjects, including a value or range of transposition probability for the ERV locus.
  • the detecting the level of activation of the ERV locus comprises detecting the level of methylation of the ERV locus, eg, the level of methylation of the LTR regions flanking it.
  • the methylation level of a particular region can be detected according to methods known in the art.
  • the enrichment levels of histone markers eg H3K9me3 or H3K36me3
  • ChIP chromatin immunoprecipitation
  • the method comprises using a primer pair comprising SEQ ID NOs: 9 and 10.
  • the transcript level of the coding sequence of the ERV, or the level of the protein it encodes is detected.
  • the coding sequence comprises the Env, Pol, Pro and/or Gag genes of the ERV.
  • the transcript levels of the coding sequences are detected by gene-specific primer pairs or probes.
  • the level of the protein encoded by the coding sequence is detected by a protein binding molecule.
  • Protein-binding molecules include any molecule capable of binding to a given protein, such as, but not limited to, antibodies, antigen-binding fragments of antibodies, and antibody derivatives.
  • antigen-binding fragments of antibodies include, but are not limited to, Fv fragments (eg, single-chain Fv and disulfide-bonded Fv) and Fab-like fragments (eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment).
  • Fv fragments eg, single-chain Fv and disulfide-bonded Fv
  • Fab-like fragments eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment.
  • the subject or animal is a mammal. In some embodiments, the subject or animal is a non-human mammal.
  • the subject or animal is a mouse and optionally the ERV is from the MMTV family; or the subject or animal is a non-human primate, preferably a monkey, and optionally the ERV is from the ERVW family .
  • the subject or animal is a human, and optionally, the ERV is from the HERVK family.
  • the subject is a human.
  • the ERV is from the HERVK family.
  • detection is performed against a HERVK coding region (HERVK-int), eg, a transcript thereof.
  • detection is performed against the MER61 coding region (MER61-int), eg, transcripts thereof.
  • the LTR region of MamGyp, eg, its transcript, is detected.
  • transcripts or translated proteins of coding sequences of HERVK are detected, including, for example, transcripts or translated proteins of Env, Pol, Pro and/or Gag genes of HERVK. It is known in the art that there are multiple HERVK loci in the human genome, and there are slight differences between different HERVK loci, wherein the Env, Pol, Pro and Gag genes of the HERVK locus HML2_1q22 are respectively as SEQ ID NO:5, 6, 7 and 8, the encoded proteins are shown in SEQ ID NOs: 1, 2, 3 and 4.
  • the transcript comprises, or has at least 80%, 85%, 90%, 95%, 96% with one of SEQ ID NOs: 5-8 , 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identical to transcripts of nucleotide sequences.
  • the protein comprises or has at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NOs: 1-4 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of the protein.
  • the method comprises using a method comprising SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20 or the primer pair of SEQ ID NOs: 21 and 22.
  • the method comprises using a probe comprising the nucleotide sequence of SEQ ID NO:60.
  • ERV particles from cells can be detected by imaging techniques, such as transmission electron microscopy (TEM) techniques.
  • imaging techniques such as transmission electron microscopy (TEM) techniques.
  • Detection of ERV particle release from cells can also be achieved by detection of ERV proteins, preferably Env proteins, for example comprising or at least 80%, 85%, 90%, 95%, 96%, 97% with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of the protein.
  • Env proteins for example comprising or at least 80%, 85%, 90%, 95%, 96%, 97% with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of the protein.
  • the method comprises using a method comprising or having at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NO: 1-4 %, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of a protein.
  • the method comprises using SEQ ID NO: 1 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, Proteins with amino acid sequences of 99.6%, 99.7%, 99.8% or 99.9% percent identity.
  • Samples useful in the methods of the present invention are samples comprising cells or tissues from the subject, eg, biopsy samples of various tissues. Samples useful in the methods of the present invention may also be samples of bodily fluids, such as urine, saliva, and blood, including whole blood, plasma, or serum.
  • bodily fluids such as urine, saliva, and blood, including whole blood, plasma, or serum.
  • the present invention also provides a method for assessing the degree of senescence of isolated animal cells or cell populations, comprising:
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the "activation level" of an ERV locus includes ERV locus methylation levels, ERV transcript levels, ERV protein levels, and/or ERV particles present in and/or released from cells. Lower methylation levels, higher transcript levels, higher protein levels, and/or higher ERV particles present in or released from cells represent higher levels of ERV locus activation.
  • the activation level of the ERV locus includes the transposition probability of the ERV locus. A higher probability of transposition represents a higher level of activation.
  • the reference value is obtained by detecting reference cells.
  • the reference cell is a cell of a specific number of passages or a specific range of times.
  • the reference value is obtained by detecting a reference cell population.
  • the reference value can be expressed in the form of "mean ⁇ standard deviation" or in the form of a range. Where a population is tested to determine reference values, the highest and lowest parts of the measured values can also be removed, eg the highest 2.5% and/or the lowest 2.5%.
  • the senescence degree of the cell or cell population is identified as higher than the senescence degree corresponding to the reference value; when the detection result (ie, the activation level of the ERV locus) is lower than a reference value, then the degree of senescence of the cell or cell population is identified as being lower than the degree of senescence corresponding to the reference value.
  • the reference value is obtained by detecting a reference cell or population of reference cells.
  • the senescence degree of the cell or cell population is identified as higher than the senescence degree corresponding to the passage number of the reference cell or cell population; when the detection result is lower than the reference value, Then the senescence degree of the cell or cell population is identified as being lower than the senescence degree corresponding to the passage number of the reference cell or cell population.
  • the reference value is a value or range that represents the activation level of the ERV locus for a reference subject or population of reference subjects, including a value or range of ERV locus methylation levels, ERV transcript levels A value or range of values, a value or range of ERV protein levels, a value or range of ERV particles present in and/or released from cells, and/or a value or range of transposition probability of an ERV locus.
  • the reference value is a value or range that represents the activation level of the ERV locus for a reference subject or population of reference subjects, including a value or range of ERV locus methylation levels, ERV transcript levels A value or range of values, a value or range of ERV protein levels and/or a value or range of amounts of ERV particles present in and/or released from cells.
  • the reference value is a value or range that represents the level of activation of the ERV locus in a reference subject or population of reference subjects, including a value or range of transposition probability for the ERV locus.
  • the detecting the level of activation of the ERV locus comprises detecting the level of methylation of the ERV locus, eg, the level of methylation of the LTR regions flanking it.
  • the methylation level of a particular region can be detected according to methods known in the art.
  • the enrichment levels of histone markers eg H3K9me3 or H3K36me3
  • ChIP chromatin immunoprecipitation
  • the method comprises using a primer pair comprising SEQ ID NOs: 9 and 10.
  • the transcript level of the coding sequence of the ERV, or the level of the protein it encodes is detected.
  • the coding sequence comprises the Env, Pol, Pro and/or Gag genes of the ERV.
  • the transcript levels of the coding sequences are detected by gene-specific primer pairs or probes.
  • the level of the protein encoded by the coding sequence is detected by a protein binding molecule.
  • Protein-binding molecules include any molecule capable of binding to a given protein, such as, but not limited to, antibodies, antigen-binding fragments of antibodies, and antibody derivatives.
  • antigen-binding fragments of antibodies include, but are not limited to, Fv fragments (eg, single-chain Fv and disulfide-bonded Fv) and Fab-like fragments (eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment).
  • Fv fragments eg, single-chain Fv and disulfide-bonded Fv
  • Fab-like fragments eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment.
  • the cells detected by the method of the present invention may be any animal cells, such as animal cells used for industrial production of recombinant proteins or vaccines, or stem cells.
  • the cells are non-human mammalian cells.
  • the cells are murine cells, such as CHO and mouse cells.
  • the cell is a mouse cell and the ERV is from the MMTV family.
  • the cells are non-human primate cells.
  • the non-human primate is an Old World monkey.
  • the cells are vero cells.
  • the ERV is from the ERVW family.
  • the cells are human cells.
  • the ERV is from the HERVK family.
  • detection is performed against a HERVK coding region (HERVK-int), eg, a transcript thereof.
  • detection is performed against the MER61 coding region (MER61-int), eg, transcripts thereof.
  • the LTR region of MamGyp, eg, its transcript, is detected.
  • transcripts or translated proteins of coding sequences of HERVK are detected, including, for example, transcripts or translated proteins of Env, Pol, Pro and/or Gag genes of HERVK.
  • the transcript comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NOs: 5-8 %, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identical to transcripts of nucleotide sequences.
  • the protein comprises or has at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NOs: 1-4 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of the protein.
  • the method comprises using a method comprising SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20 or the primer pair of SEQ ID NOs: 21 and 22.
  • the method comprises using a probe comprising the nucleotide sequence of SEQ ID NO:60.
  • the presence and/or release of ERV particles from cells can be detected by imaging techniques, such as TEM techniques.
  • Detection of ERV particle release from cells can also be accomplished by detecting, for example, an ERV protein, preferably an Env protein, in the culture medium, for example comprising SEQ ID NO: 1 or having at least 80%, 85%, 90%, 95%, SEQ ID NO: 1 Proteins with amino acid sequences that are 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical.
  • the present invention provides a method of modulating cellular senescence comprising contacting the cell with a modulator of ERV locus activation.
  • the methods of the present invention modulate cellular senescence by modulating endogenous retroviruses.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the modulator can be an inhibitor or enhancer, and the method also includes the use of an inhibitor against ERV particles.
  • Cell senescence can be delayed by inhibiting endogenous retrovirus, and cell senescence can be promoted by activating endogenous retrovirus.
  • an inhibitor of ERV locus activation involves specific inhibition (eg knocking out or knocking down the locus, knocking out or knocking down a gene in the locus, increasing locus methylation levels) and nonspecific Suppression (increase genome-wide methylation levels to promote methylation at the locus, or non-specific retroviral inhibitors, inhibit viral packaging, etc.).
  • the ERV inhibitor refers to an inhibitor against ERV viral particles.
  • the ERV locus activation inhibitor promotes ERV locus methylation, eg, by increasing methyltransferase activity.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the inhibitor of ERV locus activation is a gene therapy system targeting the ERV locus.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is an antisense nucleic acid or an interfering nucleic acid (including but not limited to siRNA, shRNA and miRNA).
  • the ERV inhibitor is a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof, that can bind to a protein of the ERV, such as the Env protein, preferably an ERV-neutralizing antibody or antigen-binding fragment thereof or Antibody Derivatives.
  • the subject is a non-human mammal.
  • the subject is a rodent.
  • the rodent is a mouse.
  • the ERV is from the MMTV family.
  • the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. Preferably, the non-human primate is an Old World monkey. Preferably, the ERV is from the ERVW family.
  • the subject is a human.
  • the ERV is from the HERVK family.
  • the inhibitor of ERV locus activation is a gene editing system that knocks out or knocks down ERV loci, including but not limited to HERVK and MMTV.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the HERVK locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the ERV locus activation inhibitor encodes the shRNA of SEQ ID NO: 51 or 52.
  • the ERV inhibitor is a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof, that binds to ERV-Env, including but not limited to HERVK-Env, HERVW-Env, and MMTV-Env .
  • the ERV inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with HERVK-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • the modulator is an ERV locus activation promoter of the cell.
  • the ERV locus activation promoter may be a specific promoter or a non-specific promoter.
  • the specific enhancer is a transcriptional activation system targeting the ERV locus.
  • the transcriptional activation system is the CRISPR-dCas transcriptional activation system.
  • the specific enhancer is a demethylation system targeting the ERV locus.
  • the demethylation system is a CRISPR-dCas-based demethylation system, eg, CRISPR-dCas-Tet.
  • the non-specific promoter is a DNA methyltransferase inhibitor, such as decitabine, Lomeguatrib, SGI-1027, or 5-azacytidine.
  • the cells are non-human mammalian cells. In some embodiments, the cells are rodent cells. In some embodiments, the cells are mouse cells. In some embodiments, the ERV is from the MMTV family.
  • the cells are non-human mammalian cells. In some embodiments, the cells are non-human primate cells. In some embodiments, the non-human primate is an Old World monkey. In some embodiments, the ERV is from the ERVW family.
  • the cells are human cells, such as tumor cells, infected cells, or cells in damaged tissue.
  • the ERV is from the HERVK family.
  • the sgRNA in the CRISPR-dCas transcriptional activation system comprises SEQ ID NO:59.
  • the method comprises contacting the human cell with HERVK particles.
  • the present invention provides a method of treating, or preventing or delaying senescence in a subject or cells, tissues or organs thereof in need thereof, comprising administering to the subject an inhibitor of ERV locus activation or an inhibitor of said ERV.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the ERV locus activation inhibitor promotes ERV locus methylation, eg, by increasing methyltransferase activity.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the inhibitor of ERV locus activation is a gene therapy system targeting the ERV locus.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is an antisense nucleic acid or an interfering nucleic acid (including but not limited to siRNA, shRNA and miRNA).
  • the ERV inhibitor is a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof, that can bind to a protein of the ERV, such as the Env protein, preferably an ERV-neutralizing antibody or antigen-binding fragment thereof or Antibody Derivatives.
  • the subject is a non-human mammal.
  • the subject is a rodent.
  • the rodent is a mouse.
  • the ERV is from the MMTV family.
  • the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. Preferably, the non-human primate is an Old World monkey. Preferably, the ERV is from the ERVW family.
  • the subject is a human.
  • the ERV is from the HERVK family.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the HERVK locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the ERV locus activation inhibitor encodes the shRNA of SEQ ID NO: 51 or 52.
  • the ERV inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with HERVK-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • a binding molecule such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • Isolated animal cells are used for industrial production of recombinant proteins or vaccines, and the viability and proliferative capacity of the cells used are important to the production.
  • Cellular senescence reduces cell viability and proliferative capacity, which adversely affects productivity. In the process of culturing cells, preventing or delaying cell senescence is beneficial for industrial production.
  • the present invention provides a method of culturing an isolated animal cell or population of cells comprising contacting said cell with an inhibitor of ERV locus activation of said cell or an inhibitor of said ERV.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the ERV locus activation inhibitor promotes ERV locus methylation, eg, by increasing methyltransferase activity.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the inhibitor of ERV locus activation is a gene therapy system targeting the ERV locus.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is an antisense nucleic acid or an interfering nucleic acid (including but not limited to siRNA, shRNA and miRNA).
  • the ERV inhibitor is a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof, that can bind to a protein of the ERV, such as the Env protein, preferably an ERV-neutralizing antibody or antigen-binding fragment thereof or Antibody Derivatives.
  • the cells are non-human mammalian cells.
  • the cells are murine cells, such as CHO and mouse cells.
  • the cell is a mouse cell and the ERV is from the MMTV family.
  • the cells are non-human primate cells.
  • the non-human primate is an Old World monkey.
  • the cells are vero cells.
  • the ERV is from the ERVW family.
  • the cells are human cells.
  • the ERV is from the HERVK family.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the HERVK locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the ERV locus activation inhibitor encodes the shRNA of SEQ ID NO: 51 or 52.
  • the ERV inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with HERVK-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • a binding molecule such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • the present invention also provides a method of preventing or delaying cellular senescence comprising contacting said cell with an inhibitor of ERV locus activation of said cell or an inhibitor of said ERV.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the ERV locus activation inhibitor promotes ERV locus methylation, eg, by increasing methyltransferase activity.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the inhibitor of ERV locus activation is a gene therapy system targeting the ERV locus.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is an antisense nucleic acid or an interfering nucleic acid (including but not limited to siRNA, shRNA and miRNA).
  • the ERV inhibitor is a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof, that can bind to a protein of the ERV, such as the Env protein, preferably an ERV-neutralizing antibody or antigen-binding fragment thereof or Antibody Derivatives.
  • the cells are non-human mammalian cells.
  • the cells are murine cells, such as CHO and mouse cells.
  • the cell is a mouse cell and the ERV is from the MMTV family.
  • the cells are non-human primate cells.
  • the non-human primate is an Old World monkey.
  • the cells are vero cells.
  • the ERV is from the ERVW family.
  • the cells are human cells.
  • the ERV is from the HERVK family.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the HERVK locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the ERV locus activation inhibitor encodes the shRNA of SEQ ID NO: 51 or 52.
  • the ERV inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with HERVK-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • a binding molecule such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • the present invention also provides a method of promoting cellular senescence comprising contacting the cell with an ERV locus activation promoter of the cell or the ERV particle.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the ERV locus activation promoter may be a specific promoter or a non-specific promoter.
  • the specific enhancer is a transcriptional activation system targeting the ERV locus.
  • the transcriptional activation system is the CRISPR-dCas transcriptional activation system.
  • the specific enhancer is a demethylation system targeting the ERV locus.
  • the demethylation system is a CRISPR-dCas-based demethylation system, eg, CRISPR-dCas-Tet.
  • the ERV loci include, but are not limited to, HERVK, HERVW, and MMTV loci.
  • the non-specific promoter is a DNA methyltransferase inhibitor, such as decitabine, Lomeguatrib, SGI-1027, or 5-azacytidine.
  • the cells are non-human mammalian cells. In some embodiments, the cells are rodent cells. In some embodiments, the rodent is a mouse. In some embodiments, the ERV is from the MMTV family.
  • the cells are non-human mammalian cells. In some embodiments, the cells are non-human primate cells. In some embodiments, the non-human primate is an Old World monkey. In some embodiments, the ERV is from the ERVW family.
  • the cells are human cells, such as tumor cells, infected cells, or cells in damaged tissue.
  • the ERV is from the HERVK family.
  • the sgRNA in the CRISPR-dCas transcriptional activation system comprises SEQ ID NO:59.
  • the method comprises contacting the human cell with HERVK particles.
  • the present invention also provides a method for preventing or delaying the degeneration of tissues and organs, treating related diseases, and delaying the aging of the body, such as preventing or delaying skin aging, treating or preventing osteoarthritis and frailty, including administering to a subject in need.
  • ERV locus activation inhibitor or ERV inhibitor include HERVK, HERVW, and MMTV loci.
  • the ERV locus activation inhibitor promotes ERV locus methylation, eg, by increasing methyltransferase activity.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the inhibitor of ERV locus activation is a gene therapy system targeting the ERV locus.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus.
  • the ERV inhibitor is a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof, that binds to ERV-Env.
  • the present invention also provides a method of preventing or delaying skin aging comprising administering to a subject in need thereof an inhibitor of HERVK locus activation or a HERVK inhibitor.
  • the inhibitor of HERVK locus activation promotes methylation of the HERVK locus by, eg, increasing the activity of a methyltransferase.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the inhibitor of HERVK locus activation is a non-specific retroviral inhibitor comprising, for example, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the inhibitor of ERV locus activation is a gene therapy system targeting the ERV locus.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the inhibitor of HERVK locus activation is a gene editing system that knocks out or knocks down the HERVK locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the HERVK locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the ERV locus activation inhibitor encodes the shRNA of SEQ ID NO: 51 or 52.
  • the HERVK inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with HERVK-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • a binding molecule such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • the administration is topical, intradermal, or subcutaneous.
  • the present invention also provides a method of treating progeria, such as HPGS or WS, and aging-related diseases, including but not limited to arthritis, premature ovarian failure, liver fibrosis, pulmonary fibrosis, and cardiovascular disease, comprising administering to a subject in need thereof ERV locus activation inhibitor or ERV inhibitor.
  • the ERV is selected from HERVK, HERVW, and MMTV.
  • the ERV locus activation inhibitor promotes ERV locus methylation, eg, by increasing methyltransferase activity.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the inhibitor of ERV locus activation is a gene therapy system targeting the ERV locus.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the ERV locus activation inhibitor encodes the shRNA of SEQ ID NO: 51 or 52.
  • the ERV inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with ERV-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • a binding molecule such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • the present invention is also a method of treating progeria, such as HPGS or WS, comprising administering to a subject in need thereof an inhibitor of HERVK locus activation or a HERVK inhibitor.
  • the inhibitor of HERVK locus activation promotes methylation of the HERVK locus by, eg, increasing the activity of a methyltransferase.
  • the inhibitors include, but are not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the inhibitor of HERVK locus activation is a gene editing system that knocks out or knocks down the HERVK locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the HERVK locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the inhibitor of ERV locus activation encodes the shRNA of SEQ ID NO: 51 or 52.
  • the HERVK inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with HERVK-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • a binding molecule such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • the present invention provides a kit for identifying the degree of aging of a subject or its cells, tissues or organs, or assessing the physiological age of a subject, or diagnosing progeria, comprising a kit for detecting endogenous retrovirus (ERV) in a sample from the subject Activation level detection agent.
  • EBV endogenous retrovirus
  • the present invention also provides a detection agent for detecting the level of activation of endogenous retrovirus (ERV) in a sample from the subject in preparation for identifying the degree of aging of a subject or its cells, tissues or organs or assessing the physiological age or diagnosis of a subject Uses in a kit for progeria.
  • ERP endogenous retrovirus
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the detection agent is used to detect the methylation level of the ERV locus, eg, the methylation level of its flanking LTR regions.
  • the detection agent comprises a primer pair comprising the polynucleotides of SEQ ID NOs: 9 and 10.
  • the detection agent is used to detect the transcript level of the coding sequence of the ERV, or the level of the protein it encodes.
  • the coding sequence comprises the Env, Pol, Pro and/or Gag genes of the ERV.
  • the detection agent is a gene-specific primer pair or probe for detecting transcript levels of the coding sequence.
  • the detection agent is a protein-binding molecule for detecting the level of the protein encoded by the coding sequence. Protein-binding molecules include any molecule capable of binding to a given protein, such as, but not limited to, antibodies, antigen-binding fragments of antibodies, and antibody derivatives.
  • antigen-binding fragments of antibodies include, but are not limited to, Fv fragments (eg, single-chain Fv and disulfide-bonded Fv) and Fab-like fragments (eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment).
  • Fv fragments eg, single-chain Fv and disulfide-bonded Fv
  • Fab-like fragments eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment.
  • the subject is a non-human mammal.
  • the subject is a rodent.
  • the rodent is a mouse.
  • the ERV is from the MMTV family.
  • the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. Preferably, the non-human primate is an Old World monkey. Preferably, the ERV is from the ERVW family.
  • the subject is a human.
  • the ERV is from the HERVK family.
  • detection is performed against a HERVK coding region (HERVK-int), eg, a transcript thereof.
  • detection is performed against the MER61 coding region (MER61-int), eg, transcripts thereof.
  • the LTR region of MamGyp, eg, its transcript, is detected.
  • the detection agent is used to detect transcripts or translated proteins of coding sequences of HERVK, including, for example, transcripts or translated proteins of the Env, Pol, Pro and/or Gag genes of HERVK.
  • the transcript comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NOs: 5-8 %, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identical to transcripts of nucleotide sequences.
  • the protein comprises or has at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NOs: 1-4 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of the protein.
  • the kit comprises a primer pair comprising SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18 , the polynucleotides of SEQ ID NOs: 19 and 20 or SEQ ID NOs: 21 and 22.
  • the kit comprises a probe comprising the nucleotide sequence of SEQ ID NO:60.
  • the kit comprises a binding partner comprising or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of the protein.
  • the binding partner comprises or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6% from SEQ ID NO:1 %, 99.7%, 99.8% or 99.9% percent identical to amino acid sequences.
  • Samples that can be detected with the kit of the present invention are samples comprising cells or tissues from the subject, such as biopsy samples of various tissues.
  • the samples that can be detected by the kits of the present invention can also be samples of bodily fluids, such as urine, saliva, and blood, including whole blood, plasma, or serum.
  • the present invention also provides a kit for assessing the degree of senescence of isolated animal cells or cell populations comprising a detection agent for detecting the level of ERV activation in said cells.
  • the present invention also provides the use of a detection agent for detecting the level of ERV activation in cells in the preparation of a kit for assessing the degree of senescence of isolated animal cells or cell populations.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the level of activation of the ERV includes ERV locus methylation levels, ERV transcript levels, ERV protein levels, presence, localization and/or release of ERV particles in cells, and/or ERV The transposition probability of the locus.
  • the activation level of the ERV includes ERV locus methylation levels, ERV transcript levels, ERV protein levels, and/or the presence and/or release of ERV particles in cells.
  • the activation level of the ERV includes the transposition probability of the ERV locus.
  • the level of activation of the ERV is manifested by the localization of ERV particles in the cell.
  • the detection agent is used to detect the methylation level of the ERV locus, eg, the methylation level of its flanking LTR regions.
  • the detection agent comprises a primer pair comprising the polynucleotides of SEQ ID NOs: 9 and 10.
