Compositions and methods for treating, preventing, or reversing age-related inflammation and disorders

Abstract

To provide methods for preventing, delaying or reversing age-associated inflammation.SOLUTION: This method includes the step of administering to a patient in need thereof a therapeutically effective amount of at least one reverse transcriptase inhibitor (RTI). The reverse transcriptase inhibitor includes censavudine or elvucitabine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention is in the field of medicinal chemistry. In particular, the present invention relates to a method for treating, preventing, or reversing age-related inflammation by administering a reverse transcriptase inhibitor (RTI) to a patient in need thereof. Age-related inflammation may be present in patients with Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, vision loss, hearing loss, peripheral degenerative disease, or cardiovascular dysfunction, frontotemporal dementia (FTD), multiple sclerosis (MS), Aicardi-Goutières syndrome, progressive supranuclear palsy (PSP), osteoarthritis, skin aging, atherosclerosis, chemotherapy-induced adverse effects, hematopoietic stem cell function, osteoporosis, physical function, and / or pulmonary fibrosis, or in patients requiring wound healing or tissue regeneration.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was developed with funding from the following: Glenn / AFAR Postdoctoral Fellowship, NIH P20 GM119943 COBRE pilot award; NIH F31 AG043189; NIH T32 AG041688; NIH F31 AG050365; Biotechnology and Sport Medicine Fellowships, School of Pharmacy, University of Bologna, Bologna, Italy; NIH R37 AG016667, R01 AG024353, P01 AG051449, Glenn-AFAR Breakthroughs in Gerontology Award; NIH R01 AG050582, P20 GM109035; NIH R37 AG016694, P01 AG051449. [Background technology]

[0003] Background of the Invention Globally, the number of people surviving to age 60 or beyond is increasing. Between 2012 and 2050, the proportion of people aged 60 and over is expected to increase from 809 million to 2 billion (or from 11% to 22% of the population). 1 Among the leading causes of death in older adults are several chronic conditions, including heart disease, cancer, diabetes, Alzheimer's disease, and infectious diseases. Importantly, many of these age-related diseases, and aging itself, are closely linked to low-level chronic inflammation. 2,3,4 Systemic chronic inflammation can accelerate aging 5 Indeed, many inflammatory markers are important predictors of mortality in the elderly. 6 .

[0004] Despite this common link between aging, inflammation, and chronic disease, progress in understanding the mechanisms that control age-related inflammation has been limited, and the causal relationship between these regulators and chronic degenerative diseases is not fully understood. A better understanding of the role of these regulators in age-related inflammation should lead to new strategies for promoting the health of older adults.

[0005] Thus, there is a need in the art for better treatment and prevention of age-related inflammation and age-related disorders. Summary of the Invention

[0006] Brief Summary of the Invention The present invention provides a better understanding of the mechanisms underlying age-related inflammation and its role in aging, as well as compositions and methods for preventing and alleviating age-related inflammation and disorders.

[0007] Retrotransposable elements (RTEs) are deleterious at multiple levels, and therefore, failure of the host surveillance system can have a negative impact. However, the contribution of RTE activity to aging and age-related diseases was unknown. This invention is based on several empirical observations, including that during cellular senescence, LINE-1 (L1) elements are transcriptionally upregulated and activate the type I interferon (IFN-I) response. The IFN-I response is a novel phenotype of late aging and contributes to the maintenance of the senescence-associated secretory phenotype (SASP). The IFN-I response is triggered by cytoplasmic L1 cDNA and antagonized by reverse transcriptase inhibitors (RTIs) that inhibit L1 reverse transcriptase (RT). Treatment of aged mice with the RTI lamivudine downregulated IFN-I activation and age-related inflammation in several tissues. Thus, RTE activation is a critical component of sterile inflammation, a hallmark of aging, and L1 RT is a relevant target for the treatment of age-related disorders.

[0008] The present invention provides methods for treating, preventing and / or reversing age-related inflammation by administering to a patient in need thereof a therapeutically effective amount of at least one reverse transcriptase inhibitor (RTI).

[0009] In a comparative evaluation of several RTI drugs in a dose-response assay for inhibition of L1 activity, two RTI drugs, censavudine and elvucitabine, demonstrated unexpectedly superior ability to inhibit L1 activity in mice and humans. The present invention further provides methods for treating, preventing, and / or reversing age-associated inflammation in a patient in need thereof by administering to the patient a therapeutically effective amount of censavudine and / or elvucitabine.

[0010] Age-related inflammation is associated with upregulation of L1, accumulation of cytoplasmic L1 cDNA, activation of the IFN-I response, and / or enhancement of the pro-inflammatory state of the SASP. The RTI drug is administered in an amount sufficient to prevent or reverse at least one of the upregulation of L1, accumulation of cytoplasmic L1 cDNA, activation of the IFN-I response, and / or enhancement of the pro-inflammatory state of the SASP.

[0011] Age-related inflammation that can be prevented, treated, or reversed using the methods of the present invention is present in patients with diseases or disorders, including, but not limited to, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, vision loss, hearing loss, peripheral degenerative disease, or cardiovascular dysfunction, frontotemporal dementia (FTD), multiple sclerosis (MS), Aicardi-Goutières syndrome, progressive supranuclear palsy (PSP), osteoarthritis, skin aging, atherosclerosis, chemotherapy-induced adverse effects, hematopoietic stem cell function, osteoporosis, physical function, and / or pulmonary fibrosis, or in patients requiring wound healing or tissue regeneration. In one embodiment, age-related inflammation is present in patients with Alzheimer's disease. In an alternative embodiment, age-related inflammation is present in patients with ALS.

[0012] Also provided are methods for slowing or reversing the progression of underlying pathology of disease disorders caused by age-related inflammation, comprising administering a therapeutically effective amount of at least one RTI to a patient in need thereof. In some embodiments, the patient has Alzheimer's disease or ALS and experiences a reduction in one or more symptoms of Alzheimer's disease or ALS compared to before the patient's first administration of an RTI. In some embodiments, the one or more symptoms of Alzheimer's disease include memory loss, misplacing things, forgetting to name places or objects, repeating questions, reduced flexibility, confusion, disorientation, obsessive-compulsive behavior, compulsive behavior, delusions, aphasia, sleep disorders, mood swings, depression, anxiety, frustration, agitation, difficulty performing spatial tasks, agnosia, gait disturbance, weight loss, loss of speech, short-term memory loss, or long-term memory loss.

[0013] In some embodiments, the reduction in one or more symptoms of Alzheimer's disease is assessed according to DSM-5. 7In some embodiments, the symptom reduction is determined using the cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-cog). In some embodiments, the symptom reduction is determined using the Clinician's Interview-Based Impression of Change (CIBIC-plus). In some embodiments, the symptom reduction is determined using an Activities of Daily Living (ADL) scale. In some embodiments, the symptom reduction is for 1 to 36 months.

[0014] In some embodiments, any change in the underlying pathology is identified by detecting a biomarker before and after administration of an RTI. In some embodiments, the biomarker is β-amyloid or tau protein. In some embodiments, the biomarker is detected by PET imaging. In some embodiments, the underlying pathology is identified by measuring β-amyloid or tau protein in cerebrospinal fluid. In some embodiments, the underlying pathology is identified by measuring brain volume before and after administration of an RTI. In some embodiments, the underlying pathology is reversed or delayed by 1 to 36 months.

[0015] In some embodiments, the at least one RTI is a nucleoside reverse transcriptase inhibitor (NRTI). In some embodiments, the at least one NRTI is selected from the group consisting of abacavir (ZIAGEN™), abacavir / lamivudine (Epzicom), abacavir / lamivudine / zidovudine (TRIZIVIR™), adefovir, alovudine, amdoxovir, apricitabine, ATRIPLA®, BARACLUDE®, BIKTARVY®, censavudine, COVIRACIL™, DAPD / DXG, D-D4FC, dexelbucitabine, didanosine (VIDEX™), didanosine extended-release (Videx EC), dOTC, EFdA, emtricitabine (EMTRIVA™), emtricitabine / tenofovir alafenamide (DESCOVY®), emtricitabine / tenofovir disoproxil fumarate (TRUVADA®), elvucitabine, fosalvudine, lamivudine / zidovudine (COMBIVIR™), EVIPLERA™, GENVOYA™, HIVID™, KIVEXA™, lamivudine ( In some embodiments, the at least one NRTI is selected from EPIVIR™, LODENOSINE™, ODEFSEY®, PREVEON®, rasibir, stampidine, stavudine (ZERIT™), STRIBILD®, TENOFOVIR™, tenofovir disoproxil fumarate (VIREAD™), TRIUMEQ®, trizivir, VEMLIDY®, and / or zidovudine (RETROVIR™). In some embodiments, the at least one NRTI is censavudine. In some embodiments, the at least one NRTI is elvucitabine.

[0016] In some embodiments, at least one RTI is a non-nucleoside reverse transcriptase inhibitor (NNRTI), hi some embodiments, at least one NNRTI is selected from delavirdine (DLV), efavirenz (EFV), etravirine-nevirapine (NVP), and / or rilvipirin.

[0017] In some embodiments, the patient has Alzheimer's disease, and the method further comprises administering at least one second therapeutic agent useful for treating the symptoms of Alzheimer's disease. In some embodiments, the at least one second therapeutic agent is selected from donepezil, galantamine, memantine, and / or rivastigmine. In some embodiments, the at least one second therapeutic agent is an antibody that binds to β-amyloid or tau protein. In some embodiments, the antibody binds to β-amyloid and is bapineuzumab. In some embodiments, the antibody binds to tau protein and is ABBV-8E12. In some embodiments, the at least one second therapeutic agent is a vaccine against β-amyloid or tau protein. In some embodiments, the at least one second therapeutic agent is an agent that reduces or alters the brain content of β-amyloid or tau. In some embodiments, the second therapeutic agent reduces or alters the brain content of β-amyloid and is a β-secretase 1 (BACE) inhibitor. In some embodiments, the BACE inhibitor is selected from CTS-21166, lanabecestat (AZD3293), LY2886721, and verubecestat (MK-8931). In some embodiments, the second agent reduces or alters brain content of tau and is nicotinamide, or MPT0G211.

[0018] In some embodiments, the patient has ALS, and the method further comprises administering at least one second therapeutic agent useful for treating the symptoms of ALS.In some embodiments, the at least one second therapeutic agent useful for treating ALS is edaravone and / or riluzole.In other embodiments, the at least one second therapeutic agent is an integrase inhibitor. In some embodiments, the integrase inhibitor is selected from aurintricarboxylic acid, derivatives of aurintricarboxylic acid, BMS-538158, caffeic acid phenethyl ester, derivatives of caffeic acid phenethyl ester, curcumin, derivatives of curcumin, chicoric acid, derivatives of chicoric acid, 3,5-dicaffeoylquinic acid, derivatives of 3,5-dicaffeoylquinic acid, GSK364735C, L-870812, and L-25 870810, MK-0518, quercetin, derivatives of quercetin, raltegravir, S-1360, tyrphostin, derivatives of tyrphostin, and / or zintevir (AR-177).

[0019] In some embodiments, the patient is evaluated for one or more symptoms or disease pathology for 1 to 36 months after the patient's first administration of the RTI.

[0020] In some embodiments, the RTI inhibits L1 reverse transcriptase activity in the patient's cells.

[0021] Also provided is a method for preventing the onset of Alzheimer's disease in a patient suspected of having mild cognitive impairment or preclinical Alzheimer's disease, comprising administering a therapeutically effective amount of at least one RTI to a patient in need thereof.

[0022] Other embodiments are also described and listed herein. [The present invention 1001] A method for treating, preventing and / or reversing age-related inflammation in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a reverse transcriptase inhibitor (RTI), wherein the RTI comprises censavudine or elvucitabine. [The present invention 1002] The method of the present invention 1001, wherein aging-associated inflammation is associated with upregulation of long interspersed repeat element 1 (L1), accumulation of cytoplasmic long interspersed repeat element 1 (L1) cDNA, activation of type I interferon (IFN-I) response, and enhancement of the senescence-associated secretory phenotype (SASP) pro-inflammatory state. [The present invention 1003] The method of claim 1002, wherein the composition is administered in an amount sufficient to prevent or reverse upregulation of L1. [The present invention 1004] The method of claim 1002, wherein the composition is administered in an amount sufficient to prevent or reverse the accumulation of cytoplasmic L1 cDNA. [The present invention 1005] The method of claim 1002, wherein the composition is administered in an amount sufficient to prevent or reverse activation of the IFN-I response. [The present invention 1006] The method of claim 1002, wherein the composition is administered in an amount sufficient to prevent or reverse the SASP pro-inflammatory state. [The present invention 1007] Any of the methods of claims 1001-1006, wherein the age-associated inflammation is present in a patient in need of wound healing or tissue regeneration, or a patient with a disease or disorder selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, vision loss, hearing loss, peripheral degenerative disease, or cardiovascular dysfunction, frontotemporal dementia (FTD), multiple sclerosis (MS), Aicardi-Goutieres syndrome, progressive supranuclear palsy (PSP), osteoarthritis, skin aging, atherosclerosis, chemotherapy-induced adverse effects, hematopoietic stem cell function, osteoporosis, physical function, and pulmonary fibrosis. [The present invention 1008] The method of any of claims 1001 to 1006, wherein the age-associated inflammation is present in a patient with Alzheimer's disease. [The present invention 1009] The method of any of claims 1001 to 1006, wherein the age-associated inflammation is present in a patient with ALS. [The present invention 1010] A method for slowing or reversing the progression of the underlying pathology of a disorder caused by age-related inflammation, comprising administering to a patient in need thereof a therapeutically effective amount of an RTI, wherein the RTI comprises censavudine or elvucitabine. [The present invention 1011] The method of claim 1010, wherein said patient has Alzheimer's disease or ALS and experiences a decrease in one or more symptoms of Alzheimer's disease or ALS compared to before the first administration to said patient. [The present invention 1012] The method of claim 1011, wherein the patient has Alzheimer's disease and one or more symptoms comprise memory loss, misplacing things, forgetting to name places or objects, repetitive questioning, decreased flexibility, confusion, disorientation, obsessive-compulsive behavior, compulsive behavior, delusions, aphasia, sleep disturbances, mood swings, depression, anxiety, frustration, agitation, difficulty performing spatial tasks, agnosia, gait disturbances, weight loss, loss of speech, short-term memory loss, or long-term memory loss. [The present invention 1013] 10. The method of claim 10, wherein said patient has Alzheimer's disease and the reduction of one or more symptoms is assessed according to DSM-5. [The present invention 1014] 10. The method of claim 10, wherein said patient has Alzheimer's disease and the reduction in symptoms is determined using the cognitive subscale of Alzheimer's Disease Assessment Scale (ADAS-cog). [The present invention 1015] 10. The method of claim 10, wherein said patient has Alzheimer's disease and symptom reduction is determined using the Clinician's Interview-Based Impression of Change (CIBIC-plus). [The present invention 1016] The method of any one of claims 10 to 11, wherein said patient has Alzheimer's disease and the reduction in symptoms is determined using the Activities of Daily Living Scale (ADL). [The present invention 1017] The method of any of claims 1011 to 1016, wherein any change in the underlying pathology is identified by detection of a biomarker before and after administration of the RTI. [The present invention 1018] 1017. The method of claim 1017, wherein the biomarker is β-amyloid or tau protein. [The present invention 1019] The method of any one of claims 1017 to 1018, wherein the biomarker is detected by PET imaging. [The present invention 1020] The method of any one of claims 1017 to 1018, wherein the biomarker is detected by measurement in cerebrospinal fluid. [The present invention 1021] The method of any of claims 1001 to 1020, further comprising the step of administering to the patient at least one second therapeutic agent. [The present invention 1022] 1022. The method of paragraph 1021, wherein the patient has Alzheimer's disease and the at least one second therapeutic agent is useful in treating a symptom of Alzheimer's disease. [The present invention 1023] 1022. The method of paragraph 1021, wherein the patient has ALS and the at least one second therapeutic agent is useful in treating ALS.