  • the detection agent is used to detect the transcript level of the coding sequence of the ERV, or the level of the protein it encodes.
  • the coding sequence comprises the Env, Pol, Pro and/or Gag genes of the ERV.
  • the detection agent is a gene-specific primer pair or probe for detecting transcript levels of the coding sequence.
  • the detection agent is a protein-binding molecule for detecting the level of the protein encoded by the coding sequence. Protein-binding molecules include any molecule capable of binding to a given protein, such as, but not limited to, antibodies, antigen-binding fragments of antibodies, and antibody derivatives.
  • antigen-binding fragments of antibodies include, but are not limited to, Fv fragments (eg, single-chain Fv and disulfide-bonded Fv) and Fab-like fragments (eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment).
  • Fv fragments eg, single-chain Fv and disulfide-bonded Fv
  • Fab-like fragments eg, Fab fragments, Fab' fragments, and F(ab') 2 fragment.
  • the cells are non-human mammalian cells.
  • the cells are murine cells, such as CHO and mouse cells.
  • the cell is a mouse cell and the ERV is from the MMTV family.
  • the cells are non-human primate cells.
  • the non-human primate is an Old World monkey.
  • the cells are vero cells.
  • the ERV is from the ERVW family.
  • the cells are human cells.
  • the ERV is from the HERVK family.
  • detection is performed against a HERVK coding region (HERVK-int), such as transcripts thereof.
  • detection is performed against the MER61 coding region (MER61-int), eg, transcripts thereof.
  • the LTR region of MamGyp, eg, its transcript, is detected.
  • the detection agent is used to detect transcripts or translated proteins of coding sequences of HERVK, including, for example, transcripts or translated proteins of the Env, Pol, Pro and/or Gag genes of HERVK.
  • the transcript comprises, or has at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NOs: 5-8 %, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identical to transcripts of nucleotide sequences.
  • the protein comprises or has at least 80%, 85%, 90%, 95%, 96%, 97% with one of SEQ ID NOs: 1-4 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of the amino acid sequence of the protein.
  • the kit comprises a primer pair comprising SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18 , the polynucleotides of SEQ ID NOs: 19 and 20 or SEQ ID NOs: 21 and 22.
  • the kit comprises a probe comprising the nucleotide sequence of SEQ ID NO:60.
  • compositions for regulating aging are provided.
  • the present invention provides a pharmaceutical composition for treating premature aging in a subject in need thereof, or cells, tissues or organs thereof, or preventing or delaying aging in a subject or cells, tissues or organs thereof, comprising ERV locus activation inhibition agent or inhibitor of the ERV and a pharmaceutically acceptable excipient.
  • the present invention also provides that the present invention provides an inhibitor of ERV locus activation or an inhibitor of said ERV in preparation for the treatment of premature aging of a subject or its cells, tissues or organs, or to prevent or delay the subject or its cells, Use in a pharmaceutical composition for tissue or organ aging.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the inhibitor of ERV locus activation includes, but is not limited to, methyltransferases or polynucleotides encoding them.
  • the ERV locus activation inhibitor is a non-specific retroviral inhibitor comprising, eg, a reverse transcriptase inhibitor, an integrase inhibitor, and/or a protease inhibitor.
  • the reverse transcriptase inhibitor includes a nucleoside reverse transcriptase inhibitor such as enofovir, abacavir, stavudine (D4T), lamivudine (3TC) and/or zidovudine, and/or non-nucleoside reverse transcriptase Inhibitors such as efavirenz, etravirine and/or nevirapine.
  • the integrase inhibitor comprises Raltegravir.
  • the protease inhibitor includes Lopinavir and/or Darunavir.
  • the retroviral inhibitor comprises lamivudine (3TC), abacavir and/or Raltegravir.
  • the ERV locus activation inhibitor is a methylation-increasing CRISPR-dCas system targeting the ERV locus.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the ERV locus, including but not limited to meganucleases, zinc finger nucleases (ZFNs), transcription activators like Effector Nucleases (TALENs) and CRISPR/Cas Systems.
  • ZFNs zinc finger nucleases
  • TALENs Effector Nucleases
  • CRISPR/Cas Systems CRISPR/Cas Systems.
  • the ERV locus activation inhibitor is an antisense nucleic acid or an interfering nucleic acid (including but not limited to siRNA, shRNA and miRNA).
  • the ERV inhibitor is a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof, that can bind to a protein of the ERV, such as the Env protein, preferably an ERV-neutralizing antibody or antigen-binding fragment thereof or Antibody Derivatives.
  • the subject is a non-human mammal.
  • the subject is a rodent.
  • the rodent is a mouse.
  • the ERV is from the MMTV family.
  • the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. Preferably, the non-human primate is an Old World monkey. Preferably, the ERV is from the ERVW family.
  • the subject is a human.
  • the ERV is from the HERVK family.
  • the ERV locus activation inhibitor is a gene editing system that knocks out or knocks down the HERVK locus. In some embodiments, the ERV locus activation inhibitor is used for knockdown or knockout comprising, or having at least 80%, 85%, 85%, 85, %, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% percent identity to the nucleic acid molecule of the nucleic acid sequence or interfering nucleic acids (including but not limited to siRNA, shRNA and miRNA). In some embodiments, the ERV locus activation inhibitor encodes the shRNA of SEQ ID NO: 51 or 52.
  • the ERV inhibitor is at least 80%, 85%, 90%, 95%, 96%, 97% with HERVK-Env (eg comprising or with SEQ ID NO:1 , 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% percent identity of amino acid sequences) to a binding molecule, such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • a binding molecule such as an antibody or antigen-binding fragment or antibody derivative thereof.
  • the pharmaceutical composition skin is used for preventing or delaying degeneration of tissues and organs, treating related diseases, delaying body aging, such as preventing or delaying skin aging, treating or preventing osteoarthritis and frailty.
  • the pharmaceutical composition is for topical, intradermal, or subcutaneous administration.
  • the pharmaceutical composition is used to treat progeria, such as HGPS or WS, and aging-related diseases, including but not limited to arthritis, premature ovarian failure, liver fibrosis, pulmonary fibrosis, and cardiovascular disease.
  • progeria such as HGPS or WS
  • aging-related diseases including but not limited to arthritis, premature ovarian failure, liver fibrosis, pulmonary fibrosis, and cardiovascular disease.
  • the pharmaceutical composition is used to prevent or delay skin aging. In some embodiments, the pharmaceutical composition is for topical, intradermal, or subcutaneous administration.
  • the pharmaceutical composition is used to treat progeria, such as HGPS or WS.
  • the present invention also provides a pharmaceutical composition for promoting cell senescence, which comprises an ERV locus activation promoter of the cell or the ERV particle.
  • the present invention also provides a cell ERV locus activation promoter or the use of the ERV particles in the preparation of a pharmaceutical composition for promoting cell senescence.
  • the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and ERVW and homologs thereof. In some embodiments, the ERV is selected from the following families: HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, ERVW and MMTV and homologs thereof.
  • the ERV includes at least an ERV from the HERVK family (including HERVK11), optionally, the ERV further includes an ERV selected from the following families: MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS -P2 and ERVW and their homologues.
  • the enhancer is a transcriptional activation system targeting the ERV locus.
  • the transcriptional activation system is the CRISPR-dCas transcriptional activation system.
  • the specific enhancer is a demethylation system targeting the ERV locus.
  • the demethylation system is a CRISPR-dCas-based demethylation system, eg, CRISPR-dCas-Tet.
  • the enhancer is a DNA methyltransferase inhibitor, such as decitabine, Lomeguatrib, SGI-1027, or 5-azacytidine.
  • the cells are non-human mammalian cells. In some embodiments, the cells are rodent cells. In some embodiments, the rodent is a mouse. In some embodiments, the ERV is from the MMTV family.
  • the cells are non-human mammalian cells. In some embodiments, the cells are non-human primate cells. In some embodiments, the non-human primate is an Old World monkey. In some embodiments, the ERV is from the ERVW family.
  • the cells are human cells, such as tumor cells, infected cells, or cells in damaged tissue.
  • the ERV is from the HERVK family.
  • the sgRNA in the CRISPR-dCas transcriptional activation system comprises SEQ ID NO:59.
  • C57BL/6J mice were purchased from Speifu (Beijing) Biotechnology Co., Ltd. and housed in ventilated cages in the animal room of the Institute of Biophysics and Institute of Zoology, Chinese Academy of Sciences, and fed standard laboratory chow and water for 12 h /12h light and dark cycle.
  • mice were sacrificed and euthanized with CO2, followed by cervical dislocation.
  • Lung, liver and skin samples were collected from 8 young and old cynomolgus monkeys with no clinical or experimental history.
  • HGPS cynomolgus monkeys were housed in the Yunnan Key Laboratory of Primate Biomedical Research (LPBR), and ethical animal experiments were performed in accordance with the International Committee for the Assessment and Accreditation of Laboratory Animals (AAALAC) (68).
  • the grip strength of the limbs was measured with a grip dynamometer (Panlab Grid Strength Meter, LE902) according to the product instructions. Briefly, the mouse was placed on top of the grip dynamometer and pulled in the direction of the grid at a constant rate until the mouse let go. Repeat 10 times, record the peak pulling force for each time, and calculate the average value as the grip strength of each mouse.
  • a grip dynamometer Panlab Grid Strength Meter, LE902
  • mice were placed in a treadmill (SANS Bio Instrument, SA101), and the exercise ability of the mice was tested according to the product instructions. Specifically, the inclination of the track was set to 5°, the electrical stimulation was 2mA, and the running speed was set to: the initial speed was 5m/min for 5 minutes; then the acceleration was 1m/min2 to the final speed of 25m/min, for a total of 20 minutes. Mice were first trained for three consecutive days and tested on the fourth day. The number of electrical stimulations, running distance and time within 25 minutes were recorded.
  • mice were placed in different channels on the rod with an initial speed of 4 rpm/min on the rotarod, and then accelerated to 44 rpm/min at an acceleration of 8 rpm/min until the mice dropped, each small. Rat practice 3 times. Mice were trained for three consecutive days and tested on the fourth day, 3 tests per mouse, and the time was recorded after the mice were dropped.
  • Micro-CT PE Quantum FX
  • the scanning parameters of Micro-CT were voltage 50Kvp, power 40W, 6-frame stacking, and field of view 4cm ⁇ 4cm.
  • three-dimensional image screenshots were taken and bone mineral density was calculated using Ansys software.
  • mice were placed on the end of any arm of the Y maze (a trisection radial maze, consisting of 3 arms of equal length (50cm ⁇ 18cm ⁇ 35cm), and the angle between each two arms was 120 degrees), and they were allowed to explore freely. After 5 minutes, the camera system recorded the behavioral changes of the animals for 5 minutes, and the spatial short-term memory ability of the mice was detected by recording the total number of arm entry times and the number of turns (alternation) of successively entering all three arms of the Y maze.
  • HEK293T cells and human fibroblasts were supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific), 2mmol/L GlutaMAX (Thermo Fisher Scientific), 0.1mmol/L non-essential amino acids (NEAA, Thermo Fisher Scientific), 1% Penicillin/Streptomycin (Thermo Fisher Scientific) in Dulbecco Modified Eagle Medium (DMEM, Thermo Fisher Scientific).
  • FBS fetal bovine serum
  • GlutaMAX Thermo Fisher Scientific
  • NEAA non-essential amino acids
  • Penicillin/Streptomycin Thermo Fisher Scientific
  • DMEM Dulbecco Modified Eagle Medium
  • Cells were lysed in IX SDS buffer (100 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS and 2% 2-mercaptoethanol) and boiled at 105°C for 10 minutes. Protein concentration was measured with BCA kit. Cell lysates were subjected to SDS-PAGE electrophoresis and then electrotransferred onto PVDF membranes (Millipore).
  • IX SDS buffer 100 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS and 2% 2-mercaptoethanol
  • Membranes were incubated with primary antibodies, washed followed by incubation with HRP-conjugated secondary antibodies, and then blotted with substrate, a mixture of 1000 ⁇ L of Substrate A and 3 ⁇ L of Substrate B (Substrate A: 0.2 mM coumaric acid, 1.25 mM luminal, 0.1 M Tris-HCl, pH 8.5; Substrate B: 3 % H2O2 ) or Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Imaging was performed using Image Lab software. Quantification was performed with ImageJ.
  • Cells were grown in hMPC medium containing microvesicle-depleted fetal bovine serum (dFBS) for 48 hours.
  • Conditioned medium was collected and filtered through a 0.2 ⁇ m filter (Pall Corporation) and pre-washed with Protein A/G Sepharose beads for 2-3 hours at 4°C.
  • the supernatant was incubated with 1/1000 volume HERVK-Env antibody and fresh Protein A/G Sepharose beads overnight at 4°C, then the supernatant was collected, the beads were washed with PBS, and western blotting was performed.
  • dFBS microvesicle-depleted fetal bovine serum
  • the medium was collected and the number of cells was counted.
  • Media from the same number of cells was used for microvesicle purification by centrifugation at 300 x g for 10 min to eliminate cellular contamination, at 15,000 x g for 20 min, filtered through a 0.2 ⁇ m filter (Pall Corporation) , followed by ultracentrifugation at 110,000 x g for 2 hours.
  • the pellets were collected as the microvesicles, dissolved in the same volume of PBS, and subjected to viral RNA isolation or Western blotting.
  • Total genomic DNA from cells was extracted using a DNA extraction kit (Tiangen) following the manufacturer's instructions.
  • TRIzol Thermo Fisher Scientific
  • samples were incubated with prepared buffer AVL and carrier RNA. After addition of ethanol, samples were purified with a QIAamp Mini column, washed, eluted, and then treated with DNase I (20 ⁇ L of viral RNA with 2 ⁇ L of DNase I in 2.4 ⁇ L of reaction buffer for 30 min at 37°C). DNase I was inactivated and removed using the inactivation reagent provided in the kit, followed by centrifugation at 10,000 x g for 1.5 min, and the supernatant was heated at 70 °C for 10 min to eliminate any residual DNase activity.
  • Viral RNA purified from microvesicles was reverse transcribed into cDNA for ddPCR using the GoScript Reverse Transcription System (Promega) following the manufacturer's instructions.
  • PCR amplification was performed in a thermal cycler (Bio-Rad Laboratories, Inc.) and samples were placed in a QX200 droplet reader (Bio-Rad Laboratories, Inc.) to analyze absolute copies of HERVK in microvesicles number.
  • the medium was collected and filtered with a 0.2 ⁇ m filter, then incubated in IL6 antibody-coated plates (BioLegend, pre-coated at 4° C. overnight before use according to the manufacturer’s instructions). The plate was then incubated with detection antibody, avidin-HRP and freshly mixed TMB substrate. After addition of stop solution, the plates were measured at 450 nm using Synergy H1 (BioTek).
  • culture medium or human serum was first concentrated 100-fold or 10-fold using Centricon Plus-70 (Millipore) or Amicon Ultra-15 (Millipore), respectively, and then treated with HERVK_7p22.1Provirus
  • the Ancestral Env Polypretein (ERVK6) ELISA Kit was measured according to the manufacturer's instructions. Briefly, concentrated medium or serum (100 ⁇ L) was added to coated plates and incubated for 2 hours at 37°C with biotin antibody, avidin-HRP followed by fresh mixing of TMB substrate and stop solution and measured at 450 nm using Synergy H1 (BioTek).
  • the level of 2'3'-cGAMP in cells was measured with a 2'3'-cGAMP ELISA kit. Briefly, 50 ⁇ L of cell lysate (lysed in RIPA buffer) from the same number of indicated cell lines was added to the plate, followed by the addition of 2'3'-cGAMP HRP tracer, 2'3' at 20°C -cGAMP polyclonal antiserum. Shake at room temperature. After incubation with TMB substrate and stop solution, plates were measured at 450 nm using Synergy H1 (BioTek).
  • Immunohistochemical staining of tissue sections was performed using the DAB Staining Kit (ZSGB-BIO) according to the manufacturer's instructions. Briefly, sections deparaffinized using xylene and ethanol were incubated with 3 % H2O2 solution to block endogenous peroxidase activity, followed by 10 mM sodium citrate buffer (pH 6.0) Antigen recovery was performed. Sections were then permeabilized, blocked, and incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 hour, then stained with DAB substrate solution and hematoxylin, dehydrated, and mounted.
  • DAB Staining Kit ZSGB-BIO
  • RNA/DNA-Fluorescence In Situ Hybridization FISH
  • HERVK gene by single-molecule DNA-FISH was completed by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University. Senescent cells were sequentially treated with RNase A (100 ⁇ g/mL, dissolved in 2 ⁇ SSC) for 1 h at 37°C, treated with 70% formamide dissolved in 2 ⁇ SSC at 72°C for 10 minutes, and treated with pre-cooled ethanol (concentration 1 minute each from 80%, 90% to 100%). Cells were then incubated with HERVK or ACTIN-specific probe pairs (10 ⁇ M each probe) in hybridization buffer (10% deionized formamide, 2 ⁇ SSC, 10% dextran sulfate and 2 mM VRC) at 42°C Breed overnight.
  • the proportion of GFP-positive cells was analyzed by flow cytometry, or GFP-positive cells were sorted out.
  • the HERVK reporter plasmid was transfected into wild-type mesenchymal stem cells early- and late-passage (P7 and P14) cells and co-transfected simultaneously.
  • the vector dsRed expressing red fluorescence obtained from the laboratory of Xu Xingzhi, School of Basic Medicine, Shenzhen University was used to calculate the transfection probability. After the cells were confluent, the cells were digested into single cells, resuspended in PBS, and analyzed by flow cytometer BD Influx. The ratio of green fluorescence positive cells to red fluorescence positive cells was the probability of HERVK transposition events.
  • wild-type mesenchymal stem cells were infected with purified HERVK virus particles labeled with green fluorescence. cell.
  • PB phosphate buffered saline
  • cells were fixed with 1% (w/v) osmium tetroxide in PB for 2 h at 4°C, dehydrated through a graded ethanol series and placed in pure acetone in a graded mixture of acetone and resin. Wetted, then embedded in neat resin containing 1.5% BDMA, and polymerized for 12 hours at 45°C and 48 hours at 60°C. Ultrathin sections (70 nm thickness) were performed with a microtome (Leica EM UC6) and double stained with uranyl acetate and lead citrate.
  • sgRNA targeting TBK1 was cloned into lenti-pMK1334 (addgene #127965) through BstXI/BlpI sites and used the hSpCas9567 expression cassette with lenti-CRISPR v2 (addgene, #52961) Co-transfection.
  • non-targeting control (NTC) or HERVK LTR-targeting sgRNA lentiviral constructs were cloned into lenti-SAM v2 (addgene#75112) through the ESP3I site and combined with lenti-MPH v2 (addgene #89308) co-transfection.
  • HERVK-targeting vector was constructed based on the Cas9KRAB-based inhibition system (CRISPRi).
  • CRISPRi Cas9KRAB-based inhibition system
  • the Cas9KRAB-expressing vector was obtained from the laboratory of Dr. Didier Trono (EPFL, Lausanne, Switzerland).
  • MMTV-targeting sgRNAs were cloned into an empty vector expressing Cas9KRAB.
  • a full-length HERVK fragment (SEQ ID NO: 150) was synthesized according to previously published sequencing results (see, Marie Dewannieux, Genome Reserch, 2006, Young Nam Lee, PLoS Pathogens, 2007) and analyzed by NheI/ The ApaI site was cloned into a mutated pEGFP-N3 vector with a KpnI enzyme site (pEGFP-N3-KpnI, obtained from the laboratory of Xu Xingzhi, School of Basic Medicine, Shenzhen University) to obtain the vector pEGFP-N3-HERVK.
  • the CMV-EGFP fragment (SEQ ID NO: 153) was amplified by PCR from pEGFP-C1 (obtained from Xingzhi Xu's laboratory, School of Basic Medicine, Shenzhen University), and the CMV-EGFP fragment (SEQ ID NO: 153) was amplified by KpnI site. - The EGFP fragment was inserted into pEGFP-N3-HERVK to constitute GFP-HERVK.
  • the env, pol, gag, pro (SEQ ID NOs: 5-8) and rev genes (SEQ ID NO: 149) of HERVK were generated by PCR amplification using pEGFP-N3-HERVK as a template, and inserted into pEGFP-N3-KpnI to obtain Plasmids pEGFP-N3-gag-pro-pol (expressing gag, pro and pol genes), pEGFP-N3-Env (expressing env gene) and pEGFP-N3-Rev (expressing rev gene).
  • HERVK transposition reporter gene vector To construct a HERVK transposition reporter gene vector, the HERVK fragment described above was first cloned into a dsRed vector with a KpnI enzyme site mutant (obtained from the laboratory of Xingzhi Xu, School of Basic Medicine, Shenzhen University).
  • the GFP reporter gene (SEQ ID NO: 152) was obtained by PCR amplification from the L1 reporter vector (gifted by Professor Wang Jichang, Sun Yat-Sen University, Guangzhou, China), and the GFP reporter gene was inserted into dsRed-HERVK through the KpnI site, which constituted the HERVK transposition reporter gene. vector.
  • HEK293T cells were transfected with the lentiviral vector using lipofectamine 3000 (Invitrogen) along with the packaging plasmids pMD2.G (addgene, #12260) and psPAX2 (addgene, #12259).
  • Lentivirus-containing supernatants were harvested 48 and 72 hours post-transfection, filtered through a 0.2 ⁇ m filter, and concentrated by ultracentrifugation at 19,400 rpm for 2.5 hours. Suspend the virus pellet and assess the virus titer.
  • HERVK virus-containing supernatants were harvested 48 and 72 hours after transfection, filtered through a 0.2 ⁇ m filter, and concentrated by ultracentrifugation at 100,000 g for 2 hours. Suspend the virus pellet and assess the virus titer.
  • the HERVK reporter plasmid was transfected into early and late passage cells, and co-transfected with a vector expressing red fluorescence to calculate the transfection probability. After the cells were confluent, the cells were digested into single cells, resuspended in PBS, and analyzed by flow cytometer BD Influx. The ratio of green fluorescence positive cells to red fluorescence positive cells was the probability of HERVK transposition events.
  • hMPCs were inoculated (passage 0 after treatment (P0)) and treated with 500 ⁇ g/ ⁇ L 5-AZA for 4 days, then passaged and cultured in fresh medium.
  • P0 passage 0 after treatment
  • treated P2 cells were used for Ki67 immunofluorescence, SA- ⁇ -gal staining and RNA/DNA extraction.
  • hMPCs were treated with 10 ⁇ M abacavir, lamivudine (3TC), and Raltegravir, respectively, with medium changes every 3 days, followed by fresh treatments.
  • Cells were processed for two passages and then used for Ki67 immunofluorescence, SA- ⁇ -gal staining and RNA/DNA extraction.
  • hMPCs For infection with HERVK RVLPs, hMPCs (P0 after treatment) were inoculated and incubated with HERVK RVLPs in the presence of polybrene (Sigma-Aldrich) for 24 hours. To increase HERVK RVLP infection efficiency, hMPCs were centrifuged at 1200 g for 2.5 hours. Two days after infection, GFP-positive cells were sorted by FACS. Cells were infected for two passages and then used for Ki67 immunofluorescence, SA- ⁇ -gal staining and RNA/DNA extraction.
  • polybrene Sigma-Aldrich
  • hMPCs were inoculated (PO after treatment) and incubated with lentivirus for 24 hours in the presence of polybrene (Sigma-Aldrich). After two passages, cells were processed for Ki67 immunofluorescence analysis, SA- ⁇ -gal staining and RNA/DNA extraction.
  • CM conditioned medium
  • 50% conditioned medium + 50% fresh medium in the presence of polybrene (Sigma-Aldrich)
  • polybrene Sigma-Aldrich
  • CM retrovirus-like particles
  • TEM analysis To detect the adhesion of retrovirus-like particles (RVLPs) from CM to the surface of young cells, cells were treated with CM for 12 hours and then subjected to TEM analysis.
  • RVLPs retrovirus-like particles
  • cytoplasmic fraction of cells was extracted after fixation and incubated with cGAS antibody and Dynabeads Protein A overnight at 4°C. DNA extraction was performed as described above. qPCR analysis of 5S rDNA (principally not present in the cytoplasmic fraction) was used to rule out nuclear genome contamination.
  • NEB NEBNext Ultra RNA Library Prep Kit for Illumina
  • Raw reads to ends were trimmed using TrimGalore (version 0.4.5, Babraham Bioinformatics, https://github.com/FelixKrueger/TrimGalore) and mapped using hisat2 (version 2.0.4) from the UCSC Genome Browser database Human (Homo sapiens) hg19 or cynomolgus monkey (Macaca Fascicularis) MacFas5.0 reference genomes obtained from . Reads mapped to each gene were then counted by HTSeq (version 0.11.0) using high-quality mapped reads (mapping quality scores over 20).
  • DEGs Differentially expressed genes
  • DESeq2R package version 1.29.8
  • the cutoff value was "
  • the critical value is "
  • SASP Senescence-associated secretory phenotype
  • GSEA Gene Set Enrichment Analysis
  • 91 HERVK provirus-specific fasta files were obtained from the NCBI database (GenBank ID: JN675007-JN675097), which were annotated in previous studies.
  • NCBI database GenBank ID: JN675007-JN675097
  • 91 HERVK provirus fasta files were linked to the hg19(GRCh37.75) cDNA fasta file annotated in the Ensembl database.
  • a mapping index was generated by Salmon (version 0.8.2) and cleaned RNA-seq reads that were mapped to the junction of hg19 and HERVK proviruses by the Salmon quantification program with parameter "-l A--gcBias" index file.
  • Genomic DNA was extracted from cells using DNeasy Blood & Tissue Kits (QIAGEN) at 2 x 106 cells per replicate and sheared to 100-300 bp with a sonicator. Then, library preparation, bisulfite treatment, quality control, and sequencing were performed by Novogene Bioinformatics Co., Ltd.
  • Raw sequencing reads were trimmed using fastp software (version 0.19.10) with default parameters.
  • the cleaned reads were then mapped to the human hg19 reference genome obtained from the UCSC Genome Browser database using bsmap (version 2.90) and the parameter "-v 0.1-g 1-R-u" (89).
  • the level of CpG DNA methylation at each cytosine site was calculated by the Center for Microsomal DNA methylation.
  • forward and reverse strand reads were combined for each CpG site, and only CpG sites with a depth greater than 5 were retained for downstream analysis.
  • the antibodies used in the examples of the present application are conventional commercial antibodies, antiserum or antibodies that come with the kit, such as the antibodies shown in Table 3.
  • Example 1 ERV activation is associated with human cell senescence
  • human mesenchymal progenitor cells of Hutchinson-Gilford progeria syndrome (HGPS) and Werner syndrome (WS) (LMNA G608G/+ hMPC) or WS hMPC (WRN ⁇ / ⁇ hMPC) ( as a cellular model of premature senescence), and hMPCs of wild-type (WT) replicative senescence (RS) to study the relationship between HERVK activation and human cellular senescence.
  • hMPC human mesenchymal progenitor cells
  • HGPS Hutchinson-Gilford progeria syndrome
  • WS Werner syndrome
  • WRN ⁇ / ⁇ hMPC WS hMPC
  • WT wild-type replicative senescence
  • RNA-seq analysis was performed in WT RS hMPC and models of premature aging including HGPS and WS hMPC.
  • HERVK-int, MER61-int, and MER61C showed significant differences in expression in all five comparisons;
  • Charlie4 and MER101B showed significant differences in comparisons of EP HGPS vs. WT and WS vs. WT and LP HGPS vs. WT and WS vs. WT;
  • MamGypLTR (LTR region of the MamGyp family) showed significant differences in comparisons of EP HGPS vs. WT and WS vs. WT, LP HGPS vs. WT and WT EP vs. LP;
  • HERVK11 showed significant differences in comparisons of EP HGPS vs. WT and WS vs. WT, LP WS vs. WT, and WT EP vs. LP;
  • HERV16 showed significant differences in comparisons of EP HGPS vs. WT, LP HGPS vs. WT and WS vs. WT, and WT EP vs. LP;
  • HUERS-P2 showed significant differences in comparisons of EP WS vs. WT, LP HGPS vs. WT and WS vs. WT, and WT EP vs. LP.
  • human ERV at HERVK (including HERVK11), MER61, MER61C, Charlie4, MER101B, MamGyp, HERV16, HUERS-P2, and HERV30 loci can be used as biological markers of aging.
  • HML2_1q22 showed significant activation in all five comparisons
  • HML2_3q12.3 showed significant activation in comparisons of EP HGPS vs. WT and WS vs. WT and LP WS vs. WT;
  • HML2_3q21.2 showed significant activation in comparisons of EP HGPS vs. WT and WT EP vs. LP;
  • HML2_4p16.1b showed significant activation in the comparison of EP WS vs. WT;
  • HML2_21q21.1 showed significant activation in the comparison of LP HGPS vs. WT;
  • HML2_12q11.1 showed significant activation in the comparison of LP HGPS vs. WT;
  • HERVK corresponding to the locus HML2_1q22 was highly activated in senescent hMPCs.
  • RT-qPCR was performed using primers (shown in Table 2) targeting different regions of the HERVK transcript, including Env, Pol and Gag, and the results are shown in Figure 4A.
  • HERVK is highly expressed during cellular senescence, showing a similar expression trend to previously reported markers, such as the senescence positive marker p21 Cip1 (which increases during senescence) and the senescence negative marker Lamin-related protein LAP2 (TMPO) (reduced during aging).
  • markers such as the senescence positive marker p21 Cip1 (which increases during senescence) and the senescence negative marker Lamin-related protein LAP2 (TMPO) (reduced during aging).
  • RNA-FISH and single-molecule RNA-FISH analysis were performed. The results are shown in Fig. 4B-C, the cytoplasmic HERVK RNA signal was increased in HGPS and WS hMPC as well as in RS hMPC. At the same time, different fluorescent probes were labeled against different regions of the HERVK transcript (including Env, Pol and LTR), showing that different regions of HERVK partially co-localized in the same virus particle (Fig. 4D).
  • epigenetic regulation of the HERVK locus is associated with HERVK transcription and is involved in cellular senescence.
  • TEM analysis showed that, consistent with the increase in HERVK reverse transcribed RNA and protein levels, TEM results showed that RVLP accumulated in the cytoplasm of prematurely senescent and RS hMPCs (as shown in Figure 8). In contrast, RVLP was rarely detected in phenotypically young early passage WT hMPCs.
  • hMPCs were transformed using the HERVK transposition reporter vector.
  • the transformed cells were analyzed by flow cytometry BD Influx, and the ratio of green fluorescence positive cells to red fluorescence positive cells was the probability of HERVK transposition events.
  • the results showed an increased incidence of retrotransposition events in aged hMPCs (as shown in Figure 10A-B).
  • Senescent hMPCs were shown to have increased HERVK DNA copy number using single-molecule DNA-FISH (Fig. 10C-D), indicating increased retrotransposition events in senescent hMPCs.
  • Example 2 HERVK expression causes innate immune response and cellular senescence
  • the transcriptional activation system comprises an activator protein complex (co-activation mediator, SAM) with sgRNA (sgHERVK-act) targeting the HERVK-LTR5HS promoter region.
  • SAM co-activation mediator
  • sgHERVK-act sgHERVK-act targeting the HERVK-LTR5HS promoter region.
  • a CRISPR-dCas9 system containing a non-targeting sgRNA was used as a control.
  • HERVK-activated cells were analyzed by RT-qPCR and Western blotting.
  • hMPCs were treated with 5-AZA, a DNA methyltransferase inhibitor (DNMTi). ChIP-qPCR and RT-qPCR analyses were performed on the treated cells, with cells not treated with 5-AZA as a control.
  • 5-AZA a DNA methyltransferase inhibitor
  • Retroviral inhibitors reduce cellular senescence
  • Ki67 staining and SA- ⁇ -gal staining were performed on the above cells. The results are shown in Figure 25.
  • Ki67 positive cells were significantly increased compared to controls, while SA- ⁇ -gal positive cells were significantly decreased compared to controls ( Figure 16D-E).
  • HERVK DNA induces innate immune responses
  • the Pol protein encoded by HERVK has reverse transcriptional activity and can reverse transcribe HERVK RNA into DNA, thereby generating additional HERVK DNA outside the genome.
  • This excess cytoplasmic DNA may be recognized by the key DNA sensor cGMP-AMP synthase (cGAS) and trigger an innate immune response (Figure 17).
  • hMPCs and fibroblasts of different senescence were also examined: the second messenger 2'3'-cGAMP , phosphorylation of RelA/NF- ⁇ B, expression of TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) and proinflammatory cytokines such as IL1B and IL6, which are classified as SASP factors.
  • TNK1 TANK-binding kinase 1
  • IRF3 IFN regulatory factor 3
  • proinflammatory cytokines such as IL1B and IL6, which are classified as SASP factors.
  • HERVK activation promotes cellular senescence (at least in part) by activating innate immune pathways.
  • the purpose of this example is to demonstrate whether HERVK RVLPs produced by senescent cells can be released extracellularly and transmit senescence signals to non-senescent cells.
  • CM conditioned medium
  • HERVK RNA was detected by ddPCR on CM harvested from wild-type and prematurely senescent hMPCs, and the amplicon was HERVK6 in Table 2.
  • TEM images showed that HERVK particles were attached to young hMPCs (FIG. 22A). Forty-eight hours after old CM treatment, an increase in HERVK RNA was found in young hMPCs as detected by qRT-PCR (Fig. 22B), implying that HERVK in old CMs may spread to target cells.
  • anti-HERVK-Env antibody was used to remove HERVK in old CM.
  • TEM analysis showed that fewer HERVK RVLPs were attached to the cell surface or into it (Figure 23A).
  • removal of HERVK from old CMs resulted in reduced HERVK RNA and a lessened senescence phenotype in young WT hMPCs compared to old CMs from which HERVK was not removed (FIG. 23B-E).
  • old CMs in senescent hMPCs activated the cGAS-STING pathway in young hMPCs and induced SASP gene expression; knockdown of STING or knockdown of TKB1 abrogated the senescent phenotype induced by old CMs (data not shown) .
  • old CM drives cellular senescence (at least in part) by activating innate immune pathways.
  • HERVK virus can infect target cells and cause cell senescence.
  • a vector for expressing and packaging HERVK virus was constructed, and the HERVK virus was labeled with green fluorescence (Figure 24A).
  • HERVK viral RNA, protein and viral particles were detected in 293T cells, as well as GFP signal ( Figure 24B-E).
  • HMPC cells were infected with purified HERVK virus labeled with green fluorescent protein, and electron microscopy showed that virus particles entered the cells, and green fluorescence could be detected in hMPC cells, indicating that HERVK virus could enter hMPC cells (as shown in Figure 25A-C).
  • HERVK virus with green fluorescence were sorted by flow cytometry (Fig. 25D-E). Compared with cells not infected with HERVK virus, the RNA and gene copy numbers of GFP were increased in cells infected with HERVK virus (Fig. 25F-G), indicating that HERVK is not only expressed in cells but also integrated into the genome.
  • Western blotting, ChIP-qPCR, RT-qPCR, SA- ⁇ -gal and Ki67 staining, and clonal expansion ability assays showed that infected HERVK was recognized by cGAS and activated the innate immune response to drive cellular senescence (Figure 25H-M) .
  • HERVK virus was neutralized using anti-HERVK antibodies.
  • Neutralized HERVK and untreated HERVK ( ⁇ HERVK in Figure 26A is the murine anti-HERFVK-Env antibody in Table 2, IgG is the murine immunoglobulin used as a control) were contacted with hMPCs, respectively, and the examples were carried out. 2. Analysis of classical aging characteristics as described. The results showed that treatment of HERVK virus with neutralizing antibodies attenuated HERVK virus-induced activation of the innate immune response and cellular senescence compared to untreated HERVK virus (FIG. 26A-F).
  • HERVK produced in senescent cells can be released in a paracrine manner and induce cellular senescence in young cells.
  • mice Since the homologous virus corresponding to HERVK in mice is MMTV, we detected MMTV in mice.
  • MMTV levels, amplicons are MMTV1 and MMTV2.
  • Figure 27A The expression of MMTV1 in 8-month-old and 24-month-old mice was significantly higher than that in 2-month-old mice; while the expression of MMTV2 in 24-month-old mice was significantly higher than that in 2-month-old and 8-month-old mice .
  • MMTV expression levels increased with age in isolated lung, liver, and skin tissues from aged mice compared with young mice by western blot and immunohistochemical staining analysis (Figure 27B-C). ). The innate immune response was also hyperactivated in naturally aged mice (FIG. 27B).
  • ERVW-Env signaling was increased in lung, liver and skin tissues of HGPS cynomolgus monkeys relative to WT cynomolgus monkeys of the same age by western blot and immunohistochemical staining analysis (Figure 28).
  • the results also showed increased innate immune responses in HGPS cynomolgus monkey tissues, eg, increased phosphorylation of RelA (FIG. 28A) and up-regulation of the SASP gene (data not shown).