[0023] For illustrative purposes, certain embodiments of the present invention are shown in the drawings described below. Like numerals in the drawings refer to like elements throughout. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and equipment shown. In the drawings: [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 shows the activation of L1, IFN-I, and SASP in senescent cells. Gene expression was assessed by RT-qPCR. Poly(A)-purified RNA was used in all L1 assays. Figure 1a: Time course of L1 activation. P values ​​were calculated relative to the EP, early-passage control. Figure 1b: Schematic of the L1 RT-PCR strategy. Blue, sense; red, antisense (AS). For primer specificity, see Figure 6f-h; for primer design, see Methods. Primers from amplicon F were used in (a) and (e). Figure 1c: Amplicons A-F were used to assess strand-specific L1 transcription. Transcription from the 5'UTR antisense promoter was also detected. SEN(L), late senescence (16 weeks). Figure 1d: Induction of IFN-α and IFN-β1 mRNA levels. Figure 1e. Temporal induction of genes related to DNA damage (p21, also known as CDKN1A), SASP (IL-1β, CCL2, IL-6, MMP3), and IFN-I response (IRF7, IFN-α, IFN-β1, OAS1). Row clustering was calculated as 1-Pearson correlation. RS, replicative senescence; OIS, oncogene-induced senescence (induced by Ha-RAS infection); SIPS, stress-induced premature senescence (gamma-irradiation). Controls: EP, early passage; EV, empty vector-infected; CTR, non-irradiated. (a, c-e), n = 3 independent biological samples, replicated in two independent experiments. (a, c, d) Data are mean ± SD. *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t-test. [Figure 2-1]Figure 2 shows the regulation of L1 activation and IFN-I induction. Figure 2a, RB1 expression and ChIP, and Figure 2b, FOXA1 expression and ChIP. Expression was measured by RT-qPCR and immunoblotting (left panel). Binding to L1 elements was assessed by ChIP-qPCR (right panel). For primer specificity, see Figure 6b. RB1: Primers for 5'UTR, ORF1, and ORF2, amplicons A, E, and F, respectively. FOXA1: Primers for amplicons A–E. qPCR was normalized to input chromatin. SEN(E), premature senescence (8 weeks). For gel source data, see Figure 16. Figure 2c–e, RB1, FOXA1, or TREX1 were overexpressed (OE) or ablated with shRNA, and the effect on L1, IFN-α, and IFN-β1 expression was determined by RT-qPCR of poly(A)-purified RNA. In all cases, lentiviral vectors were used to directly deliver interventions to senescent cells at week 12 (point D, Figure 6a), and cells were harvested for analysis 4 weeks later (point E, week 16). Controls were uninfected senescent cells harvested at the same time (point E, week 16). Two different shRNAs (a, b) were used for each gene. For L1, primers for amplicon F were used. (Figure 2f) RB1 was overexpressed as described above, and its binding to the 5'UTR was assessed by ChIP-qPCR (amplicon A). (Figure 2g) Activation of L1, IFN-α, and IFN-β1 expression after triple (3x) intervention using shRB1 (a), shTREX1 (a), and FOXA1-OE in early-passage cells. Lentiviral infections were performed sequentially with drug selection at each step (shRB1, puromycin → shTREX1; hygromycin → FOXA1-OE, blasticidin). Figure 2h. Expression of IFN-I pathway genes was determined using RT2 Profiler PCR arrays (Qiagen). Normalized mean expression is shown for all 84 genes in the array. Red symbols: significantly upregulated genes. Dashed lines indicate ±2-fold range. (a, b, h), n = 3 independent biological samples, replicated in two independent experiments. (c–g), n = 3 independent experiments. (a–g) Data are mean ± sd. *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t-test. [Figure 2-2] See the description of Figure 2-1. [Figure 3-1]Figure 3 shows that L1 removal attenuates IFN-I activation and blunts the SASP response. Figure 3a: Cells were examined by immunofluorescence (IF) microscopy using antibodies against single-stranded DNA (ssDNA) or L1 ORF1 protein. Note the bright ssDNA dots in senescent cells that colocalized with prominent ORF1 dots. The experiment was repeated three times independently with similar results. Scale bar = 10 μm. Figure 3b: Senescent cells were treated with L1 shRNA (using the lentiviral vector described in Figure 2c, e, f) or 3TC (7.5 μM) between 12 and 16 weeks of senescence. The effect on IFN-I responses was determined by RT-qPCR, ELISA, or immunoblotting. See Figure 16 for gel source data. Figure 3c: Cells were labeled with BrdU for 2 weeks (with or without 7.5 µM 3TC), the labeled DNA was immunoprecipitated, and its L1 sequence content was quantified using a TaqMan multiplex qPCR assay (Figure 1b, amplicon F). EP(qui), early-passage quiescent cells. Figure 3d, left panel: RS cells: IFNAR1 and IFNAR2 genes were mutagenized using the CRISPR / Cas9 system delivered directly into senescent cells with a lentiviral vector. Similar to shRNA intervention, cells were infected at week 12 of senescence and harvested at week 16 of senescence (Figure 1d-f, see "Methods"). Right panel: SIPS cells: CRISPR / Cas9 intervention was performed in early-passage cells, and validated clones were irradiated to induce SIPS. Figure 3e: OIS and SIPS were induced as in Figure 1d, and cells were harvested 20 days later (OIS) or 30 days later (SIPS). 3TC (7.5 μM) was present throughout. IFN-I gene expression (IFN-α, IRF7, OAS1) was measured by RT-qPCR. Figure 3f. Cells were serially passaged to replicative senescence (RS) in the presence of 3TC (10 μM) throughout, and the temporal induction of SASP-responsive genes (IL-1β, CCL2, IL-6, MMP3) was assessed. (b–d, f), n = 3 independent experiments. (e) n = 3 independent biological samples, replicated in two independent experiments. (b–f) Data are mean ± SD. *P ≤ 0.05, **P ≤ 0.01.(b, d–f) Unpaired two-tailed t test, (c) One-way ANOVA with Tukey's multiple comparison test. [Figure 3-2] See the description of Figure 3-1. [Figure 4-1]Figure 4 shows that L1 is activated in mouse tissues with aging and that the IFN-I proinflammatory response is attenuated by RTI treatment. (Figure 4a) The presence of L1 Orf1 protein in tissues was examined by IF microscopy. Quantification of ORF1-expressing cells is shown in the right panel; for each condition, three animals and at least 200 cells per animal were scored. Scale bar = 4 μm. (Figure 4b) L1 activation in senescent cells was examined by co-staining for SA-β-Gal activity and Orf1 protein by IF (male liver, 5 and 26 months old). Scale bar = 4 μm. The experiment was repeated three times independently with similar results. (Figure 4c) Mice were administered 3TC (2 mg / ml) in drinking water for 2 weeks at the indicated ages and sacrificed after treatment. Expression of p16, an IF-responsive gene (IFN-α), and a marker of a proinflammatory state (Il-6) was assessed by RT-qPCR. See Figure 14 for additional tissues and genes. Box plots show the range (whiskers), 25th and 75th percentiles (boxes), mean (dashed line), and median (solid line) of the data. Each point represents one animal. 5 months, n = 8; 26 months, n = 12; 29 months, n = 6. Figure 4d. Six-month-old mice were nonlethally irradiated, and the expression of L1, p16, and representative IFN-I response genes (Ifn-α, Oas1) was assessed by RT-qPCR at the indicated times after irradiation. Graphical representations are the same as in (c); non-irradiated, n = 3 animals at 3 months, n = 5 animals at 6 months; irradiated, n = 4 animals at 3 months, n = 5 animals at 6 months. Figure 4e. Macrophage infiltration into white adipose tissue and kidney was scored as F4 / 80-positive cells (% of total nuclei). n = 5 animals (adipose); n = 8 animals (kidney). Skeletal muscle fiber diameters were measured (see Methods for details) and plotted as clustered boxplots. n = 5 animals per group, 500 fibers in total. Glomerulosclerosis was scored as the sum of all glomeruli with a score of 3 or 4 divided by the total in Periodic Acid-Schiff (PAS)-stained sections (see Methods for details). n = 7 animals per group, 40 glomeruli per animal. Graphical representation as in (c).3TC treatment lasted for 2 weeks in white fat and 6 months (20–26 months) in other tissues. The dashed circle indicates a single glomerulus. Scale bar = 50 μm. Figure 4f. Disruption of L1 surveillance leads to chronic activation of the IFN-I response. ISD: Interferon-stimulated DNA pathway. *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t-test (a, d, e) or one-way ANOVA with Tukey's multiple comparison test (c, e) for white fat. [Figure 4-2] See the description of Figure 4-1. [Figure 5] Figure 5 is a flow chart outlining the molecular pathways of cellular senescence leading to age-associated "sterile" inflammation. [Figure 6-1]Figure 6 shows the establishment of senescent cultures and the analysis of L1 and IFN-I activation. Figure 6a: Passage regimen for obtaining long-term replicative senescent cells (detailed in "Methods"). Point A was designated as time zero for senescence. Figures 6b-d: Confirmation of the senescent status of the cultures. A representative experiment is shown; other experiments were monitored in the same manner and generated data that met these criteria. EP, early passage control; SEN(E), early senescence (8 weeks); SEN(L), late senescence (16 weeks). Figure 6b: Cells were labeled with BrdU for 6 hours. BrdU incorporation and senescence-associated β-galactosidase (SA-β-Gal) activity were determined as indicated. DNA damage foci were visualized using γ-H2AX antibody and immunofluorescence microscopy (IF). Figure 6c: Expression of p21 (CDKN1A) and p16 (CDKN2A) proteins was determined by immunoblotting. GAPDH was used as a loading control. For gel source data, see Figure 16. Figure 6d: Expression of genes characteristic of SASP was measured by RT-qPCR. Figure 6e: L1 activation during senescence of IMR-90 and WI-38 fibroblast cell lines was assessed by RT-qPCR using poly(A)-purified RNA and primers for amplicon F (Figure 1b). Figure 6f: Long-range RT-PCR was performed using primers A-forward and C-reverse (amplicon G) and primers A-forward and D-reverse (amplicon H) (Figure 1b, Table 1), and cDNA was cloned and sequenced. Several attempts using the same protocol with early-passage expanded cells failed to yield L1 clones. Sequences were mapped to an unmasked reference genome requiring 100% identity. Thus, 658 clones could be mapped; 51 additional clones contained at least one mismatch and therefore likely represented elements polymorphic in the cell lines, while 58 were cloning artifacts. Among the 658 mappable clones, 224 unique elements were represented (Table 3). Intact elements are a subset of annotated full-length elements that do not have ORF-inactivating mutations. Feature size corresponds to the number of times the element was represented among the 658 clones.Summary of long-range PCR data presented in Figure 6g, Figure 6f, and Table 3. Figure 6h, Apparent genomic copy numbers of elements detected in our amplicons (see Figure 1b for amplicon locations and Methods for primer design strategy). Prediction: In silico PCR (see Methods for details). Findings: qPCR was performed on 1 ng of genomic DNA and normalized to known single-copy loci. Figure 6i, IFN-α and IFN-β1 gene activation during senescence in WI-38 and IMR-90 cells was measured by RT-qPCR. Figure 6j, Confirmation of the senescence status of cells in OIS (day 20, Figure 6e) and SIPS (day 30, Figure 6e) by SA-β-Gal activity. EV, empty vector control; CTR, non-irradiated cells. Figure 6k, Confirmation of full-length L1 mRNA expression in all forms of senescence using RT-qPCR with primers for amplicons A and F on poly(A)-purified RNA. Delayed activation is shown by comparing days 9 and 20 for OIS and days 12 and 30 for SIPS. (b-e, i-k), n=3 independent biological samples, replicated in two independent experiments. Data are mean ± sd. *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t-test. [Figure 6-2] See the description of Figure 6-1. [Figure 6-3] See the description of Figure 6-1. [Figure 7A-1]Figure 7 shows the mapping of transcription start sites in the L1 element, which is activated during cellular senescence. 5' RACE was performed in late-senescent cells (16 weeks, point D in Figure 6a) using primers C and D (Figure 1a, Table 1). The products were cloned, and individual clones were Sanger sequenced (see Methods for details). In Figure 7a, a multiple sequence alignment of 50 mappable clones to the L1HS consensus was generated using MAFFT software. The L1HS consensus is shown at the top. The blue shading of the aligned clones indicates the degree of identity with the consensus. The green vertical line indicates the start of the L1HS consensus (position 1). The red vertical lines indicate short gaps (1–4 nucleotides) opened in the L1HS consensus by individual clones. The consensus of 50 clones is shown at the bottom and was generated in Jalview. The initiation of L1 transcription is known to be imprecise, with the majority of start sites occurring within + / - 50 bp of the consensus start site, with a subset occurring up to +180 bp. Figure 7b, Summary of mapping data and classification of clones into families of L1 elements. Relative start sites were calculated relative to the L1HS consensus start site. Clones were assigned to L1 families using RepEnrich software. [Figure 7A-2] See the description of Figure 7A-1. [Figure 7A-3] See the description of Figure 7A-1. [Figure 7A-4] See the description of Figure 7A-1. [Figure 7A-5] See the description of Figure 7A-1. [Figure 7A-6] See the description of Figure 7A-1. [Figure 7B] See the description of Figure 7A-1. [Figure 8-1]Figure 8 shows the evolution of transcriptome changes during the progression of cellular senescence. RNA-seq was performed on early proliferating LF1 cells (EP) and on cultures at weeks 8 (SEN-E) and 16 (SEN-L) of senescence (points C and D, respectively, in the extended data in Figure 6a). Data were analyzed using three-way comparisons of EP vs. SEN-E, EP vs. SEN-L, and SEN-E vs. SEN-L (see Methods for details). Figure 8a shows an area-proportional generalized Venn diagram showing the intersection of three comparisons of the following datasets: i-ii, significantly up- and down-regulated genes (row 2x in panel b); iii-iv, significant KEGG pathways identified by GSEA. Note the considerable evolution of the transcriptome in late senescence, exemplified by large changes (especially up-regulation) in differentially expressed genes and pathways; v-vi, significantly changed genes in the IFN-I and SASP gene sets (see Table 4 for gene set annotation). Note that the majority of SASP gene changes occur early, while a large proportion of IFN-I changes are specific to late aging. Figure 8b: Summary of significantly changing genes using a fixed FDR (<0.05) and variable fold-change cutoffs (2x, 1.75x, and 1.5x). Figure 8c: GSEA analysis of KEGG pathways. The heatmap display shows significantly upregulated pathways in red (see also panel e) and downregulated pathways in blue. Nonsignificant comparisons are shown in black; vertical annotations refer to the Venn diagrams in (a, iii-iv). Note that the SASP gene set is upregulated early, while the IFN-I gene set is upregulated late. Figure 8d: Heatmap of significantly changing genes in the IFN-I and SASP gene sets. Vertical annotations refer to the Venn diagrams in (a, v-vi). Figure 8e, List of significantly upregulated KEGG pathways identified using GSEA (see Table 5 for a list of all pathways). NES, normalized enrichment score. IFN-I and SASP gene sets are highlighted in yellow. Note the significant upregulation of IFN-I between early and late senescence. Red type identifies KEGG pathways indicative of cytosolic DNA sensing and type I interferon response at later stages.Figure 8f-g, GSEA profiles of IFN-I and SASP gene sets for all comparisons; FDRs are highlighted in yellow. Note that upregulation of IFN-I is significant for EP_SEN-L and SEN-E_SEN-L, but not for EP_SEN-E, and upregulation of SASP is significant for EP_SEN-E and EP_SEN-L, but not for SEN-E_SEN-L. n = 3 independent biological samples. Differential expression data were analyzed for significance using the GSEA GenePattern interface, and output was corrected for multiple comparisons by adjusting nominal p-values ​​using the Benjamini-Hochberg method (see Methods for details). [Figure 8-2] See the description of Figure 8-1. [Figure 8-3] See the description of Figure 8-1. [Figure 8-4] See the description of Figure 8-1. [Figure 9-1]Figure 9 shows characterization of L1 effectors and IFN-I responses. Figure 9a: TREX1 expression was determined by RT-qPCR and immunoblotting. See Figure 16 for gel source data. Figure 9b: RB family gene expression was compared by RT-qPCR. Primer pairs for all genes were confirmed to be equally efficient. Figure 9c: H3K9me3 and H3K27me3 enrichment at L1 elements was examined by ChIP-qPCR (using PCR primers shown in Figure 1b: 5'UTR, amplicon A; ORF1, amplicon E; ORF2, amplicon F). Figure 9d: ChIP-seq data from ENCODE was examined for transcription factors binding to the L1 consensus sequence. Log2 fold-change enrichment compared to input controls is shown for the indicated cell lines. Binding of YY1 to the L1 promoter has been documented13 and was used as a positive control. CEBPB was used as a negative control. A schematic showing L1 coordinates and associated features is shown above. Amplicons A–E are the same as those shown in Figure 1b. Figure 9e: Transcriptional activity of the intact L1 5'UTR or a UTR lacking the FOXA1 binding site (UTR-Δ) was determined using sense and antisense reporters co-transfected with either a FOXA1 expression plasmid or an empty vector (EV) into early-passage LF1 cells. Figure 9f: FOXA1 was knocked down with shFOXA1(a) in senescent cells (see also Figure 2e and Figure 10a), and binding to the L1 5'UTR (amplicon B) was determined by ChIP-qPCR. Figure 9g: Knockdown of RB1 and TREX1 and ectopic expression of FOXA1 were performed in early passage cells in all single (1X), double (2X), and triple (3X) combinations, and activation of L1, IFN-α, and IFN-β1 expression was assessed by RT-qPCR using poly(A)-purified RNA (primers for amplicon F). Three controls are shown: cells infected with an irrelevant shRNA (shGFP), an expression construct (LacZ), or uninfected early passage cells (EP). Figure 9h: L1 5'UTR occupancy of RB1 and FOXA1 in 3X cells was determined by ChIP-qPCR performed as in Figure 2a, b.Primers for amplicons A and B were used for RB1 and FOXA1, respectively. For comparison, single interventions in early-passage cells with shRB1 (a) or FOXA1 cDNA expression (EP FOXA1-OE) are also shown. Figure 9i: Confirmation of full-length L1 mRNA expression in 3X cells using RT-qPCR with primers for amplicons A and F on poly(A)-purified RNA. CTR, cells infected with an unrelated shRNA (shGFP). Figure 9j: Heatmap display showing all biological replicates for 67 genes whose expression significantly changes in SEN and / or 3X cells (Figure 2h, Table 6). Column clustering was calculated as 1-Pearson correlation. Rows are grouped into functional subsets of the IFN-I response. Figure 9k: Venn diagram showing the overlap among the 67 significantly changed genes. (a-f, h) n = 3 independent biological samples, replicated in two independent experiments. (g, i) n = 3 independent experiments. (a-i) Data are means ± sd. *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t-test. [Figure 9-2] See the description of Figure 9-1. [Figure 9-3] See the description of Figure 9-1. [Figure 10-1]Figure 10 shows the efficacy of genetic and pharmacological interventions. Figure 10a: Knockdown with two different shRNAs (a, b) or Figure 10b: Ectopic cDNA expression was performed in senescent cells as described in Figure 2d, e, and g (see also "Methods"). The efficacy of these manipulations on their targets was assessed by RT-qPCR and immunoblotting. For gel source data, see Figure 16. Figure 10c: mRNA and protein expression of RB1, TREX1, and FOXA1 after triple (3x) intervention (Figure 2f). Figure 10d: The effect of 3TC treatment on the relative abundance of L1HS sequences in senescent cells was determined by multiplex TaqMan qPCR on total DNA (primer set 6, Table 1). SEN onset, week 0 of senescence (Figure 1a; point A in Figure 6a). 3TC was administered continuously from the onset of SEN until harvest at 16 weeks. Figure 10e: The effect of 3TC administration on retrotransposition was determined using a dual-luciferase L1 reporter system. The L1 reporter was introduced into early-passage cells using a lentiviral vector (see "Methods" for details). Cells were treated with 3TC for 4 days, then harvested and assayed. JM111, a defective reporter carrying a mutation in ORF1 (absence of 3TC); L1RP, a retrotransposition-competent reporter. Figure 10f: The effect of 3TC administration on IFN-I responses. Experiment (d) above was processed by RT-qPCR to determine the expression of IFN-α and IFN-β1. Figure 10g: Knockdown of L1 was performed using two different shRNAs (a, b) in senescent cells (as in Figure 2d, e, g) or 3X cells (as in Figure 2g). The efficacy on L1 expression was assessed by RT-qPCR using poly(A)-purified RNA and primer F. Cells from the experiment in Figure 10h, (g) were examined for ORF1 protein levels by immunofluorescence (IF). Image analysis was performed using CellProfiler software (see Methods for details). >200 cells were examined for each condition (afu, arbitrary fluorescence units). L1 shRNA treatment in the experiment in Figure 10i, (g) was replaced with 3TC treatment (10 μM) for the same period. Figure 10j: Five different RTIs (or combinations) were tested for their effect on IFN-I responses.AZT (zidovudine, 15 μM), ABC (abacavir, 15 μM), FTC (emtricitabine, 10 μM), 3TC (also known as lamivudine or epivir, 10 μM), and TZV (trizivir, a combination of 15 μM AZT, 15 μM ABC, and 7.5 μM 3TC). Cells were treated for 4 weeks between weeks 12 and 16 of senescence (Figure 1a; points D and E in Figure 1a). 48 hours after completion of the last drug selection, 3X cells (Figure 2f) were treated with 3TC. IFN-α expression was measured by RT-qPCR. In Figure 10k, the native L1 reporter (pLD143) was cotransfected with shRNA plasmid vectors into HeLa cells (see Methods for details). Retrotransposition was scored as GFP-positive cells, and shL1 knockdown was normalized to the shLuc negative control. The absolute mean retrotransposition frequency (percentage of GFP-positive cells) was 4.1, consistent with the published value of 53 for the reporter used (pLD143). (Figure 10l) Knockdown of cGAS and STING was performed in senescent or 3X cells, similar to other shRNAs (Figures 2d, e, g and a, g above). (Figure 10m) Downregulation of interferon signaling after CRISPR-mediated inactivation of the IFNAR1 and IFNAR2 genes was verified by the absence of IRF9 nuclear translocation and STAT2 phosphorylation in response to interferon stimulation. Cells were infected with a lentiviral vector expressing Cas9 and gRNAs against both IFNAR1 and IFNAR2 (ΔIFNAR, see "Methods"). After infection, cells were replated on glass coverslips, treated with interferon for 2 hours, and examined by IF microscopy. Experiments were repeated three times with similar results. (a-i, l) n = 3 independent experiments. (k) n = 3 independent biological samples, replicated in two independent experiments. (a–l) Data are mean ± sd. *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t-test. [Figure 10-2] See the description of Figure 10-1. [Figure 11-1]Figure 11 shows the characterization of cytoplasmic DNA in senescent cells. In Figure 11a, quiescent and senescent cells were treated with BrdU as in Figure 3a, and the cellular localization of BrdU incorporation was visualized by IF microscopy. Proliferating cells, EP (Prol), are shown as a positive control for nuclear BrdU incorporation. Signal was quantified using CellProfiler software (right panel, see "Methods"). >200 cells were examined for each condition (afu, arbitrary fluorescence units). In Figure 11b, senescent (and EP control) cells (neither of which were labeled with BrdU) were fractionated into nuclear and cytoplasmic fractions, and the expression of L1 sequences within these compartments (as well as whole cells) was assessed by qPCR as in Figure 3a (TaqMan Multiplex qPCR Assay 16, Amplicon F, Figure 1b). Note that the units on the y-axis differ by 10-fold between the left and right panels. Figure 11c. Cells were examined by IF microscopy for the presence of ORF1 protein, RNA-DNA hybrids, and single-stranded DNA (ssDNA). For antibodies, see "Methods" and Table 2. The RNA-DNA signal in senescent cells largely colocalized with the ORF1 signal and was lost after RNase A treatment. The ssDNA signal also colocalized with the ORF1 signal and was unmasked by RNase A treatment. The experiment was repeated three times with similar results. Figure 11d. Pulled-down BrdU-containing DNA (Figure 3c, panel (a) above, see "Methods") was cloned and Sanger sequenced. Of the 96 total clones examined, 37 mapped to L1. The red boxes represent the relative positions of these clones on the L1 consensus sequence. Figure 11e: BrdU-labeled senescent cells (Figure 3c, panel (a) above) were immunoprecipitated with anti-BrdU antibodies, and the expression of L1 sequences in the pulled-down DNA was assessed using qPCR with primers spanning the entire L1 element (Figure 1b, c). Figure 11f: Senescent cells were treated with L1 shRNA (using a lentiviral vector as described in Figure 10g) between 12 and 16 weeks of senescence, and the expression of SASP genes was measured. Figure 11g: Transcription of the entire mouse L1 element was assessed in a strand-specific manner using the same strategy applied to the human L1 element (Figure 1b, c).Amplicons (designated W–Z to distinguish from human-specific primers) correspond to the 5'UTR (W), Orf1 (X), Orf2 (Y), and 3'UTR (Z). See also "Methods" and Table 1 for primer sequences (primer sets 37, 48–50). Poly(A) RNA was prepared from male white adipose tissue. A total of 12 animals (three pools of four animals each) were evaluated in three independent experiments. Figure 11h, Expression of three currently active families of mouse L1 elements. Primers were designed to distinguish between 5'UTR polymorphisms in the MdA, MdN, and Tf families (see "Methods," primer sets 51–53 in Table 1). RT-qPCR was performed as in (f) above (non-strand-specific). (a, b, e), n = 3 independent biological samples, replicated in two independent experiments. (f), n = 3 independent experiments. (a, e–h) Data are means ± SD. *P≦0.05, **P≦0.01. (a) One-way ANOVA with Tukey's multiple comparison test, (b, e–h) unpaired two-tailed t test. [Figure 11-2] See the description of Figure 11-1. [Figure 12-1]Figure 12 shows the effects of ablation of L1 activation, the cytoplasmic DNA sensing pathway, or interferon signaling on the expression of IFN-I and SASP responses. 3X cells were treated with L1 shRNA or 3TC for 48 hours as described in Figure 12a, Figure 10g, and Figure 10i. Effects on IFN-I responses were determined by RT-qPCR, ELISA, or immunoblotting. For gel source data, see Figure 16. In Figure 12b, cells were serially passaged into replicative senescence (RS) in the presence of 3TC (10 μM) throughout as in Figure 3f, and expression of Cdk inhibitors p21 and p16 was assessed by RT-qPCR. In Figure 12c, senescent cells were treated with shRNA against cGAS or STING (as described in Figure 10l) between weeks 12 and 16 of senescence, and expression of IFN-I response genes (IFN-α, IRF7, OAS1) was measured. In Figure 12d, cGAS and STING knockdown was performed in 3X cells using shRNA (as in panel (c) above), and the expression of IFN-I genes was examined by RT-qPCR. In Figure 12e, cGAS and STING were knocked down in senescent cells using shRNA (as in panel (c) above), and the expression of SASP-responsive genes (IL-1β, CCL2, IL-6, MMP3) was assayed by RT-qPCR. In Figures 12f and 12g, the activity of K-9 was compared to 3TC in senescent and 3X cells. Senescent cultures were treated for 12 to 16 weeks (as in Figure 3b), and 3X cultures were treated for 48 hours (as in panel (a) above). The effects on the expression of IFN-I genes (IFN-α, IRF7, OAS1) and SASP genes (IL-1β, IL-6, MMP3) were assessed by RT-qPCR. (a–g), n = 3 independent experiments. (a–g) Data are means ± sd. *P ≤ 0.05, **P ≤ 0.01, unpaired two-tailed t test. [Figure 12-2] See the description of Figure 12-1. [Figure 13-1]Figure 13 shows the evaluation of p16, L1 ORF1, and pSTAT1 expression in skin specimens from senescent cells and aged humans. Figure 13a: Immunofluorescence (IF) detection of p16 and ORF1 in early-passage, 3X, and senescent cells. Figure 13b: Representative images of combinatorial ORF1 and p16 or ORF1p and pSTAT1 staining in human dermis. The experiments shown in panels (a, b) were independently repeated three times with similar results. Figure 13c: Cells were plated on coverslips, stained, and quantified as described in "Methods." 200 cells in multiple fields were scored for each condition. afu, arbitrary fluorescence units. Insets show the % of cells found in each quadrant. Figure 13d, e: Abundance of ORF1 and p16 or pSTAT1 cells in human skin. Skin biopsies were cryosectioned and stained as described in "Methods." 200 dermal fibroblasts in multiple fields were scored for each subject. Collective data from four subjects (800 cells) are shown. Data from Figure 13f, (c), and (d) were recalculated to show the relative abundance of p16+ cells among all cells and ORF1+ cells among the p16+ pool of cells. Data from Figure 13g, (e) were recalculated as in (f). Figure 13h, Characteristics of human subjects used for analysis of skin fibroblasts. These specimens were collected as part of the ongoing Leiden Longevity Study. The specimens used herein were randomly selected from the remaining material. The TIF assay relies on two-parameter (color) visualization of telomeres (using FISH probes) and immunofluorescence detection of DNA damage foci (using antibodies against 53BP1). Due to limited material, it was not possible to combine p16 detection with TIF in a three-color experiment. [Figure 13-2] See the description of Figure 13-1. [Figure 14-1]Figure 14 shows the effects of 3TC or K-9 treatment on L1, p16, IFN-I, and SASP gene expression in mouse tissues. Mice at the indicated ages were treated with 3TC for two consecutive weeks (Figures 14a-c, 4c, 4e, 15d-f, and see also "Methods"). For all conditions, expression of L1 mRNA, p16, three representative IFN-I-responsive genes (Ifn-α, Irf7, Oas1), and three representative SASP genes (Il-6, Mmp3, Pai1) was assessed by RT-qPCR. Expression at 5 months of 3TC was not significantly different from drug-free controls; therefore, these data are not shown in the figures (see Table 7 for all collected data). Box plots show the range (whiskers), 25th and 75th percentiles (boxes), mean (dashed lines), and median (solid lines) of the data. Each point represents one animal. Figure 14a, Visceral white fat, male mice. 5 months, n = 8 animals; 26 months, n = 12 animals; 26 months + 3TC, n = 12 animals. Figure 14b, Visceral white fat, female mice. 5 months, n = 8 animals; 26 months, n = 12 animals; 26 months + 3TC, n = 12 animals. Figure 14c, Liver, male mice. 5 months, n = 8 animals; 26 months, n = 10 animals; 26 months + 3TC, n = 10 animals. Figure 14d, 26-month-old mice were treated with K-9 or 3TC in drinking water for 2 weeks and analyzed by RT-qPCR as described above. NT, untreated. Visceral white fat, male mice, n = 7 animals per group. Data are mean ± sd. *P ≤ 0.05, **P ≤ 0.01, one-way ANOVA with Tukey's multiple comparison test. [Figure 14-2] See the description of Figure 14-1. [Figure 15-1]Figure 15 shows the combinatorial evaluation of senescence, IFN-I, SASP, and L1 markers, and the effect of 3TC on aging-related phenotypes in mouse tissues. Figure 15a, b. Whole-mount IF was performed on white fat of 5- and 26-month-old male mice (with and without 2-week 3TC treatment). In (a), loss of lamin B1 (a marker for senescence) colocalized with IL-6 (a marker for SASP). In (b), pStat1 (an IFN-I marker) colocalized with Orf1 (a marker for L1). Figure 15c. Quantification of the experiments shown in (a) and (b). For each condition, four animals and at least 200 cells per animal were scored. Figure 15d. Neutral lipids were stained with BODIPY to visualize mature adipocytes in whole-mount preparations, and macrophages were detected by IF using the F4 / 80 antibody. Figure 15e. The effect of 2 weeks of 3TC treatment on adipogenesis was assessed by measuring mean adipocyte size (left panel) and by RT-qPCR to determine the expression of key adipogenic genes (right panel; Acaca, acetyl-CoA carboxylase 1; Cebpa, CCAAT / enhancer-binding protein alpha; Fasn, fatty acid synthase; Srebp1, sterol regulatory element-binding protein 1). Boxplots show the range (whiskers), 25th and 75th percentiles (boxes), mean (dashed lines), and median (solid lines) of the data. Adipocyte size (BODIPY-stained area) was calculated using CellProfiler and shows aggregate data from five animals and 500 total cells. For RT-qPCR data, each point represents one animal; n = 6 animals. Figure 15f. Expression of the Ucp1 gene (thermogenin) in brown adipose tissue was determined by RT-qPCR and is represented as in (e). n = 5 animals. Figure 15g. L1 mRNA expression was determined by RT-qPCR and is represented as in (e). 5 months, n = 8 animals; 26 months, n = 12 animals; 29 months, n = 6 animals. (e-g) Data are mean ± SD. *P ≤ 0.05, **P ≤ 0.01. (c, e left panels, f, g) One-way ANOVA with Tukey's multiple comparison test; (e right panel) Unpaired two-tailed t-test. [Figure 15-2] See the description of Figure 15-1. [Figure 16-1] Figure 16 shows scans of raw immunoblots. Figure 16A: Panel a shows blot 1 - RB1:1.EP; 2.SEN(L); 3.OE-SEN; 4.SEN(E); Panel b shows blot 2 - TREX1:1.EP; 2.SEN(E); 3.SEN(L); and Panel c shows blot 3 - FOXA1:1.EP; 2.STOP; 3.SEN(E); 4.SEN(L). Figure 16B: Panel d shows blot 4 - RB1:1.EP; 2.3X; Panel e shows blot 5 - TREX1:1.EP; 2.3X; and Panel f shows blot 6 - FOXA1:1.EP; 2.3X. Figure 16C: Panel g shows blot 7 - RB1: 1.SEN(L); 2.shRB1(b); 3.shRB1(a); 4.OE-RB1; panel h shows blot 8 - TREX1: 1.SEN(L); 2.shTREX1(b); 3.shTREX1(a); 4.OE-TREX1; and panel i shows blot 9 - FOXA1: 1.shFOXA1(a); 2.shFOXA1(b); 3.SEN(L); 4.OE-FOXA1. Figure 16D: Panel j shows blot 10 - STAT2:1.3X;2.shL1;3.NRTI;4.ΔIFNAR;5.EP; panel k shows blot 11 - IRF7:1.EP;2.shL1;3.3X;4.shL1;5.ΔIFNAR; panel 1 shows blot 12 - STAT2:1.SEN(L);2.ΔIFNAR;3.shL1;4.NRTI; and panel m shows blot 13 - IRF7:5.SEN(L);6.ΔIFNAR;7.shL1;8.NRTI. Figure 16E: Panel n shows blot 14-p16(CDKN2A):1.EP;2.SEN(E);3.SEN(L); and panel o shows blot 15-p21(CDKN1A):1.EP;2.SEN(E);3.SEN(L). [Figure 16-2] See the description of Figure 16-1. [Figure 16-3] See the description of Figure 16-1. [Figure 16-4] See the description of Figure 16-1. [Figure 16-5] See the description of Figure 16-1. [Figure 17] Figure 17 shows the effects of adefovir and lamivudine on senescence-induced increases in L1 sequence abundance, interferon gene expression, and SASP gene expression. Figure 17a shows the effect of 5 μM adefovir and lamivudine on L1 sequence abundance (copy number) in three different human fibroblast cell lines: LF1, IMR90, and WI38, using a qPCR assay. Figure 17b shows the effect of 5 μM adefovir and lamivudine on interferon gene expression of two interferon genes (IFN-α and IFN-β1) in two cell lines (LF1 and IMR90). Figure 17c shows the effect of 5 μM adefovir and lamivudine on two SASP genes (IL-6 and MMP3) in the LF1 cell line. FIG. 17d shows the effect of higher doses of adefovir and lamivudine (10 μM and 50 μM) on interferon gene expression (IFN-α and IFN-β1) in the LF1 cell line. [Figure 18A] Figure 18 shows the inhibition of mouse L1 activity by eight RTI compounds: lamivudine (3TC); stavudine; emtricitabine; apricitabine; tenofir disiproxil; censavudine; elvucitabine; and tenofir. In the first experiment (Figure 18A) and a second independent experiment (Figure 18B), dose responses for the retrotransposition activity of active mouse LINE-1 were obtained using HeLa cells. [Figure 18B] See the description of Figure 18A. [Figure 19] Figure 19 shows the inhibition of human L1 activity by three RTI compounds: lamivudine (3TC), censavudine, and elvucitabine. In the first experiment using lamivudine (3TC), censavudine, and elvucitabine (Figure 19A), and in the second experiment using lamivudine (3TC) and elvucitabine (Figure 19B), dose responses for the retrotransposition activity of active human LINE-1 were obtained using HeLa cells. [Figure 20]FIG. 20 shows the cell viability of HeLa cells after treatment with 10 different RTI compounds: lamivudine (3TC); stavudine; emtricitabine; apricitabine; tenofil disiproxil; censavudine; elvucitabine; tenofil; and staurosporine. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention It should be understood that certain aspects, methods, embodiments, modifications and features of the invention are described below in varying levels of detail in order to provide a substantial understanding of the invention.