  • ERVW expression levels increased with age when compared in lung, liver and skin tissues isolated from young and physiologically aged cynomolgus monkeys ( Figure 28A-C). SASP was also hyperactivated in naturally aged monkeys, similar to progeroid HGPS monkeys ( Figure 28D).
  • HERVK levels in young and aged donor primary hMPCs were analyzed by qPCR. The results showed that the expression of HERVK genes (env, pol and gag) was significantly up-regulated in aged individuals relative to younger individuals (Figure 29A). In skin tissues obtained from young and aged donors, immunohistochemical staining showed that HERVK-Env expression increased significantly with age ( Figure 29B). More importantly, ELISA analysis of human serum showed that HERVK-Env levels were significantly increased in the serum of older individuals relative to younger individuals (Figure 29C).
  • Example 5 Inhibition of ERV to delay aging of tissues and organs and individuals
  • the purpose of this example is to explore whether the purpose of delaying the aging of tissues and organs and individuals can be achieved by inhibiting ERV.
  • the whole knee joint (including the lower end of femur, upper end of tibia, patella, articular cartilage, articular ligament and synovial fluid), RNA extraction, RT-qPCR showed that the level of inflammatory factor IL1B and the expression of aging-related protein p21 were decreased after inhibiting MMTV.
  • the purpose of this example is to synthesize specific neutralizing antibodies against endogenous virus MMTV in mice, and to detect the titer and neutralization efficiency of the synthesized antibodies for in vivo treatment.
  • MMTV antibody was provided by Yiqiao Shenzhou Biotechnology Co., Ltd. Briefly, according to structural analysis, the MTV-SU subunit (SEQ ID NO: 151), plus N-signal peptide and N-his-tag, was expressed in the HEK293 system. The MMTV-SU subunit was recombinantly expressed as the immunogen, and the mice were immunized, and the antibody binding to the immunogen was screened by ELISA.
  • Monoclonal antibodies were isolated and MMTV virus was treated with the isolated monoclonal antibodies.
  • Mouse cells were infected with the treated MMTV virus, the titer of the treated MMTV virus was reduced compared to the control, and the antibodies used to treat the MMTV virus were identified as neutralizing antibodies.
  • mice were administered to aged mice, and the mice were tested for their body weight, skin, hair, arched back, cataracts, and mobility.
  • the Y-maze test was also performed on the mice, and the survival time of the mice was recorded.
  • samples from various tissues and organs including brain, lung, liver, skin, blood, joints) were collected, and each organ was verified by sequencing techniques including transcriptome sequencing, immunohistochemical staining, western blotting, and RT-qPCR Aging phenotype.
  • SEQ ID NO: 1 exemplary HERVK-Env amino acid sequence
  • SEQ ID NO: 2 exemplary HERVK-Pol amino acid sequence
  • SEQ ID NO: 3 exemplary HERVK-Pro amino acid sequence
  • SEQ ID NO:6 exemplary HERVK-Pol encoding nucleotide sequence
  • SEQ ID NO:7 exemplary HERVK-Pro encoding nucleotide sequence
  • SEQ ID NO: 8 an exemplary HERVK-Gag-encoding nucleotide sequence
  • SEQ ID NO: 149 exemplary HERVK-Rev encoding nucleotide sequence
  • SEQ ID NO: 150 an exemplary HERVK full-length encoding nucleotide sequence

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Abstract

一种以内源逆转录病毒(ERV)作为生物标记物检测对象或其细胞、组织或器官或分离的细胞的衰老程度的方法。一种以ERV作为靶点治疗、防止或延缓对象或其细胞、组织或器官或分离的细胞的衰老的方法。

Description

鉴定和/或调节衰老的方法 技术领域
本发明涉及生物医药领域。具体而言，本发明提供了鉴定和/或调节衰老的方法。更具体而言本发明涉及通过检测内源逆转录病毒活化水平来鉴定细胞、组织、器官或对象的衰老程度，或提供通过抑制或促进内源逆转录病毒活化水平来调节细胞、组织、器官或对象的衰老程度的方法。
背景技术
由于医学的发展和公共卫生的改善，人类平均寿命稳定增长，与年龄增加相伴出现的各种衰老表现因此日益凸显。衰老导致多器官乃至整个机体的生理衰退和慢性疾病发生，为应对衰老伴随的相应健康问题，老年学以及老年医学领域均开展了大量研究，以期通过介入治疗来延缓衰老速度以及相应老年慢病的发生发展。此外，由于各种胁迫(包括工作压力、代谢紊乱、环境污染等)以及遗传因素，部分个体呈现病理性的加速衰老表型。因此，老年医学覆盖较宽的区域，不仅针对老年阶段，而是面向全生命周期，目标是延长人们在更年轻的身体功能的状态下的健康时间。因此，发掘可以鉴定个体或其细胞、组织或器官的衰老程度的生物学标记物，以及用于防止或延缓个体或其细胞、组织或器官衰老的治疗靶点对于老年医学乃至整个医学领域都是非常重要的。
然而，虽然关于衰老的研究在很多方面得到了推进并对衰老的驱动力提出了若干假设，但是目前对衰老的机理没有被完全阐明，许多潜在的分子变化和机制仍知之甚少。个体衰老通常被认为至少部分与个体中细胞的衰老相关。细胞衰老可能由各种刺激因素引起，包括由于DNA末端复制导致的端粒缩短、DNA损伤、肿瘤抑制基因和癌基因的活性改变、氧化应激、炎症、化疗剂以及暴露于紫外线照射和电离辐射等。分泌蛋白质组是细胞衰老的标志之一，它是不同种类细胞因子的表达和分泌明显增加的表型，也是衰老细胞产生的最重要的环境效应，被称为衰老相关的分泌表型(senescence-associated secretory phenotype，SASP)。这些分泌因子促进与相邻细胞和免疫系统的通信，最终影响衰老细胞的命运。一方面，SASP可通过分泌促进血管生成、细胞外基质重塑或上皮-间质转化(EMT)的因子来促进肿瘤细胞发展。另一方面，衰老诱导的慢性炎症可引起系统性免疫失调，导致衰老相关的组织损伤和变性，及衰老相关疾病的发生。
当前的生物学研究多集中于基因组的蛋白质编码部分，而在非蛋白质编码部分的相关发现却很少。尤其是基因组的“暗物质”——逆转录元件在衰老中的作用仍然是未知的。逆转录元件包括长末端重复序列(LTR)和非LTR逆转座子。LTR逆转座子包括内源逆转录病毒(ERV)，其是古代逆转录病毒感染人类并整合于人类基因组的遗迹，在进化过程中固定在人类基因组中。人内源逆转录病毒(HERV)约占人类基因组的8％。
发明人发现，内源逆转录病毒基因座的活化与细胞和个体衰老相关，是一类新型的SASP分子，可以作为个体和细胞衰老的生物学标记，以及衰老及相关疾病干预的靶点。
发明内容
在第一方面，本发明提供一种鉴定对象或其细胞、组织或器官衰老程度或评估对象生理年龄，或诊断衰老相关疾病，例如早衰症和衰弱症，或评估分离的动物细胞或细胞群的衰老程度的方法，包括
a)检测来自所述对象的样品或所述分离的动物细胞中的内源逆转录病毒(ERV)活化水平；和
b)将所述检测结果与所述ERV活化水平的参考值进行比较。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、和ERVW及其同源物。在一些实施方案中，所述参考值是通过检测参考对象或参考细胞获得的。在一些实施方案中，所述参考值是通过检测参考对象群体或参考细胞群获得的。
在一些实施方案中，所述ERV的活化水平包括ERV基因座甲基化水平、ERV转录物水平、ERV蛋白质水平、ERV颗粒在细胞中的存在、定位和/或从细胞释放，和/或ERV基因座的转座概率。在一些实施方案中，所述ERV的活化水平包括ERV基因座甲基化水平、ERV转录物水平、ERV蛋白质水平和/或ERV颗粒在细胞中的存在和/或从细胞释放。在一些实施方案中，所述ERV的活化水平包括ERV基因座的转座概率。在一些实施方案中，所述ERV的活化水平通过ERV颗粒在细胞中的定位表现。
在一些实施方案中，所述对象或动物是哺乳动物。在一些实施方案中，所述对象或动物是非人哺乳动物。例如，所述对象或动物是小鼠，且任选地，所述ERV来自MMTV家族；或者所述对象或动物非人灵长类动物，且任选地，所述ERV来自ERVW家族。优选地，所述对象或动物是人，且任选地，所述ERV来自HERVK家族。
在一些实施方案中，所述样品是体液样品，包括但不限于尿液、唾液和血液。在一些实施方案中，所述血液包括但不限于全血、血浆或血清。在一些实施方案中，所述样品包含来自所述对象或动物的组织。在一些实施方案中，所述组织包括但不限于皮肤、肺、肝脏组织。在一些实施方案中，所述样品包含来自所述对象或动物的细胞。在一些实施方案中，所述细胞包括但不限于成纤维细胞和间充质干细胞。
在第二方面，本发明提供一种治疗有需要的对象或其细胞、组织或器官的过早衰老，或防止或延缓对象或其细胞、组织或器官衰老的方法。
在一些实施方案中，所述方法包括给所述对象施用ERV基因座活化抑制剂或所述ERV的抑制剂。在一些实施方案中，其中所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述ERV基因座活化抑制剂提高所述ERV基因座甲基化水平、降低所述ERV基因座转录物水平、降低所述ERV基因座蛋白质水平、降低所述ERV颗粒在细胞中存在和/或从细胞释放。
在一些实施方案中，所述对象或动物是哺乳动物。在一些实施方案中，所述对象或动物是非人哺乳动物。例如，所述对象或动物是小鼠，且任选地，所述ERV来自MMTV家族；或者所述对象或动物非人灵长类动物，且任选地，所述ERV来自ERVW家族。优选地，所述对象或动物是人，且任选地，所述ERV来自HERVK家族。
在一些实施方案中，所述方法包括给所述对象施用逆转录病毒抑制剂，例如，逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。
在第三方面，本发明提供一种调节细胞衰老的方法，包括使所述细胞与ERV基因座活化调节剂接触。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述调节剂是ERV基因座活化抑制剂或所述ERV的抑制剂。在一些实施方案中，所述ERV基因座活化抑制剂提高所述ERV基因座甲基化水平、降低所述ERV基因座转录物水平、降低所述ERV基因座蛋白质水平、降低所述ERV颗粒在细胞中存在和/或从细胞释放。
在一些实施方案中，所述调节剂是所述细胞的ERV基因座活化促进剂。在一些实施方案中，所述ERV基因座活化促进剂降低所述ERV基因座甲基化水平、提高所述ERV基因座转录物水平、提高所述ERV基因座蛋白质水平、提高所述ERV颗粒在细胞 中存在和/或从细胞释放。
在一些实施方案中，所述对象或动物是哺乳动物。在一些实施方案中，所述对象或动物是非人哺乳动物。例如，所述对象或动物是小鼠，且任选地，所述ERV来自MMTV家族；或者所述对象或动物非人灵长类动物，且任选地，所述ERV来自ERVW家族。优选地，所述对象或动物是人，且任选地，所述ERV来自HERVK家族。
在第四方面，本发明提供一种培养分离的动物细胞或细胞群或防止或延缓细胞衰老的方法。
在一些实施方案中，所述方法包括使所述细胞与所述细胞的ERV基因座活化抑制剂或所述ERV的抑制剂接触。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述ERV基因座活化抑制剂所述ERV基因座活化抑制剂提高所述ERV基因座甲基化水平、降低所述ERV基因座转录物水平、降低所述ERV基因座蛋白质水平、降低所述ERV颗粒在细胞中存在和/或从细胞释放。
在一些实施方案中，所述对象或动物是哺乳动物。在一些实施方案中，所述对象或动物是非人哺乳动物。例如，所述对象或动物是小鼠，且任选地，所述ERV来自MMTV家族；或者所述对象或动物非人灵长类动物，且任选地，所述ERV来自ERVW家族。优选地，所述对象或动物是人，且任选地，所述ERV来自HERVK家族。
在一些实施方案中，所述方法包括是所述细胞与逆转录病毒抑制剂接触。在一些实施方案中，所述逆转录病毒抑制剂包含逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。
在第五方面，本发明提供一种促进细胞衰老的方法。
在一些实施方案中，所述方法包括使所述细胞与所述细胞的ERV基因座活化促进剂或所述ERV接触。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述调节剂是所述细胞的ERV基因座活化促进剂。在一些实施方案中，所述ERV基因座活化促进剂降低所述ERV基因座甲基化水平、提高所述ERV基因座转录物水平、提高所述ERV基因座蛋白质水平、提高所述ERV颗粒在细胞中存在和/或从细胞释放。
在一些实施方案中，所述细胞是哺乳动物细胞。在一些实施方案中，所述细胞是非人哺乳动物细胞。例如，所述细胞是小鼠细胞，且任选地，所述ERV来自MMTV家族；或者所述细胞是非人灵长类动物细胞，且任选地，所述ERV来自ERVW家族。优选地，所述细胞是人细胞，且任选地，所述ERV来自HERVK家族。
在一些实施方案中，所述方法包括使所述细胞与DNA甲基转移酶抑制剂，例如地西他滨、Lomeguatrib、SGI-1027或5-氮杂胞苷接触。在一些实施方案中，所述细胞是哺乳动物细胞，例如小鼠细胞、非人灵长类动物细胞和人细胞。
在第六方面，本发明提供一种防止或延缓组织器官退行，治疗相关疾病，延缓机体衰老的方法，例如防止或延缓皮肤衰老、治疗或预防骨关节炎以及衰弱症的治疗方法。。
在一些实施方案中，所述方法包括给有需要的对象施用逆转录病毒抑制剂。在一些实施方案中，所述逆转录病毒抑制剂包含逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。
在一些实施方案中，所述方法包括给有需要的对象施用HERVK基因座活化抑制剂或HERVK抑制剂。在一些实施方案中，所述HERVK基因座活化抑制剂所述ERV基因座活化抑制剂提高所述HERVK基因座甲基化水平、降低所述HERVK基因座转录物水平、降低所述HERVK基因座蛋白质水平、降低所述HERVK颗粒在细胞中存在和/或从 细胞释放。
在一些实施方案中，所述施用是局部施用、皮内施用或皮下施用。
在第七方面，本发明提供一种治疗早衰症，例如Hutchinson-Gilford早衰综合征(HGPS)或Werner综合征(WS)的方法。
在一些实施方案中，所述方法包括给有需要的对象施用HERVK基因座活化抑制剂或HERVK抑制剂。在一些实施方案中，所述HERVK基因座活化抑制剂所述ERV基因座活化抑制剂提高所述HERVK基因座甲基化水平、降低所述HERVK基因座转录物水平、降低所述HERVK基因座蛋白质水平、降低所述HERVK颗粒在细胞中存在和/或从细胞释放。
在一些实施方案中，所述方法包括给有需要的对象施用逆转录病毒抑制剂。在一些实施方案中，所述逆转录病毒抑制剂包含逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。
附图说明
图1：左幅为早衰细胞模型的原理示意图；右幅为HERVK活化(包括转录、翻译、包装和释放)示意图。
图2：灵长类动物进化过程中，ERVW家族和ERVK家族整合入基因组的示意图。
图3A左幅：圆环图，显示了过早衰老中共享上调的重复元素(RE)和按每个重复元素类别分类的复制衰老的(RS)hMPC的组成。考虑至少在三个比较中上调的RE(放大的RE见图3C)。右幅：过早衰老和RS hMPC中LTR类的共享上调RE的家族富集分析。图3B的热图显示过早衰老和RS hMPC中RepeatMasker注释的重复元件的相对表达水平(log2(倍数变化))。图3C的条形图显示过早衰老和RS hMPC中上调的RepeatMasker注释的重复元件的重叠图。图3D的热图显示在过早衰老的hMPC中，HERVK的所有原病毒序列的相对表达水平，从蓝色到红色的颜色键代表低到高的表达水平。图3E的火山图显示在过早衰老的hMPC中HERVK的所有原病毒序列的差异表达。图3F显示在过早衰老的hMPC中HERVK在原病毒HML2_1q22处的RNA水平上调。
图4A的热图显示通过RT-qPCR检测到的HERVK的RNA水平；图4B左幅是HERVK RNA-FISH的代表性图像，右幅是荧光信号强度的定量分析。
图5的小提琴图显示HERVK的CpG DNA甲基化水平。
图6A和B是ChIP-qPCR评估WT、HGPS和WS的hMPC中的HERVK-LTR5HS区的H3K9me3和H3K36me3富集的结果，n＝3，数据以平均值±SEM表示；图6C和D是ChIP-qPCR评估RS的HERVK-LTR5HS区的H3K9me3和H3K36me3富集的结果，n＝3，数据以平均值±SEM表示。
图7A是WT、HGPS和WS的hMPC中对HERVK-Env免疫荧光染色的结果；图7B是WT、HGPS和WS的hMPC中对HERVK-Env、p16 INK4a和LAP2的蛋白质印记的结果。图7C是RS的hMPC中对HERVK-Env免疫荧光染色的结果；图7D是RS的hMPC中对HERVK-Env、p16 INK4a和LAP2的蛋白质印记的结果。
图8显示重金属染色的WT、HGPS和WS以及RS的hMPC中的RVLP，左幅为TEM图像，右幅为RVLP计数结果，HGPS、WS以及RS的hMPC中的RVLP显著增加。
图9显示针对HERVK-Env的免疫金标的TEM分析结果。图9A中左幅是WT、HGPS和WS的hMPC的TEM图像，标尺为200nm(左)和100nm(右)，虚线表示细胞膜，E：细胞外，I：细胞内；右幅为统计分析结果。图9B中左幅为不加一抗的对照TEM，标尺为200nm(上)和100nm(下)，虚线表示细胞膜，E：细胞外，I：细胞内；中幅为RS的hMPC的TEM图像，标尺为200nm(左)和100nm(右)，虚线表示细胞膜，E：细胞外，I：细胞内；右幅为统计分析结果。
图10显示针对HERVK发生的转座概率的分析结果。图10A是构建检测HERVK转座概率的载体的示意图；图10B显示利用流式细胞术分析的在RS的hMPC中HERVK的转座概率，绿色荧光与红色荧光的比例代表转座概率；图10C显示利用单分子DNA-FISH在WT、HGPS和WS的hMPC中检测的基因组中的HERVK拷贝数；图10D显示利用单分子DNA-FISH在RS的hMPC中检测的基因组中的HERVK拷贝数。
图11显示在RS的成纤维细胞中HERVK的表观遗传学脱抑制。
图12显示经CRISPR-dCas系统激活后，HERVK-Env蛋白质水平提高(12A)；qPCR热图(12B)显示HERVK-Env表达上调，并且与衰老正标记p16 INK4a表达上调和负标记LAP2表达下调；SA-β-gal染色(12C)、Ki67染色(12D)和克隆扩增能力(12E)显示细胞衰老。
图13显示在HGPS和WS的hMPC中利用shRNA敲减HERVK降低了衰老表型。
图14显示经Cas9KRAB系统抑制后，HERVK-Env蛋白质水平降低(14A)，SA-β-gal染色(14B)、Ki67染色(14C)和克隆扩增能力(14D)显示延缓了细胞衰老。
图15显示5-AZA处理导致HERVK-LTR5HS甲基化程度降低(14A)，使HERVK活化(HERVK RNA水平提高，14B)，诱导衰老(14B-E)，而敲减HERVK(14F)有效降低5-AZA诱导的衰老表型(SA-β-gal染色阳性细胞减少，14G)。
图16显示用HERVK逆转录酶抑制剂和整合酶抑制剂延缓细胞衰老。图16A是HERVK的生命周期的示意图，其中逆转录酶和整合酶可以被小分子抑制；图16B的流式细胞术分析显示逆转录酶抑制剂Abacavir降低HERVK的转座效率；图16C显示逆转录酶抑制剂Abacavir降低HERVK的DNA拷贝数；逆转录酶抑制剂Abacavir、Lamivadine以及整合酶抑制Ratelgravir降低SA-β-gal染色阳性细胞数(图16C)，且增加Ki67阳性细胞数(图16D)。
图17是诱导先天性免疫应答的cGAS途径的示意图。
图18显示通过免疫沉淀和qPCR分析细胞质HERVK DNA上的cGAS富集。
图19显示在过早衰老的hMPC中cGAS途径活化，诱导了先天性免疫应答。图19A显示WT、HGPS和WS hMPC中2'3'-cGAMP水平的ELISA分析；图19B显示WT、HGPS和WS hMPC的培养基中IL-6水平的ELISA分析；图19C显示WT、HGPS和WS hMPC中p-TBK1、p-IRF3和p-RelA的蛋白质印记分析。
图20显示敲减HERVK抑制HGPS和WS hMPC中的cGAS途径以及先天免疫应答。敲减HERVK的结果(图20A和B)表现出与敲减STING的结果(图20C-E)一致的趋势。
图21A显示通过ddPCR分析WT、HGPS和WS hMPC中HERVK DNA拷贝数；图21B显示ELISA分析WT、HGPS和WS hMPC培养基中HERVK-Env水平；图21C显示WT、HGPS和WS hMPC的HERVK-Env免疫金标TEM图像(标尺为200nm(左)和100nm(右)，虚线表示细胞膜，E：细胞外，I：细胞内)。
图22显示与老CM温育诱导年轻hMPC衰老。
图23显示去除老CM中的HERVK颗粒后，其诱导衰老的能力降低。
图24显示体外合成并纯化带有GFP的HERVK活病毒。293T细胞转染带有GFP的HERVK载体，HERVK病毒RNA、病毒蛋白、以及病毒颗粒均可以检测到(图24B-E)。
图25显示用纯化的带有GFP的HERVK活病毒感染hMPC，HERVK病毒可以进入靶细胞(图25B-G)，激活天然免疫通路和炎症因子表达上调(图25H-J)，导致hMPC衰老(图25K-M)。
图26显示用HERVK抗体中和HERVK活病毒，可以降低天然免疫反应和炎症因子的表达(图26E-F)，缓解hMPC衰老(图26B-D)。
图27A显示年轻年老小鼠肝脏组织中的MMTV的RT-qPCR分析；图27B示年轻 年老小鼠肺组织中的MMTV-Env和p-RelA的蛋白质印记分析；27C显示分别年轻年老小鼠肺、皮肤和肝组织中MMTV-Env的免疫组织化学染色的定量分析。
图28A显示HGPS和WT食蟹猴肺组织中的ERVW-Env和p-RelA的蛋白质印记分析；图22B显示分别HGPS和WT食蟹猴肺、皮肤和肝组织中ERVW-Env的免疫组织化学染色的定量分析；图22C显示年轻和年老的食蟹猴肺组织中ERVW-Env蛋白质印记分析的定量结果；图22D显示年轻和年老的食蟹猴肺、皮肤和肝组织中ERVW-Env的免疫组织化学染色的定量分析。
图29A显示来自年轻和年老的人的原代hPMC中HERVK基因的RT-qPCR结果；图23B显示来自年轻和年老的人的皮肤组织中HERVK-Env的免疫组化染色，左幅为代表性图像，右幅为统计分析结果；图23C显示来自年轻和年老的人的血清中HERVK的ELISA分析。
图30A显示用年轻和年老的人的血清以及去除HERVK的年老人血清培养来源于年轻人的原代hPMC的示意图；图30B显示在用年轻和年老的人的血清以及去除HERVK的年老的人血清培养的来源于年轻人的原代hPMC中炎症因子的RT-qPCR分析；图30C显示用年轻和年老的人的血清以及去除HERVK的年老的人血清培养的来源于年轻人的原代hPMC中SA-β-gal分析。
图31A显示向年老小鼠关节注射抑制MMTV的慢病毒(编码sgRNA的慢病毒，所sgRNA靶向MMTV)的实验流程图；图31B显示经Cas9KRAB系统抑制后，MMTV蛋白质水平降低；图31C显示注射8周后，小鼠四肢拉力水平测试；图31D显示注射10周后，利用微型核磁检测关节骨密度；图31E显示注射10周后，关节处炎症因子的RT-qPCR分析。
图32A显示向年老小鼠关节注射逆转录酶抑制剂Abacavir实验流程图；图32B显示注射逆转录酶抑制剂Abacavir 8周后，小鼠四肢拉力水平测试；图3C显示注射逆转录酶抑制剂Abacavir 13周后，小鼠跑步能力测试；图32D显示注射逆转录酶抑制剂Abacavir 14周后，利用微型核磁检测关节骨密度；图32D显示注射逆转录酶抑制剂Abacavir，关节处炎症因子的RT-qPCR分析。
图33A显示向年老小鼠通过将逆转录酶抑制剂Abacavir溶解于饮用水中每天施用的示意图；图33B显示施用逆转录酶抑制剂Abacavir 12周后，小鼠四肢拉力水平测试；图33C显示施用逆转录酶抑制剂Abacavir 24周后，小鼠的体重、皮肤、毛发、弓背、白内障以及活动能力测试；图33D显示给年老小鼠施用逆转录酶抑制剂后的生存曲线。
图34A显示向年老小鼠施用整合酶抑制剂Ratelgravir的示意图；图34B显示施用逆转录酶抑制剂Abacavir 16周后，小鼠四肢拉力水平测试，以及小鼠的体重、皮肤、毛发、弓背、白内障以及活动能力测试。
图35显示抗MMTV抗体用于小鼠中MMTV-Env蛋白的蛋白质印迹分析(图35A)和MMTV的免疫组化染色实验(图35B)结果。
发明详述
尽管将结合以下所列举的实施方案描述本发明，但是应当理解，它们并不旨在将本发明限制于那些实施方案。相反，本发明旨在覆盖可包括在如权利要求所限定的本发明的范围内的所有替代、修改和等同形式。本领域技术人员将认识到许多与本文描述的方法或材料相似或等同的方法和材料可用于实施本发明。本发明不限于所描述的方法和材料。如果所引用的文献、专利和类似材料中的一个或多个与本申请不同或相矛盾，包括但不限于所定义的术语、术语用法、所描述的技术等，则以本申请为准。除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同含义。尽管下面描述了合适的方法和材料，但是与本文描述的那些类似或等同的方法和材料也可以用于本发明的实践或测试中。
一、定义
如本文所用，术语“和/或”涵盖由该术语连接的项目的所有组合，应视作各个组合已经单独地在本文列出。例如，“A和/或B”涵盖了“A”、“A和B”以及“B”。例如，“A、B和/或C”涵盖“A”、“B”、“C”、“A和B”、“A和C”、“B和C”以及“A和B和C”。
在本文中，术语“对象”是指非人动物对象，例如非人哺乳动物、鸟类、爬行类、鱼类等，包括家畜、家禽、宠物、竞赛动物，或人对象。
对象的“衰老”通常是指生物体对环境的生理和/或心理适应能力进行性降低、逐渐趋向死亡的现象。衰老可分为生理性衰老和病理性衰老，其中生理性衰老指成熟期后出现的生理性退化过程，而病理性衰老是由于各种外来因素(包括各种疾病)所导致的老年性变化。然而，衰老是许多病理、生理和心理过程的综合作用的结果，实际上在大多数情况下无法完全区分生理性衰老和病理性衰老。
对象的“衰老程度”通常与“生理年龄”相关，是指对象表现出某一自然年龄时生理和其功能所反映出来的水平，即与某一自然年龄相对应的生理及其功能的表现程度。
对象的过早衰老是指对象的生理年龄显著大于其自然年龄，通常是由于环境、疾病、过劳等因素造成的。在某些情况下，过早衰老包括遗传性早衰症，例如Hutchinson-Gilford早衰综合征(HGPS)和Werner综合征(WS)。
细胞的“衰老”被认为是生物体衰老的重要原因，通常是指细胞保持活力和代谢活性但丧失了增殖能力。衰老细胞的显著特征包括(i)生长停滞，(ii)细胞形态增大和变平，(iii)细胞核中的DNA损伤灶，(iv)衰老相关的分泌表型(SASP)，(v)衰老相关的β-半乳糖苷酶(SA-β-gal)活性升高，(vi)肿瘤抑制因子p16 INK4a的表达增加，和(vii)PML核体的数量和大小的增加等。
导致细胞的衰老表型途径通常包括复制衰老(RS)、过早衰老和分化后的衰老(SAD)。“复制衰老”是在大量细胞分裂后发生的衰老类型。例如，当在培养物中生长时，原代细胞在大约50次细胞分裂后经历细胞衰老。这种进一步增殖的障碍被认为是由于每次连续细胞分裂导致细胞端粒缩短，导致细胞达到触发DNA损伤应答的点(所谓的“Hayflick极限”)，最终导致诱导增殖停滞和衰老。细胞的“过早衰老”是指在没有端粒缺失或功能障碍的情况下的细胞衰老。过早衰老可能由多种刺激引起，包括例如化疗、放疗、DNA损伤、氧化应激、炎症、强有丝分裂信号传导和核糖体应激。此外，细胞中的遗传缺陷也会造成过早的细胞衰老，例如，以上所提及的HGPS或WS患者的细胞会发生过早衰老。SAD是指终末分化的有丝分裂后的细胞所显示出的衰老样表型(包括SASP)，可以由各种应激物诱导，包括遗传毒性、蛋白质毒性、氧化和核糖体应激物。有研究表明，在某些疾病中观察到SAD。在本文中，细胞的“衰老程度”是指其衰老相关表型与传代特定次数的细胞的相应表型相当。
“内源逆转录病毒(ERV)”是逆转录病毒来源的基因组序列，一般认为是在进化过程中(如数百万年前)整合到种系染色体中逆转录病毒的遗迹。尽管大多数ERV有缺陷，并且在多数情况下无活性，但它们可能在某些生理和病理条件下被激活。例如，ERVW家族在距今约2,500万年前插入灵长类种系染色体中，而HERVK家族在距今约600-700万年前插入人种系染色体中。有研究表明，在某些情况下，MMTV在啮齿类动物如小鼠中保持活性。ERVW家族在非人灵长类动物，如旧世界猴中仍然保持活性，而人中的HERVK家族保持活性。ERV基因座通常包括侧翼LTR区和编码序列。
在本文中，“参考值”是指包括处于特定状态(如年龄或传代数)的正常对象或其细胞、组织或器官，或者正常的分离细胞或细胞群体的形态、功能和代谢产物等代表性生理及生化特征的指标常数，也称为正常值。参考值可以是预先确定的值，也可以是对参考对象或参考细胞进行检测获得的值。
在本文中，“参考对象”或“参考细胞”是指具有上述代表性特征的对象或细胞。例如， 本发明中的参考对象是指例如具有特定衰老程度或生理年龄的代表性特征的对象，本发明中的参考细胞是指例如具有传代达特定次数的细胞的代表性特征的细胞。
在本文中，“基因座”是指染色体上的区域，包括一个或多个基因、基因部分和/或调控序列。
在本文中，ERV的“同源物”是与其在系统发育上序列同源或是来源于同一病毒家族的同源的其他物种的ERV。例如，根据Stockinga&Kozak,Cell.Mol.Life Sci.65(2008)3383-3398，HERVK与小鼠中的MMTV是同源物。
在本文中，“ERV基因座活化”是指ERV基因座从沉默状态转变为可转录、翻译甚至包装和分泌病毒颗粒的状态。ERV基因座活化的抑制剂/促进剂涵盖能抑制/促进(包括特异性抑制/促进和非特异性抑制/促进)ERV基因座活化的任何物质。
在本文中，“非特异性逆转录病毒抑制剂”是指不特异性针对某一特定ERV基因座的逆转录病毒抑制剂。
如本文所用，术语“多核苷酸”或者“核酸分子”包括DNA分子(例如cDNA或基因组DNA)和RNA分子(例如mRNA)及使用核苷酸类似物产生的DNA或RNA的类似物。所述核酸分子可以是单链或双链的，优选双链DNA。所述核酸的合成可以使用核苷酸类似物或衍生物(例如肌苷或硫代磷酸核苷酸)。这种核苷酸可以用于，例如，制备具有改变的碱基配对能力或者增加的核酸酶抗性的核酸。
如本文所用，术语“编码”是指多核苷酸直接指定其蛋白质产物的氨基酸序列。编码序列的边界一般由开放读框确定，所述开放读框通常以ATG起始密码子或另外的起始密码子如GTG和TTG开始，以终止密码子如TAA、TAG和TGA结束。所述编码序列可以是DNA、cDNA或重组核苷酸序列。
如本文所用，“反义核酸”是指与靶核酸(例如mRNA)具有互补序列的核酸分子，通过与靶核酸进行碱基配对结合的方式，参与基因的表达调控。反义核酸包括能与特定mRNA精确互补、特异性阻断其翻译的RNA或DNA分子。
如本文所用，“干扰核酸”是指编码用于RNA干扰(RNAi)的RNA分子，包括siRNA、shRNA、miRNA等的核酸分子。
在本文中，术语“蛋白质”是指由一或多条“多肽”链组成的生物大分子。多肽是指含有十个或更多个通过肽键连接的氨基酸残基的链。本文中的所有肽和多肽化学式或序列均是从左至右书写的，表示从氨基末端至羧基末端的方向。
在肽的语境中，术语“氨基酸”、“残基”和“氨基酸残基”可以互换使用，包括蛋白质中天然存在的氨基酸和非天然氨基酸。蛋白质中天然存在的氨基酸的单字母和三字母命名采用本领域惯用名，可见于Sambrook,et al.(Molecular Cloning:A Laboratory Manual,2nd,ed.Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1989)。
如本文所用，“相同性百分比”是指比较两个多肽的氨基酸或两个核酸分子的核苷酸，当最佳比对时，所述两个多肽或两个核酸分子具有大约指定的相同氨基酸或核苷酸百分比。例如，“95％的氨基酸/核苷酸相同性”是指比较两个多肽的氨基酸或两个核酸分子的核苷酸，当最佳比对时，所述两个多肽有95％的氨基酸/核苷酸相同。
如本文所用，“分离的”细胞是指与其来源生物体分开的细胞，通常指离体培养的细胞，包括但不限于贴壁培养的细胞、悬浮培养的细胞或3D培养的细胞。
CRISPR/Cas系统是指成簇的规律间隔的短回文重复序列及其相关系统，其能在向导RNA的指导下，在特定位置切割DNA链。
CRISPR-dCas系统涉及通过修饰造成Cas的核酸酶活性丧失，从而不能切割DNA链，同时使其与转录激活结构域连接，以实现位点特异性转录激活的效果。
同样，CRISPR-dCas系统也可用于调节感兴趣的区域的甲基化水平来调节转录。具体而言，可以将dCas与促进甲基化的序列，如具有甲基转移酶活性的氨基酸序列连接 (例如CRISPR-dCas-SunTag-DNMT3A)，以提高感兴趣的区域的甲基化水平，从而减低感兴趣的区域的转录。也可以将dCas与抑制甲基化的序列，如Tet(ten-eleven translocation)催化结构域连接(CRISPR-dCas-Tet)，以降低感兴趣的区域的甲基化水平，从而增加感兴趣的区域的转录。
二、鉴定衰老程度的方法
本发明提供一种鉴定对象或其细胞、组织或器官衰老程度或评估对象生理年龄或诊断早衰症的方法，包括
a)检测来自所述对象的样品中的内源逆转录病毒(ERV)活化水平，；和
b)将所述检测结果与所述ERV活化水平的参考值进行比较。
通常，生物体通过例如表观遗传学调控ERV。但是，在某些情况下，ERV能够绕过生物体监测，这样其基因座由于表观生物学调控(如DNA甲基化)的改变而活化，从而逆转录病毒元件开始转录、逆转座、甚至产生并释放能够感染细胞的类逆转录病毒样颗粒(RVLP)。
发明人出人意料地发现，在衰老的过程中，一些ERV基因座活化。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，ERV基因座的“活化水平”包括ERV基因座甲基化水平、ERV转录物水平、ERV蛋白质水平和/或ERV颗粒在细胞中存在和/或从细胞释放。较低的甲基化水平、较高的转录物水平、较高的蛋白质水平和/或较高的ERV颗粒在细胞中存在或从细胞释放代表较高的ERV基因座活化水平。在一些实施方案中，ERV基因座的活化水平包括ERV基因座的转座概率。较高的转座概率代表较高的活化水平。
在一些实施方案中，所述参考值是通过检测参考对象获得的。在本发明中，所述参考对象是指处于特定生理年龄的对象。可以通过检测与生理年龄相关的代表性特征(例如选自大脑活性、皮肤弹性、反射动作和平衡性等的特征)来确定想要的参考对象。由于个体差异，优选地，所述参考值是通过检测参考对象群体获得的。
根据本发明，也可以选择数量具有统计学意义的处于特定自然年龄(例如20岁、30岁、40岁、50岁、60岁、70岁、80岁、90岁或更高)或自然年龄范围(例如18-22岁、28-32岁、38-42岁、48-52岁、58-62岁、68-72岁、78-82岁、88-92岁等)的正常对象作为参考对象。
所述参考值可以以“平均值±标准差”的形式表示，也可以以范围的形式表示。在对群体进行检测以确定参考值的情况下，还可以去掉所测得的值中最高和最低的部分，例如去掉最高的2.5％和/或最低的2.5％。
当所述检测结果(即所述ERV基因座的活化水平)高于参考值，则所述对象或其细胞、组织或器官衰老程度或所述对象的生理年龄鉴定为高于所述参考值所对应的衰老程度或生理年龄；当所述检测结果(即所述ERV基因座的活化水平)低于参考值，则所述对象或其细胞、组织或器官衰老程度或所述对象的生理年龄鉴定为低于所述参考值所对应的衰老程度或生理年龄
在一些实施方案中，所述参考值是通过检测参考对象或参考对象群体获得的。当所述检测结果高于参考值，则所述对象或其细胞、组织或器官衰老程度或所述对象的生理年龄鉴定为高于所述参考对象或所述参考对象的群体或其细胞、组织或器官的(平均)衰 老程度的或所述参考对象或所述参考对象的(平均)生理年龄；当所述检测结果低于参考值，则所述对象或其细胞、组织或器官衰老程度或所述对象的生理年龄鉴定为低于所述参考对象或所述参考对象的群体或其细胞、组织或器官的(平均)衰老程度的或所述参考对象或所述参考对象的(平均)生理年龄。
在一些实施方案中，所述参考值是代表参考对象或参考对象群体的所述ERV基因座的活化水平的值或范围，包括ERV基因座甲基化水平的值或范围、ERV转录物水平的值或范围、ERV蛋白质水平的值或范围、ERV颗粒在细胞中存在和/或从细胞释放的值或范围，和/或ERV基因座的转座概率的值或范围。在一些实施方案中，所述参考值是代表参考对象或参考对象群体的所述ERV基因座的活化水平的值或范围，包括ERV基因座甲基化水平的值或范围、ERV转录物水平的值或范围、ERV蛋白质水平的值或范围和/或ERV颗粒在细胞中存在和/或从细胞释放的数量的值或范围。在一些实施方案中，所述参考值是代表参考对象或参考对象群体的所述ERV基因座的活化水平的值或范围，包括ERV基因座的转座概率的值或范围。
在一些实施方案中，所述检测ERV基因座活化水平包括检测ERV基因座甲基化水平，例如其侧翼LTR区的甲基化水平。可以根据本领域已知的方法检测特定区域的甲基化水平。例如，可以通过染色质免疫沉淀(ChIP)-qPCR的方法检测特定区域的组蛋白标记(例如H3K9me3或H3K36me3)的富集水平。在一些实施方案中，所述方法包括使用包含SEQ ID NO:9和10的引物对。
在一些实施方案中，检测所述ERV的编码序列的转录物水平，或其编码的蛋白质的水平。在一些实施方案中，所述编码序列包含所述ERV的Env、Pol、Pro和/或Gag基因。在一些实施方案中，通过基因特异性引物对或探针检测所述编码序列的转录物水平。在一些实施方案中，通过蛋白质结合分子检测所述编码序列所编码的蛋白质的水平。蛋白质结合分子包括能够与给定的蛋白质结合的任何分子，例如但不限于抗体，抗体的抗原结合片段，及抗体衍生物。在一些实施方案中，抗体的抗原结合片段包括但不限于Fv片段(例如单链Fv及二硫键键合的Fv)和Fab样片段(例如Fab片段、Fab’片段及F(ab’) 2片段)。
在一些实施方案中，所述对象或动物是哺乳动物。在一些实施方案中，所述对象或动物是非人哺乳动物。例如，所述对象或动物是小鼠，且任选地，所述ERV来自MMTV家族；或者所述对象或动物是非人灵长类动物，优选猴，且任选地，所述ERV来自ERVW家族。优选地，所述对象或动物是人，且任选地，所述ERV来自HERVK家族。
在一些实施方案中，所述对象是人。优选地，所述ERV来自HERVK家族。在一些实施方案中，针对HERVK编码区(HERVK-int)例如其转录物进行检测。在一些实施方案中，针对MER61编码区(MER61-int)例如其转录物进行检测。在一些实施方案中，对MamGyp的LTR区例如其转录物进行检测。
在一些实施方案中，检测HERVK的编码序列的转录物或所翻译的蛋白质，包括例如HERVK的Env、Pol、Pro和/或Gag基因的转录物或所翻译的蛋白质。本领域知晓，在人基因组中有多个HERVK基因座，不同HERVK基因座之间有微小的差异，其中HERVK基因座HML2_1q22的Env、Pol、Pro和Gag基因分别如SEQ ID NO:5、6、7和8所示，其编码的蛋白质如SEQ ID NO:1、2、3和4所示。因此，在一些实施方案中，所述转录物包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的转录物。在一些实施方案中，所述蛋白质是包含SEQ ID NO:1-4之一或与SEQ ID NO:1-4之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。
在一些实施方案中，所述方法包括使用包含SEQ ID NO:11和12、SEQ ID NO:13 和14、SEQ ID NO:15和16、SEQ ID NO:17和18、SEQ ID NO:19和20或SEQ ID NO:21和22的引物对。在一些实施方案中，所述方法包括使用包含SEQ ID NO:60的核苷酸序列的探针。
可以通过成像技术检测ERV颗粒在细胞中存在和/或从细胞释放，例如透射电子显微镜(TEM)技术。
检测ERV颗粒从细胞释放还可以通过检测ERV蛋白质，优选Env蛋白质，例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。
还可以检测所述样品中的ERV特异性抗体。在一些实施方案中，所述方法包括使用包含SEQ ID NO:1-4之一或与SEQ ID NO:1-4之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。优选地，所述方法包括使用包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。
可用于本发明的方法的样品是包含来自所述对象的细胞或组织的样品，例如各种组织的活检样品。可用于本发明的方法的样品也可以是体液样品，例如尿液、唾液和血液，包括全血、血浆或血清。
本发明还提供一种评估分离的动物细胞或细胞群的衰老程度的方法，包括：
a)检测所述细胞中的ERV活化水平；和
b)将所述检测结果与所述ERV活化水平的参考值进行比较。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，ERV基因座的“活化水平”包括ERV基因座甲基化水平、ERV转录物水平、ERV蛋白质水平和/或ERV颗粒在细胞中存在和/或从细胞释放。较低的甲基化水平、较高的转录物水平、较高的蛋白质水平和/或较高的ERV颗粒在细胞中存在或从细胞释放代表较高的ERV基因座活化水平。在一些实施方案中，ERV基因座的活化水平包括ERV基因座的转座概率。较高的转座概率代表较高的活化水平。
在一些实施方案中，所述参考值是通过检测参考细胞获得的。在本发明中，所述参考细胞是传代特定次数或特定次数范围的细胞。是在一些实施方案中，所述参考值是通过检测参考细胞群体获得的。
所述参考值可以以“平均值±标准差”的形式表示，也可以以范围的形式表示。在对群体进行检测以确定参考值的情况下，还可以去掉所测得的值中最高和最低的部分，例如去掉最高的2.5％和/或最低的2.5％。
当所述检测结果(即所述ERV基因座的活化水平)高于参考值，则所述细胞或细胞群体的衰老程度鉴定为高于所述参考值所对应的衰老程度；当所述检测结果(即所述ERV基因座的活化水平)低于参考值，则所述细胞或细胞群体的衰老程度鉴定为低于所述参考值所对应的衰老程度。
在一些实施方案中，所述参考值是通过检测参考细胞或参考细胞群体获得的。当所述检测结果高于参考值，则所述细胞或细胞群体的衰老程度鉴定为高于所述参考细胞或细胞群体的传代次数所对应的衰老程度；当所述检测结果低于参考值，则所述细胞或细 胞群体的衰老程度鉴定为低于所述参考细胞或细胞群体的传代次数所对应的衰老程度。
在一些实施方案中，所述参考值是代表参考对象或参考对象群体的所述ERV基因座的活化水平的值或范围，包括ERV基因座甲基化水平的值或范围、ERV转录物水平的值或范围、ERV蛋白质水平的值或范围、ERV颗粒在细胞中存在和/或从细胞释放的值或范围，和/或ERV基因座的转座概率的值或范围。在一些实施方案中，所述参考值是代表参考对象或参考对象群体的所述ERV基因座的活化水平的值或范围，包括ERV基因座甲基化水平的值或范围、ERV转录物水平的值或范围、ERV蛋白质水平的值或范围和/或ERV颗粒在细胞中存在和/或从细胞释放的数量的值或范围。在一些实施方案中，所述参考值是代表参考对象或参考对象群体的所述ERV基因座的活化水平的值或范围，包括ERV基因座的转座概率的值或范围。
在一些实施方案中，所述检测ERV基因座活化水平包括检测ERV基因座甲基化水平，例如其侧翼LTR区的甲基化水平。可以根据本领域已知的方法检测特定区域的甲基化水平。例如，可以通过染色质免疫沉淀(ChIP)-qPCR的方法检测特定区域的组蛋白标记(例如H3K9me3或H3K36me3)的富集水平。在一些实施方案中，所述方法包括使用包含SEQ ID NO:9和10的引物对。
在一些实施方案中，检测所述ERV的编码序列的转录物水平，或其编码的蛋白质的水平。在一些实施方案中，所述编码序列包含所述ERV的Env、Pol、Pro和/或Gag基因。在一些实施方案中，通过基因特异性引物对或探针检测所述编码序列的转录物水平。在一些实施方案中，通过蛋白质结合分子检测所述编码序列所编码的蛋白质的水平。蛋白质结合分子包括能够与给定的蛋白质结合的任何分子，例如但不限于抗体，抗体的抗原结合片段，及抗体衍生物。在一些实施方案中，抗体的抗原结合片段包括但不限于Fv片段(例如单链Fv及二硫键键合的Fv)和Fab样片段(例如Fab片段、Fab’片段及F(ab’) 2片段)。
本发明的方法所检测的细胞可以是任何动物细胞，例如用于工业生产重组蛋白或疫苗的动物细胞，或干细胞。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是鼠细胞，如CHO和小鼠细胞。在一些实施方案中，所述细胞是小鼠细胞，且所述ERV来自MMTV家族。
在一些实施方案中，所述细胞是非人灵长类动物细胞。优选地，所述非人灵长类动物是旧世界猴。例如，所述细胞是vero细胞。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述细胞是人细胞。优选地，所述ERV来自HERVK家族。在一些实施方案中，针对HERVK编码区(HERVK-int)例如其转录物进行检测。在一些实施方案中，针对MER61编码区(MER61-int)例如其转录物进行检测。在一些实施方案中，对MamGyp的LTR区例如其转录物进行检测。
在一些实施方案中，检测HERVK的编码序列的转录物或所翻译的蛋白质，包括例如HERVK的Env、Pol、Pro和/或Gag基因的转录物或所翻译的蛋白质。在一些实施方案中，所述转录物包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的转录物。在一些实施方案中，所述蛋白质是包含SEQ ID NO:1-4之一或与SEQ ID NO:1-4之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。
在一些实施方案中，所述方法包括使用包含SEQ ID NO:11和12、SEQ ID NO:13和14、SEQ ID NO:15和16、SEQ ID NO:17和18、SEQ ID NO:19和20或SEQ ID NO:21和22的引物对。在一些实施方案中，所述方法包括使用包含SEQ ID NO:60的核苷酸序列的探针。
可以通过成像技术检测ERV颗粒在细胞中存在和/或从细胞释放，例如TEM技术。
检测ERV颗粒从细胞释放还可以通过检测例如培养基中的ERV蛋白质，优选Env蛋白质，例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。
三、调节衰老的方法
本发明提供一种调节细胞衰老的方法，包括使所述细胞与ERV基因座活化调节剂接触。本发明的方法通过调控内源逆转录病毒调节细胞衰老。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
所述调节剂可以是抑制剂或促进剂，而且所述方法也包括使用针对ERV颗粒的抑制剂。可以通过抑制内源逆转录病毒延缓细胞衰老，通过激活内源逆转录病毒促进细胞衰老。
在本文中，ERV基因座活化抑制剂涉及特异性抑制(例如敲除或敲减所述基因座，敲除或敲减所述基因座中的基因，提高基因座甲基化水平)和非特异性抑制(提高全基因组甲基化水平从而促进所述基因座的甲基化，或非特异性逆转录病毒抑制剂，抑制病毒包装等)。所述ERV抑制剂是指针对ERV病毒颗粒的抑制剂。
在一些实施方案中，所述ERV基因座活化抑制剂通过例如提高甲基转移酶的活性，促进ERV基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的基因治疗系统。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减所述ERV基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是用于敲除或敲减ERV的Env、Pol、Pro和/或Gag基因的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。
在一些实施方案中，所述ERV抑制剂是可以与所述ERV的蛋白质如Env蛋白结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物，优选ERV中和抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是啮齿类动物。优选地，所述啮齿类动物是小鼠。优选地，所述ERV来自MMTV家族。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是非人灵长类动物。优选地，所述非人灵长类动物是旧世界猴。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述对象是人。优选地，所述ERV来自HERVK家族。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减ERV基因座的基因编辑系统，所述ERV基因座包括但不限于HERVK和MMTV。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减HERVK基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述ERV抑制剂是与ERV-Env，包括但不限于HERVK-Env、HERVW-Env和MMTV-Env，结合的结合分子，，例如抗体或其抗原结合片段或抗体衍生物。在一些实施方案中，所述ERV抑制剂是与HERVK-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述调节剂是所述细胞的ERV基因座活化促进剂。在本发明中，所述ERV基因座活化促进剂可以是特异性促进剂或非特异性促进剂。
在一些实施方案中，所述特异性促进剂是靶向所述ERV基因座的转录激活系统。在一些实施方案中，所述转录激活系统是CRISPR-dCas转录激活系统。在一些实施方案中，所述特异性促进剂是靶向所述ERV基因座的去甲基化系统。在一些实施方案中，所述去甲基化系统是基于CRISPR-dCas的去甲基化系统，例如CRISPR-dCas-Tet。
在一些实施方案中，所述非特异性促进剂是DNA甲基转移酶抑制剂，例如地西他滨、Lomeguatrib、SGI-1027或5-氮杂胞苷。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是啮齿类动物细胞。在一些实施方案中，所述细胞是小鼠细胞。在一些实施方案中，所述ERV来自MMTV家族。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是非人灵长类动物细胞。在一些实施方案中，所述非人灵长类动物是旧世界猴。在一些实施方案中，所述ERV来自ERVW家族。
在一些实施方案中，所述细胞是人细胞，例如肿瘤细胞、受感染的细胞或受损伤的组织中的细胞。优选地，所述ERV来自HERVK家族。
在一些实施方案中，所述CRISPR-dCas转录激活系统中的sgRNA包含SEQ ID NO:59。在一些实施方案中，所述方法包括使所述人细胞与HERVK颗粒接触。
本发明提供一种治疗有需要的对象或其细胞、组织或器官的过早衰老，或防止或延缓对象或其细胞、组织或器官衰老的方法，包括给所述对象施用ERV基因座活化抑制剂或所述ERV的抑制剂。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、 MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述ERV基因座活化抑制剂通过例如提高甲基转移酶的活性，促进ERV基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的基因治疗系统。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减所述ERV基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是用于敲除或敲减ERV的Env、Pol、Pro和/或Gag基因的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。
在一些实施方案中，所述ERV抑制剂是可以与所述ERV的蛋白质如Env蛋白结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物，优选ERV中和抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是啮齿类动物。优选地，所述啮齿类动物是小鼠。优选地，所述ERV来自MMTV家族。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是非人灵长类动物。优选地，所述非人灵长类动物是旧世界猴。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述对象是人。优选地，所述ERV来自HERVK家族。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减HERVK基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述ERV抑制剂是与HERVK-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