[0026] The following description of specific aspect(s) is merely exemplary in nature and is in no way intended to limit the scope of the invention, its application, or uses, which may, of course, vary. The present invention is described in conjunction with non-limiting definitions and terms contained herein. These definitions and terms are not designed to serve as limitations on the scope or practice of the invention, but are presented for illustrative and descriptive purposes only. Although compositions or processes are described as using specific materials or sequences of individual steps, it is understood that the materials or steps may be interchangeable, such that the description of the invention may include multiple parts or steps arranged in numerous ways, as readily understood by one skilled in the art.

[0027] definition Definitions of certain terms used in the specification and appended claims are provided below: Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0029] The term "approximately" or "about" with respect to a value or parameter is generally interpreted as including numbers that fall within 5%, 10%, 15%, or 20% of the number in either direction (greater or less), unless otherwise specified or clear from the context (except where such number is less than 0% or more than 100% of a possible value). As used herein, a reference to "approximately" or "about" a value or parameter includes (and describes) aspects related to that value or parameter. For example, a description that refers to "about X" includes a description of "X."

[0030] As used herein, the term "or" means "and / or." As used herein in phrases such as "A and / or B," the term "and / or" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0031] Whenever an embodiment is described herein with the language "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, whenever an embodiment is described herein with the language "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[0032] It should be understood that certain features of the invention that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Furthermore, references to values ​​stated in ranges include each and every value within that range.

[0033] The term "subject" refers to a mammal, including, but not limited to, a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. A subject can be, but is not limited to, a household pet (e.g., dog, cat), agricultural livestock (e.g., cow, horse, pig, chicken, etc.), or laboratory animal (e.g., mouse, rat, rabbit, etc.). A subject includes a human subject. A human subject can be a pediatric subject, an adult subject, or an elderly subject. A human subject can be of either gender.

[0034] The terms "effective amount" and "therapeutically effective amount" include an amount sufficient to prevent or ameliorate the manifestations of a disease or medical condition, such as an age-related disorder. It will be understood that there are many methods known in the art for determining the effective amount for a given application. For example, pharmacological methods for determining dosage can be used in therapeutic situations. In the context of therapeutic or prophylactic applications, the amount of the composition administered to a subject depends on the type and severity of the disease and the subject's characteristics, such as general health, age, sex, weight, and tolerance to drugs. It also depends on the extent, severity, and type of disease. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds.

[0035] As used herein, the terms "treating" or "treatment" or "treat" or "palliating" or "palliating" refer to both (1) therapeutic measures that cure, slow, reduce symptoms, and / or halt the progression of a diagnosed disease or infection, and (2) prophylactic or preventative measures that prevent or delay the onset of a disease or infection.

[0036] As used herein, the term "long-term" administration means that therapeutic agent or drug is administered for a period of at least 12 weeks.This includes that therapeutic agent or drug is administered to be effective for more than at least 12 weeks or for at least 12 weeks, and does not necessarily mean that administration itself is carried out for 12 weeks, for example, when sustained-release composition or long-acting therapeutic agent or drug is used.Therefore, subject is treated for a period of at least 12 weeks.In many cases, long-term administration is at least 4, 5, 6, 7, 8, 9 months or more, or at least 1, 2, 3, 5, 7, or 10 years or more.

[0037] As used herein, the term "age-related inflammation" (or "age-associated inflammation") refers to inflammation, typically chronic, particularly chronic systemic inflammation that occurs with aging. Such inflammation can be observed in subjects over the age of 30, 35, or 40, but is typically found in subjects aged 45, 50, 55, or 60 or older. In many cases, this can be low-level inflammation.

[0038] As used herein, the term "chronic inflammation" refers to persistent or prolonged inflammation (e.g., an inflammatory condition) in a subject's body. Generally speaking, this refers to an inflammatory response or condition that lasts for 20, 25, or 30 days or more, or 1 month or more, more specifically, at least 2 or 3 months or more. Chronic inflammation results in progressive changes in the types of cells present at the site of inflammation. Chronic inflammation can be a contributing factor to the development of many diseases or disorders, including degenerative diseases, or diseases or conditions associated with the loss of youthful function or aging.

[0039] As used herein, the term "systemic inflammation" refers to inflammation that is not limited to a particular tissue or site or location in the body. Inflammation can spread throughout the body. Systemic inflammation typically involves the endothelium and other organ systems.

[0040] As used herein, the term "low-level inflammation" (which term is used synonymously herein with "low-grade inflammation") is characterized by a two- to three-fold increase in systemic concentrations of cytokines such as TNFα, IL-6, and CRP, for example, as measured in plasma or serum. The increase can be relative to or compared to a normal or reference concentration, such as a concentration determined in a particular reference cohort or population of subjects, e.g., young subjects (e.g., young adults) or healthy subjects, e.g., subjects not afflicted with a disease or condition, including an inflammatory disease, or subjects without inflammation. The increase can also be relative to the level of the concentration in a subject prior to the onset of inflammation. Low-level inflammation can be observed in the absence of overt signs or symptoms of disease. Thus, low-level inflammation can be subclinical inflammation. Alternatively, a subject with low-level inflammation may not have a clinically diagnosed condition or disease but may exhibit certain signs or symptoms of an inflammatory response or condition. In other words, signs or symptoms of the effects of inflammation may be present in the body, but this may not yet progress to overt or recognized disease.

[0041] As used herein, the term "cancer inflammation" refers to inflammation that occurs in the context of cancer, and can alternatively be defined as "cancer-related inflammation." Inflammation has been identified as a characteristic of cancer, and may be necessary for tumor formation and the maintenance of cancerous conditions. Cancer symptoms are associated with inflammation. Thus, subjects with cancer may have or exhibit inflammation, which may be low-level or peripheral inflammation as discussed above, or particularly chronic or systemic inflammation as discussed above.

[0042] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of the relevant art and this disclosure, and it is further understood that they should not be interpreted in an idealized or overly formal sense unless explicitly defined as such in this specification.

[0043] Pharmaceutical Compositions The compositions and methods of the present invention can be used to treat individuals in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, for example, comprising the compound of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are intended for administration to humans, particularly for invasive administration routes (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. The excipient can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition can be in the form of a dosage unit such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized preparations for reconstitution, powders, liquids, syrups, suppositories, injections, etc. The composition can also be present in a transdermal delivery system, such as a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0044] Pharmaceutically acceptable carriers can include physiologically acceptable agents that act to stabilize, increase the solubility, or enhance the absorption of compounds such as the compounds of the present invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low-molecular-weight proteins; or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying or self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be, for example, a liposome or other polymer matrix into which the compounds of the present invention can be incorporated. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to prepare and administer.

[0045] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0046] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and corn starch. (10) oils such as soybean oil and corn oil; (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer; and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

[0047] The pharmaceutical compositions (preparations) can be administered to a subject by any of several routes of administration, including, for example, orally (e.g., drenches such as aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment, or spray applied to the skin). The compounds can also be formulated for inhalation. In certain embodiments, the compounds can be simply dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Patent Nos. 6,110,973; 5,763,493; 5,731,000; 5,541,231; 5,427,798; 5,358,970; and 4,172,896, and the patents cited therein.

[0048] The formulations can be conveniently provided in unit dosage form and can be prepared by any method known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0049] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0050] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilized preparations, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each containing a predetermined amount of a compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0051] To prepare solid dosage forms for oral administration (such as capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) Disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. For capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0052] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersants. Molded tablets can be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0053] Tablets, as well as other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents and can be compositions that release the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0054] The liquid dosage form useful for oral administration includes pharmaceutically acceptable emulsion, freeze-dried preparation for reconstitution, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form can contain the inert diluent commonly used in the art, such as water or other solvent, cyclodextrin and its derivatives, solubilizer and emulsifier, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and their mixtures.

[0055] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0056] Suspensions may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0057] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any preservatives, buffers, or propellants that may be required.

[0058] The ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0059] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0060] Transdermal patch has the additional advantage of providing controlled delivery of the compound of the present invention to the body.Such dosage forms can be prepared by dissolving or dispersing active compound in suitable medium.Absorption enhancers can also be used to increase the flux of compound across the skin.The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing compound in a polymer matrix or gel.

[0061] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intraocular (including intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0062] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0063] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0064] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of drugs.This can be achieved by using a liquid suspension of crystalline or amorphous materials with poor water solubility.The absorption rate of a drug depends on its dissolution rate, which in turn depends on the crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0065] Injectable depot forms are made by forming microencapsulated matrices of the target compound in biodegradable polymers such as polylactide-polyglycolide. The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0066] For use in the methods of the present invention, the active compound can be provided per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0067] The method of introduction can also be provided by a rechargeable device or a biodegradable device.For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained release polymer devices have been developed and tested in vivo in recent years.Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release of compounds at specific target sites.

[0068] Actual dosage levels of the active ingredient in the pharmaceutical compositions may be varied to provide an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient.

[0069] The selected dosage level will depend on a variety of factors, including the activity of the particular compound or combination of compounds, or esters, salts, or amides thereof, used, the route of administration, the time of administration, the rate of excretion of the particular compound(s) used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound(s) used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0070] A physician or veterinarian skilled in the art can easily determine and prescribe the required therapeutically effective amount of the pharmaceutical composition. For example, a physician or veterinarian can start the dosage of the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of the compound sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, other types of therapeutic agents administered together with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art. 18 .

[0071] In general, a suitable daily dose of an active compound used in the compositions and methods of the present invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0072] If desired, the effective daily dose of the active compound can be administered as one, two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In certain embodiments of the invention, the active compound can be administered two or three times a day. In other embodiments, the active compound is administered once a day.

[0073] Patients receiving this treatment may be any animal in need, including primates, particularly humans, as well as other mammals such as horses, cows, pigs, sheep, cats, and dogs, poultry, and pets in general.

[0074] In certain embodiments, the compounds of the present invention can be used alone or can be administered together with other types of therapeutic agents.

[0075] The present disclosure includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the present invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, L-arginine, benethamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.In certain embodiments, contemplated salts of the present invention include 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid , d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0076] Pharmaceutically acceptable acid addition salts may also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0077] Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives and antioxidants can also be present in the composition.

[0078] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0079] The role of cellular senescence in aging and age-related diseases Figure 5 provides a flow chart outlining the molecular pathways of cellular senescence leading to age-associated sterile inflammation, which are described in more detail below.

[0080] Upregulation of retrotransposable elements (RTEs) RTE is a senescent cell 19 and several mouse tissues 20 Three regulatory factors have previously been shown to be involved in the regulation of L1: FOXA1, RB, and TREX1. FOXA1 is upregulated in senescent cells. 21 , binds to the L1 promoter 22 It has been reported that RB represses L1 elements. 23 It has been reported that complete loss of TREX1 (germline deletion) causes an autoimmune disease (Aicardi-Goutières syndrome, AGS) associated with L1 activation. 24 .

[0081] Although these three regulatory factors have been reported to have roles in regulating L1, the data in this disclosure provide the first demonstration that misregulation of these three factors in combination is sufficient to enable activation of endogenous L1 elements in normal cells (see Example 2). Furthermore, this disclosure provides the first description of late senescence as a distinct, temporally distinct, previously unknown stage of senescence, and late senescence characterized by upregulation of L1 elements and induction of an IFN-I response (see Example 1).

[0082] Accumulation of cytoplasmic L1 cDNA The existence of cytoplasmic L1 cDNA is known. One source has been shown to be mitochondrial. 25 It has been reported that the nucleus "leaks" chromosomal DNA into the cytoplasm in senescent cells. 26 These were subsequently named "cytoplasmic chromatin fragments" (CCFs). 27 , which was later described in senescent cells 28 However, none of these reports mention RTE and L1 as components of CCF.