分离的动物细胞用于工业生产重组蛋白质或疫苗，而所用的细胞的活性和增殖能力对生产是重要的。细胞衰老会使得细胞活性和增殖能力下降，从而对生产力造成不利影响。在培养细胞的过程中，防止或延缓细胞衰老对于工业生产是有益的。
因此，本发明提供一种培养分离的动物细胞或细胞群的方法，包括使所述细胞与所述细胞的ERV基因座活化抑制剂或所述ERV的抑制剂接触。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。 在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述ERV基因座活化抑制剂通过例如提高甲基转移酶的活性，促进ERV基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的基因治疗系统。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减所述ERV基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是用于敲除或敲减ERV的Env、Pol、Pro和/或Gag基因的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。
在一些实施方案中，所述ERV抑制剂是可以与所述ERV的蛋白质如Env蛋白结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物，优选ERV中和抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是鼠细胞，如CHO和小鼠细胞。在一些实施方案中，所述细胞是小鼠细胞，且所述ERV来自MMTV家族。
在一些实施方案中，所述细胞是非人灵长类动物细胞。优选地，所述非人灵长类动物是旧世界猴。例如，所述细胞是vero细胞。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述细胞是人细胞。优选地，所述ERV来自HERVK家族。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减HERVK基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述ERV抑制剂是与HERVK-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
本发明还提供一种防止或延缓细胞衰老的方法，包括使所述细胞与所述细胞的ERV基因座活化抑制剂或所述ERV的抑制剂接触。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述ERV基因座活化抑制剂通过例如提高甲基转移酶的活性，促进ERV基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的基因治疗系统。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减所述ERV基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是用于敲除或敲减ERV的Env、Pol、Pro和/或Gag基因的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。
在一些实施方案中，所述ERV抑制剂是可以与所述ERV的蛋白质如Env蛋白结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物，优选ERV中和抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是鼠细胞，如CHO和小鼠细胞。在一些实施方案中，所述细胞是小鼠细胞，且所述ERV来自MMTV家族。
在一些实施方案中，所述细胞是非人灵长类动物细胞。优选地，所述非人灵长类动物是旧世界猴。例如，所述细胞是vero细胞。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述细胞是人细胞。优选地，所述ERV来自HERVK家族。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减HERVK基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述ERV抑制剂是与HERVK-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
本发明还提供一种促进细胞衰老的方法，包括使所述细胞与所述细胞的ERV基因座活化促进剂或所述ERV颗粒接触。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在本发明中，所述ERV基因座活化促进剂可以是特异性促进剂或非特异性促进剂。
在一些实施方案中，所述特异性促进剂是靶向所述ERV基因座的转录激活系统。在一些实施方案中，所述转录激活系统是CRISPR-dCas转录激活系统。在一些实施方案中，所述特异性促进剂是靶向所述ERV基因座的去甲基化系统。在一些实施方案中，所述去甲基化系统是基于CRISPR-dCas的去甲基化系统，例如CRISPR-dCas-Tet。在一些实施方案中给，所述ERV基因座包括但不限于HERVK、HERVW和MMTV基因座。
在一些实施方案中，所述非特异性促进剂是DNA甲基转移酶抑制剂，例如地西他滨、Lomeguatrib、SGI-1027或5-氮杂胞苷。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是啮齿类动物细胞。在一些实施方案中，所述啮齿类动物是小鼠。在一些实施方案中，所述ERV来自MMTV家族。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是非人灵长类动物细胞。在一些实施方案中，所述非人灵长类动物是旧世界猴。在一些实施方案中，所述ERV来自ERVW家族。
在一些实施方案中，所述细胞是人细胞，例如肿瘤细胞、受感染的细胞或受损伤的组织中的细胞。优选地，所述ERV来自HERVK家族。在一些实施方案中，所述CRISPR-dCas转录激活系统中的sgRNA包含SEQ ID NO:59。在一些实施方案中，所述方法包括使所述人细胞与HERVK颗粒接触。
本发明还提供一种防止或延缓组织器官退行，治疗相关疾病，延缓机体衰老的方法，例如防止或延缓皮肤衰老、治疗或预防骨关节炎以及衰弱症的治疗方法，包括给有需要的对象施用ERV基因座活化抑制剂或ERV抑制剂。在一些实施方案中，所述ERV基因座包括HERVK、HERVW和MMTV基因座。
在一些实施方案中，所述ERV基因座活化抑制剂通过例如提高甲基转移酶的活性，促进ERV基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的基因治疗系统。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减所述ERV基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减ERV基因座的基因编辑系统。
在一些实施方案中，所述ERV抑制剂是与ERV-Env结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
本发明还提供一种防止或延缓皮肤衰老的方法，包括给有需要的对象施用HERVK基因座活化抑制剂或HERVK抑制剂。
在一些实施方案中，所述HERVK基因座活化抑制剂通过例如提高甲基转移酶的活性，促进HERVK基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述HERVK基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的基因治疗系统。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述HERVK基因座活化抑制剂是敲除或敲减所述HERVK基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减HERVK基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述HERVK抑制剂是与HERVK-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述施用是局部施用、皮内施用或皮下施用。
本发明还提供一种治疗早衰症如HPGS或WS，以及衰老相关疾病，包括但不限于关节炎、卵巢早衰、肝纤维化、肺纤维化以及心血管疾病的方法，包括给有需要的对象施用ERV基因座活化抑制剂或ERV抑制剂。在一些实施方案中，所述ERV选自HERVK、HERVW和MMTV。
在一些实施方案中，所述ERV基因座活化抑制剂通过例如提高甲基转移酶的活性，促进ERV基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine 和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的基因治疗系统。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减所述ERV基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减ERV基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述ERV抑制剂是与ERV-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
本发明还一种治疗早衰症如HPGS或WS的方法，包括给有需要的对象施用HERVK基因座活化抑制剂或HERVK抑制剂。
在一些实施方案中，所述HERVK基因座活化抑制剂通过例如提高甲基转移酶的活性，促进HERVK基因座甲基化。所述抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述HERVK基因座活化抑制剂是敲除或敲减所述HERVK基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减HERVK基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述HERVK抑制剂是与HERVK-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子， 例如抗体或其抗原结合片段或抗体衍生物。
四、检测衰老的试剂盒
本发明提供一种鉴定对象或其细胞、组织或器官衰老程度或评估对象生理年龄或诊断早衰症的试剂盒，其包含用于检测来自所述对象的样品中的内源逆转录病毒(ERV)活化水平的检测剂。
本发明还提供用于检测来自所述对象的样品中的内源逆转录病毒(ERV)活化水平的检测剂在制备用于鉴定对象或其细胞、组织或器官衰老程度或评估对象生理年龄或诊断早衰症的试剂盒中的用途。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述检测剂用于检测ERV基因座甲基化水平，例如其侧翼LTR区的甲基化水平。在一些实施方案中，所述检测剂包含引物对，所述引物对包含SEQ ID NO:9和10的多核苷酸。
在一些实施方案中，所述检测剂用于检测所述ERV的编码序列的转录物水平，或其编码的蛋白质的水平。在一些实施方案中，所述编码序列包含所述ERV的Env、Pol、Pro和/或Gag基因。在一些实施方案中，所述检测剂是用于检测所述编码序列的转录物水平的基因特异性引物对或探针。在一些实施方案中，所述检测剂是用于检测所述编码序列所编码的蛋白质的水平的蛋白质结合分子。蛋白质结合分子包括能够与给定的蛋白质结合的任何分子，例如但不限于抗体，抗体的抗原结合片段，及抗体衍生物。在一些实施方案中，抗体的抗原结合片段包括但不限于Fv片段(例如单链Fv及二硫键键合的Fv)和Fab样片段(例如Fab片段、Fab’片段及F(ab’) 2片段)。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是啮齿类动物。优选地，所述啮齿类动物是小鼠。优选地，所述ERV来自MMTV家族。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是非人灵长类动物。优选地，所述非人灵长类动物是旧世界猴。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述对象是人。优选地，所述ERV来自HERVK家族。在一些实施方案中，针对HERVK编码区(HERVK-int)例如其转录物进行检测。在一些实施方案中，针对MER61编码区(MER61-int)例如其转录物进行检测。在一些实施方案中，对MamGyp的LTR区例如其转录物进行检测。
在一些实施方案中，所述检测剂用于检测HERVK的编码序列的转录物或所翻译的蛋白质，包括例如HERVK的Env、Pol、Pro和/或Gag基因的转录物或所翻译的蛋白质。在一些实施方案中，所述转录物包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的转录物。在一些实施方案中，所述蛋白质是包含SEQ ID NO:1-4之一或与SEQ ID NO:1-4之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。
在一些实施方案中，所述试剂盒包含引物对，所述引物对包含SEQ ID NO:11和12、SEQ ID NO:13和14、SEQ ID NO:15和16、SEQ ID NO:17和18、SEQ ID NO:19和 20或SEQ ID NO:21和22的多核苷酸。在一些实施方案中，所述试剂盒包含探针，所述探针包含SEQ ID NO:60的核苷酸序列。
在一些实施方案中，所述试剂盒包含结合伴侣，所述结合伴侣包含SEQ ID NO:1-4之一或与SEQ ID NO:1-4之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。优选地，所述结合伴侣包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列。
可用本发明的试剂盒检测的样品是包含来自所述对象的细胞或组织的样品，例如各种组织的活检样品。可用本发明的试剂盒检测的样品也可以是体液样品，例如尿液、唾液和血液，包括全血、血浆或血清。
本发明还提供一种评估分离的动物细胞或细胞群的衰老程度的试剂盒，其包含用于检测所述细胞中的ERV活化水平的检测剂。
本发明还提供用于检测细胞中的ERV活化水平的检测剂在制备用于评估分离的动物细胞或细胞群的衰老程度的试剂盒中的用途。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。
在一些实施方案中，所述ERV的活化水平包括ERV基因座甲基化水平、ERV转录物水平、ERV蛋白质水平、ERV颗粒在细胞中的存在、定位和/或从细胞释放，和/或ERV基因座的转座概率。在一些实施方案中，所述ERV的活化水平包括ERV基因座甲基化水平、ERV转录物水平、ERV蛋白质水平和/或ERV颗粒在细胞中的存在和/或从细胞释放。在一些实施方案中，所述ERV的活化水平包括ERV基因座的转座概率。在一些实施方案中，所述ERV的活化水平通过ERV颗粒在细胞中的定位表现。
在一些实施方案中，所述检测剂用于检测ERV基因座甲基化水平，例如其侧翼LTR区的甲基化水平。在一些实施方案中，所述检测剂包含引物对，所述引物对包含SEQ ID NO:9和10的多核苷酸。
在一些实施方案中，所述检测剂用于检测所述ERV的编码序列的转录物水平，或其编码的蛋白质的水平。在一些实施方案中，所述编码序列包含所述ERV的Env、Pol、Pro和/或Gag基因。在一些实施方案中，所述检测剂是用于检测所述编码序列的转录物水平的基因特异性引物对或探针。在一些实施方案中，所述检测剂是用于检测所述编码序列所编码的蛋白质的水平的蛋白质结合分子。蛋白质结合分子包括能够与给定的蛋白质结合的任何分子，例如但不限于抗体，抗体的抗原结合片段，及抗体衍生物。在一些实施方案中，抗体的抗原结合片段包括但不限于Fv片段(例如单链Fv及二硫键键合的Fv)和Fab样片段(例如Fab片段、Fab’片段及F(ab’) 2片段)。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是鼠细胞，如CHO和小鼠细胞。在一些实施方案中，所述细胞是小鼠细胞，且所述ERV来自MMTV家族。
在一些实施方案中，所述细胞是非人灵长类动物细胞。优选地，所述非人灵长类动物是旧世界猴。例如，所述细胞是vero细胞。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述细胞是人细胞。优选地，所述ERV来自HERVK家族。在 一些实施方案中，针对HERVK编码区(HERVK-int)例如其转录物进行检测。在一些实施方案中，针对MER61编码区(MER61-int)例如其转录物进行检测。在一些实施方案中，对MamGyp的LTR区例如其转录物进行检测。
在一些实施方案中，所述检测剂用于检测HERVK的编码序列的转录物或所翻译的蛋白质，包括例如HERVK的Env、Pol、Pro和/或Gag基因的转录物或所翻译的蛋白质。在一些实施方案中，所述转录物包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的转录物。在一些实施方案中，所述蛋白质是包含SEQ ID NO:1-4之一或与SEQ ID NO:1-4之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白。
在一些实施方案中，所述试剂盒包含引物对，所述引物对包含SEQ ID NO:11和12、SEQ ID NO:13和14、SEQ ID NO:15和16、SEQ ID NO:17和18、SEQ ID NO:19和20或SEQ ID NO:21和22的多核苷酸。在一些实施方案中，所述试剂盒包含探针，所述探针包含SEQ ID NO:60的核苷酸序列。
五、调节衰老的药物组合物
本发明提供一种药物组合物，其用于治疗有需要的对象或其细胞、组织或器官的过早衰老，或防止或延缓对象或其细胞、组织或器官衰老，其包含ERV基因座活化抑制剂或所述ERV的抑制剂和药学上可接受的赋形剂。
本发明还提供本发明提供ERV基因座活化抑制剂或所述ERV的抑制剂在制备用于治疗有需要的对象或其细胞、组织或器官的过早衰老，或防止或延缓对象或其细胞、组织或器官衰老的药物组合物中的用途。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV基因座活化抑制剂包括但不限于甲基转移酶或其编码多核苷酸。
在一些实施方案中，所述ERV基因座活化抑制剂是非特异性逆转录病毒抑制剂，其包含例如逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。在一些实施方案中，所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。在一些实施方案中，所述整合酶抑制剂包括Raltegravir。在一些实施方案中，所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。在一些实施方案中，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
在一些实施方案中，所述ERV基因座活化抑制剂是靶向所述ERV基因座的增加甲基化的CRISPR-dCas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减所述ERV基因座的基因编辑系统，包括但不限于大范围核酸酶、锌指核酸酶(ZFN)、转录激活样效应因子核酸酶(TALEN)和CRISPR/Cas系统。
在一些实施方案中，所述ERV基因座活化抑制剂是用于敲除或敲减ERV的Env、Pol、Pro和/或Gag基因的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。
在一些实施方案中，所述ERV抑制剂是可以与所述ERV的蛋白质如Env蛋白结合 的结合分子，例如抗体或其抗原结合片段或抗体衍生物，优选ERV中和抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是啮齿类动物。优选地，所述啮齿类动物是小鼠。优选地，所述ERV来自MMTV家族。
在一些实施方案中，所述对象是非人哺乳动物。在一些实施方案中，所述对象是非人灵长类动物。优选地，所述非人灵长类动物是旧世界猴。优选地，所述ERV来自ERVW家族。
在一些实施方案中，所述对象是人。优选地，所述ERV来自HERVK家族。
在一些实施方案中，所述ERV基因座活化抑制剂是敲除或敲减HERVK基因座的基因编辑系统。在一些实施方案中，所述ERV基因座活化抑制剂是用于敲减或敲除包含SEQ ID NO:5-8之一，或与SEQ ID NO:5-8之一具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的核苷酸序列的核酸分子的反义核酸或干扰核酸(包括但不限于siRNA、shRNA和miRNA)。在一些实施方案中，所述ERV基因座活化抑制剂编码SEQ ID NO:51或52的shRNA。
在一些实施方案中，所述ERV抑制剂是与HERVK-Env(例如包含SEQ ID NO:1或与SEQ ID NO:1具有至少80％、85％、90％、95％、96％、97％、98％、99％、99.5％、99.6％、99.7％、99.8％或99.9％的百分比相同性的的氨基酸序列的蛋白)结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物。
在一些实施方案中，所述药物组合物皮肤用于防止或延缓组织器官退行，治疗相关疾病，延缓机体衰老，例如防止或延缓皮肤衰老、治疗或预防骨关节炎以及衰弱症。在一些实施方案中，所述药物组合物用于局部施用、皮内施用或皮下施用。
在一些实施方案中，所述药物组合物用于治疗早衰症，如HGPS或WS，以及衰老相关疾病，包括但不限于关节炎、卵巢早衰、肝纤维化、肺纤维化以及心血管疾病。
在一些实施方案中，所述药物组合物用于防止或延缓皮肤衰老。在一些实施方案中，所述药物组合物用于局部施用、皮内施用或皮下施用。
在一些实施方案中，所述药物组合物用于治疗早衰症，如HGPS或WS。
本发明还提供一种药物组合物，用于促进细胞衰老，其包含所述细胞的ERV基因座活化促进剂或所述ERV颗粒。
本发明还提供细胞的ERV基因座活化促进剂或所述ERV颗粒在制备用于促进细胞衰老的药物组合物中的用途。
在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。在一些实施方案中，所述ERV至少包括来自HERVK家族的ERV(包括HERVK11)，任选地，所述ERV还包括选自以下家族的ERV：MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2和ERVW及其同源物。在一些实施方案中，所述促进剂是靶向所述ERV基因座的转录激活系统。在一些实施方案中，所述转录激活系统是CRISPR-dCas转录激活系统。在一些实施方案中，所述特异性促进剂是靶向所述ERV基因座的去甲基化系统。在一些实施方案中，所述去甲基化系统是基于CRISPR-dCas的去甲基化系统，例如CRISPR-dCas-Tet。
在一些实施方案中，所述促进剂是DNA甲基转移酶抑制剂，例如地西他滨、Lomeguatrib、SGI-1027或5-氮杂胞苷。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是啮齿类动物细胞。在一些实施方案中，所述啮齿类动物是小鼠。在一些实施方案中， 所述ERV来自MMTV家族。
在一些实施方案中，所述细胞是非人哺乳动物细胞。在一些实施方案中，所述细胞是非人灵长类动物细胞。在一些实施方案中，所述非人灵长类动物是旧世界猴。在一些实施方案中，所述ERV来自ERVW家族。
在一些实施方案中，所述细胞是人细胞，例如肿瘤细胞、受感染的细胞或受损伤的组织中的细胞。优选地，所述ERV来自HERVK家族。在一些实施方案中，所述CRISPR-dCas转录激活系统中的sgRNA包含SEQ ID NO:59。
实施例
材料与方法
如无特别说明，本申请实施例中的实验操作根据本领域常规方法进行。以下材料和方法意在示例性说明，而非意在限制本发明。
动物和人样品
C57BL/6J小鼠购自思培福(北京)生物科技有限公司，饲养于在中国科学院生物物理研究所和动物研究所的动物所动物房的通风笼中，喂食标准实验室饲料和水，12h/12h的明暗循环。
所有小鼠实验均遵循《动物研究伦理审批申请格式原则》，并事先获得中国科学院动物研究所动物保护与使用机构委员会的批准。
小鼠牺牲用CO2实施安乐死，然后颈椎脱臼。
所有食蟹猴实验均按照非人类灵长类动物的人道对待原则进行，并得到中国科学院动物学研究所动物护理与伦理委员会的批准。
用于实验的年轻食蟹猕猴和年老食蟹猕猴均在协尔鑫生物资源研究所饲养，并获得北京实验动物护理认可机构的认可，符合所有有关动物研究的地方和国家法律。
肺、肝和皮肤样品收集自8只没有临床或实验史的年轻和年老的食蟹猴。
HGPS食蟹猴安置于云南灵长类生物医学研究重点实验室(LPBR)，并根据国际实验动物评估和认可委员会(AAALAC)的规定，进行合乎伦理的动物实验(68)。
经北京解放军306医院伦理委员会批准，从指定年龄的人个体的牙龈分离初代hMPC。
经北京协和医院医院制度审查委员会的伦理批准，从指定的健康捐赠者的眼睑整形术获得人眼睑皮肤样品。
经昆明医科大学附属第一医院研究伦理委员会的批准，收集人血清样品。
小鼠实验
1.敲减MMTV
为了评估敲减MMTV是否减轻小鼠衰老相关的关节退行性变，表达Cas9KRAB空载体(n＝12)或Cas9KRAB-sgMMTV(n＝12)的慢病毒被注射到21月龄的小鼠的关节腔中，并在4周后补充注射。在第一次注射后4周和8周测量握力测试。在第一次注射后10周进行运动协调和平衡、运动能力和Micro-CT(micro computed tomography，微计算机断层扫描技术)，并进行取样，即将小鼠后肢关节取出，其中一个液氮冻存，另外一个在4％PFA中固定。
2.用Abacavir治疗衰老相关的关节退行性变
将10μl浓度为10mg/ml的Abacavir(含有2％DMSO的PBS)注射到22月龄小鼠(n＝12)的膝关节腔中，每周注射一次。在第一次注射后4周、8周和12周测量握力测试。在第一次注射后14周进行运动协调和平衡、运动能力和显微CT，并进行取样，即将小 鼠后肢关节取出，其中一个液氮冻存，另外一个放4％PFA固定。
3.Abacavir长期口服施用实验
Abacavir以2mg/ml溶解在含有0.4％DMSO的饮用水中，每天给18月龄的小鼠进行喂水给药(n＝23)。在施用6周、12周和24周后测量小鼠的体重、弓背、毛发、皮肤、眼睛(白内障)和活动评分(表1)并测量握力和绘制存活曲线。24周后进行运动协调和平衡、运动能力和Y迷宫测试行为学测试。
表1、小鼠表型评分标准
Figure PCTCN2021140550-appb-000001
4.Ratelgravir长期注射实验
Ratelgravir以50mg/ml溶解在30％PEG300中+2％Tween80+2％DMSO中(n＝23)，给18月龄的小鼠腹腔注射，每次注射200μl，一周注射两次。在第一次注射8周、16周后测量小鼠的体重、弓背、毛发、皮肤、白内障和活动评分，并测量握力和绘制存活曲线。
5.握力测试
用握力计(Panlab Grid Strength Meter，LE902)按照产品说明书测量四肢的握力。简而言之，将小鼠置于握力计的顶部并以恒定速率沿网格方向拉动，直到小鼠松手。重复10次，记录每次的峰值拉力，并计算平均值作为每只小鼠的握力。
6.强制跑步机测试
将小鼠放置在跑步机(SANS Bio Instrument，SA101)中，按照产品说明书测试小鼠的运动能力。具体而言，跑道倾斜度设置为5°，电刺激为2mA，跑步速度设置为：初始速度为5m/min，持续5分钟；然后以1m/min 2的加速度加速到最终速度25m/min，共20分钟。首先对小鼠进行连续三天的训练，并在第四天进行测试。记录25分钟内电刺激次数、跑步距离和时间。
7.旋转棒测试
按照产品说明书使用Rota Rod系统(Yiyan Tech,YLS-4C)测量小鼠的运动协调和平衡。简而言之，将小鼠放入杆上的不同通道中，转棒的初始速度为4rpm/min，然后以8rpm/min 2的加速度加速至44rpm/min，直至小鼠掉落，每只小鼠练习3次。小鼠连续三天接受训练并在第四天进行测试，每只小鼠进行3次测试，并在小鼠掉落后记录时间。
8.Micro-CT(微计算机断层扫描)
按照产品说明书进行Micro-CT(PE Quantum FX)。简而言之，将小鼠麻醉后放入仪器内，Micro-CT的扫描参数是电压50Kvp，功率40W，6帧叠加，视野4cm×4cm。扫 描重建后进行三维图像截图和并用Ansys软件计算骨密度。
9.Y迷宫检测
将小鼠放在Y迷宫(三等分辐射式迷宫，由3等长臂组成(50cm×18cm×35cm)，每两个臂之间夹角为120度)任意一臂末端，任其自由探索5min，摄像系统记录动物5min的行为变化，通过记录小鼠总进臂次数和依次连续进入Y迷宫全部三个臂一次的轮流(交替)次数，检测小鼠的空间短期记忆能力。
细胞培养
HEK293T细胞和人成纤维细胞在补充有10％胎牛血清(FBS，Gibco，Thermo Fisher Scientific)，2mmol/L GlutaMAX(Thermo Fisher Scientific)，0.1mmol/L非必需氨基酸(NEAA，Thermo Fisher Scientific)，1％青霉素/链霉素(Thermo Fisher Scientific)的Dulbecco Modified Eagle Medium(DMEM，Thermo Fisher Scientific)中培养。
人MPC(hMPC)在含有90％MEM Alpha Medium(Thermo Fisher Scientific)，10％FBS，2mmol/L GlutaMAX，0.1mmol/L NEEA，1％青霉素/链霉素和1ng/mL bFGF2(Joint Protein Central)的hMPC培养基中在0.1％明胶(Sigma Aldrich)包被的板(CORNING)上生长。
细胞培养于在37℃，5％CO 2的培养箱(Thermo Fisher Scientific)中。
蛋白质印迹
在1×SDS缓冲液(100mM Tris-HCl，pH 6.8，10％甘油，2％SDS和2％2-巯基乙醇)中裂解细胞，并在105℃煮10分钟。用BCA试剂盒测量蛋白质浓度。细胞裂解物进行SDS-PAGE电泳，然后电转移到PVDF膜(Millipore)上。将膜与一抗温育，洗涤后再与缀合HRP的二抗温育，然后用底物进行印迹，所述底物为1000μL底物A和3μL底物B的混合物(底物A：0.2mM香豆酸，1.25mM鲁米那，0.1M Tris-HCl，pH 8.5；底物B：3％H 2O 2)或Super Signal West Femto Maximum Sensitivity Substrate(Thermo Fisher Scientific)。使用Image Lab软件进行成像。用ImageJ进行定量。
从培养基免疫沉淀(IP)HERVK
使细胞在含有微泡耗竭的胎牛血清(dFBS)的hMPC培养基中生长48小时。收集条件培养基，并通过0.2μm过滤器(Pall Corporation)过滤，并在4℃用蛋白A/G琼脂糖凝胶珠预清洗2-3小时。将上清液与1/1000体积的HERVK-Env抗体和新鲜的蛋白A/G琼脂糖凝胶珠在4℃温育过夜，然后收集上清液，用PBS洗涤珠，并进行蛋白质印迹。
从培养基纯化微泡颗粒
在收集培养基之前，使细胞在含有微泡耗竭的胎牛血清(dFBS)的hMPC培养基中生长48小时。收集培养基并计数细胞数。来自相同数目细胞的培养基用于微泡纯化，所述纯化如下进行：在300×g离心10分钟以消除细胞污染物，在15,000×g离心20分钟，通过0.2μm过滤器(Pall Corporation)过滤，然后在110,000×g超速离心2小时。收集沉淀即为所述微泡，溶解在相同体积的PBS中，并进行病毒RNA分离或蛋白质印迹。
细胞DNA/RNA分离和(逆转录)定量PCR((RT-)qPCR)
按照制造商说明，使用DNA提取试剂盒(Tiangen)提取细胞的总基因组DNA。
按照制造商说明，用TRIzol(Thermo Fisher Scientific)提取细胞总RNA；使用GoScript逆转录系统(Promega)逆转录为cDNA用于RT-qPCR；使用THUNDERBIRD qPCR Mix(TOYOBO)和CFX384实时系统(Bio-Rad Laboratories，Inc.)进行RT-qPCR。
从培养基分离病毒RNA
按照制造商的说明，使用QIAamp viral RNA mini Kit(Qiagen)从140μL微泡提取RNA。
简而言之，将样品与准备好的缓冲液AVL和载体RNA一起温育。加入乙醇后，将样品用QIAamp Mini柱纯化，洗涤，洗脱，然后用DNase I处理(在37℃下于2.4μL反应缓冲液中，用2μL DNase I处理20μL病毒RNA，30分钟)。使用试剂盒中提供的灭活试剂将DNase I灭活并去除，然后在10,000×g离心1.5分钟，并将上清液在70℃加热10分钟以消除任何残留的DNase活性。
液滴数字PCR(ddPCR)
按照制造商的说明，使用GoScript逆转录系统(Promega)，将从微泡纯化的病毒RNA逆转录为cDNA用于ddPCR。
简而言之，将包含模板、引物和QX200ddPCR EvaGreen Supermix(Bio-Rad Laboratories，Inc.)的20μL反应溶液与70μL DG Oil(Bio-Rad Laboratories，Inc.)在DG8筒(Bio-Rad Laboratories)中，由QX200液滴发生器(Bio-Rad Laboratories，Inc.)混合。将液滴转移至96孔板，并用PX1 TM PCR Plate Sealer(Bio-Rad Laboratories，Inc.)密封。在热循环仪(Bio-Rad Laboratories，Inc.)中进行PCR扩增，然后将样品置于QX200液滴读取器(Bio-Rad Laboratories，Inc.)中，以分析微泡中HERVK的绝对拷贝数。
ELISA
对于IL6的ELISA分析，收集培养基并用0.2μm过滤器过滤，然后在IL6抗体包被的板(BioLegend，根据制造商的说明，使用前在4℃下预包被过夜)中温育。然后将板与检测抗体、抗生物素蛋白-HRP和新鲜混合的TMB底物一起温育。加入终止溶液后，使用Synergy H1(BioTek)在450nm测量所述板。
为了测量培养基或血清中的HERVK-Env蛋白水平，首先分别使用Centricon Plus-70(Millipore)或Amicon Ultra-15(Millipore)将培养基或人血清浓缩100倍或10倍，然后用HERVK_7p22.1Provirus Ancestral Env Polypretein(ERVK6)ELISA Kit按照制造商的说明进行测量。简而言之，将浓缩的培养基或血清(100μL)添加到包被的板中，并在37℃温育2小时，与生物素抗体，抗生物素蛋白-HRP温育，然后是新鲜混合的TMB底物和终止溶液，并在450nm使用Synergy H1(BioTek)测量。
用2'3'-cGAMP ELISA试剂盒测量细胞中2'3'-cGAMP的水平。简而言之，将来自相同数目指定细胞系的50μL细胞裂解物(在RIPA缓冲液中裂解)添加到平板中，然后在20℃添加2'3'-cGAMP HRP示踪剂，2'3'-cGAMP多克隆抗血清。室温摇动。与TMB底物和终止溶液温育后，使用Synergy H1(BioTek)在450nm测量板。
免疫荧光、免疫组织化学染色和显微镜检查
用PBS清洗接种在盖玻片(Thermo Fisher Scientific)上的细胞，在4％多聚甲醛(PFA)中固定，在0.4％Triton X-100的PBS中透化，并用10％的驴血清封闭。在封闭缓冲液中将盖玻片与一抗于4℃温育过夜，然后与二抗在室温温育1小时。用Hoechst 33342(Invitrogen)标记核。
根据制造商的说明，使用DAB Staining Kit(ZSGB-BIO)对组织切片进行免疫组织化学染色。简而言之，将使用二甲苯和乙醇脱石蜡的切片与3％H 2O 2溶液一起温育以阻断内源性过氧化物酶活性，然后使用10mM柠檬酸钠缓冲液(pH 6.0)进行抗原恢复。然后将切片透化，封闭，并与一抗在4℃温育过夜，再与二抗温育1小时，然后用DAB底物溶液和苏木精染色，脱水并固定。
用Leica SP5共焦显微镜或LEICA Aperio CS2拍摄图像。
RNA/DNA-荧光原位杂交(FISH)
按照制造商的说明(Thermo Fisher Scientific)，使用
Figure PCTCN2021140550-appb-000002
ViewRNA ISH Cell Assay Kit进行RNA-FISH。
简而言之，将接种在盖玻片(Thermo Fisher Scientific)上的细胞在新鲜的4％甲醛溶液中固定，用去污剂溶液QC渗透，并用蛋白酶QC消化。将探针HERVK Alexa Fluor 488(Thermo Fisher Scientific)在Probe Set Diluent QF中稀释，并加入孔中，在41+1℃的HB-1000杂交仪(Gene Company Limited)中温育3小时。将盖玻片在HB-1000杂交仪中于40+1℃与Working Pre-Amplifier Mix Solution温育30分钟，与Working Amplifier Mix Solution温育30分钟，以及与Working Label Probe Mix Solution温育30分钟，在每个步骤中用PBS洗涤。细胞核用DAPI溶液标记，将载玻片固定，并用Leica SP5共聚焦显微镜成像。
通过单分子DNA-FISH检测HERVK基因的DNA的实验由华中农业大学农业微生物国家重点实验室完成。衰老细胞依次用RNase A(100μg/mL，溶于2×SSC)在37℃处理1h，用溶于2×SSC中的70％甲酰胺在72℃处理10分钟，用预冷的乙醇处理(浓度从80％、90％至100％，各处理1分钟)。然后将细胞与HERVK或ACTIN特异性探针对(每个探针10μM)在杂交缓冲液(10％去离子甲酰胺，2×SSC，10％硫酸葡聚糖和2mM VRC)中于42℃温育过夜。用洗涤缓冲液(2×SSC，30％甲酰胺，0.1％Triton-X 100和2mM VRC)洗涤3次后，将细胞与扩增探针(SFSP-001和SFTP-001，Spatial FISH，Co.，Ltd.)和荧光探针(SFFP-001，Spatial FISH，Co.，Ltd)一起温育，以单分子检测HERVK DNA。最后，将细胞用DAPI复染并通过共聚焦显微镜成像。
流式细胞荧光分选技术分析(FACS)
用流式细胞仪分析GFP阳性细胞的比例，或将GFP阳性细胞分选出来。
简而言之，为了评估衰老过程中HERVK发生的转座事件，HERVK报告质粒转染到野生型的间充质干细胞早代和晚代(P7代和P14代)细胞中，并同时共转染表达红色荧光的载体dsRed(获自深圳大学基础医学院徐兴智实验室)用来计算转染概率。待细胞长满后，将细胞消化成单细胞，并用PBS重悬，用流式细胞仪BD Influx分析，绿色荧光阳性细胞与红色荧光阳性细胞的比例即为HERVK发生的转座事件的概率。
为了分选感染HERVK病毒颗粒的细胞，将纯化的带有绿色荧光标记的HERVK病毒颗粒感染野生型间充质干细胞，48h后，用超速流式分选仪Astrios EQ分选绿色荧光阴性和阳性的细胞。
透射电子显微镜(TEM)
沉淀细胞并在室温下用溶于磷酸盐缓冲液(PB，0.1M，pH 7.4)的2.5％(v/v)戊二醛固定20分钟，然后在4℃过夜。进行常规重金属染色。
简而言之，将细胞用溶于PB的1％(w/v)四氧化锇在4℃下固定2小时，通过分级乙醇系列脱水后置于纯丙酮中，在丙酮和树脂的分级混合物中浸润，然后在含1.5％BDMA的纯树脂中包埋，并进行聚合，45℃ 12小时，60℃ 48小时。用切片机(Leica EM UC6)进行超薄切片(厚度70nm)，用乙酸铀酰和柠檬酸铅双重染色。
对于Immuno-TEM，将细胞接种在35毫米培养皿(CORNING)中，在室温下用4％甲醛固定1小时，并在4℃过夜，然后换成1％甲醛。通过乙醇梯度将样品脱水，用LR Gold树脂梯度浸润并包埋在含引发剂的LR Gold树脂中。然后使用Leica AFS2在-20℃下通过紫外线使样品聚合24小时，并使用Leica EM UC7切片。为了染色，将切片在2％BSA(Jackson ImmunoResearch)封闭，在室温下与鼠抗-HERVK-Env一起温育2小时，用含0.1％新鲜冷明胶的PB缓冲液洗涤5×2分钟，与6nm金标记的抗小鼠二抗(Jackson ImmunoResearch)一起在室温下温育1小时，然后用溶于ddH 2O中的1％戊二醛固定。 ddH2O洗涤后，将切片在室温下用2％乙酸铀酰染色30分钟，然后使用TEM Spirit 120kV(FEI Tecnai Spirit 120kV)成像。
质粒构建
为了产生HERVK和STING敲低载体，将特异的shRNA克隆到慢病毒载体pLVTHM质粒(addgene#12247)的MluI/ClaI位点中。
为了通过CRISPR/Cas9进行的TBK1敲除，将靶向TBK1的sgRNA通过BstXI/BlpI位点克隆到lenti-pMK1334(addgene#127965)中，并用hSpCas9567表达盒与lenti-CRISPR v2(addgene，#52961)共转染。
为了激活内源性HERVK，将非靶向性对照(NTC)或HERVK LTR靶向sgRNA的慢病毒构建体通过ESP3I位点克隆到lenti-SAM v2(addgene#75112)中，并与lenti-MPH v2(addgene#89308)共转染。
为了抑制内源性HERVK，基于Cas9KRAB的抑制系统(CRISPRi)构建靶向HERVK的载体。表达Cas9KRAB的载体来自Didier Trono博士的实验室(洛桑联邦理工学院(EPFL)，洛桑，瑞士)。
为了抑制内源性MMTV，将靶向MMTV的sgRNA克隆到表达Cas9KRAB的空载体中。
为了构建全长HERVK质粒，根据之前发表的测序结果(参见，Marie Dewannieux,Genome Reserch,2006,Young Nam Lee,PLoS Pathogens,2007)合成全长HERVK片段(SEQ ID NO:150)，并通过NheI/ApaI位点克隆到具有KpnI酶位点的突变的pEGFP-N3载体中(pEGFP-N3-KpnI，获自深圳大学基础医学院徐兴智实验室)，获得载体pEGFP-N3-HERVK。
为了构建带有绿色荧光标记的HERVK，从pEGFP-C1(获自深圳大学基础医学院徐兴智实验室)中通过PCR扩增CMV-EGFP片段(SEQ ID NO:153)，并通过KpnI位点将CMV-EGFP片段插入到pEGFP-N3-HERVK，构成GFP-HERVK。使用pEGFP-N3-HERVK作为模板通过PCR扩增产生HERVK的env、pol、gag、pro(SEQ ID NOs:5-8)和rev基因(SEQ ID NO:149)，并插入pEGFP-N3-KpnI获得质粒pEGFP-N3-gag-pro-pol(表达gag、pro和pol基因)、pEGFP-N3-Env(表达env基因)和pEGFP-N3-Rev(表达rev基因)。
为了构建HERVK转座报告基因载体，首先将如上所述的HERVK片段克隆到具有KpnI酶位点突变体的dsRed载体(获自深圳大学基础医学院徐兴智实验室)中。从L1报告载体(中国广州中山大学王继昌教授赠送)通过PCR扩增获得GFP报告基因(SEQ ID NO:152)，并通过KpnI位点将GFP报告基因插入dsRed–HERVK，即构成HERVK转座报告基因载体。
慢病毒生产
为了包装慢病毒构建体，使用lipofectamine 3000(Invitrogen)将慢病毒载体与包装质粒pMD2.G(addgene，#12260)和psPAX2(addgene，#12259)一起转染HEK293T细胞。转染后48小时和72小时收获含有慢病毒的上清液，用0.2μm过滤器过滤，并以19,400rpm超速离心2.5小时进行浓缩。悬浮病毒沉淀并评估病毒滴度。
HERVK病毒生产
为了包装HERVK病毒，使用lipofectamine 3000(Invitrogen)将GFP-HERVK以及质粒pEGFP-N3-gag-pro-pol、pEGFP-N3-Env和pEGFP-N3-Rev一起转染HEK293T细胞。转染后48小时和72小时收获含有HERVK病毒的上清液，用0.2μm过滤器过滤，并以100,000g超速离心2小时进行浓缩。悬浮病毒沉淀并评估病毒滴度。
转座事件
为了评估衰老过程中HERVK发生的转座事件，HERVK报告质粒转染到早晚代细胞中，并同时共转染表达红色荧光的载体用来计算转染概率。待细胞长满后，将细胞消化成单细胞，并用PBS重悬，用流式细胞仪BD Influx分析，绿色荧光阳性细胞占红色荧光阳性细胞的比例即为HERVK发生的转座事件的概率。
细胞处理
对于5-氮杂胞苷(5-AZA)处理，接种hMPC(处理后第0代(P0))，并用500μg/μL 5-AZA处理4天，然后传代并在新鲜培养基中培养。在处理后的P2(5-AZA处理后6天)，将细胞用于Ki67免疫荧光，SA-β-gal染色和RNA/DNA提取。
对于内源病毒逆转录酶抑制剂和整合酶抑制剂处理，用10μM abacavir、lamivudine(3TC)和Raltegravir分别处理hMPC，每3天更换一次培养基，然后进行新的处理。处理两代细胞，然后，将细胞用于Ki67免疫荧光，SA-β-gal染色和RNA/DNA提取。
对于HERVK RVLP的感染，接种hMPC(处理后P0)并在聚凝胺(Sigma-Aldrich)存在下与HERVK RVLP一起孵育24小时。为了增加HERVK RVLP感染效率，将hMPC以1200g离心2.5小时。感染后两天，GFP阳性细胞通过FACS进行分选。感染两代细胞，然后，将细胞用于Ki67免疫荧光，SA-β-gal染色和RNA/DNA提取。
对于慢病毒转导，接种hMPC(处理后P0)，并在聚凝胺(Sigma-Aldrich)存在下与慢病毒一起温育24小时。经过两次传代后，处理细胞用于Ki67免疫荧光分析，SA-β-gal染色和RNA/DNA提取。
对于条件培养基(CM)处理，将50％条件培养基+50％新鲜培养基(在聚凝胺(Sigma-Aldrich)的存在下)用于培养早期传代的WT hMPC，每隔两天更换一次新的条件培养基。细胞传代并用于Ki67、SA-β-gal染色和RNA/DNA提取。
为了检测来自CM的逆转录病毒样颗粒(RVLP)粘附到年轻细胞表面，将细胞用CM处理12小时，然后进行TEM分析。
为了用年轻人(18-25岁，n＝20)或老年人(65-80岁，n＝40)的合并人类血清培养细胞，将原代hMPC在正常hMPC培养基中预培养24小时，用PBS洗涤3次，然后在含有10％人血清的培养基中培养两次。
克隆扩增试验
将五千个细胞接种在覆盖有明胶(Sigma)的6孔板(Corning)的一个孔中，并培养约10天。将细胞用4％PFA固定30分钟，并用10％结晶紫染色30分钟。通过ImageJ软件计算相对细胞积分密度。
SA-β-gal染色
将细胞用含有2％(w/v)甲醛和0.2％(w/v)戊二醛的缓冲液固定5分钟，并在37℃下用含有1mg/mL X-gal的染色缓冲液处理过夜。使用光学显微镜观察染色的细胞，并通过ImageJ软件分析阳性细胞的百分比。
染色质免疫沉淀(ChIP)-qPCR
将细胞在室温下在溶于PBS中的1％甲醛中固定10分钟，然后用125mM甘氨酸淬灭。在冰上裂解细胞10分钟，并使用Covaris S220进行超声处理。将收集的上清液与预先与2.3μg指定抗体混合的Dynabeads Protein A(Life Technologies，10001D)在4℃下温育过夜。洗涤后，样品用蛋白酶K(New England Biolabs)消化，洗脱并在68℃在热混合器上交联逆转(cross-link-reverse)2小时。使用苯酚-氯仿-异戊醇提取DNA，并对洗脱的DNA进行qPCR分析。
为了免疫沉淀cGAS，固定后提取细胞的细胞质级分，将其与cGAS抗体和Dynabeads Protein A在4℃温育过夜。如上所述进行DNA提取。5S rDNA(原则上不存在于细胞质部分中)的qPCR分析用于排除核基因组污染。
RNA-seq文库的构建和测序
使用TRIzol试剂提取总RNA，每重复1×10 6个细胞。去除基因组DNA并分离mRNA。然后，由Novogene生物信息技术有限公司进行了文库的制备，质量控制和高通量测序。其中，根据制造商的指导使用NEBNext Ultra RNA Library Prep Kit for Illumina(NEB)构建文库；根据制造商的说明，在HiSeq X 10平台上进行高通量测序。
RNA-seq数据处理
使用TrimGalore(版本0.4.5，Babraham Bioinformatics，https://github.com/FelixKrueger/TrimGalore)修剪对末端的原始读取，并使用hisat2(版本2.0.4)将其映射到从UCSC基因组浏览器数据库中获得的人(Homo sapiens)hg19或食蟹猕猴(Macaca Fascicularis)MacFas5.0参考基因组。然后，使用高质量映射的读取(映射质量得分超过20)通过HTSeq(版本0.11.0)计数映射到每个基因的读取。通过DESeq2R包(1.29.8版)计算差异表达基因(DEG)，对于hMPC，临界值为“|log 2(倍数变化)|>0.5，且经调整的P值<0.05”；对于食蟹猴组织，临界值为“|log 2(倍数变化)|>0.5且P值<0.05”。GO和通路富集分析是在metascape中进行的。衰老相关的分泌表型(SASP)基因来自先前的研究。基因集富集分析(GSEA)由台式机GSEA应用程序(版本2.2.4)进行。
重复元件表达水平的分析
为了评估重复元件的表达水平，实施了Repenrich2管道。然后，使用R包edgeR(版本3.30.3)计算差异表达的重复元件，临界值为“|log 2(倍数变化)|>0.1且FDR<0.05”。重复元件的类别在RepeatMasker中进行了注释，可以分为LTR、LINE、SINE、DNA(也称为DNA转座子)、Satellite和一些小RNA重复序列，包括tRNA、rRNA、scRNA和snRNA类。使用R(版本4.0.2)中的Fisher精确检验对差异表达的重复元件进行了家族富集分析。
HERVK前病毒(HML2)表达水平分析
首先，从NCBI数据库(GenBank ID：JN675007-JN675097)中获得了91份HERVK原病毒特异性fasta文件，该文件在先前的研究中进行了注释。为了评估早衰和RS hMPC中HERVK原病毒的转录水平，我们将91个HERVK原病毒fasta文件连接到Ensembl数据库注释的hg19(GRCh37.75)cDNA fasta文件中。然后，通过Salmon(版本0.8.2)和清理过的RNA-seq读取生成映射索引，所述读取通过Salmon定量程序以参数“-l A--gcBias”映射到hg19和HERVK前病毒的连接索引文件。接下来，获取了映射到HERVK原病毒序列的读取计数，并使用R包DESeq2(1.29.8版)计算差异表达的HERVK原病毒基因座，临界值为“|log2(倍数变化)|>0.1，Benjamini-Hochberg调整的P值<0.05”。通过Salmon定量程序计算给定HERVK前病毒基因座的TPM(每千个碱基的转录每百万映射读取的转录物)，并通过R(4.0.2版)中的ggpubr R包(0.4.0版)可视化，并且通过不成对的两个样品t检验来评估每个比较中的p值。对于基因组的每个10bp的箱尺寸计算表达水平(每千个碱基的转录每百万映射读取的读取数，RPKM)，并在IGV(版本2.7.2)中可视化。
全基因组亚硫酸氢盐测序(WGBS)文库的构建和测序
使用DNeasy Blood & Tissue Kits(QIAGEN)从细胞中提取基因组DNA，每重复2×10 6个细胞，并用超声仪剪切至100-300bp。然后，由Novogene生物信息技术有限公司进 行文库制备，亚硫酸氢盐处理，质量控制和测序。
全基因组亚硫酸氢盐测序数据处理
使用默认参数的fastp软件(版本0.19.10)对原始测序读取进行了修整。然后，使用bsmap(版本2.90)和参数“-v 0.1-g 1-R-u”(89)，将经清理的读取映射到从UCSC基因组浏览器数据库中获得的人hg19参考基因组。通过bsmap提供的methratio程序计算每个胞嘧啶位点的CpG DNA甲基化水平。为确保甲基化水平检测的准确性，将每个CpG位点的正向和反向链读取相结合，并且仅保留深度大于5的CpG位点用于下游分析。为了计算重复元件基因座中的相对CpG DNA甲基化水平，我们从UCSC基因组浏览器中获得了每个RepeatMasker注释的重复元件的基因组基因座。然后，计算每个带有RepeatMasker注释的重复元件的平均CpG DNA甲基化水平，并通过R(4.0.2版)中的ggpubr R软件包(0.4.0版)进行统计分析。通过配对的两个样品t检验评估P值。
统计分析
使用PRISM版本8软件(GraphPad软件)对所有数据进行统计分析。结果表示为平均值±SEM。使用两尾学生t检验和单向方差分析进行比较。P值<0.05被认为具有统计学显着性(*)，P值<0.01被认为具有高度统计学显着性(**)，P值<0.001被认为具有高度统计学显着性(***)。
引物、shRNA和sgRNA
本申请实施例中使用的引物、shRNA、sgRNA序列和探针序列如下表2所示。
表2
Figure PCTCN2021140550-appb-000003
Figure PCTCN2021140550-appb-000004
Figure PCTCN2021140550-appb-000005
Figure PCTCN2021140550-appb-000006
Figure PCTCN2021140550-appb-000007
抗体
本申请实施例中使用的抗体为常规商业化抗体、抗血清或试剂盒自带抗体，例如表3所示的抗体。
表3
Figure PCTCN2021140550-appb-000008
实施例1、ERV活化与人类细胞衰老相关
在本实施例中，使用Hutchinson-Gilford早衰综合征(HGPS)和Werner综合征(WS)的人间充质祖细胞(hMPC)(LMNA G608G/+hMPC)或WS hMPC(WRN -/-hMPC)(作为过早衰老的细胞模型)，以及野生型(WT)复制性衰老(RS)的hMPC来研究HERVK活化与人类细胞衰老的关系。
1.1.衰老的hMPC的RNA-seq分析
在WT RS hMPC和过早衰老模型(包括HGPS和WS hMPC)中进行了RNA-seq分析。
如图3所示，衰老的hMPC中几种转座因子的表达增加，例如LTR、DNA转座子和LINE(长散布核元件，一类非LTR)。在这些元件中，ERVK家族中的HERVK在复制型衰老和过早衰老的hMPC中均上调(图3B和3C)。
具体而言，在图3B和图3C中，显示了早期传代(EP)的hMPC中HGPS vs.WT和WS vs.WT的基因转录水平比较，晚期传代(LP)的hMPC中HGPS vs.WT和WS vs.WT的基因转录水平比较，以及野生型hMPC EP vs.LP的基因转录水平比较。
图3B中显示的是根据上述基因表达水平比较结果的综合排名，结果显示：
HERVK-int、MER61-int和MER61C在所有五项比较中均体现出表达的显著差异；
Charlie4和MER101B在EP HGPS vs.WT和WS vs.WT以及LP HGPS vs.WT和WS vs.WT的比较中显示显著差异；
MamGypLTR(MamGyp家族的LTR区)在EP HGPS vs.WT和WS vs.WT，LP HGPS vs.WT以及WT EP vs.LP的比较中显示显著差异；
HERVK11在EP HGPS vs.WT和WS vs.WT，LP WS vs.WT，以及WT EP vs.LP的比较中显示显著差异；
HERV16在EP HGPS vs.WT，LP HGPS vs.WT和WS vs.WT，以及WT EP vs.LP的比较中显示显著差异；
HUERS-P2在EP WS vs.WT，LP HGPS vs.WT和WS vs.WT，以及WT EP vs.LP的比较中显示显著差异。
由此可见，HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、HERV30基因座的人ERV可以作为衰老的生物学标记。
在图3D和图3E中，显示了早期传代(EP)的hMPC中HGPS vs.WT和WS vs.WT的全病毒HERVK在不同位点处的激活程度比较，晚期传代(LP)的hMPC中HGPS vs.WT和WS vs.WT的全病毒HERVK在不同位点处的激活程度比较，以及野生型hMPC EP vs.LP的全病毒HERVK在不同位点处的激活程度比较。