[0083] The data disclosed herein not only show that L1 DNA sequences are found in the cytoplasm of senescent cells, but also that they are enriched relative to nuclear DNA sequences (see Example 3). Therefore, the previously reported simple extrusion of bulk chromosomal DNA from the nucleus to the cytoplasm cannot explain the enrichment of L1 sequences observed here.

[0084] L1 DNA is enriched in TREX1 null cells (AGS). 29 It was previously reported that L1 cDNA accumulated in TREX1 knockout cells was localized in the cytoplasm. 30 In contrast, AGS in rare autoimmune diseases is not age-related. In contrast, the data disclosed here generalize the presence of cytoplasmic L1 DNA to cellular senescence, one of the major drivers of aging and age-related diseases.

[0085] Induction of IFN-I and crosstalk with the immune system, enhancing SASP It has previously been reported that cytoplasmic DNA (and in particular CCF) is recognized by the cGAS / STING sensor pathway, thereby promoting an inflammatory state. 31 In the context of cellular senescence, this pro-inflammatory state is known as the senescence-associated secretory phenotype (SASP). These reports demonstrated that the SASP is at least partially dependent on CCFs, as knockdown of cGAS / STING pathway components reduced the SASP. The IFN-I response was also reported to be part of this pro-inflammatory cascade. 32 As mentioned above, none of these previous reports implicated L1 elements in the promotion of SASP by CCF.

[0086] The data disclosed herein demonstrate that L1 DNA not only comprises a significant proportion of CCFs enriched in cytoplasmic DNA compared with nuclear sequences, but also functionally correlates with the promotion of SASP. In particular, the data disclosed herein demonstrate that reducing the amount of cytoplasmic L1 DNA by shRNA against L1 or by blocking L1 reverse transcription with RTI drugs reduced both the IFN-I response and SASP in senescent cells. Importantly, the data disclosed herein represent the first evidence that RTI treatment can effectively reverse both IFN-I and the proinflammatory SASP after they have fully established in senescent cells. See Examples 3 and 4.

[0087] All previous therapeutically relevant studies have been limited to interfering with the cGAS / STING sensor pathway; for example, shRNAs against components of the cGAS / STING pathway have shown to downregulate IFN-I responses and SASP. 33,34 Although it may be envisioned to use small molecule inhibitors of the cGAS / STING pathway to downregulate SASP, such treatment results in increased susceptibility to viral, bacterial, and other pathogen infections.

[0088] The present approach of targeting L1 DNA synthesis with RTI drugs provides a fundamental solution to the problem, as it targets the primary causative agent (L1 DNA itself) as opposed to downstream processing events such as the cGAS / STING sensor pathway or even downstream interferon or immune signaling components. The present invention recognizes that all of these downstream components have essential cellular functions, and therefore targeting them will, in some respects, impair normal physiological processes. On the other hand, L1 DNA is a unique "non-self" component, and its pharmacological targeting is only compromised by "off-target" effects.

[0089] In recent years, it has become clear that cellular senescence is one of the major drivers of organismal aging and age-related diseases. 35 Therefore, there has been considerable interest in "senotherapy" to block the deleterious effects of senescent cells. 36 Major efforts have been directed toward "senolytic" drugs that selectively kill (and thus remove) senescent cells from tissues. "Senomorphics" are classified as small molecules that suppress the senescent phenotype without killing cells. We choose to refer to such drugs as "senostatic" drugs to emphasize that their primary effect is to arrest or block the deleterious effects of senescent cells, particularly the SASP.

[0090] The data disclosed herein demonstrate that RTIs are senostatic drugs that reverse the SASP of senescent cells and thus alleviate age-related pro-inflammatory conditions in human cells and mouse models. The data disclosed herein also explain which specific RTIs and which doses are particularly effective in reversing SASP. The broad effectiveness of RTIs as senostatic drugs that can treat multiple age-related conditions has not been previously described in the art.

[0091] Promoting age-related "sterile" inflammation Sterile inflammation, also known as inflammaging, is a hallmark of aging and contributes to many age-related diseases. 37,38 The data of this disclosure indicate that activation of L1 elements (and possibly other RTEs) promotes inflammaging and that L1 RT is a relevant target for the treatment of age-related inflammation and disorders.

[0092] The data of this disclosure provide specific examples in aging mice where age-associated pathologies can be reversed or at least downregulated by administration of an RTI. For example, the NRTI lamivudine (also known as 3TC or Epivir) has been shown to reverse or downregulate: · A panel of IFN-I and SASP markers measured by RT-qPCR in multiple tissues; · Macrophage infiltration into white adipose and kidney tissues as measured by IF microscopy; Muscle atrophy measured by muscle fiber diameter; · Renal glomerulosclerosis measured by pathological evaluation of PAS-stained sections; Adipocyte atrophy measured microscopically by RT-qPCR analysis of cell size and key adipogenic genes; and ·Thermogenesis measured by RT-qPCR analysis of Ucp1 expression.

[0093] Thus, the present invention provides that RTIs can be used as "senostatic" drugs that can halt or block the harmful effects of senescent cells, particularly the SASP, and prevent or reverse age-related inflammation and damage.

[0094] Nucleoside reverse transcriptase inhibitors (NRTIs) NRTIs are active inhibitors of the reverse transcriptase enzyme found in retroviruses such as human immunodeficiency virus (HIV). Although different nucleoside reverse transcriptase inhibitors can be activated differently, they share the same mechanism of action. NRTIs are generally activated by phosphorylation to the triphosphate form by cellular enzymes. They then compete for cellular triphosphate, a substrate for proviral DNA by viral reverse transcriptase. NRTIs were the first class of drugs available for the treatment of human immunodeficiency virus (HIV infection) and acquired immune deficiency syndrome (AIDS).

[0095] Table 8 provides a list of commonly approved NRTI drugs or NRTI combinations used to treat HIV infection and AIDS. As described above, according to the methods of the present invention, NRTIs can be used as "senostatic" drugs that can halt or block the harmful effects of senescent cells, particularly SASP, and prevent or reverse age-related inflammation and damage.

[0096] NRTI drugs that can be used in the methods of the present invention include, but are not limited to, amdoxovir, apricitabine (ATC), ATRIPLA® (efavirenz / emtricitabine / tenofovir disoproxil), BARACLUDE® (entecavir; ETV); BIKTARVY® (bictegravir / emtricitabine / tenofovir alafenamide), censavudine (INN ; BMS-986001; OBP-601; festinavir), COMBIVIR™ (zidovudine / lamivudine), COVIRACIL™ (emtricitabine; FTC), DAPD / DXG (DAPD-2,6-diaminopurine dioxolane, active metabolite), DESCOVY® (emtricitabine / tenofovir alafenamide), D-D4FC (dexelbucitabine; Levelset; INCB-8721; DPC) 817), dOTC (2'-deoxy-3'-oxa-4'-thiocytidine; BCH-10652), elvucitabine, EMTRIVA™ (emtricitabine), EPIVIR™ (lamivudine; 3TC), EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine; MK-8591), EVIPLERA™ (rilpivirine / emtricitabine / tenofovir disoproxil), GENVOYA® (elvitegravir / cobicistat / etroxithromycin), emtricitabine / tenofovir alafenamide), HIVID™ (zalcitabine; ddC), KIVEXA™ (abacavir / lamivudine), LODENOSINE™ (F-ddA), ODEFSEY® (rilpivirine / tenofovir alafenamide / emtricitabine), PREVEON® (adefovir dipivoxil), Rasivir (RCV; (+ / -) emtricitabine), RETROVIR™ (zidovudine; ZDV; azidothymidine;AZT), Stampidine, STRIBILD® (elvitegravir / cobicistat / emtricitabine / tenofovir disoproxil), TENOFOVIR™ (TDF, bis-POC PMPA), TRIUMEQ® (dolutegravir / abacavir / lamivudine), TRIZIVIR™ (abacavir / lamivudine / zidovudine), TRUVADA® (emtricitabine / tenofovir disoproxil), VEMLIDY® (tenofovir alafenamide; TAF), VIDEX™ (didanosine ddl), VIREAD™ (tenofovir disoproxil), ZIAGEN™ (abacavir; 159U89), and ZERIT™ (stavudine; d4T);

[0097] At doses used in HIV / AIDS treatment, these drugs can cause a wide range of side effects. Common side effects of NRTIs include, among others, mitochondrial toxicity (related to inhibition of mitochondrial polymerase), neuropathy, pancreatitis, hepatic steatosis and lactic acidosis, bone marrow suppression, symptomatic myopathy, and cardiomyopathy. 39NRTIs can be used at dosages approved for HIV / AIDS treatment, but lower dosages can be used for preventing or treating age-related inflammation and disorders to avoid side effects associated with higher dosages. In one embodiment, the dosage used for preventing or treating age-related inflammation and disorders is half (50%) of the dosage approved for HIV / AIDS treatment (see Table 9). In an alternative embodiment, the dosage used is 75% of the dosage approved for HIV / AIDS treatment. In yet another alternative embodiment, the dosage used is 25% of the dosage approved for HIV / AIDS treatment. In other alternative embodiments, the dosage used is 90%, 80%, 70%, 60%, 40%, 30%, 20%, or 10% of the dosage approved for HIV / AIDS treatment. In yet other alternative embodiments, the dosage used is 0.1-99.5%, 10-90%, 20-80%, 25-75%, 30-70%, 40-60%, or 45-55% of the dosage approved for the treatment of HIV / AIDS.

[0098] In some embodiments, the subject receives long-term administration of one or more RTI drugs as defined herein. In one embodiment, the subject is treated for at least 12 weeks. In many cases, the long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1, 2, 3, 5, 7, or 10 years or more.

[0099] Age-related disorders Considering that cellular senescence is one of the major drivers of organismal aging and age-related diseases, 40 The methods of the invention can be used to prevent or treat disorders or diseases associated with cellular senescence, particularly disorders or diseases in which the presence of senescent cells is likely to have a deleterious effect, by administration of one or more senostatic RTI drugs.

[0100] Disorders or diseases associated with cellular senescence include Alzheimer's disease 41 , amyotrophic lateral sclerosis (ALS), atherosclerosis42 , Huntington's disease, vision loss, hearing loss, peripheral degenerative diseases, cardiovascular dysfunction 43 , atherosclerosis, frontotemporal dementia (FTD), multiple sclerosis (MS), Aicardi-Goutières syndrome, progressive supranuclear palsy (PSP), chemotherapy-induced adverse effects (e.g., bone marrow suppression, cardiotoxicity, cancer recurrence, blood clots, fatigue) 44 , hematopoietic stem cell function 45 , osteoarthritis 46 ,osteoporosis 47 , osteoporosis, Parkinson's disease 48 , body functions 49 , pulmonary fibrosis 50 , skin aging, wound healing, and / or tissue regeneration 51 These include, but are not limited to:

[0101] How to Treat, Prevent, and Reverse Age-Related Inflammation with RTIs Provided is a method for treating, preventing, and reversing age-related inflammation in a patient in need thereof by administering a reverse transcriptase inhibitor (RTI) to the patient in need thereof. Age-related inflammation may be present in patients with Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, vision loss, hearing loss, peripheral degenerative disease, and cardiovascular dysfunction.

[0102] In one embodiment, methods are provided for slowing or reversing the progression of the underlying pathology of an age-related inflammatory disorder, comprising administering to a patient in need thereof a therapeutically effective amount of at least one reverse transcriptase inhibitor (RTI). In some embodiments, the patient experiences a reduction in one or more symptoms of Alzheimer's disease compared to before the patient's first administration of the RTI.

[0103] In another aspect, a method is provided for preventing the development of an age-related inflammatory disorder in a patient suspected of having mild cognitive impairment, the method comprising administering at least one RTI to a patient in need thereof.

[0104] In some embodiments, the NRTI is abacavir, lamivudine, zidovudine, emtricitabine, tenofovir disoproxil fumarate, tenofovir alafenamide, didanosine, stavudine, apricitabine, alovudine, dexelbucitabine, amdoxovir, fosalvudine, or elvucitabine. In other embodiments, the RTI is abacavir (Ziagen), abacavir / lamivudine (Epzicom), abacavir / lamivudine / zidovudine (Trizivir), lamivudine / zidovudine (Combivir), lamivudine (Epivir), zidovudine (Retrovir), emtricitabine / tenofovir disoproxil fumarate (Truvada), emtricitabine (Emtriva), tenofovir disoproxil fumarate (Viread), emtricitabine / tenofovir alafenamide (Descovy), didanosine (Videx), didanosine extended-release (Videx EC), or stavudine (Zerit). In another embodiment, the NRTI is censavudine.

[0105] In some embodiments, at least one RTI is a non-nucleoside reverse transcriptase inhibitor (NNRTI). In some embodiments, at least one NRTI is efavirenz (EFV), nevirapine (NVP), delavirdine (DLV), etravirine, or rilvipirin.

[0106] In a further embodiment, the RTI inhibits L1 reverse transcriptase activity in cells, eg, brain cells, of the patient.

[0107] If the RTI is an FDA-approved drug, the RTI can be administered at a therapeutically effective amount approved for therapeutic use. In other embodiments, the effective amount can be determined using only routine experimentation. For example, the effective amount can range from about 1 ng / kg to about 200 mg / kg, about 1 μg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg. The dosage of the composition can be any dosage, including, but not limited to, about 1 μg / kg. Dosages of the compositions include about 1 μg / kg, about 10 μg / kg, about 25 μg / kg, about 50 μg / kg, about 75 μg / kg, about 100 μg / kg, about 125 μg / kg, about 150 μg / kg, about 175 μg / kg, about 200 μg / kg, about 225 μg / kg, about 250 μg / kg, about 275 μg / kg, about 300 μg / kg, about 325 μg / kg, about 350 μg / kg, about 400 μg / kg, about 425 μg / kg, about 450 μg / kg, about 460 μg / kg, about 470 μg / kg, about 480 μg / kg, about 490 μg / kg, about 500 μg / kg, about 510 μg / kg, about 520 μg / kg, about 530 μg / kg, about 540 μg / kg, about 550 μg / kg, about 560 μg / kg, about 570 μg / kg, about 580 μg / kg, about 590 μg / kg, about 600 μg / kg, about 610 μg / kg, about 620 μg / kg, about 630 μg / kg, about 640 μg / kg, about 650 μg / kg, about 660 μg / kg, about 670 μg / kg, about 680 μg / kg, about 690 μg / kg, about 700 μg / kg, about 720 μg / kg, about 750 μg / kg, about 760 μg / kg, about 770 μg / kg, about 780 μg / kg, about 790 μg / kg, about 800 μg / kg g / kg, approximately 375μg / kg, approximately 400μg / kg, approximately 425μg / kg, approximately 450μg / kg, approximately 475μg / kg, approximately 500μg / kg, approximately 525μg / kg, approximately 550 μg / kg, approximately 575 μg / kg, approximately 600 μg / kg, approximately 625 μg / kg, approximately 650 μg / kg, approximately 675 μg / kg, approximately 700 μg / kg, approximately 725 μg / kg, approximately 750 The dosage may be any dosage including, but not limited to, about 775 μg / kg, about 800 μg / kg, about 825 μg / kg, about 850 μg / kg, about 875 μg / kg, about 900 μg / kg, about 925 μg / kg, about 950 μg / kg, about 975 μg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, or more. In other embodiments, the dosage is 1 mg to 500 mg. In some embodiments, the dosage is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mg. These doses may be single or divided and may be administered one or more times daily.The above dosage amounts are exemplary of the average case, but there may be individual cases in which higher or lower dosage amounts are appropriate, and such dosage amounts are within the scope of the present disclosure. In practice, a physician will determine the most suitable therapeutically effective amount and actual administration regimen for an individual subject, which may vary according to the age, weight, and response of the particular subject.

[0108] RTIs can be administered once, twice, or three times daily, from 1 day to the end of life, or from 1 day to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years or more, or until the RTI causes unacceptable side effects or is no longer useful.

[0109] Patients are monitored for changes in the symptoms of age-related inflammatory diseases. In one embodiment, there is a reduction in symptoms. In another embodiment, the symptoms remain roughly the same, with no evidence of progression. In relation to Alzheimer's disease, such symptoms include memory loss, forgetting to put things down, forgetting to name places or objects, repeating questions, reduced flexibility, confusion, disorientation, obsessive-compulsive behavior, compulsive behavior, delusions, aphasia, sleep disorders, mood swings, depression, anxiety, frustration, agitation, difficulty performing spatial tasks, agnosia, gait disturbances, weight loss, loss of speech, short-term memory loss, or long-term memory loss. Methods for monitoring and quantifying any changes in these symptoms can be carried out by routine methods or routine experiments.

[0110] In one embodiment, any change in the symptoms of mild cognitive impairment and Alzheimer's disease is determined using the criteria described in DSM-5.In another embodiment, any change in the symptoms of mild cognitive impairment and Alzheimer's disease is determined using the Clinical Inventory of Change in Dementia (CIBIC-plus).In another embodiment, any change in the symptoms of mild cognitive impairment and Alzheimer's disease is determined using the Clinical Inventory of Change in Dementia (CIBIC-plus).

[0111] Any change in symptoms may be monitored for 1 to 36 months or longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months.

[0112] In another embodiment, the patient is monitored for changes in the underlying pathology of Alzheimer's disease. In one embodiment, there is a decrease in the underlying pathology. In another embodiment, the underlying pathology remains approximately the same with no evidence of progression.

[0113] In some embodiments, any change in the underlying pathology is identified by detecting a biomarker before and after administering an RTI. In one embodiment, the biomarker is β-amyloid or tau protein. In another embodiment, the biomarker is detected by PET imaging. In another embodiment, the underlying pathology is identified by measuring brain volume before and after administering an RTI.

[0114] In some embodiments, the reduction in the underlying pathology is reversed or delayed 1 to 36 months after the first administration of the RTI, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months.

[0115] In some embodiments, the patient is also administered at least one second therapeutic agent useful for treating symptoms of age-related inflammatory disorders. In one embodiment, the patient is administered at least one second therapeutic agent useful for treating Alzheimer's disease. In some embodiments, the at least one second therapeutic agent is donepezil, galantamine, rivastigmine, or memantine. In another embodiment, the at least one second therapeutic agent is an antibody that binds to β-amyloid or tau protein. In another embodiment, the antibody binds to β-amyloid and is bapineuzumab. In another embodiment, the antibody binds to tau protein and is ABBV-8E12. In another embodiment, the at least one second therapeutic agent is a vaccine against β-amyloid or tau protein. In another embodiment, the at least one second therapeutic agent is an agent that reduces or alters the brain content of β-amyloid or tau. In another embodiment, the second therapeutic agent reduces or alters brain content of β-amyloid and is a β-secretase 1 (BACE) inhibitor. In another embodiment, the BACE inhibitor is CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293), or LY2886721. In another embodiment, the second agent reduces or alters brain content of β-amyloid or tau and is nicotinamide or MPT0G211.

[0116] The at least one RTI and the at least one second therapeutic agent can be administered separately or together as part of a single pharmaceutical composition.

[0117] When the age-related inflammatory disorder is ALS, the patient may be administered at least one second agent useful for treating the symptoms of ALS. In some embodiments, the at least one second agent is an integrase inhibitor. In some embodiments, the integrase inhibitor is raltegravir, curcumin, a derivative of curcumin, chicoric acid, a derivative of chicoric acid, 3,5-dicaffeoylquinic acid, a derivative of 3,5-dicaffeoylquinic acid, a derivative of aurintricarboxylic acid, a derivative of aurintricarboxylic acid, caffeic acid phenethyl ester, a derivative of caffeic acid phenethyl ester, tyrphostin, a derivative of tyrphostin, quercetin, a derivative of quercetin, S-1360, zintevir (AR-177), L-870812, and L-25 870810, MK-0518, BMS-538158, or GSK364735C. 52 .

[0118] Patients may be monitored for improvement of ALS symptoms, which may include one or more of the following: difficulty walking or performing normal daily activities, tripping and falling, weakness in the legs, feet, or ankles, weakness or clumsiness in the hands, slurred speech or difficulty swallowing, muscle spasms, twitching of the arms, shoulders, or tongue, inappropriate crying, cognitive changes, and behavioral changes.

[0119] Any change in symptoms may be monitored for 1 to 36 months or longer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. In some embodiments, the reduction in the underlying pathology is reversed or delayed 1 to 36 months after the first administration of the RTI, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months.

[0120] Salts, Pharmaceutical Compositions, and Kits The method of the present disclosure can be achieved by administering at least one RTI as a neat compound or as a pharmaceutical composition. The administration of the pharmaceutical composition or neat compound of the RTI can be performed before or after clinical diagnosis of a disorder associated with age-related inflammation. Typically, the pharmaceutical composition is sterile and does not contain toxic, carcinogenic, or mutagenic compounds that would cause adverse reactions when administered.

[0121] Further provided is a kit comprising at least one RTI and, optionally, at least one second therapeutic agent useful for treating or preventing disorders associated with aging-related inflammation, packaged separately or together, and an insert with instructions for using these active agents.In one embodiment, at least one RTI is packaged separately with instructions for co-administration with at least one second therapeutic agent.At least one RTI and at least one second therapeutic agent can be administered simultaneously or sequentially to achieve the desired effect.Furthermore, the RTI and at least one second therapeutic agent can be administered from a single composition or two separate compositions.

[0122] Examples of at least one second therapeutic agent useful for treating Alzheimer's disease that can be included in the kit include donepezil, galantamine, rivastigmine, and memantine.Other optional therapeutic agents that can be included in the kit include antibodies that bind to β-amyloid or tau protein.In one embodiment, the antibody binds to β-amyloid and is bapineuzumab.In another embodiment, the antibody binds to tau and is ABBV-8E12.

[0123] In another embodiment, the kit may include at least one second therapeutic agent that is a vaccine against β-amyloid or tau protein.

[0124] In another embodiment, the kit may include at least one second therapeutic agent that reduces or alters the brain content of β-amyloid or tau protein. In some embodiments, the second therapeutic agent that alters or reduces the brain content of β-amyloid is a β-secretase 1 (BACE) inhibitor. In some embodiments, the BACE inhibitor is CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293), or LY2886721, each of which is in clinical trials for the treatment of Alzheimer's disease.

[0125] In another embodiment, the kit may include a second agent that reduces or alters brain content of tau, which is nicotinamide or MPT0G211.

[0126] In some embodiments, the patient has ALS, and the kit further comprises at least one second agent useful for treating ALS. In other embodiments, the RTI is packaged separately with instructions for administering at least one second agent for treating ALS. In some embodiments, the at least one second agent for treating ALS is edaravone or riluzole.

[0127] In some embodiments, the at least one second agent is an integrase inhibitor, ie, raltegravir, curcumin, a derivative of curcumin, chicoric acid, a derivative of chicoric acid, 3,5-dicaffeoylquinic acid, a derivative of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid, a derivative of aurintricarboxylic acid, caffeic acid phenethyl ester, a derivative of caffeic acid phenethyl ester, tyrphostin, a derivative of tyrphostin, quercetin, a derivative of quercetin, S-1360, zintevir (AR-177), L-870812, and L-25 870810, MK-0518, BMS-538158, or GSK364735C. 53 .

[0128] The second therapeutic agent is administered in an amount that provides its desired therapeutic effect. Effective dosage ranges for each optional therapeutic agent are known in the art, and any therapeutic agent is administered within such established ranges to individuals in need thereof.

[0129] This disclosure encompasses the preparation and use of salts of RTIs. As used herein, "pharmaceutically acceptable salts" refers to salts or zwitterionic forms of RTIs. Salts of RTIs can be prepared during the final isolation and purification of the compounds or separately by reacting the compounds with a suitable acid. Pharmaceutically acceptable salts of RTIs can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids that can be used to form pharmaceutically acceptable salts include inorganic acids such as nitric acid, boric acid, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid. Non-limiting examples of salts of RTIs include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethanesulfonate, phosphate, hydrogenphosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, These salts include, but are not limited to, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzenesulfonate, and p-toluenesulfonate.Furthermore, available amino groups present in RTIs can be quaternized with methyl, ethyl, propyl and butyl chlorides, methyl, ethyl, propyl and butyl bromides, and methyl, ethyl, propyl and butyl iodides; dimethyl sulfate, diethyl sulfate, dibutyl and diamyl sulfate; decyl chloride, lauryl, myristyl and steryl chlorides, decyl bromide, lauryl, myristyl and steryl bromides, and decyl iodide, lauryl, myristyl and steryl iodides; and benzyl bromide and phenethyl bromide. In light of the above, any reference to an RTI herein is intended to include the RTI and its pharmaceutically acceptable salts, hydrates, or solvates.

[0130] The present disclosure encompasses the preparation and use of solvates of RTIs. Solvates typically do not significantly alter the physiological activity or toxicity of a compound and may themselves function as pharmacological equivalents. As used herein, the term "solvate" refers to a combination, physical association, and / or solvation of a solvent molecule with a compound of the present disclosure, e.g., a disolvate, monosolvate, or hemisolvate, in which the ratio of solvent molecules to the compound of the present disclosure is about 2:1, about 1:1, or about 1:2, respectively. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain cases, a solvate may be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Thus, "solvate" encompasses both solution-phase and isolatable solvates. RTIs can exist in solvated forms with pharmaceutically acceptable solvents such as water, methanol, and ethanol, and the present disclosure is intended to encompass both solvated and unsolvated forms of RTIs. One type of solvate is a hydrate. "Hydrate" refers to a specific subgroup of solvates in which the solvent molecule is water. Solvates can typically function as pharmacological equivalents. The preparation of solvates is known in the art. For example, Caira et al. (2004) describes the preparation of solvates of fluconazole with ethyl acetate and with water. 54For similar preparations of solvates, hemisolvates, hydrates, etc., see Van Tonder et al. (2004) 55 and Bingham et al. (2001) 56 is described by. A typical, non-limiting method for preparing solvates involves dissolving at least one RTI or at least one second therapeutic agent in a desired solvent (organic, water, or a mixture thereof) at a temperature above 20° C. to about 25° C., then cooling the solution at a rate sufficient to form crystals and isolating the crystals by known methods, for example, filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of solvent in the solvate crystals.

[0131] The at least one RTI and at least one second therapeutic agent are typically administered in admixture with a pharmaceutical carrier to obtain a pharmaceutical composition selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present disclosure are formulated in a conventional manner using one or more physiologically acceptable carriers containing excipients and / or adjuvants that facilitate administration of the at least one RTI and at least one second therapeutic agent.

[0132] These pharmaceutical compositions can be prepared, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. The appropriate formulation depends on the selected route of administration. When a therapeutically effective amount of at least one RTI and / or at least one second therapeutic agent is administered orally, the composition is typically in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition can further contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 0.01% to about 95%, preferably about 1% to about 50%, of at least one RTI and at least one second therapeutic agent. When administered in liquid form, a liquid carrier such as water, petroleum, or oils of animal or plant origin can be added. Liquid forms of the composition can further contain saline, dextrose or other sugar solutions, or glycols. When administered in liquid form, the composition contains from about 0.1% to about 90% by weight, preferably from about 1% to about 50% by weight, of at least one RTI and at least one second therapeutic agent.

[0133] When a therapeutically effective amount of at least one RTI and at least one second therapeutic agent is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable solution, taking into consideration pH, isotonicity, stability, etc., is within the skill of the art. A preferred composition for intravenous, cutaneous, or subcutaneous injection typically contains an isotonic vehicle.

[0134] The at least one RTI and the at least one second therapeutic agent can be readily combined with pharmaceutically acceptable carriers well known in the art. Standard pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. 57Such carriers allow the active agent to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject to be treated. Pharmaceutical preparations for oral use can be obtained by adding at least one RTI and / or at least one second therapeutic agent to a solid excipient, optionally grinding the resulting mixture, and, if desired, processing the granular mixture after adding suitable excipients to obtain tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. If desired, disintegrants can be added.

[0135] At least one RTI and at least one second therapeutic agent can be formulated for parenteral administration by injection, for example, by bolus injection, or by continuous infusion.Injectable preparations can be provided in unit dosage forms, such as ampoules or multi-dose containers, with preservatives added.The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents.

[0136] Pharmaceutical compositions for parenteral administration include aqueous solutions of water-soluble active agents. In addition, suspensions of at least one RTI and at least one second therapeutic agent can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Alternatively, the composition can be in powder form, which can be reconstituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

[0137] At least one RTI and at least one second therapeutic agent can also be formulated into a rectal composition, such as a suppository or retention enema, containing a conventional suppository base.In addition to the above-mentioned formulations, at least one RTI and at least one second therapeutic agent can also be formulated as a depot preparation.Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection.Thus, for example, at least one RTI and at least one second therapeutic agent can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion exchange resin.

[0138] In particular, at least one RTI and at least one second therapeutic agent can be administered orally, buccally, or sublingually in the form of a tablet containing excipients such as starch or lactose, or in a capsule or small egg, alone or mixed with excipients, or in the form of an elixir or suspension containing flavoring or coloring agents. Such liquid formulations can be prepared using pharmaceutically acceptable additives such as suspending agents. At least one RTI and at least one second therapeutic agent can also be parenterally injected, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, at least one RTI and at least one second therapeutic agent are typically used in the form of a sterile aqueous solution, which can contain other substances, such as salts or simple sugars such as mannitol or glucose, to make the solution isotonic with blood.

[0139] Some aspects of the technology described herein can be defined according to any of the following numbered paragraphs: 1. A method for treating, preventing and / or reversing age-related inflammation in a patient in need thereof, comprising administering a therapeutically effective amount of a reverse transcriptase inhibitor (RTI) to the patient in need thereof, wherein the RTI comprises censavudine or elvucitabine. 2. The method of paragraph 1, wherein the age-related inflammation is present in a patient with Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, vision loss, hearing loss, peripheral degenerative disease, or cardiovascular dysfunction, frontotemporal dementia (FTD), multiple sclerosis (MS), Aicardi-Goutieres syndrome, progressive supranuclear palsy (PSP), osteoarthritis, skin aging, atherosclerosis, chemotherapy-induced adverse effects, hematopoietic stem cell function, osteoporosis, physical function, and / or pulmonary fibrosis, or a patient in need of wound healing or tissue regeneration. 3. The method of paragraph 1, wherein the age-associated inflammation is present in a patient with Alzheimer's disease. 4. The method of paragraph 1, wherein the age-associated inflammation is present in a patient with ALS. 5. A method for slowing or reversing the progression of the underlying pathology of a disorder caused by age-related inflammation, comprising administering to a patient in need thereof a therapeutically effective amount of a reverse transcriptase inhibitor (RTI), wherein the RTI comprises censavudine or elvucitabine. 6. The method of paragraph 5, wherein the patient has Alzheimer's disease or ALS and experiences a decrease in one or more symptoms of Alzheimer's disease or ALS compared to before the first administration to the patient. 7. The method of paragraph 6, wherein the patient has Alzheimer's disease and the one or more symptoms include memory loss, misplacing things, forgetting to name places or objects, repetitive questioning, decreased flexibility, confusion, disorientation, obsessive-compulsive behavior, compulsive behavior, delusions, aphasia, sleep disturbances, mood swings, depression, anxiety, frustration, agitation, difficulty performing spatial tasks, agnosia, gait disturbances, weight loss, loss of speech, short-term memory loss, or long-term memory loss. 8. The method of paragraph 6, wherein the patient has Alzheimer's disease and the reduction in one or more symptoms is assessed according to DSM-5. 9. The method of paragraph 6, wherein the patient has Alzheimer's disease and the symptom reduction is determined using the Alzheimer's Disease Assessment Scale - Cognitive subscale (ADAS-Cog). 10. The method of paragraph 6, wherein the patient has Alzheimer's disease and symptom reduction is determined using the Clinical Interview Impression of Change in Dementia (CIBIC-plus). 11. The method of paragraph 6, wherein the patient has Alzheimer's disease and the symptom reduction is determined using an Activities of Daily Living (ADL) scale. 12. The method of any one of paragraphs 6-11, wherein the reduction in symptoms is for 1 to 36 months. 13. The method of any one of paragraphs 6-11, wherein any change in the underlying pathology is identified by detection of a biomarker before and after administration of the RTI. 14. The method of paragraph 13, wherein the biomarker is β-amyloid or tau protein. 15. The method of paragraph 13 or 14, wherein the biomarker is detected by PET imaging. 16. The method of paragraphs 13 or 14, wherein the biomarker is detected by measurement in cerebrospinal fluid. 17. The method of any one of paragraphs 6-11, wherein the underlying pathology is identified by measurement of brain volume before and after administration of the RTI. 18. The method of any one of paragraphs 6 to 17, wherein the reduction in the underlying pathology is reversed or delayed for 1 to 36 months. 19. A method for preventing the onset of Alzheimer's disease in a patient suspected of having mild cognitive impairment, comprising administering to a patient in need thereof a therapeutically effective amount of a reverse transcriptase inhibitor (RTI), wherein the RTI comprises censavudine or elvucitabine. 20. The method of any one of paragraphs 1-19, wherein the patient has Alzheimer's disease or mild cognitive impairment, and further comprising administering at least one second therapeutic agent useful for treating the symptoms of Alzheimer's disease. 21. The method of paragraph 20, wherein the at least one second therapeutic agent is donepezil, galantamine, rivastigmine, or memantine. 22. The method of paragraph 20, wherein the at least one second therapeutic agent is an antibody that binds to β-amyloid or tau protein. 23. The method of paragraph 22, wherein the antibody binds to β-amyloid and is bapineuzumab. 24. The method of paragraph 22, wherein the antibody binds to tau protein and is ABBV-8E12. 25. The method of paragraph 20, wherein the at least one second therapeutic agent is a vaccine against beta-amyloid or tau protein. 26. The method of paragraph 20, wherein the at least one second therapeutic agent is an agent that reduces or alters brain content of beta-amyloid or tau. 27. The method of paragraph 26, wherein the second therapeutic agent reduces or alters brain content of beta-amyloid and is a beta-secretase 1 (BACE) inhibitor. 28. The method of paragraph 27, wherein the BACE inhibitor is CTS-21166, verubecestat (MK-8931), lanabecestat (AZD3293), or LY2886721. 29. The method of paragraph 26, wherein the second agent reduces or alters brain content of tau and is nicotinamide, or MPT0G211. 30. The method of any one of paragraphs 1, 2, or 4, wherein the patient has ALS, further comprising administering at least one second agent useful in the treatment of ALS. 31. The method of paragraph 30, wherein the drug useful for treating ALS is edaravone or riluzole. 32. The method of paragraph 30, wherein at least one second agent is an integrase inhibitor. 33. The method of paragraph 32, wherein the integrase inhibitor is raltegravir, curcumin, a derivative of curcumin, chicoric acid, a derivative of chicoric acid, 3,5-dicaffeoylquinic acid, a derivative of 3,5-dicaffeoylquinic acid, aurintricarboxylic acid, a derivative of aurintricarboxylic acid, caffeic acid phenethyl ester, a derivative of caffeic acid phenethyl ester, tyrphostin, a derivative of tyrphostin, quercetin, a derivative of quercetin, S-1360, zintevir (AR-177), L-870812, and L-25 870810, MK-0518, BMS-538158, or GSK364735C. 34. The method of any one of paragraphs 1-33, wherein the patient is evaluated for one or more symptoms or disease pathology 1 to 36 months after the patient's first administration of the RTI. 35. The method of any one of paragraphs 1-34, wherein the RTI inhibits L1 reverse transcriptase activity in the patient's cells. 36. The method of any one of paragraphs 1-35, wherein the RTI is elvucitabine. 37. The method of any one of paragraphs 1 to 35, wherein the RTI is sensavidine. 38. The method of any one of paragraphs 1-35, further comprising administering to the patient at least one second therapeutic agent. 39. The method of paragraph 38, wherein the patient has Alzheimer's disease and the at least one second therapeutic agent is useful in treating the symptoms of Alzheimer's disease. 40. The method of paragraph 38, wherein the patient has amyotrophic lateral sclerosis (ALS) and the at least one second therapeutic agent is useful in treating ALS. [Example]

[0140] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are included solely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0141] method cell culture Several different lines of normal human fibroblasts were used in this study. LF1 cells were derived from embryonic lung tissue as described. 58These cells have been continuously used in our laboratory since their isolation in 1996. For this study, we retrieved and used the original samples that were frozen in 1996 and continuously stored in our laboratory. IMR-90 and WI-38 cells were obtained from ATCC. Neither of these cell lines is listed in the International Cell Line Authentication Committee (ICLAC) database. These normal fibroblast cell lines were cultured in Ham's F-10 nutrient mixture (Thermo Scientific) containing 15% fetal bovine serum (FBS, Hyclone) under physiological oxygen conditions (92.5% N2, 5% CO2, 2.5% O2). The medium was further supplemented with L-glutamine (2 mM), penicillin, and streptomycin. 59 Cell cultures were regularly tested for mycoplasma contamination using the MycoAlert® Mycoplasma Detection Kit (Lonza).

[0142] To obtain replicative senescent (RS) cells, LF1 cultures were continuously expanded until proliferation ceased. At each passage, after reaching 80% confluence, cells were trypsinized and diluted 1:4. Thus, each passage corresponds to approximately two population doublings. In early subcultures, the time between passages remained constant at approximately 3 days. As the culture approached senescence, the time between passages gradually increased. A 2-3 week interval indicated that the culture was in its penultimate passage. At this point, after reaching 80% confluence, cells were reseeded at a 1:2 dilution, and this time point was designated as the final passage (point A in Figure 2a). Some cell proliferation typically occurred over the next 2-3 weeks, but the culture did not reach 80% confluence. Under this experimental regimen, the majority of cells in the culture entered senescence within a 3-4 week window centered approximately around the time of the last passage (gray bar in Figure 2a). At point B (week 4), cultures were trypsinized and replated as described. 60 The few remaining contact-inhibited cells were removed, and the cultures were reseeded again at point C (week 8).

[0143] Oncogene-induced senescence (OIS) was induced by infecting proliferating LF1 cells with pLenti CMV RasV12 Neo (Addgene Plasmid No. 22259). The lentiviral particle production and infection procedures are described below. At the end of infection, cells were replated at 15–20% confluency and continuously maintained under selection in G418 (250 μg / ml) until the end of the experiment. The medium was changed every 3 days until the cultures were harvested at the indicated time points. Stress-induced premature senescence (SIPS) was induced by X-ray irradiation at a dose of 20 Gy delivered at a rate of 87 cGy / min in one fraction using a cesium-137 gamma source (Nordion Gammacell 40). Cells were 15–20% confluent at the time of irradiation. The medium was changed immediately after irradiation and at 3-day intervals thereafter. Lentiviral vectors were packaged using 293T cells (Clontech) and cultured at 37°C in DMEM containing 10% FBS under normoxic conditions (air supplemented with 5% CO ).

[0144] Reverse transcriptase inhibitors (RTIs) All RTIs used in this study (lamivudine, 3TC; zidovudine, AZT; abacavir, ABC; emtricitabine, FTC) were USP grade and obtained from Aurobindo Pharma, Hyderabad, India. For trizivir (TZV), its components (ABC, AZT, and 3TC) were combined in appropriate amounts.

[0145] Mouse rearing Female and male C57BL / 6J mice were housed at the NIA Aged Rodent Colonies at 5 and 18 months of age. 61Mice were obtained from the University of California, San Diego, Calif. Five-month-old animals were sacrificed after a short (1-week) acclimation period, and various tissues were harvested, flash-frozen in LN2, and stored at -80°C. Eighteen-month-old animals were maintained until they reached the desired age. Mice were housed in a specific-pathogen-free, AAALAC-certified barrier facility. Cages, bedding (Sani-chip hardwood bedding), and food (Purina Lab Chow 5010) were sterilized by autoclaving. Food and water (also sterilized) were available ad libitum. A 12-hour light / dark cycle was used (7 AM on, 7 PM off). Temperature was maintained at 70°F and humidity at 50%. All animals were observed daily and weighed weekly. In a pilot experiment, three cohorts of 10 animals each were continuously treated with 3TC dissolved in drinking water (1.5 mg / ml, 2.0 mg / ml, 2.5 mg / ml) until sacrifice at 18 to 24 months of age. A fourth (control) cohort received the same water without the drug. No significant differences in behavior, weight, or survival were observed among the four cohorts throughout the experiment. Once during the experiment (at 20 months of age), animals were subjected to a single tail bleed of approximately 70 μL. The collected plasma was sent to the University of North Carolina CFAR Clinical Pharmacology and Analytical Chemistry Core for 3TC analysis. For the 2 mg / mL cohort, the concentration of 3TC in plasma averaged 7.2 μM. This dose of drug was selected for further experiments, mimicking the human HIV therapeutic dose (300 mg / day, 5–8 μM in plasma). 62 For the experiments presented in this communication, animals were aged in a vivarium until they reached 26 months of age. They were then randomly assigned to two cohorts by technicians blinded to the animals' appearance or other characteristics. One cohort was treated with 2 mg / mL of 3TC in drinking water for 2 weeks, while the other (control) cohort received the same drug-free water in the same manner. At the end of the treatment period, all animals were sacrificed and tissues were collected as described above. All animals from both cohorts were included in all subsequent analyses. Experiments were performed on separate occasions with male and female animals. Nonlethal total-body irradiation (6 Gy) was performed as described.63 , tissue specimens were kept on dry ice.

[0146] PCR An ABI ViiA 7 instrument (Applied Biosystems) was used for all experiments. DNA qPCR was performed according to Coufal et al. (2009). 64 The experiments were performed using the TaqMan system (Applied Biosystems) as described by [100 pg of purified genomic DNA] with the indicated primers (see Table 1). RNA reverse transcription qPCR (RT-qPCR) was performed using the SYBR Green system (Applied Biosystems). Polyadenylated RNA was used in all experiments assessing L1 element transcription, while total RNA was used for all other genes. Total RNA was collected using Trizol Reagent (Invitrogen). Poly(A) RNA was isolated from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). 1 μg of total RNA or 10 ng of poly(A) RNA was reverse transcribed into cDNA in a 50 μL reaction using the TaqMan kit (Applied Biosystems). To assess strand-specific transcription, the random primer in the RT reaction was replaced with a strand-specific primer for the target RNA. 1 μL of each RT reaction was used in the subsequent qPCR reaction. GAPDH was used as a normalization control in experiments with human cells. The arithmetic mean of GAPDH and two additional controls (Hsp90 and GusB) was used for normalization of RT-qPCR experiments with mouse tissues, except for liver, which was normalized to Hsp90 and GusB. To measure L1 transcription, poly(A) RNA samples were extensively digested with RNase-free DNase (Qiagen) prior to cDNA synthesis. A control omitting the RT enzyme was used to assess the effectiveness of DNase digestion.