图3E中显示的是根据全病毒HERVK在不同位点处的激活程度的综合排名，结果显示：
HML2_1q22在所有五项比较中均体现显著激活；
HML2_3q12.3在EP HGPS vs.WT和WS vs.WT以及LP WS vs.WT的比较中显示显著激活；
HML2_3q21.2在EP HGPS vs.WT和WT EP vs.LP的比较中显示显著激活；
HML2_4p16.1b在EP WS vs.WT的比较中显示显著激活；
HML2_21q21.1在LP HGPS vs.WT的比较中显示显著激活；
HML2_12q11.1在LP HGPS vs.WT的比较中显示显著激活；
更具体地，如图3F所示，对应于基因座HML2_1q22的HERVK在衰老的hMPC中被高度激活。
1.2.hMPC中特定的HERVK转录物的水平
使用针对HERVK转录物的不同区域(包括Env，Pol和Gag)的引物(如表2所示)进行RT-qPCR，结果示于图4A。
如图4所示，在细胞衰老过程中HERVK是高表达的，显示出与先前报道的标记物的表达趋势相似，例如衰老正标记p21 Cip1(在衰老过程中增加)和衰老负标记Lamin相关蛋白LAP2(TMPO)(在衰老过程中减少)。
进一步，进行RNA-FISH以及单分子RNA-FISH分析。结果如图4B-C所示，HGPS和WS hMPC以及RS hMPC中的细胞质HERVK RNA信号增加。同时针对HERVK转录物的不同区域(包括Env，Pol和LTR)标记不同荧光探针，显示HERVK不同区域部分共定位于同一个病毒颗粒中(图4D)。
1.3.hMPC中HERVK基因座甲基化与衰老的关系
进行全基因组甲基化分析。结果显示，与衰老的hMPC中活化的HERVK转录一致，整个HERVK家族、HERVK编码序列(HERVK-int)和侧翼LTR5HS(人类特异性HERVK启动子)区域中的全局DNA甲基化水平在过早衰老的hMPC和RS hMPC中均降低(如图5所示)。
通过基因座特异性分析，还发现前病毒基因座(包括HML2_1q22)中CpG DNA甲基化信号的降低(如图5C所示)。
进行ChIP-qPCR分析。结果显示HERVK激活与衰老的hMPC中HERVK-LTR5HS处的抑制性组蛋白标记(H3K9me3)减少和转录活化的组蛋白标记(H3K36me3)升高相关(如图6所示)。
由此可见，HERVK基因座的表观遗传学调控与HERVK转录相关，并且参与细胞衰老过程。
1.4.hMPC中HERVK蛋白成分、逆转座以及RVLP的形成和细胞衰老的关系
对各hMPC进行蛋白质印迹和免疫荧光分析。结果显示，早衰和RS hMPC中的HERVK-Env蛋白水平均增加(如图7所示)。
TEM分析显示，与HERVK逆转录RNA和蛋白质水平的增加一致，TEM结果显示RVLP在过早衰老和RS hMPC的细胞质中积累(如图8所示)。相反，在表型年轻的早期传代WT hMPCs中很少检测到RVLP。
使用抗-HERVK-Env抗体的免疫TEM分析显示，HERVK在衰老细胞中形成了直径为80至120nm的RVLP(如图9所示)。
此外，使用HERVK转座报告载体转换hMPC。
通过流式细胞仪BD Influx分析经转化的细胞，绿色荧光阳性细胞与红色荧光阳性细胞的比例即为HERVK发生的转座事件的概率。结果显示，衰老的hMPC中逆转座事件发生率增加(如图10A-B所示)。
利用单分子DNA-FISH显示衰老的hMPC HERVK DNA拷贝数增加(如图10C-D)，表明衰老hMPC中逆转座事件增加。
1.5.在其他细胞中HERVK基因座活化与衰老的关系
在人成纤维细胞中进行与以上相似的实验，结果显示，与衰老的hMPC相似，在HERVK基因组位点观察到H3K9me3的减少和H3K36me3的增加，以及衰老的人类成纤维细胞中HERVK表达(在RNA和蛋白质水平上)增加(图11A-F)。此外，在衰老的成纤维细胞中RVLP的积累增加(如图11G)。
由此可见，与未衰老的细胞相比，HERVK在衰老细胞中的活化、动员和包装。
实施例2、HERVK表达引起先天性免疫应答和细胞衰老
在此实施例中，研究HERVK基因座活化与细胞衰老直接的关系。
2.1.激活HERVK转录促进细胞衰老，而抑制HERVK转录减轻细胞衰老
使用CRISPR-dCas9转录激活系统激活HERVK转录，确定内源性HERVK的激活对细胞衰老的影响。所述转录激活系统包含带有靶向HERVK-LTR5HS启动子区域的sgRNA(sgHERVK-act)的激活蛋白复合物(协同激活介导物，SAM)。使用包含非靶向性sgRNA的CRISPR-dCas9系统作为对照。
通过RT-qPCR和蛋白质印记分析HERVK激活的细胞。
结果在蛋白质和RNA水平证明了，用CRISPR-dCas9转录激活系统激活了细胞中的内源性HERVK(如图12A和图12B)。
对HERVK激活的细胞进行经典衰老特征分析，结果表明，HERVK激活导致hMPC衰老。具体而言，衰老相关的β-半乳糖苷酶(SA-β-gal)阳性细胞的相对百分比增加，Ki67阳性细胞的数量减少和克隆扩增能力降低(如图12C-12E)。并且，RT-qPCR分析显示，衰老标记p16 INK4a水平升高，LAP2(TMPO)水平降低(如图12B)。
为了进一步评估内源性HERVK激活是否是细胞衰老的驱动力，使用HERVK特异性shRNA敲减早衰hMPC中的HERVK。结果显示，内源性HERVK的蛋白质和RNA水平降低(如图13A)。
对HERVK敲减的hMPC进行以上描述的经典衰老特征分析，结果表明，抑制HERVK可以减轻hMPC衰老(如图13B-E)。
同时使用Cas9KRAB抑制系统抑制HERVK转录。结果显示，内源性HERVK的蛋白质和RNA水平降低(如图14A)。
对HERVK受抑制的hMPC进行以上描述的经典衰老特征分析，结果表明，抑制HERVK可以减轻hMPC衰老(如图14B-D)。
2.2.HERVK基因座去甲基化
使用5-AZA(一种DNA甲基转移酶抑制剂(DNMTi))处理早期传代的hMPC。对经处理的细胞进行ChIP-qPCR和RT-qPCR分析，以未经5-AZA处理的细胞作为对照。
结果显示，5-AZA处理可降低HERVK-LTR5HS的5mC占用，同时hMPC中的HERVK RNA水平和衰老标记p16 INK4a升高(如图15A-B)。
对经处理的细胞进行经典衰老特征分析。结果与HERVK激活实验一致，5-AZA处理导致细胞过早衰老。具体而言，SA-β-gal阳性细胞增加，Ki67阳性细胞的数量减少和克隆扩增能力降低(如图15C-E)，而HERVK敲减有效地降低了5-AZA诱导的衰老(如图15F-G)。这些数据支持内源性HERVK的激活是hMPC衰老的驱动力。
2.3.逆转录病毒抑制剂降低细胞衰老
在本实施例中个，研究非特异性逆转录病毒抑制剂对细胞衰老的作用。
向WS hMPC分别添加10μM lamivudine(3TC)、abacavir(0.2μM)和Raltegravir，37℃温育，每两天换新鲜的含各药物的培养基，以添加相应量的DMSO(溶解上述药物的溶剂)作为对照。
温育10天后，逆转录酶抑制剂abacavir可有效降低HERVK的转座事件和DNA拷贝数(如图16B-C)
对上述细胞进行Ki67染色和SA-β-gal染色。结果如图25所示。在经lamivudine(3TC)、abacavir或Raltegravir处理的WS hMPC中，Ki67阳性细胞与对照相比显著增加，而SA-β-gal阳性的细胞与对照相比显著降低(如图16D-E)。
以上结果表明，逆转录病毒抑制剂可以降低衰老细胞的衰老表型。
2.4.HERVK DNA诱导先天免疫应答
HERVK编码的Pol蛋白具有逆转录活性，可以将HERVK RNA逆转录为DNA，从而在基因组之外生成其他HERVK DNA。这些过量的细胞质DNA可能被关键的DNA传感器cGMP-AMP合酶(cGAS)识别并触发先天免疫应答(如图17)。
对早期传代和晚期传代的hMPC进行单分子DNA-FISH，结果显示，与增加的HERVK RNA一致，在衰老的hMPC细胞质中的HERVK DNA显著增加(如图18A)。
免疫沉淀分析证实，衰老的hMPC的细胞质HERVK DNA上的cGAS富集增加，而在早期传代的hMPC中几乎没有富集(如图18B)，表明衰老的hMPC的胞质HERVK DNA会激活cGAS-STING途径。
为了进一步研究cGAS-STING DNA传感途径的激活状态，还检测了不同的衰老(包括RS、HGPS和WS)的hMPC和成纤维细胞中的以下关键特征：第二个信使2'3'-cGAMP，RelA/NF-κB的磷酸化，TANK结合激酶1(TBK1)和IFN调节因子3(IRF3)以及促炎细胞因子(例如IL1B和IL6)的表达，这些因子被分类为SASP因子。
在过早衰老hMPC中，2'3'-cGAMP含量的增加(如图19A)；TBK1、RelA和IRF3的磷酸化上调(如图17B)以及炎性细胞因子IL6上调(如图19C)。在RS hMPC和不同的衰老的成纤维细胞中也观察到类似的现象。
敲减HERVK或STING(shSTING-2)均可降低TBK1，RelA和IRF3的磷酸化水平，并减轻细胞衰老和SASP(如图20)。
相比之下，通过CRISPR-dCas9系统或5-AZA处理激活内源性HERVK导致磷酸化的TBK1、RelA和IRF3的水平增加，并导致hMPC中SASP细胞因子的表达上调。
由此可见，HERVK活化(至少部分)通过激活先天免疫途径而促进细胞衰老。
实施例3、细胞外HERVK诱导细胞衰老
本实施例的目的在于证明衰老细胞产生的HERVK RVLP是否可以细胞外释放并将衰老信号传递给非衰老细胞。
3.1.检测条件培养基(CM)中的HERVK RVLP
对从野生型和过早衰老的hMPC收获的CM进行ddPCR，检测其中的HERVK RNA，扩增子为表2中的HERVK6。
结果显示，与野生型hMPC相比，早衰型hMPC中CM的HERVK RNA水平高5～12倍(图21A)。
另外，使用抗HERVK-Env抗体进行的ELISA分析也显示衰老的hMPCs培养基中HERVK-Env蛋白水平升高(图21B)。
根据TEM图像，hMPC中的RVLP多于野生型，直径范围在80至120nm之间，一些HERVK病毒颗粒发芽于细胞表面或邻近细胞表面，而一些颗粒包含在包被的囊泡中并释放到细胞外环境中(图21C)。Immuno-TEM显示与TEM一致的结果；还观察到衰老的人类成纤维细胞的CM中HERVK的存在增加(结果未显示)。
3.2.CM中的HERVK诱导细胞衰老
使用从HGPS、WS或RS hMPC收集的CM(统称为“老”CM)处理年轻的hMPC(WT)，使用从年轻的WT hMPC收集的CM作为对照。
TEM图像显示HERVK颗粒附着于年轻的hMPC(图22A)。老CM处理后48小时，通过qRT-PCR检测，发现年轻的hMPC中HERVK RNA的增加(图22B)，这意味着老CM中的HERVK可能会传播到靶细胞中。
此外，SA-β-gal和Ki67染色以及克隆扩增能力试验显示，与老CM温育的年轻hMPC中诱导加速的细胞衰老(图22C-E)。
进一步，使用抗HERVK-Env抗体去除老CM中的HERVK。用去除HERVK的老CM处理年轻的hMPC后，TEM分析显示较少的HERVK RVLP附着细胞表面或进入其中(图23A)。此外，与未去除HERVK的老CM相比，从老CM去除HERVK导致年轻WT hMPC的HERVK RNA减少和衰老表型减轻(图23B-E)。
另外，来自衰老的hMPC中的老CM激活了年轻hMPC中的cGAS-STING途径并诱导了SASP基因的表达；敲减STING或敲除TKB1可消除由老CM诱导的衰老表型(数据为显示)。这表明与内源性HERVK表达相似，老CM(至少部分地)通过激活先天免疫途径驱动了细胞衰老。
最后，为了进一步验证HERVK病毒可以侵染靶细胞引起细胞细胞的衰老。构建表达和包装HERVK病毒的载体，并用绿色荧光标记HERVK病毒(如图24A)。在293T细胞中检测到HERVK病毒RNA、蛋白和病毒颗粒，以及GFP信号(如图24B-E)。
用纯化的带有绿色荧光蛋白标记的HERVK病毒感染hMPC细胞，电镜显示病毒颗粒进入细胞，并在hMPC细胞中可以检测到绿色荧光，说明HERVK病毒可以进入hMPC细胞中(如图25A-C)。
进一步，通过流式细胞仪将感染HERVK病毒带有绿色荧光的细胞分选出来(如图25D-E)。与没有感染HERVK病毒的细胞相比，感染HERVK病毒的细胞GFP的RNA和基因拷贝数增加(如图25F-G)，说明HERVK不仅在细胞中表达还整合到基因组中。蛋白质印迹、ChIP-qPCR、RT-qPCR、SA-β-gal和Ki67染色以及克隆扩增能力试验显示，侵染的HERVK被cGAS识别，并激活先天免疫应答驱动细胞衰老(如图25H-M)。
此外，使用抗HERVK抗体中和HERVK病毒。使经中和的HERVK和未处理的HERVK(如图26A中的αHERVK是表2中的鼠抗HERFVK-Env抗体，IgG是用作对照的鼠免疫球蛋白)分别与hMPC接触，并进行实施例2所述的经典衰老特征分析。结果显示，与未处理的HERVK病毒相比，用中和抗体处理HERVK病毒减轻HERVK病毒引起的先天免疫应答的激活和细胞衰老(图26A-F)。
综上所述，在衰老细胞中产生的HERVK可以旁分泌的方式释放并诱导年轻细胞的细胞衰老。
实施例4、ERV与个体衰老
在本实施例中，研究ERV与动物个体衰老之间的关系。
4.1.MMTV与小鼠的衰老
由于在小鼠中HERVK对应的同源病毒为MMTV，因此我们在小鼠中对MMTV进行检测。
获取2月龄(5只)、8月龄(5只)和24月龄(5只)的小鼠肝脏活检样品，提取RNA并使用表2中的引物通过RT-qPCR检测小鼠肝脏中的MMTV水平，扩增子为MMTV1和MMTV2。结果如图27A所示。8月龄和24月龄的小鼠中MMTV1的表达量显著高于2月龄的小鼠；而24月龄小鼠中的MMTV2的表达量显著高于2月龄和8月龄的小鼠。
此外，通过蛋白质印迹和免疫组化染色分析发现，与年轻的小鼠相比，在年老的小鼠分离的肺、肝和皮肤组织中，MMTV表达水平随年龄增加而增加(图27B-C)。先天免疫应答在自然衰老的小鼠中也被过度激活(图27B)。
以上结果表明，在衰老的小鼠中内源逆转录病毒MMTV被活化。
4.2.ERVW与食蟹猴的衰老
由于在食蟹猴基因组中不存在ERVK家族(图2)，因此选择在食蟹猴中进化上最年轻的ERVW家族进行检测。
与人类衰老细胞中激活的HERVK家族相似，通过蛋白质印迹和免疫组化染色分析发现，相对于相同年龄的WT食蟹猴，HGPS食蟹猴的肺，肝和皮肤组织中的ERVW-Env信号增加(图28)。结果还显示，HGPS食蟹猴组织中的先天免疫应答增加，例如，RelA的磷酸化增加(图28A)和SASP基因的上调(数据未显示)。
此外，在从年轻和生理性衰老的食蟹猴分离的肺，肝和皮肤组织中进行比较时，ERVW表达水平随年龄增加而增加(图28A-C)。SASP在自然衰老的猴子中也被过度激活，类似于早衰的HGPS猴子(图28D)。
4.3.HERVK与人的衰老
通过qPCR分析年轻和老年供体原代hMPC中的HERVK水平。结果显示，相对于年轻个体，老年个体中HERVK基因(env、pol和gag)的表达显著上调(图29A)。在从年轻和老年供体获得的皮肤组织中，免疫组织化学染色显示HERVK-Env表达随年龄的增长而显着增加(图29B)。更重要的是，人血清的ELISA分析显示，老年个体血清中的HERVK-Env水平相对于年轻个体显著提高(图29C)。
进一步，用含有年轻或老年个体血清的培养基培养来自年轻个体的原代hMPC。进行与实施例3.2中相应的检测，结果显示，老年个体的血清(OS)加速了原代hMPC衰老并引起了衰老相关的炎性应答，而OS中HERVK的免疫去除则消除了OS对年轻的原代hMPC的促衰老作用，并使得其中的衰老相关的炎症表型降低(图30)。
实施例5、抑制ERV延缓组织器官以及个体衰老
本实施例的目的是探索是否可以通过抑制ERV达到延缓组织器官以及个体衰老的目的。
5.1.敲低MMTV延缓老年小鼠的关节老化
向老年小鼠(21月龄)的关节腔中注射用于以Cas9KRAB抑制系统抑制MMTV的慢病毒，在第一次注射4周后重复注射一次(如图31A)。(n＝12)
首先，在MEF细胞中蛋白印迹显示经CRISPR-dCas系统抑制后，MMTV蛋白质水平降低，同时先天免疫途径相关蛋白p-RelA水平降低(如图31B)；在注射8周后，测试小鼠的四肢拉力，结果显示与未抑制MMTV的小鼠相比，抑制MMTV后，小鼠的拉力显著提高(如图31C)；微型核磁显示抑制MMTV后，关节处的骨密度增加(如图31D)；取整个膝关节(包括股骨下端、胫骨上端、髌骨、关节软骨、关节韧带和关节液)，提RNA，RT-qPCR显示抑制MMTV后炎症因子IL1B水平降低，衰老相关蛋白p21表达降低。
以上结果表明，敲低MMTV延缓老年小鼠的关节老化。
5.2.逆转录病毒抑制剂Abacavir延缓老年小鼠的关节老化
向老年小鼠(23月龄)的小鼠的关节腔中注射逆转录病毒抑制剂Abacavir，浓度为10mg/ml，每周注射一次，每次10μl(如图32A)。(n＝10)
在注射逆转录病毒抑制剂Abacavir 8周后，测试小鼠的四肢拉力，结果显示与注射载剂的小鼠相比，注射逆转录病毒抑制剂Abacavir的小鼠的拉力显著提高(如图32B)；虽然由于个体差异过大，P值不显著，但是可以看出，强制跑步机测试显示施用逆转录病毒抑制剂Abacavir可以提高小鼠的跑步距离和时间，并降低被电击的次数的趋势(如图32C)；微型核磁显示注射逆转录病毒抑制剂Abacavir 15周后，关节处的骨密度增加(如图32D)；关节取材后，提RNA，RT-qPCR显示注射逆转录病毒抑制剂Abacavir后炎症因子IL1B和IL6水平减少，衰老相关蛋白p21表达降低。
以上结果表明，施用逆转录病毒抑制剂Abacavir延缓老年小鼠的关节老化。
5.3.施用Abacavir延缓老年小鼠个体衰老并提高生存率
将逆转录酶抑制剂Abacavir以2mg/ml的浓度溶解于小鼠饮用水(含有0.3％DMSO)中，(n＝23)给18月龄的小鼠每天施用(如图33A)。
在施用逆转录病毒抑制剂Abacavir 12周后，测试小鼠的四肢拉力，结果显示注射逆转录病毒抑制剂Abacavir后，小鼠的拉力显著提高(如图33B)；在施用逆转录病毒抑制剂Abacavir 24周后，对小鼠的体重、皮肤、毛发、弓背、白内障以及活动能力进行测试，结果表明逆转录酶抑制剂Abacavir可以提高小鼠的活动能力，并改善毛发和弓背情况(如图33C)。在施用逆转录病毒抑制剂Abacavir 30周后，对小鼠进行Y迷宫测试，结果表明施用逆转录酶抑制剂Abacavir小鼠的总进臂次数和依次连续进入Y迷宫全部三个臂一次的轮流(交替)次数增加，说明小鼠的空间短期记忆能力提高(如图33D)。
在施用Abacavir后24周，施用载剂的对照组死亡2只，Abacavir组死亡0只，在施用Abacavir后30周，施用载剂的对照组死亡4只，Abacavir组死亡1只，说明逆转录酶抑制剂Abacavir可以提高老年小鼠的生存率(如图33E)。发明人相信，随着继续施用Abacavir，生存率会进一步显示显著差异。
以上结果表明，逆转录病毒抑制剂Abacavir延缓老年个体衰老并提高生存率。
5.4.施用逆转录病毒整合酶抑制剂Ratelgravir延缓老年小鼠个体衰老
将逆转录病毒整合酶抑制剂Ratelgravir以50mg/ml的浓度溶解于30％PEG300+2％Tween80+2％DMSO中，并给18月龄的小鼠腹腔注射，(n＝22)每次注射200μl，每周注射两次(如图34A)。
在注射整合酶抑制剂Ratelgravi 16周后，测试小鼠的四肢拉力，结果显示与注射载剂的小鼠相比，注射逆转录病毒整合酶抑制剂Ratelgravir的小鼠的拉力显著提高(如图34B)；同时对小鼠的体重、皮肤、毛发、弓背、白内障以及活动能力进行测试，结果表明施用整合酶抑制剂Ratelgravir可以改善毛发和弓背情况(如图34B)。
以上结果表明，合酶抑制剂Ratelgravir延缓老年个体衰老。
实施例6、合成并鉴定识别ERV的抗体
本实施例的目的合成小鼠中内源病毒MMTV的特异性中和抗体，并检测合成抗体的效价以及中和效率，用于体内治疗。
MMTV抗体由义翘神州生物技术有限公司提供。简而言之，根据结构分析，MTV-SU亚基(SEQ ID NO:151)外加N-信号肽和N-his-tag，在HEK293系统中进行表达。重组表达MMTV-SU亚基作为免疫原，免疫小鼠，通过ELISA筛选出与免疫原结合的抗体。
对目前获得的16株主克隆的上清进行鉴定。蛋白质印迹显示该16株多克隆抗体都可以检测出MMTV-Env蛋白，并显示在老年的小鼠肾脏中MMTV-Env蛋白水平升高(如图35A)；免疫组化染色显示1、2、5、6、11、12、14和15号抗体可以检测到MMTV正确的细胞定位(位于细胞质)，其中第2号和14号抗体表现出最好的染色效果。
分离单克隆抗体，并用分离到的单克隆抗体处理MMTV病毒。用经处理的MMTV病毒感染小鼠细胞，与对照相比，经处理的MMTV病毒滴度降低，用于处理MMTV病毒的抗体鉴定为中和抗体。
进一步，将上述鉴定到的中和抗体施用于老年小鼠，对小鼠的体重、皮肤、毛发、弓背、白内障以及活动能力进行测试。此外，还对小鼠进行Y迷宫测试，并记录小鼠的存活时间。在实验后期，收集来自各组织和器官(包括大脑、肺、肝脏、皮肤、血液、关节)的样品，通过测序技术包括转录组测序、免疫组化染色、蛋白免疫印迹以及RT-qPCR验证各器官衰老的表型。
序列信息
SEQ ID NO:1，示例性的HERVK-Env氨基酸序列
Figure PCTCN2021140550-appb-000009
SEQ ID NO:2，示例性的HERVK-Pol氨基酸序列
Figure PCTCN2021140550-appb-000010
SEQ ID NO:3，示例性的HERVK-Pro氨基酸序列
Figure PCTCN2021140550-appb-000011
SEQ ID NO:4，示例性的HERVK-Gag氨基酸序列
Figure PCTCN2021140550-appb-000012
SEQ ID NO:5，示例性的HERVK-Env编码核苷酸序列
Figure PCTCN2021140550-appb-000013
Figure PCTCN2021140550-appb-000014
SEQ ID NO:6，示例性的HERVK-Pol编码核苷酸序列
Figure PCTCN2021140550-appb-000015
SEQ ID NO:7，示例性的HERVK-Pro编码核苷酸序列
Figure PCTCN2021140550-appb-000016
Figure PCTCN2021140550-appb-000017
SEQ ID NO:8，示例性的HERVK-Gag编码核苷酸序列
Figure PCTCN2021140550-appb-000018
SEQ ID NO:149，示例性的HERVK-Rev编码核苷酸序列
Figure PCTCN2021140550-appb-000019
SEQ ID NO:150，示例性的HERVK全长编码核苷酸序列
Figure PCTCN2021140550-appb-000020
Figure PCTCN2021140550-appb-000021
Figure PCTCN2021140550-appb-000022
Figure PCTCN2021140550-appb-000023
SEQ ID NO:151，MMTV抗原序列
Figure PCTCN2021140550-appb-000024
SEQ ID NO:152，GFP reporter序列
Figure PCTCN2021140550-appb-000025
Figure PCTCN2021140550-appb-000026
SEQ ID NO:153，CMV-GFP序列
Figure PCTCN2021140550-appb-000027

Claims (30)

  1. 一种鉴定对象或其细胞、组织或器官衰老程度，或评估对象生理年龄或诊断早衰症，或评估分离的动物细胞或细胞群的衰老程度的方法，包括
    a)检测来自所述对象的样品中或所述细胞中的内源逆转录病毒(ERV)活化水平；和
    b)将所述检测结果与所述ERV活化水平的参考值进行比较。
  2. 权利要求1的方法，其中所述参考值是通过检测参考对象获得的。
  3. 权利要求1或2的方法，其中所述参考值是通过检测参考对象群体获得的。
  4. 权利要求1-3任一项的方法，其中所述ERV的活化水平包括ERV基因座甲基化水平、ERV转录物水平、ERV蛋白质水平、ERV颗粒在细胞中的存在、定位、和/或从细胞释放，和/或ERV基因座的转座概率。
  5. 权利要求4的方法，其中所述ERV转录物选自Env、Pol、Pro和Gag基因的转录物。
  6. 权利要求4的方法，其中所述ERV蛋白质选自Env、Pol、Pro和Gag蛋白。
  7. 权利要求1-6任一项的方法，其中所述样品包括肺、肝、皮肤、心肌以及大脑样品。
  8. 一种调节细胞衰老的方法，包括使所述细胞与ERV基因座活化调节剂接触。
  9. 权利要求8的方法，其中所述调节剂是ERV基因座活化抑制剂或所述ERV的抑制剂。
  10. 权利要求8的方法，其中所述调节剂是所述细胞的ERV基因座活化促进剂。
  11. 一种治疗有需要的对象或其细胞、组织或器官的过早衰老，或防止或延缓对象或其细胞、组织或器官衰老的方法，包括给所述对象施用ERV基因座活化抑制剂或所述ERV的抑制剂。
  12. 一种培养分离的动物细胞或细胞群或防止或延缓细胞衰老的方法，包括使所述细胞与所述细胞的ERV基因座活化抑制剂或所述ERV的抑制剂接触。
  13. 一种促进细胞衰老的方法，包括使所述细胞与所述细胞的ERV基因座活化促进剂或所述ERV接触。
  14. 权利要求10或13的方法，其中所述ERV基因座活化促进剂是靶向所述ERV基因座的转录激活系统。
  15. 权利要求14的方法，其中所述转录激活系统是CRISPR-dCas转录激活系统。
  16. 权利要求1-15任一项的方法，其中所述ERV选自以下家族：HERVK(包括HERVK11)、MER61、MER61C、Charlie4、MER101B、MamGyp、HERV16、HUERS-P2、ERVW和MMTV及其同源物。
  17. 权利要求1-16任一项的方法，其中所述对象是啮齿类动物，且任选地，所述ERV来自MMTV家族。
  18. 权利要求1-16任一项的方法，其中所述对象是非人灵长类动物，且任选地，所述ERV来自ERVW家族。
  19. 权利要求1-16任一项的方法，其中所述对象是人，且任选地，所述ERV来自HERVK家族，例如位于原病毒HML2_1q22处的HERVK。
  20. 一种促进细胞衰老的方法，包括使所述细胞与DNA甲基转移酶抑制剂，例如地西他滨、Lomeguatrib、SGI-1027或5-氮杂胞苷接触。
  21. 权利要求20的方法，其中所述细胞是非人哺乳动物细胞、非人灵长类动物细胞或人细胞。
  22. 一种治疗有需要的对象或其组织或器官的过早衰老，或防止或延缓对象或其组 织或器官衰老，或防止或延缓局部组织包括关节和皮肤衰老，或治疗早衰症和衰老相关疾病的方法，包括给所述对象施用逆转录病毒抑制剂。
  23. 权利要求22的方法，其中所述逆转录病毒抑制剂包含逆转录酶抑制剂、整合酶抑制剂和/或蛋白酶抑制剂。
  24. 权利要求23的方法，其中所述逆转录酶抑制剂包括核苷型逆转录酶抑制剂如enofovir、abacavir、stavudine(D4T)、lamivudine(3TC)和/或zidovudine，和/或非核苷型逆转录酶抑制剂如efavirenz、etravirine和/或nevirapine。
  25. 权利要求23的方法，其中所述整合酶抑制剂包括Raltegravir。
  26. 权利要求23的方法，其中所述蛋白酶抑制剂包括Lopinavir和/或Darunavir。
  27. 权利要求22-26任一项的方法，所述逆转录病毒抑制剂包含lamivudine(3TC)、abacavir和/或Raltegravir。
  28. 一种防止或延缓局部组织包括关节和皮肤衰老，或治疗早衰症以及衰老相关疾病的方法，包括给有需要的对象施用ERV基因座活化抑制剂或ERV抑制剂。
  29. 权利要求9、11、12或28的方法，其中所述ERV基因座活化抑制剂促进ERV基因座甲基化，或是针对ERV基因座的基因编辑系统，或是针对ERV的Env、Pol、Pro和/或Gag基因的反义核酸或干扰核酸，或是可以与所述ERV的蛋白质如Env蛋白结合的结合分子，例如抗体或其抗原结合片段或抗体衍生物，优选ERV中和抗体或其抗原结合片段或抗体衍生物。
  30. 权利要求22-29任一项的方法，其中所述早衰症是HGPS或WS，其中所述衰老相关疾病包括关节炎、卵巢早衰、肝纤维化、肺纤维化、衰弱症以及心血管疾病。
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