[0147] PCR primer design Primer sets 1–5 for human L1 (Table 1, amplicons A–E in Figure 1b) were designed to preferentially amplify elements of the human-specific L1HS and evolutionarily recent primate-specific L1PA(2–6) subfamilies as follows: First, consensus sequences for L1HS and L1PA2–L1PA6 elements were identified in Repbase (Genetic Information Research Institute). 65 Second, the Clustal Omega multiple sequence alignment tool was used. 66,67 A consensus sequence of these six sequences was generated using the NCBI Primer-BLAST tool. 68,69 Primer design was performed based on the overall consensus with Primer3 and BLAST using the L1 primer pair, which was then subjected to in-silico PCR against the latest genome assembly (hg38). 70The target was evaluated using the tool, and the minimum perfect match at the 3' end of each primer was equal to 15. Primers for ORF2 (primer set 6, amplicon F in Figure 1b) were developed by Coufal et al. (2009) to preferentially target L1HS. Primers for evaluating transcription of active mouse L1 elements (primer set 37, Table 1) were designed by combining consensus sequences for the L1MdA and L1Tf families obtained from Repbase and validated as described above. L1 primer pairs spanning the entire length of these elements (primer sets 48–50) were designed using the same strategy. Primer pairs specific to three active families of mouse L1 elements (primer sets 51–53) were designed using polymorphisms in the 5'UTR region. RT-qPCR analysis of L1 transcription was performed on poly(A)-purified RNA using SYBR Green. For all other (non-L1) genes, whenever possible, primers are separated by at least one intron in the genomic DNA sequence (as shown in Table 1). Primers for the human IFNα family were designed against the consensus sequence of all human IFNα gene sequences (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21) generated using the Clustal Omega multiple sequence alignment tool. All primers for mouse targets were designed as above and are listed in Table 1. The sequences of primers corresponding to the consensus for all mouse IFNα family genes, as well as the IFNB1 gene, were derived from Gautier et al. 71 To quantify the relative L1 genome copy number (human cells), Coufal et al. (2009) 72 The TaqMan multiplex method developed by [the authors] was used. These primers are listed in Table 1 as Set No. 6 and Set No. 7 (along with their corresponding VIC and 6FAM probes).

[0148] Chromatin immunoprecipitation All ChIP experiments were performed using the Chromatrap spin column ChIP kit (Porvair). 6 Cells were crosslinked in their culture dishes with 1% formaldehyde (10 min, room temperature), quenched with glycine, washed twice with ice-cold PBS (containing protease inhibitors), and finally scraped into a microcentrifuge tube. The cell pellet was resuspended in 0.4 mL of hypotonic buffer and incubated on ice for 10 min. Nuclei were spun down, resuspended in 0.3 mL of lysis buffer, and sonicated for a total of 10 min using a Bioruptor UCD-200 instrument (Diagenode) set to pulse on high (30 s, followed by a 30 s pause). The extract was centrifuged in a microcentrifuge (maximum speed, 5 min, 4°C) to remove debris, and the supernatant was transferred to a new tube and stored at -80°C. An amount of extract containing 2 μg of DNA was combined with 4 μg of antibody and loaded onto a Chromatrap solid-phase protein A matrix. Immune complexes were allowed to form overnight at 4°C with gentle agitation, and then samples were washed and eluted according to the manufacturer's protocol. Rabbit IgG and 1% input were used as controls. 1 μL of immunoprecipitated DNA was used in each qPCR reaction.

[0149] BrdU pulldown To obtain quiescent cells, proliferating cells were grown to 50% confluence, the serum supplement of the medium was changed to 0.1% FBS, and incubation was continued until harvest. Quiescent and senescent cells were continuously labeled with BrdU (BrdU labeling reagent, Thermo Fisher Scientific) for 2 weeks according to the manufacturer's protocol for labeling cultured cells. Cells were harvested and counted: 5 × 10 per condition. 5Cells were processed. Genomic DNA was purified by phenol:chloroform extraction, RNase A treated, and then sheared using a Bioruptor UCD-200 instrument (pulse on low, 30 seconds on and 30 seconds off, 10 minutes total). DNA tubes were incubated in a heat block (100°C) for exactly 1 minute and then flash-frozen in liquid nitrogen. Tubes were thawed at room temperature, and 1 μg of purified anti-BrdU antibody (BD Pharmingen, catalog no. 555627) was added per tube along with magnetic protein A / G beads and ChIP dilution buffer. The immunoslurry was incubated overnight at 4°C with constant rotation. Immunocaptured BrdU-labeled DNA was purified according to the Magna ChIP™ A / G Chromatin Immunoprecipitation Kit (Millipore Sigma). Unbound DNA was retained as input. 1 μL of immunoprecipitated DNA was used in each qPCR reaction. Alternatively, to enrich for single-stranded BrdU-labeled DNA, heat-mediated denaturation was omitted, and samples were processed for BrdU pulldown as described above. The second strand of DNA was then generated by adding a mixture of random primers (Thermo Fisher), second-strand synthesis reaction buffer, dNTPs, and DNA Pol I (New England Biolabs). The reaction was incubated at 16°C for 4 hours and then purified by phenol-chloroform extraction. After second-strand synthesis, the dsDNA was end-repaired with the End-It DNA End-Repair Kit (Epicentre, catalog no. ER0720). Blunt-ended fragments were cloned using the Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher) and then used to transform One Shot TOP10 chemically competent Escherichia coli (E. coli) (Thermo Fisher, catalog no. C404010). Individual colonies were picked and subjected to Sanger sequencing using T7 promoter primers at Beckman Coulter Genomics.

[0150] RNA-seq Total RNA from early-passage, early-stage, and deep-stage senescent cells (Figure 6a) was extracted as described above. Total RNA was processed with the Illumina TruSeq Stranded Total RNA Ribo-Zero Kit and subjected to Illumina HiSeq2500 2 x 125 bp paired-end sequencing using v4 chemistry at Beckman Coulter Genomics Inc. Over 70 million reads were obtained for each sample. RNA-seq experiments were performed in three biological replicates.

[0151] Raw RNA sequencing reads were aligned to the GrCh38 build of the human genome using HiSat2 73 Counts mapping to the genome were determined using featureCounts. 74 Counts were then normalized using the trimmed mean M (TMM) method in EdgeR. 75 EdgeR was further used to derive differential expression from the normalized dataset. Differential expression data were then ranked by log2 fold change and input into the GenePattern interface of GSEA Preranked using 1000 permutations to determine enrichment of KEGG pathways, SASP, and interferon response. 76,77 The output was then corrected for multiple comparisons by adjusting the nominal p-value using the Benjamini-Hochberg method. 78 Data were displayed using GENE-E software. 79 .

[0152] In silico analysis of transcription factors binding to L1 Transcription factor profiles were generated using ChIP-seq data from the ENCODE project (GEO accession numbers GSE2961 and GSE32465). Transcription factor ChIP-seq and input control reads were aligned to the consensus sequence of L1HS using bowtie1. 80Transcription factor ChIP-seq read coverage per million mapped reads (RPM) relative to the input control RPM value was used to calculate log2 fold-change enrichment for each base pair of the L1HS consensus and smoothed by LOESS smoothing with parameter α = 0.1. The total number of mapped reads used for RPM normalization was determined from a separate bowtie1 alignment to the human genome (hg19).

[0153] Construction of FOXA1 reporter The L1 promoter reporter plasmids L1WT and L1 del(390-526) were obtained from Sergey Dmitriev, Institute of Bioorganic Chemistry, Moscow. 81,82 Both contain luciferase as a reporter cloned in the sense orientation. To determine antisense transcription from the same plasmid, EYFP was inserted in the reverse orientation upstream of the L1 5'UTR as follows: The EYFP sequence was excised from pEYFP-N1 (Clontech, Cat. No. 6006-1) with AgeI and NotI and blunt-ended. Plasmids L1WT and L1del were digested with XbaI, blunt-ended, and treated with FastAP (Fermentas). Successful insertion of antisense EYFP was confirmed by PCR using the primers TIFF0007728402000001.tif5166 and verified using Sanger sequencing. Plasmid pcDNA3.1 / LacZ was used as a co-transfection control. Luciferase and β-galactosidase assays were performed as described. 83 EYFP-N1 was used as a positive control to detect EYFP signals. Co-transfection was performed in early passage LF1 cells using Lipofectamine with Plus Reagent (Invitrogen) according to the manufacturer's instructions.

[0154] Lentiviral vectors Constructs were obtained from public depositories as described below. Virions were produced and target cells were infected as described. 84 shRNA sequences were obtained from The RNAi Consortium (TRC) 85 The vectors were obtained from the University of California, San Diego, and cloned into the third-generation pLKO.1 vector for efficacy testing. Four selectable markers were used to allow for multiple drug selection: pLKO.1 puro (2 μg / ml) and pLKO.1 hygro (200 μg / ml) (Addgene Plasmid Nos. 8453 and 24150), pLKO.1 blast (5 μg / ml) (Addgene Plasmid No. 26655), and pLKO.1 neo (250 μg / ml) (Addgene Plasmid 13425). pLKO-RB1-shRNA63 and pLKO-RB1-shRNA19 (Addgene Plasmid Nos. 25641 and 25640) were also used. 86 For FOXA1 shRNAs, TRCN0000014881(a) and TRCN0000014882(b) were used. For TREX1 shRNAs, TRCN0000007902(a) and TRCN0000011206(b) were used. For L1 knockdown, nine shRNAs were designed and tested, two of which (shL1_11 to ORF1, TIFF0007728402000002.tif3128, and shL1_44 to ORF2 TIFF0007728402000003.tif3128) showed significant knockdown (Figure 10g) and was selected for further work. The remaining seven shRNAs produced no or minimal knockdown. For cGAS shRNAs, TRCN0000128706 (a) and TRCN0000128310 (b) were used. For STING shRNAs, TRCN0000161345 (a) and TRCN0000135555 (b) were used.

[0155] All ectopic expression experiments were performed in the lentiviral vector pLX304 (blasticidin resistant, Addgene plasmid 25890) by the ORFeome Collaboration. 87 and used constructs generated by the DNASU plasmid repository 88 RB1 (ccsbBroad304_06846, HsCD00434323), TREX1 (ccsbBroad304_02667, HsCD00445909), and FOXA1 (ccsbBroad304_06385, HsCD00441689) were obtained from

[0156] All interventions in senescent cells were initiated by infecting cells at week 12 of senescence (point D in Figure 6a). After appropriate drug selection, cells were incubated until week 16 of senescence (point E in Figure 6a), at which point they were harvested for further analysis.

[0157] 3X intervention was performed by sequentially infecting early-passage LF1 cells with the vectors pLKO.1 puro shRB, pLKO.1 hygro shTREX1, and pLX304 blast FOXA1 (Figure 10c). After each infection, the resulting drug-resistant cell pool was immediately infected with the next vector. After the third infection, 48 hours after drug selection was completed, cells were harvested for further analysis. Infections were also performed with various combinations, and in all cases, they resulted in activation of L1 expression, transition to senescence, and induction of an IFN-I response. The above sequences were chosen because they provided the most efficient selection of cells for further analysis. To enable further (fourth) interventions in 3X cells (shL1, shSTING, or shCGAS), the RB1-targeting hairpin was recloned into pLKO.1 neo, thus liberating pLKO.1 puro for the fourth gene of interest. This allowed for an efficient drug selection process and sampling 48 hours after the final selection.

[0158] Retrotransposition reporter Xie et al. (2011) 89The two-vector dual luciferase reporter system reported by et al. was adapted for lentiviral delivery. The L1RP-Fluc reporter was recloned from plasmids pWA355 and pWA366 into the lentiviral backbone pLX304. pWA355 contains functional and active L1 RP pLX304-WA355 and pLX304-WA366 contained the L1RP (JM111) element, whereas pWA366 contains a mutant element, L1RP (JM111), carrying two missense mutations in ORF1 that is unable to retrotranspose. Early passage LF1 cells were infected with puromycin-resistant lentivirus expressing Rluc. Pooled drug-resistant cells were then infected with high-titer particles of the pLX304-WA355 or pLX304-WA366 constructs. Immediately after infection, cells were treated with 3TC (concentrations indicated) for 4 days. Cells were then harvested and assayed for Rluc and Fluc luciferase activity. The native L1 retrotransposition reporter pLD143 90 was co-transfected with pLKO vectors (shLuc, shL1_11, and shL1_44) into HeLa cells using FuGene HD (Promega). Cell culture, transfection, and retrotransposition assays were performed as described above. Retrotransposition activity was measured using L1 vectors co-transfected with shLuc. RP Three independent experiments were performed for each construct.

[0159] Identification of expressed L1 elements by long-distance RT-PCR and 5′ RACE Total RNA was collected from cells using Trizol Reagent (Invitrogen). RNA was further purified using the Purelink RNA Mini Kit (Invitrogen) with DNase I digestion. Poly(A) RNA was isolated from the eluted total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). A forward primer (MDL15UTRPRAF, Primer Set 1, Table 1) was used with either one of two reverse primers (MDL15UTRPRCR, Primer Set 3, amplicon size 537 bp) or MDL15UTRPRDR, Primer Set 4, amplicon size 654 bp). High-fidelity thermostable reverse transcriptase (PyroScript RT-PCR Master Mix Kit, Lucigen) was used with 10 ng of poly(A) mRNA per reaction, and amplification was performed for 10 cycles. No template and RNase A-treated samples were used as negative controls. The resulting amplicons were cloned into the TOPO-TA (Invitrogen) vector, and the resulting plasmids were used to transform One Shot TOP10 chemically competent E. coli. Individual colonies were picked and subjected to Sanger sequencing using T7 promoter sequencing primers at Beckman Coulter Genomics. For a total of 768 sequenced clones, 96 sequencing reactions (1 plate) were performed for each primer pair across four experiments. Sequencing data were trimmed to remove RT-PCR primers and BLAST was performed against the human genome (GRCh38) with a match / mismatch cost of +1, -4 to allow for species-specific repeats in Homo sapiens. Only complete hits were scored and annotated with genomic coordinates. 658 clones could be mapped to the reference genome; 51 contained at least one mismatch and therefore likely represented elements polymorphic in the cell line, while 58 were cloning artifacts. Whenever a clone displayed multiple instances of perfect identity, fractional counts were taken, dividing the count by the number of elements sharing the same sequence.L1Xplorer. 91 Each mappable clone was further analyzed using to reconfirm the taxonomic characteristics of the L1 element and whether it was intact.

[0160] Alternatively, poly(A) RNA isolated as described above was subjected to rapid amplification of cDNA ends (RACE). Each reaction contained 10 ng of poly(A) RNA and was processed using the 5' RACE System kit (Thermo Fisher, catalog number 18374-041). The two antisense gene-specific primers (GSPs) used for 5' RACE were MDL15UTRPRDR (primer set 4, Table 1) for GSP1 and MDL15UTRPRCR (primer set 3, Table 1) for the nested GSP2. The amplified products were cloned and sequenced as described above using T7 promoter sequencing primers from Beckman Coulter Genomics. A total of 94 clones were sequenced; 26 contained poly-G stretches, primarily generated by the tailing step of the RACE protocol, and 18 could not be mapped to the human genome. The remaining 50 mappable clones contained L1 sequences and were aligned to the L1HS consensus using a setting of >95% identity at positions 1–450. 92 Mappable clones were also assigned to individual L1 families using RepEnrich software. 93 Pairwise alignment to the consensus was performed using LALIGN. 94 Multiple sequence alignments were calculated using MAFFT (multiple alignment using fast Fourier transform) and the L-INS-i algorithm (accurate for alignments of less than 200 sequences). 95 Alignment visualization, % identity coloring and consensus were generated by Jalview. 96 .

[0161] Generation and analysis of CRISPR-Cas9 knockouts GeCKO v2.0 Resources (Feng Zhang Lab, MIT 97 ) 98 Three different gRNA sequences were tested for each chain of the IFNAR receptors (IFNAR1 and IFNAR2) listed in and the following were selected: TIFF0007728402000004.tif11128Cas9 and gRNA were delivered using a single lentiviral vector carrying a puromycin resistance gene (LentiCRISPR_v2, Feng Zhang Lab, MIT; Addgene Plasmid No. 52961). The efficacy of CRISPR-Cas9 mutagenesis, which was the basis for selecting the two gRNAs mentioned above, was evaluated by treating infected and drug-selected cells with interferon (universal type I interferon, PBL Assay Science, Catalog No. 11200-1) and monitoring the nuclear translocation of phospho-STAT2 and IRF9 by immunofluorescence. The absence of translocation indicates a lack of IFN-I responsiveness and, therefore, a loss of IFNAR function. The experimental procedure followed the protocol provided by the Zhang lab. 99,100 In the experiments shown in Figure 3h (RS) and Figure 10k, both IFNAR1 and IFNAR2 gRNAs were used to treat the same cells to further enhance the effect of extinguishing the INF-I response. For early-passage and senescent cells, co-infection of IFNAR1 and IFNAR2 vectors was performed, followed by selection with puromycin. For senescent cells, high-titer lentiviral particles were applied to senescent cells at week 12 of senescence (point D, Figure 6a), and the cells were assayed 4 weeks later (point E, Figure 6a). In the experiment shown in Figure 3h (SIPS), edited early-passage cells were single-cell cloned. 24 single cells were isolated and expanded using CellRaft technology (Cell Microsystems). Genomic screening for CRISPR cleavage sites was performed by the CRISPR Sequencing Service (CCIB DNA Core, Massachusetts General Hospital). 101Successful knockout of IFNAR1 and IFNAR2 was confirmed in 4 of 24 expanded clonal cell lines.

[0162] Immunoblotting Cells were harvested in Laemmli sample buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT) and boiled at 100°C for 5 min. Whole-cell extracts (60 μg of protein) were separated by SDS-PAGE and transferred to Immobilon-FL membranes (Millipore). Nonspecific binding was blocked by incubation in PBS containing 4% bovine serum albumin (BSA; Thermo Fisher Scientific) and 0.1% Tween-20 for 1 h at room temperature. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. A list of all primary antibodies is shown in Table 2. Secondary antibodies were diluted in blocking solution and incubated for 1 h at room temperature. Signals were detected using a LI-COR Odyssey infrared imaging system (LI-COR Biosciences). For signal quantification, all samples were run on the same gel. Loading standards were visualized on the same blot as the test samples using the LI-COR 2-color system. Bands were imaged and quantified using LI-COR software. All bands compared were quantified on the same image and within the linear range of detection of the instrument.

[0163] Immunofluorescence microscopy performed on cells in culture Cells were grown on glass coverslips and samples were processed as previously described 102 Primary antibodies are listed in Table 2. Staining of ssDNA was performed as described by Thomas et al. 103Briefly, cells seeded on coverslips were fixed with 4% paraformaldehyde (PFA) for 20 min on ice and then incubated overnight at -20°C in 100% methanol. Next, cells were treated with 200 mg / mL RNase A for 4 h at 37°C. Cells were blocked with 3% BSA and incubated with primary antibodies diluted in 3% BSA overnight at 4°C. Images were acquired using a Zeiss LSM 710 confocal laser scanning microscope or a Nikon Ti-S inverted fluorescence microscope. All microscope settings were set to collect images below saturation and were kept constant for all images taken in one experiment, as previously described. 104 The tissues were subjected to image analysis as described below.

[0164] PCR array Total RNA was harvested from cells as described above (quantitative PCR) and analyzed by Qiagen RT 2 Analysis was performed using the Profiler™ Human Type I Interferon Response PCR Array (Cat. No. PAHS-016ZE-4). 1 μg of total RNA was used as starting material and analyzed using Qiagen RT 2 Reverse transcription reactions were performed using the First Strand Kit (Cat. No. 330404). 102 μL of the completed reaction was transferred to 650 μL of Qiagen RT. 2 The mixture was combined with SYBR Green ROX qPCR Mastermix (Cat. No. 330521) and 548 μL of RNase-free molecular-grade water and analyzed in a 384-well block on a ViiA 7 Applied Biosystems instrument. All procedures followed the manufacturer's protocol. All conditions were performed in triplicate. Results were analyzed using the Qiagen GeneGlobe Data Analysis Center. 105 In simple terms, C t Values ​​were normalized to a panel of housekeeping genes (HKG). t The value was calculated between the gene of interest (GOI) and the average HKG value. -ΔΔCTThe fold change was calculated using the formula: The lower limit of detection was C t = 35. For a GOI to be considered significant, the following filters were set: (i) more than a 2-fold change in expression; and (ii) p-value > 0.05. Furthermore, the mean C t >32 genes were also excluded.

[0165] Enzyme-linked immunosorbent assay (ELISA) Interferon-beta levels were quantified using the VeriKine-HS Human IFN Beta Serum ELISA Kit (PBL Assay Science, Catalog No. 41415). Cell culture medium was conditioned for 48 hours before harvesting. To remove particles and debris, 1 mL aliquots were spun at 5,000 x g for 5 minutes. All incubations were performed in a sealed chamber at room temperature (22-25°C), away from temperature fluctuations. 50 μL of sample buffer was added to each well, followed by 50 μL of diluted antibody solution. Finally, 50 μL of test sample, standard, or blank was added per well. The plate was sealed and shaken at 450 rpm for 2 hours. At the end of the incubation period, the plate contents were removed and the wells were washed three times with 300 μL of diluted wash solution. 100 μL of HRP solution was added to each well and incubated for 30 minutes under constant shaking. The wells were emptied and washed four times with wash solution. 100 μL of TMB substrate solution was added to each well. The plate was incubated in the dark for 30 minutes. Finally, 100 μL of stop solution was added to each well, and the absorbance at 450 nm was recorded within 5 minutes. The value recorded for the blank control was subtracted from the standard and sample values ​​to eliminate background. Optical density (OD) units were plotted using a four-parameter fit to the standard curve and used to calculate the interferon titer in the samples.

[0166] human tissue specimens Human skin specimens were obtained from the Leiden Longevity Study 106,107The specimens were collected as part of a study and provided by Leiden University Medical Centre, Netherlands. Informed consent was obtained, and all protocols were approved by the ethics committee of Leiden University Medical Centre. Samples were collected as 4-mm-thick full-thickness punch biopsies, embedded in optimal cutting compound (OCT), flash-frozen, and stored at -80°C. The investigators were blinded to all but the subject's age and gender. OCT-embedded specimens were cryosectioned at 8 μm thickness using a Leica CM3050S cryomicrotome. Slides were fixed in 4% PFA and 0.5% Triton X-100 in PBS (preheated to 37°C) for 20 minutes at room temperature. No further permeabilization was performed. Prior to antibody incubation, a blocking step using 4% bovine serum albumin (BSA; Fraction V, Thermo Fisher Scientific), 2% donkey serum, 2% rabbit serum, and 0.1% Triton X-100 in PBS was performed for 1 hour at room temperature. Primary antibodies were diluted (1:200) in the above blocking solution and incubated overnight at 4°C with rocking in a humidified chamber. Secondary antibodies (AlexaFluor 546 and AlexaFluor 647, Life Technologies) were also diluted in blocking solution and incubated for 2 hours at room temperature. Each antibody incubation was followed by three 15-minute washes in PBS containing 0.2% Triton X-100. Nuclei were counterstained with 2 μg / mL DAPI in PBS containing 0.2% Triton X-100 for 15 minutes. Stained slides were mounted with ProLong Antifade Mountant without DAPI (Life Technologies) and imaged with a Zeiss LSM 710 confocal laser scanning microscope. A z-series covering the entire thickness of the tissue was collected for each field. All microscope settings and exposure times were set to collect images below saturation and were kept constant for all images taken in one experiment. Image analysis was performed using CellProfiler software. 108or NIH's ImageJ open source software 109 The analysis was performed using either the DAPI channel. Nuclei were defined using the DAPI channel. Cell contours were defined by radially expanding the nuclear mask using the Propagate function until the intensity thresholds for the AlexaFluor 546 and AlexaFluor 647 channels were reached. Fluorescence intensity within these regions was then recorded in both channels. For each sample, a total of 200 nuclei were recorded across multiple fields of view. Mouse tissue sections were processed and analyzed in the same manner as above.

[0167] Mouse tissue specimens Total RNA was extracted from 50 mg of visceral fat, small intestine, skeletal muscle, brown fat, or liver tissue by mincing and homogenizing in Trizol (Invitrogen) using a Power Gen 125 homogenizer (Fischer Scientific). After phase separation, RNA from the aqueous layer was purified using the Purelink RNA Mini Kit (Invitrogen) with DNase I digestion. To assess gene expression by RT-qPCR, 1 μg of total RNA was reverse transcribed as described above. In each individual experiment, all samples were processed in parallel and unblinded.

[0168] Whole-mount white adipose tissue imaging was performed by Martinez-Santibanez et al. (2014) 110 Briefly, white adipose tissue (visceral depot) was extracted from 0.5–1 cm 3The tissue was then minced into small pieces sized 1 / 4" thick and incubated in 10 mL of fresh fixation buffer (1% PFA in PBS pH 7.4) with gentle rocking for 30 minutes at room temperature. After three washes with PBS, the tissue block was cut into six equal pieces. All subsequent incubations were performed in 2 mL cylindrical microcentrifuge tubes. Prior to primary antibody incubation, a blocking step was performed in PBS containing 5% BSA and 0.1% saponin for 30 minutes at room temperature. The primary antibody was diluted (1:200) in the above blocking solution and incubated overnight at 4°C with gentle rocking. Secondary antibodies (AlexaFluor 546, AlexaFluor 594, and AlexaFluor 647, Life Technologies) were also diluted in blocking solution and incubated for 2 hours at room temperature. Following three 10-minute washes in PBS, each antibody was incubated. Antibody-independent staining of nuclei and lipids was performed after immunostaining: DAPI and BODIPY (Thermo Fisher) were diluted in PBS containing 5% BSA and incubated with the tissue specimen for 20 minutes, followed by three washing steps as described above. The stained specimen was carefully placed on a No. 1.5 borosilicate glass chamber slide optimized for confocal imaging. A small amount of PBS prevented drying. The acquired images were analyzed as described above.

[0169] Co-staining of SA-β-Gal activity and ORF1 protein in liver sections was performed by first staining SA-β-Gal as described 111 The samples were then subjected to heat-induced epitope retrieval by steaming in antigen retrieval buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween 20, pH 9.0) for 20 minutes. The samples were then processed for immunofluorescence staining as described above (human tissue specimens).

[0170] OCT-preserved kidney tissue was cryosectioned, treated with 0.5% (w / v) periodic acid for 10 minutes, and then stained with Periodic Acid Schiff (PAS) reagent (Fisher Scientific, Cat. No. SS32-500) for 10 minutes. Stained tissue sections were mounted with Shandon Aqua Mount (Fisher Scientific, Cat. No. 14-390-5) and imaged under brightfield illumination. Glomerulosclerosis was scored as described. 112 Briefly, 40 glomeruli per animal were evaluated in a blinded manner and assigned a score of 1 to 4: score 1, <25% sclerosis; score 2, 25-50% sclerosis; score 3, 50-75% sclerosis; and score 4, >75% sclerosis. The features used to evaluate sclerosis were the intensity and spread of PAS-positive lesions within the glomerulus. As exemplified in Figure 4e, sclerotic glomeruli are more shrunken and stain more intensely with PAS.

[0171] Quadriceps muscles were embedded in OCT, sectioned at 12 μm thickness, and mounted on positively charged slides. Sections were stained with H&E (hematoxylin, 3 min, followed by eosin, 30 s). Mounted slides were imaged on a Zeiss Axiovert 200M microscope equipped with a Zeiss MRC5 color camera. To measure myofiber diameter, the shortest distance across approximately 100 myofibers per animal was measured using ImageJ software as described. 113 The Kolmogorov-Smirnov test was used to assess the statistical significance of the differences between the resulting distributions.

[0172] statistical processing Common statistical analyses (e.g., means, SDs, t-tests) were performed using Excel. R software (64-bit version 3.3.2) for statistical calculations was used for one-way analysis of variance and Tukey's multiple comparison post-hoc test. For consistency of comparisons, significance in all figures is indicated as follows: *P<0.05, **P<0.01. Sample size was based on previously published experiments and previous experience where differences were observed. No statistical tests were used to predetermine sample size. No samples were excluded. All replication attempts were successful. No findings were not replicated or could not be reproduced. The nature and number of samples (defined as n) analyzed in each experiment are listed in the figure legends. The number of independent experiments is also listed in the figure legends. Researchers were blinded when quantifying immunofluorescence results. Tissue areas or sections for quantification were randomly selected and scored using the methods indicated for each experiment. Researchers were also blinded when scoring glomerulosclerosis and myofiber diameter. For RNA-seq and PCR array experiments, statistical treatments are described in those sections (above).

[0173] Example 1 L1 and interferon activation in cellular senescence RTE activity can promote aberrant transcription, alternative splicing, insertional mutagenesis, DNA damage and genomic instability 114 RTE-derived sequences comprise up to two-thirds of the human genome. 115 Most were active millions of years ago and are no longer intact. The only human RTE capable of autonomous retrotransposition is long interspersed nuclear element type 1 (LINE-1, or L1). However, the germline activity of L1 is the major source of human structural polymorphism. 116 Increasing evidence indicates RTE activation in some cancers, the adult brain, and during aging. 117,118,119,120 Cellular defenses include heterochromatinization of elements, small RNA pathways that target transcripts, and antiviral innate immune mechanisms. 121Age-related somatic activation of RTE is conserved in yeast and Drosophila, and reduced RTE activity has beneficial effects. 122 .

[0174] As shown in Figure 1a and Figure 6a-e, L1 transcription is exponentially activated during replicative senescence (RS) in human fibroblasts and increased 4-5-fold by 16 weeks after growth arrest, a stage referred to as late senescence. Multiple RT-qPCR primers were designed to detect an evolutionarily recent L1 element (L1HS-L1PA5; Figure 1b, Figure 6h). L1 polyA+ RNA levels increased 4-5-fold in late senescent cells (RS) in the sense orientation rather than the antisense orientation throughout the element (Figure 1c). Sanger sequencing of long-range RT-PCR amplicons (Figure 1b) identified 224 elements distributed throughout the genome; one-third (75, 33.5%) were L1HS, of which 19 (25.3%, 8.5% of the total) were intact (i.e., annotated as lacking ORF-inactivating mutations; Figure 6f, g). 5′ RACE was also performed using the same primers and found that the majority of L1 transcripts were upregulated in senescent cells, initiated within or near the 5′ UTR ( Fig. 7 ).

[0175] L1 elements can stimulate IFN-I responses 123 As shown in Figures 1d and 6i, interferons IFN-α and IFN-β1 were induced at high levels in late senescent cells. Cellular senescence progresses through an early DNA damage response stage followed by the SASP response. 124 The present data demonstrate a third and even later phase characterized by L1 upregulation and IFN-I response (Figure 1e), which has not previously been mentioned, likely because most studies have focused on earlier stages. Whole-transcriptome RNA-seq analysis confirmed that the SASP and IFN-I response are temporally distinct (Figure 8). L1 activation and IFN-I induction were also observed in oncogene-induced senescence (OIS) and stress-induced premature senescence (SIPS) (Figure 1e, Figure 1j, k).

[0176] Example 2 Mechanism of L1 activation To investigate how surveillance fails during senescence, we investigated three factors: TREX1, RB1, and FOXA1. TREX1 is a 3' exonuclease that degrades foreign invasive DNA, and its loss is associated with the accumulation of cytoplasmic L1 cDNA. 125 As shown in Figure 9a, TREX1 expression was significantly reduced in senescent cells. RB1 has been shown to bind to repetitive elements, including L1, and promote their heterochromatinization. 126 As shown in Figure 2a, RB1 expression was strongly reduced in senescent cells (RS; ), whereas expression of other RB family members (RBL1, RBL2) remained unchanged (Figure 9b). RB1 enrichment in the 5'UTR of L1 elements was evident in proliferating cells, decreased with early senescence, and became undetectable at later time points (Figure 2a). This coincided with a decrease in H3K9me3 and H3K27me3 marks in these regions (Figure 9c).

[0177] To identify novel factors that interact with the L1 5'UTR, we searched the ENCODE ChIP-seq database and found that the pioneering transcription factor FOXA1 binds to this region in several cell lines (Fig. 9d). FOXA1 is upregulated in senescent cells. 127 As shown in Figure 2b, FOXA1 bound to the central region of the L1 5'UTR. Using a transcriptional reporter, we found that deletion of the FOXA1 binding site reduced both sense and antisense transcription from the L1 5'UTR. 128 (Figure 9e). Thus, the misregulation of these three factors observed in senescent cells may promote L1 activation through three additional mechanisms: loss of RB1 by relieving heterochromatin repression, gain of FOXA1 by activating the L1 promoter, and loss of TREX1 by impairing removal of the L1 cDNA.

[0178] Therefore, we used lentiviral vectors to test the effects of manipulating RB1, FOXA1, or TREX1 expression in fully senescent cells (Figure 10a, b). Ectopic expression of RB1 suppressed the elevated expression of L1, IFN-α, and IFN-β1 in senescent cells, whereas its knockdown further enhanced their expression (Figure 2d). Overexpression of RB1 also restored its occupancy at the L1 5'UTR (Figure 2c). Conversely, knockdown of FOXA1 reduced its binding to the L1 5'UTR (Figure 9f) and reduced the expression of L1, IFN-α, and IFN-β1, whereas overexpression of FOXA1 increased L1, IFN-α, and IFN-β1 levels (Figure 2e). Consistent results were obtained by manipulating TREX1 (Figure 2g). Thus, each of these factors had a distinct effect on regulating L1 and IFN-I responses in senescent cells.

[0179] Single or dual interventions targeting these factors induced only minor changes in L1 and IFN-I expression in proliferating early-passage cells. Although some of these effects were statistically significant, they were dwarfed by a triple intervention (3x) of RB1 and TREX1 knockdown combined with FOXA1 overexpression, resulting in massive induction of L1 and IFN-I expression (Figure 2f, Figure 9g-i, and Figure 10c). Thus, in normal healthy cells, all three effectors must be impaired to effectively unleash L1.

[0180] Example 3 Results of L1 activation To assess IFN-I activation by L1 in more detail, we examined the expression of 84 genes in this pathway using PCR arrays. We observed a broad response, with the majority of genes being upregulated (Figure 2h, Figure 9j, k): 68% (57 / 84) were significantly upregulated in senescent cells and 52% (44 / 84) were upregulated in 3X cells. These data validate and further extend our RNA-seq transcriptome analysis (Figure 8).

[0181] Several NRTIs developed against HIV have been shown to also inhibit L1 RT activity. 129 We also developed shRNAs against L1, two of which reduced transcript levels by 40–50% and 70–90% in late senescent and 3X cells, respectively (Figure 10g). ORF1 protein levels were correspondingly reduced in late senescent cells (Figure 5h). Finally, the shRNAs also reduced retrotransposition of a recombinant L1 reporter construct (Figure 10k).

[0182] Cells lacking TREX1 exhibit cytoplasmic L1 DNA, the accumulation of which can be inhibited with NRTIs 130 While a lack of BrdU incorporation is a standard feature of senescent cells (Fig. 6b), longer-term labeling revealed DNA that was primarily cytoplasmic and highly enriched in L1 sequences (Fig. 11a, b). Synthesis of cytoplasmic L1 DNA could be almost completely blocked with the NRTI lamivudine (3TC) or shRNA against L1 (Fig. 3a, c). An antibody against DNA:RNA hybrids detected a cytoplasmic signal in senescent cells that largely colocalized with ORF1 protein and converted to a ssDNA signal after RNase digestion (Fig. 11c). Analysis of BrdU-labeled L1 sequences in senescent cells showed that they were localized throughout the L1 element (Fig. 11d, e). A relative increase in L1HS sequences in total cellular DNA could also be detected by qPCR assays. 131,132 3TC in the range of 7.5–10 μM completely blocked this increase in senescent cells and also quenched the activity of the L1 retrotransposition reporter (Fig. 10d, e).

[0183] Knockdown of L1 with shRNA or treatment of cells with 3TC significantly reduced interferon levels and more broadly reduced the IFN-I response in both late senescent and 3X cells (Figure 3b, Figure 12a). 3TC in the 7.5–10 μM range optimally inhibited the IFN-I response and was the most effective of the four NRTIs tested (Figure 10f, j). The relative efficacy of the NRTIs is consistent with their ability to inhibit human L1 RT15. 3TC also antagonized the IFN-I response in other forms of senescence, OIS and SIPS (Figure 3e).

[0184] Cells were passaged from the proliferation phase to late senescence in the continuous presence of 3TC. 3TC did not significantly affect early SASP responses, such as the timing of senescence, the induction of p21 or p16, or the upregulation of IL-β (Figure 3f, Figure 12b). However, the magnitude of late SASP responses, such as the induction of CCL2, IL-6, and MMP3, was significantly attenuated. Treatment with L1 shRNA also reduced the expression levels of IL-6 and MMP3 in late senescent cells (Figure 11f). Thus, although L1 activation and the subsequent IFN-I response are relatively slow in onset, they importantly contribute to the mature SASP and proinflammatory phenotype of senescent cells.

[0185] 3TC did not affect L1 transcript levels (Fig. S10i), suggesting that the INF-I response is driven by the L1 cDNA, as predicted by this model, and not by the cytosolic DNA-sensing pathway components cGAS or STING. 133 Knockdown of inhibited the IFN-I response in both late senescent and 3X cells (Fig. 10l and Fig. 12c, d) and also downregulated the SASP response in late senescent cells (Fig. 12e).

[0186] NRTIs alkyl-modified at the 5' ribose position cannot be phosphorylated and therefore do not inhibit the RT enzyme. However, they have intrinsic anti-inflammatory activity by inhibiting P2X7-mediated events that activate the NLRP3 inflammasome pathway. 134Trimethoxy-3TC (K-9) at 10 μM or 100 μM did not inhibit the IFN-I response in either late-senescent or 3X cells (Fig. 12f). Thus, the effect of 3TC on the IFN-I pathway requires RT inhibition. At high concentrations (100 μM), K-9 had some inhibitory activity against inflammatory markers (Fig. 12g).

[0187] To test the role of interferon signaling in the SASP, we used CRISPR / Cas9 to inactivate IFN-α / β receptors (IFNAR1 and 2). Effective loss of IFN-I signaling was achieved in both early-passage and late-senescent cells (Figure 1M). In both replicative and SIPS senescence, loss of interferon signaling antagonized late SASP markers (CCL2, IL-6, MMP3) but not early SASP markers (IL-1β) (Figure 3D). This further demonstrates that IFN-I signaling contributes to the establishment of a complete and mature SASP response in senescent cells.

[0188] Example 4 Activation of L1 in human and mouse tissues Activation of L1 expression in human cancers is detected with an ORF1 antibody 135 The same reagent showed widespread ORF1 expression in both senescent and 3X cells (Fig. 8a, c, f). In skin biopsies from normal aged human individuals, we observed that 10.7% of dermal fibroblasts were positive for the senescence marker p16, which is within the range documented in aged primates. 136 (Fig. 13b, d, f, h). A portion of p16-positive skin fibroblasts was also positive for ORF1 (10.3%). Notably, we did not observe any ORF1 in the absence of p16 expression. We also detected the presence of phosphorylated STAT1 at the single-cell level, consistent with the presence of interferon signaling in the tissue microenvironment. 137 (Fig. 13b, e, g). Thus, a proportion of senescent cells in normal human individuals exhibit L1 activation, consistent with these events accumulating during the aging process.

[0189] We next examined mice and found that L1 mRNA was gradually upregulated with age in several tissues (Fig. 15g). The detected L1 RNA sequences were predominantly sense strands, represented throughout the entire element, and all three active L1 families were detectable (Fig. 11g, h). At the protein level, the frequency of L1 Orf1-positive cells increased in tissues with age (Fig. 4a). Areas of Orf1 staining colocalized with senescence-associated β-galactosidase (SA-β-Gal) activity (Fig. 4b). Several IFN-I-responsive genes (Ifn-α, Irf7, Oas1) and proinflammatory and SASP markers (Il-6, Mmp3, Pai1, also known as Serpine1) were upregulated in tissues from aged mice (Fig. 4c, Fig. 14). Increased L1 expression and IFN-I response genes (Ifn-α, Oas1) were also observed in a model of experimentally induced cellular senescence (young animals exposed to sublethally irradiated doses; Fig. 4d).

[0190] Aged animals (26 months) were treated with 3TC (administered in water at a human therapeutic dose) for 2 weeks. We observed widespread and significant downregulation of IFN-I responses and attenuation of the SASP proinflammatory state (Figure 4c; for the complete data set, see Figure 14 and Table 7). Expression of L1 mRNA and p16 was slightly downregulated, but in most cases did not reach statistical significance. K-9 had no effect on either IFN-I or SASP responses. Immunofluorescence analysis of tissue sections confirmed that senescent cells express SASP and that Orf1-expressing cells activate IFN-I signaling (Figures 15a-c). Treatment with 3TC significantly reduced both IFN-I and SASP but did not reduce L1 expression or the presence of senescent cells. Thus, NRTIs can be classified as "senostatic" agents, as opposed to "senolytic" treatments, which remove senescent cells from tissues. 138,139 .

[0191] adipogenesis 140 and heat generation 141The decrease in α, β, and β are hallmarks of natural aging, and both were increased in aged animals by 2 weeks of 3TC treatment (Figures 15d-f). As shown in Figure 4e, longer-term treatment (from 20 to 26 months of age) was effective in counteracting several known phenotypes of aging: (i) tissue macrophage infiltration, a hallmark of chronic inflammation; 142,143 (ii) glomerulosclerosis of the kidney 144 , and (iii) skeletal muscle atrophy 145 Macrophage infiltration of white fat was particularly responsive, returning to young (5-month-old) levels after just 2 weeks of 3TC.

[0192] Activation of endogenous L1 elements and subsequent robust activation of the IFN-I response is a novel phenotype of senescent cells, including naturally occurring senescent cells in tissues. This phenotype gradually evolves during the senescence response and appears to be an important, yet previously unrecognized, component of the SASP. We show that the expression of three regulatory factors, RB1, FOXA1, and TREX1, changes during senescence and that these changes are sufficient and necessary to enable L1 transcriptional activation (Figure 4g). Thus, multiple surveillance mechanisms must be overcome to unleash L1, highlighting the importance of keeping these elements in check in somatic cells.

[0193] Activation of innate immune signaling proceeds via the interferon-stimulated DNA (ISD) pathway in response to L1 activation during cellular senescence and aging. Cytoplasmic DNA is released from stressed mitochondria, resulting in mtDNA fragments. 146 or cytoplasmic chromatin fragments (CCFs) released from damaged nuclei 147,148 These results suggest that L1 cDNA is a key inducer of IFN-I in senescent cells. Notably, NRTI treatment not only effectively antagonized the IFN-I response but also more broadly reduced age-associated chronic inflammation in multiple tissues.

[0194] Sterile inflammation, also known as inflammaging, is a hallmark of aging and contributes to many age-related diseases. 149,150 The present data indicate that activation of L1 elements (and possibly other RTEs) promotes inflammaging and that L1 RTs are relevant targets for the treatment of age-related inflammation and disorders.

[0195] Example 5 Effect of Adefovir and Lamivudine on Senescence-Induced Increases in L1 Sequence Abundance, Interferon Gene Expression, and SASP Gene Expression The effects of adefovir and lamivudine on senescence-induced increases in L1 sequence abundance, interferon gene expression, and SASP gene expression were evaluated in human fibroblast cell lines.

[0196] The abundance (copy number) of L1 sequences was assessed in three different human fibroblast cell lines: LF1, IMR90, and WI38 using a qPCR assay. Assays were normalized to the abundance of 5S rDNA. Controls (CTRL) were untreated early-passage grown cells. Drugs were applied in the culture medium from several passages before senescence through senescence and then continuously through late senescence. "Senescence" samples were harvested 4 months after the onset of senescence. Both drugs were added to the culture medium at 5 μM. Red bars for "senescence" state: drug-free cultures at 4 months of senescence. As shown in Figure 17a, L1 copy number increased during senescence in all three cell lines, and both drugs significantly prevented this increase, with adefovir being somewhat more effective than lamivudine.

[0197] The effects of 5 μM adefovir and lamivudine on interferon gene expression were evaluated in two cell lines (LF1 and IMR90) and two interferon genes (IFN-α and IFN-β1) as performed in Figure 17a, except that expression of the indicated genes was measured by RT-qPCR. As shown in Figure 17b, high interferon gene expression in senescent cells was significantly reduced in all cases by both drugs, with adefovir again being somewhat more effective than lamivudine. The red bars represent drug-free cultures at 4 months of aging.

[0198] The effects of 5 μM adefovir and lamivudine on SASP gene expression were evaluated in one cell line (LF1) using two SASP genes (IL-6 and MMP3). CTRL was the expression in normal, untreated cells prior to senescence. As shown in Figure 17c, an increase in expression with senescence (red bars) was observed, and in both cases, SASP gene expression was significantly reduced with drug treatment, with adefovir being somewhat more effective than lamivudine.

[0199] Finally, the effects of high concentrations of lamivudine and emtricitabine (10 μM and 50 μM) on interferon gene expression (IFN-α and IFN-β1) were evaluated in the LF1 cell line. These were performed by subculturing LF1 cells to senescence, maintaining them in the senescent state for 3 months, adding the drugs, maintaining the cells in the presence of the drugs for 1 month, and then harvesting them at 4 months. Interferon gene expression was assessed by RT-qPCR. Controls (CTRL) were cells treated as above but without the drugs. As shown in Figure 17d, at 10 μM, lamivudine reduced IFN-I induction and was somewhat more effective than emtricitabine. At 50 μM, lamivudine actually induced an increase in interferon expression, even exceeding the levels seen in untreated cells. This increase is likely caused by the toxicity of the drugs at these high levels. In contrast, 50 μM emtricitabine reduced interferon expression, even less than the reduction observed at 10 μM.

[0200] Thus, at high doses, the toxicity of RTIs may impair their ability to halt or block the deleterious effects of senescent cells and to prevent or reverse age-associated inflammation and disorders.

[0201] Example 6 Comparative Evaluation of Several RTIs in a Dose-Response Assay for Inhibition of L1 Activity in Mouse and Human Cells Eight RTI compounds were evaluated for their ability to inhibit LINE-1 (L1) activity in a mouse L1 retrotransposition assay: lamivudine (3TC), stavudine, emtricitabine, apricitabine, tenofil disiproxyl, censavudine, elvucitabine, and tenofil. Three RTI compounds were evaluated in a human L1 retrotransposition assay: lamivudine (3TC), censavudine, and elvucitabine.

[0202] Mouse LINE-1 retrotransposition assay The plasmid pYX016 encoding dual luciferase containing mouse L1 elements was synthesized by Xie et al., 2011 151 The chemotherapeutic agents were described in [1]. Lamivudine (3TC), stavudine (d4T), emtricitabine, apricitabine, tenofovir disoproxil, and tenofovir were purchased from AK Science. Elvucitabine was obtained by custom synthesis. Sensavudine was synthesized by Oncolys BioPharma. HeLa cervical cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM)-high glucose containing 4500 mg / L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher) at 37°C in a humidified 5% CO2 incubator.

[0203] The assay was performed as described in Xie et al., 2011, with some modifications. 151The reporter assay was performed as described in. Reporter assays were performed in 96-well white optical-bottom plates. 6,000 HeLa cells were seeded into each well 24 h before transfection and compound treatment. All compounds were resuspended in DMSO. Stock concentrations varied from 50 mM to 1.25 mM depending on the compound's solubility. Serial dilutions (1:3) were prepared in DMSO. Ten different concentrations of each compound were tested in triplicate. Media containing different concentrations of compounds were prepared by adding 2 μL of compound dilution to 1 mL of medium. The final concentration of DMSO in the medium was 0.2%. Cells were transfected with the plasmid pYX016 using FuGENE® HD Transfection Reagent (Promega). The transfection mix was prepared in OpiMEM (Thermo Fisher) using a 3.5:1 reagent-to-DNA ratio according to the manufacturer's instructions. The medium was removed from the cells and discarded. The transfection mix (5 μL) was mixed with compound-containing medium (100 μL / well) and added to the cells in each well. The cells were incubated at 37°C / 5% CO for 48 hours.

[0204] Luciferase reporter activity was quantified using the Dual-Luciferase® Reporter Assay System (Promega) according to the manufacturer's instructions for multiwell plates, with the following modification: cells were lysed directly on the multiwell plate with 30 μL of passive lysis buffer (PLB) for 20 min at room temperature with gentle shaking to ensure complete cell lysis (instead of 20 μL of PLB for 15 min). Firefly and Renilla luciferase signals were measured using a SpectraMax i3x multimode microplate reader. Integration times of 100 ms and 10 ms were used to measure firefly and Renilla signals, respectively. Relative L1 activity was calculated as firefly / renilla * 10,000. Dose-response inhibition data were fitted to a four-parameter logistic equation using nonlinear regression (using Graphpad Prism 8) to determine the IC of each inhibitor. 50The values ​​were determined. The experiments were performed independently twice.

[0205] result The inhibitory dose-response curves of eight RTI compounds in two independent experiments are shown in Figures 18A and 18B, respectively. IC 50 The values ​​are summarized in Table 10. Surprisingly, two RTI compounds, censavudine and elvucitabine, had much lower IC than the other RTI compounds. 50 value (approximately 100 nM), thus demonstrating unexpected mouse L1 inhibitory activity.

[0206] Human LINE-1 retrotransposition assay Given their unexpected ability to inhibit mouse L1 activity, censavudine and elvucitabine were tested for their ability to inhibit human L1 activity. Lamivudine was also tested for comparison.

[0207] Assays for human L1 were performed in a manner very similar to the mouse assay. Human LINE-1 sequences 151 pYX017, encoding pYX016, was used instead of pYX016. Because the human construct produces a lower signal, compounds were incubated with cells for 72 hours instead of 48 hours, and cells were seeded at a density of 2000 cells / well instead of 6000 cells / well. Additionally, the transfection reagent to DNA ratio was 2:1 instead of 3.5:1.

[0208] result The inhibitory dose-response curves of the three RTI compounds are shown in Figure 19A-B, and the IC 50 The values ​​are summarized in Table 11. Again, ensavudine and elvucitabine were significantly higher than lamivudine (IC > 800 nM). 50 ) showed remarkable human L1 inhibitory activity (IC of approximately 100 nM) compared with that of 50 ) was shown.

[0209] Cell viability assay As mentioned above, the toxicity of RTIs may impair their ability to halt or block the harmful effects of senescent cells and prevent or reverse age-related inflammation and damage. The potential toxicity of lamivudine (3TC); stavudine; emtricitabine; apricitabine; tenofibrillar disiproxyl; censavudine; elvucitabine; and tenofibrillar was evaluated in cell viability assays at doses used to generate L1 inhibition dose-response curves.

[0210] HeLa cells were treated with eight RTI compounds at different concentrations for 48 hours. Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega®) and presented as a percentage of cell viability relative to untreated cells. Staurosporine, known to induce cell death, was used as a control.

[0211] result As shown in Figure 20, the eight RTI compounds did not induce significant levels of cell death, in contrast to the control staurosporine.

[0212] In summary, both censavudine and elvucitabine have the unexpected ability to inhibit mouse and human L1 activity (IC of approximately 100 nM) without inducing toxicity in cell viability assays at doses up to 2 μM. 50 ) was shown.

[0213] The tables described in this disclosure are provided below.

[0214] Table 1. List of primers used in PCR analysis TIFF0007728402000005.tif180135TIFF0007728402000006.tif188134TIFF0007728402000007.tif186133TIFF0007728402000008.tif188134TIFF0007728402000009.tif261331All sequences are listed in the 5'→3' direction. Primer sets 1-30 are specific for the listed human genes; primer sets 31-53 are specific for mouse. 2 All LINE-1 positions are relative to the L1Hs consensus sequence (Repbase, http: / / www.girinst.org / repbase / ). 3 See Coufal,NGet al.L1 retrotransposition in human neural progenitor cells.Nature 460,1127-31(2009). 4 See Gautier, G. et al. A type I interferon autocrine-paracrine loop is involved in Toll-like receptor-induced interleukin-12p70 secretion by dendritic cells. J. Exp. Med. 201, 1435-46 (2005).

[0215] Table 2: List of antibodies TIFF0007728402000010.tif961401 Rodic, N. et al. Long interspersed element-1 protein expression is a hallmark of many human cancers. See Am.J.Pahtol.184, 1280-6 (2014).

[0216] Table 3. List of expressed L1 elements identified by long-range RT-PCR. TIFF0007728402000011.tif218166TIFF0007728402000012.tif227166TIFF0007728402 000013.tif227166TIFF0007728402000014.tif225166TIFF0007728402000015.tif48144

[0217] Table 4. List of genes used in GSEA for IFN-I (50 genes) TIFF0007728402000016.tif180128

[0218] Table 5. GSEA analysis of KEGG pathways comparing early passage to early senescence TIFF0007728402000017.tif174128TIFF0007728402000018.tif170128TIFF0007728402000019.tif158128

[0219] Table 6. Summary of Qiagen PCR array analysis TIFF0007728402000020.tif391281 All percentages are calculated relative to the total number of genes found on the array (84). Data for all 84 genes displayed as a scatter plot are shown in Figure 2h. 2 The sum of up- and down-regulated genes passing a set of significance filters, see Methods for filter definitions. 3 Altered genes passing the significance filter that are unique to either SEN(L) or 3X cells. 4 Altered genes common to (found in) SEN(L) and 3X cells that pass the significance filter. 5 Altered genes found in SEN(L) and / or 3X cells that pass the significance filter. Expanded heatmap displays for this set of genes (67) are shown in Figure 4j, k.

[0220] (Table 7) TIFF0007728402000021.tif168130

[0221] Table 8: NRTIs approved as anti-HIV therapies TIFF0007728402000022.tif150166

[0222] Table 9. Lower dose (50%) NRTIs TIFF0007728402000023.tif150166

[0223] Table 10. Inhibition of mouse L1 activity TIFF0007728402000024.tif50142IC values ​​of nine compounds that inhibit the retrotransposition activity of active mouse LINE-1 in HeLa cells 50 Determination of retrotransposition activity. Retrotransposition activity was determined using the dual-luciferase pYX016 reporter. Cells were treated with different concentrations of each compound and simultaneously transfected with pYX016. Luminescence was measured 48 hours after transfection. Experiments were performed twice independently.

[0224] Table 11. Inhibition of human L1 activity TIFF0007728402000025.tif26142IC values ​​of three compounds that inhibit the retrotransposition activity of active human LINE-1 in HeLa cells 50 Determination of retrotransposition activity. Retrotransposition activity was determined using the dual-luciferase pYX017 reporter. Cells were treated with different concentrations of each compound and simultaneously transfected with pYX017. Luminescence was measured 72 hours after transfection. Experiments were performed twice independently.

[0225] References: TIFF0007728402000026.tif58162TIFF0007728402000027.tif228165TIFF0007728402000028.tif228165TIFF00077284020 00029.tif228165TIFF0007728402000030.tif228165TIFF0007728402000031.tif223165TIFF0007728402000032.tif85161

[0226] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0227] It should be understood that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the appended claims. The Summary and Abstract sections may describe one or more exemplary aspects of the invention contemplated by the inventor(s), but not all exemplary aspects, and therefore are not intended to limit the scope of the invention and appended claims in any way.

[0228] The foregoing description of specific embodiments sufficiently reveals the general nature of the present invention so that others, by applying knowledge within the skill of the art, can readily modify and / or adapt such specific embodiments for various uses without departing from the general concept of the present invention and without undue experimentation. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It should be understood that the phrases or terms used herein are for the purpose of description, not limitation, and should be interpreted by those of skill in the art in light of the teaching and guidance.

[0229] The scope and spirit of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0230] Sequence information SEQUENCE LISTING <110> BROWN UNIVERSITY <120> COMPOSITIONS AND METHODS FOR TREATING, PREVENTING OR REVERSING AGE-ASSOCIATED INFLAMMATION AND DISORDERS <150> US 62 / 907,251 <151> 2019-09-27 <150> US 62 / 797,109 <151> 2019-01-25 <160> 114 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 aaagtttctt atggccgggc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 2 gctgaacttg tggccgttta 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 3 aagacacatg cacacgtatg t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 4 aacaggagcg atgagtctgt a 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 gtgtatatca gcctcgtgtt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 6 gccaagatgg ccgaatagga 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 7 aaatcacccg tcttctgcgt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 cgagatcaaa ctgcaaggcg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 ccggccgctt tgtttaccta 20 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 taaacaaagc ggccgggaa 19 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 agaggtggag cctacagagg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 agagagcagt ggttctccca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 13 cagtctgccc gttctcagat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 14 acctgaaagt gacggggaga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 15 cctgccttgc tagattgggg 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 16 caaacaccgc atattctcac tca 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 17 cttcctgtgt ccatgtgatc tca 23 <210> 18 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic probe <400> 18 aggtgggaat tgaac 15 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 19 ctcgtctgat ctcggaagct aag 23 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 20 gcggtctccc atccaagtac 20 <210> 21 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic probe <400> 21 agggtcgggc ctgg 14 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 22 ttgaggtcaa tgaaggggtc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 23 gaaggtgaag gtcggagtca 20 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 24 ccaggagtga gtggaagaca g 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 ctagttgcct ccccaaagca 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 ttgatggcaa ccagttccag 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 27 tcatcccaag cagcagatga 20 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 tgttcccaag cagcagatga 20 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 acgccgcatt gaccatctat 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 30 gtctcattcc agccagtgct 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 31 actctcacct cccatgttgc 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 32 atccgtgcac tcctgttctg 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 33 ccaagaagcg ctctgctgta 20 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 34 gcaggggatt ctgcatcact a 21 <210> 35 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 35 agtcgcccac ccctcagat 19 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 36 ttccctccag cgtgtagctt 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 37 tccccttcgg atcttaacac 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 38 cgaaaaagat gagggtctgc 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 39 ggaagaccgg ccagctagag 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 40 tgaaggagta gtgggggtcc 20 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 41 agccttccaa ctctggagta atgt 24 <210> 42 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 42 ccgatgatct cccctgacaa 20 <210> 43 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 43 cccaccttac atacaggatt gtga 24 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 44 cccagacttt cagagctttc tca 23 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 45 cactggcaga aaacaacctg aa 22 <210> 46 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 46 accaggcaag tctcctcatt ga 22 <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 47 gtttttgaag agggctgaga attc 24 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 48 ccctacaaca gacccacaca atac 24 <210> 49 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 49 aagaccattg tggccaagga 20 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 50 ttcggagttt gggtttgct 19 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 51 agaatgggca gaaagcttgt ct 22 <210> 52 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 52 ccttctggtc agttggattt gc 22 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 53 cgccagtgaa atgatggctt at 22 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 54 ctggaaggag cacttcatct gt 22 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 55 caactgaggc cccctttcaa 20 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 56 cgcccgttgt agatgaaggt 20 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 57 ggctggaaaa ccaacttccg 20 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 58 gttatcccgc agcatcacga 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 59 tggagaccca aagggttgga 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 60 aggaagcagg aggtctcacc 20 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 61 acgtgctgtg aaaacaaaga ag 22 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 62 gtcccactga ctgtcttgag g 21 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 63 atatctgcgg ctgatcctgc 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 64 ggtctgctgg ggcagtttat 20 <210> 65 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 65 gagactctca gggtcgaaaa cg 22 <210> 66 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 66 ttcctgtggg cggattagg 19 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 67 cggaaggtcc ctcagacatc 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 68 ccctgtagga ccttcggtga 20 <210> 69 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 69 cggccgcatc ttcttgtg 18 <210> 70 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 70 gtgaccaggc gcccaata 18 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 71 ccaccaccct gctctgtact a 21 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 72 cctctccatg gtgcacttcc 20 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 73 cgtgaccttt gtgagcaacg 20 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 74 ctgctccata ctcgctctgg 20 <210> 75 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 75 tctgatgcag caggtggg 18 <210> 76 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 76 agggctctcc agacttctgc tctg 24 <210> 77 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 77 ctcctgagcg cagccttg 18 <210> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 78 gttcttactg ctggggccat 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 79 tctgctttat ggggcttcgg 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 80 tcgactccca tactcccagg 20 <210> 81 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 81 ctgccttgca agaagagagc 20 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 82 agtgctgcgt tctgatgatg 20 <210> 83 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 83 ccagggccgt gtgcat 16 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 84 tacgtgaacg ttgcccatca 20 <210> 85 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 85 gactcaaggg tggatgctgt 20 <210> 86 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 86 ccaactgcga agatccactg 20 <210> 87 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 87 cggagaggag acttcacaga gga 23 <210> 88 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 88 tttccacgat ttcccagaga aca 23 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 89 ggaagggcaa catgaccagg 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 90 agctgctctt ggtcggaaag 20 <210> 91 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 91 ctggctgcat ccattatgtc a 21 <210> 92 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 92 tggtagactg cccgtgtgaa 20 <210> 93 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 93 ctgcgtggct atgattatgg 20 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 94 aggttgctgt cgtctgtagt 20 <210> 95 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 95 acttttcctt aacgtgggcc t 21 <210> 96 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 96 catctcggcc agtgtctgtt 20 <210> 97 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 97 ttcgggtcgc tggatctcta 20 <210> 98 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 98 tcaaggagaa accaccacgg 20 <210> 99 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 99 agaagcgagc actgcaaggt tg 22 <210> 100 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 100 ggaagatgga ctccacctgg tt 22 <210> 101 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 101 ttcgctgatg cactgcctat 20 <210> 102 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 102 ggaatgcgag tggtcttcca 20 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 103 atctgtctcc caggtctgct 20 <210> 104 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 104 tcctccgttt acctttcgcc 20 <210> 105 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 105 gcttcggtga agtagctgga 20 <210> 106 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 106 ttcgttagag tcacgccgag 20 <210> 107 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 107 agccaaatgg atggacctgg 20 <210> 108 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 108 aaggaggggc atagtgtcca 20 <210> 109 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 109 atccggacca gaggacagg 19 <210> 110 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 110 atggcgaccg ctgctg 16 <210> 111 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 111 agagaaccgg acccaatcca 20 <210> 112 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 112 gcttgtgccc ctactcagac 20 <210> 113 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 113 tccccacggg atcctaagac 20 <210> 114 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 114 ctctgcaggc aagctctctt 20

Claims

1. A pharmaceutical composition for use in the treatment, prevention, and / or reversal of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), multiple sclerosis (MS), Aicardi-Goutieres syndrome, or progressive supranuclear palsy (PSP) in a patient in need thereof, comprising a therapeutically effective amount of censavudine and a pharmaceutically acceptable carrier, wherein about 1 μg / kg to about 100 mg / kg of censavudine is administered to the patient daily.

2. The pharmaceutical composition of claim 1, wherein about 1 mg / kg to about 50 mg / kg of censavudine is administered to the patient daily.

3. The pharmaceutical composition of claim 1, wherein the patient is administered about 5 mg / kg of censavudine per day.

4. The pharmaceutical composition of claim 1, wherein the patient is administered about 10 mg / kg of censavudine per day.

5. The pharmaceutical composition of claim 1, wherein the patient is administered about 15 mg / kg of censavudine per day.

6. The pharmaceutical composition of claim 1, wherein about 20 mg / kg of censavudine is administered to the patient daily.

7. The pharmaceutical composition of claim 1, wherein 1 mg to 500 mg of censavudine is administered to the patient once daily, twice daily, or three times daily.

8. The pharmaceutical composition of claim 1, wherein 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or 150 mg of sensavidine is administered to the patient once daily, twice daily, or three times daily.

9. The pharmaceutical composition of claim 1, wherein sensavidine is orally administered to the patient in the form of a tablet, capsule, or suspension.

10. A pharmaceutical composition according to any one of claims 1 to 9 for the treatment of Alzheimer's disease.

11. A pharmaceutical composition according to any one of claims 1 to 9 for the treatment of Parkinson's disease.

12. A pharmaceutical composition according to any one of claims 1 to 9 for the treatment of ALS.

13. A pharmaceutical composition according to any one of claims 1 to 9 for the treatment of FTD.

14. A pharmaceutical composition according to any one of claims 1 to 9 for the treatment of MS.

15. A pharmaceutical composition according to any one of claims 1 to 9 for the treatment of Aicardi-Goutieres syndrome.

16. A pharmaceutical composition according to any one of claims 1 to 9 for the treatment of PSP.

17. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein at least one second therapeutic agent is administered to said patient.

18. The pharmaceutical composition of claim 17, wherein the at least one second therapeutic agent comprises a reverse transcriptase inhibitor (RTI).

19. The pharmaceutical composition of claim 18, wherein the RTI comprises 4'-ethynyl-2-fluoro-2'-deoxyadenosine, abacavir, adefovir dipivoxil, adefovir, amdoxovir, apricitabine, dexelbucitabine, didanosine, elvucitabine, emtricitabine, entecavir, fosalvudine, lamivudine, rasvir, stampidine, stavudine, tenofovir alafenamide, tenofovir disoproxil fumarate, tenofovir disoproxil, zalcitabine, or zidovudine, or a combination thereof.

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